Ecological Evaluation Technical Guidance - State of New Jersey

expects that much variability in a short-term test with lab water and reagent salts, it iswithin reason to expect that long-term, sublethal effects from complex mixtures like sitesoil will yield substantially more variability.Toxicity Test DesignSoil toxicity studies are conducted by exposing a predetermined number of organisms(e.g., four replicates of ten organisms or seeds) to a soil sample. Organisms are placed inappropriate test chambers containing the soil sample, and in test chambers containingclean laboratory soil to serve as an experimental control. Investigators make directobservations of the exposed test setup at regular intervals for the duration of the test todetermine responses such as erratic behavior and visible mortality. However,earthworms quickly bury themselves in the test soil and are not seen again until testtermination when they are removed for final enumeration. Plants are not visible untilafter the seeds germinate and the plants begin to emerge after four to seven days.A second “reference” control should also be included in the study, using soil from outsidethe influence of the site. Use of an appropriate reference area soil is more representativeof the actual field conditions. While a site soil may show a significant effect as comparedto the laboratory control, it may not show a significant effect when compared to thereference area.The laboratory control soil should be an “artificial” soil, as described in the ASTMguidance (ASTM, 2004, Appendix A2). While both the plant and invertebrate toxicitymethods allow for use of various artificial soil mixtures, potting soils or natural soils aslaboratory controls, the objective of the study is not to compare site soils to “optimum”soil samples. Comparing the growth and reproduction of worms or plants grown in sitesoils to those grown in a laboratory control composed of a rich, organic potting soil willalmost always show a significant decrease in site sample growth compared to the control,regardless of whether the site soil is toxic or not. For this reason, potting soil or any otherrich, organic soil is not appropriate for use as a laboratory control when testing site soilsamples.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 equates to greater statistical powerand more confidence in the final results. Soil studies are typically performed with four toeight replicates of ten organisms or seeds for each test soil, which ensures sufficientstatistical power for the more sensitive sublethal growth endpoints.Toxicity Test EndpointsFor acute soil toxicity studies with earthworms, the endpoints can include survival andgrowth. While survival is a definitive endpoint, earthworm growth measurements can bemisleading because the worms are measured as wet weight at test initiation and again attest termination. Earthworms are depurated for 24 to 48 hours prior to weighing, to allowthem to purge their digestive tracts of soil. The earthworms used in toxicity testingshould all be approximately uniform in size, but because of the range of soil grain sizeand organic content, different soils will be depurated at different rates. This means thatthe worms in one soil may have completely purged their guts, while the worms fromEcological Evaluation Technical Guidance Document 123Version 1.2 8/29/12