Arhiv za higijenu rada i toksikologiju-Archives of Industrial Hygiene and Toxicology by Arhiv

ISSN 0004-1254

ARHIV ZA HIGIJENU RADA I TOKSIKOLOGIJU

ARCHIVES OF INDUSTRIAL HYGIENE AND TOXICOLOGY

Arh Hig Rada Toksikol • Vol. 65 • No. 4 • pp. 347-424 • ZAGREB, CROATIA 2014

CONTENTS Review Mycotoxicoses in children

Maja Peraica, Darko Richter, and Dubravka Rašić

347

Tomislav Ivanković, Jasna Hrenović, Grigorios Itskos, Nikolaos Koukouzas, Davor Kovačević, and Jelena Milenković

365

Ahmet Topal, Muhammed Atamanalp, Ertan Oruç, Yeliz Demir, Şükrü Beydemir, and Alparslan Işık

377

In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout

Mustafa Yardimci, Yusuf Sevgiler, Eyyup Rencuzogullari, Mehmet Arslan, Mehmet Buyukleyla, and Mehmet Yilmaz

387

Sex-, tissue-, and exposure duration-dependent effects of imidacloprid modulated by piperonyl butoxide and menadione in rats. Part I: oxidative and neurotoxic potentials

Masoumeh Khalili, Mohammad Ali Ebrahimzadeh, and Yaghoub Safdari

399

Antihaemolytic activity of thirty herbal extracts in mouse red blood cells

Jerome-Philippe Garsi, Eric Samson, Laetitia Chablais, Sergey Zhivin, Christine Niogret, Dominique Laurier, and Irina Guseva Canu

407

Half-century archives of occupational medical data on French nuclear workers: a dusty warehouse or gold mine for epidemiological research?

Veljko Flego, Dubravka Matanić Lender, and Ljiljana Bulat-Kardum

417

Case report Pericardial effusion as the first manifestation of occupational tuberculosis in a health care worker

423

Letter to the Editor A survey of professional satisfaction among anaesthesiologists in Serbia

A21 A24 A25

New editions (in Croatian) Announcement (in Croatian) Report (in English)

Ljiljana Gvozdenović, Tatjana Batak, Dejan Ivanov, and Radovan Cvijanović

Original articles Alkaline disinfection of urban wastewater and landfill leachate by wood fly ash

Cover page: Yet another winter embraces the world that surrounds us. Photographed by Linda Poščić Borovac. Disclaimer: This photo is intended to evoke the content of this issue of the journal. It is not intended for instructional or scientific purposes.

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DOI: 10.2478/10004-1254-65-2014-2557

Review

Mycotoxicoses in children Maja Peraica1, Darko Richter2, and Dubravka RaĹĄiÄ&#x2021;1 Unit of Toxicology, Institute for Medical Research and Occupational Health1, Department of Paediatrics, University Hospital Centre Zagreb2, Zagreb, Croatia Received in July 2014 CrossChecked in July 2014 Accepted in November 2014

Mycotoxicoses are acute and chronic poisonings caused by mould toxins called mycotoxins. Although acute mycotoxicoses, caused by high mycotoxin levels in food are rare nowadays, they need to be described in order to inform physicians and other health care workers about their symptoms. Children are more sensitive to mycotoxins because of their lower body mass, higher metabolic rate, and underdeveloped organ functions and detoxication mechanisms. Some mycotoxicoses appear only in children, and some are more pronounced in children than in adults. Acute mycotoxicoses in children are reported poorly, mostly because they occur in the tropical regions with poor healthcare coverage. In developed countries healthcare authorities are more concerned about child exposure to low levels of mycotoxins with immunotoxic, genotoxic or carcinogenic properties. KEY WORDS: 3-NPA; aflatoxins; ergot; fumonisins; ochratoxins; trichothecenes; Ustilago maydis toxins; zearalenone

Mycotoxins are the metabolites of moulds (fungi imperfecti) that contaminate food and feed around the world. Humans are continuously exposed to mycotoxins, as not even good agricultural and production practices can entirely prevent their production. Mould growth and the production of mycotoxins depend on the genetic properties of individual mould species and on environmental conditions such as temperature, humidity, and insect infestation. Grains are usually contaminated with several mycotoxins, which may either be the consequence of contamination with several strains of moulds, each producing its own mycotoxin, or of the production of several mycotoxins by one mould species. Out of about 400 known mycotoxins, only a few have been investigated in detail in laboratory animals and even fewer have known toxic and other health-threatening effects in humans. The main route of exposure in humans is ingestion of contaminated food, but for some mycotoxins inhalation or dermal exposure are also possible. It is

very difficult to assess the health risks associated with mycotoxins in children, as reliable exposure data are seldom available, mycotoxins often occur with other mycotoxins or other toxins, other dietary elements and contaminants affect their toxicity, and many diseases induced by mycotoxins have long latency (1). For most mycotoxins there are no epidemiological data on their carcinogenicity in humans, but for some (such as aflatoxin B1) there is convincing evidence of an association with human hepatocellular carcinoma. Other mycotoxins (such as ochratoxin A, patulin, and fumonisin B1) are suspected to be human carcinogens due to positive results in experimental animals (2). Young animals and children are more sensitive to the adverse effects of mycotoxins than adults because of lower body mass, higher metabolic rate, and underdeveloped organ functions and detoxication mechanisms. Most mycotoxins may cause acute and chronic mycotoxicoses. Acute mycotoxicoses are serious diseases caused by exposure to very high mycotoxin

348 levels. They are most common in tropical regions where exposure of children to certain mycotoxins, particularly aflatoxins, is continuously high. Conversely, chronic mycotoxicoses are associated with low-level exposure. Mycotoxicoses are important differential diagnoses and should be taken into consideration when a clinical disorder affects a group of persons in whom it cannot be related to an infectious agent. Even though clinical symptoms and the outcome are more severe in children than adults, mycotoxicosis reports seldom distinguish the two groups. This article is an attempt to review what little is known about mycotoxicosis in children and the available data on mycotoxins and biomarkers of exposure to mycotoxins in various biological materials (blood of pregnant women, umbilical cord blood, blood and urine of children). Aflatoxins Aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) are mycotoxins produced by Aspergillus spp. Their names come from the colour of their fluorescence (B - blue, G - green). The most common and also the most toxic is AFB1. In the organism, AFB1 and AFG1 are metabolised to various compounds, including aflatoxin M1 (AFM1) and M2 (AFM2), which appear in human and cow milk, urine, and faeces. Aflatoxins contaminate nuts, maize, sorghum, millet, beans, cassava, and rice, whereas their metabolites AFM1 and AFM2 contaminate milk and dairy products. Animal studies show that, apart from being acutely toxic, aflatoxins are immunosuppressive, mutagenic, teratogenic, and carcinogenic (2). The target organ of their toxicity and carcinogenicity is the liver. The International Agency for Research on Cancer (IARC) has classified natural mixtures of AFs as carcinogenic in humans (Group 1) and AFM1 as potentially carcinogenic in humans (Group 2) (2). Aflatoxins are well absorbed by the gastrointestinal (GI) tract and transported to the liver, where they are metabolised by a number of competitive pathways. They are bioactivated mainly by cytochrome P450 (CYP) 3A enzymes (CYP3A4, CYP3A5, and CYP3A7) in the first oxidative phase. AF activation is also possible in the small intestine due to the presence of CYP3A4 and CYP3A5. About 1-3 % of the absorbed AFs is activated to highly mutagenic exo-epoxide (AFB1-8,9-epoxide), whose half-life (t1/2)

Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

is only one second in aqueous buffer but long enough to react with DNA to yield 98 % of the N7-guanyl adduct. Wojnowski et al. (4) have associated high levels of the mutagenic AFB1 exo-8,9-epoxide in humans exposed to dietary AFB1 with the CYP3A5 polymorphism, particularly in individuals with low CYP3A4. AFB1 8,9-epoxides hydrolyse to nongenotoxic dihydrodiol, which is further metabolised to its dialdehyde (5). The amount of AFB1-DNA adducts is greater in the liver than in other organs and generally correlates with AFB1 levels and species susceptibility to hepatocarcinogenesis (6). DNA- and albumin-AFB1 adducts (AFB1-DNA, AFB1-alb) are reliable biomarkers of long-term exposure to AFB1. The major detoxication route for AFB1 metabolites is via conjugation with glutathione (GSH) to form the AFB1-GSH conjugate excreted in urine and bile, and this reaction determines species resistance to aflatoxin toxicity (7). In some regions of Africa and Asia the risk of hepatocellular carcinoma (HCC) is very high, particularly in hepatitis B surface antigen-positive (HBsAg+) persons. Without AF exposure, the relative risk of HCC development in HBsAg+ persons is 7.3, but with AFs exposure it soars to about 60 (8). In adults, AFs cause deletional mutations in the P53 tumour-suppressing gene and therefore activate oncogenes. However, in two to five-year-old children in Guinea with high AF-albumin adduct levels Turner et al. (9) found no mutations in P53 tumoursuppressing gene, which suggests that children could be protected from high AF carcinogenicity if vaccinated against HBV infection, as it significantly increases the risk of HCC. In order to decrease the risk of HCC, South Africa has recently introduced vaccination against HBV infection in infants (receiving three shots by their 14th week). In a five-year follow up Tsebe et al. (10) reported that this vaccination decreased significantly the HBsAg+ carrier rate in children younger than five years. Exposure to AFs may be assessed by measuring parent compound or metabolites in blood and urine, or AFB1-alb and AFB1-DNA adducts in blood. The first method is only reliable for recent exposure to AFs because of their fast metabolism and therefore a tendency to underestimate longer exposure. This is probably why there were no AFB1-positive urine findings in children from Cameroon, even though 11 % of the samples showed the presence of its metabolite AFM1 (11). The second method that measures AF-alb

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and AF-DNA adducts is therefore more reliable for longer exposures (2-3 months). Aflatoxicosis is a clinical condition following exposure to aflatoxins. Acute aflatoxicosis is a severe poisoning that results in serious liver damage, often with fatal outcome. Chronic aflatoxicosis is the consequence of exposure to lower levels of AFs over a longer period and may have chronic nutritional and immunological consequences. In terms of cancer risk, the effects are cumulative. The first report of acute AF hepatotoxicity in children dates back to 1971 (12), when 20 malnourished children from 1.5 to 5 years of age were hospitalised for symptoms of acute liver damage, 13 of whom were subicteric. They had been eating a peanut protein meal contaminated with aflatoxin (30 μg kg-1) from five days to one month. At the end of the one-year follow up, 12 had gross hepatomegaly and three had palpable liver with sharp edges. Three children died from hepatic coma after 1.5 years. Another outbreak of acute aflatoxicosis in India in 1974 practically spared the children under the age of five years, who made about 8 % of the affected patients (13,14). In the most severe aflatoxicosis outbreak in Kenya in 1981, the number of affected children was never established (15), but in the outbreak of 2004, it was half of those whose age data was known (308 of 317 people). Sixty eight children were under the age of five and 90 from five to fifteen (16). Exposure to AFs in utero in Africa is alarming (Table 1). The findings of AFs in umbilical cord blood from 1988 were the first to evidence transplacental

transport of AFs in humans (17). Egyptian researchers established a significant negative correlation between birth weight and AF levels in umbilical cord blood (18, 20). In Gambia, a negative correlation was also found between AF-albumin adduct levels in mother’s blood and weight and height gain in the first year of child’s life (19). It is estimated that up to 0.43 % of AFs from consumed food is excreted as AFM1 in breast milk (21) and that mothers’ exposure to AFs depends on their socioeconomic status (22, 23). In African countries, Turkey, Iran, and United Arab Emirates the frequency of AF-positive breast milk samples is rather high (Table 2). In Egypt and Gambia the concentrations of AFM1 in breast milk showed great seasonal variations (16-96 %), peaking in the dry season (summer) (26, 41). Breastfed children in Africa are exposed to lower AF concentrations than weaning children because the staple food in the weaning period is maize and cassava, which are often contaminated with AFs (21, 42, 43). In Europe the frequency of AFM1-positive breast milk samples does not exceed 5 %, and the AFM1 levels are much lower than in Africa, but they still exceed the maximum tolerance limit for infant formula, follow-up formula, infant milk, and follow-up milk of 0.025 µg kg-1 in some European countries (44, 45). Similar to breast milk, the frequency of AFpositive blood and urine samples of African children is high, and AF concentrations peak in the summer (Tables 3 and 4). In children from Gambia AF-albumin adduct levels in the summer are twice as high as in

Table 1 Reported aflatoxin and AF-alb adduct findings in umbilical cord blood in Africa

Country

Positive samples/ analysed (N)

Positive by toxin (N, positive samples/analysed, mean and range in ng L-1, where available) B1

Method

Ref.

Ghana*

63/188 (34 %)

17/188 (9 %) (185-43,822)

17/188 (9 %) (11-925)

21/188 (11 %) (34-7,320)

21/188 (11 %) (30-572)

HPLC-FD

Nigeria

9/78 (11 %)

1/78 (1 %) 10

3/78 (4 %) (25-8,942)

5/78 (6 %) (155-378)

HPLC-FD

United Arab Emirates

110/201 (57 %)

27/201 (13 %) X=2,040 (228-15,225)

107/201 (53 %) X=1,108 (110-4,060)

31/201 (15 %) X=854 (210-3,700)

HPLC-FD

49/99 (48 %) X=10.1 Gambia** 49/99 (48 %) ELISA 19 (5.0-89.6) Median=2.5 ND–not detected; HPLC-FD–High pressure liquid chromatography with fluorescent detection; ELISA–enzyme-linked immunosorbent assay *In addition, AFG1 was detected in three samples (611, 1354, and 354 ng L-1) and AFG2 in one (37 ng L-1)

**AF-alb adducts (pg mg-1 of protein)

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Table 2 Frequency of AFM1- and AFM2-positive breast milk samples and their concentrations across the continents

No. positive/ analysed

Country

No. positive/analysed (range ng L-1) AFM1 AFM2

Method

Ref.

HPLC-FD

ELISA

HPLC-FD

ELISA

HPLC-FD

Africa Cameroon

Egypt

3/62 (4.8 %) Apr-Jun

23/37 (62 %)

Sep-Dec

16/45 (35 %)

Jan-May

27/38 (71 %)

Total

66/120 (55 %) 248/443 (56 %) 87/125 (70 %)

Ghana

90/264 (34 %)

Nigeria

41/50 (82 %) 37/99 (37 %)

Sudan*

51/94 (54 %)

Zimbabwe

6/54 (11 %)

3/62 (4.8 %) (5.0-62.5) 23/37 (62 %) X=0.35 (0.3-2.1) 16/45 (35 %) X=0.34 (0.3-1.9) 27/38 (71 %) X=0.3 (0.2-1.6)

248/443 (56 %) (4.2-889) 87/125 (70 %) X=74.4 (73-328) 59/264 (22 %) (20-1,816) 41/50 (82 %) (4.62-92.14) 26/99 (26 %) X=19.0 51/94 (54 %) X=0.41 6/54 (11 %) (0.2-50)

18/264 (7 %) (16-2,075)

24/99 (24 %) X=12.2

Middle East Jordan

100/100 (100 %)

United Arab Emirates

144/201 (71 %)

Iran

100/100** (100 %) X=68 107/201 (53 %) X=1,108 (210-4,060) Asia

31/201 (13 %) X=854 (210-3,700)

Hamadan

8/132 (6 %)

8/132 (6 %) X=9.45 (7.1-10.8)

ELISA

Sari

1/136 (0.7 %)

1/136 (0.7 %) 20***

ELISA

Tabriz (rural)

20/91 (22 %)

20/91 (22 %) X=6.96

Tabriz (urban)

ELISA

0/91

Teheran

157/160 (98 %)

ELISA

157/160 (98 %) X=8.2 (0.3-26.7)

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Europe France

0/42 Lombardi

1/231 (0.004 %)

1/231 (0.004 %) 194***

HPLC-FD

4/82 (5 %)

4/82 (5 %) X=55.3 (7-140)

HPLC-FD

75/75 (100 %)

75/75 (100 %) (60.9-300.0)

HPLC-FD

Italy

Turkey

ELISA

South America 1/50 (2 %) Brazil

2/224 (0.01 %) 2/100 (2 %)

1/50 (2 %) 24*** 2/224 (0.01 %) 5*** 2/100 (2 %) >0.3***

ND–not detected; HPLC-FD–high pressure liquid chromatography with fluorescent detection; ELISA–enzyme-linked immunosorbent assay *In 13 samples both AFM1 and AFM2 were found **In 95 % of samples the concentration of AFM1 was higher than 25 ng L-1 ***Concentration in a single or both samples

the winter (41). The highest AF-albumin adduct levels were found in children with acute hepatitis B, followed by children with chronic hepatitis, while healthy children had the lowest levels. Although the immunotoxic effects of AFs in experimental animals are well known, there are no data on their immunotoxicity in children. In adults Jiang et al. (50) found a significant negative correlation between high AFB1-albumin adduct levels and the percentage of activated T and B cells. Denning et al. (51) believe that the immunotoxicity of AFs greatly contributed to acute lower respiratory tract infections in Filipino children, eleven of whom died, whereas Oyelami et al. (52.) found significant concentrations of AFs in the lung tissues of Nigerian children post mortem. In a study of 479 blood samples taken from children from 9 months to five years in Benin and Togo, 99 % were positive for AF-alb adducts (42). In this and another longitudinal study of 400 children from Benin peak serum AF-alb adducts in fully weaned children was 2.5 times higher than in partially breast-fed children (23, 46). These authors also found negative correlation between AF-alb adducts and children weight and height (46). In another study of children from Sierra Leone, the authors suggested that the failure to thrive may have been caused by exposure

to AFs and another mycotoxin - ochratoxin A (OTA) (53). In tropical countries neonatal jaundice is very frequent. A Nigerian study of 327 neonates with jaundice and 60 controls has demonstrated that glucose-6-phosphate dehydrogenase deficiency and/ or the presence of aflatoxins in serum are the risk factors for neonatal jaundice (54). A study performed in Kenya showed that school children with hepatomegaly, which is very frequent in this country, had significantly higher levels of AF-alb adducts than children without hepatomegaly (55). Some diseases that affect children in tropical regions, such as Rey’s syndrome and kwashiorkor were erroneously attributed to AF exposure (56, 57). Frequent post-mortem findings of AFs in various tissues of children with these diseases were probably the consequence and not the cause of liver injury. It is likely that their damaged liver could not metabolise the AFs. Ochratoxin A Ochratoxin A (OTA) is the most toxic and most common ochratoxin. Ochratoxins are produced by Penicillium verrucosum and several species of Aspergillus moulds from all over the world. They contaminate foodstuffs of plant origin (cereals, coffee

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beans, raisins, wine, beer, and grape juice) and commodities of animal origin such as pork and poultry meat, eggs, milk, and dairy products due to the carryover effect. Most data on food contamination with this heat-stable mycotoxin are from Europe (58). In experimental and domestic animals the main target organs of OTA toxicity are the kidney and liver, but it also affects the heart, blood (causing aberrations in coagulation factors), GI tract, lymphoid tissue, and bone marrow. OTA is readily absorbed by the upper GI tract and persists in the circulation for a long time due to binding to plasma proteins, enterohepatic circulation, and kidney resorption, enhanced by organic anion transporters (59). Its plasma half-life in humans is 35.55 days (60), which is extremely long and makes plasma OTA a good biomarker of exposure. There are several mechanisms involved in OTA toxicity: production of reactive oxygen species, inhibition of mitochondrial respiration, disruption of calcium homeostasis, inhibition of protein synthesis, and DNA damage (61-65). IARC has classified OTA as carcinogenic in experimental animals with limited evidence for its carcinogenicity in humans (Group 2B) (66). Despite severe acute toxicity in laboratory animals, ochratoxicosis is quite rare in humans (67). So far, no ochratoxicosis has been reported in children. Some researchers believe that OTA is involved in the

development of Balkan endemic nephropathy (BEN) and otherwise rare urothelial tumours, whose incidence is high in the endemic regions of the Balkans (68, 69). In several studies performed in the endemic regions of Bulgaria and Croatia the level of OTA or the frequency of OTA-positive food and human blood samples were higher than in control regions (for a more comprehensive review see 70). Low OTA concentrations are frequently found in blood and urine of apparently healthy persons in all countries where it was looked for, with significant geographical and seasonal variations (70-72). Various studies have demonstrated that the blood levels of OTA are higher in patients with chronic renal insufficiency treated with dialysis (for a review see 73). In Europe the calculated daily human exposure to OTA ranges from 0.7 to 4.7 ng kg-1 body weight (b.w.), which is below the tolerable daily intake of 14 ng kg-1 b.w. proposed by the Joint FAO/WHO Experts Committee on Food Additives and accepted by the European Scientific Committee (ESF) (57, 74). EFSA established the tolerable daily intake of 18 ng kg-1 b.w. (75). Most data on child exposure to OTA are also from Europe. In Switzerland Zimmerli and Dick (76) reported twice as high OTA concentrations in the umbilical cord as that in maternal blood, indicating active transplacental transport of OTA.

Table 3 Aflatoxin B1-albumin adducts in blood of children

Country

Age (years) Fully weaned

Benin/Togo

Partially breast fed Total

Acute HBV infection Chronic HBV infection

Guinea

18.0 (15.2-21.3)

475/479 (99 %) 13/118 (11 %)

32.8 (5-1064) 8.7 (5.0-30.3)*

444/444 (100 %)

31.6 (2.2-495)

404/404 (100 %) 6/6

96.9 (45.2-207.7)

34/34 (100 %)

44.9 (32.3-62.5)

119/124 (96 %)

9.9 (8-8-11.0)

3.14 ±1.05**

105

3.47±0.85**

0.9-5 0.4

3-4

Taiwan

Ref.

45.6 (38.8-53.7)

2-5 HBsAg (fmol mg-1 prot.) HBsAg+ (fmol mg-1 prot.)

AFB1-alb adducts pg mg-1 albumin Mean (95 % CI)

Healthy Gambia

No. of positive/ analysed

13-15

41 47

Cl-confidence limits; HPLC-FD–high pressure liquid chromatography with fluorescent detection; ELISA–enzyme-linked immunosorbent assay *range

**standard deviation

Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

OTA is also a frequent contaminant of breast milk. Its concentrations are about one quarter of those in maternal plasma (77) and the exposure of breast-fed children often exceeds the daily limit of 14 ng kg-1 b.w. (Table 5). In colostrum OTA concentrations are much higher than in mature breast milk. Obviously, breast milk OTA contamination is related to maternal dietary habits, which may vary from country to country. In Italy, significantly higher OTA concentrations in breast milk correlate with the consumption of bread, bakery products, and pork meat (36), while in Norway they correlate with the consumption of liver paste (liverwurst, liver paté) and cakes (cookies, fruitcakes, chocolate cakes) (86). An Egyptian study (78) looked at a number of biochemical parameters in blood and urine of breast-fed children with high and low levels of OTA in maternal milk and children’s blood. Elevated OTA levels (in either milk or blood) correlated with a microglobulinuria, which was significantly greater in children with high than with low OTA concentrations and indicated initial kidney lesion. EFSA suggested that infants and children may experience higher rate of exposure than adults (75). It is not possible to link exposure to OTA during early childhood with any known human disease, although Schwartz (89) has put forward the hypothesis that mothers’ consumption of OTAcontaminated food and OTA exposure in early childhood may be the cause of testicular cancer in adulthood. This theory is based on the correlation between the incidence of testicular cancer and percapita consumption of food items contaminated with OTA (coffee and pig meat) in 19 countries. In tropical countries children are frequently exposed to OTA and AFs at the same time (44). Any health effect of this combination of mycotoxins, such as growth failure, could only be hypothesised (53). In a recent study in children in Cameroon under five years of age combined exposure to OTA and several other mycotoxins (AFs, fumonisin B1 – FB1, deoxynivalenol – DON, zearalenone – ZEA, α-zearalenol – α-ZEA and β-zearalenol β-ZEA) did not correlate with the degree of malnutrition (11). Trichothecenes Trichothecenes are a group of about 170 mycotoxins produced mostly by the moulds of the Fusarium strains that are common in mild climates. Other strains that produce them include Trichoderma, Trichothecium, Myrothecium, and Stachybotrys. Only a few types of trichothecenes are found in grains (wheat, oats, maize,

353 barley) for human and animal consumption. The most common are deoxynivalenol (DON previously called vomitoxin), nivalenol (NIV), and diacetoxyscirpenol (DAS), while T-2 toxin is rare. IARC has designated DON, NIV, and T-2 toxin not classifiable as to carcinogenicity to humans (Group 3) (66). Trichothecenes inhibit protein synthesis and activate mitogen-activated protein kinases (MAPKs) that are involved in immune response and apoptosis signalling (90). The main characteristic of trichothecenes toxicity is immunomodulation. Lower doses increase resistance to pathogens, up-regulate many immunerelated genes, and elevate serum IgA levels. Higher doses injure tissues with high cellular turnover such as bone marrow, lymph nodes, thymus, spleen, and intestinal mucosa. The consequence is a weaker immune response. In general, trichothecenes are haematotoxic and immunotoxic without genotoxic and carcinogenic properties (91-94). Large outbreaks of acute mycotoxicosis caused by T-2 toxin, called alimentary toxic aleukia (ATA), were seen in the USSR in the 1930s and 40s. This disease mostly affected people from 10 to 40 years of age from rural areas because of the ingestion of grains that remained beneath the snow in the fields over the winter (95). Breast-fed infants of mothers affected by ATA were not ill. The first symptoms were local irritation of oral mucosa and painful swallowing. They developed after a few hours of ingestion of contaminated grains and subsided after two to three days if the exposure ceased. If it continued, the disease progressed to the second, leukopoenic stage with minimal symptoms, and then to progressive leucopoenia, granulocytopoenia, relative lymphocytosis, anaemia (low RBC or haemoglobin count), and thrombocytopoenia. In case of mild or discontinued exposure, patients would recover completely. The third stage was characterised by pharyngo-haemorrhagic symptoms with severe necrotic pharyngitis and petechial rush, characteristically involving the trunk, inner sides of the arms and hips, and the inguinal fossae. These symptoms were accompanied by nose and mouth bleeds and bleeding in the stomach and intestines. This stage of ATA had a mortality rate of 50 %. The fourth stage was the recovery stage when leukocytes counts started to increase, but it was also the stage when bacterial infections such as pneumonia and purulent tonsillitis appeared. Several later outbreaks of trichothecene toxicosis were not so severe. A large outbreak in the Kashmir Valley (India) also affected

133/134 (100 %)

110/110 (100 %)

95/97 (98 %)

5-14 (Dry season/ Boys)

Dry season/ Girls

Wet season/ Boys

Wet season/ Girls ND-not detected

Sierra Leone

43/50 (86 %)

2-4

Guinea

91/93 (98 %)

19/50 (38 %)

1-2.5

Egypt

No of positive/ analysed

Age (years)

Country

Table 4 Aflatoxins in urine of children

29/50 (58 %) 5.7 (0.6-43)

8/50 (16 %) 2,682 (179-18,000)

38/93 (41 %) (0.08-127)

32/97 (33 %) (1.2-115)

53/110 (49 %) (0.04-319)

19/93 (20 %) (0.1-12)

9/97 (9 %) (0.2-48)

18/110 (17 %) (0.2-152)

40/134 (30 %) (0.1-15.5)

5/50 (10 %) 1.4 (0.8-2.2)

1/50 (2 %) 189

47/134 (35 %) (0.6-188)

AFB2

AFB1

55/93 (59 %) (0.3-124)

42/97 (43 %) (0.1-35)

48/110 (44 %) (2.3-34)

56/134 (41 %) (0.5-374)

32/50 (64 %) 97.0 (8.0-801)

4/50 (8 %) 5.5 (5.0-6.2)

66/93 (71 %) (5.1-86)

62/97 (64 %) (1.3-41.3)

48/110 (44 %) (4.5-94)

71/134 (53 %) (4.5-130)

No. of positive/analysed Mean concentration (range ng L-1) AFM1 AFM2

18/93 (19 %) (1.0-150)

27/97 (28 %) (0.8-57.4)

42/120 (39 %) (0.4-138)

51/134 (38 %) (2.9-169)

3/93 (3 %) (1.1-2.0)

2/97 (2 %) (0.2-0.7)

ND**

3/134 (2 %) (0.1-1.5)

18/50 (36 %) 19.0 (1.4-199)

12/50 (24 %) 2.2 (0.9-8.0)

2/50 (4 %) 76.6 (72.1-81.1) 1/50 (2 %) 709

AFG2

AFG1

Ref.

354 Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

355

Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

the children (96). Symptoms appeared 15 minutes to one hour after consuming contaminated bread; they were milder than in ATA and disappeared immediately after exposure ended. In the GI tract they included abdominal pain, diarrhoea, blood in the stool, and vomiting), but the most frequent were irritated throat and secondary infections of the upper respiratory tract. Except for the study by Rubert et al. (97) there are no other reports of NIV and HT-2 toxin contamination of breast milk. In animal studies trichothecenes turned out to be about 40 times more toxic when inhaled than when ingested (98). The outbreak of idiopathic pulmonary haemorrhage (IPH) in Cleveland (Ohio, US) between 1993 and 1998 involved 37 children, mostly African American, who lived in humid houses damaged by flooding or plumbing and roof leaks (99). IPH was associated with exposure to satratoxin G and H and roridin that are produced by Stachybotrys chartarum on water-soaked cellulose walls. In 12 children the outcome was fatal, and 60 % of the children that returned home had recurrent pulmonary haemorrhage. It is not clear whether the toxins of S. chartarum caused IPH, as the Center of Disease Control declared that there was not enough evidence to confirm the suspicion (100). Zearalenone Zearalenone (ZEA) (previously also called F-2 toxin) is mainly produced by Fusarium moulds. ZEA is a field contaminant of wheat, maize, oats, and barley, particularly in warm and temperate climates (101). It can also be found in surface waters close to agricultural areas (102). In humans, ZEA is rapidly absorbed by the GI tract and metabolised to α-ZEA, β-ZEA, α-zearalenol (α-ZAL), and β-zearalenol (β-ZAL), which are then conjugated with glucuronic acid and rapidly excreted in bile and urine. Zearanol, a synthetic derivative of ZEA, is used as an anabolic agent for sheep and cattle in the USA, but in Europe it has been banned since 1989. In experimental animals ZEA is hepatotoxic, haematotoxic, immunotoxic, and genotoxic (101). It has low acute toxicity, and there are no reports of acute human ZEA mycotoxicoses. At chronic levels, ZEA and its derivatives show oestrogenic effect by binding to oestrogenic receptors and by modulating the activity of aldo-keto-reductases involved in the steroid synthesis. In female experimental and domestic animals they cause infertility, reduce litter size,

increase embryonic resorption, reduce milk production, hypertrophy mammal glands, change weight of the thyroid, pituitary, and adrenal glands, and increase oestrins. In male animals they reduce testis weight, testosterone production, spermatogenesis, and mating drive and lead to feminisation. Studies on experimental animals showed that ZEA crosses the placental barrier and is also excreted in breast milk. In a recent Spanish study (97), ZEA was found in 13 out of 35 samples of human breast milk (37 %) and its metabolites α-ZAL and β-ZAL in only one sample (2 %). Historically, in Puerto Rico thousands of children experienced puberty, mainly due to environmental hormone contamination (103). Some of these children had ZEA in their blood, probably as the consequence of zearanol use as growth promoting agent in animal breeding. Since 1989, Hungary has seen an increase in the number of early telarche/mastopathy patients (104). Five out of them 36 had blood ZEA levels between 18.9 and 103.5 ng mL-1. IARC has classified ZEA as a Group 3 carcinogen (66), but recent documents classify it and its metabolites as endocrine disruptors (105). Fumonisins Fumonisins are a group of 15 mycotoxins produced by Fusarium moulds (mostly F. verticilloides and F. moniliforme). In naturally contaminated grains the most frequent is fumonisin B1 (FB1), often accompanied by small amounts of fumonisin B2 (FB2) and fumonisin B3 (FB3). Fumonisins contaminate various grains, but are most common in maize (106). They are poorly absorbed by the GI tract and quickly eliminated from plasma with low accumulation in the kidney and liver. They exert their toxic effects by inhibiting ceramide synthase, the key enzyme in the sphingolipid metabolism. This results in lower sphingolipid de novo synthesis and reuse of sphinganine. Significantly increased blood sphinganine affects the membrane transport of folic acid by binding to folate receptors, which eventually leads to lower folate uptake (107). Fumonisins target different organs in domestic and experimental animals: in horses they cause leukoencephalomalacia, in pig pulmonary oedema, in rats they are predominantly nephrotoxic, and in mice they are hepatotoxic and teratogenic, causing neural tube defects (NTD) (2). There is a only one report of acute fumonisincaused mycotoxicosis that occurred in India (108).

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Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

Symptoms appeared after ingestion of contaminated sorghum or maize and included abdominal pain, borborygmi, and diarrhoea. The disease was selflimiting, and preschool children were less affected. In some regions where maize is staple food (Transkei in Southern Africa, China, and Northern Italy) the high frequency of oesophageal cancers is believed to be related to exposure to fumonisins or their producers (F. moniliforme) (109, 110). In the early 1990s, a higher prevalence of NTD (anencephaly and spina bifida) was observed in children born along the Texan and Mexican border by Mexican-American

women, and Hendricks et al. (111) suggested that this was caused by fumonisin exposure in the first trimester of pregnancy. High prevalence of NTD was also found in the Transkei region in Southern Africa (112), northern Iran (113), and several regions of China, where it was higher in rural than in urban population (114). In a large controlled study in China (130,142 women taking folic acid vs. 117,689 controls) the incidence of NTD was significantly decreased by folic acid supplementation (115). In Ireland, B12 and folic acid fortification of breakfast cereals decreased the rate of NTD from 47 to 13 per 10,000 births (116). In

Table 5 Ochratoxin A concentrations in breast milk

Country

Egypt Sierra Leone Brazil Sao Paolo Ribeirão Preto-SP Chile

No. of positive/ analysed 43/120 (36 %) 36/50 (72 %) 40/113 (35 %) 0/224 2/50 (4 %) 66/100 (66 %) 9/9 (100 %) 40/50 (80 %)

Iran, Sari

2/136 (1 %)

Victoria

2/100 (2 %) 9/50 (18 %)

Italy

22/111 (20 %) 74/85 (75 %) 61/82 (74 %) 198/231 (86 %)

Norway Slovakia Sweden Switzerland Turkey

38/115 (33 %) 17/80 (21 %) 23/76 (30 %) 23/40 (58 %) 4/40 (10 %) 75/75 (100 )

Concentration (ng L-1) Method Mean±SD (range) Africa 21.06±13.7 HPLC-FD (5.07-45.01) NI HPLC/Mf NI HPLC-FD 200-337,000 Americas NI HPLC-FD 10, 24 HPLC-FD 4 HPLC-FD (0.3-21) 106±45 HPLC-FD (44-184) 52±56 HPLC-FD (10-186) Asia ELISA 90, 140 HPLC-FD Australia 3, 3.6 HPLC-FD Europe 4189 HPLC-FD (1700-6,600) (100-12,000) HPLC-FD NI NI 30.43±66.9 HPLC-FD (5-405) 6.01±8.31 HPLC-FD (1-57) (10-130) HPLC-FD 30 HPLC-FD (10-182) (2.3-60.3) HPLC-FD (10-40) HPLC-FD 7.2 (5-14) HPLC-FD (620-13,111) HPLC-FD

NI-not indicated *LC/Mf: liquid chromatography with microfluorimetric detection

LOD (ng L-1)

LOQ (ng L-1)

Ref.

25.

200

NI NI

0.0003 10

39 38

0.3

0.8

1.6

200

100 20

NI NI

83 84

0.5

4.8 10 NI 10

14.4 40 5 NI

87 88 76 37

357

Peraica M, et al. MYCOTOXICOSES IN CHILDREN Arh Hig Rada Toksikol 2014;65:347-363

order to decrease the incidence of NTD, the US Food and Drug Administration (FDA) mandated fortification of grain products with folic acid (117). This resulted in lower NTD incidence in the general population, but not in several ethnic groups. There are reports of a 58 % drop in NTD occurrence rate and of an even more significant (95 %) drop in NTD recurrence rate when folate supplementation was applied starting one month before conception (116). However, some studies reported that folate supplementation was not that effective or not effective at all, and the disturbance of folate metabolism by fumonisins has not been confirmed, although there is no doubt about their neurotoxicity in experimental animals (118-120). IARC has classified fumonisins as carcinogenic in experimental animals with limited evidence of its carcinogenicity in humans (Group 2B carcinogen) (66). Ergot Ergot alkaloids are a group of about 40 toxins produced mostly by the fungal species of the genus Claviceps that contaminate rye, oats, and pearl millet. They are also produced by some strains of Penicillium, Aspergillus, and Rhizopus spp. (121). These toxins may be divided in three groups: lysergic acid derivatives (e.g. ergotamine and ergocristine), isolysergic acid drivatives (e.g. ergotaminine), and dimethylergoline derivatives (e.g. agroclavine) (122). Ergot is the name of sclerotia, a dark fungal mass that replaces the seed or kernel of the infected plant. Ergot poisoning is called ergotism and was common in European history (123). The clinical presentation of acute ergotism depends on the type of toxins that are produced by particular strains of the genus Claviceps. Ergot alkaloids are absorbed by the GI tract, distributed readily in plasma, and metabolised by CYP3A4. Some of them are conjugated with glucuronic acid and eliminated by biliary excretion (124). Claviceps purpurea produces toxins from the group of lysergic acid derivatives (ergotamine and ergocristine), which cause severe vasoconstriction. Poisoning symptoms include severe pain in the legs, loss of pulse, and oedema. Paraesthesia is followed by gangrene around the tendons, with painless demarcation. In the ergotism outbreak in Wollo (Ethiopia) in 1977-78 four children lost one or both legs (125).

Clavine alkaloids produced by Claviceps fusiformis can cause the convulsive type of ergotism that occurs 1-48 hours after ingestion of contaminated food. It starts with gastrointestinal symptoms (nausea, vomiting, dizziness) and continues with the nervous system symptoms (crawling sensation in the skin, tingling in the fingers, vertigo, headache drowsiness, prolonged sleepiness, painful muscular contractions leading to convulsions, blindness, and paralysis). Mental disturbances may appear such as mania, psychosis, and delirium. One notable historical case presented by Caporeal (126) were the teenage girls accused of witchcraft in Salem in 1692. 3-nitropropionic acid 3-nitropropionic acid (3-NPA) is produced by Arthrinium moulds and causes the so called mouldy sugar cane disease (127). This disease appears in the late winter months (February and March) in 13 northern Chinese counties and affects children who consume sugar cane contaminated with Arthrinium moulds stored for at least two months. From 1978 to 1988, 884 persons were involved in a series of small epidemics (involving usually up to five children), and 88 (10 %) died (128). The first symptoms appeared two to three hours after the consumption of sugar cane and included vomiting, general fatigue, convulsions, carpopedal spasms, and coma. Dystonia appeared in 10-50 % of the cases as the consequence of basal ganglia necrosis. These last symptoms can be predicted if the basal ganglia are scanned with computerised tomography (CT) (129). In adults, the symptoms of 3-NPA poisoning include mild disturbances of the GI tract and brain lesions, but these are very rare. Since 1995, there have been no further reports on mouldy sugar cane disease in the available scientific literature. However, Chinese health authorities keep issuing warnings in daily newspapers, which suggests that this disease is still a threat. Ustilago maydis toxins Historically, mycotoxicoses that appeared in Croatia between the two world wars were suspected to be caused by corn smut (Ustilago maydis). Children, infants, and toddlers, suspected of ustilaginism fell ill between March and May after a winter of almost exclusive consumption of corn flour from fields infested with corn smut. The clinical presentation was either acute - with acropathic symptoms including hand and sole pruritus, oedema and erythema - or

358 chronic - with frequent relapses of acute symptoms and abundant skin desquamation (130, 131). At the time, it was believed that the cause were corn smut spores at a specific window of maturity. If the corn was consumed before or after that window, it was considered harmless. This corn smut hypothesis has never been confirmed, and the only argument in its favour is that the patientâ&#x20AC;&#x2122;s condition would improve as soon (several days to a fortnight) as the corn flour was removed from the diet. No similar phenomena have been reported since 1945.

CONCLUSION Acute mycotoxicoses in children are serious diseases, mostly diagnosed only when an epidemics breaks out, affecting several children. They should be suspected when the disease cannot be explained by infection with a known microorganism. Acute mycotoxicoses are more frequent in tropical regions but are no stranger to temperate climates as well. Chronic mycotoxicoses may appear all over the world and paediatricians should keep in mind that their clinical manifestations vary a lot.

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Sažetak Mikotoksikoze u djece Mikotoksikoze su akutna i kronična trovanja toksinima plijesni koji se nazivaju mikotoksini. Odrasle osobe i djeca stalno su izloženi niskim koncentracijama mješavine mikotoksina, većinom putem kontaminirane hrane. Akutne toksikoze koje uzrokuje izloženost velikim količinama mikotoksina danas su rijetke te su opisane kako bi se liječnici i drugo zdravstveno osoblje informirali o njihovim simptomima koji mogu oponašati druge bolesti. Djeca su osjetljivija na toksični učinak mikotoksina zbog toga što je njihova tjelesna masa manja, a neki sustavi za detoksikaciju nisu u potpunosti razvijeni. Neke se mikotoksikoze javljaju samo u djece ili su u djece simptomi jače izraženi. Podrobnih podataka o epidemiologiji mikotoksikoza u dječjoj dobi nema jer one pogađaju prvenstveno najsiromašnije, uglavnom u zemljama tropskoga pojasa gdje je zdravstvena služba nedostatna, a potrebe stanovništva za liječenjem velike. U tim se zemljama češće javljaju i kronične mikotoksikoze, no ima ih i u zemljama s umjerenom klimom. U razvijenim su zemljama zdravstvene vlasti više zabrinute zbog izloženosti djece niskim koncentracijama mikotoksina koji imaju imunotoksična, genotoksična i kancerogena svojstva. KLJUČNE RIJEČI: 3-NPA; aflatoksini; ergot; fumonizini; okratoksini; trihoteceni; toksini Ustilago maydis; zearalenon

CORRESPONDING AUTHOR: Maja Peraica, MD, PhD Institute for Medical Research and Occupational Health Ksaverska cesta 2, 10001 Zagreb, Croatia E-mail: mperaica@imi.hr

Ivanković T, et al. WOOD FLY ASH AND THE DISINFECTION OF WASTEWATER AND LEACHATE Arh Hig Rada Toksikol 2014;65:365-375

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DOI: 10.2478/10004-1254-65-2014-2546

Original article

Alkaline disinfection of urban wastewater and landfill leachate by wood fly ash Tomislav Ivanković1, Jasna Hrenović1, Grigorios Itskos3, Nikolaos Koukouzas4, Davor Kovačević2, and Jelena Milenković5 Department of Microbiology1, Department of Chemistry2, Faculty of Science, University of Zagreb, Zagreb, Croatia, Nazarbayev University, School of Engineering, Astana, Republic of Kazakhstan3, Centre for Research and Technology Hellas / Chemical Process and Energy Resources Institute, Ptolemais, Greece4, Innovation Centre of the Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia5 Received in June 2014 CrossChecked in June 2014 Accepted in November 2014

Wood fly ash is an industrial by-product of the combustion of different wood materials and is mostly disposed of as waste on landfills. In our preliminary experiments, wood ash exhibited antibacterial activity against urban wastewater bacteria and we focused on wood fly ash as a potential substrate for wastewater disinfection. The addition of ash at a concentration of 10 g L-1 (1 %) caused an instant increase of pH in urban wastewater and landfill leachate. High pH (10.1–12.7) inactivated bacterial populations in the wastewater and the removal of faecal coliforms and intestinal enterococci after 6 h of contact was 100 % (below the detection limit; <1 CFU per mL) with the most efficient ash sample (ash from combustion of beech) both in urban wastewater and landfill leachate. Properly chosen wood fly ash, i.e. one that tends to increase the pH to the greatest extent, proved to be a very effective disinfection substrate. Considering that water treated with wood ash has a high pH and needs to be neutralised before discharge, ash would be suitable for disinfection of leachates when smaller volumes are treated. KEY WORDS: faecal coliforms; intestinal enterococci; pH; waste management

Wood fly ash is an industrial by-product from the combustion of different wood products in energy or power plants. It is mainly disposed of as waste material on landfills (1, 2). The production of wood ash is likely to increase in the future and finding effective applications for it is vital (2). Most of the proposed applications of wood ash thus far were in agriculture and forestry where it serves as a liming agent and increases the availability of nutrients in acid soils (24); as a component in concrete (5), cement, and mortar manufacturing (4, 6, 7); and as a catalyst for biodiesel synthesis (8). At first, our aim was to test wood fly ash as a substrate for phosphate (P) removal from urban wastewater, since various types of fly ash have been

proven to serve as potent P adsorbents (9-11). In addition, we wanted to investigate the potential toxicity of wood fly ash to microorganisms present in the wastewater as, to the best of our knowledge, such a study has not yet been reported. However, preliminary experiments showed that ash exhibited strong antibacterial activity to wastewater bacteria due to the increase of wastewater pH. We therefore shifted our focus and decided to study wood fly ash as a potential substrate for alkaline wastewater disinfection. Disinfection implies the elimination of pathogenic microorganisms in water treatment systems that facilitate the safe discharge or reuse of wastewater. Alkaline disinfection implies the inactivation of pathogenic bacteria at high pH conditions, usually

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Ivanković T, et al. WOOD FLY ASH AND THE DISINFECTION OF WASTEWATER AND LEACHATE Arh Hig Rada Toksikol 2014;65:365-375

above 12 (12). We monitored the removal of typical pathogen indicators (faecal coliforms and intestinal enterococci) in effluents of urban wastewater and landfill leachate treatment plants in order to simulate disinfection by using wood fly ash as a potential tertiary treatment method. Nutrient removal (P and Chemical Oxygen Demand) was also monitored.

MATERIALS AND METHODS Wastewater The urban wastewater was the effluent of the secondary wastewater treatment plant (50,000 PE) treating the municipal wastewater of the city of Velika Gorica, Croatia. The leachate was collected from an outlet canal after passing the activated sludge treatment plant, treating the leachate from a municipal landfill of the city of Zagreb, Croatia (1 million inhabitants). Both samples were filtered through blue ribbon filter paper (Munktel, Sweden) and used in experiments within 4 h after sampling. Basic chemical parameters of wastewater and leachate are presented in Table 1. Wood fly ash Three wood fly ash samples were tested. Samples H and L were obtained from two different heating plants in Sweden (identity not disclosed for reasons of privacy) that combust wood chips and wood dust, respectively. Sample C was obtained from the Croatian cogeneration plant Moderator d.o.o., which combusts beech (Fagus sylvaticus) wood chips. Ash samples were sieved and particle size fraction <0.125 mm was used in the experiments. Table 1 Physical and chemical parameters of urban wastewater and landfill leachate

Urban wastewater

Landfill leachate

11.5

17.1

7.9

7.7

110

282

Suspended solids (mg L-1)

Total N (mg L-1)

321

38.1

287

2.9

Parameter Temperature (°C) pH COD (mg L ) -1

Ammonia-N (mg L ) -1

P-PO4 (mg L ) -1

Characterization of wood fly ash The concentrations of major oxides and selected heavy metals in ash samples were determined by Flame- and Graphite Furnace-Atomic Absorption Spectrometry (AAS, Shimadzu AA-6300, Japan), after the complete digestion of samples with an acid mixture of HCl/H2SO4 in a microwave oven (sensitivity/ detection limits mg L -1); Cr 0.001/0.0001, Cu 0.002/0.0002, Zn 0.02/0.002). For the transfer of acidic gaseous components into solution, ash samples have been decomposed in closed microwave vessels (Berghof SPEEDWAVE ΙΙ, Germany) as described in CEN/TS 15290 (method B) (13). The sulphide concentration in the receiving solution has been determined by applying the principles of ASTM D 516-02 (turbidimetric method; HACH Spectrophotometer DR 2800, USA) (14). The P concentration in the receiving solution has been determined according to the HACH method 8114 and 10127 (HACH Spectrophotometer DR 2800, USA) (15). The electrophoretic mobility of wood fly ash samples was measured in 0.05 mol L-1 NaCl solution at pH values ranging from 2-12. The pH was measured with WTW Germany SenTix 41 electrode. Ash samples at a mass concentration of 0.2 g L-1 were dispersed in an ultrasound bath (35 kHz/3 min) and allowed to stand for 5 min to allow larger particles to be settled. An aliquot taken from the supernatant was used to measure the mobility. Measurements were performed on ZetaPlus Zeta Potential Analyser, Brookhaven Instruments Corporation (USA). The instrument uses electrophoretic light scattering and the Laser Doppler Velocimetry method for determination of particle velocity and from this the zeta potential. The leaching of heavy metals from fly ash into wastewater was determined after 1, 3, and 6 h of contact in the effluent. The effluent was filtered through Sartorius, Germany, 0.2 µm nitrocellulose filters and concentrations of heavy metals were determined by flame atomic absorption spectroscopy (AAS, Shimadzu AA 55B, Japan). At least five measurements were done for each determination. The standard deviation of results was found to be below 2 % (sensitivity/detection limits mg L-1); Cr 0.05/0.005, Cu 0.1/0.01, Zn 0.02/0.002).

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Experimental design The fly ash samples (C, H, and L) were added to Schott bottles containing 100 mL of urban wastewater or leachate at mass concentrations of 1 or 10 g L-1. The control systems were bottles with urban wastewater but without the addition of fly ash. The bottles were incubated (Memmert IPP 400, Germany) at 10 and 30 °C (experiments with urban wastewater) and 22 °C (experiments with leachate) on a mechanical shaker (180 rpm/Biosan OS-10, Latvia) without additional aeration. After 1, 3, and 6 h of incubation physicochemical and bacteriological parameters were determined. The numbers of bacteria were determined as colony forming units (CFU mL-1) by aseptically taking 1 mL of sample from the bottles, after which serial dilutions (-1 to -6) in sterile saline solution (0.05 mol L-1 NaCl) were made, and 0.1 mL of samples were plated onto suitable media. The numbers of faecal coliforms (Fc) were determined by incubating the samples on m-Faecal coliform agar plates (Biolife, Italy) at 44.5 °C for 24 h. After the incubation blue colonies were counted and designated as faecal coliforms. The numbers of intestinal enterococci (Ie), bacteria of the genus Enterococcus, were determined by incubating the samples on Slanetz-Bartley agar plates (Biolife, Italy) at 35 °C for 72 h. After the incubation, the confirmation of intestinal enterococci was done by development of black colonies on Kanamycin aesculin azide agar (Oxoid, UK). In addition, the numbers of total heterotrophs (He) were determined by incubating the samples on Tryptic glucose yeast agar plates (Biolife, Italy) at 22 °C for 72 h. The P concentration in the effluent samples was measured using HACH spectrophotometer DR2500, USA (method 8114 and 10127) (15). Prior to measurements the pH of effluent was set to 6-8 with 1 mol L-1 HCl using the SenTix 41 (WTW, Germany) electrode and filtered through Sartorius nitrocellulose filters of 0.2 µm pore diameter. The chemical oxygen demand (COD) concentration was measured by HACH method 8000 (15). Data analysis The survival of bacteria was determined as the percentage of viable cells after the designated time of incubation compared to the number of cells at the start of the experiment (t0). The CFUs were logarithmically transformed to normalise distribution and equalize the variances of the measured parameters. Statistical

analysis was done using Statistica Software 10.0 (StatSoft, Tulsa, USA). The results were compared using one-way ANOVA with post hoc Duncan’s new multiple range test. Correlation between variables was done using Pearson’s linear correlation. Significant differences were considered at level of p<0.05.

RESULTS AND DISCUSSION Characterization of wood fly ash Oxide composition and heavy metal concentrations in the examined fly ash samples are shown in Table 2. In general, the examined samples seemed to possess a typical wood chip ash composition. Samples C and H were strongly calcareous while sample L was basically siliceous with an elevated alumina presence. As in the case of most biogenic ash types, all three samples were rich in alkali elements, with potassium accounting for over 10 wt.% in samples C and H and over 4 wt.% in sample L. Sodium concentrations exceeded 3 wt.% in all of the three samples. Wood fly ash is generally rich in magnesium, a tendency also displayed in the currently examined samples (mainly the calcareous ones, which could be expected), some of which demonstrate percentages as high as >4.5 wt.% (Sample C). As for iron, unlike sample C, its presence was strong in samples H and L, reaching a level of 5.31 %, which is significantly Table 2 Chemical composition of the wood fly ash samples. Major oxides (in wt.%); heavy metals (in mg kg-1)

Major oxides SiO2 Al2O3 Fe2O3 CaO Na2O K2O MgO P2O5 SO3 Heavy metals Cd Cr Cu Mn Ni Zn

Sample C 29.06 4.02 0.99 41.66 6.45 10.58 4.62 2.31 0.10

Sample H 27.7 3.79 4.09 33.42 3.35 15.12 4.02 5.86 0.10

Sample L 38.41 13.29 5.31 20.93 4.14 4.73 2.95 7.80 0.40

6 46 106 1,818 <15 121

64 62 192 16,789 <15 8,245

55 945 1,485 2,365 163 15,416

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higher than normally in typical wood chip ash samples (16, 17). The phosphorus concentration was noticeably higher in sample L than in samples C and H and the same could be said for the concentration of heavy metals, with the exception of manganese. Zinc in sample L appeared to occur in concentrations significantly higher than in the others. Disinfection of urban wastewater Without the addition of fly ash (Control 1), the numbers of heterotrophic bacteria (He), faecal coliforms (Fc), and intestinal enterococci (Ie) in the wastewater remained constant during 6 h of incubation at 10 or 30 °C (Table 3). Ash at 1 g L-1 caused a slight increase in pH (8.2-9.8), which had no significant antibacterial effect on the monitored bacterial populations. The only exception was the reduction of Fc (30 %) after 6 h of contact with ash sample C at 30 °C (Table 3). The reduction in Fc was the result of the increased pH and high incubation temperature. When ash was added at 10 g L-1, the pH of the wastewater immediately increased to 10.1-12.7 (depending on the ash sample) and remained high during the entire 6 h of incubation, which had a strong antibacterial or bactericidal effect on the monitored bacterial populations (Figure 1). The pH values correlated significantly (p<0.05; R=0.813) with a reduction in bacterial numbers. It can be concluded that the increase of pH caused the inactivation of bacteria in the wastewater. A similar conclusion was

reported by Blinova et al. (18), where the high alkalinity of oil shale combustion fly ash solutions was the key factor of its toxicity to bacterium vibrio fischeri, crustacean Daphnia magna, and microalga Pseudokirchneriella subcapitata. The reduction of bacteria correlated positively (p<0.05; R=0.252) with the time of incubation and did not correlate significantly (p>0.05; R=0.119) with the incubation temperature (10 or 30 °C). Most bacteria can live and multiply within the range of pH 5-8 and have an optimum-near neutral pH (19). Very few bacteria can grow at pH values above 10 and the highest proven pH limit for bacterial growth was 11.3-11.4, reported for Bacillus firmus and Nitrosomonas halophila (20, 21). Escherichia coli, a typical representative of faecal coliforms, can grow within the pH range of 5.5-8.0 (22), while bacteria from the genus Enterococcus tolerate highly alkaline environments; growth occurs at pH 9.6. A pH of 10.511.0 was shown to impede the growth of Enterococcus faecalis, and no growth was observed at pH 11.5 or higher (23). Our results showed that the Fc were the most susceptible to high pH values while the Ie and He were more tolerant (Figure 1). At pH above 12 (experiments with ash C), the Fc and Ie populations were completely inactivated to below detection limit (<1 CFU mL-1) (Figure 1). This is in agreement with the available literature on alkaline disinfection of biosolids. Meckes and Rhodes (24) found that Fc and E. coli populations were more susceptible to lime

Table 3 Survival of faecal coliforms (Fc), intestinal enterococci (Ie), and total heterotrophs (He) in the urban wastewater with addition of 1 g L-1 of wood ash samples C, H or L after 1, 3, and 6 h of contact at 10 and 30 °C. Control 1-wastewater without ash; t0 pH = 7.9±0.1; t0 faecal coliforms (log CFU mL-1)=4.45±0.55; t0 intestinal enterococci (log CFU mL-1)=3.76±0.25; t0 total heterotrophs (log CFU mL-1)=5.83±0.38

Experiment

Control 1

pH Fc Ie He pH Fc Ie He pH Fc Ie He pH Fc Ie He

1h 8.2 99±1 100±3 99±6 9.4 98±14 96±1 95±6 8.7 88±13 92±7 98±6 8.7 98±22 89±1 93±4

10 °C 3h 8.5 100±1 98±4 95±12 9.7 93±4 101±9 94±5 8.8 90±14 94±5 98±5 8.9 94±18 79±3 93±4

Survival (%)

6h 8.7 104±1 100±4 93±3 9.8 91±5 101±10 97±6 8.8 96±12 96±13 107±5 8.9 101±16 98±10 97±5

1h 8.2 99±1 101±2 108±5 9.6 95±4 95±9 96±5 8.5 92±22 87±2 97±5 8.6 89±16 91±2 97±6

30 °C 3h 8.3 107±1 96±2 101±7 9.7 85±4 98±17 96±5 8.5 87±14 91±1 100±3 8.5 102±25 93±7 97±6

6h 8.5 104±1 100±3 104±9 9.8 69±1 104±5 98±5 8.6 92±14 91±6 108±6 8.7 92±7 93±5 102±2

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Figure 1 Reduction of faecal coliforms (Fc), intestinal enterococci (Ie), and total heterotrophs (He) in urban wastewater with addition of 10 g L-1 of wood ash samples C, H or L after 1, 3, and 6 h of incubation. Control wastewater without ash and with pH initially set to 11.5; t0 pH=7.9±0.1; t0 faecal coliforms (log CFU mL-1)=4.8±0.5; t0 intestinal enterococci (log CFU mL-1)=3.75±0.3; t0 total heterotrophs (log CFU mL-1)=5.7±0.4. *measurements from experiments conducted at 10 and 30 °C are presented collectively

treatment than other heterotrophic bacteria and Brewster et al. (25) reported complete inactivation of Fc and 4-log reduction of Clostridium perfringens also during lime treatment. Leaching of heavy metals from wood fly ash The leaching of heavy metals from fly ash to water solutions has been covered by a substantial number of studies (26, 27). The leaching of Cr and Zn from coal fly ash was responsible for toxic effects of ash/ sewage sludge mixtures toward bacterium Vibrio fischeri (28) and heavy metals leached from oil shale combustion ash showed toxic effect in standardised tests with bacterium, crustacean, and microalgae (18). In general, the leachability from coal fly ash is

relatively low and depends on the conditions in the water system. However, the leaching of heavy metals in laboratory conditions may noticeably differ from the leaching in natural conditions. It was thus suggested that the leaching behaviour test for the investigated water system should be performed before the use of fly ash as a substrate in water treatment (27). Our results have indeed shown substantial leaching of Cr, Cu, and Zn to the tested effluent wastewater from one of the tested samples (sample L), while samples C and H leached only Zn (Table 4). The leaching enhanced the antibacterial effect of sample L, which is visible from the comparison with the control system depicted in Figure 1; the Control 2 was effluent wastewater with pH set to 11.5 at the start of

Table 4 Concentrations of heavy metals leached into effluent water and its weight percent contained in wood ash samples C, H or L in experiments with 10 g L-1 of ash after 1, 3, and 6 h of contact at 10 and 30 °C. Leaching of Cd, Co, Mn, and Ni was not detected

Heavy metal Cr Cu Zn

C H L C H L C H L

1h 0.00/0 0.00/0 0.26/3 0.00/0 0.00/0 0.04/0.3 0.01/1 0.04/0.05 0.06/0.04

10 °C 3h 0.00/0 0.00/0 0.32/3 0.00/0 0.00/0 0.05/0.3 0.11/18 0.03/0.04 0.10/0.06

Leaching (mg L-1)/wt.% 6h 0.00/0 0.00/0 0.31/3 0.00/0 0.00/0 0.05/0.3 0.09/7 0.02/0.02 0.61/0.40

1h 0.00/0 0.00/0 0.28/3 0.00/0 0.00/0 0.05/0.3 0.19/16 0.05/0.06 0.23/0.15

30 °C 3h 0.00/0 0.00/0 0.23/2 0.00/0 0.00/0 0.05/0.3 0.30/25 0.08/0.10 0.47/0.30

6h 0.00/0 0.00/0 0.31/3 0.00/0 0.00/0 0.05/0.3 0.19/16 0.27/0.33 0.31/0.20

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the experiment. After 1, 3, and 6 h of incubation, the pH values were the same, but the reduction in bacterial numbers in the system with ash L (70-100 %) was significantly higher when compared to the reduction in bacterial numbers in the Control 2 (10-60 %). The pronounced antibacterial activity of sample L was therefore ascribed to the synergistic effect of high pH and leached Cr, Cu, and Zn. Results confirmed that a proper leaching test should be mandatory for each wood fly ash sample and water sample prior to usage in treatment systems, since the discharge of wastewater with elevated concentrations of heavy metals poses an environmental hazard.

Successful disinfection of leachate can be achieved by using hydrogen peroxide and chlorine (30, 31). However, recent regulatory trends have turned to alternative disinfectants, such as UV, due to the production of hazardous disinfection by-products during chlorination (32). The problem with UV disinfection is its strong quenching of UV light owing to the unique characteristics of the leachate, which diminishes the disinfection effectiveness (32). Our results suggest that, compared to the abovementioned methods, wood fly ash is very effective and has no negative side effects apart from increasing pH in the leachate.

Disinfection of leachate wastewater

Nutrient removal from urban and leachate wastewater

Guided by the results obtained in the experiments with urban wastewater, ash sample C, which showed the strongest antibacterial activity, was chosen for further experiments with leachate wastewater and the experiment was conducted at room temperature (22 °C). The monitored bacterial populations remained the same since Fc and Ie are commonly used as indicators of pathogenic bacteria for landfill leachate (29, 30). The initial values of Fc and Ie in leachate effluent were respectively 7.1±3.6×103 CFU 100 mL-1 and 1.1±0.7×104 CFU 100 mL-1, which is comparable to results from Grisey et al. (29) and Umar et al. (30). As in the experiments with urban wastewater, the addition of ash sample C exhibited strong antibacterial effect due to pH increase and inactivated the Fc and Ie in leachate after 1 h of contact to below the detection limit (<1 CFU mL-1) (Figure 2). The number of total heterotrophs was also significantly reduced (38-45 %).

Fly ash has been suggested as a potent substrate for phosphate (P) adsorption by many studies (9-11, 33, 34). All of these studies were conducted in systems with aqueous solutions of P salts and none used real wastewater. Urban wastewater, especially from combined sewage systems as in this study, is a complex biological and chemical system containing many different anions and emerging pollutants, such as aromatic organic compounds, chlorine, detergents, dyes, heavy metals, medicals, pesticides, surfactants, and other xenobiotics (35). These components can also adsorb on fly ash and competitively diminish the P sorption (36). We monitored P removal from urban wastewater and showed that wood fly ash is indeed a promising substrate for its removal. At an ash concentration of 10 g L-1, all three ash samples removed over 90 % of the initial P (2.3–3.2 mg L-1) during 6 h of incubation

Figure 2 Reduction of faecal coliforms (Fc), intestinal enterococci (Ie), and total heterotrophs (He) in leachate with addition of 10 g L-1 of wood ash sample C, after 1, and 6 h of incubation at 22 °C. t0 pH=7.9±0.1; t0 faecal coliforms (log CFU mL-1)=1.8±0.2; t0 intestinal enterococci (log CFU mL-1)=1.9±0.2; t0 total heterotrophs (log CFU mL-1)=4.3±0.3

Ivanković T, et al. WOOD FLY ASH AND THE DISINFECTION OF WASTEWATER AND LEACHATE Arh Hig Rada Toksikol 2014;65:365-375

at 10 or 30 °C (Figure 3). At an ash concentration of 1 g L-1, the removal percentage was lower and varied depending on the sample. The P was removed from wastewater by sorption on fly ash, rather than spontaneous precipitation due to high pH. This was confirmed by monitoring P concentrations in the control system, where the pH of wastewater was initially set to 11.5. There was no significant removal (0-14 %) during 6 h of incubation at 10 or 30 °C. Since the ash samples were positively charged at very low pH values and negatively charged at neutral and alkaline pH (Table 5), the proposed mechanism of P removal by fly ash is P adsorption onto calcite surfaces and the formation of amorphous calcium phosphate precipitates (9, 33). Zeta potential values could be related to the isoelectric points (pHiep) of various metal oxides. The wood fly ash samples are a mixture of various oxides and their isoelectric point could be influenced by their composition. Since the isoelectric point of silica (pHiep≤4) is lower than the isoelectric points of iron oxides (pHiep=6-8) and aluminium oxides (pHiep≈5), it is not surprising that, e.g. in the case of sample L (which contained the highest amount of silica), the isoelectric point seemed to be lower than in the case of sample C. The removal of COD was monitored in leachate since P concentrations were below 1 mg L-1. After addition of wood ash C, the COD reduced by 25±9 % after 1 h and 73±2 % after 6 h of incubation (starting COD=282±14 mg L-1). There was also an obvious

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reduction in colour; after 1 h of incubation, the leachate treated with ash sample C was transparent, while the control reactor without ash remained as yellow/brownish as it was at the start of the experiment. The pH of the leachate was 7.7±0.4 at the start, 12.5±0.1 after 1 h, and 12.4±0.1 after 6 h of incubation. A removal of COD from real industrial wastewater (detergent company) by 39 % (starting COD was 560 mg L-1) after 2 h of contact with coal fly ash has been reported by Ragheb (37). Comparison of wood ash to other disinfection substrates In the field of wastewater disinfection, recent research has focused on novel inorganic antibacterial media such as modified zeolites and activated carbon. Activated carbon loaded with Ag (38, 39), Cu2+-treated zeolite (40-42), CuO/Cu2O-coated carbon (43), Zntreated zeolite (44), have all been shown to exhibit a satisfactory removal of bacteria. The prominent negative sides of all of the mentioned materials are its stability, high dependency on exogenous factors (salinity of wastewater, flow velocity, etc.), questionable cost-effectiveness, and leaching of metals from substrate into wastewater. Our results suggest that, compared to the mentioned substrates, along with chlorination and UV disinfection methods, wood fly ash is extremely effective for alkaline disinfection and has no negative side effects apart from increase in wastewater pH. The alkaline disinfection in closed

Figure 3 The amount of P (%) removed from urban wastewater incubated* with 1 and 10 g L-1 of wood fly ash samples C, H, and L. *measurements obtained at 1, 3, and 6 h of incubation are presented collectively; measurements from experiments conducted at 10 and 30 °C are presented collectively; t0 (P-PO4)=2.3±0.2 mg L-1 (in experiments with samples H and L); 3.2±0.1 mg L-1 (in experiments with sample C)

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Table 5 Zeta potential of the wood fly ash samples C, H, and L in 0.05 mol L-1 NaCl. Mean values of 10 measurements with corresponding standard deviation are presented. pH start-pH value of 0.05 mol L-1 NaCl solution measured before addition of fly ash; pH final-pH value of 0.05 mol L-1 NaCl solution measured 2 h after addition of 0.2 g L-1of fly ash

Sample C pH start pH final Zeta potential (mV) Sample H pH start pH final Zeta potential (mV) Sample L pH start pH final Zeta potential (mV)

1.34 1.45 17.44±3.15

3.15 10.45 -12.72±1.73

11.83 11.90 -13.63±3.30

1.96 2.05 3.52±2.01

2.99 5.03 -27.78±1.63

8.44 10.35 -23.59±2.38

1.02 0.88 18.20±3.81

2.99 8.72 -14.37±1.71

8.44 10.61 -17.13±2.39

systems has been shown to depend on pH, exposure time, temperature, total solids content, and ammonia concentration (12, 45). In our case, the disinfection was effective in both urban (low ammonia) and leachate wastewater (high ammonia), depending on exposure time (1-6 h) and irrespective of the temperature (10 or 30 °C). The antibacterial activity of the modified zeolites and activated carbon relies on bacteria/substrate contact at solid/liquid interface (40), meaning that the physicochemical surface properties of the substrate (charge, adsorption sites, ion-exchange) play a vital role in antibacterial activity. This also means that efficiency is reduced as flow velocity (40) and the bacteria concentration in the wastewater increase. The antibacterial activity of fly ash is completely independent of the bacteria/substrate contact, as the ash increases the pH of the entire wastewater – the effectiveness is the same in all parts of the vessel, extensive mixing is not required, the form of the filter is irrelevant, and effectiveness is the same regardless of the bacterial concentration in the wastewater. The antibacterial effectiveness of the modified zeolites and activated carbon depends not only on the type of wastewater, but also on the seasonal changes in the same type of wastewater (40). Wood fly ash has shown the same level of effectiveness in two very different types of wastewater (urban, leachate) and it could be reasoned that its antibacterial activity should be the same in other types of wastewater, as well. The modified zeolites and activated carbon exhibit significant leaching of metals from substrate into outflow wastewater; up to 35 mg L-1 of Cu from the Cu-zeolite (40), up to 2 mg L-1 of Ag from the Ag-

zeolite (46), and 2.36 mg L-1 of Zn from the Zn-zeolite (42). At the proposed wood ash concentration (10 g L-1), leaching of heavy metals from the ash into urban wastewater was negligible compared to the above mentioned substrates (Table 4). Along with antibacterial properties, wood fly ash showed nutrient (P and COD) and colour removal from urban wastewater and leachate. The main drawback of using wood ash is the increase in pH of wastewater, which should be neutralised prior to discharge into a natural recipient.

CONCLUSION Properly chosen wood fly ash, in our case ash from the combustion of beech wood chips, proved to be a very effective disinfection substrate for urban wastewater and landfill leachate. The inactivation of bacteria resulted from increased wastewater pH, caused by the addition of ash. The fly ashes that tend to increase the pH to the greatest extent should therefore be considered the best disinfectant substrates. Wood fly ash was also capable of nutrient and colour removal to a certain extent. Considering that water treated with wood ash has a high pH and needs to be neutralised before discharge, the ash would be suitable for leachate disinfection, when smaller volumes are treated. Acknowledgements We wish to thank Dr Stanislaw Lazarek, Join Business & Technology, Sweden and Mr Davor Zec,

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Moderator d.o.o., Croatia for providing us ash samples. We also thank Mrs. Zvjezdana Ležaić, VG Vodopskrba d.o.o. for providing urban wastewater, and Prof Goran Durn, Faculty of Mining, Geology and Petroleum Engineering in Zagreb for fractioning the ash samples.

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landfill leachate through coupling bacterial enumeration with tracer tests. Desalination 2010;261:162-8. doi: 10.1016/j. desal.2010.05.007 Umar M, Abdul Aziz H, Suffian Yusoff M. Assessing the chlorine disinfection of landfill leachate and optimization by response surface methodology (RSM). Desalination 2011;274:278-83. doi: 10.1016/j.desal.2011.02.023 Tofant A, Farkaš A, Hrenović J, Rožić M, Tisma S. Leachates disinfection at Jakuševec waste dump. In: Central European Symposium on Industrial Microbiology and Microbial Ecology; 17-22 Sep 2007; Zadar, Croatia. Book of abstracts. Zhaoa R, Guptab A, Novak JT, Goldsmith CD, Driskill N. Characterization and treatment of organic constituents in landfill leachates that influence the UV disinfection in the publicly owned treatment works (POTWs,). J Hazard Mater 2013;258-259:1-9. doi: 10.1016/j.jhazmat.2013.04.026 Ugurlu A, Salman B. Phosphorus removal by fly ash. Environ Int 1998;24:911-8. doi: 10.1016/S0160-4120(98)00079-8 Can MY, Yildiz E. Phosphate removal from water by fly ash: Factorial experimental design. J Hazard Mater 2006;B135:165-70. PMID: 16359787 Henze M, Comeau Y. Wastewater characterization. In: Henze M, van Loosdrecht MCM, Ekama GA, Brdjanovic D, editors. Biological Wastewater Treatment: Principles, Modelling and Design. London: IWA Publishing; 2008. p. 33-53. Jellali S, Wahab MA, Anane M, Riahi K, Bousselmi L. Phosphate mine wastes reuse for phosphorus removal from aqueous solutions under dynamic conditions. J Hazard Mater 2010;184:226-33. doi: 10.1016/j.jhazmat.2010.08.026 Ragheb SM. Phosphate removal from aqueous solution using slag and fly ash. HBRC J 2013;9:270-5. 10.1016/j. hbrcj.2013.08.005 Pal S, Joardar J, Song JM. Removal of E. coli from water using surface-modified activated carbon filter media and its performance over an extended use. Environ Sci Technol 2006;40:6091-7. doi: 10.1021/es800936k

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Sažetak Dezinfekcija komunalne i procjedne otpadne vode s odlagališta otpada korištenjem letećeg pepela iz drvne industrije Leteći pepeo iz drvne industrije je nusproizvod koji nastaje spaljivanjem različitog drvnog materijala i većinom završi na odlagalištima otpada. Budući da je u preliminarnim ispitivanjima leteći pepeo pokazao antibakterijsko djelovanje prema bakterijama iz otpadne vode, pokusi su usmjereni istraživanju pepela kao potencijalnog supstrata za dezinfekciju otpadne vode. Dodatak pepela u koncentraciji od 10 g L-1 (1 %) uzrokovao je trenutno povećanje vrijednosti pH komunalne i procjedne otpadne vode s odlagališta otpada. Visoki pH (10,1–12,7) uništio je bakterije u otpadnoj vodi te je korištenjem najučinkovitijega pepela (dobivenog spaljivanjem bukve) postignuto 100-postotno uklanjanje (odnosno manje od mogućnosti detekcije; <1 CFU mL-1) fekalnih koliforma i crijevnih enterokoka iz komunalne i procjedne otpadne vode nakon šest sati kontakta. Odgovarajući pepeo, odnosno onaj koji uzrokuje najveće povećanje vrijednosti pH, pokazao se kao vrlo učinkovit supstrat za dezinfekciju. Uzimajući u obzir činjenicu da otpadna voda tretirana pepelom ima povišen pH, te da je vrijednost pH potrebno neutralizirati prije ispuštanja u prirodni prijemnik, leteći drvni pepeo bio bi pogodan za dezinfekciju procjedne otpadne vode zbog manjih volumena koji zahtijevaju obradu. KLJUČNE RIJEČI: fekalni koliformi; crijevni enterokoki; pH; zbrinjavanje otpada

CORRESPONDING AUTHOR: Tomislav Ivanković Department of Microbiology, Faculty of Science, University of Zagreb Rooseveltov trg 6, 10000 Zagreb, Croatia E-mail: tomislav.ivankovic@biol.pmf.hr

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DOI: 10.2478/10004-1254-65-2014-2547

Original article

In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout Ahmet Topal1, Muhammed Atamanalp2, Ertan Oruç3, Yeliz Demir4, Şükrü Beydemir4, and Alparslan Işık1 Department of Basic Sciences1, Department of Aquaculture2, Faculty of Fisheries, Department of Pathology, Faculty of Veterinary3, Department of Chemistry, Faculty of Science4, Ataturk University, Erzurum, Turkey Received in June 2014 CrossChecked in June 2014 Accepted in November 2014

Chlorpyrifos is an organophosphate pesticide widely used in agriculture and aquaculture. This study investigated its effects on carbonic anhydrase (CA) enzyme activity and histopathology of rainbow trout gill and liver. The fish were exposed to 2.25 (25 % of 96 h LC50), 4.5 (50 % of 96 h LC50), and 6.75 µg L-1 (75 % of 96 h LC50) of chlorpyrifos for 24, 48, 72, and 96 h. CA activity was measured in liver and gills and histopathological changes were examined by light microscopy. The most common liver changes at most of the chlorpyrifos concentrations were hyperaemia and degenerative changes. Gill tissues were characterised by lamellar hyperaemia, lamellar oedemas, clumping, cellular degeneration, hyperplasia, and lamellar atrophy. CA enzyme activity in the gills decreased at all concentrations at 48, 72, and 96 h after exposure to chlorpyrifos (p<0.05). Similarly, there was a time-dependent decrease in CA activity at all of the concentrations in liver tissues (p<0.05). The present study indicated that chlorpyrifos inhibits CA enzyme activity and causes histopathological damage in gill and liver tissues. KEY WORDS: acute toxicity; fish; histology; light microscopy; organophosphate pesticides

Chlorpyrifos is an organophosphate pesticide widely used in agriculture, aquaculture, and fishery pest control (1, 2). Numerous environmental issues have arisen so far due to the excessive use of this chemical compound (3), as it, among other consequences, causes toxic effects in non-target aquatic organisms, especially fish (4). Fish are used to assess the health of aquatic environments and physiological changes occurring as a result of pollution and multiple studies have already established that chlorpyrifos has various detrimental effects on them (5-10), such as neurotoxicity via acetylcholinesterase inhibition (11), biochemical and histopathological alterations (12, 13), oxidative stress (12, 14), genotoxicity (15), and olfactory and neurobehavioral injuries (16). It therefore poses a serious threat to aquatic organisms as well as to human

health (17). Rainbow trout has been selected for this study, because it is a sensitive indicator of aquatic pollution (18) and one of the most studied fish species due to its importance as food in terms of nutritional and economic value (19). Carbonic anhydrase (CA) is a zinc metalloenzyme found in the tissues of most eukaryotes and has important physiological functions such as respiration, gas balance, lipogenesis, ureagenesis, bone resorption, or body fluid generation in various tissues (20, 21). CA plays an important role in the excretion of metabolic carbon dioxide in fish and catalyses the reversible hydration/dehydration of carbon dioxide to bicarbonate and protons (22, 23). Therefore, any inhibition of this enzyme leads to unfavourable effects for living organisms. Additionally, CA can also be used as a biomarker of toxicity (24).

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Histological techniques are used to assess the toxic effect of pollutants such as pesticides and heavy metals in the aquatic environment (25). Little information is available on chlorpyrifos toxicity and effects on CA sensitivity in fish. This study was designed to determine CA activity levels and histopathological changes in gill and liver tissues of rainbow trout after acute exposure to chlorpyrifos.

MATERIALS AND METHODS Our experiments were performed on rainbow trout, Oncorhynchus mykiss (body mass 171±5.73 g and average length 19.47±0.94 cm). They were obtained from the Ataturk University Faculty of Fisheries and Inland Water Fish Breeding and Research Center. Experiments were carried out in 4 fiberglass tanks (each 400 L) each containing 15 fish. The tanks were filled with dechlorinated tap water (temperature 1012 °C, pH 7.1±0.3, dissolved oxygen 8.2±0.5 mg L-1, water hardness 174.6±5.19 mg L -1 CaCO 3, SO 42 =0.36 mg L-1, PO4-3=trace, NO3-=1.51 mg L-1 and NO2-=trace) and acclimated to laboratory conditions for 15 days. During acclimation, the fish were fed 2.5 % body weight with commercial trout pellets (Sibal Group, Sinop, Turkey). Chlorpyrifos was at 99.2 % purity and the study used its commercial formulation [480 g L-1 chlorpyrifos, O , O - d i e t h y l - O - ( 3 , 5 , 6 - t r i c h l o r- 2 - p y r i d y l ) phosphorothioate] purchased from a distributor company (Platin Chemistry, Turkey). The stock solution of chlorpyrifos was prepared by dissolving in distilled water. Exposure to chlorpyrifos The chlorpyrifos LC50 value for rainbow trout was set at 9 µg L-1 (26). The concentrations used for this study were 25 % (2.25 µg L-1), 50 % (4.5 µg L-1), and 75 % (6.75 µg L -1 ) of the LC 50 value. These concentrations were chosen because they were lower than the lethal concentrations for rainbow trout, and also may occur in a polluted environment. The fish were exposed to these concentrations for 24, 48, 72, and 96 h. At the end of each exposure period, fish were randomly selected from the control and exposed groups and sampled. The fish were sacrificed by cervical section and the liver and gill tissues were immediately removed. A portion of the tissues was washed with physiological saline (0.9 % NaCl) and

stored at -20 °C until analysis for CA activity. The other portion of the tissues was fixed in 10 % formalin solution for histopathological examination. Determination of CA activity Liver and gill tissue samples were washed three times with 0.9 % NaCl. Each tissue was homogenised with buffer 25 mmol L-1 Tris–HCl+0.1 mol L-1 Na2SO4 (pH 8.7) by homogenizer and the supernatant was centrifuged at 4 °C, 15000 g for 60 min. Enzyme activity was assayed by following CO2 hydration according to the protocol established by Wilbur and Anderson (27). CO2-hydratase activity as an enzyme unit (EU) was calculated by using the equation (to-tc/ tc) where t0 and tc are the times for pH change of the non-enzymatic and the enzymatic reactions, respectively. Histopathology procedures After the routine histopathology process, paraffin sections were stained in 5 µ with hematoxylin and eosine (HE). Histopathological changes were semiquantitatively assessed under a light microscope (Olympus BX51 with DP72 camera attachment system, Tokyo, Japan). The scores were derived as semi-quantitative according to severity and extent of changes and are reported as follows: none:−; mild:+; moderate:++; and severe:+++. Statistical analyses All data are expressed as mean±SEM. Statistical analysis of data was done using one-way analysis of variance (ANOVA) and LSD test and analysed using SPSS version 10.0. A value of p<0.05 was considered statistically significant.

RESULTS AND DISCUSSION Aquatic ecosystems are often faced with problems caused by contaminants released into the environment (28). Various non-target organisms, especially fish, are exposed to pesticides such as chlorpyrifos and this may cause many adverse effects, including biochemical alterations (29). In order to add to risk assessment studies conducted thus far, we strived to obtain information about the effects of chlorpyrifos on rainbow trout, one of the most studied species owing to its previously mentioned importance.

Topal A, et al. CHLORPYRIFOS-INDUCED CHANGES IN CA ACTIVITY AND HISTOPATHOLOGY OF RAINBOW TROUT Arh Hig Rada Toksikol 2014;65:377-385

CA activity in liver and gill tissues A time-dependent decrease in enzyme activity was evident, as CA activity decreased at all of the applied concentrations in all liver and gill tissues after 96 h (p<0.05). Exposure to 4.5 and 6.75 µg L -1 of chlorpyrifos for 72 h also caused a statistically significant decrease in liver and gill CA activity (p<0.05). Furthermore, the 6.75 µg L-1 concentration decreased CA activity after 48 h in both tissues and only in gill tissues after 24 h, while the 4.5 µg L-1 concentration also affected only gill tissue after 24 h when compared to the controls (p<0.05) (Figures 1 and 2). There was no statistically significant difference at either of the concentrations after 24 h in liver tissues when compared to the control (Figure 1).

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Many chemical substances lead to changes in metabolism by changing enzyme activity, particularly via inhibition of a specific enzyme (30). There are many studies in the literature about the effects of pesticides on CA activity in different fish species and rainbow trout in particular (9, 23, 31, 32). For example, the pesticides deltamethrin, diazinon, propoxur, and cypermethrin were tested on rainbow trout gill CA activity by Ceyhun et al. (33) exhibiting inhibitory effects in vivo and in vitro. In another study, Dogan (23) reported that pesticides such as lambdacyhalothrin, deltametrin, diozinon, dorzolamide, and brinzolamide caused inhibitory effects on CA activity in rainbow trout blood. CA is known to play an important role in the excretion of metabolic CO2 as

Figure 1 The effects of chlorpyrifos on liver carbonic anhydrase enzyme activity of rainbow trout. Values are expressed as mean±S.E.M. Significant difference from control values *p<0.05. EU-enzyme units

Figure 2 The effects of chlorpyrifos on gill carbonic anhydrase enzyme activity of rainbow trout. Values are expressed as mean±S.E.M. Significant difference from control values *p<0.05. EU-enzyme units

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well as in CO2 exchange between tissues and blood in fish and in catalysing the reversible hydration/ dehydration of CO2 to bicarbonate and protons (22, 23, 34). Our results have shown that there was a timedependent decrease in enzyme activity after exposure to chlorpyrifos at certain concentrations. This decrease can be explained by a decrease in CO2 hydration. It has been reported that, in catalysis, CO2 hydration is defined by the attack of a Zn2+ bound hydroxide on CO2 to yield a Zn2+ bound HCO3− species. HCO3− is subsequently replaced by water to yield a Zn2+-bound water molecule (23). In our case, the supply of HCO3− decreased with H+ excretion (35). Accordingly, we can say that chlorpyrifos inhibited the enzyme at very low concentrations due to the electronegative atoms in the pesticide’s chemical structures (9). Gill and liver histopathology In the present study, no histopathological changes were established in the control liver tissues and at 2.25 µg L-1 of chlorpyrifos (Figure 3). Hyperaemia and degenerative changes were observed at 48, 72, and 96 h of exposure to 4.5 and 6.75 µg L -1 of chlorpyrifos (Figure 4) (Table 1). Fish liver histopathology is an indicator of chemical toxicity and a useful way to study the effects of the exposure of aquatic animals to toxins present in the aquatic environment (36). The effects of different pesticides on liver in various fish species

have already been reported in other studies. Chlorpyrifos caused damages such as melanomacrophage aggregations, cellular atrophy, pyknotic nucleus, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, necrosis, and nuclear and cellular hypertrophy in the liver tissues of the common carp, while phosalone induced histopathological changes such as nuclear degeneration, cytoplasmic vacuolation, hypertrophy, and congestion (37, 38). No histopathological changes were observed in the gill tissues of the control group (Figure 3). Gill tissues showed lamellar hyperaemia at 48, 72, and 96 h of exposure to 2.25 µg L-1 and at 24, 48, 72, and 96 h at the two other concentrations (Table 1). Lamellar oedemas were intensive in all of the groups (Figures 5, 6, and 7). Apart from these, there were also other histopathological changes such as lamellar atrophy, hyperplasia, cellular degeneration, and clumping to a varying degree (Table 1). Environmental pollutants cause pathological changes in fish physiology (39). Gills are especially suitable for histopathological examination to determine the effects of pollution because these tissues are frequently those that are adversely affected by contaminants in the aquatic environment; for instance through osmoregulatory function and reduced oxygen consumption (40-43). In support of our observation, Pal et al. (37) observed numerous lesions in the gill tissues of common carp exposed to chlorpyrifos. Ba-

Table 1 Histopathological comparison of control and experimental groups none:−, mild:+, moderate:++ and severe:+++

Control

2.25 µg L-1

Lesion

Hyperaemia Degenerative changes

4.5 µg L-1

Hours 72 96 24 48 Liver tissue

6.75 µg L-1

+++

Gill tissue

Lamellar hyperaemia

Lamellar oedema

Clumping

Cellular degeneration

Hyperplasia

Lamellar atrophy

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Figure 3 Normal histological appearances of gill (left) and liver (right) sections control group

Figure 4 There was no histopathological change in the liver tissues at 2.25 µg L-1 of chlorpyrifos (a and b). Dilated and hyperaemic central veins (arrow in c) and sinusoids (arrows in d) at 48, 72, and 96h of 4.5 and 6.75 µg L-1 of chlorpyrifos

Figure 5 Gill tissue sections from fish exposed to 2.25 µg L-1 of chlorpyrifos. Curled lamellas (a) at 24 h and severe oedematous changes (arrows in b-d) at 48, 72, and 96 h, respectively

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Figure 6 Gill tissue sections from fish exposed to 4.5 µg L-1 of chlorpyrifos. Curled lamellas and clumpings (arrow in a) at 24 h severe oedematous changes (arrows in b-d) at 48, 72, and 96 h, respectively

Omar et al. (44) observed hypertrophy, epithelial lifting, desquamation and lamellar fusion in the gill tissues of Aphanius dispar exposed to pesticide temephos which is known as a non-systemic organophosphorus pesticide. Our study has shown results very similar to these. In conclusion, our results generally suggest that chlorpyrifos inhibits the CA enzyme and causes histopathological damages in gill and liver tissues under in vivo conditions, which proves that fish in both cultured and natural environments are sensitive to this pesticide and that chlorpyrifos contaminations would cause fish deaths. Therefore, stricter control must be applied to the use of this pesticide.

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Figure 7 Gill tissue sections from fish exposed to 6.75 µg L-1 of chlorpyrifos. Curled lamellas and clumpings (arrow in a) at 24 h. Oedematous lamellas (arrow in b) at 48 h, hyperplastic (long arrow in c) at 72 h, and capillary dilatation (arrows in d) in shortened lamellae at 96 h

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of ethylenediaminetetraacetic acid (EDTA). Global Veterinaria 2013;10:121-7. doi: 10.5829/idosi. gv.2013.10.2.6667 Bernet D, Schmidt H, Meier W, Burkhardt-Holm P, Wahli T. Histopathology in fish: proposal to assess aquatic pollution. J Fish Disease 1999;22:25-34. doi: 10.1046/j.1365-2761.1999.00134.x Beydemir S, Ciftci M, Küfrevioglu ÖI, Büyükokuroglu ME. Effects of gentamicin sulfate on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Biol Pharm Bull 2002;25:966-9. doi: 10.1248/bpb.25.966 Ekinci D, Beydemir S. Risk assessment of pesticides and fungicides for acid–base regulation and salt transport in rainbow trout tissues. Pestic Biochem Physiol 2010;97:6670. doi: 10.1016/j.pestbp.2009.12.006 Martínez-Tabche L, Estrada B, Galar I. Parathion and salinity effects on gills and mesonephros carbonic anhydrase activity of the fish Oreochromis hornorum. Bull Environ Contam Toxicol 1992;49:929-34. doi: 10.1007/BF00203169 Ceyhun SB, Senturk M, Erdogan O, Kufrevioglu OI. In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills. Pestic Biochem Physiol 2010;97:177-81. doi: 10.1016/j. pestbp.2010.01.003 Gülçin l, Beydemir S, Büyükokuroglu ME. In vitro and in vivo effects of dantrolene on carbonic anhydrase enzyme activities. Biol Pharm Bull 2004;27:613-6. doi: 10.1248/ bpb.27.613 Paulino MG, Sakuragui MM, Fernandes MN. Effects of atrazine on the gill cells and ionic balance in a neotropical fish, Prochilodus lineatus. Chemosphere 2012;86:1-7. doi: 10.1016/j.chemosphere.2011.08.033 Boran H, Altinok I, Capkin E. Histopathological changes induced by maneb and carbaryl on some tissues of rainbow

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Sažetak Promjene u razini ugljikove anhidraze i histopatologiji škrga i jetre kalifornijske pastrve nakon izlaganja klorpirifosu Klorpirifos je organofosforni pesticid široke primjene u poljoprivredi i ribarstvu. U ovome radu istražili smo njegov učinak na aktivnost enzima ugljikove anhidraze te histopatologiju škrga i jetre u kalifornijske pastrve. Ribe su bile izložene klorpirifosu u koncentracijama 2,25 µg L-1 (25 % 96-satnog LC50), 4,5 µg L-1 (50 % 96-satnog LC50) i 6,75 µg L-1 (75 % 96-satnog LC50) tijekom 24, 48, 72 i 96 sati. Aktivnost ugljikove anhidraze mjerena je u jetri i škrgama, a histopatološke promjene promatrane su svjetlosnom mikroskopijom. Najčešće promjene u jetri pri većini koncentracija bile su hiperemija i degenerativne promjene. Na tkivu škrga primijećeni su hiperemija i edemi u škržnim listićima, sljepljivanje i degeneracija stanica, hiperplazija te atrofija škržnih listića. Aktivnost ugljikove anhidraze u škrgama smanjila se pri svim koncentracijama nakon 48, 72 i 96 sati izloženosti (p<0.05). Također je uočeno i smanjenje aktivnosti ugljikove anhidraze u jetri ovisno o duljini izloženosti pri svim koncentracijama (p<0.05). Dobiveni rezultati upućuju na to da klorpirifos inhibira aktivnost ugljikove anhidraze i izaziva značajna histopatološka oštećenja u škrgama i jetri. KLJUČNE RIJEČI: akutna toksičnost; histologija; pesticidi; ribe; svjetlosna mikroskopija

CORRESPONDING AUTHOR: Ahmet Topal, PhD Department of Basic Sciences Faculty of Fisheries, Ataturk University, Erzurum, Turkey E-mail: drahmettopal@hotmail.com

Yardimci M, et al. SEX-, TISSUE-, AND TIME-DEPENDENT EFFECTS OF IMIDACLOPRID Arh Hig Rada Toksikol 2014;65:387-398

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DOI: 10.2478/10004-1254-65-2014-2554

Original article

Sex-, tissue-, and exposure duration-dependent effects of imidacloprid modulated by piperonyl butoxide and menadione in rats. Part I: oxidative and neurotoxic potentials Mustafa Yardimci1, Yusuf Sevgiler2, Eyyup Rencuzogullari2, Mehmet Arslan3, Mehmet Buyukleyla4, and Mehmet Yilmaz4 Adiyaman University, Institute of Natural and Applied Sciences, Department of Biology1, Adiyaman University, Faculty of Science and Letters, Department of Biology, Adiyaman2, Ardahan University, Health Sciences College, Department of Nursery, Merkez, Ardahan3, Cukurova University, Institute of Natural and Applied Sciences, Department of Biology, Balcali, Adana4, Turkey Received in July 2014 CrossChecked in July 2014 Accepted in November 2014

Earlier research has evidenced the oxidative and neurotoxic potential of imidacloprid, a neonicotinoid insecticide, in different animal species. The primary aim of this study was to determine how metabolic modulators piperonyl butoxide and menadione affect imidacloprid’s adverse action in the liver and kidney of Sprague-Dawley rats of both sexes. The animals were exposed to imidacloprid alone (170 mg kg-1) or in combination with piperonyl butoxide (100 mg kg-1) or menadione (25 mg kg-1) for 12 and 24 h. Their liver and kidney homogenates were analysed spectrophotometrically for glutathione peroxidase, glutathione S-transferase, catalase, total cholinesterase specific activities, total glutathione, total protein content, and lipid peroxidation levels. Imidacloprid displayed its prooxidative and neurotoxic effects predominantly in the kidney of male rats after 24 h of exposure. Our findings suggest that the observed differences in prooxidative and neurotoxic potential of imidacloprid could be related to differences in its metabolism between the sexes. Co-exposure (90-min pre-treatment) with piperonyl butoxide or menadione revealed tissue-specific effect of imidacloprid on total cholinesterase activity. Increased cholinesterase activity in the kidney could be an adaptive response to imidacloprid-induced oxidative stress. In the male rat liver, co-exposure with piperonyl butoxide or menadione exacerbated imidacloprid toxicity. In female rats, imidacloprid+menadione co-exposure caused prooxidative effects, while no such effects were observed with imidacloprid alone or menadione alone. In conclusion, sex-, tissue-, and duration-specific effects of imidacloprid are remarkable points in its toxicity. KEY WORDS: catalase; cholinesterase; glutathione peroxidase; glutathione S-transferase; neonicotinoids; oxidative stress

Neonicotinoids account for approximately one fourth of the world insecticide market (1). It is a novel insecticide group that has been replacing organophosphate, organochlorine, and pyrethroid insecticides (2) and is highly effective against piercing-sucking pests in the crops and in flea control

in cats and dogs (3). Imidacloprid (IMI; IUPAC name: N-{1-[(6-chloro-3-pyridyl) methyl]-4,5dihydroimidazol-2-yl} nitramide; CAS no: 13826141-3) is a systemic neonicotinoid insecticide that has first been registered in 1994 (4). Turkey alone used nearly 500 tonnes of it in 2012.

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Imidacloprid and other neonicotinoids act on insect nicotinic acetylcholine receptors (nAChRs), and fundamental differences between the nAChRs of insects and mammals make them remarkably “safe” for the latter (5). However, what is not safe for mammals are IMI’s metabolites, especially nitroguanidine and aminoguanidine derivatives (6-8). Mammalian CYP450s, (CYP3A4 in particular) and aldehyde oxidase (AOX; EC 1.2.3.1) are two important enzymes in its metabolism (9). CYP450s are responsible for the conversion of IMI to 5-hydroxy, olefin, nitrosoimine, guanidine, and urea derivatives (10), while AOX converts IMI to nitrosoguanidine and aminoguanidine metabolites (11). Many studies have shown the oxidative and neurotoxic potential of IMI in a variety of animal species, including rats (12-19). However, none has investigated how biotransformation affects prooxidative and neurotoxic effects of IMI in mammals and whether it depends on sex, tissue, or exposure duration. Modulators such as piperonyl butoxide (PBO) and menadione (MEN) are very useful to understand how biotransformation affects the toxicity of a chemical under investigation (20, 21). PBO (IUPAC name: 5-[2-(2-butoxyethoxy) ethoxymethyl]-6-propyl-1,3-benzodioxole; CAS no: 51-03-6) is a methylenedioxyphenyl compound used to enhance the potency of certain pesticides by inhibiting CYP450s and esterases (22-24). As Tomizawa and Casida (5) stated in their review that PBO synergized with IMI in toxicity to insects, we wondered what would be the toxicity (oxidative stress and cholinesterase activity) of this combination in mammals. Menadione (IUPAC name: 2-Methylnaphthalene1,4-dione; CAS no: 58-27-5) is a specific inhibitor of AOX and it can increase reactive oxygen species (ROS) levels as a redox active compound (25-27). However, according to Kerver et al. (28), it also lowers superoxide radical formation by inhibiting AOXcatalysed 3,39-diaminobenzidine (DAB) oxidation in the liver and intestine of male Wistar rats and, according to Al-Omar and Al-Arifi (29), superoxide radical and hydrogen peroxide formation by inhibiting AOX-mediated oxidation in Dunkin-Hartley guinea pig liver. This is why we decided to also investigate the effects of MEN on IMI-induced oxidative stress. By determining how these two metabolic modulators affect the oxidative and neurotoxic potential of IMI in rats, we hoped to get an insight

into the mechanisms behind IMI toxicity and their relation to sex, tissue, and exposure duration.

MATERIALS AND METHODS Chemicals Pure IMI and MEN crystals were purchased from Supelco and Sigma, respectively (Sigma-Aldrich Chemie GmbH). Technical grade (90 %) PBO was supplied from Aldrich (Sigma-Aldrich Chemie GmbH). All other chemicals used were of analytical grade. Animals and treatment This study was approved by the ethics committee of the Cukurova University Medical Sciences, Experimental Research, and Application Centre (no:11, date: 2 July 2010), which also supplied young adult, male and female rats (weighing about 200 g, regardless of sex) of the Sprague-Dawley strain (Rattus norvegicus var. albinos). They were separated by sex, kept in clean plastic cages, and acclimatised to laboratory conditions for seven days. Wood shavings in cages were changed every day. Laboratory temperature was 22±1 °C and relative humidity was 65±5 %. The rats were maintained under natural photoperiod and fed ad libitum. Feeding ceased 24 h before toxicity experiments. The rats were randomly divided in six groups of six animals. One group received physiological saline (saline control) to determine the basal levels of the parameters tested. All the chemicals used in this study were dissolved in dimethylsulfoxide (DMSO) and were given intraperitoneally (i.p.). As DMSO has some biphasic effects, which can be antioxidant or prooxidant (30), we also included a solvent control, the DMSO group. Data obtained from treated groups, however, were compared with this group only instead of saline control to display the toxicity of the investigated chemicals against the solvent’s effects. This is why we refer to the DMSO control as control. One group received only IMI (IMI group) in the LC50 dose of 170 mg kg-1 (31). One group received 100 mg kg-1 of PBO only (PBO group). One received 25 mg kg-1 of MEN only (MEN group). Two groups received pre-treatment with either PBO or MEN 90 min before receiving IMI in the same doses as

Yardimci M, et al. SEX-, TISSUE-, AND TIME-DEPENDENT EFFECTS OF IMIDACLOPRID Arh Hig Rada Toksikol 2014;65:387-398

described above (IMI+PBO group and IMI+MEN group, respectively). None of the rats died during the experiment. The rats were killed by cervical dislocation after 12 h or 24 h of exposure. Liver and kidney tissues were dissected on an ice plate, washed in physiological saline to remove blood residues, blotted dry, weighed, and stored at -80 °C until biochemical analysis. Biochemical analyses All biochemical analyses were done using a Shimadzu UV1800 UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan). Liver and kidney tissues were homogenised using a glass-teflon homogeniser (WiseStir HS30E, Daihan Scientific, Seoul, Korea) in an ice-containing bucket with 50 mmol L-1 pH 7.4 phosphate buffer that contained 1.17 % KCl and centrifuged at 10500 x g at 4 °C for 30 min (Universal 320R, Hettich, Tuttlingen, Germany). The obtained supernatants from each homogenate were used to analyse glutathione peroxidase (GPx), glutathione S-transferase (GST), catalase (CAT), total cholinesterase (tChE), total glutathione (tGSH), thiobarbituric acid reactive substances (TBARS), and total protein content. Aliquots of liver or kidney homogenates were mixed with 10 % 5-sulphosalicylic acid (1:0.5 v/v), then centrifuged at 10,500 g at 4 °C for 5 min, and the obtained supernatants used to measure tGSH content based on the recycling reaction with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence of glutathione reductase (GR) (32). The incubation medium contained 0.3 mmol L-1 of NADPH in stock buffer (143 mmol L -1 of sodium phosphate/6.3 mmol L-1 of Na-EDTA, adjusted to pH 7.5), 6 mmol L-1 of DTNB, 50 U/mL of GR, and 25 µL of sample. 5-thio-2-nitrobenzoic acid formation was monitored continuously at 412 nm for 3 min. The tGSH content was determined by comparison with a standard curve generated using GSH and expressed as µmol L-1 per mg of protein. Glutathione peroxidase activity was assayed at 37°C and 340 nm using GSH and t-butylhydroperoxide as substrates (33). The reaction medium contained 100 mmol L-1 of GSH, 10 U mL-1 of GR, 2 mmol L-1 of NADPH, and 7 mmol L-1 of t-butylhydroperoxide. Specific activity was expressed as U/mg protein using the molar extinction coefficient of 6.22 mmol L-1cm-1. Catalase activity was determined according to Beutler (33) as a decrease in absorbance of 10 mmol L-1 H2O2 at 37 °C for 2.5 min. The reaction was initiated by adding the supernatant. The degradation

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rate of H2O2 by CAT was measured at 230 nm. Specific activity was expressed as U/mg protein using the molar extinction coefficient of 0.071 mmol L-1cm-1. Glutathione S-transferase activity was measured according to Habig et al. (34), using 1-chloro-2,4dinitrobenzene (CDNB) as substrate. Enzyme activity was determined by monitoring changes in absorbance at 340 nm, which reflects the rate of CDNB (1 mmol L-1) conjugation with GSH (1 mmol L-1) at 30 °C for 2 minutes. Specific activity was expressed as U/mg protein using the molar extinction coefficient of 9.6 mmol L-1cm-1. For TBARS measurement, supernatants were mixed with 10 % trichloroacetic acid, vortexed, and then centrifuged at 11 g for 10 min. After the addition of thiobarbituric acid, samples were vortexed and held in a water bath at 100 °C for 10 min. Absorbance of the samples was measured at 535 nm, and these values were converted to TBARS concentration using the standard graphic prepared with 1,1’,3,3’tetramethoxypropane (35). TBARS concentration was expressed in nmoles per mg of protein. Total protein content was measured using the Folin-phenol reagent according to Lowry et al. (36). The absorbance was measured at 750 nm and converted to concentration using bovine serum albumin as the standard. The concentration was expressed in mg in per mL of supernatant. Since no inhibitor was used to differentiate cholinesterase types, we determined the total ChE activity in the supernatants. Briefly, supernatants were incubated with 10 mmol L-1 DTNB in pH 8 phosphate buffer for 5 min. Activity was started by adding 15 mmol L-1 acetylthiocholine iodide as substrate and monitored for 5 min at 412 nm (37). Specific activity was expressed as nmol L-1 of thiocholine formed per min per mg of protein using the molar extinction coefficient of 13.6 mmol L-1cm-1. Statistical analyses The results are expressed as mean ± standard error of mean (SEM) of six samples. The groups were compared using the Mann-Whitney U-test and the Kruskal-Wallis rank test of variance. The data were analysed using the Statistical Package for Social Sciences (SPSS, SPSS Inc., Chicago, IL, USA) version 17. The level of significance was set at P<0.05. We did not perform the statistical analysis considering exposure durations in the same tissue because of the significant differences between control values.

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Therefore, the results for different exposure durations for the same tissue are given in separate tables.

RESULTS AND DISCUSSION Oxidative stress is a part of IMI toxicity that has been evidenced by earlier studies (16-19). Our study has added to this knowledge by showing that IMI toxicity varies with sex, tissue, and exposure duration. Sex- and duration-specific effects of IMI in rat liver and kidney In the first 12 h of exposure, we found no effect on oxidative stress parameters and tChE regardless of sex and tissue (Tables 1 and 2), except for a decrease in liver tGSH in male rats (Table 2). The highest oxidative toxicity and neurotoxicity was observed in the kidney of male rats after 24 h of exposure: tGSH, TBARS, and tChE increased, but protein content decreased (Table 3). The only effect found in female rats was increase in kidney GST activity after 24 h of exposure (Table 3). Kidney is the target organ for most xenobiotics, as proximal tubular cells are able to accumulate slightly acidic and basic compounds, amino acid conjugates, and quaternary compounds at toxic levels (38). The duration-dependent effect of IMI in our study is most probably related to its metabolism. Broznić et al. (39) reported that IMI was mostly cleared from the liver of BALB/C mice within 24 h and from the kidney within 48 h. In rats, kidney clearance is generally completed within 24 h after oral or intravenous administration (40). Oxidative effects of IMI were more pronounced in both tissues of the male rats regardless of exposure duration. It is likely that different metabolic rates resulted in sex-related toxicity differences in the IMIexposed rats. Induced CYP450 activities by nicotine enantiomers lead to oxidative stress conditions in Chinese hamster ovary K1 cell line (41). Kyerematen et al. (42) reported faster metabolism of nicotine in the kidney and liver of male than female SpragueDawley rats. Being a nicotine-like compound, IMI is metabolised more effectively in males than in females (40), and its faster metabolism could result in higher ROS production. Imidacloprid is metabolised mainly by CYP3A4, and by CYP2C19, CYP2A6, CYP2C9, CYP2D6, CYP2E1, CYP1A2, and CYP2B6 (10). Kammerer et al. (43) reported that protein kinase inhibitor ML3403

was metabolised in vitro by CYP450 isoenzymes (CYP3A4, CYP2C19, CYP1A2, and CYP2D6) much faster in male than in female Wistar rats. Sundseth and Waxman (44), in turn, found that clofibrate-induced liver and kidney CYP4A expression was higher in Fischer 344 male rats than in female, probably under hormonal influence. Aldehyde oxidase functions as a ROS source (45) and is affected by sex hormones such as oestradiol, which may account for sex-specific differences in IMI toxicity, even though we did not analyse this enzyme’s activity. Huh et al. (46) reported that Fe2+-induced lipid peroxidation was higher in male than in female Sprague-Dawley liver, and that oestradiol, possibly by the way of AOX inhibition, alleviated lipid peroxide formation compared to progesterone and testosterone. Ventura and Dachtler (47) suggested that androgens increase AOX activity in the liver of male and female C57BL/6J mice, and that oestradiol decreases it in males. Our results suggest that increased tChE activity could be related to increased lipid peroxidation and tGSH levels and decreased protein content in the kidney of male rats after 24 h of exposure (Table 3). In a study by Abu-Donia et al. (13), IMI increased AChE activity in newborn Sprague-Dawley rats via maternal exposure, and the authors suggested that this induction occurred due to apoptotic events via cellular Ca2+-uptake. In another study (48), neonicotinoid insecticides acetamiprid and IMI induced cellular Ca2+ uptake via α 7 nAChRs in Sprague-Dawley rat cerebellar cell line, and Ca2+ uptake was inhibited by α-bungarotoxin, a specific α7 nAChR antagonist. Yeboah et al. (49) suggest that α7 nAChRs, which are constitutively expressed in the kidney of SpragueDawley rats, are highly permeable to Ca 2+ ions. Duzguner and Erdogan (16) believe that higher plasma Ca2+ concentrations could be responsible for the induction of ROS-generating enzymes xanthine oxidase and myeloperoxidase in the liver and brain of female Wistar rats treated with IMI intravenously. Kimura-Kuroda et al. (48) have suggested that mammalian ACh receptors are affected by IMI exposure because of conformational changes of the receptor induced by ACh. We therefore believe that higher tChE activity that lowers ACh-assisted IMIbinding may be an adaptive response to oxidative stress induced by elevated Ca2+ uptake. This is supported by Bond et al. (50), who found that AChE release in rat astroglial cells increased in response to t-butylhydroperoxide-induced oxidative stress.

LIVER 12 h Sex Saline Control DMSO Control IMI PBO MEN IMI+PBO IMI+MEN Male 6.61±0.75* 4.30±0.40a 2.73±0.25b 4.68±0.28a 7.04±0.77c 0.97±0.07d 1.16±0.02e tGSH a ab b a b Female 5.63±0.45* 4.09±0.15 3.78±0.34 3.06±0.24 4.50±0.37 2.62±0.39 3.50±0.05b Male 2.45±0.22 2.47±0.08a 2.23±0.15ab 2.17±0.13ab 2.15±0.09b 1.86±0.08b 2.14±0.11b GPx Female 7.25±0.28 6.74±0.39 6.79±0.20 7.37±0.44 7.00±0.35 6.85±0.34 6.41±0.13 Male 199.17±13.61 210.57±11.84 211.71±4.12 215.12±6.03 208.49±6.54 215.57±6.62 199.55±26.85 CAT Female 170.83±5.45 142.94±10.20 136.14±9.39 141.18±9.63 142.05±6.49 134.85±13.52 116.59±4.29 Male 181.00±29.02 217.54±29.39 219.13±23.87 263.74±36.00 228.93±19.49 171.51±29.64 155.61±22.37 GST Female 236.62±13.91 239.40±20.86ab 175.00±16.92b 260.26±22.81a 252.42±27.74a 197.44±24.84ab 283.42±16.31a Male 316.32±10.77 340.30±13.90 327.88±12.58 298.55±9.65 308.38±13.17 305.19±13.42 308.57±11.59 TBARS Female 300.77±33.65 310.51±25.02a 275.58±14.24a 261.13±20.79ab 279.02±12.09a 220.35±18.19b 214.82±20.20b Total Male 23.24±0.83 24.06±0.61 24.33±0.64 24.69±0.73 24.94±0.68 24.84±0.43 23.21±0.91 Female 23.42±0.79 25.49±0.24 24.61±0.35 26.77±1.26 25.59±0.48 24.12±1.29 25.82±0.39 protein Male 1.75±0.06* 1.93±0.05a 1.64±0.11ab 1.68±0.13ab 1.61±0.08b 1.53±0.03b 1.61±0.18ab tChE Female 3.86±0.23 4.47±0.30a 4.35±0.55ab 4.71±0.40a 3.09±0.22b 3.31±0.22b 2.09±0.07c The results are presented as mean±standard error of mean. Asterisk (*) denotes significant difference between the saline and DMSO control groups. Otherwise, saline control was not compared statistically to other groups. Different superscript letters denote significant differences in the same line (P<0.05; N=6)

Table 2 Prooxidative and neurotoxic effects of IMI modulated by PBO and MEN in the liver after 12 h of exposure in male and female Sprague-Dawley rats

KIDNEY 12 h Sex Saline Control DMSO Control IMI PBO MEN IMI+PBO IMI+MEN Male 1.40±0.06 1.32±0.07 1.33±0.07 1.41±0.10 1.25±0.07 1.39±0.11 1.45±0.24 tGSH Female 2.55±0.09 2.30±0.13 2.39±0.07 2.56±0.05 2.30±0.05 2.49±0.12 2.25±0.05 Male 5.59±1.08 3.92±0.22 3.87±0.22 3.97±0.32 3.59±0.08 3.52±0.24 3.43±0.17 GPx Female 3.50±0.09 3.30±0.23 2.88±0.19 3.32±0.24 2.91±0.12 2.89±0.23 2.79±0.10 Male 149.68±1.37 163.53±7.62 159.56±4.82 148.56±6.32 153.95±5.90 159.97±7.80 132.88±12.93 CAT Female 148.95±5.70 158.50±14.49 145.61±10.43 138.98±10.80 122.99±10.51 137.47±5.81 133.46±9.81 Male 224.17±23.23 220.63±19.23 216.77±18.20 222.67±17.83 206.69±16.16 210.22±3.94 223.53±11.77 GST Female 83.51±7.76 94.33±7.94 80.27±3.94 93.25±7.56 96.53±5.66 88.26±6.88 90.58±5.34 Male 367.03±10.03* 242.85±16.93 251.03±25.82 292.63±17.99 326.60±27.22 301.50±16.55 250.68±14.29 TBARS Female 393.80±16.31 255.45±70.63 331.50±22.23 316.75±33.10 362.10±23.78 338.19±25.73 358.24±18.81 Total Male 6.64±0.17 7.22±0.25ab 7.36±0.28a 6.71±0.19b 7.24±0.29ab 7.32±0.29ab 8.15±0.18c Female 5.00±0.17 5.14±0.28 5.35±0.18 4.79±0.20 5.31±0.11 5.26±0.33 5.15±0.17 protein Male 1.84±0.13 2.26±0.23ab 1.88±0.22a 2.36±0.29ab 2.77±0.24b 3.40±0.21c 2.96±0.34bc tChE Female 2.39±0.24 2.61±0.14a 2.71±0.15a 2.87±0.09a 2.14±0.09b 2.71±0.05a 2.54±0.10a The results are presented as mean±standard error of mean. Asterisk (*) denotes significant difference between the saline and DMSO control groups. Otherwise, saline control was not compared statistically to other groups. Different superscript letters denote significant differences in the same line (P<0.05; N=6)

Table 1 Prooxidative and neurotoxic effects of IMI modulated by PBO and MEN in the kidney after 12 h of exposure in male and female Sprague-Dawley rats

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Furthermore, Yang et al. (51) found that d-tubocurarine, which is an nAChR antagonist, prevented diisopropylphosphorofluoridate to increase AChrelated ROS and lipid peroxidation in the muscle of male Sprague-Dawley rats. While the normal range of tChE activity in our study was 1.75-2.30 nmol L-1 per min per mg of protein in male liver and 3.64-3.86 nmol L-1 per min per mg of protein in female liver (Tables 2 and 4), literature reports a wide variety of inconclusive findings, and our results were not far from some of these (52-58). Obregon et al. (59) found that DMSO mixed with different brain region homogenates of male Wistar rats inhibited ChE activity at all tested concentrations. However, in our study DMSO had no effect on the liver and kidney tChE after i.p. injection, except for a transitory elevation in the liver of male rats at 12 h. Similarly, Carr and Nail (60) reported no DMSO effect on ChE activities of different brain regions in SpragueDawley pups of either sex, while the serum activity increased significantly. However, this increase did not suffice to protect against chlorpyrifos treatment, just as in our study higher tChE in the DMSO control group did not suffice to protect against IMI+PBO in male rats. At 12 h, IMI+PBO significantly lowered tChE activity compared to either saline or DMSO control. In our study, GPx activity increased and CAT activity decreased in the liver of male rats after 24 h of exposure (Table 4). CAT and GPx compete for H2O2 detoxification (61). In a study by Duzguner and Erdogan (16), IMI lowered GPx activity in the liver and increased it in the brain of female Wistar rats, while CAT was not affected. The authors suggested that GPx could be more important than CAT in countering IMI-induced ROS formation. Table 3 shows that GST activity in our study increased in the kidney of female rats after 24 h of exposure without a change in TBARS levels. GST catalyses the conjugation of GSH with xenobiotics, including lipid peroxidation products (62), and according to the WHO (31) this reaction is not important in IMI biotransformation. The level of GST subunits 3 and 4 in females are 3-4 times higher than in males, while subunits 1 and 2 are 1.5-2 times higher in the kidney of male rats (63). Gender-specific subunit levels may be related to this increase and further research should look into which GST subunit is responsible for IMI effect in the kidney of female rats.

Effects of PBO on IMI toxicity Piperonyl butoxide can be valuable to determine the effects of CYP450-mediated biotransformation on IMI toxicity (10). We observed no change in oxidative stress parameters in the kidney regardless of sex after 12 h of exposure (Table 1). However, tGSH content was more pronounced in the liver of male rats treated with IMI+PBO than IMI alone (Table 2). IMI+PBO co-exposure decreased liver GPx activity compared to control, but it did not differ significantly from IMI and PBO alone groups. This lower GPx activity may be associated with lower tGSH, which is a substrate for the enzyme. IMI+PBO decreased total protein content in the liver of male rats after 24 h of exposure (Table 4). GPx activity dropped, while CAT remained within the control range. This suggests that GPx is more involved in IMI-induced oxidative stress than CAT. Lower CAT activity in the liver of female rats also supports this hypothesis because no change was observed in tGSH, TBARS, and total protein content (Table 4). Since PBO inhibits CYP450 enzymes, the accumulation of the parent IMI compound may provide more substrate for other biotransformation pathways such as AOX. This elevation in AOX activity could explain the oxidative effects in the liver of male rats. A similar explanation was suggested by Valentovic et al. (64), who said that increased liver toxicity of 3,5-dichloroaniline given with PBO could be related to the metabolites of the parent compound produced by metabolic processes other than CYP450. Unlike the liver, the kidney of male rats was partly protected against IMI toxicity by PBO pre-treatment; it had no effect on increased tGSH and decreased total protein content in the kidney of male rats after 24 h of exposure (Table 3). However, TBARS levels, which increased in the IMI and PBO alone groups, did not increase significantly in respect to control. Baliga et al. (65) found that PBO provided protection against H2O2-induced cytotoxicity in proximal tubular cell line LLC-PK1 and they suggested that CYP450 may have served as a source of catalytic iron in ROS production pathways. Co-exposure to IMI+PBO lowered liver tGSH and TBARS in females after 12 h of exposure (Table 2); while there was no change in the IMI alone group. These changes seem to be related to PBO pretreatment. Muguruma et al. (66) reported that PBO increased microsomal ROS production in the liver.

LIVER 24 h Sex Saline Control DMSO Control IMI PBO MEN IMI+PBO IMI+MEN Male 1.10±0.11 1.06±0.11ab 0.89±0.06a 1.24±0.08b 1.02±0.05ab 1.11±0.08b 1.34±0.13b tGSH Female 3.36±0.20 3.52±0.21 2.98±0.32 3.90±0.22 3.62±0.30 3.48±0.15 3.35±0.07 Male 3.85±0.16 3.76±0.09ab 4.01±0.08c 3.47±0.25abcd 4.03±0.12ac 3.19±0.07d 3.33±0.13bd GPx Female 6.06±0.32 5.79±0.26 6.45±0.31 6.19±0.20 5.33±0.21 5.57±0.21 6.04±0.33 Male 178.03±2.67* 146.11±4.09a 122.24±2.92b 178.10±13.55c 149.37±4.88ac 167.35±7.84ac 163.97±6.96c CAT Female 165.68±7.83 144.80±8.23a 138.12±9.85ab 134.16±7.74abc 146.20±13.30ab 111.22±2.95c 119.59±2.12bc Male 208.07±25.83 209.05±24.41 215.53±9.41 240.88±16.93 215.48±21.17 221.81±14.80 201.22±18.38 GST Female 335.05±12.15 294.94±24.26 341.59±11.66 348.60±14.69 342.43±32.42 348.88±15.91 325.98±19.22 Male 472.05±49.68 409.06±17.88ab 462.78±24.02a 454.11±24.85a 386.37±14.50b 360.00±2.71b 393.14±39.57ab TBARS Female 210.55±20.94 197.68±22.46 243.37±19.45 284.11±11.88 266.85±27.59 247.82±7.70 215.72±20.94 Total Male 24.31±0.66* 27.59±0.57a 26.37±0.80ab 25.23±1.15ab 24.30±0.39bc 24.98±0.37bc 25.05±0.75ac Female 19.49±0.56 20.86±0.85 24.61±0.35 26.77±1.26 25.59±0.48 24.12±1.29 25.82±0.39 protein Male 2.30±0.14 2.17±0.04a 2.55±0.17a 2.22±0.12a 1.85±0.05b 1.72±0.07b 2.38±0.13a tChE Female 3.64±0.36 2.78±0.11a 2.72±0.23ab 3.61±0.09c 3.17±0.38abc 2.54±0.28ab 2.28±0.04b The results are presented as mean±standard error of mean. Asterisk (*) denotes significant difference between the saline and DMSO control groups. Otherwise, saline control was not compared statistically to other groups. Different superscript letters denote significant differences in the same line (P<0.05; N=6)

Table 4 Prooxidative and neurotoxic effects of IMI modulated by PBO and MEN in the liver after 24 h of exposure in male and female Sprague-Dawley rats

KIDNEY 24 h Sex Saline Control DMSO Control IMI PBO MEN IMI+PBO IMI+MEN Male 1.24±0.06 1.14±0.03a 1.49±0.02b 1.44±0.06bc 1.45±0.03b 1.41±0.11bc 1.30±0.05c tGSH Female 2.05±0.20* 1.67±0.04 1.74±0.06 1.69±0.11 1.76±0.15 1.67±0.07 1.69±0.07 Male 2.53±0.08 2.69±0.21ab 2.82±0.23abc 2.74±0.10a 2.73±0.08a 2.13±0.16bc 2.07±0.05c GPx Female 3.86±0.27* 3.01±0.09 3.41±0.19 3.81±0.27 3.58±0.17 3.50±0.17 3.86±0.26 Male 132.00±6.69 129.42±4.33a 140.58±3.44ab 153.31±6.90b 150.60±3.24b 152.21±6.87b 147.38±3.90b CAT Female 129.21±3.22 116.09±6.40 120.38±5.54 105.31±4.90 125.64±8.57 127.65±4.64 122.67±2.04 Male 68.11±3.93* 113.46±12.35 120.40±5.74 119.47±10.67 126.92±12.85 113.44±10.41 123.58±8.87 GST Female 63.13±3.62 53.97±1.64a 69.15±1.06b 36.60±0.68c 87.44±5.01d 106.74±7.35d 100.37±4.67d Male 279.16±13.38 255.72±11.26a 302.25±2.73b 308.22±19.09b 252.71±3.99a 247.87±19.05abc 203.38±11.32c TBARS ab abc b abc ac Female 465.48±29.67* 327.86±26.26 384.79±28.09 301.93±25.99 366.23±28.06 377.70±17.63 408.62±14.82c Total Male 7.98±0.20 8.37±0.23a 6.94±0.14b 7.20±0.17bc 6.88±0.11b 7.14±0.29bc 7.74±0.15ac Female 5.63±0.30* 6.68±0.14 6.34±0.24 6.77±0.21 6.42±0.29 6.28±0.23 6.14±0.19 protein Male 1.72±0.07 1.74±0.04a 2.23±0.06b 1.60±0.14a 1.65±0.13a 2.88±0.22c 2.75±0.32bc tChE Female 1.69±0.10 1.78±0.09ab 2.07±0.14a 1.95±0.08a 1.76±0.15ab 1.96±0.08a 1.65±0.06b The results are presented as mean±standard error of mean. Asterisk (*) denotes significant difference between the saline and DMSO control groups. Otherwise, saline control was not compared statistically to other groups. Different superscript letters denote significant differences in the same line (P<0.05; N=6)

Table 3 Prooxidative and neurotoxic effects of IMI modulated by PBO and MEN in the kidney after 24 h of exposure in male and female Sprague-Dawley rats

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This suggests that lower TBARS levels in the present study could be attributed to lower tGSH levels that act against ROS production. Furthermore, co-exposure to IMI+PBO displayed tissue-specific effect of IMI on tChE activity. In the kidney of male rats, it was higher than in control, IMI, and PBO groups after 12 and 24 h of exposure (Tables 1 and 3). We believe that increased tChE activity may be an adaptive response against oxidative stress, as we discussed earlier. In the liver of male rats, however, tChE activity dropped significantly compared to control, IMI, and PBO groups after 24 h of exposure (Table 4). IMI+PBO also lowered tChE activity after 12 h of exposure compared to control (Table 2). Liver tChE activity also dropped in females compared to control, IMI, and PBO groups after 12 h (Table 2), while it remained within the control range after 24 h of exposure. PBO alone increased tChE activity (Table 4). While some authors (14, 15) reported that IMI lowered ChE, tissue-specific effects of IMI on tChE activity require further investigation. Effects of MEN on IMI toxicity Interestingly, MEN as a modulator of IMI metabolism has revealed similar tissue-specific effect of IMI on tChE activity as observed in PBO coexposure. MEN lowered tChE activity in the kidney female rats (Table 1). Similarly, Torda and Wolff (67) reported that MEN decreased ChE activity in human serum. However, IMI+MEN co-exposure resulted in no significant change, probably due to the antagonistic effect of IMI against MEN. In contrast, IMI+MEN co-exposure lowered liver tChE activity in female rats compared to control, IMI, and MEN alone groups in both exposure durations (Table 2 and 4). Therefore, we are convinced that IMI, whose metabolism was inhibited by PBO or MEN, inhibits liver tChE activity. Menadione co-exposure proved to be useful against oxidative stress in the kidney of male rats after 24 h of exposure (Table 3). It countered the TBARSincreasing and protein-lowering effects of IMI. It also lowered IMI-induced tGSH, while GPx decreased and CAT increased. Kitamura et al. (68) reported that AOX catalysed fenthion metabolism to fenthion sulphoxide, and that this transformation was inhibited by MEN and β-oestradiol in the liver of male Sprague-Dawley rats. Similarly, Honda et al. (8) reported that MEN decreased AOX activity in mouse fibroblast M10 cells. These findings suggest that MEN co-exposure may have inhibited AOX-mediated biotransformation of IMI in our study and therefore the oxidative effects of

its metabolites. Elevated tChE activity in this group may also have contributed to the protection against oxidative stress as discussed above. In female rats, MEN co-exposure also revealed the oxidative effects of IMI in both tissues. Liver TBARS and tGSH levels were significantly lower than in the control, IMI, and MEN alone groups after 12 h of exposure (Table 2). Higher kidney GST activity and TBARS levels were observed in IMI+MEN group after 24 h of exposure (Table 3). In the liver of male rats, tGSH and GPx activity dropped, but total protein content and lipid peroxidation levels did not change after 12 h of exposure (Table 2). In the liver of male Wistar rats, Sidorova and Grishanova (69) found that MEN increased total CYP450 content, GST, and a variety of CYP450 enzyme activities, including CYP1A2, the enzyme that catalyses nitroimine reduction of IMI. This suggests that, CYP450mediated metabolism of IMI could be accelerated by MEN co-exposure and contribute to IMI-related oxidative effects, especially in female rats, despite possible inhibition of AOX activity. Because of the opposite effects of MEN co-exposure (AOX inhibition and CYP450 induction), further in vivo studies with the same doses of IMI and MEN should investigate the mechanisms that affect CYP450 and AOX activities.

CONCLUSIONS This is the first study that shows tissue-, exposure duration-, and sex-differences in the prooxidative and neurotoxic potential of IMI in rats. IMI’s oxidative and neurotoxic effects after 24 h of exposure were probably related to its metabolism. The most pronounced prooxidative effect was observed in the kidney of male rats, and it may be related to hormonal effects on the enzymes that participate in IMI metabolism. However, further investigation is needed to evaluate the effects of sex hormones on CYP450s and AOX activity in the presence of IMI. IMI has also shown a tissue-specific effect on tChE activity, which was clarified by metabolic modulators PBO and MEN. Interestingly, PBO and MEN co-exposure exacerbated the prooxidative effects of IMI in the liver of male rats, while no change or a decrease was found in tChE activity. This points to a synergy between IMI and PBO in the liver, which could be more toxic to mammals in terms of oxidative stress and neurotoxicity. In contrast, PBO and MEN co-exposure alleviated the

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prooxidative effects of IMI in the kidney, as evidenced by a significant rise in tChE activity in these groups. This calls for further investigation of the protective role of cholinesterase against IMI-induced oxidative stress. MEN co-exposure was also useful in revealing the oxidative effects of IMI in female rats in both tissues because IMI alone and MEN alone had no effects in females.

11. 12. 13.

Acknowledgements We wish to thank the Adiyaman University Scientific Research Commission for supporting our study through project grants no. FEFBAP2011/007 and FEFYL2011/0011. We also wish to thank Dado Čakalo for having edited the manuscript to read better. We have no conflict of interest to declare.

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Sažetak Djelovanje imidakloprida pod utjecajem piperonil butoksida i menadiona ovisno je o spolu, tkivu i trajanju izloženosti. I. dio - oksidativno i neurotoksično djelovanje Rezultati ranijih istraživanja pokazali su oksidativni i neurotoksični potencijal imidakloprida, neonikotinoidnog insekticida, u različitih životinjskih vrsta. Primarni je cilj ovog istraživanja bio utvrditi kako modulatori metabolizma piperonil butoksid i menadion utječu na nepovoljne učinke imidakloprida na jetra i bubrege muških i ženskih štakora soja Sprague-Dawley. Životinje su 12 h odnosno 24 h bile izložene samo imidaklopridu (170 mg kg-1) ili njegovoj kombinaciji s piperonil butoksidom (100 mg kg-1) odnosno menadionom (25 mg kg-1). U homogenatima jetara i bubrega spektrofotometrijski su utvrđene razine glutation peroksidaze, glutation S-transferaze, katalaze, specifične aktivnosti ukupne kolinesteraze, ukupni glutation, ukupni proteini te razine lipidne peroksidacije. Imidakloprid se pokazao prooksidativnim i neurotoksičnim uglavnom u bubrezima muških štakora nakon 24-satne izloženosti. Naši rezultati upućuju na to da su razlike u prooksidativnom i neurotoksičnom djelovanju imidakloprida povezane sa spolnim razlikama. Predtretmanom piperonil butoksidom odnosno menadionom (90 min prije davanja imidakloprida) otkriveno je da imidakloprid djeluje na ukupnu aktivnost kolinesteraze specifično za pojedina tkiva. Povišena aktivnost kolinesteraza u bubrezima mogla bi odražavati prilagodbu na oksidativni stres uzrokovan imidaklopridom. Piperonil butoksid odnosno menadion u jetrima muških štakora samo su pogoršali toksičnost imidakloprida. U ženki je djelovanje imidakloprida s menadionom bilo prooksidativno; takvo se djelovanje nije vidjelo nakon primjene samo imidakloprida odnosno samo menadiona. Vjerujemo da je promjenjivo djelovanje imidakloprida s obzirom na spol, tkivo i trajanje izloženosti važno za daljnja istraživanja njegove toksičnosti. KLJUČNE RIJEČI: glutation peroksidaza; glutation S-transferaza; katalaza; kolinesteraza; neonikotinoidi; oksidativni stres

CORRESPONDING AUTHOR: Yusuf Sevgiler Adiyaman University, Faculty of Science and Letters, Department of Biology 02040 Adiyaman, Turkey E-mail: ysevgiler@adiyaman.edu.tr, ysevgiler@gmail.com

Khalili M, et al. ANTIHAEMOLYTIC ACTIVITY OF HERBAL EXTRACTS IN MOUSE RBCs Arh Hig Rada Toksikol 2014;65:399-406

399

DOI: 10.2478/10004-1254-65-2014-2513

Original article

Antihaemolytic activity of thirty herbal extracts in mouse red blood cells Masoumeh Khalili1, Mohammad Ali Ebrahimzadeh1, and Yaghoub Safdari2 Pharmaceutical Sciences Research Center, School of Pharmacy, Mazandaran University of Medical Sciences, Sari1, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan2, Iran Received in March 2014 CrossChecked in March 2014 Accepted in November 2014

Reactive oxygen species (ROS) can lead to haemolysis and eventually to diseases such as thalassemia and sickle cell anaemia. Their action can be counteracted by the antihaemolytic activity of therapeutic agents. The aim of our study was to identify plants that most efficiently counteract ROS-caused haemolysis. From ten plants known for their antioxidant activity (Orobanche orientalis G. Beck, Cucumis melo L., Albizzia julibrissin Durazz, Galium verum L., Scutellaria tournefortii Benth, Crocus caspius Fischer & Meyer, Sambucus ebulus L., Danae racemosa L., Rubus fruticsos L., and Artemisia absinthium L.) we prepared 30 extracts using three extraction methods (percolation, Soxhlet, and ultrasound-assisted extraction) to see whether the extraction method affects antihaemolytic efficiency, and one extraction method (polyphenol extraction) to see how much of this action is phenol-related. Extract antihaemolytic activity was determined in mice red blood cells and compared to that of vitamin C as a known antioxidant. Nine of our extracts were more potent than vitamin C, of which G. verum (aerial parts/percolation) and S. tournefortii (aerial parts/polyphenol) extracts were the most potent, with an IC50 of 1.32 and 2.08 µg mL-1, respectively. Haemolysis inhibition depended on extract concentration and the method of extraction. These plants could provide accessible sources of natural antioxidants to the pharmaceutical industry. KEY WORDS: Galium verum; hydrogen peroxide; percolation; phenols; Scutellaria tournefortii; Soxhlet; ultrasound-assisted extraction

Oxidative damage and haemolysis caused by reactive oxygen species (ROS) have a major role in the development of diseases such as thalassemia, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anaemia. Red blood cells (RBCs) are the primary targets of free radicals, owing to their high membrane concentrations of polyunsaturated fatty acids (linoleic and arachidonic acids in particular) and O2 transport associated with redox active haemoglobin molecules, which are potent promoters of ROS. Oxidation depletes membrane protein content, deforms RBCs, and disturbs microcirculation (1-4). It is also implicated in haemolysis (5).

Haemolysis has long been used to measure free radical damage and counteraction by antioxidants. It is useful for screening for oxidising or antioxidising agents (6). Several herbal secondary metabolites such as flavonoids have been found to protect cells from oxidative damage. These compounds have been evidenced to stabilise RBC membrane by scavenging free radicals and reducing lipid peroxidation (3, 7). Herbs are a rich source of flavonoids, phenolic acids, and alkaloids, some of which act as antioxidants (7-10). Nabavi et al. (9) studied the antioxidant and antihaemolytic activities of Ferula foetida in RBCs and a few other interesting studies have recently been reported, indicating the protective effects of plant

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extracts against oxidative damage in intact RBC membranes (7, 9, 10). The aim of this study was to investigate the antihaemolytic activity of herbal extracts from ten plants and identify promising alternatives to treating diseases associated with haemolysis. We also wanted to see which of the three extraction methods used ultrasound-assisted extraction (UAE), Soxhlet extraction (SO), and percolation (PE) yielded more efficient extracts. Our third aim was to verify the protective effects (as claimed in literature, cf. 11, 12) of polyphenols extracted from two plants against oxidative stress. With these aims, we prepared 30 herbal extracts and evaluated their capacity to suppress RBC haemolysis. In addition, we also measured the haemolytic activity of the most potent extracts in the absence of H2O2 in order to determine their own oxidative toxicity to RBCs.

MATERIALS AND METHODS Chemicals Formic acid, 30 % hydrogen peroxide, and vitamin C, which was used as reference compound, were purchased from Merck (Darmstadt, Germany). All solvents were of analytical grade or purer. Distilled deionised water was prepared with the Ultrapure™ water puriﬁcation system. Vitamin C was used in the same concentrations as the plant extracts. Plants Ten plant species were collected and their identity verified by Dr Bahman Eslami (Islamic Azad University of Qaemshahr, Iran). The voucher specimens were deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Mazandaran University of Medical Sciences. Table 1 lists the plants, the parts, and the methods used for extraction. Plant materials were dried under dark conditions at room temperature for 2-3 weeks. The dry materials were milled, obtaining 2-3 mm particles. Extraction methods Extraction is the first important step in the recovery and purification of active ingredients from plants. The aim is to provide maximum yield and the highest quality of target compounds in the shortest time and at the lowest cost possible. Many techniques, such as

conventional solvent extraction, microwave-assisted, and ultrasound-assisted extraction, have been developed to extract active ingredients (13). In this study, we opted for the three most common extraction methods: percolation, Soxhlet extraction, and ultrasound-assisted extraction. For percolation, we used methanol to treat known amounts of each plant part at room temperature as described elsewhere (14, 15). The resulting extracts were concentrated over a rotary vacuum (Heidolph, Schwabach, Germany) at 35-40 °C until a crude solid extract was obtained, which was then freeze-dried (MPS-55 freeze-drier, Operon Co., Ltd., Gimpo, South Korea) for complete solvent removal. In Soxhlet extraction, powdered samples were treated with methanol and extracted in an ISOLAB extractor (Wertheim, Germany) for 24 h. The extracts were then concentrated in a rotary evaporator (Heidolph, Germany) until the solvent was removed. The extracts were freeze-dried for complete solvent removal (14, 15). In ultrasound-assisted extraction samples were treated with methanol and placed in an ultrasonic cleaning bath (Tecna3, Bologna, Italy) at a frequency of 100 kHz and temperature of 25±3 °C for 1 h to yield extracts, which were then separated from the residue by filtration and concentrated in a rotary evaporator until crude solid extracts were obtained. Followed freeze-drying for complete solvent removal (16). Polyphenol extraction Literature suggests that polyphenols act as antioxidants and protect RBCs against oxidative damage (11, 12, 17). These compounds were extracted from samples according to our recently published paper (16). The extraction was performed twice at 20 °C in a shaking incubator (115 W, Promax 1020, Heidolph, Germany). The extraction time was 30 min and the extracting solvent was 100 mL of methanol/ acetone/water (3.5/3.5/3) containing 1 % formic acid. Extracts were combined and filtered through two layers of cheesecloth. The collected filtrate was centrifuged at 7000 g for 15 min. The supernatant was collected and evaporated under vacuum at 35-40 °C to remove methanol and acetone. Lipophilic pigments were then eliminated from the aqueous phase by two successive extractions in a separatory funnel with a twofold volume of petroleum ether. The aqueous phase was collected and further extracted by the equal volume of ethyl acetate three times in the separatory funnel. Three ethyl acetate phases were collected and

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Table 1 Plant names, extraction methods, part used, and Herbarium numbers

Plant name

Extraction method

Part used

Herbarium number

Orobanche orientalis (broomrape)

Ultrasonic/Soxhlet

Aerial parts

MH1001

Cucumis melo (muskmelon)

Percolation/Soxhlet/Ultrasonic

Leaf/Fruit

MH1002

Albizia julibrissin (Persian silk tree)

Ultrasonic/Soxhlet/ Percolation

Leaf/Flower

MH1003

Percolation

Aerial parts

MH1004

Percolation/Soxhlet/Polyphenol

Aerial parts

MH1010

Percolation/Polyphenol/Ultrasonic

Aerial parts/Bulb

MH1008

Soxhlet/Percolation/Ultrasonic

Flower

MH1009

Soxhlet

Aerial parts

MH1011

Percolation

Leaf

MH1015

Soxhlet/Percolation/ Ultrasonic

Aerial parts

MH1020

Galium verum (lady's bedstraw) Scutellaria tournefortii (skullcap) Crocus caspius (crocus) Sambucus ebulus (danewort) Danae racemosa (Alexandrian or poet's laurel) Rubus fruticsos (blackberry) Artemisia absinthium (wormwood)

concentrated over a rotary vacuum until a crude solid extract was obtained, which was then freeze-dried for complete solvent removal. Preparation of mice RBCs A total of 30 male Swiss albino mice (20-25 g, Institute Pasteur of Iran) were used in this study. The animals were housed in standard cages with free access to food (standard laboratory rodent chow) and water. The animal house temperature was maintained at 23±3 °C with a 12-h light/12-h dark cycle (light on from 06:00 to 18:00 h). All of the experiments were conducted between 10:00 and 14:00 h. The experimental procedure was conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (18). The mice were killed under anaesthesia and their blood collected by heart puncture in heparinised tubes. RBCs were isolated and stored according to the method described by Ebrahimzadeh et al. (7). Briefly, blood samples were centrifuged (1500 g, 10 min), and RBCs separated from plasma and buffy coat and washed three times by centrifugation (1500 g, 5 min) in 10 volumes of 10 mmol L-1 phosphate buffered saline (pH 7.4; PBS). The supernatant and buffy coats of white cells were carefully removed with each wash. Washed RBCs were stored at 4 °C and used within 6 h (19). Antihaemolytic activity assay This assay is useful for screening for agents and their metabolites that have an oxidising or antioxidising

activity (11). Peroxides such as H2O2 and cumene hydroperoxide (7), protein denaturation, and heat shock-induced damage have all been extensively studied in biological membranes (7, 20, 21). The antihaemolytic activity of the extracts in our study was determined as described by Ebrahimzadeh et al. (7). Mice RBCs were diluted in PBS buffer to obtain a 4 % suspension. The plant extracts were prepared in PBS buffer at five concentrations: 0.25, 0.5, 1, 2, and 4 mg mL-1 (see our recently published paper under ref 22). To 2 mL of RBC suspension we added 1 mL of plant extract (in the above concentrations) and enough PBS to reach the final volume of 5 mL. After 5 min of incubation at room temperature, 0.5 mL of 0.3 % H 2O 2 was added to induce oxidative degradation of membrane lipids and the mixture was shaken at 37 °C for 240 min. The samples were then centrifuged at 1500 g for 10 min and the resulting supernatant was removed and used to evaluate their haemolytic activity using a spectrophotometer (UV– Visible EZ201, Perkin Elmer, Norwalk, CA, USA) at the absorbance wavelength of 540 nm. RBC lysis in the presence of H2O2 and absence of a plant extract was considered as 100 % haemolytic activity. Haemolysis in the presence extracts was calculated relative to this control haemolysis (22). Haemolysis inhibition was calculated as follows: % antihaemolysis=[(Ao−A1)/Ao]×100 where Ao was the absorbance of control (H2O2+RBC, without extract) and A1 the absorbance in the presence

Khalili M, et al. ANTIHAEMOLYTIC ACTIVITY OF HERBAL EXTRACTS IN MOUSE RBCs Arh Hig Rada Toksikol 2014;65:399-406

Total phenolic and flavonoid content Total phenolic content was determined using the Folin-Ciocalteu method (16). We first mixed sample extract (0.5 mL) with the Folin-Ciocalteu reagent (5 mL, diluted with distilled water at the 1:10 ratio) for 5 min and then added aqueous Na2CO3 (4 mL, 1 mol L-1). After 2 h of incubation at room temperature, we measured the absorbance of reaction with a doublebeam spectrophotometer (UV- Visible EZ201, Perkin Elmer, Norwalk, CA, USA) at 760 nm. The standard curve was prepared with 0, 50, 100, 150, 200, and 250 µg mL-1 solutions of gallic acid in methanol and water (50:50, v/v). Total phenol content is expressed as milligram of gallic acid equivalents (GAE) per one gram of extract. Total flavonoid content was measured using the colourimetric aluminium chloride method (16). Briefly, 0.5 mL solutions of the extract in methanol were mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 mol L-1 potassium acetate, and 2.8 mL of distilled water and left at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm. Total flavonoid content was calculated from a calibration curve which was prepared by quercetin solutions with methanol at concentrations ranging from 12.5 to 100 µg mL-1 and is expressed as milligram of quercetin equivalent (QE) per one gram of extract.

250

IC50 (µg mL-1)

200 150 100 50

ac em S. osa eb (A u A S. lus / SO . a eb (F ) bs ul / A inth us ( UL . j iu F ) ul m / A ibri (A PE) . j ss ul in / S ib ( O C. riss L / ) S. ca in SO to sp (F ) ur iu / U ne s ( L f G ort A / ) . v ii PP er (A ) um / ( A PP) V /P ita E) m in C

of the extract or vitamin C as the reference antioxidant (7, 9, 10) used in the same concentrations as the extracts (0.25-4 mg mL-1). Each set of experiments was performed in triplicate and the inhibitory activity expressed as percentage. Based on the inhibition, the plant extracts were classified into four groups: (a) neutral, with haemolysis similar to control, about 100 %; (b) weak, with haemolysis >80 % of the control; (c) potent, whose haemolysis was <80 %; and (d) reverse, whose haemolytic activity was even higher than control (>100 %). For potent extracts we calculated the half maximal inhibitory concentration (IC50), which is the antioxidant concentration required to inhibit 50 % of haemolysis caused by H2O2. We also measured the haemolytic activity of potent extracts in the absence of H2O2 in order to see if and how much oxidative damage they alone caused to RBCs. For this experiment, we only used the highest concentration, 4 mg mL-1.

402

Figure 1 IC50 of the most potent extracts with antihaemolytic activity (less is better). Vitamin C served as reference compound.

A: aerial part; F: fruit; L; leaf; SO: Soxhlet extraction; PE: percolation; UAE: ultrasound-assisted extraction; PP: polyphenol fraction

Statistical analysis The results of the antihaemolysis assays are presented as means of three replicate measurements ± standard deviation (SD). The data were analysed using the analysis of variance (ANOVA) (p<0.05) and grouped using Duncan’s multiple range test. The IC50 values were calculated using linear regression analysis.

RESULTS AND DISCUSSION Table 2 shows that the antihaemolytic activity of our extracts and extracted polyphenols varied a lot. The group with reverse effects (higher haemolysis than control) included C. melo (leaf/percolation, ultrasonic, and Soxhlet; fruit/Soxhlet and ultrasonic), A. julibrissin (leaf/ultrasonic; flower/Soxhlet), S. tournefortii (aerial parts/Soxhlet), O. orientalis (aerial parts/ultrasonic and Soxhlet), C. caspius (aerial parts/ percolation and ultrasonic; bulb/polyphenol), A. julibrissin (leaf/percolation; flower/percolation). S. tournefortii (aerial parts/percolation) was the only neutral sample, showing no inhibitory effects on H2O2. Five samples, including S. ebulus (flower/ Soxhlet), R. fruticsos (leaf/percolation), A. julibrissin (flower/ultrasonic), C. melo (fruit/percolation), and A. absinthium (aerial parts/percolation) showed weak haemolysis inhibition.

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Khalili M, et al. ANTIHAEMOLYTIC ACTIVITY OF HERBAL EXTRACTS IN MOUSE RBCs Arh Hig Rada Toksikol 2014;65:399-406

Table 2 The haemolytic activity of plants at different concentrations. Plant names (part used)

Concentration (mg mL-1) Method

0.5

0.25

Percolation

100.0±0.81

100.0±0.94

100.0±0.95

100.0±0.17

100.0±0.26

Polyphenol

35.8±0.76

46.5±0.37

48.5±0.85

49.3±0.93

97.6±0.69

Soxhlet

114.2±0.62

112.3±0.75

111.2±0.81

107.4±90

97.2±0.63

S. ebulus (flower)

Soxhlet

85.3±0.93

93.8±0.68

92.5±0.82

93.1±0.57

94.2±0.58

R. fruticsos (leaf)

Percolation

87.5±0.72

97±0.68

98.1±0.59

99.3±0.39

100.2±0.75

C. melo (fruit)

Percolation

96.4±0.94

98.2±0.83

99.2±0.07

99.5±0.09

99.6±0.59

C. melo (fruit)

Soxhlet

108.0±0.51

107.8±1.01

104.0±0.68

100.1±0.71

97.2±0.91

C. melo (fruit)

Ultrasonic

121.4±0.55

120.5±1.12

119.3±0.95

107.5±0.22

87.5±0.61

C. melo (leaf)

Percolation

121.1±0.80

119.3±0.29

114.1±0.45

112.4±0.98

111.9±0.37

C. melo (leaf)

Soxhlet

119.9±0.12

117.4±0.65

115.8±0.26

111.6±0.71

101.4±0.31

C. melo (leaf)

Ultrasonic

106.9±0.47

102.7±1.01

97.4±0.04

91.5±0.57

88.8±0.06

Percolation

87.7±0.47

83.2±0.48

88.1±0.19

91.8±0.69

91.2±0.19

Soxhlet

65.5±0.39

80±0.58

80.5±0.96

83.3±0.83

94.4±1.00

S. ebulus (flower)

Ultrasonic

79.3±0.41

84.1±0.29

86.6±0.59

91.6±0.18

98.4±0.09

S. ebulus (flower)

Percolation

72.3±0.16

78.5±0.68

86.6±0.71

98.6±0.56

99.8±0.26

Ultrasonic

75.9±1.08

88.6±0.91

90±0.99

90.7±0.08

95.2±0.83

Soxhlet

74.9±0.13

85±0.76

91.4±034

95.1±0.98

98.9±0.80

Soxhlet

62.2±0.38

83.1±0.85

86.1±0.82

92.9±0.94

100.1±0.09

A. julibrissin (flower)

Ultrasonic

82.1±0.09

89.2±0.17

92.6±0.57

94.4±0.67

98.8±0.61

A. julibrissin (flower)

Soxhlet

129.7±0.43

127.4±0.09

121.0±0.81

120.1±0.18

110.6±0.72

A. julibrissin (leaf)

Ultrasonic

123.8±0.91

118.8±0.85

116.8±0.47

103.2±0.70

95.4±0.58

A. julibrissin (leaf)

Percolation

126.2±0.63

124.4±0.45

117.0±0.94

98.7±0.51

90.6±0.09

A. julibrissin (flower)

Percolation

92.3±0.33

90.1±0.83

88.0±0.66

86.8±0.41

83.4±0.91

C. caspius (aerial parts)

Polyphenol

67.7±0.41

75.5±0.96

85.5±1.40

88.8±0.74

96.1±0.90

C. caspius (bulb)

Polyphenol

97.7±0.59

89.1±0.19

75.9±0.07

75.3±0.94

67.9±0.79

C. caspius (aerial parts)

Ultrasonic

114.0±0.41

113.9±0.73

110.8±0.84

109.4±0.69

104.8±0.05

C. caspius (aerial parts)

Percolation

130.6±0.38

112.1±0.69

109.4±0.19

104.9±0.81

100.3±0.77

G. verum (aerial parts)

Percolation

34.5±0.27

45.5±0.06

48.8±0.54

52.0±0.83

65.4±0.91

S. tournefortii (aerial parts) S. tournefortii (aerial parts) S. tournefortii (aerial parts)

A. absinthium (aerial parts) D. racemosa (aerial parts)

A. absinthium (aerial parts) A. absinthium (aerial parts) A. julibrissin (leaf)

O. orientalis (aerial Soxhlet 94.0±0.41 91.4±0.49 91.4±0.83 85.2±0.91 80.4±0.29 parts) O. orientalis (aerial Ultrasonic 128.2±1.30 117.2±1.01 112.4±0.91 110.9±0.66 86.5±0.82 parts) Vitamin C 83.3±0.11 90.6±0.10 98.7±0.26 99.3±0.38 99.8±0.51 The relative haemolysis of control was 100 %. Data are presented as %. Each experiment was performed in triplicate. Lower values indicate higher antihaemolytic activity

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The remaining nine samples, namely D. racemosa (aerial parts/Soxhlet), S. ebulus (flower/ultrasonic and percolation), A. absinthium (aerial parts/ultrasonic and Soxhlet), A. julibrissin (leaf/Soxhlet), C. caspius (aerial parts/polyphenol), S. tournefortii (aerial parts/ polyphenol), and G. verum (aerial parts/percolation) showed strong antihaemolytic activity. These nine were also more potent than the reference vitamin C (IC50=235±5.26 µg mL-1). Their IC50 is shown in Figure 1. The most potent were G. verum (aerial parts/ percolation) and S. tournefortii (aerial parts/ polyphenol), whose respective IC 50 of 1.32 and 2.08 µg mL-1 was significantly lower than that of vitamin C (p<0.001). Their antihaemolytic activity grew with concentration. The haemolytic activity of the nine potent compounds in the absence of H2O2 was below 7 % (data not shown), which suggests that they are not toxic. Unlike A. julibrissin and C. melo, whose activity in most cases intensified H2O2-induced haemolysis. C. caspius showed contradictory findings, as its polyphenol fraction extracted from the aerial parts strongly inhibited H2O2-induced haemolysis, but its extracts obtained by percolation and ultrasound intensified it. Although the Soxhlet extract of S. ebulus showed weak antihaemolytic activity, extracts obtained with other methods were potent against haemolysis. Similarly, percolation rendered A. absinthium weak while ultrasound and Soxhlet yielded potent antihaemolytic extracts. Clearly, extraction methods deserve a more comprehensive research in this respect. Various studies have recently investigated the potential of phenols against oxidative damage in RBCs, suggesting a possible interaction between flavonoids and RBC membrane lipids and proteins

that are generally targeted by lipid peroxidation (12, 17, 23). Divya et al. reported (24) considerable antihaemolytic activity of the methanolic extracts of Bombax ceiba pentandra fruit and spike (14.57 and 19.14 % haemolysis vs. 100 % for negative control, respectively) owing perhaps to their high phenol and flavonoid content and good antioxidant activity. Deepinderjeet et al. (25) also related the antihaemolytic effects of Calendula officinalis (IC50=120.5±0.4), Juglans regia (IC50=148.8±1.4), and Ficus bengalensis (IC50=214.1±1.5 µg mL-1) to high flavonoid content and good antioxidant activity. A report by Yang et al. (26) showed high haemolysis inhibition by fermented soybean meal extract at concentrations of 2-10 mg mL-1 (IC50 value 4.599 mg mL-1). The extract also had potent in vivo antioxidant activity. Our phenol and flavonoid findings are shown in Table 3. Antihaemolytic activity strongly correlated with total phenolic content (r2=0.693) but not with flavonoid content (r2=0.029). Our findings are in agreement with studies showing that polyphenols protect RBCs from oxidative stress or increase their resistance to oxidative damage (12, 27). They also confirm the correlation between total phenolic content and antioxidant activity from studies on different foodstuffs such as fruit and vegetables (28-30). To conclude, the nine extracts that showed high antihaemolytic activity in our study, G. verum (percolation) and S. tournefortii (polyphenol fraction) could serve as easily accessible sources of natural antioxidants for the pharmaceutical industry. Acknowledgements This research was supported by a grant from the Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Iran.

Table 3 Phenol and flavonoid content of the nine most potent extracts Extraction Phenol contenta Plant name Part used method / mg g-1 of extract Ultrasonic Flower 340 A. julibrissin Soxhlet Leaf 688 A. julibrissin Percolation Aerial parts 329 G. verum Polyphenol Aerial parts 735 S. tournefortii Polyphenol Aerial parts 191 C. caspius Percolation Flower 56 S. ebulus Soxhlet Aerial parts 256 D. racemosa Ultrasonic Flower 81 S. ebulus Soxhlet Aerial parts 267 A. absinthium a gallic acid equivalents of extract; b quercetin equivalent; mg g-1 of extract

Flavonoid contentb / mg g-1 of extract 136 121 56 151 82 14.5 131 52 136

Khalili M, et al. ANTIHAEMOLYTIC ACTIVITY OF HERBAL EXTRACTS IN MOUSE RBCs Arh Hig Rada Toksikol 2014;65:399-406

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Sažetak Antihemolitička aktivnost trideset biljnih ekstrakata u mišjim eritrocitima Reaktivni kisikovi spojevi (ROS) mogu dovesti do hemolize te u konačnici do bolesti poput talasemije i anemije srpastih stanica. Takvo se djelovanje može ublažiti ili spriječiti terapijskim djelovanjem antihemolitika. Cilj je ovog istraživanja bio izdvojiti biljke koje najuspješnije sprječavaju hemolizu uzrokovanu reaktivnim kisikovim spojevima. U tu smo svrhu pripremili 30 ekstrakata biljaka poznatih po svojem antioksidacijskom djelovanju: Orobanche orientalis G. Beck, Cucumis melo L., Albizzia julibrissin Durazz, Galium verum L., Scutellaria tournefortii Benth, Crocus caspius Fischer & Meyer, Sambucus ebulus L, Danae racemosa L., Rubus fruticsos L. te Artemisia absinthium L. Rabili smo tri uobičajene ekstrakcijske metode (perkolacija, Soxhlet i ultrazvučna ekstrakcija) kako bismo utvrdili utječe li metoda na anihemolitičku aktivnost ekstrakta te smo u nekoliko uzoraka ekstrahirali polifenole kako bi se vidjelo koliko je ta aktivnost povezana s njihovom razinom. Antihemolitičku smo aktivnost mjerili u mišjim eritrocitima i usporedili je s onom vitamina C, koji je poznati antioksidans. Izdvojeno je devet ekstrakata sa snažnijom aktivnosti od vitamina C, od kojih su ekstrakti G. verum (zračni dijelovi/perkolacija) odnosno S. tournefortii (nadzemni dijelovi/polifenoli) bili najsnažniji, s inhibicijskom koncentracijom (IC50) od 1,32 odnosno 2.08 µg mL-1. Inhibicija hemolize ovisila je o koncentraciji ekstrakta te o metodi ekstrakcije. Ove bi biljke mogle poslužiti farmaceutskoj industriji kao lako dostupni izvori prirodnih antioksidansa. KLJUČNE RIJEČI: fenoli; Galium verum; perkolacija; Scutellaria tournefortii; Soxhlet; ultrazvučna ekstrakcija; vodikov peroksid

CORRESPONDING AUTHOR: Mohammad Ali Ebrahimzadeh Mazandaran University of Medical Sciences School of Pharmacy Sari, Iran E-mail: zadeh20@gmail.com

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Garsi JP, et al. USABILITY OF NUCLEAR PLANT OM RECORDS IN EPIDEMIOLOGY Arh Hig Rada Toksikol 2014;65:407-416

DOI: 10.2478/10004-1254-65-2014-2465

Original article

Half-century archives of occupational medical data on French nuclear workers: a dusty warehouse or gold mine for epidemiological research? Jerome-Philippe Garsi1, Eric Samson1, Laetitia Chablais1, Sergey Zhivin1, Christine Niogret2, Dominique Laurier1, and Irina Guseva Canu1 Laboratoire d’épidémiologie, Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses1, AREVA NC, Occupational Medicine Department, Pierrelatte2, France Received in November 2013 CrossChecked in November 2013 Accepted in November 2014

This article discusses the availability and completeness of medical data on workers from the AREVA NC Pierrelatte nuclear plant and their possible use in epidemiological research on cardiovascular and metabolic disorders related to internal exposure to uranium. We created a computer database from files on 394 eligible workers included in an ongoing nested case-control study from a larger cohort of 2897 French nuclear workers. For each worker, we collected records of previous employment, job positions, job descriptions, medical visits, and blood test results from medical history. The dataset counts 9,471 medical examinations and 12,735 blood test results. For almost all of the parameters relevant for research on cardiovascular risk, data completeness and availability is over 90 %, but it varies with time and improves in the latest time period. In the absence of biobanks, collecting and computerising available good-quality occupational medicine archive data constitutes a valuable alternative for epidemiological and aetiological research in occupational health. Biobanks rarely contain biological samples over an entire worker’s carrier and medical data from nuclear industry archives might make up for unavailable biomarkers that could provide information on cardiovascular and metabolic diseases. KEY WORDS: aetiology; cardiovascular mortality; epidemiology; ionising radiation; occupational medicine

Studies able to properly assess the risk of internal contamination with radioactive compounds are scarce. This can partly be explained by the difficulty to reliably reconstruct the received internal dose or by the need of a long and robust follow-up to be able to do that. In France, the population of nuclear workers from AREVA NC’s Pierrelatte facility appears to be interesting in this respect. The main activities at this plant involved uranium hexafluoride (UF6) enrichment between 1959 and 1990 and the chemical conversion of uranium in the 1980s. The plant produced uranium compounds enriched in 235U to different degrees from

natural uranium ore but also reprocessed uranium from spent fuel turned into stable compounds for storage. Some workers were therefore potentially exposed to inhalation of uranium particles and other industrial chemicals. A retrospective pilot study in 2005 aimed at examining the effects of long-term, low-dose exposure to different uranium compounds in a cohort of plant workers (1). In the 2010 extension (2), the cohort totalled 2,897 workers who remained at the plant for at least six months between 1 January 1968 and 31 December 2006. At first, the available information regarding these workers could not be used

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to reconstruct individual occupational exposure to uranium. For this reason, an epidemiological approach consisting of a semi-quantitative exposure assessment was developed in 2007 (3) and later a specific job exposure matrix (JEM) was designed to obtain a retrospective estimate of cumulative exposure to different uranium compounds and other pollutants (4). On the basis of this exposure assessment a relationship was established for lung and hematopoietic cancer (5). Over the last decade, a potential risk of cardiovascular diseases associated with ionising radiation has become a major issue (6-11) but with inconsistent results in low-dose exposure studies (12, 13). Therefore, possible cardiovascular effects of internal uranium contamination were also studied in the Pierrelatte workers cohort (2). The results suggest that exposure to slowly soluble uranium compounds, notably reprocessed uranium compounds, may increase the risk of mortality from circulatory system diseases. The main limitations to analysing this cohort with specific internal exposure was the absence of individual biological and lifestyle data, especially about the known cardiovascular risk factors as defined by the Framingham study (14-16) or the international INTERHEART study (17, 18). Another limitation was the absence of organ-specific doses from uranium exposure. Occupational medicine (OM) in French nuclear industry began simultaneously with uranium processing at the beginning of the 1950s. The principal aim of occupational physicians was to detect workersâ&#x20AC;&#x2122; health problems at the time of employment and to monitor their health status over the entire professional life. In this new domain, the first guidelines were mainly based on the experience of physicians and medical research pioneers. Thanks to national regulations requiring that occupational medical records are archived, most cardiovascular risk parameters are still kept on record, covering a period from 1958 to the present. We studied these OM archives and collected the basic anthropometric data (height, weight), blood pressure (systolic and diastolic), and blood tests for all the monitored workers. Our intention was to test the hypothesis that low-dose ionising radiation increases the risk of cardiovascular diseases, taking into account these individual parameters. For this reason we have launched a nested case-control study that includes 394 workers (111 cases, up to 5 controls per case) from the cohort. The study is still under way.

In this preliminary report, however, we focus on the dataset collected in the above study in order to see whether the collected data could be useful for an epidemiological study or an aetiological research and to discuss other potential uses and limitations of OM records such as this. We analysed the type, quality, and variations in clinical and recording practice over time.

MEDICAL DATA COLLECTION AND ANALYSIS Study population The data presented here are related to a subsample of 394 eligible workers from the cohort of 2,897 workers employed at the AREVA NC Pierrelatte plant for at least six months between 1960 and 31 December 2006. The larger cohort was analysed for cancer and cardiovascular outcomes with unexpected results (2, 5) that called for further investigation. We collected additional data on potential risk factors for cardiovascular and metabolic diseases in order to establish their relationship with long-term exposure to low-dose uranium. In the choice of data to collect we primarily relied on the INTERHEART study (17, 18). The subsample was obtained by matching 111 cases of death from cardiovascular diseases (CVD) with controls according to sex, calendar period, fiveyear age classes, and socio-economic status at hire (clerical workers, engineering / management or technician) to account for potential confounding, especially regarding uranium exposure data which were available from the cohort study (19). Each case was matched to up to five controls whenever possible. One control could match more than one case, if the matching criteria were met. People who later died but at a certain earlier point met these matching criteria were also used as controls. To obtain the sample of controls who were alive at the time of death of the case we used an incidence density sampling method (20). Seventy cases were matched to five controls, five to four controls, another five to three controls, nine to two controls, and 13 cases to one control. No match was found for nine cases, including two women cases and cases with unmatchable combination of age and socio-economic status at hire.

Garsi JP, et al. USABILITY OF NUCLEAR PLANT OM RECORDS IN EPIDEMIOLOGY Arh Hig Rada Toksikol 2014;65:407-416

Monitoring time frame and database creation The data analysed here have been collected from OM monitoring records that span from 1959 to 2011, starting from workers’ screening at hire to the last follow-up. Medical examination by occupational physicians was annual, but some of the workers received bi- or trimestral check-up, depending on exposure conditions. Most of the paper archives were found in the Occupational Medicine Department (OMD) of the AREVA NC Pierrelatte plant. When the last employment was in another plant of the AREVA Group, we were able to track down the medical file of the worker. The files matching our population set were coded by ID numbers. For each worker we reconstructed a computer file containing full medical records by manually entering all data from the paper archive. All results had been converted to the more recent unit. The data were then split into 14 interrelated database tables: identification (including the first and last day of work at the plant, height, and full medical history); previous employments; job positions; job description; smoking (describing changes in tobacco consumption); pulmonary radiographs; external dosimetry; radio toxicological analyses results; accidental contaminations; number and causes of sick leave; capability certificates; electrocardiogram reports, medical examination findings, and blood tests. The last two tables were the subject of our analysis, as the parameters contained in them provide key information about CVD risks. The medical examination table includes data about weight, systolic and diastolic blood pressure, pulse, and cardiovascular, cancer, or other disease observations registered at each visit. The blood tests include standard haematological parameters (erythrocytes, leucocytes, haematocrit, basophils, eosinophils, lymphocytes, monocytes, and platelets) and available metabolic parameters (total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), urea, blood sugar, triglycerides, creatinine, urea and uric acid, and gamma-glutamyl-transpeptidases (GGT)). For all workers we computed body mass index (BMI) based on the standard formula:

For the body surface area (BSA) we used the Dubois and Dubois formula (21):

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BSA=0.20247*Height(m)0.725 *weight(kg)0.425 Data quality and analysis The quality of data in the database tables was assessed primarily by checking maximum and minimum values to identify and correct extreme values. Then the data of 30 randomly selected paper files were systematically reviewed to identify potential mistakes. All database values were compared to paper files. Of nearly 1,700 database values, only nineteen were wrong, indicating an error rate of about 1 %. These errors were further checked by comparing them with individual parameter means and medians and normal ranges. For all parameters of interest we calculated two main indicators: availability and completeness. Availability is the percentage of workers with at least one recorded value and completeness is the number of recorded medical examination or blood test results divided by the total number of workers.

RESULTS AND DISCUSSION The main characteristics of the collected data are given in Table 1. Among the workers, 12 are still Table 1 Main characteristics of collected data at the AREVA NC Pierrelatte uranium processing plant

Characteristics

Population

394 (100 %)

Potentially exposed to uranium compounds

330 (84 %)

Female workers Mean age at recruitment (y) (minmax) Median age at recruitment (y) Mean OM* follow-up (y) (min-max) Median OM* follow-up (y) Total number of medical examinations Mean number of medical examinations per worker (min-max) Median number of medical examination Total number of results of blood test from medical history Mean number of results of blood test from medical history per worker (min-max) Median number of results of blood test from medical history per worker *OM=Occupational Medicine

9 (2 %) 35 (20-57) 35.3 21 (1-37) 20.2 9471 25 (1-57) 22 12735 32 (1-93) 31

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employed. The mean time between the last medical examination and the occurrence of death by cardiovascular disease is 11.61 years (±8 years). Workers with more than 10 years since the last medical examination make 56.8 % of the population. Over the monitoring period, 9,471 medical examinations were performed and 12,735 blood test results obtained (Table 2). In terms of the means this translates to 25 medical examinations and 35 blood test results per person. The data show that 187 (47.10%) had 20-29 medical examinations and 286 (72.6%) at least 20 medical examinations. Only 34

(8.2%) workers have less than 20 blood test results recorded. This database clearly shows that changes in the cardiovascular system (e.g. systolic and diastolic pressure) of the workers had been carefully monitored. The number of medical examinations and blood tests is sufficient to provide reliable information about a relevant parameter for further epidemiological or aetiological research. Table 3 shows the completeness and the availability of specific variables of interest. It ranged between 1.2 % for HDL cholesterol and 99.8 % for granulocytes,

Table 2 Anthropometric and biomedical data collected at the AREVA NC Pierrelatte uranium processing plant for 394 workers

Variables

Unit

N of measurements

Range

Medical examination (N=9471) Height Weight Systolic blood pressure Diastolic blood pressure

cm kg mmHg mmHg

394i 9263 9379 9375

152-190 44-116 90-290 40-170

Body Mass Index

kg m-2

9256

16.6-37.4

Body Surface Area

9206

1.4-2.4

million mm-3

12696

4.1-6.0

g dL %

12480 11639

3.64-17.8 33.3-62.0

Blood tests from medical history (N=12735) Erythrocytes count Haemoglobin Haematocrit Mean corpuscular volume

-1

11932

32.2-199.0

Leucocytes Lymphocytes Monocytes

-3

n mm % %

12696 12709 12709

4000-25600 0-78 0-56

Thrombocytes

n mm-3

2445

91-685

% % %

12709 12709 12709

19-88 0-36 0-7

g L-1 g L-1 g L-1 g L-1 g L-1 mg L-1 IU L-1 g L-1 mg L-1

7284 993 153 7169 1273 1623 1452 11385 7169

1.0-5.1 0.2-2.4 0.4-3.6 0.1-3.3 0.3-12.5 0.2-19.0 2-112.5 0.1-0.47 0-140

Neutrophil granulocytes Eosinophil granulocytes Basophil granulocytes

Metabolic parameters Total cholesterol LDLii cholesterol HDLiii cholesterol Glycaemia Triglycerides Creatinine GGT Urea Uric acid

Height was collected once for each worker LDL= low density lipoproteins

HDL=high density lipoproteins

iii

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Table 3 Completeness and availability of worker follow-up data from medical examinations and blood tests

Variables

Completeness of data*

Medical visits Height Weight Diastolic blood pressure Systolic blood pressure Pulse BMI/BSA Results of blood test from medical history Erythrocytes Haemoglobin Haematocrits Mean corpuscular volume Leucocytes Lymphocytes Monocytes Thrombocytes Neutrophil granulocytes Eosinophil granulocytes Basophil granulocytes Metabolic parameters Total cholesterol LDL cholesterol HDL cholesterol Glycaemia Triglycerides Urea Creatinine GGT Uric acid

Availability of workers' data**

Year of first and last recorded value

99.8 % 97.7 % 99.0 % 98.9 % 90.4 % 97.6 %

99.8 % 99.2 % 99.2 % 99.2 % 99.2 % 99.2 %

1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011

99.7 % 98.0 % 91.4 % 93.7 % 99.7 % 99.8 % 99.8 % 19.2 % 99.8 % 99.8 % 99.8 %

99.2 % 99.2 % 98.7 % 99.2 % 99.2 % 99.2 % 99.2 % 92.0 % 99.2 % 99.2 % 99.2 %

1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011 1959-2011

57.2 % 7.8 % 1.2 % 56.3 % 10.0 % 89.4 % 12.7 % 11.4 % 56.3 %

98.5 % 30.0 % 8.3 % 98.5 % 42.3 % 99.0 % 69.3 % 40.8 % 98.5 %

1961-2011 1985-2011 1968-2011 1959-2011 1969-2011 1959-2011 1966-2011 1963-2011 1965-2011

*Percentage calculated as the number of collected values over the number of medical examination or results of blood test from the medical history for each workers, divided by the total number of workers with collected values **Percentage of workers with at least one value

lymphocytes, monocytes, and height. The availability of specific variables ranged from 8.3 % for HDL cholesterol to 99.8 % for height. The high completeness of blood count data provides reliable information for investigating possible effects of exposure to ionising radiation and uranium on blood count.

high cholesterol values were recorded. Routine recording started only after 1973, with the first major publications on total cholesterol as a cardiovascular risk factor (22-25). For workers hired later than 1970 the completeness rose highly, except, once again for HDL cholesterol (Figure 1).

Change of recording practice over time

The choice of parameters and their biological relevance

Workers hired before 1970 make 91 % of our population. For them, the completeness of almost every parameter of medical examination or blood test is ≥90%. The exceptions are HDL and LDL cholesterol, triglyceride, GGT, and creatinine. Until the 1960s only

The sample analysed in this study provided highly complete and available data on the risk factors for acute myocardial infarction, including the history of hypertension (almost 99 % completeness for systolic

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and diastolic pressure), abdominal obesity (by calculating BMI/BSA), total cholesterol, and diabetes (using blood sugar as an indicator). About 50 % of the workers had both blood sugar and cholesterol measured. Records on psychosocial risk factors like stress and hard physical activity at work or physical activity in free time were too scarce or imprecise to use as required by the INTERHEART study (17). Since the follow-up was no more compulsory after retirement, few data cover that period. However, because most workers were exposed to chronic lowrate exposure to ionising radiation and uranium rather than acute, high-rate accidental exposure, potential health effects would be mainly long-term, evolving from light to moderate biological and physiological changes that could lead to clinically detectable symptoms or a disease once they become irreversible. Therefore, it should be possible to identify any changes in parameters from the data collected during employment and investigate how these changes may influence the occurrence of cardiovascular disease from a mechanistic point of view. One major advantage of our approach is that for most of the parameters it can provide not only one value per individual, but the whole variation over time. For example, every worker had a mean of 23 systolic blood pressure measurements over the median followup of 20 years. The Framingham Cardiac Risk Score (22) is a well-known tool to predict 10-year cardiovascular risks for an individual. It requires seven variables to calculate the risk: sex, age, total cholesterol, HDL cholesterol, systolic and diastolic blood pressure, diabetic status, and smoking status. The availability of data for all the required parameters in our dataset

is good, with the notable exception of HDL cholesterol (which is available for only 33 workers). The diabetic status can be determined on the basis of blood sugar levels. Smoking records are not uniform (detailed information on tobacco consumption and duration of smoking is only partly known) and can only be analysed as a binary variable across the sample. Considering that blood count parameters in our sample have almost 100 % completeness and availability over time, it would be relatively easy to evaluate the effects of low-dose ionising radiation on blood counts (23, 24). Diastolic and systolic blood pressure can also be analysed in relation to internal uranium dose received before retirement. With the added benefit of blood counts and medical examination parameters, robust studies are possible. In contrast, the majority of metabolic parameters reached 50 % completeness in the 1980’s and could therefore be considered only with great caution and an extension of the workers’ follow-up. The only important variables with low availability in our dataset are creatinine and HDL cholesterol. Creatinine is a marker of renal function, fundamental in uranium internal contamination, while HDL is an important element of the Framingham score. Potential use in the upcoming nested case-control study The collected data will be used in the upcoming nested case-control study as co-variables in addition to the main exposure variables (e.g. exposure to ionising radiation and uranium compounds) to investigate how they could have affected the results of the previous cohort study by Guseva Canu et al.

100

Medical visit parameters

90 80

Blood counts parameters

Completeness

70 Total cholesterol and Glycaemia

60 50

Triglycerids, Gamma GT and Creatinine

40 30

HDL Cholesterol

20 LDL Cholesterol

10 0 Before 70

Between 71 and 80

Between 81 and 90

Between 91 and 2000

After 2000

Decade of hiring

Figure 1 Completeness of medical examination and blood test parameters data by decades

Garsi JP, et al. USABILITY OF NUCLEAR PLANT OM RECORDS IN EPIDEMIOLOGY Arh Hig Rada Toksikol 2014;65:407-416

(2). In that study, the effects of exposure to uranium compounds on cardiovascular mortality were assessed without taking into account the known cardiovascular risk factors, as they were not available at the time. Therefore, the collected variables will primarily be analysed as possible confounding or modifying factors using a conditional logistic model. These analyses should be able to handle time-dependent covariates because of the longitudinal changes in blood tests and anthropometry over time. Potential for broader use The large set of collected haematological data will allow us to address the hypotheses on the association between haematological parameters/hypertension and internal uranium contamination. For instance, Wagner et al. (25) found that exposed women ran a higher risk of elevated systolic pressure. The use of longitudinal data makes it possible to evaluate correlations within and between workers using generalised estimating equations (26). Thus, in addition to assessing the modifying or confounding effects on cardiovascular mortality, OM data can also be used independently, as an intermediate health outcome to assess the effect of uranium exposure on health parameters such as blood pressure. In that sense, OM data could also be used for aetiological research. Kathren et al. (27, 28) have already pointed to acute internal exposure to uranium compounds as the possible cause of kidney disease (27, 28). Low-dose radiation effects are less consistent in the literature, but some of the evidence calls for further studies (29, 30). In fact, the archived files we reviewed do refer to adverse renal effects, and consistently so to renal colic. We believe that data on creatinine will be useful for pending studies of the effects of radiation on the renal function in the plant workers. Furthermore, the OM data appear to be an important source of information for retrospective studies. A longitudinal follow-up of workers makes possible the use of various epidemiological and statistical models to retrospectively assess exposure risk and build a robust prospective cohort. If no specific sampling of biological material is available, the use of routinely collected OM data may be justified prior to further research. A major limitation to the use of OM data for epidemiological research are the missing or incorrect values in the database. However, this issue can be addressed with frequentist (31) or Bayesian (32) or other methods (33). If there is no

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pattern to missing data, this could be resolved by careful data management, but if the errors are systematic, Bayesian method could help to fill them in. As biobanks are very difficult to set up in France for ethical and economic reasons, the electronic storage of half a century of archived OM data on nuclear workers is a cost-effective alternative. Such data are generally directly available, accurate, and free of recall bias. Most are systematic (annual) biological measurements that span the worker’s career and follow strict medical surveillance procedures. Moreover, biobanks rarely keep biological samples for an entire career. Finally, since the OM follow-up provides epidemiologists with longitudinal data, it also provides an insight into the causal relationship between exposure to ionising radiation and symptomatic coronary artery disease through the effects of radiation on leucocyte and monocyte counts (34). The use of data already obtained by health monitoring should efficiently complement molecular epidemiology studies. These data could be used as a reference for additional data collected after retirement. Furthermore, systematic blood records for each worker make it easier to collect additional samples to analyse new biomarkers (DNA, mutations, epigenetic markers, etc…). Overall, the recording practices at the AREVA NC Pierrelatte facility provide a reliable basis for epidemiological studies, especially regarding blood counts and medical examination parameters. The completeness and availability of the data are relatively high for known major cardiovascular risk factors. The major strength of our subsample database is that it can reduce/control potential confounding and effectmodifying factors. Data on the full cohort will allow an even more precise evaluation of radiation effects by taking into account the parameters that can affect the circulatory system. Acknowledgements The authors wish to thank Olivier Laurent from the Laboratoire d’épidémiologie for carefully reading the drafts, Iris Jovanovic for her major role in creating the computer database, the personnel of the Pierrelatte Occupational Medicine Department for great help in logistics, and Dr Alain Acker (AREVA) for his help in accessing the workers’ medical records in the Pierrelatte OM department.

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Conflict of interest The authors declare no conflict of interest.

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some uranium processing sites in Egypt - health effects. J Environ Monit 2002;4:588-91. PMID:12196006 Akushevich IV, Veremeyeva GA, Dimov GP, Ukraintseva SV, Arbeev KG, Akleyev AV, Yashin AI. Modeling deterministic effects in hematopoietic system caused by chronic exposure to ionizing radiation in large human cohorts. Health Phys 2010;99:322-9. doi: 10.1097/ HP.0b013e3181c61dc1 Wagner SE, Burch JB, Bottai M, Pinney SM, Puett R, Porter D, Vena JE, HĂŠbert JR. Hypertension and hematologic parameters in a community near a uranium processing facility. Environ Res 2010;110:786-97. doi: 10.1016/j. envres.2010.09.004 Zeger SL, Liang KY, Albert PS. Models for longitudinal data: a generalized estimating equation approach. Biometrics 1988;44:1049-60. PMID: 3233245 Kathren RL, Burklin RK. Acute chemical toxicity of uranium. Health Phys 2008;94:170-9. doi: 10.1097/01. HP.0000288043.94908.1f Kathren RL, McInroy JF, Moore RH, Dietert SE. Uranium in the tissues of an occupationally exposed individual. Health Phys 1989;57:17-21. PMID: 2745077

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Sažetak Polustoljetni arhiv odjela za medicinu rada francuske nuklearne elektrane - prašnjavo skladište ili zlatni rudnik za epidemiološka istraživanja? U ovome se članku raspravlja o dostupnosti i potpunosti medicinskih podataka o radnicima iz nuklearne elektrane AREVA NC Pierrelatte te o njihovoj korisnosti za epidemiološka ispitivanja krvožilnih i metaboličkih poremećaja povezanih s izloženosti uranu. Na temelju zdravstvenih kartona 394 radnika koji su dio većeg kohortnog ispitivanja s 2897 radnika u francuskim nuklearnim elektranama stvorena je elektronička baza podataka s informacijama o ranijem zaposlenju, položaju, opisu posla, liječničkim pregledima i krvnim nalazima. Baza obuhvaća podatke iz 9.471 liječničkog pregleda te 12.735 krvnih nalaza. Dostupnost i potpunost podataka za gotovo sve parametre važne za procjenu rizika od bolesti srca i krvožilja bile su više od 90 %, ali neujednačene, i popravljale su se tek prema kraju 50-godišnjeg praćenja. U nedostatku biobanaka, prikupljanje i digitalizacija pouzdanih arhivskih podataka iz medicine rada vrijedan su alternativni izvor za epidemiološka i etiološka ispitivanja. Biobanke rijetko čuvaju biološke uzorke radnika cijeli njegov radni vijek, pa zdravstveni podaci čuvani u arhivima nuklearnih elektrana mogu kvalitetno dopuniti prazninu uslijed nedostatka biomarkera i dati uvid u nastanak i razvoj bolesti metabolizma te srca i krvožilja. KLJUČNE RIJEČI: bolesti srca i krvožilja; epidemiologija; etiologija; ionizirajuće zračenje; medicina rada; smrtnost

CORRESPONDING AUTHOR: Dominique Laurier IRSN Institut de Radioprotection et de Sûreté Nucléaire Laboratoire d’Epidémiologie des Rayonnements ionisants PRP-HOM/SRBE/LEPID BP 17 92262 Fontenay aux Roses Cedex, France E-mail: dominique.laurier@irsn.fr

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DOI: 10.2478/10004-1254-65-2014-2548

Case report

Pericardial effusion as the first manifestation of occupational tuberculosis in a health care worker Veljko Flego, Dubravka Matanić Lender, and Ljiljana Bulat-Kardum Department of Pulmonology, Clinical Hospital Centre Rijeka, Rijeka, Croatia Received in June 2014 CrossChecked in June 2014 Accepted in November 2014

Tuberculosis (TB) is an infectious disease and, apart from protecting patients, attention must be given to protecting the persons who come in contact with them, especially nurses and medical practitioners. A 43-yearold immunocompetent male nurse developed occupationally disseminated TB after contact with patients affected by active TB (culture positive) while working in a psychiatric hospital. The first manifestation of the disease was exudative pericarditis with Mycobacterium tuberculosis (MT) confirmed two months after pericardiocentesis and evacuation of 1200 mL of pericardial effusion. Many lymph nodes showed histologic findings of granulomatous inflammation with necrosis. Treatment with antituberculosis drugs caused complications, including transient short-term medication-induced toxic hepatitis, prolonged fever, left pleural nonspecific effusion, and mononeuritis of the right peroneus nerve. The treatment lasted 14 months and led to permanent consequences, including fibrothorax with restrictive ventilation disorders and reduced diffusion of the alveolar-capillary membrane. This case highlights the need to improve the protection of health care workers who are in contact with TB patients, as well as the usefulness of the tuberculin skin test and QuantiFERON-TB test, which can be used to identify early latent TB. KEY WORDS: antituberculotics; disseminated tuberculosis; extrapulmonary tuberculosis; nosocomial disease; occupational disease; tuberculous pericarditis

The incidence of active tuberculosis (TB) among health care workers (HCW) has been reported to be 0.4 % in Germany, 1.0 % in Portugal, or up to 2.5 % in Brazil (1-3), with nurses enduring the highest risk, followed by physicians. Nurses are at a greater risk of acquiring TB if they work in pulmonary medicine or human immunodeficiency virus (HIV) wards, likely due to repeated contact with infected patients (4). The risk of developing TB is highest during the first years of exposure. The predominant clinical presentations are extrapulmonary cases (48 %), followed by pulmonary (44 %) and combination cases (8 %) (5). The most common forms of extrapulmonary TB (EPTB) include pleural, lymph node, osteoarticular, and abdominal TB. Other localisations such as central nervous system, urogenital, breast, chest wall, cutaneous, middle ear, and pericardial TB are rarely observed (6, 7).

Pericarditis is a rare manifestation of TB, occurring in approximately 1-2 % of all TB cases (8). TB accounts for up to 4 % of acute pericarditis and 7 % of cardiac tamponade (5). Organisms usually spread to the pericardium from the mediastinal or hilar lymph nodes or from the lungs, and rarely as part of miliary TB. Tuberculous pericarditis (TBP) is a potentially lethal condition, and its prompt treatment can be lifesaving. Effective treatment requires a rapid and accurate diagnosis, which is often difficult. In occupationally exposed staff, pericardial effusion non-responsive to routine therapy should arouse suspicion of occupational TBP. Here, we report a case of TBP in a 43-year-old male nurse. Following a positive culture of Mycobacterium tuberculosis (MT) from the pericardial fluid, he immediately began undergoing treatment with antituberculous chemotherapy.

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Case report and discussion The 43-year-old male patient had been employed as a male nurse in a chronic psychiatric hospital for eight months before experiencing the first symptoms. He had received BCG vaccination and had a scar on his left upper arm. The patient had no history of tuberculosis, diabetes, or immunosuppression. He was not a smoker, did not drink alcohol or abuse drugs. Four months before the onset of the first symptoms, our patient was in daily contact with five patients suffering from active pulmonary TB (nosocomial TB), as shown by a positive sputum culture for MT. The patient had a negative Mantoux test at the start of his employment, as well as four months later. After an additional three months, his Mantoux test was 22×25 mm, showing hyperresponsiveness. At the same time, his QUANTIFERON-TB test result was also positive (8.19 IU mL-1). Two weeks before these positive test results, he was subfebrile for ten days. Radiological findings of the thorax were normal. Sputum was sent for microbiological examination. Ziehl-Neelsen stained smears did not show acid-fast bacilli. Six weeks later, the patient presented a fever of 39.5 °C, pain in the epigastrium, and shortness of breath with effort. He was hospitalized at the Department for Infectious Diseases, where ultrasound findings established pericardial effusion. The patient was then transferred to the Department of Cardiovascular Diseases, where he underwent pericardiocentesis and evacuation of 1200 mL of

Figure 1 A chest radiogram showing large left pleural effusion

pericardial effusion. The aetiology of pericardial effusion was not established, and the patient was treated with prednisone 40 mg at decreasing doses. Two months later, at a prednisone dose of 5 mg, the patient once again became febrile. Pericardial effusion was still found and the prednisone dose was increased to 30 mg. The patient remained febrile and fatigued. The prolonged-incubation mycobacteria growth indicator tube (MGIT) showed positive findings in the sputum collected four months earlier. Positive findings were also established in MGIT and cultures on Lowenstein-Jensen media of pericardial effusion samples from two months earlier. The patient was hospitalised at the Department of Pulmonology. Prednisone treatment was rapidly decreased and antituberculosis treatment was initiated with isoniazid 400 mg, rifampicin 600 mg, and ethambutol 1200 mg. Radiological findings of the thorax were normal. After a month of hospital treatment, the patient was discharged to his home, without a fever and in good general condition. A week later, the patient was again admitted to the Department of Pulmonology with a temperature of 39.5 °C and pain in the left side of the chest. Extensive left pleural effusion was established (Figures 1 and 2). On two occasions, thoracocentesis was performed, removing 900 and 600 mL of pleural effusion-type exudate. Treatment with piperacillin/tazobactam 4.5 g i.v. every 8 hours was started, but the fever persisted. We performed two additional thoracocenteses,

Flego V, et al. OCCUPATIONAL TUBERCULOSIS IN A HEALTH CARE WORKER Arh Hig Rada Toksikol 2014;65:417-422

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Figure 2 Axial computed tomography (CT) scan (left) and coronal reformatted CT image (right) of the chest showing left pleural effusion

removing a total of 1500 mL of additional pleural effusion. Cytological findings of extracted pleural effusion included non-specific inflammation, and bacteriological examination revealed sterility. Treatment continued with antituberculosis drugs (isoniazid 400 mg, rifampicin 600 mg, ethambutol 1200 mg, pyrazinamide 1.5 g) and prednisone 25 mg. Pleural effusion gradually regressed, but the patient remained continuously febrile. After a month of treatment, the patient’s fever subsided and prednisone treatment was suspended. A few days later, the patient’s temperature rose to 38.5 °C and would not decrease for two weeks. Multislice computed tomography (MSCT) revealed lesions consistent with active pulmonary tuberculosis and enlarged lymph nodes (neck, axilla, mediastinum, and groin), a small left pleural effusion, and a small pericardial effusion. Treatment with antituberculosis drugs was further complicated by transient shortterm medication-induced toxic hepatitis, prolonged fever of unclear aetiology, pleural effusion of unclear aetiology, and mononeuritis of the right nervus peroneus. To make sure that no other condition was causing the fever (lymphoma), positron emission tomography–computed tomography (PET/CT) scanning was performed, showing a number of metabolically active lesions in the lymph nodes and the left pleura. The patient was further investigated by cytologic puncture of three nodes (axilla and groin), and biopsy of a lymph node in the right groin. The findings showed granulomatous inflammation with

necrosis and further treatment was started with rifampicin 600 mg and isoniazid 400 mg. Two months later, an echocardiogram showed no pericardial effusion, pulmonary hypertension, or constrictive pericarditis. Spirometry indicated restrictive ventilation disorders of an intermediate level, due to a higher left side fibrothorax. It was recommended that the patient continue taking rifampicin and isoniazid. Three months later at a check-up with the pulmonologist, the patient exhibited no fever and felt well, except for the occasional stabbing pain in the left part of the chest. Further treatment with rifampicin and isoniazid was recommended. Three months later, radiological examination showed residual pleuropulmonary changes, which was a consequence of tuberculous infection. Treatment with rifampicin and isoniazid continued. After 14 months, the antituberculosis treatment was completed. The patient still felt shortness of breath, intolerance of effort, and a constant stabbing pain in the left part of the chest. As a result of excessive tuberculosis, there were residual consequences in the form of a left fibrothorax, restrictive disorders of ventilation (FVC 53.1 %), and reduced diffusion of carbon monoxide (DLCO 48.5 %) (Table 1). The patient is now employed at a location where he is no longer exposed to contact with MT and can avoid greater physical exertion. This case is important for several reasons. TB is a rare disease in the general population, but is the most common occupational infectious disease among HCWs. Tuberculous pericarditis is an uncommon

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Table 1 Time sequence of disease development

Month

Symptoms, diagnostic tests, and therapy

Employment; Mantoux test: negative.

Mantoux test: negative

Subfebrility; Chest X-ray: normal

Mantoux test: hyperresponsiveness; QUANTIFERON-TB test: positive

Pericardial effusion; Prednisone

11 13

MT positive; Chest X-ray: normal; TB therapy Fever; Left exudative pleurisy; TB therapy+piperacillin/tazobactam

Fever; Pleural effusion regressed; TB therapy+prednisone

Fever; MSCT of chest

Medicamentous toxic hepatitis

17 19 23 25

Fever; PET/CT; TB lymph nodes; TB therapy Normal echocardiogram; Left side fibrothorax; TB therapy No fever; Pleuropulmonary residues; TB therapy Completed TB therapy; Left side fibrothorax; Restrictive disorders of ventilation

MT – Mycobacterium tuberculosis TB – Tuberculosis

localization of EPTB and is rarely the first manifestation of generalized TB. The presently reported patient became very ill soon after contact with patients suffering from active TB, but the MT culture of sputum on Lowenstein-Jensen media became positive only after four months. Finally, the patient suffered notable complications during treatment with antituberculosis drugs, and severe and permanent damage after TB was cured. In 2009, Croatia reached the levels of other European countries with low incidence of TB, reaching a notification rate of below 20 TB cases per 100,000 inhabitants for the first time. The rate of occupational diseases among those working in health care and social care in Croatia was 11.78/100,000 employees (9). Occupational diseases were reported in 3 % of Croatian HCWs between 2002 and 2009, with infectious diseases comprising 68 % of such cases (10). Among these occupational infectious

diseases in HCWs, 27 % were TB (the most common). According to these data, the incidence of active TB as an occupational disease in HCWs in Croatia is 0.6 %. Among occupational diseases in HCWs, 39 % occurred in nurses and 23 % in doctors. There is mounting evidence that HCWs are at an increased risk of contracting a TB infection and developing the disease (11). Effective environmental and personal protective measures, along with education of patients and HCW are urgently needed to reduce the occupational risk of TB. EPTB occurs in 15-20 % of immunocompetent and 20-70 % of HIV-infected patients with TB (12). Studies from the Netherlands and the US show that EPTB is more often diagnosed in women and is associated with ethnic minorities and those born in other countries (13). Tuberculous pericarditis is seen in 1-8 % of EPTB patients (14). A stratification of the data by high- and low-incidence countries shows a higher proportion of EPTB in low-incidence countries. The proportion of EPTB increased during the period from 2002 to 2011 in the European Union, mainly because the notification rate of pulmonary TB decreased (15). TB is diagnosed in only 4 % of acute pericarditis cases (16), while pericarditis may be seen in 1-2 % of pulmonary tuberculosis cases. It is difficult to isolate Mycobacterium species from pericardial fluid samples and only one-third of such samples are diagnostic. The presently reported patient had generalised TB with lymph nodes, pericardium, and coexisting pulmonary TB. TBP was the first evidence of generalized TB, which is very rare (17). The significant amount of time it took to diagnose the patient contributed to the severe clinical disease condition and long duration of treatment. The disease was recognized as occupational and our patient was no longer capable of performing the work he performed before contracting the disease. It is favourable that this patient did not develop constrictive pericarditis, which is often a severe and permanent consequence of TBP (18, 19). Tuberculous pericardial effusion rarely occurs in immunocompetent persons. Our present case demonstrates the need for a higher index of suspicion for TB in any case of pericardial effusion, particularly among HCWs with previous exposure to TB. Assessment of TB as an occupational disease should be limited to occupationally exposed populations. Tuberculin skin test and QUANTIFERON-TB tests can identify early latent TB and are useful for

Flego V, et al. OCCUPATIONAL TUBERCULOSIS IN A HEALTH CARE WORKER Arh Hig Rada Toksikol 2014;65:417-422

diagnosing contact with MT in low-incidence countries, even in a vaccinated population, as well as for directing diagnoses to active TB (20).

REFERENCES

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1. Niihau’s A, Kesavachandran C, Wendeler D, Haamann F, Dulon M. Infectious diseases in healthcare workers – an analysis of the standardised data set of a German compensation board. J Occup Med Toxicol 2012;7:8. doi: 10.1186/17456673-7-8 2. Costa JC, Silva R, Ferreira J, Nienhaus A. Active tuberculosis among health care workers in Portugal. J Bras Pneumol 2011;37:636-45. doi: 10.1590/S1806-37132011000500011 3. do Prado TN, Galavote HS, Brioshi AP, Lacerda T, Fregona G, Detoni Vdo V, Lima Rde C, Dietze R, Maciel EL. Epidemiological profile of tuberculosis cases reported among health care workers at the University Hospital in Vitoria, Brazil. J Bras Pneumol 2008;34:607-13. doi: 10.1590/S180637132008000800011 4. Fronteira I, Ferrinho P. Do nurses have a different physical health profile? A systematic review of experimental and observational studies on nurses’ physical health. J Clin Nurs 2011;20:2404-24. doi: 10.1111/j.1365-2702.2011.03721 5. Wanjari K, Baradkar V, Mathur M, Kumar S. A case of tuberculous pericardial effusion. Indian J Med Microbiol 2009;27:75-7. PMID: 19172070 6. Lalić H, Kukuljan M, Đinđić Pavičić M. A case report of occupational middle ear tuberculosis in a nurse. Arh Hig Rada Toksikol 2010;61:333-7. doi: 10.2478/10004-1254-612010-2034 7. Fader T, Parks J, Khan NU, Manning R, Stokes S, Nasir NA. Extrapulmonary tuberkulosis in Kabul, Afghanistan: a hospital-based retrospective review. Int J Infect Dis 2010;14:e102-10. doi: 10.1016/j.ijid.2009.03.023 8. Bolt RJ, Rammeloo LA, van Furth AM, van Well GT. A 15-year-old girl with a large pericardial effusion. Eur J Pediatr 2008;167:811-2. doi: 10.1007/s00431-007-0559-z 9. Register of Occupational Diseases, Croatian Institute for Health Protection and Safety at Work [displayed 19 May 2 0 1 4 ] . Av a i l a b l e a t h t t p : / / h z z z s r. hr/?what=content&ID=70&cat=68 10. Bogadi-Šare A. Profesionalne bolesti zdravstvenih radnika u Republici Hrvatskoj [Occupational disease of health

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421 workers in the Republic of Croatia, in Croatian]. [displayed 19 May 2014]. Available at http://www.hzzzsr.hr/images/ documents/Skupovi%20i%20izobrazba/Skupovi%20 doma%C4%87i/4.%20SIMPOZIJ%20HRVATSKE%20 LIJE%C4%8CNI%C4%8CKE%20KOMORE/ Profesionalne%20bolesti%20zdravstvenih%20radnika.pdf Jesudas CD, Thangakunam B. Tuberculosis risk in health care workers. Indian J Chest Dis Allied Sci 2013;55:149-54. PMID: 24380222 Karstaedt AS. Extrapulmonary tuberculosis among adults: experience at Chris Hani Baragwanath Academic Hospital, Johannesburg, South Africa. S Afr Med J 2013;104:22-4. doi: 10.7196/samj.6374 Solovic I, Jonsson J, Korzeniewska- Koseła M, Chiotan DI, Pace-Asciak A, Slump E, Rumetshofer R, Abubakar I, Kos S, Svetina-Sorli P, Haas W, Bauer T, Sandgren A, van der Werf MJ. Challenges in diagnosing extrapulmonary tuberculosis in the European Union, 2011. Eurosurveillance 2013 [displayed 19 May 2014]. Available at: http://www. eurosurveillance.org/ViewArticle.aspx?ArticleId=20432 Suman A, Sarin JL, Grant SC, Bazaraa TA. A case of tuberculous pericardial effusion. Age Ageing 2003;32:450-2. doi:10.1016/S2222-1808(13)60031-6 Sandgren A, Hollo V, van der Werf MJ. Extrapulmonary tuberculosis in the European Union and European Economic Area, 2002 to 2011. Eurosurveillance 2013; [displayed 19 May 2014]. Available at: http://www.eurosurveillance.org/ ViewArticle.aspx?ArticleId=20431 Avşar A, Günay NK, Celik A, Melek M. A case of cardiac tamponade caused by tuberculous pericarditis. Turk Kardiyol Dern Ars 2008;36:482-4. PMID: 19155666 Afzal A, Keohane M, Keeley E, Borzak S, Callender CW, Iannuzzi M. Myocarditis and pericarditis with tamponade associated with disseminated tuberculosis. Can J Cardiol 2000;16:519-21. PMID: 10787468 Russell JB, Syed FF, Ntsekhe M, Mayosi BM, Moosa S, Tsifularo M, Smedema JP. Tuberculous effusive-constrictive pericarditis. Cardiovasc J Afr 2008;19:200-1. PMID: 18776964 Caroselli C, Manara F, Bruno G. Coming from the past: Tuberculous chronic constrictive pericarditis. Am J Med Sci 2011;341:233. PMID: 21446080 Targowski T, Chelstowska S, Plusa T. Tuberculin skin test and interferon-γ release assay in the detection of latent tuberculosis infection among Polish health care workers. Pol Arch Med Wewn 2014;124:36-42. PMID: 24343239

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Sažetak Perikardijalni izljev kao prva manifestacija profesionalne tuberkuloze u zdravstvenog radnika Tuberkuloza (TBC) zarazna je bolest, stoga je prijeko potrebno zaštititi ne samo bolesnike nego i osoblje koje dolazi u kontakt s njima, u prvom redu medicinske sestre i liječnike. Nakon kontakta s bolesnicima oboljelima od TBC-a (u kulturama pozitivne) 43-godišnji imunokompetentni medicinski tehničar, zaposlen u psihijatrijskoj bolnici, obolio je od profesionalnog diseminiranog TBC-a. Prva manifestacija bolesti bio je eksudativni perikarditis s dokazanim Mycobacterium tuberculosis (MT), dva mjeseca nakon perikardiocenteze i evakuacije 1200 mL perikardijalnog izljeva. Histološki nalaz limfnih čvorova na više lokalizacija pokazivao je granulomatoznu upalu s nekrozom. Liječenje antituberkuloticima bilo je praćeno komplikacijama. Došlo je do prolaznog, kratkotrajnog, medikamentozno toksičnog hepatitisa, dugotrajnog febriliteta, nespecifičnog ljevostranog pleuralnog izljeva i mononeuritisa desnog peronealnog živca. Liječenje je trajalo 14 mjeseci. Kao trajna posljedica razvio se fibrotoraks, koji je doveo do restriktivnih smetnji ventilacije i smanjene difuzije alveolarno-kapilarne membrane. Ovaj slučaj upozorava na potrebu poboljšanja zaštite zdravstvenih radnika koji su u kontaktu s oboljelima od tuberkuloze, kao i korisnost tuberkulinskog kožnog testa i QuantiFERON-TB testa, koji mogu rano otkriti latentni TBC. KLJUČNE RIJEČI: antituberkulotici; diseminirana tuberkuloza; ekstrapulmonalna tuberkuloza; nozokomijalna bolest; profesionalna bolest; tuberkulozni perikarditis

CORRESPONDING AUTHOR: Veljko Flego Department of Pulmonology Clinical Hospital Centre Rijeka Tome Strižića 3, HR-51000 Rijeka, Croatia E-mail: veljko.flego@medri.uniri.hr

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Gvozdenović LJ, et al. SATISFACTION AMONG SERBIAN ANAESTHESIOLOGISTS Arh Hig Rada Toksikol 2014;65:423-424

DOI: 10.2478/10004-1254-65-2014-2570

Letter to the Editor

A survey of professional satisfaction among anaesthesiologists in Serbia Ljiljana Gvozdenović, Tatjana Batak, Dejan Ivanov, and Radovan Cvijanović Medical University Novi Sad, Clinical Center Vojvodina, Novi Sad, Serbia

We would like to briefly address our recent findings regarding professional satisfaction among anaesthesiologists in Serbia. It is generally wellknown among physicians that the life expectancy of anaesthesiologists is smaller than in other specialties (1). Many resort to the use of cigarettes, drugs, alcohol (2). The number of suicides among anaesthesiologists is significantly larger in comparison with other physicians (3, 4). Needless to say, the level of professional satisfaction is in a direct link with the quality of patient care they are able to provide. To investigate whether there is a lack of satisfaction among anaesthesiologists in our country, we conducted a study aimed at determining if anaesthesiologists in Serbian tertiary hospitals are under chronic stress after 24-hour on-call shifts; if they endure high levels of anxiety; and whether there is a connection between their personality type and alcohol and drug abuse. The study included 60 anaesthesiologists who worked in specialised surgical tertiary health institutions 24 hours on-call. The survey was voluntary and anonymous. All of the physicians involved in the study completed a burnout questionnaire (Oldenburg Burnout Inventory). The mean age of the physicians was 43.1 years (SD=8.1, range 29-63). There was no statistically significant difference in the average age of the physicians in relation to the analysed groups (on call 44.1 years: not on call 42.2 years). Of the physicians on call, 33.3 % were over 40 years of age. Both groups comprised more women than men (on call: 67 % women; not on call: 77 % women) and the distribution by sex was not different in the groups of physicians not on call. As much as 90 % of the anaesthesiologists were on call four or more times per month. They smoked and drank alcohol more than the other doctors in the control group. Altogether 77 % of

anaesthesiologists could not rest appropriately after an on-call shift, and if their sleep was interrupted, they needed considerably more time to fall asleep again. Anxiety decreased after taking days off. Stable extraverts had an emotionally stable personality type with the lowest anxiety level. A total of 76.5 % of male and 55.8 % female physicians consumed cigarettes, alcohol, and/or drugs. This difference was not statistically significant. A total of 70.6 % of male and 34.9 % of female anaesthesiologist used cigarettes and/or alcohol. This difference was statistically significant (p=0.027). Several studies have considered the necessary strategies in detecting signs of burnout and concluded that better working conditions reduced stress on the employees; however, the support of colleagues at work and the heads of the hospitals was as equally important as family support systems (5-8). There are stress conditions and burnout amongst Serbian anaesthesiologists. The prevalence of depersonalisation was extremely high in the studied sample. In addition to effects on the health of the anaesthesiologists, burnout and depression may also affect patient care and safety.

REFERENCES 1. Katz JD. Do anesthesiologists die at a younger age than other physicians? Age-adjusted death rates. Anesth Analg 2004;98:1111-3. doi: 10.1213/01.ANE.0000105879.02463.1F 2. Bryson EO, Silverstein JH. Addiction and substance abuse in anesthesiology. Anesthesiology 2008;109:905-17. doi: 10.1097/ALN.0b013e3181895bc1 3. Nyssen AS, Hansez I, Baele P, Lamy M, De Keyser V. Occupational stress and burnout in anaesthesia. Br J Anaesth 2003;90:333-7. doi: 10.1093/bja/aeg058 4. Gaszynska E, Stankiewicz-Rudnicki M, Szatko F, Wieczorek A, Gaszynski T. Life satisfaction and work-related

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satisfaction among anesthesiologists in Poland. Scientific World J 2014;2014:601865. doi: 10.1155/2014/601865 5. Firth-Cozens J, Payne RL, editors. Stress in Health Professionals: Psychological and Organisational Causes and Interventions. Chichester: John Wiley and Sons; 1999. 6. Rama-Maceiras P, Parente S, Crank P. Job satisfaction, stress and burnout in anaesthesia: relevant topics for anaesthesiologists and healthcare managers? Eur J A n a e s t h e s i o l 2 0 1 2 ; 2 9 : 3 11 - 9 . d o i : 1 0 . 1 0 9 7 / EJA.0b013e328352816d

CORRESPONDING AUTHOR: Ljiljana GvozdenoviÄ&#x2021; Clinical Center of Vojvodina Novi Sad, Serbia E-mail: profgvozdenovic2010@hotmail.com

7. Abut YC, Kitapcioglu D, Erkalp K, Toprak N, Boztepe A, Sivrikaya U, Palsy I, Gur EK, Eren G, Bilen A. Job burnout in 159 anesthesiology trainees. Saudi J Anaesth 2012;6:4651. doi: 10.4103/1658-354X.93059 8. de Oliveira GS Jr, Chang R, Fitzgerald PC, Almeida MD, Castro-Alves LS, Ahmad S, McCarthy RJ. The prevalence of burnout and depression and their association with adherence to safety and practice standards: a survey of United States anesthesiology trainees. Anesth Analg 2013;117:18293. doi: 10.1213/ANE.0b013e3182917da9

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NEW EDITIONS Lucijan Mohorović: Socijalna komponenta zdravstvene zaštite Labina – višestoljetna bitna sastavnica zdravstvenog i općeg napretka Naslovna knjiga autora Lucijana Mohorovića tiskana je u srpnju 2014. u Rijeci u izdanju nakladnika Zigo Rijeka (239 stranica). Kao što autor uvodno navodi, tekst sadrži saznanja koja su stjecana empirijski i višegodišnjim znanstvenoistraživačkim aktivnostima. Predstavljena su i nova saznanja, široj javnosti do sada nepoznata, prikupljena u povijesnim arhivima gradova Labina, Pazina i Rijeke te u Državnom arhivu u Veneciji. U prvom se dijelu razmatra razdoblje empirijskoga stjecanja iskustava i razrada zapažanja o specifičnosti patologije u mikroregionalnim uvjetima, što prati stalna i intimna autorova potreba za stručnim i znanstvenim usavršavanjem te za prikazom rezultata epidemioloških i kliničko-laboratorijskih istraživanja. Drugo razdoblje u izboru prikazanih radova karakterizira autorov osobni doprinos istraživanju utjecaja štetnih čimbenika iz okoliša sa zdravstvenoekološkog i socijalnog aspekta zaštite humane reprodukcije. Pri tome je uvijek naglašena potreba za afirmacijom primarne prevencije povezano sa štetnim

čimbenicima iz okoliša zastupanjem primjena opreza i primjene principa održivog razvoja. U trećem dijelu autor prikazuje svoj doprinos afirmaciji uloge civilnog društva uz bitne sastavnice principa konsenzusa između ekonomskih, okolišnih i socijalno-zdravstvenih interesa lokalne i regionalne zajednice kao i države u zaštiti životnog prostora. U tom su dijelu sadržani i prikazani autorovi radovi i njegova istraživačka aktivnost objelodanjena i prepoznata i u međunarodnoj znanstvenoj zajednici. Kao liječnik specijalist ginekologije i porodništva posebno zanimanje pokazao je u istraživanju problema s mogućim učinkom iz okoliša na tijek i ishod trudnoće. Ta problematika sadržana je i u autorovoj disertaciji, koju je obranio 1991. godine, a bila je predmetom i u nekoliko zdravstvenih članaka koji su objavljeni u poznatim međunarodnim znanstvenim časopisima. S obzirom na aktivnost termoelektrane na ugljen u Plominu i planirani nastavak njena djelovanja, čak u proširenim razmjerima, problemi s kojima se između ostalog bavio dr. Mohorović ostaju i dalje aktualni. Marko Šarić

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World Health Organization. Chrysotile Asbestos. World Health Organization: 2014.

(Sažetak dostupan na URL: http://www.who.int/entity/ ipcs/assessment/public_health/chrysotile_asbestos_ summary.pdf?ua=1) Pojam azbest označava skupinu prirodnih vlaknastih serpentinskih i amfibolnih minerala, koji su zbog svojih izuzetnih svojstava kao što su čvrstoća, slaba toplinska vodljivost i relativna kemijska postojanost bili - i još su uvijek - u ekonomskoj upotrebi. Komercijalno najviše korištene vrste azbesta su krizotil (serpentinski), krocidolit, amozit, antofilit, tremolit i aktinolit, koji su amfiboli. Izloženost azbestu, uključujući krizotil, može uzrokovati rak pluća, grkljana i jajnika, mezoteliom i azbestozu. Do sada su u više zemalja poduzete mjere zabrane korištenja svih oblika azbesta. Na nacionalnoj razini cilj je ograničiti izloženost, povećati nadzor, spriječiti i konačno spriječiti bolesti izazvane azbestom. Procjenjuje se da u svijetu svake godine najmanje 107.000 ljudi umire od posljedica izloženosti azbestu. Međutim, osim onih zemalja koje su prihvatile spomenute mjere, neke ih tek trebaju provesti. Imajući to na umu, namjera je ove publikacije prije svega pomoći državama članicama Svjetske zdravstvene organizacije (WHO) u donošenju legislativnih

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odluka vezanih za upravljanje zdravstvenim rizicima osoba izloženih azbestu, s naglaskom na serpentinski mineral krizotil. Dokument se sastoji od tri dijela. U prvom dijelu dan je kratki osvrt WHO o sprečavanju bolesti izazvanih azbestom od ožujka 2014. Drugi dio dokumenta bavi se pitanjima azbestne politike: namjera je pružiti pomoć ustroju i oblikovanju azbestne regulative. Treći je dio tehnički sažetak u kojem su prvi put obuhvaćene najnovije procjene Svjetske zdravstvene organizacije o zdravstvenom učinku serpentinskog azbesta krizotila. Procjenu tih zdravstvenih učinaka obavila je Međunarodna agencija za istraživanje raka (IARC) putem Međunarodnog programa o kemijskoj sigurnosti. Tehnički sažetak sadržava i pregled rezultata ključnih istraživanja objavljenih nakon tih procjena te zaključke dobivene na osnovu tih objavljenih studija. Ova publikacija može poslužiti kao dio sveobuhvatnog nacionalnog pristupa za sprečavanje bolesti uzrokovanih azbestom. Takav bi pristup trebao biti uključen i u razvoj nacionalnih zakonodavstava radi podizanja svijesti, jačanja administrativnih kapaciteta, institucionalnog okvira i nacionalnog plana djelovanja za sprečavanje bolesti uzrokovanih azbestom. Ivan Pavičić

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World Health Organization. International Programme on Chemical Safety (IPCS). Environmental Health Criteria 242: DERMAL EXPOSURE. World Health Organization: 2014. Dostupno na URL: http://www.who.int/ipcs/ publications/ehc/ehc_242.pdf Ovdje prikazana monografija Svjetske zdravstvene organizacije, tiskana 2014., daje pregled spoznaja o izloženosti kemikalijama putem kože i o povezanoj procjeni zdravstvenih rizika. Sadržava poglavlja o izvorima i putovima izloženosti, analitičkim pristupima određivanju razine izloženosti putem kože, kožnim bolestima kože povezanima s izloženošću putem kože i mjerama za prevenciju izloženosti. Pod izloženošću kemikalijama putem kože podrazumijeva se doticaj kože s različitim tvarima tijekom određenog vremena. Izvori izloženosti mogu se naći i u radnom i u osobnom okolišu. Opisani su najčešći načini na koje kemikalije dolaze u doticaj s kožom na radnome mjestu te načini izloženosti u osobnom okolišu gdje je koža redovito izložena raznovrsnim proizvodima za kućnu upotrebu, pri čemu posebnu pažnju prilikom procjene rizika treba obratiti na izloženost djece. Kako bi se odredila razina izloženosti određenoj kemikaliji putem kože, koriste se izravne i neizravne metode. U nekim izravnim metodama rabe se dozimetri i flasteri pričvršćeni na kožu, kojima se skupljaju kemikalije, a u nekima se pak uzimaju uzorci s kože brisanjem, pranjem ili metodom ljuštenja kože (eng. tape stripping). Naposljetku, izravna metoda je i in situ video imaging, gdje se zagađenje kože posebno označenom kemikalijom izravno opaža snimanjem

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kože. Neizravne metode istražuju procese koji se događaju prije ili poslije izloženosti. Od procesa prije izloženosti proučavaju se, između ostalog, brzina i opseg otpuštanja tvari iz gotovog proizvoda u neku umjetnu tekućinu, npr. znoj, ili na površinu kože. Biomonitoringom se pak proučavaju procesi nakon izloženosti tj. mjeri se koncentracija tvari u krvi i tkivima nakon apsorpcije putem kože. Kada mjerenje izloženosti nije moguće provesti, pribjegava se računalnim modelima i alatima koji na temelju unesenih parametara daju procjenu veličine izloženosti. Monografija opisuje kontaktne dermatitise kao najčešće kožne bolesti vezane uz radnu izloženost kemikalijama putem kože, te druge rjeđe kožne poremećaje (urtikarija, akne, karcinomi, fototoksičnost). Prevencija izloženosti kemikalijama putem kože provodi se radnim propisima o granicama izloženosti na radnome mjestu, dok su za opću populaciju bitni propisi o označavanju i pakiranju proizvoda na tržištu. Daje se prednost izbacivanju ili zamjeni problematičnog proizvoda, a kada to nije moguće nastoji se izloženost smanjiti, što na radnome mjestu podrazumijeva izmjenu radnih postupaka (npr. odvajanje opasnih tvari u zasebne prostore, korištenje manje koncentracije proizvoda), edukaciju radnika i korištenje osobne zaštitne opreme. Na kraju valja reći da ovaj dokument nije sažetak svih podataka o izloženosti putem kože jer su navedeni samo podaci važni za procjenu rizika. Dodatne informacije mogu se pronaći u publikacijama na osnovi kojih je ovaj dokument nastao. Željka Babić

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ANNOUNCEMENT Arh Hig Rada Toksikol 2014;65:A24

ANNOUNCEMENT

DESETI SIMPOZIJ HRVATSKOGA DRUŠTVA ZA ZAŠTITU OD ZRAČENJA s međunarodnim sudjelovanjem; Šibenik, Solaris – Hotel Jure, 15.-17. travnja 2015. U organizaciji Hrvatskoga društva za zaštitu od zračenja (HDZZ), uz glavne suorganizatore: Institut za medicinska istraživanja i medicinu rada, Institut Ruđer Bošković i Državni zavod za radiološku i nuklearnu sigurnost, u gradu Šibeniku (Solaris – Hotel Jure) od 15. do 17. travnja 2015. održavat će se Deseti simpozij Hrvatskoga društva za zaštitu od zračenja s međunarodnim sudjelovanjem. Tim se jubilarnim simpozijem nastavlja dugogodišnja tradicija organiziranog okupljanja znanstvenika i stručnjaka različitih profila radi razmjene novih informacija i prikaza aktualnih saznanja na području zaštite od zračenja. Na skupovima koje organizira HDZZ tradicionalno sudjeluju znanstvenici i stručnjaci iz domaćih znanstvenoistraživačkih i državnih institucija, iz zdravstva i gospodarstva te sa sveučilišta, ali i inozemni predavači te izlagači. Rad simpozija odvijat će se u okviru sljedećih tema: Opće teme u zaštiti od zračenja; Dozimetrija zračenja; Biološki učinci zračenja; Izloženost stanovništva zračenju; Zaštita od zračenja u medicini; Radioekologija; Neionizirajuća zračenja te Instrumentacija i mjerne tehnike. U okviru skupa posebno će biti obilježen jubilarni 10. simpozij HDZZ-a te će se održati godišnja sjednica Zbora Društva.

Do 15. veljače 2015. kotizacija za sudjelovanje na skupu iznosi 1200 kn (nakon 15. veljače i na simpoziju 1500 kn). Plaćanjem kotizacije sudionik stječe pravo na sve tiskane materijale simpozija, Zbornik radova, koktel dobrodošlice, kavu ili čaj tijekom odmora i svečanu večeru. Prateća osoba plaća pola kotizacije. Svi sudionici simpozija s plaćenom kotizacijom dobit će potvrdu o sudjelovanju i odgovarajući broj bodova prema odluci Povjerenstva za trajno usavršavanje Hrvatske liječničke komore. Službeni jezici skupa su hrvatski i engleski (bez prevođenja). Sve informacije o rezervaciji smještaja bit će poslane sudionicima u sklopu druge obavijesti, a informacije o hotelu mogu se pronaći na poveznici http://www.solaris.hr/hotel-jure/ Svi koji žele sudjelovati u radu simpozija mogu se prijaviti elektroničkom poštom na adresu: hdzz10@imi.hr ili putem prijavnog obrasca dostupnog na mrežnoj stranici Društva, na poveznici http://www.hdzz.hr. Prihvaćeni i u roku pristigli radovi bit će tiskani u Zborniku radova prije održavanja simpozija. Rok za predaju radova u konačnom obliku (i za ranu kotizaciju) je 15. veljače 2015. Znanstveni odbor simpozija vodi dr. sc. Branko Petrinec, a Organizacijski odbor dr. sc. Tomislav Bituh iz Instituta za medicinska istraživanja i medicinu rada, Ksaverska cesta 2 (Zagreb), od kojih se mogu dobiti sve dodatne informacije o skupu (tel: +385 1 4682 653, fax: +385 1 4673 303 i e-adresa: hdzz10@imi.hr). Tomislav Bituh i Branko Petrinec

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REPORT Arh Hig Rada Toksikol 2014;65:A25-A27

REPORT

15TH EUROPEAN ALARA NETWORK WORKSHOP AND 5TH EUTERP WORKSHOP (ROVINJ, CROATIA 7-9 MAY 2014) “EDUCATION AND TRAINING IN RADIATION PROTECTION: IMPROVING ALARA CULTURE” SUMMARY AND RECOMMENDATIONS Workshop objectives and programme This joint EAN-EUTERP Workshop considered how radiation protection (RP) education and training programmes can be delivered effectively, and, in particular, how these can improve radiation protection in practice and help disseminate ALARA culture. The Workshop, which was hosted by EKOTEH Dosimetry Radiation Protection Company, was officially opened by Mr Saša Medaković, Director of the Croatian State Office for Radiological and Nuclear Safety (SORNS). There were 71 participants from 22 different countries, with half the programme devoted to presentations (including posters), and half to Working Group discussions based on the following topic areas: • Building ALARA into radiation protection training programmes • Measuring the effectiveness of training • The role of qualification and recognition schemes • Training tools and methods • National approaches to training Some key themes and issues did emerge from the workshop presentations, and these are described below. On the final day, the conclusions and recommendations from the four working groups were presented and discussed, and these are also summarised below. All of the presentations are available to download from the EAN and EUTERP websites (http://www. eu-alara.net/ and http://www.euterp.net).

THEMES AND ISSUES ARISING ALARA culture and radiation protection training As a joint EAN-EUTERP workshop it was interesting to explore the relationship between RP education and training, and ALARA culture. These are interdependent: an organisation with a strong safety culture identifies training needs and provides motivated participants; and well-designed training should, in turn, foster this culture. Through EUTERP and the ENETRAP projects, much valuable work has been done in terms of defining and agreeing the roles of the Radiation Protection Expert (RPE) and the Radiation Protection Officer (RPO), to help promote a harmonised approach in Europe through implementation of the Basic Safety Standards Directive (BSS). Through EFOMP and the MEDRAPET project similar activities were performed in relation to the Medical Physics Expert (MPE). This work has included detailed considerations of education and training requirements, especially for RPEs and MPEs. In comparison, ALARA culture is relevant to all exposure situations, and should involve all of the stakeholders. Previous EAN and EUTERP workshops have highlighted the importance of training – not just for defined roles such as the RPE/MPE, but also for workers and other stakeholders, such as managers, equipment suppliers, and competent authorities. It was agreed that a wider focus was needed in future, to encourage the development of training matrices in which the training strategies, objectives and outcomes for different groups of stakeholders are defined. Competence and culture Traditional approaches to defining education and training requirements have started from the basis of academic qualifications, which are then supplemented with RP-specific training courses. While these approaches are still relevant, it is now recognised that the goal of education and training is to produce “competence”, which is built upon acquired knowledge, understanding and skills. The Workshop

A26 strongly recommended that “soft skills”, such as leadership and communication, should also be considered. For persons such as RPEs, MPEs and RPOs, who have a role in promoting ALARA culture, these are especially important skills, and it was agreed that they should form part of the competency requirements. It was also agreed that defining training outcomes in terms of knowledge, skills and competence helps underpin the practical implementation of ALARA. It was noted, however, that ALARA culture is also defined by personal attitudes and behaviour. While these factors cannot be instilled through training alone, they can be encouraged, i.e. by training which is designed to promote reflection and a questioning attitude. This can provide a bridge between training and ALARA culture, and should form part of the training objectives. Assessing the effectiveness of training The ultimate goal of this education and training is better radiation protection. Like other protection options, training should be optimised to deliver the maximum benefit without being unduly expensive or time-consuming, i.e. it should be both effective and efficient. There was little discussion about efficiency, although it was noted that the resources generally allocated for training are increasingly limited. In contrast, assessing the effectiveness of training was a major theme throughout the workshop. Traditionally this has relied on written tests at the end of training courses; these can test knowledge and understanding, and (to some extent) how trainees might apply these in a practical scenario. It was noted that practical skills can be more directly tested using practical assessments, done under the observation of the trainers, although the quantitative marking of performance is not straightforward. Ideally, the effectiveness of training should be demonstrated by tangible improvements in radiation protection. Work-related benchmarks such as radiation doses or the frequency of incidents were discussed; however it was concluded that these were only useful in a few specific, well-defined circumstances. A better option would be to find a means of assessing individual attitudes to radiation protection, ideally before and after training. This approach is relatively unfamiliar to the radiation protection community, and it was suggested that expertise from the social sciences should be sought.

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Methods and tools Various presentations and posters gave details of different national approaches to training, which remain diverse even under the harmonising influence of the BSS. There was, however, broad agreement on the types of training methods and tools that are best suited to developing and sustaining an ALARA culture, i.e.: • Training should be interesting and engaging, and directly relevant to the trainees’ work; • It should include realistic practical exercises, designed to demonstrate the application of radiation protection theory; • Emphasis should be placed on problemsolving and trainee-to-trainee interaction (e.g. group exercises) to encourage reflection and a questioning attitude; • Practical training for incidents and emergencies should incorporate an element of stress, so that human factors can be better taken into account. The workshop also highlighted the use of computer-based “virtual reality” training tools, which can specifically consider optimisation in potentially high dose areas, and are a valuable ALARA training and planning tool in such circumstances. Other issues Many other issues were presented and discussed during the workshop, and there is not sufficient space here to describe all of these. However, to give a flavour of the proceedings, these included: • The European EQF and ECVET schemes, and approaches to mutual recognition • The competence requirements for training providers • Approaches to “train the trainers” • Continued learning and refresher training

WORKSHOP CONCLUSIONS AND RECOMMENDATIONS The four Working Groups considered many of the issues already described above, and also made some specific recommendations, which are summarised below.

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WG1: How to assess the effectiveness of training? •

• •

More work needs to be done in terms of assessing the effectiveness of training. There are several possible workplace indicators, such as monitoring results, individual doses, and reports of audits and inspections (including observation and assessment of behaviours in the workplace). These should be used to construct a framework for analysing the effectiveness of training. The above approach should be promoted by Regulatory Authorities, RPEs and professional societies. Effective training providers are critical to both the delivery and assessment of training, and there is value in exploring methods by which the quality of training providers can be assessed and recognised.

WG2: Tools to improve the effectiveness of training •

•

Training is a continuous process, involving multiple stages. It is useful to define the responsibilities of different stakeholders (employers, employees, RPEs, regulatory authorities, etc) for the different stages of this process. Training should be practical and realistic, including the use of real radiation sources, where appropriate and subject to suitable dose constraints. On-the-job training is an important component of the training cycle, and should be properly structured and involve suitably trained mentors. “Train the trainers” is an important concept, and should include information on new training techniques and technologies, and a basic understanding of the European E&T Qualification Frameworks (ECVET, ECTS, EQF, etc.).

WG3: What is achieved by recognition schemes? •

The ENETRAP project should develop guidelines for national and mutual recognition schemes, and consider whether the ECVET concepts are useful in this respect. Schemes should focus on all-round competence rather than academic qualifications.

•

In turn, Member States should aim to establish clear and transparent national schemes for the recognition of RPE competence, which are then promoted by (for example) HERCA. A similar formal system of recognition is not considered appropriate for RPOs; however a simpler system for verifying and validating that they have received suitable training should be considered.

WG4: Incorporating ALARA culture into training requirements •

•

Although risk is a factor in everyday life, the risks associated with radiation exposure are not readily understood by trainees, and not easily explained by trainers. However, persons can understand the difference between good and bad practice and the impacts in terms of increasing and decreasing the doses received; training should concentrate on practical examples of this. There is a need to develop education and training in radiation protection for the public, and this should include providing radiation protection information and data on the internet.

NEXT EAN AND EUTERP WORKSHOPS The 16th EAN workshop, on “ALARA in Industrial Radiography” is planned for March 2016, in Bern, Switzerland. Details will be announced on the EAN website. In the meantime, EAN is producing an updated version of “ALARA: from theory towards practice”. The new book will be titled “Optimisation of Radiation Protection (ALARA): A Practical Guidebook” and will hopefully be published by the end of 2014. The 6th EUTERP workshop is currently being planned and details will shortly be posted on the EUTERP website. EAN: Peter Shaw (PHE, CRCE, Leeds, UK)) and Pascal Croüail (CEPN, Paris, France) EUTERP: Richard Paynter and Michèle Coeck (SCKCEN, Mol, Belgium)