Method Development for a Short-Term 7-Day Toxicity Test with Unionid Mussels.

The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%-52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314-520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392-3409. © 2021 SETAC.

Effects of pulsed copper exposures on early life-stage Pimephales promelas.

Effects of pulsed copper exposures were investigated using Pimephales promelas aged less than 24 h in short-term chronic testing (7 or 14 d) with moderately hard synthetic water. Concentrations tested were between the species mean chronic value (22 microg/L at a hardness of 100 mg/L as CaCO3) and the 7-d continuous exposure EC50 for survival (40 microg/L) to examine exposures that were not acutely toxic and representative of actual wastewater discharge permit exceedences. Factors tested included pulse duration, recovery time between pulses, and pulse frequency. Survival was the main endpoint affected in all treatments (analysis of variance, p < 0.05). Effects on fish biomass, independent of survival effects, were observed in only 2 of 86 treatments examined. Fish survival was negatively affected at average copper concentrations between 7 and 50% of the 7-d continuous exposure EC50. Exposures having a 48- to 96-h recovery time between pulses had less effect on fish survival than did treatments with shorter (12-24 h) or longer (>120 h) recovery times. Results suggest that the criteria averaging periods used in the United States, and the averaging periods typically used in wastewater discharge permit limits for copper, may not protect against effects of certain pulsed exposures.

Toxicity models of pulsed copper exposure to Pimephales promelas and Daphnia magna.

Semiempirical models are useful for interpreting the response of aquatic organisms to toxicants as a function of exposure concentration and duration. Most applications predict cumulative mortality at the end of the test for constant exposure concentrations. Summary measures, such as the median lethal concentration, are then estimated as a function of concentration. Real-world exposures are not constant. Effects may depend on pulse timing, and cumulative analysis based only on integrated exposure concentration is not sufficient to interpret results. We undertook a series of pulsed-exposure experiments using standard toxicological protocols and interpreted the results (mortality, biomass, and reproduction) using a dynamic generalization of a Mancini/Breck--type model that includes two compartments, one for internal concentration as a function of exposure and one for site-of-action concentration or accumulated damage as a function of the internal dose. At exposure concentrations near the effects level, the model explained approximately 50% of the variability in the observed time history of survival, 43% of the change in biomass, and 83% of the variability in net reproduction. Unexplained variability may result from differences in organism susceptibility, amplified by the effects of small sample sizes in standard tests. The results suggest that response is sensitive to prior conditions and that constant-exposure experiments can underestimate the risk from intermittent exposures to the same concentration. For pulsed exposures, neither the average nor the maximum concentration alone is an adequate index of risk, which depends on both the magnitude, duration, and timing of exposure pulses. Better understanding about the impacts of pulsed exposures will require use of experimental protocols with significantly greater numbers of replicates.

Evaluation of effluent toxicity as an indicator of aquatic life condition in effluent-dominated streams: a pilot study.

The types and quality of data needed to determine relationships between chronic whole effluent toxicity (WET) test results and in-stream biological condition were evaluated using information collected over a 1.5-y period from 6 different sites across the United States. A data-quality-objectives approach was used that included several proposed measurement quality objectives (MQOs) that specified desired precision, bias, and sensitivity of methods used. The 6 facilities used in this study (4 eastern and 2 western United States) all had design effluent concentrations >60% of the stream flow. In addition to at least quarterly chronic Ceriodaphnia dubia, Pimephales promelas (fathead minnow), and Selenastrum capricornutum (green algae) WET tests, other tests were conducted to address MQOs, including splits, duplicates, and blind positive and negative controls. Macroinvertebrate, fish, and periphyton bioassessments were conducted at multiple locations upstream and downstream of each facility. The test acceptance criteria of the US Environmental Protection Agency (USEPA) were met for most WET tests; however, this study demonstrated the need to incorporate other MQOs (minimum and maximum percent significant difference and performance on blind samples) to ensure accurate interpretation of effluent toxicity. More false positives, higher toxicity, and more "failed" (noncompliant) tests were observed using no-observed-effect concentration (NOEC) as compared to the IC25 endpoint (concentration causing > or =25% decrease in organism response compared to controls). Algae tests often indicated the most effluent toxicity in this study; however, this test was most susceptible to false positives and high interlaboratory variability. Overall, WET test results exhibited few relationships with bioassessment results even when accounting for actual effluent dilution. In general, neither frequency of WET noncompliance nor magnitude of toxicity in tests were significantly related to differences in biological condition upstream and downstream of a discharge. Periphyton assessments were most able to discriminate small changes downstream of the effluent, followed by macroinvertebrates and fish. Although sampling methods were robust, more replicate samples collected upstream and downstream of each facility were needed to increase detection power. In general, macroinvertebrate and periphyton assessments together appeared to be sufficient to address project objectives.

Effects of pulsed contaminant exposures on early life stages of the fathead minnow.

Water quality standards for protecting aquatic life are based primarily on laboratory tests that use constant exposure concentrations. Typical effluent and nonpoint source exposure concentrations fluctuate in frequency, magnitude, and duration, which may result in different toxicological impacts. Current information indicates that pulsed or fluctuating exposures are generally more toxic than continuous exposures, when averaged over the applicable time period. However, few studies have evaluated chronic or sublethal effects of pulsed exposures, particularly those applicable to wastewater discharge situations. To address this issue, several pulsed exposure toxicity tests were conducted using modified fathead minnow (Pimephales promelas) early life stage (7 d) tests and several chemicals representative of those commonly encountered in wastewater effluents including copper, nitric acid, cadmium, and sodium chloride. Results suggest that survival and/or growth effects depend on the combination of frequency, magnitude, and duration, as well as the type of chemical. Nitric acid and sodium chloride pulsed treatments did not exhibit growth effects independent of survival effects, but both metals did elicit only growth effects in some treatments. Growth effects were related to pulse frequency and duration for copper and pulse duration and magnitude for cadmium. A 12-h exposure of approximately five times the 7-d continuous exposure IC(25) concentration of either metal quickly elicited mortality responses. Prolonged (>24 h) lag effects on survival were not observed in any of the experiments, regardless of the contaminant tested. Our results suggest that current water quality criteria may be underprotective if based on an average concentration over a 2-4 d exposure. For these contaminants, a short-term increase in concentration may elicit effects even though the average concentration is within nontoxic ranges for the organism.