Biological test method for measuring survival of springtails exposed to contaminants in soil: chapter 3
3.1 Facilities and Apparatus
Tests must be performed in an environmental chamber or equivalent facility having acceptable temperature and lighting control. The test facility should be well ventilated to prevent exposure of personnel to harmful fumes, and it should be isolated from physical disturbances or any contaminants that might affect the test organisms. The area used to prepare test soils should contain a fume hood and be properly ventilated.
The test facility should be isolated from the area where the springtails are cultured (Section 2.3) to avoid potential contamination. Additionally, the test facility should be removed from places where samples are stored or prepared, to prevent the possibility of contamination of test vessels and their contents from these sources. The ventilation system should be designed, inspected and operated to prevent air within the testing facility from contaminating the culturing facilities. Return air from sample handling and storage facilities or those where chemicals are processed or tested should not be circulated to the area of the laboratory where tests are conducted.
Any construction materials that might contact the organisms, water or test vessels within this facility must be nontoxic (see Section 2.3.2) and should minimize sorption of chemicals. Borosilicate glass, nylon, high-density polyethylene, high-density polystyrene, polycarbonate, fluorocarbon plastics, Teflon™, Nalgene™, porcelain, fibreglass and type 316 stainless steel should be used whenever possible to minimize chemical sorption and leaching. The use of toxic materials including copper, zinc, brass, galvanized metal, lead and natural rubber must be avoided.
The test facility must have the basic instruments required to monitor the quality (e.g., temperature, pH) of the test soil and associated test (hydration) water. Additionally, the laboratory should be equipped to facilitate prompt and accurate analysis of the moisture content of test soils. Equipment requirements include a drying oven that can be set at 105°C for drying soils, a weighing balance accurate to the nearest 0.1 mg, and a pH meter. Safety apparatus including a respirator with dust protection, gloves, laboratory clothing, and glasses for eye protection are required when preparing mixtures and aliquots of test soil.
All test vessels, equipment and supplies that might contact site soils, test soils, test (hydration) water, stock solutions or test solutions must be clean and rinsed with de- ionized or distilled water (i.e., test water) before use. All non-disposable materials should be washed after use. The following cleaning procedure is recommended (EC, 1997a, b, 2001, 2004a, 2005a):Footnote24
- soak in tap water (with or without detergent added) for 15 minutes, then scrub with detergent or clean in an automatic dishwater;
- rinse twice with tap water;
- rinse carefully with fresh, dilute (10%, v:vFootnote25) nitric (HNO3) or hydrochloric acid (HCl) (metal-free grade) to remove scale, metals and bases;
- rinse twice with de-ionized water (or other test water);
- rinse once with full-strength, pesticide-grade acetone to remove organic compounds and with reagent-grade (e.g., HPLC grade, ≥ 98.5% purity) hexane for oily residues (use a fume hood);Footnote26
- allow organic solvent to volatilize from dishware in fume hood and rewash with detergent (scrub if necessary); and
- rinse three times with de-ionized water (or other test water).
Test vessels and apparatus that might contact soil or test (hydration) water should be thoroughly rinsed with test water, before being used in the test.
3.2 Initial and Definitive Tests
3.2.1 Initial Tests
Before definitive soil toxicity tests, using the test method defined in Section 4, are performed for the first time by a testing laboratory, it is recommended that a minimum of five control performance tests with one or more samples of uncontaminated natural or artificial soil intended (or under consideration) for use in one or more definitive soil toxicity tests as negative control soil (see Section 3.3) be undertaken by laboratory personnel. Additionally, a minimum of five reference toxicity tests should be performed using one or more samples of a candidate artificial or natural negative control soil intended for routine use in conjunction with definitive soil toxicity tests (see Section 4.9). These initial tests are recommended to confirm that acceptable performance of the test species (F. candida, O. folsomi, F. fimetaria or P. minuta) can be achieved in a candidate natural or artificial negative control soil using that laboratory and the culturing conditions and procedures specified in this report (see Section 2.3).
The conditions and procedures used to perform these initial tests with negative control soil should be identical and according to Section 4, whereas the conditions and procedures used to perform the initial reference toxicity tests should be identical and according to Section 4.9. Each test with negative control soil or reference toxicant(s) should be performed using a different lot of test organisms of the same species from the same source.
Data from the control performance tests (n ≥ 5) must show that the criteria for test validity (see Section 4.4) can be met for the intended test species using a natural or artificial soil intended for use as negative control soil in a definitive soil toxicity test. Data from the initial reference toxicity tests (n ≥ 5) should be compared by calculating and appraising the magnitude of the coefficient of variation (CV) for the respective series of tests and endpoint values (see Section 4.9).
3.2.2 Definitive Tests
Test vessels to be used in definitive tests must be inert to test and reference substances or contaminant mixtures (i.e., the test or reference substances, or mixtures thereof, should not adhere to or react in any way with the test vessel). The volume of the vessel should be sufficiently large to accommodate springtail survival and reproduction for the duration of the test. Wide-mouthed glass jars (e.g., Mason canning jars), with a capacity of 100−125 mL (~5 to 8 cm in diameter), are to be used as test vessels. Each glass jar must be cleaned thoroughly before and after use, and rinsed well with de-ionized or other test water before use. Each test vessel should be covered with a plastic or metal lid (i.e., metal lid with rubber seal secured with metal screw-top ring). For tests with P. minuta, test vessels must be sealed with Parafilm® secured with a metal screw-top ring, to prevent the organisms from escaping from the test vessels.Footnote27 If at any time during the test the Parafilm® lid appears to be damaged or does not seal properly to the test vessel, it must be replaced with a new piece. Weekly replacement of the Parafilm® is recommended to ensure that the Parafilm® remains intact and the vessel remains sealed.
3.3 Negative Control Soil
Each soil toxicity test must include negative control soil as one of the experimental treatments. Negative control soil is essentially free of any contaminants that could adversely affect the performance of springtails during the test. The use of negative control soil provides a measure of test acceptability, evidence of the health and performance of the test organisms, assurance as to the suitability of the test conditions and procedures, and a basis for interpreting data derived from the test soils.
A soil toxicity test may use clean (uncontaminated) natural soil and/or artificial soil as the negative control soil. The selection of an appropriate negative control soil depends on considerations such as the study design, physicochemical characteristics of the test soil(s), and the availability of suitable clean natural soil with acceptable properties.Footnote28 For definitive tests with field-collected boreal forest and taiga soils, it is recommended that uncontaminated natural soil be used as the negative control soil. There should also be prior experimental evidence that the soil chosen for use as negative control soil will consistently and reliably meet the criteria for test validity defined herein for each test species.
The first edition of the biological test method described herein was developed and tested using five negative control soils with diverse physicochemical characteristics (AquaTerra Environmental Ltd., 1998; Stephenson et al., 1999a, b, 2000a; AquaTerra Environmental Ltd. and ESG, 2000; ESG 2000, 2001, 2002; ESG and AquaTerra Environmental Ltd., 2002, 2003; Becker-van Slooten et al., 2003, 2005; Stämpfli et al., 2005; EC, 2007a). These clean soils included one artificial soil and four natural soils (i.e., samples of sandy loam and silt loam agricultural soil from southern Ontario, a clay loam prairie soil from Alberta, and a forest loam soil from the Canadian ShieldFootnote29 in northern Ontario). The methodology described in this second edition test method document was further developed for testing boreal soils with P. minuta, using artificial soil and eight natural soils collected from Canada’s Boreal Region, including: Gleyed Humo-ferric Podzols from Newfoundland, New Brunswick and Ontario; a Dark Grey Luvisol, an Ortho Eutric Brunisol, and Eluviated Dystric Brunisol from Saskatchewan; and a Rego Humic Gleysol and Rego Dark Grey Chernozem from Alberta. These soils differed in composition with respect to the physicochemical characteristics that could potentially influence the fate and effects of contaminants. All of the field-collected soils originated from uncontaminated areas that had not been subjected to any direct application of pesticides in recent previous years, and therefore were considered to be “clean.” The origin and physicochemical characteristics of these natural soils are further described in Appendix G. The test validity criteria for F. candida, O. folsomi, F. fimetaria and P. minuta described in Section 4.4 are based on the performance data for these springtails in negative control soil that were generated for each of these diverse soils (AquaTerra Environmental Ltd., 1998; Stephenson et al., 1999a, b, 2000a; AquaTerra Environmental Ltd. and ESG, 2000; ESG, 2000, 2001, 2002; ESG and AquaTerra Environmental Ltd., 2002, 2003; Becker-van Slooten et al., 2003, 2005; Stämpfli et al., 2005; EC, 2007a, 2010, 2013b), among others (Krogh, 2004).
3.3.1 Natural Soil
Negative control soil may be natural soil collected from a clean(uncontaminated) site that is known to have been free of pesticide or fertilizer applications for at least five years. The source of this negative control soil might be the same as that where springtails were collected to establish a culture (Section 2.2). Before using a sample of clean field-collected soil as negative control soil in a definitive toxicity test, the test laboratory must have previous experimental evidence showing that natural soil from this source can meet the criteria that must be achieved for the results of a toxicity test to be considered valid (see Section 4.4). Accordingly, initial tests involving a sample of this soil must be performed using the intended Collembola test species, to confirm that the test organisms are able to meet the criteria for test validity that apply to the particular test species being used (see Section 3.2.1). Thereafter, and assuming that the preceding results for these initial bioassays are satisfactory, all samples of natural soil selected for possible use as negative control soil in soil toxicity tests (as well as samples of candidate reference soil) must be analyzed for the following physicochemical characteristics:
- particle size distribution (% sand, % silt and % clay),
- total organic carbon content (%),Footnote30
- organic matter content (%),Footnote30
- moisture content (%),
- water-holding capacity (WHC), and
- cation exchange capacity (CEC).
Additionally, the following analyses should be performed:
- major cations and anions (Na+, K+, Mg2+, Ca2+, Al3+, S2-, Cl-),
- nitrogen as total N, nitrate (NO3-), nitrite (NO2-) and ammonium (NH4+),
- phosphorus as total and/or bioavailable,
- potassium as total and/or bioavailable, and
- C:N ratio.
In order to confirm that the negative control and/or reference soils are not contaminated, the following screening analyses are recommended:
- organophosphorus insecticide suite,
- organochlorine insecticide suite,
- herbicide suite,
- metal suite,
- petroleum hydrocarbons (including PAHs), and
- other site-or area-specific contaminants of concern.
Pesticide and metal concentrations should not exceed CCME soil quality criteria, if available (see footnote 28). If indigenous organisms are present and/or problematic in the sample(s) of natural soil at any time (i.e., during storage or testing), their presence (e.g., physical description and estimated numbers) should be recorded, and they should be removed manually (e.g., by sieving), if possible. If the results of both the initial biological tests and the physicochemical analyses are satisfactory, a larger sample of this natural soil can be collected, air dried to a moisture content of between 10 and 20%, coarse-screened (4−10 mm), transferred to clean, thoroughly rinsed plastic pails, and stored in darkness at 4 ± 2°C until required. Plastic pails should not be used for collection and storage of soils if there are concerns about chemical constituents of the plastic leaching into the soil.
3.3.2 Artificial Soil
Negative control soil may be artificial soil formulated in the laboratory. The use of artificial soil offers a consistent, standardized approach and is advantageous when testing the toxicity of chemicals or chemical products spiked in negative control soil (Section 6).
The formulation of artificial soil used internationally in various soil toxicity test method documents, using springtails, is very similar. Appendix F (Tables 4, 5 and 6) provides a summary of the ingredients and preparation of artificial soil recommended in various methods (Wiles and Krogh, 1998; ISO, 1999; OECD, 2005) for use as negative control soil in laboratory tests of the effects of contaminated soil on the survival and reproduction of springtails.
In keeping with the formulation of artificial soil used by Wiles and Krogh (1998), ISO (1999), Greenslade and Vaughan (2003), OECD (2005),Footnote31 and in three other Environment Canada soil toxicity test methods (EC, 2004a, 2005a, 2013a), the following ingredients should be used to prepare artificial soil to be used in the biological test method described herein:
- 10% Sphagnum sp. peat, air dried and sieved (e.g., through a 2-mm mesh screen),
- 20% kaolin clay with particles < 40 μm, and
- 70% “grade 70” silica sand.
The ingredients should be mixed thoroughly in their dry form using a mechanical stirrer and/or gloved hands.Footnote32 Reagent-grade calcium carbonate should be added to the dry mixture in a quantity sufficient to attain a pH (measured using a calcium chloride slurry method; see Section 4.6) for the artificial soil ranging within 6.0−7.5, once it is hydrated.Footnote33 Thereafter, the mixture should be hydrated gradually using test water (i.e., de- ionized or distilled water) until its moisture content is ~20% (which is ~28% of the soil’s water-holding capacity), while mixing further until the soil is visibly uniform in colour and texture. As necessary, reagent-grade calcium carbonate should be added to the hydrated mixture in a quantity sufficient to maintain a pH ranging within 6.0−7.5. Samples of pH-adjusted artificial soil should be stored in darkness at 20 ± 2°C for a minimum of three days before being used in a toxicity test, to enable adequate time for pH equilibration.Footnote33 Thereafter, artificial soil can be stored at 4 ± 2°C. As and when required for a soil toxicity test, a suitable quantity of stored artificial soil should be hydrated further using test water until its moisture content is ~70% of the water-holding capacity.
3.4 Positive Control Soil
The use of one or more samples of positive control soil is recommended for inclusion in each series of soil toxicity tests with springtails, to assist in interpreting the test results. In choosing a positive control soil, the intent is to select a toxic soil that will elicit a response in the test organisms which is predictable based on earlier toxicity tests with this material. The positive control soil might be a sample of negative control soil that is spiked with a reference toxicant for which historic data are available on its toxicity to springtails using the specified test conditions and procedures. For the biological test method described herein, one or more reference toxicants must be used when appraising the sensitivity of the test organisms and the precision and reliability of results obtained by the laboratory for that material (see Section 4.9). A test might also include a sample of negative control soil (natural or artificial; see Section 3.3) that has been spiked experimentally (Section 6) with one or more toxic chemicals or chemical products of particular concern when evaluating the sample(s) of test soil, at a concentration toxic to the Collembola species used, and according to the biological test method described herein. In some instances, a test might include a positive control soil that is comprised of a highly contaminated sample of field-collected soil or sludge shown previously to be consistently toxic to springtails according to the biological test method described herein.Footnote34
3.5 Reference Soil
One or more samples of reference soil might be included in a soil toxicity test using springtails. The type and nature of the sample(s) of soil used as reference soil in a particular study depend on the experimental design and the study’s objectives. If the toxicity of samples of field-collected soil from a contaminated or potentially contaminated site is under investigation, the reference soil included in the study might be one or more samples of field-collected soil taken from a clean (uncontaminated) site where the physicochemical properties (e.g., organic carbon content, organic matter content, particle size distribution, texture, pH and conductivity) represent the sample(s) of test (contaminated) soil as much as possible. Ideally, the reference soil is collected from the general vicinity of the site(s) where samples of test soil are collected, but is removed from the source(s) of contamination. One or more samples of field- collected clean reference soil from sites removed from the test site(s) might also be chosen due to their known lack of toxicity in previous tests with springtails, and their possession of physicochemical characteristics similar to the samples of test soil. Boreal forest and taiga reference soils must be collected as separate soil horizons, where possible. Each soil horizon is then stored and tested individually (i.e., each horizon is treated as a separate soil sample). The sample(s) of field-collected reference soil used in a study could be tested for toxic effects as undiluted soil only, or this soil could be mixed with the sample(s) of test soil to prepare a range of concentrations to be included in a multi-concentration testFootnote35 (see Sections 3.6, 4.1 and 5.3). Samples of reference soil should not be collected from sites known to have received applications of pesticides or fertilizers within the past five years or more.
An investigator might choose to include one or more samples of artificial soil as reference soil in a particular test. For instance, these could be used in multi-concentration tests with site soils or chemical-spiked soils to investigate the influence of certain physicochemical characteristics (e.g., a number of artificial reference soils prepared to provide a range of differing values for texture and/or organic matter content (%); Sheppard and Evenden, 1998; Stephenson et al., 2002) on the toxicity of a contaminated site soil or a chemical-spiked soil. Multiple samples of clean field-collected soil collected from various sites, which differ markedly with respect to one or more physicochemical characteristics, might also be used for this purpose. For such a study, a portion of each reference soil used to prepare a series of concentrations of the test soil should be included in the test without dilution (i.e., 100% reference soil).
Each test involving one or more samples of reference soil must include a sample of negative control soil (see Section 3.3). Conversely, certain tests (e.g., one involving a series of concentrations of chemical-spiked soil prepared using artificial or natural negative control soil) need not involve a sample of reference soil. For tests with field-collected site soil, the inclusion of one or more samples of reference soil from a neighbouring site is a preferred approach for comparative purposes (see Section 5.6); a decision to dilute site soil with reference soil (rather than negative control soil) when preparing multiple concentrations for testing depends on the study objectives.
3.6 Test Soil
This biological test method is intended to measure the toxicity of one or more samples or mixtures of contaminated or potentially contaminated soil (test soil), using springtails as test organisms. The sample(s) of test soil might be either field-collected soil from an industrial or other site of concern, or industrial or municipal biosolids (e.g., dredged material, municipal sludge from a sewage treatment plant, composted material or manure) under consideration for possible land disposal. A sample of field-collected test soil might be tested at a single concentration (typically, 100%) or evaluated for toxicity in a multi- concentration test whereby a series of concentrations are prepared by mixing measured quantities with either negative control soil or reference soil (see Section 5).Footnote36
Field-collected soils collected by horizon take into account contamination stratified due, in part, to the different speciation and resultant mobility of contaminants (EC, 2012).
Therefore, for soils collected from the boreal or taiga ecozones, both reference and contaminated soils must be collected in separate horizons. Soils collected in horizons are treated as individual soil samples and tested separately (see Section 4.1). Soils without distinct soil horizons (e.g., where the surface soil horizons have been mixed or disturbed due to human activity) are collected according to depth (see Section 5.1). The test soil might also be one or more concentrations of a chemical-spiked soil, prepared in the laboratory by mixing one or more chemicals or chemical products with negative control soil, reference soil or site soil (see Section 6).
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