5.1 General:

5.1.1 Sediment provides habitat for many aquatic organisms and is a major repository for many of the more persistent chemicals that are introduced into surface waters. In the aquatic environment, most anthropogenic chemicals and waste materials including toxic organic and inorganic chemicals eventually accumulate in sediment. Mounting evidences exists of environmental degradation in areas where USEPA Water Quality Criteria (WQC; Stephan et al.(66)) are not exceeded, yet organisms in or near sediments are adversely affected Chapman, 1989 (67). The WQC were developed to protect organisms in the water column and were not directed toward protecting organisms in sediment. Concentrations of contaminants in sediment may be several orders of magnitude higher than in the overlying water; however, whole sediment concentrations have not been strongly correlated to bioavailability Burton, 1991 (68). Partitioning or sorption of a compound between water and sediment may depend on many factors including: aqueous solubility, pH, redox, affinity for sediment organic carbon and dissolved organic carbon, grain size of the sediment, sediment mineral constituents (oxides of iron, manganese, and aluminum), and the quantity of acid volatile sulfides in sediment Di Toro et al. 1991(69) Giesy et al. 1988 (70). Although certain chemicals are highly sorbed to sediment, these compounds may still be available to the biota. Chemicals in sediments may be directly toxic to aquatic life or can be a source of chemicals for bioaccumulation in the food chain.

5.1.2 The objective of a sediment test is to determine whether chemicals in sediment are harmful to or are bioaccumulated by benthic organisms. The tests can be used to measure interactive toxic effects of complex chemical mixtures in sediment. Furthermore, knowledge of specific pathways of interactions among sediments and test organisms is not necessary to conduct the tests Kemp et al. 1988, (71). Sediment tests can be used to: (1) determine the relationship between toxic effects and bioavailability, (2) investigate interactions among chemicals, (3) compare the sensitivities of different organisms, (4) determine spatial and temporal distribution of contamination, (5) evaluate hazards of dredged material, (6) measure toxicity as part of product licensing or safety testing, (7) rank areas for clean up, and (8) estimate the effectiveness of remediation or management practices.

5.1.3 A variety of methods have been developed for assessing the toxicity of chemicals in sediments using amphipods, midges, polychaetes, oligochaetes, mayflies, or cladocerans (Test Method E1706, Guide E1525, Guide E1850; Annex A1, Annex A2; USEPA, 2000 (72), EPA 1994b, (73), Environment Canada 1997a, (74), Enviroment Canada 1997b,(75)). Several endpoints are suggested in these methods to measure potential effects of contaminants in sediment including survival, growth, behavior, or reproduction; however, survival of test organisms in 10-day exposures is the endpoint most commonly reported. These short-term exposures that only measure effects on survival can be used to identify high levels of contamination in sediments, but may not be able to identify moderate levels of contamination in sediments (USEPA USEPA, 2000 (72); Sibley et al.1996, (76); Sibley et al.1997a, (77); Sibley et al.1997b, (78); Benoit et al.1997, (79); Ingersoll et al.1998, (80)). Sublethal endpoints in sediment tests might also prove to be better estimates of responses of benthic communities to contaminants in the field, Kembel et al. 1994 (81). Insufficient information is available to determine if the long-term test conducted with Leptocheirus plumulosus (Annex A2) is more sensitive than 10-d toxicity tests conducted with this or other species.

5.1.3.1 The decision to conduct short-term or long-term toxicity tests depends on the goal of the assessment. In some instances, sufficient information may be gained by measuring sublethal endpoints in 10-day tests. In other instances, the 10-day tests could be used to screen samples for toxicity before long-term tests are conducted. While the long-term tests are needed to determine direct effects on reproduction, measurement of growth in these toxicity tests may serve as an indirect estimate of reproductive effects of contaminants associated with sediments (Annex A1).

5.1.3.2 Use of sublethal endpoints for assessment of contaminant risk is not unique to toxicity testing with sediments. Numerous regulatory programs require the use of sublethal endpoints in the decision-making process (Pittinger and Adams, 1997, (82)) including: (1) Water Quality Criteria (and State Standards); (2) National Pollution Discharge Elimination System (NPDES) effluent monitoring (including chemical-specific limits and sublethal endpoints in toxicity tests); (3) Federal Insecticide, Rodenticide and Fungicide Act (FIFRA) and the Toxic Substances Control Act (TSCA, tiered assessment includes several sublethal endpoints with fish and aquatic invertebrates); (4) Superfund (Comprehensive Environmental Responses, Compensation and Liability Act; CERCLA); (5) Organization of Economic Cooperation and Development (OECD, sublethal toxicity testing with fish and invertebrates); (6) European Economic Community (EC, sublethal toxicity testing with fish and invertebrates); and (7) the Paris Commission (behavioral endpoints).

5.1.4 Results of toxicity tests on sediments spiked at different concentrations of chemicals can be used to establish cause and effect relationships between chemicals and biological responses. Results of toxicity tests with test materials spiked into sediments at different concentrations may be reported in terms of an LC50 (median lethal concentration), an EC50 (median effect concentration), an IC50 (inhibition concentration), or as a NOEC (no observed effect concentration) or LOEC (lowest observed effect concentration). However, spiked sediment may not be representative of chemicals associated with sediment in the field. Mixing time Stemmer et al. 1990b, (83), aging ( Landrum et al. 1989, (84), Word et al. 1987, (85), Landrum et al., 1992,(86)), and the chemical form of the material can affect responses of test organisms in spiked sediment tests.

5.1.5 Evaluating effect concentrations for chemicals in sediment requires knowledge of factors controlling their bioavailability. Similar concentrations of a chemical in units of mass of chemical per mass of sediment dry weight often exhibit a range in toxicity in different sediments Di Toro et al. 1990, (87) Di Toro et al. 1991,(69). Effect concentrations of chemicals in sediment have been correlated to interstitial water concentrations, and effect concentrations in interstitial water are often similar to effect concentrations in water-only exposures. The bioavailability of nonionic organic compounds in sediment is often inversely correlated with the organic carbon concentration. Whatever the route of exposure, these correlations of effect concentrations to interstitial water concentrations indicate that predicted or measured concentrations in interstitial water can be used to quantify the exposure concentration to an organism. Therefore, information on partitioning of chemicals between solid and liquid phases of sediment is useful for establishing effect concentrations Di Toro et al. 1991, (69).

5.1.6 Field surveys can be designed to provide either a qualitative reconnaissance of the distribution of sediment contamination or a quantitative statistical comparison of contamination among sites.

5.1.7 Surveys of sediment toxicity are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic data. Statistical correlations may be improved and sampling costs may be reduced if subsamples are taken simultaneously for sediment tests, chemical analyses, and benthic community structure.

5.1.8 Table 2 lists several approaches the USEPA has considered for the assessment of sediment quality USEPA, 1992, (88). These approaches include: (1) equilibrium partitioning, (2) tissue residues, (3) interstitial water toxicity, (4) whole-sediment toxicity and sediment-spiking tests, (5) benthic community structure, (6) effect ranges (for example, effect range median, ERM), and (7) sediment quality triad (see USEPA, 1989a, 1990a, 1990b and 1992b, (89, 90, 91, 92 and Wenning and Ingersoll (2002 (93)) for a critique of these methods). The sediment assessment approaches listed in Table 2 can be classified as numeric (for example, equilibrium partitioning), descriptive (for example, whole-sediment toxicity tests), or a combination of numeric and descriptive approaches (for example, ERM, USEPA, 1992c, (94). Numeric methods can be used to derive chemical-specific sediment quality guidelines (SQGs). Descriptive methods such as toxicity tests with field-collected sediment cannot be used alone to develop numerical SQGs for individual chemicals. Although each approach can be used to make site-specific decisions, no one single approach can adequately address sediment quality. Overall, an integration of several methods using the weight of evidence is the most desirable approach for assessing the effects of contaminants associated with sediment, (Long et al. 1991(95) MacDonald et al. 1996 (96) Ingersoll et al. 1996 (97) Ingersoll et al. 1997 (98), Wenning and Ingersoll 2002 (93)). Hazard evaluations integrating data from laboratory exposures, chemical analyses, and benthic community assessments (the sediment quality triad) provide strong complementary evidence of the degree of pollution-induced degradation in aquatic communities (Burton, 1991 (68), Chapman 1992, 1997 (99, 100).)

5.2 Regulatory Applications—Test Method E1706 provides information on the regulatory applications of sediment toxicity tests.

5.3 Performance-based Criteria:

5.3.1 The USEPA Environmental Monitoring Management Council (EMMC) recommended the use of performance-based methods in developing standards, (Williams, 1993 (101). Performance-based methods were defined by EMMC as a monitoring approach which permits the use of appropriate methods that meet preestablished demonstrated performance standards (11.2).

5.3.2 The USEPA Office of Water, Office of Science and Technology, and Office of Research and Development held a workshop to provide an opportunity for experts in the field of sediment toxicology and staff from the USEPA Regional and Headquarters Program offices to discuss the development of standard freshwater, estuarine, and marine sediment testing procedures (USEPA, 1992a, 1994a (88, 102)). Workgroup participants arrived at a consensus on several culturing and testing methods. In developing guidance for culturing test organisms to be included in the USEPA methods manual for sediment tests, it was agreed that no one method should be required to culture organisms. However, the consensus at the workshop was that success of a test depends on the health of the cultures. Therefore, having healthy test organisms of known quality and age for testing was determined to be the key consideration relative to culturing methods. A performance-based criteria approach was selected in USEPA, 2000 (72) as the preferred method through which individual laboratories could use unique culturing methods rather than requiring use of one culturing method.

5.3.3 This standard recommends the use of performance-based criteria to allow each laboratory to optimize culture methods and minimize effects of test organism health on the reliability and comparability of test results. See Annex A1 and Annex A2 for a listing of performance criteria for culturing or testing.

1.1 This test method covers procedures for testing estuarine or marine organisms in the laboratory to evaluate the toxicity of contaminants associated with whole sediments. Sediments may be collected from the field or spiked with compounds in the laboratory. General guidance is presented in Sections 1 – 15 for conducting sediment toxicity tests with estuarine or marine amphipods. Specific guidance for conducting 10-d sediment toxicity tests with estuarine or marine amphipods is outlined in Annex A1 and specific guidance for conducting 28-d sediment toxicity tests with Leptocheirus plumulosus is outlined in Annex A2.

1.2 Procedures are described for testing estuarine or marine amphipod crustaceans in 10-d laboratory exposures to evaluate the toxicity of contaminants associated with whole sediments (Annex A1; USEPA 1994a (1)). Sediments may be collected from the field or spiked with compounds in the laboratory. A toxicity method is outlined for four species of estuarine or marine sediment-burrowing amphipods found within United States coastal waters. The species are Ampelisca abdita, a marine species that inhabits marine and mesohaline portions of the Atlantic coast, the Gulf of Mexico, and San Francisco Bay; Eohaustorius estuarius, a Pacific coast estuarine species; Leptocheirus plumulosus, an Atlantic coast estuarine species; and Rhepoxynius abronius , a Pacific coast marine species. Generally, the method described may be applied to all four species, although acclimation procedures and some test conditions (that is, temperature and salinity) will be species-specific (Sections 12 and Annex A1). The toxicity test is conducted in 1-L glass chambers containing 175 mL of sediment and 775 mL of overlying seawater. Exposure is static (that is, water is not renewed), and the animals are not fed over the 10-d exposure period. The endpoint in the toxicity test is survival with reburial of surviving amphipods as an additional measurement that can be used as an endpoint for some of the test species (for R. abronius and E. estuarius). Performance criteria established for this test include the average survival of amphipods in negative control treatment must be greater than or equal to 90 %. Procedures are described for use with sediments with pore-water salinity ranging from >0 ^o/_oo to fully marine.

1.3 A procedure is also described for determining the chronic toxicity of contaminants associated with whole sediments with the amphipod Leptocheirus plumulosus in laboratory exposures (Annex A2; USEPA-USACE 2001(2)). The toxicity test is conducted for 28 d in 1-L glass chambers containing 175 mL of sediment and about 775 mL of overlying water. Test temperature is 25° ± 2°C, and the recommended overlying water salinity is 5 ^o/_oo ± 2 ^o/_oo (for test sediment with pore water at 1 ^o/_oo to 10 ^o/_oo ) or 20 ^o/_oo ± 2 ^o/_oo (for test sediment with pore water >10 ^o/_oo ). Four hundred millilitres of overlying water is renewed three times per week, at which times test organisms are fed. The endpoints in the toxicity test are survival, growth, and reproduction of amphipods. Performance criteria established for this test include the average survival of amphipods in negative control treatment must be greater than or equal to 80 % and there must be measurable growth and reproduction in all replicates of the negative control treatment. This test is applicable for use with sediments from oligohaline to fully marine environments, with a silt content greater than 5 % and a clay content less than 85 %.

1.4 A salinity of 5 or 20 ^o/_oo is recommended for routine application of 28-d test with L. plumulosus (Annex A2; USEPA-USACE 2001 (2)) and a salinity of 20 ^o/_oo is recommended for routine application of the 10-d test with E. estuarius or L. plumulosus (Annex A1). However, the salinity of the overlying water for tests with these two species can be adjusted to a specific salinity of interest (for example, salinity representative of site of interest or the objective of the study may be to evaluate the influence of salinity on the bioavailability of chemicals in sediment). More importantly, the salinity tested must be within the tolerance range of the test organisms (as outlined in Annex A1 and Annex A2). If tests are conducted with procedures different from those described in 1.3 or in Table A1.1 (for example, different salinity, lighting, temperature, feeding conditions), additional tests are required to determine comparability of results (1.10). If there is not a need to make comparisons among studies, then the test could be conducted just at a selected salinity for the sediment of interest.

1.5 Future revisions of this standard may include additional annexes describing whole-sediment toxicity tests with other groups of estuarine or marine invertebrates (for example, information presented in Guide E1611 on sediment testing with polychaetes could be added as an annex to future revisions to this standard). Future editions to this standard may also include methods for conducting the toxicity tests in smaller chambers with less sediment (Ho et al. 2000 (3), Ferretti et al. 2002 (4)).

1.6 Procedures outlined in this standard are based primarily on procedures described in the USEPA (1994a (1)), USEPA-USACE (2001(2)), Test Method E1706, and Guides E1391, E1525, E1688, Environment Canada (1992 (5)), DeWitt et al. (1992a (6); 1997a (7)), Emery et al. (1997 (8)), and Emery and Moore (1996 (9)), Swartz et al. (1985 (10)), DeWitt et al. (1989 (11)), Scott and Redmond (1989 (12)), and Schlekat et al. (1992 (13)).

1.7 Additional sediment toxicity research and methods development are now in progress to (1) refine sediment spiking procedures, (2) refine sediment dilution procedures, (3) refine sediment Toxicity Identification Evaluation (TIE) procedures, (4) produce additional data on confirmation of responses in laboratory tests with natural populations of benthic organisms (that is, field validation studies), and (5) evaluate relative sensitivity of endpoints measured in 10- and 28-d toxicity tests using estuarine or marine amphipods. This information will be described in future editions of this standard.

1.8 Although standard procedures are described in Annex A2 of this standard for conducting chronic sediment tests with L. plumulosus, further investigation of certain issues could aid in the interpretation of test results. Some of these issues include further investigation to evaluate the relative toxicological sensitivity of the lethal and sublethal endpoints to a wide variety of chemicals spiked in sediment and to mixtures of chemicals in sediments from contamination gradients in the field (USEPA-USACE 2001 (2)). Additional research is needed to evaluate the ability of the lethal and sublethal endpoints to estimate the responses of populations and communities of benthic invertebrates to contaminated sediments. Research is also needed to link the toxicity test endpoints to a field-validated population model of L. plumulosus that would then generate estimates of population-level responses of the amphipod to test sediments and thereby provide additional ecologically relevant interpretive guidance for the laboratory toxicity test.

1.9 This standard outlines specific test methods for evaluating the toxicity of sediments with A. abdita, E. estuarius, L. plumulosus, and R. abronius. While standard procedures are described in this standard, further investigation of certain issues could aid in the interpretation of test results. Some of these issues include the effect of shipping on organism sensitivity, additional performance criteria for organism health, sensitivity of various populations of the same test species, and confirmation of responses in laboratory tests with natural benthos populations.

1.10 General procedures described in this standard might be useful for conducting tests with other estuarine or marine organisms (for example, Corophium spp., Grandidierella japonica, Lepidactylus dytiscus, Streblospio benedicti), although modifications may be necessary. Results of tests, even those with the same species, using procedures different from those described in the test method may not be comparable and using these different procedures may alter bioavailability. Comparison of results obtained using modified versions of these procedures might provide useful information concerning new concepts and procedures for conducting sediment tests with aquatic organisms. If tests are conducted with procedures different from those described in this test method, additional tests are required to determine comparability of results. General procedures described in this test method might be useful for conducting tests with other aquatic organisms; however, modifications may be necessary.

1.11 Selection of Toxicity Testing Organisms:

1.11.1 The choice of a test organism has a major influence on the relevance, success, and interpretation of a test. Furthermore, no one organism is best suited for all sediments. The following criteria were considered when selecting test organisms to be described in this standard (Table 1 and Guide E1525). Ideally, a test organism should: (1) have a toxicological database demonstrating relative sensitivity to a range of contaminants of interest in sediment, (2) have a database for interlaboratory comparisons of procedures (for example, round-robin studies), (3) be in direct contact with sediment, (4) be readily available from culture or through field collection, (5) be easily maintained in the laboratory, (6) be easily identified, (7) be ecologically or economically important, (8) have a broad geographical distribution, be indigenous (either present or historical) to the site being evaluated, or have a niche similar to organisms of concern (for example, similar feeding guild or behavior to the indigenous organisms), (9) be tolerant of a broad range of sediment physico-chemical characteristics (for example, grain size), and (10) be compatible with selected exposure methods and endpoints (Guide E1525). Methods utilizing selected organisms should also be (11) peer reviewed (for example, journal articles) and (12) confirmed with responses with natural populations of benthic organisms.

ATL = Atlantic Coast, PAC = Pacific Coast, GOM= Gulf of Mexico

1.11.2 Of these criteria (Table 1), a database demonstrating relative sensitivity to contaminants, contact with sediment, ease of culture in the laboratory or availability for field-collection, ease of handling in the laboratory, tolerance to varying sediment physico-chemical characteristics, and confirmation with responses with natural benthic populations were the primary criteria used for selecting A. abdita, E. estuarius, L. plumulosus, and R. abronius for the current edition of this standard for 10-d sediment tests (Annex A1). The species chosen for this method are intimately associated with sediment, due to their tube- dwelling or free-burrowing, and sediment ingesting nature. Amphipods have been used extensively to test the toxicity of marine, estuarine, and freshwater sediments (Swartz et al., 1985 (10); DeWitt et al., 1989 (11); Scott and Redmond, 1989 (12); DeWitt et al., 1992a (6); Schlekat et al., 1992 (13)). The selection of test species for this standard followed the consensus of experts in the field of sediment toxicology who participated in a workshop entitled “Testing Issues for Freshwater and Marine Sediments”. The workshop was sponsored by USEPA Office of Water, Office of Science and Technology, and Office of Research and Development, and was held in Washington, D.C. from 16-18 September 1992 (USEPA, 1992 (15)). Of the candidate species discussed at the workshop, A. abdita, E. estuarius, L. plumulosus, and R. abronius best fulfilled the selection criteria, and presented the availability of a combination of one estuarine and one marine species each for both the Atlantic (the estuarine L. plumulosus and the marine A. abdita ) and Pacific (the estuarine E. estuarius and the marine R. abronius) coasts. Ampelisca abdita is also native to portions of the Gulf of Mexico and San Francisco Bay. Many other organisms that might be appropriate for sediment testing do not now meet these selection criteria because little emphasis has been placed on developing standardized testing procedures for benthic organisms. For example, a fifth species, Grandidierella japonica was not selected because workshop participants felt that the use of this species was not sufficiently broad to warrant standardization of the method. Environment Canada (1992 (5)) has recommended the use of the following amphipod species for sediment toxicity testing: Amphiporeia virginiana, Corophium volutator , Eohaustorius washingtonianus, Foxiphalus xiximeus, and Leptocheirus pinguis. A database similar to those available for A. abdita, E. estuarius, L. plumulosus, and R. abronius must be developed in order for these and other organisms to be included in future editions of this standard.

1.11.3 The primary criterion used for selecting L. plumulosus for chronic testing of sediments was that this species is found in both oligohaline and mesohaline regions of estuaries on the East Coast of the United States and is tolerant to a wide range of sediment grain size distribution (USEPA-USACE 2001 (2), Annex Annex A2). This species is easily cultured in the laboratory and has a relatively short generation time (that is, about 24 d at 23°C, DeWitt et al. 1992a(6)) that makes this species adaptable to chronic testing (Section 12).

1.11.4 An important consideration in the selection of specific species for test method development is the existence of information concerning relative sensitivity of the organisms both to single chemicals and complex mixtures. Several studies have evaluated the sensitivities of A. abdita, E. estuarius, L. plumulosus, or R. abronius, either relative to one another, or to other commonly tested estuarine or marine species. For example, the sensitivity of marine amphipods was compared to other species that were used in generating saltwater Water Quality Criteria. Seven amphipod genera, including Ampelisca abdita and Rhepoxynius abronius, were among the test species used to generate saltwater Water Quality Criteria for 12 chemicals. Acute amphipod toxicity data from 4-d water-only tests for each of the 12 chemicals was compared to data for (1) all other species, (2) other benthic species, and (3) other infaunal species. Amphipods were generally of median sensitivity for each comparison. The average percentile rank of amphipods among all species tested was 57 %; among all benthic species, 56 %; and, among all infaunal species, 54 %. Thus, amphipods are not uniquely sensitive relative to all species, benthic species, or even infaunal species (USEPA 1994a (1)). Additional research may be warranted to develop tests using species that are consistently more sensitive than amphipods, thereby offering protection to less sensitive groups.

1.11.5 Williams et al. (1986 (16)) compared the sensitivity of the R. abronius 10-d whole sediment test, the oyster embryo (Crassostrea gigas) 48-h abnormality test, and the bacterium (Vibrio fisheri) 1-h luminescence inhibition test (that is, the Microtox2 test) to sediments collected from 46 contaminated sites in Commencement Bay, WA. Rhepoxynius abronius were exposed to whole sediment, while the oyster and bacterium tests were conducted with sediment elutriates and extracts, respectfully. Microtox2 was the most sensitive test, with 63 % of the sites eliciting significant inhibition of luminescence. Significant mortality of R. abronius was observed in 40 % of test sediments, and oyster abnormality occurred in 35 % of sediment elutriates. Complete concordance (that is, sediments that were either toxic or not-toxic in all three tests) was observed in 41 % of the sediments. Possible sources for the lack of concordance at other sites include interspecific differences in sensitivity among test organisms, heterogeneity in contaminant types associated with test sediments, and differences in routes of exposure inherent in each toxicity test. These results highlight the importance of using multiple assays when performing sediment assessments.

1.11.6 Several studies have compared the sensitivity of combinations of the four amphipods to sediment contaminants. For example, there are several comparisons between A. abdita and R. abronius, between E. estuarius and R. abronius, and between A. abdita and L. plumulosus. There are fewer examples of direct comparisons between E. estuarius and L. plumulosus , and no examples comparing L. plumulosus and R. abronius. There is some overlap in relative sensitivity from comparison to comparison within each species combination, which appears to indicate that all four species are within the same range of relative sensitivity to contaminated sediments.

1.11.6.1 Word et al. (1989 (17)) compared the sensitivity of A. abdita and R. abronius to contaminated sediments in a series of experiments. Both species were tested at 15°C. Experiments were designed to compare the response of the organism rather than to provide a comparison of the sensitivity of the methods (that is, Ampelisca abdita would normally be tested at 20°C). Sediments collected from Oakland Harbor, CA, were used for the comparisons. Twenty-six sediments were tested in one comparison, while 5 were tested in the other. Analysis of results using Kruskal Wallace rank sum test for both experiments demonstrated that R. abronius exhibited greater sensitivity to the sediments than A. abdita at 15°C. Long and Buchman (1989 (18)) also compared the sensitivity of A. abdita and R. abronius to sediments from Oakland Harbor, CA. They also determined that A. abdita showed less sensitivity than R. abronius, but they also showed that A. abdita was less sensitive to sediment grain size factors than R. abronius.

1.11.6.2 DeWitt et al. (1989 (11)) compared the sensitivity of E. estuarius and R. abronius to sediment spiked with fluoranthene and field-collected sediment from industrial waterways in Puget Sound, WA, in 10-d tests, and to aqueous cadmium (CdCl₂) in a 4-d water-only test. The sensitivity of E. estuarius was from two (to spiked-spiked sediment) to seven (to one Puget Sound, WA, sediment) times less sensitive than R. abronius in sediment tests, and ten times less sensitive to CdCl₂ in the water-only test. These results are supported by the findings of Pastorok and Becker (1990 (19)) who found the acute sensitivity of E. estuarius and R. abronius to be generally comparable to each other, and both were more sensitive than Neanthes arenaceodentata (survival and biomass endpoints), Panope generosa (survival), and Dendraster excentricus (survival).

1.11.6.3 Leptocheirus plumulosus was as sensitive as the freshwater amphipod Hyalella azteca to an artificially created gradient of sediment contamination when the latter was acclimated to oligohaline salinity (that is, 6 ^o/_oo ; McGee et al., 1993 (20)). DeWitt et al. (1992b (21)) compared the sensitivity of L. plumulosus with three other amphipod species, two mollusks, and one polychaete to highly contaminated sediment collected from Baltimore Harbor, MD, that was serially diluted with clean sediment. Leptocheirus plumulosus was more sensitive than the amphipods Hyalella azteca and Lepidactylus dytiscus and exhibited equal sensitivity with E. estuarius. Schlekat et al. (1995 (22)) describe the results of an interlaboratory comparison of 10-d tests with A. abdita, L. plumulosus and E. estuarius using dilutions of sediments collected from Black Rock Harbor, CT. There was strong agreement among species and laboratories in the ranking of sediment toxicity and the ability to discriminate between toxic and non-toxic sediments.

1.11.6.4 Hartwell et al. (2000 (23)) evaluated the response of Leptocheirus plumulosus (10-d survival or growth) to the response of the amphipod Lepidactylus dytiscus (10-d survival or growth), the polychaete Streblospio benedicti (10-d survival or growth), and lettuce germination (Lactuca sativa in 3-d exposure) and observed that L. plumulosus was relatively insensitive compared to the response of either L. dytiscus or S. benedicti in exposures to 4 sediments with elevated metal concentrations.

1.11.6.5 Ammonia is a naturally occurring compound in marine sediment that results from the degradation of organic debris. Interstitial ammonia concentrations in test sediment can range from <1 mg/L to in excess of 400 mg/L (Word et al., 1997 (24)). Some benthic infauna show toxicity to ammonia at concentrations of about 20 mg/L (Kohn et al., 1994 (25)). Based on water-only and spiked-sediment experiments with ammonia, threshold limits for test initiation and termination have been established for the L. plumulosus chronic test. Smaller (younger) individuals are more sensitive to ammonia than larger (older) individuals (DeWitt et al., 1997a(7), b (26). Results of a 28-d test indicated that neonates can tolerate very high levels of pore-water ammonia (>300 mg/L total ammonia) for short periods of time with no apparent long-term effects (Moore et al., 1997 (27)). It is not surprising L. plumulosus has a high tolerance for ammonia given that these amphipods are often found in organic rich sediments in which diagenesis can result in elevated pore-water ammonia concentrations. Insensitivity to ammonia by L. plumulosus should not be construed as an indicator of the sensitivity of the L. plumulosus sediment toxicity test to other chemicals of concern.

1.11.7 Limited comparative data is available for concurrent water-only exposures of all four species in single-chemical tests. Studies that do exist generally show that no one species is consistently the most sensitive.

1.11.7.1 The relative sensitivity of the four amphipod species to ammonia was determined in ten-d water only toxicity tests in order to aid interpretation of results of tests on sediments where this toxicant is present (USEPA 1994a (1)). These tests were static exposures that were generally conducted under conditions (for example, salinity, photoperiod) similar to those used for standard 10-d sediment tests. Departures from standard conditions included the absence of sediment and a test temperature of 20°C for L. plumulosus, rather than 25°C as dictated in this standard. Sensitivity to total ammonia increased with increasing pH for all four species. The rank sensitivity was R. abronius = A. abdita > E. estuarius > L. plumulosus. A similar study by Kohn et al. (1994 (25)) showed a similar but slightly different relative sensitivity to ammonia with A. abdita > R. abronius = L. plumulosus > E. estuarius.

1.11.7.2 Cadmium chloride has been a common reference toxicant for all four species in 4-d exposures. DeWitt et al. (1992a (6)) reports the rank sensitivity as R. abronius > A. abdita > L. plumulosus > E. estuarius at a common temperature and salinity of 15°C and 28 ^o/_oo . A series of 4-d exposures to cadmium that were conducted at species-specific temperatures and salinities showed the following rank sensitivity: A. abdita = L. plumulosus = R. abronius > E. estuarius (USEPA 1994a (1)).

1.11.7.3 Relative species sensitivity frequently varies among contaminants; consequently, a battery of tests including organisms representing different trophic levels may be needed to assess sediment quality (Craig, 1984 (28); Williams et al. 1986 (16); Long et al., 1990 (29); Ingersoll et al., 1990 (30); Burton and Ingersoll, 1994 (31)). For example, Reish (1988 (32)) reported the relative toxicity of six metals (arsenic, cadmium, chromium, copper, mercury, and zinc) to crustaceans, polychaetes, pelecypods, and fishes and concluded that no one species or group of test organisms was the most sensitive to all of the metals.

1.11.8 The sensitivity of an organism is related to route of exposure and biochemical response to contaminants. Sediment-dwelling organisms can receive exposure from three primary sources: interstitial water, sediment particles, and overlying water. Food type, feeding rate, assimilation efficiency, and clearance rate will control the dose of contaminants from sediment. Benthic invertebrates often selectively consume different particle sizes (Harkey et al. 1994 (33)) or particles with higher organic carbon concentrations which may have higher contaminant concentrations. Grazers and other collector-gatherers that feed on aufwuchs and detritus may receive most of their body burden directly from materials attached to sediment or from actual sediment ingestion. In some amphipods (Landrum, 1989 (34)) and clams (Boese et al., 1990 (35)) uptake through the gut can exceed uptake across the gills for certain hydrophobic compounds. Organisms in direct contact with sediment may also accumulate contaminants by direct adsorption to the body wall or by absorption through the integument (Knezovich et al. 1987 (36)).

1.11.9 Despite the potential complexities in estimating the dose that an animal receives from sediment, the toxicity and bioaccumulation of many contaminants in sediment such as Kepone®, fluoranthene, organochlorines, and metals have been correlated with either the concentration of these chemicals in interstitial water or in the case of non-ionic organic chemicals, concentrations in sediment on an organic carbon normalized basis (Di Toro et al. 1990 (37); Di Toro et al. 1991 (38)). The relative importance of whole sediment and interstitial water routes of exposure depends on the test organism and the specific contaminant (Knezovich et al. 1987 (36)). Because benthic communities contain a diversity of organisms, many combinations of exposure routes may be important. Therefore, behavior and feeding habits of a test organism can influence its ability to accumulate contaminants from sediment and should be considered when selecting test organisms for sediment testing.

1.11.10 The use of A. abdita, E. estuarius, R. abronius, and L. plumulosus in laboratory toxicity studies has been field validated with natural populations of benthic organisms (Swartz et al. 1994 (39) and Anderson et al. 2001 (14) for E. estuarius, Swartz et al. 1982 (40) and Anderson et al. 2001 (14) for R. abronius, McGee et al. 1999 (41) and McGee and Fisher 1999 (42) for L. plumulosus).

1.11.10.1 Data from USEPA Office of Research and Development's Environmental Monitoring and Assessment program were examined to evaluate the relationship between survival of Ampelisca abdita in sediment toxicity tests and the presence of amphipods, particularly ampeliscids, in field samples. Over 200 sediment samples from two years of sampling in the Virginian Province (Cape Cod, MA, to Cape Henry, VA) were available for comparing synchronous measurements of A. abdita survival in toxicity tests to benthic community enumeration. Although species of this genus were among the more frequently occurring taxa in these samples, ampeliscids were totally absent from stations that exhibited A. abdita test survival <60 % of that in control samples. Additionally, ampeliscids were found in very low densities at stations with amphipod test survival between 60 and 80 % (USEPA 1994a (1)). These data indicate that tests with this species are predictive of contaminant effects on sensitive species under natural conditions.

1.11.10.2 Swartz et al. (1982 (40)) compared sensitivity of R. abronius to sediment collected from sites in Commencement Bay, WA, to benthic community structure at each site. Mortality of R. abronius was negatively correlated with amphipod density, and phoxocephalid amphipods were ubiquitously absent from the most contaminated areas.

1.11.10.3 Sediment toxicity to amphipods in 10-d toxicity tests, field contamination, and field abundance of benthic amphipods were examined along a sediment contamination gradient of DDT (Swartz et al. 1994 (39)). Survival of E. estuarius and R. abronius in laboratory toxicity tests was positively correlated to abundance of amphipods in the field and along with the survival of H. azteca, was negatively correlated to DDT concentrations. The threshold for 10-d sediment toxicity in laboratory studies was about 300 ug DDT (+metabolites)/g organic carbon. The threshold for abundance of amphipods in the field was about 100 ug DDT (+metabolites)/g organic carbon. Therefore, correlations between toxicity, contamination, and biology indicate that acute 10-d sediment toxicity tests can provide reliable evidence of biologically adverse sediment contamination in the field.

1.11.10.4 As part of a comprehensive sediment quality assessment in Baltimore Harbor, MD, McGee et al. (1999 (41)) conducted 10-d toxicity tests with L. plumulosus. Negative relationships were detected between amphipod survival and concentrations of select sediment-associated contaminants, whereas a very strong positive association existed between survival in laboratory exposures and field density of L. plumulosus at test sites. A field validation study of the 10- and 28-d L. plumulosus tests by McGee and Fisher (1999 (42)) in Baltimore Harbor, also indicated good agreement between acute toxicity, sediment associated contaminants and responses of the in situ benthic community. In this study, the chronic 28-d test was less sensitive to sediment contamination than the acute 10-d test; however, the feeding regime used in this evaluation is different than the one currently recommended in Annex A2 and may have influenced the test results. Field validation studies with the revised 28-d test outlined in Annex A2 have not been conducted.

1.12 Chronic Sediment Methods with Leptocheirus plumulosus:

1.12.1 Most standard whole sediment toxicity tests have been developed to produce a lethality endpoint (survival/mortality) with potential for a sublethal endpoint (reburial) in some species (USEPA 1994a (1), USEPA-USACE 2001 (2)). Methods that measure sublethal effects have not been available or have not been routinely used to evaluate sediment toxicity in marine or estuarine sediments (Scott and Redmond, 1989 (12); Green and Chandler, 1996 (43); Levin et al., 1996 (44); Ciarelli et al., 1998 (45); Meador and Rice, 2001 (46)). Most assessments of contaminated sediment rely on short-term lethality tests (for example, ≤10 d; USEPA-USACE, 1991 (47); 1998 (48)). Short-term lethality tests are useful in identifying “hot spots” of sediment contamination, but might not be sensitive enough to evaluate moderately contaminated areas. However, sediment quality assessments using sublethal responses of benthic organisms, such as effects on growth and reproduction, have been used to successfully evaluate moderately contaminated areas (Ingersoll et al., 1998 (49); Kemble et al., 1994 (50); McGee et al., 1995 (51); Scott, 1989 (52)). The 28-d toxicity test with Leptocheirus plumulosus has two sublethal endpoints: growth and reproduction. These sublethal endpoints have potential to exhibit a toxic response from chemicals that otherwise might not cause acute effects or significant mortality in a test. Sublethal response to chronic exposure is also valuable for population modeling of contaminant effects. These data can be used for population-level risk assessments of benthic pollutant effects.

1.12.2 An evaluation of the distribution of L. plumulosus in Chesapeake Bay indicates that its distribution is negatively correlated with the degree of sediment contamination (Pfitzenmeyer, 1975 (53); Reinharz, 1981 (54)). A field validation study of the 10- and 28-d L. plumulosus tests by McGee and Fisher (1999 (42)) in Baltimore Harbor, indicated good agreement between acute toxicity, sediment associated contaminants and responses of the in situ benthic community. In this study, the chronic 28-d test was less sensitive to sediment contamination than the acute 10-d test and therefore had a poorer association between sediment contaminants and benthic community health. It should be noted that the feeding regime used in this evaluation is different than the one currently recommended in Annex A2 and may have influenced the test results. Field validation studies with the revised 28-d test have not been conducted.

1.13 Limitations—While some safety considerations are included in this standard, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct sediment tests.

1.14 This standard is arranged as follows:

Section

Referenced Documents

2

Terminology

3

Summary of Standard

4

Significance and Use

5

Interferences

6

Reagents and Materials

7

Hazards

8

Facilities, Equipment, and Supplies

9

Sample Collection, Storage, Manipulation, and Characterization

10

Quality Assurance and Quality Control

11

Collection, Culturing, and Maintaining Test Organisms

12

Calculation

13

Report

14

Precision and Bias

15

Keywords

16

Annexes

A1. Procedure For Conducting A 10-d Sediment Survival Test With the Amphipods Ampelisca abdita, Eohaustorius estuarius, Leptocheirus plumulosus, , or Rhepoxynius abronius

Annex A1

A2. Procedure For Conducting A Leptocheirus plumulosus 28-d Sediment For Measuring Sublethal Effects of Sediment-Associated Contaminants.

Annex A2

References

1.15 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 8.