Biological test method for measuring terrestrial plants 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 (see Section 4.3).Footnote8 The test facility should be well ventilated to prevent personnel from being exposed 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 also be properly ventilated.

The test facility should be isolated from the area 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 sample handling and storage facilities, or those where chemicals are processed or tested, from contaminating the area of the laboratory where tests are conducted.

The mean air temperature of the test facility must be maintained at 24 ± 3 °C . However, for those test facilities with growth chambers that can accommodate a cyclical temperature change, it is recommended that the temperature be maintained at a mean air temperature of 24 ± 3 °C during the day, and 15 ± 3 °C at night. The relative humidity of the test facility should be maintained at a minimum of 50%.Footnote9

Any construction materials that might contact the organisms, water, or test vessels within this facility must be nontoxic 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 and 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 which can be set at 90 °C for drying test organisms and 105 °C for drying soils, a weighing balance accurate to the nearest 0.1 mg, a light meter, 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 being used. All nondisposable materials should be washed after use. The following cleaning procedure is recommended (EC, 1997a, b, 2001, 2004c):

  1. soak in tap water (with or without detergent added) for 15 minutes, then scrub with detergent or clean in an automatic dishwater;
  2. rinse twice with tap water;
  3. rinse carefully with fresh, dilute (10%, v:vFootnote10) or hydrochloric acid (HCl) (metal-free grade) to remove scale, metals, and bases;
  4. rinse twice with de-ionized water (or other test water);
  5. 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);Footnote11
  6. allow organic solvent to volatilize from dishware in fume hood and rewash with detergent (scrub if necessary); and
  7. 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 (hydration) water, immediately before being used in the test.

3.2 Initial and Definitive Tests

3.2.1 Initial Tests

Before definitive plant toxicity tests 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 a negative control soil (see Section 3.4) 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 each test species can be achieved in a candidate natural or artificial negative control soil using that laboratory and the procedures specified in this report.

The conditions and procedures used to perform these initial tests with negative control soil should be identical and according to Section 4. The conditions and procedures used to perform these initial tests with one or more reference toxicants should be identical and according to Section 4.9. Each set of initial tests with negative control soil or reference toxicant(s) should be performed on each terrestrial plant species intended for use in future definitive toxicity tests.

Data from the control performance tests (≥ 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 (≥ 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 Test

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 seedling growth for the duration of the test. It is important that the size, shape, colour, and composition of the vessel be appropriate for the plant species chosen. The vessels should have a sealable lid and should not interfere with light quality within. The test vessel recommended for emergence-and-growth tests with terrestrial plants described herein is a 1-L clear polypropylene container, with a clear polypropylene lid.Footnote12 Alternatively, 1-L glass jars sealed with transparent lids (e.g., hinged glass lids) may be used.Footnote13 The vessels must be covered for the first seven days of the test or until the plants reach the top of the container, whichever comes first.

3.3 Lighting

Tests should be illuminated using full-spectrum fluorescent or equivalent lighting. The light fluence rate, measured adjacent to the level of the soil surface, must be 300 ± 100 µmol/(m2 · s) (i.e., equivalent to 18 750 ± 6250 lux). Since light intensity tends to vary in a given space, it should be measured at several points within the testing area. The light fluence rate within the testing area should not vary by more than ± 15% of the selected light fluence rate.Footnote14

3.4 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 plants 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.Footnote15 There should also be prior experimental evidence that the soil chosen for use as negative control soil will sustain seedling emergence and growth that consistently and reliably meet the criteria for test validity defined herein (see Section 4.4).

The biological test method described herein has been developed and tested using five negative control soils with diverse physicochemical characteristics (Aquaterra Environmental, 1998a; Stephenson et al., 1999a, b, 2000a; Aquaterra Environmental and ESG, 2000; ESG, 2000, 2001, 2002; ESG and Aquaterra Environmental, 2002; EC, 2005b). These clean soils included one artificial soil and four natural soils (i.e., samples of sandy loam and silt loam agricultural soils from southern Ontario, a clay loam prairie soil from Alberta, and a forest loam soil from the Canadian Shield in northern Ontario). 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 were therefore considered to be “clean”. The origin and physicochemical characteristics of these natural soils are further described in Appendix G. The test validity criteria for the various plant species described in Section 4.4 are based on the performance data for these plants in negative control soil, that were generated for each of these five diverse soils (Aquaterra Environmental, 1998a; Stephenson et al., 1999a, b, 2000a; Aquaterra Environmental and ESG, 2000; ESG, 2000, 2001, 2002; ESG and Aquaterra Environmental, 2002; EC, 2005b).

3.4.1 Natural Soil

Negative control soil may be natural soil collected from a clean (uncontaminated) site which is known to have been free of pesticide or fertilizer applications for at least five years. 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 plant test species, to confirm that the test organisms are able to meet the criteria for test validity (see Section 3.2.1). Thereafter, and assuming that the preceding results for these preliminary tests are satisfactory, it is recommended that samples of natural soil selected for possible use as negative control soil in soil toxicity tests (as well as samples of candidate reference soil) be analyzed for the following physicochemical characteristics:

  • pH,
  • particle size distribution,
  • conductivity,
  • texture,
  • fertility,
  • total organic carbon content (%),
  • organic matter content (%),
  • cation exchange capacity,
  • major cations,
  • total nitrogen,
  • total phosphorus,
  • bulk density,
  • WHC,
  • metals,
  • petroleum hydrocarbons (including PAHs),
  • organophosphorus insecticides,
  • organochlorine insecticides, and
  • a suite of herbicides (e.g., atrazine and other triazine herbicides, picloram, acrolein, and sulfonylureas).

Pesticide and metal concentrations should not exceed the Canadian Soil Quality Guidelines, if available (see footnote 15). If seeds from a natural seedbank germinate in the sample(s) of natural soil at any time (i.e., during storage or testing), then these seedlings must be removed. If the results of both the preliminary 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 - 6 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 the collection and storage of soils if there are concerns about chemical constituents of the plastic leaching into the soil.

3.4.2 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).

In keeping with the formulation of artificial soil recommended by OECD (1984b, 2000b), USEPA (1989), ISO (1991, 1993b, 1998) and ASTM (1999c) for earthworm testing, and that recommended in Environment Canada’s soil toxicity tests using earthworms and springtails (EC, 2004c, 2005c), 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 through a 2-mm mesh screen (ASTM, 1999c)
  • 20% kaolin clay with particles <40 µm (ASTM, 1999c)
  • 70% “grade 70” silica sand (USEPA, 1989; ASTM, 1999c)

The ingredients should be mixed thoroughly in their dry form using a mechanical stirrer and/or gloved hands.Footnote16 Reagent-grade calcium carbonate should be added to the dry mixture in a quantity sufficient to attain a pH for the artificial soil ranging within 6.5 - 7.5 once it is hydrated.Footnote17 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 6.5 - 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. Footnote17 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.

3.5 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 terrestrial plants, 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 plants using specified test conditions and procedures. For the test method described herein, one or more reference toxicants must be used as a positive control soil 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 3.4) 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 plant species used 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 terrestrial plants according to the biological test method described herein.Footnote18

3.6 Reference Soil

One or more samples of reference soil might be included in a soil toxicity test using terrestrial plants.Footnote19 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) represent the sample(s) of test (contaminated) soil as much as possible. Ideally, the reference soil is collected near the site(s) where samples of test soil are collected, but it 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 plants, and their possession of physicochemical characteristics similar to the samples of test soil. The sample(s) of field-collected reference soil used in a study could be tested for toxic effects at full strength 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 testFootnote20 (see Sections 3.7 and 5.5, as well as the introductory comments in Section 4). 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 samples 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 percent 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 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 3.4). 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 5.5); 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.

3.7 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 terrestrial plants 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). 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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