A method for reliable quantification of mercury in occupational and environmental medical urine samples by inductively coupled plasma mass spectrometry.

Over the last years, inductively coupled plasma mass spectrometry (ICP-MS) has been applied as a method for human-biomonitoring of metals in the concentration range of occupational and environmental medicine. In large scale routine monitoring, the determination of mercury (Hg) by ICP-MS remains challenging due to several reasons. Amongst others, stability of dissolved Hg and avoiding memory effects are the key facts for reliable quantification. To address these issues, we developed a robust approach for biomonitoring of mercury in human urine samples by ICP-MS. Using a solution containing HNO3, HCl and thiourea, prepared samples and calibrators were stabilized for up to 72 h. A rinse time of only 30 seconds efficiently prevented contamination of consecutive samples with Hg concentrations up to 30 µg L-1, hence significantly reducing acquisition times compared to published methods. Recovery experiments revealed iridium as an ideal internal standard to compensate matrix effects independently from creatinine concentration. Recoveries of 95.0-104.0% were obtained for Hg levels covering the range of biomonitoring guidance values established by the German Human-Biomonitoring Commission. Excellent intra-day precision and inter-day precision of ≤3.0% for two different Hg levels were achieved. The detection and quantification limit accounted for 21.7 ng L-1 and 65.6 ng L-1, respectively, enabling reliable quantification even in the range of environmental background exposures. Additionally, the method was externally validated by successful participation in the inter-laboratory comparison program G-EQUAS. With the developed method, we hence provide a sensitive and robust tool for mercury exposure assessments in future large scale human-biomonitoring studies.

Metal exposure of workers during recycling of electronic waste: a cross-sectional study in sheltered workshops in Germany.

OBJECTIVES: In Germany, the initial step of electronic waste (e-waste) recycling frequently takes place in sheltered workshops for physically and mentally handicapped workers (Werkstätten für behinderte Menschen (WfbM), in german language). E-waste recycling involves a potential risk of exposure to toxic metals. Therefore, we assessed the occupational exposure of recycling workers to toxic metals to identify potential health risks and insufficient protective measures. METHODS: We used a combined air- and bio-monitoring approach to determine exposure of recycling workers to toxic metals. Air and urine samples were collected in five sheltered workshops in Germany and were analysed for their content of aluminium, antimony, arsenic, beryllium, cadmium, chromium, cobalt, mercury and nickel. Results were compared to German and international occupational limit values and to metal exposures of workers in conventional e-waste recycling firms. RESULTS: Exposure of recycling workers in five German sheltered workshops to the studied metals and their compounds was below German and international occupational limit values across all facilities studied considering both air and urine samples. Workers in the present study were not exposed to higher amounts of toxic metals than workers in conventional e-waste recycling firms. CONCLUSION: This is the first study on toxic metal exposure of recycling workers in sheltered workshops. The results of this study revealed a low occupational exposure of e-waste recycling workers to toxic metals in this type of enterprises. Current work methods and safety measures provide the workers with adequate protection.

Metabolites of the substitute plasticiser Di-(2-ethylhexyl) terephthalate (DEHTP) in urine of children and adolescents investigated in the German Environmental Survey GerES V, 2014-2017.

Metabolites of di-(2-ethylhexyl) terephthalate (DEHTP), a substitute for ortho-based phthalate plasticisers like di-(2-ethylhexyl) phthalate (DEHP), were analysed in 2112 first-morning void urine samples from children and adolescents aged 3-17 years, participating in the population representative German Environmental Survey on Children and Adolescents, GerES V 2014-2017. The major metabolite 5cx-MEPTP was detected in all urine samples with a geometric mean (GM) of 7.39 µg/L, with highest levels in the mg/L range. The GM for the other metabolites were 0.55 µg/L for 5OH-MEHTP, 0.54 µg/L for 5oxo-MEHTP and below the limit of quantification (LOQ) for 2cx-MMHTP. As already observed for other plasticisers and their substitutes, the youngest children (3-5 years) had 2-2.5-fold higher urinary DEHTP metabolite levels compared to 14-17 years old adolescents. High urinary levels of DEHTP metabolites were associated with high DEHTP concentrations in house dust. None of the samples analysed exceeded the toxicologically derived German human biomonitoring guidance value (HBM-I-Value) of 1.8 mg/L for 5cx-MEPTP. Comparison with DEHTP levels reported in other HBM studies worldwide confirmed a widespread exposure of children, adolescents and adults, with considerably higher exposures (2.6-7 fold) reported in the United States. In GerES V, exposure data for 12 different phthalates and the phthalate substitute DINCH were generated as well. Together with the data for DEHTP presented in this manuscript, GerES V allows a current and comprehensive overview on the concurrent exposure of German children and adolescents to common plasticisers. Further evaluation of aggregate exposure characteristics shall support efforts to reduce chemical hazard burden from plasticisers in Germany and beyond.

De-novo identification of specific exposure biomarkers of the alternative plasticizer di(2-ethylhexyl) terephthalate (DEHTP) after low oral dosage to male volunteers by HPLC-Q-Orbitrap-MS.

CONTEXT: Human exposure biomonitoring relies on the availability of specific, sensitive biomarkers. For emerging chemicals, the identification (prediction, synthesis, verification) of such biomarkers is time and cost intensive. OBJECTIVE: This study aimed to further elucidate the urinary metabolic profile of the plasticizer di(2-ethylhexyl) terephthalate (DEHTP) in search of probably additional biomarkers of exposure. MATERIALS AND METHODS: Urine samples of an oral low-dose volunteer study were analysed by HPLC-Q-Orbitrap-MS combined with a commercial data mining software. Metabolite identification was based on isotopic pattern, accurate masses of product ions and excretion profiles. RESULTS: Nine phase I metabolites of DEHTP were tentatively identified by HPLC-Q-Orbitrap-MS. Four previously described, side chain oxidized monoester metabolites were confirmed in all samples. In addition, five previously unknown downstream metabolites were tentatively identified. DISCUSSION AND CONCLUSION: The excretion profiles obtained by HPLC-Q-Orbitrap-MS were in good agreement with quantitative HPLC-QqQ-MS data. For the newly discovered metabolites, plausible excretion profiles, similar to the ones of the known metabolites, were obtained. The presented approach proved to be successful for metabolite screening in urine samples after low-dose exposure and will be applied in future human metabolism studies for a fast, reliable and cost effective identification of specific biomarkers of exposure.

Exposure to the plasticizer di(2-ethylhexyl) terephthalate (DEHTP) in Portuguese children - Urinary metabolite levels and estimated daily intakes.

Classical ortho-phthalate plasticizers are, due to their endocrine disrupting potency and reproductive toxicity, increasingly replaced by alternative plasticizers. Di(2-ethylhexyl) terephthalate (DEHTP) is one of these substitutes. In this study, we investigated DEHTP exposure in 107 Portuguese children (4-17years old) by analyzing specific DEHTP metabolites in their urine using a newly developed LC-MS/MS method. We could detect the major, specific DEHTP metabolite mono(2-ethyl-5-carboxypentyl) terephthalate (5cx-MEPTP) in 100% of the samples with levels above the limit of quantification in 96% of the samples (median concentration 4.19µg/L; 95th percentile 26.4µg/L; maximum 3400µg/L). Other minor DEHTP metabolites (5OH-MEHTP, 5oxo-MEHTP and 2cx-MMHTP) were detected at lower rates and levels. Daily DEHTP intakes calculated from urinary 5cx-MEPTP levels were generally far below the tolerable daily intake (TDI) of 1000µg/kgbw/d (median 0.67µg/kgbw/d; 95th percentile 6.25µg/kgbw/d; maximum 690µg/kgbw/d). However, for one child the biomarker-derived health-based guidance value (HBM-I value) for 5cx-MEPTP of 1800µg/L was exceeded by about a factor of two. Levels of 5cx-MEPTP and calculated daily DEHTP intakes were higher in normal/under-weight children who nourished on their usual diet compared to overweight/obese children who received nutritional guidance with fresh and unprocessed food (p=0.043 and p<0.001 respectively). This indicates to processed and fatty foodstuff as a major source of DEHTP exposure. Additionally, we found children of lower age having higher DEHTP intakes (p=0.045). Again, foodstuff as a major DEHTP source, together with other child specific DEHTP sources such as mouthing of toys or ingestion of dust might be contributing factors. With the present study, we provide a first data set on the omnipresent DEHTP exposure in children. So far, general levels of DEHTP exposure seem no cause for concern. However, due to the increasing use of DEHTP as an ortho-phthalate substitute, possible increasing exposures in the future should be followed closely.

Metabolism and urinary excretion kinetics of di(2-ethylhexyl) terephthalate (DEHTP) in three male volunteers after oral dosage.

Di(2-ethylhexyl) terephthalate (DEHTP) is used as a substitute for di(2-ethylhexyl) phthalate (DEHP), an ortho-phthalate-based plasticizer that is classified and labeled due to its toxicity to reproduction. In this study the metabolism and urinary excretion kinetics of DEHTP were investigated by single oral dosage of 50 mg DEHTP to three male volunteers (resulting in individual dosages between 0.55 and 0.59 mg/kg body weight). Separate urine samples were consecutively collected for 48 h. In analogy to DEHP, we quantified specific side-chain-oxidized monoester metabolites of DEHTP (5OH-MEHTP, 5oxo-MEHTP, 5cx-MEPTP and 2cx-MMHTP) by HPLC-MS/MS with online sample clean-up and isotope dilution. All postulated metabolites were detectable in all samples after dosage. The predominant, specific urinary metabolite was 5cx-MEPTP representing about 13.0 % of the applied dose as mean of the three volunteers (range 7.0-20.4 %) in urine, followed by 5OH-MEHTP (mean: 1.8 %; range 1.3-2.4 %) and 5oxo MEHTP (mean: 1.0 %; range 0.6-1.6 %). 2cx-MMHTP was a minor metabolite representing only 0.3 % (range 0.2-0.4 %). In total, about 16.1 % of the dose was recovered in urine as the above investigated specific metabolites within 48 h with the major share (95 %) being excreted within the first 24 h. Investigation of the glucuronidation patterns revealed that the carboxy-metabolites are excreted almost completely in their free form (>90 %), whereas for 5OH-MEHTP and 5oxo-MEHTP, glucuronidation is preferred (>70 %). With this study we provide reliable urinary excretion factors to calculate DEHTP intakes based on metabolite concentrations in environmental and occupational studies.

Determination of metabolites of di(2-ethylhexyl) terephthalate (DEHTP) in human urine by HPLC-MS/MS with on-line clean-up.

Di(2-ethylhexyl) terephthalate (DEHTP) is used as a substitute for ortho-phthalate based plasticizers like di(2-ethylhexyl) phthalate (DEHP) which are discussed and regulated due to their reproductive toxicity. We developed a fast and rugged method to quantify side chain oxidized monoesters of DEHTP in human urine, namely 5OH-MEHTP, 5oxo-MEHTP, 2cx-MMHTP and 5cx-MEPTP. Sample preparation was kept simple with enzymatic deconjugation and a two column assembly for on-line sample clean up. Metabolites were identified with authentic standards and quantified via isotope dilution LC-MS/MS. The limit of quantification was 0.2µg/L for 5cx-MEPTP and 5oxo-MEHTP, 0.3µg/L for 5OH-MEHTP and 0.4µg/L for 2cx-MMHTP. Accuracy (relative recovery: 95.8-111%) and precision (relative standard deviation: <7%) were highly acceptable. In a pilot biomonitoring study with 34 volunteers (aged 25-61 (median 42), 20 female and 14 male) not known to be occupationally exposed to DEHTP, we could detect 5cx-MEPTP above the limit of quantification in 94% of the samples (median: 0.9µg/L, maximum: 38.7µg/L). The other metabolites investigated were detected at a lower rate and at lower concentration levels (5oxo-MEHTP: 21%, maximum: 1.8µg/L; 5OH-MEHTP: 18%, maximum: 3.4µg/L; 2cx-MMHTP: 9%, maximum: 0.9µg/L). All target analytes can be regarded as promising and specific urinary biomarkers for DEHTP exposure. With this method we provide a basis for quantitatively investigating the human metabolism of DEHTP and for performing exposure and risk assessments in the general population and the working environment.