Journal of Water and Health Sample Issue by IWA Publishing

12.1

2014

Surface water isolates of hemolytic and non-hemolytic Acinetobacter with multiple drug and heavy metal resistance ability Sevilay Akbulut, Fadime Yilmaz and Bulent Icgen

ABSTRACT Acinetobacter in surface waters are a major concern because of their rapid development of resistance to a wide range of antimicrobials and their ability to persist in these waters for a very long time. Four surface water isolates of Acinetobacter having both multidrug- and multimetal-resistant ability were isolated and identified through biochemical tests and 16S rDNA sequencing. Based on these analyses, two hemolytic isolates were affiliated with Acinetobacter haemolyticus with an accession number of X81662. The other two non-hemolytic isolates were identified as Acinetobacter johnsonii and Acinetobacter calcoaceticus and affiliated with accession numbers of Z93440 and AJ888983, respectively. The antibiotic and heavy metal resistance profiles of the isolates were

Sevilay Akbulut Fadime Yilmaz Kırıkkale University, Department of Biology, 71450, Kırıkkale, Turkey Bulent Icgen (corresponding author) Middle East Technical University, Department of Environmental Engineering, 06800, Ankara, Turkey E-mail: bicgen@metu.edu.tr

determined by using 26 antibiotics and 17 heavy metals. Acinetobacter isolates displayed resistance to β-lactams, cephalosporins, aminoglycosides, and sulfonamides. The hemolytic isolates were found to show resistance to higher numbers of heavy metals than the non-hemolytic ones. Due to a possible health risk of these pathogenic bacteria, a need exists for an accurate assessment of their acquired resistance to multiple drugs and metals. Key words

| 16S rDNA, Acinetobacter species, multidrug-resistant, multimetal-resistant, river, surface waters

INTRODUCTION The control of hospital-acquired infection caused by

of multidrug resistance by these organisms highlights the

multiple-resistant Gram-negative bacilli has proved to be a

need to search for alternatives for chemotherapy (Doughari

particular problem during the past two decades. Among

et al. ). In the past, Acinetobacter spp. were considered

them, it is now well-recognized that Acinetobacter spp.

saprophytes of little clinical signiﬁcance (Bergogne-Berezin &

play a signiﬁcant role in the colonization and infection of

Towner ), but with the introduction of powerful new

patients admitted to hospitals. Acinetobacter spp. are a

antibiotics in clinical practice and agriculture and the use

major concern because of their rapid development of resist-

of invasive procedures in hospital intensive care units,

ance to a wide range of antimicrobials, ability to transform

drug-resistant hospital- and community-acquired Acineto-

rapidly, surviving desiccation, and persistence in the

bacter infections have emerged with increasing frequency

environment for a very long time. The organisms are associ-

(Doughari et al. ). A. baumannii is an important emer-

ated with bacteremia, pulmonary infections, meningitis,

ging

diarrhea, and nosocomial infections with mortality rates of

A. lwoﬁi and A. haemolyticus (Berlau et al. ; Jain &

20 to 60%. Transmission is via person-to-person contact,

Danziger ). This organism is a member of a group of

water and food contamination, and contaminated hospital

phenotypically similar species that are often grouped

equipment. The increasing virulence and rapid development

together in the A. baumannii–calcoaceticus complex

doi: 10.2166/wh.2013.304

nosocomial

pathogen

worldwide,

followed

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

(ABC). ABCs have emerged as healthcare-associated patho-

these settings can be a potential source for dissemination

gens, in part because they are resilient bacteria with a

and development of antimicrobial resistance in pathogenic

diverse natural habitat. Not only can they survive in moist

bacteria, which may ﬁnd their way back into the human

environments, but they can also survive for weeks on dry

population. Therefore, surface waters have been suggested

surfaces (Jawad et al. ; Doughari et al. ). Outbreak

to play a role in the dissemination and development of anti-

investigations have demonstrated that environmental con-

biotic and heavy metal resistance in these bacteria (Reva &

tamination with ABC can be widespread and serve as

Bezuidt ). The aim of this study was to characterize river

sources of infection (Scott et al. ).

isolates of Acinetobacter resistant to multiple drugs and

The use of drugs in human and animal healthcare has

heavy metals. Identiﬁcation of the isolates was done by

resulted in the widespread development of resistance not

using biochemical tests and 16S rDNA sequencing. After

only in humans and animals, but also in the environmental

determination of multiple antibiotic and metal resistance

reservoir. Data on the prevalence of multidrug- and heavy

proﬁles, the isolates were further characterized in order to

metal-resistant Acinetobacter in surface waters are necessary

ﬁnd out the locations of the resistance determinants.

to estimate the risk of these surface waters to humans. The risk of human exposure to multidrug-resistant bacteria outside a clinical setting is increasing (Ye et al. ; Hu et al.

MATERIALS AND METHODS

). Antibiotic-resistant bacteria excreted by humans and animals treated with antibiotics end up in the environment,

Sample collection, isolation, and puriﬁcation of

for instance with the discharge of untreated or partially trea-

Acinetobacter species

ted sewage or runoff of manure. Thus, the environment can be considered a collecting vessel of antibiotic-resistant bac-

Water samples were collected along the river Kızılırmak

teria and resistance genes from human and animal origin.

extending from 39 560 53.25″N, 33 250 04.24″E, 699.5 m

Moreover, water and soil may represent selective pressure

to 39 230 53.41″N, 33 250 18.44″E, 775 m of the city

through heavy metal polluted environments where resist-

Kırıkkale, Turkey. The samples were put into sterile screw-

ance genes can be transferred among bacteria from

capped bottles aseptically, kept in an icebox containing ice

different origins, among which are environmental bacterial

packs, and taken immediately to the laboratory. A quantity

species (Endo et al. ). This may result in the creation

of 1 mL of water from each of the collected samples was dis-

of novel combinations of bacterial species and speciﬁc anti-

solved in 9 mL sterile distilled water and serial dilutions

biotic and heavy metal resistance genes (Ozer et al. ;

were made. Each dilution was plated on Luria Bertani

Aktan et al. ; Koc et al. ). People may become

(LB) agar plates by the standard pour plate method. Plates

exposed to bacteria in surface waters and soils during recrea-

were incubated at 30 C for 3 days and colonies differing

tion in contaminated water, when drinking inadequately

in morphological characteristics were selected. After the

treated drinking water, water from unprotected sources, or

growth of different microorganisms on the plates, each

consuming fresh vegetables that have been either irrigated

bacterial colony on the basis of its morphological character-

with contaminated surface waters or grown on contami-

istics was selected and further puriﬁed by repeated streaking

nated soil (Noble et al. ). The view of the role of

on nutrient agar (NA) plates and identiﬁed with Gram stain-

introduced antibiotics and heavy metals, antimicrobial

ing. Each bacterial culture was then inoculated in nutrient

resistant bacteria and genes encoding resistance in nature

broth, incubated and glycerol stocks were made and

is changing. There is also evidence of a correlation between

frozen at 70 C. For isolation and puriﬁcation, strains

tolerance to heavy metals and antibiotic resistance (Wilson &

were routinely grown in LB medium at 30 C. The strains

Salyers ; Aktan et al. ; Koc et al. ; Ozer et al.

had biochemical properties that deﬁne the genus Acineto-

). Therefore research into how dispersal of antibiotics

bacter (Bouvet & Grimont ), with identiﬁcation at the

and heavy metals affect the bacterial community in nonclini-

genus level conﬁrmed by the transformation assay (Juni

cal settings is essential and urgent. The reason for this is that

). All suspected cultures of Acinetobacter species were

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

subjected to Gram stain and observed under a light micro-

Center for Biotechnology Information (NCBI) basic local

scope for size, shape, and cell arrangements. Presumptive

alignment search tools BLASTn program (Benson &

identiﬁcation was performed with API 20NE strips (Bio-

Karsch-Mizrachi ). A distance matrix was generated

merieux, France). Preparation of the strip was done

using the Jukes-Cantor corrected distance model. The phyloge-

following the standard procedure (Biomerieux, France).

netic trees were created using Weighbor (Weighted Neighbor

A. calcoaceticus ATCC 14987 was used as a positive control

Joining: A Likelihood-Based Approach to Distance-Based

and Pseudomonas aeruginosa ATCC 9027 was used as a

Phylogeny Reconstruction). The 16S rRNA gene sequences

negative control. The following tests were used for biotyping

have been deposited with GenBank using BankIt submission

as described previously (Gerner-Smidt et al. ): liquefac-

tool and have been assigned NCBI accession numbers.

tion of gelatin, hemolysis of sheep erythrocytes, production W

of urease, and growth at 37 and 44 C in Bacto brain heart

Determination of multimetal resistance

infusion broth (Difco). For the catalase test, pure cultures of the isolates were selected using a sterile loop from the

To determine multimetal resistance, NA plates sup-

agar slant and mixed with a drop of 3% H2O2 on a clean

plemented with heavy metal salts were used (Mergeay &

glass slide. The catalase-positive coccobacilli were con-

Nies ). Stocks of the metal salts were prepared in dis-

sidered as Acinetobacter.

tilled water, sterilized by ﬁlter membrane (0.22 μm), and W

stored at 4 C. Acinetobacter isolates were inoculated in Identiﬁcation of river isolates by 16S rDNA sequence analyses

radial streaks on NA media supplemented with increasing concentrations of each heavy metal salts AlCl36H2O, LiCl, BaCl22H2O, CrN3O99H2O, MnSO4H2O, Pb(NO3)2, Co-

Conﬁrmation of the taxonomical status of the selected

(NO3)26H2O, FeCl36H2O, Hg(NO3)2H2O, CuSO45H2O,

strains was done by molecular methods. Genomic DNA

SnCl22H2O, NiSO46H2O, ZnSO47H2O, K(SbO)C4H4O6-

was isolated and analyzed from Acinetobacter isolates by

0.5H2O, Cd(NO3)24H2O, Ag(NO3), and Sr(NO3)2 in

the method of Chen & Kuo (). Bacterial 16S rDNA

varying concentrations of 8 to 5,000 μg mL 1. For metal

was ampliﬁed by using the universal bacterial 16S rDNA pri-

resistance proﬁle, overnight-grown cultures of Acinetobacter

mers, F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and R (50 -

isolates were inoculated and incubated at 30 C. The cul-

GGTGTTTGATTGTTACGACTT-30 ). PCR was performed

tures were incubated for 24–48 h, and growth in each

with a 50 μL reaction mixture containing 1 μL (10 ng) of

concentration was recorded.

DNA extract as a template, each primer at a concentration of 5 mM, 25 mM MgCl2 and dNTPs at a concentration of

Determination of multi-antibiotic resistance

2 mM, as well as 1.5 U of Taq polymerase and buffer used as

recommended

the

manufacturer

(Fermentas, W

Germany). After the initial denaturation for 5 min at 94 C,

For antibiotic tolerance LB medium supplemented with 1.8% agar, solidiﬁed plates were utilized. Overnight-grown

cultures of Acinetobacter isolates were used for antibiotic

1 min, annealing at 55 C for 1 min, extension at 72 C for

resistance or susceptibility. A. calcoaceticus ATCC 14987

there were 35 cycles consisting of denaturation at 94 C for W

1 min, and ﬁnal extension at 72 C for 5 min. PCR was car-

was used as a reference strain. Disk diffusion method was

ried out in a gene Piko Thermal Cycler (Thermo Scientiﬁc,

used to check the resistance or sensitivity of bacterial strains

USA). The obtained PCR products were puriﬁed, using the

towards given antibiotics (Bauer & Kirby ). Acinetobacter

GeneJET™ PCR Puriﬁcation Kit (Fermentas, Germany),

isolates were incubated for 24–48 h at 30 C and the zone of

according to the instructions of the manufacturer, and

inhibition was measured in millimeters. Antibiotics disks

sequenced. The PCR product was sequenced by 3730 × 1

used in this study were amikacin (30 μg mL 1), amoxicillin/

DNA synthesizer (Applied Biosystems, USA). The two 16S

(30 μg mL 1),

ampicillin

(10 μg mL 1),

aztreonam

rRNA sequences were aligned and compared with other

(30 μg mL ), bacitracin (10 μg mL ), cefepime (5 μg mL 1),

16S rRNA genes in the GenBank by using the National

ceftazidime

(30 μg mL 1),

ciproﬂoxacin

(5 μg mL 1),

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

chloramphenicol (30 μg mL 1), erythromycin (15 μg mL 1),

electrophoresis, plasmid DNA bands were viewed by ﬂuor-

gentamicin (10 μg mL 1), imipenem (10 μg mL 1), netilmi-

escence of bound ethidium bromide under a short wave

(30 μg mL 1),

(1 μg mL 1),

peﬂoxacin

ultraviolet light trans illuminator and the photographs

(5 μg mL 1), penicillin (10 μg mL 1), piperacillin (100 μg

were taken using a digital camera. The DNA bands were

cin

oxacillin

mL ), piperacillin/tazobactam (100/10 μg mL ), rifampin

matched with those for Agrobacterium tumefaciens C58C1

(10 μg mL 1), sulbactam/cefoperazone (105 μg mL 1), tetra-

and

Lambda

DNA/EcoRI þ HindIII

digest

molecular

cycline (30 μg mL ), ticarcillin (75 μg mL ), ticarcillin/CA

weight markers. The approximate molecular weight of

(75/10 μg mL 1), trimeth-sulfa (25 μg mL 1), tobramycin

each plasmid was consequently obtained by extrapolation

(10 μg mL ), and vancomycin (30 μg mL ).

on graphical plots of molecular weight of markers against the distance traveled by the respective band.

Isolation of plasmid DNA Isolation of chromosomal DNA Plasmid extraction was carried out using the method described by Akinjogunla & Enabulele (). Pure isolates

Total DNA was isolated from all selected Acinetobacter

were inoculated on MRS broth and incubated. The grown

strains using a classical protocol for isolation (Chen &

cells were harvested and suspended in 200 μL of solution

Kuo ). 1.5 mL of a saturated culture was harvested

A (100 mM glucose, 50 mM Tris hydrochloride pH 8,

with centrifugation for 3 min at 12,000 rpm in a centrifuge

10 mM ethylenediaminetetraacetic acid (EDTA)) containing

(model 5415R; Eppendorf). The cell pellet was resuspended

10 mg of lysozyme per mL and 10 μg mL 1 mutanolysin and

and lysed in 200 μL of lysis buffer (40 mM Tris-acetate pH

incubated for 30 min at 37 C in an incubator. 400 μL of

7.8, 20 mM sodium-acetate, 1 mM EDTA, 1% sodium dode-

freshly prepared 1% sodium dodecyl sulfate in 0.2 N

cyl sulfate (SDS)) by vigorous pipetting. To remove most

NaOH was added and the samples were mixed by inverting

proteins and cell debris, 66 μL of 5 M NaCl solution was

tubes. 300 μL of a 30% potassium acetate solution (pH 4.8)

added and mixed well, and then the viscous mixture was cen-

was added and the samples were mixed by vortexing. After

trifuged at 12,000 rpm for 10 min at 4 C. After transferring the

incubating on ice for 5 min, the debris was removed by

clear supernatant into a new vial, an equal volume of chloro-

5 min at 14,000 rpm centrifugation in a centrifuge (model

form was added, and the tube was gently inverted at least 50

5415R; Eppendorf). The supernatant was removed and

times until a milky solution was completely formed. Following

extracted once with a phenol–chloroform mixture (1:1)

centrifugation at 12,000 rpm for 3 min, the extracted super-

and precipitated with an equal volume of isopropanol. The

natant was transferred to another vial and the DNA was

plasmid DNA was then dissolved in 100 μL of TE (Tris

precipitated with 100% ethyl alcohol, washed twice with

and EDTA) buffer. Electrophoresis of the DNA was carried

70% ethyl alcohol, dried in speed-vac, and redissolved

out on a 0.8% agarose gel in a 0.5X concentration of Tris-

in 50 μL TE buffer. RNA was removed by adding RNase in

Borate-EDTA (TBE) buffer. Agarose gel was prepared by

the lysis step for 30 min at 37 C. Chromosomal DNA was elec-

boiling 0.8 g of agarose powder in 100 mL of 0.5X TBE

trophoresed and separated on a 1.0% agarose gel. The gel was

buffer. After boiling, the solution was allowed to cool and

visualized under UV after staining with ethidium bromide.

10 μL of ethidium bromide was added to the cooled agarose solution. This was poured into a casting tray with a comb

Plasmid curing

placed across its rim to form wells. The gel was allowed to set for 30 min and the comb was removed. 20 μL of the plas-

Plasmid curing was carried out in order to determine the

mid DNA samples were then loaded into the wells after

location (plasmid-borne or chromosomal) of the drug resist-

mixing with 2 μL of bromophenol blue. A DNA molecular

ance marker(s). The curing (elimination) of the resistant

weight marker was also loaded into one of the wells. The

plasmids was done using a sub-inhibitory concentration

gel was thereafter electrophoresed in a horizontal tank at

of 0.10 mg mL 1 of acridine orange as described by Akinjo-

a constant voltage of 60 V for about 90 min. After

gunla & Enabulele (). Acinetobacter isolates were grown

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

for 24 h at 30 C in nutrient broth containing 0.10 mg mL 1

accession number of Z93440 and AJ888983, respectively.

acridine orange. After 24 h, the broth was agitated to hom-

The former non-hemolytic isolate showed 96%, and the

ogenize the contents and loop-fulls of the broth medium

latter non-hemolytic isolate showed 97% 16S rDNA

were then sub-cultured onto Mueller-Hinton agar plates

sequence homology with A. haemolyticus (Figures 1(b)

and antibiotic sensitivity testing and plasmid analysis were

and 1(c)). The methods used in this study yielded reliable

carried out as previously described.

results with 100% agreement in terms of the correct

classiﬁcation of Acinetobacter spp. Transformation Determination of multiple antibiotic and metal Competent cells for transformation were prepared essentially

resistance proﬁles

as described by Juni (). DNA concentration was detersaturating

Antibiotic-resistant Acinetobacter are a major public health

concentration of DNA in this transformation system is

concern since the bacteria can be easily circulated in the

approximately 10 μg of DNA mL 1. Competent cells (approxi-

environment. In the present study, the antibiotic resistance

mately 109 colony-forming units/mL in 0.9 mL of 0.1 M tris

proﬁles of Acinetobacter isolates showed that river isolates

hydroxymethyl aminomethane hydrochloride buffer at pH

were resistant to multiple antibiotics (Table 1). Resistances

7.0 containing 0.1 M CaCl2) were mixed with 0.1 mL of

to β-lactams, cephalosporins, aminoglycosides, and sulfona-

DNA. After 20 min of contact between cells and DNA at

mides were highly common. However, of the four isolates,

mined

absorbance

260 nm.

The

37 C, the mixture was centrifuged and the cells were sus-

only A. haemolyticus Zn01 did not show any resistance to

pended in tryptic soy broth (TSB) to the same volume.

β-lactams and only one of the non-hemolytic isolates

A. calcoaceticus Fe10 did not display resistance to aminoglycosides. On the other hand, both of these isolates were

RESULTS AND DISCUSSION

the only isolates resistant to quinolone-type antibiotic peﬂoxacin. All of the Acinetobacter spp. were found to be

Isolation and identiﬁcation of Acinetobacter species

resistant

sulfonamide-type

antibiotic

trimethoprim-

sulfamethoxazole. Treatment of Acinetobacter infections During this study, out of 27 bacterial isolates, only four

has conventionally involved the use of β-lactams, aminogly-

Gram-negative, non-motile, oxidase-negative, catalase-posi-

cosides, and quinolones (Bergogne-Berezin & Towner ).

tive, non-fermentative, and encapsulated coccobacilli were

However, the increased use of these antibiotics has resulted

screened as presumptive Acinetobacter spp. Of four isolates,

in the widespread emergence of antibiotic-resistant strains

the two strains identiﬁed as A. haemolyticus produced a

(Bergogne-Berezin & Towner ). Our study also

clear hemolysis on horse blood agar, and hydrolyzed gelatin.

conﬁrmed

These A. haemolyticus river isolates were designated as

Acinetobacter surface water isolates. Carbapenems, a class

Mn12 and Zn01. The two non-hemolytic isolates were ident-

of β-lactams with a broad spectrum of antibacterial activity,

iﬁed as A.johnsonii and A. calcoaceticus and designated as

have widely been used as the mainstay for treatment of

Sb01 and Fe10, respectively. Analysis of the 16S rDNA

infections caused by such antibiotic-resistant strains (Dijk-

sequences with the already available database also con-

shoorn et al. ). Unsurprisingly, Acinetobacter strains

ﬁrmed these results. Based on the phylogenetic analysis,

resistant to carbapenems have also rapidly emerged world-

the

presence

antibiotic

resistance

with

wide (Dijkshoorn et al. ). However, we did not ﬁnd

A. haemolyticus with an accession number of X81662.

any Acinetobacter isolates resistant to carbapenems. Differ-

the

strains

Mn12

and

Zn01

were

afﬁliated

Both A. haemolyticus isolates showed 98% 16S rDNA

ent levels and patterns of antimicrobial susceptibilities

sequence homology with A. lwofﬁi (Figure 1(a)). The two

have been found among different Acinetobacter spp., with

non-hemolytic isolates Sb01 and Fe10 were identiﬁed as

several studies reporting a higher occurrence of multidrug

A. johnsonii and A. calcoaceticus and afﬁliated with an

resistance in A. baumannii compared with the non-A.

S. Akbulut et al.

Figure 1

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

Phylogenic trees based on 16S rDNA gene sequence analyses of A. haemolyticus isolates Mn12 and Zn01 with an accession number of X81662 (a), A. johnsonii isolate Sb01 with an accession number of Z93440 (b), and A. calcoaceticus isolate Fe10 with an accession number of AJ888983 (c). The identiﬁed isolates and their afﬁliated numbers are shown in bold. The 16S rDNA sequences are aligned and used to construct the neighbor-joining phylogenetic tree. Scale bar indicates the genetic distance and the numbers shown next to each node indicate the bootstrap values from 1,000 replicons.

baumannii spp. (Van Looveren & Goossens ; Turton

co-trimoxazole, ciproﬂoxacin, and cefoperazone. Previously

et al. ). Furthermore, intra-species diversity of antimicro-

ampicillin, second-generation cephalosporins, quinolones,

bial susceptibilities has also been reported with speciﬁc

minocyline, colistin, amynoglycosides, impenim, sulbactam,

genotypes in the A. baumannii population demonstrating

and gentamicin were used to treat Acinetobacter infections.

higher resistance rates to antimicrobial agents compared

Resistance to these antibiotics has hindered therapeutic

with other A. baumannii genotypes (Van Looveren &

management, causing growing concern throughout the

Goossens ).

world (Doughari et al. ). A. baumannii has been devel-

The major problem with Acinetobacter spp. is their

oping resistance to all antibiotics used in treating

resistance to antibiotics (Landman et al. ). Savov et al.

infections. Currently, most A. baumannii strains are resist-

() reported that these organisms are most commonly

ant to aminoglycosides, tetracyclines, cephalosporins,

resistant

carbenicillin,

ampicillins, cefotaximes, chloramphenicols, gentamicins,

tetracycline,

and tobramycins (Prashanth & Badrinath ). The activity

gentamicin,

ampicillin, amikacin,

cephalothin,

chloramphenicol,

S. Akbulut et al.

Table 1

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

Antimicrobial resistance, plasmid and phylogenetic proﬁles of the Acinetobacter river isolates

Strain designation

16S rDNA Identiﬁcation and (percent homology)

*EMBL Access No.

Plasmid proﬁle (kb)

Mn12

Acinetobacter haemolyticus (99%) Acinetobacter lwofﬁi (98%) Acinetobacter schindleri (97%) Acinetobacter soli (96%) Acinetobacter baylyi (95%) Alkanindiges illinoisensis (94%) Acinetobacter towneri (93%) Perlucidibaca piscinae (91%) Enhydrobacter aerosaccus (91%) Moraxella caviae (90%)

X81662

Zn01

Sb01

Fe10

Antibiotic resistance proﬁle

–

Al, Li, Ba, Pb, Fe, Ag, Cu, Sn, Mn, Ni, Zn, Sb, Sr

ATM, CAZ, GEN, OXA, PIP, SXT, TZP

–

Al, Li, Ba, Mn, Ag, Cu, Sn, Ni, Zn, Sr

GEN, PEF, SXT

17, 42, 117

Li, Ba, Ag, Ni, Sb, Sr

ATM, BAC, OXA, SXT, TIM, TOB

–

CAZ, FEP, OXA, PEF, PEN, PIP, SXT, TZP

X81665 AJ278311 EU290155 AF509820 AF513979 AF509823 DQ664237 AJ550856 AF005187 X81662

Acinetobacter haemolyticus (99%) Acinetobacter lwofﬁi (98%) Acinetobacter schindleri (97%) Acinetobacter soli (96%) Acinetobacter tjernbergiae (95%) Alkanindiges illinoisensis (94%) Acinetobacter towneri (93%) Perlucidibaca piscinae (92%) Enhydrobacter aerosaccus (91%) Moraxella caviae (90%)

X81665 AJ278311 EU290155 AF509825 AF513979 AF509823 DQ664237 AJ550856 AF005187

Acinetobacter johnsonii (97%)

Z93440

Acinetobacter haemolyticus (96%) Acinetobacter gyllenbergii (96%) Acinetobacter lwofﬁi (96%) Acinetobacter schindleri (96%)

X81662

Acinetobacter calcoaceticus (99%) Acinetobacter haemolyticus (98%) Acinetobacter schindleri (97%) Acinetobacter parvus (96%) Acinetobacter tandoii (95%) Acinetobacter gerneri (94%) Acinetobacter towneri (93%) Perlucidibaca piscinae (92%) Moraxella catarrhalis (91%)

Metal resistance proﬁle

AJ293694 X81665 AJ278311 AJ888983 X81662 AJ278311 AJ293691 AF509830 AF509829 AF509823 DQ664237 AF005185

*European Molecular Biology Laboratory. Metals: aluminum, Al; antimony, Sb; barium, Ba; Copper, Cu; iron, Fe; silver, Ag; lead, Pb; lithium, Li; manganese, Mn; nickel, Ni; strontium, Sr; tin, Sn; and zinc, Zn. Antibiotics: aztreonam, ATM; bacitracin, BAC; ceftazidime, CAZ; gentamicin, GEN; oxacillin, OXA; peﬂoxacin, PEF; penicillin, PEN; piperacillin, PIP; piperacillin-tazobactam, TZP; ticarcillinclavulanic acid, TIM; tobramycin, TOB; and trimethoprim-sulfamethoxazole, SXT.

of carbapenems is further jeopardized by the emergence of

penicillin-binding proteins, low/decreased permeability of

enzymatic and membrane-based mechanisms of resistance

the outer membrane to antibiotics or increase in the active

(Peleg et al. ). Antimicrobial resistance among Acineto-

efﬂux of the antibiotics, target site mutations, and inacti-

bacter is either intrinsic or acquired via transformation.

vation via modifying enzymes have been reported (Jain &

Several

Danziger ). Mechanisms of resistance to antibiotics by

mechanisms

resistance

including

altered

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

Acinetobacter spp. vary with species, the type of antibiotics,

et al. ). Heavy metals are widespread in sewage as a

and geographical location (Jain & Danziger ). Thus,

consequence of industrial pollution and there has been con-

β-lactam antibiotics are inactivated by the production of

siderable speculation about possible genetic association

β-lactamases or alterations of penicillin-binding proteins

between bacterial tolerance for these metals and multiple

and decreased permeability of the outer membrane to

antibiotic resistance (Dhakephalkar & Chopade ). The

β-lactams (Poirel et al. ); cephalosporins by chromoso-

combined resistance to heavy metals was also reported by

mally encoded cephalosporinases, and occasionally by cell

Silver (), Enne et al. (), Ozer et al. (), Aktan

impermeability and aminoglycosides via aminoglycoside-

et al. (), and Koc et al. (). Many have speculated

modifying enzymes; quinolones by altering the target

and have even shown that a correlation exists between

enzymes DNA gyrase and topoisomerase IV through chro-

metal tolerance and antibiotic resistance in bacteria because

mosomal mutations, a decrease in permeability and

of the likelihood that resistance genes to both antibiotics

increase in the active efﬂux of the drug by the microbial

and heavy metals may be located closely together on the

cell (Bonomo & Szabo ). Treatment of Acinetobacter

same plasmid in bacteria and are thus more likely to be

infections should be individualized according to suscepti-

transferred together in the environment (Endo et al. ).

bility patterns as the carbapenems, some ﬂuoroquinolones

However, microorganisms may develop resistance at their

and doxycycline may retain activity.

source, where antibiotic concentrations might be higher,

Microorganisms resistant to antibiotics and tolerant to

or by acquisition of an antibiotic resistance gene was carried

metals appear as the result of exposure to metal contami-

on a genetic element and transferred to that organism via

nated environments which cause coincidental co-selection

exposure to a different chemical. For example, if a micro-

of resistance factors for antibiotics and heavy metals.

organism is in an environment contaminated with heavy

Heavy metal tolerance in the environment may contribute

metals, the organism may obtain a genetic element from

to the maintenance of antibiotic resistance genes by increas-

other organisms that survive because they carry a gene

ing the selective pressure of the environment. The river

responsible for resistance to those metals. If a gene respon-

isolates of Acinetobacter were also tested for their resistance

sible for antibiotic resistance was also present on the same

2þ

3þ

2þ

to heavy metals such as Al , Pb , Li , Ba , Cr , Mn ,

element, it too would be transferred to the organism.

Ag2þ, Co2þ, Fe2þ, Hg2þ, Cu2þ, Sn2þ, Ni2þ, Zn2þ, Sb2þ, Cd2þ, and Sr2þ at different concentrations from 8 to 5,000 μg mL 1.

Plasmid DNA proﬁling, curing, and transformation

The Acinetobacter isolates were found to have multiple metal resistance ability (Table 1). Resistance to multiple anti-

It is well known that antimicrobial resistance genes gener-

biotics resulted in a general tendency to be resistant to

ally reside on extrachromosomal DNA molecule like

multiple heavy metals except for the non-hemolytic isolate

plasmids. In order to ﬁnd out the resistance determinants,

A. calcoaceticus Fe10 which only showed single metal resist-

river isolates of Acinetobacter were screened for the pres-

ance to iron. The other non-hemolytic isolate, A. johnsonii

ence of plasmid DNA (Table 1). Our study showed that

Sb01, was found to be resistant to heavy metals like silver,

the isolates A. haemolyticus Mn12, Zn01, and A. calcoaceticus

lithium, barium, nickel, strontium, and antimony. Almost

Fe10 did not harbor any plasmid (Figure 2(a)). The multiple

all Acinetobacter isolates were found to be resistant to

drug and heavy metal-resistant genes of these isolates were

heavy metals like silver, lithium, barium, nickel, and stron-

found to be located on chromosomes. On the other hand,

tium. Both hemolytic isolates showed resistance to a

A. johnsonii isolate Sb01 contained three plasmids with

higher number of heavy metals than the non-hemolytic ones.

molecular weights of ca. 17, 42, and 117 kb (Figure 2(b)).

Although resistance phenotype determination is of para-

Cured derivatives of A. johnsonii Sb01 isolate became sus-

mount importance for clinical isolates, the tolerance to

ceptible to antimony, oxacillin, and bacitracin. The rest of

antimicrobial substances, even when these are below the

the resistant ability of the cured derivative was found to be

resistance/susceptibility breakpoints, may represent a selec-

retained. In order to conﬁrm these results the isolated

tive advantage for the organism in the environment (Faria

plasmids were transformed into antimony, oxacillin,

S. Akbulut et al.

Figure 2

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

Journal of Water and Health

12.1

2014

Plasmid proﬁles of A. haemolyticus isolates Mn12 (lane 1), Zn01 (lane 2), A. calcoaceticus isolate Fe10 (lane 3) (a), A. johnsonii isolate Sb01 (lane 4) (b), and transformant E. coli DH5α (lane 5) (c). M1, Lambda DNA/EcoRI þ HindIII marker; M2, Agrobacterium tumefaciens C58C1 marker.

bacitracin-sensitive, plasmid-free and kanamycin-resistant

important antibiotic-resistant genes. Several factors are

derivatives of Escherichia coli DH5α by the calcium chlor-

believed to contribute to the success of plasmid-encoded

ide method (Figure 2(c)). The transformation frequency of

antibiotic resistance as an evolutionary mechanism. The

E. coli DH5α was calculated to be approximately 2.1 ×

presence of antibiotic-resistant genes on plasmids, which

10 5 per recipient. Transformant E. coli DH5α isolates car-

are normally nonessential for the survival of the organism,

rying 117 kb plasmid were able to grow on LB medium

provides the bacterial population with a means to reduce

containing kanamycin, antimony, oxacillin, and bacitracin.

the genetic and physiological load on the majority of cells

The transformant E. coli DH5α isolates were found to be

while, through the carriage of plasmids, a minority of

susceptible to the rest of the antimicrobials used in the

cells are able to maintain the genetic diversity of the popu-

study. On the basis of these observations, we concluded

lation (Miranda et al. ). Plasmid-borne genes can

that antimony, oxacillin, and bacitracin resistance ability

undergo more radical evolutionary changes without affect-

of river isolates of A. johnsonii Sb01 was linked and

ing the viability of the cell, as would changes to

determined by 117 kb plasmid. The rest of the multiple

indispensable chromosomal genes, and established plas-

resistance ability of this isolate was thought to be

mid transfer mechanisms can provide recipient cells with

chromosome-encoded.

new genetic material which has already been reﬁned by

The success of bacterial pathogens in the environment

selective pressures elsewhere (Lyon & Skurray ). Plas-

is driven by their ability to adapt, spread, and establish eco-

mids can, however, contribute to the development of

logical reservoirs (Hanssen & Ericson Sollid ). An

chromosomal resistance in two ways. First, plasmids,

important determinant of this adaptation is the acquisition

either in part or in toto, can integrate into the bacterial

of genes that confer resistance, or increase already existing

chromosome. In the case of plasmids from Gram-negative

resistance, to antibiotics and heavy metals (Hanssen &

bacteria, this may involve short segments of DNA, termed

Ericson Sollid ; Aktan et al. ; Koc et al. ;

insertion sequences (IS), which reside on both plasmid

Ozer et al. ). Despite the recognized pressures in select-

and chromosome and provide limited regions of DNA

ing for antibiotic resistance in environmental pathogens,

sequence homology for recombination. Second, plasmids,

there is little understanding of the role of the natural

together with bacteriophages, can act as vectors for trans-

environment as a reservoir of Acinetobacter and other

posable DNA elements or transposons. Antimicrobial

potentially

resistance genes carried by transposons can be translocated

pathogenic

bacteria

that

might

harbor

S. Akbulut et al.

Surface water isolates of hemolytic and non-hemolytic Acinetobacter

either from one plasmid to another or from a plasmid to a

Journal of Water and Health

12.1

2014

CONCLUSIONS

chromosomal site, irrespective of extensive genetic homology. Moreover, events involving transposons can occur

The presence of antibiotic and heavy metal-resistant bacteria

independently of the host’s general recombination (rec)

in surface waters used for recreation may pose a health risk.

system by a mechanism(s) termed site-speciﬁc recombina-

Data on the prevalence of multidrug- and heavy metal-resist-

tion. Such genes may therefore become established in

ant Acinetobacter in surface waters are necessary to estimate

diverse species in which the vector molecules themselves

the risk of these surface waters to humans. Our study

cannot replicate. In addition to facilitating the spread of

revealed two hemolytic and two non-hemolytic surface

antimicrobial resistance, the clustering of transposons car-

water isolates. The antibiotic and heavy metal resistance

rying different resistance determinants on plasmids, or the

proﬁles of these isolates displayed resistance to β-lactams,

chromosome, provides an explanation for the emergence of

cephalosporins,

aminoglycosides,

and

sulfonamides.

multi-resistant bacteria (Lyon & Skurray ). Heavy

Almost all Acinetobacter isolates were found to be resistant

metal resistance genes are often found in pathogenicity

to heavy metals like silver, lithium, barium, nickel, and

genomic islands (PAIs) of different highly virulent microor-

strontium. Acinetobacter spp. are a major concern because

ganisms (Kamachi et al. ; Levings et al. ; Reith

of their rapid development of resistance to a wide range of

et al. ; Durante-Mangoni & Zarrilli ; Reva &

drugs and heavy metals. The emergence of multidrug and

Bezuidt ). The distribution of heavy metal resistance

multimetal resistance ability of Acinetobacter spp. also

operons might be associated with mobile genomic regions.

plays a crucial role in their in vitro and in vivo survival.

The role of the heavy metal-resistant operons in pathogen-

The antimicrobial selective pressure causes conversion of

icity remains unclear, but their prevalence in PAIs suggests

the sensitive species, either by mutation or by induced

that they may be important factors which pathogens may

expression of resistance elements. These bacteria end up

use for alternative functions such as transport and detoxiﬁ-

in surface water through the discharge of untreated or par-

cation of antibiotics and other detrimental compounds.

tially treated sewage mainly derived from industrial

Furthermore, the roles of mercury resistance genes in bac-

factories, healthcare centers, farms, slaughterhouses, and

terial resistance toward clinical disinfectants have also

wastewater treatment plants. Importantly, once in the

been reported (Han et al. ). In relation to the latter,

environment, bacteria of different origin come into physical

an acquired antiseptic and disinfectant resistance of A.

contact and may exchange resistance genes with the

baumannii was associated with the arsenic and mercury

endogenous bacterial populations. Despite the generally

resistance operons (Durante-Mangoni & Zarrilli )

believed negative impact of acquired resistance on ﬁtness

which could possibly be of plasmid origin. Plasmids may

of the bacteria, the multidrug- and multimetal-resistant

exchange transposable elements with the chromosomes

Acinetobacter spp. may remain present in the environment

or sometimes the whole plasmid may be integrated into a

for a long time and pose a health risk, and need to be

bacterial genome. Having been ﬁxed on the chromosome,

more accurately assessed.

the mobile element undergoes amelioration, a process that smoothes out differences in oligonucleotide usage patterns of the host chromosome and that of the acquired element (Lawrence & Ochman ). These bring new virulence genes or simply activate the gene exchange between potentially pathogenic microorganisms that eventually lead to the achievement of effective combination of pathogenicity determinants in new virulence genomic islands (Bezuidt et al. ). This might explain the widespread

multiple

drug-

and

heavy

Acinetobacter isolates in surface waters.

metal-resistant

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First received 26 May 2013; accepted in revised form 8 August 2013. Available online 30 September 2013

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2014

Efﬁciency of peracetic acid in inactivating bacteria, viruses, and spores in water determined with ATP bioluminescence, quantitative PCR, and culture-based methods Eunyoung Park, Cheonghoon Lee, Michael Bisesi and Jiyoung Lee

ABSTRACT The disinfection efficiency of peracetic acid (PAA) was investigated on three microbial types using three different methods (filtration-based ATP (adenosine-triphosphate) bioluminescence, quantitative polymerase chain reaction (qPCR), culture-based method). Fecal indicator bacteria (Enterococcus faecium), virus indicator (male-specific (Fþ) coliphages (coliphages)), and protozoa disinfection surrogate (Bacillus subtilis spores (spores)) were tested. The mode of action for spore disinfection was visualized using scanning electron microscopy. The results indicated that PAA concentrations of 5 ppm (contact time: 5 min), 50 ppm (10 min), and 3,000 ppm (5 min) were needed to achieve 3-log reduction of E. faecium, coliphages, and spores, respectively. Scanning electron microscopy observation showed that PAA targets the external layers of spores. The lower reduction

Eunyoung Park Cheonghoon Lee Michael Bisesi Jiyoung Lee (corresponding author) Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH, USA E-mail: lee.3598@osu.edu Jiyoung Lee Department of Food Science and Technology, The Ohio State University, Columbus, OH, USA

rates of tested microbes measured with qPCR suggest that qPCR may overestimate the surviving microbes. Collectively, PAA showed broad disinfection efﬁciency (susceptibility: E. faecium > coliphages > spores). For E. faecium and spores, ATP bioluminescence was substantially faster (∼5 min) than culture-based method (>24 h) and qPCR (2–3 h). This study suggests PAA as an effective alternative to inactivate broad types of microbial contaminants in water. Together with the use of rapid detection methods, this approach can be useful for urgent situations when timely response is needed for ensuring water quality. Key words

| Bacillus subtilis spores, Enterococcus, ﬁltration-based ATP bioluminescence, male-speciﬁc (Fþ) coliphages, peracetic acid

INTRODUCTION Effective control of pathogenic microbes in water, including

that are used for sources of drinking water. The tainted drink-

bacteria, viruses, and protozoa, has been an important issue

ing water sources increase risk of disease transmission and

for providing safe water to the public. In 2004, the World

likelihood of infection (Centers for Disease Control and Pre-

Health Organization (WHO) estimated that diarrhea associ-

vention (CDC) ). For example, after Hurricane Katrina in

ated with waterborne infectious microorganisms accounted

New Orleans in 2005, approximately 1,000 cases of gastroin-

for nearly 4.1% of the global burden of disease in disability-

testinal illness were reported, such as diarrhea and vomiting.

adjusted life years and 1.8 millions of deaths (WHO ).

In order to reduce the risk to public health from

In emergency situations, such as ﬂoods and earthquakes,

exposure to pathogens in contaminated drinking water

runoff waters often pick up large quantities of pathogenic

during emergencies, the United States Environmental Pro-

microorganisms from farms, sewage, and waste waters and

tection Agency (USEPA) and CDC have recommended

subsequently contaminate groundwater and surface water

boiling and chemical treatment for emergency water

doi: 10.2166/wh.2013.002

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

disinfection (USEPA ). The most commonly used

three different microbial types: bacteria, virus, and protozoa.

chemicals are chlorine and its derivatives, which can inacti-

The reaction of PAA on Enterococcus faecium was tested to

vate many bacteria, including enteric bacteria (De Luca

determine its effectiveness as a fecal indicator of microbial

et al. ). However, chlorination shows relatively low

water quality (USEPA ). Bacillus subtilis spores were

effectiveness against viruses, protozoan cysts, and bacterial

used as a surrogate for protozoa (Driedger et al. ; Radzi-

spores. These microbes are considerably associated with

minski et al. ; Cho et al. ) and coliphage MS2 as a

health risks and are known for low susceptibility to chlori-

representative surrogate of viral contamination (USEPA

nation (Veschetti et al. ; Koivunen & Heinonen-

). The second aim was to compare PAA disinfection efﬁ-

Tanski a; Zanetti et al. ). In addition, chlorination

ciency using three detection methods for the evaluation of

has the potential for forming carcinogenic disinfection by-

which method would be more accurate, faster and practical

products (DBPs) especially when the water contains high

in emergency situations when timely results are required. A

levels of organic matter (Kitis ; Crebelli et al. ).

ﬁltration-based ATP bioluminescence assay (Lee & Deinin-

Therefore, alternative disinfectants have been proposed for

ger ), quantitative polymerase chain reaction (qPCR),

effective inactivation of pathogens, assuring a practical

and conventional culture-based methods were conducted

application while minimizing the formation of DBPs.

using E. faecium and B. subtilis spores. For male-speciﬁc

Peracetic acid (PAA) may be a good alternative disinfec-

(Fþ) coliphage quantiﬁcation, double agar layer method

tant since it is a strong oxidant and has been approved by

(DAL) (USEPA ) and reverse transcription (RT)-qPCR

the Food and Drug Administration (FDA) in the USA for

were conducted. Additionally, the effect of PAA on B. sub-

various applications, including food, beverage, medical,

tilis spores was examined using a scanning electron

and pharmaceutical industries (Kitis ; Koivunen & Hei-

microscopy (SEM) to visualize the mode of disinfection.

nonen-Tanski b). Several studies have shown that it inactivates pathogenic bacteria, such as Vibrio cholerae (Kitis ) and indicator microorganisms in both the

METHODS

absence and presence of organic matter (Koivunen & Heinonen-Tanski b; Zanetti et al. ). It has also been

Microorganism preparation

reported that PAA produces no or low amounts of DBPs and chemical residues compared to chlorination (Kitis

E. faecium (ATCC 19434), B. subtilis (ATCC 6051), and

; Koivunen & Heinonen-Tanski a; Crebelli et al.

male-speciﬁc (Fþ) coliphage (MS2, ATCC 15597-B1) were

) because PAA is easily decomposed into non-toxic

purchased from American Type Culture Collection (ATTC,

compounds, such as acetic acid and oxygen (Kitis ; Cre-

Manassas, VA, USA).

belli et al. ). Owing to these advantages, PAA has been

A 100 μL aliquot of E. faecium was added to 100 mL of

used for wastewater disinfection in England, Finland, Italy,

tryptic soy broth supplemented with 0.6% yeast extract

Brazil and Canada (Kitis ). In the USA, the USEPA

(TSBYE; Becton, Dickinson and Company, Sparks, MD,

also included PAA as one of the recommended disinfectants

USA). This was cultured at 37 C by shaking in an Incushaker

for treating combined sewer overﬂows (USEPA ). How-

(Benchmark, Ontario, Canada) for 16–24 h. The concen-

ever, there are limited studies that investigate its disinfection

tration of cultured E. faecium was determined by serial

efﬁciency against various microbial types (bacteria, viruses,

dilution with phosphate-buffered saline (PBS; Fisher Scienti-

and protozoa) since they may have different susceptibility

ﬁc, Fair Lawn, NJ, USA) and subsequent pour plates on

to PAA (Monarca et al. ; Bailey et al. ). Furthermore,

tryptic soy agar (TSA), which were incubated for 16–24 h.

no information is available about the use of PAA for drink-

To prepare the Bacillus spores, B. subtilis was initially

ing water disinfection as an alternative for the emergency

grown on TSA supplemented with 0.6% (w/v) yeast extract

situations requiring timely results of PAA effectiveness.

(TSAYE) at 30 C for 24 h. A 100 μL aliquot of overnight culW

The purpose of this study was two-fold. The ﬁrst aim was

ture was then cultured for 10–14 days on plates of TSAYE,

to investigate the disinfection efﬁciency of PAA against

which contained 10 mg L 1 of MnSO4 (Baker Chemical

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

Co., Phillipsburg, NJ, USA) at 30 C for sporulation (Ratphi-

determined according to previous studies and CDC’s rec-

tagsanti et al. ). The spores were harvested after 95% of

ommendation (Crebelli et al. ; Majcher et al. ;

the population was sporulated, which was veriﬁed with the

Rutala et al. ): 5 ppm for E. faecium; 500 ppm and

Schaeffer-Fulton stain method (Harley & Prescott ) and

3,000 ppm for B. subtilis spores; and 50 ppm for male-

phase-contrast microscopy. The surfaces of the inoculated

speciﬁc (Fþ) coliphage. Thirty-ﬁve percent of PAA was

plates were ﬂooded with 10 mL of cold, sterile deionized

used to make 5, 50, 500, and 3,000 ppm (ﬁnal concen-

water and scraped with sterile disposable plastic spreaders.

tration) by dilution for each contamination event. The

Spores were harvested and washed using sterile deionized

responses of microorganisms against PAA disinfection treat-

water. The washing was performed by centrifugation at

ments were monitored over a 1-h contact time.

9,700 × g for 15 min at 4 C. After repeated washing (at W

least four times), spore pellets were resuspended in sterile

PAA disinfection

deionized water and sonicated for 10 min at room temperature to prevent spore clumping. Heat treatment was carried

E. faecium

out at 80 C for 15 min. Any remaining vegetative cells were removed via 0.1% lysozyme (MP Biomedicals, LLC., Solon,

To generate bacterial contamination in water, a 10 mL aliquot

OH, USA) treatment, which was carried out by shaking at a

of cultured E. faecium suspension was spiked into 990 mL of

ratio of 1:1 for 1 h at room temperature. The suspended

10% PBS and was then stirred gently with a magnetic bar. The

spores were then centrifuged at 5,800 × g for 10 min and

initial level of E. faecium in this solution was designated as

washed with sterile deionized water. This step was repeated

0 min sample. 0.0143 g L 1 of PAA was added to the E. fae-

three times. Spore concentration was determined by the

cium-contaminated solution and the ﬁnal concentration of

TSA plate count method and calculated as colony forming

PAA was 5 mg L 1 (5 ppm). The 10 mL PAA-treated samples

units (CFU) mL 1.

were collected after each contact time of 5, 10, 15, 20, 25, 30,

Male-speciﬁc (Fþ) coliphage was propagated from the

and 60 min, respectively. Sodium thiosulfate (10%; Fisher

ATCC stock according to the manufacturer’s instructions.

Scientiﬁc, Fair Lawn, NJ, USA) was added to stop PAA

Its concentration was determined by USEPA 1601 DAL

activity (Majcher et al. ). The levels of bacteria at 0 min

using Escherichia coli HS (pFamp) as a host (USEPA ).

and that of surviving bacteria after each contact time were

Ten-fold dilutions of Fþ coliphage stock were prepared

enumerated with ATP bioluminescence, plate count method,

using TSB. Each tube of molten 0.7% TSA top agar (with

and qPCR explained below, respectively.

host bacteria) was inoculated with 0.5 mL of each serial dilution of Fþ coliphage stock. Each tube was then poured

B. subtilis spores

into a 1.5% TSA bottom agar plate containing 1.5 mg L 1 of ampicillin sodium sulfate (Fisher BioReagents, Fair

To generate water contamination events, a 10 mL aliquot of

Lawn, NJ, USA) and 1.5 mg L 1 of streptomycin (Sigma,

cultured B. subtilis spore suspension was spiked into

St Louis, MO, USA). After incubation at 36 C for 16–24 h,

990 mL of distilled water and was then stirred gently with a

visible plaques were counted and viral concentration was

magnetic bar. 1.43 g L 1 and 8.58 g L 1 of PAA (35%) were

calculated as plaque forming units (PFU) mL 1 (USEPA

added into spore-contaminated water to make ﬁnal concen-

). All cultured E. faecium (1.0 × 109 CFU mL 1), B. sub-

trations of 0.5 g L 1 (500 ppm) and 3 g L 1 (3,000 ppm),

tilis spores (2.3 × 1010 CFU mL 1), and Fþ coliphages (1.0 ×

respectively. The initial level of B. subtilis spores was desig-

108 PFU mL 1) were stored at 80 C until use.

nated as 0 min sample. The PAA-treated water samples

were collected at the contact times of 5, 15, 30, and 60 min. Determination of PAA disinfection concentration

Sodium thiosulfate (10%) was added to stop PAA activity. The levels of initial bacteria and surviving bacteria after

The working range of PAA (35%, FMC, Philadelphia, PA,

each contact time were enumerated with ATP biolumines-

USA) concentration and contact time for this study was

cence, plate count method, and qPCR, respectively.

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

Male-speciﬁc (Fþ) coliphage

B. subtilis spores

To generate water contamination events, a 10 mL aliquot

Heat shock treatment was carried out ﬁrst to induce Bacillus

of Fþ coliphage suspension was spiked into 990 mL of

spores in water to germinate (Lee & Deininger ). This

10% PBS and was then stirred gently with a magnetic

step is essential because the heat shock transforms inert

bar. 0.143 g L 1 of PAA (35%) was inoculated into

spores into vegetative cells and their ATP can be readily

coliphage-contaminated water. The ﬁnal concentration

detected by luminescence assay. Lee & Deininger ()

became 0.05 g L 1 (50 ppm). The initial level of Fþ coli-

reported the fastest test conditions (germination time, temp-

phages in this solution was measured using the DAL

erature, and nutrient concentration) that showed a

method and qPCR, respectively. This sample was desig-

signiﬁcantly high signal. This method was used in the current

nated as 0 min sample. The PAA-treated water samples

study because it detects spores sufﬁciently in a short time

were collected at the contact times of 5, 15, 30, and

(within 5 min) even though there was variability in germina-

60 min. Sodium thiosulfate (10%) was added to stop

tion levels. First, 50 μL of preheated (37 C) sterile TSBYE

PAA activity. The levels of surviving bacteria after each

was added into the Filtravette as a nutrient source to trigger

contact time were enumerated with the DAL method

spore germination. The Filtravette contained concentrated

and qPCR, respectively.

spores from each sample. Each Filtravette was incubated at

37 C for 15 min, which provided the optimal condition to Determination of PAA disinfection efﬁciency using

generate the highest bioluminescence signal (Lee & Deinin-

ﬁltration-based ATP bioluminescence

ger ). After the germination step, gentle air pressure was used to ﬁlter out the liquid. At this step, the germinated

E. faecium

vegetative cells remain on the surface of the Filtravette. Next, ATP bioluminescence assay was performed as

The ﬁltration-based ATP bioluminescence was performed

described above. The differences in the RLU signal measured

using Proﬁle®-1 ATP method (New Horizons Diagnostics,

before and after the spore germination process indicated the

Columbia, MD, USA) (Lee & Deininger ). Fifty

amount of ATP that originated from the Bacillus spores pre-

microliters of sample aliquots were concentrated by ﬁl-

sent in the sample (Lee & Deininger ).

tration on a Filtravette

(New Horizons Diagnostics), a

combination of a ﬁlter and a cuvette with a 0.45 μm

Determination of PAA disinfection efﬁciency using plate

pore size. After concentration, 50 μL of somatic cell-

count method

releasing agent was added to the Filtravette to remove non-bacterial cells and adjust pH for optimizing the down-

E. faecium and B. subtilis spores

stream luminescence reaction. Next, 50 μL of bacterial cell-releasing agent (BRA) was applied for 1 min to lyse

The numbers of E. faecium and B. subtilis in the samples

the concentrated bacterial cells. The extracted ATP was

were enumerated by counting colonies grown on R2A agar

then mixed with 50 μL of luciferin/luciferase (LL). Light

(Becton, Dickinson and Company, Sparks, MD, USA)

development was initiated at this point. The integrated

(Lee & Deininger ) and TSA (Becton, Dickinson and

light signal was measured using a Microluminometer

Company), respectively. After incubation at 37 C for

(Model 3550i; New Horizons Diagnostics, Columbia,

16–24 h, colonies were counted and calculated as CFU mL 1.

MD, USA). ATP luminescence was expressed in a relative luminescence unit (RLU), which is an indicator of the

Fþ coliphage

total ATP released from the bacteria present in the sample. The activity of LL was checked with serially

The numbers of coliphages were determined using a DAL

diluted ATP standard solution (Sigma). Sterilized distilled

method (USEPA ) as described above. TSB without anti-

water was used as a negative control.

biotics was used as a diluent and a negative control.

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

Determination of PAA disinfection efﬁciency using

CT values versus log CFU. The concentrations of E. faecium

RT-qPCR

and B. subtilis spores were calculated by using these standard curves.

E. faecium and B. subtilis spores Fþ coliphage DNA was extracted from 200 μL of each sample using a DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA, USA)

Coliphage RNA (50 μL) was extracted from each sample

according to the manufacturer’s instructions and then sus-

using RNeasy Mini Kit (Qiagen) according to the manufac-

pended in 50 μL of elution buffer. The eluates were used

turer’s instructions. The eluates were used immediately or

immediately or stored at 80 C until further processing.

stored at 80 C until further processing. RT was per-

Real-time quantitative PCR (qPCR) assay was carried out

formed according to the manufacturer’s instructions

with a StepOne

Real-Time System (Applied Biosystems,

(Promega, Madison, WI, USA) with minor modiﬁcations.

Foster City, CA, USA) in a 48-well format. For E. faecium

Brieﬂy, 7.5 μL of extracted viral RNA was mixed with

real-time PCR, the reaction mix (20 μL) consisted of 5 μL of

100 nM of reverse primer in a reaction tube and RNase-

DNA template, 10 μL of TaqMan® PCR Master Mix (Applied

free water was added up to 8.5 μL. The mixture was

Biosystems, Foster City, CA, USA), 500 nM of each forward

heated at 70 C for 5 min and then immediately cooled

and reverse primer, and 250 nM of probe (Ludwig & Schleifer

on ice. The reaction mixture (11.5 μL) consisted of 4 μL

). The sequences for primers and probes were as follows:

of 5× reaction buffer (Promega), 1 μL of 10 mM dNTPs,

50 -AGAAATTCCAAACGAACTTG-30 for forward primer,

40 U of RNase Inhibitor (Invitrogen, Carlsbad, CA, USA),

50 -CAGTGC TCTACCTCCATCATT-30 for reverse primer

and 100 U of M-MLV reverse transcriptase (Promega).

and 50 -6-carboxyﬂuorescein (FAM)-TGGTTCTCTCCGAAA-

This RT mixture was incubated for 1 h at 42 C, then for

TAGCTTT AGGGCTA-minor groove binding (MGB)-30 for probe (Ludwig & Schleifer ). Thermal cycling consisted W

of a holding stage (95 C for 10 min) and a cycling stage (40

5 min at 95 C to produce cDNA. Real-time qPCR was performed as described by Ogorzaly & Gantzer () with a StepOne Real-Time System (Applied Biosystems)

cycles at 95 C for 15 s and 60 C for 1 min) (Ludwig & Schlei-

in a 48-well format. Brieﬂy, 5 μL of cDNA was added to

fer ). For spore real-time qPCR, the reaction mix (20 μL)

45 μL of reaction mixture containing 25 μL of TaqMan

consisted of 5 μL of DNA template, 10 μL of SYBR® Green

Universal PCR Master Mix (Applied Biosystems), 250 nM

PCR Master Mix (Applied Biosystems), and 500 nM of each for-

of each forward and reverse primer, and 750 nM of

ward and reverse primer (50 -CAGCCTTTGGGCAGGAAGA-

probe. Each primer pair and probe is as follows: 50 -

30 for forward primer and 50 -AGGACGCGCCTAAATCGA-30

TCAGTGGTCCATACCTTAGATGC-30

for reverse primer (Ratphitagsanti et al. ). Thermal cycling

primer, 50 -ACCCCGTTAGCGAAGFFGCT-30 for reverse

consisted of a holding stage (95 C for 10 min) and a cycling W

for

forward

primer and 50 -FAM-CTCGTCGACATGG-minor groove binding

ampliﬁcation, melting curve analysis was performed by heating

probe (Ogorzaly & Gantzer ). qPCR was performed

non-ﬂuorescent

quencher

(MGBNFQ)-30

stage (40 cycles at 95 C for 15 s and 60 C for 1 min). After

for

samples to 95 C for 15 s, cooling to 60 C for 1 min, and then

in duplicate. Thermal cycling consisted of a holding stage

heating the samples at 1.0 C s 1 to 95 C. Real-time qPCR

(50 C for 2 min and 95 C for 10 min) and a cycling stage

was performed in triplicate for each sample. A negative control

(50 cycles at 95 C for 15 s and 60 C for 1 min). The CT

(distilled water) and a positive control (puriﬁed DNA of E. fae-

values of the qPCR results were determined using auto

cium or B. subtilis) were included in each run. The threshold

threshold. Ten-fold serial dilutions (101–1010 PFU per reac-

cycle (CT) values of the real-time qPCR results were determined

tion) of coliphage RNA were used for preparation of their

using auto threshold. Ten-fold serial dilutions (10 –10 CFU

standard curves. RT-qPCR was performed in triplicate to

per reaction) of E. faecium or B. subtilis DNA were used to pre-

make standard curves by plotting the CT values versus log

pare their standard curves. Real-time qPCR assays were

PFU. The coliphage RNA concentration was calculated

performed in triplicate to make standard curves by plotting

using these standard curves.

E. Park et al.

Disinfection efﬁciency of peracetic acid

Observation of PAA effect on B. subtilis spores using SEM

Journal of Water and Health

12.1

2014

treatment: 4-log reductions within 1 h. The greatest reduction (2 log) occurred within the ﬁrst 5 min of contact time when

Round glass cover slips (5 mm; Electron Microscopy

measured with ATP bioluminescence assay. The plate count

Sciences, Hatﬁeld, PA, USA) were prepared by immersing

method showed that PAA treatment reduced the E. faecium

in the order of 10% sodium dodecyl sulfate, 99% ethanol,

level by 5 log within 5 min. The level of reduction remained

and nano pure water (18.2 MΩ cm, Thermo scientiﬁc Barn-

constant over 1 h of contact time after the ﬁrst 5-min

stead Nanopure , Dubuque, IA, USA). Each sample in the

treatment. The reduction rate of E. faecium determined

volume of 2.5 μL was loaded on each cover slip and then

with the plate count method was always higher than that

dried under pure nitrogen gas purging. The cover slip with

determined with ATP bioluminescence assay. On the other

a sample was mounted onto a metal stub with double-

hand, qPCR showed that PAA treatment resulted in only

sided carbon tape, and then coated with a thin layer of

1-log reduction over 1-h contact time (Figure 1). Thus,

gold (30 nm) using a gold sputter coater (Emitech K550X;

bacterial reduction rates measured with qPCR assay were

Emitech, Ashford, Kent, UK). Images of the samples were

always lower than those determined with ATP assay or

taken using a SEM (Hitachi S-4300, Japan). Untreated

plate count method.

samples (control) and those treated with PAA were selected for comparison.

PAA treatment of contaminated water with B. subtilis spores

RESULTS

A ﬁltration-based ATP bioluminescence with heat shock method showed that B. subtilis spores were reduced by 2 log

PAA treatment of contaminated water with E. faecium

with 500 ppm of PAA within 5 min (Figure 2), whereas 3,000 ppm of PAA reduced the spores by 4 log within 5 min

The reduction trend of E. faecium by the action of PAA over

(Figure 3). The plate count method showed that the spores

the 1-h contact time is summarized in Figure 1. Interestingly,

were reduced by 1.5 log within the ﬁrst 5 min of contact time

the reduction rates differed depending on the detection

when 500 ppm of PAA was used (Figure 2). Filtration-based

methods. Based on the ﬁltration-based ATP bioluminescence,

ATP bioluminescence with heat shock method showed 3-log

E. faecium was signiﬁcantly reduced with 5 ppm PAA

reduction of B. subtilis spores under the same PAA treatment

Figure 1

Concentration of the surviving E. faecium measured with three methods when treated with PAA (5 ppm) during 1-h contact time: ATP bioluminescence (log [RLU] mL 1), plate count (log [CFU] mL 1), qPCR (log [CFU] mL 1).

Figure 2

Concentration of the surviving B. subtilis spores measured with three methods when treated with PAA (500 ppm) during 1-h contact time: ATP bioluminescence (log [RLU] mL 1), plate count (log [CFU] mL 1), qPCR (log [CFU] mL 1).

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

Figure 4

12.1

2014

Concentration of the surviving male-speciﬁc (Fþ) coliphages measured with two methods when treated with PAA (50 ppm) during 1-h contact time: DAL (log [PFU] mL 1), RT-qPCR (log [PFU] mL 1).

Figure 3

the difference in morphology between untreated spores and Concentration of the surviving B. subtilis spores measured with three methods when treated with PAA (3,000 ppm) during 1-h contact time: ATP biolumi-

those treated with PAA (500 and 3,000 ppm) and the exter-

nescence (log [RLU] mL 1), plate count (log [CFU] mL 1), qPCR (log [CFU] mL 1).

nal layers of B. subtilis spores were damaged (Figures 5(b) and 5(c)). More severe damage on the layers was observed

conditions within 1 h of contact time (Figure 2). When spores

via SEM when the spores were treated with higher concen-

were disinfected with 3,000 ppm of PAA, both ATP biolumi-

trations of PAA (3,000 ppm; Figure 5(c)).

nescence with heat shock and plate count methods showed similar spore reduction rates (∼4 log) (Figure 3). As shown in the E. faecium results, however, both 500 and 3,000 ppm of

DISCUSSION

PPA resulted in only 1-log reduction over 1-h contact time when measured with qPCR (Figures 2 and 3).

Disinfectants are usually regarded as effective when they can reduce the targeted organism by at least 3 log (99.9%)

PAA treatment of contaminated water with

in their numbers. PAA has been demonstrated to inactivate

Fþ coliphage

the indicator organism E. coli with such high disinfection efﬁciency (Koivunen & Heinonen-Tanski a). However,

For measurement of coliphages, two methods (DAL and

the variability of microbial resistance against disinfectants

qPCR) were used because coliphages do not contain ATP,

and the lack of signiﬁcant correlation between the indicator

therefore ATP bioluminescence cannot be used. The DAL

organism, E. coli and other pathogenic microorganisms

method shows that the 50 ppm PAA treatment inactivated

have suggested the importance of assessing disinfectant efﬁ-

Fþ coliphages by ∼2.5 log within 5 min and reduced up to 4

ciency by using more than one indicator organism (Gehr

log over 1 h of contact time (Figure 4). Their RNA seemed to

et al. ; Koivunen & Heinonen-Tanski a, b; Zanetti

remain intact during the 30-min contact time, although both

et al. ). In this study, we evaluated PAA disinfection

culturable coliphages and their genetic materials were reduced

efﬁciency using three different microbial types (E. faecium,

similarly (∼4-log reduction) over the 1-h treatment (Figure 4).

B. subtilis spores, and male-speciﬁc (Fþ) coliphage). E. faecium has been used as one of the recommended

B. subtilis spores observed by SEM

indicators for bacterial water quality in fresh and marine water by the USEPA for almost three decades because of

B. subtilis spores affected by PAA were observed by SEM to

its association with gastrointestinal disease (USEPA ).

determine the mechanism of PAA on spores. Figure 5 shows

As shown in Figure 1, its reduction rates against PAA over

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

residual PAA after 25 min had been measured. However, it was speculated that it was not necessary because more than 3-log reduction of E. faecium was already achieved, which was conﬁrmed with the plate count method. A ﬁltration-based

ATP

bioluminescence

has

shown

the

potential to detect only viable cells in water samples (Lee & Deininger ). This can possibly explain why the reduction rate measured by a filtration-based ATP bioluminescence was lower than that of the plate count method. Bacterial cells may be injured by PAA, but still maintain viability during 1-h contact time even though they were unable to develop colonies on culture media. It has been reported that disinfectants can cause reversible damage on bacteria, and injured bacteria can survive in stressful environments by entering viable but non-culturable (VBNC) state (Colwell ; Oliver ). In VBNC state, bacteria are no longer culturable on conventional culture media, but the cells have been shown to display active metabolism, such as respiration, gene transcription, and membrane integrity (Lleò et al. ). Furthermore, it has been reported that pathogenic bacteria in VBNC status may retain pathogenic factors/genes, which can make them a reservoir of infectious disease transmission (Colwell ; Lleò et al. ; Camper ; Oliver ; Keep et al. ). Therefore, it would be essential to find an appropriate method to detect not only those culturable, but also those which have entered VBNC state. However, it was not clear whether these VBNC bacteria were accurately detected with the filtration-based ATP bioluminescence in our experiments. To confirm this, additional tests, such as Live/Dead kit (Lleò et al. ) and flow cytometry (Oliver ), are recommended. Our results indicated that filtration-based ATP bioluminescence may detect the VBNC population which could not be detected by plate count method. This method has limitations because it does not provide information on the identification Figure 5

SEM images of B. subtilis spores: (a) without PAA treatment (1-μm scale bar); (b) after the treatment with PAA at 500 ppm (5-μm scale bar); (c) after the

of bacteria present in water samples. However, it can be

treatment with PAA at 3,000 ppm (2.5-μm scale bar).

used as a rapid and practical evaluation tool by providing more accurate numbers of viable bacteria on site, especially

time could be calculated differently depending on the detec-

for emergency situations (Deininger & Lee ). The plate

tion methods. The concentrations of E. faecium did not

count method has been widely used because of its low

decrease any further after 25 min. It was possible that

cost, simplicity of methodology, and positive correlation

most of the PAA had been consumed during the bacterial

with health risks (Camper ). However, as shown in

cell oxidations and reached its reaction capacity after

Figure 1, the underestimated rate of surviving microbes

25 min. To conﬁrm this, it would have been ideal if the

measured by plate count method may raise a question

E. Park et al.

Disinfection efﬁciency of peracetic acid

Journal of Water and Health

12.1

2014

whether this method can sufﬁciently safeguard public health

The reduction rates of spores measured by the three

(Colwell ; Lleò et al. ; Keep et al. ). In addition,

methods varied depending on PAA concentration. Plate

conventional culture-based methods usually take 1–2 days

count method and ﬁltration-based ATP bioluminescence

or sometimes 7 days depending on species or media types

showed different spore reduction rates when 500 ppm of

(Camper ). qPCR analysis showed that PAA could

PAA was used, whereas both methods showed similar spore

only achieve 1-log reduction (Figure 1). PAA is thought to

reduction rates against 3,000 ppm of PAA (Figures 2 and 3).

act by releasing active oxygen or producing oxidized sulfhy-

Lower reduction rates of spores measured with the plate

dryl bonds, which attack bacterial cells, disrupt cell walls

count method compared to the ATP assay (500 ppm PAA)

and membranes, and target enzymes and the base of the bac-

indicated that PAA could harm spores to some extent at the

terial DNA (Kitis ; Koivunen & Heinonen-Tanski

concentration of 500 ppm. However, it did not completely

a). Therefore, since qPCR assay detects the target

inactivate the spores. In contrast, similar reduction rates

DNA sequence, regardless of their intact cell structure or

were observed with both methods when 3,000 ppm of PAA

viability, it is obvious that qPCR may overestimate the true

was used. These results indicated that this high amount of

survival rates of E. faecium, indicating that qPCR may not

PAA had totally inactivated the spores. CDC recommends

be an accurate assessment tool in this disinfection case.

that concentrations ranging from 500 ppm to 10,000 ppm

B. subtilis spores have been used as a surrogate to assess

PAA are enough to decontaminate bacterial spores in health

the disinfection efﬁciency of Cryptosporidium oocysts or

settings (Rutala et al. ). Based on our analysis, we could

Giardia cysts during water treatment processes (Facile

suggest that 500 ppm PAA may not be sufﬁcient and that a

et al. ; Bitton ). There are three main advantages

higher dose is needed to ensure a safe level of disinfection.

of using B. subtilis spores as a surrogate: their lack of patho-

Male-speciﬁc (Fþ) coliphage is a surrogate for human viral

genicity on humans, easy handling of culture, and

contamination in ground water proposed by the USEPA

straightforward quantiﬁcation (Dow et al. ). It was

(USEPA ). Because viral infection may be the causative

found that B. subtilis spores were very resistant to PAA dis-

agent of approximately 50% of all acute gastrointestinal ill-

infection, especially compared to E. faecium (Figures 2 and

nesses, it is useful to assess the disinfection efﬁciency of PAA

3). Three-log reductions were achieved with the concen-

against Fþ coliphage (USEPA ; Bitton ; Jiang et al.

tration (C ) × time (t) value of 15,000 mg × min L

for the

). Our study showed that coliphages were more resistant

was needed for the

than enterococci (Figures 1 and 4). Approximately 3- or 4-log

3-log reduction of E. faecium (Figures 1 and 3). It is not sur-

reduction of coliphages was achieved with 50 ppm PAA,

prising that spores are very much resistant to chemicals

which was 10 times higher than the amount of PAA required

(Kitis ; Setlow ). The main factors of disinfection

to achieve the same degree of E. faecium reductions. This

resistance are: (1) spore coat proteins that likely react with

observation agrees well with previous ﬁndings (Kitis ; Koi-

and detoxify chemical agents; (2) low impermeability of

vunen & Heinonen-Tanski b; Zanetti et al. ). It seems

the spore’s inner membrane; (3) the protection of spore

that PAA had a greater effect on the viability of coliphages than

spores whereas only 50 mg × min L

DNA by its saturation with α/β-type small acid-soluble

on their RNA at the beginning of contact time (15 min), but as

spore proteins; and (4) DNA repair for agents that eliminate

the time went by (60 min), it also affected the RNA of coli-

spores by damaging their DNA (Setlow ). SEM images

phages because the reduction of coliphages was measured

clearly showed empty spore shells when treated with PAA

similarly (∼4-log reduction) over the 1-h contact time with

while untreated spores remained intact (Figure 5(a)). The

both methods (Figure 4).

damage to the external layers of spores was more severe as the concentration of PAA increased, indicating its increased sporicidal activity (Figures 5(b) and 5(c)). This ob-

CONCLUSIONS

servation implied that PAA appears to cause damage to the external layers of spores, which is consistent with previous

The results from this study show that PAA has a good poten-

studies (Kitis ; Setlow ; Rutala et al. ).

tial to inactivate microbial contaminants in urgent situations

E. Park et al.

Disinfection efﬁciency of peracetic acid

within a reasonably short contact time. Its disinfection efﬁciency differed based on the type of targeted microbes. Bacteria are the most susceptible to the action of PAA, followed by viruses and spores. The results also support that ﬁltration-based ATP bioluminescence is capable of detecting bacterial cells rapidly (∼5 min) and is a more reliable method than the other two methods. SEM observation showed that PAA targeted the external layers of spores. Our study also suggests that PAA may be applied as a disinfectant in ground water contaminated with sewage, and treatment of municipal wastewater prior to discharge into receiving water bodies. Further studies with different types of water (e.g., wastewater, surface water) and mixed microbial groups are warranted to expand the applicability of PAA.

ACKNOWLEDGEMENT We thank Dr Y. Jeong at the Ohio State University for his help in getting the images using SEM.

REFERENCES Bailey, M. M., Copper, W. J. & Grant, S. B.  In situ disinfection of sewage contaminated shallow groundwater: A feasibility study. Water Res. 45 (17), 5641–5653. Bitton, G.  Microbial Indicators of Fecal Contamination: Application to Microbial Source Tracking. Report submitted to the Florida Stormwater Association, pp. 1–71. Available at: www.ﬂoridastormwater.org/Files/FSA%20Educational% 20Foundation/Research/Pathogens/FSAMicrobial SourceTrackingReport.pdf (accessed 5 April 2012). Camper, A. K.  History and use of heterotrophic plate counts in water system. In: Advances in Bioﬁlm Science and Engineering (R. Jordan, D. Willams & U. Charaf, eds). Cytergy Publishing, Bozeman, MT, USA, pp. 8–15. CDC (Centers for Disease Control and Prevention)  Guidance on Microbial Contamination in Previously Flooded Outdoor Areas, CDC. Environmental Health Service Branch. National Center for Environmental Health. Atlanta, GA. Available at: www.cdc.gov/nceh/ehs/Publications/ Guidance_Flooding.htm (accessed 20 May 2012). Cho, M., Chung, H. & Yoon, J.  Quantitative evaluation of the synergistic sequential inactivation of Bacillus subtilis spores with ozone followed by chlorine. Environ. Sci. Technol. 37 (10), 2134–2138.

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Colwell, R. R.  Viable but nonculturable bacteria: a survival strategy. J. Infect. Chemother. 6 (2), 121–125. Crebelli, R., Conti, L., Monarca, S., Feretti, D., Zerbini, I., Zani, C., Veschetti, E., Cutilli, D. & Ottaviani, M.  Genotoxicity of the disinfection by-products resulting from peracetic acid- or hypochlorite-disinfected sewage wastewater. Water Res. 39 (26), 1105–1113. Deininger, R. A. & Lee, J.  Rapid determination of bacteria in drinking water using an ATP assay. Field Anal. Chem. Tech. 5 (4), 185–189. De Luca, G., Rossella, S., Franca, Z. & Erica, L.  Comparative study on the efficiency of peracetic acid and chlorine dioxide at low doses in the disinfection of urban wastewaters. Ann. Agric. Environ. Med. 15 (2), 217–224. Dow, S. M., Barbeau, B., Von Gunten, U., Chandrakanth, M., Amy, G. & Hernandez, M.  The impact of selected water quality parameters on the inactivation of Bacillus subtilis spores by monochloramine and ozone. Water Res. 40 (2), 373–382. Driedger, A., Staub, E., Pinkernell, U., Mariñas, B., Köster, W. & Gunten, U. V.  Inactivation of Bacillus subtilis spores and formation of bromate during ozonation. Water Res. 35 (12), 2950–2960. Facile, N., Barbeau, B., Prévost, M. & Koudjonou, B.  Evaluating bacterial aerobic spores as surrogate for Giardia and Cryptosporidium inactivation by ozone. Water Res. 34 (12), 3238–3246. Gehr, R., Wagner, M., Veerasubramanian, P. & Payment, P.  Disinfection efficiency of peracetic acid, UV and ozone after enhanced primary treatment of municipal wastewater. Water Res. 37 (19), 4573–4586. Harley, J. P. & Prescott, L. M.  Laboratory Exercises in Microbiology. McGraw-Hill Education, Columbus, OH, USA, 58. Jiang, S. C., Chu, W. & He, J. W.  Seasonal detection of human viruses and coliphage in Newport Bay, California. Appl. Environ. Microbiol. 73 (20), 6468–6474. Keep, N. H., Ward, J. M., Robertson, G., Cohen-Gonsaud, M. & Henderson, B.  Bacterial resuscitation factors; revival of viable but non-culturable bacteria. Cell. Mol. Life Sci. 63 (22), 2555–2559. Kitis, M.  Disinfection of wastewater with peracetic acid: a review. Environ. Int. 30 (1), 47–55. Koivunen, J. & Heinonen-Tanski, H. a Inactivation of enteric microorganisms with chemical disinfectants, UV irradiation and combined chemical/UV treatments. Water Res. 39 (8), 1519–1526. Koivunen, J. & Heinonen-Tanski, H. b Peracetic acid (PAA) disinfection of primary, secondary and tertiary treated municipal wastewaters. Water Res. 39 (18), 4445–4453. Lee, J. & Deininger, R. A.  A rapid screening method for the detection of viable spores in powder using bioluminescence. Luminescence 19 (4), 209–211. Lee, J. & Deininger, R. A.  Real-time determination of the efficacy of residual disinfection to limit wastewater

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contamination in a water distribution system using filtrationbased luminescence. Water Environ. Res. 82 (5), 475–478. Lleò, M. M., Tafi, M. C., Signoretto, C., Boaretti, M. & Canepari, P.  Resuscitation rate in different enterococcal species in the viable but non-culturable state. J. Appl. Microbiol. 91 (6), 1095–1102. Ludwig, W. & Schleifer, K. H.  How quantitative is quantitative PCR with respect to cell counts? Syst. Appl. Microbiol. 23 (4), 556–562. Majcher, M. R., Bernard, K. A. & Sattar, S. A.  Identification by quantitative carrier test of surrogate spore-forming bacteria to assess sporicidal chemicals for use against Bacillus anthraciss. Appl. Environ. Microbiol. 74 (3), 676–681. Monarca, S., Richardson, S. D., Feretti, D., Grottolo, M., Thruston Jr., A. D., Zani, C., Navazio, G., Ragazzo, P. & Serbini, I.  Mutagenicity and disinfection by-products in surface drinking water disinfected with peracetic acid. Environ. Toxicol. Chem. 21 (2), 309–318. Oliver, J. D.  The viable but nonculturable state in bacteria. J. Microbiol. 43 (S), 93–100. Ogorzaly, L. & Gantzer, C.  Development of real-time RTPCR methods for specific detection of F-specific RNA bacteriophage genogroups: application to urban raw wastewater. J. Virol. Methods 138 (1–2), 131–139. Radziminski, C., Ballantyne, L., Hodson, J., Creason, R., Andrews, R. C. & Chauret, C.  Disinfection of Bacillus subtilis spores with chlorine dioxide: a bench-scale and pilot-scale study. Water Res. 36 (6), 1629–1639. Ratphitagsanti, W., Park, E., Lee, C. S., Wu, R.-Y. A. & Lee, J.  High-throughput detection of spore contamination in food packages and food powers using tiered approach of ATP bioluminescence and real-time PCR. LWT-Food Sci. Technol. 46 (1), 341–348. Rutala, W. A., Weber, D. J. & the Healthcare Infection Control Practices Advisory Committee (HICPAC)  Guideline for Disinfection and Sterilization in Healthcare Facilities.

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Centers for Disease Control and Prevention, Atlanta, GA. Available at: www.cdc.gov/hicpac/pdf/guidelines/ Disinfection_Nov_2008.pdf (accessed 20 May 2012). Setlow, P.  Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 101 (3), 514–525. USEPA (United States Environmental Protection Agency)  Ambient Water Quality Criteria for Bacteria. Office of Water, Washington, DC. Available at: www.epa.gov/waterscience/ beaches/files/1986crit.pdf (accessed 24 April 2011). USEPA (United States Environmental Protection Agency)  Combined Sewer Overflow Technology Fact Sheet. Chlorine disinfection. EPA 832-F-99-034. Office of Water, Washington, DC. USEPA (United States Environmental Protection Agency)  Method 1601: Male-specific (Fþ) and Somatic Coliphage in Water by Two-step Enrichment Procedure. EPA-821-R-01030. Office of Water, Washington, DC. USEPA (United States Environmental Protection Agency)  Emergency Disinfection of Drinking Water. Office of Water, Washington, DC. Available at: www.water.epa.gov/drink/ emerprep/emergencydisinfection.cfm (accessed 12 January 2012). Veschetti, E., Cutilli, D., Bonadonna, L., Briancesto, R., Martini, C., Cecchini, G., Anastasi, P. & Ottaviani, M.  Pilot-plant comparative study of peracetic acid and sodium hypochlorite wastewater disinfection. Water Res. 37 (1), 78–94. WHO (World Health Organization)  Mortality and Burden of Disease from Water and Sanitation. World Health Organization, Geneva, Switzerland. Available at: www.who. int/gho/phe/water_sanitation/burden_text/en/index.html (accessed 5 August 2012). Zanetti, F., De Luca, G., Sacchetti, R. & Stampi, S.  Disinfection efficiency of peracetic acid (PAA): inactivation of coliphages and bacterial indicators in a municipal wastewater. Environ. Technol. 28 (11), 1265–1271.

First received 27 December 2012; accepted in revised form 20 August 2013. Available online 25 September 2013

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2014

Impact of elevated Ca2þ/Mg2þ concentrations of reverse osmosis membrane desalinated seawater on the stability of water pipe materials Juan Liang, Anqi Deng, Rongjing Xie, Mylene Gomez, Jiangyong Hu, Jufang Zhang, Choon Nam Ong and Avner Adin

ABSTRACT Hardness and alkalinity are known factors inﬂuencing the chemical stability of desalinated water. This study was carried out to investigate the effect of Ca2þ and Mg2þ on corrosion and/or scale formation on the surface of different water distribution pipe materials under tropical conditions. The corrosion rates of ductile iron, cast iron and cement-lined ductile iron coupons were examined in reverse osmosis (RO) membrane desalinated seawater which was remineralised using different concentrations of Ca2þ and Mg2þ. The changes in water characteristics and the coupon corrosion rates were studied before and after the post-treatment. The corrosion mechanisms and corrosion products were examined using scanning electron microscope and X-ray diffraction, respectively. We found that the combination of Ca2þ and Mg2þ (60/40 mg/L as CaCO3) resulted in lower corrosion rates than all other treatments for the three types of pipe materials, suggesting that Ca2þ/Mg2þ combination improves the chemical stability of desalinated seawater rather than Ca2þ only. Key words

| Ca2þ/Mg2þ, corrosion, desalinated water, post-treatment, remineralisation, water distribution pipeline

Juan Liang Anqi Deng Jiangyong Hu Jufang Zhang Choon Nam Ong National University of Singapore, NUS Environment Research Institute, 5A Engineering Drive 1, T-Lab Building #02-01, Singapore 117411, Singapore Rongjing Xie (corresponding author) Mylene Gomez Technology Department, the Public Utilities Board, 80/82 Toh Guan Road East, #C4-03, Singapore 60857, Singapore E-mail: xie_rongjing@pub.gov.sg Avner Adin Department of Soil and Water Sciences, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

INTRODUCTION Pipeline corrosion in drinking water distribution systems is

In recent years, seawater desalination has become one

of special concern because of various related problems

of the most important water resources, especially in coastal

(Lee & Schwab ). First, pipe mass is lost through leach-

regions with insufﬁcient fresh water supply. However, the

ing or oxidisation to soluble species or insoluble scales,

soft product water is chemically unstable and the corrosive-

which shortens pipe lifetime and increases the maintenance

ness of the desalinated water threatens the service life of the

costs. Second, the scale can accumulate as large tubercles

water distribution pipelines. To prevent corrosion from hap-

that increase head loss, decrease water capacity, and thus

pening and to control the water quality for drinking and

increase pumping costs. Finally, while having health impacts

other purposes, post-treatment of desalinated water is

on consumers, the release of soluble or particulate corrosion

required (Hasson & Bendrihem ; Lahav & Birnhack

by-products to the water decreases its aesthetic quality and

; Birnhack et al. ). Various post-treatment methods

often leads to consumer complaints of ‘red water’ at the

include direct chemical dosage, blending with other water,

tap. Iron pipe corrosion usually results in water quality

calcite/dolomite dissolution to increase pH, alkalinity and

with a red, brown or yellow colour, or a dirty appearance.

buffering capacity. Most of them can provide calcium iron

Corrosion products also provide habitats for microbial

but few methods offer magnesium mineral.

growth and react with disinfectant residuals, preventing the disinfectant from penetration of bioﬁlm. doi: 10.2166/wh.2013.060

Traditionally, the chemical stability of drinking water is a function of the value of three factors: (1) the

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

buffering capacity or alkalinity of the water, i.e., the

Journal of Water and Health

12.1

2014

METHODOLOGY

ability of the water to withstand substantial changes in pH when a base or an acid is added to it; (2) the propen-

In this study, three types of pipeline materials (ductile iron, cast

sity of the water to precipitate CaCO3, which can

iron and cement-lined ductile iron) were explored to investigate

be described by a variety of qualitative (e.g., the

the effect of Ca2þ/Mg2þ remineralised reverse osmosis (RO)

Langelier) and/or quantitative (e.g., the calcium carbon-

membrane desalinated seawater on the stability of the pipe

ate precipitation potential, CCPP) indices; and (3) the

materials. Traditionally, cast iron was a widely existing pipe

concentration of soluble Ca2þ or other ions of multi-

material for water and wastewater transport. Ductile iron is

valences in the water.

one of the most commonly used pipe materials in modern infra-

On the other hand, calcium is an element that is vital for

structure for water distribution, while cement-lined ductile iron

human growth and health (Chiu et al. ). A minimal Ca2þ

pipe has excellent anti-corrosion properties because cement

concentration value is required for health reasons and has

lining provides a barrier between the water and the iron pipe,

been typically set at 50 to 60 mg/L as CaCO3. The maximum

reducing the pipe’s susceptibility to corrosion (Bonds ).

value is due to economic reasons attributed to the need to

Ductile iron, cast iron and cement-lined ductile iron

supply water that is not excessively hard. The range for

were selected as representative pipe materials. Rectangular

Ca2þ values thus lies roughly between 50 and 120 mg/L as

metal coupons with the dimensions of 40 mm × 13 mm ×

CaCO3 (Anthony ). Magnesium is also an element

2 mm and with a 5 mm diameter hole at one end were pre-

which is vital to health because, as research has shown, it

pared following the standard method (ASTM G- ).

maintains the heartbeat and thus prevents heart attacks. It

As shown in Table 1, chemical compositions of ductile

is estimated that each year several hundred lives would be

iron and cast iron are similar. Both were made of Fe

saved by regular consumption of magnesium in desalinated

>92% and C 3.7%. For the cement-lined ductile iron cou-

water, regular rainwater or bottled mineral water. In 2009

pons, the coupons were coated with 0.5 mm cement, of

the Israeli Ministry of Health decided to add a minimum

which the elemental compositions are CaO (52.7%), SiO2

requirement of 10 mg Mg2þ/L to the criteria due to the

(24%), Al2O3 (8.9%), Fe2O3 (3.5%), MgO (2.5%) and others.

acknowledged importance that Mg2þ ions have on both

Triplicate coupons of either ductile iron, cast iron or

crop quality and public health (Catling et al. ). The

cement-lined ductile iron were immersed in 1-L glass reactors

World Health Organization (WHO) has recently rec-

with 800 mL desalinated water in temperature-controlled

2þ

ommended a minimum concentration of 10 mg Mg /L in

incubators. The temperature was kept at 28–29 C, similar to

all drinking waters, with special emphasis on desalinated

that of water in Singapore underground water distribution

water (WHO ). This recommendation is based on

pipelines. Fifteen reactors were ﬁlled with remineralised RO

recent publications which stress the role of Mg2þ in drinking

membrane desalinated seawater. SWRO product was taken

water on the human body and the possible implications of Mg2þ deﬁciency on public health (Kozisek ; Cotruvo ). Seawater reverse osmosis (SWRO) products reminera-

Table 1

Coupon composition of ductile iron, cast iron and cement liner

Element,

Cement

lised by Ca2þ have been proven to be capable of reducing

Wt %

Ductile iron

Cast iron

Composition, Wt %

iron corrosion (Liang et al. ). However, little infor-

96.84

92.36

mation is available in the literature on the combined effect

0.98

2.13

SiO2

0.27

0.23

CaO

52.7

linated water on the surface of water pipes, especially

0.18

0.24

Al2O3

8.9

Singapore tropical underground water distribution pipelines

Fe2O3

3.5

(28–29 C). Speciﬁc attention was paid to the effect of Mg2þ

3.70

3.63

MgO

2.5

as this cation has rarely been investigated in drinking water

<0.50

K2O

distribution pipelines.

NA: not available.

2þ

of Ca /Mg

on the corrosion and scale formation of desa-

liner

2.2

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Journal of Water and Health

12.1

2014

from the Singapore variable salinity plant, Tampines. As

alkalinity, total hardness and total dissolved solids (TDS),

shown in Table 2, SWRO product was remineralised with var-

were measured before and after the treatment. Concen-

ious chemicals to a concentration level slightly above the

trations of Ca2þ and Mg2þ were analysed using inductively

minimum standard based on economic concern. The exper-

coupled plasma optical emission spectrometer (ICP-OES,

iments were carried out following the standard procedures of

Optima 7000 DV, Perkin Elmer). Coupon surface was

laboratory immersion corrosion tests (ASTM G- ;

observed using scanning electron microscope (SEM, Stereo-

ASTM G- ). The corrosion phenomenon was moni-

scan 420, Leica, Cambridge Instruments).

tored during the whole process. After 497 hours’ immersion,

Compositions of the powder samples from the corrosion

all coupons were taken out of the reactors, dried, cleaned

layer and bottom of the reactors were analysed using the

and weighed for corrosion rate calculation. According to the

D5005 Bruker powder X-Ray diffractometer. Full X-ray dif-

ASTM standard, each coupon was rinsed with a forceful

fraction patterns were recorded for the scan angles (2θ)

stream of cold tap water to remove residual test solution and

from 10 to 80 with step size 0.02 to identify iron oxides.

loose corrosion products. Subsequently, the coupons were immersed in a chemical cleaning solution (500 mL HCl þ 3.5 g (CH2)6N4) for 10 min to remove attached corroded

RESULTS AND DISCUSSION

oxide ﬁlm, and then water rinsed and oven dried. Finally, the coupons were mechanically cleaned by grit paper if persistent

Corrosion phenomena

residues remained and then weighed after rinsing with water. Water was ﬁltered using 0.8 μm ﬁlter paper to remove

As shown in Figure 1, after being immersed in the SWRO

particulates. Water characteristics, such as pH, total

membrane product water, the iron coupons were subjected

Table 2

Character of tested water qualities

Alkalinity addition Tested water

Post-treatment

pH adjustment

SWRO

None

7.25

SWRO with pH adjustment

NaOH

Remineralised SWRO

NaHCO3 þ CO2 Ca(OH)2 þ CO2 Ca(OH)2 þ MgCO3 þ CO2 þ NaHCO3

Figure 1

Ca2þ addition

Mg2þ addition

(mg/L as CaCO3)

8 8 8

100 100 100

0 100 60

0 0 40

Corrosion phenomena of different iron coupons for control and remineralised water after 497 hours of immersion tests.

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Journal of Water and Health

12.1

2014

to corrosion and a layer of red substances started forming on

where: K ¼ a constant, T ¼ time of exposure in hours, A ¼ area

the surfaces of the coupons and some crust gradually settled at the bottoms of the reactors. Water colour in all reactors

in cm2, W ¼ mass loss in grams, and D ¼ density in g/cm3. Many different units are used to express corrosion rates.

with iron coupons became greenish on the second day and

Using the above units for T, A, W and D, the corrosion rate

orange from the third day onwards (photographs not

can be calculated in a variety of units with the corresponding

shown). No red precipitate was observed in the reactors

appropriate value of K. In this study, mils per year (mpy; 1 mils

with cement-lined coupons while white precipitate was

¼ 1/1,000 inch ¼ 0.0254 mm) is used as the unit of corrosion

found in treatments with Ca2þ/Mg2þ or CO2 additions. 3

rate and the constant K is 3.45 × 106. Corrosion rate was calcu-

The white precipitate was dissolved by HCl solution (4 mL

lated for each water sample tested and presented in Figure 2.

10% HCl þ 300 mL ultra pure water) and analysed by ICP-

Comparison of the corrosion rates of different pipeline

2þ

concen-

materials showed that cement-lined ductile iron had the

tration. The white precipitate CaCO3 was not found in the

lowest while cast iron had the highest corrosion rate regardless

reactors for the controls (i.e., SWRO permeate only) and

of how the desalinated seawater was post-treated. As for the

for the SWRO membrane permeate for which only pH

post-treatment methods, remineralisation of the water using

was adjusted.

Ca2þ/Mg2þ produced the lowest corrosion rate compared

OES. CaCO3 appeared and attributed to high Ca

with all the other treatments for ductile iron, cast iron and Corrosion rate comparison

cement-lined ductile iron materials. The combined Ca2þ/ Mg2þ appeared to have effectively reduced the corrosion

The simplest and longest established method of estimating

(Figure 2). However, the reduction of corrosion by addition

corrosion losses in plant and equipment is weight loss analy-

Ca2þ/Mg2þ was signiﬁcant to cast iron, less signiﬁcant to duc-

sis. A weighed sample (coupon) of the metal or alloy under

tile iron and slightly signiﬁcant to cement-lined iron coupons.

consideration is introduced into the process, and later

Other researchers also reported similar results but with-

removed after a reasonable time interval. The coupon is

out Mg2þ addition (Chang ; Hasson & Bendrihem ;

then cleaned of all corrosion products and is reweighed.

Douglas ; Birnhack et al. ). Chang () studied

The weight loss is converted to a corrosion rate (CR) as fol-

three post-treatment processes, namely lime and carbon

lows (ASTM G- ):

dioxide, sodium bicarbonate or carbonate and alkaline media ﬁlter, to reduce the corrosiveness of the product

Corrosion rate ¼ (K × W)=(A × T × D)

Figure 2

from the desalination plant using ductile iron coupons.

Corrosion rate of different coupons as related to various post-treatments of corrosive SWRO permeate.

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Journal of Water and Health

12.1

2014

The results showed that lime and carbon dioxide were very

treatment, the surfaces of ductile iron and cast iron coupons

efﬁcient to chemically stabilise the corrosive water.

were even with some small cavities. After the treatments, the

2þ

is a recent public health concern (WHO ) and

coupon surfaces became rough with pits. The corrosion

so far most of the corrosion studies have been focused on the

mechanisms that were predominant for ductile iron and

2þ

effect of Ca . Based on our knowledge, no studies have been

cast iron appeared to be uniform and identiﬁed as pitting

conducted on the effect of Mg2þ on pipeline material cor-

corrosion (Reynaud ).

rosion. Our results revealed that the combination of Ca2þ

Cement-mortar lining provided a barrier between the

and Mg2þ signiﬁcantly reduced the coupon corrosion rate.

water and the pipe material reducing its susceptibility to cor-

Therefore, post-treatment combining Ca2þ/Mg2þ can be a

rosion conditions. Two main mechanisms could inﬂuence

preferred option instead of calcium alone. However, in the

the degradation kinetics of cement lining: (1) ion transfers

engineered post-treatment methods, only dolomite dissol-

between the bulk water and the pore water; and (2) chemi-

2þ

ution can provide small amounts of Mg

(Birnhack et al.

). Several problems are encountered when attempting to

cal reaction between the water and components of the cement (Adenot & Buil ).

dissolve dolomite rocks (MgCa(CO3)2) for this purpose. The

Figures 4 and 5 show the comparisons of cement-lined

most noticeable drawback is the dissolution kinetics of dolo-

ductile iron coupons before and after corrosion tests. The

mite, which is much slower than that of calcite (Liu et al.

surface looked smoother after testing compared against the

). As a result, either a much larger reactor volume,

rough surface before test (Figure 4), probably due to

longer hydraulic retention times or lower pH is required to

cement dissolution when the coupon was submersed in

2þ

ions. If the initial pH

water. However, when the cement liner was removed, pit-

is low, NaOH would be required to elevate the alkalinity

ting corrosion was evident owing to the presence of

dissolve an adequate amount of Mg

and CCPP values, and often the limit for pH (i.e., pH 8.5)

numerous rusty dots on the iron surface (Figure 5). Desali-

would be exceeded. Another problem associated with dolo-

nated seawater permeated the pores of the liner causing

mite is that the Ca2þ to Mg2þ ratio released during its

the pitting corrosion phenomenon observed on the iron sur-

dissolution tends not to be constant with time because exca-

face. Desalinated water permeated into the liner perhaps

vated dolomite minerals are rarely pure, and the CaCO3

because the cement liner that was used was very thin

component within the dolomite structure tends to dissolve

(0.5 mm) and thickness not being unique. In practical appli-

more rapidly than the MgCO3 component (Liu et al. ).

cations, cement lining in actual pipes is packed more

A novel post-treatment approach for desalinated water, aimed at supplying a balanced concentration of alkalinity, 2þ

2þ

SO2 4 ,

densely and thicker than what was used in this project. Figure 6 shows the SEM images of corroded ductile iron

has been developed (Birnhack &

before chemical and physical cleaning in water treated with

Lahav ; Penn et al. ; Birnhack et al. ). The pro-

Ca2þ only. For the surface covered by orange corrosion sub-

Ca , Mg

and

cess is based on replacing excess Ca

2þ

ions generated in the

stances, some composites colonised together cloudily. Some

common H2SO4-based calcite dissolution post-treatment

crystals shaped in ﬁbre or blocks were found on the surface

2þ

ions originating from seawater. The

without orange iron corroded products. The crystal precipi-

exchange is complemented using a speciﬁc ion exchange

tate was calcite or aragonite components. They might have

resin that has high afﬁnity toward divalent cations to meet

formed a layer blocking further corrosion on this portion.

process with Mg

2þ

a predetermined Ca

2þ

to Mg

ratio. The proposed process

allows for the supply of cheap Mg2þ ions, thus resulting in

Scales

higher quality water at a cost-effective price. The corrosion products formed on the iron surface have a SEM microstructure

complex morphology. The exact composition and structure of iron corrosion scales, however, varies signiﬁcantly with

The microstructure of coupons before and after the treat-

water qualities, ﬂow properties as well as other environ-

ments was observed using SEM (Figure 3). Before the

mental

parameters.

Typical

iron

corrosion

products

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Figure 3

SEM images showing comparison of ductile and cast iron before and after the corrosion test.

Figure 4

SEM images of cement lining before and after corrosion treatment.

Journal of Water and Health

12.1

2014

formed on aged pipe surfaces can be described using three

macro-layer; and (c) a top layer of red rust (mainly goethite,

different layers: (a) a macroporous layer of black rust (mag-

a-FeOOH and hematite, a-Fe2O3), which is generally porous

netite, Fe3O4) in contact with the metal; (b) a microporous

with poor adherence (Sarin et al. ; Tang et al. ;

ﬁlm of a mixture of Fe2þ and Fe3þ species that covers the

Gerke et al. ).

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Journal of Water and Health

12.1

2014

were composed of FeO(OH) [lepidocrocite], Fe(OH)3 and other iron components. In Ca(OH)2 or MgCO3 treated water, calcium compounds (CaO, CaO2, CaCO3) and MgO2 were detected in the corroded scales. A few tiny white granules, which were believed to be calcium deposits, were also found on some iron coupon surfaces. Water quality Various water quality parameters before and after remineralisation are presented in Figure 7. Total alkalinity, total hardness, pH, TDS, redox potential, conductivity and Ca2þ increased considerably with cement-lined ductile iron due to cement dissolution. In a study by Bonds (), extended contact under high-ﬂow conditions Figure 5

Comparison of the ductile iron coupons with and without corrosion after testing and the removal of cement lining.

initially accelerated leaching of cement-mortar lining but the changes in water quality were not noticeable due to the constant water dilution. In our study, however,

In our study, the corrosion products were found to be

extended contact between the water and the lining material

similar in composition to that of the outer surface layer pre-

signiﬁcantly changed the water quality inside the reactor,

viously described (Table 3). There were no obvious

including pH (not shown), alkalinity and calcium concen-

differences of the corrosion products between ductile iron

tration, because the water in the reactor was not replaced

and cast iron. Rust crusts found on the coupon surfaces

during the experiment.

Figure 6

SEM images of dust and precipitation on ductile iron coupon surface.

J. Liang et al.

Table 3

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Journal of Water and Health

12.1

2014

Corrosion products of ductile iron and cast iron identiﬁed using X-ray diffraction

Tested water

Ductile iron

Cast iron

SWRO permeate (Control)

FeO(OH), Fe(OH)3

Fe0.98O, FeO

FeOOH, Fe0.98O

Ca ¼ 0, pH ¼ 8; (NaOH)

FeO(OH), Fe(OH)3

Fe0.98O,FeO, Fe2O3

Fe0.98O, Fe2O3 , FeO

FeFe2O4 Ca ¼ 100, Alk ¼ 100, pH ¼ 8; Ca(OH)2 þ CO2 þ NaHCO3

Ca ¼ 60, Mg ¼ 40, Alk ¼ 100, pH ¼ 8; Ca(OH)2 þ MgCO3 þ CO2

Figure 7

FeO(OH), Fe(OH)3

FeOOH, Fe2O3, Fe2O3

FeOOH, Fe2O3

CaO,CaO2,CaCO3

CaO, CaCO3

FeO(OH), Fe(OH)3

FeOOH, Fe0.98O, Fe2O3,

FeOOH, Fe2O3,

CaO, CaCO3, MgO2

Changes in water quality before and after the post-treatments. (a) Total alkalinity, (b) total hardness, (c) calcium concentration, (d) magnesium concentration.

It was noted that the Mg2þ concentrations, however,

cement-lined coupons and the results are shown in

decreased from 9.42 to 0.20 mg/L. To verify these changes

Table 4. The collected Ca2þ is much higher than the original

on ion concentrations before and after the test, Ca2þ/Mg2þ

amount, which means cement dissolution releasing Ca2þ to

balance was calculated for remineralised water with

the water. For treatment with MgCO3, some substances

J. Liang et al.

Table 4

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

12.1

2014

the corrosion rates for different post-treatments, desalinated

Ca2þ and Mg2þ mass balance for selected post-treatments Ca2þ (mg)

Journal of Water and Health

water treated with Ca2þ/Mg2þ led to the lowest corrosion

Mg2þ (mg)

rate for all three pipe materials. The combination of Ca2þ

Water quality

Original

Recovered

Original

Recovered

and Mg2þ further reduced pipe corrosion in comparison

Ca ¼ 100, pH ¼ 8

28.74

151.94

0.20

0.47

with Ca2þ alone. Therefore, addition of both Ca2þ and

Ca ¼ 60, Mg ¼ 40, pH ¼ 8

18.56

102.73

8.27

5.53

Mg2þ is recommended for the post-treatment because of

Recovered: left on the ﬁlter paper þ deposit at bottle bottom þ in water.

the beneﬁts both to human health and in mitigating pipeline corrosion.

containing Mg2þ (5.53 mg) were deposited at the reactor bottom and on the filter paper even though Mg2þ concentration was close to zero in the water. The water in the reactor was filtered through 0.8 μm membrane paper, and some white suspended solids from the water remained on the filter paper surface which proved to be Mg2þ compounds by ICP-OES. At the same time, some Ca2þ/Mg2þ

ACKNOWLEDGEMENTS This research was ﬁnancially supported by Environment and Water Industry (EWI), Public Utilities Board (PUB) and National University of Singapore (NUS). The authors

deposits could be washed away during the washing stage

would also like to acknowledge the water supply from

after the coupons were taken out of the reactors. Therefore,

PUB’s variable salinity plant and NUS Environment

the quantity of recovered Mg2þ (5.53 mg) was lower than the added amount (8.27 mg). For iron coupons in the water treated with Ca(OH)2 þ

Research

Institute

(NERI)/NUS

staff

for

providing

technical support to this project.

CO2 or Ca(OH)2 þ MgCO3 þ CO2 þ NaHCO3, the water characteristics were similar before and after the remineralisation process except for hardness, alkalinity, Ca2þ and Mg2þ concentrations. Total alkalinity, total hardness, Ca2þ and Mg2þ concentrations decreased after 497-hour corrosion tests probably as a result of CaCO3/MgCO3 precipitation. Concentration of Mg2þ marginally decreased from 9.42 mg/L to ∼8 mg/L in water without cement-lined coupons (Figure 7(d)), while the difference was 9.22 (9.42 0.20) mg/L in the water with cement-lined coupons. It indicates much more Mg2þ precipitation in water with cement-lined coupons due to cement dissolution. According to Figure 7(d) and Table 4, the Mg2þ ions were precipitated as a result of reaction with the cement lining, which probably explains why their effect is slightly signiﬁcant to cementlined iron coupons which were shown in Figure 2 earlier.

CONCLUSIONS This study led to several observations with elevated Ca2þ/ Mg2þ concentration in remineralised SWRO membrane product water in comparison with the control for ductile iron, cast iron and cement-lined ductile iron pipe materials. Of

REFERENCES Adenot, F. & Buil, M.  Modelling of the corrosion of the cement paste by deionized water. Cement Concrete Res. 22 (2–3), 489–496. Anthony, W.  Options for recarbonation, remineralisation and disinfection for desalination plants. Desalination 179 (1), 11–24. ASTM G1–90  Standard practice for preparing, cleaning, and evaluating corrosion test specimens. Annual Book of ASTM Standards, Vol 03.02. ASTM G31–72  Standard practice for laboratory immersion corrosion testing of metals. Annual Book of ASTM Standards, Vol 03.02. Birnhack, L. & Lahav, O.  A new post treatment process for attaining Ca2þ, Mg2þ, SO2 4 and alkalinity criteria in desalinated water. Water Res. 41 (17), 3989–3997. Birnhack, L., Penn, R., Oren, S., Lehmann, O. & Lahav, O.  Pilot scale evaluation of a novel post-treatment process for desalinated water. Desalination Water Treat. 13, 120–127. Birnhack, L., Voutchkov, N. & Lahav, O.  Fundamental chemistry and engineering aspects of post-treatment processes for desalinated water - A review. Desalination 273 (1), 6–22. Bonds, R. W.  Cement-mortar linings for ductile iron pipe. Available at: www.dipra.org/pdf/cementMortarLinings.pdf (accessed 1 November 2012).

J. Liang et al.

Elevated Ca2þ/Mg2þ impact on pipe material corrosion in desalinated seawater

Catling, L. A., Abubakar, I., Lake, I. R., Swift, L. & Hunter, P. R.  A systematic review of analytical observational studies investigating the association between cardiovascular disease and drinking water hardness. J. Water Health 6 (4), 433–442. Chang, P. H.  The optimal control for corrosive effluent from the seawater desalination plant. Master thesis, National Cheng Kung University, Tainan City, Taiwan. Chiu, H. F., Tsai, Sh., Chen, P. Sh., Wu, Tr. N. & Yang, Ch. Y.  Does calcium in drinking water modify the association between nitrate in drinking water and risk of death from colon cancer? J. Water Health 9 (3), 498–506. Cotruvo, J.  Health aspects of calcium and magnesium in drinking water. In: Proc. Int. Symp. on Health Aspects of Calcium and Magnesium in Drink. Water, Baltimore, MD, USA, April 24–26. Douglas, S. S.  Post Treatment Alternatives for Stabilizing Desalinated Water. University of Central Florida, Orlando, FL. Gerke, T. L., Maynard, B. J., Shock, M. R. & Lytle, D. L.  Physiochemical characterization of five iron tubercles from a single drinking water distribution system: Possible new insights on their formation and growth. Corrosion Sci. 50 (7), 2030–2039. Hasson, D. & Bendrihem, O.  Modeling remineralization of desalinated water by limestone dissolution. Desalination 190 (1–3), 189–200. Kozisek, F.  Health Significance of Drinking Water Calcium and Magnesium. Available at: www.alkaway.com.au/ downloads/hard-water-report.pdf. (accessed 24 January 2013). Lahav, O. & Birnhack, L.  Quality criteria for desalinated water and introduction of a novel, cost effective and

Journal of Water and Health

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2014

advantageous post treatment process. Desalination 221 (1–3), 70–83. Lee, J. E. & Schwab, K. J.  Deﬁciencies in drinking water distribution systems in developing countries. J. Water Health 3 (2), 109–127. Liang, J., Deng, A. Q., Xie, R. J., Gomez, M., Hu, J. Y., Zhang, J. F., Ong, Ch. N. & Adin, A.  Impact of seawater reverse osmosis (SWRO) product remineralization on the corrosion rate of water distribution pipeline materials. Desalination 311, 54–61. Liu, Z. H., Yuan, D. X. & Dreybrodt, W.  Comparative study of dissolution rate-determining mechanisms of limestone and dolomite. Environ. Geol. 49 (2), 274–279. Penn, R., Birnhack, L., Adin, A. & Lahav, O.  New desalinated drinking water regulations are met by an innovative post-treatment process for improved public health. WST: Water Supply 9 (3), 225–231. Reynaud, A.  Corrosion of cast irons. In: Shreir’s Corrosion. (T. Richardson, ed.) Elsevier, Oxford, pp. 1737–1788. Sarin, P., Snoeyink, V. L., Lytle, D. A. & Kriven, W. M.  Iron corrosion scales: model for scale growth, iron release, and colored water formation. J. Environ. Eng. 4, 364–373. Tang, Z., Hong, S., Xiao, W. & Taylor, J.  Characteristics of iron corrosion scales established under blending of ground, surface, and saline waters and their impacts on iron release in the pipe distribution system. Corrosion Sci. 48, 322–342. World Health Organization  Calcium and Magnesium in Drinking Water: Public Health Signiﬁcance, 1st edn. World Health Organization, Geneva, Switzerland, p. 190.

First received 13 February 2013; accepted in revised form 20 August 2013. Available online 20 September 2013

12.1

2014

Magnesium in drinking water – a case for prevention? Ragnar Rylander

ABSTRACT Studies in many countries have demonstrated a relationship between drinking water mineral content and the risk of death in cardiovascular disease (CVD). Particularly strong relationships have been found for magnesium and it has been suggested that magnesium be added to drinking water. The

Ragnar Rylander BioFact Environmental Health Research Centre, Bjorkasvagen 21, 44391 Lerum, Sweden E-mail: envhealth@biofact.se

aim of this article is to evaluate the validity of this suggestion by reviewing information on possible causative agents. Major epidemiological studies on the drinking water content of calcium, magnesium, and hardness were analysed regarding exposure speciﬁcity, confounding factors, doseresponse relationships and biological plausibility. Intervention experiments were analysed. The risk of death in CVD was related to the content of Ca, Mg and HCO3 . The data demonstrate that Ca and Mg need to be considered together, and that HCO3 could play a role by intervening with the body acid load. There is no evidence to justify the addition of magnesium only to drinking water for preventive purposes. The data suggest that Ca and Mg could be administered together but no data are available regarding the relative proportions for an optimal effect. Key words

| acidity, calcium, hardness, magnesium

INTRODUCTION The relationship between drinking water and health has

the risk of death in cardiovascular disease (CVD). Extensive

been known for thousands of years and became estab-

reviews of the subject have been presented in previous pub-

lished in religious ceremonies like the offering of votives

lications (Rylander ; Catling et al. ; WHO ).

and coins in water springs. The real break-through in

This article will focus on the interpretation of the available

terms of prevention of disease did not, however, come

data in view of epidemiological and biological principles,

until the discovery of bacteria. The health consequences

and present a conclusion regarding prevention by adding

of a microbiological contamination of drinking water

magnesium to drinking water.

have been known since the mid-1800s and were ﬁrst applied to praxis in an epidemic of cholera in London, when John Snow turned off the famous water post in

DRINKING WATER CONSTITUENTS AND DISEASE

Broad Street in 1854. Another component of drinking water that has attached

Hardness

attention for a long time is the content of minerals. Babylonian, Greek, and Roman elites believed in the healing

The ﬁrst epidemiological study on the relationship between

resources of mineral wells. Although part of the improve-

minerals in drinking water and health was published in

ment in health and well-being in spas was certainly

Japan (Kobayashi ). He noticed a high incidence of ‘apo-

attributable to general relaxation, intake of minerals could

plexia’ (cardiovascular disease) in areas with acidic drinking

have contributed to the effect.

water. To help in further studies, assistance from the USA

The aim of this article is to highlight the relationship between the mineral composition of drinking water and doi: 10.2166/wh.2013.110

was called in but they had no methods to measure acidity and used hardness instead.

R. Rylander

Magnesium and drinking water

Since then, more than 20 epidemiological studies on the

Journal of Water and Health

12.1

2014

Magnesium

relationship between hardness and CVD have been published. An inverse relationship between hardness and CVD

Mg is the second most abundant cation in intracellular ﬂuid

was found in 11 of these. As an example, several studies

(Vormann ). It is a cofactor for more than 350 cellular

from Finland demonstrated that the eastern part of the

enzymes, regulating energy metabolism, protein and nucleic

country with soft drinking water had a high incidence of

acid synthesis, muscular tension, and insulin sensitivity. The

CVD (Punsar & Karvonen ; Luoma et al. ). A Swed-

uptake is via intake of food and drinking water. Magnesium

ish study demonstrated that hardness was related to the risk

deﬁciency is common in the Western population, due to

of CVD in both genders but that the risk of ischemic heart

inadequate intake of vegetables and the use of reﬁned

disease was related to magnesium (Rylander et al. ).

ﬂour (Bates et al. ).

Contrary to these ﬁndings, studies from the UK have not

Magnesium homeostasis can be disturbed by an insufﬁ-

found a relationship between CVD and hardness (e.g.

cient intake or an increased urinary excretion of Mg. This

Morris et al. ; Lake et al. ). It has, however, been

is induced by an increased acid load, following excessive

claimed that the use of phosphates to prevent sedimentation

exercise or the consumption of large amounts of proteins

in water pipes, which is used extensively in the UK, leads to

(Remer ; Rylander et al. , ). An increased

the formation of insoluble calcium-magnesium-phosphate

acid load will increase the acidity of the urine which

salts and thus renders human exposure assessment imposs-

decreases the reabsorption of Mg in the renal tubuli

ible (Monarca et al. ).

(Remer ). Figure 1 illustrates the inﬂuence of acidity

Hardness of water is determined by the content of cal-

on the regulation of magnesium homeostasis in the body.

cium (Ca) and magnesium (Mg) cations. The degree of hardness is measured using a number of different units, all including Ca and Mg. Hardness is not a precise exposure measure of either of these ions as the relative proportions may vary. In view of the limited information regarding a causative agent that is obtained from studies on hardness, the following will focus on the two major determinants of hardness – Ca and Mg. Calcium Ca in humans is almost exclusively found in bones and teeth. In body ﬂuids it takes part in vascular contraction, blood clotting, muscle contraction, and nerve transmission.

Epidemiological investigations Several studies from Canada, Finland, Italy, Spain, and Sweden have found relationships between the amounts of Ca and Mg in drinking water and the risk of cardiovascular mortality. In an extensive review of the epidemiological studies it was concluded that there was signiﬁcant evidence of an inverse association between Mg levels in drinking water and cardiovascular mortality and that the evidence for Ca was unclear (Catling et al. ). In Finland a relationship was found between the ratio of Ca/Mg and acute myocardial infarction among males (Kousa et al. ) and

From a nutritional point of view Ca deﬁciency is uncommon as the ordinary diet, particularly dairy products, provides for an adequate intake. Regarding the risk of CVD, some studies have shown a relationship between urinary Ca excretion and blood pressure (Kesteloot et al. ). Intervention experiments with Ca have reduced blood pressure in some studies (Cappuccio et al. ; Bucher et al. ). In drinking water, some studies show an inverse relationship between the content of Ca and CVD (Yang ; Yang & Chiu ) but others have not found a relationship.

Figure 1

Acid regulation of magnesium homeostasis in the body.

R. Rylander

Magnesium and drinking water

Journal of Water and Health

12.1

2014

A study from Serbia examined population samples in three different locations with a varying content of calcium and magnesium in the drinking water (Rasic-Milutinovic et al. ). A signiﬁcantly lower diastolic blood pressure and a higher serum magnesium level were found among inhabitants in the area with the highest total hardness. Persons in that area also had signiﬁcantly lower levels of serum triglycerides and creatinine. Although a number of relationships have been found between drinking water charateristics and CVD, these relationships are not a proof of causality. In the following Figure 2

Odds ratios for death in heart infarction among males with different amounts

the data on drinking water minerals and CVD will be ana-

of magnesium in the drinking water (Rubenowitz et al. 1996).

lysed against the criteria required for causality.

between acute myocardial infarction among females and males (Kousa et al. ). One Swedish study reported a

CRITERIA FOR CAUSALITY

decrease in deaths due to heart infarction among men in areas where the content of Mg in the drinking water was

Exposure speciﬁcity

higher (Rubenowitz et al. ), as illustrated in Figure 2. The ﬁgure illustrates a dose-response relationship with a

Exposure speciﬁcity means that the agent chosen to describe

successively lower mortality in areas with a higher level of

the exposure is unrelated to any other agent in the environ-

Mg in the drinking water. In a subsequent study of acute

ment. In nature most of the exposures involve many agents.

myocardial infarction among women, it was related to the

Depending on which agent is chosen, dose-relationships for

content of Ca and to Mg in the drinking water (Rubenowitz

CVD may be present but this could also be present for a var-

et al. ). The ﬁgure also illustrates that studies in areas

iety of other agents that co-vary with the original agent tested.

with a drinking water content of Mg at the lower end of

In the previously mentioned Finnish study, the ground water

the illustrated range do not have the power to test the

also contained Fe, Zn, Al, and Cu (Kousa et al. ). A

relationship and thus cannot be used as evidence for an

slightly increased risk (not signiﬁcant) of myocardial infarc-

absence of an effect (Rosenlund et al. ).

tion was found for Fe and Cu. In a Swedish study the content of Mg co-varied with the concentration of hydrogen

Clinical investigations

carbonate (Rylander ). This could potentially reduce the acid load and prevent the loss of magnesium in urine.

A few studies have comprised clinical investigations of indi-

Thus measurements of only calcium or magnesium in drink-

viduals from population samples. Subjects living in two

ing water do not meet the requirement of exposure speciﬁcity.

Swedish cities with different levels of Mg in the drinking water were subjected to a muscle biopsy for determination

Confounding factors

of the Mg content (Landin et al. ). A questionnaire was used to assess the intake of Mg via food and water.

The risk of CVD is not related to one speciﬁc agent but to a

There was a signiﬁcantly higher intake of Mg via drinking

number of factors such as diet, smoking, genetic predisposi-

water among the inhabitants in the city with a high level

tion, and exercise. Most of the studies on the relationship

(5.7 mg/L as compared to the low level 1.7 mg/mL). The

between drinking water quality and CVD have been of an

skeletal muscle content was higher among those in the

ecological nature. This implies that there might be other

city with a higher level of Mg in the drinking water (4.1 vs

risk factors for the disease, which could inﬂuence the results

3.9 mmol/100 g).

if they are unevenly distributed in the populations studied.

R. Rylander

Magnesium and drinking water

Journal of Water and Health

12.1

2014

in many of the studies on Ca, Mg and hardness. As other agents may co-vary (absence of exposure speciﬁcity) with the agent studied, the mere presence of a dose–response relationship is not a strong support for causality. Biological plausability A relationship found in epidemiological studies needs to be ascertained by knowledge about the biological mechanisms involved. Regarding Mg and Ca, the biological plausability for effects on the cardiovascular system and CVD is very high. In a prospective cohort of women in the USA, Figure 3

Odds ratios for heart infarction for common risk factors and for magnesium in

higher plasma concentrations and dietary magnesium

drinking water (Rubenowitz et al. 2000).

intakes were associated with a lower risk of sudden cardiac death (Chiuve et al. ). In a study on 45–64-year-old sub-

This concept is illustrated by studies from Sweden and the

jects in the USA, the highest quartile of serum Mg had a

Netherlands. In the Swedish study interviews were per-

signiﬁcantly lower risk of sudden cardiac death (Peacock

formed to assess the presence of risk factors for CVD in

et al. ). In a ﬁve-year follow-up population study in

general (Rubenowitz et al. ). Figure 3 demonstrates

Germany, low serum Mg levels were associated with a

the relative importance of such factors for the risk.

higher mortality in CVD (Reffelmann et al. ). A Swedish

The ﬁgure demonstrates that the odds ratio was higher

population study could not ﬁnd a relationship between mag-

among those with a low level of Mg in the drinking water.

nesium intake determined from questionnaires but no blood

Higher odds ratios were, however, also related to other

samples were taken (Kaluza et al. ). There was, however,

risk factors for CVD such as blood pressure, stress, and

a relationship between a low Ca intake and CVD mortality.

smoking. The ﬁgure very clearly illustrates the need to take several risk factors into consideration when assessing

Intervention

causality for drinking water Mg and death in CVD. The consumption of vegetables and fruit is a risk factor

Intervention is a critical tool for assessment of causality.

for Mg-deﬁciency. In a study from the Netherlands (Leurs

Regarding Mg in general, intervention experiments to pre-

et al. ) an extensive dietary investigation was per-

vent CVD have yielded contradictory results. In a review it

formed, in addition to calculating the amount of Mg in the

was concluded that there was a suggestion of a dose-

drinking water. Although the study has some weaknesses

dependent reduction in blood pressure from Mg interven-

as the consumption of drinking water from the tap is limited

tion but that the relationship should be conﬁrmed in larger

in the Netherlands, the results demonstrated a relationship

studies, using higher doses ( Jee et al. ).

between Mg in drinking water and stroke mortality, but

Only a few, small studies have been performed on Mg

only in a group of males with insufﬁcient intake of dietary

and Ca in drinking water. In one study healthy volunteers

Mg. The presence of such risk groups may vary depending

were given drinking water with 25 mg Mg/L for 6 weeks

on the characteristics of the population studied and thus

(Rubenowitz et al. ). The urinary excretion of Mg was

introduce methodological errors.

measured after a loading test (575 mg Mg) before and after the drinking water supply. There was a 14% increase in the

Dose–response relationships

Mg/creatinine excretion at the end, suggesting that a drinking water supply of Mg could inﬂuence the body Mg homeostasis.

One of the prerequisites for conclusions on causality is the

In an intervention study using waters that contained Mg

presence of dose–response relationships. These are found

only, or a number of naturally occurring minerals, people

R. Rylander

Magnesium and drinking water

Journal of Water and Health

12.1

2014

consuming the water containing Mg had an increased

It is not possible, however, to gain support for a hypoth-

secretion of magnesium in the urine (Rylander & Arnaud

esis regarding a single, causative mineral. On the contrary,

). A reduced blood pressure was found in the group con-

the evidence suggests that Ca and Mg in combination, as

suming water with several minerals but not in the group

they appear in nature, reduce the risk of mortality in CVD

consuming water with Mg only.

(Rylander et al. ; Kousa et al. ; Rasic-Milutinovic

In another study subjects received mineral water with

et al. ). This suggests that an intervention using a mixture

74 mg Mg, 30 mg Ca, and 0.3 mg K (Rylander et al. ).

of minerals is the most powerful tool for prevention. There

There was a signiﬁcant relationship between the acidity of

are, however, no data available to suggest the optimal con-

the urine, measured as urea, and the excretion of Mg, Ca,

centration of and relationship between Ca and Mg.

and K. Among persons with a high excretion of urea, there was an inverse relationship between the excretion of Mg and systolic blood pressure. After intervention with the min-

PREVENTION

eral water, there was a strong tendency for a decrease in systolic blood pressure among those with an initial high

A prerequisite for any kind of prevention on a population

excretion of urea and a low excretion of Mg, reﬂecting an

basis is well established evidence for causality. The data pre-

Mg deﬁciency.

sented for drinking water and CVD and the model for interaction (Figure 4) suggest that this is not the case, neither

Synthesis

for Mg, nor for Ca when given as single agents. On the other hand the concept of an intervention that mimics the real-life

The analysis of causality supports the hypothesis that minerals in drinking water are of importance for the risk of mortality in CVD, and particularly heart infarction. For Ca and Mg, physiological and cell function data document their importance in muscular contraction, muscle excitability and strength. The few intervention studies available support the hypothesis that minerals in the drinking water may inﬂuence the body homeostasis. A model for the relationship between drinking water constituents and mor-

situation in terms of several minerals together has considerable support. Owing to the lack of compelling evidence for the role of magnesium as a single contributory element in relation to CVD mortality there is little support for the concept of adding Mg to drinking water for preventive purposes on a population scale. The data suggest that a mixture of Ca and Mg is the appropriate measure but the exact proportion of the different minerals to be used is not known.

tality in CVD is presented in Figure 4.

CONCLUSIONS In summary, the review demonstrates the following: 1. Drinking water composition in terms of calcium, magnesium, and potassium is related to the risk of cardiovascular disease. 2. The available evidence is not sufﬁcient to recommend addition of magnesium to drinking water on a population basis. 3. Intervention studies to assess requirements for intervention and control of calcium and magnesium in drinking water are required. In view of the potential importance Figure 4

A model for drinking water constituents and mortality in cardiovascular disease.

from a public health point of view, such studies have a high priority.

R. Rylander

Magnesium and drinking water

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R. Rylander

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Rubenowitz, E., Axelsson, G. & Rylander, R.  Magnesium and calcium in drinking water and death from acute myocardial infarction in women. Epidemiology 10 (1), 31–36. Rubenowitz, E., Molin, I., Axelsson, G. & Rylander, R.  Magnesium in drinking water in relation to morbidity and mortality from acute myocardial infarction. Epidemiology 11, 416–421. Rylander, R.  Environmental magnesium deﬁciency as a cardiovascular risk factor. J. Cardiovasc. Risk 3, 4–10. Rylander, R.  Drinking water constituents and disease. J. Nutr. 138, 423S–425S. Rylander, R. & Arnaud, M. J.  Mineral water intake reduces blood pressure among subjects with low urinary magnesium and calcium levels. BMC J. Public Health 4, 56–65. Rylander, R., Bonevik, H. & Rubenowitz, E.  Magnesium and calcium in drinking water and cardiovascular mortality. Scand. J. Work Environ. Health 17, 91–94.

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Rylander, R., Remer, T., Berkemeyer, S. & Vormann, J.  Acidbase status affects renal magnesium losses in healthy, elderly persons. J. Nutr. 136, 2374–2377. Rylander, R., Tallheden, T. & Vormann, J.  Acid-base conditions regulate calcium and magnesium homeostasis. Magnesium Res. 22, 1–4. Rylander, R., Tallheden, T. & Vormann, J.  Magnesium intervention and blood pressure – a study on risk groups. Open J. Publ. Health 2, 23–26. Vormann, J.  Magnesium – nutrition and metabolism. Mol. Aspects Med. 24, 27–37. World Health Organization  Calcium and Magnesium in Drinking Water. World Health Organization, Geneva, pp. 1–194. Yang, C-Y.  Calcium and magnesium in drinking water and risk of death from cerebrovascular disease. Stroke 29, 411–414. Yang, C-Y. & Chiu, H-F.  Calcium and magnesium in drinking water and the risk of death from hypertension. Am. J. Hypertens. 12, 894–899.

First received 14 June 2013; accepted in revised form 22 August 2013. Available online 3 October 2013

12.1

2014

The ﬁrst recorded outbreak of cryptosporidiosis due to Cryptosporidium cuniculus (formerly rabbit genotype), following a water quality incident Richard L. Puleston, Cathy M. Mallaghan, Deborah E. Modha, Paul R. Hunter, Jonathan S. Nguyen-Van-Tam, Christopher M. Regan, Gordon L. Nichols and Rachel M. Chalmers

ABSTRACT We report the first identified outbreak of cryptosporidiosis with Cryptosporidium cuniculus following a water quality incident in Northamptonshire, UK. A standardised, enhanced Cryptosporidium exposure questionnaire was administered to all cases of cryptosporidiosis after the incident. Stool samples, water testing, microscopy slides and rabbit gut contents positive for Cryptosporidium were typed at the Cryptosporidium Reference Unit, Singleton Hospital, Swansea. Twenty-three people were microbiologically linked to the incident although other evidence suggests an excess of 422 cases of cryptosporidiosis above baseline. Most were adult females; unusually for cryptosporidiosis there were no affected children identified under the age of 5 years. Water consumption was possibly higher than in national drinking water consumption patterns. Diarrhoea duration was negatively correlated to distance from the water treatment works where the contamination occurred. Oocyst counts were highest in water storage facilities. This outbreak is the first caused by C. cuniculus infection to have been noted and it has conclusively demonstrated that this species can be a human pathogen. Although symptomatically similar to cryptosporidiosis from C. parvum or C. hominis, this outbreak has revealed some differences, in particular no children under 5 were identified and females were over-represented. These dissimilarities are unexplained although we postulate

Cathy M. Mallaghan Deborah E. Modha Health Protection Agency East Midlands, Leicester, LE3 8TB, UK Paul R. Hunter Norwich School of Medicine, University of East Anglia, Norwich, NR4 7TJ, UK Christopher M. Regan Health Protection Agency East Midlands, Nottingham, NG5 1PB, UK Gordon L. Nichols Gastrointestinal, Emerging and Zoonotic Infections Department, Health Protection Agency Colindale, London, NW9 5EQ, UK

possible explanations. Key words

Richard L. Puleston (corresponding author) Jonathan S. Nguyen-Van-Tam Epidemiology and Public Health, School of Community Health Sciences, University of Nottingham/Health Protection Agency East Midlands, Nottingham, NG5 1PB, UK E-mail: Richard.puleston@nottingham.ac.uk

| Cryptosporidium, outbreak, rabbit, water quality

Rachel M. Chalmers Cryptosporidium Reference Unit, Public Health Wales Microbiology, Singleton Hospital, Swansea, SA2 8QA, UK

INTRODUCTION We report an outbreak of Cryptosporidium cuniculus following a water quality incident in Northamptonshire, UK.

C. hominis and C. parvum predominate in causing human disease (Davies & Chalmers ). Oocysts may be found

Cryptosporidiosis is a faeco-orally transmitted diar-

in any faecally contaminated water and resist chlorine disin-

rhoeal illness, caused by species of the parasitic protozoan

fection as is commonly used in producing potable water

genus Cryptosporidium. There are at least 26 recognised

(Davies & Chalmers ; Medema et al. ; Yoder &

Cryptosporidium species (Chalmers & Katzer ), but

Beach ).

doi: 10.2166/wh.2013.097

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

In industrialised countries, symptoms are most com-

Journal of Water and Health

12.1

2014

Case deﬁnition

monly reported in young children (aged 1 to 5 years) (O’Donoghue ; Chalmers et al. a). Symptoms,

After the incident it was unclear if any cases of cryptospor-

which can be relapsing, persist for up to three weeks, and

idiosis would occur. Existing surveillance systems were used

longer in the immunocompromised (Hunter & Nichols ;

to identify possible cases using the following case deﬁnition:

Cacciò et al. ; Abubakar et al. ; Davies & Chalmers

‘cases of diarrhoea/gastrointestinal illness occurring in

). Non-C. parvum and non-C. hominis species and geno-

individuals residing in the affected area with microbio-

types cause disease mainly in the immunocompromised

logically conﬁrmed Cryptosporidium sp. (later tightened to

(Elwin et al. ).

C. cuniculus), having consumed mains water between 19

Cryptosporidiosis outbreaks have been associated with recreational and drinking water use, farm visiting, childcare facilities and contaminated foods and beverages (Baldursson & Karanis, ; Robertson & Chalmers ).

June and 06.00hours on 25 June (when the boil water notice was issued)’. A standardised, enhanced Cryptosporidium exposure questionnaire was administered by telephone, post or in

We focus on the epidemiological characteristics of cases

person to all cryptosporidiosis cases notiﬁed to the

arising from this outbreak and discuss the differences from

Health Protection Agency (HPA) in the weeks following

previous incidents.

the incident. Details of symptoms, water consumption, co-morbidities and medication history were obtained. Cryptosporidium sp. isolates were typed at the national

METHODS

Cryptosporidium Reference Unit, Singleton Hospital, Swansea (Chalmers et al. b).

On 23 June 2008, a small amount of Cryptosporidium sp.

The distance of each case’s home from the water treat-

oocyst contamination (0.0005 oocysts/L) was noted, by con-

ment

works

was

estimated

using

Microsoft

Corp.,

tinuous (but not real-time) inline water ﬁltration cartridge

MapPoint© (direct and by road (the latter chosen as water

monitoring (commenced 19 June), of the drinking water

pipes, in part, follow road routes)).

supply to approximately 258,000 people in central and western Northamptonshire (UK), provided from a surface

Statistical analysis

water reservoir and treatment works based in the county (Drinking Water Inspectorate ). Repeat sampling

All statistical analyses were undertaken in Stata (StataCorp

taken up to the evening of 24 June showed a further rise.

Inc., version 10). Case characteristics were summarised.

Local public health authorities were notiﬁed and a water

Interquartile ranges are reported for medians. The signiﬁ-

supply emergency declared at 06.00 hours on 25 June.

cance of the difference in means and proportions was

Control measures were instituted, including ‘boil water’

calculated with two group mean and proportion tests

messages to users in the locality (Drinking Water Inspecto-

respectively.

Parametric

(linear)

and

non-parametric

rate ). To assess the extent of contamination, further

(Spearman’s) regression analyses to assess the association

water sampling was undertaken (continuous ﬁltration and

between the date of onset of illness and volume of water

grab sampling) at strategic points in the distribution net-

drunk/distance from the water treatment works were esti-

work, including from end user sites. A search for

mated; p-values of 0.05 were considered statistically

biosecurity failures was instigated and remediation initiated.

signiﬁcant.

Network ﬂushing and storage reservoir decontamination was undertaken. Local health professionals were alerted and requested to

RESULTS

submit stool samples for laboratory analysis from suspected cases of cryptosporidiosis and to notify the local public

Between 09.29 hours on 19 June and 11.50 hours on 23

health authorities of such cases.

June, six oocysts in 11,848 L of treated water (0.0005

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Journal of Water and Health

12.1

2014

oocysts/L) were noted in a continuous ﬁlter cartridge

the drowned rabbit was the source of the outbreak (North-

sample, where normally none would be detected. Sampling

ampton Borough ).

repeated between 11.50 hours on 23 June and 20.00 hours

There were seven male and 16 female cases (30% male);

on 24 June showed a count of 418 oocysts in 5,064 L

this difference approached but was not statistically signiﬁ-

water (0.08 oocysts/L). Over the same period, no oocysts

cantly different (p ¼ 0.061). One of the cases and possibly

were identiﬁed in the raw water; an overwhelming of the

a second may have resulted from secondary infection. The

treatment capacity of the plant was therefore discounted.

mean age of cases was 32 (95% CI 26.6–37.4) years,

Source identiﬁcation centred on a biosecurity breach from

(males 33 years, females 32 years, p ¼ 0.90).

within the treatment works. During the incident the maxi-

All presented with diarrhoea. The ﬁrst developed symp-

mum count of oocysts noted was 1.7 oocysts/L (10 L grab

toms on 24 June and the last on 14 July. The mean date of

sample) on 26 June at a clean water storage reservoir site

diarrhoea onset was 2 July (1 July males, 2 July females,

distal to the treatment works. Sporadic oocysts were found

p ¼ 0.50).

up until 22 July from storage sites, although counts were

Monitoring data indicated that the ﬁrst possible date of

below 0.01/L by 2 July. Oocyst counts from end-user custo-

contamination was 19 June and the last 23/24 June. How-

mer taps, peaked at 0.19 oocysts/L (259 oocysts in 1,391 L)

ever, the oocysts count per litre increased 165-fold from

at one address on 25 June and 0.007 oocysts/L (9 in 1,166 L)

the sample completed on 23 June and the one commenced

at another on the same date and over a similar time frame –

on 23 June and completed on 24 June and then reduced

a 27-fold difference. Counts at customer taps dropped to

rapidly. Therefore, 23 June was assumed to be the most

below 0.01 oocysts/L by 29 June, but sporadic oocysts

likely date of contamination.

were found until 3 August. End-user monitoring continued until 5 August.

Using 23 June as the exposure date, the incubation period for C. cuniculus ranged between 1 and 21 days,

Investigations discovered (evening of 27 June) a fresh wild

mean 9.2 (95% CI 7.4–11.0) days; males 8.3 (6.3–10.2)

rabbit carcass (Oryctolagus cuniculus) immediately below the

days and females 9.6 (7.1–12.1) days, p ¼ 0.50). The

inlet pipe to a backwash granulated activated carbon tank. It

median incubation period was 8 [interquartile range (IQR)

was assumed that the oocysts had been released into the dis-

8–10] days; males 8 (IQR 7–10) days and females 8.5 (IQR

infection contact tank from the carcass. Defects in two vent

8–9.5) days. The mode was 8 days for both sexes.

covers and a granulated activated carbon tank access point

The epidemic curve was similar for men and women

had allowed the rabbit to enter the treated water (Northamp-

(Figure 1); however, there were two late presenting

ton Borough ; Drinking Water Inspectorate ). The

females who did not share an address with any of the ear-

remaining gut contents contained C. cuniculus gp60 gene sub-

lier cases, although one shared an address with a

type VaA18 oocysts (Chalmers et al. b).

symptomatic individual from whom microbiological con-

Up to the week ending 6 August, 32 microbiologi-

ﬁrmation was not obtained. The median duration of

cally conﬁrmed cases of cryptosporidiosis were notiﬁed.

diarrhoea was 13 (IQR 6–19) days, males 5.5 (IQR 5–13)

Twenty-three had C. cuniculus gp60 gene subtype VaA18

days and females 14 (IQR 9–20) days. The data distri-

and were therefore linked to the incident epidemiologically

bution and number of data points preclude reporting of

and microbiologically (Chalmers et al. b). Over the

the modal duration of diarrhoea. Other epidemiological

same period in the previous year, there were only four

cases characteristics, including other potential risk factors,

(unrelated) cases of cryptosporidiosis notiﬁed to the HPA

are shown in Table 1.

from the same geographic area as this incident. Few data were available from one (non-responder). Although oocysts

Water consumption

detected at end-user sites were not submitted for typing, seven water samples taken from other points in the net-

The median self-reported total daily mains water consump-

work had C. cuniculus gp60 gene subtype VaA18

tion was 2.3 L (IQR 1.6–3.3; mean 2.4 L); males 2.8 L (IQR

contamination, further strengthening the conclusion that

2.3–3.1; mean 2.7 L) and females 1.9 L (1.3–3.5; mean

R. L. Puleston et al.

Figure 1

Rabbit cryptosporidiosis outbreak

Journal of Water and Health

12.1

2014

Epidemic curve (percentage affected) by sex.

2.3 L). The median unboiled water consumption was 1.8 L

12.1) km by road. Neither correlated to the incubation

(IQR 1.0–2.4; mean 1.8 L); for males 2.0 L (IQR 1.8–2.4;

period; however, the duration of diarrhoea was negatively

mean 2.1 L) and females 1.6 L (IQR 0.75–2.7; mean

correlated to the direct distance from the water treatment

1.7 L). Median boiled water consumption was 0.6 L (IQR

works (Spearman’s rho 0.4847, p ¼ 0.0260).

0.3–1.0; mean 0.6 L), males 0.6 L (IQR 0.25–0.9; mean 0.6 L) and females 0.6 L (IQR 0.3–1.0; mean 0.6 L). The volume of mains water consumption (total,

DISCUSSION

unboiled or boiled) did not correlate to the incubation period or date of onset of diarrhoea. There was little correlation

between

diarrhoea

duration

and

total

Cryptosporidiosis in the UK

water

consumption (Spearman’s rho 0.02, p ¼ 0.99) or unboiled

Most human cases of cryptosporidiosis in the UK are due to

or boiled tap water (Spearman’s rho 0.07, p ¼ 0.77 and

C. parvum or C. hominis with other species appearing only

0.28, p ¼ 0.27 respectively).

occasionally (Chalmers et al. a; Davies & Chalmers, ; Elwin et al. ). Worldwide, this is the only reported

Distance from water treatment works

human outbreak of cryptosporidiosis caused by a Cryptosporidium species other than C. parvum or C. hominis (Elwin et al.

The median distance of cases from the water treatment

). Prior to this incident, only one case of C. cuniculus infect-

works was 6.24 (IQR 3.9–7.9) km direct and 9.8 (IQR 5.2–

ing a human had been reported (Robinson et al. ).

Table 1

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Journal of Water and Health

No. (%) of all

Signiﬁcance of difference

cases

between

reporting

sexes ( p)

22 (100)

–

Symptom

Vomiting Nausea

4 (18)

12.1

2014

parasites at a concentration that could affect human health.

Clinical characteristics of cases of Cryptosporidium cuniculus

Watery diarrhoea

0.91

Water companies adopt a risk management approach for controlling water supply pathogens informed by the World Health Organization Water Safety Plan for drinking water standards (World Health Organization ). Demographic characteristics of this outbreak

14 (64)

0.86

5 (23)

0.68

Abdominal paina

17 (77)

0.68

Abdominal crampsa

16 (73)

0.14

Flushes

10 (48)

0.27

Fever

10 (46)

0.10

Any history of bowel problems

10 (46)

0.22

Any other medical history including diabetes, rheumatological, immune suppression, prior radio or chemotherapy (none current)

11 (50)

0.34

Acid suppression medication/antacids

6 (27)

0.49

bility of inaccuracies as a result of recall bias, inaccuracy in

10 (46)

0.48

consumption estimates and assumptions made in the analy-

Mucousy diarrhoea

Potential clinical risk factor

On other medication of any sort (including oral contraceptive but excluding acid suppressors)

Sex ratio The predominance of females affected in this incident is unusual, although other outbreaks have shown similar patterns (MacKenzie et al. ; Mason et al. ). The difference approaches signiﬁcance. Possible explanations include: ﬁrst, although men drink more liquid per day than women (East ), women consume more unboiled tap water as a proportion of their intake possibly increasing their exposure. The water consumption data of cases do not support this; however, it is important to consider the possi-

Patient selected.

However, it is now clear that C. cuniculus is a human pathogen (Chalmers et al. b). Subsequent investigations have identiﬁed further sporadic cases in the UK (Chalmers et al. ).

sis where quantities were not clearly given (e.g. one cup). Secondly, men are less likely to seek medical advice so that positive microbiology and formal notiﬁcation may have been less available from them (Galdas et al. ; Noone & Stephens ). Thirdly, there may be a behavioural explanation, such as timing of consumption of plain water in males vs females. Finally, there may be an unexplained difference in response to infection between the sexes. The

Monitoring for Cryptosporidium oocyst contamination

outbreak was caused by C. cuniculus gp60 gene subtype

in potable water supplies

family Va (Chalmers et al. c). In subsequent investigations, it has been found that in sporadic C. cuniculus

Although not statutorily required to, the water company

cases the proportion of females affected is greater than

involved in this incident routinely checked for Cryptosporidium

males with Va subtype than Vb (Chalmers et al. ).

oocysts in both the raw and treated parts of the treatment system with continuous ﬁltration cartridges placed at strategic

Children and observed age pattern

points in the water ﬂow. These cartridges were periodically changed and examined for oocysts. Although ﬁltration analysis

Sporadic cryptosporidiosis mainly affects children aged 1 to

can detect contamination, it cannot determine viability, species

5 years in the UK. Even in waterborne outbreaks, where

type or pathogenic potential. For these reasons and because

there is often an increase in adult cases, children are

local immunity contributes to whether there is a hazard

mainly affected (Davies & Chalmers ). For example,

posed by the organism, there is no speciﬁc UK regulation stan-

in the outbreak of C. parvum in Clitheroe, Lancashire,

dard for acceptable counts of oocyst contamination of potable

UK, 52% of cases occurred in children <5 years old

water supplies. However, drinking water must not contain

(Howe et al. ).

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Journal of Water and Health

12.1

2014

This outbreak was unusual as no cases in children under

contamination at the treatment works may not have been

5 years old were microbiologically conﬁrmed, reported or

the date of exposure as the transit time of water through

epidemiologically linked. Plausible explanations include:

the distribution system is not uniform nor would water con-

the volume of water consumed by young children was insuf-

sumption behaviour be the same in all cases. A single

ﬁcient to provide an infectious dose; the alert occurred early

exposure date to calculate the incubation period is there-

in the morning, giving time for parents to protect their chil-

fore artiﬁcial. The peak oocyst counts were noted in the

dren with alternative drinking water sources or adults

network samples between 25 and 27 June with the mean

avoided giving potentially contaminated water to their chil-

onset date 2 July. It is possible that the incubation period

dren, but took less care for themselves. It is unlikely these

was therefore closer to 7 rather than the calculated mean

potential explanations would hold for all children so some

of 9.2 days. However, in the absence of more precise

cases in this age group would have been expected. It

data, other incubation period approximations would be

should be noted that the HPA’s syndromic surveillance

speculative.

system (S. Smith, Health Protection Agency, West Midlands, 2011 personal communication) showed an increase in diar-

Surveillance and attack rate

rhoea reports at the time in children in this age group from the affected area, suggesting an ascertainment bias in the

It is surprising that few conﬁrmed cases were identiﬁed

local reporting of cases.

given that approximately 258,000 people were potentially

Recent evidence on unusual cases of cryptosporidiosis

exposed (Drinking Water Inspectorate ). It is likely

indicates that the median age is older in non C. parvum/

that the number of cases identiﬁed through the active sur-

C. hominis infection in the UK (Chalmers et al. ;

veillance implemented following the incident is an

Elwin et al. ). Infection due to C. cuniculus matches

underestimate. There is evidence to support this. A report

this pattern. For some unusual Cryptosporidium spp., this

for the Consumer Council for Water interviewed individuals

may be due to differential exposure, e.g. foreign travel (how-

affected by the incident and noted that while some had been

ever, foreign travel as an explanation does not apply to this

ill, none had sought medical attention (Hunt et al. ). A

outbreak).

study examining syndromic data (NHS Direct data and GP consultations – Q Surveillance) identiﬁed a 25% excess

C. cuniculus infection characteristics, surveillance

above baseline of diarrhoea cases from the area at the

and risk factors

time of the incident (Smith et al. ) with an absolute excess of 422 cases above normal. This is compatible with

Incubation period

an established estimate of 15:1 for the true burden of disease for C. parvum and C. hominis compared with the number of

The incubation period estimated for this incident may be

conﬁrmed cases (Nichols et al. ; Smith et al. ).

under or overestimated: the continuous ﬁltering sample

Other research has suggested a lower ratio of 8.2 community

methodology does not allow precise estimation of when

cases to those notiﬁed and recorded in national surveillance

oocysts ﬁrst contaminated the ﬁnal water. Contamination

data (Tam et al. ). Nonetheless, the potential for differ-

could have occurred at any stage in that 4-day period,

ences in case ascertainment between the increased sur-

although the 165-fold increase in count obtained from the

veillance implemented as a result of the incident and that

sample taken between 23 and 24 June makes it reasonable

for routinely collected surveillance data (used to produce

to assume that the major contamination occurred on

the above ratios) may make such comparisons invalid. How-

23 June. The oocyst release was unlikely to be a single

ever, if correct, the disparity observed between notiﬁed and

‘pulse’ event; some lower level contamination may have

estimated excess cases may suggest that C. cuniculus has

occurred earlier which might explain the otherwise appar-

comparable levels of population level impact to C. parvum

ently short incubation period experienced by the ﬁrst case

and C. hominis and should therefore be viewed as a signiﬁ-

(symptoms commenced on 24 June). The date of

cant cause of waterborne disease.

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Volume of water

Journal of Water and Health

12.1

2014

possible that diarrhoea duration is consistent with oocyst excretion duration and therefore the infective dose ingested

The 2008 Phase-Two (summer) National Tap Water Survey

might explain this ﬁnding.

(NTWS) reports a mean of 1.329 L of tap water or 2.003 L total ﬂuid consumption per day – including other sources

Infective dose

(East ). The mean mains (tap) water consumption in cases arising from the incident was 2.4 L and median 2.3 L

The volume of water in the network would have diluted the

(IQR 1.6–3.3). The distribution of the consumption data

number of oocysts in any single litre of water. This supports

was not normal and could not be normalised. Formal stat-

the generally accepted view that the number of organisms

istical comparison of the mean water consumption from

required to be ingested to cause symptomatic infection is

the outbreak to that reported in the NTWS report is there-

very small (DuPont et al. ; Chalmers & Davies, ).

fore not possible. Nonetheless, the outbreak consumption

The maximum concentration of oocysts per litre of water

data possibly suggests a greater level of intake than is typi-

at the treatment works was below the former regulation

cally seen nationally. Greater dosing via higher water

treatment standard of <1 oocyst per 10 L of water (ceased

volumes consumed may provide an explanation for why

22 December 2007) (Drinking Water Inspectorate ),

these individuals were affected.

indicating the potential for infection to occur with very

Future NTWS reports could usefully describe all

low counts of C. cuniculus, as has been demonstrated in pre-

measures of central tendency as well as the mean for

vious C. parvum and C. hominis outbreaks (Mason et al.

water consumption to allow comparison to intake noted in

).

outbreaks (as in outbreak situations, small datasets are unlikely to be normally distributed).

Control measures and hazards

Distance from the water treatment works

Boil water notice

The duration of diarrhoea was statistically signiﬁcantly nega-

A boil water notice was instituted early on 25 June. A

tively correlated to the direct distance from the water

risk/beneﬁt-based decision to remove the boil water

treatment works. This may be a chance ﬁnding; however,

notice was made on 4 July. Four cases occurred after

it could be that oocyst counts were lower at points further

this date, two the day after the notice was lifted who

from the treatment works and viability could have also

would have probably been incubating the infection

declined over distance. However, there was evidence of

already and the others occurring on 13 and 14 July

some concentration of oocysts in storage reservoirs and at

respectively, who were thought to be secondary cases

customer taps but these were variable with some very low

and therefore unlikely to have acquired the infection

counts taken at similar times as higher counts at other

from consuming unboiled tap water. The boil water

locations. The complex structure of the network produces

notice removal appears appropriate despite sporadic

variable ﬂows of water over time and therefore unpredict-

oocyst detections from the network beyond that date

able pathogen distribution. Early in the incident (25 June),

(assuming the secondary cases were not independently

only four end-user points were tested. These may not have

infected from the very low residual counts rather than

been representative of oocyst load elsewhere at the same

from an infected contact and that routine surveillance

time when the loading was possibly at its highest. Nonethe-

systems did not miss other cases). Continued sporadic

less the magnitude of C. parvum infective dose inﬂuences

oocyst detections in the water network pose a dilemma

the time to and duration of oocyst excretion, but not clinical

for decision makers over what constitutes an acceptable

incubation period or severity of illness (DuPont et al. ).

count to allow for lifting a boil water notice. It cannot

However, others have found longer incubation periods with

be determined from this incident whether similar residual

lower infective doses (Chalmers & Davies ). It is

contamination parameters from a future C. hominis or

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Journal of Water and Health

12.1

2014

C. parvum incident could be used to determine when to

investigating the epidemiology of sporadic C. cuniculus

lift a boil water notice as the differing species may have

infection has corroborated these ﬁndings (Chalmers et al.

differing pathogenicity and infectivity patterns.

). It is not possible to conclude from this outbreak whether the observed epidemiological characteristics of C. cuniculus are unique to this species or artifactual. How-

Hazard

ever, other ‘unusual’ Cryptosporidium sp. differ in their This outbreak has demonstrated the hazard posed by wild-

epidemiology from C. parvum and C. hominis, although

life to the safety of mains potable water supplies. The

the numbers of cases are small and therefore conclusive

Drinking Water Inspectorate, although supportive of the

differences are difﬁcult to currently ascertain (Elwin et al.

water company’s handling of the incident, was critical of

).

their maintenance arrangements (Drinking Water Inspectorate , ). The importance of oocyst-typing to aid source identiﬁcation was highlighted. Although the source

ACKNOWLEDGEMENTS

was found early, had this not been the case, timely knowledge of the Cryptosporidium species or genotype could

The authors would like to thank the staff from the Health

have been helpful for directing investigations and control

Protection Agency East Midlands South, the Regional

measures (Drinking Water Inspectorate ).

Epidemiology team, Northamptonshire environmental health ofﬁcers and other members of the outbreak control team for assisting in data collection. They would also like to thank

CONCLUSIONS

Anglian Water for their assistance. This work was supported by the Health Protection Agency East Midlands, UK and

This outbreak was classiﬁed as being strongly associated

Public Health Wales as part of the normal reactive work of

with the consumption of mains drinking water on the

these organisations. No speciﬁc grant funding was provided.

basis that the pathogen identiﬁed in clinical cases was also

Richard L. Puleston, Cathy M. Mallaghan, Jonathan S.

found in water samples from the treatment works (Tillett

Nguyen-Van-Tam, Christopher M. Regan are now all

et al. ).

afﬁliated to Public Health England (PHE) following the

C. cuniculus has conclusively been demonstrated to be a human pathogen (Chalmers et al. b). The constellation

abolition of the Health Protection Agency and absorption of its functions into PHE. Deborah E. Modha is currently

of symptoms is similar to, but with some differences to other

afﬁliated

Cryptosporidium spp., especially the age and sex proﬁle, as

L. Nichols is now afﬁliated to European Centre for Disease

shown in Table 2 (Chalmers et al. a). Recent work

University

Hospitals

Leicester.

Gordon

Prevention and Control, Stockholm, 17183 Stockholm, Sweden.

Table 2

Epidemiological comparison between this outbreak of Cryptosporidium cuniculus and an outbreak of Cryptosporidium parvum in Clitheroe, Lancashire, UK

Clitheroe outbreak Percentage of all cases reporting

(Cryptosporidium parvum) (Howe

Epidemiological feature

from this incident

et al. 2002)

Age below 5 years

Sex ratio (M/F)

30/70

52/48

Vomiting

Abdominal pain

Abdominal cramps

–

Fever

CONFLICT OF INTEREST STATEMENT Richard L. Puleston was previously a domestic water customer of Anglian Water. Cathy M. Mallaghan and Deborah E. Modha are domestic water customers of Anglian Water. Paul R. Hunter was until 2010 chair of the science advisory board of Suez Environment and has acted as an expert medical witness in legal cases relating to waterborne outbreaks. Jonathan S. Nguyen-Van-Tam is a domestic water

R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

customer of Anglian Water. The other authors have no conﬂicts of interest to declare.

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R. L. Puleston et al.

Rabbit cryptosporidiosis outbreak

Robinson, G., Elwin, K. & Chalmers, R. M.  Unusual Cryptosporidium genotypes in human cases of diarrhea. Emerg. Infect. Dis. 14, 1800–1802. Smith, S., Elliot, A. J., Mallaghan, C., Modha, D., Hippisley-Cox, J., Large, S., Regan, M. & Smith, G. E.  Value of syndromic surveillance in monitoring a focal waterborne outbreak due to an unusual Cryptosporidium genotype in Northamptonshire, United Kingdom, June–July 2008. Euro Surveill. 15, 19643. Tam, C. C., Rodrigues, L. C., Viviani, L., Dodds, J. P., Evans, M. R., Hunter, P. R., Gray, J. J., Letley, L. H., Rait, G., Tompkins,

Journal of Water and Health

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2014

D. S. & O’Brien, S. J.  Longitudinal study of infectious intestinal disease in the UK (IID2 study): incidence in the community and presenting to general practice. Gut 61, 69–77. Tillett, H. E., De Louvois, J. & Wall, P. G.  Surveillance of outbreaks of waterborne infectious disease: categorizing levels of evidence. Epidemiol. Infect. 120, 37–42. World Health Organization  Guidelines for Drinking-Water Quality. World Health Organization, Geneva. Yoder, J. S. & Beach, M. J.  Cryptosporidium surveillance and risk factors in the United States. Exp. Parasitol. 124, 31–39.

First received 30 April 2013; accepted in revised form 24 August 2013. Available online 17 October 2013

12.1

2014

Evaluation of real-time PCR for quantitative detection of Escherichia coli in beach water Jason Tszhin Lam, Edwin Lui, Simon Chau, Cathie Show Wu Kueh, Ying-kit Yung and Wing Cheong Yam

ABSTRACT The current investigation evaluated the use of real-time polymerase chain reaction (PCR) for quantitative detection of Escherichia coli in marine beach water. Densities of E. coli in 263 beach water samples collected from 13 bathing beaches in Hong Kong between November 2008 and December 2009 were determined using both real-time PCR and culture-based methods. Regression analysis showed that these two methods had a significant positive linear relationship with a correlation coefficient (r) of 0.64. Serial dilution of spiked samples indicated that the real-time PCR had a limit of quantification of 25 E. coli colonies in 100 mL water sample. This study showed that the rapid real-time PCR has potential to complement the traditional culture method of assessing fecal pollution in marine beach water. Key words

| cyd, Escherichia coli, hybridization probes, marine beach water, real-time PCR

Jason Tszhin Lam Wing Cheong Yam (corresponding author) Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital Compound, Pukfulam Road, Hong Kong Special Administrative Region, China E-mail: wcyam@hkucc.hku.hk Edwin Lui Simon Chau Environmental Compliance Division, Hong Kong Government Environmental Protection Department, Wan Chai, Hong Kong Special Administrative Region, China Cathie Show Wu Kueh Ying-kit Yung Water Policy and Planning Division, Hong Kong Government Environmental Protection Department, Wan Chai, Hong Kong Special Administrative Region, China

INTRODUCTION Fecal indicator organisms, which reside in the gastrointesti-

Real-time polymerase chain reaction (PCR) is possibly a

nal tracts of humans and animals, are used globally to assess

quicker alternative to the traditional culture-based method.

microbial water quality of bathing beaches. In Hong Kong,

Its usability in detecting fecal indicator organisms in differ-

densities of Escherichia coli in marine beach water

ent

showed the best correlation with swimming-associated

(Haugland et al. ), agriculture watersheds (Khan et al.

illnesses

aquatic

ecosystems,

such

recreational

lakes

sewage-related,

), and beaches (Noble et al. ), has been evaluated.

water-borne pathogens, as well as fecal indicators including

The real-time PCR ampliﬁes and simultaneously quantiﬁes

enterococci (Cheung et al. , a, b). Quantitative

a speciﬁc DNA sequence of an organism. In each cycle of

detection of E. coli using traditional culture-based methods,

ampliﬁcation, a double-stranded DNA molecule is separ-

when

compared

with

other

however, cannot provide same-day notiﬁcation of beach

ated, hybridized with ﬂuorescent probes, and replicated if

conditions to the public. Before overnight culture followed

the speciﬁc DNA sequence is present. By real-time monitor-

by bacterial identiﬁcation, swimmers are still being exposed

ing of the ﬂuorescence emitted by hybridization probes

to deteriorated beach water. A reliable quantitative detec-

(HybProbes), the quantity of targeted sequences in samples

tion method with a shorter turnaround time is, therefore,

can be determined in an hour. The real-time PCR gives an

in demand.

opportunity for same-day evaluation of water quality. The

doi: 10.2166/wh.2013.038

J. T. Lam et al.

Quantitative detection of E. coli in beach water using real-time PCR

current investigation evaluated the use of real-time PCR for

Journal of Water and Health

12.1

2014

Water sampling

quantitative detection of E. coli in marine beach water from A total of 263 water samples were collected from 13 bathing

Hong Kong. Different gene candidates have been suggested for E.

beaches in Hong Kong between November 2008 and

coli detection, such as lacZ, uidA, and cyd (Bej et al. ;

December 2009. The beaches covered a wide range of

Horakova et al. ). The lacZ and uidA genes encode

water quality (ranked from ‘Good’ to ‘Very poor’), with sal-

beta-D-galactosidase and beta-D-glucuronidase, respectively,

inity in the range 0.1–34.1 parts per thousand and

which are common detection targets of biochemical assays

temperature in the range 12.4–31.9 C. The beach ranking

for E. coli (Bej et al. ). The cyd gene, which encodes

system in Hong Kong classiﬁes beaches into four different

cytochrome d terminal oxidase complex, was subsequently

ranks according the E. coli colonies per 100 mL beach

included in the detection panel to increase the speciﬁcity

water (Hong Kong Environmental Protection Department

of E. coli detection (Horakova et al. ). The current

). A beach with an E. coli count of less than 25 E. coli

investigation examined the speciﬁcity of lacZ, uidA and

colonies per 100 mL water is classiﬁed as ‘good’. The ‘fair’,

cyd genes for E. coli detection. The most speciﬁc gene can-

‘poor’ and ‘very poor’ ranks are equivalent to 25–180,

didate was later selected to quantify the densities of E. coli

181–610 and more than 610 E. coli colonies per 100 mL

in 263 samples collected from 13 bathing beaches in Hong

water, respectively. Sterilized 8 L polypropylene bottles

Kong using the real-time PCR platform. The results of real-

were ﬁlled at 1 m depth after removing the caps and rinsing

time PCR were compared with the densities enumerated

thoroughly while submerged. The ﬁlled bottles were emptied

using the traditional culture-based method in parallel. The

slightly to allow a 2.5 cm headspace. The capped bottles

limit of quantiﬁcation of real-time PCR was also determined

were then stored in a cooler packed with ice and transported

using serially diluted spiked samples.

to the laboratory for further processing. Culture methods

MATERIALS AND METHODS In the laboratory, bacteria in 1, 10 and 100 mL beach water samples were concentrated by ﬁltration using 0.45 μm mem-

Bacterial strains

brane filters (Advantec, Japan). The filters were then placed Ten different bacteria were used to examine the specificity of

on absorbent pads (AP10; Millipore, USA) soaked with

lacZ, uidA and cyd genes for E. coli detection. They

2 mL CHROMagar Liquid ECC (CHROMagar, France)

included E. coli, Enterococcus faecalis, Salmonella typhi-

and incubated at 44.5 C for 24 h. After incubation, E. coli

murium,

Shigella

ﬂexneri,

Klebsiella

pneumoniae,

colonies on ﬁlters were enumerated in accordance with

Aeromonas hydrophila, Proteus mirabilis, Pseudomonas

Ho & Tam (). E. coli appeared as blue colonies whereas

aeruginosa, Citrobacter freundii and Acinetobacter lwofﬁi.

other fecal coliform bacteria and other gram-negative bac-

E. coli, E. faecalis, S. typhimurium, S. ﬂexneri and K. pneu-

teria appeared as purple and colorless respectively. The

monia may be found in the human intestine, whereas A.

Vitek 2 system was used to conﬁrm the identiﬁcation of E.

hydrophila, P. mirabilis, P. aeruginosa, C. freundii and A.

coli on membrane ﬁlters. S. ﬂexneri in water samples were

lwofﬁi are present in the natural environment. E. coli

also detected using the American Public Health Association

(ATCC 25922), E. faecalis (ATCC 29212) and S. typhimur-

Method 9260E (American Public Health Association ).

ium

(ATCC

14028)

cultures

were

purchased

from

American Type Culture Collection (ATCC). The seven

DNA extraction

other bacterial species were clinical isolates collected by the Department of Microbiology, Queen Mary Hospital

Water samples (2 L) were ﬁltered using a 0.45 μm

of Hong Kong, as identiﬁed using the Vitek 2 system

membrane ﬁlter. The ﬁlter was then transferred into a

(bioMérieux, France).

50 mL centrifuge tube containing 10 mL STE buffer solution

J. T. Lam et al.

Quantitative detection of E. coli in beach water using real-time PCR

Journal of Water and Health

12.1

2014

[0.1 M sodium chloride, 10 mM tris(hydroxymethyl)amino-

DNA Master HybProbe, 3 mM magnesium chloride, 1 μM

methane (Tris) and 1 M methylenediaminetetraacetic acid

of each forward and reverse primers, 0.2 μM of each HybP-

(EDTA) at pH 7.6]. The centrifuge tube with the ﬁlter inside

robe and 10.4 μL extracted DNA. The primers and

was vigorously shaken for 10 min using the Vortex-Genie 2

HybProbes used in this study are shown in Table 1. Cycling

vortex mixer (Scientiﬁc Industries, USA). After removing

proﬁle was as follows: 10 min 95 C followed by 50 cycles of

the membrane ﬁlter from the tube, suspended bacterial

95 C for 10 s, 50 C for 20 s and 72 C for 15 s. After ampli-

cells in the STE buffer solution were pelleted at 10,000 rpm

ﬁcation, the instrument was cooled at 40 C for 30 s. The

for 30 min at 4 C. The bacterial pellet was resuspended

ramp rate was 20 C/s except for the annealing step, which

in 1 mL distilled water. Bacterial DNA was extracted

had a ramp rate of 5 C/s. The ﬂuorescence signal emitted

using the NucliSenseasyMAG system (bioMérieux, France)

from the HybProbes was acquired once at each annealing

according to manufacturer’s instructions.

step. Fluorescence channels used for monitoring ampliﬁca-

DNA of the 10 species of bacteria used to assess the

tion of lacZ, uidA and cyd genes were 640/530, 670/530

speciﬁcity of lacZ, uidA and cyd genes was also extracted

and 705/530 nm, respectively. Densities of E. coli in beach

using the NucliSenseasyMAG system. Samples of these bac-

water samples were determined using the absolute quantiﬁ-

teria were individually spiked into 2 L artiﬁcial seawater

cation mode in the LightCycler Software 4.0. The limit of

followed by membrane ﬁltration and DNA extraction. Simi-

quantiﬁcation of real-time PCR was estimated from assays

larly, external standards ranging from 101 to 107 E. coli

of the serially diluted external standards.

colonies in 100 mL water samples were incorporated into 2 L artificial seawater followed by membrane filtration and DNA extraction. These external standards were used to calculate the densities of E. coli in marine beach water samples during real-time PCR. In addition, a blank control was prepared by membrane filtration and DNA extraction of the artificial seawater without E. coli addition.

RESULTS AND DISCUSSION Speciﬁcity The speciﬁcity of lacZ, uidA and cyd genes for E. coli detection was evaluated by performing real-time PCR on 10

Real-time PCR

different bacterial species (see Table 2). The cyd gene was the most speciﬁc gene for E. coli detection among the

The real-time PCR was performed using the LightCycler 2.0

three genes tested, although it also exhibited a positive

Instrument and the LightCyclerFastStart DNA Master

result for S. ﬂexneri. However, no S. ﬂexneri was detected

HybProbe kit (Roche, Germany). Each 20 μL real-time

in any of the 263 beach water samples collected during

PCR reaction was kept in a LightCycler capillary tube

the study period. Therefore, the cyd gene was selected to

(Roche, Germany) and included 1 × LightCyclerFastStart

quantify the densities of E. coli subsequently.

Table 1

Gene

lacZ

uidA

cyd

Primers and HybProbes used in this study HybProbesa

Primers 0

5 -GGGTTGTTACTCGCTCACATT-3

5 -GCGCCCGTTGCACCACAG-FL

50 -CGGTTTATGCAGCAACGAG-30

50 -LC640-TGAAACGCCGAGTTAACGCCATCAA-PH

50 -GGAATGGTGATTACCGACGA-30

50 -TCCATGATTTCTTTAACTATGCCGGG-FL

50 -CGTCCACCCAGGTGTTC-30

50 -LC670-TCCATCGCAGCGTAATGCTCTACACC-PH

50 -GCGCTCTCTTTCTGGAGTGT-30

50 -GGCGAACTCGTCACTGACCGC-FL

50 -CCACCAGGAACAGGGTATACA-30

50 -LC705-GGCGATCTCATCTTCTCAATGGTGCTGA-PH

FL, ﬂuorescein; LC640, LightCycler Red 640; LC670, LightCycler Red 670; LC705, LightCycler Red 705; PH, phosphate.

Reference

Bej et al. () Bej et al. () Horakova et al. ()

Table 2

J. T. Lam et al.

Quantitative detection of E. coli in beach water using real-time PCR

12.1

2014

beach ranking system, even when they are present in the

Speciﬁcity of real-time PCR

lower detection range.

Real-time PCR resulta Bacterium

lacZ

uidA

cyd

Escherichia coli ATCC 25922

Enterococcus faecalis ATCC 29212

–

Salmonella typhimurium ATCC 14028

–

Shigella ﬂexneri

–

Klebsiella pneumoniae

–

Aeromonas hydrophila

–

Proteus mirabilis

–

Pseudomonas aeruginosa

–

Citrobacter freundii

–

Acinetobacter lwofﬁi

–

Journal of Water and Health

þ , detected; –, not detected.

Comparison between culture and real-time PCR methods Regression analysis in log-scale revealed that there was a significant positive linear relationship between the culture and the real-time PCR methods (Figure 1). The overall correlation coefficient (r) of these two methods was 0.64, which is similar to that reported in Haugland et al. () but lower than that in Noble et al. (). It is uncertain whether the lower correlation coefficients are due to sample size, because the sample sizes in the current study and Haugland and colleagues’ study were at least double that in Noble and colleagues’ study. Nevertheless,

Limit of quantiﬁcation of real-time PCR method

all the three studies showed a signiﬁcant positive linear relationship between the culture and the real-time PCR

Ampliﬁcation of serially diluted spiked samples indicated

methods.

that the real-time PCR had a limit of quantiﬁcation of 25

False-positive and false-negative rates of the real-time

E. coli colonies in a 100 mL water sample, which is similar

PCR were 5% (12 out of 263 samples) and 10% (27 out of

to the study by Khan et al. (). The ampliﬁcation efﬁ-

263 samples), respectively. The former may be due to the

ciency was 1.803. Results showed that the real-time PCR

presence of dead or viable but non-cultivable E. coli cells,

was capable of differentiating between the ‘good’ (<25

whereas the latter may be due to a low E. coli density or

E. coli colonies in 100 mL water sample) and the ‘fair’

the presence of PCR inhibitors, which all complicate the

(25–180 E. coli colonies in 100 mL water sample) ranks of

interpretation of real-time PCR.

Figure 1

Scatter plot and regression analysis of E. coli densities determined by culture and real-time PCR methods.

J. T. Lam et al.

Figure 2

Quantitative detection of E. coli in beach water using real-time PCR

Journal of Water and Health

12.1

2014

Turn-around-time of culture and real-time PCR methods.

PCR inhibition was reported to be frequently encoun-

same-day evaluation of water quality. The shorter turnaround

tered in real-time PCR by Haugland et al. (); in their

time could speed up closure or opening of beaches, thus redu-

study, bacteria were disrupted by beating with glass beads

cing health risks to many swimmers. The use of a mobile

and the released DNA was then used directly for real-time

laboratory, which analyzes water quality in situ, could even

PCR without puriﬁcation (Haugland et al. ). The current

further reduce sample delivery time, particularly for the lab-

study used the NucliSenseasyMAG system to extract and

oratories that are far from sampling sites (Lane et al. ).

simultaneously purify DNA. The DNA released from bac-

Nevertheless, a larger sample volume than for culture is

teria was bound to magnetic silica particles and it was

required for this real-time PCR protocol, which may reduce

possibly separated from inhibitory substances that may be

the overall number of samples that can be collected. Apart

present in the sample. Nevertheless, the current protocol

from a higher cost of DNA extraction and real-time PCR,

did not include an internal control to detect the presence

the real-time PCR instrument may not be easily available

of inhibitors. External DNA that is not normally present in

in many water quality laboratories. However, this protocol

the environment, such as salmon, may be incorporated in

is especially suitable for the laboratories which perform

the PCR as an internal control (Haugland et al. ).

real-time PCR on a daily basis.

More importantly, turnaround time of the real-time PCR was remarkably shorter than that of the culture-based method (Figure 2). The results of real-time PCR were available

CONCLUSIONS

within four hours after sample arrival, whereas the results of culture-based method were available a day after sample col-

The current investigation demonstrated that the real-time

lection. The real-time PCR opens up the opportunity for

PCR method had the potential to complement the traditional

J. T. Lam et al.

Quantitative detection of E. coli in beach water using real-time PCR

culture method of assessing fecal pollution in marine beach water. Regression analysis showed that a signiﬁcant positive linear relationship existed between the culture and the realtime PCR methods. Using HybProbes targeting the cyd gene, the real-time PCR was capable of quantifying as few as 25 E. coli colonies in a 100 mL beach water sample within four hours. The rapid real-time PCR makes the same-day evaluation of beach water quality possible. This shorter turnaround time could speed up closure or opening of beaches and thereby reducing health risks to swimmers.

REFERENCES American Public Health Association  Detection of pathogenic bacteria: Shigella. In Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC. Bej, A. K., McCarty, S. C. & Atlas, R. M.  Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with deﬁned substrate and plating methods for water quality monitoring. Appl. Environ. Microbiol. 57, 2429–2432. Cheung, W. H., Chang, K. C., Hung, R. P. & Kleevens, J. W.  Health effects of beach water pollution in Hong Kong. Epidemiol. Infect. 105, 139–162. Cheung, W. H., Chang, K. C. & Hung, R. P. a Variations in microbial indicator densities in beach waters and healthrelated assessment of bathing water quality. Epidemiol. Infect. 106, 329–344.

Journal of Water and Health

12.1

2014

Cheung, W. H., Chang, K. C., Hung, R. P. & Kleevens, J. W. b Epidemiological study of beach water pollution and healthrelated bathing water standards in Hong Kong. Water Sci. Tech. 23, 243–252. Haugland, R. A., Siefring, S. C., Wymer, L. J., Brenner, K. P. & Dufour, A. P.  Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Res. 39, 559–568. Ho, B. S. & Tam, T. Y.  Enumeration of E. coli in environmental waters and wastewater using a chromogenic medium. Water Sci. Tech. 35, 409–413. Hong Kong Environmental Protection Department  Ranking and grading Hong Kong’s beaches. Available from: www.epd. gov.hk/epd/misc/beach_report/2005/textonly/eng/ 4_ranking.htm (accessed 3 October 2013). Horakova, K., Mlejnkova, H. & Mlejnek, P.  Direct detection of bacterial faecal indicators in water samples using PCR. Water Sci. Tech. 54, 135–140. Khan, I. U., Gannon, V., Kent, R., Koning, W., Lapen, D. R., Miller, J., Neumann, N., Phillips, R., Robertson, W., Topp, E., van Bochove, E. & Edge, T. A.  Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds. J. Microbiol. Methods 69, 480–488. Lane, S. L., Flanagan, S. & Wilde, F. D.  Selection of equipment for water sampling (ver. 2.0): U.S. Geological Survey Techniques of Water-Resources Investigations, book 9, chap. A2. Available from: http://water.usgs.gov/owq/ FieldManual/Chapter2/Ch2_contents.html (accessed 3 October 2013). Noble, R. T., Blackwood, A. D., Griffith, J. F., McGee, C. D. & Weisberg, S. B.  Comparison of rapid quantitative PCRbased and conventional culture-based methods for enumeration of Enterococcus spp. and Escherichia coli in recreational waters. Appl. Environ. Microbiol. 76, 7437–7443.

First received 7 March 2013; accepted in revised form 28 August 2013. Available online 18 October 2013

12.1

2014

Assessing risk with increasingly stringent public health goals: the case of water lead and blood lead in children Simoni Triantafyllidou, Daniel Gallagher and Marc Edwards

ABSTRACT Previous predictions of children’s blood lead levels (BLLs) through biokinetic models conclude that lead in tap water is not a primary health risk for a typical child under scenarios representative of chronic exposure, when applying a 10 μg/dL BLL of concern. Use of the US Environmental Protection Agency Integrated Exposure Uptake Biokinetic (IEUBK) model and of the International Commission on Radiological Protection (ICRP) biokinetic model to simulate children’s exposure to water lead at home

Simoni Triantafyllidou (corresponding author) Daniel Gallagher Marc Edwards Civil and Environmental Engineering Department, Virginia Tech, Blacksburg, VA 24061, USA E-mail: striant@vt.edu

and at school was re-examined by expanding the scope of previous modeling efforts to consider new public health goals and improved methodology. Speciﬁcally, explicit consideration of the more sensitive population groups (e.g., young children and, particularly, formula-fed infants), the variability in BLLs amongst exposed individuals within those groups (e.g., more sensitive children at the upper tail of the BLL distribution), more conservative BLL reference values (e.g., 5 and 2 μg/dL versus 10 μg/dL) and concerns of acute exposure revealed situations where relatively low water lead levels were predicted to pose a human health concern. Key words

| acute, biokinetic model, chronic, distribution, reconstituted formula, variability

INTRODUCTION Lead in tap water and lead in children’s blood

CDC recently determined that lead in drinking water has been associated with US children’s EBL or with BLLs that

Plumbing materials containing lead (lead pipe, lead solder,

are higher than the geometric mean BLL (i.e., BLLs

brass and bronze plumbing components) may contaminate

>1.4 μg/dL) (US CDC a). A recent literature review

drinking water at the tap. In the USA, a mandatory lead

also highlighted several cases where contaminated tap

action level of 15 μg L

has been set for home taps and a

water was a major contributor to the BLL of US children,

voluntary lead standard of 20 μg L 1 has been set for

and further summarized epidemiological studies in the

school taps/fountains (US EPA , a). Drinking

UK, Germany, France, and Canada indicating that elevated

water has typically been considered a secondary exposure

lead in water can similarly contribute to children’s BLLs

source, accounting for up to 20% of total lead exposure

elsewhere (Triantafyllidou & Edwards ).

nationally (US EPA a), with deteriorating lead paint and contaminated dust/soil being the primary lead sources

Biokinetic models for BLL predictions in children

(Levin et al. ). In the past, although no levels of blood lead were

To explore relationships between environmental lead and

deemed safe, the US Centers for Disease Control and Pre-

blood lead of exposed children, biokinetic models are fre-

vention (US CDC) considered 10 μg/dL as the blood lead

quently used for supporting risk assessment decisions

level (BLL) of concern in children, elevations above which

when BLL data are not available (Pounds & Leggett ).

(i.e., elevated blood lead, EBL) cause detectable mental

Three biokinetic models have been commonly used in pre-

impairment and behavioral changes (US CDC ). The

vious research to predict BLLs from exposure to lead in

doi: 10.2166/wh.2013.067

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

water and other environmental media: (1) the US Environ-

mean BLL (US EPA ). This variability reﬂects differ-

mental Protection Agency (US EPA) Integrated Exposure

ences among children due to genetics and diets that result

Uptake Biokinetic (IEUBK) model for lead in children

in a different individual response (i.e., different BLL) to

(White et al. ; US EPA ); (2) the International Com-

the same lead dose.

mission on Radiological Protection (ICRP) model for lead in children and adults, also called the Leggett model (Leggett

Previous risk assessments

); and (3) the O’Flaherty model for lead in children and adults (O’Flaherty ). Evaluations/comparisons

Previous modeling research of health risks from lead in tap

among the three models are available elsewhere (US EPA

water (Table 1) has been limited. This research concluded

b; Equilibrium Environmental Inc. ).

that lead in tap water is not a primary concern for a typical

From these models, the IEUBK can additionally assess

child under representative scenarios of chronic exposure,

variability in predicted blood lead concentrations among

when applying the 10 μg/dL BLL of concern. For example,

children exposed to the same lead dose, by assuming log-

an IEUBK modeling approach which assessed lead

normality of predicted BLLs with a geometric standard devi-

exposure from tap water at an Australian home (Table 1)

ation (GSD) of 1.6 μg/dL around the predicted geometric

concluded that unless a typical child consumed water at

Table 1

Summary of previous work on the modeling of children’s BLL due to lead-contaminated drinking water and key conclusions

Previous work

Gulson et al. (1997)

Sathyanarayana et al. (2006)

Goal

Health risk assessment from elevated lead in water at a worst-case Australian home

Health risk assessment from elevated lead in water at 71 elementary schools in Seattle in 2004

Group(s) considered

Children aged 0.5–7 years, consuming all their water at home

Children aged 5–6 years, consuming half their water at a given school and half at home

Model used

EPA Integrated Exposure Uptake Biokinetic Model for Lead in Children (EPA IEUBK)

Model inputs

• •

WLL daily proﬁle (as measured in the home):100 μg/L ﬁrst-draw, 46 μg/L daily average Model default values for other background lead exposures (outdoor air, indoor air, outdoor soil, indoor dust and dietary intake)

• • •

WLLs from 71 elementary schools in Seattle obtained from Seattle Public Schools’ website preremediation:1–1,600 μg/L First Draw, 1–370 μg/L Second Draw WLLs at home assumed ﬁxed at 10.3 μg/L based on lead and copper rule sampling Soil lead content of 24 μg/g (as measured in Seattle) and model default values for other background lead exposures

Model outputs

Geometric mean BLL of children at the sampled home

Geometric mean BLL of children at each school

Scenarios considered

• • • • • •

Five WLLs at home: 4 μg/L (IEUBK default value) 46 μg/L, 50% first draw 46 μg/L, 100% first draw 100 μg/L, 50% first draw 100 μg/L, 100% first draw

50% of water consumed at a given school and 50% consumed at home: ‘Worst-case’: Exposure to 90th percentile lead-in-water concentration at a given school ‘Typical Case’: Exposure to median lead-in-water concentration at that school (i.e., 50th percentile)

Key predictions and conclusions

•

Children aged 1–2 years had geometric mean BLLs 10 μg/dL only when 100% of the consumed water contained 100 μg/L lead Hypothesized that if more than 0.5 L of ﬁrst-ﬂush water was consumed, by formula-fed infants or pregnant women, then the BLL would easily exceed the recommended level

• • • • • •

Geometric mean BLLs <10 μg/dL in all cases Drinking water exposures up to 10–15 times the EPA guideline are unlikely to result in EBL In Seattle, elevated school drinking water lead concentrations are not a signiﬁcant source of lead exposure in school-age children Further analysis needed only if water lead concentrations far exceed the EPA recommendations by ∼ 80–100 times

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

lead concentrations of approximately 100 μg/L, the BLL

CDC therefore recognizes the need to identify and protect

would not exceed the CDC’s 10 μg/dL level of concern

high-risk ends of the population distributions from environ-

(Gulson et al. ). IEUBK modeling of BLLs in students

mental contaminants, including both the most sensitive and

in Seattle (USA) exposed to high lead in school water

the most exposed individuals. The US Department of

(Table 1) also concluded that drinking water does not signiﬁ-

Health and Human Services has set the goal of eliminating

cantly contribute to high BLLs in children (Sathyanarayana

every single instance of EBL in children as part of its ‘Healthy

et al. ).

People 2020’ initiative (US DHHS ), and the CDC has also committed to that goal (US CDC c).

Children’s lead exposure and risk assessment considerations as they affect predictive BLL modeling

Such objectives increase the importance of identifying and addressing all potential lead sources especially in sensitive subpopulations. For example, infants consuming

Signiﬁcant new research/policies and risk assessment con-

reconstituted formula are considered a high-risk group,

siderations are explained below, which make it desirable

because tap water may account for more than 85% of their

to expand on the scope of previous modeling efforts.

total lead exposure (US EPA ). In addition, variations in genetics and diets produce a range of BLLs in a popu-

Public health concern and public sensitivity over lowerlevel lead exposure

lation in response to a ﬁxed lead dose (US EPA ). The few modeling efforts to assess water lead risks conventionally focused on the typical child through prediction of the

Although BLLs below 10 μg/dL in children are often con-

geometric mean BLL (corresponding to the 50th percentile

sidered ‘normal,’ they are nonetheless associated with

of the BLL distribution if the distribution is log-normal).

intellectual deﬁcits (Lanphear et al. ). In fact, evidence

They did not explicitly consider the response of more sensi-

of neurodegenerative, cardiovascular, renal, and reproduc-

tive subpopulations (i.e., the 90, 95 or 99th percentile BLL

tive effects at BLLs under 10 μg/dL, and as low as 1–2 μg/

in hypersensitive children drinking the same water dose)

dL have recently been summarized in the literature

or more exposed subpopulations (i.e., formula-fed infants

(Health Canada ). The European Union Risk Assess-

who consume greater water volumes). The IEUBK model

ment Report recently proposed 5 μg/dL as an epistemic

allows for both considerations.

BLL threshold for impacts of lead upon societal cognitive resources, and 1.2 μg/dL as a reference point for the risk

Acute health risk from lead exposure

characterization of lead when assessing intellectual deﬁcits in children measured by the full scale intelligence quotient

After two cases of severe lead-poisoning by accidental inges-

(IQ) score (EUSCHER ). The US CDC also recently

tion of lead-containing jewelry charms, one of which was

adopted a reduced ‘BLL reference value’ of 5 μg/dL (US

fatal (US CDC , ), the US Consumer Product

CDC b), lowering the previous ‘BLL of concern’ which

Safety Commission (CPSC) established 175 μg of lead in a

had been used in previous modeling efforts (e.g., in

piece of jewelry as a dose triggering acute health concerns,

Gulson et al. ; Sathyanarayana et al. ). The new

product recalls and ﬁnes, resulting in recalls of more than

reference value is based on the 97.5th percentile of the

150 million children’s jewelry pieces in 2004 alone (US

BLL distribution among surveyed children 1–5 years old in

CPSC ). In this work, it is considered reasonable that

the USA (US CDC b).

a similar dose of lead through water (a product intended for human consumption) would also be cause for acute

Risk assessment and public health policy target high-risk children (more sensitive or more exposed)

health concern. No previous studies of lead-in-water hazards have explicitly considered acute health risks in children. The ICRP allows simulating 1-day exposures through the model

Signiﬁcant progress has already been made in addressing EBL

inputs, as opposed to 1-year chronic exposures considered

for large percentages of the population (US CDC b). The

in the IEUBK model (US EPA ), and 1-month exposures

S. Triantafyllidou et al.

Water lead and blood lead in children

considered in the recent IEUBK batch run mode (Donohue et al. ).

Journal of Water and Health

12.1

2014

(1) Gulson et al. () on the IEUBK modeling of children’s BLLs from water exposure at home (Table 2).

The goal of this work was to revisit previous modeling

(2) Sathyanarayana et al. () on the IEUBK modeling of

efforts by expanding their analyses to reﬂect these additional

children’s BLLs from water exposure at a given school

considerations. Such risk assessment considerations are not

(Table 2). First-draw and second-draw water lead levels

limited to these previous cases (listed in Table 1). The cur-

(WLLs) as reported by Seattle Public Schools ()

rent work includes considerations that broadly apply to

were used for this analysis.

childhood lead exposure from water at school or at home in the USA and around the world, by attempting to better quantify health risks to children from this recognized lead source.

In both cases, water lead inputs were altered from the IEUBK model default of 4 μg/L in order to represent actual water lead measurements at those locations (see Table 2). Because other environmental media (e.g., air,

MATERIALS AND METHODS

soil, dust, diet) were not the primary focus of this study, model default values were used to represent lead levels in those environmental media (US EPA ): outdoor air at

Model simulations

0.10 μg/m3 (indoor air at 30% of outdoor air), outdoor soil The IEUBK model (win32 Version 1.1 build 9) was down-

at 200 μg/g, indoor dust at 200 μg/g, and dietary intake at

loaded from the US EPA website at http://www.epa.gov/

1.95–2.26 μg/day (depending on age).

superfund/lead/products.htm. The ICRP model (version

In addition to the chronic simulations of the IEUBK

3000, Microsoft Excel interface) was provided by the Syra-

model (Table 2), the ICRP model was also utilized to simu-

cuse Research Corporation.

late hypothetical scenarios of acute lead exposure from tap

IEUBK model simulations were undertaken to expand on the work of the following:

Table 2

water in children. Details for each of the three analyses follow.

Expanded modeling analyses undertaken in the present study to extend the scope of previous work to reﬂect modern public health goals for lead

Additional analysis (present work)

Extend previous goal to assess

Motivated by Gulson et al. (1997)

Motivated by Sathyanarayana et al. (2006)

•

• • Model water lead inputs

Model used Model predictions

• •

Output variability in children’s response to a ﬁxed lead dose (post facto application of the default GSD in IEUBK model) Health impacts in more exposed population (formulafed infants) Lower BLL cutoff values of 5, 2, and 1 μg/dL 100 μg/L WLL (worst-scenario of previous work) and other WLLs Higher water consumption for formula-fed infants, compared to model default

EPA IEUBK

• •

Distribution of BLL with emphasis on upper tail that reﬂects the most sensitive children, not just geometric mean (i.e., 50th percentile BLL) that reﬂects the typical child Percent of children with EBL at each BLL cutoff value, as a risk assessment criterion

• • • • • •

Input variability in WLLs at a given school, by evaluating specific quantiles of the observed WLL (not just 50 and 90th percentile) Lower BLL cutoff of 5 μg/dL Output variability in children’s response to a fixed lead dose (post facto application of the default GSD in IEUBK model) Variable WLLs (one fixed input at a time) from different fountains of one elementary school Inclusion of actual worst-case exposure to 100% percentile lead-in-water concentration

Distribution of BLL with emphasis on upper tail, not just geometric mean that reﬂects typical child Percentage of school children with EBL as a risk assessment criterion, not just geometric mean BLL

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

Biokinetic modeling of children’s BLLs from chronic

from different water fountains, and due to different

water exposure at home

individual patterns of lead uptake and biokinetics (US EPA ).

The IEUBK modeling undertaken by Gulson et al. () (see Table 1) was expanded by the following (Table 2):

•

Biokinetic modeling of children’s BLLs from acute water Considering the whole distribution of predicted BLL with of predicted BLL), and not just the geometric mean. While the geometric mean BLL reﬂects the potential health impact for average children, the upper tail of the BLL distribution reﬂects the more sensitive children within a given age group, due to individual genetic and

•

dietary factors affecting lead uptake and biokinetics. Considering lower BLLs, aside from the conventional 10 μg/dL BLL of concern. Speciﬁcally, 5 μg/dL, 2 μg/ dL, and 1 μg/L were examined, consistent with the lower BLLs recently proposed by the US CDC or the EU Scientiﬁc Committee on Health and Environmental

•

exposure

emphasis on the upper tail (75, 90, 95 and 99th percentile

Risks. Considering scenarios of formula-fed infants, consuming

Previous modeling efforts that utilized the IEUBK model (Gulson et al. ; Sathyanarayana et al. ) assessed chronic scenarios of lead-in-water exposures in children, encompassing the model output time step of 1-year. From the three available biokinetic models for lead in children (IEUBK, ICRP, O’Flaherty), the ICRP incorporates a daily exposure input and can thus allow exploration of shortterm lead exposures. ICRP modeling was undertaken to explore hypothetical scenarios of acute lead exposure through drinking water in children aged 5 years by the following:

•

much higher volumes of water through reconstituted for-

Predicting BLL from direct consumption of a single glass of water (250 mL) containing various levels of lead (0–5,000 μg/L). The higher levels of lead (in the order

mula milk, compared to school-aged children drinking

of hundreds, or thousands μg/L) have been reported in

the water. Speciﬁcally, water consumption of 800 mL/

some US ﬁeld investigations, mostly comprised of par-

day for 0–1-year-old infants relying on baby formula is

ticulate lead (Triantafyllidou & Edwards ). Aside

considered average (US EPA ; EU SCHER ), whereas 1,200 mL/day is considered high (Benelam &

from this one-time ingestion of the contaminated water,

Wyness ; EU SCHER ). The default daily water

all lead exposures (from soil, air, food, water) were

consumption for children who rely on water for direct consumption only, is set at a much lower volume of

•

assumed to be equal to zero. Predicting BLL from indirect consumption of the con-

200–590 mL/day in the IEUBK model, depending on

taminated water, through consumption of one portion

age (US EPA ).

of pasta cooked with the water (750 mL of water required

Biokinetic modeling of children’s BLLs from chronic water exposure at schools

for one portion) containing various levels of lead (0– 5,000 μg/L). Aside from this one-time ingestion of the contaminated food, all lead exposures (from soil, air, food, water) were assumed to be equal to zero. This scenario recognizes the potential for sorption of lead in food

The IEUBK modeling undertaken by Sathyanarayana et al. () (see Table 1) was expanded by the following (Table 2):

•

during

cooking with

large contaminated volumes

(Moore ). Speciﬁcally, concentration of the lead into food via adsorption (up to 83% lead absorption

Considering the whole distribution of observed WLL and

from water into pasta) has been demonstrated in one

corresponding predicted BLL. This approach allows esti-

experimental study (Smart et al. ), while this

mation of the percentage of the population that is

exposure pathway was recently implicated as the source

predicted to have EBL (i.e., BLL 10 μg/dL or more

of lead poisoning of two children in the USA (Copeland

recently 5 μg/dL) due to consumption of water

; Triantafyllidou & Edwards ).

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

geometric mean BLL to exceed the 10 μg/dL BLL of con-

RESULTS AND DISCUSSION

cern (Table 3, scenario 1). Stated another way, at a WLL Biokinetic modeling of children’s BLLs from chronic

of 100 μg/L, 50% of the exposed children are predicted to

water exposure at home

develop EBL (i.e., BLL 10 μg/dL).

Utilizing the IEUBK model, Gulson et al. () predicted

Children’s BLL sensitivity from chronic exposure and lower BLLs

that constant exposure to a WLL of about 100 μg/L (corresponding to ﬁrst-draw water in a sampled Australian home), resulted in exceedance of the BLL of concern for a

The extended analysis performed herein reveals that a

typical 1–2-year-old child (i.e., predicted geometric mean

lower WLL of 55 μg/L is sufﬁcient to elevate lead in blood

BLL > 10 μg/dL) (see Table 1). The authors qualiﬁed this

( 10 μg/dL) in 25% of the exposed children, whereas a

conclusion by stating that if more water was consumed in

WLL of 19 μg/L would be sufﬁcient to elevate lead in blood

drinks and formula using ﬁrst-draw water, then the BLL

in 5% of those children (Table 3, scenario 1). Children belong-

could easily exceed the recommended level (Gulson et al.

ing to these percentiles (i.e., 25, 10, 5 and 1% in Table 3)

). When the IEUBK modeling work of Gulson et al.

reﬂect more sensitive children within the population of

() for children aged 1–2 years was reproduced, a water

children, in comparison to the median response of a typical

lead level of 100 μg/L was indeed required in order for the

child (expressed by the 50th percentile in Table 3).

Table 3

Required level of lead in water for a given percentile of exposed children to exceed certain BLL cutoff values. Predictions obtained with IEUBK model under three exposure scenarios, with assumptions listed for each scenario

Predicted WLL required to exceed BLL cutoff value for:

BLL cutoff value (μg/dL)

50th percentile (50% exceed BLL)

75th percentile (25% exceed BLL)

90th percentile (10% exceed BLL)

95th percentile (5% exceed BLL)

99th percentile (1% exceed BLL)

30 μg/L

19 μg/L

3 μg/L

0 μg/Lb

SCENARIO 1: 1–2-year-old child drinking tap water Assumptions: 500 mL/day water consumption (IEUBK default value) GSD ¼ 1.6 μg/dL (IEUBK default value) Background exposures from other lead sources set to IEUBK default values 10 5

100 μg/La 24 μg/L

55 μg/L 7 μg/L

0 μg/L

SCENARIO 2: 0–1-year-old infant consuming reconstituted baby formula, average consumption Assumptions: 800 mL/day water consumption [EPA (); average consumption in EU SCHER ()] GSD ¼ 1.45 μg/dL [EPA () for formula-fed children] Exposure through diet set to 0 Background exposures from other lead sources set to IEUBK default values 10

60 μg/L

40 μg/L

28 μg/L

22 μg/L

13 μg/L

18 μg/L

11 μg/L

6 μg/L

4 μg/L

0 μg/Lb

18 μg/L

13 μg/L

SCENARIO 3: 0–1-year-old infant consuming reconstituted baby formula, high consumption Assumptions: 1,200 mL/day water consumption [high consumption in EU SCHER ()] GSD ¼ 1.6 μg/dL (IEUBK default value) Background exposures from other lead sources set to 0 50 μg/L

32 μg/L

23 μg/L

20 μg/L

14 μg/L

11 μg/L

9 μg/L

6 μg/L

7.3 μg/L

5.3 μg/L

4 μg/L

3.3 μg/L

2.5 μg/L

3.5 μg/L

2.5 μg/L

2 μg/L

1.6 μg/L

1.2 μg/L

Repetition of Gulson et al.’s (1997) work. All other results reﬂect additional analyses undertaken herein. Due to other background lead exposures (e.g. air and soil/dust), even 0 μg/L lead in water would still result in exceedance of a given BLL cutoff value.

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

If the BLL cutoff was set to the new and more stringent

with the Gulson et al. () assertion, formula-fed infants

level of 5 μg/dL, then 24 μg/L of lead in water is predicted to

(Table 3, scenarios 2 and 3) are expected to be much more

cause 50% of the population to exceed that BLL cutoff, and

vulnerable to even low-level lead contamination from tap

7 μg/L is predicted to cause 25% of the population to exceed

water, due to the large volumes of water required to recon-

the cutoff. However, no WLL is needed to cause exceedance

stitute infant formula and due to the high bioavailability

for 10% of the children’s population, due to other back-

factors of infants compared to school-aged children.

ground lead exposures assumed in the model (Table 3, scenario 1).

It should be noted that the IEUBK model assumptions of (a) log-normal BLL distribution in exposed children (US EPA ), and (b) a GSD ﬁxed at the model default value

Formula-fed infants and lower BLLs

of 1.6 μg/dL (US EPA ), or modiﬁed to 1.45 μg/dL for formula-fed children (US EPA ), are critical to these

For infants aged 0–1 years consuming the average volume of

conclusions. This is because the predicted risk (i.e., % of

baby formula milk daily in the presence of other background

children exceeding a certain BLL cutoff value) is sensitive

lead exposures, 60 μg/L of lead in water would elevate the

to the upper tail of the distribution function. The estimations

blood lead of 50% of the population, whereas 28 μg/L is pre-

for the upper tail values would be most affected if these

dicted to elevate the blood lead of 10% of the population

assumptions were not accurate. For example, some previous

(Table 3, scenario 2). Infants belonging to the upper percen-

work challenged the log-normal template of the model when

tiles (i.e., 25, 10, 5 and 1% in Table 3) reﬂect more sensitive

IEUBK predictions were compared to epidemiological data

infants within the exposed population, in comparison to the

in Polish children (Biesiada & Hubicki ). But the key

median response of a typical infant (expressed by the 50th

message would remain the same: identifying WLLs that pro-

percentile in Table 3). If the BLL cutoff was set at 5 μg/

tect the typical child is not an adequate risk assessment

dL, then much lower WLLs would achieve such percentage

approach, and quantiﬁed risks to more sensitive subpopu-

exceedances for exposed infants consuming formula

lations need to be considered, consistent with the US CDC

(Table 3, scenario 2). As expected, reducing the BLL

general policy for environmental contaminants.

cutoff lowers WLLs of concern to concentrations that were previously considered inconsequential, and below

Biokinetic modeling of children’s BLLS from chronic

20 μg/L lead (Table 3, scenario 2).

water exposure at schools

For infants aged 0–1 years consuming a high dose of baby formula in the absence of any other lead exposure

Sathyanarayana et al. () simulated ‘typical case’ and

source, even the smallest WLL is predicted to affect some

‘worst case’ scenarios of exposure (as termed in that work)

percentage of the population (Table 3, scenario 3). For

to water lead for 71 Seattle elementary schools, yielding

example, 50 μg/L of lead in water is predicted to elevate

relatively low predicted geometric mean BLLs (always

lead in blood above 10 μg/dL for 50% of that population,

<10 μg/dL) for each school, and the authors concluded that

but just 4 μg/L would be enough for 10% of the population

‘school drinking water is not likely to contribute to increased

to exceed a BLL of 2 μg/dL (Table 3, scenario 3).

BLLs in children’ (see Table 1). Reproducing this previous

While the three modeled scenarios (scenario 1, 2, and 3

work for children aged 5–6 years, exposed to the 50 and

in Table 3) are not directly comparable due to the different

90th percentile of the combined WLL distribution at one

assumed background lead exposures and GSD in each (see

elementary school and at home in Seattle (see Figure 1, com-

assumptions in Table 3), this analysis demonstrates that a

bined distribution) yielded a predicted geometric mean BLL

substantial proportion of the ‘most sensitive’ children

5.5 μg/dL. Speciﬁcally, the geometric mean BLL was

within the age group 1–2 years (those in the upper tail of

3.3 μg/dL for the previous work’s ‘typical’ water exposure

the predicted BLL distribution) may be adversely affected

scenario, and 5.5 μg/dL for the previous work’s ‘worst-case’

by WLLs much lower than the previously reported WLL

water scenario at that speciﬁc school (Figure 2, tabulated

of 100 μg/L (Table 3, scenario 1). In addition, consistent

data; all other lead inputs set to IEUBK model default values).

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

From the 71 Seattle elementary schools with publicly available WLLs (Seattle Public Schools ), this speciﬁc school was selected for this analysis due to the very high WLLs measured in 2004, which caused parental concern during that time. Remedial measures have since been undertaken in this and other Seattle schools, and post-remediation WLLs have now been reported to be much lower (Seattle Public Schools ) than the 2004 levels utilized in the historical analysis herein (Figure 1). Prior to this modeling exercise, the representative WLL distribution needed to be developed. Monitored school ﬁrstdraw and second-draw WLLs were combined with assumed Figure 1

home WLLs, using the approach of Sathyanarayana et al. Percentile lead distribution in ﬁrst draw, second draw and combined water of a Seattle elementary school in 2004 (i.e., pre-remediation, n ¼ 29). The horizontal dashed lines correspond to the 50, 90 and 99th percentile of the measured WLLs in that speciﬁc school. (Source of lead-in-water data: Seattle Public Schools (2007).)

(). That is, 50% of children’s daily water was consumed at school (comprising of 25% ﬁrst draw and 75% second draw, as measured at a given school). The remaining 50% daily water was consumed at home and was assumed ﬁxed at 10.3 μg/L, equal to the 90th percentile WLL measured in Seattle homes by the drinking water utility (Sathyanarayana et al. , see Table 1). Accounting for water lead exposure at home and at school created a combined WLL distribution (Figure 1). The range of WLL values from this combined distribution were run one-by-one through the IEUBK model, and the corresponding geometric mean BLL output was recorded. The IEUBK biokinetic component thus serves as the dose-response portion of the risk assessment, by relating WLL to BLL (Figure 2, tabulated data). Based on this geometric mean, log-normal distribution assumption, and

Figure 2

assumed GSD, the percentage of the exposed population Predicted log-normal distribution of BLLs in a population of 5–6-year-old students, exposed to the 50, 90 and 99th percentile of combined WLL

exceeding a given BLL cutoff could also be calculated by

concentration in a Seattle elementary school in 2004 (IEUBK model).

the IEUBK model (Figure 2, tabulated data). For example, if water was routinely consumed at the 50th percentile of

Overall predicted risk of EBL from water exposure at schools

the combined WLL distribution exposure (i.e., 16 μg/L in Figure 1), a child’s predicted likelihood of having EBL based on a 10 μg/dL cutoff is 1.0% (Figure 2, tabulated

While evaluating individual WLL inputs and corresponding

data). Likewise, exposure to the 90th percentile WLL (i.e.,

BLLs is informative, a more formal combination of exposure

45 μg/L) corresponds to a 10.2% likelihood of EBL, while

analysis and dose-response analysis can lead to an overall

exposure to the 99th percentile WLL (i.e., 208 μg/L) corre-

measure of risk. Children would normally drink from differ-

sponds to more than 80% predicted likelihood of EBL

ent school taps over the course of a day, so the entire WLL

(Figure 2, tabulated data). Similar outputs (i.e., % EBL)

distribution should be considered. This approach is illus-

could be obtained for other BLL cutoffs (e.g., the newly pro-

trated using the 2004 WLL distribution from one

posed 5 μg/dL). At the new BLL reference value of 5 μg/dL,

elementary school in Seattle.

the respective population exceedance would be 19.2% at

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

median water lead exposure, 58.1% at the 90% water lead

4.6% (dark-shaded area in Figure 3(a)). While such a risk

exposure and 99.1% at the 99% water lead exposure (data

is not indicative of an epidemic, parents and health experts

not shown).

would consider it worrisome that 5 out of 100 students

The percentage of exposed children’s population

attending classes in that speciﬁc school are predicted to

exceeding a BLL cutoff for the range of WLLs could then

develop EBL from drinking water consumption. For the

be plotted for two example BLL cutoff values: 10 and

new BLL reference value of 5 μg/dL, the corresponding

5 μg/dL (Figure 3(a)). By numerically integrating the area

risk was calculated to be 25.5% (the lighter gray shaded

under each curve over the entire distribution of WLLs (see

area in Figure 3(a)). This methodology was recently used

Figure 3(a)), the overall predicted risk of EBL for the stu-

by the authors to assess risk before and after remediation

dents at this school could be calculated. For the previous

of lead in drinking water at many other elementary schools

BLL of concern of 10 μg/dL this risk was calculated to be

of Seattle and Los Angeles (Triantafyllidou et al. ). This process can be extended to a range of BLL cutoffs from 2 to 15 μg/dL (Figure 3(b)). As expected, higher BLL cutoffs have a corresponding low risk, with very few children from the whole school exceeding the high BLL cutoff (Figure 3(b)). However, if the BLL cutoff is set at an even lower level of 2 μg/dL, then 83.5% of students attending classes in that school are predicted to exceed the cutoff value (Figure 3(b)). Overall, lowering the BLL cutoff resulted in non-linear increase in the estimated percentage of children with EBL at one Seattle elementary school in this 2004 historical analysis (Figure 3(b)). Biokinetic modeling of children’s BLLs from acute water exposure Simulated acute exposure to contaminated water or to food cooked with that water Preliminary modeling with the ICRP model suggests that one-time exposure of a 5-year old child to a single cup of water (i.e., 250 mL) containing high levels of lead can markedly raise the predicted lead in blood (Figure 4). Such levels of lead in water that elevate lead in blood from a one-time dose have been reported in numerous US ﬁeld investigations, as summarized by Triantafyllidou & Edwards (). These high levels were typically associated with particulate (rather than soluble) lead in water and indeed caused elevated lead in blood of some exposed individuals

Figure 3

Predicted percentage of children with elevated blood lead at distinct levels of water lead exposure observed in a Seattle elementary school in 2004 based on IEUBK model (Figure 3(a)). The combined WLL (μg/L) corresponding to each

(Triantafyllidou & Edwards ). Food that requires large volumes of water for cooking can

percentile is given on the secondary x-axis (Figure 3(a)). Integration of the

also concentrate lead from the water. This exposure pathway,

curves (i.e., calculation of highlighted area under the curves in Figure 3(a))

which has never been assessed in existing blood lead models,

estimates overall percentage exceedances depending on the chosen BLL cutoff value, for this historical case study (Figure 3(b)).

has the potential to increase blood lead of children through

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

must also be considered an acute health risk to children. Similarly, a one-time consumption of pasta cooked with 750 mL water containing 233 μg/L of lead, would result in a lead dose of 175 μg [i.e., (0.750 L) × (233 μg/L) ¼ 175 μg] and pose an acute health concern if all the lead was captured in the food. While water lead levels of 233 μg/L, 700 μg/L or higher are relatively rare, they have been detected in worstcase Seattle schools (e.g., in the school examined in this study) and other US schools or homes (Triantafyllidou & Edwards ), and could cause an acute health concern.

Figure 4

Predicted blood lead level of 5-year-old-child after direct consumption of lead-

LIMITATIONS

contaminated water or consumption of food cooked with that water. The ICRP biokinetic model was employed to predict maximum BLLs from one-time exposures.

Limitations of existing models apply not only to the work presented here, but also to results of previous research.

consumption of a single food portion, when assuming that

Speciﬁcally, potential limitations of the ICRP and IEUBK

100% of the lead present in water would sorb to the

models have been reported elsewhere (Pounds & Leggett

cooked food (Figure 4). Such a pathway was involved in iso-

; US EPA b; Equilibrium Environmental Inc.

lated cases of childhood lead poisoning due to food

). The numerical results presented in this paper are

consumption, when tap water was reportedly not consumed

based on certain modeling assumptions, and it is recognized

directly (Copeland ; Triantafyllidou & Edwards ).

that different assumptions would result in different results.

The predictions of the ICRP model from acute water

For example, Donohue et al. () proposed different

exposures (Figure 4) should be viewed with caution, because

default input values (e.g., for daily water consumption), com-

the ICRP model was shown to overestimate BLLs for similar

pared to the IEUBK default values used here. It is also

chronic exposure scenarios in children, when compared to the

recognized that the ability of the IEUBK model to predict

IEUBK model (US EPA b). In addition, it is possible that

the ‘tail’ of the BLL distribution is limited, which is why pre-

not all lead found in water will sorb to cooked food, as was

vious research was justiﬁed in restricting conclusions to the

assumed here. Nonetheless, the point of this analysis is that

typical case (i.e., predictions of geometric mean BLL only).

modeling acute exposures and cooking with contaminated

In addition, the high WLLs used in ICRP simulations

water can provide useful insights for the risk assessment of

are typically associated with particulate lead presence in

lead in water, that were not previously considered. Because

tap water, which may be less bioavailable compared to the

some lead in water may sorb onto cooked food, source-appor-

ICRP model default bioavailability factor of 30% for oral

tionment analyses should consider both direct drinking water

ingestion of lead. However, recent work by Deshommes &

consumption and indirect dietary impacts.

Prévost () on particulate lead suggests that this bioavailability

Applying the US CPSC lead acute health dose to water

factor

reasonable.

Regardless

potential

limitations, the general trends reported here reﬂect predictions based on available modeling tools.

If the US CPSC dose of acute health concern in children’s lead jewelry (175 μg lead in one piece of ingested jewelry as described in the Introduction) was applied to lead detected

CONCLUSIONS

in water, then the one-time ingestion of 250 mL of water containing 700 μg/L lead would result in an equal lead dose of

Repetition of previous modeling work is consistent with the

175 μg [i.e., (0.250 L) × (700 μg/L) ¼ 175 μg] and therefore

expectation that lead in tap water is not a major risk for a

S. Triantafyllidou et al.

Water lead and blood lead in children

Journal of Water and Health

12.1

2014

typical child under chronic exposure scenarios when apply-

and of the Robert Wood Johnson Foundation under the

ing a 10 μg/dL BLL for health concerns.

Public Health Law Research Program. Opinions and

Considering the whole predicted distribution of BLLs

ﬁndings expressed herein are those of the authors and do

(log-normal with a GSD of 1.6 μg/dL in the IEUBK

not necessarily reﬂect the views of the National Science

model) and not just the geometric mean BLL for a popu-

Foundation or the Robert Wood Johnson Foundation.

lation exposed to a ﬁxed WLL, reveals signiﬁcant health

Gary Diamond of the Syracuse Research Corporation

impacts for the most sensitive children at the upper tail of

graciously provided a user-friendly version of the ICRP

the distribution, even at low levels of water lead. From a

model (version 3000, Excel interface), and the US EPA is

risk assessment perspective, the upper tail of the BLL distri-

acknowledged for providing free on-line access to the

bution is critical in deﬁning risk. This is because it allows

IEUBK model (win32 Version 1.1 build 9 was available at

estimating the percentage of children predicted to exceed

the time this work was undertaken).

a certain BLL cutoff value, due to variations in genetics and diets that render them more sensitive compared to other children in that population.

REFERENCES

Explicit consideration of formula-fed infants, who are a high-risk group due to their small body weight and heavy reliance on water as a major component of their diet, also revealed signiﬁcant health concerns at relatively low WLLs (<50 μg/L). Investigating children’s lead exposure at one elementary school in Seattle, and acknowledging that children are exposed to an entire distribution of WLLs, led to a 4.6% overall predicted risk of BLL 10 μg/dL in children attending classes in that school in 2004 (pre-remediation), and 25.5% overall predicted risk of BLL 5 μg/dL. Expanding previous modeling analyses to consider lower BLL cutoff values (e.g., 5, 2, and 1 μg/dL versus 10 μg/dL) is consistent with increased public health concern about low level lead exposure, and indicated that relatively low WLLs (<24 μg/L) would have an adverse impact for high-risk groups (i.e., very young children and formula-fed infants). A reduction in the BLL reference value from 10 to 5 μg/dL also lowers the WLL of concern below 20 μg/L for certain high-risk subpopulations. Finally, acute exposures from direct water consumption or food cooked with contaminated water at the upper range of WLLs encountered in US school sampling events, have a potential to cause blood lead elevations 10 μg/dL or 5 μg/dL in children.

ACKNOWLEDGMENTS The authors acknowledge the ﬁnancial support of the National Science Foundation under grant CBET-0933246

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Leggett, R. W.  An age-specific kinetic model of lead metabolism in humans. Environ. Health Perspect. 101, 598–616. Levin, R., Brown, M. J., Kashtock, M. E., Jacobs, D. E., Whelan, E. A., Rodman, J., Schock, M. R., Padilla, A. & Sinks, T.  Lead exposures in US children, 2008: implications for prevention. Environ. Health Perspect. 116, 1285–1293. Moore, M.  Lead exposure and water plumbosolvency. In: Lead versus Health: Sources and Effects of Low Level Lead Exposure (M. Rutter & R. Russel Jones, eds). John Wiley and Sons Ltd, Chichester, UK. O’Flaherty, E. J.  A physiologically based kinetic model for lead in children and adults. Environ. Health Perspect. 106 (Suppl. 6), 1495–1503. Pounds, J. G. & Leggett, R. W.  The ICRP age-specific biokinetic model for lead: validations, empirical comparisons, and explorations. Environ. Health Perspect. 106(Suppl 6), 1505–1511. Sathyanarayana, S., Beaudet, N., Omri, K. & Karr, C.  Predicting children’s blood lead levels from exposure to school drinking water in Seattle, Washington, USA. Ambul. Pediatr. 6, 288–292. Seattle Public Schools, Drinking Water Quality Program, Results of water quality testing for Wedgwood elementary school, Updated January 29  Available from: www. seattleschools.org/modules/cms/pages.phtml?sessionid=75 e350d44895188cbff7ae58316ad1c6&pageid=2255 66&sessionid=75e350d44895188cbff7ae58316ad1c6 [Accessed October 7 2013]. Smart, G. A., Warrington, M., Dellar, D. & Sherlock, J. C.  Specific factors affecting lead uptake by food from cooking water. J. Sci. Food Agric. 34, 627–637. Triantafyllidou, S. & Edwards, M.  Lead (Pb) in tap water and in blood: implications for lead exposure in the United States. Crit. Rev. Environ. Sci. Tech. 42, 1297–1352. Triantafyllidou, S., Le, T., Gallagher, D. & Edwards, M.  Reduced risk estimations after remediation of lead (Pb) in drinking water at two US school districts. Sci. Total Environ. 466–467, 1011–1021.http://dx.doi.org/10.1016/j.scitotenv. 2013.07.111. US CDC  Briefreport: lead poisoning from ingestion of a toy necklace – Oregon, 2003. MMWR Morb. Mortal. Weekly Report 53, 509–511.

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US CDC  Preventing Lead Poisoning in Young Children. Atlanta, USA. US CDC (Centers for Disease Control and Prevention)  Death of a child after ingestion of a metallic charm – Minnesota, 2006. MMWR Morb. Mortal. Weekly Report 55, 340–341. US CDC a Lead in drinking water and human blood lead levels in the United States. Morb. Mortal. Weekly Report, Supplement 61, 1–9. US CDC b Response to Advisory Committee on Childhood Lead Poisoning Prevention Recommendations in ‘Low Level Lead Exposure Harms Children: A Renewed Call of Primary Prevention’. Available from: www.cdc.gov/nceh/lead/ acclpp/cdc_response_lead_exposure_recs.pdf [Accessed October 7 2013]. US CDC c Lead. Available from: http://www.cdc.gov/nceh/ lead/ [Accessed October 7 2013]. US CPSC (Consumer Product Safety Commission)  CPSC Announces New Policy Addressing Lead in Children’s Metal Jewelry. CPSC, Ofﬁce of Information and Public Affairs Available from: www.cpsc.gov/cpscpub/prerel/prhtml05/ 05097.html [Accessed October 7 2013]. US DHHS (Department of Health and Human Services)  Healthy People 2020 Summary of Objectives. Washington, DC. Available from: www.healthypeople.gov/2020/topicsobjectives2020/ pdfs/HP2020objectives.pdf [Accessed October 7 2013]. US EPA  Safe Drinking Water Act Lead and Copper Rule (LCR). Federal Register 56, 26460–26564. US EPA  User’s Guide for the Integrated Exposure Uptake Biokinetic Model for Lead in Children (IEUBK) Windows® version – 32 bit version. Publication EPA 540-K-01-005. US EPA  Memorandum on Risk of Elevated Blood Lead for Infants Drinking Formula Prepared with Tap Water. National Center for Environmental Assessment, Washington, DC. US EPA a 3Ts for Reducing Lead in Drinking Water in Schools. Revised Technical Guidance, EPA 816-B-05-008. US EPA b Air Quality Criteria for Lead-ﬁnal Report. US Environmental Protection Agency, Washington, DC, EPA/ 600/R-05/144aF-bF. White, P. D., Leeuwen, Van P., Davis, B. D., Maddaloni, M., Hogan, K. A., Marcus, A. H. & Elias, R. W.  The conceptual structure of the integrated exposure uptake biokinetic model for lead in children. Environ. Health Perspect. 106, 1513–1530.

First received 20 January 2013; accepted in revised form 8 September 2013. Available online 26 October 2013

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2014

Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography–mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay V. Ríos, I. Moreno, A. I. Prieto, M. E. Soria-Díaz, J. E. Frías and A. M. Cameán

ABSTRACT Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer7] MC-LR. In PCC7820, MC-LR, D-Asp3-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost

V. Ríos I. Moreno (corresponding author) A. I. Prieto A. M. Cameán Área de Nutrición y Bromatología, Toxicología y Medicina Legal, C/Profesor García González no. 2, 41012, Sevilla, Spain E-mail: imoreno@us.es M. E. Soria-Díaz Servicio de Espectrometría de Masas, Centro de Investigación, Tecnología e Innovación (CITIUS), C/Profesor García González, Sevilla, Spain J. E. Frías Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Avda Américo Vespucio 49, 41092, Sevilla, Spain

three-fold higher than in PCC7806 extracts. Key words

| ELISA, LC-MS, Microcystis aeruginosa, PCC7820, PCC7806, PP2A inhibition assay

INTRODUCTION Cyanobacteria blooms have been posing a worldwide

agriculture, livestock-watering and for drinking water

environmental issue in recent decades (Shao et al. ).

(Gurbuz et al. ).

Not all cyanobacterial blooms are toxic, with the mean fre-

Microcystis is one of the most common and widespread

quency of toxic bloom occurrence being 59%. The potency

genera of freshwater cyanobacteria throughout the world. In

of blooms can vary substantially according to sites, seasons,

most cases, it is the dominant component of phytoplankton

weeks, or even days. These variations could be due to

in eutrophic lakes. In temperate regions, Microcystis starts

changes in species composition, to the production of differ-

to grow in spring and, after growing exponentially during

ent toxins with varying toxicity for each clone, and to the

summer months, it moves into the stationary phase in late

inﬂuence of environmental factors (Robillot et al. ).

summer (Vasas et al. ). When a Microcystis bloom

There is a risk that blooms of these cyanobacteria contami-

occurs, a lot of biomass accumulates at the water surface for

nate water supplies that are used for recreation, ﬁsheries,

several days. The smallest Microcystis colonies may be

doi: 10.2166/wh.2013.088

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

composed of only a few individual cells, whereas the largest

for MC are highly speciﬁc, sensitive, quick to perform, inex-

ones may be composed of thousands of cells (Wu & Kong

pensive and require minimum sample processing (Gurbuz

). Microcystis aeruginosa is the most common and best-

et al. ). The most common ELISA technique used

known bloom forming Microcystis and it is the most promi-

(ELISA anti-MC-LR) is based on molecular recognition by

nent producer of cyanotoxins called microcystins (MC).

polyclonal antibodies that have good cross-reactivity with

These toxins are contained in Microcystis cells and are

MC-LR, MC-RR and MC-YR, but less reactivity with the var-

released when these cells die. Usually, cell-bound concen-

iants MC-LY and MC-LA (Ueno et al. ). This technique

trations of MC are several orders of magnitude higher than

has been applied to detect MC in water, freshwaters sedi-

extracellular toxins, whose concentrations vary from being

ments and cyanobacteria cultures (Moreno et al. ,

non-detectable to about 10% of the total (Jüttner & Lüthi

; Babica et al. ). In this ELISA technique, several

). The concentration of dissolved MC may be much

physicochemical variables (salinity, pH, etc.) can also pro-

higher in ageing or declining blooms when cell lysis is trig-

duce false positive MC results (Metcalf et al. ).

gered and the toxins are released to the environment

Moreover, an indirect competitive ELISA anti-Adda has

(Jähnichen et al. ; Jüttner & Lüthi ; Shao et al.

enabled the congener-independent detection of MC, which

; Sakai et al. ; Zegura et al. ). Currently, 80 var-

is based on recognizing Microcystis by speciﬁc antibodies

iants of MC are known and they differ widely in their

and has been used to determine these in water (Zeck et al.

abundance and toxicity (Gurbuz et al. ). These toxins

; Fischer et al. ) and in ﬁsh liver tissues (Ernst

have a structure cyclo-(D-alanine-X-D-MeAsp-Z-Adda-D-

et al. ; Moreno et al. ).

glutamate-Mdha) where Adda is (2S,3S,8S,9S)-3-amino-

The protein phosphatase inhibition assay (PPI assay)

9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid.

was developed on the basis that MC have the ability to inhi-

The toxins are named according to the two variable L-

bit serine-threonine protein phosphatase enzymes. Not all

amino acids at positions X and Z. Minor modiﬁcations can

MC variants react with protein phosphatase enzymes to a

occur on the other amino acids, such as demethylation on

similar extent and the assay is sensitive to protein phospha-

MeAsp or Mdha, or esteriﬁcation of glutamic acid. The most

tase inhibitors other than MC, such as okadaic acid (Rapala

frequently found MC include the leucine-arginine (MC-LR),

et al. ; Sevilla et al. ).

arginine-arginine (MC-RR), tyrosine-arginine (MC-YR) and

Conﬁrmatory techniques including liquid chromato-

leucine-alanine (MC-LA) variants (Gurbuz et al. ; Vasas

graphy (LC) with different detectors can be used, which

et al. ). MC-LR is the most toxic MC variant and the

allow the identiﬁcation and quantiﬁcation of individual

World Health Organization (WHO) has established a guide-

MC variants (Gurbuz et al. ). LC-mass spectrometry

line value of 1 μg/L for drinking water (WHO ).

(LC-MS) is the preferred technique to identify and quantify

MC inhibit eukaryotic protein phosphatases 1 and 2A,

MC, electrospray (ES) interface being the most applied inter-

and are recognized as potential tumour promoters and car-

face for this type of compound in environmental samples.

cinogens: in this sense, the International Agency for

This technique makes it possible to monitor precisely and

Research on Cancer classiﬁed MC-LR as a possible human

simultaneously various algae toxins in extracts from labora-

carcinogen, group 2B (http://www.iarc.fr/). In the last

tory cultures or algal blooms present in eutrophic waters

decade, evidence has been accumulating showing that MC

(Dahlmann et al. ; Cameán et al. ).

induce damage to the DNA, which means that they are gen-

M. aeruginosa PCC7806 has been frequently used for

otoxic and can act as tumour initiators. Hence, it is

different purposes, such as to study how some parameters

important to identify the production or potential synthesis

can affect the transcriptional response of some genes or

of these toxins in the environment to avoid even low-level

the MC-LR production (Dahlmann et al. ; Wiedner

exposure to humans (Zegura et al. ).

et al. ; Jähnichen et al. ; Tonk et al. ), topology

To date, several screening and analytical methods have

and toxicity of this strain (Jüttner & Lüthi ), or physio-

been developed for cyanotoxin detection and quantiﬁcation.

logical response to some chemicals (Shao et al. ). In

Enzyme-linked immunosorbent assays (ELISA) developed

these studies different analytical techniques have been

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

applied (LC coupled with photodiode-array detection LC-

The M. aeruginosa density of both cultures was obtained

DAD, ELISA, PPI assay, etc.) in order to detect cyanotoxins.

by microscopic counting of samples collected at intervals of

However, no information is available on the MC production

72 h for 21 days. Aliquots were taken and preserved in

kinetics of this strain determined by LC-MS. Robillot et al.

Lugol’s solution for cell counting. Cell density was measured

() carried out a hepatotoxin production kinetic study

on 1 mL samples using Sedgewick-Rafter chambers and an

of M. aeruginosa PCC7820 employing PP2A inihibition

inverted microscope (Olympus CKX41) according to the

assay and LC-MS technique.

Throndsen () method. After the period of 21 days, cul-

The aim of this work was to study MC production kin-

tures were harvested and concentrated by continuous

(PCC7806 and

centrifugation (23,428 g). The concentrated biomass was

PCC7820) during the growth period. Biomass and toxins

preserved at 80 C until lyophilization. The cyanobacterial

production were measured along with growth in controlled

biovolume of the samples was used to estimate the biovo-

cultures. LC-MS was used to identify and quantify the intra-

lume related to toxin concentrations of the different

cellular MC production of both strains. In the present study,

cultures.

etics of two M. aeruginosa strains

it has been detected for the ﬁrst time in M. aeruginosa

In addition, growth was monitored by measuring optical

PCC7806, a MC identiﬁed as [L-MeSer7] MC-LR. Moreover,

density at 680 nm (Wu et al. ) and chlorophyll a.

intracellular MC concentrations were measured by three

Measures of chlorophyll a were made following the

techniques: LC-MS, ELISA anti-Adda and PP2A inhibition

MacKinney () method. Growth rates were calculated

assay in order to compare them.

from OD values of the cultures measured photometrically at 680 nm. The OD was determined in 1.5 mL aliquots of culture taken at intervals of 72 h throughout the study. Speciﬁc growth rates were calculated according to the

MATERIALS AND METHODS

equation (Wu et al. ):

Cyanobacteria culture

μ ¼ ðlnOD2 lnOD1 Þ=t2 t1

M. aeruginosa PCC7806 was used in this study in order to establish for the ﬁrst time its MC production kinetics deter-

Intracellular MC extraction

mined by LC-MS since no information has been available. M. aeruginosa PCC7820 was used to compare the MC pro-

A subsample of 100 mL of a cell suspension was removed

duction between strains of the same cyanobacteria species

for analysis at each sampling time (72 h). Cells and culture

cultivated under the same conditions.

media were separated by ﬁltration through glass ﬁbre

Axenic cultures of both planktonic cyanobacteria strains

APFC 47 mm discs (Millipore). Discs obtained at each

(M. aeruginosa PCC7820 and PCC7806) were obtained from

sampling time and the concentrated biomass obtained at

the Pasteur Culture Collection. Both of them were main-

the end of the study were lyophilized prior to the extraction

tained in sterilized 250 mL Erlenmeyer ﬂasks containing

of the MC, which was carried out according to Moreno et al.

100 mL of BG11 medium (þ1 M K2HPO4·3H2O þ 5 mM

(), using 0.1 M acetic acid and a mixture of methanol/

NaNO3 þ 12 mM NaHCO3) at 30 C under continuous illu-

chloroform (1:1 v/v) solution. After sonication in an ultra-

sound bath for 15 min and stirring for 30 min at room temp-

provided by cool white ﬂuorescent tubes. Sterile air was

erature, the cell suspensions were centrifuged. For a

bubbled through the culture medium. Once cultures reached

quantitative extraction of the toxins, this procedure was per-

1.5 ± 0.2 units of optical density (OD), they were transferred

formed twice. The toxin-containing fractions were eluted in

to bottles containing 20 L of BG11 medium. An OD ¼ 0.1

0.5 mL of methanol. The ﬁnal extract was divided into three

was set (time 0) as the initial culture density for each

aliquots, one of them was directly injected into the LC-MS to

growth period, according to Preuβel et al. ().

identify and quantify the MC present in the samples, and the

mination with an intensity of 28 μmol photons m

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

other two were dried and resuspended in water in order to

with an electrospray ion source. The analytical column was

analyse the intracellular MC content by ELISA anti-Adda

a LiChroCART 250-4 reversed-phase column with a particle size of 5 μm (Merck kGaA). The ﬂow rate was 0.4 mL/min.

and PP2A inhibition assay.

Chromatographic separation was performed using a binary ELISA anti-Adda assay

gradient consisting of (A) water, and (B) acetonitrile. Both components contained 0.05% triﬂuoroacetic acid (v/v). The

For determination of the intracellular MC by ELISA, a com-

elution proﬁle was: 35% B (2 min), 35% up to 65% B

mercially available competitive anti-Adda ELISA kit

(15 min), 65% B (5 min), 100% B (3 min). Injection volume

(Abraxis LLC, USA) was used. This assay uses an antiserum

was 10 μL. Single ion monitoring (SIM) experiment was

raised against the unique C20 amino acid Adda, which is

applied for the detection of MC, where selected ions were

common to all MC variants. The main reason to employ

monitored at Q1 (m/z 995.6 for MC-LR, m/z 981.5 for

this anti-Adda ELISA assay to detect MC is because of the

[D-Asp3]MC-LR, m/z 1013.6 for [MeSer7]MC-LR, m/z 1009

high variability of compounds that might be found in the cul-

for Dglu(OCH3)MC-LR, m/z 1002.5 for MC-LY, m/z

tures. Detection based on ELISA anti-MC-LR is limited to

1025.5 for MC-LW and m/z 986.5 for MC-LF). The ion

the hepatotoxins MC-LR, MC-LA, MC-RR, MC-YR and

source was operated in positive mode. The capillary voltage

nodularin, which are known to have cross-reactivity (Wied-

and declustering potential were set to 5,500 and 105 V,

ner et al. ). Samples were analysed according to the

respectively. Curtain gas, nebulizer gas (gas 1) and heater

manufacturer’s instructions, previous calibration and veriﬁ-

gas (gas 2) were 35 psi, 60 psi and 60 psi, respectively, and

cation with a control standard. The negative control, the

the Turboprobe temperature was maintained at 350 C.

standards and samples were all analysed in duplicate. Microtitre plates were read at 450 nm and B0 values (%)

Chemicals

calculated. Results were expressed as micrograms of MC/L. All reagents were of LC grade or analytical grade. Methanol, Protein phosphatase 2A inhibition assay

acetonitrile and triﬂuoracetic acid were purchased from Merck. LC-quality water was obtained by purifying deminer-

PP2A inhibition assay was made using a commercially avail-

alized water in a Milli-Q ﬁltration system (Millipore). MC-

able kit (MicroCystest, Zeu-Inmunotec S.L.) following the

LR, MC-RR and MC-YR standards were supplied by Alexis

Sevilla et al. () method. The assay is based on the degra-

with a purity of 99%.

dation of p-nitrophenyl phosphate into p-nitrophenol. Inhibition rate is directly related to the absorbance at 405 nm measured by a plate reader (Titertek Multiskan).

RESULTS AND DISCUSSION

MC concentration was obtained by reporting the inhibition rate on a standard MC-LR calibration curve. The calibration

Cyanobacterial growth

curve was determined using inhibition percentages and logtransformed concentrations of MC-LR standards, and used

Triplicate measures of cellular biomass, chlorophyll a and

to calculate toxin concentrations (MC-LR equivalents,

OD were performed in order to study the cyanobacterial

μg/L) of unknown samples.

growth. The growth dynamics were different between both strains (Figure 1(a)). The stationary phase of PCC7806 was

Chromatographic conditions

not observed after 21 days, whereas PCC7820 reached this point after 12 days. The growth during the culture time of

Chromatographic separation was performed using a Perkin-

PCC7806 was higher than PCC7820 (1.7-fold) with a maxi-

Elmer Series 200 LC system coupled to an Applied Biosys-

mum cellular concentration of 6.22 × 107cells/mL and

tems

3.68 × 107cells/mL, respectively. Robillot et al. () also

quadrupole-linear ion trap mass spectrometer equipped

observed that PCC7820 achieved the stationary phase after

QTRAP

LC-MS/MS

system

consisting

V. Ríos et al.

Figure 1

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

Comparison of the cell growth of both strains (PCC7806 and PCC7820) during 21 days of culture (a) and quantiﬁcation of number of cells, levels of chlorophyll a and OD of both strains (b) PCC7806 and (c) PCC7806. Values are given as means ± standard deviation (n ¼ 3).

12 days reaching a maximum cellular dry weight of 60 mg,

the growth estimation as previously stated by Wang et al.

corresponding to 600 mg/L. On the other hand, the

(). In the present study, we observed that chlorophyll a

growth of PCC7806 was studied by Shao et al. ()

content showed an increasing trend with increasing growth

during 10 days of culture, obtaining a maximum level of

(number cells/mL) of the strain PCC7806 in agreement

cells of 4 × 107 cells/mL. Sevilla et al. () also studied

with the results observed by Downing et al. (). However,

the growth of this strain obtaining different growth curves

this trend was not clear in M. aeruginosa PCC7820 in our cul-

depending on the nitrogen availability. Thus, with 2 mM of

ture conditions. The maximum quantiﬁcation of cell numbers

nitrate (Pasteur Institute recommendations) the maximum

for this strain did not correspond to the highest chlorophyll a

concentration of cells was ≈4 × 107 cells/mL and with

content (Figure 1(c)).

20 mM (nitrate excess) an outstanding growth of the cells

During the study period (21 days) the maximum chloro-

(≈6 × 107 cells/mL) was observed after 15 days of culture.

phyll a level in PCC7806 culture was reached after 21 days

In our study, at 5 mM NaNO3 the quantiﬁcation of cells

(4 μg/mL), together with the highest cell number. In

after 15 days of culture was 4 × 107 cells/mL for PCC7806.

PCC7820, the highest level was reached at day 9 (2 μg/

Culture growth of M. aeruginosa PCC7806 and PCC7820

mL), whereas the maximum cell number was reached after

was followed using three different parameters: quantiﬁcation

12 days. In our culture conditions, both strains reached

of number of cells, levels of chlorophyll a and OD. The vari-

lower levels of chlorophyll a at 15 days (2.7 μg/mL

ation of M. aeruginosa PCC7806 growth is synchronous with

PCC7806, 1.37 μg/mL PCC7820) than those reported by

chlorophyll a levels (Figure 1(b)), which could be useful for

Sevilla et al. () with PCC7806 (∼5.3 μg/mL). However,

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

our results agree with the levels obtained by Wu et al. ()

previously by del Campo & Ouahid () in M. aeruginosa

who observed that the chlorophyll a concentration of

UAM 1303 strain. It should be noted that serine is the bio-

PCC7806 increased with time (≈4 μg/mL at 20 days).

synthetic precursor of dehydroalanine, so this toxin

Speciﬁc growth rates under the given conditions ranged from 0.21 to 0.033 d

and 0.25 to 0.004 d

for PCC7806

together with the desmethylated MC-LR could be biogenetic precursors of LR (Namikoshi et al. ). Moreover, the complete identiﬁcation of six different

and PCC7820 strains, respectively. The growth rates of both strains decreased during the cultivated period and

were similar to those calculated by Wu et al. () for

(Figure 2(b)). This analysis conﬁrmed the presence of MC-

Microcystis PCC7806 after 20 days of culture.

LR, D-Asp3-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW

Microcystis

PCC7820

has

been

performed

Comparing the three parameters measured, cell num-

and MC-LF. Table 1 shows the monoisotopic m/z ratios of

bers, chlorophyll a and OD, we found that chlorophyll a

[M þ H]þ ions for all these MC. The MC proﬁle of Microcys-

correlated well with OD and with cell number in the

tis PC7820 observed in this study was similar but not

PCC7806 strain. However, for the PCC7820 strain, chloro-

identical to that described by Lawton et al. (). In this

phyll a correlated better with OD rather than with cell

sense, MC-LM detected previously by these authors was

number. Thus, under our conditions, the results for these

not identiﬁed during the present work and D-Asp3-MCLR

three parameters are not always consistent with each other.

observed in the present study was not detected by these authors. The relative abundance of MC-LR (79%), the

Identiﬁcation and quantiﬁcation of MC produced by M.

major variant detected, agrees with the percentage pre-

aeruginosa PCC7806 versus PCC7820

viously found by Lawton et al. (). Moreover, Bateman et al. () identiﬁed seven MC variants, MC-LR being pre-

LC-MS was used to separate and identify MC in the cellular

dominant; MC-VF and MC-AR were also detected, but D-

extracts of both strains. Since MC are mainly accumulated

Asp3-MCLR was not identiﬁed. Robillot et al. () have

within the cell and extracellular toxins only contributed

reported the presence of three new variants, desmethylated

marginally to the total MC concentration (Jänichen et al.

MC-LW, desmethylated MC-LF and MC-LL, which we did

; Jüttner & Lüthi ; Shao et al. ), we determined

not observed. According to these authors, MC-LR, MC-LW

intracellular MC content.

and MC-LF were the major toxins, whereas in the present

The chromatogram of the Microcystis PCC7806 extract

study the main toxins were MC-LR, D-Asp3-MCLR and

shows three MC (Figure 2(a)), one of them being identiﬁed

Dglu(OCH3)-MCLR. The discrepancy between the MC pro-

for the ﬁrst time in this strain. The assignation of the ions

ﬁles could be due to differences in the methods used for MC

obtained from the fragmentation of MC is presented in

extraction, puriﬁcation, LC-MS determination, as well as in

Table 1. The majority was MC-LR at m/z 995.6, representing

culture conditions (del Campo & Ouahid ).

81% of total MC (Figure 2(a)). This toxin was identiﬁed

The total amount of MC was estimated by measuring the

using its respective commercial reference. The compound

area of the highest UV absorption peaks at 238 nm. However,

detected at 981.5 Da corresponds to a demethylated MC-

only the commercial standard of MC-LR was available imply-

LR (D-Asp3-MCLR). The presence of both MC in extracts

ing that, theoretically, only this toxin could be quantiﬁed.

from Microcystis PCC7806 has already been described in

Consequently, according to Robillot et al. (), we approxi-

previous studies; however, their objective was to study the

mated that UV absorbance coefﬁcient at 238 nm is identical

impact of some parameters in the MC production (Downing

for all MC, as it has been veriﬁed for the three available stan-

et al. ; Jähnichen et al. ; Sevilla et al. ), but no

dards (MC-LR and MC-RR/MC-YR not detected in this

MC production kinetic during growth was described. The

study). Thus, quantiﬁcations were made using MC-LR cali-

third compound was detected at m/z 1013.6, which has

bration curves, admitting a systematic error in the case of

been detected for the ﬁrst time in this strain, and could cor-

tryptophan and tyrosine containing MC.

respond to a MC-LR variant with methyl serine (MeSer) at

The quantiﬁcation of the intracellular MC identiﬁed in

position 7 ([L-MeSer7] MC-LR), which has been identiﬁed

both strains at the end of the period of culture is shown in

Figure 2

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

LC-MS chromatogram of M. aeruginosa. (a) PCC7806 cellular extracts. Electrospray mass spectra were associated with MC-LR, demethylated MC-LR and [MeSer7] MC-LR. (b) PCC7820 cellular extracts. Electrospray mass spectra were associated with MC-LR, demethylated MC-LR, Dglu(OCH3) MC-LR, MC-LY, MC-LW and MC-LF.

Table 2. The total MC content detected in M. aeruginosa

proportion increased signiﬁcantly (∼9-fold) in D-Asp3-MC-LR

PCC7820 was higher than in PCC7806 extracts (∼3-fold).

levels (92,000 ± 10,120 ng/g versus 10,600 ± 1,378 ng/g),

When MC-LR levels in both extracts are compared this pro-

the other common MC in both strains. The differences in

portion, 498,000 ± 54,780 ng MC-LR/g and 166,000 ±

the MC production between strains of the same cyanobac-

21,580 ng MC-LR/g, respectively is conﬁrmed. However, this

teria species have been previously observed by other

Table 1

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

Monoisotopic m/z ratios of [M þ H]þ ions for different MC

Molecular mass

m/z values

MC-LR

994

995.6

D-Asp3-MC-LR

980.5

981.5

1,012

1,013.6

MC-LR

994

995.6

D-Asp3-MC-LR

980.5

981.5

MC ascription

M. aeruginosa PCC7806

[MeSer ]MC-LR M. aeruginosa PCC7820

Dglu(OCH3)-MC-LR

1,008.5

1,009.5

MC-LY

1,001.5

1,002.5

MC-LW

1,024.5

1,025.5

MC-LF

985.5

986.5

Table 2

Quantiﬁcation by LC-MS of intracellular MC identiﬁed in PCC7806 and PCC7820 at the end of the study (21 days)

Intracellular concentration (ng/g) PCC7806 PCC7820

MC-LR

166,000 (21580)a

498,000 (54,780)a

MC-LF

3,600 (396)a

MC-LW

6,580 (724)a 10,000 (1,100)a

MC-LY 7

[MeSer ]MC-LR

33,600 (43,68)

D-Asp3-MC-LR

10,600 (1,378)a

Levels of MC detected by LC-MS, ELISA anti-Adda and PP2A inhibition assay in samples from (a) PCC7806 strain and (b) PCC7820 strain. Values are given as means ± standard deviation (n ¼ 3).

92,000 (10,120)a a

Dglu(OCH3)LR Total MC

Figure 3

16,460 (1,811) 210,200

626,640

Standard deviation (n ¼ 3).

assay; 347.5 ± 36.2 μg/L by LC-MS). The levels of MC calculated by ELISA anti-Adda were higher than MC detected by the other two techniques, with maximum concentrations observed at day 9 (2151.6 ± 97.4 μg/L) and at day 15

authors in cultures of different strains from M. aeruginosa (Ohtake et al. ).

(1997.7 ± 110.9 μg/L), and showing signiﬁcant decreases at days 12 (866 ± 112.65 μg/L) and 21 (853.0 ± 200.0 μg/L). In addition, the maximum concentrations obtained from

Toxin production kinetics

samples of the PCC7820 strain were similar with the PP2A inhibition assay (1086.0 ± 66.4 μg/L) and LC-MS (981.0 ±

Intracellular MC concentration was studied by LC-MS,

17.9 μg/L) techniques, and an overestimated MC concen-

ELISA anti-Adda technique and the PP2A inhibition assay

tration

during the study period (21 days). For the strain samples

(1879.0 ± 106.2 μg/L) (Figure 3(b)). The intracellular MC

of PCC7806, analysis of MC by LC-MS and PP2A inhibition

concentrations during the study period showed the same

assay gave lower values than ELISA anti-Adda (Figure 3(a)).

trend with the three techniques employed.

was

detected

with

ELISA

anti-Adda

assay

The maximum levels of MC were reached at day 15 (400.6 ±

The mean levels of intracellular MC detected by ELISA

52.1 μg/L by PP2A inhibition assay; 356.4 ± 46.3 μg/L by

were higher than those obtained by LC-MS; these differ-

LC-MS) and 21 (417.3 ± 54.2 μg/L by PP2A inhibition

ences were more marked in PCC7806 samples (4-fold)

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

than in the case of PCC7820 (2-fold). An overestimation in

Intracellular MC production measured by PP2A inhi-

the amounts of MC detected by ELISA in comparison

bition assay did not show variations as consistent as the

with LC-UV was previously reported in our laboratory in

above relating between ELISA and LC. The maximum con-

natural blooms from the Guadiana River (Moreno et al.

centration calculated by the PP2A inhibition assay in both

). Similarly, the comparison of LC-MS, ELISA and

strains is slightly higher than that measured by LC

phosphatase assay for the determination of MC in cyanobac-

(417.3 ± 54.2 μg/L versus 347.5 ± 36.2 μg/L in PCC7806 1086.0 ± 66.4 μg/L

versus

981.0 ± 17.9 μg/L

teria products revealed that the LC-MS/MS results were

and

signiﬁcantly lower than biochemical assays (Lawrence

PCC7820). To understand these slight differences, a corre-

et al. ). Rapala et al. () compared the suitability of

lation diagram was established, displaying the MC content

ELISA and LC-UV to detect different toxin variants using

from the PP2A inhibition assay as a function of the MC con-

several matrices (pure toxins, laboratory cultures, water

centration from the LC-MS analysis for both strains

and bloom samples of toxic cyanobacteria). According to

(Figure 4). The diagrams have a linear trend and the

these authors, despite the possible overestimation of toxin

slopes correspond to the ratio α calculated following this for-

concentrations with LC in standard toxins, the analyses of

mula (Robillot et al. ):

the ﬁeld samples gave lower values than ELISA assay. All these results are in agreement with those obtained by Tillmanns et al. () who reported that results measured by

α¼

[equivalent MC-LR] by PP2A inh: assay ½total MC by LC

ELISA were higher than those obtained by LC-DAD (4fold). They suggested that this difference may depend on

Its value would theoretically be equal to 1 if the sample

the number of MC variants being produced in situ. The over-

contained only hepatotoxins that inhibit PP2A as much as

estimation in MC concentrations observed with the ELISA

MC-LR (Robillot et al. ). In our study, the calculated

assay cannot be only due to the matrix effects, because

values are 1.06 ± 0.50 and 1.10 ± 0.19 for PCC7806 and

when using extracts the organic compounds are concen-

PCC7820, respectively. Consequently, such linear trends

trated and may interfere with the results. On the contrary,

suggest that α is not time-dependent, indicating that MC

other authors, such as Gurbuz et al. (), have found a

composition might be stable over the growth period in

positive correlation between ELISA and LC-DAD results

both strains. LC-MS analysis showed that the proportion

for MC-RR and MC-LR, although the ELISA results for MC-LF and LW were almost 6-fold lower than the LC results. In this sense, Mikhailov et al. () also detected lower values of MC by ELISA than those determined by LC-DAD. The correlation between the ELISA and LCDAD analyses of the MC in sediment samples was studied by Babica et al. (). Although the results from both methods were in agreement for higher MC concentrations, only weak correlation was observed below 0.1 μg/g, which may reﬂect the generally higher variability in the ELISA results. In conclusion, we can say that the ELISA assay is not a suitable method for quantiﬁcation of MC due to the larger variation observed when it is compared with another technique such as LC-DAD or LC-MS. Indeed, it would be necessary to validate the ELISA assay in order to set up a reliable cut-off for detecting MC in culture extracts to discriminate between false positives, false negatives, true positives and true negatives (Moreno et al. ).

Figure 4

Correlation between intracellular MC concentrations measured by LC-MS and by the PP2A inhibition assay in samples from (a) PCC7806 and (b) PCC7820 strains (n ¼ 3).

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

Journal of Water and Health

12.1

2014

of MC-LR in PCC7806 varied between 70.5% and 88.2%

PCC7820 strain (Figure 5(b)). The cell quota (fentogram/

during growth, and in PCC7820 the range was 79.4–80.1%.

cell) varied within this strain during culture growth. The

Our results have demonstrated that the choice of

highest concentration of toxins by cell was reached at the

analytical procedures to measure MC content can cause dis-

beginning of the exponential growth phase (6 days) with a

crepancies in the quantiﬁcation of these cyanotoxins when

value of 87 ± 6.5 fgMC/cell measured by LC-MS and 97 ±

comparing ELISA and LC-MS results. However, a good cor-

6.7 fgMC/cell with PP2A inhibition assay. During the

relation has been obtained between PP2A inhibition assay

stationary phase the MC levels by cell decreased consider-

and LC-MS. All these methods are based on different prin-

ably down to levels of approximately 17 fgMC/cell

ciples.

detected by both techniques. In the decline growth curve

The

ELISA

test

measures

the

total

toxin

concentration of the sample, while the PP2A inhibition

phase (12–21 days), an increase in the MC concentration

assay assesses its total potential toxicity. In the case of LC-

by cells was observed, reaching 70 ± 6.3 to 78 ± 7.6 fgMC/

MS, individual MC can be separated and recognized on

cell after 18 days of culture.

the basis of their retention times and mass spectra (Rapala et al. ).

Concerning the results obtained for PCC7806, slight variations in the MC levels by cell overall by PP2A inhibition assay have been detected (Figure 5(a)). The

Comparison of intracellular toxin production between

maximum concentration of fgMC/cell detected by LC-MS

both strains during culture

was ∼10 fgMC/cell during the exponential growth phase, and the minimum level was 5.3 ± 0.9 fgMC/cell at day 9,

Intracellular MC concentrations were not constant during

just at the end of the exponential phase and at the beginning

the culture period in both strains (Figures 5(a) and 5(b)).

of the stationary phase. The MC cell quota in M. aeruginosa

This fact was mainly observed in the M. aeruginosa

PCC7806 is lower than in PCC7820 (∼9-fold). In both strains, the maximum MC production took place at the beginning of the exponential growth curve in our conditions. Robillot et al. () studied the MC production kinetics of M. aeruginosa PCC7820, observing that the maximum intracellular MC concentration was reached at day 7. In this study, the stationary phase was reached after 12 days of culture. Considering these results, it can be conﬁrmed that the toxicity of toxic Microcystis cells is variable and dependent on the strains used and the culture conditions. It implies that the comparison of toxin production between different species or strains should consider their production kinetics.

CONCLUSION The comparative study of MC production during the culture period of two strains of M. aeruginosa (PCC7806 and PCC7820) has shown that the majority of intracellular MC production was observed during the exponential growth phase. In the present study, for the ﬁrst time a new MC Figure 5

Levels of MC per cell (fentogram/cell) in samples from (a) PCC7806 and (b) PCC7820 strains during the studied period. Values are given as means ± standard deviation (n ¼ 3).

has been identiﬁed ([MeSer7]) in the M. aeruginosa PCC7806 strain. The MC production and the MC cell

V. Ríos et al.

MC in M. aeruginosa PCC7820 versus PCC7806

quota during the culture period in PCC7806 were lower than in PCC7820 (3-fold and ∼9-fold, respectively). In both cases, MC-LR was the predominant toxin detected. In both strains, the maximum MC production was observed at the beginning of the exponential growth curve under our conditions. In this study, a good correlation has been demonstrated in MC levels determined by PP2A inhibition assay and LC-MS in both strains that were considered during the culture period.

ACKNOWLEDGEMENTS The authors wish to thank the CICYT (AGL 2006–06523/ ALI) for the ﬁnancial support for this study and ZeuInmunotec for the kind supply of the MicroCystest kits.

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First received 8 April 2013; accepted in revised form 11 September 2013. Available online 15 October 2013

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Incorporating potable water sources and use habits into surveys that improve surrogate exposure estimates for water contaminants: the case of bisphenol A Syam S. Andra and Konstantinos C. Makris

ABSTRACT Human biomonitoring studies for water contaminants are often accompanied by surveys relying solely on total drinking water consumption rates, thus, failing to account for specific water sources (bottled and tap water) and use habits, such as water used for preparing cold/hot beverages (coffee, tea, juice, etc.). Despite the extensive use of bisphenol A (BPA) in polycarbonate (PC)-based water contact materials, rarely do BPA biomonitoring studies focus on various PC water uses and sources. Better resolved water consumption rates could reduce the uncertainty associated with surrogate daily BPA intake estimates using fine-tuned surveys. This approach provided a proof of concept on inclusion of water consumption from various sources and uses into estimates of daily intake for water contaminants like BPA found in water-contact materials. The next steps would be in quantifying the extent of improvement in exposure assessment that adds value to refined survey designs. Key words

Syam S. Andra Konstantinos C. Makris (corresponding author) Water and Health Laboratory, Cyprus International Institute for Environmental and Public Health in association with Harvard School of Public Health, Cyprus University of Technology, Irenes 95, Limassol 3041, Cyprus E-mail: konstantinos.makris@cut.ac.cy Syam S. Andra Harvard-Cyprus Program, Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115, USA

| biomonitoring, bisphenol A, environmental epidemiology, exposure assessment, polycarbonate bottled water, water and health

INTRODUCTION In the post-Second World War era, public health ofﬁcials were

cultural differences, geographic location, etc. More than a

already witnessing dramatic decreases in waterborne illness

single source of potable water is used in developed countries

incidence rates in areas utilizing centralized drinking water

(such as tap, bottled, desalinated seawater/groundwater,

treatment with pre- and post-chlorination facilities. Since

etc.), perplexing estimates of potable water consumption

then, research focus on mortality and morbidity risk factors

(Makris et al. a, b). Water surveys have been traditionally

has shifted away from water-based health indicators, while

used in epidemiologic, marketing, or economic studies, but

only during the last decade has considerable attention for

they often do not reﬂect the multitude of water consumption,

safe access to water and sanitation issues again been documen-

source and use characteristics for the population of interest.

ted for developed countries. Unsafe water, sanitation, and

Bottled water enjoys big sales in many parts of the

hygiene represent the leading environmental risk factors inﬂu-

world, being another popular choice of potable water

encing global mortality and morbidity when compared with

(Rodwan ). However, water packaged into plastic con-

other environmental factors, such as urban outdoor air pol-

tainers could come in contact with leached plasticizers

lution, indoor smoke from solid fuels, environmental lead

and plastics additives (Andra et al. , ), such as anti-

exposure, and global climate change (Ezzati et al. ).

mony, bisphenol A, phthalates (to name a few), showing

Water consumption rates are characterized by large inter-

endocrine disrupting activity in key hormonal centers, like

population and intra-population variability reﬂecting differ-

reproduction (Wolff et al. a) and thyroid (Andra &

ences in age, sex, socioeconomic status, racial characteristics,

Makris ). Exposure assessment of water contaminants,

doi: 10.2166/wh.2013.068

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

including those in bottled water, has not gained as much

stalls selling mountain water, etc.) and water uses in surveys

attention as food contaminants enjoy in major pregnancy–

that could link biomonitoring-based exposure estimates to

birth cohorts (Makris & Andra ). With the exception

external water source(s). As an example, PC-based water con-

of disinfection by-products, potable water contaminants

sumption, rather than total (both bottled and tap) water

like endocrine-disrupting chemicals have received little, if

consumption was signiﬁcantly (p ¼ 0.017) associated with

any, attention in biologically relevant exposure assessment

urinary BPA concentrations in a small (n ¼ 35) young adult

for environmental epidemiological studies (Makris &

subpopulation group that largely relied upon PC bottled

Andra ). Exposure assessment for water contaminants

water to satisfy its potable needs (Makris et al. b). Poly-

has typically relied upon water intake survey questionnaire

ethylene terephthalate (PET)-based water consumption

information (Smith et al. ; Llop et al. ; Haugen

patterns showed a signiﬁcant (p ¼ 0.02) positive correlation

et al. ) rather than water chemical analysis (Makris &

with urinary antimony concentrations, linking the anti-

Andra ). Where available, water intake survey question-

mony-containing PET bottled water to human internal

naires tend to rely on contemporary questions such as ‘how

exposures (Makris et al. a). When total water consump-

many glasses of water do you daily consume?’, which do not

tion patterns were used (data derived from survey-based

provide explicit details that could better drive estimates of

questions like ‘how many glasses of water per day you con-

daily water intake for water pollutants such as disinfection

sume?’), no correlation was observed with either antimony

by-products (Maskiell et al. ) and perchlorate (Mendez

or BPA (Makris et al. a, b).

et al. ).

Given the distinct differences and gaps in surveys used in

The example of bisphenol A (BPA) is exempliﬁed here,

epidemiological studies monitoring endocrine disruptors’

since it is a controversial endocrine disruptor found in poly-

exposures from various environmental stressors, including

carbonate (PC)-based water contact materials (WCM),

water contamination, we aimed at further improving

among other sources (cans, baby bottles, personal care pro-

exposure assessment with the suggestion of a ﬁne-tuned ques-

ducts, etc.) (Von Goetz et al. ; Makris et al. b;

tionnaire probing into individual preferences of water source

Muncke et al. ). Typically, the source of BPA occurrence

(s) and use characteristics. Despite the small size of our study

in tap water is epoxy resin coatings or polyvinyl chloride

(n ¼ 35), it successfully showed the effect of reﬁning water-

material used in drinking water distribution system pipes

related surveys for environmental contaminants onto improv-

(Bae et al. ). However, BPA is not detected or present

ing our ability to associate speciﬁc exposure sources to body

at very low concentrations in tap water (Makris & Snyder

burden estimates. The superiority of ﬁne-tuned survey content

) because of chlorine-induced degradation of BPA to

over that of a contemporary survey was proven within the

chlorinated BPA congeners (Hu et al. ). However, BPA

same pilot study of ours. Though outcomes of this ﬁne-

in packaged water may be expected to be measured, since

tuned survey questionnaire as a relationship between per

chlorination of packaged water is not practiced unless a pre-

capita water consumption and daily intake of either antimony

viously chlorinated potable water source was used for

from PET bottled water (Makris et al. a) or BPA from PC

packaging. Traditional survey questions relying on total

bottled water (Makris et al. b) was earlier reported, the

water consumption per capita often fail to address speciﬁc

novelty of this submission is in presenting the superiority

water source(s), use and frequency characteristics (cups/

and differences in intake estimates (equivalent to exposure)

glasses per day or week), poorly reﬂecting upon biologically

compared with a contemporary approach used in parallel,

relevant exposure estimates of water contaminants. Speciﬁc

considering urinary BPA data as a case study. We also

water uses refer not only to water used for plain drinking

searched the literature to assess the extent of water (either

water purposes, but also water used for making cold

tap or bottled) contaminant studies following such reﬁned

(frappe, coffee, juice, etc.) and/or hot (tea, coffee, etc.)

water-related surveys. The overall objective was to emphasize

beverages, or water used for cooking. It is often subtle differ-

the need for detailed survey questions while assessing

ences and differentiation of water sources (various plastic

exposures to endocrine disruptors found in food and water-

types and volumes of bottled water, well water, mobile

contact materials, like BPA.

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

METHODS

Journal of Water and Health

12.1

2014

identify relations between water intake pattern and associated exposures to endocrine disrupting compounds. A

In an effort to improve the environmental exposure estimates

detailed ﬁne-tuned questionnaire was administered and

explaining the variability in chronic disease incidence rates, a

compared the outcomes with those of a contemporary

reﬁned survey content emphasizing hypothesis-driven

survey approach (detailed below). Urinary antimony and

exposure sources and pathways was applied in an exposure

BPA were used as a measure of water consumption from

assessment study for BPA (Makris et al. b). In compari-

PET and PC bottles, respectively (Makris et al. a, b).

son, we gathered human biomonitoring-based studies from

The mean [±SD (standard deviation)] age and body mass

a literature search engine that reported geometric means of

index (BMI) of the study participants was 30.5 ± 7.5 years

either creatinine-corrected or unadjusted urinary BPA

and 23.6 ± 4.3 kg m 2, respectively. The majority were

levels. A comprehensive search was performed using

females (63%) in the age group 26–35 years (57%), with a

Scopus queried from 1960 (start date) to March 1, 2013.

normal BMI of <25 kg m 2 (74%).

The keyword search ‘bisphenol AND water’ yielded 3,200 peer-reviewed publications, which narrowed down with speciﬁc keyword combinations such as ‘bisphenol AND water AND exposure’ resulted in 400 articles. About 200 articles relevant to the environmental sciences were selected, and both abstract and full length text (where necessary) were read and assessed carefully in summarizing the available studies. Thirty-six studies were identiﬁed reporting urinary

Survey approach Both contemporary and ﬁne-tuned survey questionnaires were implemented in parallel (provided together and at the same time). Graphical representation of the steps involved in these approaches is presented in Figure 1. Details are provided below as purposes and approaches.

BPA in an exposure and/or epidemiological study perspective. The majority of biomonitoring studies reported urinary BPA concentrations as geometric mean and they were retained for further analysis (28 out of 36 available studies, Table SI-1, available online at http://www.iwaponline.com/ wh/012/068.pdf). Only a few studies reported arithmetic mean and median values of urinary BPA, three (Kim et al. ; Tsukioka et al. ; Yang et al. ) and ﬁve studies

Purpose-I Determine the type and extent of use of a water source to meet daily potable water consumption requirements.

•

Pointers: Pictures of each water source should be shown to participants towards better addressing this question. A

(Yang et al. ; Schoringhumer & Cichna-Markl ;

250 mL glass or cup needs to be displayed as an indicator

Wolff et al. b; Meeker et al. ; Casas et al. ),

for cup volume water consumption. If the participant pro-

respectively. These were excluded from further consideration

vides answer for the ‘other’ source, the interviewer

to facilitate data comparison across all studies reporting urin-

should make a special note of it for further follow up.

ary BPA in the same statistical expressions (geometric mean

•

Posed questions: ○

and geometric standard deviation). Following this effort, we

Q1: Which of the following one or more sources of

evaluated how well the reﬁned survey content matched that

water do you use – a YES or NO response for: (1)

presented in the selected BPA studies relying on a contempor-

water board of city (tap water), (2) ground water

ary survey content which entailed a single question of ‘how

(well water), (3) bottled water, (4) mobile stations

much water do you consume per day’ (Table SI-1).

(water stalls on the road ﬁlled with mountainous spring water), (5) other, please specify.

Survey study and participants

○

Q2: If YES, please specify how many glasses (a glass equals 250 mL) of water per day and how many days

Study details are presented elsewhere (Makris et al. a). In brief, a pilot study of 35 adult volunteers (university postgraduate students, faculty, and staff) was conducted to

•

per week from each water source. Possible outcome: Provides an overall depiction of major water sources and an estimate of water intake from each

Figure 1

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

Steps followed in assessing water consumption via contemporary and ﬁne-tuned approach.

source. This leads to further understanding on the extent

perturbations is recommended. PET bottles are coded

of use of water sources ‘other’ than tap water, which is

1 and widely used because of their convenience to

usually considered as the predominant, and sometimes

carry, but recommended for one time use. PET material

as the one and only, source of water.

likely contains phthalates (known endocrine disrupting chemicals, EDC) and antimony (possible EDC and a car-

Purpose-II

cinogen). PC bottles are coded 7 and widely used as large water storage containers that are usually reused.

To determine which plastic type of bottled water was used.

•

PC material likely contains BPA, a known endocrine dis-

Pointers: A brief on types of plastics used in consumer

ruptor. PET and PC material is used not only for bottling

products and examples of daily sources of exposure to

water but also for packaged beverages and food. Pictures

plastics should be mentioned to each participant. It is

of both PC (19 L) and PET (0.5/0.75/1.5 L) must be

necessary to carry a schematic of plastics codes from 1

shown to the participants for a better response to the

through 7, corresponding details about each resin identi-

questionnaire. If a participant provides an unrealistic

ﬁcation code, and a mention on location of the code on

number of glasses of water consumption per day,

plastic containers. A brief note on plastics additives

the interviewer must speciﬁcally seek the correct

chemical

answers for ‘how many days per week from each bottle

nature

and

associated

human

health

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

type’ – since preferences may change from while at

•

home and when being outside home (e.g., at work). Posed questions: ○

Q1: Which one of the following plastic bottles did you

•

Journal of Water and Health

12.1

2014

Possible outcome: Describes the distribution of water source usage among daily water use types. It also presents information on some of the neglected water use purposes in a generic survey questionnaire.

use for water consumption – a YES or NO response for: (1) polyethylene terephthalate (PET), (2) polycar-

Calculations

bonate (PC), (3) both, please specify. ○

Q2: If YES for PET, please specify the bottle volume –

Per capita water consumption using the ﬁne-tuned approach

a YES or NO response for: (1) 0.5 L, (2) 0.75 L, (3)

was calculated by using the formulae:

1.5 L, (4) all. ○

Q3: Where and when a YES response from Q1 and Q2, please specify how many glasses (250 mL) of water per day and how many days per week from

•

each plastic bottle type. Possible outcome: Provides a detailed description on bottled water type preference in the survey population. This furthers helps in identifying possible exposures to

Daily water consumption rates; L day 1 person 1 X ¼ XPlain Water þ XCold Beverages þ XHot Beverages

(1)

where X ¼ glasses day 1 0:25 L glass 1 days week 1 = 7 days week 1

(2)

respective endocrine disruptors that vary with plastic type – for example phthalates and antimony used in PET

Urinary BPA analysis

bottles and BPA in PC bottles. Moreover, the effort from this step increases the awareness of study participants on

Urinary BPA (total of free and conjugated BPA) was ana-

various plastics used in consumer products, associated

lyzed using a liquid chromatography–mass spectrometry

health concerns, and recycling capabilities of each type.

method. The experimental and analytical details of the

Purpose-III To determine the use of water for which other consumption purposes other than plain drinking water.

•

method are detailed elsewhere (Makris et al. b). The primary focus of this work was to highlight the differences in daily intake estimates derived from using both contemporary and a ﬁne-tuned survey questionnaire on per capita water consumption rate and BPA intake (back calculated

Pointers: Pictures of cold and hot beverages may help in

from urinary BPA). Descriptive statistics, univariate and cor-

making better sense of posed questions by the partici-

relation analyses were performed using JMP 7.0 (SAS Inc.,

pants. Again, if the interviewer gets an unrealistic

NC, USA).

number from the participant, then it is recommended to re-pose the questions for a better breakdown of water

•

consumption pattern towards cold and hot beverages.

RESULTS AND DISCUSSION

Posed questions: ○

○

Q1: Which one of the following purposes did you use

Compiling data from 28 human biomonitoring studies that

water for consumption – a YES or NO response for:

reported geometric mean BPA concentrations helped us

(1) plain drinking water, (2) making cold beverages

evaluate whether details about water source and usage

(juice, mixed drink, etc.), (3) making hot beverages

characteristics

(tea, coffee, etc.), and (4) all of the above or a combi-

estimating daily intake estimates (Table SI-1, available at

were

taken

into

consideration

when

nation, please specify.

online

Q2: If YES, please specify how many glasses (250 mL)

With respect to water consumption per source as a question,

http://www.iwaponline.com/wh/012/068.pdf).

of water per day and how many days per week from

bottled water was surveyed in the studies by Yang et al.

each water source.

(), Carwile et al. (), Lakind & Naiman (),

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

Morgan et al. (), and Li et al. (), and tap water was

proportion (30–35% of total daily water consumption)

studied by Völkel et al. (), Becker et al. (), and Li

took the form of cold and/or hot beverages (coffee, tea,

et al. (). Makris et al. (b) included other water

juices). The observation became apparent because this

sources, such as ground water and mobile stations which is

study was conducted during the summer period of higher

water packaged in reusable PC containers. Regarding con-

water consumption and use (Table 1). The reﬁned question-

tainer type, PC bottled water consumption was surveyed by

naire indicated that 35% of per capita daily water

Carwile et al. () and Li et al. (). In addition to PC bot-

consumption would go unreported, if only plain drinking

tles, water consumption from PET bottles was assessed by

water was taken into account (Table 1). The ‘use of other

Makris et al. (b). With respect to water use character-

water source’ question did not yield any answers, while

istics, consumption in the form of plain water was taken

the intention was to seek information on alternative water

into consideration by Völkel et al. (), Carwile et al.

sources, such as surface water (lake, etc.). This indicates

(), and Li et al. (), while in the form of cold beverage

the need for going over, one by one, all water source options

by Carwile et al. () and hot beverage by Li et al. (). All

rather than having the participant think through them.

three forms of water use (plain water, and cold and hot bev-

Signiﬁcant association (p ¼ 0.02) was observed between

erages) were monitored by Makris et al. (b). Though

water consumption from PC bottles derived from the ﬁne-

urinary BPA levels were measured in the 28 studies, neither

tuned approach and urinary BPA (Figure 2(d)). No such sig-

water consumption source nor water use speciﬁcs were

niﬁcance (p > 0.05) was noted between urinary BPA and

reported by 20 out of the 28 studies listed in Table SI-1

water consumed from all sources (Figure 2(a)), all bottled

(viz., Yang et al. (), Calafat et al. (), Wolff et al.

water (Figure 2(b)), and water from both PC and PET bottles

(), Calafat et al. (), Lakind & Naiman (), Maha-

(Figure 2(c)). The ability to take into account the total use of

lingaiah et al. (), Teitelbaum et al. (), Wolff et al.

PC bottled water in a systematic manner using a ﬁne-tuned

(b), Ye et al. (), He et al. (), Bushnik et al.

approach helped in ﬁnding a meaningful association

(), Cantonwine et al. (), Galloway et al. (), Men-

between PC bottled water consumption and BPA intake

diola et al. (), Milieu en Gezondheid (), Mok-Lin

(measured as urinary BPA). Otherwise, the estimates using

et al. (), Braun et al. (), Kim et al. (), Zhang et al.

a contemporary approach yielded no such signiﬁcant associ-

(), and Nahar et al. ()). Among the 28 BPA studies

ation with BPA intake due to an inherent deﬁciency of

available, only three studies included more than one single

failing to consider the contribution of PC bottled water

water source in their questionnaire, because often consumers

towards per capita water consumption. Furthermore, no

in developed countries use two or more water sources for

such signiﬁcant linear trends (p > 0.05) were observed

potable use (Carwile et al. ; Li et al. , Makris et al.

between urinary BPA levels and water consumption from

b) (Table SI-1). These three studies were those that

PET bottles (Figure SI-1b) and tap (Figure SI-1c) that were

included questionnaire items looking into water use charac-

also derived from the ﬁne-tuned survey data. (Figure SI-1

teristics (water used for cold or hot beverages) (Table SI-1).

is available online at http://www.iwaponline.com/wh/

Indeed, most of the studies in Table SI-1 did not consider

012/068.pdf.)

the possible contribution of any water source and/or use

Most of the existing water use surveys in search of associ-

characteristics to daily BPA intake estimates back-calculated

ations with urinary BPA relied on either daily water

from urinary biomarker measurements.

consumption or an average of two consecutive days as in

About 50% higher per capita water consumption was

the United States National Health and Nutrition Examin-

reported using the proposed ﬁne-tuned approach (the break-

ation Survey (NHANES) (Lakind & Naiman ;

down resulted in a mean water consumption estimate of

Sebastian et al. ). However, the ﬁne-tuned approach in

person , while a contemporary approach indi-

this study showed that relying on water consumption infor-

cated a mean water consumption of about 1.85 L day 1

mation for a day or two may not be a good representation

3.44 L day 1

person ; Table 1). Use of plain drinking water topped the

given the variable number of hours spent weekly by individ-

potable water consumption chart, while a signiﬁcant

uals at work, leisure time and at home. For example,

Table 1

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

Development of a ﬁne-tuned questionnaire with respect to per capita water consumption data (L day 1 person 1) in comparison to a contemporary approach, for water contaminants that often show multiple exposure sources. Values represent mean ± standard deviation of 35 adult volunteers Average number of glasses day 1 (¼ glasses day 1 * days

Per capita water consumption

week 1/7 day week 1)

(L day 1 person 1)

Contemporary approach (with no additional classiﬁcations in comparison to ﬁne-tuned approach) Bottled water

3±3

0.86 ± 0.80

Tap water

3±4

0.65 ± 1.05

Ground water

0±1

0.06 ± 0.28

Mobile station water

1±4

0.27 ± 0.90

Contemporary approach: Cumulative per capita water consumption

7±5

1.85 ± 1.18

Fine-tuned approach (further classiﬁcation based on water source, use, and plastic type) Bottled water Polycarbonate (PC 19 L/big blue container)

3±2 1±1 1±1

0.63 ± 0.61 0.20 ± 0.27 0.18 ± 0.26

4±3

1.02 ± 0.79

3±4 1±2 1±1

0.74 ± 0.89 0.21 ± 0.40 0.13 ± 0.24

4±5

1.08 ± 1.24

8±6

2.10 ± 1.42

3±4

0.74 ± 0.89

Cold beverage

1±1

0.13 ± 0.25

Hot beverage

1±1

0.15 ± 0.22

Plain water Cold beverage Hot beverage

Total PC water Polyethylene terephthalate (PET 0.5/ 0.75/1.5 L)

Plain water Cold beverage Hot beverage

Total PET water Total bottled water Tap water

Plain water

3±5

0.81 ± 1.29

0.20 ± 0.9

0.05 ± 0.23

Cold beverage

0.02 ± 0.1

0.01 ± 0.03

Hot beverage

0.02 ± 0.1

0.01 ± 0.04

0.24 ± 1.0

0.06 ± 0.26

1±3

0.26 ± 0.71

Total tap water Ground water

Plain water

Total ground water Mobile station water

Plain water Cold beverage

1±3

0.17 ± 0.64

Hot beverage

0.2 ± 0.7

0.04 ± 0.18

Total mobile station water

2±6

0.48 ± 1.43

Fine-tuned approach: Cumulative per capita water consumption

14 ± 8

3.44 ± 2.02

participants usually have a wider choice of water sources and

mean water consumption and a huge standard deviation

also water use needs when at work or in school on workdays,

when calculations were based on a daily basis in comparison

while the options may differ on a weekend. Hence, we took a

with frequency (weekly) basis (Figure SI-2, available online at

‘frequency approach’ to average out water consumption over

http://www.iwaponline.com/wh/012/068.pdf). A ﬁne-tuned

a 1-week period by integrating responses from two survey

approach resulted in an average (±SD) per capita water con-

questions: (1) how many glasses (250 mL) per day from

sumption of 5.0 ± 3.1 and 3.4 ± 2.0 L day 1 person 1 from all

each water source, and (2) how many days per week from

water sources using daily and weekly based calculations,

each water source? As noted, there was an inﬂation in

respectively (Figure SI-2). Similarly, the per capita water

Figure 2

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

Creatinine-adjusted BPA concentrations in the survey study participants (n ¼ 35) in relation to the reported per capita water consumption from all drinking sources (a) and only bottled water (b) using a contemporary survey. Similar relations were graphed between urinary BPA levels and per capita water consumption from the reﬁned PC þ PET water survey (c) and only PC bottled water (d).

consumption from only plastic bottles (all bottled water)

uses of PC water that may signiﬁcantly increase both per

using a daily and weekly based calculation was 3.2 ± 2.1

capita water consumption and BPA intake estimates, which

and 2.1 ± 1.4 L day 1 person 1, respectively (Figure SI-2).

has gone unnoticed and under-considered thus far in con-

On the other hand, the PC water consumption estimate

ducting risk assessments.

based on plain drinking water purpose alone was 0.6 ± 1

0.6 L day

person , which signiﬁcantly increased to

The proposed ﬁne-tuned approach, however, suffers from limitations, because the provision of a detailed ques-

1.0 ± 0.8 L day 1 person 1 upon inclusion of PC water use

tionnaire

for cold and hot beverages (p ¼ 0.03) (Figure 3). The resulting

overestimate water consumption rates from each source

increased contribution of PC water consumption from

and use, thereby overestimating water-based BPA intake

inclusion of other water use aspects signiﬁcantly enhanced

rates. The other limitation would be lack of a ‘gold standard’

the BPA intake estimate from 24 ± 24 to 40 ± 32 ng kg

in drinking water survey content to compare and/or validate

might

induce

redundant

tendency

(p ¼ 0.04) (Figure 3). This observation further

our study questionnaire with. Similarly, higher rates of water

supports the deemed necessity in considering alternative

consumption and greater variation were reported during

day

Figure 3

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

Water use characteristics (either as plain water or beverage or both) gives a closer estimate on per capita water consumption from PC bottles and associated BPA intake by the 35 adult volunteers participating in this study. Mean values denoted by different letters are statistically signiﬁcant using Student’s t-test (α ¼ 0.05).

summer and winter periods, with an average total per capita 1

potential strength of this study was that the results indicate

(1.14

new areas of research interest in deriving ﬁner exposure esti-

and 1.02 L day 1 person 1), respectively, and ranged from 0

mates to drinking water contaminants. The ﬁndings of this

water consumption of 38.6 and 34.6 oz day 1

person

person ) (Barraj et al. ). An

validation study are of the utmost importance as a proof-

additional limitation would be the small sample size (n ¼ 35)

of-pilot-concept for further exploration and exploitation.

of the study, preventing us from making inferences for the

Optimization, validation, and rigorous testing of the pro-

general population. Hence, the results should be interpreted

posed

with a certain degree of caution. Nonetheless, given that

warranted for future application in survey-based health

there is very little information available on (1) usage of

studies.

to 396 oz (0 to 12 L day

questionnaire

are

the

necessary

steps

more than one potable water source and (2) usage for

Adult human exposures to BPA stem primarily from

more than one water use, this critical examination of ques-

dietary sources such as canned food (>90% contribution)

tionnaire readouts and the initial descriptive analyses are a

(Geens et al. ); while the contribution of non-dietary

useful ﬁrst step and contribution to the literature. The

sources is much smaller, such as dust (<1%) (Loganathan

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Journal of Water and Health

12.1

2014

& Kannan ), thermal and other paper (<5%) (Liao &

and industrial agencies with risk assessment. The next

Kannan a), currency paper (<1%) (Liao & Kannan

steps would be to quantify the extent of improvement in

b), and dental surgery (<5%) (Von Goetz et al. ).

exposure assessment of epidemiological studies that adds

Within dietary sources, PC bottled water as an exposure

value to the proposed survey design. Eventually, this exer-

source has received little attention compared to canned

cise could help identify speciﬁc sources and routes of

food and beverages, perhaps because BPA leaching is not

exposure, detailing the extent of exposure contribution

instantaneous but may be the combined kinetic inﬂuence

from each source.

of various environmental factors like UV, temperature, re-

The next phase of this work is in progress, where

use, etc. Hence, the development of a ﬁne-tuned question-

approximately 400 residents from 200 households are parti-

naire could help us in reducing uncertainty associated

cipating in a cross-sectional study in Cyprus. One of the

with water consumption, water intake and individual risk

major objectives was to ﬁnd associations between detailed

estimates for pollutants found in water, but also in other

water consumption patterns (tap and bottled water per

exposure sources (diet, air, soil, dust). The far-reaching aim

capita consumption patterns as identiﬁed in this study)

will be the development of similar questionnaires for emer-

and unintentional human exposures to a suite of drinking

ging chemicals found in consumer products for which

water contaminants (measured as urinary levels of disinfec-

biomonitoring studies are yet unavailable, such as UV ﬁlters

tion by-products and bisphenol A). The aforementioned

that have shown disruption of both reproductive (Weisbrod

study will help us in providing a wide range of individual

et al. ) and thyroid function in animal studies (Schmut-

level data on age, indicators of socio-economic, habitual,

zler et al. ).

and lifestyle factors, and medical history. Thus, this facilitates the opportunity to practice the approaches presented in this study at a much larger scale in a general population

CONCLUSIONS

with no selection bias.

Human biomonitoring studies for water contaminants are often accompanied by surveys relying solely on total drink-

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S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

Maskiell, K. E., Heyworth, J. S. & McCaul, K. A.  Validation of a water consumption questionnaire for a study of the adverse health outcomes associated with disinfection by-products. Int. J. Environ. Health Res. 16, 145–153. Meeker, J. D., Calafat, A. M. & Hauser, R.  Urinary bisphenol A concentrations in relation to serum thyroid and reproductive hormone levels in men from an infertility clinic. Environ. Sci. Technol. 44, 1458–1463. Mendez, W., Dederick, E. & Cohen, J.  Drinking water contribution to aggregate perchlorate intake of reproductiveage women in the United States estimated by dietary intake simulation and analysis of urinary excretion data. J. Expo. Sci. Environ. Epidemiol. 20, 288–297. Mendiola, J., Jorgensen, N., Andersson, A. M., Calafat, A. M., Ye, X., Redmon, J. B., Drobnis, E. Z., Wang, C., Sparks, A., Thurston, S. W., Liu, F. & Swan, S. H.  Are environmental levels of bisphenol a associated with reproductive function in fertile men? Environ. Health Perspect. 118, 1286–1291. Milieu en Gezondheid  Vlaams Humaan Biomonitoringsprogramma 2007–2011. Resultatenrapport: deel referentie biomonitoring. Available at: http://www. milieu-en-gezondheid.be/rapporten/Luik%2044/ rapportopvolgstudies_pasgeborenen_FINAL.pdf (accessed 28 September 2013). Mok-Lin, E., Ehrlich, S., Williams, P. L., Petrozza, J., Wright, D. L., Calafat, A. M., Ye, X. & Hauser, R.  Urinary bisphenol A concentrations and ovarian response among women undergoing IVF. Int. J. Androl. 33, 385–393. Morgan, M. K., Jones, P. A., Calafat, A. M., Ye, X., Croghan, C. W., Chuang, J. C., Wilson, N. K., Clifton, M. S., Figueroa, Z. & Sheldon, L. S.  Assessing the quantitative relationships between preschool children’s exposures to bisphenol A by route and urinary biomonitoring. Environ. Sci. Technol. 45, 5309–5316. Muncke, J., Wagner, M. & Makris, K. C.  A new paradigm for risk assessment of endocrine disruptors in food contact materials. Comp. Rev. Food Sci. Food Safety 12, 1–4. Nahar, M. S., Soliman, A. S., Colacino, J. A., Calafat, A. M., Battige, K., Hablas, A., Seifeldin, I. A., Dolinoy, D. C. & Rozek, L. S.  Urinary bisphenol A concentrations in girls from rural and urban Egypt: a pilot study. Environ. Health. 2, 11–20. Rodwan Jr, J. G..  ‘Bottled Water 2010’- The Recovery Begins. International Bottled Water Association, April/May 2011 Issue. Available at: www.nxtbook.com/ygsreprints/IBWA/ G19428IBWA_AprMay11Nxtbk/index.php?startid=4#/12 (accessed 29 August 2013). Schmutzler, C., Bacinski, A., Gotthardt, I., Huhne, K., Ambrugger, P., Klammer, H., Schlecht, C., Hoang-Vu, C., Grüters, A., Wuttke, W., Jarry, H. & Köhrle, J.  The ultraviolet ﬁlter benzophenone 2 interferes with the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid peroxidase. Endocrinology 148, 2835–2844.

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Schoringhumer, K. & Cichna-Markl, M.  Sample clean-up with sol-gel enzyme and immunoaffinity columns for the determination of bisphenol A in urine. J. Chrom. B 850, 361–369. Sebastian, R. S., Wilkinson Enns, C. & Goldman, J. D.  Drinking Water Intake in the U.S.: What We Eat In America, NHANES 2005–2008. Food Surveys Research Group Dietary Data Brief No. 7. September 2011. Available at: http://ars. usda.gov/Services/docs.htm?docid=19476 (accessed 29 August 2013). Smith, R. B., Toledano, M. B., Wright, J., Raynor, P. & Nieuwenhuijsen, M. J.  Tap water use amongst pregnant women in a multi-ethnic cohort. Environ. Health. 8 (Suppl 1), S7. Teitelbaum, S. L., Britton, J. A., Calafat, A. M., Ye, X., Silva, M. J., Reidy, J. A., Galvez, M. P., Brenner, B. L. & Wolff, M. S.  Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the United States. Environ. Res. 106, 257–269. Tsukioka, T., Brock, J., Graiser, S., Nguyen, J., Nakazawa, H. & Makino, T.  Determination of trace amounts of bisphenol A in urine by negative-ion chemical-ionization-gas chromatography/mass spectrometry. Analyt. Sci. 19, 151– 153. Völkel, W., Kiranoglu, M. & Fromme, H.  Determination of free and total bisphenol A in human urine to assess daily uptake as a basis for a valid risk assessment. Toxicol. Lett. 179, 155–162. Von Goetz, N., Wormuth, M., Scheringer, M. & Hungerbühler, K.  Bisphenol a: how the most relevant exposure sources contribute to total consumer exposure. Risk Analysis 30, 473–487. Weisbrod, C. J., Kunz, P. Y., Zenker, A. K. & Fent, K.  Effects of the UV filter benzophenone-2 on reproduction in fish. Toxicol.Appl. Pharmacol. 225, 255–266. Wolff, M. S., Teitelbaum, S. L., Windham, G., Pinney, S. M., Britton, J. A., Chelimo, C., Godbold, J., Biro, F., Kushi, L. H., Pfeiffer, C. M. & Calafat, A. M.  Pilot study of urinary biomarkers of phytoestrogens, phthalates, and phenols in girls. Environ. Health Perspect. 115, 116–121. Wolff, M. S., Britton, J. A., Boguski, L., Hochman, S., Maloney, N., Serra, N., Liu, Z., Berkowitz, G., Larson, S. & Forman, J. a Environmental exposures and puberty in inner-city girls. Environ. Res. 107, 393–400. Wolff, M. S., Engel, S. M., Berkowitz, G. S., Ye, X., Silva, M. J., Zhu, C., Wetmur, J. & Calafat, A. M. b Prenatal phenol and phthalate exposures and birth outcomes. Environ. Health Perspect. 116, 1092–1097. Yang, M., Kim, S. Y., Lee, S. M., Chang, S. S., Kawamoto, T., Jang, J. Y. & Ahn, Y. O.  Biological monitoring of bisphenol a in a Korean population. Arch. Environ. Contam. Toxicol. 44, 546–551. Yang, M., Kim, S. Y., Chang, S. S., Lee, I. S. & Kawamoto, T.  Urinary concentrations of bisphenol A in relation to

S. S. Andra & K. C. Makris

Daily water use patterns and bisphenol A human exposures

biomarkers of sensitivity and effect and endocrine-related health effects. Environ. Mol. Mutagen. 47, 571–578. Ye, X., Pierik, F. H., Hauser, R., Duty, S., Angerer, J., Park, M. M., Burdorf, A., Hofman, A., Jaddoe, V. W., Mackenbach, J. P., Steegers, E. A., Tiemeier, H. & Longnecker, M. P.  Urinary metabolite concentrations of organophosphorous pesticides, bisphenol A, and phthalates among pregnant

Journal of Water and Health

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women in Rotterdam, the Netherlands: the Generation R study. Environ. Res. 108, 260–267. Zhang, Z., Alomirah, H., Cho, H. S., Li, Y. F., Liao, C., Minh, T. B., Mohd, M. A., Nakata, H., Ren, N. & Kannan, K.  Urinary bisphenol A concentrations and their implications for human exposure in several Asian countries. Environ. Sci. Technol. 45, 7044–7050.

First received 7 March 2013; accepted in revised form 13 September 2013. Available online 18 October 2013

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2014

Removal of bacteria, protozoa and viruses through a multiple-barrier household water disinfection system A. C. Espinosa-García, C. Díaz-Ávalos, R. Solano-Ortiz, M. A. Tapia-Palacios, N. Vázquez-Salvador, S. Espinosa-García, R. E. Sarmiento-Silva and M. Mazari-Hiriart

ABSTRACT Municipal water disinfection systems in some areas are not always able to meet water consumer needs, such as ensuring distributed water quality, because household water management can be a contributing factor in water re-contamination. This fact is related to the storage options that are common in places where water is scarce or is distributed over limited time periods. The aim of this study is to assess the removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses. Water samples were taken from households in Mexico City and spiked with a known amount of protozoa (Giardia cyst, Cryptosporidium oocyst), bacteria (Escherichia coli), and viruses (rotavirus, adenovirus, F-specific ribonucleic acid (FRNA) coliphage). Each inoculated sample was processed through a multiplebarrier device. The efficiency of the multiple-barrier device to remove E. coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed, 99.6% of the adenovirus was removed, and the rotavirus was almost totally removed. An effect of site by zone was detected; this observation is important because the water characteristics could indicate the efficiency of the multiple-barrier disinfection device. Key words

| disinfection, drinking water, household water disinfection, microbial removal, multiple

A. C. Espinosa-García Instituto de Ecología, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior Ciudad Universitaria, Coyoacán 04510, Distrito Federal, México C. Díaz-Ávalos Instituto de Investigaciones en Matemáticas y Sistemas, UNAM, Circuito Escolar Ciudad Universitaria, Coyoacán 04510, Distrito Federal, México R. Solano-Ortiz M. A. Tapia-Palacios N. Vázquez-Salvador S. Espinosa-García Instituto de Ecología, UNAM, Circuito Exterior Ciudad Universitaria, Coyoacán 04510, Distrito Federal, México R. E. Sarmiento-Silva Facultad de Medicina Veterinaria y Zootecnia, UNAM, Circuito Exterior Ciudad Universitaria, Coyoacán 04510, Distrito Federal, México M. Mazari-Hiriart (corresponding author) Instituto de Ecología, UNAM, Circuito Exterior Ciudad Universitaria, Coyoacán 04510, Distrito Federal, México E-mail: mazari@unam.mx

barrier system

INTRODUCTION Water for human use and consumption must comply with

periods of time; in these cases, water can be supplied

regulatory quality parameters according to public health

with an acceptable quality but due to indoor storage it

standards.

can be contaminated prior to its consumption (Clasen &

The authorities that manage the water supply systems for human population centers have a responsibility to

Bastable ; Wright et al. ; Nath et al. ; Eshcol et al. ).

ensure the provision of sufﬁcient amounts of quality

Microbiological water assessment has been based on

water that is suitable for human use; however, the efﬁ-

coliform bacteria, which have been considered as an indi-

ciency of water provision is not always adequate.

cator of microorganisms for more than 100 years (Ashbolt

Furthermore, water management in households is a key

et al. ); however, it has been shown that the presence

factor related to microbiological water quality. The latter

or absence of coliform bacteria is not related to viral

is related to the various storage options that are common

agents and protozoa, both of which are relevant to public

in places where water is scarce or distributed for limited

health (Lambertini et al. ; WHO a).

doi: 10.2166/wh.2013.080

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

Disinfection at the point of use is one option for improving the drinking water quality supplied through the formal

Journal of Water and Health

12.1

2014

formed carbon ﬁlter, a chlorination unit, and ﬁnally, an activated carbon polisher to reduce excess chlorine.

distribution system. It has been shown that the disinfection

The aim of this work was to evaluate the reduction of

of domestic water or the disinfection of water at the point

rotavirus, adenovirus, coliphages, Giardia cysts, and Cryp-

of use improves drinking water quality and reduces the

tosporidium oocysts that had been added to drinking

risk of disease associated with contaminated water (Clasen

water samples by employing a popular commercial puriﬁer

et al. ; WHO b). The most relevant results have

device, Pureit® Classic, which uses a multi-barrier combi-

been obtained in systems with frequent water contamination

nation of disinfection technologies. The device was also

due to pipe breaks or discontinuous water ﬂow, or in situ-

tested using groundwater from different Mexico City areas

ations in which the contamination of the water source is

that correspond to different aquifer subsystems.

known (WHO a). The World Health Organization (WHO) highlights technologies such as the following: (i) chemical disinfection with

METHODS

chlorine, ozone, or another oxidant; (ii) filtration through membranes, ceramics, or composed filters; (iii) filtration

Study area

through granular media; (iv) solar disinfection; (v) ultraviolet light; (vi) heat disinfection; (vii) coagulation-

Seventy percent of the Mexico City Metropolitan Area

precipitation or sedimentation; and (viii) multiple barrier

(MCMA) water supply derives from groundwater, while

treatments. There is a wide range of efﬁciency levels

30% is supplied from surface sources from within its basin

among the different methods; however, it is noteworthy

or other basins, as well as from a river and a few springs.

that the production of safe water in the household depends on the presence of microorganisms in the water.

Groundwater for the MCMA is extracted from three aquifer subsystems that are denominated Ciudad de México,

Multi-barrier treatment refers to any combination of two

Texcoco, and Chalco-Xochimilco (Mazari & Mackay ).

or more technologies employed, simultaneously or sequen-

The aquifer subsystems present different hydrogeologic fea-

tially (WHO b). Some of these treatments comprise

tures that are constituted of diverse materials and that

commercial devices that combine particle removal by physical

consequently present different permeability rates, causing vary-

methods and microbial inactivation using chemicals (i.e. chlor-

ing water inﬁltration into each subsystem (DGCOH ). This

ine). It is important to consider that water features are variable

groundwater was used as the experimental matrix for the

according to the various water sources, such as groundwater or

spiked microorganisms, serving as a good approximation of

surface water, and including rainwater. There are microbiolo-

real Mexico City groundwater source conditions (Figure 1).

gical and chemical processes that take place at different

To obtain water that represents at least the three main

locations, therefore, it is not advisable to generalize the capa-

conditions of the water supply sources to Mexico City inhabi-

bilities of commercial purifying devices that have been

tants, a directory of household addresses was integrated from

evaluated under speciﬁc conditions, as these capabilities may

people who had indicated their willingness to participate in

be related to the water in which they have been tested. In Mexico, there are different commercial device options that use multi-barrier treatments, for example,

this study. From the list of addresses, the ﬁnal sites were randomly selected, obtaining ﬁve sites for each of the three Mexico City areas; all sites were sampled in triplicate.

there are commercial devices that employ ﬁltration and ultraviolet (UV) light or ﬁltration and inverse osmosis. Lim-

Sample preparation

ited information is available to the population, however, about the disinfection efﬁciency of these devices. The

Drinking water samples were collected using 10 L polypro-

speciﬁc multi-barrier puriﬁer water device utilized in this

pylene bottles that had been previously washed and

study is an integrated unit with a sequence of ﬁltrations

sterilized

through a microﬁber that retains large particles, a pre-

measurements

(APHA in

). situ

Physicochemical included

pH,

parameter temperature,

A. C. Espinosa-García et al.

Figure 1

Microbe removal through a water disinfection system

Journal of Water and Health

12.1

2014

Sampling sites in Mexico City: Northwest, Northeast and South.

conductivity, turbidity, total dissolved solids and dissolved

for recovering MS2 coliphages after the multi-barrier

oxygen using a multiparameter sonde YSI 6600-M (Love-

treatment.

land, CO). Following the HACH manual (HACH ),

The E. coli (ATCC 23631) was propagated in 50 mL of

residual chlorine was also measured, and if it was detected,

tryptone glucose yeast extract broth (TGYB) medium

sodium thiosulfate (J.T. Baker, Edo México) was added to

(Becton Dickinson, Cuautitlán, México) and incubated for

neutralize the sample. To eliminate any microorganism

12 h at 37 C (ISO ) in an aerobic incubator (Binder,

that could be present in the water, samples were sterilized

Tuttlingen, Germany). The E. coli (ATCC 700891) was pro-

(at 120 C for 20 min), leaving water samples to cool

pagated in 50 mL of Trypticase soy broth medium (Becton

overnight.

Dickinson, Cuautitlán, México) and incubated for 3 h at

Simultaneously, 1 L samples were collected in sterilized polypropylene bottles. These samples were processed to

37 C in a water bath (Daiki Science Co., Korea) under slow agitation (EPA ).

determine the presence of fecal coliform bacteria in household drinking water by means of the standard membrane

Enteric virus culture

ﬁltration method (APHA ). Rotavirus SA-11 VR-1565 (ATCC, Manassas, VA) was proPositive controls of experimental microorganisms

pagated by infecting MA104 cells (kindly donated by Dr C. Árias, IBT-UNAM). Brieﬂy, rotavirus was activated

Coliform bacteria

with 3 μg/mL of trypsin for 45 min at 37 C in 5% CO2 incuW

bator NU-4750, (NUAIRE, US). Conﬂuent MA104 cells in Two strains of Escherichia coli (ATCC 23631 and 700891),

ﬂasks were inoculated with rotavirus and incubated for 1 h

acquired from ATCC (Manassas, VA), were cultivated and

at 37 C and 5% CO2 to allow for rotavirus adsorption.

employed to spike the drinking water samples and as hosts

Non-supplemented DMEM was added to the infected

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

ﬂasks (Huang et al. ), and these ﬂasks were incubated W

for 18–20 h at 37 C and 5% CO2.

Journal of Water and Health

12.1

2014

To avoid interaction between E. coli and MS2 coliphages, the temperature of the spiked samples was

Adenovirus 41 VR-930 (ATCC, Manassas, VA) and A549 CCL-185 cells (ATCC, Manassas, VA) were obtained from

maintained at 20 C, considering that bacteria pili are presW

ent at 37 C.

ATCC and used for adenovirus propagation. Conﬂuent

Because G. lamblia cysts and C. parvum oocysts were

A549 cells (CCL-185 ATCC) were inoculated with adeno-

obtained from residual water, the entire inoculation and disin-

virus, allowing for 1 h of adsorption at 37 C and 5% CO2.

fection process by the multi-barrier device, as well as

The non-supplemented Dulbecco’s modiﬁed Eagle’s medium

concentration sampling, recovery, and quantiﬁcation, was per-

(DMEM) was added to the ﬂask with cells and viruses,

formed separately, with simultaneous sample collection. These

samples were prepared in the same way as those that were

which was incubated for 10 days at 37 C and 5% CO2. The lysates for both viruses were recuperated by freez-

inoculated with rotavirus, adenovirus, coliphages, and E. coli.

ing and thawing three times. The rotavirus titer was obtained by the TCID50 method, and the adenovirus was

Multi-barrier water puriﬁer device

quantiﬁed by quantitative real-time polymerase chain reaction (qPCR) (Xagoraraki et al. ). Both virus lysates

The Pureit® Classic (Unilever, India) multi-barrier water

comprised the stocks for spiking the sterilized drinking

puriﬁer device utilized in this study is an integrated unit

water samples.

that allows for particle reduction through a microﬁber

MS2 coliphages (ATCC 15597-B1, Manassas, VA) were

ﬁlter and a carbon ﬁlter that retains protozoan (oo)cysts

propagated in E. coli as host bacteria. The protocols followed

and chemical contaminants (i.e. pesticides, organic com-

for MS2 propagation were EPA 1602 (EPA ) and ISO

pounds). Afterward, the water ﬂows through a chlorination

10705-1 (ISO ). This procedure allowed for the detection

chamber with a chlorine feeder cartridge with a contact

of 10 plaque-forming units (PFU)/mL; this limit of detection

time of approximately 20–30 min, and the water passes

was determined by the MS2 stock serial dilutions assay.

through a silver-impregnated granular activated carbon ﬁlter. This latter treatment removes the excess chlorine

Protozoa

and chlorination byproducts, thus improving the taste of the water (Clasen et al. ; Verma & Arankalle ;

Giardia lamblia cysts and Cryptosporidium parvum oocysts

Pureit® Manual ).

were obtained from residual water that tested positive for

One part of the Pureit® device comprises a closed

both pathogens. Their identiﬁcation and quantiﬁcation in

deposit to store ﬁnished drinking water, which is dispensed

the residual water was carried out directly using ﬂuor-

through a spigot from which the water sample was collected

escence

for the analyses (approximately 10 L).

microscopy

and

monoclonal

antibodies

for

Giardia lamblia and Cryptosporidium parvum, as described in the recovery section.

Concentration of drinking water samples

Spiked drinking water samples

Once the samples were inoculated and homogenized, each was treated with the multi-barrier device following the man-

The sterilized 10 L drinking water samples were inoculated,

ufacturer’s instructions for use. During the test, it was

and the ﬁnal concentrations of each microorganism were

important to work with the whole multi-barrier device, not

E. coli (9.49 × 1013 colony-forming units (CFU)/10 L), rota-

with separate treatment stages, because we needed to

virus SA-11 (7 × 105 50% tissue culture infective dose

relate the results with the whole system, in accordance

(TCID50)/10 L), adenovirus 41 (7.8 × 10 PFU/10 L), coliph-

with WHO recommendations (WHO b). After the

age MS2 (1.5 × 1014 PFU/10 L), Giardia lamblia cysts

multi-barrier treatment, the drinking water samples were

(164.1 cysts/10 L) and Cryptosporidium parvum oocysts

concentrated by ultraﬁltration using the method developed

(22.5 oocysts/10 L).

by Polaczyk et al. ().

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

Sodium polyphosphate (NAPP) 0.1% (Sigma-Aldrich,

Journal of Water and Health

12.1

2014

Recovery of rotavirus and adenovirus

MO) was added to each sample as a disaggregant after the sample had passed through a hollow ﬁber F80A poly-

An aliquot of the concentrated drinking water sample was

sulfone ﬁlter (Fresenius, Medical Care, MA) and a

used to quantify the rotavirus and adenovirus by MA104

continuous ﬂow peristaltic Masterﬂex (Cole-Parmer Instru-

and A549 cell infection, respectively, using the TCID50

ments Co., USA) pump. Samples were concentrated at a

method. Because rotavirus and adenovirus were inoculated

rate of 1,700 mL/min. The ultraﬁltration system had pre-

together in the drinking water samples, to control the poss-

viously been disinfected using a circulating chlorine

ible co-infection of cells, rotavirus and adenovirus were

solution and was also treated with a 0.1% solution of

neutralized alternately prior to infecting the conﬂuent

NAPP (Figure 2). The sodium polyphosphate treatment of

cells. The monoclonal anti-rotavirus antibody SC-58188

the ﬁlter produced an electronegative charge to avoid

(Santa Cruz, TX) was employed to assess adenovirus infec-

adherence to the hollow fiber filter surface. The final

tivity, and the monoclonal anti-adenovirus antibody SC-

volume of each concentrated sample was approximately

58651 (Santa Cruz, TX) was utilized to evaluate rotavirus

50 mL. All concentrates were divided in aliquots and

infectivity.

stored under refrigeration according to the requirements for the recovery of each microorganism.

Figure 2

Diagram of the ultraﬁltration system used to concentrate drinking water samples.

Trypsin was added to aliquots separately to measure rotavirus infectivity (Huang et al. ), as described

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

Journal of Water and Health

12.1

2014

previously, to activate the rotavirus present in the samples in

Cruz, TX) (1:100 in PBS) and 40 ,6-diamidino-2-phenylindole

which the adenovirus had been neutralized by a monoclonal

(DAPI) (BioGenex, CA) solutions (1:100 in PBS) were

antibody. Then, 96-well plates with conﬂuent MA104 cells

added, and the samples were incubated at 37 C for 30 min

were infected with 100 μL of 10-fold serial dilutions of the

prior to visualization under ﬂuorescence microscopy.

activated concentrate. The plates were incubated for 48 h at 37 C and 5% CO2 (Huang et al. ). W

The parameters considered for detection and quantiﬁcation included the size, shape, and wall integrity of the

For adenovirus, 5-fold serial dilutions from samples in

ﬂuorescent (oo)cysts (Smith & Rose ).

which the rotavirus had been neutralized by a monoclonal

The method described allowed for the detection of

antibody were prepared and 96-well plates with conﬂuent

(oo)cysts when there were at least six (oo)cysts/10 L present,

A549 cells were infected with 100 μL of each dilution. The

determined by serial dilutions of the stocks.

plates were incubated for 10 days at 37 C and 5% CO2 (Jiang et al. ). After incubation of the rotavirus and adenovirus, 96-well plates were ﬁxed with 50 μL/well of methanol

RESULTS AND DISCUSSION

(4 C) for 5 min. The methanol was discarded, and the cells were dyed with crystal violet in ethanol (0.1%) for 20 min.

To record the main features of the drinking water supplied

Finally, the plates were washed with tap water to eliminate

in three Mexico City areas in situ, the following parameters

the excess dye; the results were reported as TCID50.

were measured: temperature, pH, conductivity, total dis-

This protocol allowed for the quantiﬁcation of rotavirus and adenovirus with a limit of detection of 7 TCID50/mL.

solved solids (TDS), and dissolved oxygen. Table 1 depicts the results for each Mexico City area related to the three aquifer subsystems.

Giardia lamblia and Cryptosporidium parvum recovery

The NE drinking water area presented the highest conductivity and TDS of all of the areas around Mexico City;

Fifty-milliliter aliquots from sample concentrates were pro-

this result must be due to the concentrations of salts and par-

cessed by a second concentration through centrifugation at

ticles typical of the water in the NE aquifer subsystem

3,000 × g for 15 min (Centra CL3R, Thermo-IEC, MA);

(Texcoco). Although NE conductivity and TDS are adequate

each pellet was then re-suspended in 3 mL of phosphate-

for drinking water, there are parameters that must be taken

buffered saline (PBS) solution (Sigma-Aldrich, MO). When

into account because salts and particles can affect the per-

required, the solution was stored with glycerol (10%) at –

formance of the multi-barrier device with regard to

20 C (Rangel-Martínez ; Tapia-Palacios ).

process time and the lifetime of consumables (microﬁber

For (oo)cyst detection, 1 mL of the solution concentrate

and carbon ﬁlter). Measurements of the remaining par-

was incubated with a mouse monoclonal IgG antibody,

ameters did not show signiﬁcant differences. However, the

either the anti-Giardia antibody SC-57743 (Santa Cruz,

drinking water samples collected, as previously described,

TX) or the anti-Cryptosporidium antibody SC-58112 (Santa

represent a better approach to the real conditions of the

Cruz, TX), at 4 C overnight. Afterward, a ﬂuorescein iso-

drinking water distributed in Mexico City.

was

Analyses for fecal coliform bacteria in drinking water

incubated at 37 C for 1.5 h to react with the previously

samples prior to sterilization were all negative. Therefore,

added anti-mouse IgG. Finally, a propidium iodide (Santa

sterilization ensures the elimination of all microorganisms

thiocyanate

(FITC)-conjugated

rabbit

antibody

Table 1

Average values of measured parameters in the drinking water of houses of three Mexico City areas W

Mexico City area

Temperature ( C)

Conductivity (mS/cm)

TDS (mg/L)

Oxygen (mg/L)

Northeast (NE)

23.29

7.53

0.633

0.395

5.73

Northwest (NW)

20.53

7.27

0.238

0.169

4.39

South (S)

22.67

7.37

0.244

0.166

6.90

100

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

that could be present in drinking water samples and avoids microbial overestimation.

Journal of Water and Health

12.1

2014

One stage of the multi-barrier Pureit® Classic device was not measured due to the integrity device, which was the

Free chlorine is a basic water quality indicator, and, in

chlorination chamber for evaluating the chlorine feeder

many countries, is the only monitoring parameter. In

operation, although 6 mg/L of free chlorine in the water

Mexico, drinking water regulations establish a free chlorine

within the chamber was reported (Clasen et al. ).

concentration of 0.2–1.5 mg/L (DOF ). The multibarrier treatment of the Pureit

Recovery analyses for each microorganism obtained

Classic device includes a

from the spiked drinking water samples showed a reduced

ﬁnal step to reduce chlorine. Free chlorine concentration

number of positive samples. The spiked bacteria were elimi-

measurements in triplicate for drinking water samples in

nated to the level of non-detection in all samples, but there

situ (households) and at the end of treatment are presented

were positive samples noted for enteric viruses and proto-

in Table 2.

zoa. However, is important to consider the limit of

A minimal free chlorine concentration (or the absence

detection of the quantitative methods used to evaluate the

of free chlorine) makes it necessary to recommend care to

removal capacity of the Pureit® device. This feature is

prevent recontamination. It is interesting to note that the

important because the capacity of the Pureit® device, and

free chlorine concentrations in household drinking water

most likely of other commercial devices, depends on the

in this study, with the exception of two samples from the

quantities and types of microorganisms, as well as on the

NE and two from the S, complied with the Mexican regu-

speciﬁc conditions of the processed water. These aspects,

lations (DOF ). This observation is interesting because

which have not been included in other studies, are con-

normally it is assumed that drinking water in Mexico City

sidered in the present study. It has been previously noted

very frequently does not meet the regulatory requirements.

that these factors should be taken into account (Clasen et al. , ; Verma & Arankalle ; WHO b).

Table 2

Average free chlorine concentrations in the household drinking water of three

The results suggest that it is necessary to analyze the

areas of Mexico City, and in drinking water after the multi-barrier household

presence of microorganisms with respect to the sampling

treatment

sites, due to a possible drinking water source effect, or to Sampler points in

Households

After multi-

Mexico City

(mg/L)

barrier mg/Lc

barrier mg/Ld

1 NE

1.2

1.23

1.75

2 NE

>2.0b

3 NE

Not measured

4 NE

1.98

0.23

<0.2a

5 NE

1.25

<0.2a

6 NW

0.78

<0.2a

7 NW

0.81

<0.2a

8 NW

0.28

0.22

0.21

9 NW

1.16

<0.2a

10 NW

Not measured

11 S

0.36

0.23

<0.2a a

12 S

0.21

<0.2

<0.2a

13 S

0.38

<0.2a

14 S

<0.2a

15 S

<0.2a

Free chlorine concentration below the detection limit (0.2 mg/L). b Free chlorine concentration above the detection limit (2.0 mg/L). c

Samples spiked with rotavirus, adenovirus, bacteriophage MS2, and E. coli.

Samples spiked with Giardia lamblia and Cryptosporidium parvum oocysts.

simply discard the site effects and consider the microorganisms’ tolerances to multi-barrier treatment. To identify the possible effect of the Mexico City site or area (Figure 1) on the presence of microorganisms, our team applied Student’s t-test and linear generalized models. The E. coli that had been spiked into the drinking water samples was removed to a non-detectable level; this result represents a removal of >10 log units, independent of the site or the water source. In Mexico, there are regulations that establish criteria, but these criteria only point out limits for bacteria reduction with which all water puriﬁer devices must comply; including a reduction capacity of 4 log units of total coliform bacteria and 95% for mesophilic aerobic bacteria (DOF ). The results of the present study show that the Pureit® Classic device meets the Mexican regulatory requirements, however, the authors of this study believe that these waterpurifying device requirements are incomplete if we focus on the necessity for safe water indoors.

101

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

Journal of Water and Health

12.1

2014

Removal of the MS2 is shown in Figure 3. Except for

were signiﬁcant for samples of areas S and NE2. In area S

sites NE5, NW3, and S2, the removal of spiked MS2 was

(S2, S3, S4, and S5), rotavirus removal was lower than aver-

>10 log units. The linear model shows signiﬁcant differ-

age (Figure 5). Both the rotavirus and adenovirus removal

ences at sites NW3 (p ¼ 0.00206) and NE5 (p ¼ 0.02512).

levels obtained in this study were similar (6.53 log units)

With respect to the removal of adenovirus, except for

to those reported for hepatitis E and hepatitis A viruses

the S2 site, removal was at least from 3 log units to 7 log

that had been spiked in the water and removed using the

units (Figure 4). However, the results showed variability

Pureit® device in India (Verma & Arankalle ).

within area S, and the linear model suggests a site effect on adenovirus removal.

The capacity of the Pureit® Classic device for removing Cryptosporidium parvum oocysts was 2 log units >87% of

Rotavirus removal at the majority of sites was >6 log

oocysts spiked, but the method’s limit of detection of six

units. However, the generalized linear model test results

oocysts/10 L must be considered. It is important to emphasize that in this case, the stock was recovered from the residual water and the spiked oocysts were only 2 log units (Figure 6). To verify whether Cryptosporidium parvum oocyst removal depends on an area or on the site, a generalized linear model was ﬁtted with a Poisson-type error and logarithmic link. The results do not show a relationship between the site and the oocyst quantity present in the drinking water after multi-barrier treatment (p > 0.05). Therefore, there is no site-based pattern that affects the removal of Cryptosporidium parvum oocysts. Giardia lamblia cyst removal was 2 log units >98% (Figure 7) because cysts were below the limit detection (six cyst/10 L) at the majority of sampling sites. The test to ident-

Figure 3

Removal of MS2 spiked in drinking water samples (10 L) processed by the

ify a relationship between removal and site was not

multi-barrier device. The boxes show the variability between replicates and dashed lines the data distribution. *MS2 removal for these samples was below

signiﬁcant (p > 0.05); therefore, any site pattern was

the method limit of detection.

Figure 4

Removal of adenovirus spiked in drinking water samples (10 L) processed by

Figure 5

Removal of rotavirus spiked in drinking water samples (10 L) processed by the

the multi-barrier device. The boxes show the variability between replicates and dashed lines the data distribution. *Adenovirus removal for these samples

multi-barrier device. The boxes show the variability between replicates and dashed lines the data distribution. *Rotavirus removal for these samples was

was below the method limit of detection.

below the method limit of detection.

102

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

Journal of Water and Health

12.1

2014

able to remove >3 log units. For the latter, it is advisable to perform this type of evaluation with an excess of (oo)cysts. This research has demonstrated the importance of efficiency assessment with a perspective concerning which differences in water sources and water characteristics (groundwater or surface) can have an effect on the microorganisms and on the water purifier performance. In this case, a site effect was detected on the reduction of enteric viruses in drinking water from three aquifer subsystems; however, an experimental design is needed to identify the main factors or parameters that must be determined, an issue to be considered by purifier device manufacturers. Figure 6

Removal of Cryptosporidium parvum oocysts spiked in drinking water samples (10 L) processed by the multi-barrier device. The boxes show the variability

Point of use water disinfection today is a real option for

between replicates and dashed lines the data distribution. *Oocyst removal for

a safe water supply in households, with the water quality

these samples was below the method limit of detection.

control in the hands of the consumer. However, it is noteworthy that this must not eliminate the responsibility of the authorities to supply the population with safe water from the integrated water management perspective, that is, from the supply source to the consumer, as suggested by the WHO (a). The maintenance and operation of pumps, disinfection systems, pipes, and tanks, to mention only a few factors, always play a major role with the purpose of improving and protecting the health of the population as related to water quality.

CONCLUSIONS The multi-barrier water puriﬁcation device showed a removal capacity of >10 log units of E. coli that had been Figure 7

Removal of Giardia lamblia cysts spiked in drinking water samples (10 L) pro-

inoculated into drinking water without differences among

cessed by the multi-barrier device. The boxes show the variability between replicates and dash lines the data distribution. *Cyst removal for these

samples from three Mexico City areas. For MS2, phage inac-

samples was below the method limit of detection.

tivation was observed in the majority of drinking water samples. The removal capacity was >10 log units, but the

discarded. For protozoan removal, Clasen et al. () evalu-

inactivation process included a site effect for all three sites.

ated the Pureit® removal capacity using microspheres as a

A site effect was also detected in the removal of adenovirus

surrogate for the protozoan cysts. Their results showed a

and rotavirus, demonstrating the importance of water source

reduction of at least 3 log units after Pureit® device treat-

characteristics to be taken into account for evaluating the dis-

ment. We could not attempt to replicate this result because

infection efﬁciency of any point of use household device.

the (oo)cyst stocks (C. parvum and G. lamblia) were inocu-

The minimum and maximum removal capacities of the

lated at only 2 log units in the experimental water samples;

multi-barrier Pureit® Classic were 3 and >7 log units for

our analysis did not allow us to determine whether, under

adenovirus, respectively, and 2 and 6 log units for rotavirus,

our experimental conditions, the multi-barrier device is

depending on the site. Rotavirus inactivation was less in the

103

A. C. Espinosa-García et al.

Microbe removal through a water disinfection system

southern area of Mexico City compared with the NE and NW areas. Cryptosporidium parvum oocysts and Giardia lamblia cysts of 2 log units were removed from drinking water without any site pattern detected. Under Mexico City-specific conditions, the multi-barrier Pureit® Classic device showed to be efficient for the reduction of E. coli, MS2 coliphages, adenovirus, and rotavirus, as well as for the Cryptosporidium parvum oocysts and Giardia lamblia cysts, all of which were spiked in real drinking water samples collected from Mexico City households that had been supplied by three aquifer subsystems. The Pureit® Classic device improved the microbial quality of the drinking water inside the house, but it is advisable that the capacity for principal chemical contaminant removal be assessed, as these contaminants have been scarcely studied in household water purifiers.

ACKNOWLEDGMENTS The authors wish to acknowledge C. Flores-Gracia and R. Castañón-Ibarra from the staff at the Coordinación de Innovación y Desarrollo, UNAM as well as UNILEVER for the Pureit® units used in this work and their support for this project. We would like to acknowledge E. Hjort for help in elaborating the ﬁgures.

REFERENCES American Public Health Association (APHA)  Standard Methods for Examination of Water and Wastewater. 21st edn. United Book Press, Washington, D.C. Ashbolt, N. J., Grabow, W. O. K. & Snozzi, M.  Indicators of microbial water quality. In: Water Quality: Guidelines, Standards and Health: Assessment of Risk and Risk Management for Water-Related Infectious Disease (L. Fewtrell & J. Bartram, eds). World Health Organization Press, Geneva, Switzerland. Clasen, T. & Bastable, A.  Faecal contamination of drinking water during collection and household storage: the need to extend protection to the point of use. J. Wat. Health. 3, 109–115. Clasen, T., Nadakatti, S. & Menon, S.  Microbiological performance of a water treatment unit designed for

Journal of Water and Health

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household use in developing countries. Trop. Med. Int. Health. 11, 1399–1405. Clasen, T., Naranjo, J., Frauchiger, D. & Gerba, C.  Laboratory assessment of a gravity-fed ultrafiltration water treatment device designed for household use in low-income settings. Am. J. Trop. Med. Hyg. 80, 819–823. Diario Oficial de la Federación (DOF)  Norma Oficial Mexicana NOM-127-SSA1-1994. Modificación. Salud ambiental, Agua para uso y consumo humano. Límites permisibles calidad y tratamientos a que debe someterse el agua para su potabilización. Diario Oficial de la Federación 20 octubre, 2000. México. Dirección General de Construcción y Operación Hidráulica (DGCOH)  Hidrología subterránea en el valle de México. Ingen. Hidrául. México 1, 90–98. DOF  NOM-244-SSA1-2008, Equipos y sustancias germicidas para tratamiento doméstico de agua. Requisitos sanitarios. Diario Oficial de la Federación 4 septiembre, 2009. México. Environmental Protection Agency (EPA)  Method 1602: Male-specific (Fþ) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. Environmental Protection Agency, Washington, DC. Eshcol, J., Mahapatra, P. & Keshapagu, S.  Is fecal contamination of drinking water after collection associated with household water handling and hygiene practices? a study of urban slum households in Hyderabad, India. J. Water Health. 7, 145–154. HACH  Water Analysis Handbook, 4th edn. HACH Co., CA, 1260. Huang, J. A., Nagesha, H. S., Snodgrass, D. R. & Holmes, I. H.  Molecular and serological analyses of two bovine rotaviruses (B-11 and B-60) causing calf scours in Australia. J. Clin. Microbiol. 30, 85–92. ISO  10705-1: Water Quality- Detection and Enumeration of Bacteriophages- Part 1: Enumeration of F-specific RNA Bacteriophages. International Organization for Standardization, Geneva, Switzerland. Jiang, S. C., Han, J., He, J. W. & Chu, W.  Evaluation of four cell lines for assay of infectious adenoviruses in water samples. J. Water Health 7, 650–656. Lambertini, E., Spencer, S. K., Kieke Jr, B. A., Loge, F. J. & Borchardt, M. A.  Virus contamination from operation and maintenance events in small drinking water distribution systems. J. Water Health 9, 799–812. Mazari, M. & Mackay, D. M.  Potential for groundwater Contamination in Mexico City. Environ. Sci. Technol. 27, 794–801. Nath, K. J., Bloomfield, S. F. & Jones, M.  Household water storage, handling and point of-use treatment. A review commissioned by the IFH; available from: www.ifhhomehygiene.org. Pureit® Manual  Available from: www.pureitwater.com/MX/ pureit-classic.

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Polaczyk, L. A., Narayanan, J., Cromeans, L. T., Hahn, D., Roberts, M. J., Amburgey, E. J. & Hill, R. V.  Ultraﬁltration-based techniques for rapid and simultaneous concentration of multiple microbe classes from 100-L tap water samples. J. Microbiol. Methods. 73, 92–99. Rangel-Martínez, C.  Identiﬁcación de Blastocytis hominis en muestras de agua obtenidas de Ciudad Universitaria en la Ciudad de México. Tesis de Licenciatura (Biología). Universidad Simón Bolivar, México, D.F. Smith, H. V. & Rose, J. B.  Waterborne Cryptosporidiosis. Parasitol. Today. 6, 8–12. Tapia-Palacios, M. A.  Detección de Cryptosporidium parvum y Giardia lamblia en agua del Río Cuitzmala, Jalisco. Tesis de Licenciatura (Biología). Facultad de Ciencias, Universidad Nacional Autónoma de México, México, D.F.

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Verma, V. & Arankalle, V. A.  Virological evaluation of domestic water puriﬁcation devicescommonly used in India emphasizes inadequate quality and need for virological standards. Trop. Med. Int. Health. 14, 885–891. World Health Organization (WHO) a Guidelines for Drinking Water Quality, 4th edn. WHO Press, Geneva, Switzerland. WHO b Evaluating Household Water Treatment Options: Health-Based Targets and Microbiological Performance Speciﬁcations. WHO Press, Geneva, Switzerland. Wright, J., Gundry, S. & Conroy, R.  Household drinking water in developing countries: a systematic review of microbiological contamination between source and point-ofuse. Trop. Med. Int. Health 9, 106–117. Xagoraraki, I., Kuo, D. H.-W., Wong, K., Wong, M. & Rose, J. B.  Occurrence of human adenoviruses at two recreational beaches of the Great Lakes. Appl. Environ. Microbiol. 73, 7874–7881.

First received 6 May 2013; accepted in revised form 15 September 2013. Available online 26 October 2013

105

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The potential of lipopolysaccharide as a real-time biomarker of bacterial contamination in marine bathing water Anas A. Sattar, Simon K. Jackson and Graham Bradley

ABSTRACT The use of total lipopolysaccharide (LPS) as a rapid biomarker for bacterial pollution was investigated at a bathing and surﬁng beach during the UK bathing season. The levels of faecal indicator bacteria Escherichia coli (E. coli), the Gram-positive enterococci, and organisms commonly associated with faecal material, such as total coliforms and Bacteroides, were culturally monitored over four months to include a period of heavy rainfall and concomitant pollution. Endotoxin measurement was performed using a kinetic Limulus Amebocyte Lysate (LAL) assay and found to correlate well with all indicators. Levels of LPS in excess of 50 Endotoxin Units (EU) mL 1 were found to correlate with

Anas A. Sattar (corresponding author) Simon K. Jackson Graham Bradley B409 Portland Square, School of Biomedical and Biological Sciences, Plymouth University, Drake Circus, Plymouth, Devon, PL4 8AA, UK E-mail: anas.sattar@plymouth.ac.uk

water that was unsuitable for bathing under the current European regulations. Increases in total LPS, mainly from Gram-negative indicator bacteria, are thus a potential real-time, qualitative method for testing bacterial quality of bathing waters. Key words

| faecal indicator bacteria, LPS, rapid method, sewage pollution, surﬁng/bathing water

INTRODUCTION The quality of beaches in the UK and Europe is governed by

in order to make an informed decision on whether to under-

the European Bathing Water Directive (EU ) which

take an activity. The term lipopolysaccharide (LPS) is often

sets legislation and deﬁnes standards that all beaches must

used interchangeably with endotoxin and is the major com-

comply with in order to be considered as a designated bathing

ponent of the outer membrane of all Gram-negative

beach. Faecal Indicator Bacteria (FIB), namely Escherichia

bacteria (Raetz ; Rietschel et al. ). LPS has been tar-

coli and enterococci, are important indicators for determin-

geted by previous studies for estimating total biomass and

ing the quality of marine bathing waters as they can

testing potable water ( Jorgensen et al. , ; Watson

eliminate the need for expensive and time consuming testing

et al. ; Evans et al. ; Haas et al. ) but not as a

for pathogenic bacteria and viruses (Lucena et al. ). The

potential biomarker for bacteria-polluted bathing water, poss-

FIB previously used were total coliforms, faecal coliforms, E.

ibly due to background levels from Gram-negative marine

coli and faecal streptococci as these microorganisms can pro-

species.

vide a general indication of faecal pollution; these were later

Studies conducted by Fiksdal et al. (), Allsop & Stickler

revised and coliforms were substituted by enterococci. How-

() and Kreader () have suggested the use of Bacteroides

ever, current quantitative culture-based FIB methods have

species as an excellent bacterial indicator candidate since it is

drawbacks (Borrego et al. ; Anderson et al. ; Field

shed in high numbers in faeces and is unlikely to reproduce in

& Samadpour ) as these indicators have a tendency to

seawaters. However, because of its anaerobic nature and conse-

multiply in bathing waters and the results are retrospective,

quent difﬁcult culture requirement, it has always been

taking at least 24–48 h to inform regulatory bodies. Marine

overlooked by legislators. Bacteroides species inhabit the intes-

recreational water users themselves really need an instant,

tines of human and warm-blooded animals where they

qualitative indication of possible bacterial pollution events

comprise 30–50% of normal faecal matter in humans (Isar

doi: 10.2166/wh.2013.142

106

A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

12.1

2014

et al. ) and they are present in bathing waters when polluted

the beach. This beach was chosen because it is divided

with poorly treated sewage from urban discharge or runoff from

into two distinct areas: bathing and surﬁng.

farms, hence Bacteroides was included in this study.

Composite samples, comprising the four stations from

Therefore, the aim of this study was to measure the total

each area, were collected on 10 occasions over the

LPS levels in seawater and correlate them with the current

summer of 2012 at Challaborough beach starting from

culture-based methods for determining this water quality

early June to the beginning of September (UK bathing

using bacterial indicators such as E. coli and Bacteroides.

season), in addition to water samples from the stream.

This was undertaken at a surﬁng/bathing beach during the

Samples were usually taken once a week with a random

UK bathing season, including a high rainfall pollution event.

timing for low and high tide, except for one sample in which heavy rainfall and high level of runoff had been observed and which was considered as the ‘polluted’

MATERIALS AND METHODS

sample (15/08/2012 sample in Figure 3). Samples were collected in 500 mL disposable, sterile, screw-capped wide-

Seawater sampling regime

mouth pots for the bacteriological investigation whilst 50 mL endotoxin-free Falcon™ tubes (BD Biosciences,

Challaborough beach was chosen as a popular bathing, body

UK) were used to collect water samples for LPS detection.

boarding and surﬁng beach located in the South Hams dis-

Shallow water samples were collected from the surface of

trict of Devon, UK (Latitude: 50.287159, Longitude:

the water whilst deeper water samples were taken from

3.899052). It is a horseshoe-shaped bay divided by a

approximately 1 m depth. Sediment samples were also col-

small stream that runs from a valley down into the sea

lected in similar pots from the shallow water area for both

(Figure 1).

bathing and surﬁng areas. Samples were immediately

Challaborough village comprises two small ﬁxed cara-

taken to the laboratory and processed within 3 h. Samples

van sites and a few private houses which end just beside

for LPS detection were aliquoted into endotoxin-free glass tubes (Lonza, UK) and preserved at 20 C until assay. W

Seawater sample filtration Water samples were filtered aseptically through a 0.45 μm membrane (Whatman, UK) and placed on appropriate media; Bacteroides Bile Esculin (BBE) (Livingston et al. ), Membrane Lauryl Sulphate Broth, and Slanetz and Bartley agar (Oxoid, UK) to isolate Bacteroides, total coliforms/E. coli and enterococci, respectively. Appropriate volumes of the samples were aseptically filtered in duplicate using a vacuum pump attached to the water filtration system. A 1:10 dilution of the sediment samples (2 g) was prepared in sterile simulated sea water (Instant Ocean, Underworld, UK) (18 mL) and placed in stomacher bags that were placed in a stomacher (Seward Lab, UK) for 2 Figure 1

Topography of Challaborough beach at low tide and the surrounding caravan site. A, B, C and D represent sampling stations for shallow water and sediment at the bathing area. E, F, G and H represent sampling stations of approximately 1 m depth water sampling in the bathing area. I, J K and L represent sampling stations for shallow water and sediment at the surﬁng area. M, N, O and P represent sampling stations of approximately 1 m depth water sampling in the surﬁng area. Q represents the sampling site for stream water.

minutes, and left to settle for 10 minutes. Then, 10 mL of the supernatant was ﬁltered in a manner similar to the method described for water sample ﬁltration above. BBE agar cultures were incubated in an anaerobic incubator W

(Don Whitley, UK) at 37 C for 48–72 h, Slanetz and Bartley

107

A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

12.1

2014

agar was incubated aerobically at 35 C for 4 h then at 44 C

according to the manufacturer’s instructions. Endotoxin

for 44 h, and Membrane Lauryl Sulphate total coliforms cul-

from E. coli O55:B5 (provided with the kit) was used as a

tures were incubated aerobically at 35 C for 24–48 h and for W

standard to calibrate the assay as described by the manufac-

E. coli incubated aerobically at 35 C for 4 h, then at 44 C

turer’s instructions. Appropriate serial dilutions were

for 44 h.

prepared from the endotoxin standard and samples from 50 to 0.005 Endotoxin Units (EU)/mL. The US Food and

Enumeration and isolation of bacterial colonies

Drug Administration (FDA) deﬁned an EU as the endotoxin activity of 0.2 ng of Reference Endotoxin Standard depend-

The numbers of colony-forming units (CFU) were calculated and numbers were expressed as CFU 100 mL

of water or

per g 1 of sediment.

ing on the source of endotoxin (Liebers et al. ). Presently, the conversion rate of the current reference standard used by the FDA (EC-6) is 10 EU/ng. Samples and standard endotoxins in duplicate were loaded into endo-

Determination of total endotoxin concentration

toxin-free 96-well plates (Costar, Corning, USA) and W

incubated for 10 minutes at 37 C in a pre-warmed plate In order to determine whether there was a correlation

reader (BioWhittaker elx808, BioTek, UK). The default tem-

between the total LPS concentration in bathing water and

plate parameters of WinKQCL Endotoxin detection and

the number of CFU of the bacterial indicators, a kinetic-

analysis software version 3.0.1 (Lonza) were set to 40 readings

QCL™ LAL assay (Lonza, UK) was conducted to determine

absorbance with a 405 nm measurement ﬁlter. Concentrations

the LPS concentration in the water samples. Brieﬂy, the

of unknown samples were calculated using the values of the

principle of the kinetic-QCL™ LAL assay is that the endo-

standard curve to give a quantitative endotoxin EU value.

toxin sample from Gram-negative bacteria is mixed with

All tips (Fisher Scientiﬁc, UK) and glass dilution tubes

Limulus Amebocyte Lysate (LAL, extracted from horseshoe

(Lonza, USA) used in each experiment were certiﬁed as endo-

crab Limulus polyphemus) substrate in appropriate con-

toxin-free. As a quality assessment of the assay, a spike and

dition and monitored over a period of time for the

recovery experiment using a known quantity of standard

appearance of a colour change due to endotoxin presence.

endotoxin was performed to investigate any inhibition/

Reaction time is the time that a colour change (yellow)

enhancement when assaying for endotoxin in seawater.

reaches an optical density absorbance of 0.2; this reaction time is inversely proportional to the concentration of endo-

Statistical analysis

toxin and the concentration of unknown samples is determined from standard curve.

Statistical analysis was performed using Microsoft Ofﬁce

A composite sample of the 10 water samples from the

Excel 2010 and Minitab® version 16.1.1 (Kruskal-Wallis

stream in addition to each area, i.e. shallow or deep (S or

test and one-way analysis of variance). LAL assay concen-

D), bathing or surﬁng (bathe or surf), was prepared. Four

trations were calculated using WinKQCL Endotoxin

dilutions (undiluted sample, 1/10, 1/100 and 1/1000) from

Detection and Analysis Software Version 3.0.1 (Lonza).

each composite sample were run for their total LPS activity using the kinetic-QCL™ method to investigate whether different sampling areas possess different LPS levels. In

RESULTS

addition, 10 individual water samples from the bathing area were assayed for their endotoxin activity to assess

Bacterial enumeration and total LPS estimation

whether the total LPS levels correlate to the number of E. coli and Bacteroides and followed a similar trend to these

The mean CFU in the 10 composite samples of the four

bacterial numbers. Water samples were diluted with endo-

areas for Bacteroides, E. coli, enterococci and total coli-

toxin-free water and assayed for their endotoxin activity

forms (n ¼ 10) over the sampling period are shown in

using a kinetic LAL assay kit (Kinetic-QCL™; Lonza, UK)

Figure 2. The number of indicator bacteria generally

108

Figure 2

A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

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2014

Mean number of CFU 100 mL 1 bacteria in 10 composite bathing water samples for four areas. (a) E. coli, (b) Bacteroides species, (c) enterococci and (d) total coliforms. S. bathe represents shallow water samples collected from the bathing area, D. Bathe represents deep water samples collected from the bathing area, S. surf represents shallow water samples collected from the surﬁng area, and D. surf represents deep water samples collected from the surﬁng area. n ¼ 10. Bars show standard error of means. * means E. coli p < versus S. surf and D. surf; Bacteroides p < 0.05 versus S bathe; Total coliforms p < 0.05 versus S. surf and D. surf.

showed that the bathing area was more contaminated than

consists of small rocks and sand grains which tend to have

the surﬁng area. There was a signiﬁcant difference in

a poor surface for bacterial attachment and also therefore

the number of CFU of E. coli (p ¼ 0.03) and total

as a bacterial reservoir (Harrison ).

coliforms (p ¼ 0.01) when comparing the surﬁng and the bathing areas. Bacteroides results only showed a signiﬁcant difference between S. surf and S. bathe areas. Enumerated enterococci

The mean LPS concentration over the sampling period for each area is shown in Figure 3 where LPS concentration varied between 34 and 135 EU mL 1. There were highly signiﬁcant differences among all the sampling sites.

showed no signiﬁcant difference between the bathing and

When examined over the time period, a distinct increase

the surﬁng areas, however, the numbers of CFUs were still

in contamination was shown for all indicators after a period

higher than in the bathing area. A 95th and 90th percentile

of heavy rainfall and concomitant runoff (data shown for

was calculated according to the recommended formula by

Bacteroides and E. coli only, Figure 4). Endotoxin levels fol-

the 2006 European directive; results showed that all the

lowed a similar trend to the number of FIB CFUs showing

water samples from both bathing and surﬁng areas were

the highest increase at the polluted run-off event (15/08/

classiﬁed as ‘poor’ (data not shown). Sediment samples

12) and returning to threshold levels within the next

showed extremely low numbers of bacteria; the sediment

sampling period (7 days).

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A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

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concentration of 0.5 EU mL 1 with appropriate controls and performing the LAL assay. The recovery of LPS was over 10-fold lower at undiluted levels but this inhibition was removed at dilutions of more than one in 10. Consequently, all seawater samples were diluted 1/100 for LPS measurements.

DISCUSSION AND CONCLUSIONS Testing the quality of marine bathing waters is an essential Figure 3

| Mean LPS concentrations of 10 composite water samples from ﬁve areas. A

procedure to ensure compliance with EU legislation and

one-way analysis of variance was conducted to evaluate the difference in LPS

the health of bathers and watersports performers. How-

concentrations among different water sampling areas using Tukey’s Multiple

ever, a real-time cost effective method is needed as

Comparison Test. *** means p <0.001 among all sampling areas. Stream represents water samples collected directly from the stream area, S. bathe represents shallow water samples collected from the bathing area, D. bathe represents deep water samples collected from the bathing area, S. surf represents shallow water samples collected from the surﬁng area, D. surf represents deep water samples collected from the surﬁng area. n ¼ 4. Error bars show standard error of means.

current culture-based methods are time consuming with retrospective results reﬂecting the water quality status at least 24–48 h previous. Results from this study have shown that total LPS can be used as a rapid, qualitative biomarker taking approximately 1.5 h and can be optimised to give qualitative results in 30 minutes to detect levels above a calculated threshold of 50 EU mL 1 of LPS in marine bacterial and faecal contamination. Studies conducted previously have explored the use of LPS as an indicator for testing contamination in drinking and other water. Jorgensen et al. () ran a pilot study to explore the possibility of using a LAL assay to estimate the concentration of endotoxins in potable and reclaimed, advanced waste treatment waters and showed that chlorination of water interferes with the assay. Watson et al. () used three techniques including the determination of LPS concentration to estimate the biomass and

Figure 4

and total endotoxin

number of bacteria in marine water. That study showed

concentration EU mL 1 in water samples (n ¼ 4) of the shallow bathing area during the sampling periods. The dotted line represents the cut-off of LPS

that LPS can be related to the number of bacteria and

concentration above which readings are considered as unsuitable water for

suggested a factor to convert LPS to bacterial carbon.

Mean number of Bacteroides and E. coli CFU 100 mL

bathing.

LPS concentration in water supplies from a stream

Good correlations between the number of indicator bac-

water correlating with the culture-based bacterial count

teria and concentrations of total LPS were obtained

of coliforms, enteric, Gram-negative and heterotrophic

(Figure 5). The European directive threshold value for E.

bacteria was investigated by Evans et al. () using the

coli is also shown in Figure 5(a) and is converted to EU of

gel clot and a spectrophotometric LAL assay. This

LPS.

showed that the gel clot method was less sensitive and

The use of undiluted seawater samples indicated that

less reproducible than the spectrophotometric assay;

there was inhibition of the LAL assay. To conﬁrm the inhi-

Evans et al. () study also suggested the continued

bition, a spike-and-recovery assessment was performed by

reﬁning of the LAL assay for implementation in water

spiking artiﬁcial seawater (Instant Ocean) to a ﬁnal

quality investigation.

110

Figure 5

A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

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Pearson correlation of 10 composite water sample means for CFU and total LPS at the shallow bathing area (S. bathe) (a) E. coli, Pearson correlation of LPS and E. coli ¼ 0.936, p < 0.001 (b) Bacteroides, Pearson correlation of LPS and Bacteroides ¼ 0.954, p < 0.001 and (c) total coliforms, Pearson correlation of LPS and total coliforms ¼ 0.852, p < 0.05. A threshold as set by the European directive and the corresponding EU/mL of endotoxin is highlighted with the dotted line where appropriate.

In the current study, a more accurate and highly sensi-

indicators in the European directive 2006 guide for coastal

tive kit was used to investigate the bacteriological LPS

bathing water quality. Using Figure 5, the authors estimate

quality of recreational bathing seawater. The kinetic-

the corresponding LPS concentration at these limits to be

QCL™ is a highly sensitive method that can detect LPS

equivalent to 50.3 EU mL 1. This level could be set for a

down to 0.005 EU mL . Results showed that total LPS con-

future

centration correlates very well with the current bacterial

development.

real-time

qualitative

bathing

water

test

kit

indicator, E. coli and with Bacteroides, and slightly less

Marine bathing water represents a sanctuary for

with total coliforms (Figure 5), all including a pollution

eukaryotes and prokaryotes, including indigenous Gram-

event after heavy rainfall. Increases in LPS also appeared

negative bacteria. When estimating the total LPS from

to dissipate from the bathing water within the time period

marine bathing waters, part of the total LPS measurement

of the next sample (7 days) and probably within the 70 h

is due to the presence of ‘background indigenous Gram-

by the methods described by Shibata et al. (). LPS mol-

negative bacteria’ which will affect the estimation of

ecules are shed from the Gram-negative bacterial cell wall

LPS using the LAL assay. However, many studies have

when the environmental factors are not suitable for these

shown that the major bacterial species present in seawater

cells or when infected with bacteriophages (Fuhrman

is characterised by low endotoxin virulence because low

; Nagata ). LPS is being constantly removed from

acylation

seawater by ﬂagellates (Shibata et al. ). The LPS

marine Gram-negative bacteria (Ramos et al. ; Krasi-

threshold at which seawater can be considered as polluted

kova et al. ; Leone et al. ). The presence of low

or unsuitable for bathing was determined based on the cur-

acylation – penta- and tetra-acyl species – and phosphoryl-

rent legislated number for a ‘sufﬁcient’ level of bacterial

ation in the lipid A structure signiﬁes low immunological

and

phosphorylation

are

encountered

111

A. A. Sattar et al.

Endotoxin, a rapid biomarker of bacterial contamination in bathing water

Journal of Water and Health

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activity compared to endotoxically active molecules

Whilst background levels of endogenous LPS appeared

(Rietschel et al. ) and is also an indication of low

to remain low and stable during this study, a speciﬁc LPS

activity with the LAL assay. Hence, even when there is

assay for one indicator would be preferable. The authors

an increase in the background levels it is likely to

are working on developing a more speciﬁc biomarker invol-

induce a low reactivity in the LAL assay. Collected

ving LPS of Bacteroides as real time biomarker using ELISA

water samples processed in this study have never shown

assay.

a false positive in the LAL assay, and low CFU numbers

In conclusion, a rapid method has been developed to

of FIB (especially E. coli) were always accompanied

screen the bacterial quality of marine bathing water and

by low LAL activity. The background level of LPS was

has the potential to be developed and used as a qualitative,

measured in an excellent quality bathing water sample

on-the-spot test by bathers and beach goers, based on an

and results showed it was 6 EU mL 1. However, there is

excellent correlation between the culture-based method

a possibility of occasionally observing false positives,

and total LPS determination.

events above 50 EU mL 1, in the absence of FIB. This may perhaps be true due to, for example, eutrophication (Nixon ) in seawater that allows marine Gram-negative bacteria to thrive. A less plausible source is from

ACKNOWLEDGEMENTS

cyanobacterial blooms and an increase in vibrio densities. Although these increases in total LPS would be related to

The authors would like to thank Dr Wondwossen Abate for

a non-faecal pollution event, this can still be useful as an

help in running the LAL assays.

indicator for the presence of these blooms which have been shown to cause serious human health problems such as gastrointestinal tract infections, cyanobacterial intoxications and even cholera (Morris & Acheson ; Dietrich et al. ). Our immunological studies (unpublished data) have shown that an increase in LPS levels in contaminated bathing water is directly linked to an increase in human cell line pro-inﬂammatory cytokines and the production of these cytokines was dose-dependent on LPS levels. Hence the LAL test has an advantage in detecting total LPS levels even in a non-faecal contamination event. The quantitative kinetic-QCL™ assay was performed in approximately 1.5 h when using a 10-fold range of LPS standards 0.005–50 EU mL 1 and approximately 30 minutes to detect 0.5 EU and above, which is equivalent to 50 EU mL 1 after 100-fold dilution. This method could potentially be optimised as an endpoint chromogenic assay, manufactured and available for untrained bathers as a real-time, qualitative, single-use kit to examine the bacterial quality of bathing waters. The LAL method is not an attempt to replace the applicability of current culture or future PCR methods as these are a valuable method used in microbial source tracking but could be used as part of a ‘tool box’ approach to water quality management.

REFERENCES Allsop, K. & Stickler, D. J.  An assessment of Bacteroides fragilis group organisms as indicators of human faecal pollution. J. Appl. Microbiol. 58, 95–99. Anderson, K. L., Whitlock, J. E. & Harwood, V. J.  Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Appl. Environ. Microbiol. 71, 3041–3048. Borrego, J. J., Arraba, F., De Vicente, A., Gomez, L. F. & Romero, P.  Study of microbial inactivation in the marine environment. J. Water Pollut. Control Fed. 55, 297–302. Dietrich, D., Fischer, A., Michel, C. & Hoeger, S. J.  Toxin mixture in cyanobacterial blooms – a critical comparison of reality with current procedures employed in human health risk assessment. In: Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs (H. K. Hudnell, ed.). Springer, New York, pp. 885–912. EU  Directive 2006/7/EC of the European parliament and of the council of 15 February 2006 concerning the management of bathing water quality and repealing directive 76/160/EEC. Ofﬁcial Journal of the European Union L64, 37–52. Evans, T. M., Schillinger, J. E. & Stuart, D. G.  Rapid determination of bacteriological water quality by using limulus lysate. Appl. Environ. Microbiol. 35, 376–382. Field, K. G. & Samadpour, M.  Fecal source tracking, the indicator paradigm, and managing water quality. Water Res. 41, 3517–3538.

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Fiksdal, L., Maki, J. S., Lacroix, S. J. & Staley, J. T.  Survival and detection of Bacteroides spp., prospective indicator bacteria. Appl. Environ. Microbiol. 49, 148–150. Fuhrman, J. A.  Impact of viruses on bacterial processes. In: Microbial ecology of the oceans. (D. L. E. Kirchman, ed.). Wiley-Liss, New York, pp. 327–350. Haas, C. N., Meyer, M. A., Paller, M. S. & Zapkin, M. A.  The utility of endotoxins as a surrogate indicator in potable water microbiology. Water Res. 17, 803–807. Harrison, D.  A Student’s Guide to the Seashore. Emerald Group Publishing Limited, Bingley, UK. Isar, J., Agarwal, L., Saran, S. & Saxena, R. K.  Succinic acid production from Bacteroides fragilis: Process optimization and scale up in a bioreactor. Anaerobe 12, 231–237. Jorgensen, J. H., Lee, J. C. & Pahren, H. R.  Rapid detection of bacterial endotoxins in drinking water and renovated wastewater. Appl. Environ. Microbiol. 32, 347–351. Jorgensen, J. H., Lee, J. C., Alexander, G. A. & Wolf, H. W.  Comparison of Limulus assay, standard plate count, and total coliform count for microbiological assessment of renovated wastewater. Appl. Environ. Microbiol. 37, 928–931. Krasikova, I. N., Kapustina, N. V., Isakov, V. V., Dmitrenok, A. S., Dmitrenok, P. S., Gorshkova, N. M. & Solov’eva, T. F.  Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119T. Eur. J. Biochem. 271, 2895–2904. Kreader, C. A.  Design and evaluation of Bacteroides DNA probes for the speciﬁc detection of human fecal pollution. Appl. Environ. Microbiol. 61, 1171–1179. Leone, S., Silipo, A., Nazarenko, E. L., Lanzetta, R., Parrilli, M. & Molinaro, A.  Molecular structure of endotoxins from Gram-negative marine bacteria: an update. Mar. Drugs 5 (3), 85–112.

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Liebers, V., Brüning, T. & Raulf-Heimsoth, M.  Occupational endotoxin-exposure and possible health effects on humans (review). Am. J. Ind. Med. 49, 474–491. Livingston, S. J., Kominos, S. D. & Yee, R. B.  New medium for selection and presumptive identification of the Bacteroides fragilis group. J. Clin. Microbiol. 7, 448–453. Lucena, F., Lasobras, J., McIntosh, D., Forcadell, M. & Jofre, J.  Effect of distance from the polluting focus on relative concentrations of Bacteroides fragilis phages and coliphages in mussels. Appl. Environ. Microbiol. 60, 2272–2277. Morris, J. G. & Acheson, D.  Cholera and other types of Vibriosis: a story of human pandemics and oysters on the half shell. Clin. Infect. Dis. 37, 272–280. Nagata, T.  Production mechanisms of dissolved organic matter. In: Microbial ecology of the oceans. (D. L. E. Kirchman, ed.). John Wiley & Sons, New York, pp. 121–152. Nixon, W.  Coastal marine eutrophication–A definition, social causes, and future concerns. Ophelia 41, 199–219. Raetz, C. R. H.  Biochemistry of endotoxins. Annu. Rev. Biochem. 59, 129–170. Ramos, J. L., Gallegos, M. A.-T., Marqués, S., Ramos-González, M.-I., Espinosa-Urgel, M. & Segura, A.  Responses of Gram-negative bacteria to certain environmental stressors. Curr. Opin. Microbiol. 4, 166–171. Rietschel, E. T., Kirikae, T., Schade, F. U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A. J., Zähringer, U., Seydel, U. & Di Padova, F.  Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8, 217–225. Shibata, A., Yasui, H. & Fukuda, H.  Fate of the bacterial cell envelope component, lipopolysaccharide, that is sequentially mediated by viruses and flagellates. Coast. Mar. Sci. 33, 39–45. Watson, S. W., Novitsky, T. J., Quinby, H. L. & Valois, F. W.  Determination of bacterial number and biomass in the marine environment. Appl. Environ. Microbiol. 33, 940–946.

First received 3 August 2013; accepted in revised form 16 September 2013. Available online 26 October 2013

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Broader incubation temperature tolerances for microbial drinking water testing with enzyme substrate tests Robert L. Matthews and Rosalind Tung

ABSTRACT Microbiological testing is an integral part of measures to ensure safe drinking water. However, testing can be restricted in low-resource settings by the requirement for specialized laboratory facilities and testing procedures. Precisely controlled incubation temperature is one example. The effect of varied incubation temperatures on the performance of two enzyme substrate tests for the detection of Escherichia coli and total coliforms has been examined. The aim was to determine

Robert L. Matthews (corresponding author) Rosalind Tung University of Bristol, Water and Health Research Centre, Bristol, BS8 1TR, UK E-mail: rob.matthews@bristol.ac.uk

whether these tests would provide consistent and comparable enumeration over a broader temperature range than currently speciﬁed. Recovery of chlorine-injured and wild type E. coli was examined over a range of non-standard incubation temperatures in comparison to 37 C ± 1. W

Colilert® and Aquatest, a new E. coli-speciﬁc detection medium, served as the two representative enzyme substrate media. Recovery of chlorine-injured E. coli in Colilert was not impaired within the W

range 33–39 C; the equivalent range in Aquatest medium was 31–43 C. Both these tests recovered E. coli without signiﬁcant loss of performance over a wider range of temperatures than currently speciﬁed. Key words

| Aquatest, coliform, Colilert, drinking water, Escherichia coli, incubation temperature

INTRODUCTION Microbiological testing of drinking water is an increasingly

One standard that is difﬁcult to adhere to in developing

important component of global efforts to reduce water-

countries is tightly controlled incubation temperature. Tests

related disease (UNICEF & WHO ; Bain et al. a).

for total coliforms and E. coli are generally incubated at

This is evident in the post-2015 targets for water, sanitation

35 C ± 0.5 in the USA and 37 C ± 1 in the UK (APHA

and hygiene, proposed recently by a UNICEF/WHO work-

; Standing Committee of Analysts ). The Inter-

ing group, which place greater emphasis on water quality,

national Organization for Standardization encompasses

mentioning speciﬁcally the measurement of Escherichia

both of these by specifying 36 C ± 2 (ISO , ).

coli in drinking water (JMP ). In many areas of develop-

There are some differences when comparing E. coli incu-

ing countries, however, the ability to conduct microbial

bation temperature standards used in other disciplines. For

testing is constrained due to a shortage of laboratory facili-

example, in the testing of dairy products using an enzyme

ties, trained personnel and essential infrastructure, such as

substrate test (Petriﬁlm™), a temperature of 35 C ± 1 is rec-

electricity supply.

ommended (APHA ). A laboratory or testing facility in a

A recent review of currently available water quality tests found many to be of limited suitability for use in

developing country may not have an incubator capable of maintaining temperature within such tight tolerances.

resource-poor settings (Bain et al. b). A contributing

In addition, the lack of a reliable electricity supply

factor is the difﬁculty of conducting tests in a manner

would limit the operation of an incubator in many low-

that complies with regulations and standard operating

resource settings. Access to electricity has been highlighted

procedures.

as a major constraint to the use of healthcare technology

doi: 10.2166/wh.2013.076

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R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

Journal of Water and Health

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in developing countries (Malkin ; Howitt et al. ).

It is well established that the maximum growth rate for

International Energy Agency ﬁgures show that, as of 2009

E. coli occurs at approximately 37–42 C (Mhor & Krawiec

approximately 1.3 billion people (20% of the global popu-

; Ingraham & Marr ). However, the inﬂuence of

lation) were without access to electricity, mostly in

temperature on recovery of E. coli is less well documented.

developing countries. Sub-Saharan Africa has the lowest

In this review, no studies could be identiﬁed which support

electriﬁcation rate of only 30.5%, with 14.2% in rural

the tolerances in any of the aforementioned standards. It is

areas (http://www.iea.org).

noted that these standards were established with reference

A review of the history of incubation temperature

to the coliform group using lactose fermentation tests

standards and tolerances demonstrates that those cur-

(lauryl tryptose and brilliant green lactose bile broths in

rently employed have been used since the development

the USA, MacConkey broth in the UK), and it is not appar-

of water testing technology in the early to mid twentieth

ent that they have been adapted for E. coli (APHA ;

century. The American standard incubation temperature

DHSS ). There appears to be an absence of published lit-

of 35 C ± 0.5 ﬁrst appears in 1955 in the 10th edition

erature examining the performance of speciﬁc tests at

of the APHA’s Standard Methods for the Examination of

different controlled temperatures, and that the development

Water, Sewage and Industrial Wastes (later to become

of new methods has been performed to conform to pre-exist-

Standard Methods for the Examination of Water and

ing standards.

Wastewater). It is applied in the context of testing for coli-

The current standards were originally established for

form bacteria using a multiple fermentation tube method

methods involving gas or acid production from the fermen-

followed by conﬁrmation on solid media (APHA ).

tation of lactose. These processes require enzymes, such as

All subsequent editions of the manual apply the same

lactose permease, that are not required in enzyme substrate

temperature and tolerances even when new test method-

media. The older standard methods also utilize inhibitory

ologies are used, such as membrane ﬁltration and,

agents such as bile salts. Consequently, shifts in incubation

latterly, enzyme substrate methods (APHA , ,

temperature may have a different impact on the recovery

, , , , , ). Previous editions

of E. coli and coliforms with traditional tests compared to

describe testing for the coliform group, or its predecessors

enzyme substrate methods.

Bacillus coli or the coli-aerogenes group, at 37 C without

If, for some tests, wider temperature tolerances were to

temperature tolerances, the exception being the 9th edi-

be deﬁned, incubators that are less accurate but cheaper

tion which recommends 35–37 C (APHA , ,

than conventional laboratory models could be used. Such

, , , ).

increased ﬂexibility could enable more widespread and regu-

The ﬁrst regulatory manual for microbiological drinking water analysis in the UK is The Bacteriological Examination

lar monitoring of microbial water quality, particularly in remote or impoverished settings (Bain et al. b).

of Water Supplies, informally known as ‘Report 71’, ﬁrst pre-

This study deﬁnes the incubation temperature range for

pared in 1934 and revised in 1939. This gives an incubation

Colilert and Aquatest media. Both tests employ enzyme sub-

temperature for coliform tests of 37 C without a tolerance

strate

range (Ministry of Health , ). Temperature toler-

temperatures to select for target organisms. Colilert was

technology

and

not

require

speciﬁc

ances appeared in the 1982 and 1994 revisions where

selected as a widely recognized and approved method.

37 C ± 0.5 was recommended for total coliform testing by

Aquatest medium is an E. coli-speciﬁc detection medium uti-

multiple tube fermentation and by membrane ﬁltration, the

lized within Aquatest, a device developed to enable reliable

latter following 4 hours prior incubation at 30 C ± 0.5

enumeration of E. coli in drinking and surface waters in low-

(DHSS ; Standing Committee of Analysts ). In the

resource settings. In parallel with the development of Aqua-

most recent revision, the temperature tolerance has been

test medium was that of a latent heat storage incubator to

relaxed to 37 C ± 1, and this also covers enzyme substrate

maximize the applicability of Aquatest in low-resource set-

techniques such as Colilert lysts ).

(Standing Committee of Ana-

tings (Aquatest ). Since incubation based on latent heat may not adhere to standard temperature tolerances, it

115

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

would be advantageous if Aquatest medium were suitable

Journal of Water and Health

12.1

2014

Chlorine injury

for use over a broad incubation temperature range. This chlorine-injury method was developed as a simpler alternative to the US Environmental Protection Agency

MATERIALS AND METHODS

(USEPA) chlorine-injury protocol (USEPA ), using a single strain of E. coli instead of wastewater samples. It is

The incubation temperature ranges for Colilert and Aqua-

a less rigorous test than the USEPA method, but gives an

test medium were established by comparing the recovery

indication of the ability of a test to recover injured organ-

of both chlorine-injured and wild type E. coli, when the

isms. The method of injury was similar to that applied by

tests were incubated at non-standard temperatures in com-

Walsh & Bissonnette () and Bolster et al. ().

parison to a reference of 37 C ± 1. Chlorine injury is used

A 20-h culture of E. coli was grown in nutrient broth

to approximate a water treatment process (USEPA ).

(Fluka No. 2) from a single Microbank bead. The resulting

It also presents a greater challenge to the test media, as

culture contained approximately 109 E. coli ml 1; 1 ml of

recovery of chlorine-injured E. coli takes longer and is

culture was added to 9 ml PBS and placed on ice. The E.

more sensitive to the test environment (McFeters et al.

coli were injured by exposure to chlorine, by adding 50 μl

). The effect of non-standard temperatures on total

of a 10% dilution of technical grade sodium hypochlorite

coliform recovery was also assessed for Colilert. Both

(Fisher) directly to the diluted culture to achieve a 2–4 log

media were used in combination with IDEXX Quanti-

reduction in number of organisms; a level of injury rec-

Trays™.

ommended

USEPA

(USEPA

).

After

5 min

exposure, the chlorine was quenched by the addition of 200 μl 20% sodium thiosulfate. Ten-fold dilutions were pre-

Microorganisms

pared in PBS to a concentration of 10 5. To ascertain the For use in the chlorine-injury study, E. coli NCTC 9001 were

level of injury, 100 μl of the 10 3, 10

obtained from the Health Protection Agency, Bristol, UK,

and 100 μl of 10

and 10

dilutions,

unchlorinated culture, were added to

80 C until

100 ml sterile deionized water containing Colilert medium

required. For testing using wild type E. coli and total coli-

and enumerated in Quanti-Trays following incubation at

forms, a 500-ml sample of Bristol harbour water was

37 C ± 1 for 24 h. Injured organisms were refrigerated at

and maintained on Microbank™ beads at

collected in a sterile container from Prince Street Bridge,

4 C until use.

Bristol, and transported to the laboratory on ice. A fresh sample was obtained prior to each test. The initial concen-

Test procedure

tration of E. coli and total coliforms in the sample was evaluated using Quanti-Tray and Colilert medium. Charac-

Tests using chlorine-injured E. coli were performed as fol-

teristics of the raw water were as follows: source

lows: if a 2–4 log reduction in population had been

temperature 9–15 C; pH 7.89–8.36; total coliform concen-

achieved by the chlorination step, the tests were performed

24–30 h following injury, otherwise the test was aborted and

tration as most probable number (MPN) 10.76 MPN ml 1

to >200.5 MPN ml ; E. coli concentration 2–65.9 MPN

the chlorination step repeated. Growth media (Colilert or

ml 1.

Aquatest) were each suspended in 1.2 L sterile water at the concentration stated by the manufacturer. This was inocu-

Growth media

lated with injured E. coli to yield a population of approximately 10–30 E. coli per 100 ml, and dispensed in

The two growth media used in this study were Colilert and

100 ml volumes into 12 Quanti-Trays; a stack of six was

Aquatest growth medium. Colilert was obtained from

placed in an incubator at 37 C ± 1 and six were placed in

IDEXX in individual test snap packs. Aquatest medium

an incubator at an alternative temperature: X C ± 1. The

was obtained from the Aquatest project.

test procedure was repeated for alternative temperatures in

116

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

the range 23–47 C at 2 C intervals. To facilitate accurate

Journal of Water and Health

12.1

2014

RESULTS

temperature measurement, an additional Quanti-Tray containing only sterile water and featuring a thermocouple

The temperature ranges over which Colilert and Aquatest

(T-type) in one central well and one peripheral well was

medium were used without loss of performance for the detec-

positioned in the centre of each stack. Temperature was

tion of wild type and chlorine-injured E. coli are presented in

recorded by means of a datalogger (Picotech TC-08, cali-

Table 1. These temperatures represent the maximum and

brated to ±0.5 C), measurements were taken once per

minimum incubation temperatures that did not result in a sig-

minute during the incubation period. The MPN E. coli per

niﬁcantly lower recovery of E. coli in comparison to a control

100 ml was recorded at 24 h ± 0.5 h.

incubation temperature of 37 C ± 1 (Student t-test, p < 0.05).

Tests using wild type E. coli were performed following

Percentage recovery at all non-standard incubation tempera-

enumeration of the sample and 24–30 h from sample collec-

tures relative to 37 C ± 1 is shown in Figure 1 for Colilert and

tion. The procedure was similar to that for injured E. coli.

Figure 2 for Aquatest medium. In general, all temperatures

The sample was diluted with sterile deionized water contain-

within the limits stated in Table 1 resulted in 80–120%

ing either Colilert or Aquatest medium, to give 1.2 L with an

E. coli recovery, none of which were signiﬁcantly lower. All

E. coli concentration of approximately 10–30 E. coli per

non-standard temperatures above and below the limits

100 ml. The remainder of the test was performed as above.

resulted in signiﬁcantly lower recovery. Aquatest medium

For raw water tests using Colilert, total coliform results

proved more resistant to non-standard incubation tempera-

were also recorded.

tures than Colilert for wild type E. coli, with a range of W

18 C in comparison to 12 C for Colilert, and chlorineW

Statistical analysis

injured E. coli, with a range of 12 C compared to 6 C for Colilert. This is particularly notable in the recovery of wild

The Student t-test was used to assess difference in recovery

type E. coli at low temperatures, where Aquatest medium dis-

between alternative temperatures and the control. A one

played good recovery even at 25 C. Recovery values for all

tailed t-test was performed between the control Quanti-

tests are presented in supplementary information (Tables

Trays incubated at 37 C ± 1 and the alternative temperature

S1 to S4, available online at http://www.iwaponline.com/

Quanti-Trays. A one tailed t-test was selected as the objective

wh/012/076.pdf).

was to determine whether the alternative temperature

Our results also suggest that temperatures exceeding the

resulted in a lower recovery than the control, that is, H0:

upper limit display a more rapid decline in recovery than

alternative temperature recovery control recovery; H1:

temperatures below the lower limit. This general trend can

alternative temperature recovery < control recovery. The

be observed in the shape of the graphs in Figures 1 and 2.

null hypothesis was rejected for p < 0.05. Unless speciﬁcally

This is in accordance with analysis of temperature and

mentioned, instances where the alternative temperature

growth rate by Ingraham & Marr (), which found a

yielded higher recovery than the control are regarded as

more rapid decline in growth rate at temperatures above

not signiﬁcant and are marked p > 0.5.

42 C than at temperatures below 37 C.

Data of this nature can be assumed to be represented better by a lognormal distribution (Cochran ). The afore-

Table 1

Maximum and minimum temperature for each test media and E. coli type not resulting in a signiﬁcant difference in recovery (p > 0.05) in comparison to

mentioned analysis was also performed on log-transformed

37 C ± 1 W

data, and in addition a non-parametric Mann–Whitney U W

Acceptable temperature range ( C)

test was conducted. In general, there was very good agreement between the three tests; whilst there were small differences in p-value, the nature of the results were consistent. For clarity, only the analysis of untransformed data is presented herein. Analysis was performed using SPSS Statistics version 19 and Microsoft Excel 2007.

Raw water E. coli

Chlorine-injured E. coli

Lower

Upper

Lower

Upper

Colilert

Aquatest

Test medium

117

Figure 1

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

Journal of Water and Health

12.1

2014

Percentage recovery of E. coli using Colilert at non-standard incubation temperature relative to a control incubated at 37 C ± 1. Error bars show combined percentage standard W

error. (a) Recovery of chlorine-injured E. coli. (b) Recovery of wild type E. coli.

Figure 2

Percentage recovery of E. coli using Aquatest medium at non-standard incubation temperature relative to a control incubated at 37 C ± 1. Error bars show combined W

percentage standard error. (a) Recovery of chlorine-injured E. coli. (b) Recovery of wild type E. coli.

Data on total coliform recovery are only available for Colilert, as Aquatest medium does not contain a total coliform substrate. Percentage recovery of total coliforms using Colilert is presented in Figure 3. The minimum temperature at which Colilert could be used without producing a W

signiﬁcantly lower recovery was 29 C; however any tempW

erature above the 37 C control, i.e. 39 C and above, gave a signiﬁcantly lower recovery (p < 0.05). In addition 31 W

and 33 C resulted in substantially higher recoveries than W

37 C, 143 and 188% respectively. To assess whether these were signiﬁcantly higher, a one tailed t-test was performed with reversed hypotheses. Both of these were found to be signiﬁcant, with p-values of 0.043 and 0.003, respectively.

Figure 3

Percentage recovery of total coliforms from a raw water sample using Colilert at non-standard incubation temperature relative to a control incubated at 37 C ± 1 C. Error bars show combined percentage standard error. W

118

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

Journal of Water and Health

12.1

2014

At these temperatures, recovery of E. coli was not signiﬁ-

differences in media composition. This suggests that it

cantly different, implying that the test was recovering more

would be possible to adjust the formulation of a test that

total coliforms that are not E. coli. Total coliform recovery

utilizes β-D-glucuronidase to optimize robustness to incu-

values using Colilert are presented in supplementary infor-

bation temperature. Previous work has shown that the

mation

(Table

S5,

available

online

http://www.

iwaponline.com/wh/012/076.pdf).

rate of β-D-glucuronidase activity for E. coli is relatively W

stable over the range 25–45 C, but potentially slightly faster at the higher temperature (Adams et al. ; Tryland & Fiksdal ).

DISCUSSION

This study did not include investigation of false-positive and false-negative rates; however, an independent evalu-

E. coli test performance

ation (University of Surrey, UK) has found Aquatest

This study demonstrates that two water quality tests can

18® in recovering E. coli from a range of water sources in

medium to be at least as sensitive and speciﬁc as Colilertrecover E. coli without loss of performance over a wider

South Africa (unpublished data). It follows that the compo-

range of temperatures than current standards specify. This

sition of Aquatest medium enables better recovery of E. coli

applies to both chlorine-injured E. coli and to wild type E.

at extreme temperatures. It is possible that E. coli have a

coli present in a raw water sample. Chlorine-injured organ-

higher growth rate and/or shorter lag phase in Aquatest

isms were more sensitive to non-standard incubation

medium than in Colilert.

temperature than wild type. The raw water sample would have contained E. coli with an array of injury severity,

Total coliform test performance

whereas those in the injury study are all subject to severe stress and were used to represent a worst-case scenario for

One of the differences between the two media is the absence

test performance.

of a total coliform test in Aquatest medium. In Colilert this is

Whether or not an organism is recovered within the test

provided by the galactosidase substrate ONPG, which

interval is dependent on both the growth rate and the lag

releases the yellow-coloured nitrophenol dye when cleaved.

phase duration of the organism. Over a temperature range

The nitrophenol has a quenching effect on the blue ﬂuor-

of approximately 21–37 C, the logarithm of the growth

escence of the MUG and as a result E. coli counts can be

rate follows a linear relationship with the inverse of temp-

more difﬁcult to read. It is possible that some Quanti-Tray

erature (Ingraham & Marr ). In addition, organisms

wells contained slow-growing E. coli that produced weak

that have been subject to stress prior to inoculation can be

ﬂuorescence, which was masked by the yellow colour

expected to have longer lag phases and also more variability

when the results were read. This could mean that stressed

in lag phase duration (Stephens et al. ; Li et al. ).

bacteria are more detectable in the Aquatest medium,

McFeters et al. () observed that a substantial proportion

which is not subject to the quenching effect of ONPG.

of E. coli subject to chlorine injury were not recovered

This offers one potential explanation as to why Colilert

within a typical 24-h test interval using both enzyme sub-

appears to be less robust to low temperatures where the E.

strate and conventional media at the standard incubation

coli can be expected to grow more slowly.

temperature. At low temperatures, the reduced growth rate

The fact that the total coliform component of the Coli-

may result in an increased number of cells with long lag

lert test appears more sensitive to high temperatures is not

phases not reaching a detectable concentration within the

surprising; the total coliform group consists of a number of

test interval.

different organisms, some of which are of environmental

Despite relying on the same enzymatic substrate for the

origin and will therefore be more acclimatized to lower

detection of E. coli, 4-methylumbelliferyl β-D-glucuronide

temperatures than enteric bacteria (Leclerc et al. ). As

(MUG), the two media exhibited different acceptable incu-

a group of organisms, it is also possible that the optimum

bation temperature ranges, which is presumably due to

temperature range for total coliforms, being a composite of

119

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

Journal of Water and Health

12.1

2014

several different temperature ranges, may be relatively

Use of these tests at temperatures outside the limits

narrow and it may also vary from source to source depend-

suggested herein is likely to result in a reduction in sensi-

ing on the relative populations.

tivity, indicated by the impaired recovery of injured organisms. This is in agreement with an analysis of ambient

Implications

temperature incubation by Brown et al. (). This may still be preferable if the alternative is not testing, especially if

In demonstrating that the incubation range can be broadened

untreated sources are being tested. In such cases the

for Colilert and Aquatest medium, the temperature require-

nature of the results for temperatures above or below the

ments for other E. coli tests based on the β-glucuronidase

limits should be considered. Progressively lower tempera-

enzyme could potentially be relaxed without compromising

tures appear to result in a gradual decline in the recovery,

the sensitivity of tests. The fact that these two tests exhibited

whereas increasing the temperature above the upper limit

differing robustness to temperature implies that it would be

results in a more severe reduction, suggesting that erring

difﬁcult to establish universal expanded incubation tempera-

on the low side is preferable. If testing for total coliforms

ture ranges for all such tests. However, even a conservative

is required then overheating is a more serious problem, as

approach based on the data presented here would suggest

our results suggest that any increase above 37 C ± 1 could

36 C ± 3 could be adopted for β-glucuronidase E. coli tests,

have a detrimental effect on recovery.

which is broader even than the range stated by the ISO. For combined E. coli and total coliform tests the picture is slightly less clear; however, our data tentatively indicate a temperature range of 35 C ± 2 would enable Colilert to operate W

without loss of performance over the current protocol. Further testing using different water types and an increased number of samples would be required to verify this. Other water quality tests should be assessed individually to establish appropriate incubation temperature ranges where performance is not compromised. The adoption of broader incubation temperature standards would beneﬁt testing in low-resource settings, as it would enable the use of less accurate but more achievable incubation methods. For example, the use of an egg incubator, as suggested by Bluewater Biosciences in conjunction with their Coliplate™ and Watercheck™ tests. A similar incubator could also be used in conjunction with Aquatest or Colilert. Novel alternatives also include use of an incubator based on phase change material, such as the ﬁeld incubator developed for use with the Aquatest device (Aquatest ). Another example is the D-lab Portatherm,

CONCLUSIONS Two enzyme substrate methods for the detection of E. coli in drinking water, Colilert and Aquatest medium, can be incubated over a broader range of temperatures than currently speciﬁed, without loss of performance in comparison to 37 C ± 1. This is standard incubation temperature for E. W

coli test media, including enzyme substrate methods, as stated by the UK Environment Agency (Standing Committee of Analysts ). Aquatest medium is more robust to nonstandard temperatures than Colilert, and for both media E. coli from a raw water sample were recovered over a wider temperature range than E. coli injured by exposure to chlorine. Relaxation of temperature tolerances would instil greater conﬁdence in the use of alternative incubation methods; expanding water quality monitoring and reducing incidence of water-related disease in remote and impoverished areas.

which has been applied to tuberculosis testing using the QuantiFERON-TB Gold In-Tube assay (Dominguez et al. ), but can also be used for water quality testing. In

CONFLICT OF INTEREST

being more robust to low temperatures and equivalent or better at high temperatures, it would appear that Aquatest

Robert Matthews is a named inventor on an international

medium is more suitable for use in conjunction with such

patent application number PCT/GB2012/050452 entitled:

incubators than Colilert.

Apparatus for testing the quality of a ﬂuid sample.

120

R. L. Matthews & R. Tung

Expanding the incubation temperature tolerances for microbial water testing

ACKNOWLEDGEMENT We would like to thank the Bill & Melinda Gates Foundation for supporting this work.

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Standing Committee of Analysts  The Microbiology of Water 1994 Part 1: Drinking Water. Her Majesty’s Stationery Ofﬁce, London, UK. Standing Committee of Analysts  The Microbiology of Drinking Water (2009) – Part 4 – Methods for the Isolation and Enumeration of Coliform Bacteria and Escherichia coli. Environment Agency, Bristol, UK. Stephens, P. J., Joynson, J. A., Davies, K. W., Holbrook, R., Lappin-Scott, H. M. & Humphrey, T. J.  The use of an automated growth analyser to measure recovery times of single heat-injured Salmonella cells. J. Appl. Microbiol. 83, 445–455. Tryland, I. & Fiksdal, L.  Enzyme characteristics of B-Dgalactosidase and B-D-gulcuronidase positive bacteria and their interference in rapid methods for detection of waterborne coliforms and Escherichia coli. Appl. Environ. Microbiol. 64, 1018–1023. UNICEF & WHO  Drinking water: Equity, Safety and Sustainability. JMP thematic report on drinking water. UNICEF and the World Health Organization. USEPA  EPA Microbiological Alternate Test Procedure (ATP) Protocol for Drinking Water, Ambient Water, Wastewater, and Sewage Sludge Monitoring Methods. United States Environmental Protection Agency, Washington, DC, USA. Walsh, S. M. & Bissonnette, G. K.  Survival of chlorineinjured enterotoxigenic Escherichia coli in an in vitro water system. Appl. Environ. Microbiol. 55, 1298–1300.

First received 19 April 2013; accepted in revised form 27 September 2013. Available online 20 December 2013

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Seasonal variation of acute gastro-intestinal illness by hydroclimatic regime and drinking water source: a retrospective population-based study Lindsay P. Galway, Diana M. Allen, Margot W. Parkes and Tim K. Takaro

ABSTRACT Acute gastro-intestinal illness (AGI) is a major cause of mortality and morbidity worldwide and an important public health problem. Despite the fact that AGI is currently responsible for a huge burden of disease throughout the world, important knowledge gaps exist in terms of its epidemiology. Speciﬁcally, an understanding of seasonality and those factors driving seasonal variation remain elusive. This paper aims to assess variation in the incidence of AGI in British Columbia (BC), Canada over an 11-year study period. We assessed variation in AGI dynamics in general, and disaggregated by hydroclimatic regime and drinking water source. We used several different visual and statistical techniques to describe and characterize seasonal and annual patterns in AGI incidence over time. Our results consistently illustrate marked seasonal patterns; seasonality remains when the dataset is disaggregated by hydroclimatic regime and drinking water source; however, differences in the magnitude and timing of the peaks and troughs are noted. We conclude that systematic descriptions of infectious illness dynamics over time is a valuable tool for informing disease prevention strategies and generating hypotheses to guide future research in an era of global environmental change. Key words

| acute gastro-intestinal illness, climate change, ecological determinants, seasonality

Lindsay P. Galway (corresponding author) Faculty of Health Sciences, Simon Fraser University, 11830 Blusson Hall, 8888 University Drive, Burnaby, BC, Canada E-mail: lpg@sfu.ca Diana M. Allen Earth Sciences, Faculty of Science, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada Margot W. Parkes Canada Research Chair in Health, Ecosystems and Society, School of Health Sciences/Cross-appointed, Northern Medical Program, University of Northern British Columbia, 3333 University Way, Prince George, BC, Canada Tim K. Takaro Faculty of Health Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada

INTRODUCTION ‘Whoever wishes to investigate medicine properly,

costs, and burden to the health care system remain high

should proceed thus: in the ﬁrst place to consider the sea-

(Payment & Riley-Buckley ; Majowicz et al. ;

sons of the year, and what effects each of them produces,

Fleury et al. ). Additionally, major waterborne out-

for they are not all alike, but differ much from themselves

breaks such as the Walkerton, Ontario example in 2001

in regard to their changes.’

are reminders of the potentially devastating population

Hippocrates (Lloyd et al. )

health impacts of waterborne AGI (Ali ; Eggertson ). Despite the fact that AGI is currently responsible

Acute gastro-intestinal illness (AGI) is a major cause of mor-

for a huge burden of disease throughout the world and is a

tality and morbidity worldwide (Prüss et al. ). Estimates

major public health problem, important knowledge gaps

suggest that AGI infections cause four million cases of diar-

exist in terms of its epidemiology. Speciﬁcally, an under-

rhea worldwide each year (WHO ). In developing

standing of seasonality, the systematic periodic occurrence

nations, diarrhea is the third leading cause of death (WHO

of illness, and those factors driving seasonal variation

). Although mortality due to AGI in developed nations

remain poorly understood (Grassly & Fraser ; Nau-

is relatively low, the morbidity, associated socio-economic

mova ; Fisman ; Lal et al. ).

doi: 10.2166/wh.2013.105

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Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

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The causes and consequences of seasonal variation in

control strategies, and improve the accuracy of early warn-

infectious disease occurrence have intrigued medical pro-

ing systems (Pascual & Dobson ; Altizer et al. ).

fessionals and epidemiologists for more than a century (i.e.

Infectious diseases that vary seasonally demonstrate some

Ransome (); Lloyd et al. ()). More recently, increas-

form of climatic dependence and are most likely to be inﬂu-

ing concerns regarding global environmental change,

enced by climate change and variability. Hence, a greater

globalization and ‘an apparent surge in infectious disease

understanding of seasonal variation can play an important

emergence’ and re-emergence have inspired a renewed inter-

role in the climate change adaptation process (McMichael

est in the seasonality of infectious illness (Fisman ; Lal

; Fleury et al. ; WHO ). Furthermore, an in-

et al. ). The dynamics of AGI are surely inﬂuenced by

depth understanding of past and current seasonal variation

a complex interaction of ecological, social, and biological

in infectious illness epidemiology can provide a baseline

determinants; however, waterborne AGI in particular is

for monitoring the early impacts of climate change on

mediated by ecological factors that inﬂuence drinking

health (Martens & McMichael ). Lastly, exploring dis-

water quality and quantity (Eisenberg et al. ; Institute

ease dynamics, and speciﬁcally how these vary by relevant

of Medicine (US) ; Lal et al. ). Ecological drivers

factors and across different settings, can generate hypoth-

have not been adequately explored due to the dominant

eses and highlight future research priorities.

public health paradigm that is largely focused on individual

This paper analyses variations in the incidence of AGI in

level risk factors (McMichael ; March & Susser ;

British Columbia (BC), Canada over the period of 1999–2010

Ruiz-Moreno et al. ; Eisenberg et al. ; Lal et al. ).

in order to answer the following questions: (1) Does the inci-

The role of hydroclimatology, a term commonly used to

dence of AGI in BC, Canada illustrate a seasonal pattern?

describe the dominant climatic drivers for watershed

(2) Does the seasonal pattern of AGI in BC differ according

responses (Allen et al. ), is one such ecological factor

to hydroclimatic regime and drinking water source? and

that has not been adequately explored to date. Recent

(3) Has the incidence of AGI in BC changed over time? Sev-

advances have been made with regards to our understanding

eral different methods are applied in order to provide a

of cholera epidemiology, in part due to increased interest and

complete picture and robust understanding of seasonality in

research pertaining to environmental drivers of seasonality,

AGI incidence. To the best of our knowledge, this is the

including hydroclimatology (Bertuzzo et al. ). The work

ﬁrst study to describe and compare seasonal patterns of

of Akanda et al. () and Bertuzzo et al. () has high-

AGI by hydroclimatic regime and drinking water source.

lighted ways in which distinct hydroclimatic regimes can inﬂuence pathogen transmission and disease occurrence in different ways, resulting in distinct seasonal patterns of illness

METHODS

at the population level. Hydroclimatology may play a role in driving the seasonality of AGI. Other potentially important

Design

ecological drivers of seasonality include drinking water source, agricultural and other land use activities, and vari-

We conducted a retrospective, population-based study to

ations in wild animal populations. Herein, we examine the

assess the seasonality of reported and laboratory conﬁrmed

dynamics of AGI over time and across different hydrocli-

AGI cases from January 1, 1999 to January 1, 2010 in tar-

matic regimes and drinking water sources to explore

geted communities in the province of BC. This study was

whether and how these factors may drive seasonality of AGI.

approved by the Simon Fraser University (SFU) Research

A better understanding of AGI seasonality, those factors

Ethics Board for the use of secondary data.

driving disease dynamics, and differences in seasonality across settings is key to disease prevention at the population

Setting

level. More speciﬁcally, this knowledge can facilitate the prediction and forecasting of longer-term changes in the

BC, Canada was selected as the setting for this work for several

risk of illness, inform effective disease prevention and

reasons. First, the province offers a rich diversity of

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L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

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hydroclimatology and drinking water sources, thus acting as a unique case study to examine the seasonal patterns of gastrointestinal illness across these factors (Allen et al. ). Second, a province-wide surveillance system, the Integrated Public Health Information System (iPHIS), operational since the late 1990s, offered access to laboratory-conﬁrmed illness data of high quality and adequate time span for the analysis for seasonal disease dynamics. Third, despite the fact that AGI rates are higher in this province than the rest of the country, a knowledge gap exists with regards to seasonality of infectious AGI in this setting (Davies & Mazumder ). The hydroclimatology of BC can be broadly classiﬁed as

Figure 1

Hydroclimatic regimes and drinking water sources across the study communities.

rainfall-dominated (pluvial) and snowmelt-dominated (nival) (Allen et al. ). Rainfall-dominated regimes are found primarily in coastal lowland areas and snowmelt-dominated

in turn report cases to the BC Centre for Disease Control

regimes occur in the interior plateau and mountain regions

through iPHIS. Five potentially waterborne AGI pathogens

(Eaton & Moore ). The hydrology of rainfall-dominated

are included in this study: Campylobacter, Salmonella, vero-

regimes is characterized by seasonal changes in rainfall,

toxigenic Escherichia coli, Giardia, and Cryptosporidium.

with peak streamﬂow and groundwater recharge occurring

The ﬁrst three are bacterial pathogens while the latter two

during the rainy winter months (November–February) and

are protozoan. Reported cases along with information on

the lowest monthly streamﬂows and groundwater levels

age at onset of illness, sex, disease type, serotype, episode

occurring in the late summer and early fall (July–September)

date, report date, address, and a unique identiﬁer were

(Eaton & Moore ). On the other hand, hydrological pro-

extracted from the iPHIS system for the selected study

cesses in snowmelt-dominated regimes are controlled

communities.

primarily by melting snowpack and glaciers. Thus, the

A total of 89 cases (3.9% of all reported cases) were

hydrology of snowmelt-dominated regimes is characterized

excluded. Cases were excluded if they were identiﬁed as

by the highest ﬂows occurring in the spring and early

travel cases (identiﬁed by disease subtypes known to be

summer, and the lowest ﬂows in the late summer and

only travel related), or were identiﬁed as clearly outside of

throughout the winter (Eaton & Moore ).

the study community after geocoding the cases (for example,

Drinking water sources in BC include groundwater, sur-

on a nearby island or more than 75 km from the center of

face water, or a mixture of groundwater and surface water

the study community). We expect that travel cases remain

(hereafter referred to as mixed water). Approximately 30%

in our dataset as the travel variable was poorly populated.

of BC residents rely on groundwater for drinking water (Government of Canada ).

Linking cases to a drinking water source

Eight study communities were selected from across the province of BC for this analysis. These eight communities

Cases were geocoded by street address and postal code of

were selected to represent different combinations of hydro-

residence using a geographic information system (GIS) in

climatic regimes and drinking water sources relevant

ArcMap10.0 (ESRI, Redlands, CA, USA). A spatial join

across the province, as illustrated in Figure 1.

function was then performed to assign each case to a speciﬁc drinking water system based on land parcel infor-

Case data

mation, ultimately linking each case to a drinking water source. More speciﬁcally, we gathered spatial data indicat-

Physicians in BC are mandated to report cases of notiﬁable

ing water service areas for the various drinking water

waterborne illness to regional public health authorities, who

systems within each study community, and province-wide

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L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

12.1

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layers indicating the location of groundwater wells and sur-

groundwater and surface water. In these cases, information

face water extraction points. Information about small

from local community ofﬁcials and water managers regard-

community systems was gathered from community water

ing service populations for all community water systems

managers and manually added to the GIS. Using these

was used to estimate the proportion of the total population

spatial datasets, we assumed that all cases linked to a resi-

serviced with each type of water source. This proportion

dential land parcel located within a drinking water system

was then applied to the total population of communities

service area were serviced by that system, and thus we

D and H to estimate the population in these two commu-

assigned the case the associated water source unless there

nities supplied with groundwater, surface water, and

was an active surface water extraction license, groundwater

mixed water. The calculations were then used to generate

well, or small community system linked to the land parcel.

total estimated population at risk for each drinking water

This method of linking case data to a drinking water

source.

system and source is adapted from the work of Jones et al. Analysis

().

We used time-series plots, monthly plots, negative binomial

Generating time-series

models, and spectral analysis as visual and statistical tools to We generated monthly times-series with rates expressed as the number of cases per month per 100,000 population for the entire dataset and for cases disaggregated by hydrocli-

characterize the seasonal and secular trends in the AGI time-series. Analysis involved the use of several graphical and statistical techniques to triangulate ﬁndings and provide a more robust understanding of disease dynamics. We

matic regime and drinking water source. The variable ‘episode date’ (deﬁned as the date of symp-

applied each of these methods to the full dataset and to

tom onset) was used to derive the day, month, and year of

the dataset disaggregated by cases occurring in each hydro-

illness occurrence, and then to generate monthly time-

climatic regime and linked to each drinking water source.

series representing the number of cases per month over

By examining seasonal variation in the times-series disaggre-

the 132-month study period. Monthly rates were then gener-

gated by these selected factors, seasonal patterns under

ated using total population at risk derived from census data

different conditions were evaluated.

as the denominator. Population at risk within each commu-

(1) Time-series plots: To reﬂect trends in AGI data, we

plotted the 11-year time-series of monthly AGI incidence.

dissemination area population totals – the smallest adminis-

The time-series was smoothed using the three-month

trative unit with census derived data. Population at risk was

moving average, a basic smoothing technique, to enhance

calculated at two points during the 11-year study period,

interpretation of trends (Zeger et al. ). By superim-

2001 and 2006, and an average population at risk over the

posing the time-series disaggregated by hydroclimatic

study period was generated.

regime and drinking water source, similarities and differ-

nity

was

estimated

spatially

using

aggregates

We also estimated population at risk totals for the

ences across these factors could be explored.

different hydroclimatic regimes and water sources of inter-

(2) Monthly plots: Total monthly incidence over the 11-year

est. To generate population totals for each hydroclimatic

study period was calculated and plotted. Again, we super-

regime, we pooled the population counts for those commu-

imposed the total monthly incidence over the 11-year

nities located in snowmelt-dominated regimes and for

study period disaggregated by hydroclimatic regime and

those communities located in rainfall-dominated regimes.

drinking water source. Also, negative binomial regression

This pooling approach could not be used to generate popu-

models using PROC GLM were used to test statistical sig-

lation at risk estimates for each drinking water source,

niﬁcance of monthly variation. A negative binomial was

because communities D and H (Figure 1) are characterized

used rather than a Poisson model due to over-dispersion

by some entirely groundwater systems, some entirely sur-

in the data as indicated by deviance factors greater than

face water systems, and others that are a mix of both

1 (Osgood ). The month with the lowest number of

126

L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

12.1

2014

cases was used as the reference month as per convention

11-years in the study period. Additionally, a negative

(Naumova et al. ).

binomial regression model was used to test the statisti-

(3) Spectral analysis: Spectral analysis is useful for detecting

cal signiﬁcance of variation across years relative to

periodicities (dominant cyclical patterns) in time-series

1999.

and to test data for seasonality (Cryer & Chan ). We

Statistical analyses were carried out using SAS software,

constructed a periodogram to identify inﬂuential periodicities present in the AGI data. We also tested the statistical signiﬁcance of seasonal patterns using two

version 9 (SAS Institute, Inc., Cary, North Carolina) and graphs were created using Microsoft Excel.

formal tests: the Fisher–Kappa (FK) test and the Bartlett Kolmogorov–Smirnov (BKS) test. The FK tests the hypothesis that the series is white noise against the alternative hypothesis that the series contains a periodic component of unspeciﬁed frequency. The BKS tests the null hypothesis that the series is white noise, or that there is no periodicity. (4) Annual plots: Finally, we examined secular trends in the data using simple plots of illness rates across the

RESULTS During the study period from January 1, 1999 to January 1, 2010 2,308 cases of AGI were reported to iPHIS after excluding known travel cases and cases outside the study communities. Of the total 2,308 cases, 1,805 cases (78%) were caused by bacterial pathogens and 458 cases (22%) were caused by protozoan pathogens (Table 1). The inci-

Table 1

dence of AGI across age categories is bimodal with one

Characteristics of all AGI cases, 1999–2010

peak among children less than 5 and another between the

No. of Cases (n ¼ 2308)

% of Total

1,805

78.2

458

21.8

Snowmelt-dominated

1,081

47.0

Rainfall-dominated

1,227

53.1

Groundwater

700

30.3

Surface water

1,011

43.8

597

25.9

Characteristic

Pathogen type Bacterial Protozoan

females (Figure 2).

Hydroclimatic regime

Water source

Mixed water

Figure 2

ages of 20–30. A similar pattern is seen for males and

AGI rates per 100,000 by sex and age, 1999–2010.

Time-series plots Visual examination of the time-series plots for all cases illustrates a clear pattern in the data characterized by annual cyclical peaks and troughs (Figure 3(a)). The timing and amplitude of the peak rates clearly vary from year to year. In some years, the time-series illustrates a bimodal peak, a large peak in the summer followed by a second smaller

127

Figure 3

L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

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Time-series of monthly AGI rates (1999â&#x20AC;&#x201C;2010); (a) for all cases, (b) by hydroclimatic regime, and (c) by water source.

peak in the fall, while in other years there is a single sum-

drinking water source (Figure 3(c)). However, it is difďŹ cult

mertime peak. There is a range of peaks across the years;

to identify any consistent patterns in the time-series plots

however, more than half of the years in the study period

across these factors. For example, in some years the seasonal

illustrate a peak incidence in July and all years show peak

cycles in Figure 3(b) show an earlier peak among those cases

incidence in the summer or fall months.

occurring in snow-dominated regimes compared to those

Cyclical patterns remain, although the amplitude and

cases occurring in the rainfall-dominated regimes, while in

timing of peaks and troughs differ when the cases are disag-

other years the opposite is the case. Aside from the consist-

gregated by a hydroclimatic regime (Figure 3(b)) and

ently higher peaks among those cases linked to a mixed

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L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

12.1

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water source, there are no clear or consistent trends looking at the time-series disaggregated by drinking water source. Monthly plots Figure 4 shows the monthly plots of AGI incidence. The monthly incidence for all cases (Figure 4(a)) illustrates a seasonal pattern with the trough occurring in April and a peak occurring from July to September (summertime). Statistically signiﬁcant peak incidences were found in July (incidence risk ratio (IRR) ¼ 2.21, 95% conﬁdence interval (CI): 1.79–2.73), August (IRR ¼ 2.00, 95% CI: 1.62–2.48) and September (IRR ¼ 2.13, 95% CI: 1.72–2.64) (Table 2). Among those cases occurring in rainfall-dominated regimes, the trough incidence occurs in April and the peak incidence occurs in September (IRR ¼ 2.37, 95% CI: 1.78– 3.17) (Table 2). In contrast, the trough among those cases in the snowmelt-dominated regime occurs in March and the peak incidence occurs in July (IRR ¼ 2.36, 95% CI: 1.75–3.20) (Table 2) with a steady decline through the fall and winter (Figure 4(b)). Monthly plots by drinking water source show that the timing of the peak occurs during different months of the summer and early fall across different drinking water sources (Figure 4(c)). Among those cases linked to a surface water source, the seasonal peak occurs in July–September (IRR-July ¼ 2.44, 95% CI: 1.77–3.38; IRR-Aug ¼ 2.16 95% CI: 1.55– 3.01; IRR-Sept ¼ 2.07 95% CI: 1.49–2.90) (Table 2). Similar patterns are seen in those cases linked to a groundwater source where the peak incidence also occurs from July through to September (IRR-July ¼ 2.03, 95% CI: 1.40–2.93; IRR-Aug ¼ 2.01, 95% CI: 1.39–2.90; IRR-Sept ¼ 2.00, 95% CI: 1.38–2.9) (Table 2). For those cases linked to a mixed water source the peak incidence occurs in September (IRR ¼ 2.41, 95% CI: 1.59–3.63), with another smaller peak in July (IRR ¼ 2.09 95% CI: 1.37–3.17) (Table 2). The magnitude of the peak of incidence is greatest among cases occurring in a rainfall-dominated hydrocli-

Figure 4

Total monthly AGI rates (1999–2010); (a) for all cases, (b) by hydroclimatic regime, and (c) by water source.

matic regime and linked to mixed water sources. spectral density at 12 months in the periodogram (Figure 5). Spectral analysis

The same annual periodicity is seen in the time-series data when disaggregated by hydroclimatic regime and drinking

The periodogram for all cases shows an annual (12-month)

water source (results not shown). The Fisher’s κ and

periodicity in the time-series as indicated by the large

Bartlett–Kolmogorov–Smirnov tests conﬁrm a statistical

129

Table 2

L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Results of negative binomial for years, all cases, 1999–2010

Table 3

95% Cl

IRR

Upper

Lower

2000

1.53a

1.26

1.85

2001

1.41a

1.16

1.72

2002

1.34a

1.10

1.64

2003

1.25a

1.02

1.53

2004

1.17

0.96

1.44

2005

1.20

0.98

1.47

2006

1.03

0.83

1.27

2007

1.05

0.85

1.30

2008

1.31

1.08

1.60

2009

1.27a

1.04

1.55

12.1

2014

FK test and BKS test results

Time-series

Year

Journal of Water and Health

BKS

All GI Illness cases

18.02

0.370b

Snowmelt-dominated cases

1.041b

0.193a

Rainfall-dominated cases

15.35

0.403b

Groundwater cases

10.72b

0.284b

Surface water cases

10.73

0.245b

Mixed water cases

10.78b

0.239b

p < 0.05.

p < 0.01.

FK test: the 5 and 1% critical values for the test are 5.93 and 6.56, respectively.

another increase in risk during 2008 and 2009 (Figure 6(a)).

The years 2004 to 2007 are not statistically signiﬁcantly

IRR is incidence risk ratio, CI is conﬁdence interval.

different from the 1999 reference period. Annual trends

1999 is the reference year.

differ somewhat across the hydrological regimes and drink-

Indicates statistical signiﬁcance, p < 0.05.

ing water sources (see Figure 6(b) and (c); Table 2).

DISCUSSION This study examined the seasonality of AGI in BC over an 11-year period using laboratory-conﬁrmed case data from eight communities across the province. To our knowledge, this is the most comprehensive examination of AGI seasonality in BC and the ﬁrst study anywhere to describe trends in AGI by hydroclimatic regime and drinking water source. Additionally, the majority of research examining the seasonality of AGI, and also Figure 5

Periodogram of monthly AGI. The peak in the periodogram indicates the dominant periodicity in the data (Fisman 2007).

research examining those factors that may drive these trends, originates from the USA, England, and Australia. There has been little research on the subject in BC. Tar-

signiﬁcant seasonal pattern (p < 0.01) for all AGI overall

geted ecological studies at the regional level are needed

(Table 3). This statistical signiﬁcance remains when the

to inform local decision-making, policy and action (Lal

cases are disaggregated by hydroclimatic regime and drink-

et al. ).

ing water source (Table 3).

We used several different visual and statistical techniques to characterize seasonal patterns and our results

Annual plots

consistently illustrate seasonality. These results are perhaps not surprising given that seasonal variation of AGI has been

A non-linear trend is evident over the 11-year study period.

documented in numerous other settings (Naumova et al.

Looking at the full dataset, there is a slight increase in risk

, ; Hall et al. ; Thomas et al. ; Zhang

relative to the reference year between 2001 and 2003, fol-

et al. ; White et al. ; Febriani et al. ; Harper

lowed by a decrease between 2004 and 2007, and then

et al. ; Lal et al. ). What is novel is consideration

130

L. P. Galway et al.

Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

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Examining the time-series data disaggregated by the hydroclimatic regime and drinking water source enables a better understanding of the seasonal dynamics of AGI and can help generate hypotheses about possible mechanisms driving seasonal variation. Differences across the hydroclimatic regime and drinking water source are most easily identified in the monthly plots. Examining monthly plots disaggregated by the hydroclimatic regime demonstrates that the illness peak for those cases occurring in a rainfall-dominated regime occurs in September, compared to July for those cases occurring in a snowmelt-dominated regime. This may reflect differences in temperatures and hydrological patterns contributing to source water contamination at different times of the year in the two distinct regimes. Research indicates that source water microbial loads (both surface water and groundwater sources) are positively correlated with rainfall and may be influenced by the timing of precipitation and runoff events as well as temperature patterns (Atherholt et al. ; Kistemann et al. ). The snowmelt-dominated hydroclimatic regime in BC is characterized by major runoff and groundwater recharge in the spring and early summer caused by warming temperatures that lead to rapid snowmelt; this is known as the spring freshet. At this time, the rivers run high and groundwater levels reach their maximum. Flooding is also a common occurrence. Runoff and groundwater recharge in the rainfall-dominated regimes is driven by precipitation, with streamflow and groundwater levels peaking through the late fall and early winter (Allen et al. ). Although the summertime peak in incidence is likely in part explained by higher temperatures which influence both human behavior and pathogen survival and replication, it is also plausible that the spring freshet in the snow-dominated watersheds causes runoff events and significant source water contamination contributing to the early summertime peak among Figure 6

Total annual AGI rates (1999–2010); (a) for all cases, (b) by hydroclimatic regime, and (c) by water source.

those cases occurring in snowmelt-dominated watersheds. Rapid snowmelt may also overwhelm drinking water treatment systems (Naumova ). ‘A rapid snowmelt,

of the seasonal patterns by different hydroclimatic regimes

resultant runoff, and ﬁltration system failure at the over-

and water source relevant in the context of BC. The season-

loaded drinking water treatment plant were implicated

ality of these diseases suggests that hydroclimatic factors

with the largest known waterborne outbreak of cryptospor-

such as temperature, precipitation, and runoff may inﬂuence

idiosis, which occurred in Milwaukee, Wisconsin in 1993’

disease occurrence.

(Naumova ). Thomas et al. () examined the

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Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

12.1

2014

association between spring freshet events and AGI out-

We expect environmental factors that vary across years are

breaks across Canada, but found no statistical signiﬁcance

more relevant in this case as the reporting of notiﬁable dis-

(Thomas et al. ). Our results suggest that any associ-

eases is required by law in BC (Hill ).

ation may have been masked by the inclusion of cases in both

snowmelt-dominated

and

rainfall-dominated

watersheds.

When interpreting these results, one must note that any effects of weather or hydrological variables on AGI occurrence would be lagged in time, likely on the scale of

Fall precipitation may play a role in source water con-

several weeks to months. Furthermore, it is a challenge to

tamination and the fall incidence peak among those cases

separate the effects of various factors driving seasonal illness

occurring in rainfall-dominated watersheds (Patz et al.

patterns. More advanced statistical analysis is needed to

). Fall rains following the summertime drought are

tease out the effects of different potential environmental dri-

characteristic of rainfall-dominated watersheds and can

vers of seasonality. Poisson time-series analyses using the

lead to more severe runoff that is highly contaminated,

generalized linear model (GLM) and the generalized addi-

thus posing a greater risk of contamination and illness

tive model (GAM), which have been used extensively in

(Charron et al. ). Additionally, high levels of rainfall

air pollution research, are good candidates for further

onto saturated soil may facilitate the movement of patho-

research. Case-crossover analysis may prove useful in exam-

gens over land and into drinking water sources (Lake

ining the inﬂuence of more extreme events such as rapid

et al. ; Britton et al. ). More research is needed to

snowmelt, extreme rainfall events, and ﬂooding.

test and better understand the possible role of the spring fre-

There are no consistent discernible patterns in seasonal-

shet in snowmelt-dominated regimes, and precipitation in

ity across those cases linked to different drinking water

rainfall-dominated regimes in driving AGI transmission

sources. Although we might expect to see some differences

and seasonality.

in patterns given that research has shown that water source

Our results indicate that seasonal patterns vary across

mediates risk of illness in BC (Uhlmann et al. ; Teschke

years. Inter-annual variability in seasonal patterns may

et al. ), our results may be limited by misclassiﬁcation of

suggest that ecological factors, such as weather and the

drinking water source exposure. We are conﬁdent with the

hydrologic response of the watershed, which are known to

accuracy of our approach linking cases to their residential

vary across years, play a role in driving seasonal patterns.

drinking water source because of high predictive values docu-

Interestingly, some evidence in settings where weather

mented in a sensitivity analysis, but it may be that using

variability is limited both within years and across years

residential drinking water source as a surrogate for all

points to limited variation in AGI rates (Araj et al. ).

source water exposure is problematic. Individuals are likely

It is also possible that longer scale climatic processes such

to drink water from a different source at work or when

as the El Nino-Southern Oscillation (ENSO), a periodic ﬂuc-

they are away from their homes in the evening or weekends,

tuation of atmospheric pressure and sea surface temperature

such that the residential drinking water source may not be a

in the equatorial Paciﬁc Ocean, could inﬂuence inter-annual

good surrogate measure for exposure. This issue has been

variation. Past research has found evidence for a relation-

highlighted in the literature, but no solutions have been ident-

ship between ENSO and multiannual cycles of cholera

iﬁed (Jones et al. ). This issue may be particularly

(Pascual et al. ; Koelle et al. ; Hashizume et al.

relevant in our dataset as 64% of our cases originate from

). Time-series data longer than 11 years are needed to

communities where there are drinking water systems sup-

adequately explore the possible role of longer scale Earth

plied by groundwater, surface water, and a mix of the two

system processes.

within the community itself. For these communities, it is

Other explanations are also possible, such as changes in

likely that individuals are exposed to different drinking

reporting behavior from year to year (Zeger et al. ). Sev-

water sources when drinking water in their residences and

eral studies have shown that reporting of infectious illness

when drinking water in other settings in the community. Fur-

can be affected by factors such as publicized outbreaks in

thermore, trends in the data across different water sources

other settings or policy changes (Naumova et al. ).

may be inﬂuenced by foodborne cases which we would not

132

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Seasonal variation of AGI by hydroclimatic regime and drinking water source

Journal of Water and Health

12.1

2014

expect to vary by water source. Although we had originally

Increasing water supply and source testing during the

hoped to focus on the protozoan cases, which are very com-

spring freshet is an example. This testing could be used to

monly waterborne pathogens, low case counts did not allow

inform early warning systems for the community and

this approach. Future research should consider an analysis

could also serve as data for future research.

focused on speciﬁc pathogen groups (bacterial and proto-

There are both strengths and weaknesses in this study.

zoan) or pathogens whenever possible. Explicit attention to

The main strength is the use of different methods for charac-

cryptosporidiosis and giardiasis, recognized by the World

terizing seasonality. Often, temporal trends are examined

Health Organization (WHO) as ‘neglected diseases’, is

using visual analysis of plotted time-series only; we have

called for (Savioli et al. ). Another limitation of this

taken this approach but have used additional methods to gen-

study, and which may warrant attention in future studies, is

erate richer and more robust ﬁndings. This study is unique

the need for attention to recreational exposures.

because we have characterized seasonality by selected hydro-

In addition to the knowledge generation and methodo-

climatic regime and drinking water source. We have used a

logical contributions of this study, our efforts to compare

GIS to link cases to the hydroclimatic regime and drinking

seasonal patterns of AGI by hydroclimatic regime and drink-

water source, allowing for analysis of seasonality across

ing water source also contribute to an enhanced conceptual

these factors. Finally, this study answers the increasingly

understanding of AGI as well as informing of practical

common call for an interdisciplinary approach to infectious

recommendations. Although the combination of hydrocli-

disease research. The study team is an interdisciplinary

matology and drinking water source is unlikely to

group bringing together knowledge from medicine, human

represent the full complexity of factors driving AGI in BC,

ecology, public health, and earth sciences.

our explicit attention to interrelated ecological determinants

There are also important limitations to consider when

indicates an approach grounded in systems thinking and

interpreting these results. Although a diverse group of com-

complexity. The ecological factors that we have examined

munities with different drinking water sources and

in this work may explain some seasonal variation, but

geographic locations across the province was selected, it is

clearly other factors also contribute to the seasonal

possible that there are important differences between our

dynamics of AGI in BC. Future research in BC and

study communities and other settings limiting the generaliz-

beyond may consider eco-bio-social approaches that have

ability of this work. Another limitation is that the AGI case

been applied to other re-emerging infectious diseases (see

data used in these analyses likely under-represent the true

for example Arunachalam et al. () and Kittayapong

number of illness cases. Under-reporting could introduce

et al. ()) and should examine the important linkages

bias into the study if those cases captured in the data are sys-

between ecological and social factors when data permit.

tematically different than those cases not captured in the

With regards to the practical implications arising from

data. Additionally, our case data do not include information

these results, we suggest two speciﬁc practice and policy

about foodborne versus waterborne transmission; such data-

oriented recommendations. First, water managers, environ-

sets do not exist in BC and are generally uncommon.

mental health ofﬁcers, and researchers should work

Unfortunately, AGI data from surveillance systems rarely,

together to identify locally-relevant conditions and times of

if ever, include information about foodborne versus water-

the year when the risk of AGI illness may be heightened.

borne transmission pathways. Additionally, we have used

In communities located in snow-dominated watersheds,

the variable ‘episode date’ from the iPHIS reporting

early summer and the spring freshet are likely to be high-

system to represent the date of onset of the gastrointestinal

risk times. For those communities located in rain-dominated

illness case. It is possible, however, that the ‘episode date’

watersheds, late summer and early fall precipitation events

in the reporting system is not reﬂective of the true date of

are likely candidates. Second, microbiological and/or tur-

onset which may introduce lag-time between case reporting

bidity monitoring programs could be adapted to account

and case occurrence. Finally, although we have excluded

for high-risk conditions and times, taking into account

those cases known to be travel related, we suspect that

local

travel-related cases remain in the dataset.

conditions

and

water

system

characteristics.

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Seasonal variation of AGI by hydroclimatic regime and drinking water source

CONCLUSIONS Seasonality represents a rich area for future research, particularly given widespread environmental degradation and our changing climate (Fisman ). These ﬁndings provide strong evidence for seasonality in general and differences in seasonality across selected ecological factors. This knowledge can provide insight into disease etiology and can contribute to public health policy-making and water resource management. Knowledge of the timing of disease peaks, for example, could allow public health programs to focus resources and preventative actions at certain times of the year. Furthermore, ‘disease forecasting and warning systems could allow public health ofﬁcials to alert the populace

when

speciﬁc

meteorological

conditions

pose

considerable risk to health’ (Naumova ). We conclude that systematic descriptions of infectious disease dynamics over time is a valuable tool for generating hypotheses for future research, establishing climate change sensitivity and potentially informing of disease prevention strategies.

ACKNOWLEDGMENTS Parkes was supported by the Canada Research Chair Program during her involvement in this study. Galway was supported by the Canadian Health Research Institute during her involvement in this study. The authors acknowledge the support of the Simon Fraser University Community Trust Endowment Fund and appreciate Sunny Mak for his editing of the paper.

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First received 17 May 2013; accepted in revised form 16 September 2013. Available online 28 October 2013

136

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Human viruses and viral indicators in marine water at two recreational beaches in Southern California, USA David C. Love, Roberto A. Rodriguez, Christopher D. Gibbons, John F. Grifﬁth, Qilu Yu, Jill R. Stewart and Mark D. Sobsey

ABSTRACT Waterborne enteric viruses may pose disease risks to bather health but occurrence of these viruses has been difﬁcult to characterize at recreational beaches. The aim of this study was to evaluate water for human virus occurrence at two Southern California recreational beaches with a history of beach closures. Human enteric viruses (adenovirus and norovirus) and viral indicators (Fþ and somatic coliphages) were measured in water samples over a 4-month period from Avalon Beach, Catalina Island (n ¼ 324) and Doheny Beach, Orange County (n ¼ 112). Human viruses were concentrated from 40 L samples and detected by nested reverse transcriptase polymerase chain reaction (PCR). Detection frequencies at Doheny Beach were 25.5% (adenovirus) and 22.3% (norovirus), and at Avalon Beach were 9.3% (adenovirus) and 0.7% (norovirus). Positive associations between adenoviruses and fecal coliforms were observed at Doheny (p ¼ 0.02) and Avalon (p ¼ 0.01) Beaches. Human viruses were present at both beaches at higher frequencies than previously detected in the region, suggesting that the virus detection methods presented here may better measure potential health risks to bathers. These virus recovery, concentration, and molecular detection methods are advancing practices so that analysis of enteric viruses can become more effective and routine for recreational water quality monitoring. Key words

| adenovirus, bathing water, coliphage, detection methods, norovirus

David C. Love (corresponding author) Roberto A. Rodriguez Christopher D. Gibbons Jill R. Stewart Mark D. Sobsey Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA E-mail: dlove@jhsph.edu John F. Grifﬁth Southern California Coastal Water Research Project, Costa Mesa, CA 92626, USA Qilu Yu Division of Geriatric Medicine and Gerontology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA Roberto A. Rodriguez Current address: Environmental and Occupational Health Sciences, School of Public Health-El Paso Regional Campus, University of Texas-Health Science Center at Houston, El Paso, TX 79902, USA

INTRODUCTION Enteric viruses can enter recreational waters from runoff or

gastrointestinal, skin, and respiratory illnesses acquired by

sewage and pose risks to human health (Okoh et al. ).

beachgoers, may not require medical attention and therefore

Some of the viruses with a history of causing human water-

go unreported to public health agencies. However, it is esti-

borne illnesses like gastroenteritis are in the Adenoviridae,

mated that beachgoers in Southern California experience

Astroviridae, Caliciviridae, Picornaviridae, and Reoviridae

between 627,800 and 1,479,200 gastrointestinal illnesses

families (Bosch ; Okoh et al. ). Exposure to even

each year (Given et al. ). Epidemiologic studies in

low doses of these viruses in water can cause infections.

Southern California have conﬁrmed infectious disease

For example, the probability of human infection from a

risks and health effects associated with water contact for

single virus is as high as 30% for rotavirus (Gerba et al.

beachgoers (Colford et al. ; ).

) and 50% for norovirus (Teunis et al. ).

To protect bathers, the United States Environmental

Los Angeles and Orange Counties, California, USA, are

Protection Agency (USEPA) has developed water quality

in a heavily urbanized region with a favorable climate and

criteria and guidelines based on the levels of fecal indicator

160 km of coastal shoreline that attracts recreational bathers

bacteria (FIB), e.g. Escherichia coli and enterococci, which

in large numbers. Self-limiting diseases, like many of the

correlate with risks of disease symptoms (Cabelli ;

doi: 10.2166/wh.2013.078

137

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

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Dufour ; USEPA ). Molecular tools to detect these

along a transect (Figure 1(a), sites: A, B, D, E) three times

bacteria have been developed (Haugland et al. ; Noble

per day (07:00 hours, 13:00 hours, and 15:00 hours), and

et al. ) and benchmarked to health outcomes (Wade

at 07:00 hours from a channelized urban stream (San Juan

et al. , ). However, the actual agents that cause dis-

Creek) (Figure 1(a), site C) that forms a lagoon behind the

ease among bathers have not been well characterized.

beach between sites B and D. The lagoon was reported as

Research suggests that human viruses may be a major contri-

a source of fecal pollution impacting the beach (Colford

butor (World Health Organization ), and a quantitative

et al. ), and during this study, the lagoon did not

microbial risk assessment has recently identiﬁed enteric

breach the berm separating it from the ocean.

viruses as potentially responsible for observed illnesses in swimmers (Soller et al. ).

At Avalon Beach on Catalina Island, water sampling was conducted daily Thursday through Sunday from July

The aim of this study was to quantify the occurrence of

26 to September 2 2007 and September 8 and 9 2007, for

human adenoviruses and human noroviruses at two

a total of 26 sampling days. Water samples at Avalon

Southern California recreational beaches. This work also applied the newly developed Nanoceram© (Argonite Corp.) positively-charged cartridge filter for viral monitoring of beach water. This filter has been demonstrated to be highly efficient for the concentration and recovery of noroviruses from large volumes of marine waters using a modified adsorption-elution method described previously (Gibbons et al. ). To our knowledge this is the first report of using a Nanoceram© cartridge filter for the detection of human adenovirus and human norovirus during a beach monitoring study. This study aimed to compare the occurrence of viruses with other microbial water parameters at beaches impacted by non-point source contamination.

METHODS Site description and sample collection Doheny and Avalon Beaches, CA, were selected because of their high summer use, history of beach postings or closures, and potential impact from non-point source pollution. Sampling events were scheduled on weekends and during summer months because these dates are most popular among beach visitors (Dwight et al. ). An intensive sampling approach allowed for the capture of transient conditions of viral contamination in beaches used for recreation. At Doheny Beach in Dana Point, water sampling was conducted one weekend day per week from May 28 to July 1 2007, and July 4 2007, for a total of eight sampling days. A total of 112 water samples were collected from Doheny Beach at a depth 0.5 m below the water surface

Figure 1

Site map of (a) Doheny Beach, CA and (b) Avalon Beach, CA.

138

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

Beach were collected along a beach transect (Figure 1(b),

precipitation. Samples were shaken at 200 rpm (Lab-Line

sites: A, B, C) three times per day (08:00 hours, 12:00

platform shaker, Barnstead International, Dubuque, IA,

hours, and 15:00 hours). Samples were collected at a

USA) at 4 C overnight. The eluents were then centrifuged

depth 0.5 m below the surface of the water at all sampling

for 1 h at 5,100 × g and 4 C, after which point the super-

times, as well as at 1 m depth at the 12:00 hours sampling

natant was discarded. The pellet was resuspended in 2 mL

time. There were 324 water samples in total. Previous

of phosphate-buffered saline (PBS, pH 7.2; 0.027 M KCl,

research has identiﬁed that fecal pollution comes from a var-

0.88 M KH2HPO4, 0.0064 M Na2HPO4, 0.14 M NaCl) and

iety of sources including a leaky sewage system (Boehm

stored at –80 C.

et al. ), which contaminates groundwater and dis-

In seawater, the efﬁciency of virus adsorption-elution

charges into Avalon Bay during outgoing tides (Boehm

recovery by the Nanoceram© ﬁlter was published previously

et al. ).

and was 99% for norovirus and 3.5% for human adenovirus (Gibbons et al. ). The efﬁciency of virus recovery by the

Adenovirus and norovirus concentration

secondary concentration step with PEG precipitation was 59% for norovirus and 39% for adenovirus. The total recov-

Adenoviruses and noroviruses were concentrated from 40 L ©

water samples by positively-charged Nanoceram

alumina

ery efﬁciency of the method was 58% for norovirus and 1.4% for adenovirus.

cartridge ﬁlters (Argonide Corporation, Sanford, FL, USA), using a previously described method with known adenovirus

Viral nucleic acid extraction

and norovirus recovery efﬁciencies (Gibbons et al. ). Filtrations were performed at a ﬁeld laboratory near the beach

A guanidinium thiocyanate (GuSCN) extraction method

by Southern California Coastal Water Research Project

was utilized for viral nucleic acid extraction of Doheny

(SCCWRP) staff, and at a ﬁltration rate of approximately

Beach and Avalon Beach sample concentrates (Boom

2.5 L per min (Emerson self-priming pump, ITT Jabsco,

et al. ; Jothikumar et al. ). Sample concentrates

St Louis, MO). To preserve the samples during shipment,

(1.2 mL for each Doheny Beach sample and 0.2 mL for

350 mL of transport medium consisting of sterile 3% (w/v)

each Avalon Beach sample) were mixed with equal volumes

beef extract (Becton, Dickinson and Company, Sparks,

of GuSCN extraction buffer containing 120 g of GuSCN,

MD) – 0.1 M glycine (BE, pH 7.3) solution was poured into

100 mL of TE Buffer (10 mM of Tris (tris(hydroxymethyl)

each ﬁlter cartridge housing. The housing was sealed, and

aminomethane) and 1 mM of EDTA (ethylenediaminete-

shipped overnight at 4 C to the University of North Carolina

traacetic acid)) pH 8.0 (Ambion, Austin, TX), 55 mM

(UNC) laboratory in Chapel Hill, NC, for analysis.

sodium chloride, 33 mM sodium acetate, and 4.4 mg of poly-

To elute viruses at UNC, an additional 150 mL of BE

adenylic acid (50 ) potassium salt for a total volume of

was added to the existing 350 mL BE transport medium sol-

240 mL. The solution was vortex mixed for 1 min then incu-

ution, and the liquid was recirculated through the cartridge

bated for 10 min at room temperature. Then, two volumes of

ﬁlters using a peristaltic pump. The sample pH was adjusted

100% ethanol were added to the extract and the mixture was

to pH 9.5 using 1 M NaOH during recirculation, and main-

vortex mixed for 15 sec. Six hundred microliters of solution

tained at pH 9.5 for 15 min (changing the ﬂow direction

was transferred to a HiBind RNA mini-column (OMEGA

every 5 min). Sample eluents were then transferred to a

Bio-Tek, Doraville, GA) and centrifuged at 16,000 × g for

1 L bottle, and the pH was adjusted to pH 7.3 using a 1 M

1 min; this step was repeated until the entire sample was

HCl solution. Between samples, the pH meter probe was dis-

passed through the column. Three separate HiBind RNA

infected with 0.1 M HCl for 2 min. Each sample eluent was

mini-columns were used to extract the 1.2 mL Doheny

then split into two Corning polypropylene 250 mL conical

Beach concentrate sample (one mini-column per 0.4 mL

bottles (Corning Inc., Corning, NY) and 9% (w/v) polyethy-

volume). Only one column was used for each 0.2 mL

lene glycol (PEG 8000, Fisher Scientiﬁc, Fair Lawn, NJ) and

Avalon Beach concentrate sample. The columns containing

0.3 M sodium chloride (NaCl) were added for PEG

viral nucleic acids were washed twice with 0.5 mL of 75%

139

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

ethanol, and then spun again in a clean collection tube to

through 2% agarose in 0.1 Tris/borate/EDTA and 5 μg/mL

remove the ethanol. Nucleic acids were eluted from the

of ethidium bromide.

column with 50 μL of nuclease-free water and stored at 80 C until further analysis. One blank control (PBS) was W

Norovirus detection by nested RT-PCR

run for every three samples. At Avalon Beach, it was initially determined that

Norovirus region A was detected using previously described

decreasing the volume of sample from 1.2 to 0.2 mL for

methods (Vinjé et al. ). A Qiagen One-Step RT-PCR Kit

nucleic acid extraction improved virus detection (data not

(Qiagen, Valencia, Ca) was used as described by the manu-

shown). These modiﬁcations reduced the amount of the

facturer. The One-step RT-PCR consisted of 5 μL of 5×

40-L water sample analyzed per nested polymerase chain

Qiagen One-Step RT-PCR buffer, 5 μL of Qsolution, 1 μL

reaction (PCR) for adenovirus from 6% at Doheny Beach

of Qiagen enzyme mix, 10 units of RNase inhibitor,

to 1% at Avalon Beach, and similarly reduced the amount

400 μM each dNTP, 2 μM MJV12 primer and 2 μM RegA

of sample analyzed per nested reverse transcriptase PCR

primer, 5 μL of the extracted viral nucleic acid, and water

(RT-PCR) reaction for norovirus from 2% at Doheny

for a reaction volume of 25 μL. The RT-PCR conditions

Beach to 1% at Avalon Beach. Some of the authors con-

were 50 C for 30 min, 94 C for 15 min, 40 cycles of 94 C

ducted additional analyses of the inhibitory effect of beach

for 15 sec, 50 C for 15 sec, and 72 C for 30 sec, then a

water, and found that Avalon Beach samples were more

ﬁnal extension step of 72 C for 7 min. A nested PCR develW

inhibitory than Doheny Beach samples for norovirus detec-

oped by Maloney () was then run on the products of the

tion by quantitative reverse transcriptase PCR (qRT-PCR)

ﬁrst reaction. The nested PCR reaction consisted of 5 μL of

(Rodríguez et al. b).

10× PCR buffer, 2 mM MgCl2, 200 μM each dNTP, 1 μM RegA primer and 1 μM MP290 primer, 1.5 units Hot Star Taq, 2 μL of the ﬁrst PCR reaction product and water for a

Adenovirus detection by nested PCR

reaction volume of 50 μL. The sequence of the MP 290 Adenovirus (species A–F) were detected using previously

primer is 50 GAY TAC TCY CS/inosine TGG GAY TC 30

described methods (Ko et al. ). The ﬁrst round PCR reac-

(degenerate base symbols: Y ¼ C/T; S ¼ C/G). Nested PCR

tion mixture consisted of a 5 μL of 10× PCR buffer, 2.0 mM

conditions were 94 C for 10 min, 40 cycles of 94 C for

MgCl2, 10 μL 5× Q solution (Qiagen, Valencia, CA, USA),

20 sec, 49 C for 30 sec, and 72 C for 1 min, and a ﬁnal

200 μM each deoxyribonucleotide triphosphate (dNTP),

extension step of 72 C for 7 min. Negative control PBS

1 μM each primer (Hex 1 and Hex 2), 1.5 units of Hot Star

blanks and positive control norovirus G2.4 genomic RNA

Taq (Qiagen), 15 μL of extracted viral nucleic acid and

were included in each PCR run, and products were analyzed

water for a reaction volume of 50 μL. For Avalon Beach

by gel electrophoresis as for adenoviruses.

samples, the volume of viral nucleic acid was decreased from 15 to 5 μL to reduce environmental inhibitors to PCR,

Coliphage detection

based upon preliminary studies characterizing the degree of sample-volume-related PCR inhibition. The nested PCR mix-

For coliphage analysis, water samples were collected in 2 L

ture was the same as described above, except the primer Hex

sterile wide-mouth bottles and shipped overnight at 4 C to

3 replaced Hex 2 (Ko et al. ) and 2 μL of the ﬁrst PCR pro-

the UNC laboratory. A modiﬁed version of the two-step

duct was added. Conditions for both PCR reactions was 94 C W

enrichment US EPA Method 1601 (USEPA ) was used

for 10 min, 40 cycles of 94 C for 30 sec, 50 C for 30 sec, and

for the quantiﬁcation and detection of male-speciﬁc (Fþ)

30 sec at 72 C, and a ﬁnal extension step of 72 C for 7 min

and somatic coliphages as described previously (USEPA

(Peltier Thermal Cycler, MJ Research, Waltham, MA,

; Rodríguez et al. a). In short, multiple volumes

USA). A negative control PBS blank and a positive control

were analyzed up to a total volume of 1 L per coliphage

of adenovirus 41 genomic DNA were included in each run.

group (Fþ and somatic) in order to calculate a most probable

PCR products were then visualized by gel electrophoresis

number (MPN) of coliphages present in each sample.

140

D. C. Love et al.

Human viruses in marine water at recreational beaches

Fecal indicator bacteria detection

Journal of Water and Health

12.1

2014

among human viruses, environmental parameters, somatic coliphages, Fþ coliphages, and FIB were also measured

FIB were assayed in water samples by SCCWRP staff at a

with a GEE model. Bivariate relationships between predic-

ﬁeld laboratory near the beach. Concentrations of total

tors and probability of human virus presence was explored.

and fecal coliforms were determined as previously described

Factors with a p value of less than 0.10 were chosen to

in Standard Methods 9222 B and D, respectively (Eaton

enter into the multivariate GEE model. Model selection

et al. ). Enterococci were quantiﬁed using previously

was then performed based on likelihood-ratio test with a

described EPA Method 1600 (USEPA ). FIB data

backward selection process. Factors with a signiﬁcance

were only used for modeling purposes and comparison to

level of 0.05 or less were kept in the model. Modeling was per-

enteric virus data, hence no FIB summary statistics are pre-

formed in SAS version 9.2 (Cary, NC).

sented in this manuscript, although portions of these FIB data have been published previously (Colford et al. ).

Quality assurance and quality controls

Environmental parameters

The UNC laboratory was set up as described in the USEPA manual for quality assurance for using PCR methods applied

Various parameters were recorded at Avalon Beach to

to environmental samples (USEPA ). Each step in the

model their association with viruses. Water temperature

processing of the sample was physically separate: separate

data were collected by Ali Boehm, Stanford University,

rooms were used for mixing PCR reagents, ﬁeld sample pro-

using a probe ﬁxed to a pier in Avalon Beach, and matched

cessing, positive control manipulation and post PCR

with sampling times. Rainfall data were collected by the Cat-

processing. The same equipment was used for the duration

alina Airport (National Climate Data Center ), and

of the study. Blank samples were included in between

coded as a binary variable as previous rainfall within the

every three ﬁeld samples for nucleic acid extraction and

past 48 h or not. A beach advisory report for beach closures

PCR. One positive control for each virus (adenovirus 41

in 2007 was collected from a State of California website that

and norovirus G2.4) was run for each master mix. The

is no longer active. Rainfall, water temperature, surf height,

manipulation of the positive controls was performed in a

sea state, water color, wind, current, odor, trash, and kelp

separate room from ﬁeld samples.

presence were collected at Doheny Beach during four of the eight sampling days in 2007, and this incomplete data set was not used in data modeling.

RESULTS

Statistical analysis and modeling

Virus detection at beaches

Fisher’s exact test was used to assess beach sampling site

Adenoviruses and noroviruses were detected in 25.5 and

effects on human virus detection frequency, while Friedman’s

22.3%, respectively, of water samples at Doheny Beach,

test and Dunn’s multiple comparison post-test determined

and in 9.3 and 0.7% of water samples at Avalon Beach,

sampling site effects on coliphage concentrations. Virus inac-

respectively, (Table 1). The larger proportion of virus-posi-

tivation rates due to sunlight were calculated using linear

tive samples at Doheny Beach may be a function of

regression in Prism (Graphpad, La Jolla, CA, USA). Chi-

differences in fecal pollution sources at the two beaches

squared analysis was performed on virus frequency at bea-

and the fact that larger water sample volumes were analyzed

ches with and without recreational water guidelines. A

by molecular methods at Doheny Beach than Avalon Beach.

generalized estimating equation (GEE) model was used to

Fþ and somatic coliphages were detected in 26.7 and

test for time-of-day effects on virus detection frequency and

82.8% of samples at Doheny Beach, and 56.9 and 41.5%

concentration for each beach, which accounted for spurious

of samples at Avalon Beach, respectively. Among samples

correlations due to repeated measures. The associations

with detectable concentrations, the median concentrations

141

Table 1

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

Detection frequency and concentration of viruses during the summer of 2007 at Doheny and Avalon Beaches

Doheny Beach, CA (May 28 to July 4 2007)

Virus type (sample volume)

% positive

Adenovirus (40 L)

25.5

Avalon Beach, CA (July 26 to September 9 2007)

Mediana (MPN /

Range (MPN /

100 mL)

n/a

Mediana (MPN /

Range (MPN /

100 mL)

9.3

291

n/a

% positive

Norovirus (40 L)

22.3

n/a

0.7

291

n/a

Fþ coliphages (1 L)

26.7

101

0.3

<0.09 to 140

56.9

318

0.3

<0.1 to 37

Somatic coliphages (0.1 L)

82.2

101

4.9

<1 to 150,000

41.5

318

3.1

<1 to > 370

Median among samples with a detectable concentration.

of Fþ coliphages were the same at the two beaches

a signiﬁcant site effect for somatic coliphage concentration

(0.3 MPN/100 mL), and median concentrations of somatic

(p ¼ 0.007) (Figure 2(f)), although this effect was not signiﬁ-

coliphages were similar (4.9 and 3.1 MPN/100 mL at

cant when measured by somatic coliphage detection

Doheny and Avalon Beaches, respectively) (Table 1).

frequency (p ¼ 0.7) (Figure 2(e)). Neither detection frequency (p ¼ 0.8) nor concentration (p ¼ 0.09) showed a

Effects of sampling location on virus detection

signiﬁcant site effect for Fþ coliphages. These results were consistent with a diffuse source of pollution to the beach.

No noroviruses (zero of seven samples) and only one adenovirus positive (one of seven samples) was detected in the

Effects of time of day

lagoon at Doheny Beach, while the viruses were readily detected in beach water at frequencies as high as 32% for

There was a signiﬁcant time of day effect, in which Fþ and

noroviruses (Figure 2(a), site D) and 41% for adenoviruses

somatic coliphage concentrations and detection frequencies

at beach sampling sites (Figure 2(a), site E). Along the

decreased throughout the day at both beaches (Figure 3(c)

beach transect (sites A, B, D, E), no signiﬁcant sampling

and (f)), presumably due to inactivation by sunlight.

site effect was observed for adenoviruses (p ¼ 0.2) or noro-

Fþ coliphage inactivation rate constants and 95% conﬁ-

viruses (p ¼ 0.8).

dence intervals were somewhat similar at Avalon and

At the Doheny Beach lagoon, frequencies were 100% for

Doheny Beaches (kobsAvalon

Fþ coliphage ¼ 1

0.097 ± 0.30 h 1;

¼ 0.069 ± 0.16 h ), as were somatic

somatic coliphage and 88% for Fþ coliphage (Figure 2(b)).

kobsDoheny

Along the beach transect, there was a signiﬁcant sampling

coliphage rate constants at both beaches (kobs Avalon somatic ¼

site effect for Fþ coliphage concentrations (p ¼ 0.03) and

0.248 ± 1.2 h 1; kobsDoheny

somatic coliphage concentrations (p ¼ 0.008) (Figure 2(c)),

coliphages were inactivated more slowly than somatic coli-

but not for somatic coliphage (p ¼ 0.7) or Fþ coliphage

phages. Linear regression models could account for most of

detection frequency (p ¼ 0.1) (Figure 2(b)). The only signiﬁ-

the variability in the data (R 2 ¼ 0.873 to 0.969), although

cant pair-wise comparisons between sites were for somatic

there were only three time points used to develop the models.

coliphage, where site E had a signiﬁcantly lower concen-

Time of day effects were variable for adenovirus and nor-

tration of somatic coliphages than site A (p < 0.05).

Fþ coliphage

somatic

¼ 0.239 ± 0.64 h 1). Fþ

ovirus. At Doheny Beach, time of day was signiﬁcant for

At Avalon Beach, sampling was conducted at three sites

norovirus detection frequency, but in the opposite direction

along the beach and there was no signiﬁcant sampling site

as expected; samples were 61% more likely to be positive

effect for either adenoviruses (p ¼ 0.6) or noroviruses (p ¼

for norovirus at 15:00 than 07:00 hours (p ¼ 0.05). For adeno-

0.7) (Figure 2(d)). Adenovirus detection frequency ranged

virus, detection was 73% more likely at 07:00 than 13:00

from 7 to 12% at the sampling sites at Avalon Beach. Noro-

hours (p ¼ 0.002), and no difference was observed between

viruses were only detected in two of 291 samples. There was

07:00 or 13:00 and 15:00 hours (p ¼ 0.33; p ¼ 0.08,

142

Figure 2

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

Virus detection frequency and concentration at sampling sites along beach transects at Doheny (Figure 2(a), (b) and (c)) and Avalon (Figure 2(d), (e) and (f)) beaches, and at the lagoon at Doheny Beach. Adenovirus and norovirus virus detection frequency (Figure 2(a) and (d)) and Fþ and somatic coliphage detection frequency (Figure 2(b) and (e)) and concentration (Figure 2(c) and (f)) are reported. Ninety-ﬁve percent conﬁdence intervals are provided for Fþ and somatic coliphage concentrations.

respectively). At Avalon Beach, no time of day effects were

was collected at noon at the same time as a ‘knee-deep’

observed for adenoviruses. Noroviruses were detected in

water sample (0.5 m depth) at Avalon Beach. For samples col-

one 07:00 hours sample and one 15:00 hours sample,

lected at the same time, there was no signiﬁcant effect of

which was not enough to perform statistics.

sample depth on coliphage concentration (Fþ coliphage, p ¼ 0.78; somatic coliphage, p ¼ 0.28) (Table 2). Furthermore,

Effect of sample depth

agreement between sampling depths was observed when comparing detection frequency using Fisher’s exact test (Fþ

To explore potential differences in exposure between wading

coliphage, p < 0.0001; somatic coliphage, p ¼ 0.002). Based

versus swimming, a ‘waist-deep’ water sample (1 m depth)

on the agreement between sample depths for coliphage

143

Figure 3

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

Time-of-day effects on virus detection frequency and concentration in samples from Doheny Beach, CA (a, b, c) and Avalon Beach, CA (d, e, f). Adenovirus and norovirus detection frequency (Figure 3(a) and (d)), Fþ and somatic coliphage detection frequency (Figure 3(b) and (e)), and concentration (Figure 3(c) and (f)) are presented. Linear regression trendlines are listed for Fþ and somatic coliphages (Figure 3(c) and (f)).

concentrations and frequencies, results of both sample

represent the frequency of positive samples (or concen-

depths were pooled for analyses. Adenovirus detection fre-

tration) detected from all sites on that day. For Fþ and

quencies at the two sample depths were not signiﬁcantly

somatic coliphages, values were reported only for morning

associated (p ¼ 1). Similarly, norovirus detection frequencies

samples, which presents the best-case scenario for their

at sampling depths were not signiﬁcantly associated (p ¼ 1).

detection, because solar inactivation occurred in afternoon samples. One notable ﬁnding at Doheny Beach was very

Day of study

high frequencies of noroviruses (54% positive) and adenoviruses (85% positive) on day 21 of the study (Figure 4(a)),

Sampling results were presented by day of study for both

with no increases in Fþ or somatic coliphage frequencies.

beaches (Figure 4). The values presented by day of study

There were no remarkable environmental conditions on

144

Table 2

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

Effects of water sample collection depth on the detection frequency and concentration of viruses at Avalon Beach Water sample deptha

Statistical tests

0.5 m

1.0 m Fisher’s n

Median b (MPN/100 mL)

Range (MPN/100 mL)

% positive

8.8

n/a

% positive

Virus type

Adenovirus Norovirus

Median b (MPN / 100 mL)

Range (MPN / 100 mL)

12.3

n/a

1.0

n/a

1.5

n/a

1.0

n/a

Fþ coliphage

55.1

0.38

<1 to 8

53.1

0.31

<0.1 to >37

Somatic coliphage

32.1

2.10

<1 to 370

32.1

1.20

<1 to 14

exact test, p value

Wilcoxon matched pairs test, p value

<0.0001

0.78

0.008

0.28

All samples collected at noon.

Median among samples with a detectable concentration.

Figure 4

Daily time-series and detection frequency of viruses in water samples from Doheny Beach, CA (Figure 4(a), (b) and (c)) and Avalon Beach, CA (Figure 4(d), (e) and (f)). Fþ and somatic coliphage detection frequencies and concentrations were based solely on the morning water samples to control for time-of-day effects described in Figure 3. Sample sizes: Doheny Beach n ¼ 4 to 13 samples/day (norovirus and adenovirus), n ¼ 4 samples/day (Fþ and somatic coliphages); Avalon Beach: n ¼ 6 to 11 samples/day (norovirus and adenovirus), n ¼ 3 samples/day (Fþ and somatic coliphages).

145

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

that day, which was overcast at 07:00 hours with calm seas

Relationship between human viruses and recreational

and a light wind, at 13:00 hours the sky was clear, and at

water guidelines

15:00 hours a moderate breeze and moderate choppy waves were recorded. At some sites and some times of the

Relationships between viruses and recreational water guide-

day kelp cover was observed, the water was green or blue/

lines were further studied using 2 × 2 tables (Tables 3 and 4).

green, and some litter was seen in the water in the morning.

At Doheny Beach, 35.5 and 34.4% of samples from sites

At Avalon Beach, noroviruses were rarely detected and

without a beach advisory were positive for norovirus and

adenoviruses were detected often, but rarely at frequencies

adenovirus, respectively (Table 3). There was a signiﬁcant

higher than 25% in any sampling day (Figure 4(d)). Com-

relationship between the absence of a beach advisory and

paring coliphage frequencies (Figure 4(b) and (e)) versus

virus frequency at Doheny Beach for norovirus (p ¼ 0.03)

continuous coliphage data (Figure 4(c) and (f)) shows

but not for adenovirus (p ¼ 0.3). At Avalon Beach, 8.7% of

the importance of quantiﬁcation for generating high-

samples from sites without a beach advisory were positive

resolution water quality data. Correlations among enteric

for adenovirus, although the relationship between advisory

viruses, colphages, and other parameters were then

and adenovirus was not signiﬁcant.

performed.

Comparing virus frequency with enterococci guidelines, for Doheny Beach there was no signiﬁcant relationship

Correlation among viruses and environmental

between

parameters

(Table 4), mainly because there were too few samples col-

virus

detection

and

enterococci

guidelines

lected with enterocci levels greater than 104 colony A GEE model assessed correlation at Doheny Beach among

forming units (CFU)/100 mL. Only one of the 25 and 21

human viruses, FIB, and coliphages, controlling for time of day. Only those model parameters that produced signiﬁcant models are reported. The probability of detecting adenovirus

Table 3

Frequency of human viruses detected in samples collected with and without a beach advisory

was greater in the absence of Fþ coliphages (p ¼ 0.002; odds Human virus detection

ratio (OR) ¼ 0.24) and enterococci (p ¼ 0.001; OR ¼ 0.95),

frequency (%)

in the presence of fecal coliforms (p ¼ 0.02; OR ¼ 1.004),

Beach

Beach advisory

No. samplesa

and no signiﬁcant associations were observed between

Avalon Beach

No Yes

115 111

8.7 8.1

0 0.9

Doheny Beach

No Yes

31 63

34.4 23.2

35.5 15.9

somatic coliphages and adenovirus. Norovirus was not signiﬁcantly associated with either type of coliphages or FIB. Environmental parameters were not consistently collected at Doheny Beach, but were at Avalon Beach. At Avalon Beach, signiﬁcant associations among adeno-

Adenovirus

Norovirus

At Avalon Beach, only samples collected at 0.5 m depth were used for comparability to

enterococci data in Table 4.

viruses, FIB, and coliphages were determined using a GEE model. The probability of detecting adenovirus was signiﬁ-

Table 4

cantly associated with higher fecal coliform concentrations

Frequency of human viruses detected in samples with different concentrations of enterococci

(p ¼ 0.01; OR ¼ 1.99) and total coliform concentrations

Human virus detection

(p ¼ 0.002; OR ¼ 1.44). The presence of Fþ coliphage (p ¼

frequency (%)

0.1; OR ¼ 1.98) was positively associated with the prob-

Beach

Enterococcia

No. samplesb

Adenovirus

ability of detecting adenovirus, although this association

Avalon Beach

<104 CFU/100 mL >104 CFU/100 mL

162 64

5.6 15.6

0 1.0

Doheny Beach

<104 CFU/100 mL >104 CFU/100 mL

79 15

30.0 6.7

25.3 6.7

was only marginally signiﬁcant. The probability of detecting adenovirus was not associated with water temperature, rainfall in the past 48 h, or the presence of municipal beach advisories. There were too few norovirus positive samples at Avalon Beach for model convergence.

Norovirus

California single sample standard.

At Avalon Beach, only samples collected at 0.5 m depth were used because enterococci data was not available for samples collected at 1 m depth.

146

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

samples positive for norovirus and adenovirus, respectively,

cumbersome and time-consuming pre-conditioning to an

also contained enterococci levels greater than 104 CFU/

acidic pH as with the negatively charged Filterite ﬁlter.

100 mL. Higher rates of virus detection were observed in

Noroviruses were detected less frequently than adeno-

samples from Doheny Beach with lower concentrations of

viruses during this study, which may be due to the smaller

enterococci. At Avalon Beach, there was a signiﬁcant

fraction of the sample analyzed for norovirus than adenovirus

relationship between the frequency of adenovirus and the

at Doheny Beach. The theoretical detection limits of the assay

presence of enterococci (p ¼ 0.01); the frequency of adeno-

were around one PCR unit in 2.5 L for adenovirus and 1.2 L

virus occurrence was higher in the samples above the

for norovirus. However, the method recovery efﬁciency of

established level for enterococci (Table 4).

norovirus in seawater samples should be greater than for adenovirus, which can compensate for the difference in extraction volumes assayed between these viruses. Furthermore, the detection frequency of each virus was greater

DISCUSSION

than previous reports from waters along the California coast and other coastal waters (Jiang ; Jiang et al. ; Silva

Human enteric viruses, speciﬁcally adenoviruses and noro-

et al. ; McQuaig et al. ). In a similar study, adeno-

viruses, were detected at two beaches with different types

viruses were detected in less than 10% of samples from

of fecal contamination: a known source at Avalon Beach

Doheny Beach and were not detected at Avalon Beach

and unknown, probably diffuse source(s) at Doheny Beach.

(McQuaig et al. ). An epidemiologic study of Mission

There have been several studies on the occurrence of

Bay resulted in only one detection of enteric virus despite ana-

human adenovirus in California beaches (Jiang et al. ,

lyses of more than 1800 water samples (Colford et al. ). A

; McQuaig et al. ). To our knowledge this is the

study of Newport Bay reported that adenoviruses and entero-

ﬁrst report on the detection of human norovirus at California

viruses were detected in approximately 5% of samples

beaches, and in general, there is limited information on the

collected during dry weather (Jiang et al. ). Other studies

occurrence of human noroviruses in recreational use bea-

have reported an association between rainfall, tides and

ches in the USA. At Doheny Beach, a contamination event

hydrogeographical factors associated with the detection of

was detected during the fourth sampling weekend when up

adenoviruses and other pathogens (Griffin et al. ; Jiang

to 80% of samples were positive for either human adenovirus

& Chu ; Ahn et al. ; Abdelzaher et al. ). Previous

and human norovirus without any increase in concentration

studies have also reported a lack of correlation between

and detection frequency of traditional fecal indicator micro-

enteric viruses and coliphages (Jiang & Chu ), although

organisms. The contamination event occurred during a

one study of runoff-impacted waters demonstrated a corre-

relatively calm, rain-free day, with no obvious cause or

lation between adenovirus and F-speciﬁc coliphage (Jiang

source.

et al. ). Therefore, the extent to which enteric virus occur-

Nanoceram©

positively-charged

cartridge

ﬁlter

rence and concentrations in coastal seawaters is associated

method was used for this study, which is the ﬁrst report of

with or predictable by FIB or coliphages or by other environ-

use of this ﬁlter to monitor the virological quality of rec-

mental

reational beach water. This cartridge ﬁlter is efﬁcient at

hydrological conditions remains uncertain and may be more

adsorbing human adenovirus and adsorbing and recovering

site-speciﬁc rather than generalizable.

variables,

such

temperature,

rainfall,

and

human norovirus from seawater, although the recovery efﬁ-

Sampling at multiple time points and sites each day pro-

ciency of human adenovirus is low (Gibbons et al. ). An

vided an opportunity to test existing theories about Avalon

advantage of using this ﬁlter is that it allows for the rapid ﬁl-

and Doheny Beach pollution sources. At Avalon Beach, ade-

tration of larger volumes of water (40 L) than the most

novirus, and Fþ coliphage frequency did not differ along the

commonly used methods using negatively charged mem-

beach transect, which is similar to ﬁndings by McQuaig and

branes or ultraﬁlters (0.5–1 L water) (Jiang et al. ;

colleagues for human polyomavirus at Avalon Beach

Sassoubre et al. ), and sample water does not require

(McQuaig et al. ). Currents within the Bay caused by

147

D. C. Love et al.

Human viruses in marine water at recreational beaches

Journal of Water and Health

12.1

2014

intermittent propeller wash from a passenger ferry (Ho et al.

day, although a comparison to solar radiation is needed to

) adds variability to the system that was not accounted

strengthen this ﬁnding. Given the presumed effects of sun-

for in our models. The city of Avalon is addressing poor

light on microorganisms, the timing of water sampling

water quality by repairs to the leaky sewage system (Barboza

(i.e., sampling in the morning instead of the afternoon) is

). Our results are consistent with an intermittent source

an approach water managers should consider.

of contamination and support these remediation actions to

While it may appear from our study that coliphages were

reduce fecal pollution loading in Avalon Bay and reduce

more susceptible than human viruses to photoinactivation,

swimmer health risks.

this is likely an artifact of the methods used. Coliphages

At Doheny Beach, an urban stream-fed lagoon has been

were measured using infectivity methods, while viral RNA

identiﬁed as a source of fecal pollution at the beach, and per-

or DNA was measured for human viruses using molecular

iodic breaks in a sand berm between the lagoon and the

methods. In sunlit seawater microcosms, the qPCR signal

ocean are associated with an increase in diseases to bathers

from RNA or DNA of viruses and bacteria persisted longer

(Colford et al. ). In our study, the lagoon was a source for

than their infectious counterparts (Walters et al. ). This

coliphages as well. Based on a concentration gradient that

is because the short sequences used for qPCR detection do

increased from sites E to A, it appears that coliphage

not allow for detecting damage in DNA that could eventually

travel from the lagoon into marine water and collect in the

affect the infectivity of the microbe (Rodríguez et al. ).

more stagnant waters near the jetty (site A), which is a pop-

When infectivity assays were used for measuring Adenovirus

ular surﬁng area. Human viruses did not follow the same

2 and coliphage MS2 in laboratory microcosms, similar

gradient along the beach transect according to the data of

photoinactivation rates were observed (Love et al. ).

this study. Human viruses were rarely detected in the

Associating enteric viruses with health risks has been

lagoon (one of seven samples positive) compared to

hampered by limitations in detection technologies (Griffin

marine water (37 of 94 samples positive) and were not

et al. ; Fong & Lipp ). Use of cell culture involves

associated with coliphages. There was no rainfall during

logistical challenges and drastically increases the cost to

the study period, so our ﬁndings may not be representative

the sampling program, while the use of PCR-based methods

of wet weather conditions. One year after our study was con-

does not provide information about the infectivity of the

ducted, McQuaig and colleagues rarely detected human

virus detected, unless integrated with a cell culture infectiv-

viruses in the lagoon but did detect human viruses in

ity assay, called integrated cell culture-polymerase chain

marine water (McQuaig et al. ). Considered together

reaction (ICC-PCR). With the tools we used, potential etiolo-

our studies suggest that sources of human fecal contami-

gical agents of disease can be identiﬁed in water prior to

nation other than the lagoon exist at Doheny Beach.

exposure, before clinical identiﬁcation of disease in bathers.

Microcosm studies with sunlight or simulated sunlight

An important limitation of this study, and many previous

(Sinton et al. ; Kohn et al. ; Kohn & Nelson ;

ones, is that water body characteristics vary greatly, and

Love et al. ) are expanding our understanding of virus

not all variations could be captured. Current monitoring

photoinactivation, and these concepts are being applied to

practices that involve single grab samples for a single type

recreational water settings. We now recognize that sunlight

of FIB may not be capturing the complexity in coastal

affects microbe survival in marine waters (Boehm et al.

beach environments or be associated with enteric virus pres-

), and may indirectly affect bather health risks. Morning

ence and concentrations. Another potential limitation of the

water samples of FIB had stronger relationships to health

study was not accounting for environmental inhibition of

outcomes than afternoon water samples at Doheny Beach

molecular analysis in several ways: by not measuring inhi-

(Colford et al. ). In the present study, the relationship

bition within individual samples, and by assuming one

between time of day (i.e., a proxy for solar radiation) and

nucleic acid extraction method would be optimal for remov-

virus survival was observed within sampling days at both

ing

beaches; coliphages were more prevalent in morning

ampliﬁcation, at both beaches, which some authors later

samples at both beaches and decreased throughout the

found not to be the case (Rodríguez et al. b).

inhibitors

both

adenovirus

and

norovirus

148

D. C. Love et al.

Human viruses in marine water at recreational beaches

CONCLUSION Noroviruses and adenoviruses were detected in the waters of two California beaches sampled during this study. The relationship between human viruses and microbial indicators of fecal contamination depended on the sources of contamination at the beaches. More agreement between human virus detection and FIB was observed in Avalon Beach where the fecal contamination source is known (leaking sewage infrastructure) than in Doheny Beach (unknown, non-point source of viral contamination). At Doheny Beach, sampling during the dry weather helped to identify sources of enteric virus contamination other than the previously identiﬁed source of FIB during rain events (San Juan Creek). Our results comparing enterococci to enteric virus detection demonstrate that beach advisories based on these FIB may not protect the beach users from exposure to human viruses at some California beaches. The direct detection of enteric virus pathogens and the use of more speciﬁc microbial source tracking methods may better assess recreational water quality and provide improved analytical tools for management decisions.

ACKNOWLEDGEMENTS We would like to thank Douglas Wait, Julianne Tajuba, and Jeremy Chemla at the University of North Carolina for assistance with sample analysis. We thank Melissa Madison and Ben Ferarro at the Southern California Coastal Water Research Project (SCCWRP) who assisted with sample collection and shipment, and Elizabeth Scott at SCCWRP who assisted with mapping sample locations in Figure 1. We thank Alexandria Boehm at Stanford University for reviewing a draft of this manuscript and providing data on Avalon Beach water temperatures.

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Maloney, A. M.  Comparison of conventional and TaqMan® realtime RT-PCR assays for the detection of human noroviruses in environmental water samples. Masters Thesis. University of North Carolina at Chapel Hill. McQuaig, S., Griffith, J. & Harwood, V. J.  Association of fecal indicator bacteria with human viruses and microbial source tracking markers at coastal beaches impacted by nonpoint source pollution. Appl. Environ. Microbiol. 78, 6423–6432. National Climate Data Center  Station 23191. Noble, R. T., Blackwood, A. D., Griffith, J. F., McGee, C. D. & Weisberg, S. B.  Comparison of rapid quantitative PCRbased and conventional culture-based methods for enumeration of Enterococcus spp. and Escherichia coli in recreational waters. Appl. Environ. Microbiol. 76, 7437–7443. Okoh, A. I., Sibanda, T. & Gusha, S. S.  Inadequately treated wastewater as a source of human enteric viruses in the environment. Int. J. Environ. Res. Public Health 7, 2620–2637. Rodríguez, R. A., Pepper, I. L. & Gerba, C. P.  Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples. Appl. Environ. Microbiol. 75, 297–307. Rodríguez, R. A., Love, D. C., Stewart, J. R., Tajuba, J., Knee, J., Dickerson, J. W., Webster, L. F. & Sobsey, M. D. a Comparison of methods for the detection of coliphages in recreational water at two California beaches. J. Virol. Methods 181, 73–79. Rodríguez, R. A., Thie, L., Gibbons, C. D. & Sobsey, M. D. b Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters. J. Virol. Methods 181, 43–50. Sassoubre, L. M., Love, D. C., Silverman, A., Nelson, K. L. & Boehm, A. B.  Comparison of enterovirus and adenovirus concentration and enumeration methods in seawater from Southern California, USA and Baja Malibu, Mexico. J. Water Health 10, 419–430. Silva, A. M., Vierira, H., Martins, N., Granja, A. T., Vale, M. J. & Vale, F. F.  Viral and bacterial contamination in recreational waters: a case study in the Lisbon bay area. J. Appl. Microbiol. 108, 1023–1031. Sinton, L. W., Finlay, R. K. & Lynch, P. A.  Sunlight inactivation of fecal bacteriophages and bacteria in sewagepolluted seawater. Appl. Environ. Microbiol. 65, 3605–3613. Soller, J. A., Bartrand, T., Ashbolt, N. J., Ravenscroft, J. & Wade, T. J.  Estimating the primary etiologic agents in recreational freshwaters impacted by human sources of faecal contamination. Water Res. 44, 4736–4747. Teunis, P. F., Moe, C. L., Liu, P., Miller, S. E., Lindesmith, L., Baric, R. S., Le Pundu, J. & Calderon, R. L.  Norwalk virus: how infectious is it? J. Med. Virol. 80, 1468–1476. USEPA  Male-specific (Fþ) and Somatic Coliphage in Water by Two-step Enrichment Procedure. United States Environmental Protection Agency, Washington, DC.

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indicators of recreational water quality are predictive of swimming-associated gastrointestinal illness. Environ. Health Perspect. 114, 24–28. Wade, T. J., Sams, E., Brenner, K. P., Haugland, R., Chern, E., Beach, M., Wymer, L., Rankin, C. C., Love, D., Li, Q., Noble, R. & Dufour, A. P.  Rapidly measured indicators of recreational water quality and swimming-associated illness at marine beaches: a prospective cohort study. Environ. Health 9, 66. Walters, S. P., Yamahara, K. M. & Boehm, A. B.  Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: implications for their use in assessing risk in recreational waters. Water Res. 43, 4929–4939. World Health Organization  Guidelines for Safe Recreational Waters. WHO, Geneva.

First received 24 April 2013; accepted in revised form 6 August 2013. Available online 1 October 2013

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Removal of ammonium ions by laboratory-synthesized zeolite linde type A adsorption from water samples affected by mining activities in Ghana Bright Kwakye-Awuah, Linus Kweku Labik, Isaac Nkrumah and Craig Williams

ABSTRACT Ammonium ion adsorption by laboratory-synthesized zeolite (linde type A; LTA) was investigated in batch kinetics experiments. Synthesized zeolite LTA was characterized by X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy and particle size analysis. Water samples were taken from the Nyam and Tano rivers in Ghana, and 0.8 g of zeolite was added to 100 ml portions of each sample. Portions of the samples were withdrawn every 30 min for 150 min and the concentration of ammonia in each sample was determined. The removal efﬁciency of zeolite LTA was evaluated by retrieving the zeolite from the water samples and adding to a fresh sample to repeat the process. Equilibrium data were ﬁtted by Langmuir and Freundlich isotherms. Maximum adsorption capacities were 72.99 mg g 1 for samples from the River Nyam and 72.87 mg g 1 for samples from the River Tano. The equilibrium

Bright Kwakye-Awuah (corresponding author) Linus Kweku Labik Isaac Nkrumah Department of Physics, Kwame Nkrumah University of Science and Technology, University Post Ofﬁce, Private Mail Bag, Kumasi, Ghana E-mail: bkwakye-awuah.cos@knust.edu.gh Craig Williams School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, United Kingdom

kinetic data were analysed using adsorption kinetic models: pseudo-ﬁrst order and pseudo-second order kinetic models. Linear regression was used to estimate the adsorption and kinetic parameters. The results showed that the adsorption followed pseudo-second order kinetics and suggest that zeolite LTA is a good adsorbent for the removal of nitrogen ammonia from water. Key words

| adsorption, ammonium ions, characterization, synthesis, zeolite LTA

INTRODUCTION Ammonia (NH3) is colourless gas that is highly soluble in

of oral exposure of humans to high concentrations of ammo-

water. In aqueous solutions, most (>90% below pH 8) of

nia and ammonium hydroxide include buccal, oesophageal

(NHþ 4)

and upper tracheal burns and oedema (Klein et al. ;

at equilibrium. Ammonia in the environment originates

Christesen ; Rosenbaum et al. ). Excess circulating

from both natural and anthropogenic sources. Nitrogen

levels of ammonia (hyperammonaemia) has been reported

ammonia is found in wastewaters, leachates, condensates

to cause serious neurological effects. Hyperammonaemia

evolved during composting processes and efﬂuents from

is a result either of inborn errors of the urea cycle enzymes

mining wastewaters discharged into rivers (Tsuno et al.

or of liver toxicity caused by ingested toxins and viral infec-

; Sanchez et al. ; Booker et al. ; Nguyen &

tion (Felipo & Butterworth ). Ammonium ions may also

Tanner ; Kučic´ et al. ). Deleterious effects of this

contribute to the adverse effects of Helicobacter pylori on

nitrogen ammonia include accelerated eutrophication of

the stomach. H. pylori produces urease, which breaks

lakes, depletion of dissolved oxygen in receiving waters

down the urea normally present in the stomach into ammo-

and toxicity to ﬁsh (Nguyen & Tanner ; Rahmani

nia (Mégraud et al. ; Tsujii et al. ). It may also trigger

et al. ; Rahmani & Mahvi ). Some of the effects

the release of cysteine proteases in the stomach that

the ammonia is in the protonated form ammonium

doi: 10.2166/wh.2013.093

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B. Kwakye-Awuah et al.

Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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contribute to the development of gastric haemorrhagic

of Obuasi, which is the river closest to the Anglo-Gold

mucosal lesions (Nagy et al. ). Neutrophils that migrate

Ashanti mine site, and from the River Tano, which is the

to the gastric mucosa in response to the presence of

river closest to the Newmont Ghana Gold Ltd Ahafo mine

H. pylori may release hypochlorous acid, which can interact

site. Zeolite A was synthesized in the Water Research Lab-

with

NHþ 4

to produce the powerful cytotoxic oxidizing agent

monochloramine (Murakami et al. ).

oratory, Kwame Nkrumah University of Science and Technology (KNUST), Ghana, and characterized in the

In recent years, ion exchange and adsorption have

School of Applied Sciences, Wolverhampton University,

become the most efﬁcient method of removing nitrogen

UK. Distilled water was obtained from the Water Research

from water and wastewater. This is because removal by bio-

laboratory, KNUST. Whatman ﬁlters No. 45 were purchased

logical nitriﬁcation–denitriﬁcation processes has been found

from Sigma Aldrich, UK. A Wagtech 7100 Photometer was

to be slow in response to shock loads of ammonia (Lahav &

obtained from the General Chemistry Laboratory, Institute

Green ; Huang et al. b). In addition, treatment of

of Renewable Natural Resources, KNUST.

ammonium nitrogen in wastewater of low carbon content by a biological process requires an additional carbon

Synthesis and characterization of zeolite LTA

source, adding to treatment costs (Booker et al. ; Karadag et al. ). Zeolites

are

Zeolite A was synthesized according to method described by crystalline

aluminosilicate

inorganic

materials whose framework structure consists of cavities

Kwakye-Awuah et al. () with some modiﬁcations. The batch composition for the synthesis was

or pores that are occupied by cations or water molecules (Occelli & Kessler ; Kwakye-Awuah ; Kwakye-

3:165 Na2 O:Al2 O3 :1:92 SiO2 :128 H2 O

Awuah et al. ). Although much research has been carried out on ammonia removal by zeolites, most of the

Eighty millilitres of distilled water was mixed with

zeolites used in such studies are of the natural type. Natural

0.723 g NaOH in a plastic beaker. The mixture was stirred

zeolites are relatively cheap compared to synthetic zeolites.

continuously until the sodium hydroxide was completely

However, synthetic zeolites are useful because of their con-

dissolved. Half of the resulting sodium hydroxide solution

trolled and known physico-chemical properties relative to

was mixed with 8.258 g of sodium aluminate and half with

natural zeolites (Mozgawa ). Furthermore, natural zeo-

15.48 g of sodium metasilicate; both mixtures were stirred

lites are mostly impure as they are mined from underlying

to obtain homogenous suspensions. The two suspensions

rocks. The aim of this study was investigate the adsorption

were then mixed together and again stirred until a hom-

potential of laboratory-synthesized zeolite (linde type A;

ogenous gel was formed. The gel was transferred in to

LTA) for the removal of ammonium from water samples

polytetraﬂuoroethylene (PTFE) bottles which were capped,

from two rivers in Ghana: the Nyam and the Tano. The

then the bottles were put in a pre-heated electric oven at

choice of these two rivers was strategic as they are closest

100 C for 4 h. The bottles were then removed and the reac-

to the efﬂuent treatment sites of two major mining

tion quenched immediately by running tap water over the

companies.

bottles. The crystallized samples were ﬁltered and washed

copiously with distilled water until the pH of the ﬁltrate was 9. The ﬁltered samples were then dried in an electric

MATERIALS AND METHODS

oven at 100 C for 12 h after which they were transferred into plastic containers and stored in a cupboard.

Materials and instrumentation Characterization of zeolite LTA Sodium hydroxide, sodium aluminate and sodium metasilicate were purchased from Sigma Aldrich, UK. Water

The zeolite was characterized using scanning electron

samples were collected from the River Nyam, in a suburb

microscopy

(SEM),

X-ray

diffractometry

(XRD),

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Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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energy-dispersive X-ray spectroscopy (EDX), thermogravi-

(6,900 × g, 6 min). The retrieved zeolite was washed

metric

infrared

copiously (ﬁve times) with de-ionized water and air-dried

spectroscopy (FTIR) and particle size analysis. The surface

in a fume cupboard and then dried at 50 C in an electric

morphology and the chemical composition were analysed

oven for 3 h. The retrieved zeolite was weighed and added

by SEM and EDX using a Zeiss EVO 50 scanning electron

to a fresh water sample with the same zeolite mass:water

microscope (Zeiss, UK). The EDX spectrophotometer was

volume ratio, and adsorption activity was investigated as

attached to the SEM microscope. The phase purity of the

before.

analysis

(TGA),

Fourier

transform

zeolite LTA was analysed with a Philips PW 1710 X-ray diffractometer

(PANalytical

Ltd,

Cambridge).

The

chemical structure was examined by FTIR analysis using a

RESULTS AND DISCUSSION

Mattson FTIR spectroscope (Thermo, Cambridge, UK) and the particle size distribution was analysed with a Master-

Characterization of zeolite

sizer long bed 2000 analyser. SEM micrographs showed that LTA was highly crystalline Adsorption and kinetic studies

and conﬁrmed phase purity and uniform particle morphology morphology (Figure 1).

The batch technique was utilized to monitor the effect of

The XRD analysis of the zeolite (Figure 2) was validated

contact time on ion exchange. One hundred millilitre

by the literature. The XRD patterns of the synthesized zeo-

water samples (100 ml) were measured into conical ﬂasks and 0.8 g of zeolite was added to each sample. The ﬂasks

lite did not have a ﬂat base as in some studies (Treacy & Higgins ; Kwakye-Awuah ). The ﬁrst three peaks

were placed on a rotary shaker at an average speed of

appeared at 6.8, 12.2 and 14.8 , in agreement with Treacy

200 rpm at room temperature. Portions of the samples

et al. (); no impurity phases were seen.

were withdrawn from the ﬂasks at 30, 60, 90, 120 and

The FTIR spectrum of zeolite LTA is given in Figure 3.

150 min and ﬁltered using Whatman ﬁlter No. 45 to

Peaks occurred at wave numbers 1,700, 997, 744, 674, 553

obtain clear solutions and also retrieve the zeolite. The con-

and 432 cm 1. According to Aronne et al. () and

centrations of ammonium ion in the samples before and

Sitarz et al. (), the peak at 997 cm 1 is attributed

after zeolite addition were measured by using a Wagtech

to the overlapping of the asymmetric vibrations of Si–O

7100 Photometer. The photometer measured the amount

(bridging) and Si–O (non-bridging). The peak at 744 cm 1

of ammonium nitrogen present in the sample and the result was multiplied by 1.2 to obtain the amount of ammonia present in the sample. Ten millilitres of the sample was put into a glass cruet and Palintest Ammonia Tablets Nos 1 and 2 were added, crushed and stirred until dissolved. The solution was allowed to stand for 10 min and then placed into the photometer. Readings were taken at room temperature and the procedure was repeated three times to obtain an average concentration. Repeated retrieval and reuse of zeolite LTA The extent to which zeolite LTA persisted in its adsorption activity was investigated according to the method given by Kwakye-Awuah et al. () with some modiﬁcations. Zeolite LTA was retrieved after 150 h by centrifugation

Figure 1

SEM micrograph of zeolite LTA.

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Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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tetrahedra (Mozgawa ; Kwakye-Awuah ; KwakyeAwuah et al. ). Our ﬁndings are in agreement with Ojha et al. (), who stated that the two most intense bands of zeolites usually occur at 860–1,230 cm 1 and 420–500 cm 1. The particle size distribution obtained is shown in Figure 3 (inset). Most particles were in the size range of 2–60 μm with a modal size of 22 μm. Figure 4(a) shows a TGA curve for the synthesized zeolite LTA. Loss of water from the zeolite did not affect its thermal stability. Figure 4(b) represents the elemental comFigure 2

position of the synthesized zeolite. The main elements XRD spectrum of zeolite LTA.

present in the zeolite were aluminium, silicon and sodium. The peak for oxygen coincides with that of iron; this overlap occurs because of the closeness of the energy levels of the backscattered electrons emitted by the two elements.

Removal of ammonia and adsorption isotherms For each treatment type, the removal of ammonium ions by zeolite LTA was initially a fast process with 85% of the removal being achieved within the ﬁrst 60 min (Figure 5). Thereafter, with increase in contact time the rate of removal slowed down (with the exception of ﬁrst addition treatment when the rate slowed down after 90 min). After 120 min the Figure 3

FTIR spectrum and (inset) particle size distribution of zeolite LTA powder.

rate was almost negligible. This trend of removal may be attributed to the rapid utilization of readily available adsorb-

is assigned to the symmetric stretching of the external T–O

ing sites in the zeolite LTA resulting in the fast diffusion and

linkages (Mozgawa ; Sitarz et al. ). Vibrations

attainment of the equilibrium (Donghui et al. ; Wen

associated with double rings (D6R) that connect the sodalite

et al. ; Kučic´ et al. 2013). After approximately

cages occurred at 553 cm 1 whilst the band at 432 cm 1 is

120 min of adsorption, sorption equilibrium begins to estab-

assigned to the vibrations due to the bending of the T–O

lish leading to the reduction in the removal rate. The trend

Figure 4

(a) TGA curve obtained and (b) EDX spectrum of the zeolite LTA.

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Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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when added to fresh samples gave similar results in all treatments for both rivers. The Freundlich isotherm assumes a multilayer adsorption with interactions between ions and is expressed as (Huang et al. a) qe ¼ kF Ce1=n

(3)

where qe (mg/g) is the amount of ammonium ion adsorbed Figure 5

Removal of ammonium in River Nyam water at different contact times. Dose of

at equilibrium, kF (measure of adsorption capacity) and

zeolite LTA dose was 8 g/L, pH ¼ 9 and initial ammonium concentration ¼

1/n measure of adsorption intensity) are Freundlich con-

3.625–3.635 mg/l. Similar results were obtained for River Tano water (data not shown).

stants. Taking natural logs on both sides of (3), we obtain

of removal rate was similar in all cases after regeneration

ln qe ¼ ln kF þ (1=n) ln Ce

(4)

and application of the zeolite to fresh water samples. The adsorption equilibrium data were analysed using

Values of Ce and kF are calculated from a plot (Figure 7)

the Langmuir and Freundlich isotherm models. The Lang-

of ln qe against ln Ce and are given in Table 1 ((a), (b)). The

muir isotherm theory assumes monolayer coverage of

Freundlich model yields a better ﬁt (R 2 ¼ 0.900 0.980)

adsorbate over a homogeneous adsorbent surface. This

than the Langmuir model (R 2 ¼ 0.948 0.966). In addition

implies that all adsorbing sites are equivalent and adsorption

a value of 1/n between 0 and 1 indicates a favourable

on an active site is independent of whether or not an adja-

adsorption. Since the values of 1/n obtained in this study

cent site is occupied. Mathematically, the model is given

for all treatments in both rivers were less than 1 it follows

by the equation (Donghui et al. ; Jafarpour et al. ):

that the Freundlich adsorption conditions were favourable.

qe ¼

MkL 1 þ kL C e

The repeated retrieval and reuse of zeolite LTA showed

(1)

that it continued in its adsorption activity (Figures 6–9). Kinetic studies

where Ce (mg/L) is the equilibrium concentration, qe (mg/g) is the amount of ammonium ion adsorbed at equilibrium, M

To understand the adsorption mechanism of ammonium ion

(mg/g) and kL (L/mg) are the maximum adsorption capacity

uptake onto the zeolite at time t, the pseudo-ﬁrst and second-

and Langmuir constants, respectively.

order kinetic models were used. The pseudo-ﬁrst-order kin-

The linearized form can be expressed as: qe ¼

MkL 1 þ kL C e

etic model is given by the equation (Kučic´ et al. 2012)

(2)

The linear plot of Langmuir isotherm is shown in Figure 6. The values of M and kL are calculated from the

dqt ¼ k1 ðqe qt Þ dt

(5)

Integrating (5) from t ¼ 0 to t ¼ t and from qt ¼ 0 to qt ¼ qt we obtain

slope and intercept of the plot of Ce/qe and Ce. Table 1 ((a), (b)) shows that the maximum amount of ammonium ion

ln(qe qt ) ¼ ln qe k1 t

(6)

adsorbed at equilibrium was 72.99 (mg/g) for the River Nyam water and 72.87 (mg/g) for the River Tano water.

where qe (mg/g) and qt (mg/g) are the amounts of ammonium

The amount adsorbed after regeneration of zeolite LTA and

ion adsorbed at equilibrium and at time t and k1 (1/min) is the

156

B. Kwakye-Awuah et al.

Figure 6

Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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Langmuir isotherms of ammonium adsorption on zeolite LTA for each treatment in (a) River Nyam water and (b) River Tano water obtained in batch

Figure 7

Freundlich isotherms of ammonium adsorption on zeolite LTA for each treatment in (a) River Nyam water and (b) River Tano water obtained in batch

experiments.

Table 1

Isotherm equation coefďŹ cients extracted from the linearized curves in Figures 6 and 7 for (a) River Nyam water and (b) River Tano water

Langmuir parameter

Freundlich parameter

Treatment M (mg/g)

kL (l/mg)

kF (mg/g)

River Nyam water First addition

72.993

4.893

0.963

62.797

29.754

0.935

First retrieval

72.464

5.308

0.964

64.136

28.736

0.910

Second retrieval

71.942

6.043

0.966

64.703

29.155

0.980

Third retrieval

71.942

6.318

0.967

64.780

28.818

0.980

River Tano water First addition

11.455

4.269

0.930

19.834

30.836

0.928

First retrieval

11.377

6.500

0.957

38.648

34.014

0.915

Second retrieval

71.942

7.655

0.964

39.146

50.505

0.900

Third retrieval

71.942

7.226

0.948

38.749

30.488

0.979

157

B. Kwakye-Awuah et al.

Figure 8

Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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Pseudo-ﬁrst-order kinetic plots of ammonium adsorption on zeolite LTA in (a) River Nyam water and (b) River Tano water obtained in batch experiments.

pseudo-ﬁrst-order rate constant. The values of qe and k1 were calculated from the slope and intercept of a plot (Figure 8) of ln(qe qt) against t. Table 2 ((a), (b)) shows that the exper-

Figure 9

Pseudo-second-order kinetic plots of ammonium adsorption on zeolite LTA in (a) River Nyam water and (b) River Tano water obtained in batch experiments.

imental value obtained for qe is far greater than that obtained from calculation. Furthermore, the R 2 value

intercept of a plot (Figure 9) of t/qt against t. Results

obtained suggests that the experimental data did not ﬁt the

obtained are shown in Table 2 ((a), (b)). As can be seen

pseudo-ﬁrst-order kinetic model. The pseudo-second-order

from Table 2 ((a), (b)), the R 2 values obtained showed high

kinetic model is given by the expression (Kučic´ et al. ,

correlation. Furthermore, the experimental and theoretical

)

values of qe and qt are in good agreement. It therefore follows that pseudo-second-order kinetics predominate over

dqt ¼ k1 (qe qt )2 dt

(7)

pseudo-ﬁrst-order kinetics. Thus the rate limiting step is likely to be chemical sorption, involving forces due to the sharing of or exchange of electrons between the adsorbent

Integrating from t ¼ 0 to t ¼ t and from qt ¼ 0 to qt ¼ qt

and the adsorbate.

we obtain Health implications of the presence of ammonia in qt ¼

t 1=(k2 q2e ) þ t=qe

(8)

relation to The need to treat contaminated water bodies is very urgent

where, again, qe (mg/g) and qt (mg/g) are the amount of

because many farmers depend on a good water supply for

ammonium ion adsorbed at equilibrium and at time t and

their activities and also because of the increasing demand

k2 (1/min) is the pseudo-ﬁrst-order rate constant. The

for potable water in urban and rural areas. Although ammo-

values of qe and k1 were calculated from the slope and

nia levels in groundwater are usually below 0.2 mg/l,

158

Table 2

B. Kwakye-Awuah et al.

Ammonium ion removal from water by zeolite LTA

Journal of Water and Health

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Kinetic equations coefﬁcients extracted from the linearized curves in Figures 8 and 9 for (a) River Nyam water and (b) River Tano water

Pseudo-ﬁrst-order

Pseudo-second-order

Treatment k1 (L/min)

qe,exp (mg/g)

qe,calc (mg/g)

k2 (L/min)

qe,exp (mg/g)

qe,calc (mg/g)

River Nyam water First addition

0.173

19.894

0.377

0.846

0.019

19.894

12.819

0.970

First retrieval

0.080

19.142

0.403

0.867

0.019

19.142

11.109

0.924

Second retrieval

0.079

19.138

0.367

0.867

0.019

19.138

11.109

0.924

0.080

19.938

0.392

0.808

0.021

19.938

14.331

0.934

Third retrieval

River Tano water First addition

0.017

14.869

0.377

0.948

0.012

14.869

6.451

0.910

First retrieval

0.020

13.404

0.403

0.959

0.019

13.404

10.274

0.890

Second retrieval

0.019

12.969

0.367

0.919

0.019

12.969

8.238

0.916

Third retrieval

0.020

13.046

0.392

0.944

0.125

13.046

11.890

0.833

ammonia may be present in drinking water as a result of dis-

nitrite as the result of catalytic action (Reichert & Locht-

infection with chloramines. Furthermore, cement mortar

mann ) or the accidental colonization of ﬁlters by

used for coating the insides of water pipes may release con-

ammonium-oxidizing bacteria. High levels of nitrate are par-

siderable amounts of ammonia into drinking water and

ticularly harmful to human health, and can cause blue-baby

compromise chlorine disinfection (Wendlandt ). For

syndrome, kidney failure and cancer. Irrespective of the

communities affected by mining activities, ammonia levels

mining method, one of the biggest and most long-term

in both underground and surface waters may even be

impacts of mining activities is on water resources. Using zeo-

higher. Mining involves the extraction of rock that contains

lite LTA is promising means of removing nitrogen ammonia

valuable materials or minerals, such as coal, diamonds, gold,

from water sources by adsorption of the ammonium ion.

platinum, vanadium and copper. This rock is abstracted by means of both opencast and underground blasting. Blasting to access the mineral ore typically involves the use of ammo-

CONCLUSIONS

nia-based explosives. Metallurgical extraction of minerals from the rock typically involves the use of nitrate-based

From the results obtained and their evaluation, it can be

acids, such as aqua regia (Yashina & Nesterova ).

concluded that zeolite LTA is a good adsorbent for remov-

Both these activities lead to increased concentrations of

ing ammonium ions from aqueous solutions. The removal

nitrates in groundwater resources as the nitrogen cycle

percentage was between 92.2 and 95.8% in the various

ﬂows between ammonia under reducing (or acidic) con-

treatments stages. Langmuir and Freundlich isotherms

ditions, and nitrite under oxidizing conditions (Moore &

were both demonstrated to provide good ﬁt for the sorp-

Luoma ). High levels of ammonia on slimes dams

tion

(which are used to retain mining waste) are oxidized as

adsorption capacity of zeolite LTA was found to be

the water from the slimes dam leaches into the groundwater,

72.99 and 72.46 mg/g for ﬁrst addition and ﬁrst retrieval

where it occur as high levels of nitrate. If present at high

and 71.94 for second and third retrieval processes for

levels in water sources, the ammonia may interfere with

water samples from the River Nyam. An adsorption

ammonium

ions

onto

zeolite

LTA.

The

removal processes of other heavy metals that are deleterious

capacity of 72.87 and 72.39 for ﬁrst addition and ﬁrst

to human health since too much oxygen is consumed by

retrieval and 71.94 for second and third retrieval pro-

nitriﬁcation. In addition, the presence of the ammonium

cesses were obtained for water samples from the River

cation in raw water may result in drinking water containing

Tano. Kinetics studies suggest that ammonium adsorption

159

B. Kwakye-Awuah et al.

Ammonium ion removal from water by zeolite LTA

on zeolite LTA could be better described by the pseudosecond-order model with the parameters better estimated by the linear method.

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Lahav, O. & Green, M.  Ammonium removal using ion exchange and biological regeneration. Water Res. 32, 2019–2028. Mégraud, F., Neman-Simha, V. & Brugmann, D.  Further evidence of the toxic effect of ammonia produced by Helicobacter pylori urease on human epithelial cells. Inf. Immun. 60, 1858–1863. Moore, J. N. & Luoma, S. N.  Hazardous wastes from large-scale metal extraction. Env. Sci. Technol. 24, 1278–1285. Mozgawa, W.  The relation between structure and vibrational spectra of natural zeolites. J. Mol. Struct. 596, 129–137. Murakami, M., Asagoe, K., Dekigai, H., Kusaka, S., Saita, H. & Kita, T.  Products of neutrophil metabolism increase ammonia-induced gastric mucosal damage. Digest Dis. Sci. 40, 268–273. Nagy, L., Kusstatscher, S., Hauschka, P. V. & Szabo, S.  Role of cysteine proteases and protease inhibitors in gastric mucosal damage induced byethanol or ammonia in the rat. J. Clin. Invest. 98, 1047–1054. Nguyen, M. L. & Tanner, C. C.  Ammonium removal from wastewaters using natural New Zealand zeolites. New Zealand. J. Agric. Res. 41, 427–446. Occelli, M. I. & Kessler, H. (eds)  Synthesis of Porous Materials: Zeolites, Clays and Nanostructures. CRC Press, New York. Ojha, K., Pradhan, N. C. & Samanta, A. N.  Zeolite from ﬂy ash: synthesis and characterization. Bull. Mater. Sci. 27, 555–564. Rahmani, A. R. & Mahvi, A. H.  Use of ion exchange for removal of ammonium: A biological regeneration of zeolite. Global NEST J. 8, 146–150. Rahmani, A. R., Mahvi, A. H., Mesdaghinia, A. R. & Nasseri, S.  Investigation of ammonia removal from polluted waters by clinoptilolite zeolite. Int. J. Environ. Sci. Technol. 1, 125–133. Reichert, J. & Lochtmann, S.  Auftreten von Nitrit in Wasserversorgungssystemen (Occurrence of nitrite in water distribution systems). Gas- und Wasserfach, WasserAbwasser 125, 442–446. Rosenbaum, A. M., Walner, D. L., Dunham, M. E. & Holinger, L. D.  Ammonia capsule ingestion causing upper aerodigestive tract injury. Otolaryngol. Head Neck Surg. 119, 678–680. Sanchez, E., Milan, Z., Borja, R., Weiland, P. & Rodriguez, X.  Piggery waste treatment by anaerobic digestion and nutrient removal by ionic exchange. Resour. Conserv. Recycl. 15, 235–244. Sitarz, M., Handke, M. & Mozgawa, W.  FTIR studies of the cyclosilicate-like structures. J. Mol. Struct. 596, 185–189. Treacy, M. M. J. & Higgins, J. B.  Collection of Simulated XRD Powder Patterns for Zeolites. Elsevier, Amsterdam. Tsujii, M., Kawano, S. & Tsuji, S.  Ammonia: a possible promotor in Helicobacter pylori-related gastric carcinogenesis. Canc. Lett. 65, 15–18.

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(Ammonium/ammonia as cause of bacterial regrowth in drinking-water pipes). Gas- und Wasserfach, WasserAbwasser 129, 567–571 (in German). Yashina, G. M. & Nesterova, S. V.  Possible methods for preliminary opening up of minerals to accelerate complex extraction of metals from balanced ores. J. Mining Sci. 28, 299–306.

First received 9 May 2013; accepted in revised form 22 September 2013. Available online 24 December 2013

2014

161

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Cryptosporidium infection, onsite wastewater systems and private wells in the arid Southwest Kristine Tollestrup, Floyd J. Frost, Twila R. Kunde, Marylynn V. Yates and Stephanie Jackson

ABSTRACT Few prior studies have examined the potential health risks from transmission of enteric parasites via aquifers contaminated by wastewater from onsite systems. A cross-sectional study of 600 residents in households served with either onsite wastewater systems and private wells or city sewer/water systems in three different sites in central New Mexico compared serological responses to Cryptosporidium, a common waterborne infections agent. Study participants completed a short selfadministered questionnaire with questions on demographic characteristics, characteristics of the onsite wastewater system and private well, and common risk factors associated with cryptosporidiosis. A sample of household tap water was collected, as well as a blood sample from each study participant to measure IgG responses to antigen groups for Cryptosporidium. Logistic regression analysis showed a signiﬁcant association between having an onsite wastewater system and private well and the 27-kDa marker for Cryptosporidium in the River Valley site after adjusting for covariates (OR ¼ 1.98; 95% CI ¼ 1.11–3.55). This study, together with one prior study, suggests that the presence of onsite wastewater systems and private wells might be associated with an increased risk of Cryptosporidium infection. Key words

| drinking water quality, groundwater, wastewater, waterborne infectious diseases, water epidemiology

Kristine Tollestrup (corresponding author) Floyd J. Frost Stephanie Jackson Department of Family and Community Medicine, 2400 Tucker NE, 1 University of New Mexico, Albuquerque, NM 87131-0001, USA E-mail: ktollestrup@salud.unm.edu Floyd J. Frost Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA Twila R. Kunde LCF Research, 2309 Renard Place SE, Suite 103, Albuquerque, NM 87106, USA Marylynn V. Yates Department of Environmental Sciences, University of California, Riverside, 900 University Avenue, Riverside, CA 92521, USA

INTRODUCTION Recently, there has been concern that onsite wastewater sys-

annually there are 168,000 viral illnesses and 34,000 bac-

tems, especially in areas that are densely populated or have

terial illnesses due to contamination of public ground

porous soils, may increase the risk of enteric disease trans-

water systems (US EPA ).

mission (Carroll et al. , ; Zarate-Bermudez ).

Approximately 20% of US households rely on onsite

Surveillance of waterborne disease outbreaks in the

wastewater systems for treatment and disposal of domestic

United States has suggested that outbreaks of gastrointesti-

sewage (US EPA ). In rapidly growing rural areas of

nal (GI) illnesses are associated with failed onsite

the USA, onsite wastewater systems are the most common

wastewater systems (Craun & Calderon ). Borchardt

method of sewage treatment and disposal. Onsite waste-

et al. () found that septic system densities were associ-

water systems include any system for the collection,

ated with endemic diarrheal risk in children in Wisconsin.

storage, treatment, neutralization or stabilization of sewage

Denno et al. () also found that Salmonella infection

that occurs on a property. A common type of onsite waste-

was also associated with use of private wells and residential

water system is a septic system that is designed to remove

onsite wastewater systems. The United States Environ-

solids in a septic tank and disperse the wastewater in a

mental Protection Agency (US EPA) has estimated that

leach ﬁeld that relies on soils to perform secondary

doi: 10.2166/wh.2013.049

162

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

treatment of liquid waste. Under optimum conditions, an

of indicator bacteria may provide little useful information

onsite wastewater system will remove 99.9% of viruses and

about the risks of parasite contamination.

other wastewater constituents (Van Cuyk et al. ). How-

Reviews of waterborne outbreaks in the United States

ever, a US EPA review of studies found failure rates ranging

have suggested that groundwater contamination is a very

from 10 to 20%, resulting in groundwater contamination

common mode of parasite and virus transmission (Barwick

(US EPA ). Furthermore, detailed information on soil

et al. ; Craun & Calderon ; Frost et al. ; Lee

characteristics in areas with onsite systems is generally

et al. ; Craun et al. ; Liang et al. ; Yoder

unavailable.

et al. ). However, outbreaks account for only a very

Data from the United States Geological Survey estimate

small fraction of all infections and illnesses. For many infec-

that groundwater supplies almost 50% of the American

tious agents, infections do not always result in disease,

population with drinking water; groundwater was the pri-

disease clusters, or severe disease. More commonly, infec-

mary source of the water for 98% of people using private

tious agents may result in only a few infections at any one

wells (Hutson et al. ). The growing use of onsite waste-

time, and, therefore, are never recognized as an epidemic.

water systems and the reliance on groundwater sources for

This is likely to be especially common in sparsely populated

drinking water has raised concerns among local and state

rural areas where few infections occur.

public health agencies, the US EPA, and the Centers for Dis-

For viral gastrointestinal illnesses, enteric viruses may be

ease Control and Prevention over the potential health effects

transmitted by either direct transmission, such as food or

of these trends and the risks to sensitive populations such as

water contamination, or via person-to-person transmission.

children and those with compromised immune systems

Since a large fraction of viral gastrointestinal agents are

(ASTHO ).

transmitted person-to-person (Morens et al. ), it is often

The increased population growth outside urban areas

difﬁcult to identify the food or waterborne sources for

has increased the pressure on rural and semi-urban areas

these viral agents. In contrast, the parasite Cryptosporidium

to absorb more housing. Because of greater distances

does not appear to be commonly transmitted person-to-

between houses in rural areas and the costs of building

person. One study of a 1993 Milwaukee, Wisconsin Cryptos-

new centralized wastewater treatment plants, developers

poridium outbreak found that only 5% of Cryptosporidium

and home-owners often rely on relatively inexpensive

infections among adults may have been due to person-to-

onsite wastewater systems. Very often the homes served by

person transmission (MacKenzie et al. ). A second

onsite wastewater systems are also served by private wells

study estimates secondary transmission rates were slightly

drilled on the same building lot. These private wells are

higher at 10% (95% CI ¼ 6–21%) (Eisenberg et al. ).

not regulated by the US EPA under the Safe Drinking

Therefore, it is possible to use serological prevalence of Cryp-

Water Act. State and local regulations may regulate well

tosporidium antibody to identify prior food, waterborne, or

construction, but not water quality. State and local regu-

other routes of Cryptosporidium infections. We believe that

lations for locating wells near sources of contamination

Cryptosporidium antibody levels, an indicator of prior infec-

may specify minimum distances (at least 50 feet) between

tion, may effectively identify populations exposed to prior

the well and onsite wastewater system. This is seldom ade-

oocyst contamination of food or water, especially when the

quate and can be especially inadequate in certain soils that

level of oocyst contamination may be low.

facilitate the rapid movement of pathogens from the onsite wastewater system to the well.

No prior studies have compared the risks of prior Cryptosporidium infection among individuals having both an

Unfortunately, soil characteristics that might predict

onsite wastewater system and a private well compared to

protection from pathogens near drinking water wells

users of municipal drinking water systems. In this study,

remain either unknown or unexamined during the well dril-

we examined whether living in a household with an onsite

ling process. Well water testing generally only examines

wastewater system and private well was associated with

bacterial indicators of contamination. Since the indicator

increased serological responses to Cryptosporidium. An

bacteria are much more fragile than parasites, the absence

elevated serological response to Cryptosporidium was used

163

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

as an indicator of exposure to and/or infection by this

vendor. Individuals who expressed interest in participating

common waterborne pathogen. A serological test for this

in the study were screened for eligibility. Criteria for eligi-

pathogen is readily available and has already been validated

bility to participate included being at least 18 years of age,

(Frost & Craun ; Frost et al. a, b, , ). We

living in the site area for at least 1 year, drinking tap water

also analyzed the drinking water for common indicators of

which was not ﬁltered or had not been treated by reverse

contamination (fecal and total coliform, enterococcus, and

osmosis, and being in overall self-reported good health. Con-

coliphage). We hypothesized that study participants who

sent was obtained from individuals eligible to participate in

were living in households with onsite wastewater systems

the study using the protocol approved by the University of

and private wells would be more likely to have elevated ser-

New Mexico Human Research Review Committee. Arrange-

ological responses compared to those living in households

ments were also made to collect blood sera, administer a

using city sewer and water systems because of increased

brief questionnaire, and collect a water sample if the partici-

exposure to Cryptosporidium oocysts.

pant lived in a household using an onsite wastewater system and private well. For the River Valley site, questionnaire data, water and blood samples were collected over a 1 year

METHODS

time period from November 2004 to October 2005. For the East Mountains site, this information was collected from

The study design was a cross-sectional study of 600 residents

May to October 2006, and from August 2006 to February

in households served with either onsite wastewater systems

2007 for the Alluvial Fan site.

and private wells or city sewer/water systems in three differ-

A short self-administered questionnaire was completed at

ent geographic sites in the greater Albuquerque, New

the time of the blood draw. Information on the questionnaire

Mexico area. The three included: (1) a semi-rural area on

included: demographic characteristics, length of time at the

the river valley ﬂoor of the Rio Grande in north and

residence, age of wastewater system and frequency of pump-

north-central Albuquerque (River Valley), (2) a more urban

ing, depth of the well, type of water supply, amount of water

area on an alluvial plain in northeast Albuquerque (Alluvial

consumed in the past 24 hours, past diagnoses of cryptospor-

Plain), and (3) a semi-rural area with limestone soils in the

idiosis, diarrhea (three or more loose bowel movements a

Sandia and Manzano Mountains foothills east of Albuquer-

day) that lasted 4 or more days in the past 2 months, and

que (East Mountains). Households were selected to

number of episodes of gastrointestinal illness (diarrhea,

participate based on whether they used an onsite wastewater

vomiting, nausea) in the past year. The questionnaire also col-

system and a private well (exposed group) or city sewer/

lected information on common risk factors associated with

water systems (unexposed group). The River Valley and

cryptosporidiosis such as having young children, having chil-

Alluvial Plain sites included a mix of households using

dren in day care, changing diapers, caring for someone with

these two types of systems. For these two sites, the exposed

diarrhea, visiting someone in hospital, handling pets, young

and unexposed groups lived in the same geographic areas.

pets, and livestock, engaging in water activities, having

Unfortunately, there were no municipal sewer and water

plumbing work done in the home and traveling outside the

systems in the East Mountains site. The River Valley unex-

USA. The information on these risk factors was used to ident-

posed households were used as the comparison group for

ify confounding variables in the regression models.

the East Mountains site because of their comparability in the rural nature of their neighborhoods that included

Water samples

relatively undeveloped lots. Study participants were recruited using neighborhood

Field staff collected a sample of the participant’s home tap

association meetings, ﬂyers, advertisements in local papers,

water in accordance with the US EPA Method 1601 for

mailings to residential addresses in the study area, and tele-

coliphage and Standard Methods for the Examination of

phone calls. Addresses and phone numbers for residences

Water and Wastewater protocols for the bacterial indicators.

in the study areas were purchased from a commercial

Two sterile water bottles, a 500 mL and a 1 L bottle, were

164

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

used for each sample. The water was collected from either

the curve of response intensity for each lane at the expected

the kitchen faucet (preferable) or an outside hose bib. The

location of the response for the antigen group. The quality

water was allowed to run for 3–5 minutes at a rate that

control procedures, involving digitizing response intensities,

ﬂushed the lines but did not cause splash back onto the

were standardized to the responses of a reference sample.

faucet. After the ﬂush time had elapsed, the bottles were

They also maintained relatively low costs to accurately

ﬁlled to within 1 inch of the top. The bottle and cap connec-

identify responses and estimate the intensity of each

tion were sealed using paraﬁlm, stored in gallon zip-lock

response.

bags, and shipped in a cooler to Dr Marylynn Yates’ laboratory at the University of California, Riverside for analysis.

The IgG result for each sample specimen was standardized by computing the ratio of the response intensity for

The water samples were tested for coliphage and other

the sample specimen to the response intensity for a positive

indicators of fecal contamination. Somatic coliphage ana-

control serum contained on each blot. A strong serological

lyses were performed using the two-step enrichment

response was deﬁned as having a ratio of at least 0.20

procedure, US EPA Method 1601. The water samples were

(20% of the response of the standard positive control). The

also tested for total coliforms (Standard Methods: Standard

IgG positive control sera were obtained from individuals

Total Coliform Membrane Filter Procedure. Sect. 9222.B),

with a strong serological response to both antigens, approxi-

fecal coliforms (Standard Methods: Fecal Coliform Mem-

mating the intensity of responses observed from several

brane Filter Procedure. Sect. 9222.D), and enterococci

individuals with laboratory-conﬁrmed cryptosporidiosis.

(Standard Methods: Fecal Streptococcus and Enterococcus

The same positive control sera were used on all blots. For

Groups Membrane Filter Techniques Sect. 9230.C).

comparison purposes as a quality control measure, we had

Once the water results were available, each household

laboratory results from hundreds of individuals from many

was sent a letter stating whether their total coliform results

countries (USA, Canada, Australia, New Zealand, South

showed a presence or absence of bacteria. If total coliform

Africa, Hungary, Italy, Russia, and UK) (Frost et al. ,

bacteria were present, the letter also included information

, , a, b; Duncanson et al. ; Egorov et al.

on how to chlorinate the well and telephone numbers for

; Kozisek et al. ).

the county environmental health ofﬁce. Data analysis Blood samples Descriptive statistical analyses used the Pearson chi-square Blood samples were collected using vacutainers to collect a

test to assess associations between the exposure (onsite

5 mL volume of whole blood. Blood was allowed to clot at

wastewater and water systems), categorical outcome (serolo-

room temperature for 30 minutes and was centrifuged

gical response), and covariates. The Mann–Whitney U test

according to American Society for Clinical Pathology proto-

was used to examine differences among continuous serologi-

cols. The serum was transferred to a cryotube for frozen

cal results (intensity of response) and other variables not

storage and shipment. Sera samples were not labeled with

having a normal distribution. Multivariate logistic regression

names of individual study participants.

models examined the association between having an onsite

Sera were analyzed by immunoblot to measure IgG ser-

wastewater system/private well and having a strong

ological response to the 15/17-kDa and 27-kDa antigen

response to the 15/17-kDa or the 27-kDa markers deﬁned

groups. This method, which uses the miniblot format, has

as at least 20% of a positive control and controlled for poten-

been described in detail elsewhere (Frost et al. a, b).

tial confounding factors that might explain elevated or

The intensities of the serological responses to the

reduced risks of prior infection. The comparison city

15/17-kDa and 27-kDa antigen groups on the immunoblots

system group for the East Mountains site was that from

were digitally analyzed by an IS-2000 Digital Imaging

the River Valley since both areas were more rural and the

System (Alpha Innotech). Serological responses to the two

lifestyle factors were more similar than the more urban Allu-

antigen groups were based on the measured area under

vial Fan site. Variables were manually selected for the ﬁnal

165

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

models, and the models always included the exposure

higher percentage of participants with onsite wastewater sys-

(having an onsite wastewater system/private well). Risk

tems/private wells living in either the River Valley site or the

factor and behavior variables, as well as the two illness

East Mountains site were men compared to those using city

measures (diarrhea lasting more than 4 days in the past

sewer/water systems (p ¼ 0.008 and p ¼ 0.010, respectively).

2 months and having at least one gastrointestinal illness in

A signiﬁcantly higher percentage of participants with onsite

the past year), were entered into the full model if they

wastewater systems/private wells living in the Alluvial Fan

were found to be associated with the outcome in the univari-

site were over the age of 40 years compared to those using

ate analyses (Pearson χ2 test p-value 0.25). Covariates were

city sewer/water systems (p ¼ 0.001).

removed from the model if they were non-signiﬁcant at the

Information on risk factors for cryptosporidiosis and

0.1 alpha level and did not change the estimate of the

gastrointestinal illness was also collected from the question-

main effect by more than 20%. Each of the three geographic

naire (Table 2). The percent of participants reporting that

sites was modeled separately.

they had a speciﬁc risk factor varied considerably. For example, only 1.0–4% of participants reported drinking untreated water away from home, whereas 85.8–93.8% of participants reported handling pets. There were signiﬁcant

RESULTS

differences among the ﬁve groups in the percent of partici-

The demographic characteristics of the study participants from the ﬁve groups are shown in Table 1. A signiﬁcantly

pants reporting handling young pets (p ¼ 0.001), handling livestock (p ¼ 0.001), swimming or wading in a lake or stream (p ¼ 0.011) and traveling outside the USA in the past 12 months (p < 0.001).

Table 1

Demographic characteristics of study participants by geographic area and type of onsite wastewater system/private well

City systems

The percent of participants reporting that they had had diarrhea lasting 4 or more days in the past 2 months ranged

Onsite wastewater system/private well

from 4.5% in Alluvial Fan participants on city systems to 11.9% in River Valley participants with onsite wastewater

River

Alluvial

Valley N ¼ 99

Fan N ¼ 134

East River Valley N ¼ 101

Alluvial Fan N ¼ 134

Mountains N ¼ 130

systems/private wells (Table 2). Approximately one-half of all participants reported that they had had at least one gastrointestinal illness in the past year, ranging from 46.9% in East

Gender (%): Male

23.2

35.1

40.6

46.3

50.8

Mountain participants to 59.6% of River Valley participants

Female

76.8

64.9

59.4

53.7

49.2

on city systems. The differences in percentages of diarrhea

and gastrointestinal illness did not differ among the ﬁve sites.

Age group (%): 18–39 yrs

20.4

23.9

10.9

40–49 yrs

22.4

20.1

24.8

50–59 yrs

24.5

27.6

32.7

60þ yrs

32.7

28.4

31.7

16.2

The results of the water analyses are reported in Table 3

36.4

25.4

as the percent of samples testing positive for total coliforms,

29.5

30.8

fecal coliforms, enterococci, and coliphage. One water

25.8

27.7

sample was collected from each household during the study

8.3b

time period. The range in colony counts is also shown in

Race/ethnicity (%): NHW

62.6

80.6

73.3

84.3

83.8

Table 4. A total of 83 samples tested positive for total

Other

36.4

19.4

25.7

15.7

16.2

coliforms, 22 tested positive for fecal coliforms, 11 tested

positive for enterococci, and 1 tested positive for coliphage.

Education d

Non-college grad

39.4

29.9

35.6

20.1

34.6

College grad

60.6

70.1

64.4

79.9

65.4

East Mountains, tested positive for total coliforms, fecal

Difference between city and onsite system participants, chi-square p-value ¼0.008. Difference between city and onsite system participants, chi-square p-value ¼0.001.

b c

Non-Hispanic white.

Two samples, one from the River Valley and one from the

Difference between city and onsite system participants, chi-square p-value ¼0.010.

coliforms, and enterococci. The sample testing positive for coliphage also tested positive for total coliforms. The percentages of samples testing positive on any of the four tests were signiﬁcantly different in the three areas: 16.8% (River

166

Table 2

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

Risk factors associated with cryptosporidiosis and gastrointestinal illness in study participants

Onsite wastewater system/Private well City systems Risk factor

Children under 5 yrs in household Child attends daycare

River Valley

Alluvial Fan

River Valley

Alluvial Fan

East Mountains

N ¼ 99

N ¼ 134

N ¼ 101

N ¼ 134

N ¼ 130

9.1%

17.9%

7.9%

11.9%

14.6%

9.1%

12.7%

10.9%

9.0%

10.8%

Handled child with diapers

21.2%

31.3%

18.8%

23.1%

20.2%

Cared for someone with diarrhea

14.1%

23.1%

16.8%

20.9%

19.7%

Visited anyone in hospital

43.4%

53.0%

48.5%

51.1%

46.9%

Handled pets

90.9%

85.8%

91.1%

88.8%

93.8%

Handled young pets

45.5%

23.9%

44.6%

34.3%

30.0%

Handled livestock, wild animals, or zoo animals

26.3%

10.4%

42.6%

17.2%

28.5%

1.0%

1.5%

4.0%

2.2%

2.3%

Swam or waded in a lake or stream

22.2%

20.1%

34.7%

26.9%

36.9%

Swam or waded in a pool, hot tub, or water park

58.6%

52.2%

45.5%

55.2%

60.8%

Had plumbing done in the home

23.2%

27.6%

29.7%

19.4%

19.2%

Traveled outside the USA

14.1%

18.7%

30.7%

38.1%

21.5%

Drank untreated water from lakes or streams

Had diarrhea lasting 4þ days in past 2 months

10.1%

4.5%

11.9%

7.5%

8.5%

Had at least 1 episode GI illness in past yr

59.6%

57.5%

57.4%

51.5%

46.9%

Table 3

Results from household water samples for the three geographic areas with

Table 4

onsite wastewater systems/private wells

Percent of participants with a strong response ( 20% of a positive control) to the serological marker for each site by type of wastewater and water system

Percent positive results

Type of test

Total coliforms Colony counts

River Valley N ¼ 101

16.8%

Percent of respondents with a strong response to the marker

Alluvial Fan N ¼ 133

24.8% 1–TNTC

Alluvial Fan

East Mountains

Onsite

City

Onsite

Onsitea

marker

City

N ¼ 99

N ¼ 134

N ¼ 101

N ¼ 134

N ¼ 130

15/17-kDa

37.4%

43.6%

35.8%

26.9%

39.2%

27-kDa

47.5%

61.4%b

57.5%

59.7%

43.8%

1–TNTC

Fecal coliforms

2.7%

4.5%

10.9%

Colony counts

1–16

1–68

Enterococci

2.0%

3.0%

3.9%

Colony counts

1–3

1–39

1–TNTC

Coliphage presence

0.0%

0.8%

Any agent

16.8%

27.8%

31.5%

River Valley Serological

25.8% a

1–35

East Mountains N ¼ 128

Too numerous to count. Only 69 wells tested for fecal coliforms.

Compared to River Valley city system participants.

Chi-square test, p ¼ 0.048, for comparison city and onsite.

Participants using onsite wastewater systems/private wells in the River Valley and Alluvial Fan sites were compared to those using city sewer/water systems in the River Valley and Alluvial Fan sites, respectively. In the East Mountains,

Valley), 27.8% (Alluvial Fan), and 31.5% (East Mountains)

the participants were compared to participants from the

(p ¼ 0.036).

River Valley using the city sewer/water systems because of

The results from the serological analyses for Cryptospor-

the comparability of the rural nature of the neighborhoods.

idium are shown in Table 4 as the percent of participants

There was a signiﬁcantly higher percentage of partici-

with a strong response (an intensity of 20% of a positive

pants using onsite wastewater systems/private wells in the

control) to the two markers (15/17-kDa and 27-kDa).

River Valley who had a strong response to the 27-kDa antigen

167

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

than participants using city sewer/water systems (p ¼ 0.048).

response to the 27-kDa marker compared to those using the

However, there were no signiﬁcant differences in the percent

city sewer and water systems after controlling for handling

of participants with a strong response to the 15/17-kDa anti-

livestock, the only risk factor variable entering the model.

gen between participants using onsite wastewater systems/

This positive association between having onsite waste-

private wells and those using city sewer/water systems in

water/private well systems and a strong response to the 27-

any of the groups. It should be noted that response to the

kDa marker was also observed for the Alluvial Fan site. The

15/17-kDa antigen is shorter lived and returns to background

odds ratio for participants using onsite wastewater systems/

levels in several months following an infection, whereas

private wells was 1.28 after controlling for several risk factors,

the response to the 27-kDa antigen remains elevated for

but it was not signiﬁcantly elevated (95% CI ¼ 0.74–2.21). In

9 months or longer (Muller et al. ). Therefore, using the

this group, several risk factors were signiﬁcantly associated

27-kDa increases the likelihood that differences will be

with an increased adjusted odds ratio: having plumbing

detected between the two exposure groups.

done in the home (OR ¼ 2.11; 95% CI ¼ 1.10–4.03), handling

The relationship between well contamination (measured

pets (OR ¼ 2.83; 95% CI ¼ 1.24–6.49), and being 60 years of

by any positive test for an indicator microorganism) and a

age or older (OR ¼ 4.20; 95% CI ¼ 1.79–9.89). In the East

strong serological response to the 15/17-kDa and 27-kDa

Mountains, this increase in the odds ratio was not observed

markers were also examined (Table 5). There was no signiﬁ-

for the overall effect, but participants in the East Mountains

cant association between having a contaminated well and

60 years of age or older were over three times more likely to

showing a strong response to either of the markers.

have a strong response (OR ¼ 3.69; 95% CI ¼ 1.61–8.46).

Logistic regression models were completed to determine

For the 15/17-kDa marker, there was no signiﬁcant

whether having an onsite wastewater system/private well

association between using onsite wastewater systems/pri-

was associated with a stronger serological response for

vate wells and having a strong response in any of the three

each of the antigen groups after controlling for other con-

groups. However, the overall adjusted odds ratio in the

founding risk factor variables (Table 6). The ﬁnal models

River Valley group was elevated (OR ¼ 1.34; 95% CI ¼

for each site are shown for each of the two markers. A signiﬁ-

0.74–2.44). In the River Valley, the adjusted odds ratio for

cant association was observed between using onsite

handling a child with diapers was signiﬁcantly increased

wastewater systems/private wells and the 27-kDa marker in

(OR ¼ 2.24; 95% CI ¼ 1.07–4.69) and being of a race/ethni-

the River Valley. Participants using onsite wastewater

city other than non-Hispanic white signiﬁcantly increased

systems/private wells were almost two times more likely

the adjusted odds ratio (OR ¼ 1.92; 95% CI ¼ 1.01–3.66).

(OR ¼ 1.98; 95% CI ¼ 1.11–3.55) to have a strong serological

Participants residing in the Alluvial Fan had signiﬁcantly

Table 5

Relationship between well contamination and serological response to Cryptosporidium antigen for participants with onsite wastewater systems/private wells

Participants with a strong

response to 15/17-kDa

response to 27-kDa

No. of private wells tested

% (N)

p-value

% (N)

p-value

Uncontaminated

40.5% (34)

0.164

61.9% (52)

0.812

Contaminated

58.8% (10)

Uncontaminated

30.2% (29)

Contaminated

18.9% (7)

Uncontaminated

39.1% (34)

Contaminated

40.0% (16)

River Valley 58.8% (10)

Alluvial Fan 0.189

60.4% (58)

0.700

56.8% (21)

East Mountains 0.922

47.1% (41) 35.0% (14)

0.200

168

Table 6

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

Results of logistic regression analysis examining the effect of having an onsite wastewater system/private well on serological responses to Cryptosporidium antigens

15/17-kDa marker

27-kDa marker

Geographic area

Variables in model

Odds ratio

95% CI

Odds ratio

95% CI

River Valley

Onsite wastewater system/private well

1.34

0.74–2.44

1.98

1.11–3.55

0.53

0.29–0.97

1.28

0.74–2.21

2.11

1.10–4.03

2.83

1.24–6.49

Handled livestock

Alluvial Fan

Handled a child with diapers

2.24

Age: 18–39 yrs

1.00

1.07–4.69

40–49 yrs

1.95

0.70–5.37

50–59 yrs

1.68

0.61–4.60

60þ yrs

4.00

1.29–9.00

Other race/ethnicity than NHW

1.92

1.01–3.66

Onsite wastewater system/private well

0.64

0.35–1.17

Child attends daycare

0.34

0.09–1.35

Plumbing work done in home

2.26

1.16–4.39

Handled pets (cats, dogs)

East Mountains

Age: 18–39 yrs

1.00

40–49 yrs

1.05

0.35–3.17

0.89

0.40–2.01

50–59 yrs

1.85

0.64–5.30

1.80

0.80–4.03

60þ yrs

7.31

2.60–20.58

4.20

1.79–9.89

College graduate

0.49

0.25–0.94

0.37

0.19–0.72

Onsite wastewater system/private well

1.07

0.62–1.84

0.85

0.49–1.46

Handled pets (cats, dogs)

0.23

0.08–0.68

Age: 18–39 yrs

1.00

40–49 yrs

1.76

0.74–4.18

50–59 yrs

1.92

0.83–4.43

60þ yrs

3.69

1.61–8.46

elevated adjusted odds ratios if they were 60 years of age or

models for the Alluvial Fan site for the 27-kDa marker

older (OR ¼ 7.31; 95% CI ¼ 2.60–20.58); adjusted odds

included both variables, but they both dropped out of the

ratios were signiﬁcantly reduced for participants who were

ﬁnal model. For the East Mountains site, the variable for

college graduates (OR ¼ 0.49; 95% CI ¼ 0.25–0.94). In par-

diarrhea was included in the initial model for the 15/17-

ticipants from the East Mountains, the odds ratio was

kDa marker and the variable for gastrointestinal illness

signiﬁcantly reduced if they had handled pets (cats, dogs)

was included in the initial model for the 27-kDa marker.

(OR ¼ 0.23; 95% CI ¼ 0.08–0.68).

Again, both dropped out of the ﬁnal analysis.

Neither of the two illness variables (diarrhea and gastrointestinal illness) was included in the ﬁnal models for the two serological markers. For River Valley participants,

DISCUSSION

they were not included in the model because they did not reach the required cutoff level in the univariate analyses of

According to the American Water Works Association

their association with the outcome variables. The initial

Research Foundation (AWWA) an American family of four

169

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

uses 400 gallons of water each day (AWWA ). Approxi-

suspected of causing illness. Based on outbreak data,

mately 70% of that is used indoors, 26.7% of which is used

groundwater contamination is a very common mode of

by the toilet. Approximately one-ﬁfth of US homes use septic

Cryptosporidium transmission (Craun & Calderon ).

systems (US EPA ). The ability of a viable pathogen to sur-

We believe that a low probability of outbreak detection

vive from septic tank discharge to entering the groundwater

should be expected, especially for populations using private

depends on a number of factors, including pathogen-speciﬁc

drinking water wells. Small outbreaks and outbreaks associ-

survival time in the soil. On the soil surface, elevated tempera-

ated with private wells are not likely to be recognized and

tures signiﬁcantly reduce oocyst survival (Jenkins et al. ).

investigated (Lee et al. ) and once protective immunity

However, if released below surface levels, such as in a septic

is developed in the small number of exposed people, the out-

tank drain ﬁeld, where the oocysts experience reduced temp-

break will not continue. However, visitors to the home who

eratures, Cryptosporidium oocysts are believed to survive

have not developed protective immunity may be at risk for

60–180 days after leaving the septic tank (Salvato ).

both infection and illness. It would be important to consider

Detecting parasites in the water is both difﬁcult and expensive. Furthermore, it is subject to very wide conﬁdence

monitoring performance of onsite wastewater systems as a public health measure at the local or state level.

intervals. Previous studies have suggested that groundwater

The absence of detectable disease outbreaks does not

contamination may be a route for diarrheal diseases

indicate that having both an onsite wastewater system and

from bacteria, viruses, and parasites (Barwick et al. ;

private well can be a health problem. Even in large cities,

Craun & Calderon ; Frost et al. ; Lee et al. ;

small to moderate sized outbreaks can remain undetected.

Craun et al. ; Liang et al. ; Yoder et al. ). How-

Furthermore, long-term residents of rural areas who drink

ever, these studies have only focused on detectable

contaminated groundwater are likely to develop a protective

outbreaks and have not examined infection risks from resi-

immunity after years of exposure to pathogens in their drink-

dential onsite wastewater systems combined with private

ing water. In our study, two measures of past illness were

wells in the absence of an outbreak. In this study, instead

collected. There was no signiﬁcant association between

of detecting parasites in drinking water, we examined

either of these two measures and a strong response for both

human sera for evidence of prior infection. A signiﬁcant

serological markers, suggesting that the participants in the

association was observed in the River Valley participants

study had developed a protective immunity. However, new

between having onsite wastewater systems/private wells

residents and individuals who develop auto-immune diseases

and having a strong response to the 27-kDa Cryptospori-

may be the ones at greatest risk of illness. The magnitude of

dium antigen. The odds ratio was increased approximately

the illness risk depends upon the size of the susceptible popu-

two-fold

variables.

lation. Thus, if a large fraction of residents in a geographic

Although this association was only observed in one of the

after

controlling

for

confounding

area have developed protective immunity, it is unlikely that

three study sites, this is one of the ﬁrst studies to suggest

a detectable disease cluster will be identiﬁed.

a relationship between having onsite wastewater systems/

It is difﬁcult to detect viruses in either infected humans

private wells and increased serological response to Cryptos-

or in drinking water near the time of an outbreak. Disease

poridium antigens. Together with a previous study in Iowa

surveillance systems are relatively insensitive to detecting

(Frost et al. ), these two studies provide suggestive evi-

mild cases of gastroenteritis. We do not know how many

dence that inadequately treated sewage from onsite

waterborne outbreaks go unrecognized and the extent to

wastewater systems may have contaminated private wells

which viral outbreaks may be underestimated in the USA

with Cryptosporidium oocysts and infected users of private

(Frost et al. ).

well water. This is not unexpected since there are no data

Coliphage testing is one analysis recommended by the

available to show onsite wastewater systems are capable of

US EPA as an indicator of groundwater contamination by

inactivating Cryptosporidium oocysts.

viruses. Unfortunately, there have been few studies actually

Unfortunately, few studies have determined the occur-

linking coliphage occurrence with increased risk of virus

rence of parasites in at-risk groundwater that is not

infection or even water contamination. The ﬁndings of this

170

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

Journal of Water and Health

12.1

2014

study are not encouraging since only one sample tested posi-

The study may also be subject to selection bias since a

tive for coliphage when using an experienced laboratory

convenience sample of volunteers was recruited into the

with state-of-the-art techniques.

study. Volunteers often differ from the sampling population

This study also found a high percentage of wells with

in underlying characteristics related to the exposure and out-

positive tests for total coliforms, fecal coliforms, or entero-

come. Although we recruited most study participants by ‘cold

cocci in all three sites. Overall, a total of 26% (94 of 361)

calling’ lists of telephone numbers in the study areas, rather

of wells tested had indicators of bacterial contamination,

than from ﬂyers and newspaper advertisements, the possi-

with the highest level being in the East Mountains site, an

bility of a biased sample still exists. For example, potential

area with limestone soils. Evidence of fecal coliforms was

participants who were worried about the quality of their

also found in 7.7% of these. This elevated prevalence of indi-

well water or who wanted their water tested because they

cators of contamination has been observed elsewhere

thought it might be contaminated might have been more

(Tuthill et al. ; Swistock & Sharpe ; Locas et al.

likely to volunteer for this study. It is not clear how this

). It is of interest to note that no association was

may affect the estimates of the odds ratio. Recall bias may

observed between well contamination and a strong response

be an issue since past history of illness and risk factors for

to either serological marker for Cryptosporidium.

cryptosporidiosis were collected using a questionnaire. How-

The results of this study may be limited since the three geographic sites were all located in the arid Southwest

ever, this type of information is usually easily remembered and identiﬁed.

where rainfall is concentrated during the monsoon season in the summer. However, each site was selected to represent a different geologic stratum (river valley, alluvial fan, and

CONCLUSIONS

limestone formations). Information on soil tests that were completed for approval of the onsite wastewater systems

This study, together with one prior study, suggests that the

by the county was also not available from the county regulat-

presence of onsite wastewater systems and private wells

ory agencies.

might be associated with an increased risk of Cryptospori-

The cross-sectional design of the study was not able to

dium infection. If a relatively large parasite can be

take into account the temporal association between

transmitted from inadequately treated sewage from an

exposure and outcome. Information on type of wastewater

onsite wastewater system to a private well, then other smal-

treatment system, source of water, and bacterial and viral

ler enteric pathogens may also be transmitted. Borchardt

contaminants in the water was collected at the same time

et al. () observed a relationship between the density of

as the blood samples. Thus, it was not possible to track

onsite septic systems and the risk of viral and bacterial diar-

the participants’ serological responses forward in time.

rhea in children residing in an area that primarily uses septic

However, the two Cryptosporidium antigens are indicative

systems for disposal of fecal waste. However, they did not

of past exposure history, rather than current history. The

ﬁnd this same relationship with respect to Cryptosporidium

15/17-kDa marker remains elevated for a relatively short

infections. Continuing surveillance of waterborne disease

period of time (several months), whereas the 27-kDa

outbreaks in the USA has also shown that outbreaks of gas-

marker remains elevated for 9 months or longer. The fact

trointestinal illnesses are associated with failed onsite

that we observed a signiﬁcantly increased odds ratio for

wastewater systems (Craun & Calderon ). Thus, there

the 27-kDa marker may be a reﬂection of a chronic, long-

is some evidence to suggest that there may be a potential

term exposure to contaminated well water. Our information

for increasing risk of enteric disease illness as more house-

on well water contamination was also based on one point-in-

holds in the USA use onsite wastewater systems and

time sample. Sequential sampling of 60 wells has shown that

private wells and the density of these systems increases.

seasonal contamination occurs frequently (unpublished

Since the economic cost of installing an onsite waste-

data). The use of the long-term elevated 27-kDa marker is

water system is a relatively high one-time expense, it may

appropriate for this situation.

be beneﬁcial to better understand the efﬁcacy of these

171

K. Tollestrup et al.

Cryptosporidium infection and onsite wastewater systems

systems to insure that the systems are properly designed and maintained to minimize adverse health effects from groundwater contamination. This is an emerging public health problem that requires additional studies to identify the environmental, behavioral, and health factors associated with increasing the risk of enteric illnesses from onsite wastewater systems and private wells.

ACKNOWLEDGMENTS We thank R. Gelting and M. Herring for their assistance as technical advisers on this project, M. A. Zarate for his extensive assistance with the manuscript, and D. Vigil for her assistance with coordinating the data collection and participant interviews. This study was funded by a grant from the Association for Prevention Teaching and Research under a cooperative agreement with the Centers for Disease Control and Prevention.

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First received 24 February 2013; accepted in revised form 14 August 2013. Available online 8 October 2013

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The challenge of global water access monitoring: evaluating straight-line distance versus self-reported travel time among rural households in Mozambique Jeff C. Ho, Kory C. Russel and Jennifer Davis

ABSTRACT Support is growing for the incorporation of fetching time and/or distance considerations in the definition of access to improved water supply used for global monitoring. Current efforts typically rely on self-reported distance and/or travel time data that have been shown to be unreliable. To date, however, there has been no head-to-head comparison of such indicators with other possible distance/time metrics. This study provides such a comparison. We examine the association between both straight-line distance and self-reported one-way travel time with measured route distances to water sources for 1,103 households in Nampula province, Mozambique. We find straight-line, or Euclidean, distance to be a good proxy for route distance (R 2 ¼ 0.98), while self-reported travel time is a poor proxy (R 2 ¼ 0.12). We also apply a variety of time- and distance-based indicators proposed in the literature to our sample data, finding that the share of households classified as having versus lacking access would differ by more than 70 percentage points depending on the particular indicator

Jeff C. Ho Kory C. Russel Jennifer Davis (corresponding author) Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega, Stanford, CA 94305, USA E-mail: jennadavis@stanford.edu Jennifer Davis Woods Institute for the Environment, Stanford University, 473 Via Ortega, Stanford, CA 94305, USA

employed. This work highlights the importance of the ongoing debate regarding valid, reliable, and feasible strategies for monitoring progress in the provision of improved water supply services. Key words

| Africa, GIS, monitoring, Mozambique, water access, water fetching

INTRODUCTION As of 2012, almost half of the world’s population must still

example, Pickering and Davis ﬁnd that a 15-minute decrease

leave their home to fetch water (WHO/UNICEF ).

in one-way walk time to water source is associated with a

Roughly three-quarters of those fetching water outside the

41% reduction in diarrhea prevalence, improved child nutri-

home are classiﬁed as having access to improved water

tional status, and an 11% reduction in under-ﬁve child

supply as per the deﬁnition used by the United Nations–

mortality. Other research has documented the effect of

World Health Organization’s Joint Monitoring Program

lengthy walks to water sources on the risk of gender-based

(JMP) (WHO/UNICEF ). As many scholars and prac-

violence (Shah ; Kirchner ; Ivens ; Sorenson

titioners have noted, however, this deﬁnition considers

et al. ; Thompson et al. ), as well as of injury from

only whether the household reports using an improved

physical stress to joints and from accidents (Ivens ;

source (WHO/UNICEF ), and is not dependent on the

Sorenson et al. ). Taken together, these ﬁndings suggest

distance between the household and the source, nor on

that incorporating time and/or distance considerations in

the time costs of water fetching for household members

the deﬁnition of access to improved water would better

(Hunter et al. ; Clasen ; WSMP Ghana ).

reﬂect the public health goals of the sector.

Recent research indicates that the time spent walking to

Whereas it has been acknowledged that incorporating

water is signiﬁcantly associated with health outcomes

distance or time to water source in the JMP’s metric

(Wang & Hunter ; Pickering & Davis ). For

would be desirable (WHO/UNICEF ; UNDG ;

doi: 10.2166/wh.2013.042

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Evaluating indicators of water fetching distance

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Hunter et al. ), feasible ﬁeld measurement capability of

distance refers to the straight-line distance measured

water fetching distances has been limited. The most accurate

between two points. In this work, we investigate the applica-

measures would require in situ ﬁeld measurement, with an

bility of straight-line distance between a household and its

enumerator either measuring household-to-source distances

water source for measuring access to water supply

manually (White et al. ) or accompanying each respon-

(Figure 1).

dent on the walk to his/her water source (Pickering et al.

Studies evaluating the efﬁcacy of straight-line distance

). These approaches are costly to apply on a large

are not uncommon. Apparicio et al. () ﬁnd that

scale. Much more commonly, researchers ask household

straight-line distance is strongly correlated with distance as

members to estimate the travel time (WHO & UNICEF

calculated based on a road network. Their ﬁndings are simi-

; ICF International ) or the distance (Demeke

lar to those of Fortney et al. (), who ﬁnd that Euclidean

) to their water source during an in-person survey.

distance explains more than 90% of travel time to health

These strategies have documented biases and limitations

providers in the United States. However, most studies eval-

(Nyong & Kanaroglou ; Buor ).

uating straight-line distance have been conducted in the

More speciﬁcally, self-reported measures of time often

context of health care access rather than access to water

suffer from recall bias (Shiffman ). Biasing processes

supply; they have also been focused in urban areas with

are

well-developed road networks. To our knowledge, there

memory is distorted by interpretation processes inﬂuenced

prominent

during

self-reported

recollections,

has been no study of the effectiveness of straight-line dis-

by the respondent’s current state (Bradburn et al. ;

tance as a proxy for distance to water source in rural areas

Gorin & Stone ). Kahneman et al. () argue that

lacking road networks.

this bias is based on the availability of instances in

This is not to say that straight-line distance is not impor-

memory when one is asked to recall speciﬁc events. A

tant for water supply planning; indeed, it is regularly used in

respondent who is asked to estimate water fetching travel

siting water points. For example, it is common to draw a

time, for example, may more easily recall instances in

circle around a planned water point location and use

which he/she explicitly took note of the travel time; these might be trips that took longer than normal, potentially biasing his/her responses. The key finding by Niemi () that the accuracy of survey measurements depends critically on factors of memory supports this assertion. We are aware of only one other published study that assesses the accuracy of self-reported water fetching time. Using global positioning system (GPS) route data to measure fetching time, Davis et al. () find that households in informal settlements in Kenya frequently overestimated their fetching time, despite spending less than 10 min total and traveling less than 400 meters on average. Together, this evidence suggests that using self-reported indicators of water fetching distance is not optimal. In other fields, distance has been estimated ex situ with a geographic information system (GIS), rather than being measured in situ or estimated with household survey data. For example, GIS-measured estimates such as Euclidean distance and shortest-route distance have been used in identifying populations with poor access to health services (Love & Lindquist ; Bamford et al. ). Euclidean

Figure 1

Straight-line and route distances for households in one sample village.

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straight-line distance to ensure that intended beneficiary households lie within the distance as specified by sector policy (e.g., 500 meters or 1 kilometer). In addition, straight-line distance was once used to monitor access to improved water supply. Surveyors would simply count the number of households that lay within the specified distance of a water point and classify them as having access. Such practices were criticized for over-estimating access, however, because they assumed that all households located near the water point made use of it. Direct interview of household members about which water source(s) they use has become the preferred method of data collection for global monitoring of access. This type of interview data is generally viewed as being valid and reliable with regard to household water source choice and water use. As discussed above, however, selfreported information about distance or travel time to a

Figure 2

water source is likely to suffer from various types of bias.

Study area in Nampula province, Mozambique. Black dots represent communities. Inset map shows Nampula’s location relative to Mozambique and Africa as a whole.

Recognizing that support is growing for the inclusion of such a distance or time measure in the deﬁnition of water access in the post-2015 period, this study explores the feasi-

Data collection

bility of using straight-line distance for global access This study uses data collected in June 2011 for a larger inves-

monitoring.

tigation into the impacts of rural and peri-urban water investments. A total of 54 clusters of households in six dis-

METHODOLOGY

tricts

were

selected

random;

1,606

households

(approximately 27 to 30 households per cluster) were surData used in this study were collected from households in

veyed in all. Enumerators followed randomly determined

rural and peri-urban communities in the province of Nam-

transects within each of the 54 communities and selected

pula, Mozambique (Figure 2). Nampula’s terrain is

every second or third household to be interviewed. Respon-

homogenous throughout the province, with 99% of the pro-

dents were male (61%) or female (39%) heads of household.

vince classiﬁed as sandy topsoil (Food and Agricultural

Informed consent was obtained from all participants, and

Organization ). This homogeneity in soil type is corro-

the study protocol was approved by the Institutional

borated by household responses describing the terrain on

Review Board of Stanford University, California, USA and

their water fetching route: 76% of households indicate that

the National Statistical Institute of Mozambique.

their route’s terrain is sandy. The slope along fetching

Each respondent was asked to identify the primary

routes also does not vary much, with a median slope in

water source used by his/her household at the time of

study areas of 2.9 as calculated from a digital elevation

survey, and enumerators took GPS coordinates of each

model (ASTER GDEM ). A study by Finley & Cody

sampled household and all shared water points in each

study community. Among sampled households, 503 (31%)

induce no change in walking pace. For this reason, and

reported using a primary water source other than a commu-

also partly due to difﬁculty in characterizing steepness of

nity water point, and/or a source for which GPS coordinates

slope along water fetching routes, we do not consider

were not obtained. Given the objectives of this study, these

slope in our analysis.

households were removed from the dataset, leaving 1,103

() of urban pedestrians found that slopes up to 4

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Evaluating indicators of water fetching distance

Journal of Water and Health

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households. In addition, in order to identify the paths used

Straight-line and route distances for one of the sample

to reach water sources in each community, enumerators col-

communities are shown in Figure 1.

lected GPS track data as they were escorted to each water point by local leaders. These GPS track data did not include

Analytical methods

all paths in each community; they therefore provide an incomplete picture of the true paths available for water

We compare the water route estimations from satellite path

fetching in these communities.

data to straight-line distances using ordinary least squares

As a result of the incomplete GPS track data, we esti-

(OLS) regression. We then use the residual value between

mated water fetching distance using paths digitized from

the two distance measures as the dependent variable in a

freely available, high-resolution satellite imagery. Satellite

second OLS model designed to identify the factors associ-

imagery has previously been used in estimating water fetch-

ated

with

comparatively

good

(or

poor)

predictive

ing routes (Davis et al. ). As of March 2012, Google

accuracy. Using the same methods (OLS regression plus

Earth provided 2.5 m resolution SPOT satellite imagery

modeling of residuals), we compare self-reported one-way

for all of the sites in our study area and sub-meter resol-

travel time with route time estimates calculated from route

ution DigitalGlobe or GeoEye imagery for 24 (44%) of

distance. We estimate route time from route distance using

our sites (Google Inc. ). The dirt paths and roads in

a typical walking pace as reported for the region in pub-

these communities were visible via satellite imagery, so

lished literature. A conversion factor of 62.5 m/min was

these paths were digitized and then used to determine

used as the typical walking pace for the region, calculated

and measure the shortest route from each household to

from the average of 41.5 m/min (2.5 km/h) (Tanser et al.

its water source. For each satellite image used to digitize

) and 83.3 m/min (5 km/h) (Calvo ). As before,

paths for a community, we recorded the landscape type

we model the residuals (i.e., absolute value of the difference

(e.g., completely forested) and the image resolution (i.e.,

between self-reported time and estimated travel time) using

high (sub-meter) or low (2.5 m)). Twenty-eight percent of

multiple OLS regression to evaluate the factors associated

sites were completely forested, 17% partially forested,

with better (or worse) prediction.

40% with sparse shrubs and 15% were peri-urban. Once

For both models, we expect the accuracy of the indicator

digitized, the paths were assessed for accuracy relative to

to depend on the robustness of the route estimation method

the travel paths captured by enumerators with GPS track

vis-à-vis variations in satellite image landscape and resol-

data. Over all study communities, the digitized paths over-

ution. We therefore include both parameters in each

lapped with 82% of the total distance within the GPS track

model to distinguish between the performance of the indi-

data. The majority of the uncaptured paths were not critical

cators and systematic errors in the route estimation

to the analysis: for example, they included the path

method. The distance model also includes straight-line dis-

between the community entrance and the local leader’s

tance as an independent variable to test whether the

house, which was often unrelated to paths between house-

prediction accuracy varies with indicator magnitude.

holds and water points.

The time model includes household and respondent

We used a least-cost path method in ArcGIS 10 to esti-

characteristics that we hypothesize may inﬂuence the accuracy

mate the route from each household to its reported

of self-reported travel time, including respondent education,

primary water point (ESRI Inc., Redlands, CA, USA). We

age, and gender, household wealth, and cell phone ownership.

created a 30-meter resolution cost raster in which path

We also include three indicators related to water fetching prac-

cells were assigned a low cost and other cells were assigned

tice: the share of total fetching time spent queuing (as opposed

an arbitrarily high cost. This approach ensured that the

to walking), whether fetching is typically undertaken alone or

identiﬁed least-cost route would extend from the household

with a companion, and the number of person-trips for water

to the closest digitized path, and then along that path to the

fetching taken each week by the respondent. Route slope and

water point. Tanser et al. () employ a similar method to

terrain may also contribute to self-reported travel time accu-

model travel time to health clinics in rural South Africa.

racy, but as noted above, the study communities are

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Evaluating indicators of water fetching distance

characterized by limited variation in slope and terrain. We have

Journal of Water and Health

12.1

2014

RESULTS

thus omitted these indicators from all models. We include respondent education, age, and gender, as

Among the 1,103 households included in our ﬁnal sample, the

well as household wealth, to test whether socioeconomic

mean number of people in the household was 4.1 (median of

factors inﬂuence the prediction ability of respondent time

4.0). Twenty-ﬁve percent of households reported owning

estimates. An array of literature suggests that such factors

both a bike and a radio while only 6% of households reported

are associated with the accuracy of recall (Ferber ;

owning a cell phone. The average respondent age was 40, and

Butler et al. ; Mathiowetz & Duncan ; Niemi

58% of respondents reported having completed at least some

; Beckett et al. ). We included a simple educational

formal schooling. Sample households use an average of 23.2

attainment dummy variable that takes the value of 1 if the

liters of water per capita per day (median of 19.9). The 577

respondent completed any formal schooling and 0 other-

respondents who fetched water for their households took an

wise. We measured wealth similarly with a dummy

average of 13 trips per week; almost half (45%) of the total

variable that takes the value of 1 if the respondent’s house-

time spent water fetching was spent queuing, as opposed to

hold owns both a bicycle and a radio, and 0 otherwise.

walking to and from the source. Of the respondents,

We include cell phone ownership in the model to test the

332 (58%) reported fetching water alone versus with others.

hypothesis that the clocks on mobile phones may make

Forty-one percent of sample households reported using more

respondents more aware of the time spent on daily activities.

than one domestic water source at the time of interview, an

We also hypothesize that increased share of fetching time

average of 1.4 sources per household overall. For 64% of

spent queuing (as opposed to walking) decreases the respon-

households, the primary source (from which the greatest

dent’s prediction ability, owing to respondents potentially

share of water was obtained) was a shallow hand-dug well

conﬂating long queue time with long walk time when recal-

(poço traditional); 22% used a deep (mechanically drilled)

ling fetching experiences. Finally, we include in our time

borewell ( furo), and 14% fetched water from a river or lake.

model the presence of a companion while fetching, expect-

Using satellite imagery and the distance estimation

ing that respondents who fetch alone may be better able to

methods described in the section ‘Data collection’, the aver-

estimate their one-way travel time. A study by Niemi ()

age one-way water fetching route distance for sample

reported that activities that are clearly distinct from others

households was 925 m (standard deviation, SD ¼ 988 m)

(i.e., with no overlap in purpose) produce more accurate

and the average straight-line distance was 726 m (SD ¼

results in time surveys. Thus, if water fetching is combined

759 m) (Table 1). The average self-reported one-way travel

with socializing, we might expect survey responses regard-

time from the survey was 48.5 min (SD ¼ 53.2 min). In con-

ing the time spent on water fetching to be less accurate.

trast, the average one-way route time as calculated from

Table 1

Summary statistics for distance and time metrics and model-dependent variables

Mean

Standard deviation

Median

Range

925

988

656

1.5–7,200

Route distance from satellite path data (m)

Straight-line distance (m)

726

759

506

1.5–5,500

One-way route time from route distance using literature conversion (62.5 m/min) (min)

14.8

15.8

10.4

0–116

Self-reported one-way travel time (min)

48.5

53.2

1–540

Self-reported queue time (min)

81.4

120

0–1,440

Distance model: A minus B (m)

202

275

115

6.3–2,600

Time model: Absolute value of D minus C (min)

25.8

40.1

14.8

0–450

Model-dependent variables

178

J. C. Ho et al.

Evaluating indicators of water fetching distance

Journal of Water and Health

12.1

2014

route distance (using the conversion factor based on typical

distance itself is also signiﬁcant; each 100 m increase is

walking rates in the literature) was 14.8 min (SD ¼ 15.8 min).

associated with a 29 m worsening in prediction on average.

Straight-line versus self-reported measures

None of the socioeconomic indicators (respondent edu-

The results of the time model are shown in Table 3. cation, gender, age, or household wealth) was found to As shown in Figure 3(a), straight-line distance performed

have a statistically signiﬁcant effect. Share of fetching time

very well as a predictor of route distance overall (R ¼

spent queuing (queue time share) is signiﬁcant (p < 0.01);

0.98). However, the slope of the regression line implies that

however, contrary to expectations a 10% increase in queue

straight-line distance under-predicts route distance by an

time share is associated with a 5 min improvement in predic-

average of 23%, which translates to a distance of 202 m

tion by respondents on average. In other words, if two

(SD ¼ 275 m). By contrast, self-reported one-way travel time

respondents each spend a total of 60 min fetching water,

is a poor predictor of route distance among sample house-

but one respondent spends 30 min queuing and the other

holds (Figure 3(b), R 2 ¼ 0.12). No systematic over- or under-

spends 36 min queuing, the respondent who spends more

estimation is observed in these data.

time queuing is 5 min better in his/her walk time prediction,

all else held constant. Three-quarters of sample households Models of indicator accuracy

spend between 4 and 85% of their total fetching time queuing, so this effect size represents a fairly signiﬁcant

Table 2 presents the results of a multiple linear regression

improvement in prediction among households in our data-

model of the difference between the two distance measures.

set. The direction of effect was the opposite of what we

Both variables used to test the robustness of the route esti-

expected, however.

mation method are signiﬁcant in the model. Using a high

The number of person-trips made by the respondent to

resolution satellite image (sub-meter versus 2.5 meter)

his/her water source each week is also signiﬁcant in the

when digitizing paths for route estimation is associated

time model (p < 0.01), with each additional trip associated

with a 77 m improvement in prediction on average

with a 45 s improvement in prediction on average. Three-

(p < 0.01). Having a landscape covered in sparse shrubbery

quarters of household respondents report between 6 and

(as compared to a peri-urban landscape) is similarly associ-

21 person-trips per week, so this effect size represents a

ated with a 59 m improvement (p < 0.01), while a

fairly small improvement in prediction over the range of

completely forested landscape is associated with a 51 m

observed values. We also ﬁnd no evidence that owning

worsening in average prediction (p < 0.01). Straight-line

a cell phone allows water fetchers to produce better

Figure 3

Ordinary least squares regressions of route distance on straight-line distance (a) and self-reported one-way walk time (b).

179

Table 2

J. C. Ho et al.

Evaluating indicators of water fetching distance

Journal of Water and Health

Ordinary least squares regression of difference (in meters) between straight-line and route distance

12.1

2014

while fetching. Finally, we ﬁnd no evidence that solitary water fetchers provide better estimates as compared to

Std. error

p-value

Intercept

37.8

15.8

0.02

Straight-line distance (100 m)

28.6

0.71

<0.01

76.6

11.4

<0.01

Completely forested landscapeb

50.6

18.0

<0.01

Partially forested landscapeb

20.3

17.8

0.26

59.5

17.3

<0.01

Sparse shrubbery landscape

We re-estimated this time model using an average walking pace for survey respondents (7.7 m/min versus 62.5 m/min from the literature) and observed no substantive change in

Satellite image characteristics High resolution (dummy)a

those who regularly fetch with one or more companions.

our results.

DISCUSSION

Adjusted R ¼ 0.65; Number of observations ¼ 1,088

Our results indicate that, for this sample of households,

Represents the effect of high resolution satellite images (e.g., GeoEye, DigitalGlobe) used

during path digitization versus using low resolution satellite images (e.g., SPOT). All landscape parameters relative to images with peri-urban landscape.

straight-line distance serves as a reasonable proxy for route distance. Whereas there is some discrepancy between straight-line and route distance, the signiﬁcant effect of satel-

Table 3

Ordinary least squares regression of difference (in minutes) between self-

lite image resolution in the distance model indicates that

reported one-way travel time and travel time estimated from route distance

systematic error during route estimation, rather than poor

using literature-based conversion factor (n ¼ 520)

predictive ability, may be the principal explanation. The Std.

error

value

60.8

11.2

<0.01

0.24

0.18

0.19

Share of fetching time spent queuing (%)

0.51

0.05

<0.01

Respondent dry season trips per week

0.73

0.25

<0.01

Fetched water alone (dummy)

4.60

3.33

0.17

Some formal education (dummy)

0.81

3.40

0.81

Gender (dummy; 1 ¼ male)

2.60

straight-line distance could be a useful proxy for water fetch-

3.54

0.46

0.63

ing distance. This result is consistent with ﬁndings from

Bike and radio ownership (dummy)

3.77

0.87

0.05

several other studies comparing straight-line and route dis-

Age (years)

0.12

0.69

tance to health care facilities (Phibbs & Luft ; Fortney

0.86

7.28

0.91

et al. ; Apparicio et al. ).

5.05

3.57

0.16

mates the actual distance that sample households travel to

3.90

5.77

0.50

fetch water by 23% on average. In addition, straight-line dis-

Partially forested landscape

1.83

5.90

0.76

Sparse shrubs landscapeb

tance does not account for variations in land cover (Noor

1.76

5.61

0.76

Intercept Route distance (100 m)

Household and respondent characteristics

Cell phone ownership (dummy)

Completely forested landscapeb b

accuracy model is less clear. For example, completely forested landscape could create more winding water fetching routes and therefore worse alignment between straightline and route distance, but it could also contribute to increased error in path digitization and less accurate route estimation. Nonetheless, the high correlation of straight-line distance with route distance (R 2 ¼ 0.98) implies that overall

Satellite image characteristics High resolutiona

interpretation of landscape type signiﬁcance in the distance

That said, we ﬁnd that straight-line distance underesti-

Adjusted R2 ¼ 0.22; Number of observations ¼ 520 a

Represents the effect of high resolution satellite images (e.g., GeoEye, DigitalGlobe) used during path digitization versus using low resolution satellite images (e.g., SPOT).

All landscape parameters relative to images with peri-urban landscape.

et al. ); nor does it capture the physical challenges associated with water fetching in mountainous regions (Perry & Gesler ) or areas with uneven or steep hillsides (Sorenson et al. ). Among sample households with the greatest discrepancy between straight-line and route dis-

self-reported estimates of travel time. This result should be

tance, many traverse winding paths through heavily

interpreted with caution, however, given the small share of

forested landscape. In the most extreme case, these factors

sample households with phones (6%) and the fact that we

add up to a two-kilometer discrepancy between straight-

do not know which respondents actually carry a phone

line and route distance. Finally, use of satellite images to

180

J. C. Ho et al.

Evaluating indicators of water fetching distance

Journal of Water and Health

12.1

2014

determine straight-line distance requires that each house-

possible explanation could be that a long queue creates a

hold’s water source(s) are known and visible in the imagery.

‘break’ in the fetching trip that allows a respondent to

By comparison, self-reported travel time is poorly corre-

better distinguish walk time from wait time. This result

lated with route distance (R 2 ¼ 0.12, Figure 3(b)), bringing

would imply that self-reported travel times may be more

into question the validity of this widely used indicator. It is

accurate for water points with substantial queue times, all

important to note that this conclusion is dependent on the

else held constant.

homogeneity of terrain found in the study area (and the

The number of water fetching trips that a respondent

resulting assignment of a single walking pace to all house-

makes per week was also found to be signiﬁcant in the

holds). In other settings, variations in terrain could lead to

time model, albeit with small effect size, consistent with

differences in the time required for water fetching for house-

our hypothesis that increased familiarity with the route

holds located at equivalent distances from their source. At

might increase the accuracy of self-reported fetching time.

the same time, our results are consistent with those of

Although not tested with our data, it may be that this

Davis et al. (), who found a similar discrepancy between

effect is greater in communities whose water source is in a

self-reported water collection times and GPS-measured trip

central location that individuals may pass by even when

times in Kenyan informal settlements.

not fetching water. Finally, whether or not the respondent

We tested two potential explanations for why self-

usually fetches water alone was not statistically signiﬁcant

reports may be poor in this work. The ﬁrst relates to selec-

in the time model, although the direction of effect indicates

tive memory bias as described above (Kahneman et al.

that respondents who collect water alone have predictions

). If this explanation were correct, we would expect

that are an average of 4.6 min more accurate as compared

socioeconomic factors such as age, education, and wealth

with those who collect water with others.

to be signiﬁcantly associated with the accuracy of self-

This ﬁnding raises another limitation of self-reported

reported travel time in our multivariate model, as suggested

fetching time, namely that water fetchers may derive some

by a large literature (Ferber ; Butler et al. ; Mathio-

beneﬁt (such as opportunities to socialize) from the activity

wetz & Duncan ; Niemi ; Beckett et al. ). We

that also increases the total time spent fetching (Short &

ﬁnd, however, that self-reported travel time is a poor predic-

Thompson ; Devoto et al. ). Disaggregating the

tor of route time for sample households regardless of

time burden of water fetching into these components for

socioeconomic and demographic characteristics, although

the purpose of global monitoring of access would seem

limited variation in these attributes could be the cause of

quite challenging. Within our sample, 58% of respondents

this result. The second possible explanation we explored

reported that they typically fetch water with one or more

was the presence of time-keeping devices in the household;

companions. These individuals reported an average one-

however, we observe no association between household cell

way walk time to their water source that is 5 min longer as

phone ownership (the most commonly used time-keeping

compared to those who fetch alone, holding straight-line dis-

device in the study area) and improved time prediction. In

tance to source constant. Whether this 5-minute increment

sum, respondent and household characteristics provided

should be considered part of the total time burden of fetching

little insight regarding the reasons that self-reported travel

is a question that, to our knowledge, has not been explicitly

time so poorly predicts route time among sample households.

considered within the global monitoring framework to date.

Features of the water fetching activity itself were more

One major limitation of this work is that it assumes

helpful in explaining variation in respondents’ ability to esti-

water carriers travel the most direct path to their source,

mate their travel time. For example, we found that the

which may not always be true. Water fetchers face many

accuracy of a respondent’s estimate improves as the share

hazards that may lead them to take indirect routes to collect

of total fetching time spent queuing increases. This result

water, including danger from road trafﬁc (Sorenson et al.

was counter to our expectations, as we hypothesized that

), abuse from wealthy landowners (Shah ), or the

respondents with a high share of queue time would be

risk of assault or animal attack (Ivens ; Sorenson

more likely to conﬂate queue time with travel time. One

et al. ). We are unsure to what extent this limits the

181

Table 4

J. C. Ho et al.

Evaluating indicators of water fetching distance

Journal of Water and Health

12.1

2014

Percentage of sample households classiﬁed as having access to water supply using alternative indicators

Access criterion Indicator Water fetching travel time <30 min

Household within 1 km of water source

Straight-line distance

79, (n ¼ 1,103)

Route distance

68, (n ¼ 1,103)

Self-reported time

Including queue time a

Without queue time

Per trip

7, (n ¼ 1,386)

31, (n ¼ 1,525)

Per day

4, (n ¼ 988)

20, (n ¼ 836)

Estimated time using literature walking pace

Including queue time a

Without queue time

Per trip

17, (n ¼ 1,011)

65, (n ¼ 1,103)

Per day

10, (n ¼ 535)

42, (n ¼ 577)

Number of households included in each indicator calculation shown in parentheses. a

All queue times are self-reported.

effectiveness of straight-line distance as an indicator for

In sum, despite general consensus that any notion of

water access, and note it as a topic for future study. In

‘access’ to water supply should incorporate some measure

addition, we limited our analysis to each household’s pri-

of associated time cost and/or physical burden, many ques-

mary water source at the time of interview, whereas

tions remain regarding what speciﬁcally should be the focus

households may use multiple water sources and/or change

of measurement, as well as the best indicators to employ for

their water source at different times of the year.

regular monitoring. Among our sample of rural Mozambi-

This work was motivated by the desire of many in the

can households, a higher percentage would be classiﬁed as

sector to incorporate a time and/or distance indicator into

having access with a straight-line distance-based indicator,

the global monitoring of access to improved water supply.

and a lower percentage with a self-reported time indicator,

Among our sample households, assuming that all other cri-

both as compared to an indicator based on route distance

teria were satisﬁed, the choice of a particular time/

from the household to its water point. Given that even

distance criterion would have substantial impact on the per-

these relative ﬁndings are unlikely to hold across different

centage classiﬁed as having versus lacking access (Table 4).

geographies and contexts, it seems important for future

For example, using a distance threshold of 1 km (WHO/

work to evaluate the conditions under which different indi-

UNICEF ) and a straight-line distance measure for

cators perform comparatively better or worse. More

each household, 79% of the sample would be deemed to

generally, this work highlights the importance of the

have access. This percentage falls to 68% if route distance

ongoing debate about valid, reliable, and feasible strategies

is used. By comparison, using a threshold of 30 min round-

for monitoring progress in the provision of improved water

trip travel time (WHO/UNICEF ; Pond & Pedley

supply services.

), between 31 and 65% of households would be classiﬁed as having access (depending on whether self-reported or estimated travel time were used). Applying the 30-minute

ACKNOWLEDGEMENTS

threshold to total fetching time, i.e., walk time and queue time, would reduce these values to 7 and 17%, respectively.

This study made use of data from a project funded by the

Finally, using a per-day (rather than per-trip) time threshold

Millennium

of 30 min, between 20 and 42% of sample households would

Evaluation Unit, Department of Policy and Evaluation.

be classiﬁed as having access if only walk time were con-

Graduate funding for Jeff C. Ho was provided by the

sidered, compared with 4 to 10% if total fetching time

Natural Sciences and Engineering Research Council of

(including queuing) were considered.

Canada. We thank two anonymous reviewers, as well as

Challenge

Corporation,

Monitoring

and

182

J. C. Ho et al.

Evaluating indicators of water fetching distance

members of the Davis research group and participants in the Stanford symposium on Water, Health & Development, for useful feedback on earlier versions of this paper. Finally, we extend a sincere thanks to the members of our enumerator team and to the households who participated in this study.

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UNDG  Indicators for Monitoring the Millennium Development Goals: Deﬁnitions, Rationale, Concepts and Sources. United Nations Publications, New York. Wang, X. & Hunter, P. R.  Short report: a systematic review and meta-analysis of the association between self-reported diarrheal disease and distance from home to water source. Am. J. Trop. Med. Hygiene 83 (3), 582–584. White, G. F., Bradley, D. J. & White, A. U.  Drawers of Water; Domestic Water Use in East Africa. University of Chicago Press, Chicago, IL. WHO & UNICEF  Core Questions on Drinking-Water and Sanitation for Household Surveys. World Health Organization, Geneva, Switzerland. WHO/UNICEF  Global Water Supply and Sanitation Assessment 2000 Report. World Health Organization, Geneva, Switzerland. WHO/UNICEF  A Snapshot of Drinking Water and Sanitation in Africa: A Regional Perspective based on new data from the WHO/UNICEF Joint Monitoring Programme for Water Supply and Sanitation. Cairo, Egypt. WHO/UNICEF  Progress on Drinking Water and Sanitation: 2012 Update. World Health Organization, Geneva, Switzerland. WSMP Ghana  The Time Factor in Ghana’s Coverage Figures for Drinking Water. Available at: http://www.wsmp.org/ downloads/4d4bd2b1c35b2.pdf (retrieved March 18, 2012).

First received 26 February 2013; accepted in revised form 15 August 2013. Available online 24 September 2013

184

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Of water and worms: Guinea worm re-emergence in Niger Nathaniel Royal

ABSTRACT This paper aims to provide a better understanding of the re-emergence of Guinea worm into the water bodies of the Tillabéri region of the Niger Sahel. It examines the period of re-emergence and subsequent decline from 2002 until 2006. Using a geographic information system combined with a statistical approach to examine the location data of lakes with Guinea worm-associated cases, it is shown that the

Nathaniel Royal Emerging Pathogens Institute, 3141 Turlington Hall, Gainesville, FL 32611-7315, USA E-mail: nroyal01@yahoo.com

locations of population centers with cases of Guinea worm disease, the locations of lakes infected with Guinea worm larva, and features of the built environment are correlated in space. Key words

| Africa, geographic information systems, Guinea worm

INTRODUCTION Dracunculus medinesis, or Guinea worm as it is more com-

This study tests the hypothesis that the distribution of

monly known, is a two-host nematode parasite affecting

features in the built environment relative to the distribution

humans who drink water contaminated with Guinea worm

of seasonal lakes impacts the spread of Guinea worm.

larvae and is notable for its dramatic clinical manifestations.

Guinea worm cases are known to be more common near

The number of cases of Guinea worm disease (dracunculia-

infected sources of water (Royal ). As those drinking con-

sis) has been in rapid decline since the early 1980s, when

taminated water are more likely to be local residents, this is

the Dracunculiasis Eradication Program (DEP) was formed

unsurprising; however, it is necessary to deﬁne precisely

and organized a coordinated worldwide eradication cam-

who drinks this water and where they live, not because

paign (Cairncross et al. ). As the number of reported

some areas of the lake are more infected with Guinea

Guinea worm cases continues to decrease worldwide, the

worm larvae than others, but because people living near

remaining regions in which it persists are those with the

infected lakes may have particularly at-risk behaviors.

few remaining water bodies on the planet that are home to

The Carter Center, a major player in the Guinea worm

Guinea worm larvae. Since Guinea worm-infected individ-

eradication community, collects location data for Guinea

uals are the primary vector for transporting Guinea worm

worm-infected individuals in order to coordinate various

from water body to water body, the extent of Guinea worm

monitoring and eradication programs. However, precise

spread is determined by the travel behavior of the few at-

geographic information regarding the bodies of water used

risk populations that live near Guinea worm-infected lakes.

by the infected individuals is not always recorded. Although,

Much to the frustration of the DEP and the populations still

in the past, bodies of water suspected of containing the para-

living in regions infested with Guinea worm, there have

site have been treated with larvicides, this method of

been several resurgences of the parasite in the past decade:

combating the spread of Guinea worm is not preferred.

Chad, Mali, Niger, Ghana, and Ethiopia have all seen the

Temaphos, the chemical most commonly used to combat

total number of cases countrywide increase at some point

Guinea worm, is an organophospate larvacide used to

during the past ten years (Callahan et al. ). If there is to

treat water infested with disease-carrying insects. Larvacides

be a resurgence of the parasite before full eradication has

of this type kill the water ﬂea (Cyclops) which serves as a

been achieved in other regions, it will inevitably stem from

host for Guinea worm larvae, effectively halting the para-

the human use of the few remaining infected water bodies.

sites reproductive cycle. Larviciding has been controversial

doi: 10.2166/wh.2013.053

185

N. Royal

Of water and worms

Journal of Water and Health

12.1

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in that, in addition to killing the Guinea worm larvae, it kills

course of the year, making them difﬁcult to map (Nicholson

other larval life and there are fears it may negatively affect

et al. ). While the ﬁll levels of these bodies of water may

those drinking the water. Additionally the lack of available

vary according to the local precipitation rates, they are also

safe drink water infrastructure causes the need for action

affected by ﬂash ﬂooding. Many bodies of water are part of

such as larvaciding, which is no more than a temporary sol-

seasonal river systems that may ﬂood hundreds of miles

ution. Eradication through larvacides does not protect

downstream when rainfall is sufﬁcient in other areas of

individuals who choose to travel beyond the watershed of

the watershed. Additionally, the desertiﬁcation of much of

a lake that has been treated with larvacides. Thus, efforts

the southern Sahel has caused changes in watershed

have focused on teaching individuals how to contain the

dynamics (Leblanc et al. ). This changing environment

spread of Guinea worm and how to avoid becoming hosts

is difﬁcult to monitor, although recent efforts to collect

themselves (Rwakimari et al. ), rather than focusing

data on water use and groundwater recovery have been

on larvaciding the water supplies.

undertaken by several countries through the actions of the

The vast majority of remaining Guinea worm cases occur in regions that endure some form of water stress

United Nations Educational, Scientiﬁc and Cultural Organization’s (UNESCO) International Hydrological Program.

during the annual cycles of rain (Lebel et al. ). Rainfall in these regions, primarily in the Sahel of sub-Saharan Africa, is seasonal; the annual pattern begins with several

STUDY AREA

dry months with little or no precipitation, followed by a rainy season that lasts from three to five months. These Sahelian regions often have a limited water infrastructure, forcing inhabitants to be reliant on the local supplies of water (Giordano ). Drinking water is often drawn from unclean surface water sources, such as ponds, streams, lakes, and sometimes even from watering holes serving livestock. Water clarity is often poor, making a visual inspection for water fleas difficult. As an alternative to surface water,

The study area for this research comprises the majority of the Tillabéri arrondissement (borough) of northeastern Niger where there was a brief resurgence of Guinea worm cases in 2003 (Table 1). The area is centered on the city of Tillabéri, the capital city of the Tillabéri arrondissement and the focus of the study is on Tillabéri city and the surrounding region. This region is at an intersection of Saharan trade networks and is at the northernmost extent

underground water is sometimes either drawn from wells

of Sahelian agriculture in eastern Niger (Figure 1). The

or, less commonly, pumped to the surface using electric or

population of approximately 200,000 in the region is divided

hand-powered water pumps. While the subsurface water

into three tribal groups: the Zarma (Djerma), who tradition-

sources are free from Guinea worm larvae, there are not

ally work as farmers or merchants, and the Fulani and the

enough existing wells and pumps to support the inhabitants

Bella- or Tomacezk-speaking people, who are nomadic

of the Sahel. Additionally, migrations and agricultural activi-

and semi-nomadic pastoralists. While Zarma is the domi-

ties regularly draw the at-risk populations away from the

nant language of the region, each tribe speaks its own

cleaner water sources (Cairncross et al. ).

dialect as well.

Detailed information on bodies of water that are capable of supporting the water ﬂea and Guinea worm larvae is com-

Even for Niger, the Tillabéri study region is very dry. Precipitation primarily occurs in June, July, and August,

plicated by the seasonality of the precipitation in Sahelian regions. Throughout most of the Sahel evapotranspiration rates of the lakes are quite high due to high temperatures, low humidity, and sparse vegetation. Rainfall often occurs in great deluges rather than moderate, intermittent rains; ﬂooding of the lowlands is an annual threat. Lakes and rivers, in some cases several kilometers long or wide, may ﬂood seasonally and then dry up completely during the

Table 1

Guinea worm cases by year

Year

2002

2003

2004

2005

2006

Cases in study region

175

152

165

Cases in Niger

293

233

183

110

54,638

32,193

15,912

10,674

25,217

Cases worldwide

186

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Journal of Water and Health

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in the study site. Roads are differentiated from trails as roads are classiﬁed as vehicle-serving transportation routes. While similar to trails in that they are often unpaved, roads have been reinforced with compacted stones to prevent vehicles from sinking into the often-sandy terrain. Only one road in the region is paved: the route connecting Tillabéri to Niamey to the south and Mali to the north. With the onset of the rainy season, pastoralists communities are obliged to journey to more remote pasture land to avoid interfering with crop cultivation. While many herding migrations will stay within or near the outskirts of a town, some will undertake much longer journeys. In the Tillabéri region of Niger, Fulani- and Tomaczek-speaking herders Figure 1

will move animals north into Mali, using a series of seasonal Extent of the study region.

lakes as watering stops on the trip (Figure 2). Population centers and farmed ﬁelds are avoided during these

averaging between 400 and 1,200 mm annually. Water is

migrations by traveling on a network of paths through

scarce during the dry months. Agricultural activities occur

unfarmed land. The number of livestock on this northward

predominantly during the rainy season, when the landscape

procession may vary from year to year, contingent on the

changes from an arid, rocky, and sandy wasteland to green

rainfall and ﬁll of each lake, as well as the rainfall in the pas-

ﬁelds of millet, sorghum, and beans. Seasonal lakes, which

ture areas in southeastern Mali, near the Niger border.

can grow to several kilometers long during the rainy

Desertiﬁcation and insufﬁcient precipitation in the Mali pas-

season, often dry up entirely during the hottest months of

turelands impedes cultivation of the local grain staples of

the year when the temperatures can top 50 C. The study

millet or sorghum (Mohamed ). However, there is

area contains 32 seasonal lakes, each of which is rep-

enough rainfall suitable for grasses, enough ground water

resented by a single point; known as ‘vanish points’, these

to keep the arriving herds of animals hydrated, and the

points were determined by the Nigerien Ministry of Hydrol-

lands are free from grazing limitations and land-use conﬂicts

ogy to be the lowest and ﬁnal points of water for each lake

with farmers. After three to four months of grazing the ani-

before it evaporates completely. There are no permanent

mals in relative isolation in the north, the pastoralists return

lakes within the study region. The water bodies these

with their herds after harvest to feed the animals on the crop

points represent are the largest surface water bodies in the

gleanings in the ﬁelds in their hometowns in Niger.

study region and were ﬁlled during every year of the study.

The locations of nomadic routes were documented

These vanish points are important for this study as they rep-

during ﬁeld research between January and June 2010.

resent the locus of congregation for people using the last of

With help from local pastoralists the 281 km of nomadic

the lake water. The concentrations of Guinea worm are at

trails were mapped. The coordinates of medical facilities

their highest immediately preceding the complete evaporation

were also identiﬁed through ﬁeld research. Medical facilities

of a body of water; thus, Guinea worm is most likely to be

in the region were of three different types: medical stations

spread during a critical period of water stress (Adekolu ).

(six), medical centers (ﬁve), and hospitals (one). Medical

Details regarding the location, type, and depth of each

stations were staffed by a single nurse and functioned as

well in the area were made available for this study by the

pharmacies and clinics. Diagnosis, provision of medicine

Ministry of Hydrology, so as to enable a complete assess-

and injections, assistance for births, and the pronouncement

ment of the available water resources. Trail and road

of death are the services provided by the nurse and staff at a

datasets were donated by the Africa Data Dissemination

medical station. Contacting the regional hospital and for-

Service, detailing 524 km of roads and 1,598 km of trails

warding ill patients to more complete medical facilities via

187

Figure 2

N. Royal

Of water and worms

Journal of Water and Health

12.1

2014

Herding paths used between the northern Tillabéri region of Niger and southeastern Mali.

ambulance is also a function of the medical station. Most

to assess their effect on the incidence of Guinea worm in

medical stations have no electricity. Medical centers are

the study area. Each feature was selected because of its

similar to medical stations except they are larger, have

role in propelling people toward Guinea worm-infected

more extensively trained nurses, have electricity, other

regions or because of its role in educating or providing

staff to aid the nurses, a vehicle, and a radio tower to com-

alternatives to the utilization of contaminated water.

municate with other medical facilities and hospitals. The

The Carter Foundation had identiﬁed Guinea worm

region had only one hospital in the capital city of Tillabéri.

cases and the locations of where those infected were

The hospital was fully equipped, and was staffed by a variety

living for the study region for the period from January

of doctors, specialists, and others. Guinea worm eradication

2002 to December 2006. This dataset was made available

efforts were managed out of the Tillabéri hospital, but all

to this study and it was placed in a geographic information

medical facilities and staff in the region were part of the era-

system along with data pertaining to the locations of fea-

dication effort.

tures of the built environment. Through interviews with health agents and previously infected individuals, it was determined which lake was presumed to infect each

METHODS

infected individual during the study period. Twenty-two of the 33 lakes in the region had no cases of Guinea

Since very few demographic statistics are available for the

worm associated with them while 11 lakes had associated

Tillabéri region, analysis of the effects the built environment

cases of Guinea worm.

has on disease spread is the next best solution. Health facili-

As each lake serves a certain population, the risk of

ties, transportation networks, and water sources all

exposure to Guinea worm larvae varies within the study

contribute to the living conditions of those at risk of

region. In order to appropriately model the relationship

Guinea-worm infection. The locations of wells, roads,

between the incidence of Guinea worm in each lake and

trails, nomadic routes, and health facilities were examined

distances to features of the environment, it is important

188

N. Royal

Of water and worms

Journal of Water and Health

12.1

2014

not only to understand how many cases of Guinea worm

Thiessen polygons centered at the location of the lakes’

are associated with each lake, but also how many people

vanish points using the study site borders as a bounding

the Guinea worm larvae in each lake could have poten-

box. The log of the area associated with each lake was

tially infected. Ideally, exposure to the Guinea worm

used rather than the actual square kilometer area to nor-

larvae in each lake could be measured by the sum of the

malize the great size differentials between the areas each

number of trips taken to each lake by the population the

lake serves (Figure 3).

lake serves over a speciﬁed time period in combination

One suitable method to analyze the relationship

with the numbers of infected Cyclops within each water-

between the locations of Guinea worm-infected water

body. However, this data is unavailable and gathering

supplies and the built environment relies on using a zero-

this data during ﬁeld work was deemed unfeasible. Instead,

altered negative binomial model, otherwise known as a

the log of the area of each lake was included in the statisti-

hurdle model with a negative binomial second stage.

cal models used in this analysis as an offset to serve as an

Hurdle models are a type of discrete mixture models used

indirect measure of exposure. This log-area method

for count data when there is an excess of zeros in the

assumes that the population density in the service area is

data. When a dependent variable contains more zeros

uniform. While this is certainly not the case, it is the pre-

than would be expected for a negative binomial distribution,

ferred method of measuring exposure for this study

these excess zeros are better managed through the use of a

region, as the population here is widely and relatively

two-model approach; if these excess zeros are not included

evenly dispersed across the terrain and engaged in agricul-

in the model, the parameter estimates and standard errors

tural work during the start of the dry season, when the risk

may be biased. The hurdle model used in this analysis exam-

of contracting Guinea worm is highest (Périès et al. ).

ines the proximate relationship between lakes with

The area associated with each lake was determined using

associated cases of Guinea worm disease, as well as the

Figure 3

Areas associated with each lake within the study region.

189

N. Royal

Of water and worms

lakes’ proximate relationship to the built environment. f zero altered negative binomial ðy; β, γ Þ 8 f binomialðy ¼ 0; γ Þ y¼0 > > < f negative binomial (y; β) ¼ ð1 f binomialðy ¼ 0; γ ÞÞ × > 1 negtive binomial ðy ¼ 0; β Þ > : y>0

Journal of Water and Health

12.1

2014

the probability of achieving a zero value; once accomplished,

the

second

stage

examines

the

dependent

variables without zero values, thus dividing the process of analysis into two specialized examinations. Any hurdle model has two stages to its analysis as it is effectively a two-model approach. The hurdle model with a negative binomial second stage used in this analysis is

where y ¼ counts of Guinea worm, β ¼ the distances from

one of two types of statistical model referred to as hurdle

vanish points to built environment features, and y ¼

models. While the ﬁrst stage of any hurdle model consists

regression parameters (features of the built environment

of a binomial generalized linear model, the second stage

used in model) (Zuur et al. ).

can be either a negative binomial model or a Poisson

The hurdle model was chosen to analyze the relation-

model. In terms of this study, a binomial generalized

ship between the associated cases of Guinea worm in each

linear model is utilized to analyze the location differences

lake and the proximity to features of the environment over

between lakes with and without Guinea worm; and either

several possible models. Considering that the over-dis-

a negative binomial or Poisson generalized linear model

persion in the data is due not to the large differences in

can be utilized to analyze the location differences between

the numbers of associated cases of Guinea worm per lake,

lakes with different counts of Guinea worm-associated

but rather to the large number of lakes without associated

cases (Figure 4). Analyzing each version of the hurdle

cases, the hurdle model is more suitable to this study’s analy-

model, including the log-area offset to account for exposure,

sis than ordinary linear or negative binomial models

the negative binomial version of the hurdle model is the pre-

(Greene ). The ﬁrst stage of a hurdle model estimates

ferred model for this analysis according to the results of an

Figure 4

Cases of Guinea worm associated with each lake within the study region.

190

N. Royal

Of water and worms

Akaike information criterion (AIC) test, which is a measure of the relative goodness of ﬁt for statistical models. A model’s AIC score is calculated using the following equation: AIC ¼ 2k 2Ln(L)

Journal of Water and Health

Table 2

12.1

2014

AIC tests comparing a hurdle model with negative binomial second stage and hurdle model with a Poisson second stage

Model type

Degrees of freedom

AIC score

207.320

145.072

581.050

Zero altered Poisson Hurdle model Zero altered negative Binomial hurdle model

where k is the number of parameters in the model and L is

Log-rate model

the maximized value of the likelihood function for the estimated model (Akaike ). The preferred model for this analysis is one with a low

binomial second stage performs similarly to the hurdle

AIC, as the lower the AIC, the less information will be lost

model with a Poisson second stage as well as the Poisson

when describing the relationship between number of associ-

model (Figure 5).

ated cases per lake and the surrounding environment. The

Poisson models can struggle to handle ancillary zero

AIC measures the balance between model ﬁt and parsi-

counts, just as negative binomial models do if the ancillary

mony. When two models with the same likelihood value

zero values are too numerous (Greene ). In the case of

(ﬁt) but with different numbers of parameter estimates are

this analysis, the ancillary zero counts represent the lakes

examined, a model selection decision using AIC will select

without associated cases of Guinea worm. As the proportion

the model with fewer parameters.

of lakes without associated cases of Guinea worm is two-

A Poisson model offers an alternative to the hurdle

thirds the total number of lakes within the study region, it

model for examining this dataset. The formula for a Poisson

is probable that a Poisson model would be more suitable

model with the aforementioned log-area offset is as follows:

for an analysis examining other regions where there were fewer lakes without associated cases of Guinea worm.

logðμi Þ ¼ log(Ai ) þ Xi β

For each model, lakes with larger residual values have higher predicted case counts, with the Poisson model ﬁtting

where the offset, log(Ai) is the log of service area of the lake,

slightly higher case counts to lakes with higher residual

μ is the numbers of associated cases, and β is the distance

values than either of the two hurdle models. However, as

between lakes and the environmental feature.

the AIC shows that the hurdle model with a negative bino-

As there is a Poisson distribution of the case count data,

mial second stage describes the relationship between the

a Poisson model is included in the AIC test in order to vali-

dependent and independent variables with the least loss of

date the use of a two-stage model.

information, the test conﬁrms it is the preferred model for this analysis. In the ﬁrst stage of either hurdle model (the binomial

RESULTS

generalized linear model), the lakes are separated into those with associated cases of Guinea worm and those with-

In comparing the two variants of the hurdle models (nega-

out. The number of cases of Guinea worm associated with

tive binomial and Poisson), the negative binomial variant

each infected lake is not examined; only the presence or

has an AIC score of 145 and the Poisson hurdle variant

absence of cases is noted. Lakes without Guinea worm are

has an AIC score of 207 (Table 2). A Poisson model with

examined to identify what it is about their location and

the log-area offset, the next most suitable regression model

their proximity to features of the built environment that dis-

for this analysis, underperforms both hurdle models with

tinguishes them from lakes with associated cases of Guinea

an AIC of 581. Examining a scatter plot of the ﬁtted

worm (Table 3). The distinction between an infected lake

values estimated for each model against the model’s

and an uninfected lake is examined in terms of distance

residuals shows that the hurdle model with a negative

(in kilometers) from the vanishing point of a lake to a

191

N. Royal

Figure 5

Table 3

Of water and worms

Journal of Water and Health

12.1

2014

Fitted values and residuals for each of the tested models.

Distance from seasonal lakes to environmental features (in kilometers)

Lake

No. of Guinea

worm cases

265

4.81

2.94

9.86

4.24

14.89

105

1.63

6.31

4.74

13.13

9.37

4.54

5.65

3.41

1.17

1.64

10.41

3.58

12.21

2.41

14.7

11.65

15.12

1.06

0.24

10.41

2.71

17.31

9.92

4.57

5.064

15.02

5.62

12.10

0.95

0.99

Wells

Nomadic

Health

Trails

Roads

built-environment feature (Figure 6). In comparing the positioning of the lakes in relation to the proximate built environment, each lakeâ&#x20AC;&#x2122;s spatial distribution within the built environment can be examined in terms of its relative isolation or nearness to features that are presumed inďŹ&#x201A;uential to the spread of Guinea worm. The second stage of a

Figure 6

Example of measuring the distances from lake vanish points to features of the built environment.

192

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Of water and worms

Journal of Water and Health

hurdle model reveals the differences within lakes with cases

12.1

2014

DISCUSSION

of Guinea worm; lakes without Guinea worm are excluded. Each infected lake’s relationship to the built environment is

Examining the locations of all the health facilities in the geo-

once again examined, this time comparing the lakes to each

graphic information system reveals that the four health

other in terms of the counts of associated Guinea worm

facilities closest to lakes were at the very center of two of

cases, rather than simply noting the presence or absence

the three most infected regions. Each of these four facilities

of Guinea worm.

is a medical station. The health facilities in these locations

The results of a hurdle model with a negative binomial

were also close to the vanishing points of the lakes, located

second stage suggest that there is a signiﬁcant correlation

on the banks of the seasonal lakes during the parts of the

between health facilities and lakes with Guinea worm and

year they were completely ﬁlled. Each of these medical

between lakes with Guinea worm and the location of trails

stations was close to a moderately sized (<1,250 persons)

(Table 4). In the analysis of lakes with and without Guinea

population center. It is worth noting that health facilities

worm, there is a negative correlation between health facili-

were not always located within the borders of large popu-

ties near a lake and the incidence of Guinea worm;

lation centers; in fact some were several kilometers from

suggesting the closer a health facility is to a lake, the

any population center. Many of the other health facilities,

higher the likelihood of Guinea worm being present in the

particularly in regions without Guinea worm, were not posi-

lake. In the second stage of the hurdle model (the negative

tioned near lakes and were located on higher terrain and

binomial count model), the health facility is again negatively

comparatively far from lakes. This neither proves the useful-

correlated, suggesting that the more cases of Guinea worm

ness of the model nor refutes its ﬁndings, although it does

associated with a lake, the nearer the health facility is to

suggest that a comparative analysis at a larger scale—with

the lake.

more health facilities in a larger area—would be insightful.

Table 4

The model results also reveal a relationship that exists Hurdle model results

between the location of trails and whether or not a lake will have associated cases of Guinea worm. Given the

Stage 1: Hurdle model (binomial with log link) Estimate

Std. Error

Z value

Pr(>|z|)

results of the model it is suggested that the farther a trail is

1.9796

0.6631

2.985

0.0028

from a lake, the more likely the lake is to have at least one

Health

0.3210

0.0768

4.180

<0.0001

associated case of Guinea worm. Conversely, infected

Wells

0.0135

0.0585

0.230

0.8177

lakes near trails also tended to have more associated cases

Trails

0.4602

0.2075

2.210

0.0266

than infected lakes that were distant. The effects of other

Roads

0.0364

0.0340

0.911

0.3623

transportation networks on the spread of Guinea worm

Nomadic

0.0378

0.0258

1.469

0.14185

and on the lakes are unclear. There is little evidence of cor-

(Intercept)

relation between wells and Guinea worm-infected lakes, Stage 2: Count model (truncated negative binomial with log link) Estimate

Std. Error

z value

Pr(>|z|)

although given the large number of the wells in the study region it could be that their statistical effects are unclear

0.043

since they are proximate to most features of the natural

2.630

0.008

and built environment.

0.0722

0.337

0.736

Human travel behavior is a critical factor for deter-

0.4354

0.2584

1.685

0.092

mining the boundary of the spread of Guinea worm

Roads

0.0922

0.0754

1.224

0.221

(Royal ). The destination choices of humans traveling

Nomadic

0.0383

0.0462

0.829

0.407

through the Tillabéri region determine the locations to

Log(theta)

0.27

0.64

0.43

0.670

which

(Intercept)

3.9263

1.9372

0.2652

0.1008

Wells

0.0243

Trails

Health

2.0270

Theta count ¼ 1.5258; log-likelihood: 59.54 on 13 degrees of freedom. Person residuals: Min: 1.4801; 1Q: 0.5777; median: 0.3972; 3Q: 0.4983; max: 2.7665.

Guinea

worm

spreads.

Local

transportation

choices appear to be just as important in determining the vulnerability to Guinea worm as more distant

193

N. Royal

Of water and worms

Journal of Water and Health

12.1

2014

locations. Whereas the disease must ﬁrst arrive in a

no data on when these medical facilities began to receive

region of re-emergence from a distant location, its

patients could be uncovered, and further ﬁeld work would

spread locally appears to result from travelers moving

be required to fully eliminate the possibility of endogeneity

along local trails. Trails passing by lakes with Guinea

in the model used for this analysis.

worm logically increase the lake’s exposure and, in turn,

Another possible relationship between the locations of

provide a community of hosts for the parasite. Those tra-

health facilities in relation to Guinea worm-infected lakes

veling by trail often travel by foot or pack animal and a

is that there is a correlation between the residential location

readily available water source may have even inﬂuenced

of infected people and their proximity to health facilities.

the trails location, as travelers through the desert often

This analysis assumes that all cases in the Tillabéri region

want for drinking water. Lakes isolated from all transpor-

are accounted for; there is little evidence to suggest that

tation networks are those which travelers are less likely to

the Guinea worm monitoring efforts have been anything

frequent. Given the farming practices in the region, it is

less than extraordinarily well organized and determined,

likely that these lakes are in remote areas and only used

although the possibility of incomplete reporting of Guinea

by hunters or by farmers at their zigi. (The zigi is the

worm cases certainly exists. Migrant populations, who are

‘far ﬁeld’, that is a ﬁeld of crops in another locale to

often absent in Niger during some seasons of the year, are

the farmer’s usual habitation.) Many of those individuals

difﬁcult to monitor and account for in the annual Guinea

who embark on exode (seasonal migration) will return

worm census. When migrant Guinea worm-infected individ-

to their homes in time to farm. Future work may beneﬁt

uals return to Niger, their proximity to health facilities is

from further investigation into whether or not lakes in

almost certainly a factor in their willingness to report them-

remote areas far from transportation networks are becom-

selves to the eradication community. Further information as

ing infected by individuals returning from exode or other

to the numbers of persons traveling in and out of the study

long-distance travels.

region as well as the numbers of people who use each

Relationships between health facilities and regions with high incidence of Guinea worm are of great importance to

health facility would allow a further understanding of this possibility.

eradication efforts. Regional health facilities monitor and

Finally, persons traveling to or from health facilities may

treat infected people, and their cooperation with health ofﬁ-

infect or become infected by the local water sources on the

cials and eradication workers has been invaluable. Finding

way. Just as health centers are the nexus for medical treat-

correlation between the locations of health centers and in

ment they may also serve as a catalyst for its spread. Other

the location of cases is clearly troubling as it supports

factors that localize trafﬁc into certain regions such as

three possible explanations as to why such a relationship

schools, jobs, religious centers, festivals, etc. would be

exists. The least contentious of these possible relationships

useful to fully understand why people travel where in the

is that health facilities were placed in regions where there

region and how these health facilities affect travel. Each

were high incidences of Guinea worm. Older maps (circa

health facility has its own administrative boundary for the

1960) of the Tillabéri region show health facilities in only

communities it serves, and Niger law is such that it

four locations in the study region, whereas in the past 50

encourages citizens to visit only their regional medical facil-

years there has been a threefold increase in their number.

ity. Any prevalent communicable disease in a town with a

Regions with Guinea worm cases are often remote and

health facility puts a traveler visiting the health facility for

deprived of basic infrastructure—a condition that would

another illness at risk of becoming infected by a local epi-

increase the relative incidence of many diseases, not just

demic. This forced the regionalized health care to become

Guinea worm. Thus, health facilities may have been built

institutionalized through the decentralization laws that

in these regions to combat poor health conditions, which

were put in place during Mamadou Tandja’s (1999–2010)

would result in endogeneity in the models. If the starting

presidential administration. They were instituted to keep

dates of operation for each of these medical facilities exist,

communicable diseases more locally contained. Quarantin-

further analysis may support this possibility. Unfortunately,

ing may also raise infection rates; having a single

194

N. Royal

Of water and worms

Journal of Water and Health

12.1

2014

destination for health care creates a highly contagious

locally traveling individuals put themselves and others at

environment. Most carriers do not know they have Guinea

risk of Guinea worm. Understanding the health needs of

worm until the parasite is at the oviposition stage and

these high-risk individuals and their behavior may provide

ready to expunge its eggs. Recovering or treated individuals

the key to ﬁnally eradicating the parasite.

leaving a health facility also risk contact with Guinea worm on the way home to other parts of the region. They are more likely to be unfamiliar with which water sources in the area are free of Guinea worm, therefore having a greater risk of

ACKNOWLEDGEMENTS

contracting the parasite. Funding for this research was provided by a Fulbright Fellowship. The research was carried out in cooperation

CONCLUSIONS

the Niger Ministry of Science, the Ministry of Hydrology, and the Ministry of Statistics. Further thanks to the Carter

While the methods of this model suffer from small sample

Center and the Ministry of Health in Niger for the use of

size, in that there are so few water bodies in the region suit-

their data.

able for Guinea worm, the evidence suggests that the relationship between health facilities and Guinea worm infection is not ideal for the model of eradication currently employed. The assumption that all cases of Guinea worm are being recorded and found is either incorrect, or health facilities (or some undiscovered spatial correlate to them) may be affecting the spread of Guinea worm and/or are ineffective in reducing the numbers of proximate cases. Both issues are clearly problematic for eradication efforts; solutions that change either the way Guinea worm cases are identiﬁed or how health facilities prevent local outbreaks of the parasite are needed. It is important to remember that Guinea worm is not merely a Sahalien disease, or one conﬁned to a limited geographic region—in the past Guinea worm was found all through North Africa, the Middle East, and Southern Asia. Guinea worm disease still has the potential to be an emerging pathogen, despite its currently limited numbers. Further research on the exact ways in which health facilities impact the spread of Guinea worm would be the next logical step. It may be that as health facilities are normally situated in larger towns and Guinea worm cases are clustering around health facilities, urban populations without clean water sources may be more at risk for Guinea worm in the late stages of eradication efforts. The destination and route choice involved in travel behaviors, once understood, can help manage and mitigate their effects in encouraging the spread of Guinea worm. This model offers some insight into which behaviors of

REFERENCES Adekolu-John, E.  The impact of lake creation on guineaworm transmission in Nigeria on the Eastern side of Kainji Lake. Int. J. Parasitol. 13, 427–432. Akaike, H.  Factor analysis and AIC. Psychometrika 52, 317–332. Cairncross, S., Muller, R. & Zagaria, N.  Dracunculiasis (Guinea worm disease) and the eradication initiative. Clin. Microbiol. Rev. 15, 223–246. Callahan, K., Bolton, B., Hopkins, D. R., Ruiz-Tiben, E., Withers, P. C. & Meagley, K.  Contributions of the Guinea Worm disease eradication campaign toward achievement of the millennium development goals. PLoS Negl. Trop. Dis. 7, e2160. Giordano, M.  Agricultural groundwater use and rural livelihoods in sub-Saharan Africa: a first-cut assessment. Hydrogeol. J. 14, 310–318. Greene, W. H.  Limdep User’s Manual. Bellport, New York. Lebel, T., Sauvageot, H., Hoepffner, M., Desbois, M., Guillot, B. & Hubert, P.  Rainfall estimation in the Sahel: the EPSATNIGER experiment. Hydrolog. Sci. J. 37, 201–215. Leblanc, M. J., Favreau, G., Massuel, S., Tweed, S. O., Loireau, M. & Cappelaere, B.  Land clearance and hydrological change in the Sahel: SW Niger. Global Planet. Change 61, 135–150. Mohamed, E. S.  Spatial assessment of desertification in north Sinai using modified MEDLAUS model. Arab J Geosci 6, 4647–4659. Nicholson, S. E., Some, B. & Kone, B.  An analysis of recent rainfall conditions in West Africa, including the rainy seasons of the 1997 El Niño and the 1998 La Niña years. J. Climate 13, 2628–2640.

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Périès, H., Rooy, C. & Nwe, Y. Y.  Monitoring and evaluation of Guinea Worm eradication. Eval. Program Plann. 21, 393–408. Royal, N.  Dracunculiasis, proximity, and risk: analyzing the location of Guinea Worm disease in a GIS. Trans. GIS. 17, 298–312.

Journal of Water and Health

12.1

2014

Rwakimari, J. B., Hopkins, D. R. & Ruiz-Tiben, E.  Uganda’s successful Guinea worm eradication program. Am. J. Tropical Med. Hyg. 75, 3–8. Zuur, A. F., Ieno, E. N., Walker, N., Saveliev, A. A. & Smith, G. M.  Mixed Effects Models and Extensions in Ecology with R. Springer Press, New York.

First received 1 April 2013; accepted in revised form 4 September 2013. Available online 29 October 2013

196

12.1

2014

Community-based wastewater treatment systems and water quality of an Indonesian village H. S. Lim, L. Y. Lee and S. E. Bramono

ABSTRACT This paper examines the impact of community-based water treatment systems on water quality in a peri-urban village in Yogyakarta, Indonesia. Water samples were taken from the wastewater treatment plants (WWTPs), irrigation canals, paddy ﬁelds and wells during the dry and wet seasons. The samples were tested for biological and chemical oxygen demand, nutrients (ammonia, nitrate, total nitrogen and total phosphorus) and Escherichia coli. Water quality in this village is affected by the presence of active septic tanks, WWTP efﬂuent discharge, small-scale tempe industries and external sources. We found that the WWTPs remove oxygen-demanding wastes effectively but discharged nutrients, such as nitrate and ammonia, into irrigation canals. Irrigation canals had high levels of E. coli as well as oxygen-demanding wastes. Well samples had high E. coli, nitrate and total nitrogen levels. Rainfall tended to increase concentrations of biological and chemical oxygen demand and some nutrients. All our samples fell within the drinking water standards for nitrate but failed the international and Indonesian standards for E. coli. Water quality in this village can be improved by improving the WWTP treatment of nutrients, encouraging more villagers to be connected to WWTPs

H. S. Lim (corresponding author) Department of Geography, National University of Singapore, Kent Ridge, Singapore 117570 E-mail: hanshe_lim@hotmail.com L. Y. Lee NUS Environmental Research Institute, 5A Engineering Drive 1, Singapore 117411 S. E. Bramono Indonesian Society of Sanitary and Environmental Engineers, (Ikatan Ahli Teknik Penyehatandan Teknik Lingkungan Indonesia/IATPI), Apartemen Menteng Square, Tower A, ﬂoor 22 unit 2, Jl. Matraman Raya 30E, Jakarta Pusat, 10320, Indonesia

and controlling hotspot contamination areas in the village. Key words

| community-based wastewater treatment, water quality

INTRODUCTION The Millennium Development Goals from the 2000 UN

tanks, is discharged into surface waters (rivers and drains) or

General Assembly Millennium Meeting aim to halve the

allowed to seep into the ground with minimal treatment.

proportion of people without sustainable access to sani-

High levels of oxygen-demanding substances, nutrients and

tation and safe drinking water by 2015 (Cornel et al. ).

faecal indicator organisms in septic tank efﬂuent pollute

About 40% of the world’s population, mostly in developing

groundwater and surface waters. This results in widespread

countries, still lack basic sanitation and suffer the conse-

health problems related to malaria, cholera and other causes

quences of water pollution on human health (Massoud

of diarrhoea because groundwater is the main source of drink-

et al. ; Cornel et al. ). In cases where government

ing water (Roma & Jeffrey ). In response to this, the

efforts to provide sanitation have failed or are lacking, com-

Indonesian government started developing community-

munity-based projects have emerged, aided by non-

based wastewater treatment projects in the 1990s, funded by

governmental organisations (NGOs) or the private sector,

the government itself and external agencies such as the

to address these sanitation and water quality/human

United Nations Children’s Foundation, the Asian Develop-

health issues (Sansom ).

ment Bank, the World Bank, the Australian Government

Indonesia has one of the lowest rates of sewerage coverage

Overseas Aid Program and the Cooperative for American

in Asia, partly a result of government policy, which assigns

Remittances to Europe (CARE). Community participation

responsibility for sanitation to households (Foley et al. ).

was encouraged in all phases of these projects: design, con-

Untreated or partially treated sewage, mostly from septic

struction, operation and maintenance. At the same time,

doi: 10.2166/wh.2013.003

197

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

villagers who were not included in these schemes started peti-

). This study hopes to contribute to the growing aca-

tioning their local governments for assistance in setting up

demic literature on community-based sanitation efforts by

their own municipal wastewater treatment systems (Mukher-

focusing on the water quality aspects of such systems (treat-

jee & Josodipoero ; Foley et al. ; Lasut et al. ).

ment efﬁciencies and efﬂuent quality) in relation to their

This paper presents a case study of a typical Indonesian

environmental water quality using a typical Indonesian vil-

village, Sukunan Village, where village activism led to the

lage, which has a community-based sanitation system, as a

construction and maintenance of wastewater treatment sys-

case study. We examined the extent of water pollution in

tems in their village. The villagers were increasingly

the village and identiﬁed the source/s of pollution to see if

concerned about the health effects of water pollution arising

the presence of the WWTPs has a positive impact on

from inadequate wastewater treatment and farming activi-

environmental quality. The water quality parameters

ties associated with cattle and fertiliser usage. The villagers

chosen for study have an impact on human health (nutrients

petitioned the government for assistance in the construction

and Escherichia coli). Finally, we hope to suggest some prac-

of municipal wastewater treatment plants (WWTPs). Five

tical measures that can improve water quality in this village

such systems were built in the village in 2008 and have

as well as other Indonesian villages.

been in operation since then. Community-based efforts similar to those in Sukunan Village are found throughout Indonesia. There are quite a

STUDY SITE

number of reports detailing these initiatives and how they have led to the success or failure of the projects (Mukherjee

Sukunan Village (area 0.2 km2) is a small village located in

& Josodipoero ; Foley et al. ; Roma & Jeffrey ).

the eastern part of Yogyakarta in the peri-urban part of the

However, the literature about the performance and impact

Special District of Yogyakarta, Java, Indonesia (Figure 1).

of community-based sanitation systems on environmental

The village population is approximately 900.

quality is scarce and often only reports treatment efﬁciencies

The climate of this region is humid equatorial but

for solids and oxygen-demanding wastes (e.g. Foley et al.

approaching monsoonal equatorial conditions according to

Figure 1

Map of Sukunan Village showing the locations of the WWTPs and the surface and subsurface sampling points.

198

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

the Köppen-Geiger climate classiﬁcation system (Kottek

use septic tanks because they could not afford the cost of

et al. ). Annual rainfall is 1,900–2,000 mm (Smith

pipeline connection to the WWTPs. The municipal

et al. ). The dry season lasts from April/May to Septem-

WWTPs include four units using the contact aeration (CA)

ber. The rainy season is from October to March/April.

process and one unit using the rotating biological contactor

Details about the hydrogeological conditions of this vil-

(RBC) process (Figure 1). The CA process provides biologi-

lage are based on Smith et al. () who studied a village

cal contact ﬁlters for the microorganisms to break down

3 km south of our study site. The topography of the village

wastewater through aerobic degradation; it uses a surface

is ﬂat and the area is underlain by porous, sandy volcanic

aerator to introduce air as an oxidant to break down munici-

deposits. The groundwater system is part of the Merapi aqui-

pal wastewater. The RBC process uses a series of parallel

fer, one of the most productive aquifers in Central Java. The

discs mounted on a rotating shaft; microorganisms attached

aquifer is approximately 150 m thick with a shallow and a

to the disc surface provide sequential aerobic and anaerobic

deep layer. At Sukunan, depth of the water table is about

degradation of the wastewater as the disc is exposed to the

10 to 11 m, which probably coincides with the shallow

air and subsequently submerged in wastewater (Metcalf &

layer of the Merapi aquifer.

Eddy ). These WWTPs are maintained by a villager

Land use consists of unpaved roads, gardens and trees;

who regularly carries out checks and simple maintenance

domestic animals (e.g. ducks, chickens and cattle) are free

tasks such as cleaning and de-sludging. The efﬂuent from

to roam about the village. Paddy farming is the main econ-

all WWTPs is discharged into the irrigation canals which

omic activity. Paddy ﬁelds are supplied with water from a

feed the paddy ﬁelds.

nearby river through a network of irrigation canals running

Drinking water for the villagers comes from wells that

within and around the village (Figure 1). Both commercial

may be located in the house or outside. All wells in this vil-

and home-made fertilisers are used. Commercial fertilisers

lage are at least 10 m from a septic tank in accordance with

include urea while home-made fertilisers are made from

Indonesian law (Pokja AMPL ). Water is disinfected by

combining vegetable waste, cow faeces and sludge from

boiling but a few wealthier villagers disinfect their well

the municipal WWTPs. There are three to four small

water by chlorination (Smith et al. ).

home-based industries in the village that produce compost bins and tempe, a soybean staple of the Indonesian diet. Tempe is produced by fermentation of soybeans. The soy-

METHODS

beans are soaked, dehulled, boiled and inoculated with the fungus Rhizopus spp.; mycelial growth knits the soybeans

To characterise water quality within the village, water samples

into a cake that has a meaty texture (Bisping et al. ).

were taken from surface and subsurface locations throughout

Nout & Rombouts () provide a detailed review of the

the village. Sampling sites include irrigation canals (11 sites),

tempe production process.

wells (13 sites), paddy ﬁelds (two sites) and wastewater efﬂuent

Cattle (about 48 cows) are kept at one location in the

points (two sites). Some irrigation sampling points are directly

southeast corner of the village to minimise their movement

downstream of WWTP efﬂuent points (three sites) while

and to centralise the collection of their faeces for compost-

others are unaffected by WWTP discharge (eight sites)

ing (Figure 1).

(Figure 1). Water samples were collected in August and

Septic tanks were previously the main wastewater treat-

December 2011 during the dry and wet seasons, respectively.

ment system in the village. Five municipal wastewater

The treatment performance of the WWTPs was exam-

treatment systems were installed in the village in 2008

ined by taking water samples from different points within

with the help of an Indonesian NGO (Yayasan Dian Desa;

the treatment process including the inﬂuent point, inside

http://diandesa.org/Home.html), local government and

the wastewater treatment chamber, and the efﬂuent point.

external sources of funding. They serve about half of the vil-

This was done only for the RBC system (Site 1) and one of

lage households (approximately 150 households, i.e. 30

the CA systems (Site 3) for the following reasons. First, the

households/system). The remaining villagers continued to

efﬂuent discharge point from the CA process at Site 2 was

199

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

inside the irrigation canal so it was not possible to get a true

The CA wastewater treatment process (Site 3, Figure 1)

efﬂuent sample. Second, the CA process at Sites 4 and 5 was

also removed TSS, BOD5 and COD most effectively. Removal

not functioning in August so it was not possible to compare

efﬁciencies were often above 85% (Table 1). The CA process

treatment performance for the two sampling periods.

managed to remove TP quite effectively (greater than 60%

The water quality parameters examined were chosen to

removal efﬁciency for both sampling periods) but failed to

reﬂect the oxygen demand in, and the nutrient and bacteria

remove nitrate and ammonia from raw sewage (Table 1).

content of, the water. Elevated nutrient content may result

This treatment process managed to remove some TN from

in eutrophication with undesirable algal growth and sub-

raw sewage but removal efﬁciencies were low (30.5 and

sequent smell. Elevated nitrate concentrations may cause

10.7% for August and December) (Table 1). The nutrient

human health problems such as methaemoglobinaemia in

removal efﬁciencies were also inconsistent between the two

babies (blue-baby syndrome), and bacteria such as E. coli

sampling periods. For example, samples taken during

cause urinary tract infections and gastroenteritic diseases. Turbidity and pH were measured in situ using portable

August had no ammonia removal whereas the removal efﬁciency for December was very high (89.6%) (Table 1).

equipment. Water samples were collected by hand in clean polyethylene bottles and kept in a cooler box before being

Comparing efﬂuent quality with water sampled from

sent for analysis by an accredited Indonesian laboratory.

the village

Samples were tested for total suspended solids (TSS), biological oxygen demand (BOD5), chemical oxygen demand

A comparison between the quality of WWTP efﬂuent and

(COD), ammonia (NH3-N), nitrate (NO3-N), total nitrogen

water samples taken from different sources in the village

(TN) and total phosphorus (TP) (Table 1). Unfortunately,

shows the degree of water pollution within the village

orthophosphate could not be tested due to equipment pro-

(Figure 2, Table 2). World Health Organization (WHO) and

blems at the Indonesian laboratory. Samples for E. coli

United States Environmental Protection Agency (USEPA)

were obtained by professional staff from another accredited

drinking water guidelines are also included in Table 2 as a

Indonesian laboratory and analysed on the same day.

reference. These drinking water guidelines cover only a limited number of water quality parameters, namely pH, NO3-N and E. coli.

RESULTS E. coli Treatment performance of the Sukunan WWTPs E. coli was present in all samples taken from the Sukunan vilThe RBC treatment process (Site 1, Figure 1) was able to

lage (Figure 2(a), Table 2). There was no clear distinction

remove TSS, BOD5 and COD from sewage quite effectively.

between the median E. coli counts found in samples from irri-

Removal efﬁciencies were above 75% for these water quality

gation canals directly downstream of WWTP efﬂuent

parameters (except BOD5 in December) and were quite con-

discharge points and unaffected irrigation sites. Overall, the

sistent for the two sampling occasions (Table 1). The

highest E. coli count (2.4 × 1010 counts/MPN (most probable

treatment performance for nutrients was lower and more

number), Figure 2(a)) was for a sample taken at a canal just

variable between the two sampling periods. For instance,

upstream of the CA system (Site 2, Figure 1) and was a bit

the removal efﬁciency of nitrate varied quite considerably

unusual as higher concentrations are expected downstream

between August (64.9%) and December (26.6%) (Table 1).

of a WWTP efﬂuent discharge point (Figure 1). This sample

Further, the RBC system failed to remove ammonia and

might have been contaminated by village animals or localised

TN for both sampling periods (i.e. negative treatment efﬁ-

sources from homes nearby and warrants further investigation.

ciencies).

The system managed to

remove TP but

E. coli was also detected in all well samples and varied

treatment efﬁciency was very low, at less than 10% efﬁ-

quite signiﬁcantly across the village (up to ﬁve orders of

ciency for both sampling periods.

magnitude, Figure 2(a), Table 2). Some wells had counts

200 H. S. Lim et al.

Water quality of wastewater at different points of treatment and ﬁnal efﬂuent for two wastewater treatment plants (WWTPs), Sukunan Village Nitrate (NO3-N)

Ammonia

TSS (mg L 1)

BOD5 (mg L 1)

COD (mg L 1)

(mg L 1)

(NH3-N) (mg L 1)

TN (mg L 1)

TP (mg L 1)

E. coli (MPN/100 ml)

RBC process (Site 1)

Aug

Dec

Aug

Dec

Aug

Dec

Aug

Dec

Aug

Dec

Aug

Dec

Aug

Dec

Aug

Dec

Inlet

125.0

242.0

146.0

246.5

447.0

561.6

2.9

0.4

0.8

0.3

168.0

137.9

5.2

4.2

Inside

47.0

41.0

50.0

84.5

102.1

123.6

1.1

0.4

1.0

0.4

211.0

192.7

5.3

5.0

Efﬂuent

8.0

37.0

40.5

88.0

84.5

133.7

1.0

0.3

0.8

0.3

173.4

167.3

4.8

3.8

5.4 × 10

1.6 × 109

Removal (%)

93.6

84.7

72.3

64.3

81.1

76.2

64.9

26.6

2.7

5.7

3.2

21.3

7.8

10.6

Inﬂuent

102.0

528.0

110.0

187.0

215.3

294.5

0.8

0.7

0.5

0.3

211.0

249.1

5.3

2.1

Aeration tank

811.0

32.0

39.0

54.5

83.3

102.0

0.6

0.3

1.2

0.0

211.0

310.7

5.3

2.1

Efﬂuent (next to biogas)

12.5

18.0

7.5

8.2

13.9

15.1

1.3

2.2

1.4

0.0

146.5

222.4

0.9

0.8

2.4 × 106

5.4 × 107

Down stream of biogas site

10.0

20.0

3.6

2.9

10.4

18.9

2.3

2.4

1.4

0.0

127.7

195.7

0.3

0.7

1.4 × 106

5.4 × 105

Removal (%)

87.7

96.6

93.2

95.6

93.5

94.9

60.0

194.8

170.9

89.6

30.5

10.7

83.2

63.6

–

Removal efﬁciency of other Indonesian WWTPa

–

Method of analysis

APHA 2540D, 2005

CA process (Site 3)

Based on eight community-based WWTPs in Indonesia (Foley et al. 2001).

APHA 5220C, 2005

SNI 06.2480, 1991

IKM/5.4.38/B LK-Y

APHA4500N.Org C

IKM/ 5.4.40/ BLK-Y

APHA 2005, Section 9221-E Journal of Water and Health

APHA 5210B, 2005

Community-based wastewater treatment systems and water quality

Table 1

| 12.1

| 2014

201

Figure 2

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

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Boxplots of (a) E. coli, (b) BOD5, (c) COD, (d) nitrate, (e) ammonia, (f) TN, (g) TP for both sampling trips sampled from WWTP effluent, groundwater samples (Wells), irrigation samples unaffected by effluent discharge (Irrigation), irrigation samples affected by effluent discharge (Irrigation down) and paddy field samples (paddy field).

that were similar to those observed for paddy ﬁeld and irri-

water from a deeper part of the aquifer that is probably

gation water samples. The highest E. coli count for a well

less contaminated (Well 4, Figure 1). The rest of the samples

sample was recorded at a well in a home that produced

were taken from shallower wells which are closer to septic

tempe (Well 10, 5.4 × 105 counts/MPN, Figure 1). The site

tanks and more likely to experience high levels of contami-

with the lowest E. coli count for both occasions draws its

nation (Goss et al. ; Krapac et al. ).

202

Table 2

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

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Water quality for wastewater treatment systems and surrounding waters, Sukunan Village, Yogyakarta

Water/wastewater quality parameters

Types of water/wastewater

TSS

BOD5

COD

NO 3 -N

NHþ 3 -N

(mg L 1)

TN (mg L 1)

TP (mg L 1)

(MPN/100 mL)

E. coli

Wastewater treatment plant efﬂuent RBC process Wet (December)

7.15

8.0

40.5

84.5

1.0

0.8

173.4

4.8

5.4 × 106

Dry (August)

7.17

133.7

0.3

167.3

4.0

1.6 × 109

7.5

13.9

1.4

146.5

0.9

2.4 × 106

8.2

15.1

2.0

0.03

222.4

0.8

5.4 × 107

100

N.A.

Turbidity

BOD5

COD

NO 3 -N

NHþ 3 -N

(NTU)*

(mg L 1)

TN (mg L 1)

TP (mg L 1)

(MPN/100 mL)

CA process Wet (December)

7.08–7.26 12.5

Dry (August) Domestic wastewater efﬂuent discharge standardsa

6–9

E. coli

Other water sources in the village Well (groundwater)

6.16–7.11 0.06–1.78 1.75–2.92 3.52–10.2 0.33–9.4

0.002–0.18

91.2–263.8

0.067–0.80

8–5.4 × 105

Irrigation canal

6.84–7.48 13.1–63.5 1.83–5.26 3.52–45.3 0.69–5.21 0.002–1.35

95.5–262.4

0.067–1.37

2.4 × 10 4 2.4 × 1010

Irrigation canal downstream of efﬂuent points

6.89–7.63 9.8–25.8

121.4–289.8 0.10–0.71

Paddy ﬁeld

–

2.13–3.74 7.04–18.9 0.64–2.66 0.006–1.39

1.7 × 10 5 1.6 × 107

27.3–77.5 2.05–3.61 7.04–30.2 1.21–1.59 0.023–0.066 109.9–164.6 0.081–0.16

5.4 × 105

Indonesian Standards for N.A. clean water qualityb

N.A.

Indonesian Standards for N.A. drinking water qualityc

N.A.

WHO drinking water guidelines

USEPA drinking water guidelines

6.5–8.5

Peraturan Menteri Kesehatan No. 416 Tahun 1990 Tentang: Syarat-Syarat dan Pengawasan Kualitas Air, Menteri Kesehatan Republik Indonesia. Lampiran II: Daftar Persyaratan Kualitas Air

Bersih, R.I. No.: 416/MENKES/PER/IX/1990. b

Peraturan Menteri Kesehatan No. 416 Tahun 1990 Tentang: Syarat-Syarat dan Pengawasan Kualitas Air, Menteri Kesehatan Republik Indonesia. Lampiran I: Daftar Persyaratan Kualitas Air

Minum, R.I. No.: 416/MEMKES/PER/IX/1990. c Peraturan Menteri Kesehatan No. 416 Tahun 1990 Tentang: Syarat-Syarat dan Pengawasan Kualitas Air, Menteri Kesehatan Republik Indonesia. Lampiran I: Daftar Persyaratan Kualitas Air Minum, R.I. No.: 416/MEMKES/PER/IX/1990. d

The Indonesian standards only make reference to total coliform (MPN/100 mL) and not Escherichia coli.

*Nephelometric Turbidity Units.

BOD5 and COD

ranges. Low BOD5 concentrations in well samples show that there is little degradable organic matter in groundwater

BOD5 is a common indicator of the presence of polluting

(Mukherjee et al. ).

organic matter in water. As expected, WWTP efﬂuent has

The pattern of COD variation amongst the various sampling

the highest BOD5 values, varying over one order of magni-

locations is similar to that for BOD5. Wastewater-treated efﬂu-

tude (Figure 2(b), Table 2). Irrigation and paddy ﬁeld

ent samples have the highest COD concentrations and the

samples had quite similar median values and interquartile

largest variability followed by irrigation canal and paddy ﬁeld

203

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

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2014

samples (Figure 2(c)). Well samples have the lowest COD

for each WWTP) and paddy ﬁeld sites (two locations) are

amongst all types of water samples in this study. The outliers

too low for meaningful comparison with other samples.

for COD and BOD5 both came from the same location (Well

For the surface water samples, BOD5, ammonia, TN and

13, Figure 1). We do not know the cause of the high organic

TP are most affected by rainfall seasonality. Median concen-

matter at Well 13 but it warrants further investigation.

trations and variability increase for the December wet season samples (Figure 3). Nitrate shows dilution for the wet season

Nutrients (nitrate, ammonia, TN and TP) As expected, WWTP effluent had highest ammonia, TN and TP concentrations compared to the environmental samples taken from the village (Figure 2(d)–(g)). Irrigation samples affected by WWTP effluent had correspondingly high levels of ammonia and TP (Figure 2(e), (g)). For nitrate, well samples showed higher median value and greater range than even WWTP effluent (Figure 2(d)). The highest nitrate concentrations in well samples came from a tempe factory (Well 9, 9.43 mg L 1) and a well in a densely populated area (Well 11, 8.37 mg L 1) (Figures 1 and 2(d), Table 2). The nitrate levels of irrigation waters and paddy field are higher than WWTP effluent, suggesting an additional source related to fertiliser use. However, the highest nitrate concentration in irrigation waters unaffected by wastewater effluent was taken from a location where irrigation waters from outside enter the village (near Well 4, 5.21 mg L 1, Figure 1). This site also had one of the highest TN concentrations recorded for all irrigation samples (262.44 mg L 1). The median concentrations for TN are quite similar for all environmental samples except well samples, which had the greatest interquartile variability (Figure 2(f)). Finally, high TP levels were recorded for well samples when compared to irrigation water samples (Figure 2(g)). Wells with highest TP concentrations are those located in areas with home-based industries (0.798 and 0.315 mg L 1, Wells 7 and 8) (Figure 1).

Comparing water quality between wet and dry

samples for sites unaffected by WWTP due to increasing runoff in irrigation canals after rainfall, but the opposite was observed for affected sites. The increase in nitrate concentration for WWTP affected sites is most likely due to the decreasing performance of both WWTPs in December especially the CA plant (Site 3) (Figure 3(d), Table 1). For ammonia, the seasonal impacts of rainfall are harder to discern due to the unstable nature of this parameter and the interactions between its sources and WWTP treatment performance. The increase in concentrations during the wet season for irrigation sites unaffected by WWTP is most likely due to washing in of animal faeces (e.g. from the centralised cattle area and village areas) via surface run-off into irrigation canals. The decrease in ammonia concentrations for irrigation sites affected by WWTP effluent could be due to oxidation of ammonia into nitrate from increased turbulence of faster flowing waters in the canals. It could also be due to improvements in WWTP treatment performance for the CA system (Site 3), which reduce ammonia loading into irrigation canals (Figure 3(e)). Well samples exhibited higher median concentrations for E. coli and all nutrients during the December wet season (Figure 3). The increased variability observed for E. coli and all nitrogen fractions indicates percolation of nitrogen-rich septic tank effluent with infiltration following rainfall. It could also be due to rising water table levels that interact with the septic tanks. This finding is consistent with Smith et al. () who recorded increased nitrate concentrations in their well samples during the rainy season.

DISCUSSION

conditions Extent and source of pollution We compared data for wells (n ¼ 13), irrigation waters affected by WWTP efﬂuent (n ¼ 3) and irrigation waters unaf-

Surface waters

fected by WWTP efﬂuent (n ¼ 8) to examine the impact of rainfall seasonality on water quality (Figure 3). The number

Water samples from irrigation canals have higher E. coli,

of samples taken from the wastewater efﬂuent point (two

BOD5, COD and ammonia concentrations than well samples.

204

Figure 3

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

Boxplots of (a) E. coli, (b) BOD5, (c) COD, (d) nitrate, (e) ammonia, (f) TN, (g) TP for the August (dry) and December (wet) samples for well samples, irrigation canals unaffected by efﬂuent discharge (irr) and irrigation canals downstream of efﬂuent discharge (irr_down).

The quality of water in paddy ﬁelds is similar to that recorded

compared to unaffected sites. Many surface water samples

in irrigation canals, especially for parameters such as BOD5,

suffer from nitrate and ammonia pollution because their con-

ammonia and TN. The discharge of WWTP efﬂuent directly

centrations are greater than the concentration values found

into irrigation canals has an impact on water quality by increas-

for natural waters (less than 1 mg L 1) (USEPA , http://

ing levels of nitrate, ammonia and TP at sites downstream

water.epa.gov/type/rsl/monitoring/vms57.cfm).

However,

205

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

they do not pose a threat to human health since nitrate levels

efﬂuent (40–100 mg L 1) (USEPA ). This suggests that

are within the WHO and Indonesian standards for drinking

wastewater is not the only source of nitrogen in our ground-

water (10 mg L , Table 2). The concentration of E. coli in irri-

water samples. The high TN values in our samples with

gation samples exceeded allowable limits. Villagers are at risk

correspondingly low nitrate and ammonia concentrations

of health problems associated with E. coli when they come into

shows that the organic form dominates TN. The high concen-

contact with the bacteria through their daily activities (e.g.

trations and variability of nitrate and TN concentrations

washing laundry in irrigation canals and working in paddy

suggest that localised sources cause some areas to be more

ﬁelds).

contaminated than others (Figures 2(d) and 2(f)). These

Surface water pollution in this village comes from exter-

sources could be active septic tanks (especially in more den-

nal and internal sources. External sources are harder to

sely populated areas of the village, Well 11) or areas where

control since they are not within the conﬁnes of the village.

fertilisers are applied and leached into the local aquifer (e.g.

Village activities such as doing laundry in irrigation canals

Long & Sun ). Further, certain hotspot contamination

contribute to elevated TP concentrations from the detergent

areas such as the tempe factories show greater groundwater

used. Free-roaming farm animals in irrigation canals and

contamination (Wells 8 and 10, Figure 1). Water samples

paddy ﬁelds contribute nutrients and bacteria. One irrigation

taken from these locations also had consistently higher TN,

sampling site near the CA system (Site 3, Figure 1) recorded

TP and COD concentrations. Liquid wastes from tempe pro-

high nitrate (2.4 mg L 1), TP (0.25 and 0.33 mg L 1) and

duction had high BOD (348 mg L 1), COD (535 mg L 1) and

COD (20.83 and 45.31 mg L 1) values on both sampling

nutrient content (e.g. carbon, nitrogen, potassium, mag-

occasions due to the presence of chickens, and of ducks

nesium and phosphorus) (Chaerun ; Wignyanto &

swimming nearby. Elevated nutrient concentrations in irriga-

Ariningrum ). These wastes are treated at the municipal

tion waters also comes from washing in of organic fertilisers

WWTPs but soil and groundwater pollution can still occur

(compost and manure) into irrigation canals, and from

via leaking pipes and spillage onto, and/or washing of, the

WWTP efﬂuent discharge (Table 1).

earthen dirt ﬂoors where production takes place. The TP concentrations found in Sukunan well samples

Subsurface waters

range between 0.067 and 0.8 mg L 1. We cannot compare our results with a benchmark since there are no drinking

Groundwater samples are contaminated with high levels of

water quality standards for TP set by international agencies

nitrate, TN and TP (Figure 2). Some samples have concen-

or the Indonesian government. But our sample concen-

trations that were comparable to WWTP efﬂuent. Current

trations fall within the range expected for WWTP efﬂuent

nitrate levels in groundwater samples range between 0.33

in the USA (0.1–1.1 mg L 1) and are much lower than typi-

and 9.4 mg L 1 and fall within Indonesian and international

cal septic tank efﬂuent (3–40 mg L 1) (USEPA ; Lowe

standards for drinking water quality, with the exception of

et al. 2007 cited in Lusk et al. ; Withers et al. ).

two wells which have values close to the limit (Wells 8

The lack of orthophosphate data precludes us from analys-

and 10, Table 2). Consuming water from these wells will

ing the effect of septic tank efﬂuent on groundwater

have negative health impacts on the villagers in the long

quality especially since the efﬂuent has high levels of ortho-

run and should be avoided. On a broader scale, nitrate con-

phosphate or soluble reactive phosphorus ( Jarvie et al. ).

tamination of groundwater in the Sukunan Village is

E. coli counts in all well samples exceed international

relatively low compared to a nearby village 3 km away

and Indonesian drinking water standards (Table 2). E. coli

where well waters had concentrations over the 10 mg L 1

in groundwater comes from septic tank efﬂuent and cattle

limit (Smith et al. ).

manure in the village (Goss et al. ; Krapac et al. ;

Groundwater TN concentrations (91.2–263.8 mg L 1)

Budisatria et al. ; Rawat et al. ). Groundwater of

from the Sukunan wells are comparable to the concentrations

this village may also be affected by regional groundwater

observed for samples from two WWTPs in the village and

pollution from the widespread use of septic tanks in the

higher than the concentrations expected from septic tank

Yogyakarta region (Smith et al. ; Budisatria et al.

206

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

). The practice of boiling well water before consumption

countries for a variety of reasons including insufﬁcient

is sufﬁcient for the time being since there have been no

knowledge and unclear roles of government agencies and

recent outbreaks of gastroenteritic diseases in the village.

the home owner (Butler & Payne ; Harrison et al. ).

Projected population increase in this village and its immedi-

In a village like Sukunan, septic tanks are still the most afford-

ate surroundings may require the villagers to use more

able means of treating domestic wastewater. The separation

sophisticated methods of water disinfection.

distance of 10 m between septic tanks and wells does little to prevent groundwater contamination in this dense housing

Impact of the WWTPs on water quality

conﬁguration, especially if septic tanks are poorly maintained and leaking (Jarvie et al. ; Habteselassie et al.

Comparison of our ﬁndings with available data from small

; Macintosh et al. ).

community-based WWTPs indicates that the two Sukunan

Pollution from septic tanks can be reduced if more village

WWTPs perform better in removing solids (TSS) and

households are connected to the ﬁve WWTPs since these sys-

oxygen-demanding wastes from wastewater (BOD5 and

tems have not reached their maximum capacity of 50 to 150

COD) (see Table 1; Foley et al. ). The differences in

households depending on design and individual household

treatment performance could be due to differences in the

population (Foley et al. ). One obstacle to increased house-

design and quality of materials used in the WWTP construc-

hold connections to WWTP is the inability of poorer villages to

tion. The Sukunan WWTPs, however, released nutrients,

meet the pipe installation costs and the monthly operation

particularly nitrate, ammonia and TN, and E. coli into sur-

charge (3000 Rupiah, approximately 0.31 US dollars). At pre-

face waters of the village (Figure 2). Although efforts by

sent, part of the cost of WWTP connection is borne by the

the villagers to move cattle to a centralised location help

wealthier villagers. Part of the money could also come from

reduce the loading of nutrients and oxygen-demanding

the proﬁts gained from selling the village municipal waste

wastes into irrigation canals, the inconsistent and poor

and compost produced in the village. Sales of these goods

nutrient removal efﬁciencies of the two WWTPs investi-

can be improved with better marketing and price negotiations

gated nullify the reductions in nutrient loading achieved

with the help of NGOs or private companies.

from relocating the cattle. The nutrients from WWTP efﬂu-

The treatment performances of the existing WTTPs have

ent discharged into irrigation waters seem to have a

to be improved in order for increased connections to have a

beneﬁcial effect on paddy yield, according to some villagers.

positive impact on surface and subsurface water quality in

Standing waters in the village receiving irrigation waters

this village. Future work in this village includes an examin-

show the negative impacts of water quality where signs of

ation of the reasons behind the current poor WWTP

eutrophication and mosquito breeding are evident.

performance so that design improvements can be made to reduce nutrient loadings into the irrigation canals.

Suggestions to improve water quality in the village

The impact of tempe industries in the village is serious and needs to be addressed by ﬁrst monitoring the quality of

The groundwater and surface waters in this village suffer

tempe wastewater from the individual home factories. It is

from pre-existing pollution caused by septic tanks and agri-

possible that the WWTP systems, such as Systems 4 and 5,

cultural activities. The role of septic tanks in groundwater

are overloaded due to the addition of tempe wastewaters

pollution in these villages cannot be ignored given an

(Figure 1). The set-up of the home industries can be improved

increase in demand for such systems with a rising population

by sealing off the ground surface to prevent seepage. Separate

in Indonesia (Smith et al. ; Budisatria et al. ; Snguon

treatment systems may be needed to pre-treat the wastewater

et al. ). In developed countries, techniques to reduce the

before channelling them to the existing WWTPs.

impacts of septic tank discharge include improvements in

It is also important to study the impact of organic fertili-

their design and careful site selection followed by rigorous

ser application on water. The chemical characteristics of

maintenance of existing systems. Implementing rigorous

commercial and home-made fertilisers used in this village

maintenance can be a major challenge even in developed

and elsewhere in the vicinity needs to be evaluated. Ways

207

H. S. Lim et al.

Community-based wastewater treatment systems and water quality

Journal of Water and Health

12.1

2014

to reduce leaching from organic fertiliser include washing the

year when pollution is higher due to variable WWTP perform-

compost artiﬁcially before application, stepwise application

ance or rainfall. Rainfall tends to increase the concentrations

of compost over time to reduce the nutrients available for

of nutrients in irrigation canals and wells but the impact of

leaching, and timing fertiliser application to dry periods to

rainfall is complicated by WWTP discharge into irrigation

reduce leaching of the fertiliser (Christensen ). We are

canals.

not sure how these methods might be adapted to suit the

Despite the construction of the WWTPs, we found that

Sukunan agricultural practices and further research in this

irrigation waters still had high levels of E. coli and oxygen-

area is needed.

demanding wastes which could originate from village ani-

For community-based efforts at Sukunan to beneﬁt the

mals and farming activities. Well samples had high E. coli

village in the long run, the villagers need strong collabor-

counts, and nitrate and TN, which originate in active septic

ations between NGOs, local governments, academic

tanks, small-scale industries and fertilisers used in the village.

institutions and even private companies. NGOs play an

Therefore, sources of pollution include both internal and

important role as information providers and act as

external sources despite efforts by the villagers to control pol-

important links between the village community, local gov-

lution within the village. External sources of pollution are

ernments and technical institutions. As the nature of the

beyond the villagers’ control and highlight the wider problem

environmental problem changes or requires improvements,

of water pollution in the Yogyakarta region.

villagers need to draw upon the expertise, networks and

All the samples fell within recommended drinking water

ﬁnancial help of these organisations (Padawangi ;

standards for nitrate and E. coli set by both international

Sansom ). There are many examples of successful collab-

agencies and the Indonesian government with the exception

orations between NGOs and villagers in setting up and

of two wells which had nitrate levels close to the drinking

managing community projects in the literature (e.g. Kyessi

water limit. The villagers are at risk from health problems

; Sansom ). In this study, the local NGO Yayasan

associated with ingesting E. coli and nitrate from well water,

Dian Desa already provides important technical advice on

and coming into contact with E. coli through their farming

the WWTPs for the villagers. The local university, Gadja

activities.

Mada University, sends students to carry out simple moni-

To improve water quality in this village, the villagers

toring of the WWTP. These activities could be extended to

should try to increase the number of households connected

include villages in monitoring activities using simple ﬁeld

to the WWTPs, while at the same time request a more

kits and facilitated by local NGOs (e.g. Bruhn & Wolfson

thorough investigation of the WWTPs to ﬁnd out the

).

reasons behind their poor nutrient removal performance. We cannot identify the cause of poor nutrient removal from two sampling occasions. Poor performance could be

CONCLUSIONS

due to design problems or due to system overloading, perhaps from the tempe industries. In this respect, there is

This study provides an initial assessment of water quality in

also a strong need to examine the role of tempe industries

the Sukunan Village by examining wastewater and water

in the village in water pollution since these home-based

samples from different locations within the village. Our ﬁnd-

industries are common in Indonesian villages.

ings show that the WWTPs have a limited effect on improving village water quality. Oxygen-demanding wastes from sewage are removed by the WWTPs but these systems

ACKNOWLEDGEMENTS

did little to remove nutrients from sewage. The RBC system failed to remove ammonia and TN on both sampling

This work was funded by the Staff Research Support

occasions while the CA system failed to remove nitrate on

Scheme 2011/12 awarded by the Faculty of Arts and Social

both sampling occasions. Removal efﬁciencies of both sys-

Sciences, National University of Singapore. We would like

tems were also variable over time. There are times in the

to thank Mr Iswanto, the Sukunan villagers and Mr Eko

208

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Community-based wastewater treatment systems and water quality

Kusumo. We are also grateful to the anonymous reviewers for their comments on this paper.

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Journal of Water and Health

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First received 9 December 2012; accepted in revised form 5 August 2013. Available online 25 September 2013