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.

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.

1.11.7 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.

1.11.8 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).

1.12.1 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.