Elsevier

Chemosphere

Volume 277, August 2021, 130323
Chemosphere

Pulsed exposure toxicity testing: Baseline evaluations and considerations using copper and zinc with two marine species

https://doi.org/10.1016/j.chemosphere.2021.130323Get rights and content

Highlights

  • Simple modifications to standard methods increase realism while still protective.

  • Sensitivity to Cu and Zn was higher for 96 h static vs. 6 or 12 h pulsed exposures.

  • Sea urchin embryos showed variable sensitivity to Zn up to 18 h post-fertilization.

  • 96 h test period with pulsed exposures of 6–26 h captured latent effects to mysids.

  • Pulsed exposure toxicity testing is an environmentally relevant alternative method.

Abstract

Methods to assess environmental impacts from episodic discharges on receiving water bodies need a more environmentally relevant and scientifically defensible toxicity test design. Many permittees are regularly required to conduct 96-h toxicity tests on discharges associated with events that are generally less than 24 h in duration. Current standardized methods do not adequately reflect these episodic discharge conditions at either the point of compliance nor as it mixes with the receiving environment. In order to evaluate more representative biological effects, an alternative toxicity approach is described incorporating pulsed exposures of effluents and subsequent transfer of test organisms to clean water for the remainder of the test. This pulsed exposure protocol incorporates a slight modification to USEPA Whole Effluent Toxicity (WET) chronic and acute methods for two marine species, purple sea urchin embryos, Strongylocentrotus purpuratus, and juvenile mysid shrimp Americamysis bahia. Tests were performed with toxicants using standard static (96 h) and pulsed (6, 12, and 26 h) exposures. Following pulsed exposures, organisms were transferred to uncontaminated seawater for the remainder of the 96-h test period. Results for these species and endpoints indicated that the sensitivity of these species to copper and zinc were up to two orders of magnitude greater using standard continuous exposures compared to shorter pulsed exposures. Additional considerations assessed included timing of the onset of a pulse and latent effects following an exposure. This modified approach requires minimal modification to current standard methods and increases the realism to more accurately assess toxic effects resulting from episodic discharges.

Introduction

Under the United States Environmental Protection Agency (USEPA) Clean Water Act, various point source discharges and associated receiving waters including oceans, lakes, rivers, and tributaries are regularly monitored for toxicity and a suite of contaminants of potential concern. In order to minimize potentially contaminated discharge or runoff, facilities or municipalities must comply with strict water quality requirements for stormwater and other episodic discharges through permits managed by the National Pollutant Discharge Elimination System (NPDES). These permits work to protect the health of water bodies by setting discharge limits for chemistry and toxicity, and require facilities to follow prescribed monitoring and reporting requirements to maintain compliance.

Whole Effluent Toxicity (WET) testing has been developed as the primary method for evaluating toxicity of continuous point source discharges in the United States (USEPA 1995, USEPA 2002a,b,c). However, these methods have since also been applied to episodic discharges (e.g. stormwater runoff) in support of newer permitting requirements that address nonpoint source runoff. Permits that require WET testing for nonpoint source runoff or other episodic discharges typically require discrete end-of-pipe samples to be collected during the first few hours of a rainfall or discharge, or up to a 24-h composite sample. These samples are then evaluated with laboratory toxicity tests using a continuous exposure of up to 7 days, depending on the species and test endpoint (USEPA 1995, 2002a,b,c). WET testing is desirable because: 1) these methods take into account contaminant bioavailability, which can vary from that in uncontaminated filtered water used to develop water quality criteria (Stephan et al., 1985); and 2) they incorporate the potential for adverse effects associated with exposure to complex mixtures (i.e. multiple contaminants), many of which may not be monitored. Toxicity tests with effluents or pure compounds are conducted to estimate the "safe" or "no effect" concentration of these substances, which is defined as the concentration which will permit normal propagation of fish and other aquatic life in receiving waters. A wide variety of endpoints may be used to evaluate adverse effects of toxicants including survival, reproduction, growth, locomotor activity, gill ventilation rate, heart rate, blood chemistry, histopathology, enzyme activity, olfactory function, endocrine disruption, bioaccumulation and terata (Amiard-Triquet 2015). Since it is not feasible to detect and/or measure all of these (and other possible) effects of toxic substances on a routine basis, observations in toxicity tests generally have been limited to only a few of the more definitive and easier to measure endpoints, such as mortality, growth, and reproduction (USEPA 2002a, 2002b, 2002c).

While the current standard WET methods may be appropriate for sites with continuous discharges (e.g. wastewater treatment plants), they are arguably inappropriate for episodic discharges, such as storm events (Burton et al., 2000; Reinert et al., 2002; Diamond et al., 2006). Due to the episodic nature of most storm events, rainfall most typically results in runoff discharging as a series of pulses, as opposed to a continuous discharge. There is increasing consensus that current WET methods do not adequately replicate pulsed exposure conditions at either the point of compliance (e.g. storm drain) or as it mixes with the receiving environment. Katz et al. (2006) comprehensively demonstrated that while 30% of end-of-pipe samples of stormwater evaluated at multiple US Naval bases bordering San Diego Bay (San Diego, CA, USA) resulted in a toxic designation, fewer than 1% of samples collected from the ambient environment before, during, and after the storm events demonstrated toxicity. These results suggest current WET methods may overestimate the risks of actual stormwater exposures in associated receiving waters, which are typically much shorter in duration (Rosen et al., 2019; Colvin et al., 2020a). It is critically important to accurately assess toxicity at a given site, as a permit exceedance due to toxicity may require further accelerated testing. A Toxicity Reduction Evaluation (TRE) might also be required including the conduct of a Toxicity Identification Evaluation (TIE). Test results may also lead to the implementation of potentially costly best management practices (BMPs) that may or may not be warranted.

In response to these concerns, it is important to develop alternative test methods that are more ecologically relevant and can be easily adapted by laboratories responsible for stormwater or other episodic discharge evaluations. Pulsed exposure toxicity testing has been suggested as an alternative means to evaluate of episodic discharges that is more environmentally realistic (Burton et al., 2000, Schiff et al., 2003, Diamond et al., 2006, Hoang et al., 2008, Angel et al., 2010, Gordon et al., 2012). Previous studies have explored several aspects associated with pulsed toxicity exposures, including pulse concentration, pulse duration, and pulse frequency (Handy 1994; Reinert et al., 2002; Diamond et al., 2006; Gordon et al., 2012; Gosset et al., 2016; Rosen et al., 2019). It is well documented that as the concentration of a contaminant and duration of exposure increase, toxic effects also often increase (Reinert et al., 2002; Diamond et al., 2006; Stransky et al., 2015; Gosset et al., 2016; Rosen et al., 2019). A predominant observation has been that algal and invertebrate organisms have an assimilative ability to tolerate short-term trace metal exposures at elevated concentrations better than longer-term continuous exposures of the same or lower concentration (Hoang et al., 2007a; Angel et al., 2015; Rosen et al., 2019). Further, Angel et al. (2010) found that the time-averaged concentration (TAC) of trace metals better predicted toxicity to a marine amphipod than concentration, pulse duration, or pulse frequency in exposures that fluctuated in metal concentration. This is particularly important when considering episodic discharges, where organisms in the receiving environment may be exposed to high contaminant loads for a relatively short time. For acute exposures, researchers have reported latent mortality in several species, including fish and several crustacean groups, that can be delayed up to several days, sometimes weeks (Brent and Herricks 1998; Reinert et al., 2002; Diamond et al., 2006; Hoang et al., 2007b; Angel et al., 2010; Gordon et al., 2012).

The present laboratory study was part of a larger effort to refine and validate a pulsed exposure protocol for echinoderm embryo development using the purple sea urchin, Strongylocentrotus purpuratus, and survival of the mysid shrimp, Americamysis bahia, both evaluated herein, for episodic exposures. This study investigated the effects of various copper or zinc concentrations, and exposure durations, on the observed toxicity for both species. Copper and zinc were selected for evaluations as these are commonly elevated constituents in urban stormwater, typical examples of episodic discharge, and often have been identified as the primary cause of observed toxicity during routine monitoring in several studies that have evaluated toxic responses in wet weather runoff from predominantly impervious roadways and industrial facilities (Katz et al., 2006; Kayhanian et al., 2008). The two marine species and methods selected for this study are standard EPA-approved protocols, commonly incorporated into monitoring programs, and include both acute and short-term chronic endpoints (USEPA 1995; 2002a). Further, both species are notably sensitive to copper and zinc. The specific objectives of this study included: 1) establishing baseline sensitivity data for pulsed exposures of copper and zinc, independently, for both species relative to standard continuous exposures; 2) assessment of latent effects following pulsed exposures for A. bahia; and 3) examining embryo sensitivity at different times post fertilization for purple sea urchins to assess whether exposure initiation time might be important to consider when using a short-term pulsed exposure for this species and sensitive endpoint. Although a few potential mechanisms of toxicity are suggested and referenced, the objectives were focused on method development as opposed to a more specific biochemical understanding of the cause of observed effects which is well documented for copper and zinc in other literature sources for the species evaluated in this study (e.g. Lussier et al., 1985, Irwin et al., 1997a,b, Phillips et al., 2003).

Section snippets

Determination of pulsed exposure period

To develop representative pulsed exposure time periods for toxicity testing, an analysis of historical rainfall was conducted for the study site in San Diego, CA, USA (Colvin et al., 2020a). Rainfall amounts have been shown to strongly correlate with the duration of discharge runoff from predominantly impervious surfaces such as the Naval Bases in San Diego (Katz et al., 2006). Experimental pulsed exposure times for this study were derived based on rainfall data recorded at the San Diego

Results

Test results for all laboratory controls met standard EPA method test acceptability criteria of a minimum of 80% normal embryo-larval development or a minimum of 90% survival for sea urchin and mysid tests, respectively (USEPA 1995; USEPA 2002; Supplemental Data). All applicable water quality parameters (salinity, dissolved oxygen, pH, and temperature) also met test acceptability criteria for all tests conducted. Statistical summaries are shown in Table 1 and Table 2 (detailed statistics for

Discussion

This study showed that pulsed duration using copper and zinc was related to organism sensitivity for both purple sea urchins and mysid shrimp, with shorter pulses resulting in less sensitivity (Figs. 4 and 6). This is consistent with other studies using metals as the contaminant (Diamond et al., 2006, Hoang et al., 2007 a, b, c, Angel et al., 2010, Stransky et al., 2015, Rosen et al., 2019). In the present study, the magnitude of the response among different exposure periods was also found to

Conclusions

This study evaluated test methods to assess impacts from episodic discharges using purple sea urchin embryos (Strongylocentrotus purpuratus) and mysid shrimp (Americamysis. bahia), two commonly used marine species for toxicity testing (USEPA 1995; USEPA 2002). A slight modification to current USEPA WET test methods involving shortening the stressor (i.e. contaminated effluent) exposure period, but including a latent observation period, provides a feasible and protective approach to assess

Author statements

Marienne A. Colvin – Project administration, Conceptualization, Investigation, Formal analysis, Data curation, Writing – Original, Writing – review & editing, Visualization, Funding acquisition. Katherine R. Kowal – Investigation, Formal analysis, Writing – original draft. Nicholas T. Hayman – Methodology, Investigation, Writing – review & editing, Formal analysis. Chris Stransky – Methodology, Writing – review & editing, Formal analysis, Supervision. Jeff VanVoorhis – Investigation, Formal

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank J. Munson-Decker and H. Kranz whose technical support was invaluable to the success of this study. This project was supported by funding from the U.S. Navy’s Environmental Sustainability Development to Integration (NESDI) Program under project #547 (PI: M. Colvin) and the U.S. Department of Defense’s Environmental Security Technology Certification Program (ESTCP) under project ER-201727 (PI: M. Colvin).

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