Ecological Evaluation Technical Guidance - State of New Jersey

chronic toxicity test, including a series of dilutions (pore water or elutriate sample mixedwith clean laboratory water).Toxicity Test DesignAquatic toxicity studies are conducted by exposing a predetermined number of organisms(e.g., four replicates of ten organisms) to an undiluted sample or to a series ofconcentrations of a pore water or elutriate sample. Aquatic organisms are placed inappropriate test chambers (e.g., beakers, flasks, test tubes) containing the samples and intest chambers containing clean laboratory water to serve as an experimental control.Investigators make direct observations of the exposed test organisms at regular intervalsfor the duration of the test to determine responses such as mortality, reduction in growthor reduced reproduction.Pore water and elutriate samples can be tested undiluted, at 100 percent strength, but iftoxicity is observed, such a test does not indicate how toxic the sample is. For surfacewater and wastewater assays, both the USEPA and NJDEP recommend using five serialdilutions of the sample water (e.g., 100%, 50%, 25%, 12.5% and 6.25%) mixed withclean laboratory water. The clean laboratory water is also used as a negative control toassess the health of the test organisms (if toxic effects are noted in laboratory water, thebatch of test organisms is considered suspect, and the test may have to be re-run using adifferent batch of organisms). A second “reference” control can also be included in thestudy, using pore water or elutriate derived from sediment samples collected outside theinfluence of the site (e.g., upstream).To perform statistical analyses to determine whether significant differences exist betweenthe laboratory controls or reference area samples, toxicity studies should be set up withmultiple replicates. As a general rule, more replicates mean greater statistical power andmore confidence in the final results. Acute toxicity studies are typically performed withtwo replicates of ten organisms for each test concentration, which is sufficient forcalculation of lethal concentrations.Short-term chronic toxicity studies should be performed with four or more replicates often organisms per exposure concentration for fish and mysid shrimp, and with tenreplicates of a single organism per exposure concentration for Ceriodaphnia. Replicationensures sufficient statistical power for the more sensitive sublethal endpoints (e.g.,growth and reproduction).Toxicity Test EndpointsFor acute toxicity studies, the typical endpoint is lethality. Lethality is generallyexpressed as the LC 50 (the concentration of test water that kills half of the exposedorganisms). Statistical calculation methods are discussed at length in the USEPAguidance manual.For short-term chronic studies, endpoints include lethality, growth or reproductionendpoints. The seven-day fathead minnow larval survival and growth assay (USEPAMethod 1000) and the seven-day sheepshead minnow larval survival and growth assay(USEPA Method 1004) assess survival at test termination as a percentage of the numberof fish exposed at test initiation. Growth is assessed as the dry weight of the survivingfish.Ecological Evaluation Technical Guidance Document 115Version 1.2 8/29/12