Palmaria palmata and Chondrus crispus were cultured in the lab at three levels of temperature and two of nitrate, representative of commercial Atlantic halibut farming conditions. Productivity and nitrogen removal by P. palmata were greatest at temperatures <10°C. Productivity of C. crispus was greatest at >10°C, while nitrogen removal was unaffected by temperature, 6-17°C. When cultured in various nitrate and ammonium combinations, nitrogen uptake was highest when available as ammonium. Both species took up 89-100% of ammonium in 24 hours, but only 23-37% and 55-87% of nitrate was taken up by P. palmata and C. crispus, respectively. When P. palmata was integrated with halibut recirculating aquaculture, productivity and nutrient removal were compromised during summer. During winter, <11°C, nitrogen removal by P. palmata was relatively stable at 2.3 gN m-2 d-1. For 50% nitrogen removal from halibut aquaculture during winter, a finfish: seaweed biomass of 1: 1 would be required.

One advantage of Integrated Multi-Trophic Aquaculture (IMTA) is the potential for bioremediation by organic-extractive organisms. In British Columbia, a number of marine invertebrate species are being considered for use in open-water IMTA with sablefish (Anoplopoma fimbria). These include both filter-feeding bivalves (e.g. cockles, mussels, oysters, scallops) which would consume the finer suspended particulates from the finfish culture component and deposit/detrital feeders (e.g. sea cucumbers, sea urchins, prawns) which would feed on the heavier-settleable solids. The following candidate species were tested for their ability to consume sablefish faeces and uneaten sablefish feed in laboratory feeding trials: green sea urchin (Strongylocentrotus droebachiensis), basket cockle (Clinocardium nuttallii), blue mussel (Mytilus edulis), spot prawn (Pandalus platyceros), and California sea cucumber (Parastichopus californicus). Whether they can remove organic material from aquaculture wastes was tested by measuring ingestion rate or clearance rate and absorption efficiency when they were fed a diet of sablefish waste, relative to those fed a natural control diet. Egestion rates in the candidate species were quantified to estimate the potential amount of waste that may be lost from the organic-extractive component. Biophysical properties including shape, size, and settling velocity were measured in faecal pellets egested by the candidate species to provide input data for models to assess dispersal of faeces from IMTA sites. Results from the laboratory feeding trials demonstrate that all candidate species are capable of consuming wastes from sablefish aquaculture and absorbing the organic material. The relative merits and drawbacks of each candidate species are discussed with respect to the results and within the broader context of IMTA. The general conclusion is that, in order to achieve efficient removal of organic material and successful bioremediation, deposit feeders should be included in the organic-extractive component, whether alone or in conjunction with suspension feeders. / Graduate

The close proximity of net-pen salmon farms and wild Pacific salmon stocks in British Columbia (BC) is an incentive for precautionary management of the environmentally and economically damaging parasites known as sea lice. Bivalves cultured as part of an integrated multi-trophic aquaculture (IMTA) system may contribute natural, preventative louse control through the ingestion of planktonic sea lice larvae. A field trial was conducted to test sea lice mitigation by bivalves at a commercial Atlantic salmon (Salmo salar) farm in BC using Pacific oysters (Crassostrea gigas). Oysters were cultured in trays around one end of the farm and at a reference site approximately 150 m away from August 2013 until August 2014. Parasitic and planktonic sea lice (Lepeophtheirus salmonis and Caligus clemensi) were monitored before and during oyster deployment, beginning in December 2012. Parasite abundance peaked in January 2013 (6.5 lice/fish, >85% C. clemensi), and the following year in February 2014 (3.3 lice/fish, >80% L. salmonis). Larval density within cages peaked in January, both in 2013 (1.28 larvae/m3) and 2014 (0.96 larvae/m3). Parasite abundance was significantly correlated with both surface salinity (r2= 0.28, p=0.04) and sea lice larval density (r2= 0.65, p=0.01). Observed densities were significantly lower (t=3.41, p=0.009) than those calculated for the site based on water temperature and salinity, the number of adult female lice present, and the approximate number of fish. Sea lice mitigation by oysters was assessed by comparing monthly sea lice larval densities inside bivalve and non-bivalve fish cages, and by analyzing preserved oyster digestive tracts from January 2014 (when larval densities were highest) for presence of L. salmonis DNA using PCR. Using these methods, no significant evidence of sea lice mitigation was detected. Oyster growth was monitored by measuring whole wet weight, soft tissue wet, dry, and ash-free dry weight, and shell length, width, and height approximately every four months. Oysters were sampled equally across different sides of the farm and at the reference site (~150 m away from the farm) at three depths: 1, 3, and 6 m. All seven measurements increased significantly over time. Effects of side and depth varied by growth parameter; in general, oysters at 1 and 3 m were significantly larger than those at 6 m, and oysters cultured at the reference site were either significantly smaller or the same size as those cultured around the farm. Oysters from select sides were consistently, significantly larger than those from other sides and from the reference site. Overall, the findings suggest that sea lice larvae quickly dispersed away from the farm after hatching and were not significantly impacted by bivalve presence around the fish cages. Bivalves grew significantly larger over time and size was significantly impacted by both depth and side of the fish cage. While no evidence of larval sea lice reduction/ingestion by cultured bivalves was detected, this study provides information on all sea lice stages present throughout an Atlantic salmon production cycle, as well as the first detailed growth analysis of Pacific oysters cultured alongside farmed Atlantic salmon in BC. / Graduate / 0792 / byrneaa@gmail.com

In integrated multi-trophic aquaculture (IMTA) production of cultured species may not align temporally. For instance, at an IMTA site in Kyuquot Sound, BC where the cultured species are Anoplopoma fimbria (sablefish), Plactopentin yesoensis (Japanese scallop) and Saccharina latissima (sugar kelp), sablefish are grown year round while the kelp culturing lasts from winter to summer. Kelp sporophytes become visible in early spring while harvest takes place in July. This indicates that at Surprise Island the time period of nutrient extraction by the kelp is limited to only a few months per year. Two potentials methods to lengthen the time in which the kelp component was on site were employed and evaluated: 1. the use of multiple kelp species with potentially differing seasonal growth strategies and; 2. outplanting kelp seed at four different times of the year. The first method involved outplanting seed of four kelp species, Saccharina latissima, Costaria costata, Alaria marginata and Saccharina groenlandica and monitoring growth parameters (blade length and yield). For the second method, a modified seed production method of Merrill and Gillingham (1991) with Luning and Dring (1973) successfully provided seed throughout the year. Seasonally out-planted seed was also monitored for growth parameters. Results were marginal for experiments and were confounded by the lack of growth rates due to infrastructure problems, grazing by naturally setting marine snails and seemingly poor environmental conditions for kelp culturing at the farm site. However, data indicated that certain species in co-culture may slightly increase the time period, and strategically entered kelp seed may do the same. In particular the co-culture of C. costaria and S. groenlandica or biannual seed outplanting in fall and spring may increase the length of growth period of kelp provided certain limitations found during this experiment are overcome (i.e. pressures of grazing). Additional potential benefits with these kelp production strategies are the diversification of final kelp products, additional kelp harvests and increased production. / Graduate

Ulva as an aquaculture crop and IMTA component species has received mixed results globally; success has been achieved in South Africa and Israel, whilst in Europe the results have been poor. This project aims to determine if Ulva lactuca is a suitable candidate as an inorganic extractive species component within marine IMTA systems in British Columbia. The inorganic extractive feasibility of U. lactuca was determined with combination of real time growth and nutrient uptake experiments, alongside a SWOT analysis and literature review to reveal the possible potentialities and pitfalls. U. lactuca was cultivated in 680 litre tanks in the effluent of Wolf Eels, Anarrhichthys ocellatus in a recirculation system at the Aquatics facility at the University of Victoria. Growth experiments of wild local U. lactuca strains attained summer growth of up to 17.43% specific daily growth rate, with winter growth of up to 4.26% specific daily growth rate. U. lactuca demonstrates a preference for Ammonia-N uptake over other forms of inorganic nitrogen and a reduced nutrient uptake capacity during dark periods. Nitrate uptake capacity up to 202µm N gDW-1 day-1 was exhibited. These figures display the excellent biological potential of local Ulva lactuca strains to act as an inorganic extractive. However currently, long term maintenance of the crop proved problematic with instability with growth rates and nutrient uptake capacity. Cultivation issues in combination with poor economic outlook will restrict the feasibility of this species to specific types of IMTA system. Beneficial steps towards the deployment of U. lactuca inorganic extractive components would include: (i) the identification of suitable sterile strains or employment of “germling” spore production, (ii) the use of a rotational, light weight, cage cultivation system, (iii) being farmed in combination with a dark period nutrient removal species, such as Chondrus crispus, (iv) being farmed in conjunction with in-situ algivorous species. / Graduate / 0792

Excess nutrients in the form of uneaten food or waste from intensive, monospecies aquaculture farms can have negative effects on the surrounding natural ecosystem, causing eutrophication and benthic habitat degradation. Biomitigative techniques such as Integrated Multi-Trophic Aquaculture (IMTA) are being investigated for their ability to reduce these negative environmental impacts. IMTA is the co-culture of multiple species from complementary trophic levels, physically orientated in such a way that excess waste nutrients from the fed component are intercepted by the extractive species. For IMTA systems to become a sustainable aquaculture design alternative, it is important to ensure that infrastructure orientation and stocking densities of the extractive species maximize the amount of excess nutrients intercepted and overall system efficiency. Previous research has shown that the majority of wastes from fed finfish are made up of large organic particulates which sink rapidly to the benthos underneath or near the fish cages and which would be available to benthic deposit-feeding species. The California sea cucumber (Parastichopus californicus) is a promising extractive species for IMTA on the west coast of Canada due to its deposit-feeding behaviour and its relatively high market price. Owing to the sea cucumber’s morphology and ability to move through restricted spaces, containment can be difficult without reducing nutrient transfer and overall IMTA system efficiency (i.e. mesh sizes needed to contain small sea cucumbers may restrict flow of farm particulates to them). The overall goal of the present work is to effectively contain juvenile sea cucumbers in such a way that maximizes benthic extraction of large-particulate nutrients within an IMTA system. / Graduate

The overall objective of the work described in this thesis was to examine the effects of both waterborne and dietary routes of exposure to fathead minnow (Pimephales promelas) when exposed to complex metal mining mixtures. This was conducted using a 21-day, multi-trophic, short-term fathead minnow (FHM) reproductive bioassay. The endpoints that were measured were used to assess the effects on multiple levels of biological organization (sub-organismal to population endpoints). The first phase of this research was conducted in situ using environmentally realistic concentrations of 3 separate metal mining effluents [20% surface water effluent (SWE), 30% mine water effluent (MWE), 45% process water effluent (PWE)] from Sudbury, Ontario, Canada. Metals were analyzed in several media (water, sediments) and tissues (biofilm, Chironomus dilutus, female fathead minnow carcass, ovaries, liver and gills). The incorporation of the biofilm (primary producers) into the bioassay also added another level of organization that was novel to this study. Significant increases in metal concentrations were observed in the water and biofilm tissues in all treatments [SWE, MWE, PWE], compared to reference. Cobalt and nickel increased significantly in C. dilutus tissues in SWE (1.4-fold and 1.5-fold respectively), and copper and selenium in PWE (5.2-fold and 3.3-fold respectively), however no significant increases occurred in MWE compared to reference. There were no significant increases in metal concentrations in female FHM tissues (carcass, liver, gonads, gills) in any of the treatments, suggesting that metal bioavailability was reduced. Cumulative number of eggs per female per day increased significantly (+127%) after exposure to SWE and decreased significantly (-33%) after exposure to PWE when compared to the reference fish. Mean total number of days to hatch was also reduced in PWE compared to reference. In order to gain a better understanding of the routes of exposure causing toxicity in FHM, the second phase of this research examined the effects of exposure through diet, through water or through both using a fully factorial food exposure design in a laboratory setting. In this experiment we pre-exposed C. dilutus to both 45% PWE and laboratory control water until they reached the 3rd-4th instar stage of development (approximately 21 days) where they were collected and frozen until the start of the FHM reproductive bioassay. We further examined the role of food quality on fish toxicity by assessing differences between multi trophic (where fish were fed both a live and frozen diet of C. dilutus) in the laboratory. This research was conducted at the Toxicology Centre in Saskatoon, Saskatchewan, Canada. The results showed that significant effects were observed when fish were fed a live diet versus a frozen diet. Condition factor and body weight increased, although inconsistent effects were observed for liver somatic index (LSI) in fathead minnows in both experiments when exposed to one or both routes of exposure. Cumulative total egg production and cumulative spawning events were both significantly affected by both waterborne and dietborne exposures with the greatest effects seen in the multi-trophic streams and particularly when fish were fed a live diet. This significance of this research has demonstrated the importance of including both routes of exposure when assessing effects of mine effluent. This research also shows that the artificial stream technology is a useful tool in isolating the effects of a particular point source input (metal mining mixtures) when a system is highly confounded. The results suggest that under environmentally relevant exposure conditions, trophic transfer and live diet may lead to greater reproductive effects and increased fish toxicity. This also suggests that trophic transfer is an important route of exposure that is virtually impossible to attain using typical laboratory bioassay techniques (food-borne study using artificial diets or waterborne exposures only).

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.

The overall objective of the work described in this thesis was to examine the effects of both waterborne and dietary routes of exposure to fathead minnow (Pimephales promelas) when exposed to complex metal mining mixtures. This was conducted using a 21-day, multi-trophic, short-term fathead minnow (FHM) reproductive bioassay. The endpoints that were measured were used to assess the effects on multiple levels of biological organization (sub-organismal to population endpoints). The first phase of this research was conducted in situ using environmentally realistic concentrations of 3 separate metal mining effluents [20% surface water effluent (SWE), 30% mine water effluent (MWE), 45% process water effluent (PWE)] from Sudbury, Ontario, Canada. Metals were analyzed in several media (water, sediments) and tissues (biofilm, Chironomus dilutus, female fathead minnow carcass, ovaries, liver and gills). The incorporation of the biofilm (primary producers) into the bioassay also added another level of organization that was novel to this study. Significant increases in metal concentrations were observed in the water and biofilm tissues in all treatments [SWE, MWE, PWE], compared to reference. Cobalt and nickel increased significantly in C. dilutus tissues in SWE (1.4-fold and 1.5-fold respectively), and copper and selenium in PWE (5.2-fold and 3.3-fold respectively), however no significant increases occurred in MWE compared to reference. There were no significant increases in metal concentrations in female FHM tissues (carcass, liver, gonads, gills) in any of the treatments, suggesting that metal bioavailability was reduced. Cumulative number of eggs per female per day increased significantly (+127%) after exposure to SWE and decreased significantly (-33%) after exposure to PWE when compared to the reference fish. Mean total number of days to hatch was also reduced in PWE compared to reference. In order to gain a better understanding of the routes of exposure causing toxicity in FHM, the second phase of this research examined the effects of exposure through diet, through water or through both using a fully factorial food exposure design in a laboratory setting. In this experiment we pre-exposed C. dilutus to both 45% PWE and laboratory control water until they reached the 3rd-4th instar stage of development (approximately 21 days) where they were collected and frozen until the start of the FHM reproductive bioassay. We further examined the role of food quality on fish toxicity by assessing differences between multi trophic (where fish were fed both a live and frozen diet of C. dilutus) in the laboratory. This research was conducted at the Toxicology Centre in Saskatoon, Saskatchewan, Canada. The results showed that significant effects were observed when fish were fed a live diet versus a frozen diet. Condition factor and body weight increased, although inconsistent effects were observed for liver somatic index (LSI) in fathead minnows in both experiments when exposed to one or both routes of exposure. Cumulative total egg production and cumulative spawning events were both significantly affected by both waterborne and dietborne exposures with the greatest effects seen in the multi-trophic streams and particularly when fish were fed a live diet. This significance of this research has demonstrated the importance of including both routes of exposure when assessing effects of mine effluent. This research also shows that the artificial stream technology is a useful tool in isolating the effects of a particular point source input (metal mining mixtures) when a system is highly confounded. The results suggest that under environmentally relevant exposure conditions, trophic transfer and live diet may lead to greater reproductive effects and increased fish toxicity. This also suggests that trophic transfer is an important route of exposure that is virtually impossible to attain using typical laboratory bioassay techniques (food-borne study using artificial diets or waterborne exposures only).