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Applicator exposure to acetochlor based on biomonitoring
Regulatory Toxicology and Pharmacology Regulatory Toxicology and Pharmacology 43 (2005) 141–149 www.elsevier.com/locate/yrtph
Applicator exposure to acetochlor based on biomonitoring Christophe A. Gustin *, Sharon J. Moran, John D. Fuhrman, Mitchell L. Kurtzweil, Joel M. Kronenberg, David I. Gustafson, Monte A. Marshall Monsanto Company, St. Louis, MO, USA Received 14 February 2005 Available online 19 September 2005
Abstract Biomonitoring was used to assess the combined dermal, oral, and inhalation exposure associated with the agricultural use of Harness Plus, an emulsifiable concentrate formulation of the herbicide acetochlor. Twenty Spanish farmers handled and applied acetochlor to maize in the spring of 2003, following the product label recommendations. Open- and closed-cabin applications were equally represented. Urine was collected during six consecutive days, starting the day prior to application. Daily composites were analyzed for 2-ethyl-6-methyl-aniline, a common chemophore representing the major urinary acetochlor metabolites. All applicators showed detectable concentrations in urine after application. Although, the open-cabin applicators treated fewer hectares, they showed significantly higher exposure compared to the closed-cabin applicators (average exposure: 0.004 and 0.002 mg/kg bw/day, respectively). Linear regression analysis suggested that untracked incidents had a significant impact on the total exposure. Other events that may have contributed to the observed exposure are repair of faulty equipment, accidental spillages, splashes, and inadequate use of protective gloves. The average margins of exposure (MOE) for farmers ranged from 23,000 (open cabin) to about 44,000 (closed cabin). For professional applicators the MOEs were 10-fold lower. These MOEs clearly indicate that no adverse health effects should be expected from agricultural acetochlor applications. 2005 Elsevier Inc. All rights reserved. Keywords: Biomonitoring; Urine; Applicators; Acetochlor; Pesticide; Exposure
1. Introduction Acetochlor (2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6methylphenyl) acetamide) is an active ingredient (a.i.) of the chloroacetanilide family, used as a broad-spectrum pre-emergent and early post-emergent herbicide in maize for the control of grasses and selected broadleaf weeds. Acetochlor is commercialized in different world areas under different brand names and in different formulation types. This paper describes the results obtained from a biomonitoring study of farmers exposed to Harness Plus, an emulsifiable concentrate (EC) formulation containing 840 g/L of acetochlor and safened with furilazole (3*
Corresponding author. Fax: +1 314 694 8774. E-mail address: [email protected] (C.A. Gustin).
0273-2300/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2005.06.015
(dichloroacetyl)-5-(2-furanyl)-2,2-dimethyl oxazolidine) to prevent phytotoxic effects on emerging maize seedlings. The study was conducted during the spring of 2003 in Catalunya, one of the major maize growing areas in Spain. Maize is treated pre- or early post-emergent with a single acetochlor application. The maximum labeled application rate for this formulation in Europe is 2.4 liters/hectare (L/ha) or 2.016 kilogram (kg) a.i./ha. The window of application varies with geography but typically runs from early March to mid-May, which coincides with the timing of this study. Biomonitoring is a method of evaluating the absorption of chemicals by measuring the chemical or its metabolites in body fluids such as urine. The analysis of urine provides a quantitative measure of the absorbed dose when used in combination with a thorough
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understanding of the metabolism and pharmacokinetics of the test product. The direct measurement of the absorbed dose is the principal advantage of biomonitoring over passive dosimetry as it determines actual, rather than potential, absorption (OECD, 1997). Indeed, the absorbed dose is a measure of the amount of chemical that becomes systemically available through dermal uptake, oral ingestion, and inhalation. The well-documented mammalian metabolism of acetochlor permitted biomonitoring (urine) to be used as a tool for quantitative exposure assessment.
2. Methods 2.1. Selection of test subjects Study participants were selected from registered pesticide users in and around the Lleida (Catalonia) area. Twenty farmers were selected for participation in this study based on well-defined criteria, primarily related to professional experience, availability of equipment, maize growing area, and willingness to participate. Suitability of the participants was determined by means of an enrollment questionnaire. Once selected, volunteers were informed in more detail about the study, received guidance on Good Agricultural Practices, and were asked to sign an informed consent form allowing their data to be used for research purposes. Participating farmers received a cash incentive after the monitoring period and were provided with the formulation used during the on-study application. 2.2. Test system The applicators mixed and applied the acetochlor formulation at 2.4 L/ha according to Spanish label recommendations, using representative, calibrated spray equipment. They wore their usual work clothing and followed the product label safety requirements, which state that protective gloves must be worn during all mixing and loading operations, when manually folding and unfolding the spray boom, and during other boom/nozzle manipulations. New, long-sleeved nitrile gloves (SolVex Catalogue number 37-900) were provided to the volunteers for this purpose. Depending on local requirements, 13 applicators drove their tractors to a central water supply point where the spray tank was filled and the spray liquid was prepared. Seven operators prepared the spray mix in the field or on the farm after filling the tank at the water supply station. In the first case, the tank was typically filled with water to about 75% of its capacity. Then the acetochlor formulation was added in precisely measured amounts to meet the prescribed application rate for the first spray operation. Applicators carefully opened the containers
by cutting the aluminum inner lid and subsequently emptied the contents into the spray tank. When needed, a measuring jug was used for accurate dose adjustment. The containers and the measuring jug were rinsed once and the rinsate was added to the tank. After the addition of the concentrate, the tank was filled to full capacity and sealed. The tank mixes were homogenized in the spray tank by mechanical or hydraulic agitation. For all in-field mixing and loading events, the spray tank was filled to capacity at the water supply point, and the pesticide formulation was added in the field. Spray tank capacities varied between 500 and 3000 L. The study personnel took tank mix samples every time a spray tank was about half empty. All applicators had a target workload of 15 ha/day. The actual area sprayed varied between 9.8 and 17.62 ha/day. Ten of the applicators used a closed cabin tractor. In the context of this study closed cabin tractors were tractors equipped with a cabin door and windows that were closed during the application. Two of these cabins were equipped with carbon air-filters, five other cabins were equipped with paper filters. The other 10 applicators used an open cabin tractor. A cabin was considered to be open when it had no windows and no doors or when the tractor was not equipped with a cabin. Two open cabin tractors and six closed cabin tractors were equipped with a hydraulic boom folding mechanism. Boom widths ranged between 10 and 16 m. 2.3. Urine collection and sample processing Urine collection started 24 h before handling the formulated product and continued during the 120 h following the initiation of the work activities. The volunteers emptied their bladder at the end of each 24 h monitoring period to assure that each sample could clearly be attributed to a given monitoring day. The applicators collected every void of urine in a separate labeled container (fluorinated polyethylene with polypropylene screw closure) and retained these samples in a cool bag (about 10 C) equipped with a temperature logger. These samples were collected at the end of every 24 h period by the study personnel and were frozen (about 20 C) until further sample processing. The applicators filled out a daily questionnaire, intended to document their activities during the monitoring period, compliance issues with urine collection, and events that could have had an impact on their exposure results. Individual voids were combined into 24 h composite samples (about 500 mL) using a proportional amount of urine from each individual void for a given sampling day. A portion of this composite sample was used for the analysis of creatinine, which was used as an indicator of composite sample completeness. A second sub-sample was used for the determination 2-ethyl-6methylaniline (EMA), a common chemophore
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representing the major acetochlor metabolite fraction in urine (see below). The remaining composite sample was stored frozen as a backup sample. 2.4. Acetochlor metabolism Mammals metabolize acetochlor rapidly and completely to an array of metabolites that are mainly recovered in urine (Monsanto unpublished reports). The largest fraction of urinary metabolites can be accounted for by chemical conversion into the common chemophore, EMA. This material was used as the basis for quantification of systemic acetochlor exposure. 2.5. Field fortifications Field fortifications of urine samples were made using 100 mL aliquots from the composite pre-exposure urine samples. The samples were fortified with an ethanol stock solution of acetochlor tertiary amide mercapturic acid, the major mammalian acetochlor metabolite in urine (Monsanto, unpublished reports). For every applicator, a complete set of field recoveries consisted of at least three blank control samples, three samples fortified at 15 parts per billion (ppb) and three samples fortified at 150 ppb, expressed as acetochlor equivalents. The field recoveries listed in this paper and used for further data processing are corrected for average background levels. 2.6. Sample analysis Residues in urine were hydrolyzed directly under sequential acidic and basic conditions. The hydrolyzed residue, as 2-ethyl-6-methylaniline (EMA), was steam distilled into dilute acid. The pH of the acidic solution was adjusted to the upper end alkaline range and the EMA was extracted into hexane. The sample was purified on a prepared 2:1 alumina/activated Florisil column and quantitation was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The concentration of EMA-derived metabolites was converted into acetochlor equivalents. As a conservative measure, concentrations of the exposure samples reported in this paper are not corrected for background levels. Other acetanilide products such as metolachlor and alachlor or their metabolites do not hydrolyze into EMA under the conditions used in this study (Monsanto unpublished data). Also, organic molecules with the same molecular mass and retention time as EMA were excluded in the analytical method through the use of a confirmation ion (MW = 135) in addition to the quantitation ion (MW = 120). Creatinine concentrations were determined using the Roche test kit for the Roche P Module Chemistry analyser. This method is a modification of Bartels et al. (1972).
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2.7. Calculation of absorbed daily dose The urine concentrations (expressed as acetochlor equivalents) were corrected for average overall field recovery as determined by analysis of the fortification samples. This corrected concentration, multiplied by the weight of the corresponding 24 h composite sample, yielded the total amount of acetochlor equivalents excreted during each day. The amounts excreted during the day of application (Day 0) and the following 4 days were summed. No correction was made for concentrations observed at Day 1. The total amount excreted was subsequently translated into an absorbed daily dose based on the results obtained from a pharmacokinetic study in the rhesus monkey (Monsanto Company unpublished report, 2003), which served as a model for the human metabolism of acetochlor. The total amount of acetochlor equivalents excreted in urine was corrected for the average fraction of urinary acetochlor metabolites that was not converted into EMA (0.337) and for the average fraction of acetochlor metabolites excreted in the faeces combined with the un-recovered fraction in the total mass balance (0.228), yielding the total absorbed amount of acetochlor. The latter was divided by the applicatorÕs body weight to yield the absorbed daily dose as specified in the following equation. Systemic dose½lg=kg=day P4 amount excreted on dayðtÞ½lg=day ¼ t¼0 . 0.663 0.772 bodyweight½kg The excretion rate was derived from the slope of the excretion curve, obtained by plotting the base-10 logarithm of the residual absorbed amounts versus time (Acquavella et al., 2004). 2.8. Use pattern To select an appropriate toxicology endpoint for the evaluation of worker exposure, the frequency, and duration of exposure of the user (farmers and commercial applicators) must be considered. 2.8.1. Farmers The annual acetochlor exposure frequency for an individual farmer under European use conditions can be estimated from German farm survey data (Janinhoff et al., 2002). The German data covers all pesticides used by growers during a 3-year period (1996–1998), with nearly 3000 active substance applications on farms ranging from 40 to 470 ha. The data suggest that 99% of all growers apply a given active ingredient for no more than 5 days per year. This survey includes active ingredients that are used many times on the same field. Acetochlor
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use is even less frequent because each field is treated only once and only during the maize-planting season. Alavanja et al. (1999) conducted a survey in the US on pesticide use and application methods including almost 36,000 applicators in Iowa (representative for the Maize Belt in the US) and North Carolina. He concluded that Iowa farmers who planted between 50 and 200 acres applied pesticides about 15 days per year. They applied on average about three different pesticides. In 1996, about 90% of US maize farms were smaller than 202 ha (Foreman, 2001). These numbers indicate that also in US the expected average use-duration of acetochlor does not exceed 5 days. 2.8.2. Professional applicators A conservative estimate for the annual average exposure frequency of a professional applicator is less than 30 days per year. This estimate was derived from the acetochlor market share in the most mature sales market, the US (in 2002, 25% of the US maize acres were treated with acetochlor; (USDA, 2003)), and the duration of the application window for acetochlor (about 80 days). Alavanja et al. (1999) demonstrated that professional applicators in Iowa applied different pesticides over a period of about 43 days per year (median). Although for professional applicators, 30 days of consecutive acetochlor use cannot be excluded, this would be unlikely because professionals apply other products and their activities are highly influenced by weather (rain, wind). 2.9. Toxicology endpoint selection Based upon the anticipated patterns of use, the most appropriate toxicology studies to use with biomonitoring data to assess occupational risks to acetochlor would be those with exposure durations ranging from several days to about a month, and for which good estimates of systemic dose levels were available. The acetochlor studies closest to meeting these criteria are developmental toxicity (teratology) studies in rats and rabbits, and 3- to 4-month subchronic toxicity studies in rats and dogs. These studies were conducted according to USEPA and/or OECD testing guidelines and Good Laboratory Practices. The No Observable Effect Levels (NOELs) and Lowest Observable Effect Levels (LOELs) from the above acetochlor toxicology studies are shown in Table 1. All of these studies were conducted using the oral route of exposure. However, since acetochlor is rapidly and essentially completely absorbed after oral administration (Unpublished data from Monsanto Company and Dow AgroSciences, LLP), no correction factor is needed when comparing these dose levels to the human systemic doses determined via biomonitoring. Four developmental toxicity (teratology) studies were conducted with acetochlor, two each in rats and rabbits.
Table 1 Selected toxicology results from studies conducted with acetochlora Study
Dose levels
NOEL
LOEL
Rat; developmental
50, 200, and 400 40, 150, and 600 15, 50, and 190 30, 100, and 300 40, 100, and 300 2, 17, and 177 25, 75, and 200 2, 10, and 60
200 150 50 100 40 17 <25 10
400 600 190 300 100 177 25 60
Rabbit; developmental Rat; 3-month feeding Dog; 4-month feeding Dog; 3-month feeding
Dose levels in mg/kg/day. NOEL, No Observable Effect Level in mg/kg/day. LOEL, Lowest Observable Effect Level in mg/kg/day. a Derived from U.S. EPA (1993).
The rabbit appeared to be the more sensitive species, with an overall NOEL of 100 mg/kg/day when both studies were evaluated together. In these studies, pregnant animals were administered acetochlor daily by gavage for 10–14 consecutive days. Therefore, the rabbit NOEL of 100 mg/kg/day was considered to be an appropriate endpoint to assess the potential risks to farmers who typically handle acetochlor for no more than a few days each year. Four subchronic toxicity studies were conducted, two each in rats and dogs, in which acetochlor was administered daily via either the diet (rats) or capsule (dogs) for 3–4 months. The dog appeared to be the more sensitive species, with a NOEL of 10 mg/kg/day. Therefore, although the duration of exposure for these studies was longer than would be encountered by professional applicators, the dog NOEL of 10 mg/kg/day was used as a conservative endpoint for assessing potential risks to those individuals who may apply acetochlor for up to several weeks each year. 2.10. Statistical methods The statistical software package JMP version 5.1.1 (JMP, 2004) was used for all data analysis. Because the sample size (10 replicates) in both scenarios was limited, no meaningful conclusion could be drawn with regards to the distribution of the exposure data and the distribution of other study variables. Therefore, the average exposure conditions (workload) and exposure results for the open- and closed-cabin scenarios were compared with an unpaired parametric (two-tailed t test) and non-parametric test (Wilcoxon rank-sum test). For the purpose of exposure comparison, the tests were run with the absorbed amounts of acetochlor (mg) and with the absorbed amounts standardized by the total amount of acetochlor handled (mg/kg a.i./day). Absorbed amounts were used rather than systemic doses to eliminate the potential (but limited) variability introduced by bodyweight in the systemic dose.
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3. Results
For the open-cabin scenario, 4 out of 10 operators repaired leaking pumps or failing nozzles during the application. Six operators did not always wear protective gloves as instructed. Accidental spillages and splashes were reported for three operators. Potential for exposure due to spray drift during application was reported for three operators. For the closed-cabin scenario, five operators repaired leaking pumps or failing nozzles during the application. In two cases, protective gloves were not always worn, and accidental spillages and splashes were reported for seven operators.
3.1. Application
3.2. Urine collection
The workload and application conditions of the openand closed-cabin scenarios are compared in Table 2 based on key study variables. The actual application rates used in the study ranged between 1.842 and 2.397 kg a.i./ha. The spray volumes varied according to local practice (75.2–428.9 L/ha), but no statistically significant difference could be demonstrated between spray volumes used in the open- vs. closed-cabin scenario. Operators in the closed-cabin scenario had a statistically significant higher workload (measured as area treated, formulation containers opened, and amount of active ingredient applied) compared to the operators in the open-cabin scenario. The total exposure duration ranged from 3.13 to 7.85 h/day, but no significant difference was observed between the open- and closed-cabin scenarios. Also, when differentiating between time spent on mixing and loading and time spent on application, the scenarios were not significantly different. Typical work clothing consisted of long pants and long sleeved shirts and/or coveralls (cotton or Tyvek) and sturdy footwear. Depending on the temperature, a coat, jersey, and/or hat were sometimes added. In four cases, the applicator wore a respiration mask during mixing and loading as part of his normal practices (two applicators in the open cabin and two applicator in the closed-cabin scenario).
The urine collection statistics are summarized in Table 3. In general Day 1 collections were rather poor, except for a few operators. In contrast, collection compliance from Day 0 till Day +4 was good. On average, 6.5 urine voids were collected per day per participant. Only three missing voids were reported. The creatinine results confirmed that the 24 h urine composites after Day 0 reflected, in most cases, representative 24 h urine samples. The typical creatinine excretion range for males is 124–230 lmol/kg bw/day (Clinical Guide to Laboratory Tests, 1995). One composite sample exceeded the upper limit of this normal range by 40% (322 lmol/kg bw/day). Four other composite samples (belonging to a single participant) showed creatinine values less than 10% below the lower limit of this range. No missing voids were reported for the five composite samples with creatinine results below the lower limit of the typical range. On the other hand, the composite samples for which missing voids were reported showed creatinine values within the typical range. The creatinine results of 14 composite samples (representing nine volunteers) exceeded the upper limit of the typical range.
As no appreciable difference in outcome was obtained with the parametric and non-parametric tests, only the p values associated with the Wilcoxon rank-sum test are reported here since it is reasonable to assume that the data are not normally distributed. To identify a sound scientific basis for exposure standardization, bi-variate associations between key study variables and exposure (amount acetochlor absorbed) were evaluated using simple linear regression analysis.
Table 2 Comparison of open- vs. closed-cabin scenarios Variable
Open-cabin mean
Closed-cabin mean
pa
Area treated (ha) Spray rate (L/ha) Total spray volume(L) a.i. applied (kg) Containers opened Mix./load operations Time spent mix/load (min) Time spent appl. (min) Total duration (min) Different plots treated Tank capacity
11.2 281.5 3110 22.6 5.9 3.6 44.3 174.5 218.8 4.9 1330
15.0 257.8 3775 30.9 7.8 3.9 48.4 184.7 233.1 5.2 1140
0.001 0.4727 0.1849 0.0004 0.001 0.7566 0.5204 0.8798 0.5452 0.9373 0.9383
a
Wilcoxon rank-sum test.
3.3. Urine concentrations and absorbed amounts Based on all laboratory fortification samples in this study, the limit of quantification (LOQ) for EMA expressed as acetochlor equivalents was 2.33 ppb. The average recovery for laboratory fortifications was 88.5%, covering a concentration range of 5–500 ppb.
Table 3 Urine collection data Minimum Day-1 Urine weight (g) Creatinine (lmol/kg/day)
90 17
Day 0–4 Urine weight (g) Creatinine (lmol/kg/day)
572 92
Maximum
Mean
SD
1449 174
637 87
467 54
3047 322
1388 194
508 44
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The average recoveries obtained for the field spikes, fortified at nominal levels of 15 and 150 ppb were 70.93 and 72.44%, respectively. The overall average recovery of 71.69% was used as a basis for correction of the urine concentrations when calculating systemic doses. Except for three operators, all Day 1 concentrations were below the LOQ. Although no activities that could have resulted in exposure to acetochlor prior to the day of application were reported, one operator showed a concentration of 85.33 ppb in his Day 1 composite sample. His Day 0 concentration was significantly lower (43.3 ppb). All 20 volunteers had detectable concentrations of acetochlor metabolites in the five subsequent 24-h composite samples following application. The maximum concentrations (lg/kg) observed on Days 0–4 for the open-cabin scenario were 141.4, 47.4, 24.1, 22.0, and 14.6 ppb, respectively. The median concentrations for this scenario were 49.1, 29.1, 13.1, 8.9, and 5.6 ppb, respectively. For the same days, the maximum concentrations observed for the closed-cabin scenario were 43.3, 45.6, 19.9, 18.5, and 28.8 ppb, respectively. The median concentrations for this scenario were 16.5, 5.9, 4.9, 5.3, and 3.2 ppb, respectively. In most cases, excretion of the highest amounts of acetochlor equivalents occurred during the day of application (Day 0). For four operators, the amount excreted gradually decreased during the days following application, approaching monotonic first order kinetics, as shown in Fig. 1 (average excretion rate constant = 0.74 d1, average r2 = 0.993). For the other operators, this pattern was disturbed by a small or substantial increase of the excreted amount during the days after application. Although the spray equipment (tractor, boom, and cabin) was washed at the end of Day 0, the increase in excretion could, in most cases, be associated with the re-use of the spray equipment during the days following application. There was a statistically significant difference (p = 0.023) between the systemically absorbed amounts
Fig. 2. Systemically absorbed amounts of acetochlor (mg).
of acetochlor (excreted amounts corrected for pharmacokinetic recovery) for the open cabin (average = 0.35 mg/day) and the closed-cabin scenario (average = 0.19 mg/day). The absorbed amounts obtained for both scenarios are shown in Fig. 2 and summarized in Table 4. Simple linear regression analysis between exposure and the study variables listed in Table 2 demonstrated that for the open-cabin scenario the slopes of the regression lines were not significantly different from zero (p > 0.05). Other variables tested for associations with exposure included: number of direct skin contact events observed, number of times equipment was checked and/ or fixed, number of compliance failures with regard to glove use, number of smoking events, number of times drift was observed in the field, number of observed direct contacts with contaminated equipment, number of re-entry events reported in the post-application questionnaire, number of reported events where study equipment (tractor) was re-used during the monitoring period. The strongest association, although not statistically significant, was obtained for the number of observed direct skin contact events (p = 0.0505). For the closed-cabin scenario, the only variable that showed a significant (inverse) linear association with systemically absorbed amount was the total spray volume (L/day) (p = 0.04). Table 4 Absorbed amounts of acetochlor (mg/day)
Fig. 1. Monotonic excretion curves [Excretion rate (k) = slope · 2.303] obtained for four applicators.
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
Open-cabin
Closed-cabin
6.60 · 102 2.99 · 101 3.48 · 101 3.44 · 101 4.15 · 101 6.11 · 101 1.60 · 101
6.53 · 102 9.17 · 102 1.92 · 101 1.64 · 101 2.96 · 101 3.83 · 101 1.16 · 101
C.A. Gustin et al. / Regulatory Toxicology and Pharmacology 43 (2005) 141–149 Table 5 Absorbed amounts of acetochlor standardized by amount applied (mg/ kg a.i./day)
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
Open-cabin
Closed-cabin
3.44 · 103 1.10 · 102 1.55 · 102 1.56 · 102 1.93 · 102 2.80 · 102 7.55 · 103
2.16 · 103 2.92 · 103 6.19 · 103 5.18 · 103 8.91 · 103 1.50 · 102 4.09 · 103
Workload (amount of product sprayed) was the only variable that was significantly different for both scenarios and is also commonly used as the basis of exposure normalization (Lundehn et al., 1992; PHED, 1995; van Golstein Brouwers et al., 1996). Therefore, although not significantly associated with the exposure measured in this study, the total amount of active ingredient handled was used to standardize exposure (Table 5). The two tailed t test for the standardized systemic amounts confirmed the significantly (p = 0.003) higher exposure for the open-cabin scenario (open cabin average exposure = 0.016 mg/kg a.i./day vs. 0.002 mg/kg a.i./day for the closed-cabin scenario).
hopper to facilitate mixing and loading. This applicator mixed and loaded three times and emptied the three tanks on four separate fields. During the first application event, he cleaned a blocked nozzle while wearing his protective gloves. During the second mixing event he fixed a leak in the hose with latex gloves instead of nitrile gloves. He did not report potential exposure events after the day of application (re-entry, other applications). The highest absorbed daily dose in the closed-cabin scenario (3.83 · 103 mg/kg bw/day) was obtained for an operator who applied a total of 25.62 kg a.i. on 10.69 ha of maize, using an average spray volume of 281 L/ha and spent 358 min for the completion of his entire application. He wore a Tshirt, long sleeved shirt, jeans, and boots. For the application he used a 600 L spray-tank and a 10 m, manually folding spray boom. This applicator mixed and loaded five times and emptied the five tanks on five separate fields. During the first application, the operator sprayed for about 3 min with the back window open to check and adjust the pressure on the sprayer. He smoked in his cabin during the third application without having washed his hands. Accidental spillages were reported during the third, fourth, and fifth mixing and loading events. 3.5. Risk characterization
3.4. Absorbed daily doses Table 6 summarizes the absorbed daily dose (mg/kg bw/day) statistics for the open- and closed-cabin scenarios. On average, the absorbed daily doses obtained for the open-cabin scenario (average = 0.004 mg/kg bw/day) were two times higher than the doses obtained for the closed-cabin scenario (p = 0.016). The maximum absorbed daily dose for the opencabin scenario (8.24 · 103 mg/kg bw/day) was obtained for an operator who applied a total of 21.7 kg a.i. on 10.67 ha of maize, using an average spray volume of 169 L/ha, and spent 188 min completing his entire application. He wore a cotton coverall over jeans and a shirt, Wellington boots and a hat. For the application he used a 600 L spray tank, a 14 m, manual folding spray boom, and an induction
The risk characterization for the open- and closedcabin exposure scenarios is presented in Table 7. The NOELÕs (10 mg/kg bw/day for professional applicators and 100 mg/kg bw/day for farmers), were divided by the average, the 75th percentile, and maximum absorbed daily doses, respectively, to obtain the margin of exposure (MOE). For short-term exposure, which represents the exposure duration for farmers applying acetochlor, the MOE based on the average systemic exposure level was 23,000 for the open-cabin scenario and 44,000 for
Table 7 Risk characterization expressed as MOE Farmers
Table 6 Absorbed daily dose (mg/kg bw/day): statistics Absorbed daily dose (mg/kg bw/day)
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
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Open-cabin
Closed-cabin
7.77 · 104 3.37 · 103 4.34 · 103 4.18 · 103 5.79 · 103 8.24 · 103 2.05 · 103
6.87 · 104 1.08 · 103 2.29 · 103 2.19 · 103 3.69 · 103 3.83 · 103 1.32 · 103
Relevant NOEL Open-cabin MOEc Average 75th Percentile Maximum Closed-cabin MOEc Average 75th Percentile Maximum a b c
a
Professional applicators
100 mg/kg/day
10b mg/kg/day
2.30 · 104 1.96 · 104 1.21 · 104
2.30 · 103 1.96 · 103 1.21 · 103
4.37 · 104 2.79 · 104 2.61 · 104
4.37 · 103 2.79 · 103 2.31 · 103
Based on rabbit developmental study. Based on 90-day dog study. NOEL/exposure.
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the closed-cabin scenario. For intermediate term exposure, which represents the exposure duration for professional applicators using acetochlor, the MOEs were 10-fold lower.
4. Discussion The purpose of this study was to obtain higher tier exposure estimates for acetochlor under realistic conditions. The work-practices monitored were representative but conservative: multiple small plots were treated and required frequent refilling of the spray tank; the maximum use rate was applied, frequent equipment failures were observed and protective gloves were not always worn as instructed. Representative 24 h urine samples were collected during the entire monitoring period after the on-study application allowing an accurate evaluation of the excretion kinetics. The average urinary excretion half-life obtained from those applicators who showed monotonic first order urinary excretion of acetochlor metabolites (0.94 days) is consistent with the excretion kinetics obtained from the pharmacokinetic study with six male rhesus monkeys, in which two groups of three animals each were dosed at 0.005 and 0.05 mg acetochlor/kg bw, respectively (Monsanto unpublished report). Based on urinary excretion only, an overall average systemic half-life of 1.2 days was observed in monkeys. Based on the total excretion balance (urine + feces), the average systemic half-life was 1.4 days. Although operators in the closed-cabin scenario sprayed on average greater than a 25% more hectares, the average amount of acetochlor absorbed under the conditions of this scenario was two times lower than the average amount absorbed during the operations in the open-cabin scenario. This suggests that the relative contribution of the tractor mounted application events to the overall exposure was significantly higher for the open-cabin scenario compared to the closedcabin applications. Although intuitive, this conclusion may underestimate the importance and relative contributions of accidental spillages and contact with contaminated (spray) equipment during mixing, loading, and manipulations of the spray boom, which are not discretely determined from these biomonitoring data. For both scenarios, no scientifically meaningful linear relationship could be demonstrated between exposure and any of the key study variables tested in this analysis (including workload). For the closed-cabin scenario, the association between total spray volume and the systemically absorbed amount was statistically significant, but additional data analysis could not help to explain the inverse relationship. Although the applicators with the highest total spray volume had the most diluted
tank mix concentration (p = 0.001), this should not necessarily impact the exposure levels for the closedcabin scenario where the highest exposure contribution is expected to come from mixing, loading, and manipulations of the spray boom. There was no association between the number of mixing and loading events (nor the time spent on mixing and loading) and the total spray volume for the closed-cabin scenario (p > 0.05). The lack of a significant linear relationship between any of the study variables and exposure may be explained by the relatively small variability in workload and the low (from a statistical point of view) number of replicates per scenario. Alternatively it could also suggest that accidental spillages and contact with contaminated (spray) equipment during mixing, loading, and manipulations of the spray boom were the main contributors to exposure. The number of such exposure events may not be directly linked to workload. Therefore care should be taken during the interpolation or extrapolation of the normalized acetochlor exposure values to other workload scenarios. The risk assessment based on the activities monitored in this study could be considered conservative because of the work practices (equipment, number of exposure events). Accidental spillages and contact with contaminated equipment during mixing and loading were presumed to contribute the most to overall exposure in this study. Furthermore, the selection of the NOELs used in this evaluation added to the conservative nature of the risk assessment. To evaluate exposure that typically lasts only 1 or 2 days every year (farmer), an endpoint was selected based on a 14-day study, and an endpoint was selected from a 90-day study to evaluate exposure that typically lasts less than 30 days every year (professional applicator). Despite the conservative nature of the risk assessment, the MOEs were relatively large. For the open-cabin scenario the average MOEs for farmers ranged from 23,000 (open-cabin scenario) to about 44,000 (closedcabin scenario). For professional applicators the MOEs were 10-fold lower. In conclusion, the risk characterization for both farmers and professional applicators demonstrates that no health effects are to be expected for either open- or closed-cabin applicators of acetochlor.
Acknowledgments The authors thank their former colleagues Dr. Mark Martens and Catherine Schmit for their contributions to this study. They also recognize the team from Inveresk Research and in particular Jeff Old, Iain Anderson, and Dr. Santiago Marti for conducting the field portion of the study.
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References Acquavella, J.F., Alexander, B.H., Mandel, J.S., Gustin, C., Baker, B., Chapman, P., Bleeke, M., 2004. Glyphosate biomonitoring for farmers and their families: results from the farm family exposure study. Environ. Health Perspect. 112 (3), 321–326. Alavanja, M.C.R., Sandler, D.P., McDonnell, C.J., Lynch, C.F., Pennybacker, M., Zahm, S.H., Mage, D.T., Steen, W.C., Wintersteen, W., Blair, A., 1999. Characteristics of pesticide use in a pesticide applicator cohort: the agricultural health study. Environ. Res. Section A 80, 172–179. Janinhoff, A., Hoemicke, E., Westphal, D., Schafer, M., Stauber, F., Wicke, H., Brennecke, R., Schultze-Rosario, C., Becker, F.A., Schrader, J., 2002. An analysis of the duration, frequency and seasonal distribution of plant protection activities on German farms. Nachrichtenbl. Deut. Pflanzenschutzd. 54 (7), S180–S183. JMP, Version 5.1.1, SAS Institute Inc., Cary, NC, 1989–2004. Lundehn, J.R., Westphal, D., Kieczka, H., Krebs, B., Locher-Bolz, S., Maasfeld, W., Pick, E.D., 1992. Uniform Principles for
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Safeguarding the Health of Applicators of Plant Protection Products (Uniform Principles for Operator Protection). Mitteilungen aus der Biologischen Bundesanstalt fur Land-und Forstwirtschaft, Berlin-Dahlem; ISSN 0067-5849–ISBN 348927700-7. Organization for Economic Co-operation and Development Environment Directorate, Document (OECD/GD(97)) 148 Series on Testing and Assessment No. 9, Guidance Document for the Conduct of Studies of Occupational Exposure to Pesticides During Aricultural Aplication. 1997. Paris, France.. U.S. EPA, 1993. Integrated Risk Information System (IRIS). Acetochlor (CASRN 34256-82-1). . Last revised 09/01/1993. van Goldstein Brouwers, Y.G.C., Marquart, J., van Hemmen, J.J., 1996. Assessment of Occupational Exposure to Pesticides in Agriculture Part IV Protocol for the Use of Generic Exposure Data. TNO Nutrition and Food Research Institute, Rijswijk, The Netherlands, report No V 96.120.
Applicator exposure to acetochlor based on biomonitoring Christophe A. Gustin *, Sharon J. Moran, John D. Fuhrman, Mitchell L. Kurtzweil, Joel M. Kronenberg, David I. Gustafson, Monte A. Marshall Monsanto Company, St. Louis, MO, USA Received 14 February 2005 Available online 19 September 2005
Abstract Biomonitoring was used to assess the combined dermal, oral, and inhalation exposure associated with the agricultural use of Harness Plus, an emulsifiable concentrate formulation of the herbicide acetochlor. Twenty Spanish farmers handled and applied acetochlor to maize in the spring of 2003, following the product label recommendations. Open- and closed-cabin applications were equally represented. Urine was collected during six consecutive days, starting the day prior to application. Daily composites were analyzed for 2-ethyl-6-methyl-aniline, a common chemophore representing the major urinary acetochlor metabolites. All applicators showed detectable concentrations in urine after application. Although, the open-cabin applicators treated fewer hectares, they showed significantly higher exposure compared to the closed-cabin applicators (average exposure: 0.004 and 0.002 mg/kg bw/day, respectively). Linear regression analysis suggested that untracked incidents had a significant impact on the total exposure. Other events that may have contributed to the observed exposure are repair of faulty equipment, accidental spillages, splashes, and inadequate use of protective gloves. The average margins of exposure (MOE) for farmers ranged from 23,000 (open cabin) to about 44,000 (closed cabin). For professional applicators the MOEs were 10-fold lower. These MOEs clearly indicate that no adverse health effects should be expected from agricultural acetochlor applications. 2005 Elsevier Inc. All rights reserved. Keywords: Biomonitoring; Urine; Applicators; Acetochlor; Pesticide; Exposure
1. Introduction Acetochlor (2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6methylphenyl) acetamide) is an active ingredient (a.i.) of the chloroacetanilide family, used as a broad-spectrum pre-emergent and early post-emergent herbicide in maize for the control of grasses and selected broadleaf weeds. Acetochlor is commercialized in different world areas under different brand names and in different formulation types. This paper describes the results obtained from a biomonitoring study of farmers exposed to Harness Plus, an emulsifiable concentrate (EC) formulation containing 840 g/L of acetochlor and safened with furilazole (3*
Corresponding author. Fax: +1 314 694 8774. E-mail address: [email protected] (C.A. Gustin).
0273-2300/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2005.06.015
(dichloroacetyl)-5-(2-furanyl)-2,2-dimethyl oxazolidine) to prevent phytotoxic effects on emerging maize seedlings. The study was conducted during the spring of 2003 in Catalunya, one of the major maize growing areas in Spain. Maize is treated pre- or early post-emergent with a single acetochlor application. The maximum labeled application rate for this formulation in Europe is 2.4 liters/hectare (L/ha) or 2.016 kilogram (kg) a.i./ha. The window of application varies with geography but typically runs from early March to mid-May, which coincides with the timing of this study. Biomonitoring is a method of evaluating the absorption of chemicals by measuring the chemical or its metabolites in body fluids such as urine. The analysis of urine provides a quantitative measure of the absorbed dose when used in combination with a thorough
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understanding of the metabolism and pharmacokinetics of the test product. The direct measurement of the absorbed dose is the principal advantage of biomonitoring over passive dosimetry as it determines actual, rather than potential, absorption (OECD, 1997). Indeed, the absorbed dose is a measure of the amount of chemical that becomes systemically available through dermal uptake, oral ingestion, and inhalation. The well-documented mammalian metabolism of acetochlor permitted biomonitoring (urine) to be used as a tool for quantitative exposure assessment.
2. Methods 2.1. Selection of test subjects Study participants were selected from registered pesticide users in and around the Lleida (Catalonia) area. Twenty farmers were selected for participation in this study based on well-defined criteria, primarily related to professional experience, availability of equipment, maize growing area, and willingness to participate. Suitability of the participants was determined by means of an enrollment questionnaire. Once selected, volunteers were informed in more detail about the study, received guidance on Good Agricultural Practices, and were asked to sign an informed consent form allowing their data to be used for research purposes. Participating farmers received a cash incentive after the monitoring period and were provided with the formulation used during the on-study application. 2.2. Test system The applicators mixed and applied the acetochlor formulation at 2.4 L/ha according to Spanish label recommendations, using representative, calibrated spray equipment. They wore their usual work clothing and followed the product label safety requirements, which state that protective gloves must be worn during all mixing and loading operations, when manually folding and unfolding the spray boom, and during other boom/nozzle manipulations. New, long-sleeved nitrile gloves (SolVex Catalogue number 37-900) were provided to the volunteers for this purpose. Depending on local requirements, 13 applicators drove their tractors to a central water supply point where the spray tank was filled and the spray liquid was prepared. Seven operators prepared the spray mix in the field or on the farm after filling the tank at the water supply station. In the first case, the tank was typically filled with water to about 75% of its capacity. Then the acetochlor formulation was added in precisely measured amounts to meet the prescribed application rate for the first spray operation. Applicators carefully opened the containers
by cutting the aluminum inner lid and subsequently emptied the contents into the spray tank. When needed, a measuring jug was used for accurate dose adjustment. The containers and the measuring jug were rinsed once and the rinsate was added to the tank. After the addition of the concentrate, the tank was filled to full capacity and sealed. The tank mixes were homogenized in the spray tank by mechanical or hydraulic agitation. For all in-field mixing and loading events, the spray tank was filled to capacity at the water supply point, and the pesticide formulation was added in the field. Spray tank capacities varied between 500 and 3000 L. The study personnel took tank mix samples every time a spray tank was about half empty. All applicators had a target workload of 15 ha/day. The actual area sprayed varied between 9.8 and 17.62 ha/day. Ten of the applicators used a closed cabin tractor. In the context of this study closed cabin tractors were tractors equipped with a cabin door and windows that were closed during the application. Two of these cabins were equipped with carbon air-filters, five other cabins were equipped with paper filters. The other 10 applicators used an open cabin tractor. A cabin was considered to be open when it had no windows and no doors or when the tractor was not equipped with a cabin. Two open cabin tractors and six closed cabin tractors were equipped with a hydraulic boom folding mechanism. Boom widths ranged between 10 and 16 m. 2.3. Urine collection and sample processing Urine collection started 24 h before handling the formulated product and continued during the 120 h following the initiation of the work activities. The volunteers emptied their bladder at the end of each 24 h monitoring period to assure that each sample could clearly be attributed to a given monitoring day. The applicators collected every void of urine in a separate labeled container (fluorinated polyethylene with polypropylene screw closure) and retained these samples in a cool bag (about 10 C) equipped with a temperature logger. These samples were collected at the end of every 24 h period by the study personnel and were frozen (about 20 C) until further sample processing. The applicators filled out a daily questionnaire, intended to document their activities during the monitoring period, compliance issues with urine collection, and events that could have had an impact on their exposure results. Individual voids were combined into 24 h composite samples (about 500 mL) using a proportional amount of urine from each individual void for a given sampling day. A portion of this composite sample was used for the analysis of creatinine, which was used as an indicator of composite sample completeness. A second sub-sample was used for the determination 2-ethyl-6methylaniline (EMA), a common chemophore
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representing the major acetochlor metabolite fraction in urine (see below). The remaining composite sample was stored frozen as a backup sample. 2.4. Acetochlor metabolism Mammals metabolize acetochlor rapidly and completely to an array of metabolites that are mainly recovered in urine (Monsanto unpublished reports). The largest fraction of urinary metabolites can be accounted for by chemical conversion into the common chemophore, EMA. This material was used as the basis for quantification of systemic acetochlor exposure. 2.5. Field fortifications Field fortifications of urine samples were made using 100 mL aliquots from the composite pre-exposure urine samples. The samples were fortified with an ethanol stock solution of acetochlor tertiary amide mercapturic acid, the major mammalian acetochlor metabolite in urine (Monsanto, unpublished reports). For every applicator, a complete set of field recoveries consisted of at least three blank control samples, three samples fortified at 15 parts per billion (ppb) and three samples fortified at 150 ppb, expressed as acetochlor equivalents. The field recoveries listed in this paper and used for further data processing are corrected for average background levels. 2.6. Sample analysis Residues in urine were hydrolyzed directly under sequential acidic and basic conditions. The hydrolyzed residue, as 2-ethyl-6-methylaniline (EMA), was steam distilled into dilute acid. The pH of the acidic solution was adjusted to the upper end alkaline range and the EMA was extracted into hexane. The sample was purified on a prepared 2:1 alumina/activated Florisil column and quantitation was accomplished by gas chromatography coupled with mass spectrometry (GC/MS). The concentration of EMA-derived metabolites was converted into acetochlor equivalents. As a conservative measure, concentrations of the exposure samples reported in this paper are not corrected for background levels. Other acetanilide products such as metolachlor and alachlor or their metabolites do not hydrolyze into EMA under the conditions used in this study (Monsanto unpublished data). Also, organic molecules with the same molecular mass and retention time as EMA were excluded in the analytical method through the use of a confirmation ion (MW = 135) in addition to the quantitation ion (MW = 120). Creatinine concentrations were determined using the Roche test kit for the Roche P Module Chemistry analyser. This method is a modification of Bartels et al. (1972).
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2.7. Calculation of absorbed daily dose The urine concentrations (expressed as acetochlor equivalents) were corrected for average overall field recovery as determined by analysis of the fortification samples. This corrected concentration, multiplied by the weight of the corresponding 24 h composite sample, yielded the total amount of acetochlor equivalents excreted during each day. The amounts excreted during the day of application (Day 0) and the following 4 days were summed. No correction was made for concentrations observed at Day 1. The total amount excreted was subsequently translated into an absorbed daily dose based on the results obtained from a pharmacokinetic study in the rhesus monkey (Monsanto Company unpublished report, 2003), which served as a model for the human metabolism of acetochlor. The total amount of acetochlor equivalents excreted in urine was corrected for the average fraction of urinary acetochlor metabolites that was not converted into EMA (0.337) and for the average fraction of acetochlor metabolites excreted in the faeces combined with the un-recovered fraction in the total mass balance (0.228), yielding the total absorbed amount of acetochlor. The latter was divided by the applicatorÕs body weight to yield the absorbed daily dose as specified in the following equation. Systemic dose½lg=kg=day P4 amount excreted on dayðtÞ½lg=day ¼ t¼0 . 0.663 0.772 bodyweight½kg The excretion rate was derived from the slope of the excretion curve, obtained by plotting the base-10 logarithm of the residual absorbed amounts versus time (Acquavella et al., 2004). 2.8. Use pattern To select an appropriate toxicology endpoint for the evaluation of worker exposure, the frequency, and duration of exposure of the user (farmers and commercial applicators) must be considered. 2.8.1. Farmers The annual acetochlor exposure frequency for an individual farmer under European use conditions can be estimated from German farm survey data (Janinhoff et al., 2002). The German data covers all pesticides used by growers during a 3-year period (1996–1998), with nearly 3000 active substance applications on farms ranging from 40 to 470 ha. The data suggest that 99% of all growers apply a given active ingredient for no more than 5 days per year. This survey includes active ingredients that are used many times on the same field. Acetochlor
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use is even less frequent because each field is treated only once and only during the maize-planting season. Alavanja et al. (1999) conducted a survey in the US on pesticide use and application methods including almost 36,000 applicators in Iowa (representative for the Maize Belt in the US) and North Carolina. He concluded that Iowa farmers who planted between 50 and 200 acres applied pesticides about 15 days per year. They applied on average about three different pesticides. In 1996, about 90% of US maize farms were smaller than 202 ha (Foreman, 2001). These numbers indicate that also in US the expected average use-duration of acetochlor does not exceed 5 days. 2.8.2. Professional applicators A conservative estimate for the annual average exposure frequency of a professional applicator is less than 30 days per year. This estimate was derived from the acetochlor market share in the most mature sales market, the US (in 2002, 25% of the US maize acres were treated with acetochlor; (USDA, 2003)), and the duration of the application window for acetochlor (about 80 days). Alavanja et al. (1999) demonstrated that professional applicators in Iowa applied different pesticides over a period of about 43 days per year (median). Although for professional applicators, 30 days of consecutive acetochlor use cannot be excluded, this would be unlikely because professionals apply other products and their activities are highly influenced by weather (rain, wind). 2.9. Toxicology endpoint selection Based upon the anticipated patterns of use, the most appropriate toxicology studies to use with biomonitoring data to assess occupational risks to acetochlor would be those with exposure durations ranging from several days to about a month, and for which good estimates of systemic dose levels were available. The acetochlor studies closest to meeting these criteria are developmental toxicity (teratology) studies in rats and rabbits, and 3- to 4-month subchronic toxicity studies in rats and dogs. These studies were conducted according to USEPA and/or OECD testing guidelines and Good Laboratory Practices. The No Observable Effect Levels (NOELs) and Lowest Observable Effect Levels (LOELs) from the above acetochlor toxicology studies are shown in Table 1. All of these studies were conducted using the oral route of exposure. However, since acetochlor is rapidly and essentially completely absorbed after oral administration (Unpublished data from Monsanto Company and Dow AgroSciences, LLP), no correction factor is needed when comparing these dose levels to the human systemic doses determined via biomonitoring. Four developmental toxicity (teratology) studies were conducted with acetochlor, two each in rats and rabbits.
Table 1 Selected toxicology results from studies conducted with acetochlora Study
Dose levels
NOEL
LOEL
Rat; developmental
50, 200, and 400 40, 150, and 600 15, 50, and 190 30, 100, and 300 40, 100, and 300 2, 17, and 177 25, 75, and 200 2, 10, and 60
200 150 50 100 40 17 <25 10
400 600 190 300 100 177 25 60
Rabbit; developmental Rat; 3-month feeding Dog; 4-month feeding Dog; 3-month feeding
Dose levels in mg/kg/day. NOEL, No Observable Effect Level in mg/kg/day. LOEL, Lowest Observable Effect Level in mg/kg/day. a Derived from U.S. EPA (1993).
The rabbit appeared to be the more sensitive species, with an overall NOEL of 100 mg/kg/day when both studies were evaluated together. In these studies, pregnant animals were administered acetochlor daily by gavage for 10–14 consecutive days. Therefore, the rabbit NOEL of 100 mg/kg/day was considered to be an appropriate endpoint to assess the potential risks to farmers who typically handle acetochlor for no more than a few days each year. Four subchronic toxicity studies were conducted, two each in rats and dogs, in which acetochlor was administered daily via either the diet (rats) or capsule (dogs) for 3–4 months. The dog appeared to be the more sensitive species, with a NOEL of 10 mg/kg/day. Therefore, although the duration of exposure for these studies was longer than would be encountered by professional applicators, the dog NOEL of 10 mg/kg/day was used as a conservative endpoint for assessing potential risks to those individuals who may apply acetochlor for up to several weeks each year. 2.10. Statistical methods The statistical software package JMP version 5.1.1 (JMP, 2004) was used for all data analysis. Because the sample size (10 replicates) in both scenarios was limited, no meaningful conclusion could be drawn with regards to the distribution of the exposure data and the distribution of other study variables. Therefore, the average exposure conditions (workload) and exposure results for the open- and closed-cabin scenarios were compared with an unpaired parametric (two-tailed t test) and non-parametric test (Wilcoxon rank-sum test). For the purpose of exposure comparison, the tests were run with the absorbed amounts of acetochlor (mg) and with the absorbed amounts standardized by the total amount of acetochlor handled (mg/kg a.i./day). Absorbed amounts were used rather than systemic doses to eliminate the potential (but limited) variability introduced by bodyweight in the systemic dose.
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3. Results
For the open-cabin scenario, 4 out of 10 operators repaired leaking pumps or failing nozzles during the application. Six operators did not always wear protective gloves as instructed. Accidental spillages and splashes were reported for three operators. Potential for exposure due to spray drift during application was reported for three operators. For the closed-cabin scenario, five operators repaired leaking pumps or failing nozzles during the application. In two cases, protective gloves were not always worn, and accidental spillages and splashes were reported for seven operators.
3.1. Application
3.2. Urine collection
The workload and application conditions of the openand closed-cabin scenarios are compared in Table 2 based on key study variables. The actual application rates used in the study ranged between 1.842 and 2.397 kg a.i./ha. The spray volumes varied according to local practice (75.2–428.9 L/ha), but no statistically significant difference could be demonstrated between spray volumes used in the open- vs. closed-cabin scenario. Operators in the closed-cabin scenario had a statistically significant higher workload (measured as area treated, formulation containers opened, and amount of active ingredient applied) compared to the operators in the open-cabin scenario. The total exposure duration ranged from 3.13 to 7.85 h/day, but no significant difference was observed between the open- and closed-cabin scenarios. Also, when differentiating between time spent on mixing and loading and time spent on application, the scenarios were not significantly different. Typical work clothing consisted of long pants and long sleeved shirts and/or coveralls (cotton or Tyvek) and sturdy footwear. Depending on the temperature, a coat, jersey, and/or hat were sometimes added. In four cases, the applicator wore a respiration mask during mixing and loading as part of his normal practices (two applicators in the open cabin and two applicator in the closed-cabin scenario).
The urine collection statistics are summarized in Table 3. In general Day 1 collections were rather poor, except for a few operators. In contrast, collection compliance from Day 0 till Day +4 was good. On average, 6.5 urine voids were collected per day per participant. Only three missing voids were reported. The creatinine results confirmed that the 24 h urine composites after Day 0 reflected, in most cases, representative 24 h urine samples. The typical creatinine excretion range for males is 124–230 lmol/kg bw/day (Clinical Guide to Laboratory Tests, 1995). One composite sample exceeded the upper limit of this normal range by 40% (322 lmol/kg bw/day). Four other composite samples (belonging to a single participant) showed creatinine values less than 10% below the lower limit of this range. No missing voids were reported for the five composite samples with creatinine results below the lower limit of the typical range. On the other hand, the composite samples for which missing voids were reported showed creatinine values within the typical range. The creatinine results of 14 composite samples (representing nine volunteers) exceeded the upper limit of the typical range.
As no appreciable difference in outcome was obtained with the parametric and non-parametric tests, only the p values associated with the Wilcoxon rank-sum test are reported here since it is reasonable to assume that the data are not normally distributed. To identify a sound scientific basis for exposure standardization, bi-variate associations between key study variables and exposure (amount acetochlor absorbed) were evaluated using simple linear regression analysis.
Table 2 Comparison of open- vs. closed-cabin scenarios Variable
Open-cabin mean
Closed-cabin mean
pa
Area treated (ha) Spray rate (L/ha) Total spray volume(L) a.i. applied (kg) Containers opened Mix./load operations Time spent mix/load (min) Time spent appl. (min) Total duration (min) Different plots treated Tank capacity
11.2 281.5 3110 22.6 5.9 3.6 44.3 174.5 218.8 4.9 1330
15.0 257.8 3775 30.9 7.8 3.9 48.4 184.7 233.1 5.2 1140
0.001 0.4727 0.1849 0.0004 0.001 0.7566 0.5204 0.8798 0.5452 0.9373 0.9383
a
Wilcoxon rank-sum test.
3.3. Urine concentrations and absorbed amounts Based on all laboratory fortification samples in this study, the limit of quantification (LOQ) for EMA expressed as acetochlor equivalents was 2.33 ppb. The average recovery for laboratory fortifications was 88.5%, covering a concentration range of 5–500 ppb.
Table 3 Urine collection data Minimum Day-1 Urine weight (g) Creatinine (lmol/kg/day)
90 17
Day 0–4 Urine weight (g) Creatinine (lmol/kg/day)
572 92
Maximum
Mean
SD
1449 174
637 87
467 54
3047 322
1388 194
508 44
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The average recoveries obtained for the field spikes, fortified at nominal levels of 15 and 150 ppb were 70.93 and 72.44%, respectively. The overall average recovery of 71.69% was used as a basis for correction of the urine concentrations when calculating systemic doses. Except for three operators, all Day 1 concentrations were below the LOQ. Although no activities that could have resulted in exposure to acetochlor prior to the day of application were reported, one operator showed a concentration of 85.33 ppb in his Day 1 composite sample. His Day 0 concentration was significantly lower (43.3 ppb). All 20 volunteers had detectable concentrations of acetochlor metabolites in the five subsequent 24-h composite samples following application. The maximum concentrations (lg/kg) observed on Days 0–4 for the open-cabin scenario were 141.4, 47.4, 24.1, 22.0, and 14.6 ppb, respectively. The median concentrations for this scenario were 49.1, 29.1, 13.1, 8.9, and 5.6 ppb, respectively. For the same days, the maximum concentrations observed for the closed-cabin scenario were 43.3, 45.6, 19.9, 18.5, and 28.8 ppb, respectively. The median concentrations for this scenario were 16.5, 5.9, 4.9, 5.3, and 3.2 ppb, respectively. In most cases, excretion of the highest amounts of acetochlor equivalents occurred during the day of application (Day 0). For four operators, the amount excreted gradually decreased during the days following application, approaching monotonic first order kinetics, as shown in Fig. 1 (average excretion rate constant = 0.74 d1, average r2 = 0.993). For the other operators, this pattern was disturbed by a small or substantial increase of the excreted amount during the days after application. Although the spray equipment (tractor, boom, and cabin) was washed at the end of Day 0, the increase in excretion could, in most cases, be associated with the re-use of the spray equipment during the days following application. There was a statistically significant difference (p = 0.023) between the systemically absorbed amounts
Fig. 2. Systemically absorbed amounts of acetochlor (mg).
of acetochlor (excreted amounts corrected for pharmacokinetic recovery) for the open cabin (average = 0.35 mg/day) and the closed-cabin scenario (average = 0.19 mg/day). The absorbed amounts obtained for both scenarios are shown in Fig. 2 and summarized in Table 4. Simple linear regression analysis between exposure and the study variables listed in Table 2 demonstrated that for the open-cabin scenario the slopes of the regression lines were not significantly different from zero (p > 0.05). Other variables tested for associations with exposure included: number of direct skin contact events observed, number of times equipment was checked and/ or fixed, number of compliance failures with regard to glove use, number of smoking events, number of times drift was observed in the field, number of observed direct contacts with contaminated equipment, number of re-entry events reported in the post-application questionnaire, number of reported events where study equipment (tractor) was re-used during the monitoring period. The strongest association, although not statistically significant, was obtained for the number of observed direct skin contact events (p = 0.0505). For the closed-cabin scenario, the only variable that showed a significant (inverse) linear association with systemically absorbed amount was the total spray volume (L/day) (p = 0.04). Table 4 Absorbed amounts of acetochlor (mg/day)
Fig. 1. Monotonic excretion curves [Excretion rate (k) = slope · 2.303] obtained for four applicators.
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
Open-cabin
Closed-cabin
6.60 · 102 2.99 · 101 3.48 · 101 3.44 · 101 4.15 · 101 6.11 · 101 1.60 · 101
6.53 · 102 9.17 · 102 1.92 · 101 1.64 · 101 2.96 · 101 3.83 · 101 1.16 · 101
C.A. Gustin et al. / Regulatory Toxicology and Pharmacology 43 (2005) 141–149 Table 5 Absorbed amounts of acetochlor standardized by amount applied (mg/ kg a.i./day)
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
Open-cabin
Closed-cabin
3.44 · 103 1.10 · 102 1.55 · 102 1.56 · 102 1.93 · 102 2.80 · 102 7.55 · 103
2.16 · 103 2.92 · 103 6.19 · 103 5.18 · 103 8.91 · 103 1.50 · 102 4.09 · 103
Workload (amount of product sprayed) was the only variable that was significantly different for both scenarios and is also commonly used as the basis of exposure normalization (Lundehn et al., 1992; PHED, 1995; van Golstein Brouwers et al., 1996). Therefore, although not significantly associated with the exposure measured in this study, the total amount of active ingredient handled was used to standardize exposure (Table 5). The two tailed t test for the standardized systemic amounts confirmed the significantly (p = 0.003) higher exposure for the open-cabin scenario (open cabin average exposure = 0.016 mg/kg a.i./day vs. 0.002 mg/kg a.i./day for the closed-cabin scenario).
hopper to facilitate mixing and loading. This applicator mixed and loaded three times and emptied the three tanks on four separate fields. During the first application event, he cleaned a blocked nozzle while wearing his protective gloves. During the second mixing event he fixed a leak in the hose with latex gloves instead of nitrile gloves. He did not report potential exposure events after the day of application (re-entry, other applications). The highest absorbed daily dose in the closed-cabin scenario (3.83 · 103 mg/kg bw/day) was obtained for an operator who applied a total of 25.62 kg a.i. on 10.69 ha of maize, using an average spray volume of 281 L/ha and spent 358 min for the completion of his entire application. He wore a Tshirt, long sleeved shirt, jeans, and boots. For the application he used a 600 L spray-tank and a 10 m, manually folding spray boom. This applicator mixed and loaded five times and emptied the five tanks on five separate fields. During the first application, the operator sprayed for about 3 min with the back window open to check and adjust the pressure on the sprayer. He smoked in his cabin during the third application without having washed his hands. Accidental spillages were reported during the third, fourth, and fifth mixing and loading events. 3.5. Risk characterization
3.4. Absorbed daily doses Table 6 summarizes the absorbed daily dose (mg/kg bw/day) statistics for the open- and closed-cabin scenarios. On average, the absorbed daily doses obtained for the open-cabin scenario (average = 0.004 mg/kg bw/day) were two times higher than the doses obtained for the closed-cabin scenario (p = 0.016). The maximum absorbed daily dose for the opencabin scenario (8.24 · 103 mg/kg bw/day) was obtained for an operator who applied a total of 21.7 kg a.i. on 10.67 ha of maize, using an average spray volume of 169 L/ha, and spent 188 min completing his entire application. He wore a cotton coverall over jeans and a shirt, Wellington boots and a hat. For the application he used a 600 L spray tank, a 14 m, manual folding spray boom, and an induction
The risk characterization for the open- and closedcabin exposure scenarios is presented in Table 7. The NOELÕs (10 mg/kg bw/day for professional applicators and 100 mg/kg bw/day for farmers), were divided by the average, the 75th percentile, and maximum absorbed daily doses, respectively, to obtain the margin of exposure (MOE). For short-term exposure, which represents the exposure duration for farmers applying acetochlor, the MOE based on the average systemic exposure level was 23,000 for the open-cabin scenario and 44,000 for
Table 7 Risk characterization expressed as MOE Farmers
Table 6 Absorbed daily dose (mg/kg bw/day): statistics Absorbed daily dose (mg/kg bw/day)
Minimum 25th Percentile Average Median 75th Percentile Maximum Standard deviation
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Open-cabin
Closed-cabin
7.77 · 104 3.37 · 103 4.34 · 103 4.18 · 103 5.79 · 103 8.24 · 103 2.05 · 103
6.87 · 104 1.08 · 103 2.29 · 103 2.19 · 103 3.69 · 103 3.83 · 103 1.32 · 103
Relevant NOEL Open-cabin MOEc Average 75th Percentile Maximum Closed-cabin MOEc Average 75th Percentile Maximum a b c
a
Professional applicators
100 mg/kg/day
10b mg/kg/day
2.30 · 104 1.96 · 104 1.21 · 104
2.30 · 103 1.96 · 103 1.21 · 103
4.37 · 104 2.79 · 104 2.61 · 104
4.37 · 103 2.79 · 103 2.31 · 103
Based on rabbit developmental study. Based on 90-day dog study. NOEL/exposure.
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the closed-cabin scenario. For intermediate term exposure, which represents the exposure duration for professional applicators using acetochlor, the MOEs were 10-fold lower.
4. Discussion The purpose of this study was to obtain higher tier exposure estimates for acetochlor under realistic conditions. The work-practices monitored were representative but conservative: multiple small plots were treated and required frequent refilling of the spray tank; the maximum use rate was applied, frequent equipment failures were observed and protective gloves were not always worn as instructed. Representative 24 h urine samples were collected during the entire monitoring period after the on-study application allowing an accurate evaluation of the excretion kinetics. The average urinary excretion half-life obtained from those applicators who showed monotonic first order urinary excretion of acetochlor metabolites (0.94 days) is consistent with the excretion kinetics obtained from the pharmacokinetic study with six male rhesus monkeys, in which two groups of three animals each were dosed at 0.005 and 0.05 mg acetochlor/kg bw, respectively (Monsanto unpublished report). Based on urinary excretion only, an overall average systemic half-life of 1.2 days was observed in monkeys. Based on the total excretion balance (urine + feces), the average systemic half-life was 1.4 days. Although operators in the closed-cabin scenario sprayed on average greater than a 25% more hectares, the average amount of acetochlor absorbed under the conditions of this scenario was two times lower than the average amount absorbed during the operations in the open-cabin scenario. This suggests that the relative contribution of the tractor mounted application events to the overall exposure was significantly higher for the open-cabin scenario compared to the closedcabin applications. Although intuitive, this conclusion may underestimate the importance and relative contributions of accidental spillages and contact with contaminated (spray) equipment during mixing, loading, and manipulations of the spray boom, which are not discretely determined from these biomonitoring data. For both scenarios, no scientifically meaningful linear relationship could be demonstrated between exposure and any of the key study variables tested in this analysis (including workload). For the closed-cabin scenario, the association between total spray volume and the systemically absorbed amount was statistically significant, but additional data analysis could not help to explain the inverse relationship. Although the applicators with the highest total spray volume had the most diluted
tank mix concentration (p = 0.001), this should not necessarily impact the exposure levels for the closedcabin scenario where the highest exposure contribution is expected to come from mixing, loading, and manipulations of the spray boom. There was no association between the number of mixing and loading events (nor the time spent on mixing and loading) and the total spray volume for the closed-cabin scenario (p > 0.05). The lack of a significant linear relationship between any of the study variables and exposure may be explained by the relatively small variability in workload and the low (from a statistical point of view) number of replicates per scenario. Alternatively it could also suggest that accidental spillages and contact with contaminated (spray) equipment during mixing, loading, and manipulations of the spray boom were the main contributors to exposure. The number of such exposure events may not be directly linked to workload. Therefore care should be taken during the interpolation or extrapolation of the normalized acetochlor exposure values to other workload scenarios. The risk assessment based on the activities monitored in this study could be considered conservative because of the work practices (equipment, number of exposure events). Accidental spillages and contact with contaminated equipment during mixing and loading were presumed to contribute the most to overall exposure in this study. Furthermore, the selection of the NOELs used in this evaluation added to the conservative nature of the risk assessment. To evaluate exposure that typically lasts only 1 or 2 days every year (farmer), an endpoint was selected based on a 14-day study, and an endpoint was selected from a 90-day study to evaluate exposure that typically lasts less than 30 days every year (professional applicator). Despite the conservative nature of the risk assessment, the MOEs were relatively large. For the open-cabin scenario the average MOEs for farmers ranged from 23,000 (open-cabin scenario) to about 44,000 (closedcabin scenario). For professional applicators the MOEs were 10-fold lower. In conclusion, the risk characterization for both farmers and professional applicators demonstrates that no health effects are to be expected for either open- or closed-cabin applicators of acetochlor.
Acknowledgments The authors thank their former colleagues Dr. Mark Martens and Catherine Schmit for their contributions to this study. They also recognize the team from Inveresk Research and in particular Jeff Old, Iain Anderson, and Dr. Santiago Marti for conducting the field portion of the study.
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References Acquavella, J.F., Alexander, B.H., Mandel, J.S., Gustin, C., Baker, B., Chapman, P., Bleeke, M., 2004. Glyphosate biomonitoring for farmers and their families: results from the farm family exposure study. Environ. Health Perspect. 112 (3), 321–326. Alavanja, M.C.R., Sandler, D.P., McDonnell, C.J., Lynch, C.F., Pennybacker, M., Zahm, S.H., Mage, D.T., Steen, W.C., Wintersteen, W., Blair, A., 1999. Characteristics of pesticide use in a pesticide applicator cohort: the agricultural health study. Environ. Res. Section A 80, 172–179
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Safeguarding the Health of Applicators of Plant Protection Products (Uniform Principles for Operator Protection). Mitteilungen aus der Biologischen Bundesanstalt fur Land-und Forstwirtschaft, Berlin-Dahlem; ISSN 0067-5849–ISBN 348927700-7. Organization for Economic Co-operation and Development Environment Directorate, Document (OECD/GD(97)) 148 Series on Testing and Assessment No. 9, Guidance Document for the Conduct of Studies of Occupational Exposure to Pesticides During Aricultural Aplication. 1997. Paris, France.