3,4 Dichloroaniline-haemoglobin adducts in humans: preliminary data on agricultural workers exposed to propanil

Propanil is one of the major herbicides used on rice-paddies and is thought to produce adverse health effects through the action of its metabolite 3,4-dichloroaniline (3,4-DCA). T he feasibility of monitoring human exposure to propanil on the basis of 3,4-DCA adducts to haemoglobin (Hb) was investigated. We developed a method based on gas chromatography negative ion chemical ionization-mass spectrometry (NICI-GC-MS) to quantify 3,4-DCA released from human Hb after alkaline hydrolysis of the protein. 3,4-DCA-Hb adducts were identified in agricultural workers exposed to propanil and were detectable even 4 months after the last herbicide application. Urine samples collected at the same time had no measurable level of 3,4-DCA. 3,4-DCA-Hb adducts might be useful for monitoring human exposure to 3,4-DCA from agricultural sources.     

Determination of dichloroanilines in human urine by GC–MS, GC–MS–MS, and GC–ECD as markers of low-level pesticide exposure

Methods for the determination of 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) as common markers of eight non-persistent pesticides in human urine are presented. 3,5-DCA is a marker for the exposure to the fungicides vinclozolin, procymidone, iprodione, and chlozolinate. Furthermore the herbicides diuron, linuron, neburon, and propanil are covered using their common marker 3,4-DCA. The urine samples were treated by basic hydrolysis to degrade all pesticides, metabolites, and their conjugates containing the intact moieties completely to the corresponding dichloroanilines. After addition of the internal standard 4-chloro-2-methylaniline, simultaneous steam distillation extraction (SDE) followed by liquid–liquid extraction (LLE) was carried out to produce, concentrate and purify the dichloroaniline moieties. Gas chromatography (GC) with mass spectrometric (MS) and tandem mass spectrometric (MS–MS) detection and also detection with an electron-capture detector (ECD) after derivatisation with heptafluorobutyric anhydride (HFBA) were employed for separation, detection, and identification. Limit of detection of the GC–MS–MS and the GC–ECD methods was 0.03 and 0.05 μg/l, respectively. Absolute recoveries obtained from a urine sample spiked with the internal standard, 3,5-, and 3,4-DCA, ranged from 93 to 103% with 9–18% coefficient of variation. The three detection techniques were compared concerning their performance, expenditure and suitability for their application in human biomonitoring studies. The described procedure has been successfully applied for the determination of 3,4- and 3,5-DCA in the urine of non-occupationally exposed volunteers. The 3,4-DCA levels in these urine samples ranged between 0.13 and 0.34 μg/g creatinine or 0.11 and 0.56 μg/l, while those for 3,5-DCA were between 0.39 and 3.33 μg/g creatinine or 0.17 and 1.17 μg/l.     

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L^?1 h^?1. Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L^?1 h^?1. At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.     

Use of haemoglobin adducts in exposure monitoring and risk assessment

Many industrial bulk chemicals are oxiranes or alkenes that are easily metabolised to oxiranes in mammalian systems. Many oxiranes may react with DNA and are therefore mutagenic in vitro. Some oxiranes have been shown to be carcinogenic in rodents in vivo as well. Despite the very limited evidence of the carcinogenicity of oxiranes in humans, they should be considered potential human carcinogens. As a consequence, exposure to these compounds should be minimised and controlled. Twenty-five years ago, Ehrenberg and co-workers suggested that exposure to oxiranes might be determined through the measurement of the adducts they form with haemoglobin (Hb). Ten years later, a modification of the Edman degradation was developed at Stockholm University that allowed determination of adducts with the N-terminal valine of Hb by GC-MS. In our laboratory, this methodology was modified and adapted for analysis on an industrial scale. Since 1987, exposure of operators in our facilities to ethylene oxide (EO) has been routinely monitored by determination of N-(2-hydroxyethyl)valine in Hb. Biological monitoring programmes for propylene oxide (PO) and 1,3-butadiene (BD) were developed later. In this review, the methodology and its results are discussed as a tool in human risk assessment of industrial chemicals. Two major advantages of Hb adduct determinations in risk assessment are (1) the qualitative information on the structure of reactive intermediates that may be obtained through the mass spectrometry, which may provide insight in the molecular toxicology of compounds such as BD, and (2) the possibility of reliable determination of exposure over periods of several months with limited number of samples for compounds such as ethylene oxide (EO), propylene oxide (PO) and BD which form stable adducts with Hb. Since good correlations between the airborne concentrations of these chemicals with their respective adducts have been established, Hb adducts can also be used to quantitate airborne exposure which is of paramount importance as exposure assessment is usually one of the weaker parameters in risk assessment.     

Characterization of novel linuron-mineralizing bacterial consortia enriched from long-term linuron-treated agricultural soils

Linuron-mineralizing cultures were enriched from two linuron-treated agricultural soils in the presence and absence of a solid support. The cultures contained linuron-degrading bacteria, which coexisted with bacteria degrading either 3,4-dichloroaniline (3,4-DCA) or N,O-dimethylhydroxylamine (N,O-DMHA), two common metabolites in the linuron degradation pathway. For one soil, the presence of a solid support enriched for linuron-degrading strains phylogenetically related to but different from those enriched without support. Most linuron-degrading consortium members were identified as Variovorax, but a Hydrogenophaga and an Achromobacter strain capable of linuron degradation were also obtained. Several of the linuron-degrading isolates also degraded 3,4-DCA. Isolates that degraded 3,4-DCA but not linuron belonged to the genera Variovorax, Cupriavidus and Afipia. Hyphomicrobium spp. were involved in the metabolism of N,O-DMHA. Whereas several isolates degraded linuron independently, more efficient degradation was achieved by combining linuron and 3,4-DCA-degraders or by adding casamino acids. These data suggest that (1) linuron degradation is performed by a group of metabolically interacting bacteria rather than by individual strains, (2) there are other genera in addition to Variovorax that degrade linuron beyond 3,4-DCA, (3) linuron-degrading consortia of different origins have a similar composition, and (4) interactions between consortium members can be complex and can involve exchange of both metabolites and other nutrients.     

Monitoring for occupational exposure to 4,4′-methylenebis(2-chloroaniline) by gas chromatographic-mass spectrometric analysis of haemoglobin adducts, blood, plasma and urine

The feasibility of using plasma, blood and haemoglobin adducts for monitoring occupational exposure to the suspected human carcinogen 4,4′-methylenebis(2-chloroaniline) (MOCA) was investigated. A method utilising capillary gas chromatography-negative-ion chemical-ionisation mass spectrometry (GC-MS) for the determination of pentafluoropropionyl (PFP) derivatives of MOCA, released by alkaline hydrolysis from protein adducts and conjugates, was both sensitive and selective. When selected ion monitoring was used, sub-femtomole amounts of PFP-MOCA could be measured. The detection limit for haemoglobin adducts of MOCA was below 10 fmol/g Hb, well below the levels found for occupationally exposed individuals. Capillary GC with electron-capture detection also had the required sensitivity for the determination of MOCA in blood and urine of five individuals who were exposed to MOCA during the manufacture of polyurethane elastomers were determined by the GC-MS method. The MOCA concentrations for the various blood fractions and urine were within the following ranges: haemoglobin adducts, 0.73–43.3 pmol MOCA/g Hb; plasma alkaline hydrolysate, 0.05–22.0 nmol/l; whole blood, 0.13–17.4nmol/l; urine, 4.5–2390 nmol/l. Because the products of MOCA in the blood reflect metabolic activation of MOCA and integrate exposure over a period of weeks, the use of blood samples for monitoring exposure to MOCA offers advantages over the currently used urinary MOCA measurements.     

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040-589 nmol g^-1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10-10 000 pmol N-(2-hydroxethyl)valine g^-1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040–589?nmol?g^?1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10–10?000 pmol N-(2-hydroxethyl)valine?g^?1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^?3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Haemoglobin adducts as biomarkers of exposure to the herbicides propanil and fluometuron

Abstract

Aromatic amine herbicides, including propanil, fluometuron, alachlor, trifluralin, and pendimethalin, were examined for their ability to form haemoglobin adducts in rats as potential biomarkers of exposure. Many aromatic amines are known to form haemoglobin adducts via conversion to the nitroso metabolite and binding of this metabolite to cysteinyl groups on haemoglobin. Since red blood cells are long lived, adducts formed with these cells may be reliable biomarkers of exposure with the potential for showing progressive accumulation. Gas chromatographic-mass spectrometric analyses of haemoglobin revealed that adducts were formed in rats treated with the rice herbicide propanil and the cotton herbicide fluometuron. Adducts were not detected with the herbicides alachlor, trifluralin, or pendamethalin.     

Cytogenetic effects of 3,4-dichloroaniline in human lymphocytes and V79 Chinese hamster cells

3,4-Dichloroaniline (3,4-DCA), an intermediate in various chemical syntheses, has been detected as an environmental contaminant in surface waters and in the effluents from dye-manufacturing plants. Tested for clastogenicity in human lymphocytes in vitro the compound was inactive in the chromosome aberration assay yet exhibited a positive sister-chromatid exchange response in the presence of a mammalian metabolic activation system. Exposure of V79 Chinese hamster cells to 3,4-DCA caused a concentration-dependent increase in the incidence of spindle disturbances, predominantly of the initial c-mitotic type. The results indicate that 3,4-DCA might induce aneuploidy in mammalian cells by interaction with the mitotic apparatus.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring.

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7-992 nmol g-1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2-250 pmol g-1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.     

Determination of hemoglobin adducts in workers exposed to 2,4,6-trinitrotoluene

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. TNT can be taken up through the skin and by inhalation. It is therefore essential to have fast and reliable methods to monitor human exposure. In rat experiments, it has been shown that TNT binds covalently to blood proteins and to tissue proteins. Hemoglobin (Hb) adducts of TNT are markers for the internal dose and possibly for the toxic effects of TNT, e.g. cataracts. In the present paper we introduce a new efficient method to quantify Hb adducts of TNT. Precipitated Hb was hydrolyzed with base in the presence of the surrogate internal standard 3,5-dinitroaniline (35DNA). The released 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT) were quantified against 35DNA by gas chromatography-mass spectrometry with negative-ion chemical ionization. Hb of 50 workers and controls from a Chinese munition factory were investigated. The Hb adduct levels ranged from 3.7 to 522 ng for 4ADNT and from 0 to 14.7 ng for 2ADNT per gram of Hb. However, in control samples from Germany no Hb adducts of 4ADNT or 2ADNT could be found.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.     

Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Effets sur le spermatozoïde humain du Diuron (3-(3,4-dichlorophényl)-1,1-diméthyl-urée) et de l’un de ses produits de transformation, la 3,4-dichloroaniline (3,4-DCA) (Etude préliminaire)

Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides.     

Monitoring of occupational exposure to epichlorohydrin by genetic effects and hemoglobin adducts

The present work is focused on the determination of in vivo doses and studies of genetic effects in workers exposed to epichlorohydrin (ECH). The studied endpoints were hemoglobin (Hb) adducts, frequencies of hprt mutants, micronuclei in cytochalasin B blocked binucleated lymphocytes, sister chromatid exchanges (SCE) and high frequency cells (HFC). Blood samples were collected from office clerks and ECH exposed factory workers at an industrial plant in Germany. The workers were exposed to 0.11–0.23 ppm ECH in the air 45 h per week and to 0.2–2.6 ppm for 3 h per week. Some Swedish non-exposed subjects were also used for Hb adduct measurements. The genetic data, HFC and SCE, showed a significant difference between exposed and unexposed donors. In contrast to earlier studies on SCE, no impact of smoking was observed. Effects on micronuclei were on the borderline of significance, whereas there was no effect for HPRT mutants. The average Hb adduct level was higher in exposed than in non-exposed donors, although the difference was only significant when the exposed group was compared to Swedish controls. Smoking gave significantly increased adduct levels. The absence of significant correlations between individual data for Hb adducts and genetic effects, may be explained by the different periods of time covered by the responses in these endpoints. Whereas Hb adducts reflect the exposure during up to 4 months (i.e. the life span of human erythrocytes), the SCE, and particularly the HFC, seem to accumulate for years in a long-lived fraction of T-lymphocytes without DNA repair. Thus, the adduct data does not reflect the exposure backwards in time unless it can be shown that exposure conditions have remained unchanged. The origin of the background adduct levels in non-smoking control persons is at present not known.     

Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring

Abstract

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7–992 nmol g^?1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2–250 pmol g^?1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.