Biological test method: fertilization assay using echinoids (sea urchins and sand dollars), chapter 10
Section 8: Specific Procedures for Testing Samples of Liquid Extracted from Sediment or Similar Material
- 8.1 General Aspects of Procedure
- 8.2 Testing Liquids Extracted from Sediments and Similar Solids
General instructions are given here for testing liquids derived from samples of sediment or similar solids such as sludge or soil. These are in addition to the general instructions provided in Section 4. In this section, the word “sediment” is used for convenience but should be taken to include other similar solid materials such as soils and industrial or municipal sludges, which might contribute pollutants to natural waters or require testing for other reasons.
8.1 General Aspects of Procedure
When the first edition of this test method document was published, assessing the toxicity of sediments was becoming widely recognized as an important part of environmental protection, notably when considering ocean dumping, such as under Part VI of the Canadian Environmental Protection Act. Since its publication in 1992, this sublethal test has been used on a regular basis on samples of sediment pore water, as part of the Disposal at Sea Program, to help evaluate the suitability of dredged material for disposal at sea (CEPA, 1999; Government of Canada, 2001). However, the potential influence of confounding factors (e.g., ammonia and sulphides) in sediment pore water, on the interpretation of results, has contributed to uncertainty with the application of this test from a regulatory perspective (Scroggins et al., 2003). As a result, Environment Canada initiated some method-improvement and method-development research which has led to further guidance for the improvement of the echinoid fertilization assay for porewater testing, described herein. Additionally, Environment Canada is currently developing a new sediment-contact embryo/larval test for measuring the toxicity of samples of solid-phase sediment to the early life stages of echinoids (Buday, 2006; Jackman and Doe, 2004, 2006; McLeay, 2007).
The echinoid fertilization assay described herein is suitable for testing the toxicity of liquids derived from sediments. It provides a rapid method for comparing extracts of contaminated sediments (Long et al., 1990). Tests on sediment-derived liquids showed that an echinoderm embryo test and the bacterial Microtox test were the most sensitive of seven sediment tests evaluated (Pastorok and Becker, 1989). Sublethal toxicity tests including a fertilization assay using Arbacia and interstitial water from sediments, were considerably more sensitive than a standard test on the whole sediment using amphipods (Carr and Chapman, 1992).
General guidance is given here on application of the echinoid fertilization assay for testing liquids derived from sediments. It is not the purpose of this report to provide instructions for carrying out a field survey of sediments, sampling them, or extracting aqueous or other material from them. Detailed guidance for the collection, handling, transport, and storage of sediment samples (Section 8.1.1) is provided in Environment Canada (1994). The same Environment Canada document provides procedural details for the extraction of liquids [i.e., pore water, elutriate, or solvent extract (Section 8.2)] from sediments for subsequent toxicity tests and chemical analyses. This guidance document should be consulted and followed, in addition to the guidance provided here. Detailed information regarding the basis of such guidance can be found in books (e.g., Mudroch and MacKnight, 1991) and in primary literature cited in Environment Canada (1994) or related reviews of this subject matter (e.g., Geisy and Hoke, 1989; McLeay and Sprague, 1991).
This fertilization assay is not suitable for testing the whole sediments themselves (i.e., the solids), but is useful for liquids derived from those solids, whether leachate, elutriate, solvent extract, or pore water.
Based on the results of an inter-laboratory study carried-out in 2008 to investigate the improvement of the porewater testing component of EPS 1/RM/27, the following sections of this second edition method document offer some additional guidance for the testing of samples of sediment pore water (Miller, 2008). These procedures may be included to help reduce the potential influence of confounding factors on test results in porewater fertilization tests. This additional guidance is based on methodologies developed at the Columbia Environmental Research Centre, Marine Ecotoxicology Research Station (Carr, Nipper, and Biendenbach, pers. comm., 2008; Carr and Chapman, 1992, 1995; Carr et al., 2006). It includes guidance on the following: the use of a control pore water in the pre-test to establish the sperm:egg ratio to be used in the definitive test (see Section 4.2.3); and the use of both a control pore water and one or more samples of reference pore water, for determining the toxicity (or lack thereof) in porewater samples from contaminated sites or sites of interest (see Sections 8.1.4, 8.2.1, 8.2.2 and 8.2.3).
8.1.1 Sample Labelling, Transport and Storage
General procedures for labelling, transporting, and storing sediment samples should be as described in Section 6.1. Temperature limits are those described in Section 6.1, and samples must not freeze or partially freeze, or be allowed to dry (ASTM, 1991b; EC, 1994).
For the liquids derived from sediments, containers and handling procedures should be the same as those given in Section 6.1 for elutriates. If a non-aqueous solvent has been used to extract substances or materials, a glass container should be used to store the liquid, so that it will not be affected by the solvent or leach substances into the sample.
Testing of samples should start as soon as possible after collection. Extraction procedures should begin within two weeks of sampling, and preferably within one week. Testing must start no later than six weeks after collection of samples (EC, 1994).Footnote 76 Testing of the liquid obtained from sediments should begin within 1 day of making such preparations (EC, 1994) and must begin within 3 days, unless specified otherwise in a regulation or other designated procedure.
8.1.2 Preparing Samples
Depending on the nature of the sample and the objectives of the test, homogenization of a sample might or might not be required before testing. If mixing is carried out, it must be thorough. Sub-samples (i.e., a sample divided between two or more containers) must be mixed together (i.e., composited). If further sample storage is required, the composited sample, or a portion of it, should be returned to the sub-sample containers and stored.
8.1.3 Observations and Measurements on Sample
Observations of the colour, turbidity, foaming, precipitation, etc. should be made on both the sediment and any liquid derived from it, during preparation of test solutions, as described in Section 6.4.
8.1.4 Liquid from Control and Reference Sediments
Liquid (pore water) extracted from one or more samples of control and/or reference sediment (unpolluted)Footnote 77 must be assessed in the same manner as that extracted from the sediment under investigation. The liquids derived from the reference sediment and the control sediment serve different purposes. Pore water extracted from a reference sediment serves as a field reference or a site reference, for a comparison of the biological effects observed in the test sediment pore water. The reference pore water should be used for comparative purposes whenever possible or appropriate, because this provides a site-specific evaluation of toxicity. Using other kinds of tests, however, Environment Canada and other laboratories have frequently recorded apparent toxic effects with pore water from unpolluted reference sediments, or the reference pore water is unidentified in the case of coded or blind samples, making it unsuitable for comparison. In such cases, it would be necessary to compare the biological effects for the test pore water with those for the control sediment pore water. Accordingly, pore water derived from one or more control (“clean”) sediments should be included as a sample, with each test of pore water(s) derived from sediment (or series of sediments), to help establish a baseline or “normal” level. It would be desirable to establish a standard, clean, “control sediment” for this purpose, or ideally a series of reference sediments of differing characteristics that could be matched with those for the sediments being tested.
Ideally, the physicochemical characteristics of a reference sediment(s) should be similar to those of the sediment(s) being investigated, and should account for the confounding factors (i.e., not the contaminants of concern) that might be present in the sample pore waters (Scroggins et al., 2003).Footnote 78 However, it is also important that some type of clean (i.e., control) pore water be used routinely in porewater tests to provide a basis for interpreting data derived from the test. A control pore water might be collected from a sediment that is low in contaminants, and contains low levels of ammonia, and sulfides. Once a good (i.e., clean, stable, previously shown to support good fertilization etc.) control sediment is located, large volumes of pore water can be collected and stored (i.e., frozen) for future porewater tests. The control sediment pore water serves a different purpose than the laboratory seawater control (i.e., control/dilution water) which provides a direct measure of the test validity. Typically, both types of controls (i.e., the laboratory seawater control and the porewater control using pore water extracted from a suitable control sediment) should be used in toxicity tests for pore water from contaminated sediments (Scroggins et al., 2003), along with a suitable porewater sample from one or more reference sites, since experience indicates that control water (i.e., laboratory seawater) alone might not be sufficient for an acceptable evaluation of porewater toxicity.
There is no single procedure for making use of the results from the control sediment and reference sediment (i.e., pore water), however, if a control pore water and a reference pore water are included in the test, are handled identically to the test pore water (see Sections 8.2.1 and 8.2.2), and run concurrently with the test pore water, tests for significant differences between the results for the reference/control and test sediments can be carried out (i.e., the control pore water can be used as a baseline for determining the presence or absence of toxicity at each treatment level [see Section 8.2.3]; and the reference pore water, if not blind or coded, can provide a site-specific evaluation of toxicity). Caution should be exercised in interpreting findings for the test substance or material, and such tests should be carried out with guidance from a statistician. Sometimes, neither a reference pore water nor a control porewater sample are available. In these situations, it might be necessary to use the laboratory water control (i.e., control/dilution water) for comparison with porewater test results for environmental samples, however this approach is less preferred.Footnote 79 If only control/dilution water is used as a basis of comparison, there is no control for confounding factors, and interpretation of test results might require an understanding of the tolerance limits of the test species for potential confounding factors in the test samples. Also, any site-specific evaluation of toxicity would be limited.
8.2 Testing Liquids Extracted from Sediments and Similar Solids
Toxicants from sediments or soils can enter an aqueous phase and affect organisms in natural waters. The aqueous phase might be a liquid derived from a soil or sediment (e.g., interstitial or pore water), or a liquid used to treat the sample and extract potential toxicants (e.g., an elutriate).
A liquid obtained from sediment for toxicity testing would be expected to fall into one of four broad categories.
- Interstitial water, which fills the spaces between particles (i.e., pore water), and could exchange with the overlying water making up the estuary, bay, etc. It is normally obtained from a sediment by centrifuging or squeezing it (ASTM, 1991b).
- Water that is essentially fresh water, used to obtain an aqueous extract of materials from the sediment (i.e., elutriate), for example by shaking a sample with added clean water. This category is not very appropriate for the echinoid fertilization assay.
- Control/dilution water or other clean water which has a salinity equivalent to seawater, used to obtain an aqueous extract as in (2).
- Solvents other than water (e.g., organic solvents), used to remove substances or materials from the sample of sediment (Schiewe et al., 1985; True and Heyward, 1990).
The water of the first three categories could be tested as a normal liquid sample, following the universal procedures given in Section 4 and the procedures for effluents, leachates, and elutriates as given in Section 6. For the fourth category (i.e., solvents), the preferred option is to have the same concentration of solvent in each of the test vessels. The control/dilution water to be used in the test is brought to the same concentration of solvent as that in the highest concentration of sample that will be tested. Lower concentrations are prepared using the modified control/dilution water, so that the solvent effect, if any, should be the same in all vessels. There should be a control without solvent, as well as the one with solvent. It would be desirable to run a separate test to determine the ICp of the solvent.
8.2.1 Preparing Test Samples
Specific guidance is given in Environment Canada (1994) for the extraction of liquids (i.e., pore water, elutriate, or solvent extract) from sediment. This guidance should be consulted and followed when preparing sediment extracts for echinoid fertilization assays, and also applies to the preparation of control and reference pore water.Footnote 80
Compositing of “sub-samples” of liquid obtained from the sediment (e.g., successive extractions) should be as described in Section 6.2. Sub-samples should not be composited if the relative toxicity of successive extractions was to be ascertained. Samples or sub-samples of elutriate or pore water should be centrifuged to remove suspended solids (EC, 1994). Only control porewater samples may be frozen for the purpose of long-term storage. Control porewater samples may be frozen at -20°C and stored at that temperature for up to one year as long as they are particle-free before freezing. To obtain particle-free pore water, control porewater samples should be centrifuged twice (e.g., centrifuge sediment sample at ~3000 × g for 15 min at 4°C, remove pore water being careful not to remove particles near sediment layer, and then centrifuge pore water at ~3000 × g for 15 min at 4°C). Samples should be thawed the day before they are to be used in a test, and should be centrifuged after thawing to remove any precipitated suspended particulate matter which can inhibit the ability of the echinoid sperm to locate and fertilize eggs (Carr and Chapman, 1995). The pH and dissolved oxygen content of the sample should be checked with regard to the limits in Sections 4.1.2 and 4.1.3.
Once the liquid has been obtained, test concentrations are prepared in the standard manner (Section 4.1.2). As in testing effluents, leachates, and elutriates, there could be a single-concentration test (plus control) for regulatory purposes, or a multi-concentration test to determine the ICp (Section 6.5). The procedures for obtaining and testing liquid from samples of control or reference sediment should be identical to those used for the test sediment.
8.2.2 Control/Dilution Water
If the sediment sample is marine or estuarial, and the water derived from it is essentially seawater in the standard range of salinity for this test (i.e., 28 to 32 g/kg), the Universal procedures of Section 4 would be followed.
For freshwater samples derived from sediment, or if the water derived from the sediment has salinity lower than that of full-strength seawater (i.e., similar to estuarial water), its salinity would normally be adjusted upwards. The standard procedure would be to adjust the salinity of all test and control solutions to the same value (within 1 g/kg of each other in the range 28 to 32 g/kg), usually accomplished by adjusting an aliquot of the sample as described in Section 4.3.2. Procedures using commercially-available dry ocean salts, reagent-grade salts, or natural or artificial hypersaline brine are recommended, as outlined in Section 2.3.4.
The same control/dilution water (e.g. laboratory seawater) must be used for the reference and/or control pore water, and the test pore water dilutions and controls in multi-concentration porewater tests. In addition, the reference and/or control pore water, and the test pore water must include the same treatments (i.e., porewater dilutions) and should include the same number of replicates for each treatment (i.e., to facilitate tests for significant differences between the results for the pore waters).
8.2.3 Endpoints and Calculations
To test for significant differences between the reference and/or control pore water and the test pore water, percent fertilization in each test porewater treatment is compared to the equivalent reference and/or control porewater treatment using Dunnett’s t-test.Footnote 81
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