A New Toxicity Test Using the Freshwater Copepod Cyclops vernalis.

The cladocerans Ceriodaphnia dubia and Daphnia magna are widely used in environmental toxicity testing and the test methodologies for these species are well developed. However, copepods are a much more abundant contributor to zooplankton in many lakes, but they are not routinely used in toxicity tests. Therefore, we propose toxicity test methods for the freshwater copepod, Cyclops vernalis assessing effects on its survival and growth. A case study is presented in which the proposed test was performed with a range of concentrations of total dissolved solids (TDS) and used as part of a test battery to develop a site-specific water quality objective. C. vernalis was less sensitive to TDS compared to D. magna and C. dubia, but similarly sensitive to an alga, a diatom, a rotifer, a chironomid, and two fish species. No adverse effects were observed on survival or growth of C. vernalis at TDS concentrations up to 1500 mg/L.

Molecular responses to 17ß-estradiol in early life stage salmonids.

Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17ß-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1 µg/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (∼90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (∼35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1 µg/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts.

Use of Toxicity Identification Evaluation Procedures to Clarify the Relationship Between Ammonium Concentrations and Phytoplankton Blooms in the San Francisco Bay Estuary, California, USA.

Phytoplankton blooms in the northern San Francisco Bay Estuary have historically supported much of the larval fish production in the estuary. In the past, blooms were limited largely by reduced light intensities and net outflows through the system, as well as dense populations of introduced clams that continuously filter the water column. Conversely, the estuary is exposed to a wide variety of contaminants that may also impact phytoplankton growth. Interestingly, previous investigations have suggested that relatively low concentrations of ammonium may inhibit development of bloom conditions by interfering with nitrate assimilation. Given the complex dynamics of the system, with multiple factors that could potentially affect algal growth, additional data to validate this hypothesis are important to identify appropriate management options. Consequently, toxicity identification evaluation (TIE) procedures were applied to ambient water samples and monitored for 72-96 h under controlled conditions to evaluate their effects on algal growth and utilization of dissolved inorganic nitrogen. The TIE treatments specifically targeted ammonium, as well as the potential contributions of metals and nonpolar organic contaminants. Notably, all samples exhibited positive growth over the exposure period with no evidence of toxicity, and TIE treatments did not further improve growth. A subsequent 72-h study evaluated the effect of ammonium up to 12 µM at a fixed concentration of nitrate was monitored at 24-h intervals and showed no inhibition of the development of bloom conditions. Collectively, there was no evidence that ammonium interfered with growth, even at concentrations well above the range of postulated effect levels. Of additional interest, the lack of increased growth in TIE treatments targeting chelatable metals and nonpolar organics suggested that these contaminant classes were not present at inhibitory concentrations. These results demonstrate the importance of validation of cause in multistressor environments, and further clarify the roles of different factors that may limit development of bloom conditions in the estuary. Environ Toxicol Chem 2023;42:178-190. © 2022 SETAC.

Using ultrahigh-resolution mass spectrometry and toxicity identification techniques to characterize the toxicity of oil sands process-affected water: The case for classical naphthenic acids.

Previous assessments of oil sands process-affected water (OSPW) toxicity were hampered by lack of high-resolution analytical analysis, use of nonstandard toxicity methods, and variability between OSPW samples. We integrated ultrahigh-resolution mass spectrometry with a toxicity identification evaluation (TIE) approach to quantitatively identify the primary cause of acute toxicity of OSPW to rainbow trout (Oncorhynchus mykiss). The initial characterization of OSPW toxicity indicated that toxicity was associated with nonpolar organic compounds, and toxicant(s) were further isolated within a range of discrete methanol fractions that were then subjected to Orbitrap mass spectrometry to evaluate the contribution of naphthenic acid fraction compounds to toxicity. The results showed that toxicity was attributable to classical naphthenic acids, with the potency of individual compounds increasing as a function of carbon number. Notably, the mass of classical naphthenic acids present in OSPW was dominated by carbon numbers ≤16; however, toxicity was largely a function of classical naphthenic acids with ≥17 carbons. Additional experiments found that acute toxicity of the organic fraction was similar when tested at conductivities of 400 and 1800 µmhos/cm and that rainbow trout fry were more sensitive to the organic fraction than larval fathead minnows (Pimephales promelas). Collectively, the results will aid in developing treatment goals and targets for removal of OSPW toxicity in water return scenarios both during operations and on mine closure. Environ Toxicol Chem 2017;36:3148-3157. © 2017 SETAC.

Application of sediment toxicity identification evaluation techniques to a site with multiple contaminants.

Sediment toxicity identification evaluations (TIEs) are conducted to determine causes of adverse effects observed in whole-sediment toxicity tests. However, in multiple contaminant scenarios, it is problematic to partition contributions of individual contaminants to overall toxicity. Using data from a site with multiple inputs and contaminants of concern, the authors describe a quantitative approach for the TIE process by tracking toxicity units to determine whether all toxicity is accounted for. The initial step established the level of toxicity associated with the whole sediment and then partitioned sources of toxicity into general contaminant classes (e.g., ammonia, metals, nonpolar organic compounds). In this case, toxicity was largely the result of nonpolar organics, so the sediments were extracted and the extracts added back into dilution water and tested to confirm recovery of toxicity. Individual fractions were then generated using a solvent gradient and tested for toxicity. Fractions of interest were evaluated with gas chromatography/mass spectrometry to identify specific constituents associated with toxicity. Toxicity units associated with these constituents were then evaluated to determine probable associations with cause and whether all toxicity was accounted for. The data indicated that toxicity was associated with 2 contaminant classes, representing legacy compounds and contaminants of emerging concern, with the contribution of each varying across the site. Environ Toxicol Chem 2016;35:2456-2465. © 2016 SETAC.

Aeration with carbon dioxide-supplemented air as a method to control pH drift in toxicity tests with effluents from wastewater treatment plants.

Environment Canada methods for acute toxicity tests with rainbow trout require continuous aeration of test solutions during exposure. Depending on the sample, this procedure can result in an increase in pH as dissolved carbon dioxide (CO2) is stripped from solution as a result of aeration. In samples that contain ammonia, the pH may increase to the point where the unionized fraction results in artifactual toxicity. Consequently, aeration with air supplemented with different CO2 concentrations was investigated as a method for maintaining pH at the level found in the original sample without adversely affecting other water quality parameters. Aeration with CO2 was an effective method for maintaining pH during exposure, depending both on the concentration of CO2 and the alkalinity of the sample. A multiple regression conducted on the data determined an equation that was effective at calculating the CO2 concentration necessary in an aeration mixture to maintain a target pH value as a function of sample alkalinity.

Identification of chlorfenvinphos toxicity in a municipal effluent in Sydney, New South Wales, Australia.

Acute toxicity in a municipal sewage treatment plant in Sydney, New South Wales, Australia, was traced to chlorfenvinphos, an organophosphorous pesticide. Toxicity identification evaluation procedures led to the tentative identification of chlorfenvinphos as the toxic contaminant in the sample. Subsequent analytical verification revealed 0.95 microg/L of chlorfenvinphos in the effluent sample, and spiking studies confirmed that it accounted for the observed toxicity. The 48-h median lethal concentration of chlorfenvinphos to Ceriodaphnia dubia averaged 0.28 microg/L (n = 4). Source-control measures were effective at eliminating chlorfenvinphos and associated toxicity from the discharge.

The effect of sulfate on selenate bioaccumulation in two freshwater primary producers: A duckweed (Lemna minor) and a green alga (Pseudokirchneriella subcapitata).

Predicting selenium bioaccumulation is complicated because site-specific conditions, including the ionic composition of water, affect the bioconcentration of inorganic selenium into the food web. Selenium tissue concentrations were measured in Lemna minor and Pseudokirchneriella subcapitata following exposure to selenate and sulfate. Selenium accumulation differed between species, and sulfate reduced selenium uptake in both species, indicating that ionic constituents, in particular sulfate, are important in modifying selenium uptake by primary producers.

Chronic toxicity of chloride to freshwater species: effects of hardness and implications for water quality guidelines.

Toxicity tests using nine freshwater species (Ceriodaphnia dubia, Daphnia magna, Oncorhynchus mykiss, Pimephales promelas, Lumbriculus variegatus, Tubifex tubifex, Chironomus dilutus, Hyallela azteca, and Brachionus calyciflorus) were conducted to evaluate their sensitivity to chloride. Acute-to-chronic ratios (ACRs) from these tests indicate the ACR of 7.59 employed by the United States Environmental Protection Agency (U.S. EPA) in deriving its water quality guideline for chloride may be conservative; a revised ACR of 3.50 is presented here. The endpoints used to calculate the ACR included 24-h to 96-h median lethal concentrations (LC50s) for acute tests, and 48-h to 54-d inhibition concentration (ICx) values for growth or reproduction for chronic exposures. Data from the present chronic toxicity tests, and other investigators, were used to propose a water quality guideline for long-term exposure to chloride using a species sensitivity distribution (SSD) approach. The 5th percentile from the SSD was calculated as 307 mg/L and proposed as the water quality guideline. Cladocerans were the most sensitive species in the dataset. Ceriodaphnia dubia was used to evaluate the relationship between water hardness and sensitivity to chloride. A strong relationship was observed and was used to establish a hardness-related equation to modify the proposed water quality guideline on the basis of water hardness, resulting in values ranging from 64 mg/L chloride at 10 mg/L hardness to 388 mg/L chloride at 160 mg/L hardness (as CaCO3). These data suggest that current water quality guidelines for chloride may be overly conservative in water with moderate-to-high hardness, and may not be sufficiently protective under soft-water conditions.

An aquatic toxicological evaluation of sulfate: the case for considering hardness as a modifying factor in setting water quality guidelines.

Elevated concentrations of sulfate occur commonly in anthropogenically impacted and natural waters. However, water quality guidelines (WQG) have not been developed in many jurisdictions, and chronic toxicity data are scarce for this anion. A variety of test organisms, including species of invertebrate, fish, algae, moss, and an amphibian, were tested for chronic toxicity to develop a robust dataset that could be used to develop WQGs. As an example of how these data might be used to establish guidelines, calculations were performed using two standard procedures: a species sensitivity distribution (SSD) approach, following methods employed in developing Canadian WQGs, and a safety factor approach, according to procedures typically used in the development of provincial WQGs in British Columbia. The interaction of sulfate toxicity and water hardness was evaluated and incorporated into the calculations, resulting in separate values for soft (10-40 mg/L), moderately hard (80-100 mg/L) and hard water (160-250 mg/L). The resulting values were 129, 644, and 725 mg/L sulfate, respectively, following the SSD approach, and 75, 625, and 675 mg/L sulfate, following the safety factor approach.