Biological test method for measuring terrestrial plants exposed to contaminants in soil: chapter 6

Section 6 - Specific Procedures for Testing Chemical-Spiked Soil

• 6.1 Sample Properties, Labelling, and Storage
• 6.2 Preparing Test Mixtures
• 6.3 Test Observations and Measurements
• 6.4 Test Endpoints and Calculations

This section gives guidance and instructions for preparing and testing negative control soil spiked experimentally with chemical(s) or chemical product(s). These recommendations and instructions apply to the biological test method described in Section 4. Guidance in EC (1995) for spiking negative control sediment with chemical(s) and conducting toxicity tests with chemical/sediment mixtures is also relevant here, for chemical-spiked soil. Further evaluation and standardization of procedures for preparing chemical-spiked soil provided herein (Section 6.2) might be required before soil toxicity tests with plants or other appropriate soil organisms are applied to evaluate specific chemical/soil mixtures for regulatory purposes.

The cause(s) of soil toxicity and the interactive toxic effects of chemical(s) or chemical product(s) in association with otherwise clean soil can be examined experimentally by spiking negative control soil (Section 3.4) with these substances. The spiking might be done with one or more chemicals or chemical products. Other options for toxicity tests with plants, performed using the procedures described herein, include the spiking of chemical(s) or chemical product(s) in reference soil (Section 3.6) or test soil (Section 3.7). Toxicity tests using soil spiked with a range of concentrations of test chemical(s) or chemical product(s) can be used to generate data that estimate statistical endpoints based on threshold concentrations causing specific sublethal effects (see Section 4.8).

In Section 6.2, procedures are described for preparing test mixtures of chemical-spiked soil. Section 6.3 describes making observations and measurements during and at the end of the toxicity test, and Sections 4.8 and 6.4 provide procedures for estimating test endpoints for multi-concentration tests. These procedures also apply to the mixing of multiple concentrations of field-collected test soil (including particulate waste material such as sludge or other dredged material intended for land disposal) in negative control soil or reference soil, and to performing multi-concentration tests and determining statistical endpoints for these mixtures (see Section 5, and especially 5.5). Multi- concentration tests with positive control soil (Section 3.5) or one or more reference toxicants spiked in negative control soil (Section 4.9) are also performed using the procedures and statistical guidance described in this section. Additionally, the influence of the physicochemical characteristics of natural or artificial negative control soil on chemical toxicity can be determined with spiked-soil toxicity tests according to the procedures and statistical guidance described in this section.

6.1 Sample Properties, Labelling, and Storage

Information should be obtained on the properties of the chemical(s) or chemical product(s) to be spiked experimentally in the negative control soil.^Footnote71 Information should also be obtained for individual chemicals or chemical products (e.g., pesticides or other commercial formulations) on their concentration of major or “active” ingredients and impurities, water solubility, vapour pressure, chemical stability, dissociation constants, adsorption coefficients, toxicity to humans and terrestrial organisms, and biodegradability. Where aqueous solubility is in doubt or problematic, acceptable procedures previously used for preparing aqueous solutions of the chemical(s) should be obtained and reported. If an acceptable procedure for solubilizing the test chemical(s) in water is not available, preliminary testing for its solubility in test water of a non-aqueous solvent should be conducted and confirmed analytically. Other available information such as the structural formulae, nature and percentage of significant impurities, presence and amounts of additives, and n-octanol:water partition coefficient, should be obtained and recorded. Any pertinent Material Safety Data Sheets (MSDSs) should be obtained and reviewed.

Chemical(s) to be tested should be at least reagent grade, unless a test on a formulated commercial product or technical grade chemical(s) is required. Chemical containers must be sealed and coded or labelled upon receipt. Required information (chemical name, supplier, date received, person responsible for testing, etc.) should be indicated on the label and/or recorded on a separate datasheet dedicated to the sample, as appropriate. Storage conditions (e.g., temperature, protection from light) are frequently dictated by the nature of the chemical.

6.2 Preparing Test Mixtures

On the day of the start of the toxicity test (i.e., Day 0), the mixture(s) of chemical(s) or chemical product(s) spiked in negative control soil should be prepared and transferred to test vessels (see Sections 4.1 and 4.2). Each batch of test soil representing a particular treatment (concentration) should be prepared in a quantity sufficient to enable all test replicates of that treatment (concentration) to be set up along with any additional replicates or quantities required for physicochemical analyses (Section 6.3) or the performance of other soil toxicity tests using earthworms or other soil organisms (e.g., those performed according to EC, 2004c or EC, 2005c).

The use of artificial soil (Section 3.4.2) to prepare each test mixture is recommended because it offers a consistent, standardized soil for comparing results for other chemicals or chemical products tested similarly in the same laboratory or by others (e.g., according to USEPA, 1989, ISO, 1993a, 1995; ASTM, 1999b; and OECD, 2000a). If used, the formulation for artificial soil provided in Section 3.4.2 should be followed. The quantity of artificial soil required for the test(s) should be prepared, hydrated to ~20% moisture content, adjusted if and as necessary to a pH within the range of 6.5 - 7.5^Footnote72, aged for a minimum three-day period, and stored at 4 ± 2 °C until required (see Section 3.4.2). The final moisture content (including that due to the addition of a measured aliquot of a test chemical or chemical product dissolved in test water, with or without an organic solvent) of any chemical-spiked soil prepared using artificial soil should be ~70% of the water-holding capacity of the final mixture (Section 3.4.2), for each treatment (concentration).^Footnote73 The final moisture content of each mixture (treatment) included in a test should be as similar as possible.

Investigators may choose to use natural control soil (Section 3.4.1) rather than artificial control soil (Section 3.4.2) as the negative control soil to be spiked with chemical(s) or chemical product(s) and for the corresponding replicates of control soil to be included in the test. Procedures described herein for artificial soil apply equally if natural soil is used. An exception is that the final moisture content of each batch of chemical-spiked soil (including control batches) prepared using field-collected soil should be adjusted to the optimal percentage of its WHC using guidance in Section 5.3. The volume of soil in each test vessel might also differ if natural soils are used, due to differences in bulk density of the various soils that might be used.

The procedure to be used for experimentally spiking soil is contingent on the study objectives and the nature of the test substance to be mixed with negative control soil or other soil. In many instances, a chemical/soil mixture is prepared by making up a stock solution of the test chemical(s) or chemical product(s) and then mixing one or more measured volumes into artificial or natural negative control soil (Section 3.4). The preferred solvent for preparing stock solutions is test water (i.e., de- ionized or distilled water); use of a solvent other than 100% test water should be avoided unless it is absolutely necessary. For test chemical(s) or chemical product(s) that do not dissolve readily in test water, a suitable water-miscible organic solvent of relatively low toxicity (e.g., acetone, methanol, or ethanol) may be used in small quantities to help disperse the test substance(s) in water. Surfactants should not be used.

If an organic solvent is used, the test must be conducted using a series of replicate test vessels containing only negative control soil (i.e., 100% artificial or natural clean soil containing no solvent and no test substance), as well as a series of replicate test vessels containing only solvent control soil (OECD, 1984a, 2000a; ISO, 1993a, 1995; ASTM, 1999b; EC, 2000). For this purpose, a batch of solvent control soil must be prepared containing the concentration of the solubilizing agent that is present in the highest concentration of the test chemical(s) or chemical product(s) in soil. Solvent from the same batch used to make the stock solution of test substance(s) must be used. Solvents should be used sparingly because they might contribute to the toxicity of the prepared test soil. The maximum concentration of solvent in the soil should be at a concentration that does not affect the emergence or growth of plants during the test. If this information is unknown, a preliminary solvent only test, using various concentrations of solvent in negative control soil, should be conducted to determine the threshold-effect concentration of the particular solvent being considered for use in the definitive test.

For tests involving the preparation of concentrations of chemical spiked in artificial soil, in which the chemical is insoluble in water but soluble in an organic solvent, the quantity of test substance needed to prepare a required volume of a particular test concentration should be dissolved in a small volume of a suitable organic solvent (e.g., acetone). This chemical-in-solvent mixture should then be sprayed onto or mixed into a small portion of the final quantity of fine quartz sand that is required when preparing each test concentration comprised of a measured amount of a particular chemical-in- solvent mixture spiked in artificial soil (see Section 3.4.2). The solvent is then removed by evaporation by placing the container under a fume hood for at least one hour, and until no residual odour of the solvent can be detected. Thereafter, the chemical- in-sand mixture (with solvent evaporated) is mixed thoroughly with the remaining quantity of pre- moistened sand and other ingredients required to make up artificial soil (Section 3.4.2). An amount of de-ionized water necessary to achieve a final moisture content of approximately 70% of the maximum water-holding capacity for this artificial soil is then added and mixed with the soil/sand/peat mixture. The chemical-spiked soil can then be added to the test vessels (OECD, 2000a).

The sample of solvent control soil to be included in the test must be prepared using the same procedure but without the addition of the test chemical. Additionally, the solvent control soil must contain a concentration of solvent that is as high as that in any of the concentrations of chemical-spiked soil included in a test.

If the test chemical to be spiked in artificial soil is insoluble in both water and any suitable (non-toxic) organic solvent, a mixture should be prepared which is comprised of 10 g of finely ground industrial quartz sand and the quantity of the test chemical necessary to achieve the desired test concentration in the soil. This mixture should then be mixed thoroughly with the remaining constituents of the pre-moistened artificial soil. An amount of de- ionized water necessary to achieve a final moisture content of ~70% of the maximum water-holding capacity is then added and mixed in. The resulting mixture of chemical-spiked soil can then be added to the test vessels (OECD, 2000a).

Concentrations of chemical(s) or chemical product(s) in soil are usually calculated, measured, and expressed as mg test substance/kg soil (or µg substance/g soil) on a dry-weight basis (OECD, 1984a; ISO, 1993a, 1995). The assessment endpoints (e.g., ICps) are similarly expressed on a dry-weight basis (Section 4.8).

Mixing conditions, including test solution:soil ratio, mixing and holding time, and mixing and holding temperature, must be standardized for each treatment included in a test. Time for mixing a spiked soil should be adequate to ensure homogeneous distribution of the chemical, and may be from minutes up to 24 h.  During mixing, temperature should be kept low to minimize microbial activity and changes in the mixture's physicochemical characteristics. Analyses of subsamples of the mixture are advisable to determine the degree of mixing and homogeneity achieved.

For some studies, it might be necessary to prepare only one concentration of a particular mixture of negative control (or other) soil and chemical(s) or chemical product(s), or a mixture of only one concentration of contaminated soil or particulate waste in negative control or other soil. For instance, a single-concentration test might be conducted to determine whether a specific concentration of chemical or chemical product in clean soil is toxic to the test organisms. Such an application could be used for research or regulatory purposes (e.g., “limit” test).

A multi-concentration test, using a range of concentrations of chemical added to negative control soil (or other soil) under standardized conditions, should be used to determine the desired endpoint(s) (i.e., EC50 and ICp; see Section 4.8) for the chemical/soil mixtures. A multi-concentration test using negative control soil spiked with a specific particulate waste might also be appropriate. At least nine test concentrations plus the appropriate control treatment(s) must be prepared for each multi-concentration test, and more (i.e., ≥ 11 plus controls) are recommended (see Sections 4.1 and 4.8). When selecting the test concentrations, an appropriate geometric dilution series may be used in which each successive concentration of chemical(s) or chemical product(s) in soil is at least 50% of the previous one (e.g., 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.63 mg/kg). Test concentrations may also be selected from other appropriate logarithmic dilution series (see Appendix H); or may be derived based on the findings of preliminary “range-finding” toxicity tests. The reader is referred to Section 4.1 for additional guidance when selecting test concentrations.

To select a suitable range of concentrations, a preliminary or range-finding test covering a broader range of test concentrations might prove worthwhile. The number of replicates per treatment (see Section 4.1) could be reduced or eliminated altogether for range-finding tests and, depending on the expected or demonstrated (based on earlier studies with the same or a similar test substance) variance among test vessels within a treatment, might also be reduced for nonregulatory screening bioassays or research studies.

Based on the objectives of the test, it might be desirable to determine the influence of substrate characteristics (e.g., particle size or organic matter content) on the toxicity of chemical/soil mixtures. For instance, the influence of soil particle size on chemical toxicity could be measured by conducting concurrent multi-concentration tests with a series of mixtures comprised of the test chemical(s) or chemical product(s) mixed in differing fractions (i.e., segregated particle sizes) or types of natural or artificial negative control soil (Section 3.4). Similarly, the degree to which the total organic carbon content (%) or organic matter content (%) of soil can modify chemical toxicity could be examined by performing concurrent multi-concentration tests using different chemical/soil mixtures prepared with a series of organically enriched negative control soils. Each fraction or formulation of natural or artificial negative control soil used to prepare these mixtures should be included as a separate control in the test.

Depending on the study objectives and design, certain soil toxicity tests using plants might be performed with samples of negative control soil or reference soil to which chemical(s) or chemical product(s) are applied to the soil surface, rather than mixing it with the soil. Surface applications can be applied in the field or the laboratory. Procedures for chemical application include the use of a calibrated track sprayer to achieve a uniform distribution of the chemical over a specific area. Concentration of chemical(s) or chemical product(s) in the soil can be determined based on the penetration depth, the surface area or swathe width, the nozzle size, the pressure, and the speed of coverage of the sprayer (G. L. Stephenson, personal communication, Aquaterra Environmental, Orton, ON, 2001). The OECD (2000a) provides some guidelines for applying test substances to the soil surface, in preparation for emergence-and-growth tests with terrestrial plants.

6.3 Test Observations and Measurements

A qualitative description of each mixture of chemical-spiked soil should be made when the test is being established. This might include observations of the colour, texture, and visual homogeneity of each mixture of chemical-spiked soil. Any change in appearance of the test mixture during the test, or upon its termination, should be recorded.

Section 4.6 provides guidance and requirements for the observations and measurements to be made at the beginning, during, and at the end of the test. These observations and measurements apply and must be made when performing the soil toxicity test described herein using one or more samples of chemical-spiked soil.

Depending on the test objectives and experimental design, additional test vessels might be set up at the beginning of the test (see Section 4.1) to monitor soil chemistry. These would be destructively sampled during or at the end of the test. Test organisms might or might not be added to these extra test vessels, depending on study objectives. Measurements of chemical concentrations in the soil within these test vessels could be made by removing aliquots of soil for the appropriate analyses, at the beginning of the test, as it progresses, and/or at its end, depending on the nature of the toxicant and the objectives of the test.

Measurements of the quality (including soil pH and moisture content) of each mixture of spiked soil being tested (including the negative control soil) must be made and recorded at the beginning and end of the test for pH and at the beginning of the test only for moisture content, as described in Section 4.6. If analytical capabilities permit, it is recommended that the stock solution(s) be analysed together with one or more subsamples of each spiked-soil mixture, to determine the chemical concentrations, and to assess whether the soil has been spiked satisfactorily. These should be preserved, stored, and analyzed according to suitable, validated procedures.

Unless there is good reason to believe that the chemical measurements are not accurate, toxicity results for any test in which concentrations are measured for each spiked-soil mixture included in the test should be calculated and expressed in terms of these measured values. As a minimum, sample aliquots should be taken from the high, medium, and low test concentrations at the beginning and end of the test^Footnote74; in which instance, endpoint values calculated (Sections 4.8 and 6.4) would be based on nominal ones. Any measurements of concentrations of the test chemical(s) or chemical product(s) should be compared, reported, and discussed in terms of their degree of difference from nominal strengths. If nominal concentrations are used to express toxicity results, this must be explicitly stated in the test- specific report (see Section 7.1.6).

6.4 Test Endpoints and Calculations

Multi-concentration tests with mixtures of spiked soil are characterized by test-specific statistical endpoints (see Section 4.8). Guidance for calculating the EC50 for emergence data is provided in Section 4.8.2, whereas that for calculating an ICp (based on data showing growth inhibition; see Section 4.8) is presented in Section 4.8.3. Section 5.5 provides guidance for calculating and comparing endpoints for single-concentration tests using samples of field-collected soil, which applies equally to single-concentration tests performed with mixtures of spiked soil. For further information on these or other appropriate parametric (or nonparametric) statistics to apply to the endpoint data, the investigator should consult the Environment Canada report on statistics for the determination of toxicity endpoints (EC, 2004a).

For any test that includes solvent control soil (see Section 6.2), the test results for plants held in that soil must be compared statistically with that for test organisms held in negative control soil. If any of the endpoints for these two control soils used to establish test validity (see Section 4.4) differ significantly according to Student’s t-test, only the solvent control soil may be used as the basis for comparison and calculation of results. If the results for the two controls are the same, the data from both controls should be combined before using it to calculate results or to assess test validity.