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Biological test method for measuring terrestrial plants exposed to contaminants in soil: chapter 2

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Section 2 - Test Organisms

2.1 Species

Test organisms to be used in this biological test method must be selected from the specified group of 12 terrestrial plant species listed here. Acceptable choices for plant species include five monocotyledons and seven dicotyledons, as follows:

monocotyledons

dicotyledons

For some plant species, specific varieties are recommended for use in this test method. The varieties, where named, are based on those that have been used successfully in the development of the test method described herein, and include: barley (var. Chapais), durum wheat (var. Durum), red fescue (var. Creeping), carrot (var. Royal Chantenay), cucumber (var. Marketmore 76 or var. Marketer), lettuce (var. Buttercrunch or var. Grand Rapid), radish (var. Champion or Cherry Belle), and tomato (var. Heinz 1439).

The selection of multiple test species should depend on the species’ sensitivity to the substance or material being tested, if known. The number and type of species selected for comprehensive studies (i.e., multi-species effects) will depend on the purpose of the study and the regulatory requirements under which the tests are being conducted. The ratio of monocotyledons to dicotyledons selected will vary depending on the study objectives.Note de bas de page2 The ecological, agricultural, and physiological significance of the 12 terrestrial plant species to be used in this biological test method are summarized in Section 1.2.

The test must be started using certified seed (i.e., seed is certified for purity and percent germination).Note de bas de page3 The plant seed used for testing must be from the same lot number for each of the individual plant species, and should be free of fungicides, insecticides, repellents, or other chemical pretreatment agents. It is preferable to use untreated seed; however, if untreated seed is unavailable and/or the objectives of a specific test would be better met with the use of treated seed, then treated seed may be used.Note de bas de page4 Details on seed purchasing, sorting, storing, and condition are provided in Sections 2.2, 2.3, 2.4, and 2.5.

Each plant species has unique characteristics that affect its performance in a toxicity test; therefore, certain test procedures and conditions (i.e., number of seeds used to initiate a test, test duration, and test validity criteria) are modified on a species-specific basis to accommodate these requirements (see Sections 4.2, 4.3, and 4.4).

2.2 Source

Seeds used to initiate a soil toxicity test should be obtained from commercial seed companies or government seed banks. When purchasing certified seed, the supplier should provide proof that the seed has not been pretreated with any substances. The following information should be obtained when purchasing seed:

The date the seed package is opened should also be recorded. Seed should generally be purchased at least annually Note de bas de page5 , preferably within one year of the date of the seed germination rating; however, a given lot of seed may be used as long as the seed can meet the control performance criteria described herein (see Section 4.4), and provided that the sensitivity of the seed does not change significantly over time as determined by the results of reference toxicity tests (see Section 4.9).

Sources that have been used to secure quality seed for toxicity testing include:

William Dam Seeds Ltd. Box 8400
Dundas, ON Canada L9H 6M1
Phone: (905) 628-6641
Fax:(905) 627-1729
Web site
Species: alfalfa, carrot, cucumber, lettuce, radish, red clover, red fescue, tomato

Rosebank Seed Farms Ltd.
7340 Perth Line 24
RR #2, Staffa, ON Canada N0K 1Y0
Phone: 1-888-289-9934
Fax: (519) 345-9930
Species: barley

Pickseed Canada Inc. Box 3230
Sherwood Park, AB Canada T8A 2A6
Phone: (780) 464-0350
Fax: (780) 464-0305
Web site
Species: northern wheatgrass

Alberta Nursery & Seeds Ltd. Box 20
Bowden, AB Canada T0M 0K0
Phone: (403) 224-3544
Fax: (403) 224-2455
Web site
Species: radish, red fescue

Prairie Moon Nursery
Route 3, Box 1633
Winona, NM USA 55987-9515
Phone: 1-866-417-8156
Fax: (507) 454-5238
Web site
Species: blue grama grass

Early’s Farm & Garden Centre Inc.
2615 Lorne Ave. Saskatoon, SK Canada S7J 0S5
Phone: 1-800-667-1159
Fax: (306) 931-7110
Web site
Species: carrot, lettuce, radish, northern wheatgrass

David T. Gehl, Seed Increase Unit Sustainable Production Systems Indian Head Research Farm Agriculture and Agri-Food Canada
P.O. Box 760
Indian Head, SK Canada, S0G 2K0
Phone: (306) 695-2274
Fax: (306) 695-3445
E-mail: gehl@agr.gc.ca
Species: durum wheat

Prairie Habitats
Box 10, Argyle
Manitoba, Canada, R0C 0B0
Web site
Species: blue grama grass

A current list of seed suppliers can be obtained by contacting:
Soil Toxicology Laboratory Environmental Technology Centre Environment Canada
335 River Road
Ottawa, ON, K1A 0H3
Phone: (613) 990-9544

2.3 Seed Sorting and Preparation

The seeds of a given species vary in size, shape, and, in some cases, colour. These differences in external features of the seed are often associated with different rates of germination or even different germination requirements (ASTM, 1999b). To minimize variance in test results, the seed should be “hand-sorted” or screenedNote de bas de page6 to ensure uniformity in size, colour, and “quality”. Quality of seed refers to those seeds without a blemished seed coat or irregular shape. Separation of broken or damaged seeds, empty hulls, and other vegetative debris from the seed lot is important. It is especially necessary to sort the seed of northern wheatgrass and barley, since for these species there might be a number of hulls without seed. Some of the test species vary in size, and it is important to use seed that is as uniform as possible to reduce the variation within and among the test vessels (Aquaterra Environmental, 1998a). Uniformity in colour of seed might be a consideration for some species such as alfalfa.Note de bas de page7 Seeds that have evidence of fungal contamination on the seed coat, or seeds that appear to be damaged, must be discarded.

2.4 Seed Storage

The seeds should be kept in their original paper packages and stored in the dark, in labelled, sealed containers (e.g., zip-lock bags) at 4 ± 2 °C. Test seed must remain refrigerated until the day of test initiation (Day 0), at which time the seed must be removed from the refrigerator and brought to room temperature. Seed must not be stored in a freezer since this could cause damage to the seeds (e.g., damage due to dehydration, or that due to expansion of moisture during freezing and in some instances the resultant splitting of the seed).

2.5 Seed Condition

The sensitivity of each new lot of seed used in a definitive soil toxicity test must be measured using a 7- or 10-day (i.e., depending on the species) reference toxicity test (see Section 4.9). Ideally, a reference toxicity test should be performed together with each definitive soil toxicity test. However, laboratories routinely undertaking soil toxicity tests may choose instead to conduct routine reference toxicity tests (i.e., at least once every two months) using a portion of each lot of seed used for definitive soil toxicity tests. All tests with a reference toxicant(s) should be performed using the conditions and procedures outlined in Section 4.9. Species- specific, test-related criteria used to judge the validity of a particular definitive soil toxicity test (and, indirectly, the condition of the lot of seed used in the test), based on the performance of the test organisms in the negative control soil, are given in Section 4.4.

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