Perfluorinated Compounds in Fish and Blood of Anglers at Lake

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Perfluorinated Compounds in Fish and Blood of Anglers at €hne, Sauerland Area, Germany Lake Mo J€urgen H€olzer,*,† Thomas G€oen,‡ Paul Just,§ Rolf Reupert,|| Knut Rauchfuss,|| Martin Kraft,|| Johannes M€uller,‡ and Michael Wilhelm† †

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Department of Hygiene, Social and Environmental Medicine, MA 1/33, Ruhr-University Bochum, Universit€atsstrasse 150, 44801 Bochum, Germany ‡ Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University Erlangen-Nuremberg, Germany § Staatliches Veterin€aruntersuchungsamt, Arnsberg, Germany North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection, Recklinghausen, Germany

bS Supporting Information ABSTRACT: Perfluorinated compounds (PFCs) were measured in fish samples and blood plasma of anglers in a cross-sectional study at Lake M€ohne, Sauerland area, Germany. Human plasma and drinking water samples were analyzed by solid phase extraction, high-performance liquid chromatography (HPLC), and tandem mass spectrometry (MS/MS). PFCs in fish fillet were measured by ion pair extraction followed by HPLC and MS/MS. PFOS concentrations in 44 fish samples of Lake M€ohne ranged between 4.5 and 150 ng/g. The highest median PFOS concentrations have been observed in perches (median: 96 ng/g) and eels (77 ng/g), followed by pikes (37 ng/g), whitefish (34 ng/g), and roaches (6.1 ng/g). In contrast, in a food surveillance program only 11% of fishes at retail sale contained PFOS at detectable concentrations. One hundred five anglers (99 men, 6 women; 14
88 years old; median 50.6 years) participated in the human biomonitoring study. PFOS concentrations in blood plasma ranged from 1.1 to 650 μg/L (PFOA: 2.1
170 μg/L; PFHxS: 0.4
17 μg/L; LOD: 0.1 μg/L). A distinct dose-dependent relationship between fish consumption and internal exposure to PFOS was observed. PFOS concentrations in blood plasma of anglers consuming fish 2
3 times per month were 7 times higher compared to those without any fish consumption from Lake M€ohne. The study results strongly suggest that human internal exposure to PFC is distinctly increased by consumption of fish from PFC-contaminated sites.

’ INTRODUCTION Perfluorocompounds (PFCs) have been produced since the 1960s as surface active additives in commercial and consumer product applications. The world’s former largest manufacturer, 3M Company, phased out the production of perfluorooctane sulfonate (PFOS) by 2002, and the European Union has set out a ban on the use of PFOS in a number of goods in 2006. PFCs are still produced and released into the environment. For a global production, emission, and environmental inventory see ref 1. Numerous reports of detectable PFC concentrations in environmental media are indicating their worldwide distribution. PFCs have been observed in oceans,2
4 rivers,5
7 lakes,8,9 drinking water,7,10 and biota.11
14 Human biomonitoring studies demonstrate global human exposure with regional differences. PFOS concentrations from North American populations seem to be slightly higher than those from European, Asian, and Australian populations studied.15 The reduction of PFOS production is reflected by a decrease in the PFOS concentrations in the American general population, which has been reported from the National Health and Nutrition Examination Survey (NHANES 2003/4).16 r 2011 American Chemical Society

Dietary intake is supposed to be an important path of exposure for the general population to PFOS and perfluorooctanoate (PFOA).17,18 Contamination of drinking water led to significantly increased PFOA concentrations, in blood samples of the affected populations in Little Hocking, Ohio, USA,10 Arnsberg, Germany,19 and Minnesota, USA.20 Fish is an important part of the diet and recently the significance of fish consumption on the internal exposure to PFOS was emphasized.21,22

’ SITE-SPECIFIC BACKGROUND: LOCATION AND HISTORY OF PFC CONTAMINATION The river M€ohne is a 65-km-long tributary to the river Ruhr. Surface water is processed by 16 waterworks along these two rivers to produce drinking water by bank filtration and artificial Special Issue: Perfluoroalkyl Acid Received: December 31, 2010 Accepted: April 15, 2011 Revised: April 14, 2011 Published: May 02, 2011 8046

dx.doi.org/10.1021/es104391z | Environ. Sci. Technol. 2011, 45, 8046–8052

Environmental Science & Technology recharge for approximately 5 million residents in the Ruhr Region. In the early 20th century, a dam was built to form the Lake M€ohne, a water reservoir (134.5 million m3) to regulate the water flow of the river M€ohne and to supply a storage power plant. The lake has a water surface of about 10 km2. The lake and surrounding area are popular recreational resorts. Angling is one of the leisure activities supported by the water management company Ruhrverband and local communities. As reported earlier,23 surface waters along the river M€ohne including the Lake M€ohne became contaminated with PFCs mainly by application of polluted soil conditioner on agricultural lands, which drained into tributaries of the river M€ohne. Soil conditioner (53,000 tons) has been distributed on more than 1300 farm lands between 2000 and 2006. Today, environmental authorities estimate that the pollution of soil conditioner had begun in 2004. PFC concentrations (given as sum of PFOA and PFOS) in soil samples from these areas in Northrhine-Westfalia (N = 919) ranged from below LOQ (57%) to >100 μg/kgdw (10%) (dw = dry weight). In 33% of all samples, concentrations between LOQ and 100 μg/kgdw were measured.24 Maximum PFOA and PFOS concentrations were 2400 and 33 000 μg/kgdw, respectively. Subsequently, not only surface waters, but also drinking water became contaminated. Whereas soil samples contained more PFOS than PFOA, PFOA was the predominant compound observed in drinking water (2006: 500
640 ng PFOA/L). A human biomonitoring study was implemented, which revealed 4
8-fold increased PFOA blood concentrations in residents exposed to contaminated drinking water compared to a reference population.19 In the mentioned study multivariate analysis revealed that self-reported fish consumption from local water bodies was statistically associated with PFOS concentrations in blood plasma. It was the aim of this study to (i) measure the concentrations of PFOS and 9 other PFCs in fish fillet from Lake M€ ohne, (ii) quantify the internal exposure of a cohort of 105 anglers at the Lake M€ohne to PFOS and 5 other PFCs by assessing the two main routes of exposure (fish, drinking water), and (iii) assess the association between fish consumption and the PFOS body burden at Lake M€ohne in a prospective cross-sectional study.

’ MATERIALS AND METHODS Study Population and Sampling (Blood Plasma, Fish, Tap Water). Every holder of a fishing license for the Lake M€ ohne valid

for the years 2006 or 2007 (n = 1161) was informed in writing and invited to take part in the study. One hundred sixty-seven (14%) anglers responded to the invitation letter. Telephone interviews were conducted with all responders to allow stratification by fish consumption. One hundred five study participants were finally selected and classified into 6 groups according to their average fish consumption from Lake M€ohne between 2000 and 2008. Blood collection began on July 1 and ended on September 4, 2008. Standard materials for venipuncture and blood sampling were used. Blood samples were processed on the same day (centrifugation at 3300g for 10 min, separation of plasma). Plasma samples were stored at
20 °C and transported frozen to the analytical-toxicological laboratories in Erlangen, Germany. Tap water samples were collected from all residents’ homes at the kitchen. Sampling vessels (polypropylene, 50 mL, Greiner) were thoroughly rinsed with methanol prior to use. On the date of blood sampling tap water samples were taken by the study participants following a written instruction. Water samples were

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frozen, stored at
20 °C, and transported to the laboratories of the North Rhine-Westphalian State Agency for Nature, Environment and Consumer Protection in D€usseldorf, Germany, for further processing. One water sample per day and place of residence of the participants was analyzed. Both laboratories (blood analyses, water analyses) were blinded with respect to the origin of the samples and performed the analyses after the end of the field phase. Fishes from Lake M€ohne and river M€ohne were caught by electric fishing or net fishing. After sacrifice, head, skin, and viscera were removed and the fillet samples were stored at
20 °C. Fish samples for food monitoring were taken from retail trade, wholesale trade, supermarkets, and producers, frozen at
20 °C, and stored until analysis. Questionnaire. A questionnaire for self-completion was sent to the study participants to obtain information on characteristics such as body height, body weight, school education, smoking habits, exposure to environmental tobacco smoke, and environmental or occupational exposures to PFC. On the date of blood sampling trained interviewers administered a standardized questionnaire on fishing, fish consumption, drinking water consumption, and diet. To account for temporal changes in consumers’ behavior, current as well as past fish and drinking water consumptions were recorded. Analysis of Perfluorinated Compounds in Blood Plasma. Human plasma samples were analyzed for PFOA, PFOS, perfluorohexanoate (PFHxA), perfluorohexane sulfonate (PFHxS), perfluoropentanoate (PFPA), and perfluorobutane sulfonate (PFBS). After spiking of isotope-labeled internal standards compounds the analytes were extracted from interfering matrix compounds by solid phase extraction.25 After elution, the analytes were chromatographically separated by high-performance liquid chromatography (HPLC) and detected by a tandem mass spectrometer with electrospray ionization source (LC-ESI-MS/ MS). The detection of the analytes was performed by multiple reaction monitoring (MRM). External standards were used for calibration of the method. The results were calculated using the quotients of MRM results of the analyte and MRM results of the selected internal standard compound. Within-series imprecision was determined by analyzing a pooled human plasma sample spiked with PFHxS, PFBS, PFHxA, and PFPA (approximately 5 μg/L each). For PFOS and PFOA, the native concentrations were measured (approximately 9.5 and 5.5 μg/L, respectively). The coefficients of variation (CVs) were acquired to be between 4.4 and 10.9% at 10 repetitions. The between-day imprecision was determined by analyzing the same material 49 times on different days (2
3 times a day). The CVs thereby ranged from 6.9 to 12.7%. Depending on the analyte, mean relative recoveries were found to be between 101.9 and 122.0% at a spiked concentration of 10 μg/L using 5 different plasma samples. Limits of detection were estimated to be 0.1 μg/L for PFOS, PFHxS, PFBS, and PFOA and 1.0 μg/L for PFHxA and PFPA based on a 3-fold signal-to-noise-ratio. Quality control of the analytical results was performed by the analyses of quality control samples alongside the analyses of the study samples as well as by the successful participation in the proficiency tests of the German External Quality Assessment Scheme (G-EQUAS, Erlangen, Germany). More details are provided in the Supporting Information. Analysis of PFCs in Fish. Fish muscle (fillet) samples were analyzed for PFDoA (perfluordodecanoate), PFUnA (perfluorundecanoate), PFDA (perfluordecanoate), PFNA (perfluornonaoate), PFOA, PFOS, PFHpA (perfluorheptanoate), 8047

dx.doi.org/10.1021/es104391z |Environ. Sci. Technol. 2011, 45, 8046–8052

Environmental Science & Technology PFHxA, PFHxS, and PFBS. PFCs in muscle of fish were measured by ion pair extraction followed by HPLC/MS/MS.26,27 Quality control was performed according to DIN EN ISO/EC 17025.28 The limits of detection ranged between 0.1 (PFHxS), 0.2 (PFOS), and 2.5 (PFNA) ng/g and the recoveries ranged between 86 and 110%. Coefficients of variation (CVs) varied between 5.1 and 13.0% (10 samples) and between 2.4 and 9.2% (1 sample, 10 repeats).The method was successfully tested by an interlaboratory study on PFC in fish. More details are provided in the Supporting Information. Analysis of PFCs in Drinking Water. Drinking water samples were analyzed for PFBA (perfluorobutanoate), PFBS, PFPA, PFHxA, PFHxS, PFOA, PFOS, PFHpA, PFNA, and PFDA by solid phase extraction followed by HPLC, MS/MS according to German standard method DIN 38407-42.29 The limit of quantification (LOQ) was 10 ng/L (signal-to-noise >10:1, peak to peak mode). The recovery rate for the internal substances varied between 70% and 100%. The measurement uncertainty was determined by standard addition method. It was less than 15% for the carboxy acids and 20
25% for the sulfonic acids at a concentration level of 100 ng/L. More details are provided in the Supporting Information. Reverse Dosimetry. The PFOS intake from fish consumption of each participant was calculated from interview data (amount and species of fish caught and eaten, individual body weight) and the median PFOS concentrations of corresponding fish species in Lake M€ohne. Additionally, PFOS intake was estimated from biomonitoring data, based on the description of Harada et al.30 and the parametrization of Fromme et al.17 Daily intake (D [ng/kgbw]) was estimated as the product of plasma concentration in steady-state (C [μg/L]), volume of distribution (Vd [220 mL/kg], 31) and the first order rate constant for PFOS elimination (0.693/t1/2 [1661 days] 32): D¼

0:693  C  Vd t1=2

For a discussion of this approach, see Vestergren and Cousins.33 Data Description, Statistical Methods, and Approval of the Ethical Commission. For PFC concentrations below the limit of detection (LOD), an imputed value equal to 1/2 the LOD was used. Differences of log10(PFOA, PFOS, PFHXs) concentrations between groups of different fish consumption behavior were tested for statistical significance by analysis of variance, followed by pairwise multiple comparison tests (Gabriel’s procedure). Multivariate analyses were performed through linear regression models. Dependent variables were log10(PFOA, PFOS, PFHXs) concentrations in blood plasma [ng/L]. Regressors were chosen based on the study hypothesis: self-reported sum of fish from Lake M€ ohne eaten by each participant between 2004 and 2008 (fish consumption 2004
8 [Σ kg]), PFOS or PFOA concentration in tap water sample at home, and results of former studies (residence in Arnsberg, age). PFC concentrations have been rounded to two significant figures. All data were analyzed using the statistical software package SAS v. 9.2 (SAS Institute Inc., Cary, NC). The study was approved by the ethical commission of the Ruhr-University of Bochum and was conducted in accordance with the ethical principles for medical research involving human subjects as defined by the Helsinki Declaration. Written informed consent was obtained from each participant.

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Table 1. PFOS Concentration in Fish Species from Lake M€ ohne Compared to Those in Fish Sampled during Food Monitoring N