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 USEPA Water Quality Criteria for aquatic organisms (Guide E1241).

5.5 Results of acute toxicity tests (for example, 24- to 96-h tests) might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most predictions take into account the results of acute toxicity tests, and so the usefulness of the results of a chronic toxicity test is greatly increased by reporting also the results of an acute toxicity test conducted with a similar life stage of the same species under the same conditions (Guide E729).

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

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

5.8 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.

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

5.8.2 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.

5.8.3 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.

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

5.8.5 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. Future revisions to this standard may describe methods for conducting toxicity tests with (1) adult freshwater mussels and (2) contaminated sediments using various life stages of freshwater mussels.

5.8.6 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 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 exposures (Annex A1). Future revisions to this standard may describe methods for conducting toxicity tests with (1) adult freshwater mussels and (2) contaminated sediments using various life stages of freshwater mussels.

1.2 Many factors are cited as potentially contributing to the decline of freshwater mussel populations in North America. Of the nearly 300 taxa of freshwater mussels in North America, 70 species (23 %) are listed as endangered or threatened and another 40 species (14 %) are candidates for possible listing (Williams et al 1993 (1); Neves 1997, 2004 (2, 3)).² Habitat alteration, introduction of exotic species, over-utilization, disease, predation and pollution are considered causal or contributing factors in many areas of the United States (Neves et al 1997) (4). Over the past decade, there have been over 75 published studies conducted that have evaluated the role of contaminants in the decline of populations of freshwater mussels (Kernaghan et al 2005) (5). In these studies, early life stages of mussels of several species are highly sensitive to some metals and ammonia in water exposures when compared to many of the most sensitive species of other invertebrates, fish, or amphibians that are commonly used to establish U.S. Environmental Protection Agency Water Quality Criteria (WQC; Augspurger et al 2003 (6) , Keller et al 2005 (7), Kernaghan et al 2005 (5); USGS (2005a,b) (8, 9) section 1.5). Importantly, results of these previous studies indicate WQC for individual chemicals established for the protection of aquatic organisms may not be adequately protective of sensitive stages of freshwater mussels.

1.3 Summary of Life History of Freshwater Mussels:

1.3.1 Freshwater mussels are bivalve mollusks belonging to the family Unionidae or Margaritiferidae (section 10.1). Adults are sedentary animals, spending their entire lives partially or completely burrowed in the bottoms of streams, rivers, or lakes. Adult mussels are filter feeders, using their gills to remove suspended particles from the water column. The microscopic, juvenile stage uses foot (pedal) feeding to some degree for the first several months of their lives, feeding on depositional materials in pore water of sediment, including bacteria, algae, and detritus. Freshwater mussels have an unusual and complex mode of reproduction, which includes a brief, obligatory parasitic stage on fish or other host organisms called glochidia (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. 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. Encystment on the host occurs by overgrowth of host tissue. Metamorphosis of juvenile mussels on the fish host occurs within days or weeks, depending on species and temperature. Host fish specificity varies among mussels. While some mussel species appear to require a single host organism, other species can transform their glochidia into juvenile mussels on several species of host fish. Following proper host infestation, glochidia transform into microscopic juveniles and excyst (drop off) and settle into suitable habitat to survive. The transformation of glochidia to juveniles results in the development of internal organs necessary for self-sustained existence as a benthic organism.

1.3.3 Newly-transformed juvenile mussels have a life style different from adult mussels. Transformed juvenile mussels may be at the sediment-water interface or may burrow several centimeters into sediment and rely on water percolating between substrate particles of sediment for food and oxygen. Newly-transformed juvenile mussels feed using ciliary currents on the foot and mantle. Older juvenile and adult mussels likely use different food types when living in different microenvironments. Given that glochidia and juvenile mussels are ecologically and physiologically different from adult mussels, protection of habitat quality of adult life stages may not be protective of glochidia or juvenile life stages of freshwater mussels. Distributions of adult mussels are dependent both on the presence of host fish and on microhabitat conditions. Efforts to assess effects of contaminants on mussels need to evaluate potential exposure to host fish in addition to exposure to each unique life stage 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 toxicity tests with glochidia and juvenile mussels. Recommended test conditions for conducting these toxicity tests are based on various published methods outlined in Table A1.1 and Table A1.4 in Annex A1 and are based on the conditions used to conduct an inter-laboratory toxicity test with glochidia and juvenile mussels (section 16.5). Glochidia and juvenile mussels are only available on a seasonal basis. Section 10 describes procedures for collecting adult female mussels from the field to obtain glochidia for conducting toxicity tests or for obtaining glochidia to propagate juvenile mussels using a host organism.

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 E724 describes procedures for conducting acute 48-h toxicity tests with embryos or larvae of saltwater bivalve mollusks. Endpoints measured in Guide E724 include survival or shell deposition. Procedures outlined in Guide E724 may be useful in helping to design studies for conducting toxicity tests with freshwater mussels as outlined in Annex A1.

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 Summary of Results of Toxicity Tests Conducted with Freshwater Mussels:

1.5.1 Keller et al (2005) (7) summarized results of acute laboratory toxicity tests conducted with glochidia and juvenile mussels described in 16 published studies. Freshwater mussels tended to be less sensitive in exposures to some pesticides and other organic compounds compared to other commonly-tested aquatic organisms. In contrast, Keller et al (2005) (7) concluded that U.S. Environmental Protection Agency (USEPA) water quality criteria (WQC) for some metals and ammonia may not be protective of freshwater mussels.

1.5.2 Augspurger et al (2003) (6) evaluated ammonia toxicity data generated for glochidia and juvenile of freshwater mussels in laboratory toxicity tests. Specifically, these toxicity data were used to estimate concentrations that would not likely be harmful to mussels in acute and chronic exposures and were used to evaluate the protectiveness of the WQC for ammonia. Results of acute toxicity tests (24 to 96 h) for 10 species in 8 genera were used to calculate genus mean acute values (GMAVs) ranging from 2.56 to 8.97 mg/L (total ammonia as N at pH 8 at 25°C). The freshwater mussels are at the sensitive end of the range when added to the GMAVs from the database used to derive the acute WQC for ammonia. Recalculation of the criteria maximum concentration (CMC) including these mussel data resulted in a CMC 75 % lower than the CMC of 5.62 mg/L total ammonia as N at pH 8 at 25°C (for application when salmonids absent). No chronic ammonia toxicity data (for example, 21 to 28-d exposures) were available for freshwater mussels; however, when a range of acute to chronic ratios were used to estimate a criteria continuous concentration (CCC), the estimated CCC for mussels was 20 to 75 % less than the CCC of 1.24 mg/L total ammonia as N at pH 8 and 25°C. Hence, Augspurger et al (2003) (6) concluded that the acute and chronic WQC for ammonia may not be protective of freshwater mussels.

1.5.3 Milam et al (2005) (10) conducted a series of 24-h acute toxicity tests with glochidia of six freshwater mussel species, Leptodea fragilis, Utterbackia imbecillis , Lampsilis cardium, Lampsilis siliquoidea, Megalonaias nervosa, and Ligumia subrostrata, and with two commonly-tested organisms, Ceriodaphnia dubia and Daphnia magna. Chemicals selected for testing (carbaryl, copper, 4-nonylphenol, pentachlorophenol, permethrin, and 2,4-dichlorophenoxyacetic acid [2,4-D]) represented different chemical classes and different toxic modes of action (Dwyer et al 2005a,b) (11, 12). No single chemical elicited consistently high or low toxicity; however, carbaryl and 2,4-D were generally the least toxic to the species tested. Milam et al (2005) (10) concluded that the toxicity data generated with C. dubia and D. magna were relatively protective of the range of sensitivities exhibited by glochidia of the mussels species tested. However, toxicity data generated with the commonly-tested U. imbecillis were not always protective of the range of sensitivities exhibited by the other mussel species tested.

1.6 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

1.7 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.

1.8 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 and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7.