ENVIRONMENTAL RESEARCH53, 62-75 (1990)
Effects of the Organophosphorus Pesticide Fenitrothion on Behavior and Reproduction in Zebra Finches STEPHEN B . HOLMES *'1 AND PETER T. B O A G t
*Forestry Canada, Forest Pest Management Institute, Sault Ste. Marie, Ontario, Canada P6A 5M7; and ?Queen's University, Department of Biology, Kingston, Ontario, Canada K7L 3N6 Received April 12, 1990 Activity was significantly altered in zebra finch pairs orally dosed with fenitrothion insecticide at rates of 1.04 and 3.80 mg/kg. Both the level and the diurnal pattern of activity were affected. Recovery of normal activity required 1-2 days, depending on the dosage administered. Fenitrothion had only a slight effect on reproduction when parents were dosed at the egg-laying stage of the breeding cycle. The implications of these results for the interpretation of field monitoring studies are discussed. © 1990AcademicPress,Inc.
INTRODUCTION
Fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) is a broad spectrum organophosphorus (OP) insecticide that is widely used to control forest insect pests. On the basis of the results of laboratory toxicity tests, fenitrothion has been classified as moderately to extremely toxic to birds (Smith, 1987). Hudson et al. (1984) reported acute oral LDso'S for this compound ranging from 23.6 mg/kg for bobwhites to 1662 mg/kg for mallards. Variable results have been obtained from field monitoring studies of the effects of fenitrothion on forest passerines. Pearce et al. (1976, 1979) reported a substantial reduction in singing activity in operational spruce budworm spray blocks treated with fenitrothion at 175-280 g/ha and interpreted this result as indicating adult mortality. Others have found much less severe, or negligible, impacts at similar dosage rates (e.g., Buckner, 1974; Buckner et al., 1976; Kingsbury and McLeod, 1981; Spray et al., 1987). Recent fenitrothion monitoring studies have concentrated on the measurement of brain cholinesterase (ChE) inhibition in samples of birds collected from spray blocks (Peterson, 1976; Hamilton et al., 1981; Busby et al., 1981, 1983a, 1987a; Anonymous, 1988, 1989). This technique has also been used to monitor forest spraying with aminocarb (Busby et al., 1982, 1983b; Peterson, 1976), mexacarbate (Busby et al., 1987b), acephate (Julin and Gramlich, 1978; Zinkl et al., 1979, 1980), carbaryl (Zinkl et al., 1977, 1979; Gramlich, 1979), and trichlorfon (Peterson, 1976; Zinkl et al., 1977, 1979). Although the ChE technique has proven to be a useful method for measuring exposure of birds to ChE inhibitors, its general suitability for assessing pesticide impacts has been questioned. Because the relationships between sublethal brain ChE inhibition and effects on bird behavior and physiology are poorly known, the results of ChE monitoring studies may be difficult to interpret (Grue and Shipley, 1981; Grue et al., 1983). Also, a bias in the To whom correspondence should be addressed. 62 0013-9351/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
F E N I T R O T H I O N E F F E C T S ON ZEBRA F I N C H E S
63
method used to obtain birds for ChE analysis may affect estimates of pesticide impacts (Busby et al., 1981; Peakall and Bart, 1983; Mineau and Peakall, 1987). Zebra finches have been used in laboratory studies to model the effects of pesticides on wild songbirds (Evans and Rosenbaum, 1984; Evans and Batty, 1986; Holmes and Boag, 1990; Mineau et al., 1990). The zebra finch is a good test species because it is prolific, easy to breed, and habituates quickly to new surroundings (Mineau et al., 1990). It is also about the same size as the small forest songbirds that are exposed to fenitrothion when forests are sprayed (Mineau et al., 1990). Fenitrothion has been showen to kill zebra finches at dosages as low as 3.80 mg/kg when administered orally, with 60% mortality occurring in 12 hr at 13.22 mg/kg (Holmes and Boag, 1990). In the present study, behavioral and reproductive effects were measured in zebra finches exposed to fenitrothion in the laboratory. Our objectives were (1) to obtain information which would aid in the interpretation of results of field studies using the ChE monitoring technique, particularly those involving fenitrothion, and (2) to test an automated system for collecting behavioral data on caged birds to determine its usefulness in evaluating neurobehavioral effects resulting from exposure to pesticides. MATERIALS AND METHODS
Experimental Animals Zebra finches (Poephila guttata Gould) were purchased from a commercial supplier (Flikkema Aviaries, Fenwick, Ontario). Birds were less than 1 year old and had no breeding experience. Pairs were maintained in individual breeder cages (62.5 x 27.5 x 35 cm; Rolf C. Hagen, Inc., Scarborough, Ontario) on a 14L: 10D light cycle and provided with finch seed mix (Essex Topcrop Sales, Ltd., Essex, Ontario) and water (containing L. K. Avitron liquid vitamin supplement; Rolf C. Hagen) ad libitum. Once each week, birds were given boiled egg (fortified with L. K. Theralin vitamin mineral powder; Rolf C. Hagen) and either shredded lettuce or alfalfa sprouts. A 3-month period of acclimation allowed time for the birds to recover from any previous exposure to ChE-inhibiting agents, such as pest strips containing dichlorvos, which are sometimes used to control flies in commercial aviaries. Dosing Procedure Technical grade fenitrothion (Sumithion, Sumitomo Chemical Co., Ltd., Osaka, Japan; assayed at 95.6% sample purity) was used in all experiments. The insecticide was dissolved in soybean oil and administered to individual birds via a catheter inserted down the esophagus to the crop. Birds were fasted for 10-12 hr prior to treatment. Forty-eight pairs of breeding birds were randomly assigned to three dosage groups (control, 1.04 mg/kg fenitrothion, 3.80 mg/kg fenitrothion) of 16 pairs each. Pairs were matched among groups on the basis of their breeding success (the number of eggs, young, and fledglings produced by the pair) during the 3-month acclimation period. The individual members of each pair were separated and they were not reunited until the day before they were dosed. Feni-
64
HOLMES A N D BOAG
trothion dosages were selected on the basis of the results of a previous experiment (Holmes and Boag 1990). The expected mean maximum brain ChE depression was 50% for the lower dosage rate and 70% for the higher dosage rate. Different dosages were obtained by adjusting the concentration of fenitrothion in prepared standards. Birds received either pure soybean oil (controls) or a fenitrothionsoybean oil mixture at a volume rate of 0.5 ixl/g of body weight. An automated micro-doser (Houston Atlas, Inc., Houston, TX) was used to deliver the small volumes of material. Birds were deprived of food and water for a further 10-15 rain after being dosed. Birds were dosed on either 18 or 19 September 1987 between 0830 and 0930 hr. Half of the birds in each treatment group (3 dosages x 8 replicates = 24 pairs) were dosed on the first day (Group A) and half on the second day (Group B). Group B served as an untreated control for Group A on the first treatment day.
Activity Profiles Effects on behavior were measured as changes in perch-hopping activity. The activity of each pair of birds was monitored continuously using a computer-based automated activity recorder. Two perches in each cage were mounted on microswitches, which were connected to an external terminal board linked via a serial cable to a custom I/O board installed in a Zenith Z-140 microcomputer (Zenith Data Systems Corp., St. Joseph, MI). Whenever, a bird hopped onto a perch, a circuit was closed. The status of each perch, open or closed circuit, was polled by the microcomputer once every second throughout the experiment. The number of perch closures for each cage was summed hourly and stored on disk.
Reproductive Success Nests were monitored daily, and numbers of eggs, nestlings and fledglings were counted. Nestlings were weighed on the day they hatched. Fledglings were weighed and the lengths of their wing chords were measured on the first day they left the nest.
Statistical Analyses Analysis of variance (ANOVA) and Bonferroni t tests (Milliken and Johnson, 1984) were used to identify differences among means. Levene's test for equal variances were used to test for heteroscedasticity in the data (Brown and Forsythe, 1974a). Data that failed Levene's test were transformed (Box and Cox, 1964) to equalize variances. In those cases where a suitable transformation could not be found, Welch tests were used (Welch, 1951; Brown and Forsythe, 1974b). Tests were conducted at a = 0.05 unless specified otherwise. RESULTS
Activity Profiles Prior to treatment, there were no significant differences in the activities of birds in the different day of treatment and dosage groups (repeated measures ANOVA, P > 0.05 for Tday and Dosage; Table 1). Activity was significantly depressed in
65
FENITROTHION EFFECTS ON ZEBRA FINCHES
TABLE 1 CHANGES IN ACTIVITY PATTERNS OF ZEBRA FINCHES REVEALED BY REPEATED MEASURES ANALYSIS OF VARIANCE APPLIED TO PERCH HOPPING ACTIVITY DATA COLLECTED BEFORE AND AFTER ORAL DOSING WITH FENITROTHION Day b Source a
Pre
0
1
2
Tday Dosage TD Hour HT HD HTD
0.12 ¢ 0.15 0.31
Effects of the Organophosphorus Pesticide Fenitrothion on Behavior and Reproduction in Zebra Finches STEPHEN B . HOLMES *'1 AND PETER T. B O A G t
*Forestry Canada, Forest Pest Management Institute, Sault Ste. Marie, Ontario, Canada P6A 5M7; and ?Queen's University, Department of Biology, Kingston, Ontario, Canada K7L 3N6 Received April 12, 1990 Activity was significantly altered in zebra finch pairs orally dosed with fenitrothion insecticide at rates of 1.04 and 3.80 mg/kg. Both the level and the diurnal pattern of activity were affected. Recovery of normal activity required 1-2 days, depending on the dosage administered. Fenitrothion had only a slight effect on reproduction when parents were dosed at the egg-laying stage of the breeding cycle. The implications of these results for the interpretation of field monitoring studies are discussed. © 1990AcademicPress,Inc.
INTRODUCTION
Fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) is a broad spectrum organophosphorus (OP) insecticide that is widely used to control forest insect pests. On the basis of the results of laboratory toxicity tests, fenitrothion has been classified as moderately to extremely toxic to birds (Smith, 1987). Hudson et al. (1984) reported acute oral LDso'S for this compound ranging from 23.6 mg/kg for bobwhites to 1662 mg/kg for mallards. Variable results have been obtained from field monitoring studies of the effects of fenitrothion on forest passerines. Pearce et al. (1976, 1979) reported a substantial reduction in singing activity in operational spruce budworm spray blocks treated with fenitrothion at 175-280 g/ha and interpreted this result as indicating adult mortality. Others have found much less severe, or negligible, impacts at similar dosage rates (e.g., Buckner, 1974; Buckner et al., 1976; Kingsbury and McLeod, 1981; Spray et al., 1987). Recent fenitrothion monitoring studies have concentrated on the measurement of brain cholinesterase (ChE) inhibition in samples of birds collected from spray blocks (Peterson, 1976; Hamilton et al., 1981; Busby et al., 1981, 1983a, 1987a; Anonymous, 1988, 1989). This technique has also been used to monitor forest spraying with aminocarb (Busby et al., 1982, 1983b; Peterson, 1976), mexacarbate (Busby et al., 1987b), acephate (Julin and Gramlich, 1978; Zinkl et al., 1979, 1980), carbaryl (Zinkl et al., 1977, 1979; Gramlich, 1979), and trichlorfon (Peterson, 1976; Zinkl et al., 1977, 1979). Although the ChE technique has proven to be a useful method for measuring exposure of birds to ChE inhibitors, its general suitability for assessing pesticide impacts has been questioned. Because the relationships between sublethal brain ChE inhibition and effects on bird behavior and physiology are poorly known, the results of ChE monitoring studies may be difficult to interpret (Grue and Shipley, 1981; Grue et al., 1983). Also, a bias in the To whom correspondence should be addressed. 62 0013-9351/90 $3.00 Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
F E N I T R O T H I O N E F F E C T S ON ZEBRA F I N C H E S
63
method used to obtain birds for ChE analysis may affect estimates of pesticide impacts (Busby et al., 1981; Peakall and Bart, 1983; Mineau and Peakall, 1987). Zebra finches have been used in laboratory studies to model the effects of pesticides on wild songbirds (Evans and Rosenbaum, 1984; Evans and Batty, 1986; Holmes and Boag, 1990; Mineau et al., 1990). The zebra finch is a good test species because it is prolific, easy to breed, and habituates quickly to new surroundings (Mineau et al., 1990). It is also about the same size as the small forest songbirds that are exposed to fenitrothion when forests are sprayed (Mineau et al., 1990). Fenitrothion has been showen to kill zebra finches at dosages as low as 3.80 mg/kg when administered orally, with 60% mortality occurring in 12 hr at 13.22 mg/kg (Holmes and Boag, 1990). In the present study, behavioral and reproductive effects were measured in zebra finches exposed to fenitrothion in the laboratory. Our objectives were (1) to obtain information which would aid in the interpretation of results of field studies using the ChE monitoring technique, particularly those involving fenitrothion, and (2) to test an automated system for collecting behavioral data on caged birds to determine its usefulness in evaluating neurobehavioral effects resulting from exposure to pesticides. MATERIALS AND METHODS
Experimental Animals Zebra finches (Poephila guttata Gould) were purchased from a commercial supplier (Flikkema Aviaries, Fenwick, Ontario). Birds were less than 1 year old and had no breeding experience. Pairs were maintained in individual breeder cages (62.5 x 27.5 x 35 cm; Rolf C. Hagen, Inc., Scarborough, Ontario) on a 14L: 10D light cycle and provided with finch seed mix (Essex Topcrop Sales, Ltd., Essex, Ontario) and water (containing L. K. Avitron liquid vitamin supplement; Rolf C. Hagen) ad libitum. Once each week, birds were given boiled egg (fortified with L. K. Theralin vitamin mineral powder; Rolf C. Hagen) and either shredded lettuce or alfalfa sprouts. A 3-month period of acclimation allowed time for the birds to recover from any previous exposure to ChE-inhibiting agents, such as pest strips containing dichlorvos, which are sometimes used to control flies in commercial aviaries. Dosing Procedure Technical grade fenitrothion (Sumithion, Sumitomo Chemical Co., Ltd., Osaka, Japan; assayed at 95.6% sample purity) was used in all experiments. The insecticide was dissolved in soybean oil and administered to individual birds via a catheter inserted down the esophagus to the crop. Birds were fasted for 10-12 hr prior to treatment. Forty-eight pairs of breeding birds were randomly assigned to three dosage groups (control, 1.04 mg/kg fenitrothion, 3.80 mg/kg fenitrothion) of 16 pairs each. Pairs were matched among groups on the basis of their breeding success (the number of eggs, young, and fledglings produced by the pair) during the 3-month acclimation period. The individual members of each pair were separated and they were not reunited until the day before they were dosed. Feni-
64
HOLMES A N D BOAG
trothion dosages were selected on the basis of the results of a previous experiment (Holmes and Boag 1990). The expected mean maximum brain ChE depression was 50% for the lower dosage rate and 70% for the higher dosage rate. Different dosages were obtained by adjusting the concentration of fenitrothion in prepared standards. Birds received either pure soybean oil (controls) or a fenitrothionsoybean oil mixture at a volume rate of 0.5 ixl/g of body weight. An automated micro-doser (Houston Atlas, Inc., Houston, TX) was used to deliver the small volumes of material. Birds were deprived of food and water for a further 10-15 rain after being dosed. Birds were dosed on either 18 or 19 September 1987 between 0830 and 0930 hr. Half of the birds in each treatment group (3 dosages x 8 replicates = 24 pairs) were dosed on the first day (Group A) and half on the second day (Group B). Group B served as an untreated control for Group A on the first treatment day.
Activity Profiles Effects on behavior were measured as changes in perch-hopping activity. The activity of each pair of birds was monitored continuously using a computer-based automated activity recorder. Two perches in each cage were mounted on microswitches, which were connected to an external terminal board linked via a serial cable to a custom I/O board installed in a Zenith Z-140 microcomputer (Zenith Data Systems Corp., St. Joseph, MI). Whenever, a bird hopped onto a perch, a circuit was closed. The status of each perch, open or closed circuit, was polled by the microcomputer once every second throughout the experiment. The number of perch closures for each cage was summed hourly and stored on disk.
Reproductive Success Nests were monitored daily, and numbers of eggs, nestlings and fledglings were counted. Nestlings were weighed on the day they hatched. Fledglings were weighed and the lengths of their wing chords were measured on the first day they left the nest.
Statistical Analyses Analysis of variance (ANOVA) and Bonferroni t tests (Milliken and Johnson, 1984) were used to identify differences among means. Levene's test for equal variances were used to test for heteroscedasticity in the data (Brown and Forsythe, 1974a). Data that failed Levene's test were transformed (Box and Cox, 1964) to equalize variances. In those cases where a suitable transformation could not be found, Welch tests were used (Welch, 1951; Brown and Forsythe, 1974b). Tests were conducted at a = 0.05 unless specified otherwise. RESULTS
Activity Profiles Prior to treatment, there were no significant differences in the activities of birds in the different day of treatment and dosage groups (repeated measures ANOVA, P > 0.05 for Tday and Dosage; Table 1). Activity was significantly depressed in
65
FENITROTHION EFFECTS ON ZEBRA FINCHES
TABLE 1 CHANGES IN ACTIVITY PATTERNS OF ZEBRA FINCHES REVEALED BY REPEATED MEASURES ANALYSIS OF VARIANCE APPLIED TO PERCH HOPPING ACTIVITY DATA COLLECTED BEFORE AND AFTER ORAL DOSING WITH FENITROTHION Day b Source a
Pre
0
1
2
Tday Dosage TD Hour HT HD HTD
0.12 ¢ 0.15 0.31
