Toxicity of Chemicals in Unconventional Gas Wastewaters to Selected Australian Freshwater Invertebrates

Unconventional gas extraction utilises hydraulic fracturing involving high-pressure water, silica sands, and other additives to extract natural gas from underground reservoirs. This process produces large volumes of chemically complex wastewaters, which must be managed or treated to reduce toxicity. Spills and leaks from storage and transporting these waters are potential avenues of exposure for freshwater invertebrates. The mechanisms and magnitude of toxicity of these wastewaters have become an emerging research field to address this knowledge gap. Subsequently, the knowledge gained provides greater insight into potential environmental implications that enable better management and informed decisions. The research presented in this thesis attempted to evaluate and further understand the toxicity of these wastewaters by first comprehensively reviewing and evaluating common chemicals found in these wastewaters (Chapter 2) and selecting the least studied and/or those predicted to have effects on native freshwater species, especially in combination, to study experimentally. The selected chemicals were barium, o-Cresol and sodium chloride. The chemicals barium (ensuring confounding effects of sulfate reacting with barium were addressed) and sodium chloride were evaluated for their chronic toxicity as single and binary chronic exposures to measure survival (parents) and reproductive effects (number of neonates) using the water flea Ceriodaphnia dubia (Chapter 3). Results indicated barium and sodium chloride at concentrations found in onshore unconventional gas wastewaters and further diluted in impacted surface waters reduced the population of C. dubia. In a surface water environment, there could be a potential shift in species composition to those that are more tolerant to barium and sodium chloride. The acute toxicity (survival) of all three chemicals was also assessed as single chemical exposures, and binary and ternary mixtures using the freshwater shrimp Paratya australiensis, in combination with sub-lethal assessments using the enzyme assays glutathione S- transferase, acetylcholinesterase and sodium-potassium adenosine triphosphatase (Chapter 4). These enzyme assays provided a more sensitive indicator for sub-lethal perturbations from these chemical exposures. These enzymes have roles in detoxification, nerve signalling, energy and osmoregulation. Adverse changes to enzyme activity have implications for reducing shrimps’ overall fitness. Shrimp in the environment may become more susceptible to illness or increased predation pressures. The concentrations of barium and sodium chloride tested (Chapters 3 and 4) were environmentally relevant, particularly to enclosed surface water environments impacted by inorganically/metal-rich wastewaters from shale gas (Chapter 2). Reproduction was adversely affected in C. dubia at ≤16 mg/L of dissolved barium. However, parent C. dubia survival was unaffected at these concentrations, suggesting that dissolved barium caused a shift in energy or cellular resources away from reproduction. Relatively small additions of sodium chloride (approximately 200 mg/L) increased reproduction, but further additions (approximately 400 mg/L) were detrimental to reproductive output and parent survival. A portion of the binary barium and sodium chloride combinations suggested some antagonistic interactions (Chapter 3), though a more comprehensive assessment is needed. Shrimp acute (96-hours) survival was reduced from 10 mg/L dissolved barium, 5.4 mg/L o-Cresol and 3100 mg/L sodium chloride. Mortality occurred more gradually across the concentrations tested for dissolved barium (10 to 105 mg/L) and sodium chloride (approximately 1000 to 6000 mg/L), while a sharp increase in mortality occurred from o-cresol exposure from 5.4 to 21.8 mg/L. Synergism was observed in binary mixtures of barium and o-Cresol. The o-Cresol and sodium chloride binary combinations showed greater synergistic responses than barium and sodium chloride exposures. Ternary mixtures were highly synergistic. However, the three enzyme assays (glutathione S- transferase, acetylcholinesterase and sodium-potassium adenosine triphosphatase) generally showed inconclusive trends. This was thought to result from high sample variability since wild-collected shrimp were used for the experiments, and replication of samples was reduced due to the high mortality of shrimp in higher concentration treatments. After investigating select and prominent chemicals in unconventional gas wastewaters, research progressed to investigating chronic toxicity of a shale gas flowback wastewater sample using the water flea Daphnia carinata (Chapter 5) and Paratya australiensis (Chapter 6) to provide further environmental context to the previous work on the toxicity of select chemicals within these wastewaters (Chapter 3 and 4). Daphnia carinata showed significantly increased reproduction at 0.5% dilution of this water sample. Still, reproduction was reduced at lower and higher test concentrations, likely due to an interaction between this wastewater sample's salinity and water hardness. Increased reproduction was linked with increased size (length) of individuals, which was generally promoted by the wastewater compared to its major ion salinity controls and control group across different aged D. carinata. It was interpreted that the chemically complex inorganic-rich wastewaters at these dilutions provided additional nutrients that supported growth. Sub-lethal oxidative stress activity was measured using the biochemical assays of glutathione S-transferase, lipid peroxidation, reactive oxygen species and superoxide dismutase. Generally, activity in the wastewater treatments was either reduced non-significantly or significantly compared to the control and major ion salinity control, with a clear trend observed with reactive oxygen species activity decreasing with exposure to increasing wastewater concentrations. Metabolomics and lipidomics assessment showed the wastewater sample caused greater perturbations in both lipid and metabolite compounds in D. carinata compared to those in the salinity control. Paratya australiensis (Chapter 6) was not adversely impacted regarding increased mortality from exposure, except at the highest 1% wastewater. At these dilutions, the toxicity shifted to a suspected iron-oxyhydroxide particle in the wastewater, clogging and damaging gill tissues, reducing respiration, causing hunger and eventually death. These same shrimps showed greater metabolite and lipid perturbations, and central carbon metabolism metabolites were generally enriched in this treatment compared to other more dilute wastewater samples, as well as the salinity controls and control. Key metabolic pathways were enriched in shrimp exposed to the wastewater, indicating a shift towards fat and protein degradation. In conclusion, the results of this research present substantial evidence that unconventional gas wastewater chemicals or whole untreated samples from this industry can be toxic to freshwater invertebrates and, importantly, at dilutions relevant to surface waters impacted by spills or leaks of these wastewaters. These wastewaters can also be highly variable in chemical composition and thus vary in toxicity. Further research on the toxicity of these wastewaters could involve a mesocosm approach using freshwater biota. This would further increase environmental relevance as predator- prey interactions are considered and abiotic conditions are better simulated to those in surface water ecosystems.