Arch. Environ. Contam. Toxicol. 48, 118–126 (2004) DOI: 10.1007/s00244-004-0229-8 Effects of Azinphos-Methyl on Cholinergic Responses and General Health in Zebra Finches (Taeniopygia guttata) After Previous Treatment with p,p¢-DDE H. Gill,1 T. D. Williams,2 C. A. Bishop,2,3 K. M. Cheng,1 J. E. Elliott1,3 1 AgroecologyGroup, Facultyof Agricultural Sciences, Universityof British Columbia, 2357 Main Mall, Vancouver, British Columbia V6T 1Z4, Canada 2 Centre for Wildlife Ecology, Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada 3 Canadian Wildlife Service, Pacific Wildlife Research Center, Environment Canada, Delta, British Columbia V4K 3N2, Canada Received: 2 November 2003 /Accepted: 15 July2004 Abstract. Although organochlorine (OC) pesticides were Like all OP insecticides, azinphos-methyl acts by inhibiting replaced with organophosphates (OPs) in the early1970s, they the cholinesterase (ChE) enzymes (Chambers and Levi 1992). continue to persist in orchard environments today. Extensive Those enzymes are involved in the breakdown of the neuro- research has been conducted to determine the effects of cur- transmitter acetylcholine to acetate and choline, thus providing rentlyused OPs on cholinesterase (ChE) activity;however, a keyrole in the removal of excess acetylcholine.ChE en- although OCs continue to be prevalent in areas of previous use, zymes are critical to the normal function of the nervous system few studies have looked at the toxicityof a combination of in both vertebrates and invertebrates, and therefore the po- residual OC compounds with currentlyused OP pesticides. tential exists for nontarget effects whenever OPs are released The focus of our studywas to determine the effects of azin- into the environment (Grue et al. 1991). Measurement of these phos-methyl (a common OP used in apple orchards today) on enzymes is commonly used in field situations to determine the ChE activityand general health in zebra finches ( Taeniopygia cause of mortalityor extent of exposure to OPs (Zinkl et al. guttata) previouslyexposed to p,p¢-DDE (a commonlyde- 1980; Busby et al. 1981). Several field-monitoring studies tected metabolite of DDT). The main results of our studywere have measured ChE inhibition to examine the response of as follows: (1) azinphos-methyl alone caused a dose-dependent songbirds residing in orchards after azinphos-methyl treatment inhibition of plasma and brain ChE activity; (2) p,p¢-DDE in (Graham and DesGranges 1993; Burgess et al. 1999; Gill et al. combination with azinphos-methyl did not change azinphos- 2000). methyl inhibition of ChE activity; and (3) there were sugges- Although no longer applied, organochlorine (OC) pesti- tions of immunostimulation in birds dosed 1 year previously to cides, particularlyDDT [1,1,1-trichloro-2,2- bis(p-chlorophe- p,p¢-DDE and of anemia when p,p¢-DDE was combined with nyl)ethane]–related compounds, persist at high concentrations azinphos-methyl; however, there was no dose–response for in areas of past intensive use, such as fruit orchards (Harris these parameters in birds subsequentlydosed with p,p¢-DDE. et al. 2000). Songbirds residing in Ontario and British Columbia fruit orchards have been reported to contain high bodyburdens of DDE [2,2- bis(4-chlorophenyl)-1,1-dichloro- ethylene, a persistent metabolite of DDT]. Eastern bluebird (Sialia sialis) eggs from Ontario orchards had p,p¢-DDE resi- Azinphos-methyl (O,O-dimethyl S-[(4-oxo-1,2,3-benzo-tria- dues as high as 105 lg/g (Bishop et al. 2000) and DDE con- zin-3(4H)-yl)methyl] phosphorodithioate) is a broad-spectrum centrations in American robin (Turdus migratorius) eggs in organophosphate (OP) insecticide used extensivelythroughout Okanagan orchards (British Columbia) were as high as 302 lg/ the world to control insect pests (Chemagro Division Research g (Gill et al. 2003). Persistence of DDT and its metabolites in Staff 1974). In Canada, azinphos-methyl is registered for orchard soils has been suggested to be caused byhigh previous control of insects on agricultural and vegetable crops, pri- application rates and minimal tillage or other soil disturbance, marilyapples, peaches, potatoes, and cherries. In apple orch- which limit loss byvolatilization and erosion (Harris et al. ards, azinphos-methyl is commonly used to control infestation 2000). of the insect pest, coddling moth (Cydia pomonella), and is Because of the persistence of DDE in the environment, sprayed one to three times per season (Province of British songbirds residing in orchards commonlyhave high DDE Columbia 1991). (particularly p,p¢-DDE) bodyburdens when OP sprayingoc- curs. However, few avian studies have examined the joint toxicityof OC and OP pesticides. In mammals, previous dosing or exposure to OC compounds appears to decrease the effects of subsequent OP exposure byincreasing activityof the Correspondence to: J. E. Elliott; email: [email protected] ChE enzymes (Ball et al. 1954; Triolo and Coon 1966; Menzer Effects of Azinphos-Methyl on Zebra Finches 119 1970). However, it appears this protection is not seen in avian Table 1. Summaryof pesticide historyand treatment in male zebra species. For example, Ludke (1977) found that DDE exposure finches used to determine cholinesterase activities in birds exposed to in coturnix quail (Coturnix coturnix japonica) resulted in a a combination of p,p¢-DDE and the OP azinphos-methyl dose-related increase in plasma ChE activity. After treatment This Experiment: This Experiment: with the OP parathion, plasma ChE activitydecreased in Previous Exposure p,p¢-DDE Dosage Azinphos-methyl control and DDE-pretreated birds to approximatelythe same to p,p¢-DDEa n (mg/kg) Exposure levels. Because DDE-pretreated birds had relativelygreater No 10 No No plasma ChE activitybefore parathion dosage, dosing with No 10 No Yes parathion resulted in a relativelygreater ChE inhibition in Yes 10 No Yes DDE pretreated birds than untreated individuals. Thus, in Yes 10 Yes quail, previous feeding of DDE did not decrease the effect of Yes 10 Yes subsequent parathion exposure (see also Dieter 1974). Yes 10 Yes In addition to enzyme effects, the general health of animals a Previous exposure to p,p¢-DDE occurred at 34 mg/kg approximately exposed to environmental stressors such as pesticides maybe 1 year before the start of the experiment. negativelyimpacted. One of the first field studies to examine OP = organophosphate. immune parameters in wild birds exposed to pesticides found a general trend of immunostimulation in tree swallows (Bishop et al. 1998). Basal hematologic parameters, such as percent section), we subsequentlydosed additional groups of birds at 14.6, hematocrit and leucocrit, are easilyobtainable and are useful 25.5, and 45.3 mg/kg per the same methodology. Three zebra finches for detecting the effects of environmental, infectious, parasitic, were dosed per treatment, except for the highest dose (45.3 mg/kg) in or toxicologic stresses on animals. Theyhave been widely which 5 birds were dosed. To determine the effect that duration of used in studies of health (Ots et al. 1998), reproduction fasting had on brain ChE activity, we dosed 9 birds (3 birds/treatment) (Morton 1994; Merila and Svensson 1995), and adaptation at the following dosages after overnight fast: 1.5, 4.8, and 8.5 mg/kg. To determine anybehavioral effects, an additional 12 birds (3 birds/ (Carpenter 1975). treatment) were fasted overnight and dosed at 1.5, 4.8, 8.5, and 14.6 The objectives of this studywere (1) to determine the dose– mg/kg. Those birds were observed for 1 week after dosing and response relationship between azinphos-methyl and brain and checked dailyfor mortalityor abnormal behavior (such as tremors, plasma ChE activityin the zebra finch ( Taeniopygia guttata) convulsions, or lethargy). and (2) to determine the effects of azinphos-methyl on ChE activityand hematologic and immune-function parameters in The effect of azinphos-methyl exposure on plasma ChE activity birds pre-exposed to p,p¢-DDE at low, medium, and high was also determined. For this study, an additional 55 zebra finches (5 birds/treatment) were dosed with azinphos-methyl (or only plain corn doses. Our studywas part of a larger laboratoryinvestigation oil for controls) after 1 hour of fasting, and plasma samples were of the sublethal effects of orchard pesticides on songbird collected for ChE activitymeasurement. Birds received the following reproduction. dosages of azinphos-methyl: 0.27, 0.48, 0.85, 1.5, 2.7, 4.8, 8.5, 14.6, 25.5, and 45.3 mg/kg. One treatment group was also exposed to plain corn oil containing no azinphos-methyl. Three hours after dosing, birds were anesthetized, and blood samples were collected (200 ll) Methods from the jugular vein followed byexsanguination. Animals and Husbandry Experiment 2. Effects of p,p¢-DDE and azinphos-methyl on ChE activityand general health. Sixtyadult male zebra finches were di- This studywas conducted on a captive colonyof zebra finches vided into cages with 5 birds cage (total of 12 cages). Because of maintained at the Simon Fraser UniversityAnimal Care Facilitylo- limitations in bird numbers, 40 of the birds used were from a previous cated in Burnaby, British Columbia. Zebra finches were housed in a experiment in which theywere exposed to p,p¢-DDE (34 mg/kg) controlled environment (temperature 19°Cto23°C; humidity35 % to approximately 1 year previously. There was a total of 6 treatment 55%; photoperiod 14 hours light to 10 hours dark; lights on at 0700). groups with 10 birds/treatment (Table 1). Birds exposed to a low, All birds were provided with mixed seed (panicum and white millet medium, and high dose of p,p¢-DDE were treated with 19, 34, and 60 1:2; 11.7% protein, 0.6% lipid, and 84.3% carbohydrate by dry mass), mg/kg p,p¢-DDE (log-scale dilution), respectively, daily for 4 weeks, water, grit, and cuttlefish bone (calcium) ad libitum plus a multivi- and the remaining birds were exposed to plain corn oil dailyfor 4 tamin supplement in the drinking water once per week.