Toxicity of Chemicals in Unconventional Gas Wastewaters to Selected Australian Freshwater Invertebrates
Creators: name: Willems, Daniel
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 410201
subject: Bioavailability and ecotoxicology
classificationCode: 410404
subject: Environmental management
classificationCode: 410402
subject: Environmental assessment and monitoring

description: 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.