Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 8

Section 6: Specific Procedures for Testing Samples of Effluent, Leachate, and Elutriate

• 6.1 Sample Collection, Labelling, Transport, and Storage
• 6.2 Preparing Test Solutions
• 6.3 Control/Dilution Water
• 6.4 Test Observations and Measurements
• 6.5 Test Endpoints and Calculations

This section gives particular instructions for the collection, preparation, and testing of effluents, leachates, and elutriates, in addition to the procedures listed in Section 4.

6.1 Sample Collection, Labelling, Transport, and Storage

Containers for transportation and storage of samples of effluent, elutriate, or leachate must be made of nontoxic material. Collapsible polyethylene or polypropylene containers manufactured for transporting drinking water (e.g., Reliance^TM plastic containers) are recommended. The volume of these containers can be reduced to fit into a cooler for transport, and the air space within kept to a minimum when portions are removed in the laboratory for the toxicity test or for chemical analyses. The containers must either be new or thoroughly cleaned, and rinsed with uncontaminated water. They should also be rinsed with the sample to be collected. Containers should be filled to minimize any remaining air space.

Most tests with effluent, leachate, or elutriate will be performed “off-site” in a controlled laboratory facility. Testing of effluents and leachates should commence within 1 day of sampling whenever possible, and must commence no later than 3 days after sampling. Samples of sediment or other solid material collected for extraction and subsequent testing of the elutriate should also be tested as soon as possible. Extraction procedures followed by testing should begin within two weeks of sampling (preferably within one week), and testing must start no later than six weeks after collection (EC, 1994). Procedures given in Environment Canada (1994) for the preparation of elutriates should be followed. Testing of elutriates must commence within 3 days of their preparation, or as specified in a regulation or protocol.

Generally, a two-litre sample is adequate for an off-site multi-concentration test, associated routine chemical analysis, and any necessary adjustments or repeat tests. Smaller amounts are required for single-concentration tests (see Section 4.5.3). Upon collection, each sample container must be filled, sealed, and labelled or coded. Labelling should include at least sample type, source, date and time of collection, and name of sampler(s). Unlabelled or uncoded containers arriving at the laboratory should not be tested, nor should samples arriving in partially filled containers be routinely tested, because volatile toxicants escape into the air space. However, if it is known that volatility is not a factor, such samples might be tested at the discretion of the investigator.

An effort must be made to keep samples of effluent or leachate cool (1 to 7 °C, preferably 4 ± 2 °C) throughout their period of transport. Upon collection, warm (>7 °C) samples must be cooled to 1 to 7 °C with regular ice (not dry ice) or frozen gel packs. As necessary, ample quantities of regular ice, gel packs, or other means of refrigeration must be included in the transport container in an attempt to maintain sample temperature within 1 to 7 °C during transit. Samples must not freeze during transport or storage.

Upon arrival at the laboratory, the temperature of the sample must be measured and recorded. An aliquot of effluent or leachate required at that time may be adjusted immediately or overnight to the test temperature and used in the test. Any remaining portion(s) of sample held for possible additional testing must be stored in darkness in sealed containers, without air headspace, at 4 ± 2 °C.

Temperature conditions should also be as previously indicated for transportation and storage of elutriates, as well as for samples intended for aqueous extraction and subsequent testing of elutriate, unless otherwise specified.

6.2 Preparing Test Solutions

Each sample in a collection container must be agitated thoroughly just before pouring, to ensure the re-suspension of settleable solids. Subsamples (i.e., a sample divided between two or more containers) must be mixed together to ensure their homogeneity. The dissolved oxygen content and pH of each sample must be measured just before its use. As necessary, the sample should be pre-aerated (see Section 4.3.3) before the test solutions are prepared and distributed to replicate test chambers.

The salinity of each test sample should be measured before the test is started. If it is outside the range considered acceptable for the test (i.e., 28 to 32 g/kg), the salinity of the sample or each test solution should be adjusted to within this range using natural or artificial hypersaline brine, commercially-available dry ocean salts, reagent-grade salts (see Sections 2.3.4 and 4.3.2), or deionized water. Nominal test concentrations must be prepared and reported in consideration of any such salinity adjustments.

Filtration of samples is normally not required nor recommended. However, if a sample of effluent or leachate contains debris or indigenous organisms which might be confused with or attack gametes or fertilized eggs, the sample must be filtered through a sieve with 60-µm mesh openings, before use (USEPA, 1994). Such filtration could remove some suspended or settleable solids that are characteristic of the sample and might otherwise contribute to part of the toxicity or modify the toxicity. For instance, high concentrations of biological solids in certain types of treated wastewater might contribute to sample toxicity due to ammonia and/or nitrite production (Servizi and Gordon, 1986). Alternatively, the presence of high concentrations of suspended solids in a sample might inhibit fertilization or damage the gametes directly. In instances where concern exists regarding the effect of this filtration on sample toxicity, a second test should be conducted concurrently using an unfiltered portion of the sample.

6.3 Control/Dilution Water

Tests conducted with samples of effluent or leachate for monitoring and regulatory compliance purposes should use, as the control/dilution water, either a supply (source) of the laboratory seawater shown previously by the testing laboratory to routinely enable valid test results, or a sample of the receiving water. Because results could be different for the two sources of water, the objectives of the test must be decided before a choice is made. Shipping difficulties and costs should also be considered; the use of receiving water for dilutions and controls increases the volume of liquid to be shipped, although that might not be a major factor for this small-scale assay.

The use of uncontaminated receiving water as the control/dilution water can be desirable if site-specific information is required on the potential toxic impact of an effluent, leachate, or elutriate on a particular receiving water (see rationale in Section 4.3.2). An important example of such a situation would be testing for sublethal effect at the edge of a mixing zone, under site-specific regulatory requirements. Conditions for the collection, transport, and storage of such receiving-water samples should be as described in Section 6.1. Any sample of receiving water used as the control/dilution water for testing effluents or leachates should be filtered according to the standard recommendation for natural control/dilution water, through a 60-µm mesh sieve (USEPA, 1994; Section 3.4). If a sample of receiving water is to be used as control/dilution water, a separate control solution must be prepared using the laboratory seawater that is normally used for performing fertilization tests (i.e., holding water or other suitable laboratory water; see Section 4.1.1).

Tests requiring a high degree of standardization should use reconstituted (artificial) seawater as control/dilution water (see Section 3.4). An example of such a situation might be a test intended to compare the toxicity of a particular effluent, leachate, or elutriate with that of samples collected and/or tested elsewhere.

If any artificial or natural hypersaline brine (HSB; Section 2.3.4) is added to the sample or test solutions (see Section 6.2), the toxicity test must include a set of controls (i.e., HSB controls) which is prepared and adjusted to the test salinity (i.e., 30 ± 2g/kg) using only this HSB and deionized water. Likewise, if any commercially-available dry ocean salts or reagent-grade salts are added to the sample or test solutions, the toxicity test must include a set of controls (i.e., salt controls) which is prepared using the same source, batch, and concentration of dry salts as that added to the test sample. A second set of controls (i.e., dilution-water controls), comprised of 100% dilution water, is required if any water used to dilute the sample differs in any respect from the HSB controls or salt controls (Section 4.1.1). The salinity of all test concentrations should be within 1 g/kg of the controls.

If it is desired to assess the total effect of the wastewater including its low (or high) salinity, for regulatory compliance or other (e.g., research) purposes, the test could be run without adjusting the salinity of the control/dilution water or the sample/test solutions to 30 ± 2 g/kg. For instance, salinity could be adjusted to that of the receiving water, or the control/dilution water adjusted to 30 ± 2 g/kg without adjusting the salinity of sample or test solutions. Such an evaluation should include a second, salinity-adjusted test and/or a set of salinity controls (Section 4.1.4).

6.4 Test Observations and Measurements

Success of fertilization should be observed as in Section 4.4.

Colour, turbidity, odour, and homogeneity (i.e., the presence of floatable or settleable solids), should be observed in the sample of effluent, leachate, or elutriate at the time of preparing test solutions. A record should be made of any reactions or overt changes upon dilution with water or during the test, such as precipitation, flocculation, foaming, odour, and change in colour or turbidity.

For effluent samples having appreciable solids content, it is desirable to measure total suspended and settleable solids (APHA et al., 1989, 2005) upon receipt, as part of the overall description of the effluent, and as sample characteristics that might influence the results of the toxicity test.

6.5 Test Endpoints and Calculations

Tests for monitoring and for compliance with regulatory requirements should normally include, as a minimum, three or more replicates per concentration (including the control) if a multi-concentration design is used and an ICp is calculated. A test which compares full-strength solutions to a control (single-concentration) via hypothesis testing should normally include, as a minimum, four or more replicates. Test procedures for regulatory compliance might specify that a single concentration (100% sample unless otherwise specified) be used, or might require determination of the ICp.

Toxicity tests can have other objectives such as determination of in-plant sources of toxicity, or toxicity changes resulting from waste treatment or process changes. Such tests might be multi-concentration tests or single-concentration tests (100% or an appropriate dilution, plus a control). Single-concentration tests are often cost-effective for determining the presence or absence of measurable toxicity or as a method for screening a large number of samples for relative toxicity. Endpoints for these tests would again depend on the objectives of the undertaking, but could include arbitrary “pass” or “fail” ratings, or percent reduction in fertilization at a specified concentration. Items in Section 4.5.3 provide instructions that are relevant here, on statistical analysis and reporting of results from a set of tests on different samples, each tested at only one concentration.