Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 9

Section 7: Specific Procedures for Testing Receiving-Water Samples

Instructions for testing samples of receiving waters, additional to those provided in Section 4, are given here.

7.1 Sample Collection, Labelling, Transport, and Storage

Procedures for the collection, labelling, transportation, and storage of samples of receiving water should be as described in Section 6.1. The test should commence within 1 day of sampling, whenever possible, and must start within 3 days after sampling.

7.2 Preparing Test Solutions

Samples in the collection chambers should be agitated before pouring to ensure their homogeneity. Compositing of sub-samples, preparation and use of controls, and adjustment of the salinity of sample, test solutions, and/or control/dilution water should be as described in Sections 4.1.1, 4.3.2, 6.2, and 6.3.

Each receiving-water sample should be filtered through a sieve with a 60-µm mesh opening before use, to enable the removal of potential predators or suspended material which might otherwise interfere with the test results (USEPA, 1994). If there is concern about the contribution of suspended solids to sample toxicity or of toxicity reduction due to sample filtration, a second test, without sample filtration, should be carried out concurrently, as described in Section 6.2.

7.3 Control/Dilution Water

For receiving-water samples collected in the vicinity of a wastewater discharge, chemical spill, or other point-source of possible contamination, “upstream” water may be sampled concurrently and used as control water and diluent for the “downstream” samples. Discussion in Section 4.1.1 is relevant here, on the implications and possible effects of using such water for the control and for dilution. This control/dilution water should be collected as close as possible to the contaminant source(s) of concern, but outside its zone of influence. Water current or dispersal tracer studies might be necessary to establish an acceptable sampling location. All control/dilution water from a natural source should be filtered (Section 3.4).

If uncontaminated receiving water is used as control/dilution water, a separate control solution must be prepared using the laboratory seawater shown previously by the testing laboratory to routinely enable valid test results in an echinoid fertilization test. Test conditions and procedures for preparing and evaluating each control solution should be identical, and as described in Sections 4.1 and 5.3.

Logistic constraints, lack of on-site information, expected toxic effects, or other site-specific practicalities might prevent or rule against the use of “upstream” water as the control/dilution water. In such cases, a suitable laboratory seawater supply (i.e., holding water or a laboratory seawater known to routinely achieve valid test results using this biological test method) should be used as control/dilution water. This water may be adjusted in salinity to partially simulate “upstream” water (Section 4.1.1), but the salinity limitations of this echinoid assay preclude major manipulations.

7.4 Test Observations and Measurements

Observations and measurements of test samples and solutions for colour, turbidity, foaming, precipitation, etc. should be made as described in Section 6.4, both during the preparation of test solutions and subsequently during the tests. These are in addition to the primary toxicity observations described in Section 4.4.

7.5 Test Endpoints and Calculations

Statistical endpoints for tests using samples of receiving water should be consistent with the options and approaches identified in Sections 4.5 and 6.5, and would again be based on success in fertilization compared to the control(s).

Testing of each receiving-water sample should include a minimum of three replicates per concentration (including the control) if a multi-concentration design is used and an ICp is calculated. Endpoints for tests with receiving-water samples might often be restricted to data on fertilization of gametes exposed to sample of full-strength receiving water in single-concentration tests (see Section 4.5.3). In this case, a minimum of four replicates should be used.

If toxicity of receiving-water samples is likely, and information is desired concerning the degree of dilution necessary to permit normal fertilization in echinoids, a multi-concentration test to determine the ICp should be conducted as outlined in Section 4. The undiluted (100%) sample should be included in the test as the highest concentration of the series.

Certain sets of tests might use a series of samples such as seawater from a number of locations, each tested at full strength only. Statistical testing and reporting of results for such tests should follow the procedures outlined in Section 4.5.3.

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