A r ' c h l v e s of
E nviPonmenC.al ntamination c oTo"
Arch. Environ. Contain. Toxicol. t9,782-788 (1990)
9 1990 Springer-Verlag New York Inc.
Biological Monitoring of Human Exposure to Acephate M. Maroni .1, G. Catenacci**, D. Galli*, D. CavaUo*, and G. Ravazzani*** *Institute of Occupational Health, University of Milano, Via S. Bamaba 8, 20122 Milano, Italy, **Department of Occupational, Preventive, and Community, University of Pavia, and ***SIPCAMResearch Center, Salerano, Italy Abstract. Acephate is a water-soluble organophosphate insecticide whose action on insects has been related to its conversion to methamidophos, a very potent anticholinesterase agent which has caused delayed neuropathy in man. Inhalation and skin exposure to acephate was evaluated in four workers engaged in 8-day campaigns with the formulation of the 97%-pure technical product. Before, during, and after exposure, the workers were monitored for the urine content of acephate and methamidophos, and for erythrocyte (ACHE) and plasma (PChE) cholinesterase levels. Median air concentrations (8-hr TWA) ranged from 0.278 to 2.170 mg/m3; median total-body skin deposition ranged from 26.1 to 41.9 mg/day. Based on these values, daily workers' absorption of acephate was estimated to be in the order of 10-20 mg. Urinary excretion of unchanged acephate followed a pattern consistent with exposure, showing peak values of excretion during the workshift or in the eight hr after the end of the workshift. The urine levels of unchanged acephate were found to vary from 1 to 10 mg/L. Methamidophos was not detected in any urine sample (detection limit: 30 Ixg/L). High correlation (r = 0.78) was found between skin exposure level and urine acephate elimination. No changes in AChE or PChE were observed for the workers whose urinary concentrations of acephate were 1 or 2 mg/L. One subject who had urinary acephate excretion between 3 and 8 mg/L, showed slightly decreased values of PChE during exposure and of AChE after exposure.
Acephate (O,S-dimethyl-acetylphosphoramidothioate or Nethoxy(methylthio)phosphinoylacetamide) is an insecticide active on lepidopterans and aphids, used for the protection of vegetables and fruits. In soil and plants, acephate is converted into metabolic products, primarily methamidophos (O,S-dimethyl-phosphoramidothioate) which is a very potent anticholinesterase agent (Bull 1979). The insecticidal action of acephate has been related to its conversion to methamidophos or to a combined anticholinesterase effect
i To whom correspondence should be addressed.
of acephate and methamidophos (Kao and Fukuto 1977; Eto et al. 1977; Magee 1982; Hussain et al. 1984). Methamidophos, besides acute intoxication, has caused delayed neuropathy in man through the mechanism of "neuropathy target esterase" (NTE) inhibition (Senanayake and Johnson 1982). In mammals, acephate shows low to moderate acute toxicity, but, in view of the limited toxicological information available, a temporary ADI as low as 0.0005 mg/kg bw was established in 1984 (FAO 1985a). Studies in man have been very limited. Summarized reports made available in the FAO literature would indicate that methamidophos was present in the urine together with other metabolites in subjects exposed to acephate in formulating plants (Swencicki and Hartz 1972 quoted in FAO 1977). In a survey of agricultural field workers exposed to acephate, low levels of acephate were detected in urine in the absence of methamidophos (Pack 1972 quoted in FAO 1977). Rodent metabolism studies would suggest that acephate is not degraded to methamidophos, but rather excreted unchanged with the urine (Lee 1972, quoted in FAO 1977). The present study was undertaken to evaluate acephate exposure of formulation workers and to identify reliable procedures for human biological monitoring. Questions to be answered included the presence or not of methamidophos in biological samples, which has implication for the assessment of toxicological risk, the existence of a correlation between exposure and biological indicators, and the relationship between internal levels in the organism and inhibition of blood cholinesterase.
Materials and Methods Plant and Formulation Process The investigations were conducted in a small plant where several pesticides were formulated. The factory was divided into two departments, one for formulation of liquid products, and the other for formulation of solids. Production took place in campaigns, each one dealing with a single product. In the formulation of acephate, the 97%-pure technical product was formulated to produce a dust containing 42.5% of the active
Human Exposure to Acephate ingredient. The formulating process was divided in tw• subsequent steps: dilution, in which pure acephate was adsorbed on the solid support and temporarily stocked in 1000 t containers, and packaging, in which the product was packed into the final small-size commercial boxes. The process was carried out by two workers, one in charge of loading, control-board operation, and assistance for failures, and the other in charge of packaging. The main sources of dust emission in the working environment were represented by loading of pure acephate, loading of the formulated product to the bagging machine, packaging and extemporary maintenance activities needed in case of malfunction and plugging. Dust emission at the workplaces for loading and packaging was controlled by an aspiration system.
Campaigns Two campaigns of acephate formulation, each lasting for two weeks, were investigated. In the first one, two workers were surveyed, obtaining airborne acephate concentrations by personal sampling, acephate concentrations in urine, and blood cholinesterase assay before, during, and after exposure. In the second campaign, the same program was followed but detTaal exposure assessment was also made.
Assessment of Exposure Airborne concentration of acephate was monitored in the breathing zone by personal samplers throughout the daily workshift of each worker. Sampling was performed on Sartorius cellulose nitrate membranes by ZS/4 personal samplers (Zambelll, Italy) operating at an air flux of 4 L/rain for 4-hr sampling cycles. Acepliate was extracted from filter dust by sonication of the filter membrane in a 3:1 water/methanol solution, followed by filtering the brei through a 0.5 ~ filter. Recovery of extraction was around 95%. The filtrate was analyzed with a Hewlett-Packard model 1084 HPLC equipped with 4.6 m m x 25 cm 10 tx-RP-18 column (Brownlee Labs, Applied Biosystem Inc, Santa Clara, CA, USA) and a model HP 79850B-LC Terminal. Acephate was eluted with water-methanol 3:1 at a flow rate of 0.7 ml/min. A UV detector set at 226 nm was used. Elution time for acephate was 5 min. Acephate standard curves were analyzed at concentrations from 0.001 to 1 mg/ml; lower detection limit of the method was 0.001 mg/ml. Skin exposure was assessed only during the second campaign, using the method recommended by WHO (WHO 1982), with the following modifications. Each worker wore three gauze pads, one on the working suit in proximity of the neck, representative of face exposure, and the other two pads underneath the suit in contact with the skin, positioned one on the chest and the other on the back, in corresponding places. The surface of these pads in contact with the skin was covered by a thin layer of aluminum foil. At the end of the work, skin pads were extracted with acetone, the solution dried and redissolved in 3:1 water/methanol solution, and analyzed by HPLC as above described. Recovery from extraction was 95%. The hands were washed with water accompanied by gentle brushing with a small teflon brush. The washings were collected and analyzed by HPLC.
Urine Sampling and Storage Urine samples were collected from the workers as spot specimens during the first campaign and as 8-hr or 24-hr samples during the
783 second campaign. Urine was stored frozen at --18~ before analysis.
Determination of Acephate in Urine Ten ml of urine were saturated with NaC1 and loaded onto a 10 red liquid-Iiquid extraction column (Chem-Elut-CE 1010 Anatytichem International, Harbor City, CA, USA). Efficiency of extraction was 90%. Elution was made with 20 ml dichtoromethane, the solvent was evaporated under nitrogen flow, and the residue redissoived in 0.1 ml acetone; 0.5 ~1 of this sample were injected into a gas-chromatograph (Perkin Elmer, rood. 8320-NPD with model LCI 100) equipped with a 25 m capillary amorphous silica SE52 column (i.d. 0.23 ix), operating under the following conditions: carrier gas: Helium 20 psi; injector and detector temperature: 250~ oven temperature: 12O~ for 2', 20~ up to 200~ 200~ for 6'. The nitrogenphosphorus detector was set at 280 ml/min air flow and 40 ml/min hydrogen flow. The lower limit of detection of the method was 30 ~g/L for acephate and methamidophos.
Blood Cholinesterase Assay Venous blood samples were obtained in the morning from the subjects. Erythrocyte cholinesterase activity was assayed by the Michel method (Michel 1949). Plasma cholinesterase assay was performed by a kit (IATRON Laboratories Inc, Tokyo; distributed by Poll S.p.A., Milano) based on the Trinder kinetic method (Okabe et al. 1977). According to this method, choline which is liberated from benzoyl-choline as substrate by cholinesterase, is oxidized by choline-oxidase to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximal absorbance at 500 nm.
Results Air and urine concentrations of acephate measured during the first production campaign are shown in Table 1. The median airborne exposure level during the campaign was in the order of 0.3-0.4 mg/m 3 as a t/me-weighted average on a 8 hr base. The worker in charge of packaging (MAB) showed a median exposure level slightly lower than the control-board operator (RV), although the highest peak exposure values were measured at packaging, Urine was analyzed for acephate and methamidophos, but only the first compound was present in detectable amounts. Median acephate excretion was found to be between 0.4 and 1.1 mg/L at the end of the workshift. The control board operator showed urinary levels 2 - 3 fold higher than the packager in the spot urine sample collected at the end of the workshift, but the difference was less pronounced in the other urine samples~ Cholinesterase activity 'was measured before exposure, at days 2, 7, and 13 after the beginning of exposure and 7 days after the end of the campaign. The results, shown in Table 2, did not indicate any significant change during or after exposure, as compared with individual pre-exposure values. During the second campaign of acephate formulation, workers' exposure was tested by determining beth air concentration and skin deposition of acephate. The results are
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M. Maroni et al.
Table 1. Air concentration and urinary excretion of acephate of two workers (MAB and RV) during the first campaign of formulation
Day
Subject
Air concentration (mg/m3) sampling time 8-12 13.30-17.30
MON
MAB RV MAB RV MAB RV MAB RV MAB RV
0.086 0.083 2.050 0.277 0.549 0.338 nda 0.574 2.168 0.498
0.432 1.208 0.265 0.547 0.144 1.341 nd 0.251 1.223 0.487
0.259 0.645 1.157 0.412 0.346 0.839 nd 0.412 1.695 0.492
0.044 0.228 0.165 0.596 0.453 0.668 0.642 0.697 0.849 0.537
0.051 0.460 0.282 0.545 0.728 0.853 0.666 0.658 0.579 0.899
0.180 0.829 0.476 1.094 0.737 1.267 0.424 1.000 0.755 1.359
MAB RV MAB RV MAB RV
0.345 0.331 0.087 0.502 0.236 0.291
0.211 0.220 0.109 0.351 0.078 0.194
0.278 0.275 0.098 0.426 0.157 0.242
0.279 0.221 0.444 0.588 0.770 1.002
0.312 0.487 0.251 0.566 0.371 1.015
0.333 0.788 0.291 1.246 0.554 1.402
MAB
min max median
0.086 2.168 0.345
0.078 1.223 0.211
0.098 1.695 0.278
0.044 0.849 0.448
0.051 0.728 0.327
0.180 0.755 0.433
RV
rain max median
0.083 0.574 0.333
0.194 1.341 0.364
0.242 0.839 0.413
0.221 1.002 0.591
0.460 1.015 0.576
0.788 1.402 1.104
TUE WED THU FRI MON TUE WED
TWA 8 hr
Urine concentration (rag/L) collection time 8 12
17
nd = not determined Table 2. Activity of erythrocyte (ACHE) and plasma (PChE) cholinesterase measured in the two workers engaged in the first campaign of formulation Subject
Pre-exposure
During exposure days: 2
7
13
7 days after the end of exposure
MAB
AChEa PChEa
0.596 4279
0.594 3940
ndb ndb
0.648 4099
0.594 4348
RV
AChE a PChEa
0.539 3932
ndb 4027
0.621 3638
0.652 3699
0.622 3947
Units: ACHE: ~ pH/ml erythrocyte/hr PChE: mU/L b n d = not determined shown in Table 3. The median level of air concentration was around 1.2 mg/m3 as a time-weighted average for the packager (MRL) and 2.1 mg/m3 for the control-board operator (CN). Assuming a ventilation rate of 10 m 3 per workshift, the total inhalation loads would be at levels of 12 and 21 mg/day, respectively. The same exposure ratio between the subjects was observed for skin contamination that gave a median overall amount of 26.1 and 41.9 mg per day, respectively. It is interesting to note that the amount recovered from the hands accounted for 90% of total skin exposure. In order to estimate the amount of acephate absorbed by the workers, it is necessary to hypothesize the fraction absorbed through the airway system and through the skin. Assuming 3 0 - 5 0 % a b s o r p t i o n of the inhalation dose and 10-20% absorption of the skin dose, the median intake for these workers would lie in the order of 10-20 mg per day. Urinary excretion of acephate was determined in these
workers by the third day of the campaign and followed-up for one week. Methamidophos again was not detected in any of the urine samples which, however, contained measurable amounts of acephate (Figure 1). The analytical results showed that the control-board operator had a remarkable acephate excretion with daily peaks between 3 and 8 mg/L, while the excretion of the packager was lower, with the exception of a single-day value exceeding 9 mg/L. Thus, urinary values were to some extent concordant with external exposure assessment, providing a median value of acephate excretion during the week higher for the control-board operator than for the packager. The peak excretion observed for the latter on Wednesday occurred after the workshift in which the highest skin contamination was measured, even though the differences with the preceding or following days were not as remarkable for skin exposure as were for urinary excretion values.
785
Human Exposure to Acephate Table 3. Exposure to acephate of two workers (MRL and CN) during the second campaign of formulation Inhalation total load (estimated) mg
Day
Subject
WED
MRL CN
0.54 3.15
5.4 31.5
7.8 8.6
1.32 0.71
nd ~ nd
9.12 9.31
14.52 40.81
THU
MRL CN
0.66 0.72
6.6 7.2
23.9 35.1
1.65 1.56
nd nd
25.55 36.66
32.15 43.86
FRI
MRL CN
1.22 0.75
12.2 7.5
29.3 33,6
1.73 0.52
0.03 0.06
31.06 34.18
43.26 4! ,68
MON
MRL CN
1.67 1.81
16.7 18.1
32.8 46.0
0.96 1.71
nd nd
33.76 47.71
50.46 65o81
TUE
MRL CN
2.34 2.50
23.4 25.0
20.0 44.8
1,13 2.45
0.07 nd
21.2 47.25
44.60 72.25
WED
MRL CN
1.51 2.16
15.1 21.6
41.0 50.3
0.56 2.16
0.04 0.05
41.60 52.51
56,7 74.11
THU
MRL CN
0.8 3.47
8.0 34.7
30.8 36.2
1,36 5.45
0.05 nd
32.21 41.65
49.21 76,35
FRI
MR L CN
1.26 4.36
12.6 43.6
22,6 60,6
1.27 3.23
0.19 0.07
24.06 63.9
36,66 107.5
12.5 21.7
24.5 36.7
1.29 1.78
0.05 0.06
26.1 41.9
40.9 66.2
MRL median CN median
Skin exposure Hands Face mg mg
Body nag
Skin total (estimated) rag
Total estimated exposure mg
Inhalation exposure TWA mg/m 3
and = not determined
Fig, 1. Urinary excretion of acephate of the workers MRL and CN during the second campaign. Shadowed bars indicate exposure periods
W h e n correlating e x p o s u r e m e a s u r e m e n t s with urinary excretion, significant P e a r s o n ' s coefficients of correlation w e r e found b e t w e e n total individual e x p o s u r e (as estimated in Table 3) and urinary acephate concentration in the 8 hr
following the w o r k s h i f t (r = 0.717; P = 0.001) or in the 24-hr refine collected since the beginning of the w o r k s h ~ t (r = 0.788; P = 0.001). If these same urinary parameters are tested against inhalation or skin e x p o s u r e values separately,
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M. Maroni et
al.
Total exposure
(rag) 110-
10090807060509 o9
40r =
30-
.79
20~o I 0
I 1000
i 2000
I 3000
I
I
I
I
4000
5000
6000
7000
URINARY
ACEPHATE
the coefficients of correlation are as follows: r = 0.465 for inhalation exposure vs 8-hr after-work urine; r = 0.518 for inhalation exposure vs 24-hr urine; r = 0.720 for skin exposure vs 8-hr after-work urine; r = 0.777 for skin exposure vs 24-hr urine. The scatter diagrams showing the correlation between total exposure or skin exposure and 24-hr urinary excretion of acephate are shown in Figures 2 and 3. Cholinesterase activity in these workers had been measured for four consecutive months before exposure; thus, mean and variation of the individual baseline were accurately estimated for each subject (Table 4). Cholinesterase assay during and after exposure did not show any significant change in the packager. In the control-board operator, erythrocyte ChE activity was slightly depressed (P < 0.05) 1 week and 4 months after exposure. Plasma cholinesterase showed a transient decrease, at borderline level of significance, only during exposure.
Discussion Acephate shows low to moderate acute toxicity in mammals, with oral LDso values of 361 mg/kg bw in the mouse and 866-945 mg/kg bw in the rat (Worthing and Walker 1987). 15o values of acephate for human plasma and bovine erythrocyte cholinesterase are one or two orders of magnitude higher than those of methamidophos (Tucker 1972 quoted in F A t 1977). In man, 150 values for brain ACHE, erythrocyte ACHE, and plasma cholinesterase are in order of decreasing magnitude, with values of, respectively, 5.4 mM, 2.7 mM, and 1.8 mM (Bennet and Morimoto 1982 quoted in F A t 1985). Biotransformation of acephate has been studied in the rat
Fig, 2. Correlation between total exposure and urinary excretion of acephate
(IJg~24h) after oral as well dermal treatment with 5-methyl-14C-acephate. After oral administration, excretion of radioactivity was rapid, with 82-95% of the administered dose recovered in urine, 1-4% exhaled, and 1% found in the faeces. Urinary metabolites were identified as unchanged acephate (73-77%), O-S-dimethylphosphorothioate (3-6%), and Smethyl-acetylphosphoramidothioate (3-4%). Methamidophos was not detected (Lee 1972, quoted in F A t 1977). After a single dermal application at a level of 10 mg/kg bw, the 14C dose was rapidly absorbed into the blood, with max:imum concentration occurring after one to three hr and a secondary peak observed at 48 hr. Urinary excretion of radioactivity showed a maximum between 6 and 24 hr. Within 3 days, about 30% of the dermal dose was excreted as unchanged acephate and 1% as methamidophos (Tucker 1974, quoted in F A t 1985b). Studies in man have been very limited and none of them has been published in the scientific literature. Some information is available in abstracted form in the Reports from the Joint Meeting on Pesticide Residues published by the FAt. No adverse effects were reported for workers having urinary acephate concentrations up to 5 mg/L in a survey on subjects exposed to acephate in pilot plant and formulation processes. The urine of the workers of the pilot plant were reported to contain other metabolites or technical products of the production besides acephate. The subjects exposed to technical acephate during formulation were reported to show methamidophos and trimethylphosphate as urinary metabolites ( F A t 1977). Based on observations on the acephate decay in human blood added with the compound, it has been proposed that blood may contain certain enzymes capable of metabolizing acephate to methamidophos (Singh 1984). The present study does not support the concept that acephate in the human
787
Human Exposure to Acephate Skin exposure (rag) 70-
60-
50|
Q
400 | | 0
30-
0
r
O
20-
= .78
Fig. 3. Correlation between skin exposure and urinary excretion of acephate
10- , I 0
1000
2000
3000
4000
5000
6000
7000
URINARY ACEPHATE (~g/24hl
Table 4. Activity of erythrocyte (ACHE) and plasma (PChE) cholinesterase measured for the workers MRL and CN Subject
Jan.
Pre-exposure values Febr. Apr.
May
Mean
lower 5th percentile
Exposure June 3
1week after
4 months after
MRL AChE a PChE a
0.929 3055
0.962 3204
0.921 2378
0.952 3148
0.941 2946
0.901 2178
0.969 2407
0.966 2592
ndb ndb
CN
0.765 3350
0.757 3795
0.647 3119
0.760 3855
0.732 3530
0.618 2820
0.681 2826r
0.600c 3446
0.613 ~ 3845
AChE a PChE a
a = Units: see Table 2 b = n d = not determined c = Values close or inferior to the 5th percentile of the distribution of the pre-exposure values
organism is substantially converted into methamidophos, at least as far as this can be evaluated by the study of its urinary excretion. Our workers were estimated to have absorbed daily doses of about 10-20 nag per day and showed urinary concentrations of unchanged acephate from 1 to 10 mg/L. The presence in urine of methamidophos at a concentration ratio greater than 1:100 so acephate was excluded. Urinary excretion of acephate followed a pattern consistent with exposure, showing peak values of excretion in the samples collected during the workshift or in the 8 hr after the end of the workshift. Urinary elimination was quite rapid; urine concentrations were undetectable within 48 hr after the end of exposure. In the formulation process under study, skin exposure was remarkable, with the main contribution given by hand contamination. Skin exposure as assessed by the conventional method of the skin pads, provided an estimate of individual exposure highly correlated with urinary excretion of acephate. This issue deserves attention in the light of experi-
mental data pointing to a substantial penetration of acephate through rat skin (Tucker t984 quoted in FAO 1985b). Total exposure calculated by combining inhalation and skin e x p o s u r e was well c o r r e l a t e d with e x c r e t i o n , accounting for about 60% of the variation of the biological results. The evaluation of the biological effects of acephate was made in this study by the assay of red cell and plasma cholinesterase activity. No changes with respect to pre-exposure values were observed for those subjects whose urinary acephate excretion averaged 1 - 2 mg/L or less. The subject having u r i n a r y e x c r e t i o n values b e t w e e n 3 and 8 mg/L showed a slightly decreased value of ptasma cholinesterase during exposure ( - 2 0 % ) and of erythrocyte cholinesterase afterwards ( - 1 8 % ) . These changes were just outside the limits of analytical and biological variability of the measurements and remained very far from the threshold at which clinical symptoms of anticholinesterase poisoning are expected.
788
References Bennet EL, Morimoto H (1982) The comparative in vitro activity of acephate technical on brain, erythrocyte and plasma cholinesterases from the human, monkey, and rat. Unpublished report quoted in FAO, 1985 Bull DL (1979) Fate and efficacy of acephate after application to plants and insects. J Agric Food Chem 27:268-272 Eto M, Okabe S, Ozoe Y, Maekawa K (1977) Oxidative activation of O,S-dimethyl phosphoramidothiolate. Pestic Biochem Physiol 7:367-377 FAO (1977) 1976 Evaluation of some pesticide residues in food. The monographs. ACEPHATE. FAO Plant Production and Protection Series n. 8:l-54-Rome (1985a) Pesticide residues in food, 1984. Report of the joint meeting on Pesticides residues. Rome 24 Sept, 3 Oct 1984 (PAPER 62) - (1985b) Pesticide residues in food, 1984. The monographs-ACEPHATE--Fao Plant Production and Protection Paper 67:527-536-Rome Hussaln MA, Mohamad RB, Olofts PC (1984) Toxicity and metabolism of acephate in adult and larval insects. J Environ Sci Health B19(3):355-377 Kao TS, Fukuto TR (1977) Metabolism of O,S dimethyl propionyland hexanoylphosphor-amidothioate in the housefly and white mouse. Pestic Biochem Physiol 7:83-95 Lee H (1972) Metabolism of ORTHENE in rats. Unpublished report quoted in FAO, 1977 Magee PS (1982) Structure-activity relationships in phosphorami-
M. Maroni et al. dates. In: Coats JR (ed) Insecticide mode of action. Academic Press, New York Michel HO (1949) Electrometric method for determination of red blood cell and plasma cholinesterase activity. J Lab Clin Med 34:1564-1568 Okabe H, Sagesaka K, Nakajima N, Noma A (1977) New enzymatic assay of cholinesterase activity. Clin Chim Acta 80:87-94 Pack D (1972) Orthene insecticide occupational exposure. Field station personnel. Unpublished study quoted in FAO, 1977 Senanayake N, Johnson MK (1982) Acute polyneuropathy after poisoning by a new organophosphate insecticide. New Engl J Med 306:155-157 Singh AK (1984) Improved analysis of acephate and methamidophos in biological samples by selective ion monitoring gas chromatography-mass spectrometry. J Chromatogr 301:465469 Swencicki R, Hartz W (1972) Orthene human exposure study. Clinical evaluation. Unpublished study quoted in FAO, 1977 Tucker B (1972) Acetylcholinesterase inhibition of orthene and ortho 9006. Unpublished report quoted in FAO, 1977 WHO (1982) Field Surveys of exposure to pesticides: Standard Protocol. WHO, Division of Vector Biology and Control. Document 82.1 Worthing CR, Walker SB (1987) The pesticide manual. 8th ed British Crop Protection Council, Thornton Heath, UK
Manuscript received February 15, 1989 and in revised form August 8, 1989.
E nviPonmenC.al ntamination c oTo"
Arch. Environ. Contain. Toxicol. t9,782-788 (1990)
9 1990 Springer-Verlag New York Inc.
Biological Monitoring of Human Exposure to Acephate M. Maroni .1, G. Catenacci**, D. Galli*, D. CavaUo*, and G. Ravazzani*** *Institute of Occupational Health, University of Milano, Via S. Bamaba 8, 20122 Milano, Italy, **Department of Occupational, Preventive, and Community, University of Pavia, and ***SIPCAMResearch Center, Salerano, Italy Abstract. Acephate is a water-soluble organophosphate insecticide whose action on insects has been related to its conversion to methamidophos, a very potent anticholinesterase agent which has caused delayed neuropathy in man. Inhalation and skin exposure to acephate was evaluated in four workers engaged in 8-day campaigns with the formulation of the 97%-pure technical product. Before, during, and after exposure, the workers were monitored for the urine content of acephate and methamidophos, and for erythrocyte (ACHE) and plasma (PChE) cholinesterase levels. Median air concentrations (8-hr TWA) ranged from 0.278 to 2.170 mg/m3; median total-body skin deposition ranged from 26.1 to 41.9 mg/day. Based on these values, daily workers' absorption of acephate was estimated to be in the order of 10-20 mg. Urinary excretion of unchanged acephate followed a pattern consistent with exposure, showing peak values of excretion during the workshift or in the eight hr after the end of the workshift. The urine levels of unchanged acephate were found to vary from 1 to 10 mg/L. Methamidophos was not detected in any urine sample (detection limit: 30 Ixg/L). High correlation (r = 0.78) was found between skin exposure level and urine acephate elimination. No changes in AChE or PChE were observed for the workers whose urinary concentrations of acephate were 1 or 2 mg/L. One subject who had urinary acephate excretion between 3 and 8 mg/L, showed slightly decreased values of PChE during exposure and of AChE after exposure.
Acephate (O,S-dimethyl-acetylphosphoramidothioate or Nethoxy(methylthio)phosphinoylacetamide) is an insecticide active on lepidopterans and aphids, used for the protection of vegetables and fruits. In soil and plants, acephate is converted into metabolic products, primarily methamidophos (O,S-dimethyl-phosphoramidothioate) which is a very potent anticholinesterase agent (Bull 1979). The insecticidal action of acephate has been related to its conversion to methamidophos or to a combined anticholinesterase effect
i To whom correspondence should be addressed.
of acephate and methamidophos (Kao and Fukuto 1977; Eto et al. 1977; Magee 1982; Hussain et al. 1984). Methamidophos, besides acute intoxication, has caused delayed neuropathy in man through the mechanism of "neuropathy target esterase" (NTE) inhibition (Senanayake and Johnson 1982). In mammals, acephate shows low to moderate acute toxicity, but, in view of the limited toxicological information available, a temporary ADI as low as 0.0005 mg/kg bw was established in 1984 (FAO 1985a). Studies in man have been very limited. Summarized reports made available in the FAO literature would indicate that methamidophos was present in the urine together with other metabolites in subjects exposed to acephate in formulating plants (Swencicki and Hartz 1972 quoted in FAO 1977). In a survey of agricultural field workers exposed to acephate, low levels of acephate were detected in urine in the absence of methamidophos (Pack 1972 quoted in FAO 1977). Rodent metabolism studies would suggest that acephate is not degraded to methamidophos, but rather excreted unchanged with the urine (Lee 1972, quoted in FAO 1977). The present study was undertaken to evaluate acephate exposure of formulation workers and to identify reliable procedures for human biological monitoring. Questions to be answered included the presence or not of methamidophos in biological samples, which has implication for the assessment of toxicological risk, the existence of a correlation between exposure and biological indicators, and the relationship between internal levels in the organism and inhibition of blood cholinesterase.
Materials and Methods Plant and Formulation Process The investigations were conducted in a small plant where several pesticides were formulated. The factory was divided into two departments, one for formulation of liquid products, and the other for formulation of solids. Production took place in campaigns, each one dealing with a single product. In the formulation of acephate, the 97%-pure technical product was formulated to produce a dust containing 42.5% of the active
Human Exposure to Acephate ingredient. The formulating process was divided in tw• subsequent steps: dilution, in which pure acephate was adsorbed on the solid support and temporarily stocked in 1000 t containers, and packaging, in which the product was packed into the final small-size commercial boxes. The process was carried out by two workers, one in charge of loading, control-board operation, and assistance for failures, and the other in charge of packaging. The main sources of dust emission in the working environment were represented by loading of pure acephate, loading of the formulated product to the bagging machine, packaging and extemporary maintenance activities needed in case of malfunction and plugging. Dust emission at the workplaces for loading and packaging was controlled by an aspiration system.
Campaigns Two campaigns of acephate formulation, each lasting for two weeks, were investigated. In the first one, two workers were surveyed, obtaining airborne acephate concentrations by personal sampling, acephate concentrations in urine, and blood cholinesterase assay before, during, and after exposure. In the second campaign, the same program was followed but detTaal exposure assessment was also made.
Assessment of Exposure Airborne concentration of acephate was monitored in the breathing zone by personal samplers throughout the daily workshift of each worker. Sampling was performed on Sartorius cellulose nitrate membranes by ZS/4 personal samplers (Zambelll, Italy) operating at an air flux of 4 L/rain for 4-hr sampling cycles. Acepliate was extracted from filter dust by sonication of the filter membrane in a 3:1 water/methanol solution, followed by filtering the brei through a 0.5 ~ filter. Recovery of extraction was around 95%. The filtrate was analyzed with a Hewlett-Packard model 1084 HPLC equipped with 4.6 m m x 25 cm 10 tx-RP-18 column (Brownlee Labs, Applied Biosystem Inc, Santa Clara, CA, USA) and a model HP 79850B-LC Terminal. Acephate was eluted with water-methanol 3:1 at a flow rate of 0.7 ml/min. A UV detector set at 226 nm was used. Elution time for acephate was 5 min. Acephate standard curves were analyzed at concentrations from 0.001 to 1 mg/ml; lower detection limit of the method was 0.001 mg/ml. Skin exposure was assessed only during the second campaign, using the method recommended by WHO (WHO 1982), with the following modifications. Each worker wore three gauze pads, one on the working suit in proximity of the neck, representative of face exposure, and the other two pads underneath the suit in contact with the skin, positioned one on the chest and the other on the back, in corresponding places. The surface of these pads in contact with the skin was covered by a thin layer of aluminum foil. At the end of the work, skin pads were extracted with acetone, the solution dried and redissolved in 3:1 water/methanol solution, and analyzed by HPLC as above described. Recovery from extraction was 95%. The hands were washed with water accompanied by gentle brushing with a small teflon brush. The washings were collected and analyzed by HPLC.
Urine Sampling and Storage Urine samples were collected from the workers as spot specimens during the first campaign and as 8-hr or 24-hr samples during the
783 second campaign. Urine was stored frozen at --18~ before analysis.
Determination of Acephate in Urine Ten ml of urine were saturated with NaC1 and loaded onto a 10 red liquid-Iiquid extraction column (Chem-Elut-CE 1010 Anatytichem International, Harbor City, CA, USA). Efficiency of extraction was 90%. Elution was made with 20 ml dichtoromethane, the solvent was evaporated under nitrogen flow, and the residue redissoived in 0.1 ml acetone; 0.5 ~1 of this sample were injected into a gas-chromatograph (Perkin Elmer, rood. 8320-NPD with model LCI 100) equipped with a 25 m capillary amorphous silica SE52 column (i.d. 0.23 ix), operating under the following conditions: carrier gas: Helium 20 psi; injector and detector temperature: 250~ oven temperature: 12O~ for 2', 20~ up to 200~ 200~ for 6'. The nitrogenphosphorus detector was set at 280 ml/min air flow and 40 ml/min hydrogen flow. The lower limit of detection of the method was 30 ~g/L for acephate and methamidophos.
Blood Cholinesterase Assay Venous blood samples were obtained in the morning from the subjects. Erythrocyte cholinesterase activity was assayed by the Michel method (Michel 1949). Plasma cholinesterase assay was performed by a kit (IATRON Laboratories Inc, Tokyo; distributed by Poll S.p.A., Milano) based on the Trinder kinetic method (Okabe et al. 1977). According to this method, choline which is liberated from benzoyl-choline as substrate by cholinesterase, is oxidized by choline-oxidase to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximal absorbance at 500 nm.
Results Air and urine concentrations of acephate measured during the first production campaign are shown in Table 1. The median airborne exposure level during the campaign was in the order of 0.3-0.4 mg/m 3 as a t/me-weighted average on a 8 hr base. The worker in charge of packaging (MAB) showed a median exposure level slightly lower than the control-board operator (RV), although the highest peak exposure values were measured at packaging, Urine was analyzed for acephate and methamidophos, but only the first compound was present in detectable amounts. Median acephate excretion was found to be between 0.4 and 1.1 mg/L at the end of the workshift. The control board operator showed urinary levels 2 - 3 fold higher than the packager in the spot urine sample collected at the end of the workshift, but the difference was less pronounced in the other urine samples~ Cholinesterase activity 'was measured before exposure, at days 2, 7, and 13 after the beginning of exposure and 7 days after the end of the campaign. The results, shown in Table 2, did not indicate any significant change during or after exposure, as compared with individual pre-exposure values. During the second campaign of acephate formulation, workers' exposure was tested by determining beth air concentration and skin deposition of acephate. The results are
784
M. Maroni et al.
Table 1. Air concentration and urinary excretion of acephate of two workers (MAB and RV) during the first campaign of formulation
Day
Subject
Air concentration (mg/m3) sampling time 8-12 13.30-17.30
MON
MAB RV MAB RV MAB RV MAB RV MAB RV
0.086 0.083 2.050 0.277 0.549 0.338 nda 0.574 2.168 0.498
0.432 1.208 0.265 0.547 0.144 1.341 nd 0.251 1.223 0.487
0.259 0.645 1.157 0.412 0.346 0.839 nd 0.412 1.695 0.492
0.044 0.228 0.165 0.596 0.453 0.668 0.642 0.697 0.849 0.537
0.051 0.460 0.282 0.545 0.728 0.853 0.666 0.658 0.579 0.899
0.180 0.829 0.476 1.094 0.737 1.267 0.424 1.000 0.755 1.359
MAB RV MAB RV MAB RV
0.345 0.331 0.087 0.502 0.236 0.291
0.211 0.220 0.109 0.351 0.078 0.194
0.278 0.275 0.098 0.426 0.157 0.242
0.279 0.221 0.444 0.588 0.770 1.002
0.312 0.487 0.251 0.566 0.371 1.015
0.333 0.788 0.291 1.246 0.554 1.402
MAB
min max median
0.086 2.168 0.345
0.078 1.223 0.211
0.098 1.695 0.278
0.044 0.849 0.448
0.051 0.728 0.327
0.180 0.755 0.433
RV
rain max median
0.083 0.574 0.333
0.194 1.341 0.364
0.242 0.839 0.413
0.221 1.002 0.591
0.460 1.015 0.576
0.788 1.402 1.104
TUE WED THU FRI MON TUE WED
TWA 8 hr
Urine concentration (rag/L) collection time 8 12
17
nd = not determined Table 2. Activity of erythrocyte (ACHE) and plasma (PChE) cholinesterase measured in the two workers engaged in the first campaign of formulation Subject
Pre-exposure
During exposure days: 2
7
13
7 days after the end of exposure
MAB
AChEa PChEa
0.596 4279
0.594 3940
ndb ndb
0.648 4099
0.594 4348
RV
AChE a PChEa
0.539 3932
ndb 4027
0.621 3638
0.652 3699
0.622 3947
Units: ACHE: ~ pH/ml erythrocyte/hr PChE: mU/L b n d = not determined shown in Table 3. The median level of air concentration was around 1.2 mg/m3 as a time-weighted average for the packager (MRL) and 2.1 mg/m3 for the control-board operator (CN). Assuming a ventilation rate of 10 m 3 per workshift, the total inhalation loads would be at levels of 12 and 21 mg/day, respectively. The same exposure ratio between the subjects was observed for skin contamination that gave a median overall amount of 26.1 and 41.9 mg per day, respectively. It is interesting to note that the amount recovered from the hands accounted for 90% of total skin exposure. In order to estimate the amount of acephate absorbed by the workers, it is necessary to hypothesize the fraction absorbed through the airway system and through the skin. Assuming 3 0 - 5 0 % a b s o r p t i o n of the inhalation dose and 10-20% absorption of the skin dose, the median intake for these workers would lie in the order of 10-20 mg per day. Urinary excretion of acephate was determined in these
workers by the third day of the campaign and followed-up for one week. Methamidophos again was not detected in any of the urine samples which, however, contained measurable amounts of acephate (Figure 1). The analytical results showed that the control-board operator had a remarkable acephate excretion with daily peaks between 3 and 8 mg/L, while the excretion of the packager was lower, with the exception of a single-day value exceeding 9 mg/L. Thus, urinary values were to some extent concordant with external exposure assessment, providing a median value of acephate excretion during the week higher for the control-board operator than for the packager. The peak excretion observed for the latter on Wednesday occurred after the workshift in which the highest skin contamination was measured, even though the differences with the preceding or following days were not as remarkable for skin exposure as were for urinary excretion values.
785
Human Exposure to Acephate Table 3. Exposure to acephate of two workers (MRL and CN) during the second campaign of formulation Inhalation total load (estimated) mg
Day
Subject
WED
MRL CN
0.54 3.15
5.4 31.5
7.8 8.6
1.32 0.71
nd ~ nd
9.12 9.31
14.52 40.81
THU
MRL CN
0.66 0.72
6.6 7.2
23.9 35.1
1.65 1.56
nd nd
25.55 36.66
32.15 43.86
FRI
MRL CN
1.22 0.75
12.2 7.5
29.3 33,6
1.73 0.52
0.03 0.06
31.06 34.18
43.26 4! ,68
MON
MRL CN
1.67 1.81
16.7 18.1
32.8 46.0
0.96 1.71
nd nd
33.76 47.71
50.46 65o81
TUE
MRL CN
2.34 2.50
23.4 25.0
20.0 44.8
1,13 2.45
0.07 nd
21.2 47.25
44.60 72.25
WED
MRL CN
1.51 2.16
15.1 21.6
41.0 50.3
0.56 2.16
0.04 0.05
41.60 52.51
56,7 74.11
THU
MRL CN
0.8 3.47
8.0 34.7
30.8 36.2
1,36 5.45
0.05 nd
32.21 41.65
49.21 76,35
FRI
MR L CN
1.26 4.36
12.6 43.6
22,6 60,6
1.27 3.23
0.19 0.07
24.06 63.9
36,66 107.5
12.5 21.7
24.5 36.7
1.29 1.78
0.05 0.06
26.1 41.9
40.9 66.2
MRL median CN median
Skin exposure Hands Face mg mg
Body nag
Skin total (estimated) rag
Total estimated exposure mg
Inhalation exposure TWA mg/m 3
and = not determined
Fig, 1. Urinary excretion of acephate of the workers MRL and CN during the second campaign. Shadowed bars indicate exposure periods
W h e n correlating e x p o s u r e m e a s u r e m e n t s with urinary excretion, significant P e a r s o n ' s coefficients of correlation w e r e found b e t w e e n total individual e x p o s u r e (as estimated in Table 3) and urinary acephate concentration in the 8 hr
following the w o r k s h i f t (r = 0.717; P = 0.001) or in the 24-hr refine collected since the beginning of the w o r k s h ~ t (r = 0.788; P = 0.001). If these same urinary parameters are tested against inhalation or skin e x p o s u r e values separately,
786
M. Maroni et
al.
Total exposure
(rag) 110-
10090807060509 o9
40r =
30-
.79
20~o I 0
I 1000
i 2000
I 3000
I
I
I
I
4000
5000
6000
7000
URINARY
ACEPHATE
the coefficients of correlation are as follows: r = 0.465 for inhalation exposure vs 8-hr after-work urine; r = 0.518 for inhalation exposure vs 24-hr urine; r = 0.720 for skin exposure vs 8-hr after-work urine; r = 0.777 for skin exposure vs 24-hr urine. The scatter diagrams showing the correlation between total exposure or skin exposure and 24-hr urinary excretion of acephate are shown in Figures 2 and 3. Cholinesterase activity in these workers had been measured for four consecutive months before exposure; thus, mean and variation of the individual baseline were accurately estimated for each subject (Table 4). Cholinesterase assay during and after exposure did not show any significant change in the packager. In the control-board operator, erythrocyte ChE activity was slightly depressed (P < 0.05) 1 week and 4 months after exposure. Plasma cholinesterase showed a transient decrease, at borderline level of significance, only during exposure.
Discussion Acephate shows low to moderate acute toxicity in mammals, with oral LDso values of 361 mg/kg bw in the mouse and 866-945 mg/kg bw in the rat (Worthing and Walker 1987). 15o values of acephate for human plasma and bovine erythrocyte cholinesterase are one or two orders of magnitude higher than those of methamidophos (Tucker 1972 quoted in F A t 1977). In man, 150 values for brain ACHE, erythrocyte ACHE, and plasma cholinesterase are in order of decreasing magnitude, with values of, respectively, 5.4 mM, 2.7 mM, and 1.8 mM (Bennet and Morimoto 1982 quoted in F A t 1985). Biotransformation of acephate has been studied in the rat
Fig, 2. Correlation between total exposure and urinary excretion of acephate
(IJg~24h) after oral as well dermal treatment with 5-methyl-14C-acephate. After oral administration, excretion of radioactivity was rapid, with 82-95% of the administered dose recovered in urine, 1-4% exhaled, and 1% found in the faeces. Urinary metabolites were identified as unchanged acephate (73-77%), O-S-dimethylphosphorothioate (3-6%), and Smethyl-acetylphosphoramidothioate (3-4%). Methamidophos was not detected (Lee 1972, quoted in F A t 1977). After a single dermal application at a level of 10 mg/kg bw, the 14C dose was rapidly absorbed into the blood, with max:imum concentration occurring after one to three hr and a secondary peak observed at 48 hr. Urinary excretion of radioactivity showed a maximum between 6 and 24 hr. Within 3 days, about 30% of the dermal dose was excreted as unchanged acephate and 1% as methamidophos (Tucker 1974, quoted in F A t 1985b). Studies in man have been very limited and none of them has been published in the scientific literature. Some information is available in abstracted form in the Reports from the Joint Meeting on Pesticide Residues published by the FAt. No adverse effects were reported for workers having urinary acephate concentrations up to 5 mg/L in a survey on subjects exposed to acephate in pilot plant and formulation processes. The urine of the workers of the pilot plant were reported to contain other metabolites or technical products of the production besides acephate. The subjects exposed to technical acephate during formulation were reported to show methamidophos and trimethylphosphate as urinary metabolites ( F A t 1977). Based on observations on the acephate decay in human blood added with the compound, it has been proposed that blood may contain certain enzymes capable of metabolizing acephate to methamidophos (Singh 1984). The present study does not support the concept that acephate in the human
787
Human Exposure to Acephate Skin exposure (rag) 70-
60-
50|
Q
400 | | 0
30-
0
r
O
20-
= .78
Fig. 3. Correlation between skin exposure and urinary excretion of acephate
10- , I 0
1000
2000
3000
4000
5000
6000
7000
URINARY ACEPHATE (~g/24hl
Table 4. Activity of erythrocyte (ACHE) and plasma (PChE) cholinesterase measured for the workers MRL and CN Subject
Jan.
Pre-exposure values Febr. Apr.
May
Mean
lower 5th percentile
Exposure June 3
1week after
4 months after
MRL AChE a PChE a
0.929 3055
0.962 3204
0.921 2378
0.952 3148
0.941 2946
0.901 2178
0.969 2407
0.966 2592
ndb ndb
CN
0.765 3350
0.757 3795
0.647 3119
0.760 3855
0.732 3530
0.618 2820
0.681 2826r
0.600c 3446
0.613 ~ 3845
AChE a PChE a
a = Units: see Table 2 b = n d = not determined c = Values close or inferior to the 5th percentile of the distribution of the pre-exposure values
organism is substantially converted into methamidophos, at least as far as this can be evaluated by the study of its urinary excretion. Our workers were estimated to have absorbed daily doses of about 10-20 nag per day and showed urinary concentrations of unchanged acephate from 1 to 10 mg/L. The presence in urine of methamidophos at a concentration ratio greater than 1:100 so acephate was excluded. Urinary excretion of acephate followed a pattern consistent with exposure, showing peak values of excretion in the samples collected during the workshift or in the 8 hr after the end of the workshift. Urinary elimination was quite rapid; urine concentrations were undetectable within 48 hr after the end of exposure. In the formulation process under study, skin exposure was remarkable, with the main contribution given by hand contamination. Skin exposure as assessed by the conventional method of the skin pads, provided an estimate of individual exposure highly correlated with urinary excretion of acephate. This issue deserves attention in the light of experi-
mental data pointing to a substantial penetration of acephate through rat skin (Tucker t984 quoted in FAO 1985b). Total exposure calculated by combining inhalation and skin e x p o s u r e was well c o r r e l a t e d with e x c r e t i o n , accounting for about 60% of the variation of the biological results. The evaluation of the biological effects of acephate was made in this study by the assay of red cell and plasma cholinesterase activity. No changes with respect to pre-exposure values were observed for those subjects whose urinary acephate excretion averaged 1 - 2 mg/L or less. The subject having u r i n a r y e x c r e t i o n values b e t w e e n 3 and 8 mg/L showed a slightly decreased value of ptasma cholinesterase during exposure ( - 2 0 % ) and of erythrocyte cholinesterase afterwards ( - 1 8 % ) . These changes were just outside the limits of analytical and biological variability of the measurements and remained very far from the threshold at which clinical symptoms of anticholinesterase poisoning are expected.
788
References Bennet EL, Morimoto H (1982) The comparative in vitro activity of acephate technical on brain, erythrocyte and plasma cholinesterases from the human, monkey, and rat. Unpublished report quoted in FAO, 1985 Bull DL (1979) Fate and efficacy of acephate after application to plants and insects. J Agric Food Chem 27:268-272 Eto M, Okabe S, Ozoe Y, Maekawa K (1977) Oxidative activation of O,S-dimethyl phosphoramidothiolate. Pestic Biochem Physiol 7:367-377 FAO (1977) 1976 Evaluation of some pesticide residues in food. The monographs. ACEPHATE. FAO Plant Production and Protection Series n. 8:l-54-Rome (1985a) Pesticide residues in food, 1984. Report of the joint meeting on Pesticides residues. Rome 24 Sept, 3 Oct 1984 (PAPER 62) - (1985b) Pesticide residues in food, 1984. The monographs-ACEPHATE--Fao Plant Production and Protection Paper 67:527-536-Rome Hussaln MA, Mohamad RB, Olofts PC (1984) Toxicity and metabolism of acephate in adult and larval insects. J Environ Sci Health B19(3):355-377 Kao TS, Fukuto TR (1977) Metabolism of O,S dimethyl propionyland hexanoylphosphor-amidothioate in the housefly and white mouse. Pestic Biochem Physiol 7:83-95 Lee H (1972) Metabolism of ORTHENE in rats. Unpublished report quoted in FAO, 1977 Magee PS (1982) Structure-activity relationships in phosphorami-
M. Maroni et al. dates. In: Coats JR (ed) Insecticide mode of action. Academic Press, New York Michel HO (1949) Electrometric method for determination of red blood cell and plasma cholinesterase activity. J Lab Clin Med 34:1564-1568 Okabe H, Sagesaka K, Nakajima N, Noma A (1977) New enzymatic assay of cholinesterase activity. Clin Chim Acta 80:87-94 Pack D (1972) Orthene insecticide occupational exposure. Field station personnel. Unpublished study quoted in FAO, 1977 Senanayake N, Johnson MK (1982) Acute polyneuropathy after poisoning by a new organophosphate insecticide. New Engl J Med 306:155-157 Singh AK (1984) Improved analysis of acephate and methamidophos in biological samples by selective ion monitoring gas chromatography-mass spectrometry. J Chromatogr 301:465469 Swencicki R, Hartz W (1972) Orthene human exposure study. Clinical evaluation. Unpublished study quoted in FAO, 1977 Tucker B (1972) Acetylcholinesterase inhibition of orthene and ortho 9006. Unpublished report quoted in FAO, 1977 WHO (1982) Field Surveys of exposure to pesticides: Standard Protocol. WHO, Division of Vector Biology and Control. Document 82.1 Worthing CR, Walker SB (1987) The pesticide manual. 8th ed British Crop Protection Council, Thornton Heath, UK
Manuscript received February 15, 1989 and in revised form August 8, 1989.
