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Human biomonitoring of polychlorinated biphenyls (PCBs) in plasma of former underground miners in Germany – A case-control study
International Journal of Hygiene and Environmental Health 221 (2018) 1007–1011
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Human biomonitoring of polychlorinated biphenyls (PCBs) in plasma of former underground miners in Germany – A case-control study
T
Thomas Schettgen∗, A. Alt, C. Schikowsky, A. Esser, T. Kraus Institute for Occupational, Social and Environmental Medicine, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, D-52074, Aachen, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Biological monitoring PCB Mining age distribution Exposure assessment
Polychlorinated biphenyls (PCBs) are very persistent organic pollutants of severe environmental concern due to their toxic properties. Former underground miners might have been exposed to this substance group due to the widespread use of PCBs in hydraulic oils from the late 1960s to the mid 1980s. We have conducted a blinded case-control study in order to evaluate the possibility of retrospective exposure assessment of PCBs using human biomonitoring in former underground miners decades after the last possible exposure. We have identified n = 34 male former underground miners and n = 136 age-matched male control persons from the database of patients of our occupational outpatient clinic aged between 47.9 and 83.7 years at the time of sampling (June 2006–June 2016). These archived plasma samples have been blinded and analysed for 21 different PCB-congeners using a validated and quality controlled procedure using GC/MS (LOQ: 0.01 μg/L). Highly significant differences between cases and age-matched controls were only found for the PCB-congeners PCB 74 and PCB 114. The median (95th percentile) levels of PCB 74 in cases and controls were 0.126 μg/L plasma (0.899 μg/L plasma) vs. 0.058 μg/L plasma (0.368 μg/L plasma) and the 95th percentile levels for PCB 114 were 0.039 μg/L plasma vs. 0.017 μg/L plasma. Linear regression models revealed that this difference in plasma levels was unequivocally attributed to the underground mining activity. Thus, retrospective exposure assessment for underground miners by use of human biomonitoring seems feasible and further studies with a particular focus on this special group of workers should be performed.
1. Introduction Polychlorinated biphenyls (PCBs) are technical mixtures of 209 possible theoretical congeners with varying chlorine content that have been extensively used in the past due to their excellent technical properties, e.g. flame resistance, thermal stability and low electrical conductivity (Frame et al., 1996). PCBs are highly persistent in the environment and especially the higher chlorinated congeners were shown to bioaccumulate in the food chain, leading to an age-dependent body burden in the general population, mainly in industrialised countries (Becker et al., 2002; Xue et al., 2014; Schettgen et al., 2015). This led to a ban of the production and use of PCBs (or PCB-containing products) in Germany in 1989 (in the late 1970s in the United States) and finally worldwide with the Stockholm Convention, which came into force in 2004 (Stockholm convention on persistent organic pollutants (POPs)). More recently, inadvertent formation and distribution of some PCB-congeners (e.g. PCB 11) has been reported in the production of paints and pigments (Vorkamp, 2016). PCBs are considered to be carcinogenic to humans (Group 1) by the
∗
International Agency for Research on Cancer (IARC) (Lauby-Secretan et al., 2013). The Deutsche Forschungsgemeinschaft (DFG) has classified them in category 4 of the carcinogenic substances (non-genotoxic carcinogen) (DFG, 2017). There are numerous reports on the toxicological effects of exposure to PCBs (IARC, 2013). One of the longest known effects of PCBs are skin alterations in persons with direct contact to PCB-containing oils (Rogan et al., 1988). However, there are strong hints that PCBs might also have immunotoxic, neurotoxic and endocrine disrupting properties (Grandjean et al., 2001; Seegal, 2000; Harper et al., 1995; Heilmann et al., 2010; Gaum et al., 2016). A possible link between the development of diabetes and internal exposure to PCBs is also currently under discussion (Lee et al., 2006; Esser et al., 2016) as well as a causal relationship between exposure to PCBs and non-hodgkin-lymphoma (Kramer et al., 2012). Furthermore, especially lower chlorinated PCBcongeners (and their metabolites) are discussed to have genotoxic and cancer initiating properties and are responsible for shortened telomere lengths in lymphocytes of exposed workers (Lehmann et al., 2007; Ludewig and Robertson, 2013; Ziegler et al., 2017).
Corresponding author. E-mail address: [email protected] (T. Schettgen).
https://doi.org/10.1016/j.ijheh.2018.06.006 Received 16 January 2018; Received in revised form 25 June 2018; Accepted 25 June 2018
1438-4639/ © 2018 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
International Journal of Hygiene and Environmental Health 221 (2018) 1007–1011
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Large amounts of PCBs had been used since the late 1960s in underground mining after a severe accident in a Belgian coal mine in 1956, where mineral oils caused a fire and death of 267 miners. They were mainly used in hydraulic fluids that were for example applied to run the large coal cutters. Due to the flame retarding properties of PCBs, their use was especially advantageous in the potential explosive underground atmosphere (Reichel, 1973). In the mid 1980s PCBs were replaced by other less persistent flame retardants until the final ban of PCB usage in 1989 in Germany. Many of these coal mines are now no longer operative in Germany; however, many machines and PCB-containing devices are still underground. In combination with the mining companies' plan to slowly reduce the water pumping in these abandoned mines (leading to a rise in groundwater level), the (unknown) load of PCBs in these mines and the potential release into rivers and groundwater is currently under intensive discussion in Germany. Underground miners might have been exposed to these hydraulic fluids not only in repair tasks, but also in regular operation, as they often report leakages in the hydraulic pipes or alignments with intensive skin contact to the oils at these damp and narrow working places in their anamnesis. Consequently, a considerable amount of underground miners might have been exposed to PCBs during their work in the time between the late 1960s and the mid 1980s, when PCBs had been gradually replaced by Ugilec®, a structurally similar, but far less persistent flame retardant. With respect to the toxic potency of PCBs and the long latency of potential diseases, a quantitative exposure assessment for these workers would be favourable. However, retrospective exposure assessment for these workers using human biomonitoring is connected with several difficulties: first, the PCB-mixture formerly used in these hydraulic fluids (Clophen A30 – Clophen A60; Aroclor 1016 – Aroclor 1254) is widely unknown (even for the mining company and producers of hydraulic fluids) and therefore, a specific congener pattern that could be used to retrospectively quantify the exposure to these mixtures is also unknown. Secondly, the time since last exposure is extremely long. At best, the last exposure occurred more than 30 years ago. Although the plasma half-life for some higher chlorinated PCBs has been reported to be in the range of 15–25 years, it is questionable whether the internal exposure of former underground miners might still be significantly above the continuously decreasing background exposure of the general population. Furthermore, it is not known whether miners have indeed been exposed to these higher chlorinated congeners. Thirdly, these former underground miners are now very old, aged between 60 and 85 years. There are hardly any data available on internal exposure to PCBs of a non-exposed comparison group, as most studies have only collected data from persons to the age of 65 years. In order to overcome these problems and to clarify the question whether it is feasible to retrospectively quantify the potential PCB-exposure of underground miners in Germany, we decided to conduct a blinded case control study using archived plasma samples from patients of our occupational outpatient clinic. To our knowledge, this is the first study that assesses the internal exposure to PCBs in former underground miners in Germany.
Table 1 Age distributions for the group of former underground miners and controls in our study.
n mean age median age std. deviation minimum age maximum age percentiles
25 50 75 95
Former underground miners
Controls
34 71.16 72.36 8.29 47.92 83.77 66.04 72.36 77.86 82.53
136 71.10 72.26 8.20 47.85 83.73 66.53 72.26 78.04 82.45
exposed to PCBs in the past. In addition to age, the date of blood sampling for cases and controls was also matched with a subordinated priority in order to account for the decreasing background exposure to PCBs and potential effects of long-term storage of the samples in this period of time. The age of the control persons ranged from 47.9 to 83.7 years (median: 72.3 years). The age distribution of both groups is summarised in Table 1. All of those persons have attended our outpatient clinic between June 2006 and June 2016. Plasma samples were collected at the visit, immediately aliquoted and stored at −80 °C in Eppendorf caps until analysis. All persons gave written consent about the donation of blood samples for scientific purposes. An approval of the ethics committee of the RWTH Aachen University (EK 206/09) is available for the collection of these blood samples. The laboratory staff including the corresponding author (TS) has been blinded with respect to the information on group membership until all analyses were finished. After that, results were decoded and evaluated by AE and CS.
2.2. Analysis of PCBs in plasma These plasma samples were not only analysed for the 6 indicator congeners (PCB 28, 52, 101, 138, 153 and 180) and the 12 dioxin-like congeners (PCB 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, 189), but also for three additional congeners (PCB 66, 74, 99) that were previously found in elevated concentrations in plasma of former capacitor workers (Seegal et al., 2011) as well as in persons with high indoor exposure to PCBs (Meyer et al., 2013). We used a slightly modified method approved by the Deutsche Forschungsgemeinschaft (DFG) described previously (Schulte et al., 1993; Schettgen et al., 2011). A 2 ml sample of the plasma was deproteinised using formic acid. The PCBs were then extracted with n-hexane (containing PCB 54 as internal standard for the non-dioxin-like congeners as well as 13C12labelled PCBs for all 12 dioxin-like congeners), cleaned up on a silica gel column and analysed by GC/EI-MS in Selected Ion Monitoring-Mode (SIM). We used a matrix-matched calibration curve prepared in bovine serum in the range of 0.04–3 μg/L. The limit of quantification (based on a signal-to-noise ratio of 6) was determined to be 0.01 μg/L plasma for all analytes investigated. For quality control purposes, bovine serum was spiked with all analytes at a concentration of 0.4 μg/L and included in every analytical series. The between-day imprecision in this period of time (August 2016–November 2016, n = 23) has been determined to be in the range of 3.2%–9.9% for all analytes. Furthermore, a reagent blank was included in every series. Due to an accurate cleaning of reagents and glassware, no PCBs could be detected in these reagent blanks. Accuracy of our results is checked by biannual successful participation in a round robin for the determination of the indicator-PCBs in plasma in the environmental concentration range organised in Germany (www.g-equas.de).
2. Materials and methods 2.1. Study groups From the database of patients in our occupational outpatient clinic, n = 34 male patients (= cases) were identified who reported to have formerly worked in underground mining during their anamnesis. The age of these persons at the visit of our clinic ranged between 47.9 and 83.8 years (median: 72.4 years). From the same database, two of the authors (AE and CS) have compiled a age-matched control group (n = 136, meaning a 1:4 match) from male patients of the same age who did not report to have worked in underground mining or as electrician where they could have been 1008
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industry). The full dataset of our study is available as Excel-File in the supplemental information to this manuscript.
2.3. Statistical analysis All examinations were conducted with SPSS.21 (IBM, 2013). Initial alpha level was set at 0.05. The normal-distribution of PCB-values was examined by using Komolgorov-Smirnov-Test, Shapiro-Wilk-Test and histograms. Even residuals of each PCB congener were examined by QQ-plots and histograms. In addition, all PCB-values were log-transformed to reach an approximate normal distribution of the residuals. This allowed using parametric analysis procedures. First, the logtransformed PCB values for each congener were used to perform an independent samples t-test with the group of former miners and controls. The t-test was selected because of its lower sensitivity and higher specificity than non-parametric tests. To avoid an accumulation of the alpha error, a Bonferoni-Holm correction was performed. In a second step, Spearman's rank correlation was used to determine whether there is an influence of age on the raw PCB-values. In order to quantify the influence of age due to bioaccumulation in comparison to a binary dummy variable for miner (dummy = 1) versus non-miner (dummy = 0) we used a linear regression model with the log-transformed PCB-values as independent variable. The figures were prepared using Microcal ™ Origin® Version 6.0.
4. Discussion Our investigation revealed highly significant differences between former underground miners and age-matched controls only for the PCBcongeners PCB 74 and PCB 114, even more than 30 years after the last possible exposure underground. This result was unexpected, as we initially expected stronger differences especially for the higher chlorinated congeners that were already described to have long plasma halflifes (e.g. 16.3 and 27.5 years for PCB 138 and 153, respectively) possibly allowing us to differentiate between former miners and controls (Yakushiji et al., 1984). However, a previous study by Seegal et al. already described halflives for different PCB-congeners in plasma of former capacitor workers by re-analysis of blood samples 28 years after last exposure. In this study, the serum half-live for PCB 74 has been described to be as long as 15.9 years, while 4.6 years for PCB 28, 13.7 years for PCB 105, 13.8 years for PCB 118 and 41 years for PCB 156 were calculated (Seegal et al., 2011). Thus, the fact that we only found significant differences for PCB 74 might be explained by the considerably long half-life of this (tetrachlorinated) congener. On the other hand, the differences between both groups for PCB 105, PCB 118 and PCB 156 or the higher chlorinated PCB 138, PCB 153 and PCB 180 (with described equal or even longer half-lifes) did not reach statistical significance in our study (see Table 3). This gives implications that the PCB exposure in underground mining might have been dominated by the lower chlorinated congeners that usually are less bioaccumulative. Our (scarce) information about the formerly used hydraulic fluids from statements of the manufacturers Mobil®, Bayer® and BP® is summarised in the supplemental information to this manuscript (Table S1). Available data on the congener composition of the different commercial PCB-mixtures are also given in the supplement to this manuscript (Schulz et al., 1989). Interestingly, PCB 74 was found in all of these mixtures, while PCB 114 was only described as a component of Clophen A 40 (see Supplement Tables S1 and S2). In summary, it seems that in Germany lower chlorinated PCB-mixtures (e.g. Clophen A30, Clophen A40 or Aroclor 1242) might have been used for hydraulic oils in underground mining. This is underlined by the fact that we found no significantly increased levels of higher chlorinated PCBs in plasma of the miners compared to the control group, which is different to our previous investigation on plasma levels of workers in the transformer recycling industry (Schettgen et al., 2012a). In addition, it has already been shown in in-vitro experiments that skin permeability of PCBs is dependent on the degree of chlorination with lower chlorinated PCBs showing far better permeation than the higher chlorinated congeners and thus contributing to a larger extent to the internal body burden in dermal exposure situations
3. Results The results for the different PCB congeners in plasma of former underground miners and control persons are given in Table 2 (for the non-dioxin-like congeners) and Table 3 (for the dioxin-like congeners). As the dioxin-like congeners PCB 77, 81, 123, 126 and 169 have not been detected in any sample, Table 3 only contains the results for the remaining dioxin-like congeners. In summary, we found highly significant differences between former underground miners and age-matched controls only for the PCB-congeners PCB 74 and PCB 114 while the differences for all other congeners failed to reach significance. Fig. 1 shows the boxplots of the plasma levels for both congeners as well as the sum of the indicator congeners in both collectives. As expected, the higher chlorinated congeners showed a strong correlation with age, which is also the case for the tetrachlorinated PCB 74 and 66 as well as the pentachlorinated congener PCB 99 at the nonparametric correlation analysis (see Supplemental information, Figure S1). The linear regression model for PCB 74 and PCB 114 showed higher and significant standardized regression coefficients for the group membership than for age. Details on this analysis are shown in Table 4. Fig. 2 shows the correlation between the plasma levels of PCB 74 and the sum of the indicator congeners for all persons investigated, clearly enabling the identification of formerly exposed miners. Consequently, we consider these two congeners as highly selective “marker” congeners able to distinguish former underground miners from persons of the general population (at least for the German underground mining
Table 2 Results for the non-dioxin-like PCBs in plasma of former underground miners and controls. p-values for differences between groups were calculated by independent samples t-test. PCB 28 (μg/L plasma)
PCB 52 (μg/L plasma)
PCB 101 (μg/L plasma)
PCB 138 (μg/L plasma)
PCB 153 (μg/L plasma)
PCB 180 (μg/L plasma)
∑ PCB (μg/ L plasma)
PCB 66 (μg/L plasma)
PCB 74 (μg/L plasma)
PCB 99 (μg/L plasma)
Former underground miners (n = 34)
% > LOQ Median 95th perc. Max. value
20.6 < 0.01 0.019 0.023
0 < 0.01 < 0.01 < 0.01
8.8 < 0.01 0.015 0.015
100 0.865 3.266 3.782
100 1.380 4.950 6.031
100 1.629 4.886 7.822
4.057 12.794 17.663
32.4 < 0.01 0.019 0.023
100 0.126 0.899 1.035
100 0.056 0.186 0.300
Controls (n = 136)
% > LOQ Median 95th perc. Max. value p-values
25 < 0.01 0.031 0.053 0.211
0 < 0.01 < 0.01 < 0.01 –
8.8 < 0.01 0.013 0.024 0.855
100 1.100 2.383 3.933 0.639
100 1.825 3.663 6.394 0.495
100 1.843 3.979 5.183 0.939
4.645 9.712 15.572 0.738
33.8 < 0.01 0.025 0.057 0.296
100 0.058 0.141 0.368 < 0.001
100 0.046 0.110 0.210 0.194
significance
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Table 3 Results for the dioxin-like PCBs in plasma of former underground miners and controls (note that PCBs 77, 81, 123, 126 and 169 were not detectable in both groups). p-values for differences between groups were calculated by independent samples t-test. PCB 118 (μg/L plasma)
PCB 114 (μg/L plasma)
PCB 105 (μg/L plasma)
PCB 167 (μg/L plasma)
PCB 156 (μg/L plasma)
PCB 157 (μg/L plasma)
PCB 189 (μg/L plasma)
Former underground miners (n = 34)
n > LOQ Median 95th perc. Max. value
100 0.110 0.511 0.567
50 < 0.01 0.039 0.078
73.5 0.018 0.071 0.088
97 0.037 0.157 0.188
100 0.162 0.537 0.729
94 0.027 0.090 0.091
100 0.028 0.095 0.165
Controls (n = 136)
n > LOQ Median 95th perc. Max. value p-values
100 0.099 0.246 0.799 0.656
15.4 < 0.01 0.017 0.036 < 0.001
66.9 0.015 0.036 0.116 0.447
97.8 0.049 0.112 0.213 0.355
100 0.186 0.349 0.526 0.279
94.9 0.032 0.058 0.078 0.906
93.4 0.034 0.067 0.094 0.822
significance
(Dennerlein et al., 2013). Hence, our results show that it is in principle possible to specifically differentiate between underground miners with former exposure to PCBs and unexposed persons using human biomonitoring, even when exposure has ceased for more than 30 years. In this way, a retrospective exposure assessment might be possible for those workers. Currently, there are only few studies available on the toxicity of PCB 74 and PCB 114, as mostly mixtures have been investigated in the past. Recently, Benson et al. found an association between plasma levels of PCB 74 and levels of thyroid hormones (TT3) in a community health survey (Benson et al., 2018). With respect to their long half-life and their obvious relevance for occupational exposures, a more detailed toxicological investigation of these congeners (and their metabolites) would be desirable. Concerning the results for the indicator congeners in plasma, our results are well in accordance with previous studies. The plasma levels for the lower chlorinated PCB 28, 52 and 101 in both collectives are not significantly different and confirm previous results, as they have hardly been detectable in those persons and only a few persons exceed the reference values (BAR, PCB 28: 0.02 μg/L plasma; PCB 52, PCB 101: < 0.01 μg/L plasma) previously derived for non-exposed people by the Deutsche Forschungsgemeinschaft (DFG, 2017). This can be explained by the comparable shorter half-lifes of these congeners. While reported half-lifes for PCB 28 were 4.6 years and 4.5 years (Seegal et al., 2011), the half-lifes of PCB 52 and PCB 101 were determined to be considerably shorter with values of 1.3 years and 2.8 years, respectively (Schettgen et al., 2012b). Consequently, the potential internal exposure of former underground miners to these lower chlorinated congeners is now no longer detectable after this time - in contrast to PCB 74. On the other hand, the detectable increment for PCB 74 in the miners now 30 years after last possible exposure means that the levels of other, more short-lived lower chlorinated congeners was considerably higher when the miners were still actively working underground. With respect to the higher chlorinated indicator congeners, the 95th percentile plasma levels of both study groups even exceed the previously freshly revised reference values of the human biomonitoring commission (age group 60–69 years), which is mainly attributed to the
Table 4 Linear regression model with log-transformed PCB values as depended variable and age (continuous) and group membership (case = 1, controls = 0) as predictors. PCB 74
Age Case v. control Adj. R2
PCB 114
B
SE(B)
β
p
B
SE(B)
β
p
0.019 0.930
0.007 0.135
0.188 0.462
0.006 < 0.001
0.014 0.492
0.005 0.093
0.209 0.370
0.003 < 0.001
0.240
0.171
B: regression coefficient. SE: standard error. β: standardized regression coefficient. p: threshold for significance = 0.05.
Fig. 2. Correlation between plasma levels of PCB 74 and the sum of the indicator congeners in both groups.
Fig. 1. Boxplots for the plasma levels of PCB 74 (A), PCB 114 (B) and ∑ PCBs (C) in former underground miners and controls. 1010
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higher age of the persons investigated in our study (German Human Biomonitoring Commission, 2016). Likewise, the results for the dioxinlike PCBs in this study are considerably higher than in the highest age group of our previous pilot study (Schettgen et al., 2011). However, it is remarkable that two persons (one in each group) even exceeded the biological limit value (BAT-value, ∑ (indicator)PCBs = 15 μg/L plasma) for PCBs previously derived for occupational exposures by the DFG (DFG, 2017). Our study has clear strengths and limitations. A major strength of this study is the good match of both study groups with regard to age (see Table 1) and examination date as well as the blinded study design that allowed us to unequivocally relate the differences in plasma levels of both groups for PCB 74 and PCB 114 to the underground mining activity of the participants. However, one of the major limitations in our study is the lack of specific information on the workplace of those former miners and the intensity and duration of possible exposure to PCB-containing oils, as the general workplace anamnesis has not been that detailed and we only had the information “has worked in underground mining”. Consequently, our results for the mining group are far from representative and the differences we found in this pilot study probably rather underestimate the real situation. Further studies with a special focus on former underground miners and specific anamnestic information are necessary.
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5. Conclusion In conclusion, we have performed the first pilot study to investigate the potential internal exposure of former underground miners in Germany to PCBs decades after the last potential exposure. Due to the unique study design, our results revealed a highly significant difference between underground miners and age-matched controls in the plasma levels of the congeners PCB 74 and PCB 114 that was unequivocally attributed to the underground mining activity. Thus, retrospective exposure assessment for underground miners by use of human biomonitoring (e.g. under consideration of pharmacokinetic models) seems possible and further studies with a particular focus on this special group of workers should be performed. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.ijheh.2018.06.006. References Becker, K., Kaus, S., Krause, C., Lepom, P., Schulz, C., Seiwert, M., Seifert, B., 2002. German Environmental Survey 1998 (GerES III): environmental pollutants in blood of the German population. Int. J. Hyg Environ. Health 205 (4), 197–308. Benson, K., Yang, E., Dutton, N., Sjödin, A., Rosenbaum, P.F., Pavuk, M., 2018. Polychlorinated biphenyls, indicators of thyroid function and thyroid autoantibodies in the Anniston Community Health Survey I (ACHS-I). Chemosphere 195, 156–165. Dennerlein, K., Kilo, S., Göen, T., Korinth, G., Zschiesche, W., Drexler, H., 2013. Penetration von polychlorierten Biphenylen (PCB) in und durch exzidierte Humanhaut. Dokumentationsband zur 53. wissenschaftlichen Jahrestagung der Deutschen Gesellschaft für Arbeits- und Umweltmedizin (DGAUM) in Bregenz, S. pp. 693–695 (in German). ISBN 978-3-9811784-8-7. DFG (Deutsche Forschungsgemeinschaft), 2017. List of MAK- and BAT-values 2017. Report No. 53. Wiley-VCH, Weinheim. Esser, A., Schettgen, T., Gube, M., Koch, A., Kraus, T., 2016. Association between polychlorinated biphenyls and diabetes mellitus in the German HELPcB cohort. Int. J. Hyg Environ. Health 219 (6), 557–565. Frame, G.M., Cochran, J.W., Boewadt, S.S., 1996. Complete PCB congener distributions for 17 Aroclor mixtures determined by 3 HRGC systems optimized for comprehensive, quantitative, congener-specific analysis. J. High Resolut. Chromatogr. 19, 657–668. Gaum, P.M., Lang, J., Esser, A., Schettgen, T., Neulen, J., Kraus, T., Gube, M., 2016. Exposure to polychlorinated biphenyls and the thyroid gland - examining and discussing possible longitudinal health effects in humans. Environ. Res. 148, 112–121. German Human Biomonitoring Commission, 2016. Aktualisierung der Referenzwerte für polychlorierte Biphenyle (PCB) im Blut. Bundesgesundhbl 59, 1020–1027 (in German). Grandjean, P., Weihe, P., Burse, V.W., Needham, L.L., Storr-Hansen, E., Heinzow, B.,
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Human biomonitoring of polychlorinated biphenyls (PCBs) in plasma of former underground miners in Germany – A case-control study
T
Thomas Schettgen∗, A. Alt, C. Schikowsky, A. Esser, T. Kraus Institute for Occupational, Social and Environmental Medicine, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, D-52074, Aachen, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Biological monitoring PCB Mining age distribution Exposure assessment
Polychlorinated biphenyls (PCBs) are very persistent organic pollutants of severe environmental concern due to their toxic properties. Former underground miners might have been exposed to this substance group due to the widespread use of PCBs in hydraulic oils from the late 1960s to the mid 1980s. We have conducted a blinded case-control study in order to evaluate the possibility of retrospective exposure assessment of PCBs using human biomonitoring in former underground miners decades after the last possible exposure. We have identified n = 34 male former underground miners and n = 136 age-matched male control persons from the database of patients of our occupational outpatient clinic aged between 47.9 and 83.7 years at the time of sampling (June 2006–June 2016). These archived plasma samples have been blinded and analysed for 21 different PCB-congeners using a validated and quality controlled procedure using GC/MS (LOQ: 0.01 μg/L). Highly significant differences between cases and age-matched controls were only found for the PCB-congeners PCB 74 and PCB 114. The median (95th percentile) levels of PCB 74 in cases and controls were 0.126 μg/L plasma (0.899 μg/L plasma) vs. 0.058 μg/L plasma (0.368 μg/L plasma) and the 95th percentile levels for PCB 114 were 0.039 μg/L plasma vs. 0.017 μg/L plasma. Linear regression models revealed that this difference in plasma levels was unequivocally attributed to the underground mining activity. Thus, retrospective exposure assessment for underground miners by use of human biomonitoring seems feasible and further studies with a particular focus on this special group of workers should be performed.
1. Introduction Polychlorinated biphenyls (PCBs) are technical mixtures of 209 possible theoretical congeners with varying chlorine content that have been extensively used in the past due to their excellent technical properties, e.g. flame resistance, thermal stability and low electrical conductivity (Frame et al., 1996). PCBs are highly persistent in the environment and especially the higher chlorinated congeners were shown to bioaccumulate in the food chain, leading to an age-dependent body burden in the general population, mainly in industrialised countries (Becker et al., 2002; Xue et al., 2014; Schettgen et al., 2015). This led to a ban of the production and use of PCBs (or PCB-containing products) in Germany in 1989 (in the late 1970s in the United States) and finally worldwide with the Stockholm Convention, which came into force in 2004 (Stockholm convention on persistent organic pollutants (POPs)). More recently, inadvertent formation and distribution of some PCB-congeners (e.g. PCB 11) has been reported in the production of paints and pigments (Vorkamp, 2016). PCBs are considered to be carcinogenic to humans (Group 1) by the
∗
International Agency for Research on Cancer (IARC) (Lauby-Secretan et al., 2013). The Deutsche Forschungsgemeinschaft (DFG) has classified them in category 4 of the carcinogenic substances (non-genotoxic carcinogen) (DFG, 2017). There are numerous reports on the toxicological effects of exposure to PCBs (IARC, 2013). One of the longest known effects of PCBs are skin alterations in persons with direct contact to PCB-containing oils (Rogan et al., 1988). However, there are strong hints that PCBs might also have immunotoxic, neurotoxic and endocrine disrupting properties (Grandjean et al., 2001; Seegal, 2000; Harper et al., 1995; Heilmann et al., 2010; Gaum et al., 2016). A possible link between the development of diabetes and internal exposure to PCBs is also currently under discussion (Lee et al., 2006; Esser et al., 2016) as well as a causal relationship between exposure to PCBs and non-hodgkin-lymphoma (Kramer et al., 2012). Furthermore, especially lower chlorinated PCBcongeners (and their metabolites) are discussed to have genotoxic and cancer initiating properties and are responsible for shortened telomere lengths in lymphocytes of exposed workers (Lehmann et al., 2007; Ludewig and Robertson, 2013; Ziegler et al., 2017).
Corresponding author. E-mail address: [email protected] (T. Schettgen).
https://doi.org/10.1016/j.ijheh.2018.06.006 Received 16 January 2018; Received in revised form 25 June 2018; Accepted 25 June 2018
1438-4639/ © 2018 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Large amounts of PCBs had been used since the late 1960s in underground mining after a severe accident in a Belgian coal mine in 1956, where mineral oils caused a fire and death of 267 miners. They were mainly used in hydraulic fluids that were for example applied to run the large coal cutters. Due to the flame retarding properties of PCBs, their use was especially advantageous in the potential explosive underground atmosphere (Reichel, 1973). In the mid 1980s PCBs were replaced by other less persistent flame retardants until the final ban of PCB usage in 1989 in Germany. Many of these coal mines are now no longer operative in Germany; however, many machines and PCB-containing devices are still underground. In combination with the mining companies' plan to slowly reduce the water pumping in these abandoned mines (leading to a rise in groundwater level), the (unknown) load of PCBs in these mines and the potential release into rivers and groundwater is currently under intensive discussion in Germany. Underground miners might have been exposed to these hydraulic fluids not only in repair tasks, but also in regular operation, as they often report leakages in the hydraulic pipes or alignments with intensive skin contact to the oils at these damp and narrow working places in their anamnesis. Consequently, a considerable amount of underground miners might have been exposed to PCBs during their work in the time between the late 1960s and the mid 1980s, when PCBs had been gradually replaced by Ugilec®, a structurally similar, but far less persistent flame retardant. With respect to the toxic potency of PCBs and the long latency of potential diseases, a quantitative exposure assessment for these workers would be favourable. However, retrospective exposure assessment for these workers using human biomonitoring is connected with several difficulties: first, the PCB-mixture formerly used in these hydraulic fluids (Clophen A30 – Clophen A60; Aroclor 1016 – Aroclor 1254) is widely unknown (even for the mining company and producers of hydraulic fluids) and therefore, a specific congener pattern that could be used to retrospectively quantify the exposure to these mixtures is also unknown. Secondly, the time since last exposure is extremely long. At best, the last exposure occurred more than 30 years ago. Although the plasma half-life for some higher chlorinated PCBs has been reported to be in the range of 15–25 years, it is questionable whether the internal exposure of former underground miners might still be significantly above the continuously decreasing background exposure of the general population. Furthermore, it is not known whether miners have indeed been exposed to these higher chlorinated congeners. Thirdly, these former underground miners are now very old, aged between 60 and 85 years. There are hardly any data available on internal exposure to PCBs of a non-exposed comparison group, as most studies have only collected data from persons to the age of 65 years. In order to overcome these problems and to clarify the question whether it is feasible to retrospectively quantify the potential PCB-exposure of underground miners in Germany, we decided to conduct a blinded case control study using archived plasma samples from patients of our occupational outpatient clinic. To our knowledge, this is the first study that assesses the internal exposure to PCBs in former underground miners in Germany.
Table 1 Age distributions for the group of former underground miners and controls in our study.
n mean age median age std. deviation minimum age maximum age percentiles
25 50 75 95
Former underground miners
Controls
34 71.16 72.36 8.29 47.92 83.77 66.04 72.36 77.86 82.53
136 71.10 72.26 8.20 47.85 83.73 66.53 72.26 78.04 82.45
exposed to PCBs in the past. In addition to age, the date of blood sampling for cases and controls was also matched with a subordinated priority in order to account for the decreasing background exposure to PCBs and potential effects of long-term storage of the samples in this period of time. The age of the control persons ranged from 47.9 to 83.7 years (median: 72.3 years). The age distribution of both groups is summarised in Table 1. All of those persons have attended our outpatient clinic between June 2006 and June 2016. Plasma samples were collected at the visit, immediately aliquoted and stored at −80 °C in Eppendorf caps until analysis. All persons gave written consent about the donation of blood samples for scientific purposes. An approval of the ethics committee of the RWTH Aachen University (EK 206/09) is available for the collection of these blood samples. The laboratory staff including the corresponding author (TS) has been blinded with respect to the information on group membership until all analyses were finished. After that, results were decoded and evaluated by AE and CS.
2.2. Analysis of PCBs in plasma These plasma samples were not only analysed for the 6 indicator congeners (PCB 28, 52, 101, 138, 153 and 180) and the 12 dioxin-like congeners (PCB 77, 81, 105, 114, 118, 123, 126, 156, 157, 167, 169, 189), but also for three additional congeners (PCB 66, 74, 99) that were previously found in elevated concentrations in plasma of former capacitor workers (Seegal et al., 2011) as well as in persons with high indoor exposure to PCBs (Meyer et al., 2013). We used a slightly modified method approved by the Deutsche Forschungsgemeinschaft (DFG) described previously (Schulte et al., 1993; Schettgen et al., 2011). A 2 ml sample of the plasma was deproteinised using formic acid. The PCBs were then extracted with n-hexane (containing PCB 54 as internal standard for the non-dioxin-like congeners as well as 13C12labelled PCBs for all 12 dioxin-like congeners), cleaned up on a silica gel column and analysed by GC/EI-MS in Selected Ion Monitoring-Mode (SIM). We used a matrix-matched calibration curve prepared in bovine serum in the range of 0.04–3 μg/L. The limit of quantification (based on a signal-to-noise ratio of 6) was determined to be 0.01 μg/L plasma for all analytes investigated. For quality control purposes, bovine serum was spiked with all analytes at a concentration of 0.4 μg/L and included in every analytical series. The between-day imprecision in this period of time (August 2016–November 2016, n = 23) has been determined to be in the range of 3.2%–9.9% for all analytes. Furthermore, a reagent blank was included in every series. Due to an accurate cleaning of reagents and glassware, no PCBs could be detected in these reagent blanks. Accuracy of our results is checked by biannual successful participation in a round robin for the determination of the indicator-PCBs in plasma in the environmental concentration range organised in Germany (www.g-equas.de).
2. Materials and methods 2.1. Study groups From the database of patients in our occupational outpatient clinic, n = 34 male patients (= cases) were identified who reported to have formerly worked in underground mining during their anamnesis. The age of these persons at the visit of our clinic ranged between 47.9 and 83.8 years (median: 72.4 years). From the same database, two of the authors (AE and CS) have compiled a age-matched control group (n = 136, meaning a 1:4 match) from male patients of the same age who did not report to have worked in underground mining or as electrician where they could have been 1008
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industry). The full dataset of our study is available as Excel-File in the supplemental information to this manuscript.
2.3. Statistical analysis All examinations were conducted with SPSS.21 (IBM, 2013). Initial alpha level was set at 0.05. The normal-distribution of PCB-values was examined by using Komolgorov-Smirnov-Test, Shapiro-Wilk-Test and histograms. Even residuals of each PCB congener were examined by QQ-plots and histograms. In addition, all PCB-values were log-transformed to reach an approximate normal distribution of the residuals. This allowed using parametric analysis procedures. First, the logtransformed PCB values for each congener were used to perform an independent samples t-test with the group of former miners and controls. The t-test was selected because of its lower sensitivity and higher specificity than non-parametric tests. To avoid an accumulation of the alpha error, a Bonferoni-Holm correction was performed. In a second step, Spearman's rank correlation was used to determine whether there is an influence of age on the raw PCB-values. In order to quantify the influence of age due to bioaccumulation in comparison to a binary dummy variable for miner (dummy = 1) versus non-miner (dummy = 0) we used a linear regression model with the log-transformed PCB-values as independent variable. The figures were prepared using Microcal ™ Origin® Version 6.0.
4. Discussion Our investigation revealed highly significant differences between former underground miners and age-matched controls only for the PCBcongeners PCB 74 and PCB 114, even more than 30 years after the last possible exposure underground. This result was unexpected, as we initially expected stronger differences especially for the higher chlorinated congeners that were already described to have long plasma halflifes (e.g. 16.3 and 27.5 years for PCB 138 and 153, respectively) possibly allowing us to differentiate between former miners and controls (Yakushiji et al., 1984). However, a previous study by Seegal et al. already described halflives for different PCB-congeners in plasma of former capacitor workers by re-analysis of blood samples 28 years after last exposure. In this study, the serum half-live for PCB 74 has been described to be as long as 15.9 years, while 4.6 years for PCB 28, 13.7 years for PCB 105, 13.8 years for PCB 118 and 41 years for PCB 156 were calculated (Seegal et al., 2011). Thus, the fact that we only found significant differences for PCB 74 might be explained by the considerably long half-life of this (tetrachlorinated) congener. On the other hand, the differences between both groups for PCB 105, PCB 118 and PCB 156 or the higher chlorinated PCB 138, PCB 153 and PCB 180 (with described equal or even longer half-lifes) did not reach statistical significance in our study (see Table 3). This gives implications that the PCB exposure in underground mining might have been dominated by the lower chlorinated congeners that usually are less bioaccumulative. Our (scarce) information about the formerly used hydraulic fluids from statements of the manufacturers Mobil®, Bayer® and BP® is summarised in the supplemental information to this manuscript (Table S1). Available data on the congener composition of the different commercial PCB-mixtures are also given in the supplement to this manuscript (Schulz et al., 1989). Interestingly, PCB 74 was found in all of these mixtures, while PCB 114 was only described as a component of Clophen A 40 (see Supplement Tables S1 and S2). In summary, it seems that in Germany lower chlorinated PCB-mixtures (e.g. Clophen A30, Clophen A40 or Aroclor 1242) might have been used for hydraulic oils in underground mining. This is underlined by the fact that we found no significantly increased levels of higher chlorinated PCBs in plasma of the miners compared to the control group, which is different to our previous investigation on plasma levels of workers in the transformer recycling industry (Schettgen et al., 2012a). In addition, it has already been shown in in-vitro experiments that skin permeability of PCBs is dependent on the degree of chlorination with lower chlorinated PCBs showing far better permeation than the higher chlorinated congeners and thus contributing to a larger extent to the internal body burden in dermal exposure situations
3. Results The results for the different PCB congeners in plasma of former underground miners and control persons are given in Table 2 (for the non-dioxin-like congeners) and Table 3 (for the dioxin-like congeners). As the dioxin-like congeners PCB 77, 81, 123, 126 and 169 have not been detected in any sample, Table 3 only contains the results for the remaining dioxin-like congeners. In summary, we found highly significant differences between former underground miners and age-matched controls only for the PCB-congeners PCB 74 and PCB 114 while the differences for all other congeners failed to reach significance. Fig. 1 shows the boxplots of the plasma levels for both congeners as well as the sum of the indicator congeners in both collectives. As expected, the higher chlorinated congeners showed a strong correlation with age, which is also the case for the tetrachlorinated PCB 74 and 66 as well as the pentachlorinated congener PCB 99 at the nonparametric correlation analysis (see Supplemental information, Figure S1). The linear regression model for PCB 74 and PCB 114 showed higher and significant standardized regression coefficients for the group membership than for age. Details on this analysis are shown in Table 4. Fig. 2 shows the correlation between the plasma levels of PCB 74 and the sum of the indicator congeners for all persons investigated, clearly enabling the identification of formerly exposed miners. Consequently, we consider these two congeners as highly selective “marker” congeners able to distinguish former underground miners from persons of the general population (at least for the German underground mining
Table 2 Results for the non-dioxin-like PCBs in plasma of former underground miners and controls. p-values for differences between groups were calculated by independent samples t-test. PCB 28 (μg/L plasma)
PCB 52 (μg/L plasma)
PCB 101 (μg/L plasma)
PCB 138 (μg/L plasma)
PCB 153 (μg/L plasma)
PCB 180 (μg/L plasma)
∑ PCB (μg/ L plasma)
PCB 66 (μg/L plasma)
PCB 74 (μg/L plasma)
PCB 99 (μg/L plasma)
Former underground miners (n = 34)
% > LOQ Median 95th perc. Max. value
20.6 < 0.01 0.019 0.023
0 < 0.01 < 0.01 < 0.01
8.8 < 0.01 0.015 0.015
100 0.865 3.266 3.782
100 1.380 4.950 6.031
100 1.629 4.886 7.822
4.057 12.794 17.663
32.4 < 0.01 0.019 0.023
100 0.126 0.899 1.035
100 0.056 0.186 0.300
Controls (n = 136)
% > LOQ Median 95th perc. Max. value p-values
25 < 0.01 0.031 0.053 0.211
0 < 0.01 < 0.01 < 0.01 –
8.8 < 0.01 0.013 0.024 0.855
100 1.100 2.383 3.933 0.639
100 1.825 3.663 6.394 0.495
100 1.843 3.979 5.183 0.939
4.645 9.712 15.572 0.738
33.8 < 0.01 0.025 0.057 0.296
100 0.058 0.141 0.368 < 0.001
100 0.046 0.110 0.210 0.194
significance
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Table 3 Results for the dioxin-like PCBs in plasma of former underground miners and controls (note that PCBs 77, 81, 123, 126 and 169 were not detectable in both groups). p-values for differences between groups were calculated by independent samples t-test. PCB 118 (μg/L plasma)
PCB 114 (μg/L plasma)
PCB 105 (μg/L plasma)
PCB 167 (μg/L plasma)
PCB 156 (μg/L plasma)
PCB 157 (μg/L plasma)
PCB 189 (μg/L plasma)
Former underground miners (n = 34)
n > LOQ Median 95th perc. Max. value
100 0.110 0.511 0.567
50 < 0.01 0.039 0.078
73.5 0.018 0.071 0.088
97 0.037 0.157 0.188
100 0.162 0.537 0.729
94 0.027 0.090 0.091
100 0.028 0.095 0.165
Controls (n = 136)
n > LOQ Median 95th perc. Max. value p-values
100 0.099 0.246 0.799 0.656
15.4 < 0.01 0.017 0.036 < 0.001
66.9 0.015 0.036 0.116 0.447
97.8 0.049 0.112 0.213 0.355
100 0.186 0.349 0.526 0.279
94.9 0.032 0.058 0.078 0.906
93.4 0.034 0.067 0.094 0.822
significance
(Dennerlein et al., 2013). Hence, our results show that it is in principle possible to specifically differentiate between underground miners with former exposure to PCBs and unexposed persons using human biomonitoring, even when exposure has ceased for more than 30 years. In this way, a retrospective exposure assessment might be possible for those workers. Currently, there are only few studies available on the toxicity of PCB 74 and PCB 114, as mostly mixtures have been investigated in the past. Recently, Benson et al. found an association between plasma levels of PCB 74 and levels of thyroid hormones (TT3) in a community health survey (Benson et al., 2018). With respect to their long half-life and their obvious relevance for occupational exposures, a more detailed toxicological investigation of these congeners (and their metabolites) would be desirable. Concerning the results for the indicator congeners in plasma, our results are well in accordance with previous studies. The plasma levels for the lower chlorinated PCB 28, 52 and 101 in both collectives are not significantly different and confirm previous results, as they have hardly been detectable in those persons and only a few persons exceed the reference values (BAR, PCB 28: 0.02 μg/L plasma; PCB 52, PCB 101: < 0.01 μg/L plasma) previously derived for non-exposed people by the Deutsche Forschungsgemeinschaft (DFG, 2017). This can be explained by the comparable shorter half-lifes of these congeners. While reported half-lifes for PCB 28 were 4.6 years and 4.5 years (Seegal et al., 2011), the half-lifes of PCB 52 and PCB 101 were determined to be considerably shorter with values of 1.3 years and 2.8 years, respectively (Schettgen et al., 2012b). Consequently, the potential internal exposure of former underground miners to these lower chlorinated congeners is now no longer detectable after this time - in contrast to PCB 74. On the other hand, the detectable increment for PCB 74 in the miners now 30 years after last possible exposure means that the levels of other, more short-lived lower chlorinated congeners was considerably higher when the miners were still actively working underground. With respect to the higher chlorinated indicator congeners, the 95th percentile plasma levels of both study groups even exceed the previously freshly revised reference values of the human biomonitoring commission (age group 60–69 years), which is mainly attributed to the
Table 4 Linear regression model with log-transformed PCB values as depended variable and age (continuous) and group membership (case = 1, controls = 0) as predictors. PCB 74
Age Case v. control Adj. R2
PCB 114
B
SE(B)
β
p
B
SE(B)
β
p
0.019 0.930
0.007 0.135
0.188 0.462
0.006 < 0.001
0.014 0.492
0.005 0.093
0.209 0.370
0.003 < 0.001
0.240
0.171
B: regression coefficient. SE: standard error. β: standardized regression coefficient. p: threshold for significance = 0.05.
Fig. 2. Correlation between plasma levels of PCB 74 and the sum of the indicator congeners in both groups.
Fig. 1. Boxplots for the plasma levels of PCB 74 (A), PCB 114 (B) and ∑ PCBs (C) in former underground miners and controls. 1010
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higher age of the persons investigated in our study (German Human Biomonitoring Commission, 2016). Likewise, the results for the dioxinlike PCBs in this study are considerably higher than in the highest age group of our previous pilot study (Schettgen et al., 2011). However, it is remarkable that two persons (one in each group) even exceeded the biological limit value (BAT-value, ∑ (indicator)PCBs = 15 μg/L plasma) for PCBs previously derived for occupational exposures by the DFG (DFG, 2017). Our study has clear strengths and limitations. A major strength of this study is the good match of both study groups with regard to age (see Table 1) and examination date as well as the blinded study design that allowed us to unequivocally relate the differences in plasma levels of both groups for PCB 74 and PCB 114 to the underground mining activity of the participants. However, one of the major limitations in our study is the lack of specific information on the workplace of those former miners and the intensity and duration of possible exposure to PCB-containing oils, as the general workplace anamnesis has not been that detailed and we only had the information “has worked in underground mining”. Consequently, our results for the mining group are far from representative and the differences we found in this pilot study probably rather underestimate the real situation. Further studies with a special focus on former underground miners and specific anamnestic information are necessary.
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5. Conclusion In conclusion, we have performed the first pilot study to investigate the potential internal exposure of former underground miners in Germany to PCBs decades after the last potential exposure. Due to the unique study design, our results revealed a highly significant difference between underground miners and age-matched controls in the plasma levels of the congeners PCB 74 and PCB 114 that was unequivocally attributed to the underground mining activity. Thus, retrospective exposure assessment for underground miners by use of human biomonitoring (e.g. under consideration of pharmacokinetic models) seems possible and further studies with a particular focus on this special group of workers should be performed. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.ijheh.2018.06.006. References Becker, K., Kaus, S., Krause, C., Lepom, P., Schulz, C., Seiwert, M., Seifert, B., 2002. German Environmental Survey 1998 (GerES III): environmental pollutants in blood of the German population. Int. J. Hyg Environ. Health 205 (4), 197–308. Benson, K., Yang, E., Dutton, N., Sjödin, A., Rosenbaum, P.F., Pavuk, M., 2018. Polychlorinated biphenyls, indicators of thyroid function and thyroid autoantibodies in the Anniston Community Health Survey I (ACHS-I). Chemosphere 195, 156–165. Dennerlein, K., Kilo, S., Göen, T., Korinth, G., Zschiesche, W., Drexler, H., 2013. Penetration von polychlorierten Biphenylen (PCB) in und durch exzidierte Humanhaut. Dokumentationsband zur 53. wissenschaftlichen Jahrestagung der Deutschen Gesellschaft für Arbeits- und Umweltmedizin (DGAUM) in Bregenz, S. pp. 693–695 (in German). ISBN 978-3-9811784-8-7. DFG (Deutsche Forschungsgemeinschaft), 2017. List of MAK- and BAT-values 2017. Report No. 53. Wiley-VCH, Weinheim. Esser, A., Schettgen, T., Gube, M., Koch, A., Kraus, T., 2016. Association between polychlorinated biphenyls and diabetes mellitus in the German HELPcB cohort. Int. J. Hyg Environ. Health 219 (6), 557–565. Frame, G.M., Cochran, J.W., Boewadt, S.S., 1996. Complete PCB congener distributions for 17 Aroclor mixtures determined by 3 HRGC systems optimized for comprehensive, quantitative, congener-specific analysis. J. High Resolut. Chromatogr. 19, 657–668. Gaum, P.M., Lang, J., Esser, A., Schettgen, T., Neulen, J., Kraus, T., Gube, M., 2016. Exposure to polychlorinated biphenyls and the thyroid gland - examining and discussing possible longitudinal health effects in humans. Environ. Res. 148, 112–121. German Human Biomonitoring Commission, 2016. Aktualisierung der Referenzwerte für polychlorierte Biphenyle (PCB) im Blut. Bundesgesundhbl 59, 1020–1027 (in German). Grandjean, P., Weihe, P., Burse, V.W., Needham, L.L., Storr-Hansen, E., Heinzow, B.,
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