Biological test method for toxicity tests using early life stages of rainbow trout: chapter 6

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

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

Particular instructions for testing samples of effluent, elutriate, and leachate, in addition to the procedures listed in Section 4, are given in this section.

6.1 Test Options

Periodic tests with effluent, elutriate, or leachate (i.e., wastewater) samples for monitoring and compliance with regulatory requirements might use either the E, EA, or EAF test option (Section 4.3.1). Before one of these test options is adopted for periodic or frequent use (e.g., as part of an environmental effects monitoring program) with a particular wastewater, comparative assessment of these test options 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.

At the time that an EA or EAF test is set up, it is recommended that a multi-concentration E test be established and run concurrently, using the samples or subsamples of wastewater 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 multi-concentration E tests might also be performed weekly with the samples or subsamples of wastewater 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).

Regulatory testing programs might require test designs and endpoints other than the standard ones described herein. For example, regulations might require a single-concentration test with three or more undiluted portions of the sample, and three or more replicate control solutions. A required endpoint might be based on the results for a single concentration, usually 100% wastewater. See Section 6.6 for further guidance.

The requirements for volume of wastewater sample should be given serious consideration before undertaking any program. Approximately equal amounts of sample would be required for static-renewal and flow-through tests, but the amount might differ considerably for the different test options.^Footnote 40 Appreciable savings in the required volumes of wastewater could be achieved in the EA and EAF tests by starting with the lower daily volumes of new testwater required at first, and increasing the volume in phases, as required by the actual biomass in a test chamber. Given the requirements for large volumes of sample in certain EA or EAF tests, especially those with ≥50 alevins per replicate and/or large individuals, it might be preferable to undertake such tests at the source of the wastewater, using a mobile laboratory. Any strategy for minimizing the sample volume requirements must, of course, keep replicate groups intact and separate from other replicates.

Samples of effluent, leachate, or elutriate are normally not filtered or agitated during the test. However, the presence of suspended or settleable inorganic or organic solids in a sample can impair the development of embryos, alevins, or swim-up fry, and can cause stress responses, decreased growth, or other sublethal effects in fry and older life stages at concentrations ≤100 mg/L (Noggle, 1978; McLeay et al., 1987; Servizi and Martens, 1987). High concentrations of biological solids in certain types of treated effluent can also contribute to sample toxicity due to ammonia and/or nitrite production (Servizi and Gordon, 1986). An additional test should be conducted simultaneously if there is concern about elevated concentrations of suspended or settleable solids in samples of effluent, elutriate, or leachate contributing to toxicity, and if the intent of the study is to quantify the degree to which sample solids contribute to toxicity. The second test should use a portion of the sample, treated by filtering or decanting to remove solids, but procedures should be otherwise identical.

Measurement of acute lethality to rainbow trout fry or fingerlings is recommended upon receipt of each sample to be used in an EA or EAF test. The lethal test should determine the 96-h LC50 or mortality in 100% sample during 96 hours, following the methods of Environment Canada (1990b, with 1996 amendments). Monitoring each sample for acute lethality might detect atypical variations in toxicity from chemical spills or other incidents, in-plant process changes, performance of an effluent treatment plant, or temporal environmental changes (if leachate). Information from concurrent acute toxicity tests will be useful in interpreting time-related toxic effects that occur during the EA or EAF tests.

6.2 Sample Collection, Labelling, Transport, and Storage

Containers for transportation and storage of samples or subsamples of effluent, leachate, or elutriate must be made of nontoxic material. Collapsible polyethylene or polypropylene containers manufactured for transporting drinking water are recommended (e.g., Reliance^TM), since their volume can be reduced to fit into a cooler for transport, and 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.

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 or, if collected, one of the subsamples (with the remaining subsamples left unopened and sealed), must be measured and recorded. An aliquot of effluent or leachate required at that time may be adjusted immediately or overnight to 14°C, and used in the test. The remaining portion(s) of sample or subsamples required for subsequent solution renewals must be stored in darkness in sealed containers, without air headspace, at 4 ± 2°C. For elutriates, as well as for samples intended for aqueous extraction and subsequent testing of elutriate, transport and storage conditions should be as indicated for effluents and leachates.

Tests with effluent, elutriate, or leachate may be performed "off-site" in a controlled laboratory facility. Each off-site E, EA, or EAF test must be conducted using one of the following two collection procedures and approaches.

1. A single sample of wastewater may be used for performing an E test, provided that it is divided into three separate containers upon collection, for transport and storage. If this collection procedure is followed, each of the three subsamples must be used to prepare all test solutions of the sample during two or three consecutive days of the test.^Footnote 41 Similarly, for an off-site EA or EAF test, a single sample of wastewater may be used for each 7-day interval provided that it is subdivided into three full, sealed containers upon collection and used in the same manner.
2. If changes in toxicity of the wastewater are known or anticipated during 7 to 10 days of storage before use, fresh samples for an off-site E test should be collected on at least three separate occasions with sampling intervals of two to three days or less. These samples must be used consecutively during the test.^Footnote 42 Similarly, for off-site EA or EAF tests involving wastewater samples known or anticipated to be unstable during storage, this sampling and testing regime (involving ≥3 discrete samples per week) should be implemented throughout each week of the test.

An alternative approach for unstable wastewater is to perform these tests on-site, using fresh wastewater and either flow-through or static-renewal conditions (see Sections 4.3.2 and 6.1, including footnote 40).

Testing of effluent and leachate samples should commence as soon as possible after collection. Use of any sample in a test should begin within one day whenever possible, and must begin no later than three days after sampling. If effluents or leachates are tested in on-site laboratories, samples should be used in the test within one day or less following their collection^Footnote 43 (USEPA, 1989).

Samples of sediment, soil, or other solid material collected for aqueous extraction and subsequent testing of the elutriate should be extracted and tested as soon as possible following their collection, and no later than ten days following receipt in the laboratory. For the derived elutriates, aliquots of the prepared sample should be used on the same schedule as indicated for samples of effluent or leachate, if possible. The prolonged storage of elutriate samples is undesirable because the toxicity of the sample might not be stable. Elutriate tests must commence within three days of sample preparation, unless specified otherwise in a regulation or prescribed method.

6.3 Preparing and Aerating Test Solutions

Each sample or subsample in a collection or storage container must be agitated thoroughly just before pouring, to ensure the re-suspension of settleable solids. The dissolved oxygen content and pH of each sample or subsample must be measured just before its use. As necessary, each test solution should be pre-aerated (see Section 4.3.4) before aliquots are distributed to replicate test chambers.

Filtration of samples or subsamples is normally not required nor recommended. However, if they contain organisms which might be confused with the test organisms, attack them, or compete with them for food, the samples or subsamples must be filtered through a sieve with 60µm mesh openings before use (USEPA, 1989; 1994). Such filtration could remove suspended solids that are characteristic of the sample or subsample, and might otherwise contribute part of the toxicity or modify the toxicity. If there is such a concern, a second and concurrent test should be conducted using an unfiltered portion of the sample/subsample.

During E, EA, or EAF tests with samples of effluent, each solution including the controls should normally be gently aerated within the chamber. A decision to test without aeration might be made, however, because of regulatory requirements, a very low oxygen demand of the wastewater, or a particular test objective such as including the oxygen demand as part of the overall toxic effect. In such a case, use of a flow-through test is recommended (see Sections 3.3, 4.3.2, and 4.3.4).

6.4 Control/Dilution Water

Tests with samples of effluent or leachate, intended to assess compliance with regulations, should use either the laboratory water or a sample of the receiving water as the control/dilution 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. Given the volume requirements, the use of receiving water for dilutions and as control water might be impractical for off-site tests.

The use of receiving water as the control/dilution water might be desirable for some on-site tests, if site-specific information were desired. An important example would be testing for sublethal effect at the edge of a mixing zone, under site-specific regulatory requirements. Collection, transport, and storage of such receiving-water samples should be as described in Section 6.2.

If a sample of upstream receiving water is to be used as control/dilution water, a separate control solution must be prepared using the laboratory water supply that is normally used for rearing and testing fish. Measured biological endpoints (e.g., embryo viability, mortality rates for alevins or swim-up fry, incidence of deformed fish, weight of fry at test end) of fish in the laboratory control water must be compared to that in the sample of upstream receiving water (Section 4.5).

Tests requiring a high degree of standardization may be undertaken with reconstituted water for the control and for dilution. This requires relatively large volumes of water, but might be feasible and desirable in some cases. In such a case, the use of soft reconstituted water is recommended (hardness 40 to 48 mg/L as CaCO₃, pH 7.2 to 7.5, see Section 5.4). For example, the use of soft reconstituted water would be worthwhile if it were desired to minimize any modifying influence of the dilution water. Such situations might include studies intended to interrelate toxicity data from various types and sources of wastewater, from a number of test facilities, or from a single facility where water quality was variable. It is not recommended that the hardness of the reconstituted water be adjusted higher than values typical of the waterbody receiving a particular wastewater, nor that pH be adjusted outside the normal range, since such practice can reduce (or increase) the toxicity of the test substance and provide a misleading test result.

6.5 Test Observations and Measurements

Observations of the number of obviously dead (E, EA, and EAF tests) and deformed individuals (EA and EAF tests) in each replicate should be recorded daily. Complete counts for the relevant stages, including "missing" individuals, should be made at the end of the major stage in each test (i.e., seven days after fertilization in the E test, seven days after 50% hatch in the control in the EA test, and at the time of 50% control swim-up in the EAF test). For the EAF test, there are measurements of mortality and average dry weight of surviving fry after 30 days of exposure with feeding (see Sections 4.4 and 4.5). Observations on the number hatched (EA and EAF tests) and the number exhibiting swim-up behaviour (EAF test) in each replicate should also be recorded daily, as should abnormal behaviour.

Various measurements besides those specified in Sections 4.3.3, 4.3.4, 4.3.5, and 4.4 should be done on the characteristics of the wastewater and conditions during the test. When solutions are prepared from the sample of wastewater, there should be observations of its colour, turbidity, odour, and homogeneity (i.e., presence of floatable material or settleable solids). Upon dilution with water, records should be made of precipitation, flocculation, colour change, odour, or other reactions. During the test, observations should be made on any changes in appearance of solutions, such as foaming, settling, flocculation, increase or decrease in turbidity, and colour change.

For tests with highly coloured or opaque solutions, or for samples producing foam in one or more test chambers, the embryos and alevins should be inspected by briefly lifting the incubation unit out of each solution. If necessary, the incubation unit could be moved briefly to a container of clear control/dilution water while observations were made on mortality and aberrant appearance or behaviour. All replicates, including controls, must be treated identically for any such inspection manoeuvres.

For effluent samples with appreciable solids content, it is desirable to measure the total suspended and settleable solids upon receipt (APHA et al., 1995), as part of the overall description of the effluent, and as sample characteristics that might influence the results of the toxicity test. Additional measurements that would help to characterize each sample of effluent, leachate, or elutriate should also be made. These could include pH, conductivity, hardness, alkalinity, colour, chemical oxygen demand, biochemical oxygen demand, dissolved oxygen, and concentrations of specific toxic contaminants (e.g., resin acids, chlorophenolic compounds, dissolved metals, chlorine, chloramine, ammonia).

6.6 Test Endpoints and Calculations

The endpoints for tests performed with samples of wastewater will usually be the standard ones, 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. Other narrative statements on delayed development, deformities, and behaviour are required in the longer tests, and additonal (optional) observations can be detailed, as described in Section 4.5.

Tests for monitoring or regulating effluents, leachates, or elutriates must use the standard options and endpoints defined in Section 4. In the EAF test, with three standard endpoints, the most sensitive effect would be taken as the definitive indication of toxicity. The standard methods of analysis would apply (see Section 4.5).

Tests for monitoring and compliance with regulatory requirements should normally include, as a minimum, three or more replicates of the undiluted sample/subsamples (or a specified dilution thereof), and three or more replicate control solutions. Depending on regulatory requirements, tests for compliance might be restricted to a single concentration (100% wastewater unless otherwise specified), or might require a series of concentrations (i.e., a multi-concentration test) including 100% wastewater (see Section 4.5). Single-concentration tests are often cost-effective for determining the presence of measurable toxicity, and also for screening a large number of samples.

Specific adaptations of the standard toxicity test could be adopted for special purposes such as locating in-plant sources of toxicity, or assessing the effectiveness of in-plant process changes or of effluent treatment. The tests could be multi-concentration or single-concentration (100% or an appropriate dilution, plus a control). Endpoints would depend on the objectives of the undertaking, but could include arbitrary "pass/fail" limits such as a maximum percent nonviable embryos or maximum percent mortality of alevins at a suitable time period. Section 4.5 provides relevant instructions on statistical analysis and reporting for sets of tests on different samples, each tested at only one concentration.