UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects of p,p’-DDE and current-use pesticides on reproduction and health in zebra finches (taeniopygia… Gill, Harpreet 2003

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2003-0229.pdf [ 4.67MB ]
Metadata
JSON: 831-1.0090939.json
JSON-LD: 831-1.0090939-ld.json
RDF/XML (Pretty): 831-1.0090939-rdf.xml
RDF/JSON: 831-1.0090939-rdf.json
Turtle: 831-1.0090939-turtle.txt
N-Triples: 831-1.0090939-rdf-ntriples.txt
Original Record: 831-1.0090939-source.json
Full Text
831-1.0090939-fulltext.txt
Citation
831-1.0090939.ris

Full Text

THE EFFECTS OF p,p '-DDE AND CURRENT-USE PESTICIDES ON REPRODUCTION AND HEALTH IN ZEBRA FINCHES (TAENIOPYGIA GUTTATA) by HARPREET GILL B.Sc, Simon Fraser University, 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Department of Animal Science, Faculty of Agricultural Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 2003 ® Harpreet Gill, 2003 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of AUM f\L 6W&H(£ The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada 1 ii ABSTRACT Until the early 1970's, organochlorine (OC) insecticides were used primarily to control a wide range of pests in North America. Following several restrictions on the usage, OCs were replaced with more acutely toxic, but less persistent insecticides, primarily organophosphates and carbamates. However, many OCs, particularly DDT-related compounds, persist at high concentrations in areas of past intensive usage, such as fruit orchards. Organophosphates, such as azinphos-methyl, and fungicides, such as mancozeb, are commonly used insecticides in fruit orchards, applied alone or as tank mixtures. There are no data available on the toxicity of this common mixture or whether these currently used pesticides can have synergistic effects with DDE (a persistent metabolite of DDT). This study examined effects of DDE alone and in combination with current-use pesticides, using the Zebra finch (Taeniopygia guttata) as a laboratory model. The objectives were to determine 1) the dose-response relationship of azinphos-methyl on brain and plasma cholinesterase (ChE) activity in Zebra finches, 2) the effects of DDE, mancozeb and azinphos-methyl exposure on egg production and yolk precursor levels, and 3) the effects of DDE, mancozeb and azinphos-methyl exposure on passerine reproduction on the post-hatch period. In the first study, although, Zebra finches experienced ChE inhibition following exposure to azinphos-methyl, they appeared to be less sensitive compared to other songbird species. In addition, p,p '-DDE did not appear to affect the degree of ChE inhibition following subsequent azinphos-methyl exposure, however, it did appear to result in stimulation of the immune system. In the second study we found little evidence that dosing 6f breeding females with current-use pesticides, including azinphos-methyl and mancozeb, either alone or in combination with p,p'-DDE, had significant negative effects on reproductive traits (timing of laying, egg size and number, yolk precursor levels) or immune statues (percent hematocrit and leucocrit and H/L I l l ratio) in female Zebra finches. However, birds with previous exposure to p,p '-DDE (one year prior to the start of the experiment) may be negatively impacted in terms of yolk precursor levels and immune response. In the final study, we found no evidence that dosing of adult Zebra finches with current-use pesticides, including azinphos-methyl and mancozeb, either alone or in combination with p,p '-DDE, had significant negative effects on reproductive traits or immune status in female Zebra finches. Future studies to determine potential effects of in ovo exposure to pesticides on reproduction post-fledging as well as later reproductive success would be beneficial in explaining effects of pesticides on songbirds species. In addition, the possibility of long-term exposure ofp,p '-DDE on immune function should be further studied. These data are essential for developing strategies to sustain healthy songbird populations in orchards and will allow for excellent comparative data for a complimentary field study being conducted in Ontario, Canada. iv TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables v List of Figures vii Acknowledgements viii Dedication x CHAPTER 1 Introduction 1 CHAPTER 2 Effects of exposure to p,p '-DDE and azinphos-methyl on brain and plasma cholinesterase activity and general health in Zebra finches (Taeniopygia guttata) 6 2.1 Introduction 6 2.2 Methods 8 2.3 Results 15 2.4 Discussion 17 CHAPTER 3 Effects of p,p '-DDE and current-use pesticides on egg production and yolk precursor levels in the Zebra finch (Taeniopygia guttata) 31 3.1 Introduction 31 3.2 Methods 33 3.3 Results 40 3.4 Discussion 43 CHAPTER 4 Effects of chronic p,p '-DDE exposure in combination with current-use pesticides in breeding Zebra finches {Taeniopygia guttata) 63 4.1 Introduction 63 4.2 Methods : 65 4.3 Results 71 4.4 Discussion 73 CHAPTER 5 Conclusions 84 / Literature cited 88 V LIST OF TABLES 2.1 Summary of pesticide history and treatment in male Zebra finches used to determine cholinesterase activity 23 2.2 Concentrations of p,p '-DDE (mg/kg, wet weight) in contaminated egg food used to dose Zebra finches 24 2.3 Organochlorine concentrations (mg/kg, wet weight) in male Zebra finch livers exposed to p,p '-DDE at 19 mg/kg, 34 mg/kg and 60 mg/kg 25 2.4 Comparison of phytohemagglutinin skin test response in adult male Zebra finches treated with corn oil or p,p '-DDE at 19 mg/kg, 34 mg/kg and 60 mg/kg 26 2.5 Percent hematocrit and leucocrit in male Zebra finches exposed to corn oil or p,p '-DDE at 19 mg/kg, 34 mg/kg and 60 mg/kg and the organophosphate azinphos-methyl 27 3.1 Summary of pesticide history and treatment in male and female Zebra finches used to determine the effects of p,p '-DDE and current-use pesticides on egg production and yolk precursor levels 50 3.2 Organochlorine concentrations (mg/kg, wet weight) in female Zebra finch livers exposed to p,p '-DDE approximately one year prior to tissue collection 51 3.3 Comparison of reproductive output up to clutch completion in Zebra finch pairs exposed to a combination of p,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb 52 3.4 Comparison of reproductive out put up to clutch completion in Zebra finch pairs exposed to p,p '-DDE approximately one year prior to the start of the experiment 54 3.5 Comparison of reproductive output up to clutch completion in Zebra finch pairs exposed to the current-use pesticides azinphos-methyl and mancozeb 55 3.6 Hematological parameters of female Zebra finches exposed top,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb 56 3.7 Hematological parameters of female Zebra finches exposed to p,p '-DDE approximately one year prior to the start of the experiment 57 3.8 Hematological parameters of female Zebra finches exposed to the current-use pesticides azinphos-methyl and mancozeb 58 4.1 Summary of pesticide treatment in male and female Zebra finches used to determine the effects of p,p '-DDE and current-use pesticides on reproductive success and general health 78 vi 4.2 Comparison of reproductive output up to clutch completion in Zebra finch pairs exposed to p,p '-DDE 79 4.3 Comparison of reproductive output following clutch completion in Zebra finch pairs exposed to a combination of p,p '-DDE and current-use pesticides 80 4.4 Comparison of haematological parameters and phytohemagglutinin skin test response in adult female Zebra finches exposed to various combinations of p,p '-DDE and current-use pesticides , 82 4.5 Comparison of mass, haematological parameters and phytohemagglutinin skin test response in chicks of adult Zebra finches exposed to various combinations of p,p'-DDE and current-use pesticides 83 V l l LIST OF F I G U R E S 2.1 Plasma and brain cholinesterase activity in adult male Zebra finches orally dosed with the organophosphate azinphos-methyl 28 2.2 Plasma and brain cholinesterase activity in adult male Zebra finches exposed to corn oil orp,p '-DDE and the organophosphate azinphos-methyl 29 3.1 Vitellogenin yolk precursor levels in plasma of female Zebra finches exposed to p,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb 59 3.2 Very-low-density-lipoprotein yolk precursor levels in plasma of female Zebra finches exposed top,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb.. ..60 3.3 Vitellogenin and very-low-density-lipoprotein yolk precursor levels in plasma of female Zebra finches exposed to p,p '-DDE approximately one year prior to the start of the experiment 61 3.4 Vitellogenin and very-low-density-lipoprotein yolk precursor levels in plasma of female Zebra finches exposed to the current-use pesticides azinphos-methyl and mancozeb 62 ACKNOWLEDGEMENTS V l l l First and foremost, I would like to thank my supervisor Tony Williams. Not only did Tony provide me with insightful comments on the study design and the results of the thesis work, but he always had a open-door policy and was very supportive throughout the process. In addition, my supervisory committee of John Elliott, Christine Bishop and Kim Cheng were also a great source of encouragement and ideas. In particular, John trusted me to partake in this endeavor and provided encouragement and support throughout. I would like to thank the institutions and staff of the National Wildlife Research Centre (Hull, Quebec) and the Great Lakes Institute for Environmental Research (Windsor, Ontario) for conducting laboratory analyses that were an integral part of this thesis. The Toxic Substance Research Initiative, Health Canada and Canadian Wildlife Service, Environment Canada provided funding for this study. The Animal Care Facility (Simon Fraser University, Burnaby, British Columbia) provided housing for the Zebra finches. A big thanks goes to the Williams Lab, mostly for putting up with me and keeping me company Friday afternoons. The Williams Lab rocks! You have definitely made these last 2.5 years memorable for me. And of course, thanks to the Cooke Lab and the Farrell Lab (plus honorary member Michelle) for putting up with the Pink Lab. Next I would like to thank two people that helped me through the most stressful periods. Firstly, my sister Mini, who sat through the tough times with me (literally). The sacrifices you made were indeed noticed and valued. Secondly, Kat - you were my rock!!! What more can I say? The support you showed me my last 6 weeks (and throughout) will be everlasting in my memory. You definitely helped make my thesis defense a lot more memorable, fulfilling and, of course, such a success. So, to both Mini and Kat thanks so much for putting up with my "drama" but mostly, for understanding. In addition, I want to thank my family - mom, dad, Jas, Karen, ix and Jovan - for letting me be a pain when the stress level increased. I agree -1 am high maintenance. Lastly, the following people helped with laboratory work, in the writing and presenting of the study or just plain stress relief: G. Baker (my paparazzi), M. Benhamida, N. Bhandal, J. Christensen, J. Dale, K. Gorman (my "twin"), G. Kardosi, E. Kramer, S. Lee, P. Martin, G. Mayne, A. Pomeroy, Kan-Shan Sangha, the Segec's, M. Tolksdorf S. Weech and L. Wilson. DEDICATION TO ALL THOSE WHO BELIVED IN ME 1 CHAPTER 1 Introduction There are certain regions within Canada that contain a high density of apple orchards. In particular this occurs in the Carolinian forest in Ontario and the valley-bottom land in the Okanagan Valley, British Columbia. Approximately 12,500 ha of land in Ontario is occupied by apple orchards, while in British Columbia, apple orchards occupy approximately 7,400 ha of land. It has been suggested that these areas are one of the few in Canada that has the specific combination of soil type, climate and heat units suitable for the production of fruit (Kerr et al. 1985). These orchards are optimal habitat for bird species, especially during the breeding season. A study conducted to determine species composition of the bird community in Okanagan orchards found up to 54 species utilizing these orchards, of which 10 species were found nesting (Sinclair and Elliott 1993). Until the mid-1970s, Canadian orchards were treated heavily with organochlorines (OCs), in particular DDT [l,l,l-trichloro-2,2-*w(/?-chlorophenyl)ethane], in order to control insect pests. Restrictions on the usage of DDT were brought about due to concerns over adverse effects on wildlife and human health. Although discontinued 25 years ago, DDT, mostly in the form of its major metabolite DDE [2,2- &/'s(4-chlorophenyl)-l,l-dichloroethylene] is prevalent both in the soils and organisms inhabiting orchard environments. Elevated levels of DDT metabolites (particularly DDE) are still detected in American kestrel (Falco sparverius), Eastern bluebird (Sialia sialis) (Hebert et al. 1994) and American robin (Turdus migratorius) (Harris et al. 2000b) food chains (i.e. soil, earthworms, songbirds). Many characteristics of orchards have been suggested to encourage the persistence of DDT in the environment, including 1) extremely high past application rates in orchards compared with other crops (Harris and Sans 1971) which results in higher residual concentrations of DDT (Edwards 1966, Beyer and Gish 1980), and 2) 2 minimal tillage or other soil disturbance in this environment limiting the loss by volatilization and erosion (Bailey et al. 1974). Due to this increased persistence of DDT in orchard environments, Harris et al. (2000b) suggested that wildlife using these habitats may be more vulnerable to associated reproductive effects than wildlife in other locations. Some of the most well published effects of DDT and its principal toxic metabolite DDE are those on avian wildlife. Population decline in numerous species of predatory and fish-eating bird species were attributed to these chemicals (Hickey and Anderson 1968, Newton 1986, Wieymer et al. 1993) with exposure in some species being strongly correlated with eggshell thinning (Wiemeyer and Porter 1970, Cooke 1973, Elliott and Martin 1994) and reduced productivity (Ratcliffe 1980, Wieymer et al. 1988). Mass songbird mortalities were recorded immediately following spray events of DDT (Wallace 1959, Carson 1962, Wurster et al. 1965), however limited literature exists on the long-term effects of DDT (and its metabolite DDE) on productivity and health of those species. Songbird species have been suggested to be at reduced risk to the effects of persistent OCs on reproduction due to their feeding lower on the food chain, and to the lack of evidence showing the effects of DDE on shell quality of songbirds (Blus 1996). However, studies of captive Bengalese finches (Lorichura striata) have shown subtle effects of p,p -DDT and p,p '-DDE such as delayed ovulation resulting in decreases in reproductive success (Jefferies 1967). In addition, OC contamination has been associated with a decrease in reproductive success in Eastern bluebirds (Bishop et al. 2000a) and American robins (Johnson et al. 1976) nesting in contaminated fruit orchards. In general, however, there are few studies documenting reproductive effects of DDT-related compounds in songbird populations. Gill et al. (2003) and Elliott et al. (1994) both found that American robins nesting in Okanagan orchards were not negatively impacted in terms of reproductive success. In fact, birds had a higher nesting success when compared to those nesting in non-orchard habitat. It was suggested by Gill et al. (2003) that this may be due to an ecological advantage that orchard habitat may 3 have when compared to the non-orchard habitat chosen for that study (i.e. an increase in food availability or a decrease in the number of predators present). It was also suggested that birds choosing to utilize orchard habitats might possibly have some physiological advantage compared to those nesting in uncontaminated sites. These confounding variables make it difficult to interpret and conclude the effects of OC compounds on songbirds breeding in orchard habitats. Following severe restrictions on usage, most OC pesticides have been replaced with more acutely toxic, but less persistent insecticides, primarily organophosphates (OPs) and carbamates. Azinphos-methyl (Guthion™), an OP, is used primarily to control Coddling moths (Cydia pomonella), a primary pest on fruit in orchards in Canada. Azinphos-methyl is one of the top 20 reportable pesticides sold in British Columbia. In 1995, 21,804 kg of azinphos-methyl was sold in British Columbia, 20,698 kg (95 %) of which was used in the Southern Interior (principally the Okanagan Valley) (Norecol Dames & Moore 1997). Azinphos-methyl is considered extremely toxic (Class I pesticide; Hill et al. 1975) with an LD50 value of 8.5 mg/kg in Red-winged blackbirds (Agelaiusphoeniceus) (Smith 1987). Cholinesterase (ChE) enzyme inhibition is common following exposure to OP insecticides including azinphos-methyl (Chambers and Levi 1992). Cholinesterase enzymes are critical to the normal function of the nervous system in both vertebrates and invertebrates as they are involved in the breakdown of the neurotransmitter acetylcholine to acetate and choline, which is key in the removal of excess acetylcholine. Due to inhibition of this critical enzyme following OP exposure, the potential exists for non-target effects whenever OPs are released into the environment (Grue et al. 1991). Mortality in birds following exposure to azinphos-methyl has been recorded iri Washington (Stinson and Bromley 1991) and in the Okanagan valley (Gill et al. 2000) when it was applied to apple orchards. Organophosphate pesticides continue to be used in combination with fungicides on fruit crops. Previous orchard field studies have revealed that the ethylene bisdithiocarbamate (EBDC) fungicide, mancozeb, is used presently in apple orchards, applied alone or as tank mixtures with 4 azinphos-methyl (Sinclair and Elliott 1993, Norecol Dames & Moore 1997). Mancozeb is primarily used to control scabs, cedar apple rust and quince rust on apple crops (Province of British Columbia 1991). In 1995, 41,907 kg of mancozeb was sold in British Columbia, 15,796 kg (38 %) of which was used in the Southern Interior (Norecol Dames & Moore 1997). Less information is available on the toxicity of mancozeb to birds. The LD50 value in Mallards (Anas platyrhynchos) is >6400 mg/kg (Harris et al. 2000a). It is'doubtful that wild birds would be exposed to these levels of the fungicide, therefore it alone is unlikely to cause mortality in wild populations. However, ethylene thiourea (ETU), a principal breakdown product of EDBCs (de Snoo 1986), has suspected carcinogenic and teratogenic properties (Harris et al. 2000a) and has been seen to produce high chronic toxicity in lab animals (Houeto 1995). Due to the persistence of DDE (particularly p,p -DDE) in the environment, songbirds residing in orchards commonly have high p,p '-DDE body burdens when spraying of current-use pesticides occurs (Harris et al. 2000b, Gill et al. 2003). Few avian studies have examined the joint toxicity of OCs with these current-use pesticides, but for those studies that take into account the effects of multiple pesticide exposure on songbirds, reproductive success and general health of the bird (i.e. immune function) appears to be negatively impacted with exposure (Bishop et al. 1998, Bishop et al. 2000a, Johnson et al. 1976). Both Eastern bluebirds and Tree swallows (Tachycineta bicolor) nesting in Ontario orchards experienced a decline in chick survival with increasing toxicity score (where toxicity score is used to describe the exposure of multiple pesticides for each nest), with swallows appearing to be more sensitive than bluebirds (Bishop et al. 2000b). In one of the first field studies conducted to examine immune parameters in wild birds exposed to pesticides, a general trend of immunostimulation in Tree swallows was noted (Bishop etal. 1998). In addition, prior OC exposure appears to affect the extent of ChE inhibition. In mammals, prior exposure to OC compounds appears to reduce the effects of subsequent OP exposure by increasing the activity of the ChE enzymes (Ball et al. 1954, Triolo 5 and Coon 1966, Menzer 1970), while in avian species it was suggested that prior OC exposure causes a greater relative decrease in ChE inhibition when subsequent OP exposure occurs (Ludke 1977). The research described in this thesis has three main objectives, each of which comprises one of the three main chapters. All experiments were conducted in a laboratory setting using the Zebra finch (Taeniopygia guttata) as a model species to represent songbirds. These birds were chosen as they breed well in captivity, are amenable to handling and experimental manipulation during breeding and have a short generation time, as they reach sexual maturity at three months of age. In Chapter 2,1 determine the dose-response relationship of azinphos-methyl on brain and plasma ChE activity in the Zebra finch. This is necessary as a baseline to see how sensitive the model species is to exposure. In addition, this chapter attempts to determine the effects of azinphos-methyl on ChE enzyme activity, and hematological and immune function parameters in birds pre-exposed to p,p '-DDE at low, medium and high doses. In Chapter 3,1 determined the sub-lethal effects on Zebra finches following chronic exposure to p,p '-DDE, and single-dose treatment of azinphos-methyl and mancozeb during the pre-laying period. Here, I assessed the general health of the bird by measuring various hematological and immunological parameters, and early reproductive effort (up to clutch completion). Finally, in Chapter 4,1 assessed the effects ofp,p '-DDE exposure on the early reproductive effort (up to clutch completion) and then determined the effects ofp,p '-DDE in combination with current-use pesticides. This was assessed by measuring the general health of the bird (including various hematological and immunological parameters), and later reproductive effort (from clutch completion to fledging). These data are essential for developing strategies to sustain healthy songbird populations in orchards and will allow for excellent comparative data for a complimentary field study being conducted in Ontario, Canada. CHAPTER 2 Effects of exposure to p,p '-DDE and azinphos-methyl on brain and plasma cholinesterase activity and general health in Zebra finches (Taeniopygia guttata) 2.1 Introduction Azinphos-methyl (0,0-Dimethyl S-[(4-oxo-l,2,3-benzo-triazin-3(4H)-yl)methyl] phosphorodithioate) is a broad spectrum organophosphate (OP) insecticide that is used extensively throughout the world to control insect pests (Chemagro Division Research Staff 1974, Grady 1999). In Canada, azinphos-methyl is registered for control of insects on agricultural and vegetable crops, primarily apples, peaches, potatoes and cherries. In apple orchards, azinphos-methyl is commonly used to control infestation of the insect pest, Coddling moth (Cydia pomonella), and is sprayed one to three times per season (Province of British Columbia 1991). Agricultural surveys estimated that in 1993, the total amount of azinphos-methyl used in Ontario was 71,983 kg (Hunter and McGee 1999) while in 1995 in British Columbia the total used was 21,804 kg (Norecol Dames and Moore 1997). Like all OP insecticides, azinphos-methyl acts by inhibiting the cholinesterase (ChE) enzyme (Chambers and Levi 1992). These enzymes are involved in the breakdown of the neurotransmitter acetylcholine to acetate and choline, providing a key role in the removal of excess acetylcholine. Thus, ChE enzymes are critical to the normal function of the nervous system in both vertebrates and invertebrates and, therefore, the potential exists for non-target effects whenever OPs are released into the environment (Grue et al. 1991). Measurement of this enzyme is commonly used in field situations to determine cause of mortality or extent of exposure to OPs (Zinkl et al. 1980, Busby et al. 1981). Several field-monitoring studies have measured ChE inhibition to examine the response of songbirds residing in orchards following azinphos-methyl treatment (Graham and DesGranges 1993, Burgess et al. 1999, Gill et al. 2000). 7 However, in the field there are many factors that could influence the potency of ChE inhibitors, including breakdown products, the persistence and fate of ChE inhibitors in the environment, the potential for exposure of sensitive species and the general health and physiological condition of non-target organisms exposed to ChE inhibitors (Holmes and Boag 1990). In addition, other contaminants present in the environment may influence the biochemical response to OPs in avian species (Johnston et al. 1994). Although no longer applied, organochlorine (OC) pesticides, particularly DDT [1,1,1-trichloro-2,2-iz'5(p-chlorophenyl)ethane] related compounds, persist at high concentrations in areas of past intensive usage, such as fruit orchards (Harris et al. 2000b). Songbirds residing in Ontario and British Columbia fruit orchards have been reported to contain high body burdens of DDE [2,2- £/'s(4-chlorophenyl)-l,l-dichloroethylene; a persistent metabolite of DDT]. Eastern bluebird (Sialia sialis) eggs from Ontario orchards had p,p '-DDE residues as high as 105 pg/g (Bishop et al. 2000a) while DDE concentrations in American robin (Turdus migratorius) eggs in Okanagan orchards (British Columbia) had DDE residues as high as 302 mg/kg (Gill et al. 2003). Persistence of DDT compounds in orchard soils is suggested to be due to high previous application rates and minimal tillage or other soil disturbance, which limits loss by volatilization and erosion (Harris et al. 2000b). Due to the persistence of DDE in the environment, songbirds residing in orchards commonly have high DDE (particularly p,p -DDE) body burdens when OP spraying occurs. However, few avian studies have examined the joint toxicity of OC and OP pesticides. In mammals, prior dosing or exposure to OC compounds appears to reduce the effects of subsequent OP exposure by increasing the activity of the ChE enzymes (Ball et al. 1954, Tfiolo and Coon 1966, Menzer 1970). However, it appears this protection is not seen in avian species. For example, Ludke (1977) found that DDE exposure in Coturnix quail (Coturnixcoturnix japonica) resulted in a dose-related increase in plasma ChE activity. Following treatment with . • 8 the OP parathion, plasma ChE activity decreased in control and DDE-pretreated birds to about the same levels. As DDE-pretreated birds had relatively greater plasma ChE activity prior to the parathion dosage, dosing with parathion resulted in a relatively greater ChE inhibition in DDE pretreated birds than untreated individuals. Thus in quail, prior feeding of DDE did not reduce the effect of subsequent parathion exposure (see also Dieter 1974). In addition to enzyme effects, animals exposed to environmental stressors such as pesticides, may be negatively impacted in terms of general health. One of the first field studies conducted to examine immune parameters in wild birds exposed to pesticides found a general trend of immunostimulation in Tree swallows (Bishop et al. 1998). Basal hematological parameters, such as percent hematocrit and leucocrit, are easily obtainable and are useful for detecting the effects of environmental, infectious, parasitic, or toxicological stresses on animals. They have been widely used in studies of health (Ots et al. 1998), reproduction (Morton 1994, Merila and Svensson 1995) and adaptation (Carpenter 1975). The objectives of this chapter were to determine a) the dose-response relationship between azinphos-methyl and brain and plasma ChE activity in the Zebra finch (Taeniopygia guttata), and b) the effects of azinphos-methyl on ChE activity, hematological and immune function parameters, in birds pre-exposed Xop,p '-DDE at low, medium and high doses. This study was part of a larger laboratory investigation of the sub-lethal effects of orchard pesticides on songbird reproduction. 2.2 Methods 2.2.1 Animals and husbandry This study was conducted on a captive colony of Zebra finches maintained at the Simon Fraser University Animal Care Facility located in Burnaby, British Columbia. Zebra finches were housed in a controlled environment (temperature 19-23°C; humidity 35-55%; photoperiod = 14 9 hr L: 10 hr D, lights on at 0700). All birds were provided with mixed seed (panicum and white millet, 1:2; 11.7% protein, 0.6% lipid and 84.3% carbohydrate by dry mass), water, grit and cuttlefish bone (calcium) ad libitum plus a multivitamin supplement in the drinking water once per week. Experiments and animal husbandry were carried out under a Simon Fraser University Animal Care Committee permit, in accordance with guidelines from the Canadian Committee on Animal Care (CCAC). 2.2.2 Experiment 1 - Dose-response of plasma and brain cholinesterase (ChE) activity to azinphos-methyl To determine the response of brain ChE activity to azinphos-methyl, a dose-response study was conducted. A series of dilutions for azinphos-methyl were prepared on a log scale and the following dosages were administered: 0.27, 0.48, 0.85, 1.5, 2.7, and 4.8 mg/kg. One group was also exposed to plain corn oil, containing no azinphos-methyl. Due to the low response over this range (see Results) we subsequently dosed additional groups of birds at 14.6, 25.5 and 45.3 mg/kg. Three Zebra finches were dosed per treatment, except for the highest dose (45.3 mg/kg) in which five birds were dosed. In order to determine the effect of starvation on brain ChE activity, we dosed nine birds (three per treatment) at the following dosages with an overnight fast of food: 1.5, 4.8 and 8.5 mg/kg. To determine any behavioral effects, an additional twelve birds (three birds per treatment) were fasted overnight and dosed at 1.5, 4.8, 8.5 and 14.6 mg/kg. These birds were observed for 1 week after dosing and checked daily for mortality or abnormal behavior (such as tremors, convulsions or lethargy). The effect of azinphos-methyl exposure on plasma ChE activity was also determined. For this study, an additional 55 Zebra finches (five birds per treatment) were dosed with azinphos-methyl and plasma samples were collected for ChE activity measurements. Birds received the following dosages of azinphos-methyl: 0.27, 0.48, 0.85, 1.5, 2.7, 4.8, 8.5, 14.6, 25.5 10 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 pi) from the jugular vein followed by exsanguination. 2.2.3 Experiment 2 - Effects of p,p '-DDE and azinphos-methyl on cholinesterase (ChE) activity and general health Sixty adult male Zebra finches were divided into cages with five birds per cage. Forty birds used were from a previous experiment in which they were exposed to p,p '-DDE (34 mg/kg) approximately 1 year previously. There was a total of six treatment groups with 10 birds per treatment (Table 2.1). Birds that were exposed to a low, medium and high dose ofp,p '-DDE, were treated at 19, 34, and 60 mg/kgp,p '-DDE (log-scale dilution) respectively for four weeks. The remaining birds were exposed to plain corn oil. Birds were offered p,p '-DDE contaminated egg food for four weeks and were weighed (±0.1 g, initial mass) prior to exposure and within 24 hours at the end of the third week of dosing. The phytohemagglutinin (PHA) immune function test was conducted after three weeks of p,p '-DDE dosing. The PHA immune function test is now being recognized as a valuable tool to study cell-mediated immunity of wild animals (Smits et al. 1996, Smits and Williams 1999). This test measures the response to the plant lectin PHA, in terms of T-lymphocyte proliferation. Zebra finches required that a 1 cm patch of skin on the mid-patagium of both wings be plucked of feathers. Three measurements of patagium thickness of the wings were measured to 0.01 mm using a gauge micrometer (The Dyer Company, Lancaster, PA). Each bird was then injected with 30 pi of PHA lectin solution in the left wing and 30 pi of phosphate buffered saline (PBS) in the right wing. Twenty-four hours following injection, thickness of both wings was measured at the injection site. The response was considered to be the difference between the change in thickness of the PHA-injected site and the PBS-injected site in each bird. 11 Twenty-four hours following their last p,p '-DDE (or corn oil) dose (after four weeks), birds were exposed to azinphos-methyl at 18.4 mg/kg. This dosage was predicted to cause 20-25% brain ChE inhibition (see Section 2.3.2) which is indicative of OP exposure (Ludke et al. 1975, Busby et al. 1981). Three hours after dosing, birds were anesthetized and plasma and brain samples were collected for determination of ChE activity. In addition, blood samples (40 pi) were collected to determine percent hematocrit and leucocrit. Hematocrit, or packed cell volume (PCV), measures the relative amount of red blood cells in total blood volume and reflects the extent and efficiency of oxygen uptake and transfer to tissues (Ots et al. 1998). Measurement of the leucocrit (or 'buffy coat' layer) is indicative of the number of white blood cells in a sample (Wardlaw and Levine 1983), which is one measure of humoral immune function. Within four hours of collection, blood was centrifuged for 3 minutes at 5,000 r.p.m. after which the height of the buffy layer, hematocrit and total sample volume was immediately measured, using a digital caliper (± 0.01mm). The measurer stayed consistent throughout all samples. To determine residues remaining in birds from prior DDE exposure, six male Zebra finches were sacrificed at the start of the experiment, which had been exposed to p,p '-DDE approximately one year previously at 34 mg/kg. Livers were collected using acetone rinsed utensils and stored in acetone / hexane triple-rinsed jars. Tissues were immediately frozen at -20°C until analysis. 2.2.4 Dosing procedure Technical grade azinphos-methyl (Guthion™, Supelco, Bellefont, Pennsylvania, 100 % purity, NEAT) was used in all experiments. The insecticide was dissolved in corn oil and administered via intubation to individual birds. Each bird received 0.1 ml of the insecticide - corn oil solution (or plain corn oil for controls). Birds were fasted for one hour prior to dosing. In order to determine the effect of starvation, we dosed an additional subsample of birds following an 12 overnight fast. Dosages were calculated based on the average weight per bird, which was 15.0 ± 0.3 grams. Birds were given food and water 10 to 15 minutes after dosing. Technical grade DDE [p,/?-DDE; 2,2-bis(4-chlorophenyl)-l,l-dichloroethelene, Sigma-Aldrich, Oakville, Ontario, 99 % purity] was used for the second study (see Section 2.2.3) where birds were exposed to p,p '-DDE prior to OP dosing. Birds in this study received a supplemental diet of egg food, which was the vehicle for the insecticide. Egg food consisted of a mixture of hard-boiled eggs (3 eggs at approximately 60 grams / egg), breadcrumbs (40 grams) and corn meal (40 grams), which the birds readily consumed (20.3% protein: 6.6% lipid). The pesticide was initially dissolved in corn oil and then the insecticide - corn oil mixture (or plain corn oil for controls) was incorporated into the egg food. Three grams of prepared egg food was offered daily per bird, most of which was consumed following 24 hours. Dosages prepared contained 19, 34 and 60 mg/kgp,p '-DDE (log-scale dilution). In order to validate the methodology used to prepare the contaminated egg food, two food samples from each dosage (excluding 34 mg/kg) were collected randomly throughout the study and analyzed for p,p '-DDE content. Two food samples from a previous study prepared at 34 mg/kg/?,/? '-DDE were also analyzed and levels reported in this study as the preparation methodology was the same. Upon collection, food samples were immediately frozen at -20°C until analysis. 2.2.5 Tissue sample collection, preparation and storage Three hours after dosing with azinphos-methyl or corn oil, birds were anesthetized via an intra-muscular injection of 50 pi ketamine and xylazine solution (50:50 by volume; Associated Veterinary Products, Abbotsford, British Columbia) followed by exsanguination. Blood and brain samples were collected within two minutes of death. Blood samples were collected via the jugular vein using heparinized pipettes and transferred to heparinized 0.6 ml centrifuge tubes. Heads were separated from bodies and stored in sterile plastic bags. Both blood and heads were 13 immediately stored on ice for up to six hours. Plasma was separated from the red blood cell component by centrifugation at 5,000 r.p.m. for 15 minutes and immediately frozen at -20°C. Heads were frozen at -20°C within six hours from collection. 2.2.6 Cholinesterase (ChE) assay Head and plasma samples were sent to the National Wildlife Research Centre (NWRC) in Hull, Quebec for measurement of ChE activity under the supervision of Suzanne Trudeau. Upon arrival, brains were removed from the cranium, the whole brain was homogenized in buffer / Triton-X-100 at a ratio of 250 mg/ml and 10 pi of the homogenate were analyzed. Cholinesterase activity was determined based on Hill and Flemming's (1982) modification of the method of Ellman et al. (1961). The cuvette contained 3 ml of DTNB (5,5'-dithiobis-(nitrobenzoic acid); 2.5 x 10-4 M) in phosphate buffer (0.05 M, pH 7.9), 10 pi of brain homogenate (or 20 pi of plasma) and 100 pi of acetylthiocholine iodide (0.032 M for brain and 0.156 M for plasma). Cholinesterase activity was determined with a spectrophotometer (Hewlett Packard Diode Array HP8452, S/N 2610A00276) at 30°C. The change in absorbance at 406 nm over one minute was recorded, with readings every 15 seconds. Duplicate analyses were performed for most samples. For each sample, the average of the duplicate assays was used in the data analyses. Quality assurance procedures included the analysis of Precinorm E (control serum from Boehringer Mannheim) with each series of samples and conducting duplicate analyses. Three plasma samples from Experiment 2 could not be analyzed because of their high lipid content. 2.2.7Organochlorine (OC) analysis Food and liver samples were sent to the Great Lakes Institute for Environmental Research (GLTER) at the University of Windsor in Windsor, Ontario for measurement of OC content. Due 14 to insufficient sample size, percent moisture was not determined in liver samples. All solvents used during sample extractions were of high purity suitable for gas chromatography/pesticide analysis. 1,3,5-Tribromobenzene used as a surrogate standard for analyte recovery during sample extractions was obtained from Accu Standards, CT, USA. Organochlorine pesticide standards containing 1,2,4,5-tetrachorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, a-hexachlorocyclohexane, p-hexachlorocyclohexane, y-hexachlorocyclohexane, octachlorostyrene, oxy-chlordane, gamma(trans)-chlordane, alpha(cis)-chlordane, trans-nonachlor, cis-nonachlor, p,p'-DDE, -DDE), p,p'-DDT, mirex, photomlrex were donated to GLEER by the Canadian Wildlife Service, Environment Canada, PQ, Canada. The organic and metals laboratory at GLEER is a member of the Canadian Association for Environmental Analytical Laboratories (CAEAL) and is accredited to perform OC pesticide analyses in sediment, water and biological tissue samples. As a CAEAL certified laboratory, GLEER actively participates in interlaboratory comparisons and routine audits to ensure quality control of techniques and procedures. GLEER also participates in interlaboratory comparisons of OC analyses in tissue homogenates supplied by the Canadian Wildlife Service, Environment Canada, Hull, PQ. All methodologies and procedures are documented in the GLEER Quality Manual of the Laboratory. Organochlorines analyzed for include 1,2,4,5-tetrachorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, a-hexachlorocyclohexane, P-hexachlorocyclohexane, y-hexachlorocyclohexane, octachlorostyrene, heptachlor epoxide, oxy-chlordane, gamma(trans)-chlordane, alpha(cis)-chlordane, trans-nonachlor, cis-nonachlor, p,p -DDE, p,p -DDD, p,p -DDT, dieldren, mirex, photomirex. 2.2.8 Statistical Analyses Statistical analyses were performed using IMP software program (SAS Institute Inc 2000). All parameters were tested for normality (Shapiro-Wilk W Test) prior to analysis. Plasma and brain 15 ChE activity and response to the lectin PHA did not meet the requirements and were therefore logio transformed prior to analysis. To determine if time of starvation (one hour versus overnight) had an effect on brain ChE response, an analysis of covariance (ANCOVA) was conducted, with time of starvation as a covariate. Cholinesterase activities were similar in birds starved for one hour to those starved overnight prior to administration of the OP (Fi)3g=1.12, P=0.2969), therefore these data were pooled for subsequent analysis. A regression analysis correcting for body mass was conducted to determine the relationship between increasing dosages of azinphos-methyl and plasma and brain ChE activity. To determine if a difference existed in plasma and brain ChE response to azinphos-methyl exposure in birds exposed to low, medium and high levels ofp,p '-DDE, an ANCOVA was conducted, with body mass as a covariate. If a difference existed, Tukey's Honestly Significantly Different (HSD) test was used for multiple comparisons. As body mass was not correlated with the remaining parameters tested (PHA response or percent hematocrit and leucocrit) a least squares analysis of variance (ANOVA) was conducted to see if a difference existed amongst the treatments. If a difference existed, this was followed by Tukey's HSD for multiple comparison. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analysis. Further statistical analyses were also conducted to determine the individual pesticide effects on the hematological and immune function parameters (i.e. birds previously exposed top,p '-DDE one year prior to the start of the experiment, and those exposed to azinphos-methyl). 2.3 Results 2.3.1 Confirmation ofpesticide contaminated egg food preparation and liver residues in p,p '-DDE pre-exposed Zebra finches Measured concentrations of p,p '-DDE in treated egg food were within an average of 15% of the target doses (Table 2.2) confirming preparation ofp,p '-DDE contaminated egg food. 16 Organochlorine liver residues were generally low in males dosed with p,p '-DDE one year prior to sampling (range: 1.25-4.14 mg/kgp,p '-DDE; Table 2.3). In addition to p,p '-DDE, some birds also had traces of y-hexachlorocyclohexane residues in their liver. All other OC compounds tested for (see Section 2.2.7) were non-detectable. Organochlorine contaminants in livers of males after exposure to p,p '-DDE for four weeks at 19 mg/kg, 34 mg/kg and 60 mg/kg are summarized in Table 2.3. 2.3.2 Experiment 1 - Dose-response of plasma and brain cholinesterase (ChE) activity to azinphos-methyl Abnormal behavior (i.e. tremors, convulsions, lethargy) was not observed in any birds throughout this study, including those observed for one week after dosing with azinphos-methyl. One bird died immediately after dosing in the 1.5 mg/kg dosage group and therefore was not analyzed for ChE activity. Plasma and brain ChE activity were seen to decrease linearly with increasing dosage of azinphos-methyl (plasma ChE activity: Fi;5i=69.8, PO.0001; brain ChE activity: Fi,38=105.9, PO.0001; Figure 2.1). From these results it was calculated that approximately 20-25% brain ChE inhibition would occur at 18.4 mg/kg. 2.3.3 Experiment 2 - Effects of p,p '-DDE and azinphos-methyl on cholinesterase (ChE) activity and general health The PHA test was conducted following three weeks of exposure to oil or p,p '-DDE. There was no difference in response to the lectin in the five treatment groups (range: 0.31-1.23 mm; F4;53=2.50, P^ O.0535; Table 2.4). However, birds exposed one year previously top,p '-DDE (mean: 0.65 ± 0.03 mm, n=38) had a significantly higher response to the plant lectin compared to those not previously exposed (mean: 0.52 ± 0.04 mm, n=20) (Fi,56=7.86, P=0.0069). 17 After four weeks of exposure to oil or DDE, birds were orally dosed with the OP azinphos-methyl. Three hours following this dosage, hematocrit values were higher in the control birds (corn oil + corn oil) than those exposed to the corn oil + azinphos-methyl (range: 40.3-63.5%; F5>5i=3.41, P-0.0098; Table 2.5). In addition, significantly lower hematocrit values were observed in birds exposed one year previously top,p '-DDE (mean: 53.6 ± 0.8, n=37) compared to those not previously exposed (mean: 56.8 ± 1.0, n=20) (Fi>55=6.40, P=0.0143). Current azinphos-methyl exposure also showed lower hematocrit values (mean: 54.1 ± 0.7, n-47) when compared to those not exposed to the azinphos-methyl (57.7 ± 1.55, n=10) (Fi,55=5.12, P=0.0276). Percent leucocrit was higher in birds exposed to azinphos-methyl (0.87 ± 0.06, n=47) compared to those not exposed (0.55 ± 0.14, n=10) (Fi)55=4.60, P=0.0363). There was no difference in percent leucocrit amongst the combination of treatments (F5,5i=1.76, P=0.1384; Table 2.5) and those exposed top,p '-DDE one year previously (meanp,p '-DDE: 0.86 ± 0.07, n=37; mean corn oil: 0.74 ±0.10, n=20; Fi,55=0.82, P=0.3679). Both plasma (F5,48=22.2, PO.0001) and brain (F5,5i=18.1, P<0.0001) ChE activities were inhibited following exposure to azinphos-methyl, however there was no difference in extent of inhibition in birds exposed to low, medium and high dosages ofp,p '-DDE (Figure 2.2). 2.4 D i s c u s s i o n The main results of this chapter were as follows: 1) azinphos-methyl resulted in a dose-dependent inhibition of plasma and brain ChE activity, 2) Zebra finches were a relatively less sensitive species to azinphos-methyl exposure compared with other songbirds, 3) p,p '-DDE in combination with azinphos-methyl did not change azinphos-methyl inhibition of ChE activity, and 4) pesticide exposure caused immunostimulation and possibly anemia in Zebra finches. These results will be compared to those for other songbirds and discussed below. 18 2.4.1. Validation of methodology and dosing Validation of egg food preparation containing p,p '-DDE was confirmed with the random sampling of prepared egg food for OC content. Results showed that the contaminated egg food contained relatively close amounts of p,p '-DDE to the desired dosage and minimal variation existed among samples of the same dosage. As many birds used in this experiment were dosed with p,p '-DDE one year previously, it was important to determine the extent of the contaminant remaining prior to the start of the current experiment. Approximately one year following dosing, Zebra finches had generally lowp,p '-DDE residues in liver (range: 1.25-4.14 mg/kg). Few data are available for liver DDE residues remaining after long-term dosing in birds. However, in the lower mainland, American robins residing in habitat with no known previous DDT exposure had 0.3-3.2 mg/kg DDE in eggs (Gill et al. in press). Assuming a similar egg to liver ratio as that reported in Herring gulls (Larus argentatus) (Braune and Norstrom 1989), robins with DDE in the eggs ranging from 0.3-3.2 mg/kg DDE would be associated with liver residues of 0.34-3.64 mg/kg DDE. These levels are comparable to levels found in birds used in our study thereby suggesting minimal significance of these residues (i.e. they are typical of birds from non-contaminated areas). In addition to p,p '-DDE, Zebra finches also had low levels of y-i hexachlorocyclohexane residues in their liver. This pesticide is commonly used as a seed treatment so this is the likely route of exposure. However, since all birds used for the experiments, including controls, received the same seed it is therefore unlikely that any significant results were due to possible effects of this pesticide. 2.4.2. Effects of azinphos-methyl on cholinesterase (ChE) activity Zebra finches exposed to azinphos-methyl generally showed an increase in brain and plasma ChE inhibition with increasing dosage of the pesticide. This is a typical response seen in avian species following exposure to an OP (Holmes and Boag 1990, Anam and Maitra 1995, Westlake i I 19 et al. 1981). For example, Zebra finches dosed with the OP fenitrothion showed increasing inhibition of brain and plasma ChE activity with increasing dosage (Holmes and Boag 1990). A similar trend was seen in the brain ChE activity in Roseringed parakeets (Psittacula krameri) where increasing exposure to the OP quinalphos resulted in a decrease in brain ChE activity (Anam and Maitra 1995). Maximum brain ChE inhibition at our highest dose of 45.3 mg/kg resulted in 42.9% inhibition of the enzyme. Brain ChE inhibition of 50% is used as an indicator of OP exposure resulting in death (Ludke et al. 1975, Busby et al. 1981). However birds can have inhibition >50% and still survive. Our birds were approaching brain ChE inhibition of 50% and did not succumb to the exposure or behave abnormally. Other studies have shown that Zebra finches exposed to fenitrothion have survived following dosages which resulted in >50% brain ChE inhibition (Holmes and Boag 1990). Although abnormal behavior was not noticed in birds exposed to the varying doses of azinphos-methyl, one bird died immediately after being dosed with azinphos-methyl at 1.5 mg/kg. However, it is unlikely that this bird died from OP exposure, as others given much higher doses did not succumb to the exposure. Thus, Zebra finches appear to be more resistant to azinphos-methyl exposure when compared with other songbird species based on previous studies. Our birds were administered the pesticide at dosages as high as 45.3 mg/kg and no birds at this dosage suffered mortality. In comparison, other songbird species have much lower LD50 values (or the lethal dose which will result in 50 % mortality in the group of test animals). For the Red-winged blackbird (Agelaiusphoeniceus), the LD50 value is 8.5 mg/kg and for European starlings (Sturnus vulgaris) it is 27 mg/kg (Smith 1987). Zebra finches in our study exposed to doses much higher than this appeared td behave normally and did not succumb to the exposure. Amongst the songbirds, this shows that there is a high variation in sensitivity to the OP azinphos-methyl, with Zebra finches appearing to be least sensitive. 20 2.4.3. Effects of p,p '-DDE and azinphos-methyl on cholinesterase (ChE) activity Earlier studies showed that OC compounds reduce the response to subsequent OP exposure due to an increase in ChE enzyme activity in both mice and rats (Ball et al. 1954, Triolo and Coon 1966, Menzer 1970). Rats given a single oral dose of aldrin, chlordane or lindane were protected four days later against subsequent parathion exposure; that is, plasma ChE activity was less inhibited with pre-exposure to the chlorinated hydrocarbons, than those with no pre-exposure (Ball et al. 1954). Isomers of DDT have been reported to accelerate several detoxification processes of rat liver microsomes, including the hydrolysis of paraoxon (the anticholinesterase form of the OP parathion) and dealkylation of some OPs (Crevier et al. 1954). Lower mortality is also seen in mice pretreated with chlorinated hydrocarbons followed by OP exposure than those exposed to the OP alone (Triolo and Coon 1966). In the present study in Zebra finches, although azinphos-methyl exposure consistently resulted in a decrease in both plasma and brain ChE activity, there was no difference in enzyme activities in birds pretreated with p,p '-DDE compared to those with no p,p '-DDE pretreatment. Other avian studies have similarly shown that OC exposure does not reduce the effects of subsequent OP exposure, but in contrast to mammals, have suggested that DDE actually increases the toxicity of OPs. Coturnix quail exposed to DDE showed an increase in plasma ChE activity related to dosage (Dieter 1974, Ludke 1977). However, Ludke (1977) showed that this increase in plasma ChE activity did not minimize the effects of subsequent exposure to the OP parathion as was seen in laboratory mammals. Birds with no pretreatment to DDE displayed relatively less plasma ChE inhibition that did DDE pretreated birds when they were subsequently challenged with parathion suggesting that any protection was offset by a net difference resulting from some other pretreatment effect. In addition, quail exposed to a combination of DDE and the OP parathion experienced higher mortality than those exposed to the OP alone (Ludke 1977). As no birds in our study suffered mortality, including those exposed to p,p -DDE plus the OP 21 azinphos-methyl, this suggests that Zebra finches were not as susceptible to p,p '-DDE pretreatment. 2.4.4. Direct effects of p,p '-DDE and azinphos-methyl on general health Measures of immune function are becoming increasingly common when studying toxicology to evaluate the immune status of individuals (Dethloff and Bailey 1998, Smits et al. 1996, Smits and Williams 1999). Previous studies on occupationally pesticide-exposed humans and captive animals dosed with pesticides showed the most common impact on the immune system to be immunosuppression (Vos et al. 1989, Dean and Murray 1991, Pruett et al. 1992, Pruett 1994). The PHA immune function test is becoming more common as a determinant of cell-mediated immune function. In our study, the PHA response was higher in birds with previous exposure to p,p '-DDE (approximately one year prior to the start of the experiment) suggesting immunostimulation in p,p '-DDE exposed birds. This immuno stimulation was further supported by the increase in leucocrit values in birds exposed to azinphos-methyl. These findings are consistent with results obtained in a Ontario field study where Tree swallows exposed to orchard pesticides including azinphos-methyl and p,p '-DDE showed immunostimulation (Bishop et al. 1998). The study conducted by Bishop et al. (1998) is the first field study conducted to examine the immune parameters in wild birds exposed to pesticides. Our study conducted in a laboratory setting further supports this suggestion of immunostimulation in songbirds exposed to orchard pesticides. Hematocrit values were lower in birds exposed to corn oil + azinphos-methyl (with previous exposure to p,p '-DDE approximately one year ago) than the control birds (corn oil + corn oil). However, other groups with pesticide exposure did not show any difference in hematocrit response, therefore it is likely that this may be a spurious relationship. However, when looking at individual pesticide effects, birds exposed to p,p '-DDE previously 22 (approximately one year prior to the start of the experiment) and those exposed to azinphos-methyl had lower hematocrit values than those with no pesticide exposure suggesting an anemic response to pesticides. Bishop et al. (1998) found the same trend in Tree swallows exposed to orchard pesticides in which pesticide exposed birds also had lower hematocrits. 2.4.5. Conclusions In conclusion, although Zebra finches experienced ChE inhibition following exposure to azinphos-methyl, they appeared to be less sensitive compared to other songbird species. Although p,p '-DDE did not appear to affect the degree of ChE inhibition following subsequent azinphos-methyl exposure, it did appear to result in stimulation of the immune system and possible anemia. Further studies are warranted to determine pesticide effects on the immune system in order to further understand and interpret these results. i 23 Table 2.1. Summary of pesticide history and treatment in male Zebra finches (Taeniopygia guttata) used to determine cholinesterase activities in birds exposed to a combination of p,p '-DDE and the organophosphate (OP) azinphos-methyl. Previous exposure to p,p '-DDE1 n This experiment -p,p '-DDE dosage This experiment - Azinphos-methyl exposure No 10 No No No 10 No Yes Yes 10 No Yes Yes 10 19 mg/kg Yes Yes 10 34 mg/kg Yes Yes 10 60 mg/kg Yes Previous exposure to p,p '-DDE occurred at 34 mg/kg approximately one year previous to the start of the experiment. 24 Table 2.2. Concentrations o?p,p '-DDE (mg/kg, wet weight) in contaminated egg food used to dose Zebra finches (Taeniopygia guttata). No other organochlorine compounds tested for (see Section 2.2.7) were detected. Sample % lipid % moisture p,p'-DDE 19 mg/kg egg food 16.7 72 16.4 19 mg/kg egg food 17.7 54 17.2 34 mg/kg egg food 15.2 46 36.8 34 mg/kg egg food 14.7 46 39.4 60 mg/kg egg food 15.2 55 63.8 60 mg/kg egg food 15.5 63 56.8 25 Table 2.3. Organochlorine concentrations (mg/kg, wet weight) in male Zebra finch (Taeniopygia guttata) livers exposed to p,p '-DDE at 19 mg/kg, 34 mg/kg and 60 mg/kg. In addition livers from controls (exposed to corn oil) were sent in for comparison. All birds were additionally exposed to p,p '-DDE (34 mg/kg) approximately one year prior to tissue collection. No other organochlorine compounds tested for (see Section 2.2.7) were detected. Sample p,p '-DDE dosage % lipid % moisture p,p'-DDE Y-HCH 1 Corn oil 118 n.d. 3.77 n.d. 2 Corn oil 1.74 n.d. 1.25 n.d. 3 Corn oil 2.56 n.d. 4.14 0.003 4 19 mg/kg 3.72 n.d. 8.12 0.57 5 19 mg/kg 5.77 n.d. 7.59 0.03 6 34 mg/kg 7.98 n.d. 159.16 6.16 7 34 mg/kg 1.87 n.d. 36.16 0.56 8 60 mg/kg 8.33 n.d. 60.67 1.80 9 60, mg/kg 8.11 n.d. 125.72 1.00 n.d. = not determined 26 Table 2.4. Comparison of phytohemagglutinin (PHA) skin test response in adult male Zebra finches (Taeniopygia guttata) treated with corn oil or p,p '-DDE in low, medium or high concentrations (19, 34 and 60 mg/kg, respectively). Values are expressed as the least squared mean ± standard error. Statistical analysis was conducted using a least squared analysis of variance (ANOVA). Dosage group n PHA response (mm) Corn oil 20 0.52 ±0.04 Corn oil (DDE) 9 0.77 ±0.06 19 mg/kg p,p '-DDE (DDE) 10 0.63 ±0.06 34 mg/kgp,p '-DDE (DDE) 10 0.60 ±0.06 60 mg/kg p,p '-DDE (DDE) 9 0.61 ±0.06 P-value 0.0535 ^hose with DDE in parentheses indicate birds that were previously exposed to p,p '-DDE (34 mg/kg) approximately one year ago. 27 Table 2.5. Percent hematocrit and leucocrit in male Zebra finches (Taeniopygia guttata) exposed to corn oil or p,p '-DDE in low, medium and high doses and the organophosphate, azinphos-methyl. Values are expressed as the least squared mean ± standard error with the sample size in parentheses. Statistical analysis was conducted using a least squared analysis of variance (ANOVA) followed by Tukey's HSD for multiple comparisons. Mean values with same letters indicate values that are not significantly different. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analysis. Dosage group Hematocrit (%) Leucocrit(%) Corn oil + corn oil 57.7 ± 1.4 (10) a 0.55 ±0.14 (10) Corn oil + azinphos-methyl 55.8 ± 1.4(10)ab 0.94 ±0.14 (10) Corn oil + azinphos-methyl (DDE) 50.7 ± 1.5 (9)b 0.70 ±0.15 (8) 19 mg/kg p,p '-DDE + azinphos-methyl (DDE) 55.6 ± 1.4(10)ab 0.73 ±0.14(10) 34 mg/kgp,p '-DDE + azinphos-methyl (DDE) 52.1 ± 1.5 (9)ab 1.02 ±0.14 (10) 60 mg/kg p,p '-DDE + azinphos-methyl (DDE) 55.6 ± 1.5 (9)ab 0.96 ±0:14 (9) P-value 0.0098 0.1384 1Thosei with DDE in parentheses indicate birds that were previously exposed to p,p '-DDE (34 mg/kg) approximately one year ago. 28 ^ 2000 -I £ o 0 10 20 30 40 50 Dosage (mg/kg) ^ 0 H 1 1 1 . 1 0 10 20 30 40 50 Dosage (mg/kg) Figure 2.1. Plasma and brain cholinesterase (ChE) activity in adult male Zebra finches (Taeniopygia guttata) orally dosed with the organophosphate (OP) azinphos-methyl. Both plasma (P<0.0001, adjusted R2=0.57) and brain (PO.0001, adjusted R2=0.72) ChE activity were seen to decrease linearly with increasing dosage of azinphos-methyl. 29 1600 -i Corn oil + Corn oil + Corn oil + 19 mg/kgp,p'- 34 mg/kgp,p'- 60 mg/kgp,p'-corri oil azinphos- azinphos- DDE + azinphos- DDE + azinphos- DDE + azinphos-methyl methyl (DDE) methyl (DDE) methyl (DDE) methyl (DDE) Com oil + Corn oil + Com oil + 19 mg/kgp,p'- 34 mg/kgp,p'- 60 mg/kgp,p '-com oil azinphos- azinphos- DDE'+ azinphos- DDE + azinphos- DDE + azinphos-methyl methyl (DDE) methyl (DDE) methyl (DDE) methyl (DDE) Figure 2.2. Plasma and brain cholinesterase (ChE) activity in adult male Zebra finches (Taeniopygia guttata) exposed to corn oil or p,p ' -DDE and the organophosphate, azinphos-methyl. Those with D D E in parentheses indicate birds that were previously exposed to p,p '-D D E (34 mg/kg) approximately one year ago. Results shown are the least squared mean ± standard error with the sample size presented above each bar. Statistical analyses were conducted using a least squared analysis of covariance (ANCOVA), with weight of the bird as a 30 covariate. This was followed by Tukey's HSD for multiple comparisons. Bars with an asterisks are seen to be statistically different that those with no asterisks (P<0.0001 for both plasma and brain ChE activity). 31 C H A P T E R 3 Effects of p,p ' -DDE and current-use pesticides on egg production and yolk precursor levels in the Zebra finch (Taeniopygia guttata) 3.1. Introduction Songbirds commonly use apple orchards during the breeding season for feeding and nesting (Sinclair and Elliott 1993, Fleutsch and Sparling 1994) and during this time, many orchards receive repeat applications of pesticides to control various insect and plant pests (Sinclair and Elliott 1993, Norecol Dames and Moore 1997, Hunter and McGee 1999, Bishop et al. 2000a). Prior to the 1970s, the organochlorine (OC) pesticide DDT [l,l,l-trichloro-2,2-A7'5(p-chlorophenyl)ethane] was commonly used in orchards to control insect pests. However, due to its persistent nature, this was replaced with the less persistent but more acutely toxic organophosphates (OPs). Today OPs continue to be used in combination with fungicides on fruit crops. However, even though DDT has not been used in Canadian orchards for over 20 years, high concentrations of DDT and DDE (2,2- 6/s(4-crdorophenyl)-l,l-dichloroethylene; a persistent metabolite of DDT) still persist in orchard environments and resident wildlife (Hebert et al. 1994, Elliott et al. 1994, Harris et al. 2000b, Bishop et al. 2000a). DDT exposure has been associated with population declines in numerous species of predatory and fish-eating bird species (Hickey and Anderson 1968, Newton 1986, Wieymer et al. 1993) and with mass songbird mortalities (Wallace 1959, Carson 1962, Wurster et al. 1965). Chronic effects of DDT and DDE exposure on avian species include eggshell thinning (Wiemeyer and Porter 1970, Cooke 1973, Elliott and Martin 1994) and reduced productivity (Jefferies 1971, Ratcliffe 1980, Wieymer et al. 1988). Although some of the most well published effects of DDT and its principal toxic metabolite DDE are on avian wildlife, most are on raptorial and fish-eating 32 species. Among the songbirds, American robins (Turdus migratorius) have suffered mortality immediately following DDT spraying (Wurster et al. 1965, Wallace 1959). Few studies exist on the reproductive effects of chronic exposure to these compounds in songbirds, with variable results having been reported. In American robins (Johnson et al. 1976) and Eastern bluebirds (Sialia sialis) (Bishop et al. 2000a) nesting in orchards, OC contamination has been associated with a decrease in clutch size and hatching success, respectively. However, other orchard studies have reported the opposite result where American robins containing OC burdens actually showed a higher clutch size (Gill et al. 2003). DDT also has been shown to induce the production of vitellogenin (VTG), a complex phbspholipoprotein yolk precursor produced by the liver (Celius and Walther 1998). Plasma VTG is a useful indicator of reproductive state in adult avian females (Mitchell and Carlisle 1991, Challenger et al. 2001) and has been suggested to be a useful bioindicator of estrogenic effects (Palmer et al. 1998). Changes in circulating VTG levels may be used to characterize reproductive adaptation to change in physiological status or environmental change (Phillips et al. 1985). Very low-density lipoprotein (VLDL), the other main avian yolk precursor produced by the liver, is the primary source of yolk lipid and increases markedly during egg production (Walzem et al. 1999, Challenger et al. 2001). In addition to residual OC contaminants in the environment, songbirds residing in orchards are continuously exposed to current-use pesticides throughout the breeding season. Previous orchard field studies have revealed that the OP, azinphos-methyl (0,0-dimethyl S-[(4-oxo-1,2,3-benzo-triazin-3(4H)-yl)methyl] phosphorodithioate), and the ethylene bisdithiocarbamate (EBDC) fungicide, mancozeb, are two common pesticides used presently, applied alone or as tank mixtures (Hunter and McGee 1999, Norecol Dames & Moore 1997). Azinphos-methyl has been associated with a decrease in reproductive success in songbirds when applied alone (Gill et al. 2000) or with other OPs (Fleutsch and Sparling 1994, Bishop et al. 1998). Less information is 33 available on the toxicity of mancozeb to birds but the LD50 value in Mallards (Anas platyrhynchos) is > 6400 mg/kg (Harris et al. 2000a). In comparison, the LD50 value of azinphos-methyl is 8.5 mg/kg for the Red-winged blackbird (Agelaiusphoeniceus) and 27 mg/kg for European starlings (Sturnus vulgaris) (Smith 1987) suggesting mancozeb to be a lot less toxic than azinphos-methyl. In reality, birds nesting in orchards contain residual OC burdens prior to the spraying of current-use pesticides (Elliott et al. 1994, Gill et al. 2003). Few studies exist which look at the effects of combined pesticides on avian species. In order to provide a relevant assessment of the avian risks inherent in the orchard environment, it is necessary to consider the effects of multiple pesticide exposure. The purpose of this chapter was to determine the sub-lethal effects on Zebra finches (Taeniopygia guttata) following chronic exposure to p,p '-DDE, and single-dose treatment of azinphos-methyl and mancozeb during the pre-laying period in a controlled environment. Pesticides were administered alone and in combination with one another. We then assessed a) the general health of the bird by measuring various hematological and immunological parameters, and b) early reproductive effort, including the laying interval, clutch size, egg mass, shell thickness and circulating levels of the yolk precursors VTG and VLDL. 3.2 Methods 3.2.1. Animals and Husbandry This study was conducted on a captive colony of Zebra finches maintained at the Simon Fraser University Animal Care Facility located in Burnaby, British Columbia. Zebra finches were housed in a controlled environment (temperature 19-23°C; humidity 35-55%; photoperiod = 14 hr L: 10 hr D, lights on at 0700). All birds were provided with fresh mixed seed (panicum and white millet, 1:2; 11.7% protein, 0.6% lipid and 84.3% carbohydrate by dry mass), water, grit and 34 cuttlefish bone (calcium) ad libitum plus a multivitamin supplement in the drinking water once per week. Experiments and animal husbandry were carried out under a Simon Fraser University Animal Care Committee permit, in accordance with guidelines from the Canadian Committee on Animal Care (CCAC). 3.2.2. Experimental design This experiment was carried out in two trials in June 2001 and February 2002. Birds were assigned to a treatment group at random (see Table 3.1 for treatments). Four of the seven treatment groups contained females which had previously been exposed to p,p '-DDE at 34 mg/kg approximately one year ago. A subsample of livers (n=6) from birds that were exposed to p,p '-DDE approximately one year previously were analyzed to determine p,p '-DDE residues remaining prior to the start of the current experiment. Initially, Zebra finches were divided into cages separating males from females with five to seven birds per cage. Birds were given at least 24 hours to acclimatize to their environment. Zebra finches in this study received a supplemental diet of egg food (20.3% protein: 6.6% lipid). This consisted of a mixture of hard-boiled eggs, breadcrumbs and corn meal, which the birds readily consumed. Birds were exposed to corn oil or p,p '-DDE (34 mg/kg) for four weeks with the egg food being the vehicle for the insecticide. After three weeks ofp,p '-DDE dosing, birds were paired and given a single oral dose of corn oil or azinphos-methyl or mancozeb, depending on treatment group (see Table 3.1 for treatment groups; see Section 3.2.3 and 3.2.4 for dosage rationale and dosing procedure, respectively). Dosing of corn oil or p,p '-DDE continued for one more week for breeding pairs (four weeks total). Following the discontinuation ofp,p '-DDE exposure, birds were offered clean egg food daily (approximately three grams per bird) up to the completion of the experiment. 35 Each breeding pair was placed in a separate cage (61 X 46 X 41 cm) with a external nest box (11.5X11.5X11.5 cm). Birds were weighed (± 0.1 g) at the time of pairing and at clutch completion. Following pairing, birds were observed and reproductive output up to clutch completion was assessed. Nest boxes were monitored daily to determine the timing of laying. Reproduction was measured by: 1) proportion of Zebra finch pairs which laid a minimum of one egg, 2) clutch size (number of eggs laid), 3) laying interval (days to lay first egg from pairing), 4) mean egg mass of total clutch, and 5) mean shell thickness of the second egg. Blood samples were collected from female Zebra finches at the one-egg stage (see Section 3.2.5). Eggs were numbered in consecutive order of laying and the second egg was collected from each nest and archived (see Section 3.2.6). Each egg removed was substituted with a replacement egg. Birds that did not lay eggs two weeks following pairing were discontinued and classed as non-breeders. Upon clutch completion (or two days after the last egg was laid), birds were separated and discontinued. 3.2.3. Determination of Pesticide Dosage To determine the amount of pesticide that should be administered for this experiment, we mimicked exposure in songbirds residing in Canadian orchards. Average DDE in American robin eggs from the Okanagan Valley in 1993-95 was 85.1 ± 10.8 mg/kg, wet weight, with levels reaching 232 ±61.0 mg/kg (Harris et al. 2000b). Eastern bluebird eggs contained levels of DDE as high as 105.1 pg/g, wet weight (Bishop et al. 1998). From these examples, 100 mg/kg was chosen as the value of DDE that should be present in egg contents at the time of egg laying. The mean egg-to-whole-body ratio for DDE in Herring gulls (Larus argentatus) is 0.56 (Braune and Norstrom 1989). Therefore, if eggs have 100 mg/kg of DDE residues, the whole body burden we would like to achieve in birds is 178 mg/kg. Zebra finches generally lay their first egg six days 36 after pairing. Birds were dosed for 3 weeks prior to pairing and then one week following pairing. One week after pairing they should have started laying. If birds were dosed for four weeks (three weeks prior to pairing plus one week after pairing), body burdens should have DDE residue levels of approximately 178 mg/kg. Therefore assuming a daily additive effect when ingesting DDE, it was determined that 34 mg/kg should be ingested daily in order to have a body burden level of 178 mg/kg following four weeks of exposure. Therefore each bird was offered three grams of egg food daily containing 34 mg/kg p,p '-DDE for four weeks. To determine the dosage of azinphos-methyl that should be administered to Zebra finches that would produce 20-25% brain cholinesterase (ChE) inhibition (which is indicative of exposure; Ludke et al. 1975, Busby et al. 1981), a range finding experiment was initially conducted (see Chapter 2). This study found that approximately 18.4 mg/kg of azinphos-methyl resulted in this brain ChE inhibition; therefore this dosage was used throughout the study. Mancozeb was administered at 4.82 times the amount of azinphos-methyl, based on the concentration sprayed relative to azinphos-methyl in orchards, as the two are commonly applied as tank mixtures. Therefore, the dosage of mancozeb administered for this study was 88.7 mg/kg. 3.2.4. Dosing procedure Birds were dosed with technical gradep,p '-DDE [2,2-bis(4-chlorophenyl)-l,l-dichloroethelene, Sigma-Aldrich, Oakville, Ontario, 99 % purity]. The pesticide was initially dissolved in corn oil. The insecticide - corn oil mixture (or plain corn oil for controls) was incorporated into the egg food and three grams were offered daily per bird, most of which was consumed following 24 hours. Dosage prepared contained 34 mg/kg p,p '-DDE. In order to validate the methodology used to prepare the contaminated egg food, two food samples from a previous experiment were analyzed for p,p '-DDE content and confirmed to have the desired dosage (36.8 and 39.4 mg/kg 37 p,p '-DDE; see Chapter 2). In addition one contaminated and one uncontaminated sample were chosen randomly and sent in from this study to confirm consistent preparation of the egg food. Birds were orally dosed with technical grade azinphos-methyl (Guthion™, Supelco, I Bellefont, Pennsylvania, 100 % purity, NEAT) or mancozeb (Sigma-Aldrich, Oakville, Ontario, > 75% purity). The insecticide was dissolved in corn oil and administered via intubation to individual birds. Each bird received 0.1 ml of the insecticide - corn oil solution (or plain corn oil for controls). Dosage was calculated based on the average weight per bird, which was 15.0 ± 0.3 grams. Birds were fasted for one hour prior to dosing and were given food and water ten to 15 minutes after dosing. 3.2.5. Blood sampling Blood samples (200 ul) were collected from the brachial vein from female Zebra finches at the one-egg stage. Upon collection, blood was centrifuged for ten minutes at 5,000 r.p.m. Following centrifugation, the plasma component was removed and stored at -20°C until analysis. In addition 40 u.1 of blood was collected using heparinized capillary tubes to determine percent hematocrit and leucocrit. Once collected, the capillary tubes were sealed with critoseal at the bottom and left to stand in a vertical position. Blood was centrifuged for three minutes at 5,000 r.p.m. after which the height of the leucocrit, hematocrit and total sample volume was immediately measured, using a digital caliper (± 0.01mm). The measurer stayed consistent throughout all the samples. In addition, blood smears were made for each bird upon collection of blood for differential white blood cell counts. Each blood sample was smeared on two individually marked microscope slides, fixed in methyl alcohol, and stained with Hemacolor stain (VWR Canlab). Slides were used to estimate the proportion of different leucocytes. The proportion of different types of leucocytes was assessed on the basis of an examination of a total of 100 leucocytes under oil 38 immersion. The total leucocyte count included heterphils, eosinophils, lymphocytes and monocytes. In the analyses, only data for heterophils and lymphocytes, as the most numerous immune cells, are used and the ratio reported (hereon referred to as the H/L ratio). This ratio is widely used as a stress estimator in poultry and is known to increase in response to various stressors (Gross and Siegel 1983, Maxwell 1993). 3.2.6. Egg sampling The second egg was removed from each nest and replaced with a substitute egg to potentially reduce the risk of nest abandonment. Each egg was weighed and the contents emptied into an acetone / hexane rinsed jar and immediately frozen at -20°C and archived. A subsample of three eggs (one from a control nest and two from contaminated nests) from a preliminary experiment was analyzed forp,p '-DDE content. In addition one egg for a contaminated nest was chosen randomly from this study and sent in to confirm exposure to p,p '-DDE. Shell thickness of each second egg was determined. Eggshells were air-dried for approximately three months prior to measuring thickness. Shell thickness, including intact membrane, was measured using a gauge micrometer (± 0.005 mm; The Dyer Company, Lancaster, Pennsylvania). Three measurements were taken around the midline of the egg and averaged. The measurer stayed consistent throughout all the samples. 3.2.7. Analysis ofplasma for egg-yolk precursors Plasma samples were assayed for VTG using the zinc method developed for the domestic hen (Zinc kit - Wako Chemicals, Virginia, USA; Mitchell and Carlisle 1991), and validated for passerines (Williams and Martinylek 2000, Challenger et al. 2001). This assay measures total plasma zinc, and then separates the zinc bound to serum albumin from that bound to VTG and 39 VLDL by depletion of the yolk precursors from the plasma sample by precipitation with dextran sulphate. The depleted plasma sample is then assayed for zinc. Vitellogenic zinc (VTG-Zn) is equal to the difference between total and depleted zinc; VLDL accounts for only 2% of total plasma zinc (Mitchell and Carlisle 1991). Inter-assay coefficient of variation for total plasma zinc was 11.1% and for depleted zinc was 3.7% using a laying hen plasma pool. Intra-assay coefficient of variation for total plasma zinc was 2.2 % and for depleted zinc was 5.9 %. All assays were run using a 96-well microplate and measured using a Biotek 340i microplate reader. VLDL was assayed using a triglyceride kit (Triglyceride E kit - Wako Chemicals, Virginia, USA) according to the method of Mitchell and Carlisle (1991). Validation of this method for passerines was described by Williams and Christians (1997). This method measures total VLDL by cleaving the fatty-acid chains off the triglyceride molecules (which is made up of non-laying, generic VLDL and estrogen-dependent, yolk-targeted VLDL) resulting in free glycerol molecules. The concentration of plasma glycerol is proportional to the plasma concentrations of triglyceride and VLDL. Inter-assay coefficient of variation was 14.8 % using 19-week hen plasma pool and intra-assay coefficient of variation was 6.1 %. All assays were conducted using 96-well microplates, and measured using a Biotek 340i microplate reader. 3.2.8. Organochlorine (OC) analysis Egg, food and liver samples were sent to the Great Lakes Institute for Environmental Researh (GLIER) at the University of Windsor in Windsor, Ontario for measurement of OC content. Due to insufficient sample size, percent moisture was not determined in all of the samples. Methodology used to determine OC concentrations is summarized in Chapter 2. Organochlorines analyzed for include 1,2,4,5-tetrachorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, a-hexachlorocyclohexane, p-hexachlorocyclohexane, y-40 hexachlorocyclohexane, octachlorostyrene, heptachlor epoxide, oxy-chlordane, gammaftrans)-chlordane, alpha(cis)-chlordane, trans-nonachlor, cis-nonachlor, p,p '-DDE,p,p '-DDD,p,p -DDT, dieldren, mirex, photomirex. 3.2.9. Statistical Analysis Statistical analyses were performed using TMP software program (SAS Institute Inc 2000). All parameters were tested for normality (Shapiro-Wilk W Test) prior to analysis. Those not meeting the requirements were login transformed prior to analysis. These variables included: female mass at time of bleed, VLDL and H/L ratio. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analysis. Prior to further analyses, a bivariate analysis was conducted to determine if the parameters chosen for this study were correlated with female mass. This was only true for mean egg weight. Therefore, to compare the mean egg weight among treatments, an analysis of covariance (ANCOVA) was used, with female mass as a covariate. A least squared analysis of variance (ANOVA) was conducted to see if a difference existed amongst the remaining seven treatments with the following parameters: clutch size, laying interval, mean shell thickness, VTG, VLDL, H/L ratio and % hematocrit and leucocrit. When a difference existed amongst treatments, Tukey's HSD for multiple comparisons was applied to determine which treatment differed. Further statistical analyses were also conducted to determine the individual pesticide effects on reproductive, hematological and yolk precursor parameters (i.e. birds previously exposed to p,p '-DDE one year prior to the start of the experiment, and those exposed to azinphos-methyl or mancozeb). 3.3. Results 3.3.1 Validation of methodology and dosing 41 Residues ofp,p '-DDE in contaminated egg food collected from this study was 24.0 mg/kg. In addition one control sample was analyzed and contained minimal residues ofp,p '-DDE (0.2 mg/kg). Egg residues ofp,p '-DDE from contaminated parents ranged from 36.4-43.6 mg/kg, while the control egg had minimal levels (0.1 mg/kg). An additional egg sample collected from this experiment contained 23.4 mg/kg ofp,p '-DDE. Organochlorine liver residues in female Zebra finches exposed to p,p '-DDE approximately one year previously were generally low (1.96 ± 0.26 mg/kg; range: 0.44-4.92 mg/kg; Table 3.2). In addition to p,p '-DDE, birds also had low levels (<0.5 mg/kg) of y-hexachlorocyclohexane residues in their liver (0.17 ± 0.02 mg/kg; ). All other OC compounds tested for (see Methods) were non-detectable. 3.3.2. Egg production, yolk precursor levels and hematological parameters Table 3.3 summarizes the reproductive output of Zebra finch pairs amongst the different treatment groups up to clutch completion. In all treatment groups, the proportion of nests in which eggs were laid was high (60.0 - 85.7 %) with no difference between treatments (G-test, P=0.9540). Clutch size ranged from one to nine eggs with no difference between treatments (F6,35=0.28, P=0.9407) and laying interval ranged from one to 14 days with no difference between treatments (F6,35=0.34, P=0.9110). In an overall analysis of mean egg mass, there were few significant treatment effects, in particular with azinphos-methyl and mancozeb exposure. However, mean egg mass was significantly higher in birds exposed to p,p '-DDE + corn oil (with previous exposure to p,p -DDE) as opposed to corn oil + corn oil and corn oil + mancozeb, both of which had no previous pesticide exposure (F6,33=3.92, P=0.0046). While the overall model for shell thickness suggested a significant effect of treatments (F6)35=2.52, P=0.0392), when Tukey's HSD for analysis of multiple comparisons was applied there was no significant difference noted 42 amongst treatments (range: 0.052-0.058 mm). The yolk precursor VTG ranged from 1.036-1.715 pg/ml, zinc, with no difference amongst treatments (F6,3o=0.85, P=0.5455; Figure 3.1). Circulating levels of VLDL ranged from 3.907-13.247 mg/ml, triglyceride. In an overall analysis of VLDL, there were few significant treatment effects, however levels were significantly lower in birds exposed to p,p '-DDE + corn oil (with previous exposure to p,p -DDE) when compared to those exposed to corn oil + corn oil and corn oil + azinphos-methyl (both with no previous exposure top,p -DDE) (F6,33=3.75, P=0.0059; Figure 3.2). There was no difference in the hematological parameters tested in all the treatment groups (P>0.05 for all; Table 3.6). The H/L ratio ranged from 0.15-1.20. Percent hematocrit and leucocrit ranged from 42.5-48.1% and 0.35-0.87%, respectively. Some birds were exposed to p,p '-DDE one year prior to the start of this experiment. Additional statistical analyses were conducted to determine the effect of this pretreatment on the various parameters measured. Of the reproductive parameters measured, only egg weight showed a significant difference in birds with pretreatment of p,p '-DDE when compared to those with no pretreatment (Table 3.4). We found that those with pretreatment had heavier eggs than those with no pretreatment (F1,38=10.29, P=0.0027). Circulating levels of both VTG and VLDL were lower in birds with pretreatment ofp,p '-DDE than those with no pretreatment (VTG: FU5=5.40, P=0.0260; VLDL: FU8=8.48, P=0.0060) (Figure 3.3). Although the H/L ratio and percent hematocrit were not different in the two groups (P>0.05 for both), percent leucocrit was higher in birds pretreated withp,p '-DDE than those with no pretreatment (Fi,38=4.82, P=0.0343; Table 3.7). There was no effect of the second dose administered (oil, azinphos-methyl or mancozeb) on the reproductive parameters tested for (Table 3.5), circulating levels of VTG and VLDL (Figure 3.4), and hematological parameters measured (Table 3.8) (P>0.05 for all). 43 3.4 Discussion The purpose of this study was to determine the sub-lethal effects on Zebra finches following exposure to p,p '-DDE, azinphos-methyl and mancozeb administered alone and in combination with one another by measuring both the reproductive output up to clutch completion and general health of the bird (using hematological parameters as an index). We chose to study the effects of pesticides on the pre-hatch period as songbirds nesting in orchards (i.e. Eastern bluebirds, American robins) commonly lay second clutches later in the season and during this time (i.e. during egg formation and early stages of clutch initiation) birds can be exposed to a single spray event. This study attempted to mimic field exposure of songbirds to the pesticides and exposure was confirmed as follows: a) residues of p,p '-DDE in eggs of Zebra finches were similar to levels found in eggs of wild songbirds nesting in Canadian orchards, b) azinphos-methyl exposure occurred at the dosage resulting in 20-25 % cholinesterase activity inhibition (see Chapter 2) with this level of inhibition being indicative of OP exposure (Ludke et al. 1975, Busby et al. 1981), while the mancozeb dosage was administered relative to the field exposure of the fungicide in comparison to azinphos-methyl, and c) dosing of the current-use pesticides occurred during the time of egg formation, which is when the second clutch laid by songbirds in a single breeding season would be exposed. The main results of this chapter suggest that current exposure to p,p, '-DDE and current-use pesticides had no effect on the reproductive or hematological parameters measured. However, additional analyses suggested that birds exposed to p,p '-DDE approximately one year previous to the start of the experiment have 1) heavier mean egg mass, 2) lower levels of circulating VTG and VLDL, and 3) higher percent leucocrit values suggesting immunostimulation. These results will be further discussed below. 44 3.4.1. Validation of methodology and dosing Birds in our study were confirmed to be orally exposed to concentrations of p,p '-DDE similar to songbirds in Canadian orchards. Harris et al. (2000b) showed that earthworms in the Okanagan Valley orchards contained average levels as high as 52 mg/kg of DDE. Our birds were dosed at 34 mg/kg, which was confirmed by analysis of diet which contained p,p '-DDE in the range 24.0-39.4 mg/kg. In addition, eggp,p '-DDE residues in Zebra finches from our study were similar to levels found in songbirds breeding in Canadian orchards. Robins in Ontario orchards had from 30 to 44 mg/kg DDE in eggs from 1993 to 1995 (Harris et al. 2000b) and average DDE in Okanagan orchards from 1993-95 and 1997-98 was 39.3 mg/kg (range: 4.8-302 mg/kg) (Gill et al. 2003). Of the subsample collected from a preliminary Zebra finch laboratory study where dosing followed the same regime as our study, eggs from p,p '-DDE exposed nests contained from 23 to 44 mg/kg. 3.4.2. Effects of current exposure to p,p '-DDE and current-use pesticides on early reproductive output and hematological parameters In an overall analysis of current pesticide exposure, there were few significant treatment effects. In our study, mean egg mass was 15-18% higher in thep,p'-DDE + corn oil (range: 1.131-1.329 g) treatment group when compared with the true controls (corn oil + corn oil; range: 0.925-1.095 g) and those exposed to oil + mancozeb (range: 0.945-1.315 g). Williams (1996) showed that unmanipulated Zebra finches had a mean egg mass of 1.133 ± 0.012 g. As this is lighter than the mean egg mass in the p,p '-DDE + corn oil treatment group, this further supports the finding of heavier eggs in our study. This is in contrast to a laboratory study conducted by Jefferies (1971) in a similar songbird species, which showed that Bengalese finches (Lonchura striata) dosed with p,p '-DDE laid significantly lighter eggs. In addition, our study also showed that birds exposed to 45 p,p '-DDE + corn oil had lower VLDL levels when compared to birds exposed to corn oil + corn oil and corn oil + azinphos-methyl, however the remaining treatments did not differ, and there was no effect on the second yolk precursor, VTG. The lack of systematic differences in mean egg mass and VLDL levels suggest that a possible confounding factor may be driving these results. In our study we found no difference amongst the various treatment groups for clutch size, i.e. azinphos-methyl, mancozeb orp,p '-DDE alone or in combination did not affect this parameter. Mean clutch size in our study was also comparable to previous studies. For example, Salvante and Williams (2002) found that unmanipulated Zebra finches had a mean clutch size of 5.4 ± 0.2 eggs and in our study, mean clutch size ranged from 4.8 to 5.8 eggs amongst the treatments. Other studies have similarly found no effect on clutch size following pesticide exposure (Powell 1984, Busby et al. 1990, Elliott et al. 1994). American robins nesting in Okanagan Valley orchards in British Columbia from 1990-91 (containing up to 103 mg/kg DDE) did not show a difference in clutch size when compared to non-orchards sites (Elliott et al. 1994). In a follow-up study, robins nesting in orchards (containing up to 302 mg/kg DDE) actually showed an increase in clutch size when compared with those nesting in non-orchard sites (Gill et al. 2003). It was suggested that this might be due to an ecological advantage of orchard habitat for robins compared to the non-orchard habitat chosen for that particular study or the possibility of development of resistance to DDT and related chemicals after twenty-five generations of exposure. Another orchard study looking at the effects of spraying with the OP azinphos-methyl found that although spraying had no effect on clutch size, the proportion of nests with unhatched eggs was significantly higher in sprayed orchards in American robins (Gill et al. 2000). Field studies conducted looking at exposure effects to a combination of pesticides also showed no relationship with clutch size (Fluetsch and Sparling 1994, Bishop et al. 2000b). American robins exposed to organophosphates (including azinphos-methyl), carbamates and organochlorine pesticides in apple 46 orchards in Pennsylvania showed no difference in clutch size (Fluetsch and Sparling 1994). Bishop et al. (2000b) used toxicity scores to quantify pesticide exposure (including OCs and OPs) to Tree swallows and Eastern bluebirds nesting in Ontario orchards and showed no evidence of declining clutch size with increasing toxicity score. Our study further supports the results of the above studies and thus pesticide exposure does not appear to negatively impact clutch size in songbird species. Historically, negative effects of DDT-related compounds on eggshell quality are common in raptorial and fish-eating avian species, for example Brown pelicans (Pelecanus occidentalis), Ospreys (Pandion haliaetus), Bald eagles (Haliaeetus leucocephalus) and Prairie falcons (Falco mexicanus) (Anderson et al. 1975, Fyfe et al. 1988, Wiemeyer et al. 1988, Wiemeyer et al. 1993, Blus 1996). It has been suggested that this is due to their feeding higher up on the food chain, which increases sensitivity to DDE effects on eggshell quality. In contrast, songbird species have been considered at reduced risk to the effects of persistent chlorinated hydrocarbons on reproduction due to their feeding lower on the food chain, and to a lack of evidence showing effects of DDE on shell quality of songbirds (Blus 1996). Our results are consistent with this idea in that p,p '-DDE exposed Zebra finches did not produce eggs with thinner shells than those with no p,p '-DDE exposure. In addition, further exposure to the current-use pesticides azinphos-methyl or mancozeb in combination with p,p '-DDE did not cause any eggshell thinning effect in Zebra finches. Hematological parameters were not different amongst the treatment groups tested and were comparable to values from other studies. The H/L ratios in our study ranged from 0.15-1.20 amongst the treatments. Percent hematocrit and leucocrit in our study ranged from 42.5-48.1 and 0.35-0.87, respectively. Williams and Christians (1997) showed female Zebra finches to have an average of 46% for hematocrit and 0.6% for leucocrit, which are close to the values in our study. These results suggest that current exposure to residual p,p '-DDE and the current-use pesticides 47 azinphos-methyl and mancozeb are not negatively impacting the health of songbirds in terms of the parameters measured in this study. 3.4.3. Effects of p,p '-DDE pre-treatment on early reproductive output and hematological parameters As some treatment groups for this study contained Zebra finches that were exposed to p,p '-DDE approximately one year previously, additional analyses were conducted to determine its effects. We found that pre-treatment with p,p '-DDE resulted in a) heavier mean egg mass, b) lower circulating levels of both VTG and VLDL in the plasma of laying females, and c) an increase in percent leucocrit. The finding of lower circulating levels of yolk precursors contradicts previous studies that show that short-term exposure to DDT resulted in induction of the yolk precursors. In fish, DDT exposure at environmentally relevant levels induced VTG production (Donohoe and Curtis 1996, Metcalfe et al. 2000); however, this induction has only been reported in avian species following exposure to extremely high concentrations (above environmentally significant levels) (Lorenzen et al. 2001). Although, several previous studies have suggested that plasma VTG may be a useful bioindicator of estrogenic effects of xenobiotics, this has mainly been through expression of VTG in immature, non-breeding females or males that normally have non-detectable levels of VTG (Palmer et al. 1998, Donohoe and Curtis 1996, Metcalfe et al. 2000). Our study used breeding females that already have elevated levels of the yolk precursors. As few studies exist looking at VTG levels in avian species following DDT exposure and the usage of breeding females with already elevated levels of yolk precursors in our study, the results discussed below warrant further study for conclusive remarks. 48 It is not too surprising that egg mass increased even though VTG and VLDL levels decreased. Previous studies have shown a weak negative relationship between egg mass and plasma yolk precursor levels in Zebra finches fed a high quality diet (Salvante and Williams 2002). Lower circulating levels of VTG and VLDL in laying females following pre-treatment with p,p '-DDE could be due to greater uptake of the yolk precursors by the egg during its formation (Christians and Williams 2001). It has been seen previously that yolk mass is positively correlated with the rates of yolk precursor uptake in female Zebra finches (Christians and Williams 2001). Heavier egg mass seen in birds with pre-treatment of p,p '-DDE exposure further supports this hypothesis. Alternatively, the decrease in circulating yolk precursors in pretreated birds may be due to a decrease in the livers' ability to produce VTG and VLDL one year following exposure to p,p '-DDE. However, the mass and activity of the livers were not determined following exposure to the contaminant in this study. Pre-treatment with p,p '-DDE resulted in an increase in percent leucocrit suggesting immunostimulation in Zebra finches. This is consistent with the earlier study (see Chapter 2) where Zebra finches exposed to p,p '-DDE one year previously had a greater immune response to those not pretreated. These findings are consistent with a study of Tree swallows residing in Ontario orchards, which reported that Tree swallows exposed to orchard pesticides, including azinphos-methyl and p,p '-DDE, were immunostimulated (Bishop et al. 1998). Our study conducted in a laboratory setting further supports this suggestion of immunostimulation in songbirds exposed to orchard pesticides, which contradicts the immunosuppression that has been seen previously in mammalian studies (Vos et al. 1989, Dean and Murray 1991, Pruett et al. 1992, Pruett 1994). 49 3.4.4. Conclusions In conclusion, we found little evidence that dosing of breeding female Zebra finches with current-use pesticides, including azinphos-methyl and mancozeb, either alone or in combination with p,p '-DDE, had significant negative effects on early reproductive traits (timing of laying, egg size and number, yolk precursor levels) or immune function (percent hematocrit and leucocrit and H/L ratio). However, birds with previous exposure to p,p '-DDE (approximately one year ago) may be impacted in terms of egg mass, yolk precursors and immune response. 50 Table 3.1. Summary of pesticide history and treatment in male and female Zebra finches (Taeniopygia guttata) used to determine the effects ofp,p '-DDE and current-use pesticides on egg production and yolk precursor levels in the Zebra finch. Dosage group Previous exposure to p,p '-DDE1 n Corn oil + corn oil No 12 Corn oil + corn oil Yes 6 Corn oil + azinphos-methyl No 10 Corn oil + mancozeb No 10 p,p '-DDE + corn oil Yes 7 p,p '-DDE + azinphos-methyl Yes 5 p,p'-DDE + mancozeb Yes 7 Previous exposure to p,p '-DDE (34 mg/kg) occurred approximately one year ago in females Table 3.2. Organochlorine concentrations (mg/kg, wet weight) in female Zebra finch (Taeniopygia guttata) livers exposed to p,p '-DDE (34 mg/kg) approximately one year prior to tissue collection. No other organochlorine compounds tested for (see Section 3.2.8) were detected. Sample Days since exposure % lipid % moisture1 p,p'-DDE y-HCH2 1 317 2.08 n.d. 1.37 0.08 2 362 2.74 n.d. 4.92 0.37 3 363 1.92 n.d. 0.44 0.08 4 152 3.33 n.d. 1.78 0.19 5 316 2.03 n:d. 1 91 0.24 6 152 1.69 n.d. 1.33 0.06 ln.d. = not determined 2Y-HCH=y-hexachlorocyclohexane 52 Table 3.3. Comparison of reproductive output up to clutch completion in Zebra finch (Taeniopygia guttata) pairs exposed to a combination ofp,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb. Statistical analysis to determine the proportion of nests lay was conducted using the G-test. Mean egg mass was analyzed using an analysis of covariance (ANCOVA) with female weight as a covariate. Remaining analyses were conducted using a least i squares analysis of variance (ANOVA). Values given (excluding proportion of nests lay) are the presented as the least squared mean ± standard error with the sample size in parentheses. Different letters indicate significantly different treatments (P<0.05 with Tukey's HSD for multiple comparisons). Dosage group1 Prop, of Clutch Laying Mean egg Shell thickness nests lay size interval weight (g) (mm) Corn oil + 75.0% 4.8±0.7 8.0+1.2 1.02 + 0.03 0.0546 + 0.0004 corn oil (12) (9) (9) (9) a (8) Corn oil + 83.3 % 5.2 + 0.9 8.4+1.6 1.16 + 0.04 0.0556 + 0.0005 corn oil (DDE) (6) (5) (5) (5) ab (5) Corn oil + 70.0 % 5.6 + 0.7 8.9+1.3 1.10 + 0.03 0.0546 ± 0.0004 azinphos-methyl (10) (7) (7) (7)ab (7) Corn oil + 80.0 % 5.8 + 0.7 7.1 ± 1.2 L04±0.03 0.0562 + 0.0004 mancozeb (10) 1 (8) (8) (8) a (8) p,p'-DDE + 85.7 % 5.6 + 0.8 6.8+1.6 1.20 + 0.04 0.0558 + 0.0005 Corn oil (DDE) (7) (5) (5) (5)b (5) p,p '-DDE + 60.0 % 5.7 + 1.1 7.7 + 2.0 1.08 + 0.05 0.0543 ± 0.0006 53 azinphos-methyl (5) (3) (3) (3)ab (3) (DDE) p,p'-DT>E + 71.4% 5.6 + 0.9 6.6 ± 1.6 1.09 ±0.04 0.0550 ± 0.0005 mancozeb (DDE) (7) (5) (5). (5) ab (5) P-value 0.9540 0.9407 0.9110 0.0046 0.0392 ^ D E in parentheses indicates those with previous exposure to p,p '-DDE (34 mg/kg) approximately one year ago. 54 Table 3.4. Comparison of reproductive output up to clutch completion in Zebra finch (Taeniopygia guttata) pairs exposed to p,p '-DDE approximately one year prior to the start of the experiment. Statistical analysis to determine the proportion of nests lay was conducted using the chi-squared test. Mean egg mass was analyzed using an analysis of covariance (ANCOVA) with female weight as a covariate. Remaining analyses were conducted using a least squares analysis of variance (ANOVA). Values given (excluding proportion of nests lay) are the presented as the least squared mean + standard error with the sample size in parentheses. Dosage group Prop, of Clutch Laying Mean egg Shell thickness nests lay size interval weight (g) (mm) Corn oil 75.0 % 5.3+0.4 8.0 + 0.7 1.05 + 0.02 0.0551 +0.0003 (32) (24) (24) (24) (23) p,p'-DDE 76.0 % 5.5 + 0.4 7.3 + 0.8 1.14 + 0.02 0.0553 ± 0.0003 (25) (18) (18) (18) (18) P-value 0.6553 0.7571 0.5565 0.0027 0.7327 55 Table 3.5. Comparison of reproductive output up to clutch completion in Zebra finch (Taeniopygia guttata) pairs exposed to the current-use pesticides azinphos-methyl and mancozeb. Statistical analysis to determine the proportion of nests lay was conducted using the G-test. Mean egg mass was analyzed using an analysis of covariance (ANCOVA) with female weight as a covariate. Remaining analyses were conducted using a least squares analysis of variance (ANOVA). Values given (excluding proportion of nests lay) are the presented as the least squared mean ± standard error with the sample size in parentheses. Dosage group Prop, of Clutch Laying Mean egg Shell thickness nests lay size interval weight (g) (mm) Corn oil 80.0 % 5.1 ±0.4 7.8 ±0.8 1.11 ±0.02 0.0552 ±0.0003 (25) (19) (19) (19) (18) Azinphos-methyl 66.7 % 5.6 ±0.5 8.5± 1.1 1.10 ±0.03 0.0545 ± 0.0004 (15) (10) (10) (10) (10) Mancozeb 76.5 % 5.7 + 0.5 6.9 ± 0.9 1.06 + 0.03 0.0558 + 0.0003 (17) (13) (13) (13) (13) P-value 0.9900 0.5893 0.5387 0.2731 0.0501 56 Table 3.6. Hematological parameters of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage exposed to various combinations of p,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb. Statistical analyses were conducted using a least squared analysis of variance (ANOVA). The heterophil: lymphocyte ratio (H/L ratio) was login transformed prior to analysis. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analyses. Values are expressed as the least squares mean + standard error with the sample size in parentheses. Dosage group H/L ratio Hematocrit (%) Leucocrit (%) Corn oil + corn oil 0.23 ± 0.29 (8) 47.8 ± 1.7(8) 0.51 ±0.11 (8) Corn oil + corn oil (DDE) 0.19 ±0.37 (5) 42.5 ±2.1 (5) 0.64 ±0.14 (5) Corn oil + azinphos-methyl 0.30 ±0.31 (7) 47.0 ± 1.8 (7) 0.47 ±0.12 (7) Corn oil + mancozeb 1.20 ±0.29 (8) 46.8 ± 1.7(8) 0.35 ±0.13 (6) p,p '-DDE + corn oil (DDE) 0.15 ±0.37 (5) 48.1 ±2.1 (5) 0.50 ±0.14 (5) p,p '-DDE + azinphos-methyl (DDE) 0.30 + 0.48 (3) 45.6 ±2.8 (3) 0.58 ±0.18 (3) p,p '-DDE + mancozeb (DDE) 0.42 ± 0.34 (6) 47.6 ±2.0 (6) 0.87 ±0.13 (6) P-value 0.4567 0.5438 0.1608 *DDE in parentheses indicates those with previous exposure to p,p '-DDE (34 mg/kg) approximately one year ago. 57 Table 3.7. Hematological parameters of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage exposed to p,p '-DDE approximately one year prior to the start of the experiment. Statistical analyses were conducted using a least squared analysis of variance (ANOVA). The heterophil: lymphocyte ratio (H/L ratio) was login transformed prior to analysis. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analyses. Values are expressed as the least squares mean ± standard error with the sample size in parentheses. Dosage group H/L ratio Hematocrit (%) Leucocrit (%) Corn oil p,p '-DDE P-value 0.59 + 0.18 (23) 47.2 + 1.0(23) 0.45 +0.07 (21) 0.27 + 0.20(19) 46.1 + 1.1 (19) 0.67 + 0.07(19) 0.2617 0.4673 0.0343 58 Table 3.8. Hematological parameters of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage exposed to the current-use pesticides azinphos-methyl and mancozeb. Statistical analyses were conducted using a least squared analysis of variance (ANOVA). The heterophil: lymphocyte ratio (H/L ratio) was login transformed prior to analysis. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analyses. Values are expressed as the least squares mean ± standard error with the sample size in parentheses. Dosage group H/L ratio Hematocrit (%) Leucocrit (%) Corn oil 0.23 ±0.29 (8) 46.4 ±1.1 (18) 0.54 ±0.08 (18) Azinphos-methyl 0.30 + 0.31 (7) 46.6 + 1.5 (10) 0.50 + 0.10(10) Mancozeb 1.20 + 0.29 (8) 47.1 ± 1.3 (14) 0.61+0.10(12) P-value 0.4567 0.9002 0.7625 59 2.5 -, Corn oil + Com oil + Com oil + Com oil + p,p '-DDE p,p '-DDE p,p '-DDE + com oil com oil azinphos- mancozeb + com oil + azihphos- mancozeb (DDE) methyl (DDE) . methyl (DDE) Figure 3.1. Vitellogenin (VTG) yolk precursor levels in plasma of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage (P=0.5455). Birds were exposed to p,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb, either singly or in combination. Those with DDE in parentheses indicate birds that were previously exposed to p,p '-DDE (34 mg/kg) approximately one year ago. Bars represent the least squared mean + standard error with the value above each bar representing the sample size. 60 Corn oil + Corn oil + Corn oil + Corn oil + p,p '-DDE /?,p'-DDE p,p'-DDE + com oil com oil azinphos- mancozeb + com oil + azinphos- mancozeb (DDE) methyl (DDE) methyl (DDE) (DDE) Figure 3.2. Very-low-density-lipoprotein (VLDL) yolk precursor levels in plasma of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage (P=0.0059). Birds were exposed to p,p '-DDE and the current-use pesticides azinphos-methyl mancozeb, either singly or in combination. Those with DDE in parentheses indicate birds that were previously exposed to p,p '-DDE (34 mg/kg) approximately one year ago. Bars represent the least squared mean ± standard error with the value above each bar representing the sample size. Different letters indicate significantly different treatments (P<0.05 with Tukey's HSD for multiple comparisons). 61 Corn oil p,p '-DDE Figure 3.3. Vitellogenin (VTG; P=0.0260) and very-low-derisity-lipoprotein (VLDL; P=0.0060) yolk precursor levels in plasma of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage in birds exposed to p,p '-DDE (34 mg/kg) approximately one year prior to the start of the experiment. Bars represent the least squared mean ± standard error with the value above each bar representing the sample size. 62 Figure 3.4. Vitellogenin (VTG; P=0.9346) and very-low-density-lipoprotein (VLDL; P=0.0719) yolk precursor levels in plasma of female Zebra finches (Taeniopygia guttata) bled at the one-egg stage in birds exposed to current-use pesticides. Bars represent the least squared mean ± standard error with the value above each bar representing the sample size. 63 CHAPTER 4 Effects of chronic p,p '-DDE exposure in combination with current-use pesticides in breeding Zebra finches (Taeniopygia guttata) 4.1 Introduction Twenty years following its discontinuation, organochlorine (OC) compounds, such as DDT [EEl-trichloro j^Z-i/'s^ -chloropheny^ethane] and its metabolite DDE [2,2- bis(4-chlorophenyl)-l,l-dichloroethylene] still exist in orchard environments (Harris et al. 2000b). Today, these orchards are regularly sprayed with organophosphates (OPs) and fungicides throughout the growing season of the fruit crops. During this time, orchards are used by various songbird species for breeding habitat, including American robins (Turdus migratorius), Tree swallows (Tdchycineta bicolor), House wrens (Troglodytes aedon) and Eastern bluebirds (Sialia sialis) (Elliott et al. 1994, Bishop et al. 2000a). In addition to accumulating body burdens of the persisting chemicals of DDT and its metabolites (Hebert et al. 1994, Gill et al. 2003), songbirds breeding in the orchard environments are therefore also exposed to OPs and fungicides that are currently used today. DDT and DDE exposure has been strongly correlated with eggshell thinning (Elliott and Martin 1994, Wieymeyer and Porter 1970, Cooke 1973, Jefferies 1971, Longcore et al. 1971) and reduced productivity (Wieymeyer et al. 1988, Ratcliffe 1980, Jefferies 1971, Longcore and Stendell 1977) in raptor and fish-eating species. Songbird species appear to be more tolerant to residual OC contaminants in the environment. In a study in the Okanagan Valley, British Columbia, American robins had higher productivity when compared to robins nesting in non-orchard habitat in the lower mainland, British Columbia (Gill et al. 2003). Organophosphate exposure effects on reproductive success in songbirds have also been examined and exposure has been associated with decreased productivity in Tree swallows, Eastern bluebirds (Bishop et al. 64 1998) and American robins (Gill et al. 2000, Fluetsch and Sparling 1994). Grue et al. (1982) found that European starlings (Sturnus vulgaris) made fewer trips to young after one OP (dicrotophos) spray event. Of the few studies that exist which take into account the effects of multiple pesticide exposure (including residual OCs and current-use OPs and fungicides) on songbirds, reproductive success and general health of the bird (i.e. immune function) appears to be negatively impacted with exposure (Bishop et al. 1998, Bishop et al. 2000a). However, it is difficult to discern whether the impact of specific pesticide exposure is significant alone or in combination with other pesticides. The aim of this study was to mimic spray regimes of orchard field sites in a controlled laboratory setting to determine the effects of previous and current-use pesticides on the reproduction and health of songbirds. Zebra finches (Taeniopygia guttata) were used as a model songbird species and were exposed to p,p '-DDE in combination with the current-use OP azinphos-methyl and fungicide mancozeb. The latter two pesticides are commonly used in orchards today, applied either alone or as tank mixtures (Sinclair and Elliott 1993, Norecol Dames & Moore 1997). We first assessed the effects ofp,p '-DDE exposure on the early reproductive effort (up to clutch completion), including clutch size, laying interval, egg mass and shell thickness (since at this point birds are not routinely exposed to OP sprays). Following this, the effects of p,p '-DDE in combination with current-use pesticides was determined by assessing a) the general health of the bird by measuring various hematological and immunological parameters, and b) subsequent reproductive effort (from clutch completion to fledging), including hatch rate, brood size and fledge rate. 65 4.2 Methods 4.2.1. Animals and Husbandry This study was conducted on a captive colony of Zebra finches maintained at the Simon Fraser University Animal Care Facility located in Burnaby, British Columbia. Zebra finches were housed in a controlled environment (temperature 19-23°C; humidity 35-55%; photoperiod = 14 hr L: 10 hr D, lights on at 0700). All birds were provided with fresh mixed seed (panicum and white millet, 1:2; 11.7% protein, 0.6% lipid and 84.3% carbohydrate by dry mass), water, grit and cuttlefish bone (calcium) ad libitum plus a multivitamin supplement in the drinking water once per week. Experiments and animal husbandry were carried out under a Simon Fraser University Animal Care Committee permit, in accordance with guidelines from the Canadian Committee on Animal Care (CCAC). 4.2.2. Experimental design This experiment was carried out in two trials in December 2001 and May 2002. Initially, 68 Zebra finches were divided into cages (61 X 46 X 41 cm) separating males from females with five to seven birds per cage. Upon grouping, the birds were given at least 24 hours to acclimatize to their environment and assigned to a treatment group at random (see Table 4.1 for treatments). Birds in this study received a supplemental diet of egg food (20.3% protein: 6.6% lipid) during the egg-laying and chick-rearing stages. This consisted of a mixture of hard-boiled eggs, breadcrumbs and corn meal, which the birds readily consumed. Fifty-one birds of each sex were chronically dosed with p,p '-DDE at 34 mg/kg. The remaining 17 of each sex were dosed with corn oil only. The egg food was the vehicle for the insecticide / corn oil and three grams were offered daily per bird, most of which was consumed within the following 24 hours. Four females and one male being dosed with p,p '-DDE were discontinued prior to the completion of dosing due to death or harassment by cage-mates. Three 66 weeks into p,p '-DDE dosing, birds were randomly paired. Dosing of corn oil or p,p '-DDE continued for one more week for breeding pairs (four weeks total). Following the discontinuation of p,p '-DDE exposure, birds were offered clean egg food daily (approximately three grams per bird) until clutch completion (or two days after the last egg was laid) and then again during the chick-rearing stage. Each breeding pair was weighed (± 0.01 g) and placed into separate cages with a nest box (11.5X11.5X11.5 cm): Birds were given a single dose of corn oil or azinphos-methyl or mancozeb, depending on treatment group (see Table 4.1 for treatment groups; see Section 4.2.3 and 4.2.4 for dosage rationale and dosing procedure, respectively) two times after pairing in order to simulate the field spray events. This was done during mid-incubation (six days into incubation) and mid-chick rearing (chick day 12). Following pairing, birds were observed throughout a 12-week reproductive cycle and productivity was assessed. Nest boxes were monitored daily to determine timing of laying and to predict hatching date. Birds that did not lay eggs two weeks following pairing were separated and classified as "non-breeders". Eggs were numbered in consecutive order of laying and the second egg was collected from each nest and archived (see Section 4.2.5). Later, nest boxes were again checked daily to determine actual hatching dates and brood size. Clutches in which no eggs hatched three weeks following the start of incubation were discontinued. All nestlings were banded and weighed at eight days of age and weighed again at 23 days of age. The phytohemagglutinin (PHA) immune function test was conducted on adult females and chicks when chicks were 21 days old (see Section 4.2.6). Adult females and chicks were bled at the completion of the experiment (i.e. when chicks were 23 days old) (see Section 4.2.7). 67 4.2.3 Determination of Pesticide Dosage A complete explanation of pesticide dosage determination is reported in Chapter 3. However, in summary to determine the amount of pesticide exposure required, we mimicked exposure in songbirds residing in Canadian orchards. It was determined that daily dosing of p,p '-DDE at 34 mg/kg would yield body burdens of contamination similar to American robins and Eastern bluebirds nesting in Canadian orchards (Harris et al. 2000b). Chapter 2 determined that azinphos-methyl exposure of 18.4 mg/kg would result in 20-25% brain cholinesterase inhibition which is indicative of exposure (Ludke et al. 1975, Busby et al. 1981) and therefore this is the dosage used in this study. Mancozeb was administered at 4.82 times the amount of azinphos-methyl (or 88.7 mg/kg), based on the concentration sprayed relative to azinphos-methyl in orchards. 4.2.4. Dosing procedure Birds were dosed with technical gradep,p '-DDE [2,2-bis(4-chlorophenyl)-l,l-dichloroethelene, Sigma-Aldrich, Oakville, Ontario, 99 % purity]. The pesticide was initially dissolved in corn oil. The insecticide - corn oil mixture (or plain corn oil for controls) was incorporated into the egg food and three grams were offered daily per bird, most of which was consumed in the following 24 hours (the dosage prepared contained 34 mg/kgp,p -DDE). In order to validate the methodology used to prepare the contaminated egg food, two food samples from a previous experiment were analyzed for p,p '-DDE content and confirmed to have the required dosage (36.8 and 39.4 mg/kgp,p '-DDE; see Chapter 2). In addition one contaminated food sample and one uncontaminated food sample was chosen randomly and sent in from this study to confirm consistent preparation of the egg food. 68 Birds were orally dosed with technical grade azinphos-methyl (Guthion™, Supelco, Bellefont, Pennsylvania, 100 % purity, NEAT) or mancozeb (Sigma-Aldrich, Oakville, Ontario, > 75% purity) in all experiments. The insecticide was dissolved in corn oil and administered via intubation to both male and female adult finches. Each bird received 0.1 ml of the insecticide -corn oil solution (or plain corn oil for controls). Dosage was calculated based on the average weight per bird, which was 15.0 ± 0.3 grams. Birds were fasted for one hour prior to dosing and were given food and water ten to 15 minutes after dosing. 4.2.5. Egg sampling The second egg from each clutch was collected and replaced with a substitute egg to potentially reduce the risk of nest abandonment. Upon collection, eggs were weighed and the contents emptied into acetone/hexane rinsed jars and archived at -20°C. A subsample of three eggs (one from a control nest and two from contaminated nests) from a preliminary experiment was analyzed for p,p '-DDE content. An additional egg was chosen randomly and sent in from this study. Shell thickness of each second egg was determined. Eggshells were air-dried for approximately three months prior to measuring thickness. Shell thickness, including intact membrane, was measured using a gauge micrometer (± 0.005 mm; The Dyer Company, Lancaster, Pennsylvania). Three measurements were taken around the midline of the egg and averaged. Measurements were conducted by the same individual throughout. 4.2.6. Phytohemagglutinin (PHA) immune function test The PHA immune function test is now being recognized as a valuable tool to study cell-mediated immune function of wild animals (Smits et al. 1996, Smits and Williams 1999, Smits et al. 1999). This test measures the response to the plant lectin PHA, in terms of T-lymphocyte 69 proliferation. Zebra finches required that a 1 cm patch of skin on the mid-patagium of both wings be plucked of feathers. Three measurements of patagium thickness of the wings were measured to 0.01 mm using a gauge micrometer (The Dyer Company, Lancaster, PA). Each bird was then injected with 30 pi of PHA lectin solution in the left wing and 30 pi of phosphate buffered saline (PBS) in the right wing. Twenty-four hours following injection, thickness of both wings was measured at the injection site. The response was considered to be the difference between the change in thickness of the PHA-injected site and the PBS-injected site in each bird. 4.2.7. Blood sampling Blood samples (200 pi) were collected from the brachial vein from adult females and chicks at the completion of the experiment (chick day 23). Upon collection, blood was centrifuged for ten minutes at 5,000 r.p.m. Following centrifugation, the plasma component was removed and stored at -20°C until analysis. In addition, 40 ul of blood was collected using heparinized capillary tubes to determine percent hematocrit and leucocrit. Once collected, the capillary tubes were sealed with critoseal at the bottom and left to stand in a vertical position. Blood was centrifuged for three minutes at 5,000 r.p.m. after which the height of the leucocrit, hematocrit and total sample volume was immediately measured, using a digital caliper (± 0.01mm). The measurer stayed consistent throughout all samples. In addition, blood smears were made for each chick upon collection of blood for differential white blood cell counts. Each blood sample was smeared on two individually marked microscope slides, fixed in methyl alcohol, and stained with Hemacolor stain (VWR Canlab). Slides were used to estimate the proportion of different leucocytes. The proportion of different types of leucocytes was assessed on the basis of an examination of a total of 100 leucocytes under oil immersion. The total leucocyte count included heterphils, eosinophils, lymphocytes and monocytes. In the analyses, only data for heterophils and lymphocytes, as the 70 most numerous immune cells, are used and the ratio reported (hereon referred to as the H/L ratio). This ratio is widely used as a stress estimator in poultry and is known to increase in response to various stressors (Gross and Siegel 1983, Maxwell 1993). 4.2.8. Organochlorine (OC) analysis 1 Egg and food samples were sent to the Great Lakes Institute for Environmental Researh (GLEER) at the University of Windsor in Windsor, Ontario for measurement of OC content. Due to insufficient sample size, percent moisture was not determined in all of the samples. Methodology used to determine OC concentrations is summarized in Chapter 2. Organochlorines analyzed for include 1,2,4,5-tetrachorobenzene, 1,2,3,4-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, a-hexachlorocyclohexane, P-hexachlorocyclohexane, y-hexachlorocyclohexane, octachlorostyrene, heptachlor epoxide, oxy-chlordane, gamma(trans)-chlordane, alpha(cis)-chlordane, trans-nonachlor, cis-nonachlor, p,p -DDE, p,p'-DDD,p,p -DDT, dieldren, mirex, photomirex. 4.2.9. Statistical analysis 1 Statistical analyses were performed using JMP software program (SAS Institute Inc 2000). All parameters tested for were tested for normality (Shapiro-Wilk W Test) prior to analysis. Those not meeting the requirements were login transformed prior to analysis. These variables included: female weight at pairing and at time of bleed and injection, H/L ratio in chicks, and the change in response in the PHA test in adult females. As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analysis. Prior to further analysis, a bivariate analysis was conducted to determine if the parameters chosen for this study were correlated with female mass. This was true for the following parameters: laying interval and mean egg mass. Therefore to compare laying interval and mean egg mass among treatments, an analysis of 71 covariance (ANCOVA) was used, with female mass at pairing as a covariate. In chicks, percent leucocrit was correlated with chick mass and therefore an ANCOVA was used to determine the effect of treatment on percent leucocrit, with chick mass as a covariate. A least squared analysis of variance (ANOVA) was conducted to determine if a difference existed amongst the four treatments with the remaining parameters (unless otherwise stated). 4.3 Results 4.3.1. Validation of methodology and dosing Residues ofp,p '-DDE in contaminated egg food from a preliminary experiment ranged from 36.8-39.4 mg/kg (see Chapter 2). Contaminated egg food collected from this experiment contained 27.8 mg/kg p,p '-DDE while uncontaminated egg food contained 0.002 mg/kg p,p '-DDE. Preliminary experiments showed that egg residues ofp,p '-DDE levels from contaminated parents ranged from 36.37-43.59 mg/kg, while the control egg had minimal contamination (0.1 mg/kg, see Chapter 3). The egg collected for contaminated parents in this experiment contained 12.4 mg/kg p,p '-DDE 4.3.2. Effects of p,p '-DDE and current-use pesticides on reproductive parameters At completion of egg laying, birds had only been exposed to either p,p '-DDE or the corn oil (i.e. not OP or fungicide at this stage). Table 4.2 summarizes the reproductive output up to clutch completion in birds with and without p,p '-DDE exposure. In both treatments, there was no significant difference in all the parameters measured. The proportion of nests in which eggs were laid was high (77.3 - 86.7 %; chi-squared test, P=0.4351). Clutch size ranged from four to 10 (Fi,45=0.1278, P=0.7224) and laying interval (defined as the number of days between pairing and the day the first egg was laid) ranged from one to 14 days (Fi,44=5.7687, P=0.1633). Mean egg mass ranged from 0.89 - 1.28 g (Fi;44=0.0866, P=0.7699) and shell thickness ranged from 0.0507 - 0.0579 mm (FMi=l .5470, P=0.2206). The remaining reproductive parameters through incubation and chick-rearing include the potential combined effects ofp,p '-DDE and OP or fungicide exposure. Table 4.3 summarizes the reproductive output following clutch completion amongst the four treatment groups. There was no difference amongst treatments in any of the parameters measured. The proportion of nests with hatched eggs ranged from 33.3 - 63.6 % (G-test, P=0.8870). Hatch rate ranged from 19.5 to 24.1 % (G-test, P=0.9970). Brood size ranged from one to four, with one nest hatching seven chicks where all but one survived to fledging (DDE + corn oil group; F 3 j 9 = 1.2406, P=0.3227). The proportion of nest with chicks that reached fledging age ranged from 71.4 - 100 % (G-test, P=0.6730). Fledge rate ranged from 75.0 to 93.3 % (G-test, P=1.000). In addition, chick mass at day 8 ranged from 6.61 to 9.04 g (F3,16=1.4571, P=0.2636; Table 4.5) and at day 23 chick mass ranged from 10.0 to 13.8 g (F3,,6=2.3270, P=0.1134; Table 4.5). 4.3.3. Effects ofp,p '-DDE and current-use pesticides on hematological and immune function parameters Table 4.4 summarizes the hematological parameters and PHA response in adult female Zebra finches amongst the four different treatments. There was no difference between treatments for any of the parameters measured in females that successfully fledged a minimum of one chick. Percent hematocrit ranged from 42.5 to 58.6 % (F3,i6=0.2350, P=0.8706) and percent leucocrit ranged from 0.13 to 1.14 % (F3,i6=l 0500, P=0.3976). The response to PHA ranged from 034 to 0.99 mm (F3,i6=0.9381, P=0.4453). Additional analyses were conducted to determine response of the above parameters (i.e. percent hematocrit and leucocrit and change in response to PHA) including adult females that successfully laid a minimum of one egg and those which 73 successfully had a minimurn of one egg hatch, however, there was no significant difference seen (P>0.05, for all parameters). Table 4.5 summarizes the hematological parameters and PHA response in chicks of adult Zebra finches dosed with p,p '-DDE and current-use pesticides. Response of chicks did not differ between treatments for all the parameters measured. The H/L ratio in chicks ranged from 0.06 to 0.83 (F3,i5=0.3437, P=0.7941), while percent hematocrit (F3,i6=0.1265, P=0.9430) and leucocrit (F3,i5=1.6667, P=0.2166) ranged from 41.1 - 53.9 % and 0.13 -1.32 %, respectively. The response to PHA ranged from 0.41 - 1.23 mm (F3,i6=2.3792, P=0.1080). 4.4 D i s c u s s i o n The purpose of this study was to determine the sub-lethal effects on Zebra finches following exposure to p,p '-DDE, azinphos-methyl and mancozeb administered alone and in combination. Sub-lethal effects were measured by determining reproductive output and the general health of the birds. Songbirds nesting in orchards (i.e. Eastern bluebirds and American robins) commonly lay two clutches in one year. It is during the first clutch that songbirds may be exposed to spray events during the incubation and chick-rearing stages. Upon entering the orchard breeding grounds, songbirds tend to accumulate burdens of OC compounds, in particular p,p '-DDE (Gill et al. 2003). Therefore, when spray events of OPs and fungicides occur, songbirds already contain OC contamination. This study mimicked this field exposure to pesticides by initially exposing Zebra finches to p,p '-DDE at a concentration commonly found in orchard environments and then exposing the birds to azinphos-methyl and mancozeb at ecologically relevant dosages. Dosing of the latter two pesticides occurred mid-incubation and mid-chick-rearing in order to simulate when the first clutch in songbirds would be exposed to the spray events in the field. The main results of this chapter suggest that exposure to residual p,p '-DDE had no effect on reproductive success up to clutch completion. In addition, subsequent exposure 74 to azinphos-methyl and mancozeb has no effect on later reproductive success and the general health of both the adults and chicks. These results will be further discussed below. 4.4.1. Validation of methodology and dosing Egg and food samples collected from a preliminary study (see Chapter 2 and 3) and the present study confirmed exposure of Zebra finches to p,p '-DDE at ecologically relevant doses as food preparation and dosing remained the same for this study. 4.4.2. Effects of p,p '-DDE on early reproductive output Up to the end of clutch completion, Zebra finches were exposed to either corn oil or p,p '-DDE. This allowed us to determine the effect ofp,p '-DDE treatment up to clutch completion, prior to the exposure of the current-use pesticides used in this study. In this study, early reproductive output (up to clutch completion) was not negatively impacted following chronic exposure to p,p '-DDE and the parameters tested for (proportion of nests lay, clutch size, laying interval, mean egg mass and shell thickness) were comparable to other studies. For clutch size, Salvante and Williams (2002) looked at reproductive effort in unmanipulated Zebra finches that were maintained with similar conditions and diet as our experiment (i.e. supplemental egg food during the incubation and chick rearing stages). They found that the clutch size in unmanipulated Zebra finches ranged from one to 11 eggs (mean: 5.4 ± 0.2 eggs) and similarly, in our study, clutch size ranged from four to 10 eggs. Laying interval reported by Salvante and Williams (2002) ranged from one to 13 days (mean: 5.7 ± 0.4 days) and similarly, in our study, ranged from one to 14 days. Other studies conducted in the field have also noticed no difference in reproductive success when exposed to p,p '-DDE. American robins nesting in Okanagan Valley orchards did not appear to be negatively impacted in terms of reproductive success even thought their eggs contained high OC burdens including p,p '-DDE (Gill et al. 2003). Shell thickness in our study 75 was not affected by p,p '-DDE exposure. This is consistent with the literature in which there is a lack of evidence showing DDE effects on eggshell quality of songbirds (Blus 1996) as is commonly seen in birds higher on the food chain (Anderson et al. 1975, Wieymer et al. 1988). 4.4.3. Effects of p,p '-DDE and current-use pesticides on later reproductive success Later reproductive success (including hatch rate, brood size and fledge rate) was not negatively impacted following exposure to p,p '-DDE and the current-use pesticides, azinphos-methyl and i mancozeb. Values of these reproductive traits were again comparable to unmanipulated Zebra finches housed and maintained in a similar environment (Williams unpublished data). Williams (unpublished data) found mean brood size to be 3.8 ± 0.4 and in our study it mean brood size ranged from 2.3 to 3.8 amongst treatments. In nests that had a minimum of one egg hatch, the mean number of chicks that fledged in our study ranged from 2.2 to 3.3 amongst treatments. Williams (unpublished data) had a similar fledge success in unmanipulated Zebra finches, which averaged 3.7 ± 0.4. In contrast to our study, field studies conducted in Ontario orchards have found that both Eastern bluebirds and Tree swallows faced a decline in chick survival with increasing toxicity score (where toxicity score is used to describe the exposure of pesticides for each nest) (Bishop et al. 2000a) with swallows appearing to be more sensitive than bluebirds. In this study, Bishop et al. (2000a) suggested that a combined negative effect of residual OC concentrations and current-use pesticides in Tree swallows is possible however our study did not suggest either single exposure or combined exposure effects. Toxicity scores were also used by Patnode and White (1991) where they looked at reproduction in Northern mockingbirds (Mimus polyglottus), Northern cardinal (Cardinalis cardinalis) and Brown thrashers (Toxostoma rufum) and also found that chick survival was lower with higher toxicity scores. It has been suggested by Patnode and White (1991) that the OP and carbamate compounds (which are also a class of pesticides commonly used in orchards today) are the most toxic compounds and attributed the 76 effects seen to these compounds. However, Bishop et al. (2000a) indicates that although OPs and carbamates are potentially highly toxic to birds, the negative impact on reproduction is likely due to OC compounds, in particular DDE. Reduced hatching success and chick survival have been seen in captive Bengalese finches (Lonchura striata) and Ringed doves (Streptopelia risoria) dosed with p,p '-DDT or p,p '-DDE (Jefferies 1967, Keith and Mitchell 1993, Jefferies 1971), however this was not supported in our study. In addition to the above parameters, chick mass at 8 days did not differ amongst treatments in our study, which is consistent with Bishop et al. (2000b) where they also found no relation in masses of chicks and pesticide use in Ontario orchards. It must be considered that our study followed reproduction only until fledging, and not long-term success. As previously suggested, pesticide contamination effects may hot manifest themselves until the organism reaches sexual maturity (Fry 1995). Significant alteration of reproductive behavior in highly exposed individuals has been suggested in further studies conducted to determine effects of in ovo exposure to DDE on later reproduction (Smith et al. 2001). Future studies in a controlled environment should be conducted to determine effects of pesticides on later reproductive success in which the reproductive success of offspring of exposed adults are observed. 4.4.4. Effects of p,p '-DDE and current-use pesticides on hematological and immune function parameters Exposure to p,p '-DDE and the current-use pesticides used in this study did not result in a difference in hematological and immune function parameters tested in Zebra finch adults and chicks. Earlier studies with Zebra finches (see Chapter 2 and 3) showed that exposure to p,p '-DDE one year previously had a greater immune response to those not exposed. However, birds used in the present study were not exposed to p,p '-DDE one year previously. One of the first field studies conducted to determine the effects of pesticides (including azinphos-methyl and 77 p,p -DDE) on immune function showed that Tree swallows were immunostimulated following exposure to pesticides (Bishop et al. 1998). It is possible that Tree swallows from this study contained DDE burdens from the previous breeding season, which may be resulting in the stimulation of the immune response. This suggestion is further supported by this study where we saw that current p,p '-DDE exposure did not stimulate the immune response. However, future studies to determine effect of long term exposure to p,p '-DDE on immune function should be conducted. 4.4.5. Conclusions In conclusion, we found no evidence that dosing of adult Zebra finches with current-use pesticides, including azinphos-methyl and mancozeb, either alone or in combination with p,p '-DDE, had significant negative effects on reproductive traits or immune statues in female Zebra finches. Future studies to determine potential effects of in ovo exposure to pesticides on reproduction post-fledging as well as later reproductive success would be beneficial in explaining effects of pesticides on songbirds species. In addition, the possibility of long-term exposure ofp,p '-DDE on immune function should be further studied. Table 4.1. Summary of pesticide treatment in male and female Zebra finches (Taeniopygia guttata) used to determine the effects ofp,p '-DDE and current-use pesticides on reproductive success and general health. Dosage group n Corn oil + corn oil 17 p,p '-DDE + corn oil 15 p,p '-DDE + azinphos-methyl 17 p,p '-DDE + mancozeb 15 79 Table 4.2. Comparison of reproductive output up to clutch completion in Zebra finch (Taeniopygia guttata) pairs exposed to p,p '-DDE at 34 mg/kg. Statistical analysis to determine the proportion of nests lay was conducted using the chi-squared test. Laying interval and mean egg mass was analyzed using an analysis of covariance (ANCOVA), with female weight as a covariate. Remaining analyses was conducted using a least squares analysis of variance (ANOVA). Values given (excluding proportion of nests lay) are the presented as the least squared mean ± standard error with the sample size in parentheses. Dosage group Prop, of Clutch Laying Mean egg Shell thickness nests lay size1 interval mass (g) (mm) Corn oil 86.7 % 5.9 + 0.4 6.0 + 0.9 1.07 + 0.03 0.0556 ± 0.0004 (15) (13) (13) (13) (12) p,p '-DDE 77.3 % 5.9 + 0.4 7.4 + 0.5 1.08 + 0.02 0.0551 +0.0002 (44) (34) (34) (34) (31) P-value 0.4351 0.7224 0.1633 0.7699 0.2206 including those that laid a minimum of one egg only 80 Table 4.3. Comparison of reproductive output following clutch completion in Zebra finch (Taeniopygia guttata) pairs exposed to a combination of p,p '-DDE and current-use pesticides. Statistical analysis to determine the effect of treatment on brood size was conducted using a least squared analysis of variance (ANOVA). Remaining analyses was conducted using a G-test. Brood size is presented as the least squared mean ± standard error with the sample size in parentheses, while the remaining are presented as percentages. Dosage group Prop, of Hatch Brood Prop, of Fledge No. of nests rate2 size3 nests rate5 chicks hatch1 fledge4 fledge6 Corn oil + 46.2 % 19.5 % 2.5 ±0.5 100 % 93.3 % 2.3 ±0.6 corn oil (13) (77) (6) (6) (6) (6) p,p'-T)DE + 63.6% i 23.5 % 2.3 ±0.5 71.4% 75.0 % 2.4 ±0.6 corn oil (11) (68) (7) (7) (5) (5) p,p'-DDE + 33.3 % 21.4% 3.8 ±0.7 100 % 86.7 % 3.3 ±0.7 azinphos-methyl (12) (70) (4) (4) (4) (4) p,p '-DDE + 54.5 % ' 24.1 % 2.3 ±0.5 83.3 % 78.6 % 2.2 ±0.6 mancozeb (11) (58) • (6) (6) (5) (5) P-value 0.8870 0.9970 0.3227 0.6730 0.880 0.6764 ^nly nests that had a minimum of one egg laid were included in the analyses. Total number of eggs hatched divided by the total number of eggs laid. Mean taken of those nests in which a minimum of one egg hatched. 4Only nests that had a minimum of one egg hatch were included in the analyses. 5Total number of chicks fledged divided by the total number of chicks hatched. 'Mean taken of those nests in which a minimum of one chick fled Table 4.4. Comparison of hematological parameters and phytohemagglutinin (PHA) skin test response in adult female Zebra finches (Taeniopygia guttata) exposed to various combinations of p,p '-DDE and current-use pesticides. Only those with nests in which a minimum of one chick fledged were included in the analyses. Statistical analyses were conducted using a least squared analysis of variance (ANOVA). As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analyses. Values are expressed as the least squares mean ± standard error with the sample size in parentheses. Dosage group Hematocrit (%) Leucocrit (%) PHA response (mm) Corn oil + corn oil 49.8 + 1.9(6) 0.37 + 0.12(6) 0.72 + 0.07 (6) p,p '-DDE + corn oil 50.3 +2.1 (5) 0.54 + 0.13 (5) 0.58 + 0.08 (5) p,p '-DDE + azinphos-methyl 50.6 + 2.3 (4) 0.40 + 0.15 (4) 0.61 + 0.09 (4) p,p'-DDE + mancozeb 52.0 ± 2.1 (5) 0.66 ± 0.13 (5) 0.63 + 0.08 (5) P-value 0.8706 0.3976 0.4453 83 Table 4.5. Comparison of day 8 mass, hematological parameters and phytohemagglutinin (PHA) skin test response in chicks of adult Zebra finches (Taeniopygia guttata) exposed to various combinations of p,p '-DDE and current-use pesticides. Percent leucocrit was analyzed using an analysis of covariance (ANCOVA) with chick weight as a covariate. Remaining parameters were analyzed using a least squared analysis of variance (ANOVA). As hematocrit and leucocrit values were converted to percentages, data was arc sine transformed prior to analyses. Values are expressed as the least squares mean ± standard error with the sample size in parentheses. Dosage Chick day 8 Chick day 23 H/L Hematocrit Leucocrit PHA response group mass (g) mass (g) ratio (%) (%) (mm) Corn oil + corn 8.46 ±0.31 11.73 ±0.34 0.28 ±0.09 49.3 ± 1.6 0.48 + 0.09 0.70 ± 0.08 oil (6) (6) (6) (6) (6) (6) p,p '-DDE + corn 7.84 + 0.34 12.06 ±0.37 0.33 ±0.11 50.0 ± 1.7 0.47 ±0.10 0.89 ± 0.09 oil (5) (5) (4) (5) (5) (5) p,p -DDE + 7.60 ±0.38 11.16 ±0.42 0.20 ±0.11 49.1 ± 1.9 0.26 ±0.12 0.65 ±0.10 azinphos-methyl (4) (4) (4) (4) (4) (4) p,p'-DDE + 7.66 ±0.34 12.59 ±0.37 0.26 ±0.10 48.6 ± 1.7 0.65 ±0.12 0.58 ±0.09 mancozeb (5) (5) (5) (5) (5) (5) P-value 0.2636 0.1134 0.7941 0.9430 0.2166 0.1080 84 C H A P T E R 5 Conclusions The primary goal of the research described in this thesis was to determine the potential synergistic effects ofp,p '-DDE and the current-use pesticides azinphos-methyl and mancozeb on reproduction and the general health of songbirds using the Zebra finch (Taeniopygia guttata) as a model species for songbirds. This research resulted in four major findings. Firstly, reproductive success was not negatively impacted with simultaneous exposure to p,p '-DDE followed by subsequent exposure to current-use pesticides. However, in birds that were exposed to p,p '-DDE one year previously, reproductive success was negatively impacted in terms of circulating yolk precursor levels and egg mass. Secondly, long term p,p '-DDE exposure appears to be having the greatest impact on the general health of Zebra finches, while current exposure to pesticides appear to have minimal impact. Thirdly, p,p '-DDE exposure followed by subsequent azinphos-methyl exposure did not appear to affect the degree of cholinesterase (ChE) enzyme inhibition in Zebra finches. And finally, Zebra finches are a suitable model species to use in laboratory studies to determine effects of pesticides on reproduction and the general health of the birds. However, they appear to be less sensitive to OP exposure when compared with other songbird species and this must be taken into account when conducting future laboratory studies and extrapolating the findings to wild species. There is plenty of evidence for DDT and DDE effects on predatory and fish eating species on reproduction. However, few studies exist looking at effects oh songbird species. In addition, the available studies did not consider previous exposure to OC compounds that accumulate in the body prior to current-use pesticide spraying. There was minimal evidence that reproductive success of songbirds entering the breeding grounds would be negatively impacted due to current exposure (Chapter 3 and 4). However, songbirds, which may routinely return to 85 contaminated habitat to breed, may be negatively impacted due to previous contaminant exposure. In Chapter 3, birds that were exposed to p,p '-DDE approximately one year previously experienced a decrease in the circulating levels of the yolk precursors vitellogenin (VTG) and very-low density lipoprotein (VLDL). In addition, those birds laid heavier eggs when compared with those not exposed to pesticides previously. Future studies should be conducted to further explain long-term effects of pesticide exposure especially by contaminants such as DDE that are persistent and accumulate both in the environment and in living organisms. In addition, studies to determine in ovo pesticide exposure effects on reproduction post-fledging and later reproductive success would be beneficial for extrapolating findings to songbirds breeding in contaminated orchards. General health of animals exposed to environmental stressors such as pesticides, may be negatively impacted. To evaluate the immune status of individuals, measures of immune function are becoming increasingly common. However, there are few published studies which report the impact of contaminant exposure on immune function in avian species. Our study found that current exposure to pesticides did not result in a negative impact in terms of immune function; however, birds exposed one year previously were immuno stimulated. In Chapter 2, birds exposed to p,p '-DDE one year previously had a greater response to the plant lectin phytohemagglutinin l(PHA) and in Chapter 3, birds with previous p,p '-DDE exposure had greater percent leucocrit values (which is indicative of the number of white blood cells present) when compared with birds with no pesticide exposure. These laboratory studies further support the suggestion of immunostimulation in songbirds exposed to orchard pesticides, which contradicts reports of immunosuppression seen previously in mammalian studies. In addition to immunostimulation, birds exposed to p,p '-DDE one year previously also had lower percent hematocrit values suggesting anemia in these birds. As few studies exist looking at pesticide effects on the general health (including immune function and hematological parameters) in 86 songbirds, this topic warrants further studies for proper extrapolation and interpretation of results. As with all OP insecticides, azinphos-methyl exposure is expected to inhibit the activity of the cholinesterase (ChE) enzyme. Cholinesterase enzymes are critical to the normal function of the nervous system in both vertebrates and invertebrates. Therefore when OPs are released into the environment, the potential exists for non-target effects. The dose-dependent increase in plasma and brain ChE inhibition was typical of OP exposure in other avian species (Chapter 2). As songbirds in orchard habitats present during the spray event already contain p,p ' -DDE burdens it was important to determine if this affected the degree of ChE inhibition. Chapter 2 suggested that exposure to p,p ' -DDE followed by subsequent azinphos-methyl exposure did not affect the degree of ChE inhibition as suggested with other mammalian and avian studies. In order to better understand and interpret possible pesticide exposure effects in field situations, it is important to conduct complementary laboratory studies. This minimizes confounding factors that may otherwise be present in field situation. When conducting laboratory studies, it is imperative to have a suitable model to represent the species in question. Generally toxicological studies use mammalian (i.e. mice and rats) or avian species (i.e. quail). However, this limits extrapolation and interpretation of results when referring to songbird species. Zebra finches have been used extensively for experiments conducted in laboratory settings, however their use in toxicological studies remains limited. Our study found Zebra finches to be an extremely tolerant species to handling and experimental manipulation (i.e. oral dosing, collection blood samples). In addition, they bred well in captivity and had a relatively short generation time. It is suggested that Zebra finches are a suitable songbird model when conducting laboratory studies to determine toxicological responses to contaminants. However, they did appear to be more resistant to pesticide exposure when compared with other songbird species. Zebra finches were extremely tolerant to azinphos-methyl exposure when compared 87 with other songbird species in terms of degree of ChE inhibition (Chapter 2). In addition, no birds were concluded to have succumbed from pesticide exposure in any of the studies conducted as seen with previous studies. Therefore, the sensitivity of Zebra finch species must be considered when extrapolating findings to wild species. The implication of this laboratory study suggests that Zebra finches exposed to contaminants present in orchard environments are at an increased risk to their health and survival. Although OP pesticides are more acutely toxic, it is suggested that chronic exposure to residual p,p ' -DDE is having a more sub-lethal effect on Zebra finches. Songbirds breeding in orchard environments commonly return year after year to breed in these habitats, and it is this previous years' exposure to p,p ' -DDE which appears to be impacting health and reproduction in Zebra finches. This laboratory approach to mimic more closely field exposure of songbirds to contaminants in orchard environments are essential to better understand the effects of contaminants on wild species of songbirds. Understanding these effects are critical in developing management options for pesticide use in apple orchards that will be effective for pest control and in addition protect the health of songbirds. The finding of significant results justifies rationalization for further testing of the effect of individual and combined pesticides used in apple orchards both today and previously. L I T E R A T U R E C I T E D 88 Anam KK, Maitra SK (1995) Impact of Quinalphos, on blood glucose and acetylcholinesterase (AChE) activity in brain and pancreas in a Roseringed parakeet (Psittacula krameri borealis: Newmann). Arch Environ Contam Toxicol 29(l):20-23 Anderson DW, Jehl JR, Risebrough RW, Woods Jr, LA, Deweese LR, Edgecomb WG (1975) Brown Pelicans: Improved reproduction off the Southern California Coast. Science 90:806-808 Bailey S, Bunyan PJ, Jennings DM, Norris JD, Stanley PI, Williams JH (1974) Hazards to wildlife from the use of DDT in orchards: II. A further study. Agro-Ecosys 1:323-338 Ball WL, Sinclair JW, Crevier M, Kay K (1954) Modification of parathion's toxicity for rats by pretreatment with chlorinated hydrocarbon insecticides. Canad J Biochem Physiol 32:440-445 Beyer WN, Gish CD (1980) Persistence in earthworms and potential hazards to birds of soil applied DDT, dieldrin and heptachlor. JApplEcol 17:295-307 Bishop CA, Boermans HJ, Ng P, Campbell GD, Struger J (1998) Health of Tree swallows (Tachycineta bicolor) nesting in pesticide-sprayed apple orchards in Ontario, Canada. I. Immunological parameters. J Toxicol Environ Health Part A 55:531-559 Bishop CA, Collins B, Mineau P, Burgess NM, Read WF, Risley C (2000a) Reproduction of cavity-nesting birds in pesticide-sprayed apple orchards in southern Ontario, Canada, 1988-1994. Environ Toxicol Chem 19(3):588-599 Bishop CA, Ng P, Mineau P, Quinn JS, Struger J (2000b) Effects of pesticide spraying on chick growth, behavior, and parental care in Tree swallows (Tachycineta bicolor) nesting in an apple orchard in Ontario, Canada. Environ Toxicol Chem 19(9):2286-2297 Blus LJ (1996) DDT, DDD, and DDE in birds. In Environmental contaminants in wildlife. 89 Interpreting tissue concentrations. Beyer WN, Heinz GH, Redmon-Norwood AW (eds). Boca Raton, Florida. CRC press, Inc. Braune BM, Norstrom RJ (1989) Dynamics of organochlorine compounds in Herring gulls: III. Tissue distribution and bioaccumulation in Lake Ontario gulls. Environ Toxicol Chem 8:957-968 Burgess NM, Hunt KA, Bishop C, Weseloh DV (1999) Cholinesterase inhibition in Tree swallows (Tachycineta bicolor) and Eastern bluebirds (Sialis sialis) exposed to organophosphorus insecticides in apple orchards in Ontario, Canada. Environ Toxicol Chem 18(4): 708-716 Busby DG, Pearce PA, Garrity NR. (1981) Brain cholinesterase response in songbirds exposed to experimental fenitrothion spraying in New Brunswick, Canada. Bull Environm Toxicol 26:401-406 Busby DG, White LM, Pearce PA (1990) Effects of aerial spraying of fenitrothion on breeding White-throated sparrows. JApplEcol 27:743-755 Carpenter FL (1975) Bird hematocrits: effects of high altitude and strength of flight. Comp Biochem Physiol 50A:415-417 Carson R (1962) Silent Spring. Boston: Houghton Mifflin Celius T, Walther BT (1998) Differential sensitivity of zonagenesis and vitellogenesis in Antlantic salmon (Salmo salar) to DDT pesticides. J Exp Zool 281:346-353 Challenger WO, Williams TD, Christians JK Vezina F (2001) Follicular development and plasma yolk precursor dynamics through the laying cycle in the European starling (Sturnus vulgaris). Physiol Biochem Zool 74(3):356-365 Chambers JE, Levi PE (1992) Organophosphates. Chemistry, fate and effects. Academic Press, Inc. San Diego, California Chemagrd Division Research Staff (1974) Guthion (azinphos-methyl): Organophosphorus 90 insecticide. Res Rev 51:123-180 Christians JK, Williams TD (2001) Interindividual variation in yolk mass and the rate of growth of ovarian follicles in the zebra finch (Taeniopygia guttata). J Comp Phys B 3: 255-261 Cooke AS (1973) Shell thinning in avian eggs by environmental pollutants. Environ Pollut 4:85-152 Crevier M, Ball WL, Kay K (1954) Observations on toxicity of aldrin. II. Serum esterase changes in rats following administration of aldrin and other chlorinated hydrocarbon insecticides. A.M.A. Arch IndHyg Occup Med 9:306-314 Dean JH, Murray MJ (1991) Toxic responses of the immune system. In Toxicology, 4th Amdur MO, Doull J, Klaassen CD (eds). New York: Pergamon Press Pp. 282-333 de Snoo G (1986) Effects of dithiocarbamates on birds. Report of the Ecotoxicology Work Group / Information Centre for Biology, Free University of Amsterdam, Amsterdam, Netherlands Dethloff GM, Bailey HC (1988) Effects of copper on immune system parameters of rainbow trout (Onchorhynchus mykiss). Environ Contam Toxicol 17:1807-1814 Dieter MP (1974) Plasma enzyme activities in Coturnix quail fed graded doses of DDE, polychlorinated-biphenyl, malathion, and mercuric chloride. Toxicol Appl Pharmacol 27:86-98 i Donohoe RM, Curtis LR (1996) Estrogenic activity of chlordecone, o,p '-DDT and o,p '-DDE in juvenile rainbow trout: induction of vitellogenesis and interaction with hepatic estrogen binding sites. Aquatic Tocicol 36:31-52 i Edwards CA (1966) Insecticide residues in soils. Residue Rev 13:82-132 i • Elliott JE, Martin PA (1994) Chlorinated hydrocarbons and shell thinning in eggs of (Accipiter) hawks in Ontario, 1986-1989. Environ Pollut 86:189-200 Elliott JE, Martin PA, Arnold TW, Sinclair PH (1994) Organochlorines and reproductive success of birds in orchard and non-orchard areas of central British Columbia, Canada, 1990-1991. Arch Environ Contam Toxicol 26:435-443 Ellman GL, Courtney KD, Andres VJr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmac 7:88-95 Fluetsch KM, Sparling DW (1994) Avian nestling success and diversity in conventionally and organically managed apple orchards Environ Toxicol Chem 13:1651-1659 Fry DM (1995) Reproductive effects in birds exposed to pesticides and industrial chemicals. Env Health Perspect 103:165-71 Fyfe RW, Risebrough RW, Monk JG, Jarman WM, Anderson DW, Kiff LF, Lincer JL, Nisbet ICT, Walker III W, Walton BJ (1988) DDE, productivity, and shell thickness relationships within the genus Falco. In Pergrine falcon populations: their management and recovery. Cade TJ, Enderson JH, Thelander CG, White CM. Boise Idaho (eds). The Pergrine Fund Inc. Gill H, Wilson LK, Cheng KM, Elliott JE (2003) An assessment of DDT and other chlorinated compounds and the reproductive success of American robins (Turdus migratorius) breeding in fruit orchards. Ecotox 12:113-123 Gill H, Wilson LK, Cheng KM, Trudeau S, Elliott JE (2000) Effects of azinphos-methyl on American robins breeding in fruit orchards. Bull Environ Contam Toxicol 65:756-763 Grady T (1999) Saving Guthion. Fruit Grower 119(5):20-22 Graham DJ, DesGranges JL (1993) Effects of the organophosphate azinphos-methyl on birds of potatoe fields and apple orchards in Quebec, Canada. Agri Eco Environ 43:183-199 Gross WB, Siegel HS (1983) Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens. Avian Dis 27:972-979 Grue CE, Hart ADM, Mineau P (1991) Biological consequences of depressed brain 92 cholinesterase activity in wildlife. In Cholinesterase-inhibiting insecticides. Mineau P (ed). Elsevier Science Publisher BV, Amsterdam. Pp. 151-209 Grue CE, Powell GVN, McChesney MJ (1982) Care of nestlings by wild female starlings exposed to an organophosphorus pesticide. JAppl Ecol 19:327-335 Harris CR, Sans WW (1971) Insecticide residues in soils on 16 farms in southwestern Ontario -1964, 1966, and 1969. PesticMonit J5:259-267 Harris ML, van den Heuvel MR, Rouse J, Martin PA, Struger J, Bishop CA, Takacs P (2000a) Pesticides in Ontario: A critical assessment of potential toxicity of agricultural products to wildlife, with consideration for endocrine disruption. Canadian Wildlife Service 2000, Environmental Conservation Branch, Ontario Region. 102 pp Harris ML, Wilson LK, Elliott JE, Bishop CA, Tomlin AD, Henning KV (2000b) Transfer of DDT and metabolites from fruit orchard soils to American robin (Turdus migratorius) twenty years after agricultural use of DDT in Canada. Arch Environ Contam Toxicol 39:205-220 Hebert CE, Weseloh DV, Kot L, Glooschenko V (1994) Organochlorine contaminants in a terrestrial food web on the Niagra Peninsula, Ontario, Canada 1987-1989. Arch Environ Contam Toxicol 26:356-366 Hickey JJ, Anderson DW (1968) Chlorinated hydrocarbons and eggshell changes in raptorial and fish-eating birds. Science 162:271 Hill EF, Fleming WJ (1982) Anticholinesterase poisoning of birds: Field monitoring and diagnosis of acute poisoning. Environ Toxicol Chem 1:27-38 Hill EF, Heath RG, Spann JW, Williams JD (1975) Lethal dietary toxicities of environmental pollutants to birds. US Fish Wildl Serv Spec Sci Rept Wildl 191 Holmes SB, Boag PT (1990) Inhibition of brain and plasma cholinesterase activity in Zebra finches orally dosed with fenitrothion. Environ Toxicol Chem 9:323-334 93 Houeto P, Bindoula G, Hoffman JR (1995) Ethylenebisdithiocarbamates and ethylenethiourea: possible human health hazards. Environ Health Perspect 103(6):568-573 Hunter C, McGee B (1999) Survey of pesticide use in Ontario, 1998. Estimates of pesticides used on field crops, fruit and vegetable crops, and other agricultural crops. Ontario Ministry of Agriculture, Food and Rural Affairs. Policy Analysis Branch, Guelph, ON, Canada Jefferies DJ (1967) The delay in ovulation produced in p,p'-DDT and its possible significance in the field. Ibis 109:266-272 Jefferies DJ (1971) Some sublethal effects of pp'-DDT and its metabolite pp'-DDE on breeding passerine birds. Overdruk uit: Mededelingen fakulteit Landbouw - Wetenschappen Gent 36(l):34-42 Johnson EV, Mack G.L, Thompson DQ (1976) The effects of orchard pesticide applications on breeding robins. Wilson Bull 88:16-35 Johnston G, Walker CH, Dawson A (1994) Interactive effects between EBI fungicides (prochloraz, propiconazole and penconazole) and OP insecticides (dimethoate, chlorpyrifos, diazinon and malathion) in the hybrid Red-legged partridge. Environ Toxicol Chem 13(4):615-620 Keith JO, Mitchell CA (1993) Effects of DDE and food stress on reproduction and body conditions of ringed turtle doves. Arch Environ Contam Toxicol 25:192-203 Kerr MA, Mannings EW, Seguin J, Pelton LJ. (1985) Okanagan fruitlands: land use change dynamics and the impact of federal programs. No. 26. Environment Canada Land Use in Canada Series. Lands Directorate, Ottawa, Canada Longcore JR, Samson FB, Whittendale TW Jr (1971) DDE thins eggshells and lowers reproductive success of captive Black ducks. Bull Environ Toxicol 6(6):485-490 Longcore JR, Stendell RC (1977) Shell thinning and reproductive impairment in Black ducks 94 after cessation of DDE dosage. Arch Environ Contam Toxicol 6:293-304 Lorenzen A, Casley WL, Moon TW (2001) A reverse transcription-polymerase chain reaction bioassay for avian vitellogenin mRNA. Toxicol Appl Pharmac 176:169-180 Ludke JL (1977) DDE increases the toxicity of parathion to Coturnix quail. Pestic Biochem Physiol 7:28-33 Ludke JL, Hill EF, Dieter MP (1975) Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch Environ Contam Toxicol 3(1): 1-21 Maxwell MH (1993) Avian blood leucocyte responses to stress. World's Poul Sci J49:34-43 Menzer RE (1970) Effect of chlorinated hydrocarbons in the diet on the toxicity of several organophosphorous insecticides. Toxicol Appl Pharmacol 16:446-452 Media J, Svensson E (1995) Fat reserves and health state in migrant Goldcrest (Regulus regulus). FuncEcol 9:842-848 Metcalfe TL, Metcalfe CD, Kiparissis Y, Niimi AJ, Foran CM, Benson WH (2000) Gonadal development and endocrine responses in Japanese medaka (Oryzias latipes) exposed to o,p -DDT in water or through maternal transfer. Environ Toxicol Chem 19(7): 1893-1900 Mitchell MA, Carlisle AJ (1991) Plasma zinc as an index of vitellogenin production and reproductive status in the domestic fowl. Comp Biochem Physiol 100:719-724 Morton ML (1994) Hematocrits in Montane Sparrows in relation to reproductive schedule. Condor 96:119-126 Newton I (1986) The Sparrowhawk. Calton: Poyser Norecol Dames & Moore (1997) Survey of pesticide use in British Columbia: 1995. DOE FRAP # Technical Report. British Columbia Ministry of Environment, Lands and Parks. Vancouver, BC, Canada Ots I, Murumagi A, Horak P (1998) Haematological health state indices of reproducing Great Tits: Methodology and sources of natural variation. FuncEcol 12:700-707 Palmer BD, Huth LK, Pieto DL, Selcer KW (1998) Vitellogenin as a biomarker for xenobiotic estrogens in an amphibian model system. Environ Toxicol Chem 17(l):30-36 Patnode KA, White DH (1991) Effects of pesticides on singbird productivity in conjunction with pecan cultivation in southern Georgia: A multiple-exposure experimental design. Environ Toxicol Chem 10:1479-1486 Phillips JG, Butler PJ, Sharp PJ (1985) Thermoregulation. In Physiological strategies in avian biology. Glasgow : Blackie, New York. Pp. 56-80 Powell GVN (1984) Reproduction by an altricial songbird, the Red-Winged Blackbird, in fields treated with the organophosphate insecticide fenthion. J Appl Ecol 21:83-95 Province of British Columbia Ministry of Agriculture and Fisheries (1991) Tree fruit production guide for Interior Districts. 1991-1992 edition for commercial growers. Victoria, BC Pruett SB (1994) Immunotoxicity of agrochemicals, an overview of currently available information. Toxicol Ecotoxicol News 10(2):49-54 Pruett SB, Barnes DB, Han Y, Munson A (1992) Immunotoxicological characteristics of sodium methyldithiocarbamate. Fundam Appl Toxicol 18:40-47 Ratcliffe DA (1980) The Peregrine Falcon. Calton: Poyser Salvante KG, Williams TD (2002) Vitellogenin dynamics during egg-laying: daily variation, repeatability and relationship with egg size. J Avian Biol 33(4):391-398 SAS Institute Inc (2000) JMP Statistics and Graphics Guide, Version 4 of JMP. Cary, NC, USA. Sinclair PH, Elliott JE (1993) A survey of birds and pesticide use in orchards in the south Okanagan/Similkameen region of British Columbia, 1991. Canadian Wildl Serv Tech. Rep. No. 185 Smith GJ (1987) Pesticide use and toxicology in relation to wildlife: Organophosphorus and carbamate compounds. US Dept Int Fish & Wildl Serv Res Pub 170. Washington, DC Smith LK, Wilson LK, Elliott JE, Cheng KM (2001) The effects of DDT exposure on American 96 robins from orchards of the Okanagan Valley, British Columbia. Society of Environmental Toxicology and Chemistry 22nd Annual Meeting. Poster PWO15 Smits JE, Blakley BR, Wobeser GA (1996) Immunotoxicity studies in mink (Mustela visori) chronically exposed to dietary bleached kraft pulp mill effluent. J Wild!Dis 32:199-208 Smits JE, Bortolotti GR, Telia JL (1999) Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence. FuncEcol 13:567-572 Smits JE, Williams TD (1999) Validation of immunotoxicology techniques in passerine chicks exposed to oil sands tailings water. Ectox Environ Saf44:105-112 Stinson ER, Bromley PT (1991) Pesticides and wildlife: A guide to reducing impacts on animals and their habitat. Virginia Dept Game Inland Fish Publ 420-004 Triolo AJ, Coon JM (1966) Toxicological interactions of chlorinated hydrocarbon and organophosphate insecticides. JAgr Food Chem 14:549-555 Vos J, Van Loveren H, Wester P, Vethaak D (1989) Toxic effects of environmental chemical on the immune system. Trends Pharmacol Sci 10:289-292 Wallace GJ (1959) Insecticides and birds. Audubon 61:10-12 Walzem RL, Hansen RJ, Williams DL, Hamilton RL (1999) Estrogen induction of VLDLy assembly in egg-laying hens. JNutr 129:467S-472S Wardlaw SC, Levine RA (1983) Quantitative buffy coat analysis: A new laboratory tool functioning as a screening complete blood cell count. J Amer Med Assoc 249:617-620 Westlake GE, Bunyan PJ, Martin AD, Stanley PI, Steed LC (1981) Organophosphate poisoning. Effects of selected organophosphate pesticides on plasma enzymes and brain esterases of Japanese quail (Coturnix coturnix japonica). JAgr Food Chem 29:772-778 Wieymeyer SN, Bunck CM, Krynitsky AJ (1988) Organochlorine pesticides, polychlorinated biphenyls and mercury in osprey eggs - 1970-79 - and there relationships to shell-thinning and productivity. Arch Environ Contam Toxicol 17:767-787 Wieymer SN, Bunck CM, Stafford CJ (1993) Environmental contaminants in Bald eagle eggs -1980-84 - and further interpretations of relationships to productivity and shell thickness. Arch Environ Contam Toxicol24:213-27 Wieymeyer SN, Porter RD (1970) DDE thins eggshells of captive American kestrels. Nature 227:737-738 Williams TD (1996) Intra- and inter-individual variation in reproductive effort in captive-breeding Zebra finches (Taeniopygia guttata). Can J Zool 74:85-91 Williams TD, Christians JK (1997) Female reproductive effort and individual variation: Neglected topics in environmental endocrinology? In Proceedings of the 13th International Congress of Comparative Endocrinology. Kawashima S and Kikuyama S (eds). Monduzzi Editore, Yokohama, Japan. Pp. 1669-1675 Williams TD and Martyniuk CJ (2000) Tissue mass dynamics during egg-production in female Zebra finches Taeniopygia guttata: dietary and hormonal manipulations. J Avian Biol 31:87-95 Wurster DH, Wurster CF Jr, Strickland WN (1965) Bird mortality following DDT spray for Dutch elm disease. Ecology 46:488-499 Zinkl JG, Roberts RB, Henny CJ, Lenhart DJ (1980) Inhibition of brain cholinesterase activity in forest birds and squirrels exposed to aerially applied acephate. Bull Environ Contam Toxicol 24:676-683 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0090939/manifest

Comment

Related Items