Mine water effluent contains high levels of nitrogen due to residues from undetonated ammonium- nitrate based explosives. Excess nitrogen in aquatic ecosystems can cause eutrophication. Within a mining area, tailings and clarification ponds have the potential to reduce nitrogen levels by biological uptake of nitrogen into growing algae and denitrification in pond sediments. A previous study at the LKAB Kiruna mine investigated the potential nitrogen removal within the tailings and clarification ponds. The study showed that about 1-10 tonnes of nitrogen were removed each year, and that the removal by denitrification was limited by carbon.  The aim of this master thesis was to investigate if additions of different carbon compounds could improve the denitrification in sediment from the clarification pond at the LKAB Kiruna mine site. It was also of interest to see if the composition of the edogenous microbial community involved in nitrogen reduction changed after the treatments. Samples of sediment and pond water were collected in January 2014 and a laboratory experiment was set up where sediment and water was incubated with carbon additions under anoxic conditions. Three different carbon sources were tested: sodium acetate, hydroxyethyl cellulose and green algae. Pond water without additional carbon was used as a control. The sediment was incubated eight weeks at 20 °C with weekly water exchange and carbon addition. The removed water was analyzed to determine the amount of nitrogen removed. At start and after ending the incubation, potential denitrification in the sediment was determined with an enzymatic assay and the size of the genetic potential of nitrogen reduction was determined.  At start, the enzymatic assay showed that the potential denitrification rate in the sediment of the clarification pond at the LKAB Kiruna mine was not immediately enhanced by addition of carbon. However, during the incubation the removal of nitrate was enhanced by external carbon sources. Algae were a good carbon source, since the denitrifying community grew, the potential denitrification increased four times after incubation and the removal of nitrate was next to complete in the end of the incubation. The addition of cellulose also enhanced the denitrification activity to some extent and the abundance of genes coupled to denitrification increased. Further studies are needed to assess the practical use of external carbon sources like algae and plant material and how they would function in and potentially also affect a large, cold and complex system like the LKAB mining site.

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).

Metal mines release treated effluents that contain a variety of metals, metalloids, and organics into the aquatic environment. A number of metal mine effluents (MMEs) have been found to contribute to adverse effects in fish and benthic invertebrates, such as decreased diversity and density, however the specific causal factors of toxic responses during chronic exposures to the MMEs are often unknown. Therefore, the overall objective of this dissertation was to explore causative and modifying factors of MME toxicity to a resident fish species, the fathead minnow (Pimephales promelas), during chronic, multi-trophic exposures. The representative MME used in this study was the process water effluent (PWE) of a Canadian metal mine, which is released into Junction Creek in Sudbury, Ontario, Canada. Chronic exposure to the MME has been a source of decreased reproductive output in fathead minnows in several previous studies, however, these same studies were not able to determine the potential causal factors of the reproductive impairment. In order to address the overall objective, several laboratory mesocosm studies were conducted, which consisted of three separate components. The first component included exploring several metals (Cu, Ni, and Se; alone and in mixture) that are consistently present in the MME and are known to cause toxicity at fairly low concentrations as potential causes for decreased egg production in fathead minnows. The second component included evaluating the role of decreased food availability (a possible indirect effect of MME in the receiving environment) as a potential cause of decreased egg production in fathead minnows. The third and final component included examining the role of water chemistry [(increased alkalinity and dissolved organic carbon (DOC)] as potential modifying factors of chronic MME toxicity to fathead minnows. In general, my results suggest that the metals present in the MME likely do not contribute directly to decreased reproductive performance in fathead minnows during chronic exposures, under the conditions examined. Instead, the MME appears to decrease food availability, therefore indirectly influence fathead minnow egg production. Furthermore, water chemistry modifications tested in this thesis were not able to entirely mitigate the reproductive effects in fish induced by the MME, although they did improve egg production relative to unmodified MME. Metal concentrations in fish tissues were not influenced by increases to alkalinity or DOC level in the exposure water, suggesting that bioavailability of metals during chronic exposure to metal-mixtures cannot be fully explained based on our understanding of metal complexation with abiotic ligands (inorganic and organic) during single metal or acute exposures. From a regulatory perspective, water chemistry modifications may somewhat improve fathead minnow reproductive performance during chronic exposure to the MME, however the MME would still not be entirely free of effects relative to the uncontaminated water. Future studies should focus on understanding the factors responsible for decreased food availability in MME-impacted aquatic ecosystems, and further explore potential approaches for ameliorating effluent quality.

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).

Three streams (Parker Run, Little Leading Creek and Thomas Fork) in the Leading Creek watershed, Meigs County, Ohio were impacted by active coal mining, agricultural and abandoned mined land sedimentation and acid mine drainage (AMD), respectively. An ecotoxicological evaluation was performed using physical (water chemistry and sediment depth analyses), toxicological (acute water column, chronic sediment and 35-day in situ toxicity tests) and ecological (benthic macroinvertebrate community sampling) parameters. Persistent acute toxicity (mean 48-hr LC50 of 30.3% to C. dubia) due to low pH (mean of 5.4) and high concentrations of dissolved metals (ex: Al ~ 10 mg/L) were responsible for the significantly depressed benthic macroinvertebrate community sampled in Thomas Fork. Heavy sedimentation (>30 inches), with no associated toxins, significantly decreased both abundance and diversity of benthic macroinvertebrates in Little Leading Creek. High concentrations of sodium (mean of 910 mg/L), TDS (mean of 3,470 mg/L), and periodic acute water column toxicity (mean C. dubia survival of 62% in 100% sample) were most likely responsible for the depressed benthic macroinvertebrate community observed in Parker Run. In ranking the severity of impacts, AMD was first followed by non-toxic sedimentation, and active coal mining ranked last. A catastrophic coal slurry spill significantly impacted the benthic macroinvertebrate community in Parker Run in April 1997. Six sampling stations were established to monitor the recovery of the stream's benthic community and evaluate any impact the active coal mine effluent had on the recovery time of the community. The effluent, characterized by high concentrations of TDS (~4,200 mg/L), significantly hindered benthic macroinvertebrate community recovery in Parker Run. The benthic community at the initial spill site, which was above the active mine effluent, recovered to levels measured at an upstream reference within 4-9 months. Benthic communities impacted by both the slurry spill and the effluent still had not recovered 16 months after the spill. Concentrations of TDS measured in the stream were significantly correlated (r = -0.765 and -0.649 respectively) with both EPT richness and percent C. dubia survival in water column toxicity tests. Laboratory analysis of synthetic coal mine effluent, similar in composition to that of the Parker Run effluent, was performed to determine toxicity thresholds for sodium, sulfate, TDS and conductivity. Acute toxicity thresholds were found for sodium (between 900 and 1,000 mg/L), TDS (4,200 and 6,400 mg/L), and conductivity (5,000 and 6,200 µmhos/cm). It was also determined that any toxic contribution of sulfate in solution with high concentrations of sodium (~1,000 mg/L) and/or TDS (~4,200 __ 6,400 mg/L) was secondary to that of the toxic effect of sodium or TDS in that solution. / Master of Science