Analytical Methods

Toxicity tests with whole sediment are designed to determine whether the dredged material is likely to produce unacceptable adverse effects on benthic organisms. In benthic toxicity tests, the test animals are exposed to the whole sediment and any effects recorded.

11.2.1 Species Selection

Species representing three life history strategies are recommended for use in the whole sediment toxicity tests, one each representing a filter feeder, deposit feeder and a burrowing organism where possible (Table 11-2). The rationale for testing more than a single species is to cover the range of differing species sensitivities and to be environmentally protective. No single species is adequately protective of the broad range of possible chemical contaminants nor of the equally broad range of possible biological responses. Of the species tested, at least one sensitive benchmark (starred) species needs to be used in all cases except as provided below; however, this does not preclude the use of benchmark species representative of all three required categories. If only two different species are being tested they should, together, cover the following three life history strategies: filter feeder, deposit feeder, burrower. Since amphipods are excellent organisms for short term toxicity, they are recommended as one of the species to be tested. Non-benchmark species listed in Table 11-2 can be used if a summary of test conditions and test acceptability criteria similar to the starred benchmark species are established and data from reference toxicity tests (see Appendix G.2.10.5.2) are provided on the sensitivity of the species. In order to be technically justified, species proposed for use regionally and not listed in Table 11-2 need to meet the species characteristics criteria provided later in this section and proponents need to provide the following supporting information:

• data from toxicity tests using a set of reference chemicals with differing modes of action demonstrating that the proposed species is as sensitive or more sensitive than the species in Table 11-2
• summary of test conditions and test acceptability criteria.

If species proposed for use regionally are tested in conjunction with a benchmark species, the above supporting information is desirable but not needed. However, if the region substitutes all species, the above information is needed.

Examples are not presented in order of importance; however, the asterisks indicate sensitive recommended benchmark species. Benchmark species comprise a substantial data base, represent the sensitive range of a variety of ecosystems, and provide comparative data on the relative sensitivity of local test species. Other species may be designated in future as benchmark species by EPA and the USACE when the data on their response to contaminants are adequate. Only benthic species should be tested. Although sediment dwellers are preferable, intimate contact with sediment is acceptable. Note that testing with all recommended taxa is not required; however, at least one starred amphipod taxon must be tested.

 [f = filter feeder; d = deposit feeder; b = burrower]. Note that A. abdita, L. plumulosus, C. tentans, and H. limbata are not direct filter feeders, but are suspension feeders.

 a -- These species can also be used in sublethal, chronic testing (methods for such testing are available but not detailed in this manual).

 b -- This species can be used in estuarine waters down to appropriate low levels of salinity (see Appendix E).

 For the purposes of this manual, related to the tolerances of the test animals, (F) = Freshwater, salinity less than or equal to 1‰ (N) = Near Coastal, salinity greater than or equal to 25‰ (E) = Estuarine, salinity 1-25‰. It is recognized that the commonly accepted salinity range for estuaries is 1-35‰ and near coastal water is usually greater than 30‰ salinity.

Benthic organisms are used to evaluate the potential benthic impact of dredged material disposal. Testing of contaminated sediments (e.g., Word et al., 1989; Gentile et al., 1988; Rogerson et al., 1985) and regulatory program experience since 1977 under the Marine Protection, Research, and Sanctuaries Act and the Clean Water Act have shown that different species have various degrees of sensitivity to the physical and chemical composition of sediments.

To accurately evaluate potential benthic impact, appropriately sensitive toxicity test species should be related as closely as possible, both phylogenetically and ecologically, to benthic organisms in the disposal site area. Commercially important but possibly less sensitive benthic species in the vicinity of the disposal site may also be considered for testing.

Sediment grain size is likely to vary substantially between the dredged material, the reference sediment, and the control sediment. If candidate test species are overly sensitive to the different grain sizes (for instance, excessive mortality in the reference sediments attributable to grain size and not to other factors), either this must be taken into account (e.g., DeWitt et al., 1988) or other, more grain-size tolerant species should be considered for the project.

Final selection of test species for a particular dredged material disposal project should be made in consultation with regional regulatory and scientific personnel. Two phylogenetically and ecologically different species are recommended to account for different sensitivities to contaminants. The following is a list, not necessarily in order of importance, of characteristics to consider for species selection:

• readily available year-round
• preferably ingest sediments
• tolerate grain sizes of dredged material and control and reference sediments equally well or differences should be accounted for
• give consistent, reproducible response to toxicants
• tolerate handling and laboratory conditions
• related phylogenetically and/or by ecological requirements to species characteristic of the benthic environment of the disposal site area in the season of the proposed disposal
• standardized test protocols are available
• important ecologically, economically, and/or recreationally
• appropriately sensitive.

Infaunal amphipods are excellent organisms for short term toxicity tests with whole sediment (Swartz et al., 1979, 1985; Mearns and Word, 1982; Rogerson et al., 1985; Nebeker et al., 1984; Gentile et al., 1988; Scott and Redmond, 1989; Word et al., 1989; Burton, 1991), and are strongly recommended as appropriate test species for acute toxicity bioassays in marine/estuarine/fresh waters. Guidance on available testing procedures (static, 10-d exposures) provided in ASTM (1994d,e) may be followed on all points that do not conflict with this manual. Infaunal amphipods are:

• sensitive
• readily available
• as a group, tolerant of a wide range of grain sizes and laboratory exposure conditions
• ecologically relevant to most dredged material disposal sites.

The identity of all species should be verified by experienced taxonomists, particularly for animals collected in the field. If the toxicity test animals are also to be used in estimating bioaccumulation potential, the factors discussed in Section 12.1.1 for species selection should also be considered.

11.2.2 Laboratory Procedures

General Test Procedures

Acceptable water quality parameters during testing include but are not necessarily restricted to:

• the correct temperature and pH range
• adequate oxygen levels
• proper lighting
• the correct salinity range (near coastal and estuarine organisms)
• the correct hardness range (fresh water organisms)
• the absence of, or insignificant concentrations of, toxicants such as ammonia.

Amphipod and other small organism tests are often, but not always, conducted in 1 L containers understatic conditions (Appendix E). Static renewal or even flow-through methods such as those described by Redmond et al. (1989) or Benoit et al. (1993) may be required for certain tests or where static non-renewal conditions would result in unacceptable build-up of, for instance, ammonia and/or sulfides (see second and third paragraphs, Ammonia and Sulfide toxicity, this section).

Before use, all glassware should be washed with detergent, rinsed with acetone, five times with tap water, placed in a clean 10% HCl acid bath for a minimum of 4 h, rinsed five times with tap water, and then thoroughly flushed with either distilled or deionized water. Equipment and facilities must provide acceptable lighting requirements and temperature control. An environmental incubator or a water-bath system that allows temperature control within +/-1^oC is recommended.

Dilution water should not be stressful to the test organisms, and should be stable throughout the exposure period. Salinity for marine/estuarine organisms should be stable within +/-2‰ and, for all organisms, temperature should be stable within +/-2^oC throughout the exposure period. Dissolved oxygen concentration should not be allowed to fall below an absolute minimum of 40% saturation for warm water species and 60% for cold water species. The flow to the exposure chamber should be directed to achieve good mixing without disturbing the sediment on the bottom of the chamber.

A minimum of five replicate exposure chambers for the dredged material, reference, and control is recommended. The standard test duration is 10 d.

The quantity of sediment needed depends on the size of the exposure chambers. The sediment should be deep enough to meet the biological needs of the test organisms, i.e., allow organisms to burrow in their normal position, etc. Overcrowding of organisms must be avoided.

Prior to use in toxicity tests, sediments must be thoroughly homogenized. Very small amounts of clean diluent water may be added to facilitate mixing. If separation into liquid and solid phases occurs in posthomogenization storage, remixing will be required prior to usage.

The reference and control sediments, as well as the dredged material being tested, may contain live organisms. If necessary, macrobenthic organisms can be removed by press-sieving the sediments through an appropriately sized screen immediately prior to testing. The material remaining on the screen should be noted and discarded.

The experimental procedure described in ASTM (1994d) should be followed for preparing the exposure chambers for amphipod toxicity tests. For larger exposure chambers, sediment should be placed on the bottom of the exposure chamber and covered with clean diluent water; any sediment suspended during placement should be allowed to settle for 24 h before introducing the test organisms. In continuous-flow tests, the flow should be established after most of the suspended sediment has settled, usually 12 to 24 h, but at least 1 h before introducing the test organisms.

During the exposure period, daily records should be kept of obvious mortalities, emergence of infaunalorganisms, formation of tubes or burrows, and any other or unusual behavior. Daily records of water quality (e.g., dissolved oxygen, salinity (if appropriate), ammonia, temperature, pH) should be maintained using test containers appropriate for this purpose. In flow-through or static-renewal systems, water quality may be kept within acceptable bounds by increasing the flow rate or frequency of water changes.

After the exposure period, live organisms are removed to clean diluent water, which may include sieving the sediments, and then counted. If greater than acceptable mean mortality occurs in the control, as defined in the procedures for proper conduct of that test, the test must be repeated. Organisms which show any response to gentle probing of sensitive parts or gentle swirling of the water should be considered alive. Sediment dwellers (e.g., amphipods) not recovered at the end of the test have to be considered dead. If organisms from these toxicity tests are to be used in estimating bioaccumulation potential, the survivors are gently and rapidly counted and then treated as described in Section 12.

Ammonia and Sulfide Toxicity

Whether ammonia is or is not a contaminant of concern depends on the disposal site. In order to identify elutriate or solid phase dredged material toxicity due to ammonia, it is essential to make routine measurements of ammonia on appropriate test fractions. These measurements are compared to water-only toxicity data for the same species used in the dredged material test (see Appendix F). The water-only toxicity data generated separately should be generated under conditions (e.g., pH, test length) reasonably similar to those in the test with the dredged material. If ammonia concentrations are too low to have potentially caused the observed toxicity in the dredged material sample, other contaminants are responsible for the toxicity. If ammonia concentrations are high enough to have caused the observed toxicity, toxicity identification evaluation (TIE) procedures should be used to confirm this suspicion. When there is no TIE confirmation that ammonia is responsible for sediment toxicity, it must be assumed that persistent contaminants other than ammonia are causing toxicity. Full details of procedures to identify ammonia as a toxicant in toxicity tests with dredged material are provided in Appendix F.

Whenever chemical evidence of ammonia is present at toxicologically important levels, i.e. ammonia concentrations exceed the species-specific acceptability ranges shown below (or 20 mg/L for freshwater organisms), and ammonia is not a contaminant of concern at the disposal site, the laboratory analyst should set up one or more beakers explicitly for the purpose of measuring interstitial ammonia. Ammonia in the sediment interstitial water should be reduced to below the species-specific level shown below (or to below 20 mg/L for freshwater organisms) before adding the benthic test organisms. Ammonia concentrations in the interstitial water can be reduced by sufficiently aerating the sample at saturation and replacing two volumes of water per day. The analyst should measure interstitial ammonia each day until it reaches a concentration below the appropriate species-specific level (or less than or equal to 20 mg/L for freshwater organisms). After placing the test organisms in the sediment, the analyst should ensure that ammonia concentrations remain within an acceptable range by conducting the toxicity test with continuous flow or volume replacement not to exceed two volumes per day. Peer-reviewed papers that deal with ammonia in sediments include: Dewitt et al. (1988), Scott and Redmond (1989), Burton (1991), EPA (1992, 1994c, 1994d), Benoit et al. (1993), Ankley et al. (1991, 1992a, 1992c, 1994).

The chemistry and toxicology of sulfides is less well-understood than that of ammonia. However, sulfides are not likely to be a problem in most open-water situations, or in bioassays where adequate oxygen levels are maintained in the overlying water.

11.2.3 Chronic/Sublethal Tests

Chronic/sublethal responses to sediment are presently only available, in addition to the end-point of survival, for a very few toxicity tests, for example: the amphipods Hyalella azteca, Ampelisca abdita and Leptocheirus plumulosus; the midges Chironomus tentans and C. riparius; the oligochaetes Tubifex tubifex and Lumbriculus variegatus, and the polychaete Neanthes arenaceodentata. [Note: EPA has recently developed chronic sediment toxicity test methods for freshwater organisms (C. tentans and H. azteca). EPA and USACE are jointly developing a chronic sediment toxicity test method manual for marine and estuarine organisms (L. plumulosus). These documents are currently under review and will be published as standard methods manuals.] Unlike acute toxicity tests, there is presently no consensus as to what level of chronic/sublethal effects (e.g., reduction of growth, reproduction, fecundity, survival of young) is cause for concern. Further, there is also no consensus as to when such effects would preclude disposal or would constitute unacceptable adverse effects requiring some type of management action. Hence, chronic/sublethal tests are not presently part of Tier III in this national manual. However, regional testing manuals may apply appropriate chronic/sublethal tests to sediments in advance of their inclusion in this national manual provided this is done with a benchmark species (e.g., C. tentans) or in addition to the benchmark testing.

Guidance for conducting the above tests may be found in publications including Nebeker and Miller (1988), Nebeker et al. (1984), Johns and Ginn (1990), Johns et al. (1990), Ingersoll and Nelson (1990), Dillon et al. (1993), Phipps et al. (1993), McGee et al. (1993). Burton (1991) provides a comprehensive review of freshwater sediment toxicity tests. Survival and growth are the endpoints of all of these tests. In addition, some tests also measure reproductive end-points.

Criteria for control acceptability for chronic/sublethal tests are specific to the test and organism. If control criteria are exceeded, the test must be repeated.

11.2.4 Data Presentation and Analysis

Data Presentation

The data for each test species should be presented in separate tables that include the following information:

• scientific name of the test species
• number of organisms in each treatment at the start of the test
• number of organisms recovered alive and/or in normal health from each chamber at the end of the test (including positive and negative controls)
• information regarding emergence, burrowing, tube building, behavioral abnormalities, growth, reproduction, and any other observations
• water-quality data for each test chamber for each day.

Data Analysis

It is possible that neither mortality nor other effects will be observed in any of the treatments or that survival in the dredged material will be equal to or higher than survival in the reference or control sediments. In either of these situations, there is no need for statistical analysis and no indication of adverse effects due to the dredged material. Similarly, if survival is higher in test sediments than in the control, but lower than in the reference area, and control survival is at acceptable levels (i.e., 90% or greater survival), there is no need for statistical analysis and no indication of benthic toxicity due to the dredged material. However, if survival in the reference sediment is higher than in the dredged material treatments and exceeds the allowable percent difference between the two treatments, the data have to be analyzed statistically to determine whether there is a significant difference between the reference and dredged material. Statistical procedures recommended for analyzing benthic acute toxicity data are described in detail in Appendix D. Local guidance must be developed to interpret chronic/sublethal tests.

11.2.5 Conclusions

Guidance on the use of the results to reach a determination is provided in Section 6.2.

11.3 Tier IV: Chronic/Sublethal Effects Evaluations

At present, it is not appropriate to incorporate sediment chronic/sublethal effects testing in this national manual (see Sections 6.0 and 11.2.3). When standardized chronic effects tests are approved, they will be incorporated in Tier III. Until then, such non-standard tests should be used in Tier IV except where regional testing manuals apply such tests in advance of their inclusion in future revisions of this national manual, provided this is done with a benchmark species or in addition to the benchmark testing.

11.4 Tier IV: Case Specific Evaluations

Biological effects tests in Tier IV should be used only in situations that warrant special investigative procedures. They may include chronic/sublethal tests, field studies such as benthic infaunal studies (EPA, 1992), experimental studies such as in situ toxicity tests or toxicity identification evaluation (Ankley et al., 1992a), risk assessments and/or no effects levels for aquatic life. In such cases, test procedures have to be tailored for specific situations, and general guidance cannot be offered. Such studies have to be selected, designed, and evaluated as the need arises, with the assistance of administrative and scientific expertise from EPA and USACE, and other sources as appropriate.

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