OCCURRENCE OF FLAME RETARDANTS IN WATER, SLUDGE AND SEDIMENT IN THE CZECH REPUBLIC by International Educational Scientific Research Journal

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SERVICES, INC., PRAGUE, CZECH REPUBLIC - 110 00. CENTRAL INSTITUTE FOR SUPERVISING AND TESTING IN AGRICULTURE, BRNO, CZECH REPUBLIC – 603 00. 3 NATIONAL INSTITUTE OF PUBLIC HEALTH, PRAGUE, CZECH REPUBLIC - 100 42. 4 NORWEGIAN INSTITUTE FOR WATER RESEARCH, OSLO, NORWAY - NO-0349. 5 BIOCEV, 1ST MEDICINAL FACULTY CHARLES UNIVERSITY, CZECH REPUBLIC - 252 50. 2

ABSTRACT Flame retardants (FR) are compounds added in large quantities to the commercially sold products in order to increase their fire safety. Brominated (BFR) and organophosphate (PFR) FR represent the most frequently used FR and at the same time, the most frequently monitored due to their bioaccumulative properties and consequent health risks. In order to assess these risks, it is necessary to clarify the ways that FR are spread from products into the environment and human organism. Therefore, broad scale of methods for products screening (reported elsewhere) in various environmental matrices was carried out in the Czech Republic for a large variety of BFR and PFR (in 226 samples mostly 39 substances were analysed). As one identified pathway of human exposure to FR is from municipal sewage sludge, via soil and sediments, into plants and food, the current study will focus on the first steps in this. Measurements were performed at the following locations: at the output from municipal wastewater treatment plants (WWTPs) using passive sampling devices (PSDs), in sewage sludge at these WWTPs, in soils, and in pond sediments PBDE were detected at low concentrations in pond sediments. The behaviour of individual congeners in the soil varied, depending on the number of bromines. The future development of the environmental contamination by FR is difficult to predict because of the replacement of previously widespread FR, such as PBDEs, hexabromocyclododecane, or polybrominated biphenyls, due to the banning or restriction of production by legislation. Although the production of some FR has been terminated, the contamination may continue and increase significantly, as products with a high content of FRs begin to be disposed of. For these reasons, it is necessary to continue with environment monitoring of "classical", and newly produced FR. Keywords: Flame Retardants; Soil Contamination; WWTP; Sludge; Sediment, Czech Republic. polystyrene (EPS), extruded polystyrene (XPS) and 1. Introduction industrial textiles (Kočí 2012). It has been industrially manufactured since the 1980s, with a significant increase Flame retardants in production in the 1990s. Due to its lipophilic character FRs are added to a broad range of materials for fire and negligible degradability, HBCDD accumulates in the protection reasons in order to slow down their food chain and has a negative impact on human health and combustion or for the prevention of ignition. In many ecosystems. cases, these fire resistant properties are a legal It is one of the most hazardous persistent organic requirement. FR are added to combustible materials such pollutants and it is controlled by the Stockholm convention as plastics, electronics, thermal insulation in the (SC). Moreover, this compound was labelled as a substance construction industry, thermoplastics, electric cables, of very high concern (so-called SVHC) by the European textiles and fabrics, and many other articles (de Wit Chemical Agency (ECHA) in 2008. 2002). FRs may often constitute a considerable part of the weight of the product, for example plastics may contain up Polybrominated diphenyl ethers to 15% PBDE and polyurethane foam up to 30% PBDE PBDEs are widely used in textiles, plastics, building (European Flame Retardants Association. Flame retardant materials and electronics (Alaee 2003). They include 209 types and applications. 2004). From an environmental congeners which differ in the number (from 1 to 10) and perspective, most attention has been paid to brominated the position of bromine atoms on the benzene retardants (BFR) and phosphorus containing retardants rings. Commercially produced PBDEs can be divided into (PFR). For these reasons, the present study was mainly three groups according to the number of present bromine dedicated to the isomers of HBCDD and PBDEs. atoms i.e. penta, octa and deca BDE. Penta-BDEs were Hexabromocyclododecane produced in Israel, Japan, USA and EU. However, in 2001 98% of the demand (mainly used in polyurethane foam) HBCDD is a compound used as a FR in expandable

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originated from North America (Hale R. C. 2003). The mostly universally applied BFR is Deca-BDE that is also known as BDE209. In 2001 Deca-BDE represented more than 83% of the world demand of PBDEs. As the chemical structure of PBDEs is similar to PCB (polychlorinated biphenyls), DDT (dichlorodiphenyltrichloroethane) and PBB (polybrominated biphenyls), their chemical and environmental properties are comparable as well. They are both hydrophobic substances which bond to lipids and bioaccumulate. They are persistent and resistant to chemical and biological degradation. Moreover, they are toxic to living organisms and they are transportable to long-distance with proven findings (Hites 2004) in human adipose tissue (e.g. breast milk and blood). PBDEs represent the first group of brominated FR which were detected in the environment (de Carlo 1979). As a consequence of that initial detection, many studies, e.g.(Law R. J. 2006), have documented their accumulation in the environment. Considerable experimental data show that penta-BDE can disrupt the endocrine system and affect the thyroid gland, ovaries, and androgen functions (Legler 2008). Exposure to PBDEs in prenatal and neonatal period may have implications for genetic expression of hepatic enzymes (Szabo et al. 2009), endocrine disruption (Kuriyama S. N. 2007), immunotoxicity (Arkoosh M. R. 2010) and neurotoxicity (Eriksson P. 2001). Animal-testing on mice and rats proved that deca-BDE affects thyroid hormones, causing hyperthyroidism, and is hepatotoxic (Lee E. 2010). PBDEs are often bound to solid particles (e.g. soil, sediments, and dust particles) due to their hydrophobic nature. Inhalation of so contaminated dust is together with the consumption of fatty fish the major source of PBDEs in human body. PBDE can be transported from soil and sewage sludge into plants. This has been described as another possible pathway of these substances entry into the food chain (Hites 2004).

Phosphate flame retardants Since the ban of some brominated flame retardants (BFRs) (van der Veen and de Boer 2012), phosphorus flame retardants (PFRs), which represented only 20% of the European FR consumption in 2006, are proposed as alternatives. However, limited information is available on the occurrence of PFRs in the environment. A number of studies aimed on the occurrence of tricresylphosphate (TCP), tris(2-chloroethyl)phosphate (TCEP), tris(chloropropyl)phosphate (TCPP), and several other PFRs in air, water and sediment have been performed. Nevertheless, limited data were found on their occurrence in biota. Concentrations of these PFRs detected in air were up to 47 µg/m3, in sediment up to 24 mg/kg and in surface water up to 379 ng/L. TCPP was dominant in all the mentioned matrices. The only exception were concentrations found in dust, which were up to 67 mg/kg and TDCPP was dominant. Reported PFR concentrations were often higher than

polybrominated diphenylether (PBDE) concentrations (van der Veen and de Boer 2012). Human exposure due to PFR concentrations appears to be higher than exposure due to PBDE concentrations in indoor air. The Cl-containing PFRs are proven to be carcinogenic, and severe negative human health effects were found for Cl-containing PFRs as well as for TCP. At the same time, TCEP are considered to be toxic to (aquatic) organisms and/or (potentially) carcinogenic. Phosphorus compounds have some advantages compared to BFRs. During a fire, toxic by-products are created from BFRs. Due to the char, which is formed when using PFRs, emission of gases is reduced and in that way the release of toxic gases from phosphorus-based compounds is far lower than of BFRs(McPherson 2004). In addition, when using PFRs, the combustion gases are not contaminated with additional corrosive gases (HCl, HBr) from the FR (Hörold 1999).

Implementation of the Stockholm Convention in the Czech Republic As the Czech Republic is a signatory of the SC it is obliged to carry out regularly updated national inventory (NI) of substances listed in the SC annexes. The NI summarizes, for each group of substances, whether the country manufactured, imported, traded, or used them in manufacturing, agriculture or consumer articles, and what are their concentration levels in the environment, or if they are accumulated in biological matrices (particularly human tissues) (Stockholm Convention). The NI is then the basis for updating the National Implementation Plan (NIP) of the SC, thus identifying needs and formulating a plan about how to respond to these needs. This includes the creation of the necessary national capacities for monitoring of these substances. In addition, the European Commission (Directive 2008/105/EC and Directive 2013/39/EU) and subsequently the Government regulation has determined the maximum permitted concentration limits for surface water (maximum concentration 0.14 µg/L) and aquatic biota (annual average concentration 0.0085 µg/kg) (the sum of 6 PBDEs congeners: BDE28, 47, 99, 100, 153 and 154).

FRs transport to solid matrices The overall aim of the present study is to provide an evaluation of the concentrations of brominated and organophosphate FR in waste water and in solid matrices, as an important step towards providing an NI for these substances in the Czech Republic, in accordance with the SC. The transport of FR from the every-day-use products containing added FRs is examined via wastewater sludge to solid matrices in the environment. In addition, an advanced information analysis (AIA) will be provided in order to explain the transfer of FR and determine correlations of PBDEs.

Materials and Methods Measurements on WWTPs were performed as part of a NI

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screening project in the Czech Republic in 2016 (Ocelka et al. 2017). The main task of the mentioned project was to determine the occurrence and concentration levels of FRs in different matrices. Therefore, a large number of samples with a large set of analytes were examined. The total number of samples was 226, including: every-day-use products, human tissues and blood, foodstuffs, air (n=81, 53, 20 and 52, respectively), and that from WWTPs (20). In most of them 39 analytes were analysed organophosphates (PFR), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCDD) and polybrominated biphenyls (PBB).

Numbers of samples In order to clarify the transfer FR from WWTPs into solid matrices the following samples were taken:  Waste water in the outlet from WWTPs: 11 samples (E&H Services, Inc.) - 39 BFR and PFR, sampled 2016  Sludge from WWTPs: 10 samples (E&H Services, Inc., NIVA) - 39 PFR and BFR, sampled 2016  Sludge from WWTPs sludge - regular annual measurements: 64 samples each time from the different WWTPs (CISTA) - 9 PBDE congeners, sampled 2010-2015  Pond sediments - 31 samples (CISTA) - 9 PBDE congeners, sampled 2012-2015  Soils, 64 samples (CISTA) - 9 PBDE congeners, sampled 2011-2015. All 64 analytes were below the determination limit, therefore results have not been taken into consideration.

Waste water sampling The waste water was sampled by integrative PSDs, which is an innovative way to measure FR in water and enables overcoming of the potentially highly fluctuating concentrations associated with WWTPs. For non-polar substances BFR, PBDE, HBCDD and PBB, the Semi Permeable Membrane Device (SPMD) (Bergqvist 2000) was used. SPMDs consist of a membrane made of low density polyethylene (LDPE) with the following dimensions; length approx. 91 cm, width 2.5 cm, giving an area of 460 cm2. The LDPE is approximately 75 to 100 microns thick and is filled with approximately 1ml of triolein a neutral triacylglyceride (1,2,3-tri(cis-9-octadecenoyl)glycerol. The analyte molecules pass diffuse through the LDPE into the triolein sampling medium, where they accumulate proportionally to the sampling period. The SPMDs were spiked with deuterated PAH as performance reference compounds (PRCs) which diffuse out of the SPMDs during deployment. As this dissipation is equally affected by environmental factors that also influence uptake, then the PRCs can be used to establish SPMD sampling rates (Rs, equivalent amount of water extracted in L/d), which in turn allow the calculation of water concentrations (Huckins et al. 1999), (Booij et al. 2002). SPMDs have been applied to a wide

range of sampling media and scenarios(Ferenčík et al. 2016). For the polar PFR compounds the Polar Organic Chemical Integrative Sampler (POCIS) was applied, e.g. (Alvarez et al. 2004), (Zhang et al. 2008). The POCIS system consists of a body of two stainless steel rings which hold a sandwich of polyethersulphone (PES) membrane containing a sorbent material as the receiving phase. The sorbent chosen was OASIS HLB, as this is the most commonly applied to POCIS(Harman C 2012 ). Dissolved polar compounds pass through the PES membrane and accumulate in the sorbent, which is subsequently analysed. Both PSD types were constructed in house at the laboratory of E&H services in the Czech Republic. Both PSDs were placed in protective cages, which among other things help to mitigate for large fluctuations in medium flow rates. Two SPMD samplers and one POCIS, were placed at 11 WWTPs, in the location of final discharge (mostly drain gutter).

Sampling of sludge at WWTPs In 2016 municipal sewage sludge was taken at the same WWTPs (10) where wastewater was sampled, except one. Triplicate samples were taken using a metal spoon and were homogenized before being placed in an ALU container. The container was inserted in a PE bag, cooled, transported (the temperature was <10°C) and stored in a freezer at -18°C until the analysis.

Sampling WWTPs sludge - regular measurements According to the Czech legislation, PBDEs are monitored in sludge by CISTA (Florián 2016) as sludge may represent one of the possible inputs of FR to soil. This monitoring is mainly focused on larger WWTPs and on those, where is assumed that a significant part of the sludge production was ultimately directed to the agricultural use. The concentration of 9 PBDE in samples (from 10 to 12) was analysed by the CISTA National reference laboratory every year. Different WWTPs were chosen every year.

Sampling of soil - regular measurements The National reference laboratory CISTA started PBDE screening of agricultural soils in 2012 under basal monitoring. The purpose of this monitoring was to provide the information for the Czech Republic administration about the status and development of soil properties, as a response to the PBDE inclusion under SC. The second purpose was to meet the requirements of the European food safety authority (EFSA) monitoring these substances in food. 10 samples from the topsoil horizons on the monitoring areas were analysed every year, in order to determine organic pollutants.

Sampling of pond sediment - regular measurements Between 2011 - 2015 CISTA analysed 31 pond sediment samples for the same 9 PBDE congeners which were measured in the sludge and soils. The ponds sediment is formed by sedimentation of eroded solid particles that are naturally carried by water. Depending on the principle of

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sedimentation process, a significant difference of grain composition occurs in these sediments, influencing the chemical composition as well. Organic and inorganic substances are attached mainly on the surface of soil particles flushed out of the finest topsoil layer of the farmland. In the Czech Republic, these sediments are legislatively defined as waste. Using the sediments on agricultural land resources is not prohibited and is provided by Czech law about using the sediments on agricultural land resources and about their protection. A critical indicator for the possibility of using sediments is the rate of hazardous elements and organic pollutants contamination.

Analytes 1. Organophosphates (PFR) There were 16 substances (CAS given in brackets): 1. TCEP Tris(2-chloroethyl)phosphate (115-96-8), 2. TPrP Tripropylphosphate (513-08-6), 3. TCPP tris(chloropropyl)phosphate (13674-84-5), 4. TiBP Triisobutyl phosphate (126-71-6), 5. BdPhP Butyl diphenyl phosphate (2752-95-6), 6. TPP Triphenylphosphate (115-86-6), 7. DBPhP Dibutylphenyl phosphate (2528-36-1), 8. TnBP Tributylphosphate (126-73-8), 9. TDCPP Tris(1,3-dichloro-2-propyl)phosphate (13674-87-8), 10. TBEP Tris(2-butoxyethyl) phosphate (78-51-3), 11. TCP tricresylphosphate (1330-78-5), 12. EHDP 2-Ethylhexyl diphenyl phosphate (1241-94-7), 13. TEHP Tris(2-ethylhexyl) phosphate (78-42-2), 14. DCP Diphenylcresyl phosphate (26444-49-5), 15. T35DMPP Tris(3,5-dimethylphenyl) phosphate (25653-16-1), 16. TBPP Tris(2,3-dibromopropyl)phosphate (126-72-7). Information on these substances: their properties, applications, manufacturing, commercial mixtures names, bioaccumulation, toxicity, risk classification and legislative restrictions can be found on the ECHA, e.g. information about TCPP at(European Chemical Agency).

Brominated retardants (BFR)

- 8 PBDEs congeners: BDE28, 47, 54, 99, 100, 163, 183, and 209 - 3 HBCDD isomers (α, β, γ) - TBBA - 3,3',5,5'-Tetrabromobisphenol A (79-94-7) - not detected on WWTPs above determination limit - 11 PBB congeners (3, 15, 18, 52, 101, 153, 180, 194, 206, 209, and HBB). PBB were not found above the determination limit in any WWTP sample, (nor in any of the products and other environmental samples within whole FR screening project (Ocelka et al. 2017)). Therefore, these compounds are further not considered. 9 PBDEs congeners were measured in sludge, soils and pond sediments in CISTA National reference laboratory: BDE28, 47, 66, 85, 99, 100, 153, 154 and 183. The heaviest congener BDE209 was not determined here.

Chemical analysis Extraction with organic solvents (mixture of hexane/acetone – 3:1) was used as the first step of sample

processing. In case of PFR and BFR, aliquots of C13 labeled PBDE, C13 labeled PBB, C13 labeled HBB, C13 labeled TBBA, deuterated gamma HBCDD, deuterated TBP were spiked. PFR were cleaned-up using column with silicagel and hexane/ethyl-acetate eluting system and were concentrated to 200 µl of methanol. For BFR, aliquots were purified using acidic and basic columns filled with silicagel, alumina and charcoal and final volume of the sample ranged from 40 to 100 µl of nonane. Samples prepared by these procedures were measured using GC-HRMS or GC-ITQ (ion trap) analytical method or LC-MS/MS with similar conditions (van der Veen and de Boer 2012), (Van den Eede et al. 2012), (McGoldick 2014), (Martínez-Carballo 2007).

Method for the determination of PBDE and PBB: Analyses were performed on GC-MS/MS Polaris Q or GC-HRMS MAT95 with these chromatographic conditions: column Thermo Scientific TG-XLBMS, 30 m, ID 0.25 mm, film 0.25 m, flow rate of helium 1.2 ml/min, temperature programme: 150°C for 1 min, rate 20°C/min to 220, rate 3°C/min to 286 holding 1 min, rate 10°C to 310°C holding 20 min, total analysis 50 minutes; MS transfer line 275°C, inlet temperature 270°C. All of these instrumentations were manufactured by Thermoquest Finnigan, San Jose, USA.

Method for the determination of BFR and HBCDD: Analyses were performed on Quantum LC-MS/MS with these chromatographic conditions in MS/MS regime: HBCDD (column Phenomenex Kinetex 50x2.1 mm 2.6 m, mobile phase: A=water, B=methanol, isocratic elution 0-15 min, 20 % A and 80 % B, flow rate 0.3 ml/min) BFR (column Phenomenex Kinetex 50x2.1 mm 2.6 m, mobile phase: A=water+0.1 % formic acid, B=methanol+0.1 % formic acid, gradient elution 0-18 min, 0-4 min 80 % A and 20 % B, 4-9 min 30 % A and 70 % B, 9-18 min 80 % A and 20 % B flow rate 0.2 ml/min. The 50 samples, including 10 sewage sludge from WWTPs were simultaneously analysed in by the Norwegian Institute for Water Research (NIVA, Oslo) by using high-resolution LC-HR ToFMS. The regular determination of PBDEs in sludge soils and pond sediments was carried out by National reference laboratory CISTA under basal monitoring.

Advanced Information Analysis Advanced Information analysis means (i) marginal analysis that includes exploratory analysis of univariate data, (ii) multivariate analysis, which precedes exploratory analysis of multidimensional data. In the current data set was a high occurrence of results below the LOQ. Used software tools were: QC Expert™ (Trilobyte Company, Ltd.) and frequently used complement: MS Excel ™ XLSTAT (Addinsoft Company). A Spearman correlation for data processing was applied, using the following assumptions: • results below LOQ were replaced by the LOQ value

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• the same uncertainty for sample preparation and analytical techniques have been taken into account for all measurements, corrected during the analytical process with recovery ratio of surrogate internal standards • for the data evaluation the estimated points were used (usually median, especially if a priori assumptions of data normality were not confirmed). Correction of the uncertainty of the method, e.g. by the weight determination, was not performed • normality, if it was assessed in a level of one-dimensional case, than each variable separately, not as a normality of all variables.

Results and discussions Waste water Substances that were positively detected in PSDs (as ng/sampler) are depicted in Table 1. The following PFRs were demonstrated; TCPP and TDCPP in all of 11 WWTPs and TCEP in 10 WWTPs. The highest values were for TCPP which was found in 9 WWTPs at µg/POCIS levels with a maximum value of 4.5 µg/POCIS. The maximum found concentration of TDCPP was 5.6 µg/POCIS, the values for the other compounds were typically hundreds of ng/POCIS. The maximum for TCEP was 220 ng/POCIS. For BFRs which were over LOQ (0.5 ng/SPMD) there were just two PBDE congeners: BDE47 and BDE99 which were positively detected in all WWTPs apart from one. The detected maximum was 9.7 and 3.4 ng/SPMD (BDE47 and BDE99, respectively). In all WWTPs, the BDE47 found concentration was approximately 2.5 times higher than the one of BDE99. The proportion of individual congeners is completely different compared to the products on the market or sludge. In consumer products (Ocelka et al. 2017) and in sludge the dominant congener was BDE209, which was not measured by the passive samplers nor in the wastewater itself. BDE209 molecule is the heaviest and most hydrophobic PBDE congener and is only very sparingly soluble in water. The ratio of the congeners was fundamentally changing during the transport from the sources - materials with added FRs - to treated wastewater.

Table 1: Wastewater in 11 WWTPs measured by passive samplers POCIS and SPMD (ng/sampler) CAS number

Substance

Mean

LOQ

Positive samples

PFR – Passive sampler POCIS (unit ng/POCIS) TCEP

115-96-8

TCPP TDCPP

13674-84-5

2300

13674-87-8

820

Sum PFR

3205

PBDE – Passive sampler SPMD (unit ng/SPMD) BDE47

5436-43-1

4,0

0,5

BDE99

60348-60-9

1,6

0,5

Sum PBDE

Sludge at WWTPs

5,6

Results from the sludge sampled at 10 WWTPs simultaneously with wastewater are shown in Table 2. 10 of the PFRs analysed, 10 of 16 were positively detected. PFRs diversity is not surprising because they can concentrate on the sludge from various sources. The presence of FR in wastewater sludge can be used as an indicator of presence in the environment, as due to the accumulation they can be easily detected. HBCDD was positive in all 10 samples. Isomers α and γ were present in approximately equal amounts. β isomer (LOQ=1 ng/g) was not detected in any sample, as in the commercial mixtures the proportion of this isomer is too low. Regarding BFRs, no PBBs were detected (similar to other matrices). From the 8 BDEs analysed, at least 7 congeners were positively detected in all of WWTPs. In this solid matrix, as well as in the commercial products analysed within an associated study (Ocelka et al. 2017), congener 209 was completely dominant.

Table 2: FRs in sludge at 10 WWTPs in year 2016 (µg/kg of dry sample) Substance

Positive CAS number Mean LOQ samples PFRs

TCPP

13674-84-5

1700

TiBP

126-71-6

180

TPP

115-86-6

140

TnBP

126-73-8

460

TDCPP

13674-87-8

TBEP

78-51-3

210

TCP

1330-78-5

130

EHDP

1241-94-7

TEHP

78-42-2

360

DCP

26444-49-5

790

Sum PFR

3700

HBCDD αHBCD

134237-50-6

βHBCD

134237-51-7

γHBCD

134237-52-8

Sum HBCDD

PBDE BDE47

5436-43-1

7.5

0.5

BDE100

189084-64-8

1.7

0.5

BDE99

60348-60-9

7.7

0.5

BDE154

207122-15-4

1.0

0.5

BDE153

68631-49-2

1.4

0.5

BDE183

207122-16-5

2.0

0.5

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0.5

760

10 10

PBDE in WWTPs sludge - regular measurements The concentration of 9 PBDE in samples (from 10 to 12) is analysed by the CISTA National reference laboratory every year. Levels of the sum of PBDE were relatively stable over the study period, with a decline from 38 µg/kg in 2010 to 31 µg/kg in 2015. The congeners BDE99 and BDE47 have the highest proportional of the PBDE sum (approximately 2/3 of total). These are also the congeners which reach the highest levels in living organisms (Hites 2004),(Kazda et al. 2004).

PBDEs in soils - regular measurements As the concentration levels of all PBDE congeners in these 40 soil samples in years 2012-2015 were below the LOQ (0.1 µg/kg dw), CISTA stopped the PBDE screening in 2016. For the same reason the soil samples are not evaluated further in the current study. The screening studies of background levels of PBDEs in soils in both the UK and Norway were described by Hassanin and co-workers who found total concentrations ranging from 65 to 12 000 ng/kg dry weight (Hassanin A. 2004). The most frequent congeners were BDE47, BDE99, BDE100, BDE153, and BDE154, which are the main congeners of penta-BDE and are not too influenced by alteration processes, such as oxidation and degradation during the air transport. BDE183, which is a marker of octa-BDE, was detected in concentrations from 9 to 7 000 ng/kg, and the median value was 50 ng/kg. Higher concentrations of PBDE were in forest soils than pastureland soils, with different composition of congeners also detected between different soil types. The highest concentrations were in the northern regions, where BDE47 concentrations increased with distance from the source, whereas the concentration of BDE99 and higher congeners decreased.

PBDE in pond sediment - regular measurements Between 2011-2015 CISTA analysed 31 pond sediment samples for the same 9 PBDE congeners which were measured in the sludge and soils: BDE 28, 47, 66, 85, 99, 100, 153, 154 and 183. Positively detected (over LOQ = (0.1 µg/kg dry weight)) were only three congeners, in 5 samples; 3 times for BDE47 (maximum concentration 0.2 µg/kg dw), 5 times for BDE99 (max 0.3 µg/kg dw), and 3 times by congener 183 (max 0.3 µg/kg dw). The total average sum for all congeners was 0.48 µg/kg and a median value was 0.45 µg/kg. PBDE was measured in samples from the field, forest and village ponds, but was not detected in rivers and pond flow water. The representation of individually measured congeners was relatively uniform, with BDE99 being the most represented (15%). The representation of other BDEs ranged from 10 to 12%.

Saturation bulky molecules of congener BDE209 with 10 bromine atoms rapidly sedimented in the aquatic environment. BDE209 was not detected neither in the aquatic environment not in human blood and adipose tissue in this project. On the contrary, the main source for entering into the environment (in the Czech Republic) was in the sum of PBDEs dominant. BDE209 occurs negligibly in surface waters due to its hydrophobicity. The BDE209 human health protection in the US is described in the study (Lucio G. Costa 2011). It was proved that PDBE209 in human organism may be quite inert and have a relatively short half-life as it is excreted and hardly permeating cell walls. However, the legislative ban was based on the detection of relatively high concentrations in infants and children with exposure pathways through breast milk and dust inhalation in households. The acute and chronic toxicity is relatively low, when the liver and thyroid are the most influenced organs (Kuriyama S. N. 2007),(Dishaw et al. 2014). Although BDE209 is highly stable, a study (An Exposure Assessment of Polybrominated Dipheyl Ethers 2010) has focused on the possibilities of the transformation of BDE209 to less brominated PBDE with potentially greater toxicity in the environment and in the human body. It was estimated that in the USA (An Exposure Assessment of Polybrominated Dipheyl Ethers 2010) due to inhalation and ingestion of the sum of PBDE people receive approximately from 400 to 700 ng/day, and from 8 to 50 ng/kg/day adjusted for body weight. 28% (from 2.2 to 14 ng/kg/day) of this total is represented by BDE209. These concentrations are much lower than the proposed and still discussed (by the Environmental Protection Agency in the USA (US EPA)) conservative limit of 7000 ng/kg/day.

Advanced Information Analysis In order to evaluate the possible transfer of PBDEs from WWTPs sludge to agricultural land resources, a methodology of discriminatory and canonical correlation analysis was used. Clustering analysis of PBDE in samples of wastewater, sludge and pond sediments was performed for 116 samples. Seven congeners were included: BDE28, 47, 54, 99, 100, 163, 183. BDE209, which dominated in the sludge during the sampling campaign in 2016, was not included in the analysis, because it was not detected in the wastewater, even not during the regular annual measurements in soils and sediment ponds.

Clustering analysis of PBDE Clustering analysis divides PBDE congeners measured data in 4 clusters, as is described in Graph 1. This division was confirmed by linear discriminatory analysis.

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Graph 1: Clustering PBDE analysis

below the LOQ. It should be mentioned, that its low level in water can be caused by a low diffusivity into the SPMD passive sampler, due to the size and shape of the molecules. The publication (Jhon F. Narvรกez Valderrama 2016) states that low diffusion coefficients of PBDEs and in particular of BDE 209 in LDPE imply that the polymer diffusion coefficients need to be taken into account.

BDE 209 Saturation bulky molecules of congener BDE209 with 10 bromine atoms rapidly sedimented in the aquatic environment. BDE209 was not detected neither in the aquatic environment not in human blood and adipose tissue in this project. On the contrary, the main source for entering into the environment (in the Czech Republic) was in the sum of PBDEs dominant. BDE209 occurs negligibly in surface waters due to its hydrophobicity.

Correlation dependence among PBDE congeners was carried out, for PFRs such correlations were not demonstrated. In the performed analysis, 116 samples were included, where 7 PBDE congeners were measured in all samples. The correlation among analysed PBDE congeners was significant for the solid matrices. All congeners exclude BDE183 show high correlation dependence. The correlation of wastewater was significant only for congeners BDE47 and BDE99. Heavier congeners with more bromine atoms have a propensity for accumulation on solid phases. The transfer of contaminants from wastewater to the environment is dependent on the phase. The cluster analysis leads to split into 4 clusters. No cluster has correlation with the sampling locations. Cluster 2 was mostly represented by the pond sludge, had the lowest values of all PBDE congeners, and included 46% of all samples. Cluster 1 was characterized by a uniform distribution of the congeners proportionally, unlike cluster 2, where the samples are at levels that are close to LOQ. Cluster 4 contained the highest values, represented 9% of all samples, and contained samples of the sludge with the highest concentrations from the annual CISTA monitoring. At the WWTPs the congeners of the lower brominated PBDE as BDE47 or BDE99 are almost completely transferred into the liquid phase, contrary to higher brominated congeners (153 and higher) which are present in the solid sludge phase. The heaviest congener 209 was not measureable in the aqueous phase at all. Mainly due to the absence of BDE209 in the regularly annually measured samples, we failed to make a clear conclusion about behaviour of this congener. It was concentrated in sewage sludge, where its concentration was nearly 40 times higher than the sum of all the other congeners. It follows that the risk of its transfer to the land resources is the highest of all the other congeners. Its concentration in wastewater in the WWTPs output is

The BDE209 human health protection in the US is described in the study (Lucio G. Costa 2011). It was proved that PDBE209 in human organism may be quite inert and have a relatively short half-life as it is excreted and hardly permeating cell walls. However, the legislative ban was based on the detection of relatively high concentrations in infants and children with exposure pathways through breast milk and dust inhalation in households. The acute and chronic toxicity is relatively low, when the liver and thyroid are the most influenced organs (Kuriyama S. N. 2007),(Dishaw et al. 2014). Although BDE209 is highly stable, a study (An Exposure Assessment of Polybrominated Dipheyl Ethers 2010) has focused on the possibilities of the transformation of BDE209 to less brominated PBDE with potentially greater toxicity in the environment and in the human body. It was estimated that in the USA (An Exposure Assessment of Polybrominated Dipheyl Ethers 2010) due to inhalation and ingestion of the sum of PBDE people receive approximately from 400 to 700 ng/day, and from 8 to 50 ng/kg/day adjusted for body weight. 28% (from 2.2 to 14 ng/kg/day) of this total is represented by BDE209. These concentrations are much lower than the proposed and still discussed (by the Environmental Protection Agency in the USA (US EPA)) conservative limit of 7000 ng/kg/day.

Clustering analysis of PBDE Clustering analysis divides PBDE congeners measured data in 4 clusters, as is described in Graph 1. This division was

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confirmed by linear discriminatory analysis.

Graph 1: Clustering PBDE analysis

concentration in wastewater in the WWTPs output is below the LOQ. It should be mentioned, that its low level in water can be caused by a low diffusivity into the SPMD passive sampler, due to the size and shape of the molecules. The publication (Jhon F. Narváez Valderrama 2016) states that low diffusion coefficients of PBDEs and in particular of BDE 209 in LDPE imply that the polymer diffusion coefficients need to be taken into account.

Conclusion

This study focused on clarifying the transfer of FRs to environmental matrices from WWTPs. In the current and also past studies, usied BFR and PFR were analysed in the sludge at the WWTPs. PBDEs were determined in agricultural soils, and unlike other widely studied POPs such as PCBs (polychlorinated biphenyls), PAHs (polycyclic aromatic hydrocarbons) and OCP (persistent chlorinated pesticides), PBDE was not detected over the LOQ in any sample. In pond sediments, PBDEs were found only in 5 samples and none of them exceed the value of 1 mg/kg (sum PBDE). As expected, even in the pond sediments were not detected significant concentrations of FR. Concerning PFRs at WWTPs, there were detected 10 substances in the sludge and 3 in the aquatic phase from the PSDs. As far as HBCDD in sludge is concerned, there were positive detections of α and γ isomers almost in all samples. The ration of α, β and γ isomers does not exactly follow the commercially described composition as also from the literature is known, that this ratio may be changed considerably during transfer to different mediums. In wastewater and sludge, as expected, PBBs were not detected, which is in contrast to the long-term monitoring of other POPs, which were detected in the sludge in the past. Concerning PBDEs, the behaviour of individual congeners in the environment varied depending on the number of bromines. The smaller BDE molecules occurred in the aqueous phase and in the treated waste water, when BDE47 was the most dominant. The fully substituted congener BDE209 was not detected in the same water at the WWTPs outlet, although it was dominant in the sludge. After the legislative ban PBDEs were not commercially produced as FR anymore, the contamination is therefore historical. The most widely produced PBDE was Deca-BDE (BDE209), which was often detected in various matrices. It is probably the most-proportional congener in agricultural land resources. The development of FRs concentrations in solid matrices is difficult to predict as effective legislative action including the ban of some BFRs has been conducted but on the contrary, historically applied FR will continue to be released to wastewaters from already manufactured products. The current study has shown that substitutes of BFRs, organophosphates, whose toxicity and bioaccumulative properties are not currently thoroughly examined, occur

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widely in the environment, at quantities exceeding previously used BFRs such as HBCDD, PBDE and PBB. Therefore, the authors recommend continuing screening of FRs in sludge exported from the WWTPs, especially those destined for agricultural uses, as a useful indicator of their overall contamination levels in the environment. Due to previously detected PCBs and PAHs, an application of WWTPs sludge to agricultural land resources cannot be recommended without a detoxification. The dehalogenation can be performing for example by methods with subsequent transfer of the inorganic phase of the bromine atom into toxicologically more neutral phase.

Acknowledgement Acknowledgments the project "Flame retardants in products and internal environment in the Czech Republic" - TB030MZP003 supported by Technical Agency of the Czech Republic in the BETA program.

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