Numerous contaminants are present in mixtures in the aquatic environment. Among these are the azoles, a group of chemicals that includes both pharmaceuticals and pesticides. Azole fungicides are designed to inhibit lanosterol 14-demethylase (cytochrome P450 (CYP) 51), while other azoles are intended to inhibit aromatase (CYP19), i.e. the enzyme catalyzing biosynthesis of estrogens. In fish, a variety of CYP enzymes are involved in biotransformation of waterborne contaminants, and in metabolism of endogenous compounds including steroidal hormones. The induction of CYP1A protein and 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers for exposure to aryl hydrocarbon receptor (AhR) agonists in fish. We developed an assay to measure inhibition of CYP1A activity (EROD) in three-spined stickleback and rainbow trout gill tissue ex vivo. Several azole fungicides were found to be potent inhibitors of CYP1A activity. A wastewater effluent containing high concentrations of pharmaceuticals was also shown to inhibit CYP1A activity. Further, several azoles inhibited CYP19 activity in rainbow trout brain microsomes in vitro. Azole mixtures reduced both CYP1A and CYP19 activity monotonically and in an additive way. Given the additive action of the azoles, studies to determine adverse effects of azole mixtures on CYP-regulated physiological functions in fish are needed. Induction of EROD and of gene expression of CYP1 in several organs was observed in an in vivo exposure with the same effluent shown to inhibit EROD. This finding could imply that there was a mixture of AhR agonists and CYP1A inhibitors in the effluent. Finally, wastewater treatment technologies were evaluated using biomarker responses in rainbow trout exposed to effluents of different treatments. The results from chemical analysis together with the biomarker results show that ozone and granulated active carbon treatment removed most pharmaceuticals, as well as AhR agonists and other chemicals present in the regular effluent. This part of the thesis demonstrates that biomarkers in fish such as induction of CYP1 gene expression are applicable to evaluate the efficiency of different treatment technologies for wastewater.

No description available.

Flodpärlmusslan (Margaritifera margaritifera. L) har en obligatorisk fas i sin komplexa livcykel där den lever som parasit på antingen öringens (Salmo trutta, L) eller laxens (Salmo salar, L) gälar. För värdfisken skulle detta kunna innebära en ökad energikostnad när den födosöker i rinnande vatten. Öringens furagering består i att den finner en utgångspunkt (eng. focal point) i strömmen utifrån vilken den fångar sina byten. I denna studie undersöks det om en infektion av flodpärlmusslans glochidielarver på öring påverkar öringens födointag och hantering av födan. Sextio årsungar av öring infekterades med glochidielarver av känd koncentration, och deras födosökning mättes över en 10-veckors period för att bedöma effekten av den växande mussellarven i jämförelse med en icke-infekterad (kontrollgrupp) grupp fiskar. Individuella födoförsök, tio försök per mätningstillfällena, utfördes i ett strömakvarium där en enskild öring matades med drivande föda i fem minuter. Bytet släpptes ned i strömmen med två sekunders intervall, antalet byten räknades och furageringsbeteendet observerades. Födoförsöken genomfördes med två veckors intervall. Vid försöken hittades inga signifikanta skillnader med avseende på: 1) antalet fångade byten, 2) proportionen av fångade byten av försökets första tre minuter, eller 3) de rumsliga aspekterna av öringens utgångspunkt. Vidare uppmättes ingen signifikant effekt av tid på furageringsframgången på den infekterade gruppen jämfört med kontrollgruppen. Däremot var det ett högre antal infekterade individer som spottade ut byten jämfört med kontrollgruppen. Skillnaden i utspottade byten pekar på att infektionen har en negativ påverkan på fiskarnas furageringsförmåga. Dock visade försöken inga resultat som stödjer tidigare studier där signifikant skillnad erhållits med avseende på födointag. Förklaring till detta kan finnas i metodskillnader mellan denna och tidigare studier, inklusive skillnad i typ av byte, strömakvarier, ursprungsström för musslorna och öringen och infektionsgrad hos de infekterade fiskarna samt kontrollgruppen. / The freshwater pearl mussel (Margaritifera margaritifera, L) has a mandatory phase of its complex life cycle where it lives as a parasite on the gills of either trout (Salmo trutta, L) or salmon (Salmo salar, L). For the host fish this could mean an increased cost when it forages in running water. The trout´s foraging consists of finding a focal point in the stream from where it catches its prey. This study examines whether an infection of the freshwater pearl mussel´s glochidium larva on the gills of brown trout affect trout foraging. Sixty juvenile (0+) brown trout were infected with glochidium larva of a known concentration, and trout foraging behaviour was measured over a 10-week period to assess the effects of the growing mussel larvae of the fish compared to non-infected (control) fish.  Individual feeding trials, ten replicates for each session, were conducted in a stream aquarium where a single trout was fed drifting prey for five minutes. The prey were released into the stream every two seconds, the number of prey captured was counted, and the foraging behavior was characterized. Feeding trials were conducted at two-week intervals. No significant differences between the infected and control groups were found for: 1) the number of prey eaten, 2) the proportion of prey eaten during the first three minutes of the trial, or 3) location of the focal points. Furthermore, there was no significant effect of time on foraging success of the infected group compared to the control group. However, there was a higher number of infected individuals who spat out the prey compared with the control group. The difference in rejected prey suggests that the infection has a negative impact on the fish’s foraging ability. However, the results of this experiment do not support previous studies, where a significant difference was obtained with respect to food intake. This might be explained by differences in experimental protocol between this and previous studies, including differences in prey type, stream aquaria, stream origins of mussels and trout, and the infection status of infected and control fish.

Selenium (Se) is known to cause chronic toxicity in aquatic species. In particular, dietary exposure of fish to selenomethionine (SeMet), the primary form of Se in the diet, is of concern. Previous studies reported that chronic exposure to elevated dietary SeMet altered swimming performance, aerobic metabolism, and energy and endocrine homeostasis in adult fish. However, little is known about the direct effects of dietary SeMet exposure in juvenile fish. Therefore, the overall objective of this thesis was to investigate sublethal pathophysiological effects of subchronic dietary SeMet exposure in two juvenile fish species, fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss). In the first experiment, 20 days post hatch (dph) juvenile fathead minnow were exposed to different measured concentrations (2.8, 5.4, 9.9, 26.5 µg Se/g dry mass [dm]) of Se in food in the form of SeMet for 60 days. In the second experiment, 14 dph juvenile rainbow trout were exposed for 37 days to different measured concentrations (1.0, 4.1, 11.2, 26.1 µg Se/g dm) of Se in food in the form of SeMet. Following exposure, samples were collected for Se analysis and fish were subjected to a swimming performance challenge to assess critical swim speed (Ucrit), tail beat frequency and tail beat amplitude, oxygen consumption (MO2), cost of transport (COT), standard metabolic rate (SMR), active metabolic rate (AMR), and factorial aerobic scope (F-AS). Dietary SeMet exposure impaired swimming ability in both fathead minnow and rainbow trout. Juvenile fathead minnow showed alterations in aerobic metabolism with increased MO2, COT and AMR at the 9.9 and 26.5 µg Se/g diets, while dietary SeMet exposure did not appear to affect aerobic metabolism in juvenile rainbow trout. After swim performance experiments, swam fish were considered fatigued and metabolic and energy storage endpoints were compared to non-swam (non-fatigued) fish. Energy storage capacity was measured via whole body (fathead minnow) and liver and muscle (rainbow trout) triglyceride and glycogen concentrations. For fathead minnow, triglyceride concentrations in non-swam fish were significantly elevated in the 5.4 µg Se/g group relative to controls, and swam fish had significantly lower whole body triglycerides than non-swam fish. All non-swam SeMet exposure groups had significantly decreased whole body glycogen concentrations compared to controls while the 5.4 and 26.5 µg Se/g exposure groups had significantly greater whole body glycogen concentrations in swam versus non-swam fish. In juvenile rainbow trout, liver triglyceride concentrations were significantly lower in all SeMet exposed groups compared to controls in non-swam fish. Swimming decreased liver and muscle triglycerides in the control and 11.2 µg Se/g treatment groups. Liver glycogen concentrations were greater in swam trout in the 4.1 µg Se/g dm exposure group. Muscle glycogen concentrations in non-swam fish, were significantly decreased in the 4.1 and 11.2 µg Se/g exposed groups compared to controls, while muscle glycogen in swam fish was unaffected by dietary SeMet exposure. For the swim status factor, muscle glycogen concentrations were significantly greater in swam versus non-swam trout in all treatment groups. Therefore, dietary SeMet exposure caused impaired swimming performance and metabolic alterations in both juvenile fathead minnow and juvenile rainbow trout. Species differences were apparent, especially in the patterns of altered energy status between swam and non-swam fish exposed to Se. Overall, the pathophysiological implications of these sublethal effects are unclear, but suggest that dietary SeMet exposure may negatively influence juvenile fish survivability in natural habitats.

This thesis investigates the influence of predator movements upon habitat selection and foraging success. It deals with two very distinct datasets one from a marine system, the snow crab (Chionoecetes opilio) fishery, and the second from a freshwater system, an experimental rainbow trout (Oncorhynchus mykiss) aquaculture operation. Deriving a standardized measure of catch from logbook data is important because catch per unit effort (CPUE) is used in fisheries analysis to estimate abundance, but it some cases CPUE is a biased estimate. For the snow crab fishery, a relative abundance measure was developed using fisher movements and logbook data that reflected commercially available biomass and produced an improved relative abundance estimate. Results from the aquaculture dataset indicate that escaped farmed rainbow trout continue to use the cage site when waste feed is available, while native lake trout do not interact with the cage. Once access to waste feed is removed, both lake trout and escaped rainbow trout do not use the cage site. This thesis uses methods to identify patterns and behaviours using movement tracks to increase our understanding of predator habitat usage.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

β2-microglobulin (β2m) exists free of the major histocompatibility complex class I (MHC I) receptor in many bodily fluids. The amount of protein present in these fluids has been found to be a useful prognostic marker for various diseases but outside of its practical value not much is known about this form of β2m. In fish, soluble β2m has not been studied at all. Another unknown in fish is the effects that plants lectins might have on naturally carnivorous species in aquaculture. These plant proteins which bind to specific sugar groups found on cells have been shown to have a multitude of gastrointestinal and immune effects in mammals and can be found in the plant products being fed to carnivorous, cultured fish making them possible toxicants. The two studies of this thesis set out to pioneer knowledge on these subjects using rainbow trout as a model. The first investigation inspected the various body fluids of these fish for their free β2m content. Soluble β2m was found to be present in the plasma, the seminal fluid, ovarian fluid, and the mucus of the skin and intestines. This distribution shows that β2m could indeed make a good biomarker, not only for disease but also for pheromone release and alludes to some possible functions of soluble β2m while opening the way for future research on this form of the protein. The second study looked at the effects of lectins on the gut of rainbow trout by treating RTgutGC, an intestinal epithelial cell line derived from trout, with plant lectins from wheat (WGA) and soybean (SBA), among others. This study found WGA to be a potent inducer of morphological and cytotoxic effects in these cells while other lectins and plant factors were not. WGA was also observed to effect the expression of β2m and the α-chain of the MHC I receptor. This work suggests WGA ingested by trout through the wheat in their diet might be causing them harm and should be studied further. It is also interesting that both studies related β2m to the intestines of trout. This could allow soluble β2m to serves as a marker of WGA’s effect or for WGA to aid in the study of free β2m.

L'article 2 de la tesi enviat a la revista Conservation Biology, finalment va ser publicat a la revistat Conservation Genetics 3:385:400, 2002 / L'estudi de la diversitat i la diferenciació genètiques de les poblacions de truita comuna (Salmo trutta L.) a la Península Ibèrica ha confirmat l'elevada diferenciació observada en treballs previs i la divergència, ja descrita, entre les poblacions de la vessant atlàntica i la mediterrània. El resultats obtinguts, però, ens permeten observar patrons d'estructura poblacional tant en les poblacions atlàntiques com les mediterrànies. A l'Atlàntic s'observa un marcat patró hidrogràfic en la distribució de la diferenciació genètica, que contrasta fortament amb la distribució d'aquesta diferenciació en les poblacions mediterrànies, caracteritzades pels contactes secundaris entre llinatges durant les expansions pleniglacials i una forta divergència local conseqüència de la seva marginalitat i aïllament en els períodes interglacials. El manteniment d'aquesta diferenciació i individualitat descrites en les poblacions de truita de la Península, es veu seriosament compromès per les contínues repoblacions dels rius amb exemplars exògens d'origen nord europeu. La substitució dels genomes autòctons per la introducció de gens al.lòctons provoca una erosió dels patrimonis genètics natius i una homogeneïtzació de les poblacions, destruint els patrons de diferenciació existents. Al mateix temps, els nostres resultats indiquen que les conseqüències de les repoblacions no són sempre les mateixes. Concretament, es constata un fracàs de les repoblacions en rius intensament repoblats i sotmesos a pesca intensiva, que contrasta amb una enorme erosió de les poblacions quan les repoblacions s'efectuen sobre àrees protegides i sense cap mena de pressió pesquera. Això suggereix que múltiples factors com la gestió dels rius posterior a les repoblacions, l'estat de les poblacions o les condicions de l'hàbitat són determinants de la introducció efectiva dels exemplars alliberats; fet que dificulta la predicció sobre actuacions particulars. Malgrat aquesta introgressió de gens exògens que es detecta en moltes de les poblacions analitzades, els gens natius predominen en gairebé tots els rius de la Península. La conservació d'aquesta elevada riquesa genètica que encara resta en les poblacions de truita de la Península Ibèrica ha de ser l'objectiu final de qualsevol programa de gestió. Per això, defensem una gestió basada en el propi riu mitjançant una pesca sostinguda per la reproducció natural de les poblacions salvatges, acompanyada d'una millora i recuperació d'hàbitats adequats per la truita, i evitant, per sobre de tot, la introducció en els rius d'exemplars exògens, degut als efectes nocius i incontrolables que comporta aquest procés. / Our study on the genetic diversity and differentiation of brown trout (Salmo trutta L.) population of the Iberian Peninsula confirmed the high levels of differentiation and the divergence between Atlantic and Mediterranean populations previously described. However, additional substructuring among Atlantic rivers and among Mediterranean ones has been detected. In the Atlantic, genetic diversity is related with the river network, producing a marked hydrographical pattern in sharp contrast with a mosaic distribution of genetic variation among the Mediterranean collections. In this later area, genetic resources distributed as a result of distinct colonization episodes during pleniglacial expansions, secondary contacts, and local divergence due to its marginality and isolation in the interglacial periods. Continuous stocking with hatchery-reared fish of north European origin, seriously compromises the conservation of this differentiation and individuality described in brown trout populations of the Iberian Peninsula. Replacement of the autochthonous genome due to introduction of exogenous genes is leading to an erosion of the native gene pools, a homogenisation of populations, and a destruction of the ancestral patterns of differentiation. At the same time, our results indicated that successful stocking appears to be limited to wild populations subjected to occasional releases in protected or unfished areas. Extensive stocking in fished areas result in a more limited impact on the recipient native population. These results suggest that stocking success is unpredictable and controlled by different factors including habitat and population conditions at the time of stocking, as well as post-release management actuations. Although many of the analysed populations are introgressed with exogenous genes, native gene pools predominate in almost all the Iberian rivers. Conservation of this high genetic richness that still remains in brown trout Iberian populations must be the final goal of any management program. So, our management proposal is based on fisheries sustained by natural reproduction of the respective wild populations, coupled with an improvement and recovery of habitat suitable for brown trout; avoiding stocking with exogenous fish due to the harmful and incontrollable risks that this process involves for the genetic resources of the species.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 6 uppsatser.