5.1 Protection of a species requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species. Toxicity tests can be used provide information about the toxicity of a test material to a specific life stage of a particular species of mussel. The primary adverse effects studied are reduced survival or growth.

5.2 Results of toxicity tests might be used to predict effects likely to occur on mussels in field situations as a result of an exposure under comparable conditions.

5.3 Results of toxicity tests might be used to compare the sensitivities of different mussel species and the toxicity of different test materials, and to study the effects of various environmental factors on results of such tests.

5.4 Results of toxicity tests conducted with mussels might be an important consideration when assessing the risks of test materials to aquatic organisms or when deriving environmental guideline values for toxicants.

5.5 An acute toxicity test is conducted to obtain information concerning the immediate effects on mussels of a short exposure to a test material under specific experimental conditions. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, might provide such information (Guide E729).

5.6 Results of chronic (at least 28 d) toxicity tests with mussels might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions.

5.7 Short-term chronic toxicity tests are conducted for 7 d, a complementary test duration in the USEPA shot-term methods for estimating the chronic toxicity of effluents and receiving waters to fathead minnow (Pimephales promelas; USEPA 2002) (31) and provides a more direct estimate of the safe concentrations of effluents and receiving waters than acute toxicity tests, at a slightly lower level of effort compared to chronic 28 d toxicity test.

5.8 Results of toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.

5.9 Results of toxicity tests will depend on temperature, composition of the dilution water, condition of the test organisms, and other factors.

5.10 Interferences—A number of factors can impede or prevent selection and use of freshwater mussels for toxicity testing (Guide E1850). The following should be considered when selecting a test species and measuring the sensitivity of the test species during toxicity tests.

5.10.1 Handling of field-collected adult mussels resulting from collection or transport to the laboratory might cause excessive mortality or sublethal effects.

5.10.2 The age, health, and physical condition of adult mussels (for example, the presence of parasites, bacteria, and disease) collected from a resident population might not be adequately known.

5.10.3 The physical characteristics of the testing environment (such as water quality, temperature, water flow, light) and food requirements might affect the ability of the test organisms to acclimate, recover from handling, or adapt to the laboratory environment conditions.

5.10.4 The degree of contamination and the history of contamination at the collection of the adult mussels might not be adequately known.

5.10.5 In the field, mussels may be exposed to contaminants in water, sediment, or food. This standard only addresses effects associated with exposure of mussels to contaminants in water. Methods for conducting sediment toxicity tests with juvenile mussels are included in Guide E1706.

5.10.6 There are insufficient data available to determine if juvenile mussels are able to avoid exposure to chemicals by valve closure. If it is suspected that juvenile mussels are avoiding exposure to a chemical in a toxicity test, it may be desirable to place the suspected live test organisms into dilution water that does not contain any added test material for 1 d to 2 d after the end of the toxicity test to determine whether these test organisms are alive or dead (section A1.4.7; Guide E729).

1.1 This standard guide describes methods for conducting laboratory toxicity tests with early life stages of freshwater mussels including glochidia and juvenile mussels in water-only and effluent exposures (Annex A1). Future revisions to this standard may describe methods for conducting toxicity tests with endpoints of reproduction, behaviors, and biomarkers.

1.2 Freshwater mussels (order Unionida) are one of the most imperiled groups of animals in the world, and environmental contamination has been linked as a contributing factor to the decline of mussel populations (Lydeard et al. 2004 (1); Strayer et al. 2004 (2); Haag 2012 (3); Lopes-Lima et al. 2017 (4)).2 Three critical life stages (glochidia, juvenile mussels, and adults) have been used in toxicity assessments and the toxicity studies are separated according to the medium of exposure (water, sediment, and host fish (Ingersoll et al. 2007 (5)). Recent studies on early life stages of mussels have demonstrated that the mussels are among the most sensitive freshwater species to a variety of contaminants, including ammonia, some metals (for example, aluminum, copper, nickel, and zinc), and major ions (for example, chloride, nitrate, potassium, and sulfate) (Bringolf et al. 2007 (6); Newton et al. 2007 (7); Wang et al. 2007ab, 2010, 2011ab, 2016, 2017ab, 2018abc, 2020ab (8-20); Cope et al. 2008 (21); Gillis et al. 2008, 2010, 2011, 2021 (22-25); Miao et al. 2010 (26); Salerno et al. 2020 (27)). These studies indicate that environmental guideline values for individual chemicals established for the protection of aquatic organisms may not be adequately protective of sensitive stages of freshwater mussels. For example, when freshwater mussel toxicity data were included in an update to the United States Environmental Protection Agency (USEPA) ambient water quality criteria (WQC) for ammonia, the acute criterion decreased by about a 1.4 fold and the chronic criterion decreased by 2.4 fold (USEPA 2013) (28).

1.3 Summary of Life History of Freshwater Mussels: 

1.3.1 Freshwater mussels are bivalve mollusks belonging to the taxonomic Order Unionida (section 10.1). Like most bivalves, mussels are totally aquatic, relatively sedentary, filter-feeding animals, and spend most of their lives partially or completely burrowed in the substrate of streams, rivers, or lakes. Freshwater mussels have an unusual and complex life cycle that includes a larval stage, the glochidium, that is briefly parasitic on fish (Fig. 1).

1.3.2 The successful transfer of mature glochidia to a suitable host constitutes a critical event in the life cycle of most freshwater mussels (Haag 2012) (3). Once the glochidia are released from the female, the glochidia need to attach to the gills or the fins of an appropriate fish host and encyst to complete development. Although glochidia may survive for months during brooding in the female mussel, glochidia typically survive for only a few days after release unless the glochidia reach a compatible host. Host fish specificity varies among mussels. While some mussel taxa appear to require a single host species, others can utilize several species of host fish. Encapsulation on the host occurs by overgrowth of host tissue. Within the capsule, glochidia obtain nutrition from the host, continue their development, and metamorphose within days to weeks. Metamorphosis is followed by excapsulation (drop-off) and transition to self-sustained existence as a benthic organism.

1.3.3 Both juvenile and adult mussels live embedded in sediment and feed using ciliary mechanisms to capture fine particles (Haag 2012) (3). However, young juvenile mussels (~0.2 mm –10 mm) may bury several centimeters in sediment where they feed and respire from interstitial water, while larger adult mussels can access the water column. Water column and substrate conditions suitable for adult life stages may not be protective of juvenile life stages of freshwater mussels.

1.4 Summary of Toxicity Testing Conditions: 

1.4.1 Section 4 provides a summary of conditions for conducting toxicity tests with glochidia and juvenile mussels. Annex A1 provides guidance for conducting water-only laboratory toxicity tests with glochidia and juvenile mussels. Recommended test conditions for conducting these laboratory toxicity tests are based on various published methods and are based on the conditions used to conduct an intra- or inter-laboratory toxicity test with glochidia and juvenile mussels (section 16.5). Female mussels brooding matured glochidia are only available on a seasonal basis. Section 10 describes procedures for collecting brooding female mussels from the field and holding them in the laboratory to obtain glochidia for conducting toxicity tests or for obtaining glochidia to propagate juvenile mussels using a host fish. Juveniles propagated using host-free (in vitro) methods may or may not produce comparable results (section 10.5.4.2).

1.4.2 In the field, mussels may be exposed to contaminants in water, sediment, or food. This standard only addresses effects associated with exposure of mussels to contaminants in water.

1.4.3 Guide E1706 provides guidance for conducting sediment toxicity tests with juvenile mussels. Guide E2122 provides guidance for conducting in situ field exposures with caged mussels.

1.4.4 Results of tests, even those with the same species, using procedures different from those described in Annex A1 may not be comparable. Comparison of results obtained using modified versions of these procedures might provide useful information concerning new concepts and procedures for conducting toxicity tests with aquatic organisms. If tests are conducted with procedures different from those described in this standard, additional tests are required to determine comparability of results. General procedures described in this standard might be useful for conducting tests with other aquatic organisms; however, modifications may be necessary.

1.5 This standard is arranged as follows:

Section

Scope

1

Referenced Documents

2

Terminology

3

Summary of Guide

4

Significance and Use

5

Apparatus

6

Hazards

7

Dilution Water

8

Test Material

9

Test Organisms

10

Quality Assurance and Quality Control

11

Experimental Design

12

Analytical Methodology

13

Calculation of Results

14

Report

15

Precision and Bias

16

Keywords

17

Guidance for Conducting Water-only Toxicity Tests with Early Life Stages of Freshwater Mussels

Annex A1

References

1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7.

1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.