Biological test method for measuring terrestrial plants exposed to contaminants in soil: chapter 1


1.1 Background

The Method Development and Applications Section (MDAS) of Environment Canada is responsible for the development, standardization, and publication (see Appendix A) of a series of biological test methods for measuring and assessing the toxic effect(s) on single species of terrestrial or aquatic organisms, caused by their exposure to samples of test materials or substances under controlled and defined laboratory conditions. In 1994, MDAS, the Canadian Association of Petroleum Producers (CAPP), and the federal Panel for Energy Research and Development (PERD) initiated a multi-year program to research, develop, validate, and publish a number of standardized biological test methods for measuring the toxicity of samples of contaminated or potentially contaminated soil, using appropriate species of terrestrial test organisms. The goal was to develop new biological test methods that were applicable to diverse types of Canadian soil using terrestrial species that were representative of Canadian soil ecosystems. The initial phase of this multi-year program involved a comprehensive review of existing biological test methods, used globally to evaluate the toxicity of contaminated soils to plants and soil invertebrates. The resulting report recommended that Environment Canada support the development, standardization, and publication of a number of single-species biological test methods for measuring soil toxicity, including those using terrestrial plants (Bonnell Environmental Consulting, 1994). This recommendation was endorsed by both the headquarters and regional offices of Environment Canada (Appendix B) and the Inter-Governmental Environmental Toxicity Group (IGETG) (Appendix C).

Since 1994, several years of research have been completed under the direction of the MDAS on the selection of suitable and sensitive test organisms for measuring soil toxicity to meet Canadian regulatory and monitoring requirements, and on the development of appropriate biological test methods. A technical report was produced describing species selection criteria and processes, as well as the results of testing associated with the development of a terrestrial plant toxicity test for the assessment of contaminated soils (Aquaterra Environmental, 1998a). Other technical reports written concurrently describe tests for assessing the toxicity of soils; specifically a test for mortality and reproductive inhibition of a small soil-dwelling arthropod (Collembola: Onychiurus folsomi; Aquaterra Environmental, 1998b) and tests for mortality, avoidance behaviour, and reproductive inhibition of earthworms (Aquaterra Environmental, 1998c).

Numerous soil toxicity tests have been coordinated or supported by Environment Canada, using various terrestrial plant species exposed to samples of soil contaminated with pesticides, metals, petrochemical wastes, volatile hydrocarbons, or prospective reference toxicants. These studies (Aquaterra Environmental, 1998a; Stephenson et al., 1999a, b, 2000a; Aquaterra Environmental and ESG, 2000; ESG, 2000, 2001, 2002; ESG and Aquaterra Environmental, 2002) focussed on the development and standardization of biological test methods for determining the sublethal toxicity of samples of contaminated soil to plants. Based on the results of these studies, together with the findings of a series of interlaboratory method validation studies (EC, 2005a); Environment Canada proceeded with the preparation and finalization of a biological test method for conducting soil toxicity tests that measure emergence and growth inhibition of terrestrial plant species, as described in this report.

A Scientific Advisory Group (see Appendix D) of international experts experienced with the design and implementation of soil toxicity tests using terrestrial plants provided key references which were reviewed and considered as part of this undertaking. These individuals also served actively in providing a critical peer review of the initial draft of this methodology document. The experience of the international scientific community when performing similar soil toxicity tests using terrestrial plants (see Appendices E and F) was relied on heavily when preparing this biological test method.

Detailed procedures and conditions for preparing and performing this biological test method are defined herein. Universal procedures for preparing and conducting soil toxicity tests using selected species of agricultural crop, market-garden, or grassland plants are described. Options for test species include: alfalfa (Medicago sativa), barley (Hordeum vulgare), blue grama grass (Bouteloua gracilis), carrot (Daucus carota), cucumber (Cucumis sativus), durum wheat (Triticum durum), lettuce (Lactuca sativa), northern wheatgrass (Elymus lanceolatus; formerly named Agropyron dasystachyum), radish (Raphanus sativus), red clover (Trifolium pratense), red fescue (Festuca rubra), and tomato (Lycopersicon esculentum). Guidance is also provided for specific sets of conditions and procedures which are required or recommended when using this biological test method for evaluating different types of substances or materials (e.g., samples of field-collected soil or similar particulate waste, or samples of one or more chemicals or chemical products experimentally mixed into or placed in contact with natural or formulated soil). The biological endpoints for this method are: (a) seedling emergence, and (b) plant growth (measured as live shoot and root length and shoot and root dry mass) measured at the end of the test.

The flowchart in Figure 1 illustrates the universal topics covered herein, and lists topics specific to testing samples of field-collected soil, similar particulate waste (e.g., sludge, drilling mud, or dredged material), or soil spiked experimentally with chemical(s) or chemical product(s).

This biological test method is intended for use in evaluating the sublethal toxicity of samples of material such as:

  1. field-collected soil that is contaminated or potentially contaminated;
  2. soils under consideration for removal and disposal or remediation treatment;
  3. dredged material destined or under consideration for land disposal after dewatering;
  4. industrial or municipal sludge and similar particulate wastes that might be deposited on land; and
  5. clean or contaminated soil (natural or artificial), spiked with one or more chemicals or chemical products (e.g., for risk assessment of new or current-use chemicals).

In formulating this biological test method, an attempt has been made to balance scientific, practical, and cost considerations, and to ensure that the results will be sufficiently precise for most situations in which they will be applied. It is assumed that the user has a certain degree of familiarity with soil toxicity tests. Explicit instructions that might be required in a regulatory protocol are not provided in this report, although it is intended as a guidance document useful for that and other applications.

For guidance on the implementation of this and other biological test methods, and on the interpretation and application of endpoint data for soil toxicity, the reader should consult Sections 4.12, 5.5, and 5.6.4 in Environment Canada’s “Guidance Document on Application and Interpretation of Single-Species Tests in Environmental Toxicology” (EC, 1999a).

1.2 Selection of Test Species

Phase I (Bonnell Environmental Consulting, 1994) of the soil toxicity test method development program (see Section 1.1) produced a list of potential species to be investigated for inclusion in a future Canadian terrestrial plant soil toxicity test method. This list includes both the traditional agricultural crop species and some more “ecologically relevant” species.

During Phase II (1995 1998) of the project, 30 plant species were screened to assess their suitability for use in toxicity tests.Note de bas de page1.1 Preliminary screening was performed with two negative control soils (a formulated artificial soil and a field- collected natural soil) which were spiked with boric acid (Aquaterra Environmental, 1998a; Stephenson et al., 1997; Stephenson, 2003a). The results of these reference tests were used in conjunction with predetermined selection criteria to focus or reduce the list of 30 candidate test species, while ensuring that some of the biological and ecological diversity of plants was incorporated into test species selection (Stephenson, 2003a). A priori selection criteria included plant characteristics that were considered:

  1. extremely important [e.g., type of germination (epigeal or hypogeal), class of angiosperm (monocotyledon vs. dicotyledon), and phenology and life history traits (biennial, perennial, annual)];
  2. moderately important (e.g., time to germination, crop vs. non-crop species, and nature of the photosynthetic system); and
  3. less important (e.g., above vs. below-ground crop species).

Figure 1: Diagram of approach taken in delineating test conditions and procedures appropriate to various types of materials

Figure 1: Diagram of approach taken in delineating test conditions
Description

This figure divides the methods contained in this document into two specialized testing categories in addition to listing universal procedures which are common to the testing of any substance. The two specialized categories are procedures specific for the testing of chemical-spiked soil and field-collected soil or particulate waste.

Each species was also assessed according to six criteria used to evaluate its amenability to the prospective new biological test method and its associated procedures (e.g., test duration, ease of root separation, sufficient biomass at the end of a test, seed size, time to emergence, and effect of soil on early seedling emergence and growth), in addition to its relative sensitivity to boric acid in soil. Species recommended for definitive plant tests were those with a known range of sensitivities to boric acid; including those considered to be sensitive (alfalfa, northern wheatgrass, carrot, cucumber, and radish), moderately sensitive (lettuce, timothy, red fescue, and grama grass), and insensitive or tolerant (canola and corn) to the toxicants tested (Aquaterra Environmental, 1998a). Timothy was dropped from further investigation because its fragile roots made it difficult to work with, and corn was chosen over canola to represent the “tolerant” species (Stephenson, 2003a).

In February 2003, Environment Canada hosted a workshop in Vancouver, BC, on the toxicological assessment of Canadian soils and development of standardized test methods. One of the recommendations of the workshop participants was to expand the current battery of test organisms in Environment Canada’s draft plant method to include more species (EC, 2004b). As a result, numerous soil toxicity tests have been undertaken by Environment Canada to further expand the list of potential test species described herein (EC, 2005b). In addition, corn was dropped from earlier drafts of this test method because it is one of the least sensitive test species, and workshop participants agreed that this “tolerant species” should not be included as an option in the test method document (EC, 2004b).

The twelve terrestrial plant species (and varieties, where applicable) selected for use in this test method are described in detail in the following subsections and summarized in Table 1.

1.2.1  Alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.), also called lucerne, is one of the oldest cultivated forage crops in North America and is one of the most palatable and nutritious (i.e., rich in protein, vitamins, and minerals). It has a very high yield compared with that of other crops and is an integral component of many crop rotations because of its ability to fix nitrogen, improve soil structure, and control weeds in subsequent crops (Sullivan, 1992). The distribution of alfalfa is worldwide. In Canada, alfalfa is grown mainly as forage and fodder for livestock; however, alfalfa seeds are also sprouted for human consumption as a vegetable (Munro and Small, 1997). Alfalfa is the most important forage legume in Canada, and is grown in almost all provinces. Most of the Canadian crop is used as bailed hay (2 × 106 hectares), and some is used as silage and pasture (2 - 3 × 106 hectares) (Munro and Small, 1997). Alfalfa has also naturalized in many areas throughout Canada.

Alfalfa is a long-lived, perennial, dicotyledenous legume that exhibits epigeal germination. It belongs to the family Fabaceae (pea family, also known as Leguminosae) and is classified as an above-ground agricultural crop with a C3 photosynthetic system. Seeds are a bright olive-green to yellow, and are medium-sized ~2.6 × 1.5 mm) (see Table 1). Alfalfa seed must be placed in contact with moist soil to germinate, and for best seedling survival, seeds should be planted approximately 0.6 cm deep. Seedlings are unable to emerge from the soil if planted too deep and emergence is greatly reduced when seeds are planted deeper than 1.3 cm. Seedlings emerge 3 - 4 days after being planted. Alfalfa has demonstrated a range of emergence in control soils of approximately 70 - 90% ( ESG and Aquaterra Environmental, 2002; Stephenson, 2003a; EC, 2005b).

Alfalfa typically has a deep taproot, although some varieties have different root systems. The roots form nodules in association with Rhizobium spp. bacteria, which fix atmospheric nitrogen. Alfalfa is tolerant of drought and exhibits winter hardiness, and although it grows best in loamy, well-drained soils, it is tolerant of soils having a variety of textures. The response of alfalfa in toxicity tests with boric acid appears to be unaffected by soil type (Stephenson, 2003a). Alfalfa is intolerant of flooding, waterlogging, or poor soil drainage (Sullivan, 1992). In toxicity tests, well-defined concentration-response relationships were observed for exposures to boric acid, copper sulphate, diuron, and petroleum hydrocarbons such as crude oil, condensates and amines in soils (Aquaterra Environmental, 1998a; Aquaterra Environmental and ESG, 2000; ESG, 2001; ESG and Aquaterra Environmental, 2002; EC, 2005b).

Table 1 - Characteristics of Plant Species
Plant Seed Size
(mm)
Germination Monocot vs.
DicotFootnote1.1
Seedling EmergenceFootnote2 (days) Life cycle Soil Type
Preference
Tolerance
Alfalfa 2.6 × 1.5 epigeal dicot 3-4 perennial loamy, well drained tolerates drought; winter hardiness;
intolerant of flooding, waterlogging, poor soil drainage
Barley 9.0 × 3.4 hypogeal monocot 2-3 annual well drained, fertile loams and lighter clay soils; loamy to heavy soils tolerated tolerates saline soil, heat and drought;
does not grow well at pH < 6.0; intolerant of waterlogging
Blue Grama Grass 4.9 × 1.0 hypogeal monocot 3-5 perennial fine- to coarse- textured including clay, site, fine loams, sandy loams, sand and gravelly soils tolerant of cold, drought,
and shade; intolerant of salt; seed viability greater at higher temperatures
Carrot 3.6 × 1.5 epigeal dicot 4-5 biennial all soil types; grows best  in medium- to - light loose, sandy loam soils with good WHC tolerates a wide pH range (4.2  8.7) but grows best at pH 6.5  7.8; intolerant of drought
Cucumber 7.7 × 3.6 epigeal dicot 3-4 annual most well-drained soils; grows best in heavier clay loam or salty loam soils high in organic matter requires pH at or near neutral with high amount of nitrogen
Durum
Wheat
8.0 × 4.0 hypogeal monocot 2-3 annual tolerates sandy, loamy and clay soils, but requires well- drained conditions prefers dry conditions, hot days, cool nights;
tolerates wide pH range intolerant of cold and long winters
Lettuce 3.8 × 1.3 epigeal dicot 3  4 annual will grow in fine sandy loams, clay soils and muck soils, but prefers soil high in organic matter requires cool temperatures for germination; optimal growing temp. 15  18 °C; prefers pH 6.0  8.0
Northern
Wheatgrass
7.5 × 1.3 hypogeal monocot 4-5 annual/
perennial
tolerates a range of soil types, but prefers medium- to coarse- textured tolerant of moderate flooding, but is known for its drought tolerance; prefers basic soils (pH
6.0  9.5)
Radish 2.9 diameter epigeal dicot 2-3 biennial grows well in a variety of soil types tolerant of low fertile soil; prefers cooler temperatures; prefers neutral soil (pH
6.0-7.0), but can tolerate slightly acidic soils (pH
5.5-6.8);
poor salt tolerance
Red Clover 2.0 × 1.5 epigeal dicot 3  4 perennial/
biennial
well drained highly fertile loam soil; loams, silt loams and heavy soils are better than light sandy or gravelly soils tolerant of wide pH range (4.5  8.2), but prefers near neutral pH of 6.6  7.6; better than alfalfa at tolerating soils of low pH, low fertility and/or poor drainage; moderately drought tolerant
Red Fescue 6.6 × 0.9 hypogeal monocot 4-5 perennial can grow on clay loam, and sandy soils provided moisture is adequate tolerant of soils that are saline, acidic  (pH 4.5), and low in fertility; tolerates moist soils, some waterlogging, cold winters, and some drought
Tomato 3.0 × 2.4 epigeal dicot 4-5 perennial light, warm, sandy soils and heavier soils tolerant of of pH 5.5  7.5;
prefers warm days
(21  28 °C) and cool nights
(15  20 °C);
sensitive to low light and adverse temperatures; intolerant of waterlogging or high humidity (over 80%)

1.2.2  Barley (Hordeum vulgare L.)

Barley (Hordeum vulgare L.) is a cereal crop harvested for its grain (beer, food, and fodder) and for straw. It is one of the most ancient of the cultivated grains, with evidence of its cultivation dating back more than 5000 years (Magness et al., 1971). Barley is cultivated extensively throughout Canada and is considered to be a highly significant agricultural crop species in both Canada and the United States (US) (Duke, 1983; Bonnell Environmental Consulting, 1994). Hordeum vulgare L. is a six-rowed barley with a tough rachis or spiked stem (Magness et al., 1971). Barley is a plant species commonly used in Canadian toxicity testing laboratories.

Barley, a C3, monocotyledonous annual with hypogeal germination, is a member of the Poaceae (formerly named Graminae) family, also known as the family of “true grasses”. Barley is a fast growing, above-ground crop with a seed size of 9.0 × 3.4 mm (see Table 1). Seeds are sown at a depth of 1.3 - 4.5 cm (no greater than 5 cm). Seedlings are vigorous and emerge within 2 - 3 days of planting. Barley is reported to be tolerant of saline soils, heat, and drought (Duke, 1983). It is also tolerant of a soil pH range of 4.5 - 8.3; however, it does not grow well in very acid soils (i.e., below pH 6.0) (McLeod, 1982; Duke, 1983; Stoskopf, 1985). Barley can be grown on many soil types including well-drained, fertile loams and lighter, clay soils. Loamy to heavy soils are tolerated, but waterlogging is not (Valenzuela and Smith, 2002).

Although the varieties might differ with respect to their efficacy of germination, of the three varieties tested (var. CDC Buck-huskless, Bedford, and Chapais) var. Chapais consistently germinated and emerged at >96% in both the artificial soil and a field-collected negative control soil (ESG and Aquaterra Environmental, 2002; Stephenson, 2003a; EC, 2005b). Barley roots are strong and fibrous and can be easily separated from the soil with minimal breakage. Growth is rapid and plants quickly produce large amounts of phytomass, which makes barley a good choice for use in soil toxicity tests. Plant metrics (e.g., shoot/root lengths, shoot/root wet/dry masses) generally exhibit a classic concentration-response relationship in soil toxicity tests with petroleum hydrocarbons (e.g., motor gasoline), metals, and pesticides (Aquaterra Environmental and ESG, 2000; ESG, 2000, 2001; ESG and Aquaterra Environmental, 2002).

1.2.3  Blue Grama Grass [Bouteloua gracilis (HBK) Lag. ex Steud.]

Blue grama grass is a densely tufted prairie grass, native to much of North America. It is common in Alberta, east to Manitoba and south through the Rocky Mountains, Great Plains, and Midwest States to Mexico. It is uncommon in the Northwest Territories, British Columbia, and the northeastern United States. Blue grama grass is a valuable and highly palatable forage for domestic livestock as well as deer and elk. It can form dense cover, and as such, is an important soil-building grass (Anderson, 2003).The use of blue grama grass in laboratory soil toxicity tests is virtually unknown; however, its value as an ecologically relevant species to Canadian ecosystems is evident by its vast distribution.

Blue grama grass, a perennial monocot with C4 metabolism and hypogeal germination, is another “true grass”, belonging to the Poaceae (formerly named Graminae) family. It has a medium-sized seed (4.9 × 1.0 mm), from which plants are readily established (see Table 1). Seed viability appears to be temperature dependent and has been shown to be greater at higher temperatures (Aquaterra Environmental, 1998a; Anderson, 2003). Emergence occurs within 3 - 5 days, with seedlings developing rapidly. This grass emerges at rates of 70 - 89% in various control soils (EC, 2005b). Seedlings develop a single seminal root that is short-lived, and therefore, survival depends on the development of adventitious roots, which occur approximately 14- days after the seedling emerges. The fibrous root system is dense and shallow, and there are conflicting reports of rhizome formation (Anderson, 2003). Grama grass reproduces primarily by tiller and tufts (e.g., cespitose) formation. It has a high water use efficiency, which increases under warm climatic conditions and might decrease with increasing water availability (Anderson, 2003).

Grama grass occupies a range of soil types from fine- to coarse-textured, including: clay, silt, fine loams, sandy loams, sand, and gravelly soils. Good growth occurs in the well-drained soils found in open plains, foothills, and mesas (Aquaterra Environmental, 1998a; Anderson, 2003). Grama grass is cold, drought, and shade tolerant; however, it is fairly intolerant of salt. Because of its wide adaptation, ease of establishment, and economic value, grama grass is used extensively for conservation purposes, rangeland seeding, and landscaping (Anderson, 2003). Grama grass exhibited a strong concentration- response relationship with boric acid and copper sulphate when studied for use as a potential test species for inclusion in this test method (Aquaterra Environmental, 1998a; Aquaterra Environmental and ESG, 2000; and EC, 2005b). However, when using this species as a test organism, maximum test temperature and duration are recommended in order to yield adequate phytomass for endpoint measurements.

1.2.4  Carrot (Daucus carota L.)

Daucus carota contains both wild and domesticated forms of carrot and has numerous variants. There is little agreement on the most appropriate nomenclature for the many forms that have been described. All of the domesticated forms, however, are in the subspecies sativus (Hoffm.) Arcangeli (Munro and Small, 1997). The domesticated carrot is a below-ground, market-garden, crop species that is harvested annually. It is one of the most important of cool-climate root crops worldwide, and is one of the most valuable crops in Canadian vegetable production (Munro and Small, 1997). The carrot is cultivated primarily for its enlarged fleshy taproot that is widely consumed raw, cooked, or as juice. The carrot is sometimes used as fodder, as oil, as a sweetening agent, as a coffee substitute and/or in liqueurs (Munro and Small, 1997).

The carrot, a C3 biennial dicotyledon, is a member of the Apiaceae family (previously named Umbelliferae). It has a medium-sized seed (3.6 × 1.5 mm) that should be planted at depths of 1 - 2 cm (see Table 1). It does well in all soil types; however, it grows best in medium-to-light, loose, sandy loam soils with good water-holding capacity. The carrot is extremely sensitive to soil conditions, and good single, thick tap roots can only be produced in soils that permit their easy penetration. The carrot tolerates a wide pH range (4.2 - 8.7), but grows best in soils with a pH ranging from 6.5 - 7.8 (Duke, 1983) and mean temperatures of 16 - 21 °C (Huxley et al., 1992; Munro and Small, 1997). The carrot is intolerant of drought. This dicotyledon reproduces biennially by seed, with epigeal germination. In the laboratory, carrot seedlings emerged in approximately 4-5 days and percent emergence ranged from 64 - 87% and 76 - 86% in reference and artificial control soils, respectively (Aquaterra Environmental, 1998a; EC, 2005b). The toxicity of boric acid to the carrot appears to be unaffected by soil type (Stephenson, 2003a). Concentration response relationships for both root and shoot growth were classic (i.e., the severity of effect increased with increasing exposure concentration), in tests with boric acid and tests with copper sulphate (Aquaterra Environmental, 1998a; Aquaterra Environmental and ESG, 2000; EC, 2005b).

1.2.5  Cucumber (Cucumis sativus L.)

The cucumber (Cucumis sativus) is an ancient Old World vegetable that likely originated in India. It is an important crop worldwide and is one of the most important vegetables in Canada, representing about 5% of the value of the fresh vegetable industry (Bonnell Environmental Consulting, 1994; Munro and Small, 1997). The cucumber has a wide distribution in Canada, and is grown in Nova Scotia, central New Brunswick, eastern and western Ontario, central Manitoba, southern British Columbia, and northern Alberta.

The cucumber is a rapid-growing, above-ground, C3 crop species that is harvested annually. This dicotyledon belongs to the family Cucurbitaceae or the “gourd family”. It grows well in most well- drained soils; however, it does best in heavier clay loam or salty loam soils that are high in organic matter (Munro and Small, 1997). The cucumber prefers higher temperatures, and develops deep root systems. It requires soils with a pH at or near neutral and with high amounts of nitrogen (Munro and Small, 1997). The seed of a cucumber is relatively large (mean seed size of approximately 7.7 × 3.6 mm; see Table 1), enabling the plant to produce more phytomass in a short period of time, by relying on internal energy reserves. Seedlings exhibit epigeal germination and emerge in 3 - 4 days. In toxicity tests with boric acid, cucumbers (var. Marketer) exhibited a concentration-response relationship for both root and shoot growth in terms of length and wet mass measurements (Aquaterra Environmental, 1998a; EC, 2005b). Root growth was significantly reduced in response to exposure to boric acid, but the above-ground biomass was affected to a lesser degree. Percent emergence in toxicity tests in both reference and artificial control soils ranged from 90 - 98% and the nature of the soil had no effect on the observed toxicity of boric acid (Aquaterra Environmental, 1998a; EC, 2005b). The cucumber has been shown to be relatively sensitive to both organic and inorganic contaminants in soil (Aquaterra Environmental and ESG, 2000; ESG and Aquaterra Environmental, 2002).

1.2.6  Durum Wheat [Triticum durum (Desf.) or Triticum turgidum L. subsp. durum (Desf.) Husn. or Triticum pyramidal (Percival)]

Durum wheat, also known as “hard wheat”, is an annual grass that is planted in the spring and harvested in late summer. It is the only tetraploid species of wheat cultivated today, and is the hardest of all wheats. Compared to Triticum aestivum (i.e., bread wheat), far less durum wheat is grown in North America. It accounts for roughly 8% of global wheat production, and leading producers include the European Union, Canada, and the US (Small, 1999). Canada produces some of the highest-quality amber durum wheat in the world, and has an Annual Production Average (APA) of about 4.09 × 106 tonnes. Approximately 80% of the durum wheat grown in Canada between 1992 and 1995 was exported (AFBMI, 1998). On average, durum wheat has a higher protein content than bread wheat. It is grown primarily for the production of pasta products, such as spaghetti and macaroni, and for couscous and bulgar (Small, 1999; Vaughan and Geissler, 1997).

Durum wheat is a monocotyledon belonging to the family Poaceae (formerly named Graminae) (i.e., grass family). It is an above-ground cereal crop with a C3 photosynthetic system, hypogeal germination like most cereals, and a fibrous root system. Durum wheat is suited to a dry climate with hot days and cool nights, does well under dry conditions, and has a low resistance to cold and to long winters (Vaughan and Geissler, 1997). Durum wheat is tolerant of many soil types including light (sandy), medium (loamy), and heavy (clay) soil, but requires well-drained conditions. It tolerates a wide range of soil pH. Durum wheat is characterized by its large (8.0 × 4.0 mm), ovate-shaped, amber-coloured seed (see Table 1) which should be planted at a depth of about 2.5 cm (OMAF, 2002). Seedlings emerge in 2 - 3 days, and germination ranged from 83 - 92% in toxicity tests involving both artificial and field- collected soils. In addition, durum wheat has demonstrated acceptable concentration-response relationships in toxicity tests with boric acid (EC, 2005b).

1.2.7  Lettuce (Lactuca sativa L.)

Lettuce is an above-ground, market-garden, crop plant that is harvested annually. It is the Western World’s most popular salad plant with year-round demand. Canada grows over 50 000 tons of lettuce yearly, mostly in Quebec; however, this represents less than 1/5 of the lettuce actually consumed in Canada (Munro and Small, 1997).

Lettuce, a C3 dicotyledonous annual, with epigeal germination, is a member of the sunflower family (i.e., Asteraceae, formerly named Compositae). It has a medium-sized seed of 3.8 × 1.3 mm, and seedlings begin to emerge in 3 - 4 days (see Table 1). Lettuce is a cool-season crop, requiring cooler temperatures for germination. Optimal growing temperatures for lettuce range from 15 - 18 °C (minimum 7 °C/maximum 24 °C) (Munro and Small, 1997). Lettuce germinates and grows best when water is not limited and when the appropriate light is provided. Care must be taken in choosing varieties for use in toxicity testing, because various cultivars of lettuce have substantially different light intensity requirements for optimal emergence and growth. For example, the seed germination of some cultivars of lettuce is negatively photoblastic (i.e., seeds germinate only in the dark, and white light inhibits germination) (Stephenson, 2003a). Lettuce prefers soil high in organic matter, but will grow in various soils including fine sandy loams, loams, clay soils, and muck soils. The ideal soil pH for lettuce ranges between 6.0 and 8.0 (Munro and Small, 1997). The initial tap root can become quite fibrous when the plant is mature. In whole soil toxicity tests, emergence ranged from 75 - 88% for artificial soil and from 79 - 94% for field-collected soils (Aquaterra Environmental, 1998a; EC, 2005b). For the Grand Rapid and Butter Crunch varieties used in these studies, toxicity tests with boric acid exhibited acceptable concentration-response relationships for both shoot and root metrics. Lettuce is sensitive, however, to differences in certain physicochemical characteristics of artificial or natural clean soil used in a test. A “soil effect” was observed in seedling emergence tests using samples of both artificial soil and clean field-collected soil (Stephenson, 2003a).

1.2.8  Northern Wheatgrass [Elymus lanceolatus (Scribn. & J.G. Sm.) Gould; formerly Agropyron dasystachyum (Hook.) Scribn.]

Northern wheatgrass (Elymus lanceolatus), also known as thickspike wheatgrass, is widely distributed throughout North America from Alaska, south through Canada, into Northern California. It is common in the northern Rocky Mountains and in the prairies from British Columbia to Ontario (Scher, 2002). Northern wheatgrass is a long-lived, cool- season native grass that is highly beneficial to soil systems. The deep root system provides excellent soil stabilization and strong sod formation (Bonnell Environmental Consulting, 1994; Scher, 2002). This species of grass is capable of forming “tall-grass prairies” at well-drained sites. It is valued as forage for livestock and wildlife and is commonly used in re-vegetation of oil and gas well-sites, pipeline construction areas, roadsides, and other construction sites. Northern wheatgrass is an important re- vegetation species because it forms tight sod under dry conditions, has good seedling strength, and does well in low-fertility soils and at eroded sites (Scher, 2002).

Northern wheatgrass is a non-crop, C3, annual and perennial monocotyledon that belongs to the family Poaeceae. It is another of the optional species of “true grasses” that may be used in this biological test method. Northern wheatgrass is very winter hardy because of its three-way root system (i.e., creeping roots that reproduce asexually or by vegetative propagation, dense shallow roots to 25 cm, and deep roots to at least 60 cm). This grass can also reproduce sexually by seeds. It is strongly rhizomatous, but develops via hypogeal germination from a slender seed (7.5 × 1.3 mm) (see Table 1). The seeds generally have good viability (e.g., 95% emergence; Stephenson, 2003a); however, emergence can be low (67 - 77%) with some batches of seed (EC, 2005b). Seedlings have good vigour, emerging in 4 - 5 days, and under good conditions, they can experience rapid development. Northern wheatgrass grows on a wide range of soil types, but prefers medium- to coarse-textured soils. It will tolerate moderate flooding, but is known for its drought tolerance (Scher, 2002). It also prefers basic soils (pH of 6.0 - 9.5). Although little data exist regarding the sensitivity of this species to contaminants, it has been shown to exhibit a strong concentration-response relationship when exposed to boric acid, copper sulphate, diuron, or petroleum hydrocarbons (e.g., condensates, crude oil, motor gasoline) in soil (Aquaterra Environmental, 1998a; Aquaterra Environmental and ESG, 2000; ESG, 2001; ESG and Aquaterra Environmental, 2002; EC, 2005b).

1.2.9  Radish (Raphanus sativus L.)

The radish (Raphanus sativus) is a minor agricultural crop, but is important and popular in gardens and markets (Bonnell Environmental Consulting, 1994). In Canada, 6000 tons are produced annually, mostly in Ontario, Quebec, and British Columbia (Munro and Small, 1997). Although it is used primarily as a salad vegetable in North America, radishes are used in other parts of the world for the production of soap and a drying oil, and as livestock feed.

The radish is a below-ground, cool-season crop species that is harvested annually for its bulbous edible tap root. It is a C3 biennial dicotyledon belonging to the mustard family (Brassicaceae or Cruciferae). The medium-sized seed (2.9 mm in diameter) germinates rapidly and seedlings begin to emerge within 2 - 3 days under moist soil conditions (see Table 1). The seed undergoes epigeal germination and produces a strong root that is easily separated from the soil (Stephenson, 2003a). Radishes are not demanding as to the soil type, and grow well in a variety of soils (Munro and Small, 1997). Stephenson (2003a), however, found that the nature of the control soil had a significant effect on the performance of the species. In screening tests, 98% emergence of radishes (var. Cherry Belle, Champion) was observed in the artificial soil, but only 65% emerged in the field-collected negative control soil (Stephenson, 2003a). In other tests, however, emergence was shown to be consistently high (i.e., >92%) in all soil types (i.e., artificial as well as sandy, silt, and clay loam soils) (ESG and Aquaterra Environmental, 2002; EC, 2005b). Radish is tolerant of soils with low fertility. It prefers cooler temperatures and neutral soil (pH 6.0 - 7.5), but can tolerate slightly acidic soils (pH 5.5 - 6.8). Radish has a low tolerance of salty soils. Stephenson (2003a) found that a concentration-response relationship for either shoot or root length was not observed in screening tests with boric acid, and that both of these metrics were influenced significantly by the nature of the control soil. The radish proved to be relatively sensitive to metals and pesticides in other studies (Aquaterra Environmental and ESG, 2000; ESG and Aquaterra Environmental 2002). This is one of the two test species (lettuce is the other species) currently recommended in most regulatory test protocols for measuring soil toxicity.

1.2.10 Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense) is grown widely across North America and occurs from coast-to-coast in Canada, both as a cultivated crop and naturally (Bonnell Environmental Consulting, 1994). It is extensively grown for pasturage, hay, and green manure, and is the most commonly planted forage legume, after alfalfa. Compared to alfalfa, however, red clover has less digestible protein, slightly more total digestible nutrients, and a slightly higher net energy value (Duke, 1983; USDA-NRCS, 2000). Red clover is also valued as a highly significant species for maintaining soil structure, and is used frequently in reclamation studies (Bonnell Environmental Consulting, 1994).

Red clover is a short-lived, C3, perennial dicotyledonous legume that can be grown under conditions which are either too wet or too acidic for alfalfa (OMAF, 2002). Under some conditions (i.e., warmer climates) red clover is grown as a biennial. Red clover, like alfalfa, is a member of the pea family (Fabaceae). It has a relatively small seed (2.0 × 1.5 mm; see Table 1) has good seedling vigour, and is relatively easy to establish (USDA-NRCS, 2000). Red clover has epigeal germination and has demonstrated good emergence under laboratory conditions in tests using either artificial soil (68 - 90%) or samples of clean field-collected soils (88 - 92%) (EC, 2005b). Emergence typically begins 3 - 4 days after planting. It grows best on well- drained, highly fertile loam soil, but has also adapted to wetter soils. For red clover, loams, silt loams, and even heavy soils are better than light sandy or gravelly soils (Duke, 1983). It is tolerant of a wide pH range (4.5 - 8.2); however, this plant species prefers a near-neutral pH for nodulation and is most productive on soil that is within a pH range of 6.6 - 7.6. Red clover is better than alfalfa at tolerating and growing on soils of low pH or those with low fertility and/or poor drainage. Red clover has a deep tap root and is moderately drought tolerant (USDA-NRCS, 2000). Roots nodulate naturally from free-living rhizobia. Red clover is not commonly used in laboratory toxicity tests as yet, but it has demonstrated good concentration-response relationships in tests with boric acid (EC, 2005b).

1.2.11 Red Fescue (Festuca rubra L.)

Red fescue has a wide range across the Northern Temperate Zone, occurring throughout Canada from British Columbia to Newfoundland (Bonnell Environmental Consulting, 1994). It is a valuable species of forage grass in Alberta, where it grows better in poor soils than bluegrass or timothy; it can also out-compete alfalfa. In particular, Creeping (var.) red fescue is a dense, sod-forming grass that establishes and spreads vigorously on most soil types (OMAF, 2002). Its solid root system and thick top-growth make it an excellent grass for stream-bank or grass waterway protection. It is noted for its extended growth period and its retained nutritional value in the fall (OMAF, 2002). It is considered a valuable stabilizer, and an excellent soil-and-sod builder.

Red fescue is a long-lived, C3, monocotyledonous species, that is a member of the grass family (Poaceae, formerly named Graminae). Red fescue is a cool-season, perennial, ground cover that is drought resistant, saline tolerant, tolerant of acidic soils, and hardy of cold winters (Walsh, 1995). It reproduces by a relatively large seed (6.6 × 0.9 mm), and exhibits hypogeal germination (see Table 1). Red fescue can also grow vegetatively by rhizome formation and can develop deep, extensive systems of fibrous roots. Seed viability is good, ranging from 78 - 95% (Aquaterra Environmental 1998a; EC, 2005b); however, vigour is moderate. Seedlings typically emerge 4 - 5 days after planting. Red fescue tolerates moist soils and some waterlogging. It can grow on clay, loam, and sandy soils, provided that the moisture is adequate; however, this species can tolerate some drought. It is also tolerant of low fertility and low pH (4.5) in soils (Walsh, 1995). A concentration-response relationship for shoot and root metrics was demonstrated for red fescue in screening test with boric acid (Aquaterra Environmental, 1998a; EC, 2005b).

1.2.12 Tomato (Lycopersicon esculentum Mill.)

The tomato (Lycopersicon esculentum) is probably Canada’s most popular home-garden vegetable. It is the second-most consumed vegetable worldwide per capita, next to the potato (Munro and Small, 1997). About 2 × 106 hectares of tomatoes are planted annually worldwide. The Canadian domestic supply represents about 60% of all tomatoes used in the country and more than 10% of the commercial value of fresh vegetables consumed in Canada (Munro and Small, 1997).

The tomato is a C3, tropical perennial that is grown as an annual in temperate parts of the world. It is a dicotyledon belonging to the family Solanaceae (also known as the nightshade or potato family). Tomatoes are grown in light, warm, sandy soils for early crops; however, heavier soils are best for maximum production. The tomato is intolerant of waterlogging or high humidity (over 80%), both of which promote disease, but it will tolerate a pH range of 5.5 - 7.5 (Munro and Small, 1997). Its medium-sized seed (3.0 × 2.4 mm) demonstrates epigeal germination and has a fairly good viability rate (74 - 95%) (Aquaterra Environmental, 1998a; EC, 2005b). Seedlings begin to appear 4 - 5 days after planting (see Table 1). When grown from seed, the tomato forms a strong taproot. However, injury to the tap root during transplanting or potting of seedlings tends to modify the natural taproot into a more fibrous one. The tomato needs moderately high daytime temperatures (21 - 28 °C) and moderately cool nighttime temperatures (15 - 20 °C) for optimal growth (Munro and Small, 1997). The tomato is sensitive to low light and adverse temperatures, and has demonstrated a good concentration-response relationship response in toxicity tests with boric acid (EC, 2005b).

1.3 Historical Use of Terrestrial Plants in Toxicity Tests

The development of biological test methods for soil toxicity testing lags behind that for other media (e.g., water and sediment) (Bonnell Environmental Consulting, 1994). This delay is partially due to the fact that research and regulators have been focussed on the aquatic environment, and partially due to the fact that soil is a complex medium with many problems inherent in its lack of homogeneity. The variety of exposure routes available to investigators (e.g., via pore water, soil vapours, or direct contact with soil particles), coupled with the high cost of running soil toxicity tests, have often led practitioners to rely on extrapolations from aquatic test methods to soil-based exposures (Bonnell Environmental Consulting, 1994).

The use of pesticides in agriculture began in the late 1940s, and by the late 1960s and early 1970s, became routine. This led to the need to assess the effects of organic chemical pesticides on commercial agricultural crop species (Kaputska et al., 1995; Boutin and Rogers, 2000). Assessment of soil quality before the 1980s primarily involved evaluating the physicochemical properties of soil, and not until the 1980s did the initial use of standardized biological test methods for measuring soil toxicity emerge from agencies responsible for pesticide registration and application [e.g., the United States Environmental Protection Agency (USEPA), and the Office of Pesticides Programs (Holst and Ellanger, 1982)].

The first standardized whole-soil toxicity test with terrestrial plants, applicable to both pesticide and non-pesticide exposures in artificial soil, was a seedling emergence test guideline (#208) published by the Organization for Economic Co-operation and Development (OECD, 1984a). This method, however, was developed to assess chemical-spiked soils only. In 1989, the USEPA recommended test methods for the toxicity assessment of contaminated site soils, whereby the contaminated soil was amended with a clean control soil in a dilution series (USEPA, 1989). Since the establishment of the joint European Economic Community (EEC)/OECD guidelines, several other agencies such as the International Standards Organisation (ISO) and, in the US, the American Society for Testing and Materials (ASTM) have also developed whole-soil toxicity test methods for selected species of terrestrial plants exposed to samples of chemical- spiked soil and/or contaminated site soil (ISO, 1993a, 1995; ASTM, 1999b).

The toxicity of site soils became a “new” concern in the mid 1980s, and regulatory programs such as SUPERFUND in the United States, and the National Contaminated Sites Remediation Program (NCSRP) in Canada, were established to address the urgent need for guidance on the assessment and remediation of high-priority contaminated sites. Under the NCSRP, a review of existing whole-organism bioassays for soil, freshwater sediment, and fresh water (Keddy et al., 1995) was conducted to lead to the establishment of a suite of tests that could be used immediately for contaminated-site assessment in Canada (Bonnell Environmental Consulting, 1994). Keddy et al. (1995) concluded that most of the existing methods or procedures for measuring the toxicity of samples of soil from contaminated sites were inadequate for proper ecotoxicological assessment, and recommended that attempts be made to develop a suite of standardized biological test methods for soil that used test species and conditions applicable to Canadian soil ecosystems. The Canadian Council of Ministers of the Environment (CCME) published a framework for ecological risk assessment (ERA) in 1994 (CCME, 1994) which had a subsequent impact on the management of contaminated sites (CCME, 1996, 1997). The ERA approach, which relied on the results of single- species toxicity tests, led to the need to develop reliable, reproducible, and realistic soil toxicity tests with ecologically relevant terrestrial test species for the assessment of contaminated site soils (Bonnell Environmental Consulting, 1994). In the late 1990s, biological assessments in the form of toxicity testing were becoming a useful complement to chemical analyses, especially when applied to site-specific risk assessments.

Today, plants are widely used as test organisms in single-species toxicity tests intended to measure the toxicity of pure chemicals, chemical products, or samples of soil contaminated or potentially contaminated with chemicals in the field or (for experimental purposes) in the laboratory. In Canada, results of soil toxicity tests are used to:

  1. derive national soil quality criteria,
  2. establish site-specific, risk-based, cleanup objectives (e.g., remediation targets), and
  3. assess the efficacy of remediation technologies (Stephenson et al., 2002).

Extensive reviews on the use of plant toxicity tests as “ecological assessment tools” for appraising the toxicity of contaminated or potentially contaminated soils have been carried out (Wang, 1991, 1992; Wang and Freemark, 1995; Kaputska, 1997; Meier et al., 1997; Saterbak et al., 1999). In some cases, standard methods have been modified or unique methods have been developed in order to obtain relevant data (Pfleeger et al., 1991; Sheppard, 1994; Chaineau et al., 1997). Data-base reviews have been summarized in reports discussing trends of plant toxicity to various contaminants (Kenaga, 1981; Miller et al., 1985; Boutin and Rogers, 2000). Toxic effects of plant exposure to contaminated soils have been documented in laboratory studies involving samples of soil spiked or contaminated with:

  • pesticides (Fletcher et al., 1995, 1996; Boutin et al., 2000, 2004),
  • metals (Godbold and Hüttermann, 1985; Kaputska et al., 1995; Kjaer and Elmegaard, 1996; Rader et al., 1997; Kjaer et al., 1998; Redente et al., 2002; Lock and Janssen, 2003),
  • petroleum hydrocarbons (Chaineau et al., 1997; Wong et al., 1999; ), and
  • other chemicals (Siciliano et al., 1997; Kalsch and Römbke, 1999).

Various plant species have been recommended for phytotoxicity testing by different agencies (See Appendices E and F; and ASTM, 1999b). The test species most commonly recommended among international agencies include: lettuce, cabbage, cucumber, soybean, oat, perennial ryegrass, corn, tomato, rice, and carrot. Fletcher et al. (1985; 1988) reviewed the PHYTOTOX data base and provided a summary of the most commonly used terrestrial plants. These plants included wheat, pea, tomato, oats, beans, apple, soybean, corn, and barley.

The number of species recommended for use in a test battery depends primarily on the purpose of the study and the regulatory requirements. The ISO (1995) recommends a minimum of two species, OECD (1984a) recommends a minimum of three species, and ASTM (1999b) recommends a minimum of five species. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substances Control Act (TSCA), as well as the latest USEPA Office of Prevention, Pesticides and Toxic Substances (OPPTS) draft (1996) all recommend 10 species of terrestrial plants for inclusion in a test battery. The OECD (2000) draft test method recommends three species for testing general chemicals, and 6 - 10 species for testing crop protection products. The proposed Canadian guidelines for non-target plant testing with chemical pesticides (Boutin et al., 1995) suggest 10 species for non-herbicide testing and 30 species for herbicide testing. The recommended ratio of monocotyledons to dicotyledons to be used in a test battery is generally 1:2, and is fairly consistent among international agencies. Boutin and Rogers (2000) conducted an extensive review of Canadian and American data bases and found that monocotyledons show similar sensitivities, whereas dicotyledons vary in their sensitivities. In a test battery, therefore, it might be appropriate to test fewer monocotyledons than the commonly recommended monocotyledon to dicotyledon ratio of 1:2 (Boutin and Rogers, 2000). Cole et al. (1993) and Brown and Farmer (1991) also provide rationale for selecting a variety of test species for testing the effects of pesticides on non-target plant species.

Many plant species and numerous phytotoxic assessment endpoints have been used to characterize the effects of toxicants on vegetation (Markwiese et al., 2000). To date, the seedling germination test and the root elongation test are the acute phytotoxicity tests most widely used (Kaputska, 1997). Unfortunately, the germination test is relatively insensitive to many substances, primarily because the embryonic plant survives using the nutritional reserves stored in the seed and is therefore effectively isolated from the environment (Kaputska, 1997). In typical root elongation tests, roots are exposed to water extracts and soluble test soil constituents, which do not involve any exposure to whole soil.

The seedling emergence test differs from seedling germination tests, in that different endpoints are measured. Most seedling emergence tests have been modelled after the OECD Terrestrial Plant Growth Test (OECD, 1984a), in which seeds of recommended test species are exposed to potentially contaminated site soils, or to a dilution series (i.e., site soils amended with control soils), followed by the measurement of the number of seedlings that emerge from the soil to a minimum height of 3 mm. Generally, seedling emergence is not as sensitive an endpoint as growth metrics (e.g., shoot and root lengths and weights) that can be obtained from early seedling growth tests. These early seedling emergence-and-growth tests overcome some of the deficiencies of the seed germination and root elongation tests discussed earlier (Kaputska, 1997; Stephenson et al., 2002). The ASTM (1999b) has developed an early seedling growth test with a test duration that is relatively longer than the seedling emergence test (i.e., >14 days). Its measurement endpoints include shoot and root length, shoot and root wet and dry mass, and seedling emergence and seedling survival at the end of the test (Stephenson et al., 2002). The OECD is currently revising their biological test method to include both a test for seedling emergence and growth, as well as a test for vegetative vigour whereby the test substance is applied to the leaves and above-ground portions of the test organisms (OECD, 2000a). The ASTM has also included a life-cycle test with Brassica rapa (a variety of turnip, that has been genetically modified for rapid assessment) in an annex of their standard guide (ASTM, 1999b). It is a test that goes from seed-to-seed, thereby covering the complete life cycle of the test organism.

The methodology documents summarized in Appendix E have been used as guidance in developing Environment Canada’s standardized biological test method for performing a test that measures the toxic effects of prolonged exposure to chemical-spiked soil or site soil on the emergence and growth of terrestrial plants. This (Environment Canada’s) new biological test method, is defined herein.

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