Biological test method for measuring terrestrial plants exposed to contaminants in soil: appendix G

Appendix G - Natural and Artificial Negative Control Soils Used for Method Development and the Establishment of Test Validity Criteria

Negative control soil must be included as one of the experimental treatments in each soil toxicity test. This treatment requires a soil which is essentially free of any contaminants that could adversely affect the performance of plants during the test (see Section 3.4).  Before applying the test method described in this document as a standardized test to be conducted according to Environment Canada, it was necessary to first assess the performance of test organisms in different types of negative control soil representative of an array of clean soils found within Canada. Five types of negative control soils were used to develop the biological test method described herein and to further assess the robustness of the test method with samples of soil that varied considerably in their physical and chemical characteristics. These soils were also used to establish reasonable criteria for valid test results, based on control performance. The five soils tested include an artificial soil (see Section 3.4.2) and four natural soils (see Section 3.4.1) (Aquaterra Environmental, 1998a; Stephenson et al., 1999a, b, 2000a, b; Aquaterra Environmental and ESG, 2000; ESG, 2001, 2002; ESG and Aquaterra Environmental, 2002; EC, 2005b). The artificial soil was formulated in the laboratory from natural ingredients. The four natural soils included two agricultural soils from southern Ontario, a prairie soil from Alberta, and a forest soil from northern Ontario. The physicochemical characteristics of all five soils are summarized in Table G-1.

The artificial control soil (AS) used in this series of performance evaluation studies with diverse soil types was the same as that recommended for use herein (see Section 3.4.2). It consists of 70% silica sand, 20% kaolin clay, 10% Sphagnum sp. peat, and calcium carbonate (10 - 30 g CaCO₃/kg peat). The soil was formulated by mixing the ingredients in their dry form thoroughly, then gradually hydrating with de-ionized water, and mixing further until the soil was visibly uniform in colour, texture, and degree of wetness.

The four natural soils used as negative control soil while developing this biological test method and establishing the test validity criteria herein (see Section 4.4) do not represent all Canadian soil types. However, they do vary greatly in their physicochemical characteristics and include agricultural soils with diverse textures as well as a forest soil (see Table G-1). The soils originated from areas that had not been subjected to any direct application of pesticides in recent years. They were collected with either a shovel or a backhoe, depending on the location and the amount of soil collected. Sampling depth depended on the nature of the soil and the site itself.

The sample of clay loam soil, classified as a Delacour Orthic Black Chernozem, was collected in May 1995 from an undeveloped road allowance east of Calgary, Alberta. The soil beneath the sod was air dried to about 10 - 20% moisture content, sieved (4 or 9 mm), placed into 20-L plastic pails, and shipped to the University of Guelph (Guelph, ON) where it was kept in cold storage (4 °C) until needed. The soil was determined to be virtually free of any contaminants (Komex International, 1995). The physicochemical characteristics of the soil show that it is a moderate-to-fine clay loam, with a relatively high organic content and cation exchange capacity compared to the other clean soils used during the development of this biological test method and the establishment of test validity criteria (see Table G-1).

A large (~3000 L) sample of sandy loam soil was collected in June 1999 from Beauchamp Farms, Eramosa, Ontario, from a site that had been cultivated regularly for crop production but not subjected to pesticide application. The soil was air-dried and sieved (2 or 5 mm), placed into 20-L plastic buckets, and kept in cold storage (4 °C) until needed. This soil was analyzed for common organic and inorganic contaminants, and its physicochemical characteristics established to determine if any unusual soil characteristics (e.g., high conductivity or anomalous nutrient levels) were present. The sample was found to be virtually free of both contaminants and anomalies.  This soil is a fine sandy loam with a moderate organic content and a moderate cation exchange capacity compared to the other clean soils included in these studies (see Table G-1).

The sample of silt loam soil was collected in June 1999 from the University of Guelph Elora Research Station, in Nichol Township, Ontario. The topsoil had been removed several years ago when the research facility was built, and had been stockpiled beside a field. Soil collected for these method development studies was removed from the interior of the pile to avoid collecting soil that might have been inadvertently contaminated with pesticide or fertilizer spray drift from the adjacent field. The soil was air-dried and sieved (2 or 5 mm), placed into 20-L plastic buckets, and kept in cold storage (4 °C) until needed. The soil was also analyzed and found to be free of both organic and inorganic contaminants and anomalies. The measured physicochemical characteristics of this silt loam soil showed that it had a moderate organic content and a moderate cation exchange capacity, compared to the other four soils included in these method development studies (see Table G-1).

A 400-L sample of forest soil, classified as Orthic Humo-Ferric Podzols, was collected in June 2001 from a forested area located on the Canadian Shield, approximately 40 km east of Sudbury, Ontario. The leaf litter was gently raked away and a hand trowel was used to remove soil to a depth ranging from 5  10 cm. The soil was placed without sieving into 20-L plastic-lined buckets, and transported to ESG International at Guelph, Ontario. It was air-dried for 48 hours to no less than -10% moisture content, homogenized, and then sieved through 6-mm mesh. Once the sample was sieved, it was thoroughly homogenized and stored in the same 20-L plastic buckets until used. This soil was stored at room temperature (20 °C) until used. The physicochemical characteristics of the forest soil show that it is a loam with a moderate cation exchange capacity, and the highest total organic carbon content (11.9%) and highest percentage of organic matter (19.9%) of the five soils used in the method development studies (see Table G-1).