MSc Thesis - The effect of wastewater effluent on the gut microbiome of rainbow darter / The microbiome plays an important role in host physiology and can be influenced by species, diet, and environment. Municipal wastewater effluent contains a mixture of chemicals including antibiotics and antimicrobials that may affect the gut microbiome of fish living downstream of these discharges. Thus, this study examines the effect of wastewater treatment plant (WWTP) effluent on the gut microbiome of wild rainbow darter (Etheostoma cearuleum), and examines how the gut microbiome of wild fish changes in the lab. Fish were collected from sites upstream and downstream of 2 major WWTPs along the central Grand River and gut contents were aseptically sampled. After extracting gDNA, nested PCR of the V3-V4 region of the 16S rRNA gene, and Illumina sequencing were performed. The gut microbiome of exposed fish had increased bacterial diversity and was dominated by Proteobacteria, which has been linked to altered health outcomes in mammals. Next, rainbow darters were collected from a reference site on the Grand River. Fish were sampled in the field, after a 14 day lab acclimation, and after a 28 day exposure to environmental stressors (WWTP effluent or triclosan, an antimicrobial found in WWTP effluent). Surprisingly, there were no changes in the microbiome after exposure to environmental stressors. Major changes were observed between the field and laboratory fish suggesting that environment and diet are important factors influencing the gut microbiome. Changes in the gut microbiome continued up to 42 days in the lab, indicating longer acclimation periods may be needed. This study showed that effluents altered the gut microbiome of fish in the field, but not in the laboratory for unknown reasons. Laboratory studies indicated that transitioning to a new environment may require greater than 14 days before achieving a stable microbiome. / Thesis / Master of Science (MSc) / Wastewater is the largest source of pollution affecting Canada’s aquatic ecosystems; effluents contain antibiotics and antimicrobials that can affect fish and other aquatic life. The gut microbiome of fish is influenced by host species, its diet, and the environment, and thus contaminants released via wastewater effluents may alter the gut microbiome of fishes in receiving waters. This study found that the gut microbiota of rainbow darter fish exposed to wastewater effluents in the central Grand River (Waterloo/Kitchener, Ontario) were dominated by Proteobacteria and had increased diversity. Wild fish transitioned to the lab were dominated by Firmicutes and had decreased bacterial diversity in the gut compared to those in the wild. Altogether, these results suggest that wild fish exposed to wastewater effluents had altered gut microbiomes; transitions to new environments and laboratory acclimation periods are important considerations when studying the fish gut microbiome.

The present research examined respiratory responses of blue crabs to long term (4, 13, and 21 days) hypercapnic hypoxia in freshwater at 23 C. Hypoxic conditions (50-60 & 75-85 mmHg O2) were induced by allowing the crabs to consume their oxygen supply, resulting in a hypercapnic induced decrease in pH that remained through the exposure. Postbranchial hemolymph responses to hypoxia/hypercapnia in freshwater demonstrate decreases in PO2, increases in PCO2, and decreases in pH. Lactate levels decreased over time, but hemocyanin concentration was highly variable with no trends. PH, lactate, and hemocyanin observations also demonstrated high variability and a variety of different responses in individual crabs. There was no evidence of improving oxygen transport abilities. Despite varying responses high mortality rates were observed. The high mortality rate suggests blue crabs are not able to survive the multiple stress of hypoxia/hypercapnia along with the stress of living in freshwater. The mortality rates observed are much greater than previous blue crab hypoxic studies in saltwater. Elevated mortality may result from a failure of oxygen transport, acid-base balance or ion regulation.

Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.

Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.

The purpose of this research was to develop and standardize a novel feeding bioassay with Macrobrachium rosenbergii for use in the laboratory and allowing it to be easily deployed under field conditions. Standardization of the test aimed to minimize feeding rate variations and to ensure that subsequent statistical analyses have sufficient power to consistently detect changes in feeding rates. These were accomplished through the development of a post-exposure feeding toxicity test under laboratory, microcosm and in situ/field conditions. This procedure was proven to be repeatable and economical. M. rosenbergii as test animals were available in terms of quantity and uniformity in sizes. The standard guidelines and procedures for M. rosenbergii bioassay developed from this study include the size of test animals (9-10 mm), density in exposure containers (10 animals in 500 mL of medium in the laboratory, 10 animals in field chambers with 98.6 mL volume), exposure time (24 hours), feeding period for post-exposure feeding (4 hours) and number of replicates for the feeding test (10 replicates for individual measurements). The tiered approach used in the preliminary risk assessment of pesticide using TOXSWA was capable of screening the risk level of pesticide in the study area, identifying profenofos and dimethoate as test chemicals for the lethal and sub-lethal experiments. This model was beneficial in the preliminary risk assessment of pesticides in the tropics, since it was not necessary to set up laboratory work. This method could also provide preliminary data to support the environmental planner and decision/policy maker. This is an alternative way to develop a cost efficient model to inform and warn the risk of pesticide use. The effects of pH, temperature and hardness on control post-exposure feeding rates of M. rosenbergii were assessed and indicated that M. rosenbergii was very sensitive to acidic and basic conditions. The use of post-exposure feeding inhibition as the endpoint under laboratory conditions revealed that prawns were sensitive to pesticides (chlorpyrifos, dimethoate and profenofos) and a heavy metal (zinc). Post-exposure feeding rate inhibition could be used as a sublethal endpoint as the EC50 values obtained for chlorpyrifos and zinc were lower than their lethal levels. Mortality of prawn was also another endpoint used to define the toxicity of pesticides such as carbendazim, in which mortality occurred during exposure, but post-exposure feeding rate of the surviving animals did not decrease. The microcosm experiments were able to link the laboratory toxicity tests and the effects observed in the field. Microcosm studies provided another dimension to studies looking at pesticide effects on aquatic systems. In this research, carbendazim affected feeding and survival rates in the microcosm set-up but in the laboratory only mortality showed a significant difference (P < 0.05). In situ bioassays were able to show the effects of pesticides on post-exposure feeding rates using the methods developed. Post-exposure feeding rates were significantly lower than control in farms using pesticides while in uncontaminated sites (pesticide-free), the post-exposure feeding rates did not decrease. However, mortality was observed even in the uncontaminated sites which could be attributed to other factors such as low dissolved oxygen and presence of some other unidentified chemical substances. The degree of mortality and the effect on feeding rates depends not only on the type and concentration of the known pesticide but also on water quality parameters. The basic methods developed for in situ bioassay from this research is a simple, easy and fast way to determine the effect of pesticides because the results can be seen in the field. The procedures developed and results obtained from this study can be used as a basis for further toxicity studies on M. rosenbergii and other potential tropical species.

The goal of this study was to examine the potential health effects of organochlorine (OC) and polycyclic aromatic hydrocarbon (PAH) exposure on Atlantic stingray populations in Florida’s St. Johns River (SJR). Special emphasis was placed on identifying OC- and/or PAH-related effects in stingrays from areas of the lower (LSJR) and middle (MSJR) basins shown to possess elevated levels of these compounds, as well as characterizing baseline levels of pollutant exposure in the SJR shipping channel, which may be subjected to dredging in the near future, potentially resuspending and redistributing contaminated sediments and increasing pollutant-associated effects. To accomplish this, we measured OC and PAH biomarker levels in stingrays collected from contaminated and reference sites. We specifically examined the phase I detoxification enzyme, cytochrome P4501a1 (CYP1a1); the phase II detoxification enzymes, glutathione-S-transferase (GST) and uridine 5’-diphosphate glucuronosyltransferase (UGT); fluorescent aromatic compounds, PAH bile metabolites; and lipid peroxidation (LPO), cell membrane damage. Biomarker values collected between 2014 and 2016 were compared by site. Detoxification enzyme activity and LPO values from individuals collected from the three MSJR lakes between 2002 and 2005 were compared to those collected between 2014 and 2016. The data suggested that biomarker values from the SJR were variable, with elevated levels from Lake Jesup. Compared to reference estuaries, the LSJR has low biomarker values. This indicates that residing in certain portions of the MSJR is detrimental to stingray health, while residing in the LSJR is not. Lake Monroe and Lake George biomarker levels indicated reduced contaminant input over time, whereas Lake Jesup biomarker levels suggested the opposite. This study has developed a baseline for biomarker levels in the LSJR, allowing for the identification of dredging-induced changes to the system, and has identified temporal changes in biomarker levels from three MSJR lakes.

Research efforts in aquatic toxicology have historically centered on the chemical analyses and toxic effects of waters to aquatic organisms. More recently, sediment-source toxicity has been explored, with efforts concentrated on establishing sensitive and accurate methodologies. This study focused on the toxicity of trivalent chromium, hexavalent chromium, and fluoranthene to Pimephales promelas, Ictalurus punctatus, and Lepomis macrochirus. Test fish were exposed to both water-borne and sediment-source toxicants for 96 hours (h) and 30 days (d). A 96-h and 30-d LC50 (mg/L Cr, ug/L Fluoranthene) was determined for each fish species exposed to aqueous toxicants. In addition, 96-h and 30-d LC50s were determined for each fish species from sediment chromium concentrations (mg/kg) and sediment fluoranthene concentrations (ug/kg). Although lethality endpoints were used throughout this research, acute effects other than mortality were determined for Lepomis macrochirus exposed to hexavalent chromium. Lethal toxicity values (96-h and 30-d LC50 and their 95% confidence limits) for trivalent chromium could not be determined since trivalent chromium concentations above 6.0 mg/L could not be obtained at water pHs compatible with these fish species. Trivalent chromium addition to test waters at pHs compatible with fish survival resulted in a chromium precipitate that was not lethal to test fish. In contrast, fathead minnows, channel catfish, and bluegill sunfish exposed to hexavalent chromium in water and sediments experienced mortality. Fathead minnows exposed to fluoranthene in water for 96h demonstrated a maximum mortality of 69%, while 100% mortality was achieved with channel catfish in similar tests. Sediment tests with fluoranthene resulted in 100% mortality with both fathead minnows and channel catfish.

Research in the multidisciplinary science of ecotoxicology is crucial to assess injuries to ecosystem resources from chemical spills or other stressors used to support environmental decision-making. Established guidelines recommend the use of non-standard native species in toxicity investigations. This work focused on the use of native species for aquatic toxicity assessment to make more relevant conclusions on the potential for adverse biological effects to occur as a result to single chemical exposures or exposures to a complex mixture like oil. We apply these studies to investigate petroleum product impacts from the Deepwater Horizon incident and concerns for metal toxicity in estuarine environments using a new model organism. Data generated from comprehensive toxicity testing programs were used in the first probabilistic risk assessment of Deepwater Horizon oil toxicity highlighting a lack of appropriate data and representative phyla. Novel toxicity study methods and a stress-response index were developed and demonstrated sensitivity and success in using the starlet anemone in ecotoxicology studies. Swim performance was used as new method to investigate sublethal indicators of stress resulting in varied responses from sheepshead minnows and Florida pompano. These studies further our ability for better laboratory-to-field extrapolation and for decision-making. The use of native species and complex mixtures like oil presented novel challenges in conducting aquatic toxicity studies. Special emphasis is placed on the necessity to understand the appropriate laboratory conditions for native species not typically held in the laboratory and maintaining study parameters to obtain quality data for more accurate interpretation and replication.

Endocrine-disrupting compounds (EDCs) interfere with the physiology of hormone systems. Traditionally, steroidogenic pharmaceuticals have been studied as EDCs however there has been growing evidence that non-steroidogenic pharmaceuticals can alter sex steroid levels and impair reproductive functions in fish. This is of concern as pharmaceuticals are detected in surface waters at the ng L-1 to µg L-1 range. Zebrafish (Danio rerio) were exposed to 10 µg L-1 of the pharmaceuticals carbamazepine and gemfibrozil for 6 weeks. Male-biased sex ratios were observed in the sexually mature offspring after paternal exposure, suggesting that sexual differentiation may be impacted in juveniles. Currently, the ability of pharmaceuticals to interfere with sexual differentiation of parentally exposed offspring is unknown. This thesis examined the gonad histology of juvenile zebrafish to understand how sexual differentiation was affected in the offspring of exposed parents. Paternal, but not maternal, exposure to carbamazepine resulted in a significantly faster sexual differentiation of the gonads and led to a male-biased sex ratio; these effects were not observed when both parents were exposed. Combined paternal and maternal exposure to gemfibrozil resulted in significantly faster sexual differentiation and paternal, but not maternal, exposure to gemfibrozil led to male-biased sex ratios. Interestingly, sex ratios observed in the juveniles did not always reflect those found in the same lineage at sexual maturity, suggesting a sex reversal, including a male to female transition, occurred past the juvenile sexual differentiation period in some fish. This thesis demonstrates that pharmaceuticals have the ability to disrupt sexual differentiation in the F1 offspring of exposed parents and that paternal exposure is most relevant for offspring effects. / Thesis / Master of Science (MSc) / Parental exposure to the environmentally-relevant pharmaceuticals carbamazepine or gemfibrozil led to male-biased sex ratios in adult offspring of zebrafish (Danio rerio), a common model organism. The development of the gonads in juveniles was investigated to determine how this process was impacted. Predominately, paternal exposure was found to result in a faster development of the testes and male-biased sex ratios. Interestingly, sex ratios in juveniles did not always reflect those in adults, suggesting a sex reversal may have occurred in adulthood. This study demonstrates the ability of pharmaceuticals to alter gonad development in offspring of exposed parents.

The 2010 Deepwater Horizon oil spill resulted in the accidental release of millions of barrels of crude oil into the Gulf of Mexico (GoM). Photo-induced toxicity following co-exposure to ultraviolet (UV) radiation is one mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Blue crab (Callinectes sapidus) are an important commercial and ecological resource in the Gulf of Mexico and their largely transparent larvae may make them sensitive to PAH photo-induced toxicity. Mahi-mahi (Coryphaena hippurus), an important fishery resource, have positively buoyant, transparent eggs. These characteristics may result in mahi-mahi embryos being at particular risk from photo-induced toxicity. Red drum (Sciaenops ocellatus) and speckled seatrout (Cynoscion nebulosus) are both important fishery resources in the GoM. They spawn near-shore and produce positively buoyant embryos that hatch into larvae in about 24 h. The goal of this body of work was to determine whether exposure to UV as natural sunlight enhances the toxicity of crude oil to early lifestage GoM species. Larval and embryonic organisms were exposed to several dilutions of water accommodated fractions (WAF) from several different oils collected in the field under chain of custody during the 2010 spill and two to three gradations of natural sunlight in a factorial design. Here, we report that co-exposure to natural sunlight and oil significantly reduced larval survival and embryo hatch compared to exposure to oil alone.