A comprehensive evaluation of the effects of an nC24 agricultural mineral oil (AMO) on tritrophic interactions between French bean Phaseolus vulgaris cv. Redlands Pioneer [Fabales : Fabaceae ], two spotted mite (TSM) Tetranychus urticea Koch [Acari: Tetranychidea] and the predatory mite Phytoseiulus persimilus Atheus-Henriot [Acari: Phytoseiidae] was conducted under laboratory conditions. / Doctor of Philosophy (PhD)

La pollution diffuse des nappes phréatiques par les pesticides pose un problème environnemental critique. Les approches visant à moduler spatialement la dose de pesticide apportée en fonction des risques de transfert des molécules phytosanitaires à travers le sol sont a priori une solution séduisante. Dans un cadre de recherche, ces approches requièrent une connaissance spatialisée des caractéristiques hydropédologiques du sol, de l'adsorption et de la dégradation des molécules phytosanitaires, afin de pouvoir modéliser le comportement des molécules. Le travail de thèse présenté ici porte sur la première étape d'une telle démarche, la caractérisation des propriétés du sol et d'une molécule herbicide - l'isoproturon - à l'échelle d'un secteur agricole de 2600 ha de Beauce chartraine. Les objectifs de la thèse se décomposent en 3 parties : [i] Caractériser la variabilité spatiale des propriétés pédologiques, physico-chimiques et hydriques du sol (rétention en eau, conductivité hydraulique KSAT, fraction d'eau immobile fIMM), ainsi que les propriétés d'adsorption (KD) et de dégradation (DT50) de l'isoproturon dans l'horizon de surface du secteur agricole étudié ; [ii] Identifier les déterminismes agronomiques et/ou pédologiques des propriétés importantes pour le comportement des pesticides dans les sols ; [iii] Établir et tester des classes et des fonctions de pédotransfert permettant de prédire ces propriétés déterminantes à partir de propriétés agronomiques ou pédologiques plus simples. Les sols de ce secteur sont des sols moyennement épais développés dans un limon éolien reposant sur un calcaire gélifracté et cryoturbé. L'horizon de surface de 34 sites a été caractérisé, avec une détermination systématique des propriétés du lit de semence, et une détermination partielle du reste de l'horizon travaillé. Sur les 50 couches / échantillons de sol caractérisés, un tiers se trouvent dans des parcelles en travail simplifié (sans labour). À l'exception de la masse volumique apparente, les propriétés physico-chimiques du sol sont peu variables (teneur en argile et en CaCO3 notamment). Si les propriétés de rétention en eau et le KD de l'isoproturon sont, elles aussi, modérément contrastées, la fIMM, la KSAT, et la DT50 de l'isoproturon sont très variables. Un

Les galactoglycérolipides (i.e. monogalactosyldiacylglycérol, MGDG et digalactosyldiacylglycérol, DGDG) sont les lipides majeurs des membranes des chloroplastes, représentant jusqu'à 80% de la composition membranaire plastidiale. La synthèse de ces galactolipides est catalysée par deux types de galactosyltransférases, les MGDG synthases (ou MGD) et les DGDG synthases (ou DGD). Lors de cette synthèse, un groupement ß-galactosyl provenant d'un UDP- aGal, est transféré sur la position sn-3 d'un diacylglycérol (DAG). Les DGD utilisent le MGDG produit par les MGD comme substrat pour la synthèse du DGDG. La synthèse du MGDG est donc essentielle pour la biogenèse du chloroplaste et par conséquent pour la survie de la cellule végétale. Les Apicomplexes sont des parasites intracellulaires obligatoires d'importance médicale et vétérinaire. Ils possèdent un plaste vestigial non-photosynthétique, nommé apicoplaste, acquis par endosymbiose secondaire d'une algue rouge. L'apicoplaste est impliqué dans de nombreuses voies métaboliques de type végétal. De plus, il est essentiel à la survie du parasite. La synthèse de galactolipides aux propriétés chromatographiques indiscernables de celles du MGDG et du DGDG a été mesurée chez Toxoplasma gondii, le parasite Apicomplexe responsable de la toxoplasmose. Nous avons entrepris une étude comparative du métabolisme et de la dynamique des galactolipides chez les plantes et des lipides structurellement apparenté chez les parasites Apicomplexes, par des analyses de biochimie, biologie structurale, moléculaire et cellulaire chez A. thaliana et T. gondii. Nous avons tout d'abord établi un modèle structural de la MGDG synthase chloroplastique basée sur son homologie avec la glycosyltransférase bactérienne MURG. Ce modèle structural a été validé par mutations ponctuelles rationalisées. Ce modèle indique les résidus impliqués dans la liaison de l'UDP-Gal et permet d'avancer dans notre compréhension du mécanisme catalytique. Il permet de proposer certaines régions comme participant à la fixation du DAG et à l'association à la membrane du chloroplaste. La structure présentée dans ce manuscrit constitue un outil pour l'étude d'inhibiteurs de la MGDG synthase et la compréhension de l'évolution complexe des enzymes de galactosylation. Nous avons montré que des inhibiteurs des MGDG synthases de plantes, identifiés par criblage pharmacologique d'une chimiothèque de 23360 composés, avaient des propriétés herbicides, algicides et anti-parasitaire sur A. thaliana, C. reinhartdii et T. gondii respectivement. Nous avons par la suite engagé un programme d'optimisation par synthèse chimique et mesuré les effets de plus de 200 dérivés des molécules initiales. Cette stratégie nous a permis d'optimiser certaines de ces molécules dans l'objectif de développer des candidats herbicides/médicaments. Grâce à l'utilisation de serums de lapin et de rat obtenus après immunisation avec du DGDG, nous avons détecté chez T. gondii un lipide, désigné DGLE (digalactolipid-like epitope), structuralement apparenté au digalactolipide. Ce lipide est localisé au niveau des membranes de la pellicule du parasite. De plus, la localisation du DGLE dépend du stade de vie du parasite, redistribué lors de l'invasion dans une cellule hôte. Le DGLE semble lié aux protéines associées au cytosquelette d'actine et/ou à des domaines membranaires. La détermination du lipidome de la pellicule par spectrométrie de masse montre que des dihexosyl-lipides mineurs dérivés de diacylglycérol, alkyl-acyl ou de céramide ont une masse compatible avec le DGLE. La question de la structure du DGLE, pour sa partie hydrophobe, reste donc irrésolue. Ce lipide a été détecté chez d'autres Apicomplexes tels que Plasmodium falciparum (agent de la malaria) et Cryptosporidium parvum (agent de la cryptosporidiose). Le fait d'avoir pu induire une réponse immunitaire par injection du DGDG chez le lapin et le rat conduisant à la détection d'un épitope à la surface de T. gondii, nous a amené à étudier l'antigène digalactolipidique et ses applications éventuelles dans des visées diagnostiques et / ou thérapeutiques. Nous avons montré que le serum provoquait l'agglutination du parasite et empêchait son invasion dans la cellule hôte in vitro. Le marquage de la surface du parasite par le serum (i.e. l'opsonisation) active la réponse immunitaire inné in vitro. La phagocytose par les macrophages et l'activation du complément est accrue par rapport au contrôle. Ces résultats préliminaires suggèrent que des outils diagnostics et/ou des stratégies vaccinales nouveaux, basés sur la reconnaissance de l'antigène digalactolipidique de surface des parasites Apicomplexes, pourraient être envisagés. Les perspectives de ces travaux incluent une poursuite du développement des inhibiteurs des MGDG synthases pour d'une part des recherches fondamentales par des approches de génétique chimique (invalidation chimique, utilisation de dérivés couplés à la biotine), et d'autre part des applications éventuelles en tant que candidat herbicides et / ou antiparasitaires. L'étude du rôle et de la synthèse des galactolipides chez T. gondii nécessitera la résolution de la structure du DGLE et la recherche des enzymes impliquées dans sa synthèse. Les effets remarquables du serum anti- DGDG mesuré in vitro devront être approfondis sur modèle animal.

Ce travail de recherche s'inscrit dans le contexte de contamination des sols par les pesticides utilisés en agriculture. Cette problématique est un enjeu majeur et actuel puisque l'étendue spatio-temporelle de la contamination est méconnue. Il s'agit donc ici de mieux comprendre la dynamique des herbicides dans les écosystèmes pour mieux en évaluer les risques a priori et plus précisément de savoir dans quelle mesure et par quels mécanismes les organismes édaphiques régulent leurs dynamiques physiques et chimiques. Deux hypothèses alternatives ont été testées avec la molécule herbicide modèle, l'atrazine : i) la macrofaune lombricienne contribue à la dégradation partielle ou totale de l'atrazine en stimulant l'activité de la microflore dégradante et ii) la macrofaune lombricienne favorise la liaison de l'atrazine avec les complexes argilo-humiques du sol, au sein de microsites favorisant la rétention et la rémanence de l'herbicide. Des expérimentations en présence et en absence de Lumbricus terrestris et Aporrectodea caliginosa ont été effectuées à différentes échelles spatiales et temporelles, avec de l'atrazine-14C. S'agissant du rôle de la macrofaune lombricienne, les résultats expérimentaux obtenus ont montré que : • l'ingestion du sol par la macrofaune lombricienne favorise l'adsorption de l'atrazine et conduit à un redistribution des quantités d'atrazine et de ses résidus liés dans les biostructures (avec dans l'ordre : parois de galeries - déjections excrétées dans les galeries – déjections en surface ou turricules – contenus intestinaux ou endentères) par rapport au sol non ingéré et due à un enrichissement en carbone organique conjugué à une plus forte microporosité. • inversement, la minéralisation de l'atrazine n'est pas stimulée voire ralentie en présence de vers. C'est le passage du sol dans le tractus digestif, au cours duquel la structuration des communautés bactériennes initiales du sol est modifiée, qui limite la dégradation totale de l'atrazine par voie microbienne. • une diminution significative du potentiel génétique dégradant (gènes atz) consécutive à l'ingestion a été mise en évidence, suggérant une forte déplétion de la population bactérienne dégradante Pseudomonas sp. ADP. De ces résultats originaux, il est conclu que l'activité de la macrofaune lombricienne dans les sols tendrait à augmenter le temps de résidence du polluant et donc sa rémanence mais s'opposerait à son élimination par biodégradation naturelle par les bactéries.

Graduation date: 2004

Glyphosate-based herbicides are the dominant pesticide on the market and are utilized worldwide in both the agricultural and forestry industries. Their prevalence comes at a time when concern over the potential effects of pesticide application in amphibian spawning grounds is growing. The primary goal of this thesis was to determine if the glyphosate-based herbicide WeatherMax® has the potential to disrupt sexual differentiation and growth in the wood frog (Lithobates sylvaticus) in a pulse exposure at the predicted maximal environmental concentration (PMEC) of 2.88 mg acid equivalent per liter. This was carried out in laboratory, mesocosm and in-situ field exposures, in an attempt to determine how a potential disruption might vary between experimental environments. In this study, tadpoles from three split-wetlands targeted at the PMEC for WeatherMax were found to display no significant change in survival or growth, however gene expression of several genes involved in steroidogenesis during sexual differentiation (cyp19, cyp17, star, foxl2) were found to be affected. The effects on these genes appeared to be dependant on the exposure concentration of WeatherMax in each wetland, which varied even though all three wetlands were meant to target the PMEC. The wetland that was measured as having the highest herbicide concentration (PMEC 13) was found to have a female biased sex ratio. The results found in the field varied from those found in the more artificial exposures. In the laboratory the PMEC of WeatherMax experienced complete mortality, whereas in the mesocosms survival was not significantly affected. Sex ratios were unaffected in the laboratory, however at the PMEC there was a significant male bias in the mesocosms. The discrepancies in the results obtained from the different exposure types highlights the importance of real world exposures. That the same concentration that caused complete mortality in the laboratory caused sublethal effects in the field is of importance as it denotes that these endpoints may not be easily investigated in these synthetic exposures. This project is a part of the Long Term Experimental Wetlands Area (LEWA) and contributes to the body of information amassed therein on the impact of a glyphosate-based herbicide on amphibians in a wetland ecosystem.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.

The objective of this study was to determine if the presence of two ALS inhibiting herbicide residues in three Saskatchewan soils would result in an additive, synergistic, or antagonistic interaction. This was determined through field trials where herbicides were applied sequentially over the course of two years and through dose-response modelling. The herbicides examined in these experiments were imazamethabenz, flucarbazone-sodium, sulfosulfuron, and florasulam, each in combination with imazamox/imazethapyr. The phytotoxicity and persistence of the herbicides in soil was assessed using an Oriental mustard root inhibition bioassay. The determination of herbicide interaction was made through the comparison of the experimentally observed values to theoretically expected values derived from a mathematical equation.<p>The dose response curves created by placing incremental concentrations of these herbicides in soil were compared using the I50 parameter, which is the concentration resulting in a 50% reduction in root length. It appeared that soil organic matter followed by soil pH had the greatest effect in reducing herbicide residue phytotoxicity in the tested soils. Based on the bioassay analysis of sequentially applied ALS inhibiting herbicides, it is proposed that the phytotoxic effect of herbicide residues in soil result in additive injury effects rather than synergistic or antagonistic interactions.

The objective of this study was to determine if the presence of two ALS inhibiting herbicide residues in three Saskatchewan soils would result in an additive, synergistic, or antagonistic interaction. This was determined through field trials where herbicides were applied sequentially over the course of two years and through dose-response modelling. The herbicides examined in these experiments were imazamethabenz, flucarbazone-sodium, sulfosulfuron, and florasulam, each in combination with imazamox/imazethapyr. The phytotoxicity and persistence of the herbicides in soil was assessed using an Oriental mustard root inhibition bioassay. The determination of herbicide interaction was made through the comparison of the experimentally observed values to theoretically expected values derived from a mathematical equation.<p>The dose response curves created by placing incremental concentrations of these herbicides in soil were compared using the I50 parameter, which is the concentration resulting in a 50% reduction in root length. It appeared that soil organic matter followed by soil pH had the greatest effect in reducing herbicide residue phytotoxicity in the tested soils. Based on the bioassay analysis of sequentially applied ALS inhibiting herbicides, it is proposed that the phytotoxic effect of herbicide residues in soil result in additive injury effects rather than synergistic or antagonistic interactions.

Agriculture is a valuable economic resource in western Canada, but for decades farmers have focused on intensive production practices while ignoring the long-term health and maintenance of the land. In recent years, the use of conservation agricultural techniques has been encouraged in an effort to conserve prairie landscape while sustaining cropland productivity. Sustainable agricultural practices that promote soil and water conservation and benefit wildlife and prairie biodiversity include conservation tillage and planting of winter cereal crops. Many species of wild birds nest in the ground cover provided by minimum tillage and fall seeded cropland in the spring. Although habitat quality in conservation areas is superior for birds, there is potential for eggs of ground nesting birds to be exposed to herbicides during spring weed control operations. Herbicides commonly used on the prairies to control weed growth in conservational systems include 2,4-D and Buctril-M®. Since the subtlethal effects of exposure to these herbicides may include DNA damage and immunomodulation, the overall goal of this study was to assess whether <i>in ovo</i> exposure to the herbicides 2,4-D and Buctril-M® adversely affects genetic material and/or immune system function in newly hatched domestic chickens (<i>Gallus gallus</i>) and ducks (<i>Anas platyrhynchos</i>), as surrogates for wild bird species.<p>Study design attempted to reproduce actual field exposures by use of an agricultural field spray simulator to apply formulated herbicides (as opposed to pure active ingredients) at recommended crop application rates. In three separate experiments, fertile chicken eggs were sprayed with 2,4-D ester formulation or with Buctril-M® formulation, and fertile duck eggs were sprayed with 2,4-D ester formulation, during either an early (embryonic day 6) or late (embryonic day 15 for chickens or embryonic day 21 for ducks) stage of incubation. Genotoxicity and immune system function were evaluated in the hatchlings as the main toxicological endpoints to assess potential subtle effects from herbicide exposure, but additional measures of general health and development were also evaluated. Two endpoints were used to assess subtle changes to genetic integrity. The comet assay was used to detect structural damage (strand breaks) in avian lymphocyte DNA, as an index of acute genotoxic effects. Flow cytometry was used to examine potential clastogenic effects of the herbicides, by determining if chromosomal changes resulted in variability in the DNA content of avian erythrocytes. Several endpoints were examined to evaluate potential exposure-induced effects on the immune system. Immunopathological assessment of chicks and ducklings included differential lymphocyte counts, as well as immune organ weights and histopathology. The cell-mediated and humoral immune responses in hatchlings were assessed using the delayed-type hypersensitivity test and measurement of systemic antibody production in response to immunization, respectively. Exposure of fertile chicken and duck eggs to Buctril-M® or 2,4-D had no effects on the biomarkers of genetic integrity in this study. Differences in herbicide treatment (high and low concentrations) and times of exposure (early and late incubation stages) did not translate into noticeable factor effects in final model analyses for any of the genotoxicity assay variables evaluated in newly hatched chickens exposed in ovo to 2,4-D. Similarly, comet assay outcomes in chicks exposed to Buctril-M® were not significantly associated with either herbicide treatment or time of exposure as fixed effect factors. Results of the comet assay using peripheral lymphocytes from ducklings provided evidence of potential primary genetic damage associated with the time of spray exposure in ovo. Comet tail DNA content was significantly associated (P = 0.0269) with exposure times, suggesting that ducks may be increasingly sensitive to spray exposure conditions at an early stage of embryological development. Effects of exposure timing were not attributable to herbicide treatment. Although 2,4-D exposure time was associated with DNA strand breakage in ducklings, there was no evidence of chromosomal damage. However, an association between the HPCV values (a measure of DNA content variability) and time of spray exposure was observed in the experiment where 21-day-old chickens were treated in ovo with Buctril-M®. The mean HPCV value for the early exposure group (E6) was significantly greater (P = 0.0210) than that of the group treated later in incubation (E15). However, Buctril-M® the concentration of herbicide did not have any influence on this outcome, and the reason for the difference between exposure times is uncertain, but may be attributed to stress associated with manipulations during spraying. An increase in HPCV, reflecting greater intercellular DNA variability, is indicative of increased incidence of chromosomal damage, which may be an effect of disturbance during early periods of incubation as a result of exposure conditions.<p>Among the panel of immunotoxicity tests conducted to evaluate the effects of <i>in ovo</i> exposure to 2,4-D and Buctril-M® on the developing avian immune system, only heterophil/ lymphocyte (H/L) ratios and relative immune organ weights were significantly associated with either herbicide treatment or time of spray exposure in all three experiments. In 21-day-old chicks exposed in ovo to 2,4-D, relative bursa weight was associated with the different herbicide treatments (P = 0.0006). Relative bursa weights were significantly lower in chicks in the low dose group, while the opposite effect was observed in the high dose chicks, compared with the controls. It is unlikely that the observed decrease in bursa weight in the low dose group is causally related to herbicide exposure because a consistent dose-response effect was not observed, but this outcome may be explained by a compensatory immune response. The relative spleen weights of newly hatched chickens exposed in ovo to Buctril-M® exhibited a significant association with herbicide treatment (P = 0.0137). Relative spleen weights for birds in the low dose treatment groups were significantly different than both the control (P = 0.0179) and high dose groups (P = 0.0125). However, there was no significant difference between high dose and control groups, and this outcome reduces the likelihood of a causal relationship between spleen weight and herbicide exposure. In the parallel experiment involving in ovo exposure to 2,4-D to ducklings, relative bursa weight was associated with time of spray exposure (P = 0.0434). Ducklings that hatched from eggs exposed to spray on day 6 of incubation exhibited greater mean relative bursa weights than the birds exposed to spray at a later incubation stage (E21). This result implies that spray exposure during earlier stages of development may result in conditions which affect the humoral immune response, if increased bursal weight is associated with increased B lymphocyte and antibody production. In the same experiment, mean H/L ratios in peripheral blood samples from 21-day-old ducklings were significantly different between the groups treated with the high concentration of 2,4-D and water (control) (P = 0.0395). Although ratios from the birds in the low dose groups were not significantly different from the control groups, changes in H/L ratio values demonstrate a dose dependent relationship with increasing herbicide exposure.<p>Residue analysis of chicken and duck eggs in this study measured transfer of herbicide through the shell and into the embryo 24 hours and up to 5 days (chickens only) after spraying. Mean 2,4-D residue concentrations were higher in both chicken and duck eggs from the high dose (10X) groups than in eggs exposed to the recommended field rate of herbicide application (1X). Embryo residue concentrations in the chicken eggs increased from the day following exposure to 5 days after spraying, in both low and high dose groups. This observation indicates that the risk of contaminant-induced adverse effects may continue to increase for at least several days after exposure, thereby influencing the concentration of herbicide to which the developing embryo is exposed.<p>On the Canadian prairies, wild bird eggs are potentially to be exposed to 2,4-D and Buctril-M® during various stages of embryonic development. The present study examined effects of herbicide exposure at two distinct times during incubation, and demonstrated the potential for subtle impacts on genetic integrity and the immune system. Results indicate that spray exposure during earlier stages of organogenesis may cause more significant adverse effects. Given the possible harmful consequences of the observed changes on the long-term health of wild birds, further research is needed in order to better characterize the risks of in ovo agrochemical exposure in prairie ecosystems.