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Biological monitoring of exposure to organophosphate pesticides
Toxicology Letters 134 (2002) 97 – 103 www.elsevier.com/locate/toxlet
Biological monitoring of exposure to organophosphate pesticides J. Cocker *, H.J. Mason, S.J. Garfitt, K. Jones Health and Safety Laboratory, Broad Lane, Sheffield S3 7 HQ, UK Received 21 September 2001; accepted 27 February 2002
Abstract Organophosphates (OPs) are readily absorbed through the skin and biological monitoring is an essential component of any comprehensive assessment of exposure. This paper presents a summary of our experience in a wide range of occupational studies. Additionally, we have conducted studies of non-occupational exposure and human volunteer studies looking at the kinetics of chlorpyrifos, propetamphos, diazinon and malathion. In non-occupationally exposed people, 95% of urinary alkyl phosphates do not exceed 72 mmol/mol creatinine. In occupationally exposed people, the corresponding 95th percentile of total urinary alkyl phosphates is 122 mmol/mol creatinine. In volunteer studies with 1 mg oral doses of chlorpyifos, diazinon and propetamphos the mean peak values were 160, 750 and 404 mmol/mol creatinine, respectively, and were not associated with any reduction in blood cholinesterase activity. The levels of OP metabolites seen in urine from workers potentially exposed to OPs are generally low and unlikely to cause significant reduction in blood cholinesterase activity. Crown Copyright © 2002 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Biological monitoring; Organophosphate metabolites; Occupational exposure; Cholinesterase
1. Introduction Organophosphate pesticides (OPs) are widely used and readily absorbed through the skin. Occupational exposure limits (ACGIH, 2001; HSE, 2001; DFG, 2001) for OPs usually have ‘skin’ notations to warn of the potential for dermal absorption to contribute to systemic toxicity. As a consequence, biological monitoring is an essential component of any comprehensive assessment of * Corresponding author. Tel.: + 44-114-289-2691; fax: + 44-114-289-2850 E-mail address: [email protected] (J. Cocker).
exposure. Historically, this was biological effect monitoring (BEM) by measurement of the reduction of blood cholinesterase activity. This involved the measurement of plasma cholinesterase(ChE) and erythrocyte acetyl cholinesterase (AChE), reflecting the influence of absorbed OPs to inhibit these blood enzymes as surrogates of AChE in neural tissue and neuromuscular junctions. However, it is well recognised that this is a relatively insensitive indicator of an absorbed dose of OP (Reid and Watts, 1981; Drevenkar et al., 1991; Nutley and Cocker, 1993; Hardt and Angerer, 2000). Blood cholinesterase activity
0378-4274/02/$ - see front matter. Crown Copyright © 2002 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(02)00168-6
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needs at least 15% depression from an individual’s normal level of plasma or erythrocyte enzyme activity to be considered indicative of pesticide over-exposure. Furthermore, due to the large inter-individual variability in cholinesterase activity this approach requires the collection of both postexposure and baseline samples from an individual. It also requires long term precision of the methods as this directly influences the level of ChE or AChE depression that can be considered significant (Mason and Lewis, 1989). In addition, the collection of blood samples is sometimes considered as invasive and in some occupational settings is logistically difficult. An alternative, or complementary, approach to biological monitoring for OPs is based on the analysis of metabolites in urine. These methods can either use metabolites specific to the OP under study or the dialky phosphate metabolites (DAPs) that are common to a large number of different OPs. The measurement DAPs has been used for many years to study exposure to a wide range of OPs. Studies have looked at single OPs like malathion (Bradway and Shafik, 1977; Fenske, 1988; Muan and Skare, 1989), gluthion (Franklin et al., 1981), azinphos methyl (Franklin et al., 1983, 1986), Terbufos (Peterson et al., 1985), chlorpyrifos-methyl (Aprea et al., 1997), or mixtures of OPs (Lores et al., 1978; Davies et al., 1979; Saito et al., 1984; Griffith and Duncan, 1985; Duncan and Griffith, 1985; Drevenkar et al., 1991; Nutley and Cocker, 1993; Aprea et al., 1996; Gompertz, 1996; Hardt and Angerer, 2000; Aprea et al., 2001). This paper reflects our ideas and experience gained from the area of occupational health, where on a routine and research basis we are involved in monitoring the levels of exposure in workers to chemicals including OPs. Some of the research studies have involved ethical human volunteer experiments and some have been field studies of occupational exposure.
2. Biological effect monitoring for organophosphate pesticides Measurements of plasma cholinesterase (ChE)
and erythrocyte acetylcholinesterase (AChE) have been used for a number of years in cases of clinical poisoning and accidental OP exposure, and in monitoring of workers with high risk of exposure. Depression of the plasma ChE enzyme activity is not necessarily associated with symptoms of anti-cholinergic toxicity and large depressions in ChE have been noted in the absence of any effect on erythrocyte AChE. Decreases in the red cell enzyme activity have been suggested to have closer relations to these symptoms. Therefore, in both clinical toxicology and monitoring high-risk occupational activities, the measurement of both enzymes has been recommended (HSE, 2000; Heath and Vale, 1992). In cases of severe OP exposure the level of depression of both plasma ChE and erythrocyte AChE leaves no ambiguity. But in interpreting more subtle changes in the enzymes’ activities or to detect lower levels of OP absorption, several factors need to be understood for proper interpretation. There is wide inter-individual variation in normal, unexposed subjects, particularly in plasma ChE activity and therefore, comparison with a result against a ‘population normal range’ is not a sensitive means of detecting excessive OP absorption in an individual. It is therefore, necessary to have a baseline or unexposed ChE activity for the individual. In on-going workplace biological monitoring schemes this causes no problems. However, in cases of accidental exposure, a definitive interpretation on a measurement entails a considerable delay until a true unexposed baseline measurement can be taken. This period has been suggested as being 60 days (HSE, 2000), when using both enzymes, and our work supports this. Regular monitoring of a formulation worker with two high exposure incidents (Fig. 1) clearly shows the long recovery period especially for AChE. Although OPs are often considered together, there are substantial differences between the OPs in their ability to inhibit either ChE or AChE. In-vitro human enzyme studies at HSL have looked at the OP concentration that causes a 50% inhibition after a 1 h incubation with the respective OP. We found 100–200-fold differences in
J. Cocker et al. / Toxicology Letters 134 (2002) 97–103
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Fig. 2. General structure of OP pesticide.
Fig. 1. Depression of blood cholinesterases in a worker overexposed to OP pesticides.
potency between OPs (Table 1). For example, the active product of malathion (malaoxon) is less potent at phosphorylating human cholinesterase than chlorpyrifos. These data are taken under conditions where metabolic detoxification by Aesterases in human blood are not active. In-vivo, malathion is susceptible to such metabolism rendering it less likely to phosphorylate and inactivate key enzymes and therefore less toxic. Thus, blood cholinesterase measurements, although relatively insensitive as markers of general OP absorption in comparison with urinary alkyl phosphates measurements, are better at reflecting
Table 1 Concentration of OP inhibiting human plasma cholinesterase and erythrocyte acetylcholinesterase by 50% IC50 after 1 h incubation Direct inhibitory form of OP used to inhibit
IC50 Plasma Che (mM)
Chlorpyrifos-oxon 0.12 (Chlorpyrifos) Malaoxon 12.3 (Malathion) Chlorfenvinphos 0.28 Azinphosmethyloxon 0.06 (Azinphosmethyl)
IC50 Erythrocyte AChe (mM)
1 7.5 3.3 0.07
the likely potency of the absorbed dose of OP (or OP mixture) to cause a significant anticholinergic effect. The major problem with cholinesterase measurements is their reliance on trying to measure a small decrease in enzyme activity from the normal level of activity in that individual.
3. Biological monitoring of organophosphate pesticides The general structure of OPs is given in Fig. 2. In the majority of OP pesticides the R1 and R2 groups are represented by either methyl or ethyl groups, whereas, the chemical structure of X moiety in the OP structure largely defines the majority of the structural differences between individual OP pesticides. OP pesticides are often used in the ‘thio’ form (PS), where metabolic oxidative desulphuration is necessary to produce an OP with anti-cholinesterase activity (PO form). This toxicological activation step is carried out by cytochrome P450s, including CYP 3A4, after absorption of the OP and inter-individual variation in activity of these enzymes may have implications for individual suceptibility (Sams et al., 2000; Mason et al., 2000). A major metabolic route in humans is hydrolysis of the PX bond, producing dialkyl phosphate metabolites that are excreted in urine. Therefore there is the potential to measure the X moiety that would be specific to a single OP (e.g. trichloropyridinol for chlorpyrifos) or to measure the phosphate moiety (as one or more of six alkylphosphates). The advantage of the former approach is the specificity of the assay but it has the disadvantage of requiring many analytical methods to detect the range of OPs that workers may be exposed to. Whereas, the analysis of six
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alkylphosphates allows the detection of absorption of about 85% of all OP pesticides in a single assay but without the ability to identify which OP has been absorbed. The measurement of urinary alkylphosphate metabolites in urine has been a routine assay within our laboratory (Nutley and Cocker, 1993) for over 10 years and the data shown in Fig. 3 below represent the monitoring carried out of various work activities and firms. While the measurement of total urinary alkyl phosphate allows investigation of general exposure to OPs, further information can be gained from identifying the level of specific groupings within the six metabolites. For example, segregation of (DMP, DMTP and DMDTP) and (DEP, DETP DEDTP) gives
Fig. 3. Total urinary alkylphosphate measurements in various occcupational groups; Pest control workers (n=7) use OPs in short duration spraying to control inset pest; ‘formulators1 and 2’ reflect monitoring at specific firms (n =118 and 24) which produce pesticide products for amateur and professional use. ‘Control’ reflects levels found in a non-occupationally exposed UK population (n=463). ‘Sheep dipping’ (n= 478) samples from farmers using diazinon based sheep dips. ‘Sheep handlers’ describes samples (n= 21) from workers on sheep farms from activities for about two weeks post-sheep dipping with an OP product. ‘Wool handlers’ (n= 102) reflects measurements in staff within a single firm cleaning and processing wool from fleeces. Orchard (n= 43), hop sprayers (n= 84) and nursery (n =40) are from studies of workers undertaking agricultural and horticultural tasks with OP pesticides.
some idea of the class of OPs causing exposure, discriminating between the dimethoxy OPs, such as dichlorvos or malathion which produce DMP, DMTP and, say, diazinon which produces DEP and DETP. Also the level of DMP and DEP reflects exposure to OPs which could have potentially inhibited acetylcholinesterase, whereas DMTP, DMDTP, DETP and DEDTP reflect detoxification process that protects against any internal level of active OP. The total number of samples from exposed workers is 917. The maximum value found was 915 mmol/mol creatinine, the mean and median were 33 and 15 mmol/mol creatinine, respectively. About 90% of the results in the exposed group were B 77 mmol/mol creatinine and 95% were less than 122 mmol/mol creatinine. The group with the lowest levels of alkyl phosphates in urine was pest control workers. These workers are well trained with appropriate protective equipment and only use OPs for short periods to eradicate insect pests. The groups with the highest levels of alkyl phosphates were the formulators and, to a lesser extent, wool handlers who have prolonged exposure each day and repeated daily exposure. In formulators 90% of the results were B 188 mmol/mol creatinine. The next highest urinary alkyl phosphate levels were in sheep dippers who tended to have 4–8 h exposures on only a few days a year but under difficult conditions. Other groups e.g. agricultural and horticultural workers had intermittent exposures but in conditions where better controls were possible. Many of the samples above were collected in studies that also looked at blood cholinesterase activity. These were samples collected as part of investigations of exposure during sheep-dipping, hop spraying, orchard spraying, wool handling and some of the formulators and there were no exposure related significant depression of blood cholinesterase. The control group shown in the Fig. 3 are from 463 samples collected in a number of different studies of people who had no occupational exposure to OPs and had not recently used OP-containing garden products. The data confirms that the sensitivity of the urinary alkyl phosphate
J. Cocker et al. / Toxicology Letters 134 (2002) 97–103
method is sufficient to investigate environmental exposure. Results showed that the mean and median ‘total’ urinary alkyl phosphate were 22 and 16 mmol/mol creatinine, respectively. The 90– 95% percentile value of ‘total’ urinary alkyl phosphate levels for this unexposed population was between 51 and 72 mmol/mol creatinine, respectively. These values are similar to values reported by Aprea et al. (1996), Hardt and Angerer (2000) in non-occupationally exposed Italian and German populations, respectively. Currently we have no data to show whether levels of alkylphopshate metabolites in non-occupationally exposed people reflects absorption of the intact OP pesticide or of alkylphosphate residue themselves. In an attempt to put the occupational studies into perspective we have also carried out three separate volunteer studies with chlorpyrifos, diazinon and propetamphos under the approval of the Research Ethics Committee of the UK Health and Safety Executive (Griffin et al., 1999; Garfitt et al., 2002a,b). Volunteers (four or five) were given a maximum of 1 mg oral dose and, on separate occasions, dermal doses of 100 mg (28 mg for chlorpyrifos). Urine samples were collected for up to 48 h and blood samples were taken at intervals for cholinesterase determination. No significant depressions of blood cholinesterase were observed and the urine alkyl phosphate levels are shown in Fig. 4. The overall picture shows the occupational levels of alkyl phosphates similar to those found in volunteer studies, which were not associated with reduction in blood cholinesterase activity. We have also been part of a study looking at blood cholinesterase and urine metabolites during and after use of preparations based on malathion to treat head-lice. The preparations used were those generally available to the public and were used in accordance with the instructions in the packet. Exposure was dermal only and gives another perspective on levels of urinary alkyl phosphates found in occupational studies. Fig. 5 shows urinary levels of dimethoxy alkylphosphates (DMP, DMTP, DMDTP) found in samples collected for up to 48 h after the appropriate use of malathion containing headlice shampoo products.
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Fig. 4. Urinary alkyl phosphate levels from occupationally exposed workers (n =917) and volunteers given oral and dermal doses of chlorpyrifos, diazinon and propetamphos. For the occupational group the values are the total of 6 dialkyl phosphates, for the chlorpyrifos and diazinon studies the values are the total of the diethyl phosphate metabolites and for propetamphos are the total MEPT (a mono alkyl phosphate) after hydrolysis. Urine values in the volunteer studies are from samples collected up to 24 h after an oral dose or 48 h after a dermal dose from 5 volunteers.
Malathion is an OP that will only produce dimethoxy alkyl phosphate metabolites (DMP and DMTP). Although the levels of urinary metabolites found were much higher than those in occupational studies there was no depression in blood cholinesterase activity. As noted in Table 1 malathion is a much less potent inhibitor of cholinesterase than other OPs.
4. Conclusions Both biological monitoring by determination of urinary alkyl phosphates and BEM by determination of blood cholinesterase activity have important roles in assessing the exposure of workers to OPs. The traditional approach of blood cholinesterase measurement has a role in reassuring most workers that their exposure is unlikely to
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in urine from workers potentially exposed to OPs is generally low and unlikely to cause significant reduction in blood cholinesterase activity.
References
Fig. 5. OP metabolites in occupational and volunteer studies and in subjects treated with malathion-containing headlice shampoo and controls.
result in any toxicity. It has well defined guidance values to help interpret results and is directly related to risk. Measurement of urinary metabolites is less invasive and logistically simpler. Although there are few guidance values for urinary metabolites, this approach can be used to assess the efficacy of control procedures and help to reduce exposure. It has been widely used in occupational, clinical and environmental studies and there are data to aid the assessment of exposure to OPs. As a perspective on urinary alkyl phosphates the results of studies over the last 10 years can be summarised as follows: In non-occupational exposed people 90% of the total urinary alkyl phosphates are B50 mmol/mol creatinine and 95% are B 72 mmol/mol creatinine. In occupationally exposed people 90% of the total urinary alkyl phosphates are B77 mmol/mol creatinine and 95% are B122 mmol/mol creatinine. In volunteer studies with 1 mg oral doses of chlorpyifos, diazinon and propetamphos the mean peak values were 160, 750 and 404 mmol/mol creatinine, respectively, and were not associated with any reduction in blood cholinesterase activity. Overall, our experience over the last 10 years has shown that the levels of OP metabolites seen
ACGIH (2001) Threshold limit values for chemical substances and physical agents and Biological Exposure Indices. Published by the American Conference of Governmental Industrial Hygienists Cincinnati OH USA ISBN 1-882417-40-2. Aprea, C., Sciarra, G., Orsi, D., Boccalon, P., Sartorelli, P., Sartoreli, E., 1996. Urinary excretion of alkylphosphate in the general population (Italy). Sci. Total Environ. 177, 37 – 41. Aprea, C., Sciarra, G., Sartorelli, P., Sartoreli, E., 1997. Biological monitoring of exposure to chlorpyrifos methyl by assay of urinary dialkyphospahtes and 3,4,6trichloropyridinol. J. Toxicol. Environ. 50, 581 – 594. Bradway, D.E., Shafik, T.M., 1977. Malathion exposure studies, determination of mono and dicarboxylic acids and alkyl phosphates in urine. J. Agric. Food Chem. 25 (6), 1342 – 1344. Aprea, C., Sciarra, G., Lunghini, L., Centi, L., Ceccarelli, 2001. Evaluation of respiratory and cutaneous doses and urinary excretion of alkylphosphates by workers in greenhouses treated with omethoate, fenitrothion and tolclofosmethyl. Am. Ind. Hyg. Assoc. J. 62, 87 – 95. Davies, J.E., Henry, M.P.H., Enos, H.F., Barquet, A., Morgade, C., Danauskas, J.X., 1979. Pesticide monitoring studies. The epidemiologic and toxicologic potential of urinary metabolites. Dev. Toxicol. Environ. Sci. 4, 369 – 378. DFG (2001) Deutsche Forschungsgemeinschaft List of MAK and BAT values Commision for the investigation of health hazards of chemical compounds in the work area report no 36. Published by Wiley-VCH. Duncan, R.C., Griffith, J., 1985. Monitoring study of urinary metabolites and selected symptomatology among Florida citrus workers. J. Toxicol. Environ. Health 16, 509 – 521. Drevenkar, V., Radic, Z., Vasilic, Z., Reiner, E., 1991. Dialkyl phosphate metabolites in the urine and activities of esterases in the serum as biochemical indices for human absorption of organophosphorus pesticides. Arch. Environ. Contamin. Toxicol. 20, 417 – 422. Fenske, R.A., 1988. Correlation of fluorescent tracer measurements of dermal exposure and urinary metabolite excretion during occupational exposure to malathion. Am. Indust. Hyg. J. 49 (9), 438 – 444. Franklin, C.A., Fenske, R.A., Greenhalgh, R., Mathieu, L., Denley, H.V., Leffingwell, J.T., Spear, R.C., 1981. Correlation of urinary pesticide metabolite excretion with estimated dermal contact in the course of occupational exposure to gluthion. J. Toxicol. Environ. Health 7, 715 – 731.
J. Cocker et al. / Toxicology Letters 134 (2002) 97–103 Franklin, C.A., Greenhalgh, R., Maibach, H.I., 1983. Corelation of urinary dialkyl phosphate metabolite levels with dermal exposure to azinphos methyl. In: Miyamoto, Kearney, PC. (Eds.), Pesticide Chemistry: Human welfare and the environment, vol. 4. Pergamon, New York, p. 1983. Franklin, C.A., Muir, N.I., Moody, R.P., 1986a. The use of biological monitoring in the estimation of exposure during the application of pesticides. Toxicol. Lett. 33, 127 –136. Garfitt, S.J., Jones, K., Mason, H.J., Cocker, J., Oral and dermal exposure to propetamphos: a human volunteer study, Biomarkers, (submitted to) Garfitt, S.J., Jones, K., Mason, H.J., Cocker, J., Oral and dermal exposure to diazinon: a human volunteer study, Toxicol. Lett., (submitted to) Gompertz, D., 1996. Organophosphorus pesticides. In: Biological Monitoring of Chemical Exposure in the Workplace, Guidelines, vol. 1. World Health Organisation, Geneva Switzerland, p. 1996. Griffin, P., Mason, H., et al., 1999. Oral and dermal absorption of chlorpyrifos: a human volunteer study. Occup. Environ. Med. 56 (1), 10 –13. Griffith, J.G., Duncan, R.C., 1985. Alkyl phosphate residue values in the urine of Florida cirtus field workers compared to the national health and nutrition examination survey (HABES) sample. Bull. Environ. Toxicol. 34, 210 –215. Hardt, J., Angerer, J., 2000. Determination of dialkyl phosphates in human urine using gas chromatography-mass spectrometry. J. Anal. Toxicol. 24, 678 –684. HSE (2000). Medical Aspects of work-related exposure to organophosphates Guidance note MS17: Health and Safety Executive; 2000 HSE Books PO Box 1999 Sudbury Suffolk CO10 2WA. HSE (2001) EH40/2001 Occupational Exposure limits 2001. Published by HSE Books PO Box 1999 Sudbury Suffolk CO10 2WA ISBN 0 7176 1977 X. Heath, A., Vale, J., 1992. Clinical presentation and diagnosis of acute orgnophosphate insecticide and carbamate poison-
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ing. In: Ballantyne, B, Marrs, T (Eds.), Clinical and Experimental Toxicology of Organophosphates and Carbamates. Butterworth, Oxford, pp. 513 – 519. Lores, E.M., Bradway, D.E., Moseman, R.F., 1978. Organophosphorus pesticide poisonings in humans: determination of residues and metabolites in tissues and urine. Arch. Environ. Health 27, 276. Mason, H.J., Lewis, P., 1989. Intra-individual variation in plasma and erythrocyte cholinesterase activities and the monitoring of uptake of organophosphorus pesticides. J. Soc. Occup. Med. 39, 121 – 124. Mason, H.J., Sams, C., Stevenson, A.J., Rawbone, R., 2000. Rates of spontaneous reactivation of acetylcholinesterase in human erythrocytes after inhibition of pesticides. Human Exp. Toxicol. 19, 511 – 516. Muan, B., Skare, J.U., 1989. A method for the determination of the main metabolites of malathion in biological samples. J. Agric. Food Chem. 37, 1081 – 1085. Nutley, B., Cocker, J., 1993. Biological monitoring of workers occupationally exposed to organophosphorus pesticides. Pesticide Sci. 38, 315 – 322. Peterson, R.P., Picard, G.L., Devine, 1985. Methods for the determination of total terbufos- [phosphorodithioic acid, S-(tert-butylthio)methyl O,O-diethyl ester] related residues in dermal exposure pads and air collection tubes and related alkyl phosphate metabolites in urine. J. Agric. Food Chem. 33, 1041 – 1044. Reid, S.J., Watts, R.R., 1981. A method for the determination of dialkyl phosphate residues in urine. J. Anal. Chem. 5, 126 – 132. Saito, I., Hisanaga, N., Takeuchi, Y, et al., 1984. Assessment of the exposure of pest control operators to organophosphorus pesticides. Jpn. J. Ind. Health 26, 15 – 21. Sams, C., Mason, H.J., Rawbone, R., 2000. Evidence for the activation of organophosphate pesticides by cytochrome P450 3A4 and 2D6 in human liver microsomes. Toxicol. Lett. 116, 217 – 221.
Biological monitoring of exposure to organophosphate pesticides J. Cocker *, H.J. Mason, S.J. Garfitt, K. Jones Health and Safety Laboratory, Broad Lane, Sheffield S3 7 HQ, UK Received 21 September 2001; accepted 27 February 2002
Abstract Organophosphates (OPs) are readily absorbed through the skin and biological monitoring is an essential component of any comprehensive assessment of exposure. This paper presents a summary of our experience in a wide range of occupational studies. Additionally, we have conducted studies of non-occupational exposure and human volunteer studies looking at the kinetics of chlorpyrifos, propetamphos, diazinon and malathion. In non-occupationally exposed people, 95% of urinary alkyl phosphates do not exceed 72 mmol/mol creatinine. In occupationally exposed people, the corresponding 95th percentile of total urinary alkyl phosphates is 122 mmol/mol creatinine. In volunteer studies with 1 mg oral doses of chlorpyifos, diazinon and propetamphos the mean peak values were 160, 750 and 404 mmol/mol creatinine, respectively, and were not associated with any reduction in blood cholinesterase activity. The levels of OP metabolites seen in urine from workers potentially exposed to OPs are generally low and unlikely to cause significant reduction in blood cholinesterase activity. Crown Copyright © 2002 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Biological monitoring; Organophosphate metabolites; Occupational exposure; Cholinesterase
1. Introduction Organophosphate pesticides (OPs) are widely used and readily absorbed through the skin. Occupational exposure limits (ACGIH, 2001; HSE, 2001; DFG, 2001) for OPs usually have ‘skin’ notations to warn of the potential for dermal absorption to contribute to systemic toxicity. As a consequence, biological monitoring is an essential component of any comprehensive assessment of * Corresponding author. Tel.: + 44-114-289-2691; fax: + 44-114-289-2850 E-mail address: [email protected] (J. Cocker).
exposure. Historically, this was biological effect monitoring (BEM) by measurement of the reduction of blood cholinesterase activity. This involved the measurement of plasma cholinesterase(ChE) and erythrocyte acetyl cholinesterase (AChE), reflecting the influence of absorbed OPs to inhibit these blood enzymes as surrogates of AChE in neural tissue and neuromuscular junctions. However, it is well recognised that this is a relatively insensitive indicator of an absorbed dose of OP (Reid and Watts, 1981; Drevenkar et al., 1991; Nutley and Cocker, 1993; Hardt and Angerer, 2000). Blood cholinesterase activity
0378-4274/02/$ - see front matter. Crown Copyright © 2002 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-4274(02)00168-6
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needs at least 15% depression from an individual’s normal level of plasma or erythrocyte enzyme activity to be considered indicative of pesticide over-exposure. Furthermore, due to the large inter-individual variability in cholinesterase activity this approach requires the collection of both postexposure and baseline samples from an individual. It also requires long term precision of the methods as this directly influences the level of ChE or AChE depression that can be considered significant (Mason and Lewis, 1989). In addition, the collection of blood samples is sometimes considered as invasive and in some occupational settings is logistically difficult. An alternative, or complementary, approach to biological monitoring for OPs is based on the analysis of metabolites in urine. These methods can either use metabolites specific to the OP under study or the dialky phosphate metabolites (DAPs) that are common to a large number of different OPs. The measurement DAPs has been used for many years to study exposure to a wide range of OPs. Studies have looked at single OPs like malathion (Bradway and Shafik, 1977; Fenske, 1988; Muan and Skare, 1989), gluthion (Franklin et al., 1981), azinphos methyl (Franklin et al., 1983, 1986), Terbufos (Peterson et al., 1985), chlorpyrifos-methyl (Aprea et al., 1997), or mixtures of OPs (Lores et al., 1978; Davies et al., 1979; Saito et al., 1984; Griffith and Duncan, 1985; Duncan and Griffith, 1985; Drevenkar et al., 1991; Nutley and Cocker, 1993; Aprea et al., 1996; Gompertz, 1996; Hardt and Angerer, 2000; Aprea et al., 2001). This paper reflects our ideas and experience gained from the area of occupational health, where on a routine and research basis we are involved in monitoring the levels of exposure in workers to chemicals including OPs. Some of the research studies have involved ethical human volunteer experiments and some have been field studies of occupational exposure.
2. Biological effect monitoring for organophosphate pesticides Measurements of plasma cholinesterase (ChE)
and erythrocyte acetylcholinesterase (AChE) have been used for a number of years in cases of clinical poisoning and accidental OP exposure, and in monitoring of workers with high risk of exposure. Depression of the plasma ChE enzyme activity is not necessarily associated with symptoms of anti-cholinergic toxicity and large depressions in ChE have been noted in the absence of any effect on erythrocyte AChE. Decreases in the red cell enzyme activity have been suggested to have closer relations to these symptoms. Therefore, in both clinical toxicology and monitoring high-risk occupational activities, the measurement of both enzymes has been recommended (HSE, 2000; Heath and Vale, 1992). In cases of severe OP exposure the level of depression of both plasma ChE and erythrocyte AChE leaves no ambiguity. But in interpreting more subtle changes in the enzymes’ activities or to detect lower levels of OP absorption, several factors need to be understood for proper interpretation. There is wide inter-individual variation in normal, unexposed subjects, particularly in plasma ChE activity and therefore, comparison with a result against a ‘population normal range’ is not a sensitive means of detecting excessive OP absorption in an individual. It is therefore, necessary to have a baseline or unexposed ChE activity for the individual. In on-going workplace biological monitoring schemes this causes no problems. However, in cases of accidental exposure, a definitive interpretation on a measurement entails a considerable delay until a true unexposed baseline measurement can be taken. This period has been suggested as being 60 days (HSE, 2000), when using both enzymes, and our work supports this. Regular monitoring of a formulation worker with two high exposure incidents (Fig. 1) clearly shows the long recovery period especially for AChE. Although OPs are often considered together, there are substantial differences between the OPs in their ability to inhibit either ChE or AChE. In-vitro human enzyme studies at HSL have looked at the OP concentration that causes a 50% inhibition after a 1 h incubation with the respective OP. We found 100–200-fold differences in
J. Cocker et al. / Toxicology Letters 134 (2002) 97–103
99
Fig. 2. General structure of OP pesticide.
Fig. 1. Depression of blood cholinesterases in a worker overexposed to OP pesticides.
potency between OPs (Table 1). For example, the active product of malathion (malaoxon) is less potent at phosphorylating human cholinesterase than chlorpyrifos. These data are taken under conditions where metabolic detoxification by Aesterases in human blood are not active. In-vivo, malathion is susceptible to such metabolism rendering it less likely to phosphorylate and inactivate key enzymes and therefore less toxic. Thus, blood cholinesterase measurements, although relatively insensitive as markers of general OP absorption in comparison with urinary alkyl phosphates measurements, are better at reflecting
Table 1 Concentration of OP inhibiting human plasma cholinesterase and erythrocyte acetylcholinesterase by 50% IC50 after 1 h incubation Direct inhibitory form of OP used to inhibit
IC50 Plasma Che (mM)
Chlorpyrifos-oxon 0.12 (Chlorpyrifos) Malaoxon 12.3 (Malathion) Chlorfenvinphos 0.28 Azinphosmethyloxon 0.06 (Azinphosmethyl)
IC50 Erythrocyte AChe (mM)
1 7.5 3.3 0.07
the likely potency of the absorbed dose of OP (or OP mixture) to cause a significant anticholinergic effect. The major problem with cholinesterase measurements is their reliance on trying to measure a small decrease in enzyme activity from the normal level of activity in that individual.
3. Biological monitoring of organophosphate pesticides The general structure of OPs is given in Fig. 2. In the majority of OP pesticides the R1 and R2 groups are represented by either methyl or ethyl groups, whereas, the chemical structure of X moiety in the OP structure largely defines the majority of the structural differences between individual OP pesticides. OP pesticides are often used in the ‘thio’ form (PS), where metabolic oxidative desulphuration is necessary to produce an OP with anti-cholinesterase activity (PO form). This toxicological activation step is carried out by cytochrome P450s, including CYP 3A4, after absorption of the OP and inter-individual variation in activity of these enzymes may have implications for individual suceptibility (Sams et al., 2000; Mason et al., 2000). A major metabolic route in humans is hydrolysis of the PX bond, producing dialkyl phosphate metabolites that are excreted in urine. Therefore there is the potential to measure the X moiety that would be specific to a single OP (e.g. trichloropyridinol for chlorpyrifos) or to measure the phosphate moiety (as one or more of six alkylphosphates). The advantage of the former approach is the specificity of the assay but it has the disadvantage of requiring many analytical methods to detect the range of OPs that workers may be exposed to. Whereas, the analysis of six
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alkylphosphates allows the detection of absorption of about 85% of all OP pesticides in a single assay but without the ability to identify which OP has been absorbed. The measurement of urinary alkylphosphate metabolites in urine has been a routine assay within our laboratory (Nutley and Cocker, 1993) for over 10 years and the data shown in Fig. 3 below represent the monitoring carried out of various work activities and firms. While the measurement of total urinary alkyl phosphate allows investigation of general exposure to OPs, further information can be gained from identifying the level of specific groupings within the six metabolites. For example, segregation of (DMP, DMTP and DMDTP) and (DEP, DETP DEDTP) gives
Fig. 3. Total urinary alkylphosphate measurements in various occcupational groups; Pest control workers (n=7) use OPs in short duration spraying to control inset pest; ‘formulators1 and 2’ reflect monitoring at specific firms (n =118 and 24) which produce pesticide products for amateur and professional use. ‘Control’ reflects levels found in a non-occupationally exposed UK population (n=463). ‘Sheep dipping’ (n= 478) samples from farmers using diazinon based sheep dips. ‘Sheep handlers’ describes samples (n= 21) from workers on sheep farms from activities for about two weeks post-sheep dipping with an OP product. ‘Wool handlers’ (n= 102) reflects measurements in staff within a single firm cleaning and processing wool from fleeces. Orchard (n= 43), hop sprayers (n= 84) and nursery (n =40) are from studies of workers undertaking agricultural and horticultural tasks with OP pesticides.
some idea of the class of OPs causing exposure, discriminating between the dimethoxy OPs, such as dichlorvos or malathion which produce DMP, DMTP and, say, diazinon which produces DEP and DETP. Also the level of DMP and DEP reflects exposure to OPs which could have potentially inhibited acetylcholinesterase, whereas DMTP, DMDTP, DETP and DEDTP reflect detoxification process that protects against any internal level of active OP. The total number of samples from exposed workers is 917. The maximum value found was 915 mmol/mol creatinine, the mean and median were 33 and 15 mmol/mol creatinine, respectively. About 90% of the results in the exposed group were B 77 mmol/mol creatinine and 95% were less than 122 mmol/mol creatinine. The group with the lowest levels of alkyl phosphates in urine was pest control workers. These workers are well trained with appropriate protective equipment and only use OPs for short periods to eradicate insect pests. The groups with the highest levels of alkyl phosphates were the formulators and, to a lesser extent, wool handlers who have prolonged exposure each day and repeated daily exposure. In formulators 90% of the results were B 188 mmol/mol creatinine. The next highest urinary alkyl phosphate levels were in sheep dippers who tended to have 4–8 h exposures on only a few days a year but under difficult conditions. Other groups e.g. agricultural and horticultural workers had intermittent exposures but in conditions where better controls were possible. Many of the samples above were collected in studies that also looked at blood cholinesterase activity. These were samples collected as part of investigations of exposure during sheep-dipping, hop spraying, orchard spraying, wool handling and some of the formulators and there were no exposure related significant depression of blood cholinesterase. The control group shown in the Fig. 3 are from 463 samples collected in a number of different studies of people who had no occupational exposure to OPs and had not recently used OP-containing garden products. The data confirms that the sensitivity of the urinary alkyl phosphate
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method is sufficient to investigate environmental exposure. Results showed that the mean and median ‘total’ urinary alkyl phosphate were 22 and 16 mmol/mol creatinine, respectively. The 90– 95% percentile value of ‘total’ urinary alkyl phosphate levels for this unexposed population was between 51 and 72 mmol/mol creatinine, respectively. These values are similar to values reported by Aprea et al. (1996), Hardt and Angerer (2000) in non-occupationally exposed Italian and German populations, respectively. Currently we have no data to show whether levels of alkylphopshate metabolites in non-occupationally exposed people reflects absorption of the intact OP pesticide or of alkylphosphate residue themselves. In an attempt to put the occupational studies into perspective we have also carried out three separate volunteer studies with chlorpyrifos, diazinon and propetamphos under the approval of the Research Ethics Committee of the UK Health and Safety Executive (Griffin et al., 1999; Garfitt et al., 2002a,b). Volunteers (four or five) were given a maximum of 1 mg oral dose and, on separate occasions, dermal doses of 100 mg (28 mg for chlorpyrifos). Urine samples were collected for up to 48 h and blood samples were taken at intervals for cholinesterase determination. No significant depressions of blood cholinesterase were observed and the urine alkyl phosphate levels are shown in Fig. 4. The overall picture shows the occupational levels of alkyl phosphates similar to those found in volunteer studies, which were not associated with reduction in blood cholinesterase activity. We have also been part of a study looking at blood cholinesterase and urine metabolites during and after use of preparations based on malathion to treat head-lice. The preparations used were those generally available to the public and were used in accordance with the instructions in the packet. Exposure was dermal only and gives another perspective on levels of urinary alkyl phosphates found in occupational studies. Fig. 5 shows urinary levels of dimethoxy alkylphosphates (DMP, DMTP, DMDTP) found in samples collected for up to 48 h after the appropriate use of malathion containing headlice shampoo products.
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Fig. 4. Urinary alkyl phosphate levels from occupationally exposed workers (n =917) and volunteers given oral and dermal doses of chlorpyrifos, diazinon and propetamphos. For the occupational group the values are the total of 6 dialkyl phosphates, for the chlorpyrifos and diazinon studies the values are the total of the diethyl phosphate metabolites and for propetamphos are the total MEPT (a mono alkyl phosphate) after hydrolysis. Urine values in the volunteer studies are from samples collected up to 24 h after an oral dose or 48 h after a dermal dose from 5 volunteers.
Malathion is an OP that will only produce dimethoxy alkyl phosphate metabolites (DMP and DMTP). Although the levels of urinary metabolites found were much higher than those in occupational studies there was no depression in blood cholinesterase activity. As noted in Table 1 malathion is a much less potent inhibitor of cholinesterase than other OPs.
4. Conclusions Both biological monitoring by determination of urinary alkyl phosphates and BEM by determination of blood cholinesterase activity have important roles in assessing the exposure of workers to OPs. The traditional approach of blood cholinesterase measurement has a role in reassuring most workers that their exposure is unlikely to
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in urine from workers potentially exposed to OPs is generally low and unlikely to cause significant reduction in blood cholinesterase activity.
References
Fig. 5. OP metabolites in occupational and volunteer studies and in subjects treated with malathion-containing headlice shampoo and controls.
result in any toxicity. It has well defined guidance values to help interpret results and is directly related to risk. Measurement of urinary metabolites is less invasive and logistically simpler. Although there are few guidance values for urinary metabolites, this approach can be used to assess the efficacy of control procedures and help to reduce exposure. It has been widely used in occupational, clinical and environmental studies and there are data to aid the assessment of exposure to OPs. As a perspective on urinary alkyl phosphates the results of studies over the last 10 years can be summarised as follows: In non-occupational exposed people 90% of the total urinary alkyl phosphates are B50 mmol/mol creatinine and 95% are B 72 mmol/mol creatinine. In occupationally exposed people 90% of the total urinary alkyl phosphates are B77 mmol/mol creatinine and 95% are B122 mmol/mol creatinine. In volunteer studies with 1 mg oral doses of chlorpyifos, diazinon and propetamphos the mean peak values were 160, 750 and 404 mmol/mol creatinine, respectively, and were not associated with any reduction in blood cholinesterase activity. Overall, our experience over the last 10 years has shown that the levels of OP metabolites seen
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