Biological test method for toxicity tests using early life stages of rainbow trout: chapter 7

Section 7: Specific Procedures for Testing Receiving Water Samples

• 7.1 Test Options
• 7.2 Sample Collection, Labelling, Transport, and Storage
• 7.3 Preparing and Aerating Test Solutions
• 7.4 Control/Dilution Water
• 7.5 Test Observations and Measurements
• 7.6 Test Endpoints and Calculations

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

7.1 Test Options

Periodic tests with receiving water, for monitoring and compliance with regulatory requirements, would normally use the embryo (E) or embryo/alevin (EA) test options (Section 4.3.1). Tests to measure effects on diverse life stages of developing salmonid fish would use the EA or embryo/alevin/fry (EAF) test option. Definitive tests including effects on survival and growth of developing fry would use the EAF test option. Before the E or EA test is adopted for periodic or frequent use (e.g., as part of an environmental effects monitoring program), comparative assessment of either of these test options with the more comprehensive EAF test is recommended, to quantify differences in sensitivity. Any of the three test options might be conducted as either static-renewal or continuous-flow assays, depending on the objectives, nature of the sample, volume needed, etc.^Footnote 44 A test and its associated endpoints might be restricted to three or more undiluted portions of the sample and three or more replicate control solutions (i.e., a single-concentration test), or might involve three or more replicates of each of a series of concentrations of the sample including 100%. See Section 7.6 for further guidance.

At the time that an EA or EAF test is set up, it is recommended that an E test be established and run concurrently, using the samples or subsamples of receiving water used for the first week of the test, and fertilized eggs from the same pool of test organisms. The findings of this E test will provide insight into the fertilization success rate for controls in the EA or EAF test, and will be useful in appraising the relative sensitivity to the test substance for the acute (E) and longer (EA or EAF) test options. A series of E tests might also be performed weekly with the samples or subsamples of receiving water as the EA or EAF test progresses, to provide information on the relative toxicity of the test substance used for each week of the test (Fennell et al., 1998).

The routine assessment of acute lethality of each sample which is to be used in an EA or EAF test might or might not be warranted. This would be appropriate if it were suspected or anticipated that the undiluted receiving water might be lethal at any time during the early life-stage test. The information should be useful for interpreting toxic effects that occurred at particular times during the EA or EAF tests. The lethal test should be done on a portion of the sample, upon receipt, to determine the acute lethality to rainbow trout fry or fingerlings (96-h LC50 or mortality in 100% sample during 96 hours), following the methods of Environment Canada (1990b, with 1996 amendments).

7.2 Sample Collection, Labelling, Transport, and Storage

Procedures for labelling, transporting, and storing samples are found in Section 6.2. Toxicity tests should commence as soon as possible, preferably within 24 hours of sampling, and no later than three days.

7.3 Preparing and Aerating Test Solutions

Samples in the collection containers should be agitated before pouring to ensure their homogeneity. Compositing of subsamples should be as described in Section 6.3.

Samples that might contain organisms which could affect developing salmonids should be filtered through a sieve with 60 µm mesh openings (Section 6.3) before use. If there is a concern that such filtering might change toxicity, a second unfiltered test should be run concurrently.

Dissolved oxygen content of each test solution including the control(s) must be measured upon preparation. Thereafter, either the organisms should be exposed to the solutions, or else each test solution should be pre-aerated (before organisms are exposed). In most instances, pre-aeration or aeration during the test will not be necessary or warranted (see Section 3.3, including footnotes 6 to 8), and should be avoided. A test performed without aeration should use a flow-through setup, to create a continuous circulation of test solutions around the developing embryos or alevins (see Section 3.3 including Figure 3C, and Section 4.3.2). If dissolved oxygen is below 60% of air saturation or above 100% saturation, pre-aeration or aeration should be used in either a static-renewal or flow-through test, following the guidance in Section 4.3.4.

7.4 Control/Dilution Water

For samples of surface water collected near a wastewater discharge, chemical spill, or other point-source of contamination, "upstream" water may be sampled concurrently and used as control water and diluent for the downstream samples (see Section 5.4). This control/dilution water should be collected as close as possible to the contaminant source, but upstream of it or outside its zone of influence. Surface water should be filtered to remove organisms, as described in Section 7.3.

If "upstream" water is used as control/dilution water, there must be a separate control solution of the laboratory water normally used for rearing and testing fish. Procedures for preparing and evaluating each control solution should be identical, and as described in Sections 4.1 and 5.4. Results of test exposures should be compared with those of the control in which receiving water was used (Section 4.5).

It might be unreasonable to use upstream water for a control because of logistical constraints, expected toxic effects, or other site-specific practicalities. In such cases, the laboratory water normally used for rearing fish should be used as control water and for all dilutions. It could be adjusted to partially simulate upstream water (see Section 5.4).

7.5 Test Observations and Measurements

The primary observations on test organisms should be as described in Section 4.4.

In addition, there should be observations of sample and solution colour, turbidity, foaming, precipitation, etc., as described in Section 6.5, both during preparation of solutions and subsequently during the test.

Each receiving-water sample should be characterized chemically. Depending on the suspected nature of the toxicants, measurements might include pH, conductivity, hardness, alkalinity, colour, chemical oxygen demand, biochemical oxygen demand, and concentrations of specific toxicants (e.g., resin acids, chlorophenolic compounds, dissolved metals, chlorine, chloramine, ammonia).

7.6 Test Endpoints and Calculations

Endpoints for tests with receiving water should normally be the standard ones described in Section 4.5. The use of options and approaches should be consistent with those identified in Sections 4.5 and 6.6.

The tests could be multi-concentration or single-concentration (100% or an appropriate dilution, plus a control). Tests of regulatory compliance would often include three or more undiluted portions of the sample, and three or more replicate control solutions. Regulatory tests might use only a single concentration, usually the undiluted receiving water. Single-concentration tests are often cost-effective for determining the presence of measurable toxicity, and also for screening a large number of samples (e.g., from various locations within the receiving water). Statistical testing and reporting of results for such tests should follow the procedures outlined in Section 4.5.

If toxicity of receiving-water samples is likely, and information is desired concerning the degree of dilution necessary to permit normal growth and development of rainbow trout, a multi-concentration test should be conducted as outlined in Sections 4.1 and 4.5, to determine the appropriate standard endpoints; i.e., the EC50 and EC25 for nonviability at various stages of development in the E, EA, and EAF tests, and additionally in EAF, the 30-day LC50 and 30-day IC25 for average attained weight of swim-up fry. The series tested should include one or more undiluted samples. Narrative statements on delayed development, deformities, and behaviour (EAF test only) are required when reporting the findings of an EA or EAF test, and additional (optional) observations can be detailed (see Section 4.5).