Gabriele Leng

Biological monitoring of pyrethroids in blood and pyrethroid metabolites in urine: applications and limitations

The objective of this study was to perform biological monitoring of subjects who are occupationally exposed to pyrethroids. The study group consisted of 30 pest control operators exposed to cyfluthrin, cypermethrin or permethrin. After exposure, 24-h urine samples were collected and 20 ml of blood was drawn. The pyrethroid metabolites cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzoic acid and fluorophenoxybenzoic acid were determined in the urine samples (limit of detection: 0.5 micrograms/l) by GC MS and the pyrethroids in plasma (limit of detection: 5 micrograms) GC-ECD. The concentrations of metabolites in the urine of the pest control operators ranged between < 0.5 micrograms/l and 277 micrograms/l urine. The concentrations of cyfluthrin, cypermethrin and permethrin in the plasma were below the limits of detection (< 5 micrograms/l). To test if the metabolites are specific for pyrethroid exposure, they were determined in the urine of non-exposed subjects (n = 40). In no case could pyrethroid metabolites be detected. A cyfluthrin elimination experiment showed that cyfluthrin metabolites are eliminated following first-order kinetics (t 1/2 = 6.4 h). Storage experiments demonstrate that frozen urine samples (-21 degrees C) show no significant losses of metabolites within a year. In contrast, pyrethroids stored in plasma are susceptible to further biodegeneration.

Human dose-excretion studies with the pyrethroid insecticide cyfluthrin : urinary metabolite profile following inhalation

1. Nine male volunteers were exposed to the pyrethroid insecticide cyfluthrin. The study was performed in an exposure room, where an aerosol containing cyfluthrin was sprayed to obtain atmospheres with mean cyfluthrin concentrations of 160 and 40 micrograms/m3. Four volunteers were exposed for 10, 30 and 60 min at 160 micrograms/m3 and another five volunteers were exposed for 60 min at 40 micrograms/m3. For 160 micrograms/m3 exposure urine samples were collected before and immediately after exposure as well as for the periods 1-2, 2-3, 3-4, 4-5, 5-6, 6-12 and 12-24 h after exposure. For 40 micrograms/m3 exposure urine samples were collected before and 2 h after exposure. 2. The main urinary cyfluthrin metabolites, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropane carboxylic acid (DCCA) and 4-fluoro-3-phenoxybenzoic acid (FPBA), were determined. The limit of detection (LOD) for all metabolites was 0.0025 microgram in an urine sample of 5 ml (0.5 microgram/l). After inhalative exposure of 40 micrograms cyfluthrin/m3 air for 60 min, the amount of metabolites in urine collected in the first 2 h after exposure was less than the LOD, namely 0.14 microgram for cis-DCCA, 0.15-0.28 microgram for trans-DCCA and 0.12-0.23 microgram for FPBA. 3. Of the metabolites, 93% was excreted within the first 24 h (peak excretion rates between 0.5 and 3 h) after inhalative exposure of 160 micrograms/m3. The mean half-lives were 6.9 h for cis-DCCA, 6.2 h for trans-DCCA and 5.3 h for FPBA. 4. The mean trans-:cis-DCCA ratio was 1.9 for the time course as well as for each subject. 5. The amount of metabolites in urine depends on the applied dose, on the exposure time and shows interindividual differences.

Biological monitoring of pyrethroid metabolites in urine of pest control operators

Due to their low mammalian toxicity but high insectical activity, pyrethroids are increasingly used for pest control. The objective of the present study was the development of a biological monitoring program to determine exposure to pyrethroids. A diastereoselective detection of the major pyrethroid metabolites cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzoic acid and fluorophenoxybenzoic acid by capillary gas chromatography in combination with mass selective detection was applied. The limits of determination ranged between 0.5 and 1 microgram/l urine, depending on the metabolite concerned. It was demonstrated that pyrethroid metabolites were detectable in urine for a period of elimination up to 3.5 days after exposure to cyfluthrin. Fluorophenoxybenzoic acid was shown to be a suitable biomarker for exposure to cyfluthrin. The presented method was adequate for monitoring pyrethroids in occupationally exposed subjects.

Immunological parameters in humans exposed to pesticides in the agricultural environment

Immune parameters were examined in 224 sera of non-exposed controls and in 304 sera of pesticide applicators in the agricultural environment. In comparison to the control group pesticide applicators showed significant increased odds ratios for neopterin and soluble tumor necrosis factor receptor (sTNF RII) and a decreased odds ratio for immunoglobulin M. Obtained results indicate an enhanced macrophage activation and an impaired humoral defense. These alterations have been found to correlate with exposure duration in the group of pesticide applicators in agriculture. For subjects who worked in indoor pest control an inverse correlation for sTNF RII with exposure duration was obtained indicating impairment of cell mediated immune function. It can be concluded that exposure to pesticides in the agricultural environment may contribute to modulation of the immune system. Since immune modulating agents can potentially lead to adverse health consequences the involvement of immune biomarkers in pesticide-related health studies seems to be of considerable value for risk assessment studies.

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

SummaryAn analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 μg L−1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.

The influence of individual susceptibility in pyrethroid exposure

The aim of this study was to find a suitable biomarker for pyrethroid adverse effects. It was shown that there is a correlation between the half-life time (t(1/2)) of pyrethroids in plasma and the clinical findings. We hypothized that this finding indicates an interindividual different amount of total esterase activity or even a polymorphism. By in vitro experiments it was demonstrated that pyrethroids are cleaved by carboxylesterases. After it turned out that carboxylesterase activity in human plasma is too low for detection, a method for specific determination of carboxylesterase activity in human isolated lymphocytes was developed. As a substrate for carboxylesterase activity, cyfluthrin was added to the lymphocyte suspension. As a proof for cyfluthrin degradation by carboxylesterases the produced hydrocyanic acid was determined by GC/MS. First hints for interindividual differences in carboxylesterase activity in lymphocytes were found.

Induction of mitotic cell division disturbances and mitotic arrest by pyrethroids in V79 cell cultures

Five pyrethroids (fenvalerate, deltamethrin, cypermethrin, permethrin, cyfluthrin) differing in their chemical purity were investigated on their cytotoxic effects, especially on their ability to induce mitotic cell division disturbances using Chinese hamster lung cells of line V79. The colony forming ability (CFA) resulted in distinct differences of the cytotoxic effect of the tested pyrethroids, whereby permethrin was found to be most toxic. With the exception of fenvalerate all tested pyrethroids gave rise to inhibition of cell cycle progression as shown by G2/M-arrest of synchronized V79 cells by flow cytometry as well as by the increase of the mitotic index as evaluated by light microscopy. The mitotic arresting activity could be attributed to the occurrence of abnormal mitotic figures such as initial and full C-metaphases. The results however indicate, that pyrethroids per se do not contribute to the cytotoxic effects but that other factors such as chemical impurities, source as well as manufacturing process and isomer composition may be responsible for the observed cytotoxic effects.

Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers - Characterization of suitable biomarkers for human biomonitoring

Di(2-propylheptyl) phthalate (DPHP), a high molecular weight phthalate, is primarily used as a plasticizer in polyvinyl chloride and vinyl chloride copolymers for technical applications, as a substitute for other phthalates currently being scrutinized because of endocrine disrupting effects. We determined urinary excretion fractions of three specific DPHP metabolites (mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP), mono-2-(propyl-6-oxoheptyl)-phthalate (oxo-MPHP) and mono-2-(propyl-6-carboxy-hexyl)-phthalate (cx-MPHxP)) after oral dosing of five volunteers with 50mg labelled DPHP-d4 and subsequent urine sampling for 48 h. These excretion fractions are used to back calculate external intakes from metabolite measurements in spot urine analysis. Following enzymatic hydrolysis to cleave possible conjugates, we determined these urinary metabolites by HPLC-NESI-MS/MS with limits of quantification (LOQ) between 0.3 and 0.5 μg/l. Maximum urinary concentrations were reached within 3-4h post dose for all three metabolites; elimination half-lives were between 6 and 8h. We identified oxo-MPHP as the major oxidized metabolite in urine representing 13.5±4.0% of the DPHP dose as the mean of the five volunteers within 48 h post dose. 10.7±3.6% of the dose was excreted as OH-DPHP and only 0.48±0.13% as cx-MPHxP. Thus, within 48 h, 24.7±7.6% of the DPHP dose was excreted as these three specific oxidized DPHP metabolites, with the bulk excreted within 24h post dose (22.9±7.3%). These secondary, oxidized metabolites are suitable and specific biomarkers to determine DPHP exposure. In population studies, however, chromatographic separation of these metabolites from other isomeric di-isodecyl phthalate (DIDP) metabolites is warranted (preferably by GC-MS) in order to distinguish DPHP from general DIDP exposure. Palatinol(®), Hexamoll(®) and DINCH(®) are registered trademarks of BASF SE, Germany.

Analytical method for the sensitive determination of major di-(2-propylheptyl)-phthalate metabolites in human urine.

Di-(2-propylheptyl)-phthalate (DPHP) is a specific phthalic acid ester of isomeric C10 alcohols. It is classified as high molecular weight phthalate and marketed as plasticizer for polyvinyl chloride (PVC). The increase of its production volume and its wide field of application suggest a possible background exposure of the human population as found for other phthalates, making suitable analytical methods necessary. The aim of the presented analytical report is the sensitive and selective determination of the three major DPHP metabolites mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP), mono-2-(propyl-6-oxoheptyl)-phthalate (oxo-MPHP) and mono-2-(propyl-6-carboxy-hexyl)-phthalate (cx-MPHxP) in human urine. Most of the published analytical methods for phthalate metabolites use high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The methods presented here allow a comparison of chromatographic separation between HPLC-MS/MS and gas chromatography high resolution mass spectrometry (GC-HRMS), which is useful to distinguish between DPHP and DIDP. The enhanced detection limits range between 0.05-0.1μg/L for GC-HRMS and 0.1-0.2μg/L for HPLC-MS/MS.

Bis-(2-propylheptyl)phthalate (DPHP) metabolites emerging in 24h urine samples from the German Environmental Specimen Bank (1999-2012).

Bis-(2-propylheptyl)-phthalate (DPHP) has been introduced as a substitute for other high molecular weight phthalates primarily used in high temperature applications (e.g. cable wires, roofing membranes). The aim of this study was to investigate how the increased usage of DPHP is reflected in urine samples collected over the last 14 years and to evaluate the current extent of exposure. We analyzed 300 urine samples (24h voids) from the German Environmental Specimen Bank collected in the years 1999, 2003, 2006, 2009 and 2012, 60 samples per year, from 30 male and 30 female volunteers (age: 20-30 years) for three specific, secondary oxidized DPHP metabolites (with hydroxy, oxo and carboxy modifications of the alkyl side chain). We determined DPHP metabolites with a previously developed GC-HRMS method, enabling us to unambiguously distinguish DPHP metabolites from co-eluting, structurally isomeric di-iso-decyl phthalate (DIDP) metabolites. All samples were blinded before analysis. We detected no DPHP metabolites in urine samples from the years 1999, 2003 and 2006. Thereafter, detection rates increased from 3.3% in 2009 to 21.7% in 2012. Mono-oxo-propylheptylphthalate (oxo-MPHP) was the most abundant metabolite, with concentrations between <LOQ and 0.96μg/l. Extrapolating from oxo-MPHP concentrations in the 24h urine samples we calculated a maximum daily DPHP intake of 0.32μg/kg body weight. Our results show that the general German population is increasingly exposed to DPHP. However, exposure is considerably lower than for DIDP or other high molecular weight phthalates. Future measurements will enable us to monitor the development of DPHP exposure and advise risk management steps, if warranted.