Biological test method for measuring survival of springtails 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 that is essentially free of any contaminants that could adversely affect the performance of test organisms during the test (see Section 3.3).  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 first edition of this biological test method and to further assess its robustness 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 3.3.2) and four natural soils (see Section 3.3.1) (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). 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 of this appendix.

The artificial control soil used in this series of performance evaluation studies with diverse soil types was the same as that recommended for use herein (see Section 3.3.2). 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. This artificial soil is much the same as that described by ISO (1999) and OECD (2005).

The four natural soils used as negative control soil while developing the first edition of this biological test method and establishing the test validity criteria for F. candida, F. fimetaria and O. folsomi (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 upon 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, Ontario) 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, in 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).

For this second edition test method document, the performance of P. minuta was assessed in different types of negative control soil representative of an array of clean soils collected from the boreal and taiga ecozones within Canada. Nine types of negative control soils were used to develop the biological test method described herein for use with P. minuta 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 using P. minuta, based on control performance. The nine soils tested included an artificial soil (see Section 3.3.2) and eight natural soils and soil horizons (see Section 3.3.1) (EC, 2010, 2013b). The artificial soil was formulated in the laboratory from natural ingredients. The eight natural boreal forest soils included one from Newfoundland, one from New Brunswick, one from Ontario, three from Saskatchewan and two from Alberta. The physicochemical characteristics of the artificial soil and eight (including horizons) forest soils are summarized in Table G-2.

The artificial control soil used in the P. minuta series of performance evaluation studies with boreal and taiga soil types (EC, 2013b) was the same as that recommended for use herein (see Section 3.3.2) and the same as that used for the other three test species (F. candida, F. fimetaria and O. folsomi), described earlier in this section. It consists of 70% silica sand, 20% kaolin clay, 10% Sphagnum sp. peat and calcium carbonate (10 to 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 eight natural soils used as negative control soil while developing this second edition biological test method and establishing the test validity criteria for P. minuta (see Section 4.4) do not represent all Canadian soil types. However, they do vary greatly in their physicochemical characteristics and include boreal and taiga ecozone soils with diverse textures (see Table G-2). The soils originated from areas that had not been subjected to any direct application of pesticides in recent years. Bulk soils were collected as separate horizons, where possible. Sampling depth depended on the nature of the soil and the site itself. Once collected, all soil horizons were air-dried, sieved (4 to 8 mm), homogenized, and stored at room temperature (23°C), until required.

The Newfoundland soil (NL Podzol) was classified as a Gleyed Humo-ferric Podzol, developed on a stony, loamy-to-sandy, non-calcareous glacial till (EcoDynamics Consulting Inc., 2011b). The main canopy within the site was dominated by balsam fir and scattered black spruce. The understory consisted of sheep laurel (Kalmiaangustifolia) and creeping snowberry (Gaulteria hispidula), regenerating trees, bunchberry (Cornus canadense), with lesser amounts of spinulose woodfern (Dryopteris spinulosa), cinnamon fern (Osmunda cinnamomea), two-leaved solomonseal (Maianthemum canadense) and blue bead lily (Clintonia borealis). The ground surface was dominated by feathermosses (e.g., Shreber's moss [Pleurozium schreberi], stair-step moss [Hylocomium splendens], and knight's plume [Ptilium crista-castrensis]). Prior to sampling, woody debris and leaf litter were removed, and the underlying organic F and H horizons were collected together, followed by the separate collection of the Ahe (to a depth of 3 cm), Ae (to a depth of 25 cm), and Bf horizons.

The New Brunswick soil (NB Podzol) was classified as an imperfectly drained Gleyed Humo-ferric Podzol, developed in non-calcareous, medium to moderately fine-textured basal or lodgement till (EcoDynamics Consulting Inc., 2008a). The main canopy consisted of a mixed-wood forest, consisting of beech (Fagus grandifolia), red maple (Acer rubrum), yellow birch (Betula alleghaniensis) and sugar maple (Acer saccharum), underlain by balsam fir (Abies balsamea), with an understory of hazel (Corylus cornuta) and regenerating maple and balsam fir (EcoDynamics Consulting Inc., 2008a). The forest litter (L horizon) was removed, and the underlying FH and Ahe-Aegj horizons were collected separately and placed into 25-L pails. The underlying Bf horizon was then collected; however, given the variation and wavy nature of the soil horizon boundaries, the collection of some BCgj material was unavoidable.

The Ontario soil (ON Podzol) was classified as a Gleyed Humo-ferric Podzol developed within a non-calcareous fluvial-lacustrine deposit (EcoDynamics Consulting Inc., 2011a). The site was a coniferous-dominant mixed-wood forest, with a mixture of both coniferous and deciduous species. The upper canopy consisted mainly of red pine (Pinus resinosa) and eastern white pine (Pinus strobus), with scattered sugar maple (Acer saccharum), with a lower canopy consisting of a mixture of white birch (Betula papyrifera), eastern white cedar (Thuja occidentalis), black spruce (Picea mariana), white spruce (Picea glauca), red maple (Acer rubra) and eastern hemlock (Tsuga canadensis). The understory was dominated by regenerating tree species, with lesser amounts of speckled alder (Alnus incana), beaked hazelnut (Corylus cornuta), eastern leatherwood (Dirca palustris), wild raisin (Viburnum nudum), velvet blueberry (Vaccinium myrtilloides) and twinflower (Linnaea borealis). The ground surface was dominated by bunch berry (Cornus canadensis) and goldthread (Coptis trifolia). Three horizons were collected following the removal of the forest litter: the Ahe (to a depth of 2 cm), Ae (to a depth of 7 cm), and Bf horizons (to a depth of 20 cm).

Three soils were collected from Saskatchewan. The first soil (SK01 Luvisol) was classified as a well-to-moderately well-drained Dark Grey Luvisol, developed on stone-free, loamy-to-clayey glaciolacustrine materials (EcoDynamics Consulting Inc., 2007). The forest cover was a mixture of white spruce (Picea glauca) and trembling aspen (Populus tremuloides), with an understory of aspen suckers, rose (Rosa sp.), willow (Salix spp.), bunchberry (Cornus canadensis) and twinflower (Linnaea borealis). Three horizons were collected: LFH (10 cm depth), Ahe (10 cm depth), and Bt (to a depth of 19 cm).

The second soil (SK02 Brunisol) was classified as a rapidly drained, Orthic Eutric Brunisol, developed in a stone-free, sandy glaciofluvial materials (EcoDynamics Consulting Inc., 2007). The forest cover consisted of pure jack pine (Pinus banksiana), with an understorey dominated by aspen (Populus tremuloides), green alder (Alnus crispa), bearberry (Arctostaphylos uva-ursi) and reindeer lichens (Cladinaspp.). The leaf litter was removed, and the FH was collected to a depth of approximately 6 cm; the Ah and Bm horizons were collected together to a depth of approximately 25 to 30 cm, as the Ah was discontinuous and thin (2 cm).

The third soil (SK03 Brunisol) was representative of the Taiga Shield Ecozone and the Selwyn Lake Upland Ecoregion, and was classified as an Eluviated Dystric Brunisol (EcoDynamics Consulting Inc., 2011c). The upland vegetation was dominated by a black spruce (Picea mariana) and jack pine (Pinus banksiana), with an understory of reindeer lichens (mostly Cladina mitis) and feather mosses (mostly Pleurozium schreberi), Labrador tea (Ledum groenlandicum), bog cranberry (Vaccinium vitis-idaeus), blueberry (Vaccinium myrtilloides), bog bilberry (Vaccinium uliginosum) and crowberry (Empetrum nigrum). The surface woody debris and leaf litter were removed to expose the F and H horizons, which were then collected and placed into 25-L pails. Subsequently, the underlying A (Ae) and B (Bfj and Bm) mineral horizons were collected together as their combined depth was approximately 10 cm thick.

Two soils were collected from Alberta. The first soil (AB01 Gleysol) was collected from a bog and consisted of a poorly drained Rego Humic Gleysol (Peaty Phase), with soil texture varying from loam to clay loam near the surface, and becoming clay-rich with depth (EcoDynamics Consulting Inc., 2007). The site was dominated by black spruce (Picea mariana), with an understory dominated by peat mosses (Sphagnumspp.) and haircap mosses (Polytrichumspp.). Two horizons were collected: a mixture of Of/Oh horizons, and the Ahg horizon (to a depth of 17 cm).

The second soil (AB02 Chernozem) was collected on a river floodplain terrace, and was characterized as a well-to-moderately well-drained Rego Dark Gray Chernozem (EcoDynamics Consulting Inc., 2007). The texture of the organic-rich Ah horizon was classified as a silt loam, with a very fine sand/loamy very fine sand-to-very-sandy loam texture occurring with depth. The dominant vegetation consisted of smooth brome (Bromus inermis Leyss.), interspersed with small amounts of rose (Rosasp.), northern bedstraw (Galium boreale L.) and fireweed (Epilobium angustifolium L.). Forested areas close to the river valley slopes, contained an aspen over-story, with scattered white spruce. Two horizons were also collected: the Ah horizon to a depth of 11 cm, and the Ckgj horizon to a depth of approximately 25 to 30 cm; there was no defined B horizon.

Table G-2 Physicochemical characteristics of candidate artificial and natural negative control boreal soils and soil horizons^Footnote5

Soil type: AB02 Chernozem

Source: Rego dark grey chernozem

Soil classification: Rego humic gleysol