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Hazard evaluation for northern bobwhite quail (Colinus virginianus) exposed to chlorpyrifos-treated turf and seed
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 60 (2005) 176–187
Hazard evaluation for northern bobwhite quail (Colinus virginianus) exposed to chlorpyrifos-treated turf and seed Gary M. Booth,a,* Spencer R. Mortensen,b Melvin W. Carter,a and Bruce G. Schaaljea a
Departments of Statistics and Integrative Biology, Brigham Young University, 697 WIDB, Provo, UT 84602, USA b Syngenta, 410 Swing Road, Greensboro, NC 27409, USA Received 10 July 2003; received in revised form 16 December 2003; accepted 29 January 2004
Abstract This study evaluated the toxicity effects of chlorpyrifos on bobwhite quail (Colinus virginianus) kept in 27 field-exposed large pens arranged over turf in a randomized block design with nine blocks of three pens (16 adult birds per pen). Nine pens were treated with one application of 3.4 kg active ingredient (ai) per hectare followed by a second 3.4-kg ai/ha application 2 weeks later, nine pens with one 6.7-kg ai/ha application, and nine pens with formulation blank. In addition, the seed fed to the birds in the two chemically treated pens was also treated with chlorpyrifos. Mean residue in the grass samples from the first 3.4-kg treatment pens ranged from 306795 ppm on day 0 to 1878 ppm on day 14 after treatment. The second 3.4-kg ai/ha treatment grass residues ranged from 3617167 ppm on day 0 to 38724 ppm on day 14 after treatment. Grass residues from the 6.7-kg treatment pens ranged from 9037310 ppm on day 0 to 978 ppm on day 30 after treatment. Half-lives were B2 days and 10 days for grass and seeds, respectively. Whereas the incidence of behavioral deficits was significantly (P ¼ 0:0156) higher in the 6.7-kg pens (five females, one male), two of the females could have been the same bird because they were both seen in the same pen on days 23 and 24 after treatment. There was no significant difference in mortality, brain acetylcholinesterase activity, or any other measured parameter among any of the treatments. We conclude that application of chlorpyrifos to turf at 3.4 and 6.7 kg ai/ha is not expected to have chronic deleterious effects on populations of bobwhite quail grazing on treated grass or seeds, provided there is an abundant supply of seeds for the quail to eat. r 2004 Elsevier Inc. All rights reserved. Keywords: Chlorpyrifos; Pen study; Bobwhite quail; Pyrinex 4E; Turf
1. Introduction Pyrinex 4E, which contains the active ingredient (ai) chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is used to control a variety of pests on turf as well as many crops (Makhteshim-Agan Inc., 1988). Dosages of Pyrinex 4E per 93 m2 of turf range from 22 mL (11.1 kg ai/ha) to 89 mL (4.5 kg ai/ha). The 4E formulation contains 48.9% chlorpyrifos, equivalent to 1.2 kg ai/L. Since the anticipated maximal seasonal use rate of Pyrinex 4E is 6.7 kg ai/ha, this rate represents the greatest risk to turf-grazing birds and thus was used as the maximum application rate in this study. Toxicity of chlorpyrifos to birds is moderate to high. Considering both the acute (LD50=13.3–32 mg/kg) and dietary toxicity (LC50=283–1100 mg/kg) to bobwhite *Corresponding author. Fax: +1-801-378-7423. E-mail address: gary [email protected] (G.M. Booth). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.011
quail, it appears that this bird is one of the more sensitive avian species that has been extensively evaluated (Rexrode, 1984; Smith, 1987; Hudson et al., 1984; Johnson et al., 2001). Because of its sensitivity to chlorpyrifos and also because it grazes on grass (Wiseman, 1977), the bobwhite quail was chosen as an indicator species for the turf study. Field effects of chlorpyrifos on birds are not at all decisive. For example, there have been at least two reports from the Ecological Effects Branch of the US EPA (1981) of dead geese found on golf courses that had been treated with chlorpyrifos. Additionally, Hurlbert (1977) reported significant mortality of young mallard ducks following treatment with chlorpyrifos. However, Kenaga (1974) sprayed field-confined birds with 4– 36 kg ai/ha with no apparent ill effects, whereas Booth et al. (1984) found no effects on waterfowl exposed to chlorpyrifos sprayed on winter wheat. Hence, conclusions about wildlife mortality in the field resulting from
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chlorpyrifos spraying are equivocal. For this reason, additional wildlife studies on this compound have been strongly recommended (Odenkirchen, 1988; Smith, 1987). Of particular interest to the current study was the potential impact of chlorpyrifos on upland game birds exposed to a contaminated food source resulting from direct spraying of two food substrates. Dietary exposure of insecticides under field-exposed large-pen conditions using a large number of birds and a large number of replicates represents an opportunity to study the lethal and sublethal effects of agrochemicals on selected sensitive birds under controlled field conditions of maximum exposure (Booth et al., 1980a, b). Accordingly, the objectives of the present study were to (1) monitor mortality effects of chlorpyrifos associated with dietary and environmental exposure to bobwhite quail under field-exposed large-pen enclosures over turf, (2) assess the potential of chlorpyrifos to induce behavioral deficits in quail foraging on treated turf; (3) investigate the residue profile of chlorpyrifos in treated grass and, if possible, relate these residues to bird mortality, behavioral deficits, and brain acetylcholinesterase (AChE) levels, and (4) determine whether chlorpyrifos induces body weight loss, reduced organ weight, brain AChE depression, and decreased egg production in surviving birds.
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included behavior, mortality, brain AChE determinations, environmental residues, body and organ weight determinations, and egg laying. All of the appropriate data were analyzed statistically. 2.4. Pens Hardware cloth-covered pens were constructed from modular panels to cover a minimum of 0.005 ha (50 m2 per pen). The pen dimensions were 3.1 15.2 m, with the top cover at a height of B1.8 m. A 0.91- by 1.8-m piece of 1.9-cm plywood was placed on the windward (south) side of each pen to reduce violent wind exposure. Rooftop shading was provided in one end of each pen using a nylon mesh shading material. A total of 28 pens was assembled from 3.1-m sections, with 9 pens for the formulation blank controls, 9 pens for the Pyrinex 4E treatment at 6.7 kg ai/ha, and 9 pens for the Pyrinex 4E treatment at 3.4 kg ai/ha+3.4 kg ai/ha, with each of the two 3.4-kg treatments separated by 14 days. The 28th pen was used as a holding pen for extra birds. The 27 treatment pens were placed in a randomized block design with nine blocks (Fig. 1). Randomization of the treatments and assignment of birds to each pen was done according to a random number generator. 2.5. Birds
2. Materials and methods 2.1. Chemicals Pyrinex 4E was supplied by Makhteshim Agan Inc. (Israel) via the Pennwalt Company (Philadelphia, PA), which shipped 11 L of the chemical (batch no. B23-11) directly to Brigham Young University (BYU; Provo, UT). This batch contained 48.9% of the ai chlorpyrifos. The vehicle control consisted of Pyrinex minus chlorpyrifos, which was also supplied by Makhteshim Agan Inc.
Bobwhite quail (Colinus virginianus) adults (16-weekold flight-adapted) were obtained from a pen-reared stock from Oakridge Game Farm (Gravette, AK). All birds were flight-conditioned and reared on Gro-scratch feed beginning May 10, 1988 until they were shipped by truck to Provo arriving on May 26, 1988. Each pen was stocked with 16 adult bobwhite quail (8 males+8 females) 15 days prior to the treatment application. Each bird was weighed, banded (with a numbered leg band), and randomly assigned to one of the 27 pens. 2.6. Bird feed
2.2. Test site The study was conducted in Goshen, UT, at a turf farm located B50 km west of Springville, UT. 2.3. Time table The investigation was divided into three phases: (1) An acclimation period of 15 days for the birds began when they arrived on May 26, 1988. (2) A chemical application phase of 30 days began on June 11, 1988 for the 6.7-kg ai/ha treatment and the first 3.4-kg ai/ha treatment. The second 3.4-kg ai/ha treatment was applied on June 25, 1988. The field portion of the study was terminated on July 11, 1988, when all surviving birds were sacrificed. (3) An analysis phase (60 days)
All feed used in this experiment was Gro-scratch (9% protein containing wheat, milo, sorghum, and cracked corn), a commercial feed obtained from Intermountain Farmers Association (Provo, UT). This feed had been used successfully in a previous large-pen study (Booth et al., 1980a, b). 2.7. General test conditions and procedures The pens were placed on turf with 15 in one row (end to end) and 12 in another (end to end). The rows were assembled in an east–west direction. The pens were separated east and west (end-wise) by B1.5 m, and north and south by B0.9 m. The pens were arranged so that five blocks were located on the north and four
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EXPERIMENTAL DESIGN 9 Blocks x 3 TRT/Block = 27 Pens 27 Pens x 16 Birds/Pen = 432 Birds
Pen Numbers Control
3.4 kg
6.7 kg
1 5 8 11 13 16 21 22 26
2 6 9 12 15 17 19 24 25
3 4 7 10 14 18 20 23 27
Fig. 1. Experimental design, showing arrangement of pens and holding pen. See text for details.
blocks on the south (Fig. 1). During the 15-day acclimation phase, the birds were fed daily with nontreated Gro-scratch scattered onto the turf of each pen to stimulate field-foraging behavior of quail on the treated grass. Using this procedure, B40 g/bird/day were fed to the birds based upon the average adult bobwhite feed consumption rates compiled from our laboratory (Booth et al., 1980a, b). This feeding rate was approximately twice what the birds ate in the laboratory because of (1) the bird’s inability to find all the feed in the pens and (2) greater bird activity in the pens compared to laboratory conditions, thus necessitating more feed. During the acclimation phase, feeding amounts were carefully monitored to assure that the quantity of feed available was not limiting. Water contained in 4-L poultry jars was supplied ad libitum into each pen. Water jars were emptied and refilled each day. Amoxicillin antibiotic was supplied in the drinking water at the rate of 250–500 mg/L during 10 days of the acclimation phase. This antibiotic treatment helped reduce stress and potential enteritis problems associated with the long journey to Utah (Crawley, personal communication). The turf was watered on a weekly basis or according to normal practices followed by the turf farm. In addition, the grass inside and outside the pens was mowed about once per week after treatment began. Mowing on the inside of the pens was accomplished using a mechanical reel-type push-mower so that the grass clippings remained inside the pen. 2.8. Calibration of the sprayers Calibration of each sprayer was accomplished by spraying as much as possible of 2000 mL of water from
an Erlenmeyer flask over the 50 m2 of test turf. On the average, B80 mL were left in the sprayer for priming and B120 mL were left in the flask that could not be drained. Thus, to adjust for this loss of 200 mL, instead of spraying 64 g Pyrinex 4E/1900 mL over 50 m2 (equivalent to 6.7 kg ai/ha), 71 g Pyrinex 4E/2,100 mL was sprayed over each of the nine plots for the 6.7-kg treatment; instead of spraying 32 g/1900 mL over each 50 m2 for the 3.4-kg ai/ha treatment, 36 g Pyrinex 4E/ 2100 mL was sprayed over each of these nine plots. Finally, instead of spraying 64 g formulation blank/ 1900 mL over each 50 m2 for the controls, 71 g formulation blank/2100 mL was sprayed over each of these nine plots. After each pen had been sprayed, the volume left behind in the flask was recorded to determine the nominal amount of chlorpyrifos that was actually sprayed on the plots (Table 1). 2.9. Chemical application to the grass Following the acclimation phase of the experiment, each set of 16 birds from each pen was caught, reweighed, and placed by pen number into separate bird boxes until treatment of the turf and seed was completed. All birds spent B3 h in the holding boxes. Any birds appearing unhealthy, overly stressed, or that had lost X15% of body weight were replaced (total of 28 birds) by one of the birds selected at random from the holding pen. On June 11, 1988, nine pens received 6.7 kg ai/ha sprayed by a hand-held sprayer at a rate of 1 L of water/ 23 m2, nine pens received 3.4 kg ai/ha using a handsprayer at the rate of 1 L of water/23 m2 (on June 25, 1988, these nine pens also received another 3.4 kg ai/ha),
ARTICLE IN PRESS G.M. Booth et al. / Ecotoxicology and Environmental Safety 60 (2005) 176–187 Table 1 Chlorpyrifos residues contained in Gro-scratch seed used in feeding bobwhite quaila Days after treatment
0 1 2 3 4 5 6 10 13 15 20 29 Mean residues
Chlorpyrifos (ppm) First 3.4 kg ai/ ha treatment
Second 3.4 kg ai/ha treatment
6.7 kg ai/ha treatment
12 11 11 18 12 12 9 6 10 No sample No sample No sample 11.2273.19
21 20 17 14 17 17 15 15 No sample 18 No sample No sample 17.1172.32
30 27 24 25 25 23 27 15 No sample No sample 12 11 2176.72
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Aliquots of 45 kg of seed were treated in an industrial mixer for each treatment until there were 180 kg of treated feed for each of the three treatments. Each of the treatment samples was air-dried, sampled for residues, returned to the original containers, and stored in the dark at 25 C until the next morning, when the field experiment began. At the end of 14 days after treatment, the seed used in the 3.4-kg treatment pens was re-treated at the same rate as the first time. Thus, the birds on the 3.4-kg treatment fed during the next 16 days (4 weeks total) were getting the residue from the previous treatment plus the residue from the second treatment. This paradigm simulates what would be expected in a natural field situation, in which native forage material may be double-treated under this treatment regime. In fact, frequent mowing probably created a greater hazard by allowing more of the seed to be exposed to treatment than would occur under ‘natural field conditions’.
a
Seed blanks were spiked with 480 ppm chlorpyrifos standard and found to give 490% recovery.
and nine pens received formulation blank equivalent in weight to the 6.7-kg treatment. Following drying, the grass in each pen was sampled for residues according to a randomized sampling regime, and 16 birds were randomly placed into each of the 27 pens. 2.10. Chemical application to the feed All of the feed scattered throughout the pens was treated either with formulation blank (control pens) or Pyrinex 4E (3.4-kg and 6.7-kg treatment pens). However, treatment of the feed with the proper dose posed a problem because the actual concentration of chlorpyrifos on seeds under these field conditions was unknown and difficult to estimate because of such variables as the uneven distribution of scattered seeds and variation in the density of the grass. Therefore, a seed calibration experiment was designed to estimate empirically the actual concentration of chlorpyrifos on seeds during treatment. A 0.7 0.7 m section of turf (0.5 m2) taken from the Goshen turf farm was used as the experimental unit. A total of 6.5 g of Gro-scratch (equivalent to 640 g/50 m2) was scattered over the turf surface similar to the feeding procedure used in the pens. Then, 647 mg of Pyrinex 4E (equivalent to 6.7 kg ai/ha) was placed in 19.2 mL water (equivalent to 1 L of water/23 m2) and sprayed on the turf. After a few minutes of drying, the turf section was tipped upside down over a white pan, the seeds collected first by dislodgement, and then the remaining seeds picked out using a pair of tweezers and a magnifying glass, placed in a vial, frozen, and sent to the laboratory for analysis. This seed calibration experiment was replicated three times.
2.11. Residue sampling The grass was sampled from each of the 3.4-kg and 6.7-kg treatment pens and from three control pens according to a randomized sampling schedule. For the 6.7-kg treatment, samples were taken at 0 (immediately following treatment), 1, 2, 3, 4, 5, 6, 10, 20, and 30 days after treatment. For the first and second 3.4-kg treatments, samples were taken at 0 (immediately following treatment), 1, 2, 3, 4, 5, 6, 10, and 14 days after treatment, and 0 (immediately following the second treatment), 1, 2, 3, 4, 5, 6, 10, 14, and 16 days after treatment, respectively. Three control pens were randomly selected and sampled only at time 0 (immediately following treatment). 2.12. Residue analysis All residue analyses were completed at the Agricultural Experiment Station–Chemistry Station and Analytical Laboratory (McCall Hall, Montana State University, Bozeman, MT). Residues were analyzed by following a standard method (US FDA, 1986) using gas chromatography coupled with an electron capture detector. The separation column was packed with Supelco Port packing material (100/120 mesh) coated with 1.5% SP-2250 and 1.95% SP-2401. The method detection limit was 0.01 ppm for the parent compound, and the carrier gas flow was 30 mL/min. 2.13. Weather measurements Temperature, rainfall, and wind speed were recorded during the application phase of the study at Goshen, UT, and at the Widtsoe Building at BYU where the treated seed was being exposed to the environment.
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2.14. Biological observations and measurements During the application phase, behavior, mortality, and egg laying were monitored on a daily basis for each pen. Behavioral deficits were noted, and these included isolated birds that were apparently inactive (not feeding), birds isolated with eyes shut, live but immobile birds, birds with drooping wings and/or tail fanned, salivation, staggering gait, and birds failing to respond to auditory stimuli. All surviving birds were caught on the final day of the study (July 11) and placed, according to each numbered pen, into separate numbered boxes. The birds were sacrificed by each statistical block using CO2 asphyxiation. After each bird had been weighed and the sex and bird number recorded, the brain, liver, crop, and gonads were removed and weighed. All of the brain tissue was analyzed for AChE activity using published methods (Ellman et al., 1961). 2.15. Statistical methods The variables of eggs per pen and eggs per bird day were measured for each pen, and were analyzed using weighted complete block ANOVA, with weights equal to the inverses of the respective variables. Mortality was calculated for groups of birds of each sex in each pen, and was analyzed using logistic regression with overdispersion adjustment. AChE, final body weight, brain weight, liver weight, crop weight, and gonad weight were measured for each bird, and were analyzed using split-plot ANOVA for whole plots arranged in complete blocks. Chlorpyrifos treatments composed the wholeplot factor, and sex was the sub-plot factor. For all variables except AChE, the initial weight of the bird was used as a covariate. For all of these analyses, leastsquares means for chlorpyrifos treatments, sexes, and their interaction were obtained and compared using unadjusted Student’s t-tests for contrasts. The number of behavioral deficits was compared among chlorpyrifos treatments using the sign test (Steel et al., 1997). The observations were paired by day of observation. Statistical calculations were done using the standard SAS, MINITAB, and RUMMAGE packages.
3. Results and discussion 3.1. Residue data As a quality control measure, residue samples were taken from the Pyrinex 4E formulation used in the 3.4kg and 6.7-kg treatment pens and the formulation blank used in the control pens. These analyses showed that Pyrinex 4E contained B49% chlorpyrifos (B100% of the expected amount), whereas the formulation blank
contained a trace amount (0.000026%) of chlorpyrifos. In addition, on each of the spray days, we determined how much of the spray solution actually went onto the plots by keeping track of the spray volumes left over. The 3.4-kg ai/ha spray solution used on the 3.4-kg treatment pens on day 0 (June 11, 1988) ranged in nominal concentration from 3.3 to 3.5 kg ai/ha. Nominal concentrations associated with the 6.7-kg ai/ha spray solution (day 0) were 6.4–6.9 kg ai/ha. Following the second treatment of the 3.4-kg application (June 25), nominal concentrations ranged from 3.3 to 3.4 kg ai/ha. The average chlorpyrifos concentration found in the treated Gro-scratch seeds in the calibration experiments from the three turf sections was 25.4374.60 ppm. To make sure there was an adequate amount of chlorpyrifos on the feed, the seed for the 6.7-kg treatment was treated with 33 ppm and the 3.4-kg treatment seed was treated with 16.5 ppm. This intentional increase in the treatment of the seeds from 25 to 33 ppm (about a 32% increase) was done to account for possible losses and errors (note the standard deviation) during the mixing and spraying process to insure about 20 to 30 ppm in the 6.7-kg treatment and B10 to 17 ppm in the 3.4-kg treatments. Sampling of the residues from the treated feed through time clearly shows that this was achieved. The seeds treated with the first 3.4-kg ai/ha application averaged 11.2273.19 ppm chlorpyrifos, seeds treated for the second application of 3.4 kg ai/ha averaged 17.1172.32 ppm chlorpyrifos, and seeds treated with 6.7 kg ai/ha averaged 21.9076.72 ppm chlorpyrifos (Table 1). Spiked recoveries of Gro-scratch seed blanks were 4 90%, and residues on the control seed samples were undetectable. Small amounts (0.04–3.0 ppm) of chlorpyrifos residues were detected from the grass of the three control pens (Table 2), probably as a result of drift; since each treatment had its own sprayer, cross-contamination between sprayers was not an issue. No chlorpyrifos was detected on the untreated seed used in the control pens. Fig. 2 shows the 3.4-kg ai/ha (first application) pen-topen variability in grass residues through 14 days after treatment, and Figs. 3 and 4 represent the pen-to-pen variability in grass residues from the second application of 3.4 kg/ha and a single application of 6.7 kg/ha, respectively. For the first application of the 3.4-kg ai/ ha treatment, the residues averaged 306795 ppm on day 0 (June 11, 1988) and 1878 ppm on day 14 (June 25, 1988) after treatment (Fig. 5). Mean residues from grass samples analyzed from the second 3.4-kg ai/ha application ranged from 3617167 ppm on day 0 to 38724 ppm on day 14 (Fig. 6). Pen-to-pen variability over time (coefficient of variation, CV ¼ 4778%) in grass residues sampled from the second application of 3.4 kg ai/ha (Fig. 4) were quite similar to those (CV ¼ 41717%) from the first application (Fig. 2) and did not show any indication of a cumulative effect from consecutive
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Table 2 Summary of chlorpyrifos analysis from control (untreated) seed and selected grass samples Sample type
Laboratory no.
Sample description
Chlorpyrifos concentration (ppm)
Grass Grass Grass Seed Seed
2155-1 2155-2 2155-3 2182-1 2182-21
Grass control: pen 11 Grass control: pen 13 Grass control: pen 22 control seed control seed
3.0 0.04 3.0 N.D.a N.D.a
a
N.D., not detectable.
Fig. 3. Pen-to-pen variability in grass chlorpyrifos residues for the second 3.4-kg ai/ha application through 16 days after treatment. Chlorpyrifos residues (ppm) in grass samples treated with 6.7 kg ai/ha
ppm Chlorpyrifos
1400 1200 1000 800 600 400 200 0 0
1
2
3
4
5
6
10
20
30
Days Post-treatment pen 3
pen 4
pen 10
pen 14
pen 7
Chlorpyrifos resudes (ppm) in grass samples treated with 6.7 kg ai/ha
Fig. 2. Pen-to-pen variability in grass chlorpyrifos residues for the first 3.4-kg ai/ha application through 14 days after the application.
ppm Chlorpyrifos
1200 1000 800 600 400 200 0
0
1
2
3
4
5
6
10 20 30
Days Post-treatment
applications. The 6.7-kg treatment also showed surprisingly similar pen-to-pen variability (Fig. 7; CV ¼ 44720%) over time compared to the first and second application of the 3.4-kg treatment. Mean grass residues from the 6.7-kg treatment ranged from
pen 18
pen 20
pen 23
pen 27
Fig. 4. Pen-to-pen variability in grass chlorpyrifos residues for the single application of 6.7 kg ai/ha through 30 days after treatment.
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Fig. 5. Chlorpyrifos residues (ppm) in grass samples for the first 3.4kg ai/ha application through 14 days after the application.
Fig. 6. Chlorpyrifos residues (ppm) in grass samples for the second 3.4-kg ai/ha application through 14 days after the application.
9037310 ppm on day 0 to 978 ppm on day 30 after treatment (Fig. 7). The half-lives for chlorpyrifos residues on grass for all three applications were always within 1–2 days, consistent with data from Kenaga (1974) but shorter than those reported by Kuhr and Tashiro (1978), who demonstrated a half-life of B1 week on sprayed turf. 3.2. Quail behavior, mortality, brain AChE, body weights, organ weights, and egg laying During the 30-day observation period, none of the control birds showed any behavioral anomalies (Table 3). A total of six birds with behavioral deficits (six females and one male) were observed in the 6.7-kg treatment (4% of the 6.7-kg treatment birds), and only one male showed abnormal behavior in the 3.4-kg
Fig. 7. Chlorpyrifos residues (ppm) in grass samples for the single application of 6.7 kg ai/ha through 30 days after treatment.
treatment (o1% of the 3.4-kg treatment birds). It was clear that three of the five females with behavioral deficits in the 6.7-kg treatment were different birds, because each was observed in a different pen, but it could not be determined whether the one female with behavioral deficits seen on day 23 after treatment was the same female seen on day 24 after treatment, since both observations occurred in pen 4. Analyses of these data show that the number of behavioral deficits in the 6.7-kg treatment was significantly higher than the control (P ¼ 0:0156). It is of interest that three of the six behavioral abnormalities were observed on days 22–24 after treatment, when the grass residues were fairly slight (33722 ppm, day 20 after treatment; Fig. 7). No other treatment comparisons were significant, although the differences between the two Pyrinex 4E treatments approached statistical significance (control vs. 3 lb, P ¼ 0:5; 3.4-kg treatment vs. 6.7-kg treatment, P ¼ 0:0625). Tables 4–6 summarize bird mortality for each treatment by days after treatment, bird number, sex, pen number, and brain AChE activity. Nine control birds died (Table 4; 6.25%), 11 birds receiving the 3.4-kg treatment (Table 5; 7.64%), and 14 birds receiving the 6.7-kg treatment (Table 6; 9.72%). A logistic regression analysis with overdispersion adjustment of the mortality data showed no significant differences among treatments (Table 7; P ¼ 0:6992). Although significantly more females died overall (11.57%) compared with males (4.17%) (P ¼ 0:023), the sex-by-treatment interactions were not significant. Even though some studies have shown significantly increased upland game mortalities associated with chlorpyrifos spraying on turf (Stone, 1979), other data in the literature suggest that mortality is related only to higher doses than those used in our study, and may be related to the foraging behavior of the bird. For example, Schom et al. (1973) showed no effect on adult bobwhite quail mortality when Dursban
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was fed in the diet at 100 ppm, but when the diet concentrations were raised to 200, 500, and 600 ppm they reported significant mortalities of 11%, 17%, and 39%, respectively. It is clear from our study that grass residues can be much higher than this (as high as 1400 ppm in one
replication, Fig. 6) with no significant mortality, provided that seeds are abundant. The rationale for lack of mortality in our data is that foraging quail prefer to feed on seeds, which has far less chlorpyrifos residues than does grass, when a choice between seeds and grass is available; this rationale is supported by the feeding ecology of bobwhite quail (Wiseman, 1977). Hence, from a practical standpoint, constant dietary concentrations 4100 ppm under normal turf treatment conditions and normal foraging behavior are not likely. In addition, grass residue levels drop off rapidly during the first week after treatment regardless of the treatment rate. Martin (1990) showed that, although immersing Japanese quail eggs in field concentrations of chlorpyrifos resulted in some abnormalities (foot malformations and scoliosis), the author concluded: ‘‘Overall, effects were small and the three insecticides did not represent a significant hazard to quail embryos at conditions up to two times field levels of application.’’ When pheasants (Phasianus colchicus) were exposed to chlorpyrifos-treated vegetation in pens for 48 h (Martin et al., 1996), there was no difference in mortality among treatment and control groups. Clements et al. (1992) have also shown that two species of grazing wild geese were not negatively affected (neither mortality nor behavior) when chlorpyrifos was sprayed at the recommended rate of 0.72 kg ai/ha. Furthermore, repeated applications of chlorpyrifos to ornamental trees containing robin nests (Turdus migratorius) showed no significant adult or juvenile mortality despite depressed plasma cholinesterase levels (Decarie et al., 1993). Sensitivity of aquatic upland game birds to fieldapplied chlorpyrifos also seems to be related to higher doses, age of the birds, and cold temperatures. For example, whereas 8 ppm in mallard duck diets in an open-pond design did not influence reproduction or mortality, 80 ppm (Meyers and Gile, 1986) reduced hatchability and apparently allowed no ducklings to survive on the treatment ponds to 7 days. However, Martin and Forsyth (1998) have shown no effects on
Table 3 Summary of behavioral deficits observed in quail during the 30 days after treatment Days after treatment a
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14b 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Total
Number and sex of birds with behavioral deficits Control
3.4 kg ai/ha
6.7 kg ai/ha
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1 male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 male
1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 1 1 0 0 0 0 0 0 5
183
female
male
female
female female female
females, 1 male
Table 4 Control bird mortality by days after treatment, bird number, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
13 16 19 26 26 26 27 28 30 Summary
339 253 252 282 132 257 353 255 316 9 birds total (6.25% mortality)
Female Female Female Female Female Female Female Male Female 8 females, 1 male
11 1 1 22 22 22 5 22 5 4 different pens
5.871 6.806 6.306 4.851 6.576 6.265 7.748 5.139 5.574 6.1370.89 (mean)
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Table 5 Bird mortality from the 3.4-kg ai/ha treatment by days after treatment, bird number, sex, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
11 (1st appl.) 4 (2nd appl.) 12 (2nd appl.) 12 (2nd appl.) 13 (2nd appl.) 14 (2nd appl.) 14 (2nd appl.) 14 (2nd appl.) 15 (2nd appl.) 15 (2nd appl.) 16 (2nd appl.) Summary
439 299 525 5 194 352 40 453 279 334 62 11 birds total(7.64% mortality)
Female Male Female Female Female Male Female Female Male Male Female 8 females, 3 males
2 6 25 25 25 6 12 25 12 25 12 5 different pens
4.949 5.494 4.646 5.340 7.673 6.739 5.220 5.088 6.374 6.579 5.462 5.7870.93 (mean)
Table 6 Bird mortality for the 6.7-kg ai/ha treatment by days after treatment, bird number, sex, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
6 13 13 17 18 18 20 22 23 23 26 27 29 30 Summary
455 217 220 338 157 375 155 54 222 183 374 443 370 206 14 total birds (9.72% mortality)
Female Male Female Male Female Female Male Female Female Male Female Female Male Female 9 females, 5 males
3 14 23 23 14 14 23 23 10 23 10 18 23 23 5 different pens
5.314 4.534 6.465 7.227 Not measured 5.466 6.232 Not measured 5.428 5.232 5.144 7.544 4.384 4.978 5.6671.00 (mean)
behavior of ducklings feeding and swimming in ponds treated with 420 g ai/ha. In addition, thermally stressed bobwhite quail seemed to be more sensitive to organophosphorus compounds. In fact, synergistic effects have been demonstrated in juvenile bobwhites simultaneously exposed to chlorpyrifos and cold stress (Maguire and Williams, 1987). Analysis of the brain AChE of birds that had died in the pens in our study showed no significant treatment differences (Table 7; P ¼ 0:532). However, bird no. 370 from the 6.7-kg treatment pen died while being videotaped and showed many symptoms of AChE depression. This bird also had the lowest AChE level among the birds that died on the plots (Table 6; 4.384 mmol/min/g), which represented an inhibition of B28%. All of the surviving birds were sacrificed by CO2 asphyxiation, and a summary of the analysis of their brain AChE activity, body weight, brain/body weight ratio, liver/body weight ratio, crop/body weight ratio, gonad/body weight ratio, eggs per pen, and eggs per day
is shown in Table 7. A total of 271 quail brains were analyzed as nonfrozen (fresh) tissue for AChE, and none of the treatments differed significantly from each other (Table 7, P ¼ 0:150). The remaining 113 birds were sacrificed by block, the brain dissected, frozen, and then analyzed for AChE the next day. Results for these frozen-brain enzyme data also were not statistically significant (Table 7, P ¼ 0:949). The lack of AChE inhibition in this study is not surprising given that the quail fed mainly on seeds that had greatly reduced chlorpyrifos residues relative to grass, grass residue halflives were 1–2 days, and bobwhite quail brain AChE activity has been shown to recover within 8 days of acute exposures (Soler-Rodriguez et al., 1998; Cairns et al., 1991). Average weight losses for the control, 3.4-kg, and 6.7kg treatment birds were 12.01 g (6%), 13.14 g (7%), and 18.15 g (9%), respectively. Even though these losses indicate a trend in concentration response, the differences were not significant (Table 7, P ¼ 0:375). Bennett
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Table 7 Percent mortality (logistic regression analysis), body weight, AChE, organ weight/body weight ratios, eggs per pen, and eggs per bird day Parametera
Treatment
% Mortality
Control 3.4 kg ai/ha 6.7 kg ai/ha
18 18 18
Body weight
Control 3.4 kg ai/ha 6.7 kg ai/ha
144 143 144
AChE (fresh nonfrozen brain tissue)
Control 3.4 kg ai/ha 6.7 kg ai/ha
92 89 90
AChE (frozen brain tissue)
Control 3.4 kg ai/ha 6.7 kg ai/ha
AChE(brain tissue from birds dying in pens)
SE
P value
1.91 2.54 3.11
0.6992
3.1 3.2 3.1
0.375
5.516 5.311 5.186
0.113 0.129 0.115
0.150
40 39 34
4.045 4.007 3.993
0.120 0.153 0.133
0.949
Control 3.4 kg ai/ha 6.7 kg ai/ha
9 11 12
6.130 5.780 5.660
0.320 0.290 0.170
0.532
Brain/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
144 144 142
5.378 5.424 5.491
0.073 0.073 0.074
0.566
Liver/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
142 142 136
25.824 26.197 25.407
1.021 1.025 1.045
0.856
Crop/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
136 139 131
4.890 5.382 4.961
0.473 0.466 0.483
0.729
Gonad/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
141 142 134
9.015 10.330 8.942
0.751 0.747 1.021
0.395
Eggs/pen
Control 3.4 kg ai/ha 6.7 kg ai/ha
9 9 9
15.79 20.448 16.893
2.481 2.820 2.560
0.463
Eggs/bird day
control 3.4 kg ai/ha 6.7 kg ai/ha
9 9 9
0.0104 0.0117 0.0109
0.541
a
n
Mean 3.77 4.69 6.13 189.7 188.3 183.5
0.0663 0.0837 0.0714
None of the pairwise differences was significant.
(1989) demonstrated that 2-week old bobwhite quail could discriminate between treated and untreated feed and also that there was no relationship between mortality and the amount of active ingredient ingested per bird day. However, foraging data from our study suggest that adult birds did not show an aversion to treated feed. In any case, quail mortality may have little to do with the amount of ingested active ingredient. Price et al. (1971) also have shown that bobwhite quail do not avoid chlorpyrifos-treated areas. Altered foraging behavior and anorexia have been suggested as sublethal affects associated with anticholinesterase compounds (Grue et al., 1997), but these sublethal
effects did not appear in our study. Moreover, ducks fed 80 ppm chlorpyrifos did not have the weight losses that were expected (Meyers and Gile, 1986). Domestic hens orally dosed with chlorpyrifos at 10 mg/kg body weight lost 25% of their body weight compared with the controls but recovered to 87% of the controls by day 28 after treatment (Richardson et al., 1993). All of the organ weight/body weight ratios were unaffected by treatment (see Table 7 for P values). Egg laying was also unaffected by the Pyrinex 4E treatments, whether it was calculated as mean eggs per pen (Table 7, P ¼ 0:463) or as the mean eggs per bird day (Table 7, P ¼ 0:541).
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4. Conclusions Under the conditions of these experiments, Pyrinex 4E sprayed on turf at 3.4 kg ai/ha (two treatments 2 weeks apart) and 6.7 kg ai/ha, and on seed at B12 and 30 ppm chlorpyrifos, respectively, did not significantly affect bobwhite quail mortality, brain AChE levels, body weight, brain/body weight ratio, liver/body weight ratio, crop/body weight ratio, gonad/body weight ratio, eggs per pen, or eggs per bird day. However, a significantly larger number of birds (five females, one male) in the 6.7-kg ai/ha treatment showed behavioral deficits. Three of the five females were clearly different birds, but the other two females could have been the same bird, because both were observed in the same pen on days 23 and 24 after treatment. All birds with behavioral deficits did not show significantly lower brain AChE activity relative to control birds. The two consecutive 3.4-kg ai/ha treatments did not show appreciable accumulation of residues in grass and did not show significantly more abnormal behaviors compared to the control. Regardless of application rates, chlorpyrifos half-lives were B2 and 10 days for grass and seeds, respectively. In light of these data, application of chlorpyrifos to turf at 3.4 and 6.7 kg ai/ha is not expected to have chronic deleterious effects on populations of bobwhite quail grazing on treated grass or seeds, provided there is an abundant supply of seeds for the quail to eat.
References Bennett, R.S., 1989. Role of dietary choices in the ability of bobwhite to discriminate between insecticide-treated and untreated food. Environ. Toxicol. and Chem. 8, 731–738. Booth, G.M., Woolsey, G., Alder, D., Galli, R., Carter, M., 1980a. A Simulated Field Study on the Effect of Dyfonate 10G and Dyfonate 15G on Bobwhite Quail. A final report submitted to Stauffer Chemical Company, Farmington, CT. Booth, G.M., Johnson, S.L., Human, D., Hilton, H.G., Larsen, J.R., 1980b. The Effect of Diflubenzuron on the Reproduction of Bobwhite Quail. Final report submitted to Thompson-Hayward Chemical Company, Kansas City, KS. Booth, G.M., Carter, M.W., Whitmore, R.C., Jorgensen, C. 1984. The Effect of Endrin and Chlorpyrifos to Waterfowl Associated with Small Grains in Montana. Final report submitted to the US Environmental Protection Agency, Corvallis, OR. Cairns, M.A., Maguire, C.C., Williams, B.A., Bennett, J.K., 1991. Brain cholinesterase activity of bobwhite acutely exposed to chlorpyrifos. Environ. Toxicol. and Chem. 10, 657–664. Clements, R.O., Murray, P.J., Tyas, C.J., 1992. The short-term effects on wild goose behaviour of chlorpyrifos application to permanent pasture. Ann. Appl. Biol. 120 (1), 17–23. Decarie, R., Desgranges, J.L., Lepine, C., Morneau, F., 1993. Impact of insecticides on the American robin (Turdus migratorius) in a suburban environment. Environ. Pollut. 80 (3), 231–238. Ellman, G.L., Courtney, K.D., Andres Jr, V., Featherstone, B.C., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Grue, C.E., Gibert, P.L., Seeley, M.E., 1997. Neurophysiological and behavioral changes in non-target wildlife exposed to organophosphate and carbamate pesticides: themoregulation, food consumption, and reproduction. Am. Zool. 37 (4), 369–388. Hudson, R.H., Tucker, R.K., Haegele, M.A., 1984. Handbook of Toxicity of Pesticides to Wildlife, 2nd Edition. Resource Publication 153, US Department of the Interior, Fish and Wildlife Service. Hurlbert, S.H., 1977. Toxicity of chlorpyrifos to mallard ducks. Bull. Environ. Contam. Toxicol. 17 (1), 105–107. Johnson, B.L., Calabrese, E.J., Callahan, B.G., Pastorok, R.A., Chapman, P.M., 2001. Chlorpyrifos: ecotoxicological risk assessment for birds and mammals in corn agroecosystems. Hum. Ecol. Risk Assess. 7 (3), 473–637. Kenaga, E.E., 1974. Evaluation of the safety of chlorpyrifos to birds in areas treated for insect control. In: Gunter, F.A. (Ed.), Residue Reviews, Vol. 50. Springer, New York. Kuhr, R.J., Tashiro, H., 1978. Distribution and persistence of chlorpyrifos and diazinon applied to turf. Bull. Environ. Contam. Toxicol. 20, 652–1656. Maguire, C.C., Williams, B.A., 1987. Response of thermally stressed bobwhite to organophosphorus exposure. Environ. Pollut. 47, 25–39. Makhteshim-Agan Inc., 1988. Pyrinex 4E Insecticide Composite Label. Makhteshim-Agan Inc., New York. Martin, P.A., 1990. Effects of carbofuran, chlorpyrifos, and deltamethrin on hatchability, deformity, chick size and incubation time of Japanese quail (Coturnix japonica) eggs. Environ. Toxicol. Chem. 9 (4), 529–534. Martin, P.A., Forsyth, D.J., 1998. Effects of exposure to vegetation sprayed with dimethoate or chlorpyrifos on mallard ducklings (Anas platyrhynchos). Ecotoxicology 7 (2), 81–87. Martin, P.A., Johnson, D.L., Forsyth, D.J., 1996. Effects of grasshopper-control insecticides on survival and brain acetylcholinesterase of pheasant (Phasianus colchicus) chicks. Environ. Toxicol. Chem. 15 (4), 518–524. Meyers, S.M., Gile, J.D., 1986. Mallard reproductive testing in a pond environment: a preliminary study. Arch. Environ. Contam. Toxicol. 15 (6), 757–761. Odenkirchen, E.W., 1988. Chlorpyrifos hazards to fish, wildlife, and invertebrates: a synoptic review. Gov. Reports Announce. Index, Issue 14. Price, M.A., Radeleff, R., Kunz, S.E., Everett, R.E., 1971. Toxicity of soil applications of Dursban to bobwhite quail. Texas Agricultural Experiment Station Program 3000. July 3. Rexrode, M., 1984. Chlorpyrifos Registration Standard. US Environmental Protection Agency, Washington, DC. Richardson, R.J., Moore, T.B., Kayyali, U.S., Randall, J.C., 1993. Chlorpyrifos assessment of potential for delayed neurotoxicity by repeated dosing in adult hens with monitoring of brain acetylcholinesterase, brain and lymphocyte neurotoxic esterase. Fund. Appl. Toxicol. 21 (1), 85–96. Schom, C.B., Abbott, U.K., Walker, N., 1973. Organophosphorus pesticide effects on domestic and game bird species: Dursban. Poultry Sci. 52 (5), 2083. Smith, G.J., 1987. Pesticide Use and Toxicology in Relation to Wildlife: Organophosphorus and Carbamate Compounds. Resource Publication 170, US Department of the Interior, Fish and Wildlife Service. Soler-Rodriguez, F., Santiyan-Miguez, M.-P., Sanchez-Reja, A., Cordero-Roncero, V., Cambero-Garcia, J-P., 1998. Recovery of brain acetycholinesterase and plasma cholinesterase activities in quail (Coturnix coturnix) after chlorpyrifos administration and effect of pralidoxime treatment. Environ. Toxicol. Chem. 17 (9), 1835–1839.
ARTICLE IN PRESS G.M. Booth et al. / Ecotoxicology and Environmental Safety 60 (2005) 176–187 Steel, R.G.D., Torrie, J.H., Dickey, D.A., 1997. Principles and Procedures of Statistics: A Biometrical Approach, 3rd Edition. McGraw-Hill, New York, pp. 568–569. Stone, W.B., 1979. Poisoning of wild birds by organophosphate and carbamate pesticides. N Y Fish Game J. 26 (1), 37–47. US EPA, 1981. Office of Pesticide Programs. Hazard Evaluation Evaluation Division. Pesticide Incident Monitoring System—
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Summary Reports 1978–81. US Environmental Protection Agency, Washington, DC. US FDA, 1986. Pesticide Analytical Manual, Vol. I. US Department of Health and Human Services, Food and Drug Administration, p. 212.3b. Wiseman, D.S., 1977. Food habits and weights of the bobwhite from northeastern Oklahoma tall grass prairie. Proc. Oklahoma Acad. Sci. 57, 110–117.
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Hazard evaluation for northern bobwhite quail (Colinus virginianus) exposed to chlorpyrifos-treated turf and seed Gary M. Booth,a,* Spencer R. Mortensen,b Melvin W. Carter,a and Bruce G. Schaaljea a
Departments of Statistics and Integrative Biology, Brigham Young University, 697 WIDB, Provo, UT 84602, USA b Syngenta, 410 Swing Road, Greensboro, NC 27409, USA Received 10 July 2003; received in revised form 16 December 2003; accepted 29 January 2004
Abstract This study evaluated the toxicity effects of chlorpyrifos on bobwhite quail (Colinus virginianus) kept in 27 field-exposed large pens arranged over turf in a randomized block design with nine blocks of three pens (16 adult birds per pen). Nine pens were treated with one application of 3.4 kg active ingredient (ai) per hectare followed by a second 3.4-kg ai/ha application 2 weeks later, nine pens with one 6.7-kg ai/ha application, and nine pens with formulation blank. In addition, the seed fed to the birds in the two chemically treated pens was also treated with chlorpyrifos. Mean residue in the grass samples from the first 3.4-kg treatment pens ranged from 306795 ppm on day 0 to 1878 ppm on day 14 after treatment. The second 3.4-kg ai/ha treatment grass residues ranged from 3617167 ppm on day 0 to 38724 ppm on day 14 after treatment. Grass residues from the 6.7-kg treatment pens ranged from 9037310 ppm on day 0 to 978 ppm on day 30 after treatment. Half-lives were B2 days and 10 days for grass and seeds, respectively. Whereas the incidence of behavioral deficits was significantly (P ¼ 0:0156) higher in the 6.7-kg pens (five females, one male), two of the females could have been the same bird because they were both seen in the same pen on days 23 and 24 after treatment. There was no significant difference in mortality, brain acetylcholinesterase activity, or any other measured parameter among any of the treatments. We conclude that application of chlorpyrifos to turf at 3.4 and 6.7 kg ai/ha is not expected to have chronic deleterious effects on populations of bobwhite quail grazing on treated grass or seeds, provided there is an abundant supply of seeds for the quail to eat. r 2004 Elsevier Inc. All rights reserved. Keywords: Chlorpyrifos; Pen study; Bobwhite quail; Pyrinex 4E; Turf
1. Introduction Pyrinex 4E, which contains the active ingredient (ai) chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is used to control a variety of pests on turf as well as many crops (Makhteshim-Agan Inc., 1988). Dosages of Pyrinex 4E per 93 m2 of turf range from 22 mL (11.1 kg ai/ha) to 89 mL (4.5 kg ai/ha). The 4E formulation contains 48.9% chlorpyrifos, equivalent to 1.2 kg ai/L. Since the anticipated maximal seasonal use rate of Pyrinex 4E is 6.7 kg ai/ha, this rate represents the greatest risk to turf-grazing birds and thus was used as the maximum application rate in this study. Toxicity of chlorpyrifos to birds is moderate to high. Considering both the acute (LD50=13.3–32 mg/kg) and dietary toxicity (LC50=283–1100 mg/kg) to bobwhite *Corresponding author. Fax: +1-801-378-7423. E-mail address: gary [email protected] (G.M. Booth). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.01.011
quail, it appears that this bird is one of the more sensitive avian species that has been extensively evaluated (Rexrode, 1984; Smith, 1987; Hudson et al., 1984; Johnson et al., 2001). Because of its sensitivity to chlorpyrifos and also because it grazes on grass (Wiseman, 1977), the bobwhite quail was chosen as an indicator species for the turf study. Field effects of chlorpyrifos on birds are not at all decisive. For example, there have been at least two reports from the Ecological Effects Branch of the US EPA (1981) of dead geese found on golf courses that had been treated with chlorpyrifos. Additionally, Hurlbert (1977) reported significant mortality of young mallard ducks following treatment with chlorpyrifos. However, Kenaga (1974) sprayed field-confined birds with 4– 36 kg ai/ha with no apparent ill effects, whereas Booth et al. (1984) found no effects on waterfowl exposed to chlorpyrifos sprayed on winter wheat. Hence, conclusions about wildlife mortality in the field resulting from
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chlorpyrifos spraying are equivocal. For this reason, additional wildlife studies on this compound have been strongly recommended (Odenkirchen, 1988; Smith, 1987). Of particular interest to the current study was the potential impact of chlorpyrifos on upland game birds exposed to a contaminated food source resulting from direct spraying of two food substrates. Dietary exposure of insecticides under field-exposed large-pen conditions using a large number of birds and a large number of replicates represents an opportunity to study the lethal and sublethal effects of agrochemicals on selected sensitive birds under controlled field conditions of maximum exposure (Booth et al., 1980a, b). Accordingly, the objectives of the present study were to (1) monitor mortality effects of chlorpyrifos associated with dietary and environmental exposure to bobwhite quail under field-exposed large-pen enclosures over turf, (2) assess the potential of chlorpyrifos to induce behavioral deficits in quail foraging on treated turf; (3) investigate the residue profile of chlorpyrifos in treated grass and, if possible, relate these residues to bird mortality, behavioral deficits, and brain acetylcholinesterase (AChE) levels, and (4) determine whether chlorpyrifos induces body weight loss, reduced organ weight, brain AChE depression, and decreased egg production in surviving birds.
177
included behavior, mortality, brain AChE determinations, environmental residues, body and organ weight determinations, and egg laying. All of the appropriate data were analyzed statistically. 2.4. Pens Hardware cloth-covered pens were constructed from modular panels to cover a minimum of 0.005 ha (50 m2 per pen). The pen dimensions were 3.1 15.2 m, with the top cover at a height of B1.8 m. A 0.91- by 1.8-m piece of 1.9-cm plywood was placed on the windward (south) side of each pen to reduce violent wind exposure. Rooftop shading was provided in one end of each pen using a nylon mesh shading material. A total of 28 pens was assembled from 3.1-m sections, with 9 pens for the formulation blank controls, 9 pens for the Pyrinex 4E treatment at 6.7 kg ai/ha, and 9 pens for the Pyrinex 4E treatment at 3.4 kg ai/ha+3.4 kg ai/ha, with each of the two 3.4-kg treatments separated by 14 days. The 28th pen was used as a holding pen for extra birds. The 27 treatment pens were placed in a randomized block design with nine blocks (Fig. 1). Randomization of the treatments and assignment of birds to each pen was done according to a random number generator. 2.5. Birds
2. Materials and methods 2.1. Chemicals Pyrinex 4E was supplied by Makhteshim Agan Inc. (Israel) via the Pennwalt Company (Philadelphia, PA), which shipped 11 L of the chemical (batch no. B23-11) directly to Brigham Young University (BYU; Provo, UT). This batch contained 48.9% of the ai chlorpyrifos. The vehicle control consisted of Pyrinex minus chlorpyrifos, which was also supplied by Makhteshim Agan Inc.
Bobwhite quail (Colinus virginianus) adults (16-weekold flight-adapted) were obtained from a pen-reared stock from Oakridge Game Farm (Gravette, AK). All birds were flight-conditioned and reared on Gro-scratch feed beginning May 10, 1988 until they were shipped by truck to Provo arriving on May 26, 1988. Each pen was stocked with 16 adult bobwhite quail (8 males+8 females) 15 days prior to the treatment application. Each bird was weighed, banded (with a numbered leg band), and randomly assigned to one of the 27 pens. 2.6. Bird feed
2.2. Test site The study was conducted in Goshen, UT, at a turf farm located B50 km west of Springville, UT. 2.3. Time table The investigation was divided into three phases: (1) An acclimation period of 15 days for the birds began when they arrived on May 26, 1988. (2) A chemical application phase of 30 days began on June 11, 1988 for the 6.7-kg ai/ha treatment and the first 3.4-kg ai/ha treatment. The second 3.4-kg ai/ha treatment was applied on June 25, 1988. The field portion of the study was terminated on July 11, 1988, when all surviving birds were sacrificed. (3) An analysis phase (60 days)
All feed used in this experiment was Gro-scratch (9% protein containing wheat, milo, sorghum, and cracked corn), a commercial feed obtained from Intermountain Farmers Association (Provo, UT). This feed had been used successfully in a previous large-pen study (Booth et al., 1980a, b). 2.7. General test conditions and procedures The pens were placed on turf with 15 in one row (end to end) and 12 in another (end to end). The rows were assembled in an east–west direction. The pens were separated east and west (end-wise) by B1.5 m, and north and south by B0.9 m. The pens were arranged so that five blocks were located on the north and four
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EXPERIMENTAL DESIGN 9 Blocks x 3 TRT/Block = 27 Pens 27 Pens x 16 Birds/Pen = 432 Birds
Pen Numbers Control
3.4 kg
6.7 kg
1 5 8 11 13 16 21 22 26
2 6 9 12 15 17 19 24 25
3 4 7 10 14 18 20 23 27
Fig. 1. Experimental design, showing arrangement of pens and holding pen. See text for details.
blocks on the south (Fig. 1). During the 15-day acclimation phase, the birds were fed daily with nontreated Gro-scratch scattered onto the turf of each pen to stimulate field-foraging behavior of quail on the treated grass. Using this procedure, B40 g/bird/day were fed to the birds based upon the average adult bobwhite feed consumption rates compiled from our laboratory (Booth et al., 1980a, b). This feeding rate was approximately twice what the birds ate in the laboratory because of (1) the bird’s inability to find all the feed in the pens and (2) greater bird activity in the pens compared to laboratory conditions, thus necessitating more feed. During the acclimation phase, feeding amounts were carefully monitored to assure that the quantity of feed available was not limiting. Water contained in 4-L poultry jars was supplied ad libitum into each pen. Water jars were emptied and refilled each day. Amoxicillin antibiotic was supplied in the drinking water at the rate of 250–500 mg/L during 10 days of the acclimation phase. This antibiotic treatment helped reduce stress and potential enteritis problems associated with the long journey to Utah (Crawley, personal communication). The turf was watered on a weekly basis or according to normal practices followed by the turf farm. In addition, the grass inside and outside the pens was mowed about once per week after treatment began. Mowing on the inside of the pens was accomplished using a mechanical reel-type push-mower so that the grass clippings remained inside the pen. 2.8. Calibration of the sprayers Calibration of each sprayer was accomplished by spraying as much as possible of 2000 mL of water from
an Erlenmeyer flask over the 50 m2 of test turf. On the average, B80 mL were left in the sprayer for priming and B120 mL were left in the flask that could not be drained. Thus, to adjust for this loss of 200 mL, instead of spraying 64 g Pyrinex 4E/1900 mL over 50 m2 (equivalent to 6.7 kg ai/ha), 71 g Pyrinex 4E/2,100 mL was sprayed over each of the nine plots for the 6.7-kg treatment; instead of spraying 32 g/1900 mL over each 50 m2 for the 3.4-kg ai/ha treatment, 36 g Pyrinex 4E/ 2100 mL was sprayed over each of these nine plots. Finally, instead of spraying 64 g formulation blank/ 1900 mL over each 50 m2 for the controls, 71 g formulation blank/2100 mL was sprayed over each of these nine plots. After each pen had been sprayed, the volume left behind in the flask was recorded to determine the nominal amount of chlorpyrifos that was actually sprayed on the plots (Table 1). 2.9. Chemical application to the grass Following the acclimation phase of the experiment, each set of 16 birds from each pen was caught, reweighed, and placed by pen number into separate bird boxes until treatment of the turf and seed was completed. All birds spent B3 h in the holding boxes. Any birds appearing unhealthy, overly stressed, or that had lost X15% of body weight were replaced (total of 28 birds) by one of the birds selected at random from the holding pen. On June 11, 1988, nine pens received 6.7 kg ai/ha sprayed by a hand-held sprayer at a rate of 1 L of water/ 23 m2, nine pens received 3.4 kg ai/ha using a handsprayer at the rate of 1 L of water/23 m2 (on June 25, 1988, these nine pens also received another 3.4 kg ai/ha),
ARTICLE IN PRESS G.M. Booth et al. / Ecotoxicology and Environmental Safety 60 (2005) 176–187 Table 1 Chlorpyrifos residues contained in Gro-scratch seed used in feeding bobwhite quaila Days after treatment
0 1 2 3 4 5 6 10 13 15 20 29 Mean residues
Chlorpyrifos (ppm) First 3.4 kg ai/ ha treatment
Second 3.4 kg ai/ha treatment
6.7 kg ai/ha treatment
12 11 11 18 12 12 9 6 10 No sample No sample No sample 11.2273.19
21 20 17 14 17 17 15 15 No sample 18 No sample No sample 17.1172.32
30 27 24 25 25 23 27 15 No sample No sample 12 11 2176.72
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Aliquots of 45 kg of seed were treated in an industrial mixer for each treatment until there were 180 kg of treated feed for each of the three treatments. Each of the treatment samples was air-dried, sampled for residues, returned to the original containers, and stored in the dark at 25 C until the next morning, when the field experiment began. At the end of 14 days after treatment, the seed used in the 3.4-kg treatment pens was re-treated at the same rate as the first time. Thus, the birds on the 3.4-kg treatment fed during the next 16 days (4 weeks total) were getting the residue from the previous treatment plus the residue from the second treatment. This paradigm simulates what would be expected in a natural field situation, in which native forage material may be double-treated under this treatment regime. In fact, frequent mowing probably created a greater hazard by allowing more of the seed to be exposed to treatment than would occur under ‘natural field conditions’.
a
Seed blanks were spiked with 480 ppm chlorpyrifos standard and found to give 490% recovery.
and nine pens received formulation blank equivalent in weight to the 6.7-kg treatment. Following drying, the grass in each pen was sampled for residues according to a randomized sampling regime, and 16 birds were randomly placed into each of the 27 pens. 2.10. Chemical application to the feed All of the feed scattered throughout the pens was treated either with formulation blank (control pens) or Pyrinex 4E (3.4-kg and 6.7-kg treatment pens). However, treatment of the feed with the proper dose posed a problem because the actual concentration of chlorpyrifos on seeds under these field conditions was unknown and difficult to estimate because of such variables as the uneven distribution of scattered seeds and variation in the density of the grass. Therefore, a seed calibration experiment was designed to estimate empirically the actual concentration of chlorpyrifos on seeds during treatment. A 0.7 0.7 m section of turf (0.5 m2) taken from the Goshen turf farm was used as the experimental unit. A total of 6.5 g of Gro-scratch (equivalent to 640 g/50 m2) was scattered over the turf surface similar to the feeding procedure used in the pens. Then, 647 mg of Pyrinex 4E (equivalent to 6.7 kg ai/ha) was placed in 19.2 mL water (equivalent to 1 L of water/23 m2) and sprayed on the turf. After a few minutes of drying, the turf section was tipped upside down over a white pan, the seeds collected first by dislodgement, and then the remaining seeds picked out using a pair of tweezers and a magnifying glass, placed in a vial, frozen, and sent to the laboratory for analysis. This seed calibration experiment was replicated three times.
2.11. Residue sampling The grass was sampled from each of the 3.4-kg and 6.7-kg treatment pens and from three control pens according to a randomized sampling schedule. For the 6.7-kg treatment, samples were taken at 0 (immediately following treatment), 1, 2, 3, 4, 5, 6, 10, 20, and 30 days after treatment. For the first and second 3.4-kg treatments, samples were taken at 0 (immediately following treatment), 1, 2, 3, 4, 5, 6, 10, and 14 days after treatment, and 0 (immediately following the second treatment), 1, 2, 3, 4, 5, 6, 10, 14, and 16 days after treatment, respectively. Three control pens were randomly selected and sampled only at time 0 (immediately following treatment). 2.12. Residue analysis All residue analyses were completed at the Agricultural Experiment Station–Chemistry Station and Analytical Laboratory (McCall Hall, Montana State University, Bozeman, MT). Residues were analyzed by following a standard method (US FDA, 1986) using gas chromatography coupled with an electron capture detector. The separation column was packed with Supelco Port packing material (100/120 mesh) coated with 1.5% SP-2250 and 1.95% SP-2401. The method detection limit was 0.01 ppm for the parent compound, and the carrier gas flow was 30 mL/min. 2.13. Weather measurements Temperature, rainfall, and wind speed were recorded during the application phase of the study at Goshen, UT, and at the Widtsoe Building at BYU where the treated seed was being exposed to the environment.
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2.14. Biological observations and measurements During the application phase, behavior, mortality, and egg laying were monitored on a daily basis for each pen. Behavioral deficits were noted, and these included isolated birds that were apparently inactive (not feeding), birds isolated with eyes shut, live but immobile birds, birds with drooping wings and/or tail fanned, salivation, staggering gait, and birds failing to respond to auditory stimuli. All surviving birds were caught on the final day of the study (July 11) and placed, according to each numbered pen, into separate numbered boxes. The birds were sacrificed by each statistical block using CO2 asphyxiation. After each bird had been weighed and the sex and bird number recorded, the brain, liver, crop, and gonads were removed and weighed. All of the brain tissue was analyzed for AChE activity using published methods (Ellman et al., 1961). 2.15. Statistical methods The variables of eggs per pen and eggs per bird day were measured for each pen, and were analyzed using weighted complete block ANOVA, with weights equal to the inverses of the respective variables. Mortality was calculated for groups of birds of each sex in each pen, and was analyzed using logistic regression with overdispersion adjustment. AChE, final body weight, brain weight, liver weight, crop weight, and gonad weight were measured for each bird, and were analyzed using split-plot ANOVA for whole plots arranged in complete blocks. Chlorpyrifos treatments composed the wholeplot factor, and sex was the sub-plot factor. For all variables except AChE, the initial weight of the bird was used as a covariate. For all of these analyses, leastsquares means for chlorpyrifos treatments, sexes, and their interaction were obtained and compared using unadjusted Student’s t-tests for contrasts. The number of behavioral deficits was compared among chlorpyrifos treatments using the sign test (Steel et al., 1997). The observations were paired by day of observation. Statistical calculations were done using the standard SAS, MINITAB, and RUMMAGE packages.
3. Results and discussion 3.1. Residue data As a quality control measure, residue samples were taken from the Pyrinex 4E formulation used in the 3.4kg and 6.7-kg treatment pens and the formulation blank used in the control pens. These analyses showed that Pyrinex 4E contained B49% chlorpyrifos (B100% of the expected amount), whereas the formulation blank
contained a trace amount (0.000026%) of chlorpyrifos. In addition, on each of the spray days, we determined how much of the spray solution actually went onto the plots by keeping track of the spray volumes left over. The 3.4-kg ai/ha spray solution used on the 3.4-kg treatment pens on day 0 (June 11, 1988) ranged in nominal concentration from 3.3 to 3.5 kg ai/ha. Nominal concentrations associated with the 6.7-kg ai/ha spray solution (day 0) were 6.4–6.9 kg ai/ha. Following the second treatment of the 3.4-kg application (June 25), nominal concentrations ranged from 3.3 to 3.4 kg ai/ha. The average chlorpyrifos concentration found in the treated Gro-scratch seeds in the calibration experiments from the three turf sections was 25.4374.60 ppm. To make sure there was an adequate amount of chlorpyrifos on the feed, the seed for the 6.7-kg treatment was treated with 33 ppm and the 3.4-kg treatment seed was treated with 16.5 ppm. This intentional increase in the treatment of the seeds from 25 to 33 ppm (about a 32% increase) was done to account for possible losses and errors (note the standard deviation) during the mixing and spraying process to insure about 20 to 30 ppm in the 6.7-kg treatment and B10 to 17 ppm in the 3.4-kg treatments. Sampling of the residues from the treated feed through time clearly shows that this was achieved. The seeds treated with the first 3.4-kg ai/ha application averaged 11.2273.19 ppm chlorpyrifos, seeds treated for the second application of 3.4 kg ai/ha averaged 17.1172.32 ppm chlorpyrifos, and seeds treated with 6.7 kg ai/ha averaged 21.9076.72 ppm chlorpyrifos (Table 1). Spiked recoveries of Gro-scratch seed blanks were 4 90%, and residues on the control seed samples were undetectable. Small amounts (0.04–3.0 ppm) of chlorpyrifos residues were detected from the grass of the three control pens (Table 2), probably as a result of drift; since each treatment had its own sprayer, cross-contamination between sprayers was not an issue. No chlorpyrifos was detected on the untreated seed used in the control pens. Fig. 2 shows the 3.4-kg ai/ha (first application) pen-topen variability in grass residues through 14 days after treatment, and Figs. 3 and 4 represent the pen-to-pen variability in grass residues from the second application of 3.4 kg/ha and a single application of 6.7 kg/ha, respectively. For the first application of the 3.4-kg ai/ ha treatment, the residues averaged 306795 ppm on day 0 (June 11, 1988) and 1878 ppm on day 14 (June 25, 1988) after treatment (Fig. 5). Mean residues from grass samples analyzed from the second 3.4-kg ai/ha application ranged from 3617167 ppm on day 0 to 38724 ppm on day 14 (Fig. 6). Pen-to-pen variability over time (coefficient of variation, CV ¼ 4778%) in grass residues sampled from the second application of 3.4 kg ai/ha (Fig. 4) were quite similar to those (CV ¼ 41717%) from the first application (Fig. 2) and did not show any indication of a cumulative effect from consecutive
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Table 2 Summary of chlorpyrifos analysis from control (untreated) seed and selected grass samples Sample type
Laboratory no.
Sample description
Chlorpyrifos concentration (ppm)
Grass Grass Grass Seed Seed
2155-1 2155-2 2155-3 2182-1 2182-21
Grass control: pen 11 Grass control: pen 13 Grass control: pen 22 control seed control seed
3.0 0.04 3.0 N.D.a N.D.a
a
N.D., not detectable.
Fig. 3. Pen-to-pen variability in grass chlorpyrifos residues for the second 3.4-kg ai/ha application through 16 days after treatment. Chlorpyrifos residues (ppm) in grass samples treated with 6.7 kg ai/ha
ppm Chlorpyrifos
1400 1200 1000 800 600 400 200 0 0
1
2
3
4
5
6
10
20
30
Days Post-treatment pen 3
pen 4
pen 10
pen 14
pen 7
Chlorpyrifos resudes (ppm) in grass samples treated with 6.7 kg ai/ha
Fig. 2. Pen-to-pen variability in grass chlorpyrifos residues for the first 3.4-kg ai/ha application through 14 days after the application.
ppm Chlorpyrifos
1200 1000 800 600 400 200 0
0
1
2
3
4
5
6
10 20 30
Days Post-treatment
applications. The 6.7-kg treatment also showed surprisingly similar pen-to-pen variability (Fig. 7; CV ¼ 44720%) over time compared to the first and second application of the 3.4-kg treatment. Mean grass residues from the 6.7-kg treatment ranged from
pen 18
pen 20
pen 23
pen 27
Fig. 4. Pen-to-pen variability in grass chlorpyrifos residues for the single application of 6.7 kg ai/ha through 30 days after treatment.
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Fig. 5. Chlorpyrifos residues (ppm) in grass samples for the first 3.4kg ai/ha application through 14 days after the application.
Fig. 6. Chlorpyrifos residues (ppm) in grass samples for the second 3.4-kg ai/ha application through 14 days after the application.
9037310 ppm on day 0 to 978 ppm on day 30 after treatment (Fig. 7). The half-lives for chlorpyrifos residues on grass for all three applications were always within 1–2 days, consistent with data from Kenaga (1974) but shorter than those reported by Kuhr and Tashiro (1978), who demonstrated a half-life of B1 week on sprayed turf. 3.2. Quail behavior, mortality, brain AChE, body weights, organ weights, and egg laying During the 30-day observation period, none of the control birds showed any behavioral anomalies (Table 3). A total of six birds with behavioral deficits (six females and one male) were observed in the 6.7-kg treatment (4% of the 6.7-kg treatment birds), and only one male showed abnormal behavior in the 3.4-kg
Fig. 7. Chlorpyrifos residues (ppm) in grass samples for the single application of 6.7 kg ai/ha through 30 days after treatment.
treatment (o1% of the 3.4-kg treatment birds). It was clear that three of the five females with behavioral deficits in the 6.7-kg treatment were different birds, because each was observed in a different pen, but it could not be determined whether the one female with behavioral deficits seen on day 23 after treatment was the same female seen on day 24 after treatment, since both observations occurred in pen 4. Analyses of these data show that the number of behavioral deficits in the 6.7-kg treatment was significantly higher than the control (P ¼ 0:0156). It is of interest that three of the six behavioral abnormalities were observed on days 22–24 after treatment, when the grass residues were fairly slight (33722 ppm, day 20 after treatment; Fig. 7). No other treatment comparisons were significant, although the differences between the two Pyrinex 4E treatments approached statistical significance (control vs. 3 lb, P ¼ 0:5; 3.4-kg treatment vs. 6.7-kg treatment, P ¼ 0:0625). Tables 4–6 summarize bird mortality for each treatment by days after treatment, bird number, sex, pen number, and brain AChE activity. Nine control birds died (Table 4; 6.25%), 11 birds receiving the 3.4-kg treatment (Table 5; 7.64%), and 14 birds receiving the 6.7-kg treatment (Table 6; 9.72%). A logistic regression analysis with overdispersion adjustment of the mortality data showed no significant differences among treatments (Table 7; P ¼ 0:6992). Although significantly more females died overall (11.57%) compared with males (4.17%) (P ¼ 0:023), the sex-by-treatment interactions were not significant. Even though some studies have shown significantly increased upland game mortalities associated with chlorpyrifos spraying on turf (Stone, 1979), other data in the literature suggest that mortality is related only to higher doses than those used in our study, and may be related to the foraging behavior of the bird. For example, Schom et al. (1973) showed no effect on adult bobwhite quail mortality when Dursban
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was fed in the diet at 100 ppm, but when the diet concentrations were raised to 200, 500, and 600 ppm they reported significant mortalities of 11%, 17%, and 39%, respectively. It is clear from our study that grass residues can be much higher than this (as high as 1400 ppm in one
replication, Fig. 6) with no significant mortality, provided that seeds are abundant. The rationale for lack of mortality in our data is that foraging quail prefer to feed on seeds, which has far less chlorpyrifos residues than does grass, when a choice between seeds and grass is available; this rationale is supported by the feeding ecology of bobwhite quail (Wiseman, 1977). Hence, from a practical standpoint, constant dietary concentrations 4100 ppm under normal turf treatment conditions and normal foraging behavior are not likely. In addition, grass residue levels drop off rapidly during the first week after treatment regardless of the treatment rate. Martin (1990) showed that, although immersing Japanese quail eggs in field concentrations of chlorpyrifos resulted in some abnormalities (foot malformations and scoliosis), the author concluded: ‘‘Overall, effects were small and the three insecticides did not represent a significant hazard to quail embryos at conditions up to two times field levels of application.’’ When pheasants (Phasianus colchicus) were exposed to chlorpyrifos-treated vegetation in pens for 48 h (Martin et al., 1996), there was no difference in mortality among treatment and control groups. Clements et al. (1992) have also shown that two species of grazing wild geese were not negatively affected (neither mortality nor behavior) when chlorpyrifos was sprayed at the recommended rate of 0.72 kg ai/ha. Furthermore, repeated applications of chlorpyrifos to ornamental trees containing robin nests (Turdus migratorius) showed no significant adult or juvenile mortality despite depressed plasma cholinesterase levels (Decarie et al., 1993). Sensitivity of aquatic upland game birds to fieldapplied chlorpyrifos also seems to be related to higher doses, age of the birds, and cold temperatures. For example, whereas 8 ppm in mallard duck diets in an open-pond design did not influence reproduction or mortality, 80 ppm (Meyers and Gile, 1986) reduced hatchability and apparently allowed no ducklings to survive on the treatment ponds to 7 days. However, Martin and Forsyth (1998) have shown no effects on
Table 3 Summary of behavioral deficits observed in quail during the 30 days after treatment Days after treatment a
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14b 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Total
Number and sex of birds with behavioral deficits Control
3.4 kg ai/ha
6.7 kg ai/ha
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1 male 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 male
1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 1 1 0 0 0 0 0 0 5
183
female
male
female
female female female
females, 1 male
Table 4 Control bird mortality by days after treatment, bird number, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
13 16 19 26 26 26 27 28 30 Summary
339 253 252 282 132 257 353 255 316 9 birds total (6.25% mortality)
Female Female Female Female Female Female Female Male Female 8 females, 1 male
11 1 1 22 22 22 5 22 5 4 different pens
5.871 6.806 6.306 4.851 6.576 6.265 7.748 5.139 5.574 6.1370.89 (mean)
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Table 5 Bird mortality from the 3.4-kg ai/ha treatment by days after treatment, bird number, sex, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
11 (1st appl.) 4 (2nd appl.) 12 (2nd appl.) 12 (2nd appl.) 13 (2nd appl.) 14 (2nd appl.) 14 (2nd appl.) 14 (2nd appl.) 15 (2nd appl.) 15 (2nd appl.) 16 (2nd appl.) Summary
439 299 525 5 194 352 40 453 279 334 62 11 birds total(7.64% mortality)
Female Male Female Female Female Male Female Female Male Male Female 8 females, 3 males
2 6 25 25 25 6 12 25 12 25 12 5 different pens
4.949 5.494 4.646 5.340 7.673 6.739 5.220 5.088 6.374 6.579 5.462 5.7870.93 (mean)
Table 6 Bird mortality for the 6.7-kg ai/ha treatment by days after treatment, bird number, sex, pen number, and brain AChE activity Days after treatment
Bird no.
Sex
Pen no.
AChE (mmol/min/g)
6 13 13 17 18 18 20 22 23 23 26 27 29 30 Summary
455 217 220 338 157 375 155 54 222 183 374 443 370 206 14 total birds (9.72% mortality)
Female Male Female Male Female Female Male Female Female Male Female Female Male Female 9 females, 5 males
3 14 23 23 14 14 23 23 10 23 10 18 23 23 5 different pens
5.314 4.534 6.465 7.227 Not measured 5.466 6.232 Not measured 5.428 5.232 5.144 7.544 4.384 4.978 5.6671.00 (mean)
behavior of ducklings feeding and swimming in ponds treated with 420 g ai/ha. In addition, thermally stressed bobwhite quail seemed to be more sensitive to organophosphorus compounds. In fact, synergistic effects have been demonstrated in juvenile bobwhites simultaneously exposed to chlorpyrifos and cold stress (Maguire and Williams, 1987). Analysis of the brain AChE of birds that had died in the pens in our study showed no significant treatment differences (Table 7; P ¼ 0:532). However, bird no. 370 from the 6.7-kg treatment pen died while being videotaped and showed many symptoms of AChE depression. This bird also had the lowest AChE level among the birds that died on the plots (Table 6; 4.384 mmol/min/g), which represented an inhibition of B28%. All of the surviving birds were sacrificed by CO2 asphyxiation, and a summary of the analysis of their brain AChE activity, body weight, brain/body weight ratio, liver/body weight ratio, crop/body weight ratio, gonad/body weight ratio, eggs per pen, and eggs per day
is shown in Table 7. A total of 271 quail brains were analyzed as nonfrozen (fresh) tissue for AChE, and none of the treatments differed significantly from each other (Table 7, P ¼ 0:150). The remaining 113 birds were sacrificed by block, the brain dissected, frozen, and then analyzed for AChE the next day. Results for these frozen-brain enzyme data also were not statistically significant (Table 7, P ¼ 0:949). The lack of AChE inhibition in this study is not surprising given that the quail fed mainly on seeds that had greatly reduced chlorpyrifos residues relative to grass, grass residue halflives were 1–2 days, and bobwhite quail brain AChE activity has been shown to recover within 8 days of acute exposures (Soler-Rodriguez et al., 1998; Cairns et al., 1991). Average weight losses for the control, 3.4-kg, and 6.7kg treatment birds were 12.01 g (6%), 13.14 g (7%), and 18.15 g (9%), respectively. Even though these losses indicate a trend in concentration response, the differences were not significant (Table 7, P ¼ 0:375). Bennett
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Table 7 Percent mortality (logistic regression analysis), body weight, AChE, organ weight/body weight ratios, eggs per pen, and eggs per bird day Parametera
Treatment
% Mortality
Control 3.4 kg ai/ha 6.7 kg ai/ha
18 18 18
Body weight
Control 3.4 kg ai/ha 6.7 kg ai/ha
144 143 144
AChE (fresh nonfrozen brain tissue)
Control 3.4 kg ai/ha 6.7 kg ai/ha
92 89 90
AChE (frozen brain tissue)
Control 3.4 kg ai/ha 6.7 kg ai/ha
AChE(brain tissue from birds dying in pens)
SE
P value
1.91 2.54 3.11
0.6992
3.1 3.2 3.1
0.375
5.516 5.311 5.186
0.113 0.129 0.115
0.150
40 39 34
4.045 4.007 3.993
0.120 0.153 0.133
0.949
Control 3.4 kg ai/ha 6.7 kg ai/ha
9 11 12
6.130 5.780 5.660
0.320 0.290 0.170
0.532
Brain/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
144 144 142
5.378 5.424 5.491
0.073 0.073 0.074
0.566
Liver/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
142 142 136
25.824 26.197 25.407
1.021 1.025 1.045
0.856
Crop/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
136 139 131
4.890 5.382 4.961
0.473 0.466 0.483
0.729
Gonad/body weight ratio
Control 3.4 kg ai/ha 6.7 kg ai/ha
141 142 134
9.015 10.330 8.942
0.751 0.747 1.021
0.395
Eggs/pen
Control 3.4 kg ai/ha 6.7 kg ai/ha
9 9 9
15.79 20.448 16.893
2.481 2.820 2.560
0.463
Eggs/bird day
control 3.4 kg ai/ha 6.7 kg ai/ha
9 9 9
0.0104 0.0117 0.0109
0.541
a
n
Mean 3.77 4.69 6.13 189.7 188.3 183.5
0.0663 0.0837 0.0714
None of the pairwise differences was significant.
(1989) demonstrated that 2-week old bobwhite quail could discriminate between treated and untreated feed and also that there was no relationship between mortality and the amount of active ingredient ingested per bird day. However, foraging data from our study suggest that adult birds did not show an aversion to treated feed. In any case, quail mortality may have little to do with the amount of ingested active ingredient. Price et al. (1971) also have shown that bobwhite quail do not avoid chlorpyrifos-treated areas. Altered foraging behavior and anorexia have been suggested as sublethal affects associated with anticholinesterase compounds (Grue et al., 1997), but these sublethal
effects did not appear in our study. Moreover, ducks fed 80 ppm chlorpyrifos did not have the weight losses that were expected (Meyers and Gile, 1986). Domestic hens orally dosed with chlorpyrifos at 10 mg/kg body weight lost 25% of their body weight compared with the controls but recovered to 87% of the controls by day 28 after treatment (Richardson et al., 1993). All of the organ weight/body weight ratios were unaffected by treatment (see Table 7 for P values). Egg laying was also unaffected by the Pyrinex 4E treatments, whether it was calculated as mean eggs per pen (Table 7, P ¼ 0:463) or as the mean eggs per bird day (Table 7, P ¼ 0:541).
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4. Conclusions Under the conditions of these experiments, Pyrinex 4E sprayed on turf at 3.4 kg ai/ha (two treatments 2 weeks apart) and 6.7 kg ai/ha, and on seed at B12 and 30 ppm chlorpyrifos, respectively, did not significantly affect bobwhite quail mortality, brain AChE levels, body weight, brain/body weight ratio, liver/body weight ratio, crop/body weight ratio, gonad/body weight ratio, eggs per pen, or eggs per bird day. However, a significantly larger number of birds (five females, one male) in the 6.7-kg ai/ha treatment showed behavioral deficits. Three of the five females were clearly different birds, but the other two females could have been the same bird, because both were observed in the same pen on days 23 and 24 after treatment. All birds with behavioral deficits did not show significantly lower brain AChE activity relative to control birds. The two consecutive 3.4-kg ai/ha treatments did not show appreciable accumulation of residues in grass and did not show significantly more abnormal behaviors compared to the control. Regardless of application rates, chlorpyrifos half-lives were B2 and 10 days for grass and seeds, respectively. In light of these data, application of chlorpyrifos to turf at 3.4 and 6.7 kg ai/ha is not expected to have chronic deleterious effects on populations of bobwhite quail grazing on treated grass or seeds, provided there is an abundant supply of seeds for the quail to eat.
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