An estimation of the daily intake of di(2-ethylhexyl)phthalate (DEHP) and other phthalates in the general population.

We analyzed 85 urine samples of the general German population for human specific metabolites of phthalates. By that we avoided contamination with the parent phthalates being omnipresent in the environment and for the first time could deduce each individual's internal exposure to phthalates without contamination. Determined were the secondary metabolites mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) of di(2-ethylhexyl)phthalate (DEHP) and the primary monoester metabolites of DEHP, di-noctylphthalate (DnOP), di-n-butylphthalate (DnBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Based on these internal exposure values we calculated the daily intake of the parent phthalates using urinary metabolite excretion factors. For DEHP we determined a median intake of 13.8 micrograms/kg body weight/day and an intake at the 95th percentile of 52.1 micrograms/kg body weight/day. The tolerable daily intake (TDI) value settled by the EU Scientific Committee for Toxicity, Ecotoxicity and the Environment (CSTEE) is 37 micrograms/kg body weight/day. Twelve percent of the subjects (10 out of 85 samples) within our collective of the general population are exceeding this value. Thirty-one percent of the subjects (26 out of 85 samples) had values higher than the reference dose (RfD) of 20 micrograms/kg body weight/day of the U.S. Environmental Protection Agency (EPA). For DnBP, BBzP, DEP and DnOP intake values at the 95th percentile were 16.2, 2.5, 22.1 and 0.42 micrograms/kg body weight/day respectively. Our results unequivocally prove that the general German population is exposed to DEHP to a much higher extent than previously believed. This is of greatest importance for public health since DEHP is not only the most important phthalate with respect to its production, use, occurrence and omnipresence but also the phthalate with the greatest endocrine disrupting potency. DEHP is strongly suspected to be a developmental and reproductive toxicant. We are not aware of any other environmental contaminant for which the TDI and RfD are exceeded to such an extent within the general population. The transgressions of the TDI and RfD for DEHP are accompanied by considerable ubiquitous exposures to DnBP and BbzP, two phthalates under scrutiny for similar toxicological mechanisms.     

Metabolites of the plasticizer di (2-ethylhexyl) phthalate in urine samples of workers in polyvinylchloride processing industries

Summary Little is known about occupational exposure to the plasticizer di(2-ethylhexyl)phthalate (CAS number 117-81-7), a compound widely used in polyvinylchloride (PVC) plastics. We have studied the uptake of DEHP in workers by determining the concentrations of four metabolites of DEHP in urine samples, i.e., mono(2-ethylhexyl)phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)phthalate. In addition DEHP concentrations in the air were determined by personal air sampling. Nine workers in a PVC boot factory exposed to a maximum of 1.2 mg/m³ DEHP showed an increase in the urinary concentrations of all four metabolites over the workshift. These results were obtained on both the first and the last day of the workweek. With the exception of MEHP, the increases in the concentrations of the metabolites during a workday were statistically significant. Six workers from a PVC cable factory exposed to a maximum of 1.2 mg/m³ DEHP showed a one-to fourfold increase in the concentrations of the four metabolites over the workshift, but these increases were not statistically significant. These results indicate that measurement of DEHP metabolites in urine samples may be of use for monitoring the occupational exposure to DEHP.     

Mono(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human exposure assessment to di-(2-ethylhexyl) phthalate

Exposure to di-(2-ethylhexyl) phthalate (DEHP) is prevalent based on the measurement of its hydrolytic metabolite mono-(2-ethylhexyl) phthalate (MEHP) in the urine of 78% of the general U.S. population studied in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). However, despite the high level of production and use of DEHP, the urinary MEHP levels in the NHANES samples were lower than the monoester metabolites of phthalates less commonly used than DEHP, suggesting metabolic differences between phthalates. We measured MEHP and two oxidative DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) to verify whether these other metabolites account for a greater proportion of DEHP metabolic products in 127 paired human urine and serum samples. We found that the urinary levels of MEHHP and MEOHP were 10-fold higher than levels of MEHP; concentrations of urinary MEOHP and MEHHP were strongly correlated (r = 0.928). We also found that the serum levels of MEOHP and MEHHP were comparatively lower than those in urine. Furthermore, the glucuronide-bound conjugates of the oxidative metabolites were the predominant form in both urine and serum. MEOHP and MEHHP cannot be formed by serum enzymes from the hydrolysis of any contamination from DEHP potentially introduced during blood collection and storage. Therefore, concentrations of MEHHP and MEOHP in serum may be a more selective measure of DEHP exposure than is MEHP. However, additional data on the absorption, distribution, metabolism, and elimination of these oxidative metabolites are needed to completely understand the extent of DEHP exposure from the serum concentrations of oxidative DEHP metabolites.     

Internal phthalate exposure over the last two decades--a retrospective human biomonitoring study

In a retrospective human biomonitoring study we analyzed 24h urine samples taken from the German Environmental Specimen Bank for Human Tissues (ESBHum), which were collected from 634 subjects (predominantly students, age range 20-29 years, 326 females, 308 males) in 9 years between 1988 and 2003 (each n >or= 60), for the concentrations of primary and/or secondary metabolites of di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP). Based on the urinary metabolite excretion we estimated daily intakes of the parent phthalates and investigated the chronological course of the phthalate exposure. In over 98% of the urine samples metabolites of all five phthalates were detectable indicating a ubiquitous exposure of the German population to all five phthalates throughout the last 20 years. The median daily intakes in the subsets between 1988 and 1993 were quite constant for DnBP (approx. 7 microg/kg bw/d) and DEHP (approx. 4 microg/kg bw/d). However, from 1996 the median levels of both phthalates decreased continuously until 2003 (DnBP 1.9 microg/kg bw/d; DEHP 2.4 microg/kg bw/d). By contrast, the daily intake values for DiBP were slightly increasing over the whole time frame investigated (median 1988: 1.1 microg/kg bw/d; median 2003: 1.4 microg/kg bw/d), approximating the levels for DnBP and DEHP. For BBzP we observed slightly decreasing values, even though the medians as of 1998 levelled off at around 0.2 microg/kg bw/d. Regarding daily DiNP exposure we found continuously increasing values, with the lowest median being 0.20 microg/kg bw/d for the subset of 1988 and the highest median for 2003 being twice as high. The trends observed in phthalate exposure may be associated with a change in production and usage pattern. Female subjects exhibited significantly higher daily intakes for the dibutyl phthalates (DnBP p=0.013; DiBP p=0.004). Compared to data from US National Health and Nutrition Examination Surveys (NHANES) exposure levels of the dibutyl phthalates were generally higher in our German study population, while levels of BBzP were somewhat lower. Overall, for a considerable 14% of the subjects we observed daily DnBP intakes above the tolerable daily intake (TDI) value deduced by the European Food Safety Authority (EFSA) (10 microg/kg bw/d). However, the frequency of exceedance decreased during the years and was beneath 2% in the 2003 subset. Even though transgressions of the exposure limit values of the EFSA and the US Environmental Protection Agency (US EPA) occurred only in a relatively small share of the subjects, one has to take into account the cumulative exposure to all phthalates investigated and possible dose-additive endocrine effects of these phthalates.     

Biological monitoring of carbon disulphide and phthalate exposure in the contemporary rubber industry

Objectives: We studied the range in urinary levels of 2-thiothiazolidine-4-carboxyl acid (TTCA), a metabolite of CS₂ and phthalic acid (PA), a common metabolite of phthalates, across factories and departments in the contemporary rubber manufacturing industry. Methods: Spot urine samples from 101 rubber workers employed in nine different factories were collected on Sunday and during the workweek on Tuesday, Wednesday and Thursday at ∼4 pm. In total, 386 urine samples were successfully analyzed. Results: Levels of both biomarkers increased significantly by a factor 2 (paired t-test P-value <0.05) during the working week as compared to the Sunday biomarker levels with absolute increases of approximately 70 μg/l and 5 μmol/mol creatinine for PA and TTCA, respectively. Levels in both biomarkers did not differ markedly between working days. Increases seemed to be restricted to specific factories and/or departments (e.g. molding and curing). Conclusions: The results of this study demonstrate that rubber workers in the contemporary rubber industry are exposed to phthalates and low levels of CS₂ (∼0.05 ppm) as measured by PA and TTCA, respectively. Exposures to both compounds are largely driven by specific circumstances in factories. Therefore, when estimating exposures to phthalates and CS₂ detailed information should be collected on the type and amount of phthalate containing ester plasticizers, dithiocarbamates and thiurams used. Preferably, personal exposure data should be collected. In this case, biological monitoring seems a reasonable approach. However, in the case of PA attention should be given to individual background levels as this could lead to a substantial overestimation of the occupational contribution to total phthalate exposure.     

Urinary excretion of phthalate metabolites in 129 healthy Danish children and adolescents: estimation of daily phthalate intake

Background

Phthalates are a group of chemicals with widespread use in the industrial production of numerous consumer products. They are suspected to be involved in male reproductive health problems and have also been associated with several other health problems in children including obesity and asthma.

Objectives

To study the urinary excretion of phthalate metabolites in Danish children recruited from the general population, and to estimate the daily intake of phthalates in this segment of the population.

Method

One 24 h urine sample and to consecutive first morning urine samples were collected from 129 healthy Danish children and adolescents (range 6–21 yrs). The concentrations of 11 phthalate metabolites of 5 different phthalate diesters were analyzed by liquid chromatography–tandem mass spectrometry.

Results

The analyzed metabolites were detectable in almost all 24 h urine samples. The median concentrations of monoethyl phthalate (MEP), monobenzyl phthalate (MBzP) and the sums of the two monobutyl phthalate isoforms (∑MBP(i+n)), metabolites of di-(2-ethylhexyl) phthalate (∑DEHPm) and of di-iso-nonyl phthalate (∑DiNPm) were 29, 17, 111, 107 and 31 ng/mL, respectively. The youngest children were generally more exposed to phthalates than older children and adolescents (except diethyl phthalate (DEP)). Boys were more exposed than girls. The median estimated daily intake of phthalate diesters was: 4.29 (dibutyl phthalate isoforms (DBP(i+n))), 4.04 (DEHP), 1.70 (DiNP), 1.09 (DEP) and 0.62 (butylbenzyl phthalate (BBzP)), all calculated as μg/kg body weight/24 h. Between 40% and 48% of the absolute amount of phthalate metabolites excreted over 24 h were excreted in first morning urine voids.

Conclusion

Danish children are exposed simultaneously to multiple phthalates. The highest exposure levels were found for DBP(i+n) and DEHP, which in animal models are the known most potent anti-androgenic phthalates. The combined exposure to the two isoforms of DBP, which have similar endocrine-disrupting potencies in animal models, exceeded the TDI for di-n-butyl phthalate (DnBP) in several of the younger children.     

Urinary phthalate metabolite concentrations among workers in selected industries: a pilot biomonitoring study

Phthalates are used as plasticizers and solvents in industrial, medical and consumer products; however, occupational exposure information is limited. We sought to obtain preliminary information on occupational exposures to diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) by analyzing for their metabolites in urine samples collected from workers in a cross-section of industries. We also obtained data on metabolites of dimethyl phthalate (DMP), benzylbutyl phthalate (BzBP), di-isobutyl phthalate and di-isononyl phthalate. We recruited 156 workers in 2003-2005 from eight industry sectors. We assessed occupational contribution by comparing end-shift metabolite concentrations to the US general population. Evidence of occupational exposure to DEHP was strongest in polyvinyl chloride (PVC) film manufacturing, PVC compounding and rubber boot manufacturing where geometric mean (GM) end-shift concentrations of DEHP metabolites exceeded general population levels by 8-, 6- and 3-fold, respectively. Occupational exposure to DBP was most evident in rubber gasket, phthalate (raw material) and rubber hose manufacturing, with DBP metabolite concentrations exceeding general population levels by 26-, 25- and 10-fold, respectively, whereas DBP exposure in nail-only salons (manicurists) was only 2-fold higher than in the general population. Concentrations of DEP and DMP metabolites in phthalate manufacturing exceeded general population levels by 4- and >1000-fold, respectively. We also found instances where GM end-shift concentrations of some metabolites exceeded general population concentrations even when no workplace use was reported, e.g. BzBP in rubber hose and rubber boot manufacturing. In summary, using urinary metabolites, we successfully identified workplaces with likely occupational phthalate exposure. Additional work is needed to distinguish occupational from non-occupational sources in low-exposure workplaces.     

Levels of phthalate metabolites in urine among mother-child-pairs - results from the Duisburg birth cohort study, Germany

Phthalates are used ubiquitously and human exposure is widespread. Some phthalates are anti-androgens and have to be regarded as reproductive and developmental toxicants. In the Duisburg birth cohort study we examine the associations between hormonally active environmental agents and child development. Here we report the concentrations of 21 primary and secondary phthalate metabolites from seven low molecular weight (LMW) phthalates (DMP, DEP, BBzP, DiBP, DnBP, DCHP, DnPeP) and five high-molecular weight (HMW) phthalates (DEHP, DiNP, DiDP, DPHP, DnOP) in 208 urine samples from 104 mothers and their school-aged children. Analysis was performed by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS), using internal isotope-labeled standards. In both children and mothers, 18 out of 21 phthalate metabolites were detected above the limits of quantification (between 0.2 and 1.0 μg/l) in nearly all urine samples. Among the LMW phthalates, the excretion level (geometric mean) of the ΣDiBP metabolites was most prominent in children (103.9 μg/l), followed by ΣDnBP (56.5 μg/l), and MEP (39.1 μg/l). In mothers ΣDiBP (66.6 μg/l) was highest, followed by MEP (50.5 μg/l), and ΣDnBP (36.0 μg/l). Among the HMW phthalates, ΣDEHP was highest in children and mothers (55.7/28.9 μg/l). Compared to reference values derived from the German Human Biomonitoring Commission, children's metabolite concentrations were within background levels, whereas for mothers considerably higher exposure to the LMW phthalates DnBP and DiBP, and the HMW phthalate DEHP was detected (MiBP: 10.7%; MnBP: 11.7%; ΣDEHP: 23.3% of the samples were above the reference values). The LMW metabolites from DMP, DiBP, and DnBP, and the HMW metabolites from DEHP and DiNP were correlated between the mothers and children, probably indicating shared exposure in the immediate surrounding environment. Children showed higher excretion levels for most of the secondary metabolites than mothers, confirming previous findings on higher oxidized metabolite levels in children. The LMW metabolites ΣDiBP, ΣDnBP, and MMP, and the HMW metabolites ΣDEHP were negatively associated with children's age. The LMW metabolites ΣDiBP, ΣDnBP, and MBzP were inversely associated with body mass index of the children. The LMW ΣDiBP metabolites revealed a significant association with nicotine metabolites in urine from both children and mothers. Further analyses are ongoing to study long-term phthalate exposure and the associations with puberty outcome in these children.     

Prenatal exposures to phthalates among women in New York City and Krakow, Poland

Experimental evidence has shown that certain phthalates can disrupt endocrine function and induce reproductive and developmental toxicity. However, few data are available on the extent of human exposure to phthalates during pregnancy. As part of the research being conducted by the Columbia Center for Children's Environmental Health, we have measured levels of phthalates in 48-hr personal air samples collected from parallel cohorts of pregnant women in New York, New York, (n = 30) and in Krakow, Poland (n = 30). Spot urine samples were collected during the same 48-hr period from the New York women (n = 25). The following four phthalates or their metabolites were measured in both personal air and urine: diethyl phthalate (DEP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), and butyl benzyl phthalate (BBzP). All were present in 100% of the air and urine samples. Ranges in personal air samples were as follows: DEP (0.26-7.12 microg/m3), DBP (0.11-14.76 microg/m3), DEHP (0.05-1.08 microg/m3), and BBzP (0.00-0.63 microg/m3). The mean personal air concentrations of DBP, di-isobutyl phthalate, and DEHP are higher in Krakow, whereas the mean personal air concentration of DEP is higher in New York. Statistically significant correlations between personal air and urinary levels were found for DEP and monoethyl phthalate (r = 0.42, p < 0.05), DBP and monobutyl phthalate (r = 0.58, p < 0.01), and BBzP and monobenzyl phthalate (r = 0.65, p < 0.01). These results demonstrate considerable phthalate exposures during pregnancy among women in these two cohorts and indicate that inhalation is an important route of exposure.     

Identifying sources of phthalate exposure with human biomonitoring: Results of a 48 h fasting study with urine collection and personal activity patterns

Human biomonitoring studies measuring phthalate metabolites in urine have shown widespread exposure to phthalates in the general population. Diet is thought to be a principle route of exposure to many phthalates. Therefore, we studied urinary phthalate metabolite patterns over a period of strict fasting and additionally recorded personal activity patterns with a diary to investigate non-dietary routes of exposure. Five individuals (3 female, 2 male, 27–47 years of age) fasted on glass-bottled water only over a 48-h period. All urine void events were captured in full, and measured for metabolites of the high molecular weight (HMW) di-(2-ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP) and di-isodecyl phthalate (DiDP), and the low molecular weight (LMW) di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP), butylbenzyl phthalate (BBzP), dimethyl phthalate (DMP), and diethyl phthalate (DEP). In all, 21 metabolites were measured in a total of 118 urine events, including events before and after the fasting period. At the onset of the study all phthalate metabolite concentrations were consistent with levels found in previous general population studies. Metabolites of the HMW phthalates (DEHP, DiNP and DiDP) showed a rapid decline to levels 5–10 times lower than initial levels within 24 h of the fast and remained low thereafter. After food consumption resumed, levels rose again. By contrast, metabolites of the LMW phthalates including DMP, DEP, BBzP, DnBP and DiBP showed a cyclical pattern of rising and declining concentrations suggestive of ongoing non-food exposures. Furthermore, metabolites of most of the LMW phthalates (BBzP, DnBP and DiBP) tracked each other remarkably well, suggesting concurrent exposures. Diary entries could not help explain exposure sources for these phthalates, with one exception: rises in MEP concentrations around males’ showers suggest personal care products as a major source of DEP. Exposure to HMW phthalates in this cohort appears to be driven by dietary intake, while non-dietary routes such as use of personal care products and ubiquitous sources including dust and indoor air appear to explain exposure to LMW phthalates.     

Temporal variability of urinary phthalate metabolite levels in men of reproductive age

Phthalates are a family of multifunctional chemicals widely used in personal care and other consumer products. The ubiquitous use of phthalates results in human exposure through multiple sources and routes, including dietary ingestion, dermal absorption, inhalation, and parenteral exposure from medical devices containing phthalates. We explored the temporal variability over 3 months in urinary phthalate metabolite levels among 11 men who collected up to nine urine samples each during this time period. Eight phthalate metabolites were measured by solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry. Statistical analyses were performed to determine the between- and within-subject variance apportionment, and the sensitivity and specificity of a single urine sample to classify a subject's 3-month average exposure. Five of the eight phthalates were frequently detected. Monoethyl phthalate (MEP) was detected in 100% of samples; monobutyl phthalate, monobenzyl phthalate, mono-2-ethylhexyl phthalate (MEHP), and monomethyl phthalate were detected in > 90% of samples. Although we found both substantial day-to-day and month-to-month variability in each individual's urinary phthalate metabolite levels, a single urine sample was moderately predictive of each subject's exposure over 3 months. The sensitivities ranged from 0.56 to 0.74. Both the degree of between- and within-subject variance and the predictive ability of a single urine sample differed among phthalate metabolites. In particular, a single urine sample was most predictive for MEP and least predictive for MEHP. These results suggest that the most efficient exposure assessment strategy for a particular study may depend on the phthalates of interest.     

Estimating the contribution of inhalation exposure to di-2-ethylhexyl phthalate (DEHP) for PVC production workers,using personal air sampling and urinary metabolite monitoring

Because of troubling reports of high urinary metabolite levels and adverse reproductive health effects in workers exposed to di(2-ethylhexyl)phthalate (DEHP) in occupational settings, concern about exposure to DEHP in occupational settings is increasing. However, the contributions of different routes of exposure to DEHP are unclear. We used personal air sampling and biomonitoring to determine the contribution of inhalation exposure to the body burden of DEHP in the workplace. Eighty-nine workers (high-exposure group: 66 raw-materials workers; low-exposure group: 23 administrative workers) were recruited from three polyvinyl chloride (PVC) factories. Urinary levels of mono(2-ethylhexyl) phthalate (MEHP), (mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) were measured in pre-shift and post-shift samples. The geometric means of airborne concentrations of DEHP were 5.3 μg/m³ (low-exposure group) and 32.7 μg/m³ (high-exposure group) (P < 0.01). Correlation analysis showed a consistently significant association between airborne DEHP concentration and urinary DEHP metabolite levels in the high-exposure group. Calculating daily DEHP intake based on total urinary metabolite levels showed that the geometric means of total daily urinary metabolite levels of DEHP were 9.2 μg/kg/day (low-exposure group) and 15.5 μg/kg/day (high-exposure group) (P < 0.01). A quartile analysis of all workers showed a significant trend toward an association between the individual contribution of inhalation exposure to DEHP and urinary DEHP metabolite levels, for which the mean inhalation contribution was 46.7% in the highest quartile. We conclude that inhalation-absorbed airborne DEHP significantly increased the total body burden of DEHP in these occupationally exposed workers.     

Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China

BACKGROUND: Observations of adverse developmental and reproductive effects in laboratory animals and wildlife have fueled increasing public concern regarding the potential for various chemicals to impair human fertility. OBJECTIVE: Our objective in this study was to assess the effect of occupational exposure to high levels of phthalate esters on the balance of gonadotropin and gonadal hormones including luteinizing hormone, follicle-stimulating hormone, free testosterone (fT), and estradiol. METHODS: We examined urine and blood samples of 74 male workers at a factory producing unfoamed polyvinyl chloride flooring exposed to di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) and compared them with samples from 63 male workers from a construction company, group matched for age and smoking status. RESULTS: Compared to the unexposed workers, the exposed workers had substantially and significantly elevated concentrations of mono-n-butyl phthalate (MBP; 644.3 vs. 129.6 microg/g creatinine, p < 0.001) and mono-2-ethylhexyl phthalate (MEHP; 565.7 vs. 5.7 microg/g creatinine, p < 0.001). fT was significantly lower (8.4 vs. 9.7 microg/g creatinine, p = 0.019) in exposed workers than in unexposed workers. fT was negatively correlated to MBP (r = -0.25, p = 0.03) and MEHP (r = -0.19, p = 0.095) in the exposed worker group. Regression analyses revealed that fT decreases significantly with increasing total phthalate ester score (the sum of quartiles of MBP and MEHP; r = -0.26, p = 0.002). CONCLUSION: We observed a modest and significant reduction of serum fT in workers with higher levels of urinary MBP and MEHP compared with unexposed workers.     

Internal exposure of the general population to DEHP and other phthalates--determination of secondary and primary phthalate monoester metabolites in urine

A number of phthalates and their metabolites are suspected of having teratogenic and endocrine disrupting effects. Especially the developmental and reproductive effects of di(2-ethylhexyl)phthalate (DEHP) are under scrutiny. In this study we determined the concentrations of the secondary, chain oxidized monoester metabolites of DEHP, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) in urine samples from the general population. The utilization of the secondary metabolites minimized any risk of contamination by the ubiquitously present phthalate parent compounds. Included in the method were also the simple monoester metabolites of DEHP, dioctylphthalate (DOP), di-n-butylphthalate (DnBuP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Automated sample preparation was performed applying a column switching liquid chromatography system enabling online extraction of the urine on a restricted access material (RAM) and separation on a reversed phase analytical column. Detection was performed by negative ESI-tandem mass spectrometry in multiple reaction monitoring mode and quantification by isotope dilution. The excretion of DEHP and the other phthalates was studied by analyzing first morning urine samples from 53 women and 32 men aged 7-64 years (median: 34.2 years) living in northern Bavaria (Germany) who were not occupationally exposed to phthalates. Phthalate metabolites, secondary and primary ones, were detected in all specimens. Concentrations were found to vary strongly from phthalate to phthalate and subject to subject with differences spanning more than three orders of magnitude. Median concentrations for excretion of DEHP metabolites were 46.8 microg/L for 5OH-MEHP (range 0.5-818 microg/L), 36.5 microg/L for 5oxo-MEHP (range 0.5-544 microg/L), and 10.3 microg/L for MEHP (range:<0.5 (limit of quantification, LOQ) to 177 microg/L). A strong correlation was found between the excretion of 5OH-MEHP and 5oxo-MEHP with a correlation coefficient of r=0.991, indicating close metabolic proximity of those two parameters but also the absence of any contaminating interference. Median concentrations for the other monoester metabolites were for mono-n-butylphthalate (MnBuP) 181 microg/L, for monobenzylphthalate (MBzP) 21.0 microg/L, for monoethylphthalate (MEP) 90.2 microg/L and for mono-n-octylphthalate (MOP)<1.0 microg/L (LOQ). These results will help to perform health risk assessments for the phthalate exposure of the general population.     

Trends of the internal phthalate exposure of young adults in Germany--follow-up of a retrospective human biomonitoring study

The exposure of the general population to phthalates is of increasing public health concern. Variations in the internal exposure of the population are likely, because the amounts, distribution and application characters of the phthalate use change over time. Estimating the chronological sequences of the phthalate exposure, we performed a retrospective human biomonitoring study by investigating the metabolites of the five most prominent phthalates in urine. Therefore, 24 h-urine samples from the German Environmental Specimen Bank (ESB) collected from 240 subjects (predominantly students, age range 19–29 years, 120 females, 120 males) in the years 2002, 2004, 2006 and 2008 (60 individuals each), were analysed for the concentrations of mono-n-butyl phthalate (MnBP) as metabolite of di-n-butyl phthalate (DnBP), mono-iso-butyl phthalate (MiBP) as metabolite of di-iso-butyl phthalate (DiBP), mono-benzyl phthalate (MBzP) as metabolite of butylbenzyl phthalate (BBzP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono-(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP) and mono-(2-carboxymethyl hexyl) phthalate (2cx-MMHxP) as metabolites of di(2-ethylhexyl) phthalate (DEHP), monohydroxylated (OH-MiNP), monooxidated (oxo-MiNP) and monocarboxylated (cx-MiNP) mono-iso-nonylphthalates as metabolites of di-iso-nonyl phthalates (DiNP). Based on the urinary metabolite excretion, together with results of a previous study, which covered the years 1988–2003, we investigated the chronological sequences of the phthalate exposure over two decades. In more than 98% of the urine samples metabolites of all five phthalates were detectable indicating a ubiquitous exposure of people living in Germany to all five phthalates throughout the period investigated. The medians in samples from the different years investigated are 65.4 (2002), 38.5 (2004), 29.3 (2006) and 19.6 μg/l (2008) for MnBP, 31.4 (2002), 25.4 (2004), 31.8 (2006) and 25.5 μg/l (2008) for MiBP, 7.8 (2002), 6.3 (2004), 3.6 (2006) and 3.8 μg/l (2008) for MBzP, 7.0 (2002), 5.6 (2004), 4.1 (2006) and 3.3 μg/l (2008) for MEHP, 19.6 (2002), 16.2 (2004), 13.2 (2006) and 9.6 μg/l (2008) for 5OH-MEHP, 13.9 (2002), 11.8 (2004), 8.3 (2006) and 6.4 μg/l (2008) for 5oxo-MEHP, 18.7 (2002), 16.5 (2004), 13.8 (2006) and 10.2 μg/l (2008) for 5cx-MEPP, 7.2 (2002), 6.5 (2004), 5.1 (2006) and 4.6 μg/l (2008) for 2cx-MMHxP, 3.3 (2002), 2.8 (2004), 3.5 (2006) and 3.6 μg/l (2008) for OH-MiNP, 2.1 (2002), 2.1 (2004), 2.2 (2006) and 2.3 μg/l (2008) for oxo-MiNP and 4.1 (2002), 3.2 (2004), 4.1 (2006) and 3.6 μg/l (2008) for cx-MiNP. The investigation of the time series 1988–2008 indicates a decrease of the internal exposure to DnBP by the factor of 7–8 and to DEHP and BzBP by the factor of 2–3. In contrast, an increase of the internal exposure by the factor of 4 was observed for DiNP over the study period. The exposure to DiBP was found to be stable. In summary, we found decreases of the internal human exposure for legally restricted phthalates whereas the exposure to their substitutes increased. Future investigations should verify these trends. This is of increasing importance since the European Commission decided to require ban or authorization from 1.1.2015 for DEHP, DnBP, DiBP and BzBP according to REACh Annex XIV.     

Comparisons of urinary phthalate metabolites and daily phthalate intakes among Japanese families

We measured urinary phthalate metabolites, including di-n-butyl phthalate (DnBP), di-isobutyl phthalate, benzyl butyl phthalate (BBzP), and di(2-ethylhexyl) phthalate (DEHP), from 178 school-aged children and their 284 family members using gas chromatography-mass spectrometry, and we calculated daily phthalate intakes. The highest median levels of phthalate metabolites were for mono-isobutyl phthalate in all participants except schoolchildren, where the highest levels were for mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP). Comparing the schoolchildren with their parents, the schoolchildren had significantly higher urinary metabolites for MEOHP, mono-(2-ethyl-5-carboxypentyl) phthalate, and ΣDEHP. Regarding daily intakes, the schoolchildren had significantly higher daily intakes of DnBP, BBzP, and ΣDEHP. All phthalate metabolite and sums of metabolite levels in the schoolchildren were positively correlated with their mothers’ levels, except for MEHP, whereas fathers were less correlated with their children. The DEHP intake in this study was higher than that of most other studies. Moreover, 10% of the children and 3% of the adults exceeded the Reference Dose (RfD) value (20 μg/kg/day) of the U.S. Environmental Protection Agency, which indicates that it is important to focus on children's DEHP exposure because the children exceeded the RfD more than adults among the same families who shared similar exposure sources. Our results will contribute to considerations of the regulations for some phthalates and the actual phthalate exposure levels in the Japanese population.     

Fetal exposure to phthalates – a pilot study

The fetus is considered to be the most sensitive stage of life to the potential developmental and reproductive toxicity of the phthalates. But, data on human fetal exposure to phthalates is still scarce. In this pilot study we collected 11 pairs of amniotic fluid (AF) and corresponding maternal urine (MU) samples during Caesarean section and analysed them for several phthalate metabolites by LC-MS/MS. In all AF samples, metabolites of di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP) were detectable. For the first time, we were able to detect also oxidative phthalate metabolites in AF, with two carboxy metabolites of DEHP showing the highest abundance. In the MU samples, the concentrations of the phthalate metabolites were generally much higher than in the AF samples. There was a statistically significant linear correlation for the DiBP monoester (MiBP) (r=0.93; p<0.001) in the AF and MU samples. We also found a significant correlation for the DEHP monoester (MEHP) (r=0.91; p<0.001), although there was a most likely external contamination with MEHP in the MU samples. Our results suggest that several phthalates or their metabolites, respectively, reach the human fetus, which might be able to affect fetal health. Further research is needed to elucidate fetal metabolism of phthalates and to evaluate the in utero phthalate exposure and the potential effects on fetal reproductive development. Due to the continuous turn over of AF, urinary levels may be most appropriate for assessing both maternal and fetal phthalate exposure.     

Levels of seven urinary phthalate metabolites in a human reference population

Using a novel and highly selective technique, we measured monoester metabolites of seven commonly used phthalates in urine samples from a reference population of 289 adult humans. This analytical approach allowed us to directly measure the individual phthalate metabolites responsible for the animal reproductive and developmental toxicity while avoiding contamination from the ubiquitous parent compounds. The monoesters with the highest urinary levels found were monoethyl phthalate (95th percentile, 3,750 ppb, 2,610 microg/g creatinine), monobutyl phthalate (95th percentile, 294 ppb, 162 microg/g creatinine), and monobenzyl phthalate (95th percentile, 137 ppb, 92 microg/g creatinine), reflecting exposure to diethyl phthalate, dibutyl phthalate, and benzyl butyl phthalate. Women of reproductive age (20-40 years) were found to have significantly higher levels of monobutyl phthalate, a reproductive and developmental toxicant in rodents, than other age/gender groups (p < 0.005). Current scientific and regulatory attention on phthalates has focused almost exclusively on health risks from exposure to only two phthalates, di-(2-ethylhexyl) phthalate and di-isononyl phthalate. Our findings strongly suggest that health-risk assessments for phthalate exposure in humans should include diethyl, dibutyl, and benzyl butyl phthalates.     

Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers

Phthalates are a group of industrial chemicals with many commercial uses, such as solvents, additives, and plasticizers. For example, di-(2-ethylhexyl) phthalate (DEHP) is added in varying amounts to certain plastics, such as polyvinyl chloride, to increase their flexibility. In humans, phthalates are metabolized to their respective monoesters, conjugated, and eliminated. However, despite the high production and use of DEHP, we have recently found that the urinary levels of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in 2,541 persons in the United States were lower than we anticipated, especially when compared with urinary metabolite levels of other commonly used phthalates. This finding raised questions about the sensitivity of this biomarker for assessing DEHP exposure. We explored the utility of two other DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), as additional DEHP biomarkers. These metabolites are formed by oxidative metabolism of MEHP. In urine from 62 people, both the range and the mean urinary levels of MEOHP and MEHHP were on average 4-fold higher than those of MEHP; the mean of the individual ratios of MEHHP/MEOHP, MEHHP/MEHP, and MEOHP/MEHP were 1.4, 8.2, and 5.9, respectively. These data suggest that MEOHP and MEHHP are more sensitive biomarkers of exposure to DEHP than is MEHP. These findings also suggest a predominant human metabolic route for DEHP hydrolysis to MEHP followed by oxidation of MEHP; they also imply that a similar mechanism may be relevant for other high-molecular-weight phthalates, such as di-n-octyl, di-isononyl, and di-isodecyl phthalates.     

Determination of four metabolites of the plasticizer di (2-ethylhexyl) phthalate in human urine samples

Summary A method for biological monitoring of exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) is described. In this method the four main metabolites of DEHP [i.e., mono (2-ethylhexyl) phthalate (MEHP), mono (5-carboxy-2-ethylpentyl)phthalate, mono(2-ethyl-5-oxohexyl)phthalate, and mono(2-ethyl-5-hydroxyhexyl)-phthalate] are determined in urine samples. The procedure includes enzymatic hydrolysis, ether extraction, and derivatization with triethyloxonium tetrafluoroborate. Analysis is performed by gas chromatography electron impact mass spectrometry. The detection limit for all four metabolites is less than 25 g/l urine. The coefficient of variation based on duplicate determinations of urine samples of workers occupationally exposed to DEHP was 16% for MEHP (mean concentration 0.157 mg/l) and 6% -9% for the other three metabolites (mean concentrations 0.130-0.175 mg/1). The method described here was used to study DEHP metabolism in man. Most persons excrete mono(2-ethyl-5-oxohexyl)-phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate as a (glucuronide) conjugate. Mono (5-carboxy-2-ethyl-pentyl)phthalate is mainly excreted in free form, while for MEHP a large interindividual variation in conjugation status was observed. Of the four metabolites quantified, 52% are products of a ((-l)-hydroxylation reaction of MEHP [i.e., mono (2-ethyl-5-oxohexyl)phthalate and mono (2-ethyl-5-hydroxyhexyl)phthalate], 22% is the product of a -hydroxylation reaction of MEHP [i.e., mono (5-carboxy-2-ethylpentyl)phthalate], and 26% is not oxidized further (i.e., MEHP). A good correlation is obtained when the amount of MEHP -hydroxylation products is compared with the amount of MEHP (-1)hydroxylation products in urine samples. When the internal dose of DEHP has to be established we recommend that the levels of all four metabolites of DEHP be studied in urine samples.