Name of Testing Laboratory: Northwestern Aquatic Sciences Yaquina Bay Road P.O. Box 1437 Newport, OR 97365. 2.4 Test Location: _________________________________ 2.5 Laboratory's Personnel to be Assigned to the Study: Study Director: __________________________ Quality Assurance Officer: ____________________________ Aquatic Biologist: ______________________________ Aquatic Biologist: ______________________________ 2.6 Proposed Testing Schedule: Tests are to begin within 14 days of sample collection. Reference toxicant test to be run concurrently. (Test sediments may be held under nitrogen for up to eight weeks for PSDDA and COE work.) 2.7 Good Laboratory Practices: The test is conducted following the principles of Good Laboratory Practices (GLP) as defined in the EPA/TSCA Good Laboratory Practice regulations revised August 17, 1989 (40 CFR Part 792). 3. TEST MATERIAL The test materials are marine or estuarine sediments. The collected sediments are placed in solvent and acid cleaned 1 L glass jars fitted with TFE-lined screw caps. The jars are filled completely so that there is no air space. At the laboratory, the samples may be stored at 4°C in the dark in the original sealed containers for up to 14 days prior to testing. A negative control sediment is from a clean site. In addition, a reference sediment, a clean sediment with physical characteristics similar to the test sediments, may also be employed as a control. 4. TEST WATER Test water is filtered Yaquina Bay seawater adjusted to a salinity of 28 ppt. The water is pumped daily from Yaquina Bay into a 6000 gal seasoned fiberglass reservoir from which it is supplied under pump pressure to the laboratory. Filtration is accomplished using a sand filter followed by a medium porosity (10-25 µm) cartridge filter. A fine (0.45µm) cartridge filter is then used. An alternative seawater supply of similar quality may be used. 5. TEST ORGANISMS 5.1 Organisms: Marine amphipod, Ampelisca abdita. A. abdita is a tube-dwelling amphipod found in fine sand and mud to silt from Maine to Florida and the Gulf of Mexico; it is also present in San Francisco Bay, California. 5.2. Source: This organism may be field collected or purchased from commercial suppliers. 5.3. Laboratory Handling: Amphipods should arrive in containers (usually gallon jars) with a few cm of sediment. The bottom portions of these containers are immersed in a laboratory seawater tray for temperature control. In each container, overlying seawater is partially renewed with laboratory seawater mixed with a microalgal food suspension, and gentle aeration is supplied. Animals are held for at least two days, but no longer than 10 days prior to the beginning of the test. During the holding period, static renewal conditions are maintained in each container, and amphipods are supplied with cultured microalgae (Phaeodactylum tricornutum) daily. Amphipod density will not exceed 350 amphipods/holding jar. 5.4 Age at Study Initiation: Immature amphipods are preferred for the test. If mature animals must be used, such as during times of the year when juveniles are unavailable, mature males must not be used. Males are active swimmers and die shortly after mating. The organism identification may be confirmed by reference to Mills (1964), Mills (1967), and Bousfield (1973). 6. DESCRIPTION OF TEST SYSTEM 6.1 Test Chambers and Environmental Control: Test chambers used in the toxicity test are 1000 ml glass beakers. The beakers are covered with lids to minimize evaporation of volatile compounds. Test chambers are maintained at constant temperature by partial immersion in a temperature-controlled water bath or by placement in a temperature-controlled room. Minimal aeration is supplied through a glass pipet with the tip placed not closer than 2 cm above the sediment surface. The test is performed under continuous illumination. 6.2 Cleaning: All laboratory glassware, including test chambers, is cleaned as described in EPA/600/4-90/027. New glassware and test systems are soaked 15 minutes in tap water and scrubbed with detergent (or cleaned in automatic dishwasher); rinsed twice with tap water; carefully rinsed once with fresh, dilute (10%, V:V) hydrochloric or nitric acid to remove scale, metals, and bases; rinsed twice with deionized water; rinsed once with acetone to remove organic compounds (using a fume hood or canopy); and rinsed three times with deionized water. Test systems and chambers are rinsed again with dilution water just before use. 7. EXPERIMENTAL DESIGN AND TEST PROCEDURES 7.1 Experimental Design: An experimental design is used consisting of exposure of test organisms to a number of test sediments, one or more reference sediments, and to the control sediment. Each treatment consists of five replicate test chambers each containing 20 animals. Complete randomization of test containers and blind monitoring of response criteria may be used. 7.2 Preparation of Test Sediments: Interstitial salinities of test and control sediments are measured. In some cases, it may be necessary or desirable to press sediments through a 2.0 mm screen to remove predator organisms or A. abdita indigenous to the test site. ASTM (1993) permits this. EPA/COE (1991) directs the laboratory to press-sieve all sediments (control, test reference) through a 1.0 mm screen. Each test sediment is mixed thoroughly using a non-contaminating implement, then an aliquot (175 ml) sufficient to make a 2-cm-deep layer is added to each test beaker, and the surface is smoothed. Bubbles are removed from the sediment by gently tapping each beaker against the palm of the hand. The sediment layer in each beaker is temporarily covered with a circular disc of plastic sheeting to protect the sediment layer during addition of seawater. Seawater at the test temperature and salinity is carefully poured into the beaker to the 750 ml mark, and the disc is then gently removed. Alternatively, seawater may be added by pouring into the test chamber through a plastic beaker with holes punched in the bottom. The beakers are then placed into the temperature-controlled area and covered with watchglasses. Constant illumination is provided by overhead lights. Water in the test beakers is aerated without disturbing the sediments. The test system is then allowed to temperature equilibrate overnight. 7.3 Beginning the Test: Amphipods are wet sieved, using a 0.5 mm sieve, from the holding sediment and impartially distributed to a series of containers each holding 20 amphipods. Enough amphipods are counted out at a time to add animals to one replicate set (e.g. replicate 1 of all test sediments, controls, etc.). For a very large experiment where this is not possible, addition to beakers is random. Once amphipods are added to a replicate, animals that do not burrow into the test sediment within a few minutes may be removed and replaced with healthy amphipods. Animals are added sequentially to the remaining replicates in the same manner. Following addition of amphipods to the test chambers, additional water is added to achieve a final volume of 950 ml. 7.4 Effects Criteria: Effect criteria are 1) mortality after 10 days, and 2) daily emergence of amphipods from the test sediments. Death is defined as no visible appendage movement or response to tactile stimulation. 7.5 Test Conditions: Test containers are maintained at a constant 20 ± 1°C. The test salinity is 28 ± 1 ppt. The dissolved oxygen concentration in each test container must be greater than 60% saturation throughout the 10-day test. Each beaker is supplied with oil-free compressed air provided at a rate of 120-180 bubbles per minute through disposable glass pipettes positioned with their tips not closer than 2 cm above the sediment surface. Each beaker is covered by a glass watchglass to minimize evaporation and the possibility of cross contamination between beakers. Because amphipods are known to migrate into the water column for the purposes of reproduction and dispersion under dark conditions, the test is conducted under constant illumination. 7.6 Feeding: Animals are not fed at any time during the test. During holding and acclimation, amphipods have microalgae available to them daily. 7.7 Test Duration, Type and Frequency of Observations, and Methods: The duration of the sediment toxicity test is 10 days. The type and frequency of observations to be made are summarized as follows: Type of Observation Times of Observation Biological Data Emergence from sediment daily Survival end of test Physical and Chemical Data Sediment interstitial salinity test beginning & end Salinity, dissolved oxygen, pH and temperature of overlying water (in all beakers) test beginning & end Ammonia and sulfides in overlying water (optional) test beginning & end Salinity, dissolved oxygen, pH and temperature of overlying water (one repl.) days 1 through 9 Temperature (in separate control beaker) daily Check air and lights daily The presence of amphipods that have emerged from the sediments is recorded daily. Any other unusual observations are recorded. No amphipods are removed at any time until the termination of the test. The bioassay is terminated after 10 days of sediment exposure. Floating or dead amphipods are recorded after which the sediment is wet sieved through a 0.5 mm screen to recover buried amphipods. The numbers of surviving and dead recovered amphipods are recorded. Dissolved oxygen is measured directly in test beakers using a polarographic oxygen probe calibrated in according to the manufacturer's recommendations. The pH is measured using a properly calibrated pH meter with scale divisions of 0.1 pH units. Temperature is measured using a calibrated mercury thermometer or a telethermometer. Salinity is measured using a refractometer. Ammonia-N is measured using the HACH Model FF-3 test kit (ammonia -N detection limit 0.1 mg/L). Any observed changes in sediment color or the formation of a sediment discontinuity layer is also recorded. 7.8 Criteria of Test Acceptance: For the test to be considered acceptable, the minimum survival of organisms in the control treatment at the end of the test must be 90%. Also, mortality in the reference sediments must be £ 20% over the negative control. 8. DATA ANALYSIS The mean and standard deviation are calculated for each endpoint employed (e.g. mortality) and for each treatment (i.e. test sediment). Between-treatment comparisons for each biological endpoint may be made using Student's t-test, Wilcoxon's Two-Sample test, or Mann-Whitney U test, where each treatment is compared to the control or the reference sediment. An arcsine-square root transformation of proportional data, and tests for normality and heterogeneity of variances, are performed prior to statistical comparison. 9. REPORTING A report of the test results must include the following information: name and identification of the test; the investigator and laboratory; sediment holding temperature data; information on the test sediment including the interstitial salinity for control, reference and test sediments; information on the source of seawater used; detailed information about the test organisms including acclimation conditions; a description of the experimental design and test chambers and other test conditions including water quality; information about any aeration that may have been required; definition of the effects criteria and other observations; unusual responses, if any, in the control treatment; daily emergence for each beaker and the 10-day mean and S.D. for each treatment; 10-day mortality in each beaker and the mean and S.D. for each treatment; 96-hr LC50 with reference toxicant; a description of data analysis methods employed and documentation of statistical test results; any unusual information about the test or deviations from procedures. 10. STUDY DESIGN ALTERATION Amendments made to the protocol must be approved by the sponsor and study director and should include a description of the change, the reason for the change, the date the change took effect, and the dated signatures of the study director and sponsor. Any deviations in the protocol must be described and recorded in the study raw data. 11. REFERENCE TOXICANT Reference toxicant testing should be included with each study or at regular intervals as defined in the Quality Assurance Program of the laboratory. A 96-hr reference toxicant test is normally run concurrently with this test. 12. REFERENCED PROCEDURES ASTM. 1990. Standard guide for conducting sediment toxicity tests with freshwater invertebrates. ASTM Standard Method No. E 1383-90. Am. Soc. Test. Mat., Philadelphia, PA. ASTM. 1993. Standard guide for conducting 10-day static sediment toxicity tests with marine and estuarine amphipods. E1367-92. In: Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. pp. 1138-1163. Bousfield, E.L. 1973. Shallow-water gammaridean amphipoda of New England, Cornell University Press, Ithaca, New York. Mills, E.L. 1964. Ampelisca abdita, a new amphipod crustacean from eastern North America. Canadian J. Zool. 42:559-575. Mills, E.L. 1967. The biology of an ampeliscid amphipod crustacean sibling organism pair. J. Fish. Res. Bd. Canada 24(2):305-355. Puget Sound Estuary Program. 1991. Interim final recommended guidelines for conducting laboratory bioassays on Puget Sound sediments. Prepared for U.S. Env. Prot. Agency, Region 10, Office of Puget Sound, Seattle, WA. Redmond, M.S. and K.J. Scott. 1989. Amphipod predation by the infaunal polychaete Nephtys incisa. Estuaries 12(3):205-207. Snedecor, G.W. and W.G. Cochran. 1967. Statistical methods. Sixth Ed., The Iowa State Univ. Press. Ames, Iowa., 593 pp. U.S. EPA/U.S. COE. 1991. Evaluation of dredged material proposed for ocean disposal. Testing manual. ("Green Book") EPA-503-8-91/001. Washington, D.C. 13. APPROVALS _____________________________________ for ______________________________ Name Date _____________________________________ for Northwestern Aquatic Sciences Name Date Washington 1.11 Data Set Moss Landing Marine Lab (California), Oregon Department of Environmental Quality, WA DOE (Washington) used USGS Porewater (Urchin) and GTCN Marine Pollution Studies Laboratory (Amphipod)