Use of scanning and image recognition technology to semi-automate larval development assessment in toxicity tests with a tropical copepod.

There is a high demand for the development of reliable chronic toxicity tests using tropical marine species for subsequent use in tropical risk assessment. However, many chronic test endpoints can be laborious and time-consuming to assess, particularly if the endpoints require measurements of individuals (e.g. growth, size) or advanced taxonomic expertise (e.g. differentiating between larval development stages). In this study, we used scanning and image recognition (SIR) technology to develop and validate a chronic toxicity test with larvae of the tropical euryhaline copepod, Acartia sinjiensis. Optimisation steps are described, and included egg age, and effect of algal food type and salinity on toxicity. Comparisons were made between traditional endpoints measured using microscopy and those measured using SIR. Traditional endpoints of larval development ratio (LDR) and survival achieved using microscope examination and SIR were almost identical (R2 = 0.96-0.97). Additional endpoints made possible by SIR included larval development index (LDI; based on the number of animals at different stages of development), and a range of size measurements (e.g. surface area, perimeter and length) for individual animals and for total populations (i.e. a proxy for biomass). The SIR-derived endpoints were based on measurements that had concentration-dependant responses to tested toxicants (copper, nickel, ammonia), and were a sub-set of the full range of metrics provided by the software. Toxicity values based on SIR-measurements were similar to or more sensitive than the traditional LDR endpoint. SIR technology provides a major opportunity to improve and modernise larval development tests for a range for species, but comes at a cost of increased data size and complexity. Therefore, as a research tool, SIR has significant advantages over traditional microscope methods, but for routine toxicity testing, SIR incorporation into invertebrate toxicity testing will benefit from further improvements to the associated software and data management systems.

Adult Corals Are Uniquely More Sensitive to Manganese Than Coral Early-Life Stages.

Manganese (Mn) is an essential element and is generally considered to be one of the least toxic metals to aquatic organisms, with chronic effects rarely seen at concentrations below 1000 µg/L. Anthropogenic activities lead to elevated concentrations of Mn in tropical marine waters. Limited data suggest that Mn is more acutely toxic to adults than to early life stages of scleractinian corals in static renewal tests. However, to enable the inclusion of sufficient sensitive coral data in species sensitivity distributions to derive water quality guideline values for Mn, we determined the acute toxicity of Mn to the adult scleractinian coral, Acropora muricata, in flow-through exposures. The 48-h median effective concentration was 824 µg Mn/L (based on time-weighted average, measured, dissolved Mn). The endpoint was tissue sloughing, a lethal process by which coral tissue detaches from the coral skeleton. Tissue sloughing was unrelated to superoxidase dismutase activity in coral tissue, and occurred in the absence of bleaching, that is, toxic effects were observed for the coral host, but not for algal symbionts. We confirm that adult scleractinian corals are uniquely sensitive to Mn in acute exposures at concentrations 10-340 times lower than those reported to cause acute or chronic toxicity to coral early life stages, challenging the traditional notion that early life stages are more sensitive than mature organisms. Environ Toxicol Chem 2023;42:1359-1370. © 2023 Commonwealth Scientific and Industrial Research Organisation. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Effects of Nickel and Copper on Tropical Marine and Freshwater Microalgae Using Single and Multispecies Tests.

Microalgae are key components of aquatic food chains and are known to be sensitive to a range of contaminants. Much of the available data on metal toxicity to microalgae have been derived from temperate single-species tests with temperate data used to supplement tropical toxicity data sets to derive guideline values. In the present study, we used single-species and multispecies tests to investigate the toxicity of nickel and copper to tropical freshwater and marine microalgae, including the free-swimming stage of Symbiodinium sp., a worldwide coral endosymbiont. Based on the 10% effect concentration (EC10) for growth rate, copper was two to four times more toxic than nickel to all species tested. The temperate strain of Ceratoneis closterium was eight to 10 times more sensitive to nickel than the two tropical strains. Freshwater Monoraphidium arcuatum was less sensitive to copper and nickel in the multispecies tests compared with the single-species tests (EC10 values increasing from 0.45 to 1.4 µg Cu/L and from 62 to 330 µg Ni/L). The Symbiodinium sp. was sensitive to copper (EC10 of 3.1 µg Cu/L) and less sensitive to nickel (EC50 >1600 µg Ni/L). This is an important contribution of data on the chronic toxicity of nickel to Symbiodinium sp. A key result from the present study was that three microalgal species had EC10 values below the current copper water quality guideline value for 95% species protection in slightly to moderately disturbed systems in Australia and New Zealand, indicating that they may not be adequately protected by the current copper guideline value. By contrast, toxicity of nickel to microalgae is unlikely to occur at exposure concentrations typically found in fresh and marine waters. Environ Toxicol Chem 2023;42:901-913. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Applicability of Chronic Multiple Linear Regression Models for Predicting Zinc Toxicity in Australian and New Zealand Freshwaters.

Bioavailability models, for example, multiple linear regressions (MLRs) of water quality parameters, are increasingly being used to develop bioavailability-based water quality criteria for metals. However, models developed for the Northern Hemisphere cannot be adopted for Australia and New Zealand without first validating them against local species and local water chemistry characteristics. We investigated the applicability of zinc chronic bioavailability models to predict toxicity in a range of uncontaminated natural waters in Australia and New Zealand. Water chemistry data were compiled to guide a selection of waters with different zinc toxicity-modifying factors. Predicted toxicities using several bioavailability models were compared with observed chronic toxicities for the green alga Raphidocelis subcapitata and the native cladocerans Ceriodaphnia cf. dubia and Daphnia thomsoni. The most sensitive species to zinc in five New Zealand freshwaters was R. subcapitata (72-h growth rate), with toxicity ameliorated by high dissolved organic carbon (DOC) or low pH, and hardness having a minimal influence. Zinc toxicity to D. thomsoni (reproduction) was ameliorated by both high DOC and hardness in these same waters. No single trophic level-specific effect concentration, 10% (EC10) MLR was the best predictor of chronic toxicity to the cladocerans, and MLRs based on EC10 values both over- and under-predicted zinc toxicity. The EC50 MLRs better predicted toxicities to both the Australian and New Zealand cladocerans to within a factor of 2 of the observed toxicities in most waters. These findings suggest that existing MLRs may be useful for normalizing local ecotoxicity data to derive water quality criteria for Australia and New Zealand. The final choice of models will depend on their predictive ability, level of protection, and ease of use. Environ Toxicol Chem 2023;42:2614-2629. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The Influence of Oil-in-Water Preparations on the Toxicity of Crude Oil to Marine Invertebrates and Fish Following Short-Term Pulse and Continuous Exposures.

Following an oil spill, accurate assessments of the ecological risks of exposure to compounds within petroleum are required, as is knowledge regarding how those risks may change with the use of chemical dispersants. Laboratory toxicity tests are frequently used to assess these risks, but differences in the methods for preparation of oil-in-water solutions may confound interpretation, as may differences in exposure time to those solutions. In the present study, we used recently developed modifications of standardized ecotoxicity tests with copepods (Acartia sinjiensis), sea urchins (Heliocidaris tuberculata), and fish embryos (Seriola lalandi) to assess their response to crude oil solutions and assessed whether the oil-in-water preparation method changed the results. We created a water-accommodated fraction, a chemically enhanced water-accommodated fraction, and a high-energy water-accommodated fraction (HEWAF) using standard approaches using two different dispersants, Corexit 9500 and Slickgone NS. We found that toxicity was best related to total polycyclic aromatic hydrocarbon (TPAH) concentrations in solution, regardless of the preparation method used, and that the HEWAF was the most toxic because it dispersed the highest quantity of oil into solution. The TPAH composition in water did not vary appreciably with different preparation methods. For copepods and sea urchins, we also found that at least some of the toxic response could be attributed to the chemical oil dispersant. We did not observe the characteristic cardiac deformities that have been previously reported in fish embryos, most likely due to the use of unweathered oil, and, as a consequence, the high proportion of naphthalenes relative to cardiotoxic polycyclic aromatic hydrocarbon in the overall composition. The present study highlights the need to characterize both the TPAH composition and concentration in test solutions when assessing oil toxicity. Environ Toxicol Chem 2022;41:2580-2594. © 2022 SETAC.

Exposure duration and composition are important variables to predict short-term toxicity of effluents to a tropical copepod, Acartia sinjiensis.

Predicting the toxicity of effluent exposures, which vary in duration, composition, and concentration, poses a challenge for ecological risk assessments. Effluent discharges may frequently result in the exposure of aquatic organisms to high concentrations of mixed contaminants for short durations. In the receiving environment effluents will undergo dilution and physical or chemical processes that further reduce contaminant concentrations at varying rates. To date, most studies comparing toxicity risks of continuous and pulsed contaminant exposures have focused on individual contaminants. In this study, the toxicity to the tropical euryhaline copepod Acartia sinjiensis of two complex effluents was assessed, comparing 6- and 18-h pulses and 78-h continuous exposures. Observations of larval development success and population size were completed after a 78-h incubation period, to observe for latent effects after pulse exposures. The chemical compositions of the effluents were assessed over time and different contaminants (i.e., metals, ammonia or organics) declined at differing rates. These were characterized as either a minimal, steady, or rapid decline. Nauplii development and population after 78 h were more impacted by effluent exposures following an 18-h pulse, compared to a 6-h pulse. Based on pulse-exposure concentrations, the 50% effect concentrations (EC50) were similar for continuous and 18-h exposures but up to 3-fold greater (lower toxicity) for the shorter 6-h exposures. Time-weighted average concentrations did not accurately predict toxicity from pulse exposures of the effluents. Concentration-addition toxicity modelling using toxicity data from pulse exposures of single contaminants was useful for predicting the toxicity of chemical mixtures exposed for varying durations. Recommendations for modified approaches to assessing risks of short-term effluent discharges are discussed.

The influence of salinity on the chronic toxicity of shale gas flowback wastewater to freshwater organisms.

The potential environmental risk associated with flowback waters generated during hydraulic fracturing of target shale gas formations needs to be assessed to enable management decisions and actions that prevent adverse impacts on aquatic ecosystems. Using direct toxicity assessment (DTA), we determined that the shale gas flowback wastewater (FWW) from two exploration wells (Tanumbirini-1 and Kyalla 117 N2) in the Beetaloo Sub-basin, Northern Territory, Australia were chronically toxic to eight freshwater biota. Salinity in the respective FWWs contributed 16% and 55% of the chronic toxicity at the 50% effect level. The remaining toxicity was attributed to unidentified chemicals and interactive effects from the mixture of identified organics, inorganics and radionuclides. The most sensitive chronic endpoints were the snail (Physa acuta) embryo development (0.08-1.1% EC10), microalga (Chlorella sp. 12) growth rate inhibition (0.23-3.7% EC10) and water flea (Ceriodaphnia cf. dubia) reproduction (0.38-4.9% EC10). No effect and 10% effect concentrations from the DTA were used in a species sensitivity distribution to derive "safe" dilutions of 1 in 300 and 1 in 1140 for the two FWWs. These dilutions would provide site-specific long-term protection to 95% of aquatic biota in the unlikely event of an accidental spill or seepage.

Probabilistic risk assessment of mine-derived copper in the Ok Tedi/Fly River, Papua New Guinea.

The Ok Tedi mine discharges waste rock and tailings into the Ok Tedi River in Papua New Guinea. This has resulted in elevated copper concentrations throughout the Ok Tedi/Fly River system, which can potentially impact aquatic biota. Ten years of measured copper and toxicity monitoring data were used to assess the risk of chronic effects from the mine-derived copper. Cumulative probability plots of dissolved and labile copper were compared to a species sensitivity distribution (SSD) of published copper toxicity data for four regions of the river. The Cu-SSD was used to estimate the risk of chronic effects to aquatic organisms in the Ok Tedi/Fly River at a range of potential copper exposure scenarios. The risk to species at the median labile copper concentration for each region showed a gradient effect with distance downstream from the mine and only the most sensitive (0.2-11%) species were at risk. There were copper exceedances of the region-specific guideline values (GV) and default guideline value (DGV) 88% and 74% of the time, respectively, in the Ok Tedi region (closest to the mine) and this is considered a high risk of chronic effects. Measured copper concentrations in the middle Fly River, lower Fly River (farthest downstream of the mine) and the river at Kiunga (reference site) exceeded the region-specific GVs and DGVs less frequently to rarely and present a lower risk of chronic effects from copper. The risk was supported using toxicity tests with the local microalgal species Chlorella sp. Comparison of recent (2010-2020) and historical (1996-2004) copper monitoring data from the Ok Tedi/Fly River indicates a decrease in the labile copper concentrations (30-76%) at key sites from impacted regions and a subsequent decrease in risk. This coincides with improved mining practices aimed at reducing the copper load into the Ok Tedi/Fly River.

The effects of pulse exposures of metal toxicants on different life stages of the tropical copepod Acartia sinjiensis.

Effluent discharges can potentially result in high concentrations of metals entering aquatic environments for short durations, ranging from a few hours to days. The environmental risks of such exposures are challenging to accurately assess. Risk assessment tools for effluent discharges include comparison of toxicant concentrations with guideline values and the use of direct toxicity assessments, both of which were designed to assess continuous, rather than pulse, contaminant exposures. In this study, a chronic pulse-exposure toxicity test was developed using the tropical euryhaline calanoid copepod Acartia sinjiensis. This copepod has a rapid life cycle and is highly sensitive to metal contaminants, with 50% effect concentrations (chronic EC50) for larval development of 1.7, 8.6 and 0.7 µg L-1 for copper, nickel and zinc, respectively. The toxicities of copper and nickel were assessed as a continuous exposure (78 h) and as pulses (3, 6 and 18 h) initiated at varying life stages, from egg to copepodite, and measured larval development over 78 h. Generally, 24-h old nauplii were more sensitive or of similar sensitivity to copper and nickel pulses than 48-h old nauplii. The 78-h test duration enabled observations of chronic effects following pulse exposures, which frequently occurred in the absence of acute effects. The EC50 values for pulse exposures were higher than those of continuous exposure by up to approximately 16-fold and 15-fold for copper and nickel, respectively. When metal-pulse exposure concentrations were expressed using the time-weighted averaged concentration (TAC), resultant concentration response curves were similar to those in continuous exposures to the same metal, suggesting that thresholds based on continuous exposures were also protective for pulse exposures to these metals. This research improves our understanding of the toxicity of pulse contaminant exposures and assists with developing improved approaches to for the risk assessment and regulation of short-term contaminant discharges.

Application of Bioavailability Models to Derive Chronic Guideline Values for Nickel in Freshwaters of Australia and New Zealand.

There has been an increased emphasis on incorporating bioavailability-based approaches into freshwater guideline value derivations for metals in the Australian and New Zealand water quality guidelines. Four bioavailability models were compared: the existing European biotic ligand model (European Union BLM) and a softwater BLM, together with 2 newly developed multiple linear regressions (MLRs)-a trophic level-specific MLR and a pooled MLR. Each of the 4 models was used to normalize a nickel ecotoxicity dataset (combined tropical and temperate data) to an index condition of pH 7.5, 6 mg Ca/L, 4 mg Mg/L, (i.e., approximately 30 mg CaCO3 /L hardness), and 0.5 mg DOC/L. The trophic level-specific MLR outperformed the other 3 models, with 79% of the predicted 10% effect concentration (EC10) values within a factor of 2 of the observed EC10 values. All 4 models gave similar normalized species sensitivity distributions and similar estimates of protective concentrations (PCs). Based on the index condition water chemistry proposed as the basis of the national guideline value, a protective concentration for 95% of species (PC95) of 3 µg Ni/L was derived. This guideline value can be adjusted up and down to account for site-specific water chemistries. Predictions of PC95 values for 20 different typical water chemistries for Australia and New Zealand varied by >40-fold, which confirmed that correction for nickel bioavailability is critical for the derivation of site-specific guideline values. Environ Toxicol Chem 2021;40:100-112. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Empirical Bioavailability Corrections for Nickel in Freshwaters for Australia and New Zealand Water Quality Guideline Development.

Bioavailability-based approaches have been developed for the regulation of metals in freshwaters in several countries. Empirical multiple linear regression (MLR) models have been developed for nickel that can be applied to aquatic organisms. The MLR models have been compared against the use of previously developed biotic ligand models (BLMs) for the normalization of an ecotoxicity dataset compiled for the derivation of a water quality guideline value that could be applied in Australia and New Zealand. The MLR models were developed from data for a number of specific species and were validated independently to confirm their reliability. An MLR modeling approach using different models for algae, plants, invertebrates, and vertebrates performed better than either a pooled MLR model for all taxa or the BLMs, in terms of its ability to correctly predict the results of the tests in the ecotoxicity database based on their water chemistry and a fitted species-specific sensitivity parameter. The present study demonstrates that MLR approaches can be developed and validated to predict chronic nickel toxicity to freshwater ecosystems from existing datasets. The MLR approaches provide a viable alternative to the use of BLMs for taking account of nickel bioavailability in freshwaters for regulatory purposes. Environ Toxicol Chem 2021;40:113-126. © 2020 SETAC.

Toxicity of dissolved and precipitated forms of barium to a freshwater alga (Chlorella sp. 12) and water flea (Ceriodaphnia dubia).

Barium is present at elevated concentrations in oil and gas produced waters, and there is no international water quality guideline value to assess the potential risk of adverse effects to aquatic biota. Sulfate concentration largely controls the solubility of barium in aquatic systems, with insoluble barium sulfate (barite) assumed to be less bioavailable and less toxic than dissolved barium. We exposed aquatic biota to dissolved barium only and to a mixture of dissolved and precipitated barium. The chronic dissolved barium 48-h growth rate inhibition effect concentrations, (EC10 and EC50) for the tropical freshwater alga Chlorella sp. 12 were 40 mg/L (27-54 mg/L 95% confidence limits [CL]), and 240 mg/L (200-280 mg/L 95% CL), respectively. The acute EC10 and EC50 values for 48-h immobilization of the water flea (Ceriodaphnia dubia) by dissolved barium were 14 mg/L (13-15 mg/L 95% CL) and 17 mg/L (16-18 mg/L 95% CL), respectively. Chlorella sp. 12 was significantly more sensitive to precipitated barium than to dissolved barium, whereas the opposite seemed likely for C. dubia. Ceriodaphnia dubia was predicted to be chronically sensitive to dissolved barium at concentrations measured in produced waters and receiving waters, based on a predicted chronic EC10 of 1.7 mg/L derived from the acute EC50/10. Further chronic toxicity data that account for barium toxicity in dissolved and precipitated forms are required to derive a barium guideline for freshwater biota. Environ Toxicol Chem 2018;37:1632-1642. © 2018 SETAC.