Fish are exposed to multiple stressors in their environment. The interactive effects of pesticide exposure and increased temperature on the physiological stress response were investigated in a comparative field study with cold-water (whitefish, Prosopium williamsoni) and cool-water (sucker, Catostomus) fish from the Oldman River, Alberta, Canada, and in a laboratory study with rainbow trout, Oncorhynchus mykiss. Physiogical stress indicators were measured, and exposure to pesticides was estimated using acetylcholinesterase (AChE) inhibition. Species-specific differences in AChE activities and responses of the physiological stress axis were detected in whitefish and suckers, suggesting that whitefish are a more sensitive species to temperature and pesticide stress. In vivo Dimethoate exposure inhibited AChE activity in various tissues and disrupted the physiogical stress response. Commercial Dimethoate, in vitro, caused a decrease in viability and cortisol secretion while pure grade Dimethoate did not. The results from this study can be used in predictions of fish vulnerability to stress. / ix, 137 leaves : ill. ; 29 cm.

Austroglanis gilli and A. barnardi are endemic to the Clanwilliam-Olifants System in the Western Cape, South Africa. The populations of each of these species are considered to be threatened by various anthropogenic activities, which include inappropriate agricultural practice and impacts of alien invasive fish species. The purpose of this thesis was to assess the life-history of these two endangered species in order to contribute to understanding their biology. Such information is vital for the development of strategies for their conservation. Marginal zone and marginal increment analyses from sectioned lapilliar otoliths of both A. gilli and A. barnardi showed a unimodal peak, suggesting a single annulus formation. The oldest specimens of A. gilli and A. barnardi were 12+ and 14+ years, respectively. The growth of A. gilli and A. barnardi was relatively slow and was best described by the von Bertalanffy growth curve as: L, = 131.56(1 - exp(- 0.27(t - 1.18 ))) for male and L, = 113.86(1 - exp(- 0.43(1- 0.74))) for female A. gilli from the Rondegat River. In the Noordhoeks River, growth was L, = 99.67(1 - exp(- 0.53(t - 0.35))) for male and L, = 96.60(1 - exp(- 0.64(t - 0.11 ))) for female A. gilli, and L, =71.02(1-exp(-0.26(1-3.07))) and L, = 69.50(1-exp(-0.36(1 - 1.88))) for male and female A. barnardi, respectively. The average natural mortality for the combined sexes was estimated using catch curve analysis at 0.37 ± 0.12 per year for A. gilli from the Rondegat River, and at 0.71 ± 0.05 and 0.39 ± 0.04 per year for A. gilli and A. barnardi from the Noordhoeks River, respectively. For A. gilli in the Rondegat River, the first maturity was estimated at 3.3 years (97.3 mm SL) for males and 3.1 years (94.3 mm SL) for females. In the Noordhoeks River, the first maturity for A. gilli was estimated at 2.0 years (71.9 mm SL) for males and 1.7 (66.4 mm SL) for females, and for A. barnardi at 2.0 (55.0 nun SL) for males and 2.9 years (58.9 mm SL) for females. Maturity corresponded closely to the asymptotic sizes from the von Bertalanffy curves, suggesting a shift in energy use from somatic growth to gonad development. Macroscopic assessment of the state of gonads, the Ganado-somatic index and histological examinations revealed that both A. gilli and A. barnardi have a single spawning season. The presence of oocytes in different stages of development in each of the ovaries of mature females collected between November and January suggested asynchronous, iteroperous serial spawning. The resorption of yolk was observed from ovaries collected between February and March and no vitellogenic oocyte was visible from ovaries collected between April and August. A similar trend was observed for males, with spermatozoa filling the lumen between November and January and some residual spermatozoa being present in the lumens soon after the breeding season. Spermatocytes and spermatids were dominant in the testes until October. An Index of Relative Importance revealed that A. gilli feeds predominantly on the benthic macroinvertebrate larvae of Ephemeroptera (particularly Baetidae), Diptera (particularly Chironomidae and Simuliidae) and Trichoptera. Austroglanis barnardi feeds predominantly on dipteran larvae (particularly Chironornidae and Simuliidae). Chi square contingency tables showed a significant difference in the dominant prey items of A. gilli and A. barnardi in the Noordhoeks River (X₂= 53.79, d.f. = 4, p > 0.001) and A. gilli between Rondegat and Noordhoeks rivers (x₂ = 34.74, d.f. = 4, p > 0.001). The Spearman's rank correlation test showed no shifts in the diet of A. barnardi from Noordhoeks River and A. gilli from Rondegat River with a change in size and season (p>O.OS). However, there was a shift in the diet of A. gilli from the Noordhoeks River which could suggest a distinct patchiness of benthic macroinvertebrates between the riffle feeding areas used by juveniles and other biotopes used by adults. The occurrence, in stomach contents, of other prey items from a wide variety of taxa and the presence of allochthonous material from the terrestrial environment could suggest an opportunistic feeding guild for both Austroglanis species. The life-history traits of A. gilli and A. barnardi, which are charaterized by slow growth, long life span and low relative fecundity, indicate that both species are relatively precocial and K-selected. The population of a precocial species is relatively stable and if population numbers were to be greatly reduced, they would require a long time to rebuild. An urgent conservation intervention is therefore recommended for Austroglanis spp. so as to maintain the diversity of populations within these species. The creation of protected river reserves and raising public conservation awareness may minimise activities that result in altered river hydrology and the destruction of complex benthic habitats.

Thesis (PhD (Botany and Zoology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The use of organophosphate pesticides is still an integral part of commercial farming activities and these substances have been implicated as a major source of environmental contamination in South Africa. Evidence exists that many non target animals in and around agricultural areas are at risk of being affected due to the mobile nature of pesticides and the intermittent nature of pesticide application. The extent to which non-target animals are affected by exposure to two organophosphates (azinphos-methyl and chlorpyrifos) was investigated through monitoring selected biomarker responses and life cycle effects under laboratory conditions in two selected test species. A representative species from both the aquatic and terrestrial environment was used as these two compartments of the environment are inevitably linked due to the mobility of pesticides from the area of application to surrounding areas. The earthworm Eisenia fetida was used as test organism in the terrestrial environment while the fish Oreochromis mossambicus served as representative of the aquatic environment. Juvenile life stages of both species were subjected to standard acute toxicity tests which showed that for both species, juvenile life stages were more sensitive to both pesticides than adults. It was also illustrated that azinphos-methyl is more toxic than chlorpyrifos to both species. Both test species were also subjected to an intermittent exposure regime in order to assess the effects of repeated pesticide application on biomarker, life-cycle and behaviour responses. The results indicated that for similar exposure regimes, azinphos-methyl was more toxic to E. fetida than chlorpyrifos and detrimentally affected all endpoints investigated. The present study suggests that exposure concentration may have a more pronounced effect in inducing a toxic response than exposure interval, irrespective of the pesticide used. In addition to this, E. fetida was unable to avoid the presence of these pesticides in soil, even at concentrations as high as 50% of the LC50 value, indicating that the presence of pesticides in the soil pose a realistic threat to earthworms and other soil dwelling organisms. Biomarker responses, morphological effects and feeding behaviour was assessed for O. mossambicus and similar to the terrestrial toxicity experiments, there was evidence to suggest that in the case of an intermittent exposure scenario, azinphos-methyl was more hazardous than chlorpyrifos to this species. For the majority of endpoints that were investigated, it appeared that exposure interval played a more important role in inducing an effect than exposure concentration. At a shorter exposure interval, the majority of endpoints showed no difference between higher and lower exposure concentrations, while at a longer exposure interval the effects of exposure concentration became evident. In addition, feeding behaviour was affected by pesticide exposure in a dose-dependent manner. The present study yielded important results that improve the understanding of biological impacts of pesticide pollution on the environment. This can aid in optimising farming practices such as pesticide application not only in terms of eradicating the pest organisms, but also in terms of mitigating the environmental effects associated with large-scale pesticide use, thereby ensuring sustained biodiversity in these areas. / AFRIKAANSE OPSOMMING: Die gebruik van organofosfaat plaagdoders is ‘n integrale deel van kommersiële landbou aktiwiteite maar hierdie middels is ook ‘n prominente bron van omgewingsbesoedeling in Suid-Afrika. Daar is bewys dat verskeie nie-teiken diere in en om landbouareas geaffekteer word weens die nie-statiese aard van plaagdoders in die omgewing, sowel as die herhalende aard van plaagdodertoediening. Die graad waartoe nie-teiken diere geaffekteer word deur die plaagdoders azinphos-metiel en chlorpyrifos is ondersoek deur die monitering van verskeie biomerkerresponse en lewenssiklus-effekte in geselekteerde toetsspesies binne ‘n beheerde laboratoriumomgewing. ‘n Verteenwoordigende spesie van beide die akwatiese en die terrestriële omgewing is gebruik aangesien hierdie twee dele van die omgewing onlosmaaklik verbind is weens die beweging van plaagdoders vanaf die area van toediening na omringende areas. Die erdwurm Eisenia fetida is gekies as toetsorganisme vir die terrestriële omgewing en die varswatervis Oreochromis mossambicus het gedien as verteenwoordigende spesie vir die akwatiese omgewing. Onvolwasse diere van beide spesies is onderwerp aan standaard akute toksisiteitstoetse en daar is gevind dat, vir beide spesies, onvolwasse diere meer sensitief vir die betrokke plaagdoders is as volwasse diere. Dit is ook gevind dat azinphosmetiel giftiger is as chlorpyrifos vir beide spesies. Beide toetsspesies is ook onderwerp aan ‘n chroniese blootstellingsregime om die effek van herhaalde plaagdodertoediening op biomerker-, lewenssiklus- en gedragsresponse te ondersoek. Die resultate van die herhaalde blootstelling het aangedui dat vir soortgelyke blootstellingsregimes, azinphos-metiel giftiger is as chlorpyrifos vir E. fetida en dat beide middels alle eindpunte wat ondersoek is, nadelig affekteer. Die huidige studie toon ook bewyse dat blootstellingskonsentrasie ‘n meer prominente effek as blootstellingsinterval kan hê in die teweegbringing van ‘n toksiese respons. Verder was E. fetida nie in staat om die teenwoordigheid van die plaagdoders in grond te vermy nie, self nie by konsentrasies so hoog as 50% van die LC50 waarde nie. Laasgenoemde resultaat dui dus aan dat die aanwesigheid van plaagdoders in die grondomgewing ‘n realisitese bedreiging inhou vir erdwurms en ander grondorganismes. Soortgelyk aan die terrestriële toksisiteitseksperimente, was daar getuienis vir die verhoogde toksisiteit van azinphos-metiel relatief tot chlorpyrifos vir O. mossambicus. Dit blyk dat blootstellingsinterval ‘n meer prominente rol as blootstellingskonsentrasie speel in die teweegbringing van effekte vir die meerderheid van die eindpunte wat ondersoek is. In die geval van ‘n korter blootstellingsinterval het die meerderheid van eindpunte wat ondersoek is geen verskille getoon tussen ‘n hoër en ‘n laer konsentrasie nie, terwyl met ‘n langer blootstellingsinterval daar ‘n aanduiding was dat blootstellingskonsentrasie ‘n meer prominente rol gespeel het. Verder is gevind dat voedingsgedrag in O. mossambicus geaffekteer is op ‘n konsentrasie verwante manier. Die huidige studie toon resultate wat ‘n belangrike bydrae kan lewer tot die begrip van die biologiese impakte van organofosfaat plaagdoders op die omgewing. Die resultate kan gebruik word vir die optimisering van boerderypraktyke soos plaagdodertoediening, sodat laasgenoemde effektief is vir die beheer van pes-organismes, maar ook die impakte van grootskaalse plaagdodertoediening kan minimaliseer en sodoende die biodiversiteit binne hierdie areas sal beskerm.

This thesis presents an evaluation of the dispersive solid-phase extraction (d-SPE) method referred to as the quick, easy, cheap, effective, rugged and safe (QuEChERS) method for the determination of four organochlorine pesticide residues in fish samples. The pesticides investigated in this study were o, p′-DDT, p, p′-DDE, aldrin and dieldrin. The combined use of Gas Chromatography with an Electron Capture Detector (GC-ECD) and sensitive Time of Flight (TOF) mass detector facilitated the identification of the target analytes. In the absence of certified reference material, the overall analytical procedure was validated by systematic recovery experiments on spiked samples at three levels of 2, 5 and 10 ng/g. The targeted compounds were successfully extracted and their recovery ranged from 76 to 96% with relative standard deviations of less than 13%. The optimum QuEChERS conditions were 2g of fish powder, 10 ml acetonitrile and 1 min shaking time. The optimal conditions were applied to assess the levels of chlorinated pesticides in blunt-tooth catfish (Clarias ngamensis), tigerfish (Hydrocynus vittatus), Oreochromis andersonii and red-breasted tilapia (Tilapia rendalli) from the Okavango Delta, Botswana. Dieldrin, p, p′-DDE and aldrin were detected in all the analysed samples with a concentration range of 0.04 – 0.29, 0.07 – 0.33, 0.04 – 0.28 and 0.03 – 0.24 ng/g per dry weight in O. andersonii, C. ngamensis, T. rendalli and H. vittatus respectively. These concentrations were below the US-EPA 0.1 μg/g allowable limit in edible fish and the Australian Maximum Residue Limit (MRL) of 50 - 1000 ng/g in fresh fish. DDT was not detected in all the fish species investigated. The mean lipid content recorded in the fish samples were 1.24, 2.16, 2.18 and 4.21% for H. vittatus, T. rendalli, O. andersonni and C. ngamensis respectively. No systematic trend was observed between fish age and pesticide levels in fish. Acetylcholinesterase (AChE) activity assays were performed to assess the effects of organochlorine pesticides in Clarias ngamensis. The enzyme activity recorded in Clarias ngamensis from the Okavango Delta and the reference site was 12.31 μmol of acetylcholine iodide hydrolysed/min/g brain tissue. The enzyme activity remained the same indicating no enzyme inhibition. The conclusions drawn from this study are that the QuEChERS method is applicable for the determination of organochlorine pesticide residues in fish matrices. The fish from the Okavango Delta are safe for human consumption.