Development of tropical freshwater mussel toxicity tests and an assessment of key contaminants from the Ranger uranium mine
Creators: name: Tybell, Linda
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 4104
subject: Environmental management
classificationCode: 410404
subject: Environmental management
classificationCode: 410499
subject: Environmental management not elsewhere classified

description: Many aquatic species are at risk from the toxic effects of anthropogenic stressors. Benthic invertebrate species may be more vulnerable to water-borne and sediment-bound contaminants due to their sedentary lifestyle and feeding habits, which may result in multiple routes of exposure. One such vulnerable species is the freshwater mussel, which is both a filter feeder and a deposit feeder in its early life stages. Mussel larvae (glochidia) are exposed to contaminants in the water column or during their obligate parasitic attachment stage on the host fish, while juvenile mussels are exposed to dissolved and suspended contaminants in surface waters, sediment and pore water through their filtering activity or ingestion via pedal feeding.   <br><br>The contamination of mussel habitats is one of many natural and anthropogenic stressors implicated in the increasing global decline of freshwater mussel species, particularly in North America. Research continues but is still lacking on the taxonomic status and sensitivity to contaminants of Australasian species, particularly those in tropical habitats. Freshwater mussels are widely distributed throughout tropical Northern Australia, including creeks and billabongs of high conservation value surrounding the Ranger uranium mine lease. Here, mussels play important roles both ecologically and culturally, and extensive research focusing on the biology and ecology of the most common species, Velesunio angasi, has been previously undertaken. A monitoring program for bioaccumulation of radionuclides and metals has been carried out annually using V. angasi since 2000 due to the importance of mussels in the diet of Aboriginal communities downstream of mine water discharges. After 40 years of operation, the Ranger mine is scheduled to cease operations in 2021. However, the risk of contamination from mining waste is expected to continue following rehabilitation activities, through mobilisation and leaching from contaminant sources on the mine site. Contaminants of potential concern (COPCs) from the Ranger mine include ammonia, magnesium, and uranium, and a toxicity testing program is in place to predict the toxicity of these COPCs and inform the water quality objectives for the mine. Toxicity data for the COPCs are generated primarily using local tropical species, but have also incorporated other suitable international toxicity data, to derive site-specific water quality guideline values for the slightly acidic and extremely soft waters of the catchments downstream of the mine. Such water quality generally causes increased metal bioavailability and toxicity. The water quality objectives aim to protect the World Heritage-listed downstream ecosystems, and the local Aboriginal people that rely on them, from the effects of uranium mining. However, to date, freshwater mussels have not been included as a test species due to culturing difficulties and a lack of standardised toxicity test methods. <br><br>A preliminary and secondary aim of this thesis was to confirm the genetic identity of freshwater mussels collected from populations both within and outside of the Alligator Rivers Region (ARR), Northern Territory, that could potentially be used for toxicity testing. Adult mussels were sampled from 13 different sites, and a 710-bp fragment of the mitochondrial COI gene was amplified and sequenced from the mantle tissue of each mussel (~15 mussels per site). Phylogenetic analysis revealed three distinct clades containing three known species and three to four unknown, or undescribed, species. The billabong (or lentic) water-body sourced species matching to Velesunio angasi were placed in Clade A, and diverged by 11.1% from the lentic creek-sourced mussels in Clade B that did not match to any known species. The lotic V. angasi and the lentic unknown species, referred to as Velesunio sp. throughout this thesis, were subsequently selected for the toxicity testing research. Mussel collection sites were chosen based on factors such as accessibility of the two species, proximity, and relevance to the Ranger mine.<br><br>The primary aims of this thesis were to optimise acute and chronic toxicity testing protocols for use with tropical mussel species, and conduct toxicity testing using the optimised test protocols. Test optimisation was undertaken using guidance from previous work with V. angasi and an ASTM International standard guide. Acute toxicity test optimisation involved testing different variables including glochidia selection, pH control, test volume, test vessels, number of glochidia per replicate, age of glochidia, and test duration. Following test optimisations, 24-h acute, water-only toxicity tests were undertaken using mussel glochidia from both species with ammonia, magnesium, copper, and uranium, using survival as the endpoint. Development and optimisation of the chronic test protocol involved refinement of a host fish exposure method (in order to produce juvenile mussels) using two different fish species, trialling different test volumes, the addition of fine sediment &lt;63 µm (silt), food and feeding frequency, and biological endpoints. Using the final protocol, 14-d chronic toxicity tests with ammonia, magnesium, and uranium were undertaken on newly-released (&lt;24-h) juvenile mussels that had been metamorphosed from glochidia in the laboratory using the host fish, Mogurnda mogurnda, using growth rate as the sub-lethal endpoint. All toxicity tests were conducted at pH 6.0 ± 0.3 and 27 ± 0.5 °C to represent local environmental conditions.  <br><br>Velesunio spp. glochidia and juveniles were found to be similarly and highly sensitive to ammonia, with mean median lethal (LC50) acute toxicity estimates ranging from 7.4 to 11.9 mg/L total ammonia nitrogen (TAN), and mean median effect (EC50) chronic toxicity estimates ranging from 7.0 to 11.3 mg/L TAN. When toxicity estimates were normalised to pH 7 and 20°C, Velesunio spp. were among the most sensitive species to ammonia in comparison to temperate freshwater mussel species and other tropical taxa. Velesunio spp. glochidia were also highly sensitive to copper, with LC50s ranging from 5.2 to 8.4 µg/L, and similarly sensitive in comparison to temperate freshwater mussel species. Acute and chronic exposures to magnesium indicated that Velesunio spp. were moderately sensitive, with LC50s ranging from 284 to 300 mg/L, and mean EC50s ranging from 232 to 241 mg/L. For acute uranium exposures, LC50s ranged from 227 to 375 µg/L, indicating moderate sensitivity in comparison with acute data for other tropical taxa. When developing the chronic uranium exposure method, it was demonstrated that chronic uranium exposures using pre-spiked sediment did not significantly reduce the loss of uranium from the water column, and control growth was reduced in comparison with tests using un-spiked sediments. Addition of un-spiked sediments produced satisfactory control growth in the chronic exposures, but nonetheless, water chemistry measurements indicated a significant loss of U to the sediment, with filtered U concentrations reduced by up to 90% between the start and end of tests. Median EC50s for the filtered and total fractions of the three un-spiked sediment tests ranged from 64 to 126 µg/L U, and 276 to 506 µg/L U, respectively. When comparing LC50s and EC50s for each contaminant tested, intra- and interspecies variability was low (≤2-fold), indicating similar sensitivity within and between V. angasi and Velesunio sp.  <br><br>This thesis contributes new knowledge of freshwater mussel genetic diversity in Northern Australia, and provides efficient and robust toxicity testing methods for tropical freshwater mussels. The acute and chronic toxicity data generated using the optimised test methods are a valuable contribution to the tropical datasets for the contaminants of concern. The data can be used to inform site-specific and regionally relevant water management practices for the protection of Velesunio spp., and the freshwater ecosystems they inhabit.