Degradation of bis(2-ethylhexyl) phthalate constituents under methanogenic conditions

The degradation of bis(2-ethylhexyl) phthalate (DEHP) and its intermediary hydrolysis products 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) was investigated in a methanogenic phthalic acid ester-degrading enrichment culture at 37°C. 2-Ethylhexanoic acid (2-EHA), a plausible degradation product of 2-EH, was also studied. The culture readily degraded 2-EH via 2-EHA to methane which was formed in stoichiometric amounts assuming complete degradation of 2-EH to methane and carbon dioxide. MEHP was degraded to stoichiometric amounts of methane with phthalic acid as a transient intermediate. DEHP remained unaffected throughout the experimental period (330 days).Abbreviations 2-EH 2-ethylhexyl alcohol - 2-EHA 2-ethylhexanoic acid - BBP butylbenzyl phthalate - Be-CoA benzoyl Coenzyme A - CoA Coenzyme A - DEHP bis(2-ethylhexyl) phthalate - MEHP mono(2-ethylhexyl) phthalate - MSW municipal solid waste - PA phthalic acid - PAE phthalic acid ester - TMS trimethylsilyl derivative     

电子垃圾拆解地区土壤和植物中邻苯二甲酸酯分布特征

近年来,电子垃圾不当拆解带来的环境问题引起国际社会的极大关注.本研究对浙江省台州市不同电子垃圾拆解地区土壤和植物样品中5种邻苯二甲酸酯类（PAEs）污染物进行了测定分析.结果表明：土壤（以干质量计）中PAEs类污染物的浓度为12.566~46.669 mg·kg^-1,其中邻苯二甲酸二异辛酯（DEHP）、邻苯二甲酸二丁酯（DBP）和邻苯二甲酸二乙酯（DEP）相对含量较高,约占PAEs总量的94%以上.拆解地区蚕豆（Vicia faba L.）中PAEs总量明显高于同地区其他植物,且土壤和所有植物体内PAEs浓度相关性均不显著(P>0.05).与美国国家环保局制定的土壤PAEs治理标准比较,台州电子垃圾拆解地区土壤PAEs污染较为严重.     

Degradation of environmental endocrine disruptor di-2-ethylhexyl phthalate by a newly discovered bacterium, Microbacterium sp. strain CQ0110Y

In this study di-2-ethylhexyl phthalate (DEHP)-degradation strain CQ0110Y was isolated from activated sludge. According to the biophysical/biochemical characteristics and analysis of 16S rDNA, the strain was identified as Microbacterium sp. The results of this study showed the optimal pH value and optimal temperature which influenced the degradation rate in wastewater: pH 6.5–7.5, 25–35°C. Kinetics of degradation reaction had been performed at different initial concentrations and different time. Analyzed with SPSS10.0 software, the DEHP degradation can be described as the same exponential model when the initial DEHP concentration was lower than 1,350 mg/l. The kinetics equation was ln C = −0.4087t + A, with the degradation half life of DEHP in wastewater (1.59 days). To the best of our knowledge, this is the first reported case of DEHP degradation by Microbacterium sp. strain. Xiang Li and Ji-an Chen contributed equally to this work.     

Anaerobic degradation of phthalic acid esters during digestion of municipal solid waste under landfilling conditions

Anaerobic microorganisms in municipal solid waste samples from laboratory-scale landfill reactors and a pilot-plant biogas digestor were investigated with the aim of assessing their ability to transform four commercially used phthalic acid esters (PAEs) and phthalic acid (PA). The PAEs studied were diethyl phthalate (DEP), butylbenzyl phthalate (BBP), dibutyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP). No biological transformation of DEHP could be detected in any of the experiments. Together with waste samples from the simulated landfilling conditions, the PAEs (except DEHP) were hydrolytically transformed to their corresponding monoesters. These accumulated as end products, and in most cases they were not further degraded. During incubation with waste from the biogas digestor, the PAEs (except DEHP) were completely degraded to methane and carbon dioxide. The influence of the landfill development phase on the transformations was investigated utilizing PA and DEP as model substances. We found that during both the intense and stable methanogenic (but not the acidogenic) phases, the microoganisms in the samples had the potential to transform PA. A shorter lag phase was observed for the PA transformation in the samples from the stable methanogenic phase as compared with earlier phases. This indicates an increased capacity to degrade PA during the aging phases of the municipal solid waste in landfills. No enhancement of the DEP transformation could be observed as conditions in the methanogenic landfill model changed over a year's time. The results indicate that microorganisms developing in a methanogenic landfill environment have a substantially lower potential to degrade PAEs compared with those developing in a biogas reactor.Abbreviations BBP butylbenzyl phthalate - DEHP bis(2-ethylhexyl) phthalate - CoA coenzyme A - DBP dibutyl phthalate - DEP diethyl phthalate - DS dry solids - MBeP monobenzyl phthalate - MBuP monobutyl phthalate - MEP monoethyl phthalate - MSW municipal solid waste - PA phthalic acid - PAE(s) phthalic acid ester(s) - VFA volatile fatty acids     

Isolation of Microorganisms Growing on Phthalate Esters and Degradation of Phthalate Esters by Pseudomonas acidovorans 256–1

Many microorganisms (17 strains of gram-positive bacteria, 8 strains of gram-negative bacteria, 2 strains of fungi) capable of assimilating di-2-ethyl hexyl phthalate (DEHP) were isolated from soil and other natural sources. When Pseudomonas acidovorans 256–1 among these microorganisms was aerobically cultured in media containing 0.5 % of DEHP, DEHP disappeared completely in 72 hr when assayed gaschromatographically. Most of phthalate esters could be assimilated, regardless of their side-chain types. In addition, branched-alkyl phthalate was assimilated better than n-alkyl phthalate. Based on degradation rate of n-alkyl phthalate in relation to its side chain and carbon number, two peaks were observed in n-alkyl phthalate with four and seven carbon number on its side-chain.     

Genotoxicity studies of di(2-ethylhexyl)phthalate and its metabolites in CHO cells

The widely used plasticizer di(2-ethylhexyl)phthalate (DEHP), its hydrolysis products mono(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol, and also phthalic acid have been tested for clastogenic activity in cultured Chinese hamster ovary (CHO) cells. Only MEHP was found to cause chromosome damage. MEHP was without effect in the SCE and HGPRT mutation test in CHO cells. The clastogenicity of MEHP suggests a role for this compound in the observed carcinogenicity of DEHP and its positive effect in the dominant lethal assay.     

Elucidation of the toxic mechanism of the plasticizers,phthalic acid esters,putative endocrine disrupters: effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats

We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan-niacin metabolism.     

Removal of Phthalate Esters in Soil Column Inoculated with Microorganisms

The removal of phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) was efficiently effected by inoculating and retaining viable cells of Nocardia erythropolis, a bacterium known capable of rapidly degrading phthalate esters, in soil column. When an influent containing 3000 ppm of DEHP was passed through a column inoculated with Nocardia erythropolis, the eluent from the column was gas-chromatographically free of DEHP after 1 day. Residual DEHP on the support after 32 days in the column inoculated with Nocardia erythropolis was only 0.14% against the total amount of DEHP fed, whereas it was 5.2% in the uninoculated column. Microorganisms capable of utilizing DEHP were isolated from the inoculated and un- inoculated columns after 32 days operation and identified. The DEHP utilizing microorganisms in the inoculated column were found to belong to Nocardia erythropolis, Nocardia restricta and Pseudomonas putida (Biotype B), and those in the uninoculated column to Nocardia erythropolis, Pseudomonas putida (Biotype A and B) and Pseudomonas acidovorans. In particular, strain 1-1 of Nocardia erythropolis isolated from the inoculated column was morphologically and biochemically identical with the inoculated Nocardia erythropolis S-l. Ratio of all Nocardia erythropolis to total cells recovered increased from 10.8% immediately after inoculation to 27.2% after 32 days in inoculated column.     

A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K_m and V_max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

Identification of a toxic mechanism of the plasticizers,phtahlic acid esters,which are putative endocrine disrupters: time-dependent increase in quinolinic acid and its metabolites in rats fed di(2-ethylhexyl)phthalate

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.     

A mono-2-ethylhexyl phthalate hydrolase from a Gordonia sp. that is able to dissimilate di-2-ethylhexyl phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K(m) and V(max) values for mono-2-ethylhexyl phthalate were 26.9 +/- 4.3 microM and 18.1 +/- 0.9 micromol/min . mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

Removal of Phthalate Esters by Activated Sludge Inoculated with a Strain of Nocardia erythropolis

Phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) and di-n-butyl phthalate (DBP), were efficiently removed from wastewater by inoculating viable cells of Nocardia erythropolis, a bacterium capable of rapidly degrading phthalate esters, in activated sludge. When the wastewater containing 1500 ppm of DEHP was treated with the activated sludge inoculated with Nocardia erythropolis, the DEHP was found to be removed at a rate of 98.2% in 1 day and to be gas-chromatographically free on and after the 3rd day. Activated sludges, in particular, when high concentration of substances was used, were efficiently prevented from deflocculation of sludge by inoculation of Nocardia erythropolis, and moreover, the deflocked sludge was restored and recovered by the addition of Nocardia erythropolis.     

Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [¹⁴C]PA and [¹⁴C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [¹⁴C]DEHP to ¹⁴CO₂ increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of ¹⁴C-labelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [¹⁴C]PA and [¹⁴C]DEHP. The ¹⁴C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable ¹⁴C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the ¹⁴CO₂ produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [¹⁴C]PA or [¹⁴C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).     

Identification of a Toxic Mechanism of the Plasticizers,Phtahlic Acid Esters,which are Putative Endocrine Disrupters: Time-dependent Increase in Quinolinic Acid and Its Metabolites in…

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.     

邻苯二甲酸酯在不同品种通菜-土壤系统中的累积效应研究

在邻苯二甲酸(2-乙基己基)酯(DEHP)不同污染程度的水稻土上盆栽不同品种通菜,应用GC／MS联机检测技术研究了通菜-土壤系统中DEHP的环境行为与归宿．结果表明,通菜中DEHP的含量为0．335～12．710mg·kg^-1,与土壤的污染程度呈正相关．不同品种通菜之间对DEHP的吸收累积效应存在显著差异,与其叶子大小存在一定的正相关关系．种植不同品种通菜后土壤中DEHP的残留量(1．424～19．834mg·kg^-1)存在显著差异．通菜对土壤中DEHP的BCFs都小于1．0,与土壤的污染程度呈负相关．不同通菜品种的BCFs之间存在显著差异,中等叶子通菜品种的BCFs相对较大。     

Phthalates Are Metabolised by Primary Thyroid Cell Cultures but Have Limited Influence on Selected Thyroid Cell Functions In Vitro

Phthalates are plasticisers added to a wide variety of products, resulting in measurable exposure of humans. They are suspected to disrupt the thyroid axis as epidemiological studies suggest an influence on the peripheral thyroid hormone concentration. The mechanism is still unknown as only few in vitro studies within this area exist. The aim of the present study was to investigate the influence of three phthalate diesters (di-ethyl phthalate, di-n-butyl phthalate (DnBP), di-(2-ethylhexyl) phthalate (DEHP)) and two monoesters (mono-n-butyl phthalate and mono-(2-ethylhexyl) phthalate (MEHP)) on the differentiated function of primary human thyroid cell cultures. Also, the kinetics of phthalate metabolism were investigated. DEHP and its monoester, MEHP, both had an inhibitory influence on 3''-5''-cyclic adenosine monophosphate secretion from the cells, and MEHP also on thyroglobulin (Tg) secretion from the cells. Results of the lactate dehydrogenase-measurements indicated that the MEHP-mediated influence was caused by cell death. No influence on gene expression of thyroid specific genes (Tg, thyroid peroxidase, sodium iodine symporter and thyroid stimulating hormone receptor) by any of the investigated diesters could be demonstrated. All phthalate diesters were metabolised to the respective monoester, however with a fall in efficiency for high concentrations of the larger diesters DnBP and DEHP. In conclusion, human thyroid cells were able to metabolise phthalates but this phthalate-exposure did not appear to substantially influence selected functions of these cells.     

Responses of soil microbial community and enzymes during plant-assisted biodegradation of di-(2-ethylhexyl) phthalate and pyrene

Abstract

A pot experiment was conducted to explore the plant-assisted degradation efficiency of di-(2-ethylhexyl) phthalate (DEHP) and pyrene. Three plant species: Ceylon spinach, sunflower, and leaf mustard were cultivated in co-contaminated soils under three contamination levels: control (T0), 20?mg kg^?1 (T20), and 50?mg kg^?1 (T50). The results showed that a higher DEHP and pyrene degradation efficiency was observed evidently in planted cases, increasing from 42 to 53–59% (T0), 61 to 65–76% (T20) and 52 to 68–78% (T50) for DEHP, and from 22 to 30–49% (T0), 58 to 62–72% (T20), and 54 to 57–70% (T50) for pyrene. Under T20 contamination level, soil phospholipid fatty-acid analysis depicted the increased microbial biomass in rhizosphere, especially the arbuscular mycorrhizal fungus that is effective for the degradation of organic pollutants. The study also revealed that the activities of dehydrogenase, acid phosphomonoesterase, urease, and phenol oxidase negatively correlated with pollutant concentration. In general, the removal rate of DEHP and pyrene was highest in the soil planted with leaf mustard for each contamination level considered. For soils at T20 level, sunflower and leaf mustard appeared as interesting phytoremediation plants due to the improved removal rates of organic pollutants and the soil microbial activity.     

Plant Uptake and Enhanced Dissipation of Di(2-Ethylhexyl) Phthalate (DEHP) in Spiked Soils by Different Plant Species

This study was conducted to investigate the uptake, accumulation and the enhanced dissipation of di(2-ethylhexyl) phthalate (DEHP) spiked in soil (with a concentration of 117.4 ± 5.2 mg kg^?1) by eleven plants including eight maize ( Zea mays) cultivars and three forage species (alfalfa, ryegrass and teosinte). The results showed that, after 40 days of treatment, the removal rates of DEHP ranged from 66.8% (for the control) to 87.5% (for the maize cultivar of Huanong-1). Higher removal rate was observed during the first 10 days than the following days. Plants enhanced significantly the dissipation of DEHP in soil. Enhanced dissipation amount in planted soil was 13.3–122 mg pot^?1 for DEHP, and a net removal of 2.2%–20.7% of the initial DEHP was obtained compared with non-plant soil. The contribution of plant uptake to the total enhanced dissipation was <0.3%, and the enhanced dissipation of soil DEHP might be derived from plant-promoted biodegradation and sorption stronger to the soil. Nevertheless, the capability in accumulation and enhanced dissipation of DEHP from spiked soils varied within different species and cultivars.     

Thermal and enzymatic pretreatment of sludge containing phthalate esters prior to mesophilic anaerobic digestion

The present study aimed at investigating the effect of thermal pretreatment of sludge at 70 degrees C on the anaerobic degradation of three commonly found phthalic acid esters (PAE): di-ethyl phthalate (DEP), di-butyl phthalate (DBP), and di-ethylhexyl phthalate (DEHP). Also, the enzymatic treatment at 28 degrees C with a commercial lipase was studied as a way to enhance PAE removal. Pretreatment at 70 degrees C of the sludge containing PAE negatively influenced the anaerobic biodegradability of phthalate esters at 37 degrees C. The observed reduction of PAE biodegradation rates after the thermal pretreatment was found to be proportional to the PAE solubility in water: the higher the solubility, the higher the percentage of the reduction (DEP > DBP > DEHP). PAE were slowly degraded during the pretreatment at 70 degrees C, yet this was probably due to physicochemical reactions than to microbial/biological activity. Therefore, thermal pretreatment of sludge containing PAE should be either avoided or combined with a treatment step focusing on PAE reduction. On the other hand, enzymatic treatment was very efficient in the removal of PAE. The enzymatic degradation of DBP, DEP, and DEHP could be one to two orders of magnitude faster than under normal mesophilic anaerobic conditions. Moreover, the enzymatic treatment resulted in the shortest half-life of DEHP in sludge reported so far. Our study further showed that enzymatic treatment with lipases can be applied to raw sludge and its efficiency does not depend on the solids concentration.     

Biological monitoring of the five major metabolites of di-(2-ethylhexyl)phthalate (DEHP) in human urine using column-switching liquid chromatography-tandem mass spectrometry

We present a fast and reliable on-line clean-up HPLC-method for the simultaneous determination of the five major urinary metabolites of di-(2-ethylhexyl)phthalate (DEHP) namely mono-(2-ethyl-5-carboxypentyl)phthalate (5carboxy-MEPP), mono-[2-(carboxymethyl)hexyl]phthalate (2carboxy-MMHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono-(2-ethylhexyl)phthalate (MEHP). These metabolites represent about 70% of an oral DEHP dose. We for the first time succeeded to reliably quantify 5carboxy-MEPP and to identify 2carboxy-MMHP as major metabolites in native urines of the general population. The analytical procedure consists of an enzymatic hydrolysis, on-line extraction of the analytes from urinary matrix by a restricted access material column (RAM), back-flush transfer onto the analytical column (betasil phenylhexyl), detection by ESI-tandem mass spectrometry and quantification by isotope dilution (limit of detection (LOD) 0.25 microg/l). Median concentrations of a small collective taken from the general population (n=19) were 85.5 microg/l (5carboxy-MEPP), 47.5 microg/l (5OH-MEHP), 39.7 microg/l (5oxo-MEHP), 9.8 microg/l (MEHP) and about 37 microg/l (2carboxy-MMHP). The presented method can provide insights into the actual internal burden of the general population and certain risk groups. It will help to further explore the human metabolism of DEHP-an occupational and environmental toxicant of great concern.