A Level III fugacity-type model was applied to southern Belize (Stann Creek and Toledo Districts) to predict which of the pesticides most heavily used in banana and citrus farms are most likely to end up in streams and coastal waters via surface runoff. Concentrations of all target pesticides in coastal waters of southern Belize were then measured during two sampling campaigns (dry season and rainy season) in 2008. Several pesticides were measured in significant levels, including some as far out as waters overlying coral reefs. The presence of these pesticides in the coastal waters indicates that agricultural activities in southern Belize may have a potential impact on coral reefs offshore. Results of the predictive model were compared with the empirical data to determine how well the model works in a tropical region such as southern Belize. Overall, there is considerable agreement between the two, indicating that the model employed herein can be applied to other tropical areas. Concentrations of mercury and lead were also measured in the same study area. Mercury levels were uniform and low, suggesting natural sources. Lead levels varied and are most likely explained by the presence of unregulated and illegal waste dumps near streams in the region. An analysis was carried out to examine government policy on pesticide use. Findings indicate a lack of a coherent governmental policy on the sale and use of pesticides, most likely because of too many agencies/ministries being involved and the lack of national standards for these pesticides in the environment.

Les Inuit sont de par leur lieu et mode de vie fortement exposés aux pesticides organochlorés notamment au DDT et à son métabolite principal, le DDE. La littérature scientifique rapporte des effets délétères de l’exposition prénatale à ces molécules sur le développement des fonctions cognitives des enfants. Aucune étude ne s’est intéressée aux conséquences de l’exposition in utéro au DDT et au DDE sur le développement des fonctions sensorielles des enfants. Le premier objectif de cette thèse était donc d’évaluer si l’exposition prénatale à ces molécules pouvait être associée aux fonctions visuelles des enfants Inuit. Le DDT et le DDE ont été mesurés dans le cordon ombilical ainsi que dans le sang des enfants à 5 et 11 ans. Les potentiels évoqués visuels des enfants ont été enregistrés à 11 ans. Des analyses de régressions multiples ont révélé une augmentation de l’amplitude de la composante N150 en lien avec les niveaux de DDE mesurés dans le cordon ombilical ainsi qu’une diminution de l’amplitude de la N75 en lien avec les concentrations sériques de DDE mesurées à 5 ans. Ces données suggèrent que l’exposition périnatale au DDE pourrait avoir un impact sur l’intégrité des fonctions visuelles des enfants. La seconde partie de la thèse portait sur les pesticides organophosphorés dont l’usage a remplacé celui des organochlorés dès les années 1970. Différentes études réalisées auprès de populations défavorisées ont montré que l’exposition prénatale à ces molécules pouvait altérer les fonctions cognitives des enfants. Le second objectif du présent travail était donc d’évaluer si l’exposition aux organophosphorés pouvait être associée aux fonctions cognitives d’enfants issus de la population générale en France. Les métabolites communs aux organophosphorés ont été mesurés dans les urines des mamans durant la grossesse (i.e. DAP, DM et DE). Le WISC a été administré aux enfants à 6 ans par une neuropsychologue afin d’obtenir deux indices de développement cognitif, un indice de mémoire de travail et un indice de compréhension verbale. Les analyses de régression multiple réalisées n’ont pas permis de mettre en évidence d’association entre les DAP mesurés dans les urines des mamans durant la grossesse et les fonctions cognitives des enfants. A l’inverse les niveau urinaires de DE ont été associés à de meilleurs scores de compréhension verbale. Ces données suggèrent que certaines populations pourraient être plus vulnérables que d’autres aux propriétés de neurotoxicité développementale des pesticides organophosphorés, notamment les populations issus de milieux défavorisés. Du fait de l’exposition commune aux pesticides organophosphorés il parait important de mieux caractériser les conséquences neuro-développementale de l’exposition prénatale à ces molécules. L’étude de l’intégrité des processus visuels peut parfois permettre de mettre en évidence des altérations neuro-développementales non décelables avec les outils standards d’évaluation neuropsychologique. Le troisième objectif du présent travail était donc d’évaluer si l’exposition prénatale aux pesticides organophosphorés pouvait être associée aux fonctions visuelles des enfants issus de la population générale en France. Des analyses de régression multiple ont permis de mettre en relation les niveaux urinaires de DAP, DM et DE mesurés chez les mamans durant la grossesse et les scores de sensibilité au contraste obtenus chez les enfants à 6 ans à l’aide du Functional Acuity Contrast Test. Bien qu’aucune association significative ne soit mise en évidence chez l’ensemble des sujets, de plus faibles scores sont obtenus chez les garçons en lien avec les niveaux urinaires de DE mesurés durant la grossesse. Ces données suggèrent que l’exposition prénatale aux organophosphorés pourrait influencer le développement des fonctions visuelles des enfants et plus particulièrement des garçons. / Prenatal organochlorine and organophosphate pesticides exposure and children cognitive and sensory function at school age

The chemical fate and biological impacts of cyfluthrin in aquatic ecosystems were investigated using microcosms (1.9 m^3 concrete tanks) during 1989. Results were compared to a concurrent pesticide registration study using mesocosms (634.7 m^3 earthen ponds). Ten spray drift and five soil runoff simulations were conducted. Pesticide loadings were scaled by system volume, with the same experimental design in ponds and microcosms. Aqueous cyfluthrin concentrations and sediment residue values were generally higher in microcosms, while aqueous half-life was shorter in the smaller systems.

Organochlorine pesticides (OCPs) were extensively applied in agriculture, industry and public health programs for decades. Based on the persistence and the lipophilicity of OCPs, these chemicals are ubiquitous in the environment and can be accumulated in fatty tissues of animals through the food chain. Even being restricted for years, OCPs are still detected in human bodies. In this thesis, analytical methods for the determination of OCPs were developed and applied for the analysis of cord serum samples. The evaluation of prenatal exposure to OCPs and its effects on birth outcomes as well as the postnatal growth were investigated. Due to the toxicology and carcinogenesis, biomonitoring of the OCP exposure to human is needed. Therefore, an analytical method with high sensitivity and specificity is required to detect OCPs at trace levels in serum. We compared two data acquisition modes of mass spectrometry (MS), namely selected ion monitoring (SIM) and multiple reaction monitoring (MRM). Higher sensitivity and selectivity were achieved by MRM because the background noise was reduced by lowering the matrix effects. Different ionization techniques, including electron ionization (EI), chemical ionization (CI) and atmospheric pressure chemical ionization (APCI) were evaluated. The EI source is a universal ionization technique available with the MS library for the compound identification. The negative chemical ionization (NCI) is more suitable to analyze compounds with high electronegativity. The novel ionization technique APCI was coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS). The APCI source was evaluated by terms of ionization and fragmentation performance. APCI was a soft ionization technique generating molecular ions with high intensity. The selectivity and sensitivity of APCI were comparable or better than the EI source. As one of the largest consumers and producers of OCPs, China has suffered severe OCP pollution. Previous monitoring studies reported detectable levels of OCPs in human bodies. However, studies concerning the prenatal exposure to OCPs in China are limited. Due to the vulnerability of fetuses, the effects of prenatal exposure to OCPs could be more severe than those of adults. We collected cord serum samples during the delivery period in Wuhan, China and measured the OCP concentrations to assess the prenatal exposure by using GC-MS/MS. Compared with other areas in China, the OCP levels in Wuhan were comparable in this population. The identified predominant OCPs were β-HCH and p,p'- DDE, with geometric means of 8.67 and 33.9 ng/g lipid, respectively. Slight positive associations were found between α-HCH and β-HCH, and between o,p′- DDT and p,p′-DDT, which indicated similar exposure source of these chemicals. The obtained results showed that HCH levels were associated with maternal age, body mass index (BMI) before pregnancy, education levels, and passive smoking. Associations between the prenatal exposure of OCPs and birth outcomes were investigated. The sex-specific relationships between the OCP exposure and birth size were indicated. Concentrations of β-HCH were inversely associated with birth weight and ponderal index for boys, while for girls these associations were not significant. Our results suggested that the prenatal exposure to OCPs exerted negative effects on the fetal growth, and precautions should be taken even though the OCP levels were relatively low.

The Sacramento River watershed provides important rearing habitat for key aquatic species, including juvenile Chinook salmon (Oncorhynchus tshawytscha). Salmon rearing in the watershed may inhabit the mainstem river channel or a corresponding floodplain, the Yolo Bypass, before migrating to the ocean. Studies of juvenile salmon have indicated that floodplain rearing may be beneficial in terms of growth and survival, likely related to different trophic pathways of the river and floodplain. However, fish also encounter many anthropogenic stressors in these habitats, such as pesticides, which have well-documented use and environmental presence in the region. Rearing individuals are potentially exposed to pesticides via trophic transfer, which may vary based on utilized food webs due to hydrophobic pesticide fate and transport. To examine the food web structure of each system and the potential for pesticide exposure through dietary routes, a two-year field study was completed. First, to characterize dietary contributions, a three-tiered approach incorporating stable isotopes (δ13C, δ15N and δ34S), essential fatty acids and gut content analyses was employed. Subsequently, pesticides were extracted from prey items and salmon and analyzed to determine contaminant residues. Stable isotope analyses indicated that critically important components of juvenile Chinook diet were amphipoda and adult diptera in 2019 and 2020, respectively. Amphipoda groups had higher concentrations of the fatty acid docosahexaenoic acid (DHA), an important component for fish development, than juvenile diptera or oligochaeta. Diptera (larvae and adults) were frequently found in juvenile Chinook stomachs from both areas and years. Throughout the pesticide examination, organochlorines including the DDT group (p,p’-DDT, p,p’-DDD and p,p’-DDE) were prevalent in all examined biota. There were a significantly greater number of pyrethroid and phenylpyrazole detections and concentrations in zooplankton as compared to macroinvertebrates (Poisson regression, p < 0.05) across regions and years. Additionally, significantly higher concentrations of organochlorines were exhibited in floodplain rearing fish as compared to the Sacramento River (ANOVA, p < 0.05). These findings suggest that juvenile Chinook feeding primarily on zooplankton may be exposed to a greater range of pesticides than those exhibiting benthic feeding, but these pelagic prey were not demonstrated as a major dietary item during the two-year study. Additionally, the previously inferred benefits of floodplain rearing may come at a cost of increased organochlorine exposure. This research has allowed for a robust assessment of potential trophic transfer of pesticides to juvenile salmon, which may help inform future floodplain restoration efforts.

Epidemiological studies implicate pesticide exposure as a possible etiologic factor in idiopathic Parkinson's Disease, which results from degeneration of nigrostriatal neurons, along with reduced levels of the neurotransmitter, dopamine. Behavioral and neurochemical analyses in C57BL6 mice were performed following a subchronic dosing regime with the organochlorine insecticide heptachlor or the pyrethroid deltamethrin. Results were compared to those induced by the established parkinsonian neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the end of the treatment period, mice were assessed for effects on behavior, as well as levels of striatal dopamine, nerve terminal respiration, and synaptosomal dopamine transport. The primary behavioral effect of deltamethrin was incoordination, while heptachlor caused hyperexcitability and increased locomotion. The major neurochemical effect observed for both compounds was upregulation of the presynaptic dopamine transporter (DAT) by 70% and 100% for deltamethrin and heptachlor, respectively. The insecticides exerted only modest effects on striatal levels of dopamine and its metabolite, dihydroxyphenylacetic acid. However, doses of heptachlor higher than those which caused induction of DAT (e.g. greater than or equal to 25 mg/kg), when administered subchronically, were found to cause convulsions in some animals and caused marked, dose-dependent depression of basal striatal tissue respiration rates. No synergism was observed between the effects of insecticides and MPTP. Enhanced transport was thought to be a compensatory effect from increased release of transmitters by the insecticides, <i>in vivo</i>. Striatal dopamine, GABA and glutamate nerve terminals were differentially sensitive to the releasing effects of heptachlor compared to cortical serotonin terminals, and responded in the following rank order of sensitivity: dopamine > GABA > glutamate > serotonin. Additional experiments to characterize the mechanism(s) by which cyclodienes facilitate release of neurotransmitters in synaptosomes demonstrated a lack of distinct Ca²⁺ component and no involvement of retrograde DAT activity, suggesting that released label was of vesicular origin, but did not require Ca²⁺. Insecticidal toxicants, such as organochlorines and pyrethroids, which augment dopamine release and increase the maximal rate of dopamine uptake, may inundate the cytosol of nigrostriatal neurons with high concentrations of free dopamine, which has been shown by other researchers to induce apoptosis and may thereby contribute to the development of Parkinson's disease. Funding for this work was provided under grant number HHHREP 94-01 by the Hawaii Heptachlor Foundation, a non-profit organization. The Hawaii Heptachlor Foundation may be contacted at the following address: Ocean View Center PH#3, 707 Richards St., Honolulu, HI 96813. / Ph. D.

Germination of mung bean, Phaseolus mungo L. , and wheat, Triticum aestivum L., seeds was used for bioassay to demonstrate the toxic effects of selected pesticides--menazon, disulfoton, and GS-14254-- and allergens-- alantolactone and usnic acid. The ability of gibberellic acid to counteract the toxic effects of these chemicals on germination and seedling growth was studied. Chemical composition of the treated and untreated seeds was made with special attention to starch and protein degradation. Effect of these toxicants on the synthesis of amylase, ATPase, and protease enzymes during germination was studied since these enzymes are synthesized de novo during germination. To ascertain their effect on protein synthesis in storage tissue of the germinating seeds, uptake and incorporation of 14C-L-leucine into protein was studied in potato tuber slices and germinating mung beans. Correlation of biochemical data and histochemical changes in the treated and untreated seeds of mung bean was obtained with menazon and usnic acid. Furthermore, ultrastructural changes were studied in order to relate functional and structural changes in the seeds .in conjunction with phytotoxic actions of these chemicals. Among the insecticides, menazon (250 ppm) was found to be more toxic to both species than was disulfoton. GS-14254 (100 ppm) also was equally inhibitory to seed germination and seedling growth of mung bean and wheat seeds. When a solution of the herbicide GS-14254 (100 ppm) was added to either of the insecticides at their maximum concentrations the inhibitory effect of the combined pesticides on seed germination and seedling growth was more pronounced, especially with wheat. Usnic acid (50 to 250 mg/1) and alantolactone (100 mg/1) significantly inhibited germination and root and shoot growth in both mung bean and wheat seeds. These two compounds appeared to be more phytotoxic than the pesticides. Gibberellic acid partially counteracted the inhibitory effects of the pesticides and allergens, thus these chemicals showed no antiauxin activity. Before any growth is observed there is a marked increase in respiration during germination that releases energy from food materials already present in usable form in the cells. At their maximum concentrations, menazon, disulfoton, GS-14254, alantolactone, and usnic acid significantly blocked the respiration of the germinating seeds at the end of 72 h after treatment. In all cases except alantolactone respiration of wheat seeds was considerably more affected than that of the mung beans. Compared to control seeds, pesticide chemicals as well as allergenic compounds caused significant reduction in the amounts of soluble reducing sugars and free amino acids after 72 h germination period. Similarly, starch degradation was less in the treated seeds. Among the species of seeds, considerably less amounts of reducing sugars and amino acids were formed in the pesticide-treated wheat seeds than in the mung beans as compared to their respective controls. Such differences in the inhibitory effects were not observed in seeds treated with allergenic compounds. The development of amylase and ATPase activity in the seeds treated with maximum concentrations of pesticides tended to be lower than that in the control seeds. In case of menazon, inhibition of amylase activity was more pronounced than that of disulfoton or GS-14254. Proteoiytic activity in control and disulfoton- and menazon-treated seeds was not significantly different during germination period, but in case of GS-14254, it was considerably lower. Usnic acid at highest concentration tested completely inhibited the development of amylase activity in mung beans whereas it was significantly lower in seeds treated with the maximum concentration of alantolactone. The inhibition of amylase activity in wheat seeds treated with these compounds was more or less similar. ATPase inhibition in seeds treated with usnic acid was more severe than that in alantolactone treated seeds. However, proteolytic activity in control and treated seeds showed almost the same trend during the germination period. The activity per se of amylase isolated from mung bean and wheat seeds germinated for 3 days was not significantly inhibited by the presence of the pesticides or allergens in the reaction mixture indicating that these chemicals do not inhibit already synthesized amylase enzyme. Observations with potato tissue and germinating mung beans indicated that both total uptake and incorporation of 14c-L-leucine into protein were significantly inhibited by menazon, disulfoton, GS-14254, and alantolactone. On the other hand, the uptake in germinating mung bean treated with usnic acid was not affected although both uptake and incorporation were inhibited in potato tissue. Menazon and usnic acid were then selected as the representative chemicals for pesticides and allergens, respectively, and their toxic effects were studied histochemically in 3-day germinating mung beans. It was observed that total nucleic acid content and RNA content in seeds treated with these chemicals were considerably less than that in the control seeds. Similarly, treated seeds showed more starch grains and protein bodies indicating less metabolic activity in these seeds. At the ultrastructural level, menazon- or usnic acid-treated mung bean cotyledons at day 3 of germination contained no vacuoles but many undigested protein bodies were observed. In contrast, fully developed mitochondria, endoplasmic reticulum with ribosomes, and vacuoles were seen in control cells indicating protein (enzyme) synthesis and digestion of the food reserves.

The effect of the following pesticides on DC electrical conductivity of lecithin-cholesterol membranes has been studied: endothall, paraquat, diquat, 2,4-D, 2,4-DB, 2,4,5-T, 2,4-DCP. It has been found that the ions of endothall, paraquat and diquat are essentially membrane impermeable and that they do not bind to the membrane surface. In contrast, 2,4-D, 2,4-DB, 2,4,5-T and 2,4-DCP induce electrical conductivity in lecithincholesterol membranes and in addition they also cause an increase in the nonactin-K+ membrane conductivity. The compounds 2,4-D, 2,4-DB, 2,4,5-T and 2,4-DCP basically behave as class II uncouplers. The kinetic scheme of charge transfer across the membrane, based on the assumption that the membrane is permeable both to the negatively charged dimers and to the neutral molecules of pesticides, satisfactorily explains the basic features of the experimental results: the concentration dependence of pesticide-induced membrane conductance, effect of proton concentration on membrane conductance, and the effect of pesticide concentration on the voltage dependence of membrane conductance. It fails to predict the effect of proton concentration on the voltage dependence of membrane conductance. The enhancement of nonactin-K+ membrane conductance by the pesticide is presumably due to the adsorption of the ionized form of the pesticide at the membrane surface. It was found that the Gouy-Chapman diffuse double layer theory was not applicable for the calculation of surface membrane potential due to the adsorbed ions.

Leptinotarsa decemlineata (Say) is the most important defoliating pest of potato Solanum tuberosum L., in North America and Europe.  Management of this pest relies heavily on chemical control and insecticide resistance is a persistent problem.  This phenomenon has increased the need for developing novel insecticides, resistance evaluation, and the development of alternative control strategies regarding this insect pest.  From 2010 to 2013, field and lab experiments were conducted to evaluate the efficacy of a novel insecticide tolfenpyrad on L. decemlineata.  In leaf-dip assays, tolfenpyrad was highly toxic to L. decemlineata with LC50 values of 0.013 and 0.164 g ai/L for larvae and adults, respectively.  Tolfenpyrad was also toxic to eggs with 0% hatching after being dipped in a field rate concentration.  In field efficacy trials, potato plots treated with tolfenpyrad at rates as low as 153 g ai/ha effectively controlled L. decemlineata. In 2012, populations of L. decemlineata were collected from the Eastern Shore of VA and subjected to toxicity assays to determine current susceptibility to permethrin and oxamyl.  The toxicity assays indicated an increase in toxicity to permethrin in L. decemlineata larvae (LC50 = 3.931 g ai/L) and an increase in toxicity to oxamyl in adult beetles (LC50 = 9.695 g ai/L) compared with LC50 values previously reported in 1990.  In 2012, populations of L. decemlineata from Cheriton, VA, New Church, VA, Painter, VA, and Plymouth, NC were also evaluated for enzyme activity after exposure to sub-lethal concentrations of permethrin, oxamyl, and tolfenpyrad.  Adult beetles were subjected to enzyme assays to measure the activity of cytochrome P450 mono-oxygenase (P450), glutathione-S-transferase (GST), general esterases, and protein content.  Results from the enzyme assays indicated significantly greater esterase activity in beetles from Painter, VA exposed to permethrin [±-naphthol (F= 11.66, df= 4, 20, P<0.0001) and "-naphthol (F= 11.86, df= 4, 20, P<0.0001)], oxamyl [±- naphthol (F= 10.64, df= 4, 20, P<0.0001) and "-naphthol (F= 6.94, df= 4, 20, P=0.0011)], tolfenpyrad [±- naphthol (F= 407.62, df= 1, 8, P<0.0001) and "- naphthol (F= 28.15, df= 1, 8, P= 0.0007)], and the untreated control [±- naphthol (F= 28.14, df= 3, 16, P<0.0001) and "- naphthol (F= 28.86; df= 3, 16, P<0.0001)] compared to most of the other populations tested.  GST activity was significantly greater in tolfenpyrad exposed beetles compared to the non-treated beetles from Painter VA (F= 17.66, df= 5, 24, P< 0.0001). Through laboratory assays and field experiments in potato, the efficacy of a new bio-pesticide derived from the bacterium Chromobacterium subtsugae was evaluated for the control of L. decemlineata.  Results from the laboratory assays showed L. decemlineata feeding was inhibited by the bio-pesticide derived from C. subtsugae.  However, field efficacy trials in 2010, 2011, and 2012, indicated no control of L. decemlineata. Methyl salicylate is an organic compound produced by potato and other plants in response to insect herbivory.  Abundance of predatory arthropods and L. decemlineata life stages were measured in plots treated with and without 5 g slow-release packets of methyl salicylate (95% methyl salicylate (Predalure")).  Methyl salicylate treatment had no impact on predator recruitment or cumulative mortality of L. decemlineata in potatoes. This research has provided us with a new tool for L. decemlineata management, as well as more information about resistance trends and alternative control strategies from which we can build on to reduce resistance development in L. decemlineata and ultimately formulate a stronger integrated pest management strategy for this insect pest. / Ph. D.

Genetic engineering tools are under continuous development. However, hesitation by consumers and governments regarding consumption of genetically modified organism (GMO) affects taking advantage of developments in biotechnology. While being a complicated issue to address, this challenge inspired us to investigate whether it is possible to engineer organisms without altering their wild-type genomes, but with the same customizability level offered by genetic engineering; that is, having the capacity of expressing foreign proteins not codified by the wild-type genome. I used B. subtilis spores as a model organism for this purpose. I took advantage of the sporulation process during which two compartments with differential expression, or different gene expression patterns co-exist, the mother cell and the forespore, and I programmed a single designer plasmid to behave differently in each compartment: the plasmid in the mother cell modifies the spore phenotype, while the plasmid in the forespore undergoes self-digestion. At the end of sporulation, the mother cell lyses and releases the final product — a plasmid-free engineered spore. Following this, I incorporated the forespore-specific "self-digestion" gene circuit into a variety of plasmids with different purposes, including the generation of spores expressing GFP on their protective coats and the artificial induction of sporulation, both of them as a proof-of-concept of genetically intact bioengineered organisms. Production of the different types of genetically intact bioengineered spores resulted in an average of nearly 90% of them free of detectible plasmid or genome alterations. Spores of B. subtilis and other species overall continue to gain attention in the biotechnology sector, with potential applications ranging from biopesticides, probiotics, and vaccines to energy-converting materials, self-healing concrete, and whole-cell biocatalysts. While spores represent a special case of multiple-compartment organisms among bacteria, most eukaryotic organisms possess multiple compartments, structures, or tissues with differential expression, including plants and animals. Therefore, our results in this study could serve as a starting point for new ideas and methods for the genetic modification-free engineering of complex organisms or parts of them.