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Urinary concentrations of pyrethroid metabolites in the convenience sample of an urban population of Northern Poland
International Journal of Hygiene and Environmental Health 216 (2013) 295–300
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Urinary concentrations of pyrethroid metabolites in the convenience sample of an urban population of Northern Poland夽 Bartosz Wielgomas a,∗ , Wacław Nahorski b,1 , Wojciech Czarnowski a,2 a
Department of Toxicology, Medical University of Gda´ nsk, Al. Gen. J. Hallera 107, 80-416 Gda´ nsk, Poland Department of Tropical and Parasitic Diseases, Interfaculty Institute of Maritime and Tropical Medicine in Gdynia, Medical University of Gda´ nsk, ul. Powstania Styczniowego 9B, 81-519 Gdynia, Poland b
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 30 August 2012 Accepted 3 September 2012 Keywords: Synthetic pyrethroids Exposure assessment Urinary biomarker Poland
a b s t r a c t Urinary concentrations of pyrethroid metabolites were measured in the first void urine samples col´ lected from 132 healthy people living in the Gdansk region of Northern Poland in 2010 and 2011. Four metabolites of synthetic pyrethroids: cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane1-carboxylic acids (cis-, trans-Cl2 CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br2 CA) and 3-phenoxybenzoic acid (3-PBA) were simultaneously liquid-liquid extracted, derivatized with hexafluoroisopropanol and analyzed by a gas chromatography ion-trap mass spectrometry. All the analytes were detected and quantified in the samples with various frequency, 3-phenoxybenzoic being the most often (80%) and the others less frequently (7–11%). Distribution of 3-PBA concentrations followed log-normal model, the mean concentration of 3-phenoxybenzoic acid: 0.393 g/L (0.327 g/g creatinine) is similar to those of the other general populations in various regions of the world. Neither sex nor age were predictors of urinary 3-PBA. Our findings suggest wide exposure to pyrethroid insecticides in the Polish general population. There is a continuous need to further study the exposure to synthetic pyrethroids among the general population since there is a strong, increasing trend in their usage. © 2012 Elsevier GmbH. All rights reserved.
Introduction Synthetic pyrethroids are among the most frequently used insecticides in the world. The current annual use of pyrethroids in Poland is estimated to be over 80,000 kg of active ingredient (www.bip.minrol.gov.pl, 2011). Due to its relatively low acute mammalian toxicity, this chemical group is readily used instead of organophosphates and carbamates in agriculture and public health. The majority of Poland’s household insecticide formulations used against pests also contain synthetic pyrethroids. Thirteen compounds from this chemical group are officially registered in Poland (Table 1) and are available on the market (bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, etofenprox, imiprothrin, permethrin, phenothrin, prallethrin, tetramethrin, and transfluthrin) in numerous products. From results of published epidemiological studies it appears
夽 This work was supported by grants ST-5 and MN-954 from Medical Univer´ The study protocol was approved by the Local Ethics Committee sity of Gdansk. (NKEBN/158/2010). ∗ Corresponding author. Tel.: +48 58 349 16 72; fax: +48 58 349 16 75. E-mail addresses: [email protected] (B. Wielgomas), [email protected] (W. Nahorski), [email protected] (W. Czarnowski). 1 Tel.: +48 58 699 84 55. 2 Tel.: +48 58 349 16 70; fax: +48 58 349 16 75. 1438-4639/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijheh.2012.09.001
that food products of plant origin play significant role in human non-occupational exposure (Fortin et al., 2008a; Kimata et al., 2009; Riederer et al., 2008; Schettgen et al., 2002a). Pyrethroid residues are not usually found in animal tissues (Niewiadowska et al., 2010). Although organophosphates still dominate on the overall pesticide market in Poland there is an increasing trend in synthetic pyrethroid market volume, which has doubled in the last 5 years from approximately 36,000 kg in 2005 to 87,000 kg in 2010 year (in relation to active substance). It appears that amount of pyrethroids introduced into the environment will continuously grow to replace more toxic organophosphorous insecticides (www.bip.minrol.gov.pl, 2011). Chemically, pyrethroids are esters of vinyl cyclopropane carboxylic acid and alcohol part (in most cases 3-phenoxybenzyl alcohol). In mammals they are readily detoxified by ester bond cleavage by carboxylesterases to acidic and alcoholic moieties and the latter undergoes further oxidation to 3-phenoxybenzoic acid, and both metabolites partially conjugated are eliminated with urine (Choi et al., 2002; Eadsforth and Baldwin, 1983; Takaku et al., 2011; Woollen et al., 1992). Excretion is rapid; half-lives for metabolites are expressed in a few hours, thus urinary levels only reflect a very recent exposure. Human studies demonstrate quite rapid clearance after oral, dermal or inhalation exposure (Eadsforth et al., 1988). For most of the pyrethroids over 90% of the dose is excreted with urine in 48 h with the peak occurring in 6–12 h after
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Table 1 Synthetic pyrethroids authorized in Poland with their corresponding urinary biomarkers of exposure. Parent compound
Metabolite Cl2 CAa
Bifenthrinb Cyfluthrin Cyhalothrin Cypermethrin Deltamethrin Esfenvalerate Etofenprox Imiprothrin Permethrin Phenothrin Prallethrin Tetramethrin Transfluthrin
Br2 CAa
3-PBAa
•
• • • • •
•
4F3PBA
CDCA
•
•
• •
• • • •
•
Br2 CA, cis-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid; Cl2 CA, cis-/trans-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid; 4F3PBA, 4fluoro-3-phenoxybenzoic acid; CDCA, chrysanthemumdicarboxylic acid. a Measured in this study. b Urinary metabolites of bifenthrin (2-methyl-3-phenylbenzoic acid or cis-2,2-dimethyl-3-(2,2-chloro-3,3,3-trifluoro-1-propenyl)cyclopropanecarboxylic acid) were not measured yet in non-exposed subjects.
exposure (Leng et al., 1997). Currently, for biomonitoring purposes only urine is used as a biological material for the study. There is no relevant data on the use of other, alternative matrices to assess recent or past exposure due to the rapid biotransformation. Urinary concentrations of metabolites integrates all routes of exposure and also exposure to both parent compounds and its degradation products/metabolites which after absorption are also excreted as conjugates in urine. The method for biomarker of long term exposure is still sought, however some attempts have been performed to use albumin adducts for this purpose (Noort et al., 2007). Pyrethroids act primarily on voltage-sensitive sodium channels in the nervous system (Soderlund, 2012). This mechanism of action is similar in insects and mammals but due to the effective and fast metabolism, mammals remain almost resistant to acute effects. Recent studies confirm the hypothesis that mixtures of pyrethroids act in a dose-additive model (Cao et al., 2011; Wolansky et al., 2009). Current studies also suggest other, additional modes of action on living organisms including endocrine disruption observed in vitro and in vivo, developmental neurotoxicity and reproductive toxicity (Jin et al., 2012; Meeker et al., 2009; Orton et al., 2011). Exposure to pyrethroids was assessed in the general population in a rather small number of countries, like: Germany (Heudorf and Angerer, 2001; Leng and Gries, 2005; Schettgen et al., 2002a), Italy (Saieva et al., 2004), Canada (Fortin et al., 2008a), France (Le Grand et al., 2012) and USA (Barr et al., 2010). Up to date there are no published results of pyrethroid exposure in Eastern Europe countries. The aim of this study was to assess exposure to synthetic pyrethroids in the convenience sample of a population living in Northern Poland by measuring specific urinary metabolite concentrations in the first void urine samples. Materials and methods
and 2,2-dibromovinyl-2,2-dimethylcyclopropanecarboxylic acid (Br2 CA) were obtained from Russel-Uclaf (France). cis-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cisCl2 CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1carboxylic acid (trans-Cl2 CA) were obtained from Dr. Ehrenstorfer Laboratories, Germany. All other reagents were of analytical grade. Standard stock solutions of 1 mg/mL were prepared in acetonitrile and stored at −20 ◦ C, protected from light. Working standard solutions were prepared in acetonitrile and were stored at +4 ◦ C. Stock and working standard solutions were replaced every 6 and 3 months respectively. Subjects and sample collection Urine samples were obtained from 132 subjects with age median 35 years (range 5–77 years, including 8 children under 14 years), 69 women (median age 34 years) and 63 men (median age ´ 37 years) living in the Gdansk region, Northern Poland, recruited in 2010 and 2011. Only basic sociodemographic data was collected using a questionnaire. All study participants lived in an urban area and none of the subjects were occupationally exposed to pesticides. All the procedures were approved by the Ethics Committee of the ´ Poland. Prior to enrolment in the Medical University of Gdansk, study, each subject signed the informed consent form. First morning void samples were collected in polypropylene containers and then were frozen and stored until analysis at −20 ◦ C. Chemical analysis The concentrations of cis-,trans-Cl2 CA, Br2 CA and 3-PBA were measured by gas chromatography–mass spectrometry (GC–MS) using electro-ionization operated in selected ion storage detection mode. Extraction step was adopted from previously established method (Schettgen et al., 2002b) described below.
Chemicals Sample preparation The following chemicals were obtained from Sigma–Aldrich (Germany): 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), diisopropylocarbodiimide (DIIC) and 2-phenoxybenzoic acid (2-PBA), which was used as an internal standard (IS). 3-Phenoxybenzoic acid
Briefly, 3 mL of thawed urine were transferred into a 10 mL screw-top glass tube and 25 L of IS solution (2-PBA, 1 g/mL of acetonitrile) along with 0.6 mL concentrated hydrochloric acid
B. Wielgomas et al. / International Journal of Hygiene and Environmental Health 216 (2013) 295–300 3,0 2,5 2,0 1,5
Normal value
were added. Samples were incubated at 95 ◦ C in the oven for 90 min. After bringing to room temperature, 4 mL of hexane were added and the tubes were shaken for 15 min. Following centrifugation, the hexane layer was collected to the next screw-top glass tube, and extraction with the same volume of hexane was repeated. Extracts were combined and then reextracted with 0.5 mL of 0.1 M NaOH. Hexane was discarded, while 0.1 mL of concentrated HCl and 2 mL of hexane were added to the remaining aqueous phase. Samples were again shaken, centrifuged and the resulting supernatant was evaporated to dryness under the stream of nitrogen at 45 ◦ C. The residue was treated with 10 L of HFIP, 15 L of DIIC and 250 L of hexane. Samples were mixed for 10 min at room temperature and then 1 mL of 5% K2 CO3 added. After vigorous shaking, the tubes were centrifuged and the hexane layer was transferred to an autosampler vial. Two microliters of the final extract were analyzed by GC–MS.
297
1,0 0,5 0,0 -0,5 -1,0 -1,5 -2,0 -0,5
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
3-PBA concentration [ng/ml]
Gas chromatography–mass spectrometry
Fig. 1. Distribution of the 3-PBA concentrations in the studied samples.
Analyses were performed using Varian GC-450 gas chromatograph equipped with 220-MS ion-trap mass spectrometer working in the selected ion storage (SIS) mode. Derivatives were separated on VF-5ms (Varian, Palo Alto, USA) low bleed column (30m × 0.25 mm I.D., 0.25 m film thickness) using the following oven program: 60 ◦ C to 1 min, 60–150 ◦ C (8 ◦ C/min), 150–280 ◦ C (30 ◦ C/min), 280 ◦ C to 11 min. Selected ions were monitored: 323 m/z (cis-Cl2 CA and trans-Cl2 CA), 369 m/z (Br2 CA) and 364 m/z (2-PBA and 3-PBA). Two microliters of the final extract were injected (injector temperature 280 ◦ C) in splitless mode with 25 psi pulse pressure lasting 1.0 min. The chromatograph was equipped with 1079 programmable temperature vaporizing injector and SGE focusliner glass inlet. Quality control Quality of the assay was monitored in two ways. At the beginning of the study large volume of the low concentration pool of urine was prepared from previously analyzed samples, exhibited undetectable contents of the metabolites. That pool was divided into four portions and three of them were spiked with standards (cis-Cl2 CA, trans-Cl2 CA, Br2 CA, 3-PBA) to achieve fortification levels: 1, 5 and 10 ng/mL, the fourth portion was left unfortified. Enriched samples were aliquoted into polypropylene tubes (volume of 3.2 mL) and stored along with samples (−20 ◦ C) for further quality control assessment. We also used a ClinCal® Urine Calibrator (Cat. No. 9969), lyophilized, for Toxic Organic Compounds acquired from Recipe Chemicals and Instruments GmbH (Munich, Germany). The calibrator with declared concentrations of pyrethroid metabolites served as a quality control material (3.6, 3.3, 2.5, 6.6 ng/mL respectively for cis-Cl2 CA, trans-Cl2 CA, Br2 CA, 3PBA). In each analytical batch we analyzed 30 samples including: 25 unknown, 4 spiked at different levels and one ClinCal® Urine Calibrator. A typical calibration curve was constructed on 8 points prepared based on matrix matched calibration. The analytical method applied in this work showed very good performance. Limits of detection for all metabolites were in the range 0.05–0.1 ng/mL with between day imprecision of 4.6–8.3%. Urinary creatinine was measured with spectrophotometric Jaffe method. Statistical analysis Data distribution was examined using Kolmogorov–Smirnov test. Concentrations in samples below limit of detection (LOD) were calculated according to formula: LOD value/square root of 2. Statistica ver. 9 (Statsoft, USA) software was used for
statistical evaluation of results, significance was considered when p-value <0.05. Results Concentrations of determined metabolites are presented in Table 2. The values are expressed in ng/mL (g/L) or adjusted to creatinine (g/g of creatinine). The calculation of mean and geometric mean was performed only for compounds with detectivity over 60% and values below LOD were expressed as LOD divided by the square root of two before mathematical operations. The most frequently detected compound was 3-PBA, which was present in concentrations higher than LOD in 106 of 132 analyzed samples, that represents 79.7% of all samples. The mean concentration was 0.393 and the geometric mean was 0.260. The highest concentration of 3-PBA was 3.03 ng/mL. The distribution of the 3-PBA concentrations fitted the log-normal model (Fig. 1). The remaining metabolites: cis-Cl2 CA, trans-Cl2 CA and Br2 CA were detected in 11 (8%), 9 (7%) and 15 (11%) of 132 samples. Maximum concentrations of cis-Cl2 CA, trans-Cl2 CA and Br2 CA were: 0.256, 0.409 and 3.336 ng/mL respectively. Discussion In this work 132 urine samples obtained from people living ´ in the Gdansk region, located in the Northern part of Poland were tested, and to our knowledge this is the first biomonitoring study for those metabolites in Poland. Among 13 synthetic pyrethroids authorized in Poland, 9 of them are metabolized to compounds determined in this work. Other metabolites: 4-fluoro3-phenoxybenzoic acid and chrysanthemumdicarboxylic acids are less frequently detected in non-exposed population (Leng and Gries, 2005). The liquid–liquid extraction technique was used for isolation of acidic metabolites of pyrethroids from urine hydrolysates according to Schettgen et al. (2002b). Nevertheless, the SPE protocols (using Supelco HLB cartridges) and liquid-liquid extraction with tert-butyl-methyl-ether (Leng and Gries, 2005) were also tested, but the final extracts were much cleaner when Schettgen’s method was applied. The same observation was noticed by Le Grand et al. (2012) using HPLC–MS/MS as an analytical technique. The back extraction procedure seems to be very effective in removing matrix thus the risk of ion-trap contamination is reduced and downtime of the GC–MS system is minimized. Thorough validation procedure applied in this study (reference material, in house prepared quality
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Table 2 Mean ± standard deviation (SD), geometric mean (GM) and percentiles of urinary metabolites (g/L) and creatinine adjusted (g/g creatinine) among studied population. Metabolite
% > LOD (n > LOD) n = 132
Concentration Mean ± SD
GM
Min
Not adjusted (g/L) 3-PBA 80 (106) 8 (11) cis-Cl2 CA 7 (9) trans-Cl2 CA 11 (15) Br2 CA
0.393 ± 0.427 – – –
0.260 – – –
Creatinine adjusted (g/g creatinine) 3-PBA 80 (106) cis-Cl2 CA 8 (11) 7 (9) trans-Cl2 CA 11 (15) Br2 CA
0.327 ± 0.310 – – –
0.220 – – –
control samples) proved this method as very reproducible, robust and reliable. With detection limits for all metabolites at 0.1 ng/mL it is possible to detect and quantify urinary biomarkers in the population exposed to the environmental levels of pyrethroids. Only in a very few papers, did authors declared detection limits lower than for the method that was used in the current work (Arrebola et al., 1999; Barr et al., 2007; Le Grand et al., 2012). Urine as a matrix for biomonitoring purposes posses many advantages over blood or serum, like higher metabolite concentration and ease of collection especially in the case of non-persistent chemicals which are easily transformed and eliminated from blood (Leng et al., 1997). Urinary concentration of pyrethroid metabolites served in many studies as a biomarker of recent exposure, however the amount of metabolites present in urine aggregates the total exposure to both the parent compounds and also their own metabolites and degradation products adsorbed through all possible routes. It was demonstrated in the case of organophosphate insecticides that their degradation products and metabolites – dialkylphosphates, present in food account for the majority of the dose excreted with urine (Zhang et al., 2008). A similar scenario is also possible also for synthetic pyrethroids, but they are more resistant to spontaneous hydrolysis than organophosphates. However up to date none of the studies dealt with that problem in the case of synthetic pyrethroids. Biomonitoring, does not allow for the differentiation of the route of exposure, with one exception when the fate of the chemical is different when it enters the body through the dermal or oral route. One should be very cautious when drawing conclusions about sources of exposure and chemical identity of the compound to which humans are exposed. In biomonitoring studies of other populations, mainly spot urine samples were collected in contrast to our study where all volunteers collected first void urine samples. Since morning urine usually contains more creatinine than random samples throughout the day, recalculation of volumetric concentration expressed in ng/mL to concentration per gram of creatinine decreased the value of mean concentration. For comparative purposes the adjustment to creatinine is sometimes questioned due to the significant variations of creatinine excretion rates in various isolated groups of the general population, which is age, sex and race dependent (Barr et al., 2005; Fortin et al., 2008b). Neither age nor sex were predictors of urinary 3-PBA in this study, no differences were observed in the mean concentration of 3-PBA for women and men and no relation to the age of donors (Figs. 2 and 3). Barr et al. (2010) reported significantly higher concentrations of 3-PBA only in the youngest group included in NHANES covering the range of 6–11 years old children. In the remaining population aged 12–59 years old no relationship between age and 3-PBA concentration was identified.
Max
Percentiles 5
25
50
75
90
95
3.030 0.256 0.409 3.336
0.141
0.255
0.471
0.887
1.154 0.153 0.122 0.314
1.832 0.508 0.265 1.725
0.121
0.190
0.418
0.722 0.139 0.148 0.170
0.950 0.170 0.170 0.222
Fig. 2. Creatinine adjusted urinary concentrations of 3-PBA in population subgroups of different age. No significant difference.
In this study, for the first time performed in Poland, we have found wide exposure to pyrethroids (high detection rate of 3PBA) and the urinary level of main metabolite is similar to those observed in populations of other countries (Table 3). The geometric mean concentration of 3-PBA is lower than German reference value 2 g/L (Heudorf et al., 2006). However, for the on-going
Fig. 3. Creatinine adjusted concentrations of 3-PBA in females and males. No significant difference.
B. Wielgomas et al. / International Journal of Hygiene and Environmental Health 216 (2013) 295–300
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Table 3 Urinary concentrations of 3-PBA measured in population based studies in different regions of the world. Country (study)
n
Age range
Sampling period
Population
3-PBA (g/g creatinine) (GM)
Reference
Germany, (GerES IV-pilot study) Japan
395 143 66 448 87 1998 3046 132
2–17 64a 49a 39–85 40–85 6–59 6–59 5–77
2001–2002 2005–2007
General, children Rural Suburban Rural, non-farmers Rural, farmers General population General population Convenience sample, urban, non-farmers
0.240 0.320 0.490 0.400 0.450 0.261 0.324 0.220
Becker et al. (2006) Kimata et al. (2009)
Japan USA (NHANES 1999–2002) Poland
a
2005 1999–2000 2001–2002 2010–2011
Ueyama et al. (2009) Barr et al. (2010) Present study
Mean value.
studies lowering of the detection limit is recommended by converting the method used in this work to the MS/MS technique. Because the metabolites of synthetic pyrethroids are detected in the vast majority of urine samples, it is important to estimate the health risks of exposure. Although those pesticides have a low acute toxicity to humans, actually more attention is paid to the effects of low doses especially on the reproductive system. Some animal studies shown pyrethroids as chemicals with potential to affect sperm quality (Elbetieha et al., 2001; Kumar et al., 2004; Mani et al., 2002). There are few studies demonstrating existence of the relationship between the concentrations of metabolites of synthetic pyrethroids in the urine of persons not exposed occupationally, and the lower semen quality. Meeker et al. (2008) found associations between concentration of urinary metabolites of pyrethroids and reduced semen quality as well as sperm DNA damage among 161 patients of infertility clinic. It is important to note that concentrations measured in Meeker’s study are in a range of those found in our study and the US general population in the third report of NHANES (CDC, 2005) and German population (Heudorf and Angerer, 2001). Recently, Toshima et al. (2012) observed lower sperm motility in subjects with higher urinary 3-PBA among 42 Japanese males. Han et al. (2008) in a study on 212 adult men observed that urinary 3-PBA levels were associated with increased luteinizing hormone and reduced estradiol levels. Surprisingly in comparison to other studies the detection rate of the specific deltamethrin metabolite – Br2 CA – is higher than for cisCl2 CA and trans-Cl2 CA in the studied population, and higher than other authors reported previously. Dietary sources appear to be the most important pathways of exposure in the general population, as intuitively suggested by American and German investigators (Barr et al., 2010; Schettgen et al., 2002a) however, more studies on dietary exposure should be performed to clarify those suppositions. This observation suggests a different pattern of exposure in Poland and encourages further cohort studies to be carried out to understand the sources and intensity of exposure in the general population of Poland. Acknowledgments This work was financed by grants ST-5 and MN-954 from ´ Medical University of Gdansk. The authors wish to thank Teresa Pawłowska and Hanna Lemanowicz for their excellent technical assistance. References Arrebola, F.J., Martinez-Vidal, J.L., Fernandez-Gutierrez, A., Akhtar, M.H., 1999. Monitoring of pyrethroid metabolites in human urine using solid-phase extraction followed by gas chromatography–tandem mass spectrometry. Anal. Chim. Acta 401, 45–54. Barr, D.B., Leng, G., Berger-Preiss, E., Hoppe, H.W., Weerasekera, G., Gries, W., Gerling, S., Perez, J., Smith, K., Needham, L.L., Angerer, J., 2007. Cross validation of multiple
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Mani, U., Islam, F., Prasad, A.K., Kumar, P., Suresh Kumar, V., Maji, B.K., Dutta, K.K., 2002. Steroidogenic alterations in testes and sera of rats exposed to formulated fenvalerate by inhalation. Hum. Exp. Toxicol. 21, 593–597. Meeker, J.D., Barr, D.B., Hauser, R., 2008. Human semen quality and sperm DNA damage in relation to urinary metabolites of pyrethroid insecticides. Hum. Reprod. 23, 1932–1940. Meeker, J.D., Barr, D.B., Hauser, R., 2009. Pyrethroid insecticide metabolites are associated with serum hormone levels in adult men. Reprod. Toxicol. 27, 155– 160. Ministry of Agriculture and Rural Development – Report About Annual Pesticide Market Volume, 2011. Available from: www.bip.minrol.gov.pl (accessed 04.10.11). ˙ Niewiadowska, A., Kiljanek, T., Semeniuk, S., Zmudzki, J., 2010. Determination of pyrethroid residues in meat by gas chromatography with electron capture detection. Bull. Vet. Inst. Pulawy 54, 595–599. Noort, D., van Zuylen, A., Fidder, A., Hulst, A.G., 2007. Biomonitoring of exposure to permethrin based on adducts to proteins. In: Defense Against the Effects of Chemical Hazards: Toxicology, Diagnosis and Medical Countermeasures, pp. 24-1–24-10. Available from: http://ftp.rta.nato.int/public// PubFullText/RTO/MP/RTO-MP-HFM-149///MP-HFM-149-24.pdf Orton, F., Rosivatz, E., Scholze, M., Kortenkamp, A., 2011. Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens. Environ. Health Perspect. 119, 794–800. Riederer, A.M., Bartell, S.M., Barr, D.B., Ryan, P.B., 2008. Diet and nondiet predictors of urinary 3-phenoxybenzoic acid in NHANES 1999–2002. Environ. Health Perspect. 116, 1015–1022. Saieva, C., Aprea, C., Tumino, R., Masala, G., Salvini, S., Frasca, G., Giurdanella, M.C., Zanna, I., Decarli, A., Sciarra, G., Palli, D., 2004. Twenty-four-hour urinary excretion of ten pesticide metabolites in healthy adults in two different areas of Italy (Florence and Ragusa). Sci. Total Environ. 332, 71–80.
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International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh
Urinary concentrations of pyrethroid metabolites in the convenience sample of an urban population of Northern Poland夽 Bartosz Wielgomas a,∗ , Wacław Nahorski b,1 , Wojciech Czarnowski a,2 a
Department of Toxicology, Medical University of Gda´ nsk, Al. Gen. J. Hallera 107, 80-416 Gda´ nsk, Poland Department of Tropical and Parasitic Diseases, Interfaculty Institute of Maritime and Tropical Medicine in Gdynia, Medical University of Gda´ nsk, ul. Powstania Styczniowego 9B, 81-519 Gdynia, Poland b
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 30 August 2012 Accepted 3 September 2012 Keywords: Synthetic pyrethroids Exposure assessment Urinary biomarker Poland
a b s t r a c t Urinary concentrations of pyrethroid metabolites were measured in the first void urine samples col´ lected from 132 healthy people living in the Gdansk region of Northern Poland in 2010 and 2011. Four metabolites of synthetic pyrethroids: cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane1-carboxylic acids (cis-, trans-Cl2 CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br2 CA) and 3-phenoxybenzoic acid (3-PBA) were simultaneously liquid-liquid extracted, derivatized with hexafluoroisopropanol and analyzed by a gas chromatography ion-trap mass spectrometry. All the analytes were detected and quantified in the samples with various frequency, 3-phenoxybenzoic being the most often (80%) and the others less frequently (7–11%). Distribution of 3-PBA concentrations followed log-normal model, the mean concentration of 3-phenoxybenzoic acid: 0.393 g/L (0.327 g/g creatinine) is similar to those of the other general populations in various regions of the world. Neither sex nor age were predictors of urinary 3-PBA. Our findings suggest wide exposure to pyrethroid insecticides in the Polish general population. There is a continuous need to further study the exposure to synthetic pyrethroids among the general population since there is a strong, increasing trend in their usage. © 2012 Elsevier GmbH. All rights reserved.
Introduction Synthetic pyrethroids are among the most frequently used insecticides in the world. The current annual use of pyrethroids in Poland is estimated to be over 80,000 kg of active ingredient (www.bip.minrol.gov.pl, 2011). Due to its relatively low acute mammalian toxicity, this chemical group is readily used instead of organophosphates and carbamates in agriculture and public health. The majority of Poland’s household insecticide formulations used against pests also contain synthetic pyrethroids. Thirteen compounds from this chemical group are officially registered in Poland (Table 1) and are available on the market (bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, esfenvalerate, etofenprox, imiprothrin, permethrin, phenothrin, prallethrin, tetramethrin, and transfluthrin) in numerous products. From results of published epidemiological studies it appears
夽 This work was supported by grants ST-5 and MN-954 from Medical Univer´ The study protocol was approved by the Local Ethics Committee sity of Gdansk. (NKEBN/158/2010). ∗ Corresponding author. Tel.: +48 58 349 16 72; fax: +48 58 349 16 75. E-mail addresses: [email protected] (B. Wielgomas), [email protected] (W. Nahorski), [email protected] (W. Czarnowski). 1 Tel.: +48 58 699 84 55. 2 Tel.: +48 58 349 16 70; fax: +48 58 349 16 75. 1438-4639/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijheh.2012.09.001
that food products of plant origin play significant role in human non-occupational exposure (Fortin et al., 2008a; Kimata et al., 2009; Riederer et al., 2008; Schettgen et al., 2002a). Pyrethroid residues are not usually found in animal tissues (Niewiadowska et al., 2010). Although organophosphates still dominate on the overall pesticide market in Poland there is an increasing trend in synthetic pyrethroid market volume, which has doubled in the last 5 years from approximately 36,000 kg in 2005 to 87,000 kg in 2010 year (in relation to active substance). It appears that amount of pyrethroids introduced into the environment will continuously grow to replace more toxic organophosphorous insecticides (www.bip.minrol.gov.pl, 2011). Chemically, pyrethroids are esters of vinyl cyclopropane carboxylic acid and alcohol part (in most cases 3-phenoxybenzyl alcohol). In mammals they are readily detoxified by ester bond cleavage by carboxylesterases to acidic and alcoholic moieties and the latter undergoes further oxidation to 3-phenoxybenzoic acid, and both metabolites partially conjugated are eliminated with urine (Choi et al., 2002; Eadsforth and Baldwin, 1983; Takaku et al., 2011; Woollen et al., 1992). Excretion is rapid; half-lives for metabolites are expressed in a few hours, thus urinary levels only reflect a very recent exposure. Human studies demonstrate quite rapid clearance after oral, dermal or inhalation exposure (Eadsforth et al., 1988). For most of the pyrethroids over 90% of the dose is excreted with urine in 48 h with the peak occurring in 6–12 h after
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Table 1 Synthetic pyrethroids authorized in Poland with their corresponding urinary biomarkers of exposure. Parent compound
Metabolite Cl2 CAa
Bifenthrinb Cyfluthrin Cyhalothrin Cypermethrin Deltamethrin Esfenvalerate Etofenprox Imiprothrin Permethrin Phenothrin Prallethrin Tetramethrin Transfluthrin
Br2 CAa
3-PBAa
•
• • • • •
•
4F3PBA
CDCA
•
•
• •
• • • •
•
Br2 CA, cis-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid; Cl2 CA, cis-/trans-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid; 4F3PBA, 4fluoro-3-phenoxybenzoic acid; CDCA, chrysanthemumdicarboxylic acid. a Measured in this study. b Urinary metabolites of bifenthrin (2-methyl-3-phenylbenzoic acid or cis-2,2-dimethyl-3-(2,2-chloro-3,3,3-trifluoro-1-propenyl)cyclopropanecarboxylic acid) were not measured yet in non-exposed subjects.
exposure (Leng et al., 1997). Currently, for biomonitoring purposes only urine is used as a biological material for the study. There is no relevant data on the use of other, alternative matrices to assess recent or past exposure due to the rapid biotransformation. Urinary concentrations of metabolites integrates all routes of exposure and also exposure to both parent compounds and its degradation products/metabolites which after absorption are also excreted as conjugates in urine. The method for biomarker of long term exposure is still sought, however some attempts have been performed to use albumin adducts for this purpose (Noort et al., 2007). Pyrethroids act primarily on voltage-sensitive sodium channels in the nervous system (Soderlund, 2012). This mechanism of action is similar in insects and mammals but due to the effective and fast metabolism, mammals remain almost resistant to acute effects. Recent studies confirm the hypothesis that mixtures of pyrethroids act in a dose-additive model (Cao et al., 2011; Wolansky et al., 2009). Current studies also suggest other, additional modes of action on living organisms including endocrine disruption observed in vitro and in vivo, developmental neurotoxicity and reproductive toxicity (Jin et al., 2012; Meeker et al., 2009; Orton et al., 2011). Exposure to pyrethroids was assessed in the general population in a rather small number of countries, like: Germany (Heudorf and Angerer, 2001; Leng and Gries, 2005; Schettgen et al., 2002a), Italy (Saieva et al., 2004), Canada (Fortin et al., 2008a), France (Le Grand et al., 2012) and USA (Barr et al., 2010). Up to date there are no published results of pyrethroid exposure in Eastern Europe countries. The aim of this study was to assess exposure to synthetic pyrethroids in the convenience sample of a population living in Northern Poland by measuring specific urinary metabolite concentrations in the first void urine samples. Materials and methods
and 2,2-dibromovinyl-2,2-dimethylcyclopropanecarboxylic acid (Br2 CA) were obtained from Russel-Uclaf (France). cis-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cisCl2 CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1carboxylic acid (trans-Cl2 CA) were obtained from Dr. Ehrenstorfer Laboratories, Germany. All other reagents were of analytical grade. Standard stock solutions of 1 mg/mL were prepared in acetonitrile and stored at −20 ◦ C, protected from light. Working standard solutions were prepared in acetonitrile and were stored at +4 ◦ C. Stock and working standard solutions were replaced every 6 and 3 months respectively. Subjects and sample collection Urine samples were obtained from 132 subjects with age median 35 years (range 5–77 years, including 8 children under 14 years), 69 women (median age 34 years) and 63 men (median age ´ 37 years) living in the Gdansk region, Northern Poland, recruited in 2010 and 2011. Only basic sociodemographic data was collected using a questionnaire. All study participants lived in an urban area and none of the subjects were occupationally exposed to pesticides. All the procedures were approved by the Ethics Committee of the ´ Poland. Prior to enrolment in the Medical University of Gdansk, study, each subject signed the informed consent form. First morning void samples were collected in polypropylene containers and then were frozen and stored until analysis at −20 ◦ C. Chemical analysis The concentrations of cis-,trans-Cl2 CA, Br2 CA and 3-PBA were measured by gas chromatography–mass spectrometry (GC–MS) using electro-ionization operated in selected ion storage detection mode. Extraction step was adopted from previously established method (Schettgen et al., 2002b) described below.
Chemicals Sample preparation The following chemicals were obtained from Sigma–Aldrich (Germany): 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), diisopropylocarbodiimide (DIIC) and 2-phenoxybenzoic acid (2-PBA), which was used as an internal standard (IS). 3-Phenoxybenzoic acid
Briefly, 3 mL of thawed urine were transferred into a 10 mL screw-top glass tube and 25 L of IS solution (2-PBA, 1 g/mL of acetonitrile) along with 0.6 mL concentrated hydrochloric acid
B. Wielgomas et al. / International Journal of Hygiene and Environmental Health 216 (2013) 295–300 3,0 2,5 2,0 1,5
Normal value
were added. Samples were incubated at 95 ◦ C in the oven for 90 min. After bringing to room temperature, 4 mL of hexane were added and the tubes were shaken for 15 min. Following centrifugation, the hexane layer was collected to the next screw-top glass tube, and extraction with the same volume of hexane was repeated. Extracts were combined and then reextracted with 0.5 mL of 0.1 M NaOH. Hexane was discarded, while 0.1 mL of concentrated HCl and 2 mL of hexane were added to the remaining aqueous phase. Samples were again shaken, centrifuged and the resulting supernatant was evaporated to dryness under the stream of nitrogen at 45 ◦ C. The residue was treated with 10 L of HFIP, 15 L of DIIC and 250 L of hexane. Samples were mixed for 10 min at room temperature and then 1 mL of 5% K2 CO3 added. After vigorous shaking, the tubes were centrifuged and the hexane layer was transferred to an autosampler vial. Two microliters of the final extract were analyzed by GC–MS.
297
1,0 0,5 0,0 -0,5 -1,0 -1,5 -2,0 -0,5
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
3-PBA concentration [ng/ml]
Gas chromatography–mass spectrometry
Fig. 1. Distribution of the 3-PBA concentrations in the studied samples.
Analyses were performed using Varian GC-450 gas chromatograph equipped with 220-MS ion-trap mass spectrometer working in the selected ion storage (SIS) mode. Derivatives were separated on VF-5ms (Varian, Palo Alto, USA) low bleed column (30m × 0.25 mm I.D., 0.25 m film thickness) using the following oven program: 60 ◦ C to 1 min, 60–150 ◦ C (8 ◦ C/min), 150–280 ◦ C (30 ◦ C/min), 280 ◦ C to 11 min. Selected ions were monitored: 323 m/z (cis-Cl2 CA and trans-Cl2 CA), 369 m/z (Br2 CA) and 364 m/z (2-PBA and 3-PBA). Two microliters of the final extract were injected (injector temperature 280 ◦ C) in splitless mode with 25 psi pulse pressure lasting 1.0 min. The chromatograph was equipped with 1079 programmable temperature vaporizing injector and SGE focusliner glass inlet. Quality control Quality of the assay was monitored in two ways. At the beginning of the study large volume of the low concentration pool of urine was prepared from previously analyzed samples, exhibited undetectable contents of the metabolites. That pool was divided into four portions and three of them were spiked with standards (cis-Cl2 CA, trans-Cl2 CA, Br2 CA, 3-PBA) to achieve fortification levels: 1, 5 and 10 ng/mL, the fourth portion was left unfortified. Enriched samples were aliquoted into polypropylene tubes (volume of 3.2 mL) and stored along with samples (−20 ◦ C) for further quality control assessment. We also used a ClinCal® Urine Calibrator (Cat. No. 9969), lyophilized, for Toxic Organic Compounds acquired from Recipe Chemicals and Instruments GmbH (Munich, Germany). The calibrator with declared concentrations of pyrethroid metabolites served as a quality control material (3.6, 3.3, 2.5, 6.6 ng/mL respectively for cis-Cl2 CA, trans-Cl2 CA, Br2 CA, 3PBA). In each analytical batch we analyzed 30 samples including: 25 unknown, 4 spiked at different levels and one ClinCal® Urine Calibrator. A typical calibration curve was constructed on 8 points prepared based on matrix matched calibration. The analytical method applied in this work showed very good performance. Limits of detection for all metabolites were in the range 0.05–0.1 ng/mL with between day imprecision of 4.6–8.3%. Urinary creatinine was measured with spectrophotometric Jaffe method. Statistical analysis Data distribution was examined using Kolmogorov–Smirnov test. Concentrations in samples below limit of detection (LOD) were calculated according to formula: LOD value/square root of 2. Statistica ver. 9 (Statsoft, USA) software was used for
statistical evaluation of results, significance was considered when p-value <0.05. Results Concentrations of determined metabolites are presented in Table 2. The values are expressed in ng/mL (g/L) or adjusted to creatinine (g/g of creatinine). The calculation of mean and geometric mean was performed only for compounds with detectivity over 60% and values below LOD were expressed as LOD divided by the square root of two before mathematical operations. The most frequently detected compound was 3-PBA, which was present in concentrations higher than LOD in 106 of 132 analyzed samples, that represents 79.7% of all samples. The mean concentration was 0.393 and the geometric mean was 0.260. The highest concentration of 3-PBA was 3.03 ng/mL. The distribution of the 3-PBA concentrations fitted the log-normal model (Fig. 1). The remaining metabolites: cis-Cl2 CA, trans-Cl2 CA and Br2 CA were detected in 11 (8%), 9 (7%) and 15 (11%) of 132 samples. Maximum concentrations of cis-Cl2 CA, trans-Cl2 CA and Br2 CA were: 0.256, 0.409 and 3.336 ng/mL respectively. Discussion In this work 132 urine samples obtained from people living ´ in the Gdansk region, located in the Northern part of Poland were tested, and to our knowledge this is the first biomonitoring study for those metabolites in Poland. Among 13 synthetic pyrethroids authorized in Poland, 9 of them are metabolized to compounds determined in this work. Other metabolites: 4-fluoro3-phenoxybenzoic acid and chrysanthemumdicarboxylic acids are less frequently detected in non-exposed population (Leng and Gries, 2005). The liquid–liquid extraction technique was used for isolation of acidic metabolites of pyrethroids from urine hydrolysates according to Schettgen et al. (2002b). Nevertheless, the SPE protocols (using Supelco HLB cartridges) and liquid-liquid extraction with tert-butyl-methyl-ether (Leng and Gries, 2005) were also tested, but the final extracts were much cleaner when Schettgen’s method was applied. The same observation was noticed by Le Grand et al. (2012) using HPLC–MS/MS as an analytical technique. The back extraction procedure seems to be very effective in removing matrix thus the risk of ion-trap contamination is reduced and downtime of the GC–MS system is minimized. Thorough validation procedure applied in this study (reference material, in house prepared quality
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Table 2 Mean ± standard deviation (SD), geometric mean (GM) and percentiles of urinary metabolites (g/L) and creatinine adjusted (g/g creatinine) among studied population. Metabolite
% > LOD (n > LOD) n = 132
Concentration Mean ± SD
GM
Min
Not adjusted (g/L) 3-PBA 80 (106) 8 (11) cis-Cl2 CA 7 (9) trans-Cl2 CA 11 (15) Br2 CA
0.393 ± 0.427 – – –
0.260 – – –
Creatinine adjusted (g/g creatinine) 3-PBA 80 (106) cis-Cl2 CA 8 (11) 7 (9) trans-Cl2 CA 11 (15) Br2 CA
0.327 ± 0.310 – – –
0.220 – – –
control samples) proved this method as very reproducible, robust and reliable. With detection limits for all metabolites at 0.1 ng/mL it is possible to detect and quantify urinary biomarkers in the population exposed to the environmental levels of pyrethroids. Only in a very few papers, did authors declared detection limits lower than for the method that was used in the current work (Arrebola et al., 1999; Barr et al., 2007; Le Grand et al., 2012). Urine as a matrix for biomonitoring purposes posses many advantages over blood or serum, like higher metabolite concentration and ease of collection especially in the case of non-persistent chemicals which are easily transformed and eliminated from blood (Leng et al., 1997). Urinary concentration of pyrethroid metabolites served in many studies as a biomarker of recent exposure, however the amount of metabolites present in urine aggregates the total exposure to both the parent compounds and also their own metabolites and degradation products adsorbed through all possible routes. It was demonstrated in the case of organophosphate insecticides that their degradation products and metabolites – dialkylphosphates, present in food account for the majority of the dose excreted with urine (Zhang et al., 2008). A similar scenario is also possible also for synthetic pyrethroids, but they are more resistant to spontaneous hydrolysis than organophosphates. However up to date none of the studies dealt with that problem in the case of synthetic pyrethroids. Biomonitoring, does not allow for the differentiation of the route of exposure, with one exception when the fate of the chemical is different when it enters the body through the dermal or oral route. One should be very cautious when drawing conclusions about sources of exposure and chemical identity of the compound to which humans are exposed. In biomonitoring studies of other populations, mainly spot urine samples were collected in contrast to our study where all volunteers collected first void urine samples. Since morning urine usually contains more creatinine than random samples throughout the day, recalculation of volumetric concentration expressed in ng/mL to concentration per gram of creatinine decreased the value of mean concentration. For comparative purposes the adjustment to creatinine is sometimes questioned due to the significant variations of creatinine excretion rates in various isolated groups of the general population, which is age, sex and race dependent (Barr et al., 2005; Fortin et al., 2008b). Neither age nor sex were predictors of urinary 3-PBA in this study, no differences were observed in the mean concentration of 3-PBA for women and men and no relation to the age of donors (Figs. 2 and 3). Barr et al. (2010) reported significantly higher concentrations of 3-PBA only in the youngest group included in NHANES covering the range of 6–11 years old children. In the remaining population aged 12–59 years old no relationship between age and 3-PBA concentration was identified.
Max
Percentiles 5
25
50
75
90
95
3.030 0.256 0.409 3.336
0.141
0.255
0.471
0.887
1.154 0.153 0.122 0.314
1.832 0.508 0.265 1.725
0.121
0.190
0.418
0.722 0.139 0.148 0.170
0.950 0.170 0.170 0.222
Fig. 2. Creatinine adjusted urinary concentrations of 3-PBA in population subgroups of different age. No significant difference.
In this study, for the first time performed in Poland, we have found wide exposure to pyrethroids (high detection rate of 3PBA) and the urinary level of main metabolite is similar to those observed in populations of other countries (Table 3). The geometric mean concentration of 3-PBA is lower than German reference value 2 g/L (Heudorf et al., 2006). However, for the on-going
Fig. 3. Creatinine adjusted concentrations of 3-PBA in females and males. No significant difference.
B. Wielgomas et al. / International Journal of Hygiene and Environmental Health 216 (2013) 295–300
299
Table 3 Urinary concentrations of 3-PBA measured in population based studies in different regions of the world. Country (study)
n
Age range
Sampling period
Population
3-PBA (g/g creatinine) (GM)
Reference
Germany, (GerES IV-pilot study) Japan
395 143 66 448 87 1998 3046 132
2–17 64a 49a 39–85 40–85 6–59 6–59 5–77
2001–2002 2005–2007
General, children Rural Suburban Rural, non-farmers Rural, farmers General population General population Convenience sample, urban, non-farmers
0.240 0.320 0.490 0.400 0.450 0.261 0.324 0.220
Becker et al. (2006) Kimata et al. (2009)
Japan USA (NHANES 1999–2002) Poland
a
2005 1999–2000 2001–2002 2010–2011
Ueyama et al. (2009) Barr et al. (2010) Present study
Mean value.
studies lowering of the detection limit is recommended by converting the method used in this work to the MS/MS technique. Because the metabolites of synthetic pyrethroids are detected in the vast majority of urine samples, it is important to estimate the health risks of exposure. Although those pesticides have a low acute toxicity to humans, actually more attention is paid to the effects of low doses especially on the reproductive system. Some animal studies shown pyrethroids as chemicals with potential to affect sperm quality (Elbetieha et al., 2001; Kumar et al., 2004; Mani et al., 2002). There are few studies demonstrating existence of the relationship between the concentrations of metabolites of synthetic pyrethroids in the urine of persons not exposed occupationally, and the lower semen quality. Meeker et al. (2008) found associations between concentration of urinary metabolites of pyrethroids and reduced semen quality as well as sperm DNA damage among 161 patients of infertility clinic. It is important to note that concentrations measured in Meeker’s study are in a range of those found in our study and the US general population in the third report of NHANES (CDC, 2005) and German population (Heudorf and Angerer, 2001). Recently, Toshima et al. (2012) observed lower sperm motility in subjects with higher urinary 3-PBA among 42 Japanese males. Han et al. (2008) in a study on 212 adult men observed that urinary 3-PBA levels were associated with increased luteinizing hormone and reduced estradiol levels. Surprisingly in comparison to other studies the detection rate of the specific deltamethrin metabolite – Br2 CA – is higher than for cisCl2 CA and trans-Cl2 CA in the studied population, and higher than other authors reported previously. Dietary sources appear to be the most important pathways of exposure in the general population, as intuitively suggested by American and German investigators (Barr et al., 2010; Schettgen et al., 2002a) however, more studies on dietary exposure should be performed to clarify those suppositions. This observation suggests a different pattern of exposure in Poland and encourages further cohort studies to be carried out to understand the sources and intensity of exposure in the general population of Poland. Acknowledgments This work was financed by grants ST-5 and MN-954 from ´ Medical University of Gdansk. The authors wish to thank Teresa Pawłowska and Hanna Lemanowicz for their excellent technical assistance. References Arrebola, F.J., Martinez-Vidal, J.L., Fernandez-Gutierrez, A., Akhtar, M.H., 1999. Monitoring of pyrethroid metabolites in human urine using solid-phase extraction followed by gas chromatography–tandem mass spectrometry. Anal. Chim. Acta 401, 45–54. Barr, D.B., Leng, G., Berger-Preiss, E., Hoppe, H.W., Weerasekera, G., Gries, W., Gerling, S., Perez, J., Smith, K., Needham, L.L., Angerer, J., 2007. Cross validation of multiple
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