Jürgen Angerer

Assessing exposure to phthalates – The human biomonitoring approach

Some phthalates are developmental and reproductive toxicants in animals. Exposure to phthalates is considered to be potentially harmful to human health as well. Based on a comprehensive literature research, we present an overview of the sources of human phthalate exposure and results of exposure assessments with special focus on human biomonitoring data. Among the general population, there is widespread exposure to a number of phthalates. Foodstuff is the major source of phthalate exposure, particularly for the long-chain phthalates such as di(2-ethylhexyl) phthalate. For short-chain phthalates such as di-n-butyl-phthalate, additional pathways are of relevance. In general, children are exposed to higher phthalate doses than adults. Especially, high exposures can occur through some medications or medical devices. By comparing exposure data with existing limit values, one can also assess the risks associated with exposure to phthalates. Within the general population, some individuals exceed tolerable daily intake values for one or more phthalates. In high exposure groups, (intensive medical care, medications) tolerable daily intake transgressions can be substantial. Recent findings from animal studies suggest that a cumulative risk assessment for phthalates is warranted, and a cumulative exposure assessment to phthalates via human biomonitoring is a major step into this direction.

Phthalate exposure in pregnant women and their children in central Taiwan

Phthalate exposure was found to be associated with endocrine disruption, respiratory effects, reproductive and developmental toxicity. The intensive use of plastics may be increasing the exposure to phthalates in Taiwanese population, particularly for young children. We studied phthalate metabolites in pregnant women and their newborns in a prospective cohort from a medical center in Central Taiwan. One hundred maternal urine samples and 30 paired cord blood and milk samples were randomly selected from all of participants (430 pregnant women). Eleven phthalate metabolites (MEHP, 5OH-MEHP, 2cx-MEHP, 5cx-MEPP, 5oxo-MEHP, MiBP, MnBP, MBzP, OH-MiNP, oxo-MiNP, and cx-MiNP) representing the exposure to five commonly used phthalates (DEHP, di-isobutyl phthalate (DiBP), DnBP, BBP, DiNP) were measured in urine of pregnant women, cord serum and breast milk after delivery, and in urine of their children. Exposure was estimated with excretion factors and correlation among metabolites of the same parent compound. Thirty and 59 urinary samples from 2 and 5 years-old children were randomly selected from 185 children successfully followed. Total urinary phthalate metabolite concentration (geometric mean, μg L⁻¹) was found to be higher in 2-years-olds (398.6) and 5-years-olds (333.7) than pregnant women (205.2). Metabolites in urine are mainly from DEHP. The proportion of DiNP metabolites was higher in children urine (4.39 and 8.31%, ages 2 and 5) than in adults (0.83%) (p<0.01). Compared to urinary levels, phthalate metabolite levels are low in cord blood (37.45) and milk (14.90). DEHP metabolite levels in womens urine and their corresponding cord blood are significantly correlated. Compared to other populations in the world, DEHP derived metabolites in maternal urine were higher, while phthalate metabolite levels in milk and cord blood were similar. The level of phthalate metabolites in milk and cord blood were comparable to those found in other populations. Further studies of health effects related to DEHP and DiNP exposure are necessary for the children.

Levels of metabolites of organophosphate pesticides, phthalates, and bisphenol A in pooled urine specimens from pregnant women participating in the Norwegian Mother and Child Cohort Study (MoBa)

Concerns about reproductive and developmental health risks of exposure to organophosphate (OP) pesticides, phthalates, and bisphenol A (BPA) among the general population are increasing. Six dialkyl phosphate (DAP) metabolites, 3,5,6-trichloro-2-pyridinol (TCPy), BPA, and fourteen phthalate metabolites were measured in 10 pooled urine samples representing 110 pregnant women who participated in the Norwegian Mother and Child Birth Cohort (MoBa) study in 2004. Daily intakes were estimated from urinary data and compared with reference doses (RfDs) and daily tolerable intakes (TDIs). The MoBa women had a higher mean BPA concentration (4.50 microg/L) than the pregnant women in the Generation R Study (Generation R) in the Netherlands and the National Health and Nutrition Examination Survey (NHANES) in the United States. The mean concentration of total DAP metabolites (24.20 microg/L) in MoBa women was higher than that in NHANES women but lower than that in Generation R women. The diethyl phthalate metabolite mono-ethyl phthalate (MEP) was the dominant phthalate metabolite in all three studies, with the mean concentrations of greater than 300 microg/L. The MoBa and Generation R women had higher mean concentrations of mono-n-butyl phthalate (MnBP) and mono-isobutyl phthalate (MiBP) than the NHANES women. The estimated average daily intakes of BPA, chlorpyrifos/chlorpyrifos-methyl and phthalates in MoBa (and the other two studies) were below the RfDs and TDIs. The higher levels of metabolites in the MoBa participants may have been from intake via pesticide residues in food (organophosphates), consumption of canned food, especially fish/seafood (BPA), and use of personal care products (selected phthalates).

Exposure to phthalates in 5–6 years old primary school starters in Germany—A human biomonitoring study and a cumulative risk assessment

We determined the internal exposure of 111 German primary school starters by analyzing urinary metabolites of six phthalates: butyl benzyl phthalate (BBzP), di-iso-butyl phthalate (DiBP), di-n-butyl phthalate (DnBP), di (2-ethylhexyl) phthalate (DEHP), di-iso-nonyl phthalate (DiNP) and di-iso-decylphthalate (DiDP). From the urinary metabolite levels, we calculated daily intakes and related these values to Tolerable Daily Intake (TDI) values. By introducing the concept of a relative cumulative Tolerable Daily Intake (TDI(cum)) value, we tried to account for the cumulative exposure to several of the above-mentioned phthalates. The TDI(cum) was derived as follows: the daily intake (DI) calculated from the metabolite level was divided by the TDI for each phthalate; this ratio was multiplied by 100% indicating the TDI percentage for which the DI accounted. Finally the % TDIs of the different phthalates were totalled to get the TDI(cum). A TDI(cum) above 100% is a potential cause for concern. We confirmed the ubiquitous exposure of the children to all phthalates investigated. Exposures were within range of levels previously reported for GerES, albeit slightly lower. Regarding daily intakes, two children exceeded the TDI for DnBP, whereas one child closely approached the TDI for DEHP. 24% of the children exceeded the TDI(cum) for the three most critical phthalates: DEHP, DnBP and DiBP. Furthermore, 54% of the children had total exposures that used up more than 50% the TDI(cum). Therefore, the overall exposure to a number of phthalates, and the knowledge that these phthalates (and other anti-androgens) act in a dose-additive manner, urgently warrants a cumulative risk assessment approach.

Determination of human urinary organophosphate flame retardant metabolites by solid-phase extraction and gas chromatography–tandem mass spectrometry

Organophosphorus flame retardants (OPFR), phosphorus triesters, are widely used chemicals with a high share of the worldwide flame retardant market. In animal experiments, dialkyl- and diarylphosphates are the main metabolites of OPFR. Therefore we elaborated a GC-MS/MS-method for the detection of OPFR-metabolites in human urine after solid phase extraction and derivatization with pentafluorobenzylbromide. The limits of detection range from 0.1 to 1 microg/l. Interday imprecision were 2-8%. The applicability of the method is shown by determination of the internal burden of 30 persons of the German general population. OPFR-metabolite concentrations range from <LOD to 27.5 microg/l for bis-(2-chlorethyl)-phosphate and <LOD to 4.1 microg/l for diphenylphosphate. Di-m-cresylphosphate and di-p-cresylphosphate cannot be detected in any of the native urine samples.

3-Hydroxybenzo[a]pyrene in the urine of workers with occupational exposure to polycyclic aromatic hydrocarbons in different industries.

Objectives: This study was conducted to assess external and internal exposure of workers to polycyclic aromatic hydrocarbons (PAHs). In this context, the analytical and diagnostical reliability of 3-hydroxybenzo[a]pyrene (3OH-BaP) as a biomarker of internal exposure to PAHs was established. Methods: Ambient and biological monitoring was carried out of 225 PAH-exposed employees of different industries. External exposure was determined by personal air sampling and analysis of 16 EPA-PAH. Internal exposure was examined by the urinary metabolites 3OH-BaP, 1-hydroxypyrene (OH-Pyr) and monohydroxylated phenanthrenes (OH-Phens). Results: Benzo[a]pyrene (BaP) was detected at all workplaces. Concentrations in the breathing zone of the workers ranged from below the limit of detection up to 44.3 μg/m3. In biological monitoring, urinary 3OH-BaP was found in median concentrations of 0.8 ng/g creatinine (crea) and the 95th percentile of 6.7 ng/g crea. The results ranged from the limit of detection up to 19.5 ng/g crea. Only 1% of the analysed samples showed concentrations below the limit of detection (0.05 ng/l). Regarding median concentrations, workers in coking plants showed lower 3OH-BaP concentrations (0.5 ng/g crea) than those employed in the production of fireproof material in refractories (1.1 ng/g crea), converter infeed (1.2 ng/g crea) and graphite electrode production (1.3 ng/g crea). Strong correlations of 3OH-BaP with OH-Pyr and the sum of OH-Phens were found for the workplaces converter infeed, coking plants and graphite electrode production (rPearson ranging from 0.618 to 0.867, p<0.001). The poor correlation of BaP in the air and 3OH-BaP in urine is most probably caused by routes of uptake other than via air—for example, dermal uptake. Conclusion: 3OH-BaP as a metabolite of the carcinogenic BaP could be shown to be a diagnostically specific and sensitive biomarker for determining the internal exposure of workers in different industries. Using this method, the estimation of health risks for workers can be fundamentally improved, because the 3OH-BaP represents the group of carcinogenic PAHs. The procedure for analysing 3OH-BaP is complex, but it is robust and produces reliable results.

First steps toward harmonized human biomonitoring in Europe: demonstration project to perform human biomonitoring on a European scale.

Background For Europe as a whole, data on internal exposure to environmental chemicals do not yet exist. Characterization of the internal individual chemical environment is expected to enhance understanding of the environmental threats to health. Objectives We developed and applied a harmonized protocol to collect comparable human biomonitoring data all over Europe. Methods In 17 European countries, we measured mercury in hair and cotinine, phthalate metabolites, and cadmium in urine of 1,844 children (5–11 years of age) and their mothers. Specimens were collected over a 5-month period in 2011–2012. We obtained information on personal characteristics, environment, and lifestyle. We used the resulting database to compare concentrations of exposure biomarkers within Europe, to identify determinants of exposure, and to compare exposure biomarkers with health-based guidelines. Results Biomarker concentrations showed a wide variability in the European population. However, levels in children and mothers were highly correlated. Most biomarker concentrations were below the health-based guidance values. Conclusions We have taken the first steps to assess personal chemical exposures in Europe as a whole. Key success factors were the harmonized protocol development, intensive training and capacity building for field work, chemical analysis and communication, as well as stringent quality control programs for chemical and data analysis. Our project demonstrates the feasibility of a Europe-wide human biomonitoring framework to support the decision-making process of environmental measures to protect public health. Citation Den Hond E, Govarts E, Willems H, Smolders R, Casteleyn L, Kolossa-Gehring M, Schwedler G, Seiwert M, Fiddicke U, Castaño A, Esteban M, Angerer J, Koch HM, Schindler BK, Sepai O, Exley K, Bloemen L, Horvat M, Knudsen LE, Joas A, Joas R, Biot P, Aerts D, Koppen G, Katsonouri A, Hadjipanayis A, Krskova A, Maly M, Mørck TA, Rudnai P, Kozepesy S, Mulcahy M, Mannion R, Gutleb AC, Fischer ME, Ligocka D, Jakubowski M, Reis MF, Namorado S, Gurzau AE, Lupsa IR, Halzlova K, Jajcaj M, Mazej D, Snoj Tratnik J, López A, Lopez E, Berglund M, Larsson K, Lehmann A, Crettaz P, Schoeters G. 2015. First steps toward harmonized human biomonitoring in Europe: demonstration project to perform human biomonitoring on a European scale. Environ Health Perspect 123:255–263; http://dx.doi.org/10.1289/ehp.1408616

Fish consumption patterns and hair mercury levels in children and their mothers in 17 EU countries

The toxicity of methylmercury (MeHg) in humans is well established and the main source of exposure is via the consumption of large marine fish and mammals. Of particular concern are the potential neurodevelopmental effects of early life exposure to low-levels of MeHg. Therefore, it is important that pregnant women, children and women of childbearing age are, as far as possible, protected from MeHg exposure. Within the European project DEMOCOPHES, we have analyzed mercury (Hg) in hair in 1799 mother-child pairs from 17 European countries using a strictly harmonized protocol for mercury analysis. Parallel, harmonized questionnaires on dietary habits provided information on consumption patterns of fish and marine products. After hierarchical cluster analysis of consumption habits of the mother-child pairs, the DEMOCOPHES cohort can be classified into two branches of approximately similar size: one with high fish consumption (H) and another with low consumption (L). All countries have representatives in both branches, but Belgium, Denmark, Spain, Portugal and Sweden have twice as many or more mother-child pairs in H than in L. For Switzerland, Czech Republic, Hungary, Poland, Romania, Slovenia and Slovakia the situation is the opposite, with more representatives in L than H. There is a strong correlation (r=0.72) in hair mercury concentration between the mother and child in the same family, which indicates that they have a similar exposure situation. The clustering of mother-child pairs on basis of their fish consumption revealed some interesting patterns. One is that for the same sea fish consumption, other food items of marine origin, like seafood products or shellfish, contribute significantly to the mercury levels in hair. We conclude that additional studies are needed to assess and quantify exposure to mercury from seafood products, in particular. The cluster analysis also showed that 95% of mothers who consume once per week fish only, and no other marine products, have mercury levels 0.55 μg/g. Thus, the 95th percentile of the distribution in this group is only around half the US-EPA recommended threshold of 1 μg/g mercury in hair. Consumption of freshwater fish played a minor role in contributing to mercury exposure in the studied cohort. The DEMOCOPHES data shows that there are significant differences in MeHg exposure across the EU and that exposure is highly correlated with consumption of fish and marine products. Fish and marine products are key components of a healthy human diet and are important both traditionally and culturally in many parts of Europe. Therefore, the communication of the potential risks of mercury exposure needs to be carefully balanced to take into account traditional and cultural values as well as the potential health benefits from fish consumption. European harmonized human biomonitoring programs provide an additional dimension to national HMB programs and can assist national authorities to tailor mitigation and adaptation strategies (dietary advice, risk communication, etc.) to their countrys specific requirements.

A systematic approach for designing a HBM Pilot Study for Europe

The objective of COPHES (Consortium to Perform Human biomonitoring on a European Scale) was to develop a harmonised approach to conduct human biomonitoring on a European scale. COPHES developed a systematic approach for designing and conducting a pilot study for an EU-wide cross-sectional human biomonitoring (HBM) study and for the implementation of the fieldwork procedures. The approach gave the basis for discussion of the main aspects of study design and conduct, and provided a decision making tool which can be applied to many other studies. Each decision that had to be taken was listed in a table of options with their advantages and disadvantages. Based on this the rationale of the decisions could be explained and be transparent. This was important because an EU-wide HBM study demands openness of all decisions taken to encourage as many countries as possible to participate and accept the initiative undertaken. Based on this approach the following study design was suggested: a cross-sectional study including 120 children aged 6-11 years and their mothers aged up to 45 years from each participating country. For the pilot study the children should be sampled in equal shares in an urban and a rural location. Only healthy children and mothers (no metabolic disturbances) should be included, who have a sufficient knowledge of the local language and have been living at least for 5 years at the sampling location. Occupational exposure should not be an exclusion criterion. Recruitment should be performed via inhabitant registries or schools as an alternative option. Measures suitable to increase the response rate should be applied. Preferably, the families should be visited at home and interviewed face-to-face. Various quality control measures to guarantee a good fieldwork performance were recommended. This comprehensive overview aims to provide scientists, EU officials, partners and stakeholders involved in the EU implementation process full transparency of the work carried out in COPHES. Thus this report presents the discussion and consensus in COPHES on the main aspects of designing and conducting fieldwork of a human biomonitoring study. Furthermore, it provides an example for a systematic approach that may be useful to other research groups or pan-European research initiatives. In the study protocol that will be published elsewhere these aspects are elaborated and additional aspects are covered (Casteleyn et al., 2012). Meanwhile the respective pilot study DEMOCOPHES had been conducted and assessed. The results and lessons learned will be published elsewhere.

Urinary di(2-ethylhexyl)phthalate (DEHP)--metabolites and male human markers of reproductive function.

INTRODUCTION Phthalates are suspected to act as endocrine modulators in humans and exert reproductive toxicity. The general population is exposed to phthalates through nutrition, consumer products, medications and medical devices. The aim of the present study is to explore whether internal phthalate exposure represented by metabolites of di(2-ethylhexyl) phthalate (DEHP) can be related to human markers of reproductive function (i.e. semen concentration, motility and morphology). METHODS We recruited 349 men who were part of subfertile couples and were referred for fertility work-up between April 2004 and November 2005. Semen analysis was performed according to recommendations of the World Health Organization (WHO). Parameters were dichotomized based on 1999 WHO reference values for sperm concentration (<20million/ml) and motility (<50% sperm with progressive motility), as well as Tygerberg strict criteria for morphology (<4% normal forms). We analyzed internal DEHP exposure in single spot urine samples by determining its secondary metabolites mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP), mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and 5carboxy-mono(2-ethylhexyl)phthalate (5cx-MEPP) next to the monoester metabolite mono(2-ethylhexyl)phthalate (MEHP). Logistic regression was performed for the three semen parameters (concentration, motility, and normal morphology) to estimate their dependence on the sum of the four DEHP metabolites (DEHP-4) under consideration. Adjustment was performed for age, duration of abstinence, and smoking status. RESULTS DEHP metabolites of n=349 men (age: median=34ys) were analysed. Median concentrations [microg/l] were MEHP (n=337) 4.35, 5OH-MEHP (n=341) 12.66, 5oxo-MEHP (n=341) 9.02, and 5cx- MEPP (n=292) 14.53. Semen parameters of n=349 men were analysed by logistic regression. Semen concentration (<20mio/ml: 35%) or sperm motility (WHO A+B <50%=20%) were not found to be associated statistically significantly with the sum the DEHP metabolites (DEHP-4). DISCUSSION Metabolites of DEHP and other phthalates analyzed in urine are very specific for determining recent internal phthalate exposure. According to our evaluation human reproductive parameters from semen analyses do not show significant associations with concentrations of DEHP metabolites determined in spot urine sampled at the day of andrological examination.