Dermal exposure and urinary metabolite excretion in farmers repeatedly exposed to 2,4-D amine

Toxicology Letters, 33 (1986) 73-83 Elsevier

73

TOXLett. 16383

DERMAL EXPOSURE AND URINARY METABOLITE EXCRETION FARMERS REPEATEDLY EXPOSED TO 2,4-D AMINE (Herbicide; ground rig application; dermal deposition; monitoring; single and multiple exposures)

IN

handwash; biological

R. GROVER=, C.A. FRANKLINb, N.I. MUIRb, A.J. CESSNA” and D. RIEDELb ‘Environmental Chemistry Section, Agriculture Canada, Research Station, 5000 Wascana Parkway, Regina, Sask. (S4P 3A.2, Canada) and ‘Bureau of Chemical Hazards, Environmental Health Directorate, Health and Werfare Canada, Environmental Health Centre, Tunney’s Pasture, Ottawa, Ont. (KIA 0L2, Canada) (Received 24 July 1986)

SUMMARY Following multiple exposures to 2,4-D amine salts under normal field conditions in which ground rigs were used, farmers who sprayed once or twice had an average dermal exposure of 655 fig 2,4-D acid equivalents (a.e.)/kg a.e. sprayed and an average cumulative urinary metabolite excretion of 10.9 pg 2,4-D a.e./kg a.e. sprayed. One very high outlier value was excluded from these averages (F). Following 4 to 7 exposures, the average cumulative dermal exposure was 1077 pg 2,4-D a.e./kg a.e. sprayed and the average cumulative urinary metabolite excretion was 21.5 cg 2,4-D a.e./kg a.e. sprayed. The amount of 2,4-D metabolite in urine was significantly correlated with the amount of herbicide sprayed and the time required for the urinary metabolite excretion to return to background concentration was related to the amount sprayed and the number of exposures.

INTRODUCTION

It has been reported that the amount of 2,4-D metabolite excreted in the urine of farmers applying 2,4-D for a single work day was correlated with both the daily hours of exposure and the amount of 2,4-D applied. The elimination half-life (Es) for the 2,4-D (acid) excretion ranged from 35 to 48 h [l]. This paper reports on a study in which farmers were exposed under typical field conditions on multiple occasions in a short time to amine salts of 2,4-D. Farmers from the Regina, Saskatchewan vicinity were chosen only on the basis of their willingness to participate in the program and provide the required monitoring samples. Abbreviation:

a.e., acid equivalents.

0378-42741861.303.50 @ Elsevier Science Publishers B.V. (Biomedical Division)

74

The study was designed to interfere as little as possible with the manner in which they normally carried out spray operations such as tank filling, container rinsing, and sprayer maintenance, and the farmers used their own tractors and ground rigs with no restrictions on boom or tank size, nozzle type, or whether tractors were equipped with cabs. The farmers used boom heights and spray volumes and pressures appropriate for the nozzle type used, and application rates of 2,4-D which were appropriate to the crop and the weed infestation present; they sprayed under climatic conditions they considered appropriate. STUDY PARAMETERS

Study group and control group Four farmers participated in the study the first summer (1981) and 5 the next (1982), 2 of whom were involved both summers. One farmer sprayed only once in the season, but the others sprayed 2,4-D from 2 to 7 times in a I-ldday period and not always on consecutive days. The control group consisted of 8 volunteers who were not at all involved in the spray operations. Spray operations Spray operations were conducted from mid-June to early July, with all subjects following their normal mixing and spraying procedures. All used tractor-pulled ground rigs which consisted of trailer-mounted spray tanks equipped with booms at the rear and operated with pumps powered by tractor power take-offs. A single spray operation was defined as an uninterrupted work period which included tank filling, spraying the herbicide, and breaks for rest and meals. When spray operations were stopped due to poor weather conditions, mechanical breakdown or other reasons, the resumption of spraying was considered a second spray operation. Urine sampling and analysis Total 24-h urine samples were collected during the spray operation(s), and then for 4-7 days after the last spray operation. Urine samples were taken on the day before or the day of the first spraying to estimate background values. The samples were collected such that the first urine void each day was included as part of the previous day’s sample. The urine voids were collected and stored at - 10°C until extraction. A lOO-ml aliquot was analyzed by gas chromatography [2]. Previous experiments [2] had shown that recoveries of 2,4-D from urine fortified at the 50 pg/l level and similarly stored prior to extraction were in the order of 80%. Dermal exposure (patches and handwashes) A standardized set of laundered clothing which covered most of the body with two layers of cotton was issued to all subjects at the start of each spray operation [3, 41. None of the subjects used respirators in either year. Four dermal samplers

75

(patches) were pinned to the outside of the farmers’ clothing of which the chest patch was used to estimate face exposure and the back patch to estimate neck and head exposure. Nine patches were placed underneath the clothing to estimate skin exposure by absorption of 2,4-D through the clothing [3]. Immediately after each spray operation was completed, the subjects’ hands were washed with a sodium bicarbonate solution, the dermal patches were removed and stored, and the contaminated clothing was removed [4]. Each completed spray operation was considered an ‘exposure’ and the number of exposures per subject varied from 1 to 7 in a given season. In total, there were 30 such single exposures resulting in 30 patch and handwash data sets which were analyzed to assess the correlation of the estimated dermal contact exposure with such parameters as amount sprayed, spray time, tank fills and area sprayed [4]. The residue values are expressed in terms of a.e. of 2,4-D. RESULTS AND DISCUSSION

Variability of spray operations The ages of farmers participating in this study varied from 24 to 57 years, indicating large differences in years of farming experience and previous exposure to 2,4-D and other herbicides. The variability in application parameters in a field study such as this is shown in Table I. The farmers used 6 different nozzle types, resulting in wide ranges of spray volumes (45-l 15 l/ha), pressures (172-310 kPa), and boom heights (34-76 cm). Sprayer-tank capacity, which varied by more than a factor of three (680-2270 l), would directly determine the number of tank fills required per spray season, Boom length, which varied from 12 to 22 m, together with the sprayertank capacity, would affect the time required for spraying in a spray season. TABLE I OPERATIONAL

PARAMETERS

FOR APPLICATORS

APPLYING 2,4-D BY GROUND RIG

Exposure subject

Number of exposures

Number of tank fills

Time sprayed Area sprayed Total amount Application rate sprayed (h) (ha) (g a.e./ha) (kg a.e.)

A B BLlb B” D E F G

4 1 2 2 4 7 2 6

5 3 2 4 13 10 15 23

1.5 4.4 5.1 9.0 17.8 19.8 20.5 37.5

aBoth application rates were used. bSecond spray 2 weeks after the first application. ‘Two applications the following year.

163 71 101 141 241 170 281 443

102.6 24.8 35.3 49.3 89.9 71.3 127.9 186.0

420/560” 350 350 350 6301350” 420 455 420

Metabolite excretion Urine samples were collected every day over the entire spray operation and for 4 to 7 days after the last exposure. Farmer B sprayed 2,4-D once (24.8 kg a.e.) and then did not spray again for 14 days. This operation was considered to be a single exposure, since the urinary metabolite excretion returned to background concentration before he sprayed again. The next time he sprayed (35.3 kg a.e.), he had two exposures (B”) and the following year he also had 2 exposures in which he sprayed 49.3 kg a.e. (B’). One other farmer (F) sprayed twice (127.9 kg a.e.). There were 2 farmers (D and A) who each sprayed 4 times (89.9 and 102.6 kg a.e., respectively). Farmer G sprayed a total of 186 kg a.e. on 6 days and farmer E, 71.3 kg a.e. on 7 days. The patterns of urinary excretion of 2,4-D a.e. for all these farmers are shown in Figs. 1 and 2. The variation in the number of exposures over differing periods of time precluded any averaging of the metabolite data. However, it can be seen that in all farmers the excretion of 2,4-D a.e. in the urine increased after spraying. The excretion data show that the daily amounts of 2,4-D a.e. excreted were a function of (a) the number of consecutive exposures, (b) the number of days between exposures, and (c) the time elapsed after the last exposure. In the case of the single exposure (B), the maximum daily urinary excretion of 2,4-D a.e. occurred on the 3rd day after exposure. Maximum concentrations of phenoxy herbicides in urine following a single exposure have been observed at similar times in other studies, for example, after aerial applications of 2,4-D (25-45 h) [3], ground boom applications of 2,4-D (48 h) [5], and B

B

8”

625 4-

2-x

40

I

oc. 1

16

6

24

Day

Fig. 1. Urinary excretion of 2,4-D a.e. following the kg a.e. sprayed at each exposure time.

/k

‘4

1

16

0

24

Day

one or two exposures;

numbers

on the graphs

represent

6

A

D

2-

OE

G

2-

oc I

1

16

6 Day

24

1

16

6

24

Day

Fig.2. Urinary excretion of 2.4-D a.e. following 4, 5 or 7 exposures; numbers on the graphs represent the kg a.e. sprayed at each exposure time.

dermal exposures of volunteers to MCPA (24-48 h) [6]. Consecutive daily exposures (A,G) resulted in a continuous rapid increase in daily amounts excreted. After cessation of exposure there was a gradual decline in urinary excretion of 2,4-D. A break of 2 or more days between exposures resulted in 2 maxima in the daily excretion plot for D (Fig. 2). E, who sprayed only small amounts on 7 days, showed a small gradual increase in 2,4-D a.e. excretion level. In all spray situations, detectable concentrations of 2,4-D a.e. were still present in urine samples collected 4 to 7 days after the last exposure. Other investigators have also observed prolonged urinary excretion of phenoxy herbicides after the last exposure [1,3,5,7]. Only in the case of the single exposure were the 2,4-D a.e. excretion levels observed to decrease to background amounts (on day 8 after exposure). It should be noted that all workers had detectable levels of 2,4-D a.e. in their urine (l-39 pg) prior to initiation of the spraying for this study. These residues probably resulted from some initial contact with 2,4-D during transport and handling of the herbicide containers, sprayer maintenance or nozzle calibration. As indicated above, the urinary 2,4-D excretion patterns reported here in male farmers applying 2,4-D amine salt (Figs. 1 and 2) generally resemble those seen in other studies on farmers and spray applicators similarly exposed to 2,4-D or other chlorophenoxy herbicides [5, 61. They can also be compared to the corresponding patterns seen in two small groups of male volunteers who ingested single doses of 5 mg of 2,4-D (acid)/kg body wt [8, 91. The men in the first study excreted an average of 76.5% (SD. 8.4Vo) of this dose in 48 h; those in the second study ex-

78

creted 87.6-106.3% of the dose in 144 h with an elimination half time of 10.2-28.4 h. The complete to nearly complete recovery of the dose in the second study also suggested that little if any 2,4-D was excreted by a route other than urine. Studies with rats receiving multiple peroral doses of 2,4-D [lo] showed that the urinary excretion peaks tend to become higher and broader with either increased doses or repeated doses. These comparisons indicate that the general features of the urinary 2,4-D excretion patterns of the mainly dermally exposed farmers show similarities with those seen in perorally dosed men and animals. Moreover, as the results of at least one peroral study in man [9] suggested that most of the absorbed 2,4-D is excreted in the urine, one can also conclude that only a small percentage of the 2,4-D amine salt deposited on the skin of the farmers was absorbed [4]. Exposure estimation using cumulative data (a) Urinary metabolites Since the intervals between exposures in this study were short, excretion of 2,4-D absorbed during a previous exposure would not have been completed before the next exposure occurred. Therefore, excretion of 2,4-D for all the days between the first and last exposure and for the 4 days immediately after the last day of exposure was summed and called 2,4-D a.e. excretion. The cumulative amounts of 2,4-D a.e. excreted per subject ranged from 215-6258 pg (Table II). As determined by the Pearson Correlation Coefficients, there was a positive correlation between the total amount (kg a.e.) of 2,4-D applied and the cumulative amount of 2,4-D a.e. excreted (Table III). The identity correlation, in which the raw data are correlated, was significant at the 0.01 level. The log-log and rank transformation were also considered since some extreme 2,4-D exposure values existed in the response variables; both of these were significant at the 0.05 level. The cumulative amount of 2,4-D a.e. excreted was also significantly correlated with area sprayed, spray time, and number of tank fills [4]. When the excretion data were normalized to the amount sprayed, the average value for 1 or 2 exposures was 9 pg/kg a.e. sprayed and more than double that amount for 4 to 7 exposures (average 21 pg/kg a.e. sprayed). It would appear that the number of exposures has an effect on the amount excreted per kg applied, at least under the conditions of this study. This possibility should be taken into consideration when extrapolating from studies in which there have been only single exposures. (b) Dermal exposure (patches) All sampling patches (4 outside, 9 inside) contained detectable amounts of 2,4-D and could be separated into 3 categories: those under 2 layers of clothing, those which were on exposed areas, and those which were on exposed areas and close to the mixing zone [4]. The cumulative dermal 2,4-D a.e. deposition estimates for

4803 203 976 12899

127.9

8%9

102.6

186.0

71.3

D

A

G

E

B’ ‘” B’, see note Table 1.

*%taf amount excreted during expwwe

34 407

period and for 4 days after fast eqwsure.

54909

40790 88 400

4290 85 950

1694971

8431

F

B’ 66 940

JO

10790

I873

24*8 35.3 49.3 3004

Body

“__vv-.-Total &kg sprayed

951.

67 808

1245

1763 6258

444

I 5’12

45 593 292 376

f 339

432 1586

13286

120357

289 215 734

J69926J

1529

75 377

IS,% 391

1883 13794

I@ 2,4-D a.e,

11.5

33.6

17,2

17.6

3.4

14.9

6.1

JJ.7

P13& sprayed

of 2&D a.eB -._,. -~.-

Hands

Cumutaiive urinary excretion

&g 2,4-D a-e.)

-

l’%?R

Catcuia~d demal ~~p~~j~~~~

OF 2.4-D BY GROUND RIO

;I,

subject

IN SPRAYING

ESTJMATES OF DERMAL 2,4-D a.e. DEPOSITION (WJTH AND WJTWOUT HANDS) AND URINARY 2,4-D a.e. EXCRETION

FARMERS INVOLVED

CUMULATIVE

TABLE? II

80

TABLE III PEARSON CORRELATION COEFFICIENTS BETWEEN AMOUNT SPRAYED AND DERMAL DEPOSITION (BODY OR HANDS) OR URINARY EXCRETION -____ Urine Hands Data transformation* Body identity Log-log Rank

0.41 0.78b 0.83’

0.47 0.63b 0.48

0.8T 0.78b 0.74”

‘Log-log examines correlations between log (amount sprayed) and log (dermal deposition) or log (urinary excretion). Rank examines correlations between rank (amount sprayed) and rank (dermal deposition) or rank (urinary excretion). bSignificantly different from zero at P
body, hands and the total (body + hands) for each subject are given in Table II. When the data were analyzed using Pearson Correlation Coefficients, there was a positive but not statistically significant correlation between the amount sprayed and the amount deposited on the body or the hands (Table III). Following log-log transformation of the data, significance was observed at the 0.05 level for the hand and for the body. With a rank transformation, the correlation between amount sprayed and body deposition became significant. It is apparent that the variability of the data necessitates log-log or rank transformation and that the amount sprayed vs. body deposition correlation is stronger than the amount sprayed vs. hand deposition correlation. This observation is not surprising, as the unprotected hands are susceptible to accidental or random contact with contaminated containers, equipment and clothing. The unpredictable nature of exposure to the hands and the large contribution of hand exposure to total exposure emphasizes the need for reliable hand protection when minimization of worker exposure is essential. When the cumulative deposition data were normalized to the amount sprayed (Table II), the values had a wide range: 75.9 to 13 286 pg deposited/kg 2,4-D a.e. sprayed (average 2448 pg/kg a.e. sprayed). There was no obvious relationship between the total deposition &g a.e.), normalized deposition &g/kg a.e. sprayed) and the number of spray operations. When normalized on the basis of the amount sprayed, the range was much narrower for the normalized urine values (3.4-33.6 pg/kg a.e. sprayed) than the normalized deposition values (75.9-13 286 p&/kg a.e. sprayed). Correlation of dermai and metabolite data The association between the amount of 2,4-D a.e. amount of 2,4-D a.e. deposited on the body, hands hands was examined by the Pearson Correlation transformed as described earlier and the correlations

excreted in the urine and the or the total on the body and Coefficients. The data were are presented in Table IV.

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For the urinary excretion and the hand deposition, all correlations were positive and significant at the 0.05 level for all three transformations. The total 2,4-D a.e. deposition (body and hand) was not significantly correlated with the urinary excretion. This observation is not surprising, given the wide variability in the 2,4-D a.e. residues on the patches and the great influence that these values have on the calculation of total body deposition. Even with data transformation, the correlations were still strongly influenced by several very high deposition values for workers who had very low urine excretion values. It has been argued that passive dosimetry techniques and biological monitoring cannot be conducted concurrently, the reason being that patches and handwashes or sampling gloves would reduce the amount of pesticide available for absorption and excretion. In this study, it is unlikely that the use of patches on the workers would have an important effect on the availability and absorption of dermally deposited residues, since the surface area which was covered by the patches placed next to the skin was very small. In addition, 7 of the 9 patches that were under 2 layers of clothing had insignificant amounts of residue when compared to patches at other locations demonstrating that there was negligible penetration of the active material through the clothing. Therefore, the interference from the patches relative to the total body surface area available for contact and absorption would be negligible. The manner in which the hand exposure is measured could potentially cause greater problems since this site is frequently heavily contaminated. There are two factors in this study which suggest that the manner in which the hand exposure was measured would have minimal impact on the urine data. The first was that the deposited 2,4-D was available for absorption for the full working day, since the handwashes were done at the end of the day (1 to 14.5 h) and at a time similar to when the worker would normally wash his hands with soap and water, after completion of his daily tasks. The second was that sodium bicarbonate solution was selected for the handwash TABLE IV PEARSON CORRELATION COEFFICIENTS BETWEEN THE URINARY 2,4-D a.e. EXCRETION AND DERMAL 2,4-D a.e. DEPOSITION (BODY, HANDS OR TOTAL) Data transformation”

Body

Hands

Total (body + hands)

Identity Log-log Rank

-0.12 0.34 0.48

0.67b 0.63b 0.76b

- 0.08 0.44 0.45

aLog-Iog examines correlations between log (amount sprayed) and log (dermal deposition) or log (urinary excretion). Rank examines correlations between rank (amount sprayed) and rank (dermal deposition) or rank (urinary excretion). bSignificantly different from zero at PcO.05 (one-sided).

82

due to its ability to dissolve 2,4-D. Given the pH of a sodium bicarbonate solution (1% solution, pH 9.5) and the pH of a soap-and-water solution (pH 9-lo), it is likely that either method would remove a similar amount of 2,4-D from the skin. Unfortunately, little research has been carried out on the ability of solvent washes to remove pesticides from the surface of contaminated hands and the issue cannot be fully resolved without it. The lack of correlation between the cumulative deposition estimates and the urinary excretion of 2,4-D is likely more attributable to the variable nature of the dermal deposition, influencing the outcome of the correlation analysis, rather than interference from the passive dosimetry techniques. FURTHER WORK

It would be useful to have controlled studies to more precisely measure the percentage of herbicide not excreted in the urine at the end of the collection period. The urine data from field studies could then be corrected for incomplete urinary excretion. Controlled laboratory studies would probably be necessary to determine the effects on the 2,4-D excretion patterns exerted by age, body weight to body surface ratios, environmental temperature, activity level, fluid consumption, and other factors which might affect urinary 2,4-D excretion. The frequency and extent of ‘outlier’ values deserve to be examined in future studies because these most highly exposed individuals may be the ones most at risk from adverse effects of the applied herbicide mixtures. ACKNOWLEDGEMENT

The authors wish to thank Dr. R. Burnett and Ms. J. Kearney for statistical analysis of the data and Ms. K. Nesbitt for typing the manuscript. REFERENCES 1 R.G. Nash, PC. Kearney, J.C. Maitlen, C.R. Sell and S.N. Fertig, Agricultural applicators exposure to 2,4-dichlorophenoxyacetic acid, in J.R. Plimmer (Ed.), Pesticide Residues and Exposure, Am. Chem. Sot. Symp. Ser. No. 182, 1982, 119. 2 R. Grover, A.J. Cessna and L.A. Kerr, Procedure for the determination of 2.4-D and dicamba in inhalation, dermal, hand-wash and urine samples from spray applicators, J. Environ. Sci. Hlth., B20 (1985) 113-128. 3 C.A. Franklin, R. Grover, J.W. Markham, A.E. Smith and K. Yoshida, Effect of various factors on exposure of workers involved with the aerial application of herbicides, Trans. Am. Conf. Ind. Hyg., 43 (1982) 97-l 17. 4 R. Grover, A.J. Cessna, N.I. Muir, D. Riedel, C.A. Franklin and K. Yoshida, Factors affecting the exposure of ground applicators to 2,4-D dimethylamine, Arch. Environ. Contam. Toxicol., (1986) (in press).

83 5 W.M. Draper and J.C. Street, Applicator exposure to 2,4-D, dicamba, and a dicamba isomer, J. Environ. Sci. Hlth, B17 (1982) 321-339. 6 B. Kolmodin-Hedman, S. Hoghmd, A. Swensson and M. Akerblom, Studies on phenoxy acid herbicides, II. Oral and dermal uptake and elimination in urine of MCPA in humans, Arch. Toxicol., 54 (1983) 267-273. 7 M. Akerblom, B. Kolmodin-Hedman and S. Hoghmd, Studies of occupational exposure to phenoxy herbicides, in R. Greenhalgh and N. Drescher (Eds.), Pesticide Residues and Formulation Chemistry, Pergamon, New York, 1983, pp. 4-227. 8 J.D. Kohli, R.N. Khanna and B.N. Gupta, Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man, Xenobiotica, 4/2 (1974) 97-100. 9 M.W. Sauerhoff, W.H. Braun, G.E. Blau and P.J. Gehring, The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man, Toxicology, 8 (1977) 3-11. 10 M.T. Shafik, H.C. Sullivan and H.F. Enos, A method for determination of low levels of exposure to 2,4-D and 2,4,5-T, Int. J. Environ. Anal. Chem., 1 (1971) 23-33.