Biological test method for measuring terrestrial plants exposed to contaminants in soil: appendix E
Appendix E - Procedural Variations for Tests of Emergence and Growth in Soil Using Terrestrial Plants, as Described in International Methodology Documents
The following source documents are listed chronologically, by originating agency rather than by author(s).
OECD 1984a - the standard guideline for testing the effect of chemicals on the growth of terrestrial plants, published by the Organization for Economic Cooperation and Development (Paris, France) in 1984.
USEPA 1989 - the protocol for performing seed germination tests with the lettuce seed (Lactuca sativa), published in February 1989 by the United States Environmental Protection Agency (co-authors, J.C. Greene,
C.L. Bartels, W.J. Warren-Hicks, SL.R. Parkhurst, G.L. Linder, S.A. Peterson, and W.E. Miller) as one of several protocols for short-term toxicity screening of hazardous waste sites.
ISO 1993a - an international standard test method for determining soil toxicity using terrestrial plants and the inhibition of root growth, published in 1993 by the International Organization for Standardization in Geneva, Switzerland.
ISO 1995 - an international standard test method for testing the effects of chemicals on the emergence and growth of higher plants, published by the International Organization for Standardization in Geneva, Switzerland.
ASTM-94 - is the standard practice (E 1598-94) for conducting early seedling growth tests to assess soil toxicity, written for the American Society for Testing and Materials (ASTM) under the jurisdiction of ASTM Subcommittee E47.11 on plant toxicity and published in February 1998. In 2003, this method was withdrawn as a separate standard and was included as an annex to E 1963-98. See ASTM 1999b in References.
ASTM-98 - the standard guide (E 1963-98) for conducting terrestrial plant toxicity tests (specifically, Annex A1: Seedling emergence) to assess soil toxicity, written for the American Society for Testing and Materials (ASTM) under the jurisdiction of ASTM Subcommittee E47.11 on plant toxicity and published in February 1998. See ASTM 1999b in References.
EC 2000 - the standard operating procedure for conducting early seedling growth toxicity tests using terrestrial plants, prepared in April 2000 by D. Moul for Environment Canada’s Pacific Environmental Science Centre, North Vancouver, British Columbia.
| Document1 | Test Type | Test Duration | Test Facility |
|---|---|---|---|
| OECD (1984a) | static | ≥14 jours aafter 50% of seedlings have emerged in controls | phytotrons, glasshouses, plant growth chambers |
| USEPA (1989) | static | 120 h | environmental chamber5,6,7 |
| ISO (1993a) | static | 36 to 48 h seed pre-germination 5-day exposure2,3 | growth cabinet |
| ISO (1995) | static | 14-21 days after 50% of seedlings have emerged in controls | phytotrons, greenhouse, plant growth room |
| ASTM-94 | static | ≥21 days after 50% of control plants have emerged; 28 days total | greenhouse, growth chamber, phytotron 5,6,7 |
| ASTM-98 | static | double the time required to achieve acceptable percentage germination levels, adjusted to the nearest whole week4 | greenhouse, growth chamber, phytotron5,6,7 |
| EC (2000) | static | 7 days 8 | environmental chamber5,6,7 |
Table Notes
1 See preceding pages for complete citation information.
2 Test duration may be adjusted to accommodate other species.
3 Test duration should be that which is known to produce roots no longer than 80% of the depth of the soil in the pot.
4 For example: lettuce is 90% germinated in 4 days, therefore the test would be 7 days long.
5 Free from toxic contamination and vapours.
6 Maintains recommended temperature.
7 Has reasonable humidity control and supplemental lighting.
8 May be 10 days in length for some test species.
| Document | Description of Organisms at Start of Test | Species | Number of Species for Battery | Criteria for Selection of Battery | Other Species?1 |
|---|---|---|---|---|---|
| OECD (1984a) | seeds of same size class, not imbibed | see Table 1, Appendix F | ≥5 | ≥1 from each category 2 | Yes3 |
| USEPA (1989) | seeds of same size class, untreated | lettuce (Lactuca sativa) | N.A.4 | N.A. | No |
| ISO (1993a) | pre-germinated seeds, undressed5 | barley (Hordeum vulgareL.)6 | N.A. | N.A. | Yes7 |
| ISO (1995) | seeds in uniform in size, undressed, not imbibed | see Table 2, Appendix F | ≥2 | ≥1 from each category8 | NI9 |
| ASTM-94 | certified seeds uniform in size, from same batch/lot, untreated | see Table 3, Appendix F | ≥5 | ≥3 dicots10 ≥2 monocots11 |
Yes12 |
| ASTM-98 | seeds uniform in size and colour, from same batch/lot, preferably untreated, may be field-collected | any species: lists those commonly used in other test methods (FIFRA, TSCA, FDA, OECD, APHA/AWWA, ASTM) | NI | NI | Yes |
| EC (2000) | certified seeds, uniform in size, colour and shape from same batch/lot, untreated | any species | 5 | 3 dicots3 2 monocots14 |
Yes |
Table Notes
1 Indicates whether species other than those specified in the method may be used in a test.
2 At least one species is selected from each of three categories; see Table 1 in Appendix F.
3 Other species may be used if the rationale for their selection is justified in the test report.
4 NA = not applicable.
5 Seeds are germinated in a petri dish on filter paper moistened with distilled water until the radicle has just emerged (radicle < 2 mm in length); for barley, seed germination takes 36-48 h at 20 °C in the dark.
6 Barley variety CV Triumph is recommended; however, other varieties may be used.
7 Method may be adapted for use with other dicotyledonous species with straight roots that are easily measurable.
8 At least one species is selected from each of 2 categories (dicotyledons and monocotyledons); see Table 2 in Appendix F.
9 NI = not indicated.
10 3 species from 2 families (one legume and one root crop) from list of dicotyledons; see Table 3 in Appendix F.
11 2 species from 1 family, including corn, from list of monocotyledons; see Table 3 in Appendix F.
12 If species selection guidance is followed.
13 3 species from 2 families (one legume and one root crop); recommend lettuce (Lactuca sativa).
14 2 species from 1 family, including corn; recommend barley (Hordeum vulgare).
| Document | Seed Sorting | Seed Planting | Seed Culling | Seed Storing |
|---|---|---|---|---|
| OECD (1984a) | NI1 | NI | NI | NI |
| USEPA (1989) | size grading using wire mesh screens; visually inspected to remove trash, empty hulls, and damaged seeds | space seeds 1.27 cm (0.5 in) from edige of test vessel; press seeds into test soil with bottomo of clean beaker; cover sand on top of test soil | None | in airtight waterproof containers at 4 °C |
| ISO (1993a) | NI | 10 mm beneath surface of test medium | None | NI |
| ISO (1995) | NI | NI | after assessing emergence, thin seedlings to give a total of 5 evenly spaced, representative specimens | NI |
| ASTM-94 | NI | use template for maing holes; 1.0-1.5 cm deep for small seeds; 2.5-4.0 cm for large seeds2; tap pots lightly to cover seeds; use of microbial inocula is optional | None | à 4 ± 2 °C |
| ASTM-98 | size grading may be done using sieves or screens; visually inspected to remove broken or damaged seeds | template for maing holes; 1.0-1.5 cm deep for small seeds; 2.5-4.0 cm for large seeds2; tap pots lightly to cover seeds; use of microbial inocula is optional | None | au dessiccateur, à 4 ± 2 °C |
| EC (2000) | seeds are "hand-sorted" or screened to ensure uniformity of size, colour, and shape; damaged seeds are discarded | template for small seeds; manual for larger seeds; gently cover seeds with surrounding soil | None | 4 ± 2 °C3 |
Table Notes
1 NI = not indicated.
2 Seeds should be planted at a soil depth 1.5 to 2 times the seed diameter.
3 For storage 2 months, replicate germination tests are performed to check viability; seeds germinating < certified % germination rate are discarded.
| Document | Test Vessel | Cover | Type of Test Soil1,2 | Amount of Soil/Container |
|---|---|---|---|---|
| OECD (1984a) | non-porous plastic or glazed pots of adequate size to allow unrestricted growth | NI3 | NI | NI |
| USEPA (1989) | bottom halves of 150 × 15 mm plastic petri dishes | 35 × 35 cm (12 × 12 in) polyethylene resealable bags | AS, SWMm and mixtures thereof; ASS | 100 g, dry weight artificial or test soil; 90g cover sand |
| ISO (1993a) | cylindrical pots, 8cm diameter × 11 cm high; parallel sides (not tapered); base of pot perforated (lined with filter paper) | watch-glass | AS, SS, RS, SWN and mixtures thereof; ASS, SRS | 500 g (dry weight) |
| ISO (1995) | non-porous plastic or glazed pots with top internal diameter of 85-95 mm | NI | AS, SAS, SRS, RS4 | 500 g (dry weight) |
| ASTM-94 | glass, stainless steel, or paper containers with drainage holes recommended; polyethylene or other material may be used if free from toxic materials5; large enough so as not to restrict seedling growth for test duration6 | NI | AS, SWM, SAS, SRS | NI |
| ASTM-98 | glass, stainless steel, or paper containers with drainage holes recommended; polyethylene or other material may be used if free from toxic materials5; large enough so as not to restrict seedling growth for test duration6 | récipient muni d'un couvercle pendant la prégermination; couvercle retiré dès la levée des plantules | AS, RS, SWM, SS and mixtures thereof: SL, EL, SAS, SRS | 100-300 g (nominal dry weight) |
| EC (2000) | uncovered plastic petri dish (100 × 15 mm), placed in an 18 × 20 cm, plastic zip lock freezer bag (GladTM that is placed inside a sealed 15 × 23 cm (6 × 9 in) all-glass jar | Yes | AS, SS, SL, RS et mélanges de ces matériaux; SAS, SRS | 50 g (dry weight) |
Table Notes
1 See Table 3 in this appendix for a description.
2 AS = artificial soil; SWM = solid waste material; SS = site soil; RS = reference soil; SL = sludge/slurry; EL = eluates; SAS = spiked artificial soil; SRS = spiked reference soil.
3 NI = not indicated.
4 Method can be adapted for use with solid waste material, site soils, and spiked site soils.
5 Suitability of soil medium for particular test species and conditions determined before testing.
6 Test vessels (e.g., plant pots) are inert to test and control substances (e.g., test substance does not adhere to or react with vessel).
| Document | Description of Test Soil(s) | Description of artificial soil1 |
|---|---|---|
| OECD (1984a) | solids incorporated into soils; aquious chemical substances mixed into soil; not necessarily sterile; <1.5% carbon content (3% organic matter); 10-20% fine particle (<20 µm) | NI2 |
| USEPA (1989) | solid hazardous waste (contamined soil) or aqueous chemical substances mixed in artificial soil | 20-mesh washed silica sand; cover-sand is 16 mesh sand (sieved to remove fines; 20 mesh) |
| ISO (1993a) | reference or potentially toxic site soils; solids incorporated into soils; waste residues, or aqueous chemical substances mixed into soil; alternatively, soil diluated with reference or artificial soil | washed industrial sand or similar; particle size distribution: 10% >0,6 mm, 80% 0.2-0.6 mm, 10% <0.2 mm |
| ISO (1995) | reference or potentially toxic site soils; solids incorporated into soils; waste residues, or aqueous chemical substances mixed into soil; alternatively, soil diluated with reference or artificial soil | sterile or non-sterile sieved (4-5 mm) artificial soil; carbon content ≤1,5% (3% organic content); fine particles (<0.02 mm) ≤20% or dry mass3 |
| ASTM-94 | solids incorporated into soils; aqueous chemical substances applied to or mixed into soil; "standard soil" with <5% organic matter reocmmended | synthetic soil mixes (sieved, 2.0 mm), glass beads, or washed quartz sand |
| ASTM-98 | reference or potentially toxic site soils; solids incorporated into soils; aqueous chemical substances or sludge applied to or mixed into soil; alternatively, soil diluated with reference or artificial soil | synthetic soil mixes or washed quartz sand |
| EC (2000) | reference or potentially toxic site soil; domestic or industrial sludge; soil spiked with chemicals or soil diluated with reference or articial soil | 10% sieved (2.36-mm), 20% kaolinite clay, and 70% "grade 70" silica sand; adjust pH to 7.0 with CaCO3 |
Table Notes
1 Percentages are expressed on a dry-mass basis.
2 NI = not indicated.
3 Sand should be added to natural soils to bring the organic or fine particle content to within approved limits.
| Document | Description of Control Soil | Description of Reference Soil |
|---|---|---|
| OECD (1984a) | NI1 | N.A.2 |
| USEPA (1989) | 100 % artificial soil3 | NI |
| ISO (1993a) | reference soil and/or artificial soil, where applicable3 | soil of same texture class and as similar as possible (without toxicants) to the test soil |
| ISO (1995) | reference soil and/or artificial soil, where applicable3 | soil of same texture class and as similar as possible (without toxicants) |
| ASTM-94 | reference soil and/or artificial soil, where applicable3 | natural soil (free of chemical contaminants), sieved (e.g., 2.0 mm); modified to specific soil characteristics (% clay, silt, sand, and organic matter), if necessary |
| ASTM-98 | reference soil and/or artificial soil, where applicable3 | natural soil (free of chemical contaminants) |
| EC (2000) | reference soil and/or artificial soil, where applicable3 | field-collected soil from an area that has not been cultivated or treated with pesticides or fertilizers in the past 25 years |
Table Notes
1 NI = not indiciated
2 NA = not applicable.
3 See Table 5, this appendix
| Document | Storage Conditions | Soil Characterization |
|---|---|---|
| OECD (1984a) | NI1 | NI |
| USEPA (1989) | seal in plastic (twice) and then in a pail; chill to 4 °C, ship on ice, store at 4 °C; initiate test within 24h of collection | moisture content of site soils; water-holding capacity of aritificial and site soils; pH at start and end of test |
| ISO (1993a) | NI | NI |
| ISO (1995) | if non-sterile, store in accordance with ISO 10381-6 | NI |
| ASTM-94 | NI | standard soil characterized: organic matter, pHw2, soil texture and type, cation exchange capacity, and major nutrients |
| ASTM-98 | seal in plastic (twice) and then in a pail | water-holding capacity |
| EC (2000) | in the dark at <8 °C | moisture content and pH |
Table Notes
1 NI = not indicated
2pHw = pH in water
| Document | Mixing | Sample Holding Time | Hydration | pH Adjustment |
|---|---|---|---|---|
| OECD (1984a) | screened (0-5-cm mesh); use any mixing method resulting in even dispersion of test substance throughout soil; surfactants should not be used; solvent may be used1 | test to begin <24 h after mixing test substance into soil | NI2 | NI |
| USEPA (1989) | homogenize solid test material with artificial soil using a blender; or hydrate artificial soil with aqueous test samples | not to exceed 36 h; test should begin ≤24 h after sample collection | hydrate to 85% of WHC with de-ionized water3 | if pH <4 or >104 |
| ISO (1993a) | artificial soil/test soil dried at 30 ± 2 °C for 16 h; sieved (4-mm sieve); homogenize solid test material with artificial soil or reference soil or hydrate test soil with aqueous test samples; solvents may be used5,6 | NI | maintain at 70 ± 5% of WHC with de-ionized water | NI |
| ISO (1995) | any method ensuring even distribution of chemical throughout soil; homogenize solid test material with artificial or reference soil, or hydrate soil with aqueous test samples; solvents may be used5,6 | test to begin <24 h after mixing (24 h is solvent used) | as required with de-ionized water7 | NI |
| ASTM-94 | test substance added8 | NI | au début, sans saturer le sol | facultatif, si le pH se situe à l'extérieur de la fourchette 6,0-7,59 |
| ASTM-98 | preferable to mix test substance or contaminated soil directly with test medium; stock solution10 may be prepared and added to test medium; solvents may be used8,11 | NI | hydrate to WHC of test soil with de-ionized water, at beginning of test | optional, if pH outside 6.0-7.5range9 |
| EC (2000) | screen (4-9 mm) if required, in which case dry to 10-20% moisture; mix; hydrate; solvents may be used | NI | hydrate to -35% of dry weight for each test soil, only while mixing or preparing test soils; once seeds are planted, hydrate to saturation. | NI |
Table Notes
1 For solvent use: dissolve chemical in a volatile solvent; mix the solution with sand; let the solvent evaporate; mix the sand with soil; maintain a constant sand-soil ratio for all treatments including control.
2 NI = not indicated.
3 WHC = water-holding capacity.
4 If pH range outside 4-10, results might reflect pH toxicity; altering the pH of the soil can increase or decrease (depending on contaminant) the toxicity of contaminants therein.
5 For chemicals with low water solubility, dissolve chemical in water; mix with sand; mix treated sand with soil.
6 If solvent required, dissolve chemical in a volatile solvent and mix with sand; dry sand with air flow and continuous mixing; mix the sand with the soil; ensure same quantity of solvent and sand are used for all treatments including control.
7 The appropriate water-holding capacity should be predetermined and maintained throughout test (e.g., 80% for Avena sativa and 60% for Brassica rapa).
8 Test substances with low aqueous solubility might require being dissolved in an organic solvent such as acetone. The solvent/chemical substance stock solution can be added to quartz sand or glass beads and allowed to dry. The sand and/or glass beads can then be mixed with soil for testing, or seeds can be placed in the sand or glass beads with nutrient solution.
9 pH raised with calcium carbonate; pH lowered with sulphuric acid, gypsum, ammonium sulphate.
10 If a stock solution is used, the concentration and stability of the test substance in the stock should be determined before the beginning of the test.
11 The concentration of solvent in test solutions should be kept to 1% volume-to-volume or weight-to-volume (this does not apply to any ingredients or a formulated mixture or a commercial product).
12 If solvent concentration is not the same in all test solutions, then a solvent test must be run or results of a previous solvent test must be available.
| Document | Number of Seeds per Vessel | Number of Replicates per Treatment or Concentration | Number of Concentrations per Sample or Test Material | Recommended Dilution Factor/Application Rate |
|---|---|---|---|---|
| OECD (1984a) | 5 | 4 | 3, plus control | 0, 1.0, 10.0, 100 mg/kg soil d.w.1 |
| USEPA (1989) | 40 | 3 | 5, plus control | 0.5 (e.g., 100%, 50%, 25%); d.w. hazardous waste/d.w. artifical soils, plus control (100% artificial soil) |
| ISO (1993a) | 6 | 3 | highest concentration for test substance 1000 mg/kg d.w. | geometric series, 0.5 |
| ISO (1995) | 20 (réduire pour obtenir 5 plantules)2 | 4 | concentration la plus élevée pour la substance d'essai ≤1000 mg/kg d.w. | geometric series, 0.5 |
| ASTM-94 | ≥15 per concentration | NI | ≥5, plus control | NI3 |
| ASTM-98 | 5-204 | 5 | number based on goal of study | NI |
| EC (2000) | 5-104 | 5 | ≥9, plus control | NI |
Table Notes
1 d.w. = dry weight.
2 See Table 3, this appendix.
3 NI = not indicated.
4 Number of seeds per vessel depends on size of seeds and seedling, and on test requirements.
| Document | Temperature (°C) | Lighting Conditions | Humidity | pH Range | Watering |
|---|---|---|---|---|---|
| OECD (1984a) | suitable for test species | appropriées aux espèces d'essai | suitable for test species | 5.0-7.5 | as needed |
| USEPA (1989) | 24 ± 2 °C | dark for 48 h; then 16 h light: 8 h dark, 4300 ± 430 lux; fluorescent | NI1 | 4.0-10.0 | none |
| ISO (1993a) | 20 ± 2 °C, day; 16 ± 2 °C, night | 12-16 hlight; 8-12 h dark; 25000 lm/m2 | 60 ± 5 % | NI | maintain à 70% WHC2 |
| ISO (1995) | suitable for test species | suitable for test species3 | suitable for test species | 5.0-7.5 | daily adjustment to pre-determined WHC |
| ASTM-94 | température de l'air 20-30 °C | ≥14 h light; fluorescent/ incandescent or sun; ≥300 µmol/(m2 · s) [300- 400 µmol/ (m2 · s) recommanded] |
>30 %; ≥50 % recommandé | 6.0-7.5 | as needed; solution used weekly if quartz sand, glass beads, or soil low in nutrients are used as soil medium |
| ASTM-98 | suitable for test species; temperature de l'air 20-30 °C | 16 h light; 8 h dark; incandescent; 100-200 µmol/ (m2 · s) (RPE 400-700 rim)4 |
>30 %; 50 % recommanded | 6.0-7.5 | once covers are removed, water as required (at least daily) to saturation or less (e.g., 85% WHC) |
| EC (2000) | 24 ± 2°C | 16 h light; 8 h dark5 full spectrum (Duro-testMD); 4300 ± 430 lux [765 µmol/(m2 · s)] |
NI | >4. <10 | hydrated to saturation; de-ionized water sprayed onto soil surface |
Table Notes
1 NI = not indicated.
2 WHC = water-holding capacity.
3 Method recommends 16 h daylight and a minimum of 7000 lux light intensity in the photosynthetic wavelength.
4 photosynthetically active radiation.
5 Small seeds planted at the surface should be kept in the dark for the first 48 h.
| Document | Measures1 | Biological Observations |
|---|---|---|
| OECD (1984a) | NI2 | percent emergence per replicate3; wet or dry weight per repliacte, expressed on per- plant basis4 |
| USEPA (1989) | pH of soil at start and end; soil temperature beginning of each 24-h exposure period in each test concentration and control (1 replicate) | percent germination in each replicate5 |
| ISO (1993a) | NI | length of longest root for each plant at test end6 |
| ISO (1995) | percent emergence in each replicate3 | mean wet or dry weight of seedling shoots per replicate, at test end8 |
| ASTM-94 | photoperiod; light intensity daily; continuous measurements for air temperature and relative (soil temp. of representative pot); pH9) when test medium is prepared and at test end | number of seedlings that emerge 10 time to emergence during 1st week; humidity percent survival; plant height; radicle (root) (pH length; dry weight of above-ground vegetation and roots; severity of phytotoxicity (qualitative observations) |
| ASTM-98 | light irradiance levels at start and end of test; continuous (or at least once daily) measurements air temperature, relative humidity and barometric pressure; soil temperature of representative pot; soil pH or pHw9 when test soil medium is prepared and at test end | number of emerged seedlings;11; qualitative abnormalities in growth/ for development, or morphology at test end; option for both shoot/root length12 and shoot/root dry weight13 |
| EC (2000) | continuous temperature and light; pH and conductivity at start and end, each treatment; percent moisture at start | number of seedlings emerged14; shoot length, and length of longest root; quantitative observations of phytotoxic effects |
Table Notes
1 Measurements include pH (hydrogen-ion concentration), temperature, light, humidity, etc.
2 NI = not indicated.
3 Emergence = appearance of seedling above soil surface.
4 Measure wet weight of plant immediately after harvest, or dry weight after oven drying at 70°C.
5 Germination = seedling protrudes above soil surface.
6 Shoot length may also be measured.
7 Record of temperature and humidity recommended.
8 Fresh mass weighed immediately after cutting shoots above soil surface or dry mass after oven drying at 70- 80 °C for 16 h.
9 pHw = pH in water.
10 Emergence = the hypocotyl hook or first true leaves (coleoptile) are observed above the surface of the soil medium.
11 Emergence = epicotyl above soil surface.
12 Shoot measurements are made from the transition point between the hypocotyl and root to the tallest point of the shoot; root measurements are made from the transition point between the hypocotyl and root to the tip of the root.
13 Oven dried at 70°C until constant weight achieved (recommend 24 h).
14 Emergence = shoot height of 3 mm above soil surface.
| Document | Termingating Test | Biological Endpoints | Statistical Endpoints |
|---|---|---|---|
| OECD (1984a) | count the number of plants that emerge per replicate, and determine average weight of plants | percent emergence; percent growth inhibition at test end | EC50 for emergenc; EC50 pour la croissance |
| USEPA (1989) | count seedlings protruding above soil surface | percent mortality (lack of seed germination) | EC501 |
| ISO (1993a) | lay pot on its side in a trough of water 5-cm deep; wash soil out of pots and wash each plant; measure the longest root to the nearest 0.5m mm | mean root length | NOEC/LOEC |
| ISO (1995) | count the number of plants that emerge per replicate, and determine the total mass of shoots of seedlings per replicate | percent ermergence; mean mass2 | EC50 |
| ASTM-94 | count number of plants that emrged; yield for each plant species is determined by harvesting the portion of each seedling above ground or below ground for roots and then oven-drying | percent emergence; mean time to emergence; mean heights and/or root lenths; mass; scores for qualitative phytotoxic effects | mean, 95% CL, and SD for each quantitative date set; NOEC/LOEC, EC501 |
| ASTM-98 | count seedlingts above soil surface; conduct qualitative observations and optional quantitative measurements | percent emergence; optional mean shoot/root length; mean dry weight | mean, 95% CL, and SD for each quantitative date set; NOEC/LOEC, EC50, ECx et ICp1 |
| EC (2000) | count number of emerged seedlings; photograph pots to show above ground phytomass; conduct qualitative observations; separate plants from soil and wash roots to dislodge soil; measure shoot length and longest root length | percent emergence; mean shoot/root length; scores for qualitative phytotoxic effects | LC501 , IC50, IC25, NOEC/LOEC |
Table Notes
1 Including the 95% confidence limits (CL).
2 Dry mass preferred; see Table 11, this appendix.
| Document | Requirements for Valid Test | Reference Toxicant(s) | Procedurs and Conditions for Reference Toxicity Test |
|---|---|---|---|
| OECD (1984a) | 80% of control seeds produce healthy seedlings; control seedlings exhibit normal growth throughout test | no reference substance recommended | if reference substance tested, results should be given |
| USEPA (1989) | mean control survival 90% | SDS, NaPCP or CdCl21 | using 100% artificial soil and test concentrations of reference toxicant diluted in the de-ionized water used to hydrate the soil, determine 12-h LC50 on each batch of seed; plot results on control chart; invalid if mean control survival <90% |
| ISO (1993a) | NI2 | NI | NI |
| ISO (1995) | 5 healthy seedlings per pot | sodium trichloroacetate | reference toxicant test conducted control if any major changes in procedure (e.g., test chamber, soil watering regime) |
| ASTM-94 | mean control seedling growth does not exhibit phytotoxicity or developmental effects; 90% control survival through the exposure duration | no reference chemical required or recommended | NI |
| ASTM-98 | mean control seedling growth does not exhibit phytotoxicity or developmental effects; survival during expsoure period meets minimum standards for that species (80% mean control survival unlessa a lower criterion is established for the species) | boron as boric acid | a watering solution of boric acid at desired concentration is added to the test soil (0.5 dilution series with 7 concentrations from 10-640 mg/kg soil dry weight)3 alternate substances may be used; test invalid if mean control survival is <80% |
| EC (2000) | 90% control survival (pooled replicates); 90% control plants showing no stress (e.g., chlorosis, deformity) | potassium chloride | conduct every 2 months, with seed lots being used in tests and after the acquisition of any new seeds; plot results on warning chart |
Table Notes
1 SDS = sodium dodecylsulphate; NaPCP = sodium pentachlorophenate; CdCl2 = cadmium chloride.
2 NI = not indicated.
3 Fewer concentrations may be used once the range of sensitivity for a given test species is established.
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