Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• Environmental conditions should be monitored and reported during the course of the study. The plants should be grown in non-porous plastic or glazed pots with a tray or saucer under the pot. The pots may be repositioned periodically to minimize variability in growth of the plants (due to differences in test conditions within the growth facilities). The pots must be large enough to allow normal growth.

• 23.Soil nutrients may be supplemented as needed to maintain good plant vigour. The need and timing of additional nutrients can be judged by observation of the control plants. Bottom watering of test containers (e.g. by using glass fiber wicks) is recommended. However, initial top watering can be used to stimulate seed germination and, for soil surface application it facilitates movement of the chemical into the soil.

• 24.The specific growing conditions should be appropriate for the species tested and the test chemical under investigation. Control and treated plants must be kept under the same environmental conditions, however, adequate measures should be taken to prevent cross exposure (e.g. of volatile chemicals) among different treatments and of the controls to the test chemical.

Testing at a single concentration/rate

• 25.In order to determine the appropriate concentration/rate of a chemical for conducting a single-concentration or rate (challenge/limit) test, a number of factors must be considered. For general chemicals, these include the physical/chemical properties of the chemical. For crop protection products, the physical/chemical properties and use pattern of the test chemical, its maximum concentration or application rate, the number of applications per season and/or the persistence of the test chemical need to be considered. To determine whether a general chemical possesses phytotoxic properties, it may be appropriate to test at a maximum level of 1 000 mg/kg dry soil.

Range-finding test

• 26.When necessary a range-finding test could be performed to provide guidance on concentrations/rates to be tested in definitive dose-response study. For the range-finding test, the test concentrations/rates should be widely spaced (e.g. 0,1, 1,0, 10, 100 and 1 000 mg/kg dry soil). For crop protection products concentrations/rates could be based on the recommended or maximum concentration or application rate, e.g. 1/100, 1/10, 1/1 of the recommended/maximum concentration or application rate.

Testing at multiple concentrations/rates

• 27.The purpose of the multiple concentration/rate test is to establish a dose-response relationship and to determine an ECx or ERx value for emergence, biomass and/or visual effects compared to un-exposed controls, as required by regulatory authorities.

• 28.The number and spacing of the concentrations or rates should be sufficient to generate a reliable dose-response relationship and regression equation and give an estimate of the ECx. or ERx. The selected concentrations/rates should encompass the ECx or ERx values that are to be determined. For example, if an EC50 value is required it would be desirable to test at rates that produce a 20 to 80 % effect. The recommended number of test concentrations/rates to achieve this is at least five in a geometric series plus untreated control, and spaced by a factor not exceeding three. For each treatment and control group, the number of replicates should be at least four and the total number of seeds should be at least 20. More replicates of certain plants with low a germination rate or variable growth habits may be needed to increase the statistical power of the test. If a larger number of test concentrations/rates are used, the number of replicates may be reduced. If the NOEC is to be estimated, more replicates may be needed to obtain the desired statistical power (23).

Observations

• 29.During the observation period, i.e. 14 to 21 days after 50 % of the control plants (also solvent controls if applicable) have emerged, the plants are observed frequently (at least weekly and if possible daily) for emergence and visual phytotoxicity and mortality. At the end of the test, measurement of percent emergence and biomass of surviving plants should be recorded, as well as visible detrimental effects on different parts of the plant. The latter include abnormalities in appearance of the emerged seedlings, stunted growth, chlorosis, discoloration, mortality, and effects on plant development. The final biomass can be measured using final average dry shoot weight of surviving plants, by harvesting the shoot at the soil surface and drying them to constant weight at 60 °C. Alternatively, the final biomass can be measured using fresh shoot weight. The height of the shoot may be another endpoint, if required by regulatory authorities. A uniform scoring system for visual injury should be used to evaluate the observable toxic responses. Examples for performing qualitative and quantitative visual ratings are provided in references (23) (24).

DATA AND REPORTING

Statistical analysis

Single concentration/rate test

• 30.Data for each plant species should be analyzed using an appropriate statistical method (21). The level of effect at the test concentration/rate should be reported, or the lack of reaching a given effect at the test concentration/rate (e.g., < x % effect observed at y concentration or rate)

Multiple concentration/rate test

• 31.A dose-response relationship is established in terms of a regression equation. Different models can be used: for example, for estimating ECx or ERx (e.g. EC25, ER25, EC50, ER50) and its confidence limits for emergence as quantal data, logit, probit, Weibull, Spearman-Karber, trimmed Spearman-Karber methods, etc. could be appropriate. For the growth of the seedlings (weight and height) as continuous endpoints ECx or ERx and its confidence limits can be estimated by using appropriate regression analysis (e.g. Bruce-Versteeg non-linear regression analysis (25)). Wherever possible, the R² should be 0,7 or higher for the most sensitive species and the test concentrations/rates used encompass 20 % to 80 % effects. If the NOEC is to be estimated, application of powerful statistical tests should be preferred and these should be selected on the basis of data distribution (21) (26).

Test report

• 32.The test report should present results of the studies as well as a detailed description of test conditions, a thorough discussion of results, analysis of the data, and the conclusions drawn from the analysis. A tabular summary and abstract of results should be provided. The report must include the following:

Test chemical:

• — chemical identification data, relevant properties of the chemical tested (e.g. log Pow, water solubility, vapour pressure and information on environmental fate and behaviour, if available);

• — details on preparation of the test solution and verification of test concentrations as specified in paragraph 18.

Test species:

• — details of the test organism: species/variety, plant families, scientific and common names, source and history of the seed as detailed as possible (i.e. name of the supplier, percentage germination, seed size class, batch or lot number, seed year or growing season collected, date of germination rating), viability, etc.;

• — number of mono- and di-cotyledon species tested;

• — rationale for selecting the species;

• — description of seed storage, treatment and maintenance.

Test conditions:

• — testing facility (e.g. growth chamber, phytotron and greenhouse);

• — description of test system (e.g., pot dimensions, pot material and amounts of soil);

• — soil characteristics (texture or type of soil: soil particle distribution and classification, physical and chemical properties including % organic matter, % organic carbon and pH);

• — soil/substrate (e.g. soil, artificial soil, sand and others) preparation prior to test;

• — description of nutrient medium if used;

• — application of the test chemical: description of method of application, description of equipment, exposure rates and volumes including chemical verification, description of calibration method and description of environmental conditions during application;

• — growth conditions: light intensity (e.g. PAR, photosynthetically active radiation), photoperiod, max/min temperatures, watering schedule and method, fertilization;

• — number of seeds per pot, number of plants per dose, number of replicates (pots) per exposure rate;

• — type and number of controls (negative and/or positive controls, solvent control if used);

• — duration of the test.

Results:

• — table of all endpoints for each replicate, test concentration/rate and species;

• — the number and percent emergence as compared to controls;

• — biomass measurements (shoot dry weight or fresh weight) of the plants as percentage of the controls;

• — shoot height of the plants as percentage of the controls, if measured;

• — percent visual injury and qualitative and quantitative description of visual injury (chlorosis, necrosis, wilting, leaf and stem deformation, as well as, any lack of effects) by the test chemical as compared to control plants;

• — description of the rating scale used to judge visual injury, if visual rating is provided;

• — for single rate studies, the percent injury should be reported;

• — ECx or ERx (e.g. EC50, ER50, EC25, ER25) values and related confidence limits. Where regression analysis is performed, provide the standard error for the regression equation, and the standard error for individual parameter estimate (e.g. slope, intercept);

• — NOEC (and LOEC) values if calculated;

• — description of the statistical procedures and assumptions used;

• — graphical display of these data and dose-response relationship of the species tested.

• Deviations from the procedures described in this test method and any unusual occurrences during the test.

LITERATURE

• (1)Schrader G., Metge K., and Bahadir M. (1998). Importance of salt ions in ecotoxicological tests with soil arthropods. Applied Soil Ecology, 7, 189-193.

• (2)International Organisation of Standards. (1993). ISO 11269-1. Soil Quality – Determination of the Effects of Pollutants on Soil Flora — Part 1: Method for the Measurement of Inhibition of Root Growth.

• (3)International Organisation of Standards. (1995). ISO 11269-2. Soil Quality – Determination of the Effects of Pollutants on Soil Flora — Part 2: Effects of Chemicals on the Emergence and Growth of Higher Plants.

• (4)American Standard for Testing Material (ASTM). (2002). E 1963-98. Standard Guide for Conducting Terrestrial Plant Toxicity Tests.

• (5)U.S. EPA. (1982). FIFRA, 40CFR, Part 158.540. Subdivision J, Parts 122-1 and 123-1.

• (6)US EPA. (1996). OPPTS Harmonized Test Guidelines, Series 850. Ecological Effects Test Guidelines:

• — 850.4000: Background — Non-target Plant Testing;

• — 850.4025: Target Area Phytotoxicity;

• — 850.4100: Terrestrial Plant Toxicity, Tier I (Seedling Emergence);

• — 850.4200: Seed Germination/Root Elongation Toxicity Test;

• — 850.4225: Seedling Emergence, Tier II;

• — 850.4230: Early Seedling Growth Toxicity Test.

• (7)AFNOR, X31-201. (1982). Essai d'inhibition de la germination de semences par une substance. AFNOR X31-203/ISO 11269-1. (1993) Determination des effets des polluants sur la flore du sol: Méthode de mesurage de l'inhibition de la croissance des racines.

• (8)Boutin, C., Freemark, K.E. and Keddy, C.J. (1993). Proposed guidelines for registration of chemical pesticides: Non-target plant testing and evaluation. Technical Report Series No.145. Canadian Wildlife Service (Headquarters), Environment Canada, Hull, Québec, Canada.

• (9)Forster, R., Heimbach, U., Kula, C., and Zwerger, P. (1997). Effects of Plant Protection Products on Non-Target Organisms — A contribution to the Discussion of Risk Assessment and Risk Mitigation for Terrestrial Non-Target Organisms (Flora and Fauna). Nachrichtenbl. Deut. Pflanzenschutzd. No 48.

• (10)Hale, B., Hall, J.C., Solomon, K., and Stephenson, G. (1994). A Critical Review of the Proposed Guidelines for Registration of Chemical Pesticides; Non-Target Plant Testing and Evaluation, Centre for Toxicology, University of Guelph, Ontario Canada.

• (11)Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and Soil Sc. Soc. Amer. Proc. 26:305 (1962).

• (12)Audus, L.J. (1964). Herbicide behaviour in the soil. In: Audus, L.J. ed. The Physiology and biochemistry of Herbicides, London, New York, Academic Press, NY, Chapter 5, pp. 163-206.

• (13)Beall, M.L., Jr. and Nash, R.G. (1969). Crop seedling uptake of DDT, dieldrin, endrin, and heptachlor from soil, J. Agro. 61:571-575.

• (14)Beetsman, G.D., Kenney, D.R. and Chesters, G. (1969). Dieldrin uptake by corn as affected by soil properties, J. Agro. 61:247-250.

• (15)U.S. Food and Drug Administration (FDA). (1987). Environmental Assessment Technical Handbook. Environmental Assessment Technical Assistance Document 4.07, Seedling Growth, 14 pp., FDA, Washington, DC.

• (16)McKelvey, R.A., Wright, J.P., Honegger, J.L. and Warren, L.W. (2002). A Comparison of Crop and Non-crop Plants as Sensitive Indicator Species for Regulatory Testing. Pest Management Science vol. 58:1161-1174

• (17)Boutin, C.; Elmegaard, N. and Kjær, C. (2004). Toxicity testing of fifteen non-crop plant species with six herbicides in a greenhouse experiment: Implications for risk assessment. Ecotoxicology vol. 13(4): 349-369.

• (18)Boutin, C., and Rogers, C.A. (2000). Patterns of sensitivity of plant species to various herbicides — An analysis with two databases. Ecotoxicology vol. 9(4): 255-271.

• (19)Boutin, C. and Harper, J.L. (1991). A comparative study of the population dynamics of five species of Veronica in natural habitats. J. Ecol. 9:155-271.

• (20)Boutin, C., Lee, H.-B., Peart, T.E., Batchelor, S.P. and Maguire, R.J.. (2000). Effects of the sulfonylurea herbicide metsulfuron methyl on growth and reproduction of five wetland and terrestrial plant species. Envir. Toxicol. Chem. 19 (10): 2532-2541.

• (21)OECD (2006). Guidance Document, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. Series on Testing and Assessment No 54, Organisation for Economic Co-operation and Development, Paris.

• (22)Hatzios, K.K. and Penner, D. (1985). Interactions of herbicides with other agrochemicals in higher plants. Rev. Weed Sci. 1:1-63.

• (23)Hamill, P.B., Marriage, P.B. and G. Friesen. (1977). A method for assessing herbicide performance in small plot experiments. Weed Science 25:386-389.

• (24)Frans, R.E. and Talbert, R.E. (1992). Design of field experiments and the measurement and analysis of plant response. In: B. Truelove (Ed.) Research Methods in Weed Science, 2nd ed. Southern weed Science Society, Auburn, 15-23.

• (25)Bruce, R.D. and Versteeg, D. J.(1992). A Statistical Procedure for Modelling Continuous Toxicity Data. Environmental Toxicology and Chemistry 11, 1485-1492.

• (26)Chapter C.33 of this Annex: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei).

Appendix 1

Definitions

Active ingredient (a.i.) (or active substance (a.s.)) is a material designed to provide a specific biological effect (e.g., insect control, plant disease control, weed control in the treatment area), also known as technical grade active ingredient, active substance.

Chemical means a substance or a mixture.

Crop Protection Products (CPPs) or plant protection product (PPPs) or pesticides are materials with a specific biological activity used intentionally to protect crops from pests (e.g., fungal diseases, insects and competitive plants).

ECx. x % Effect Concentration or ERx. x % Effect Rate is the concentration or the rate that results in an undesirable change or alteration of x % in the test endpoint being measured relative to the control (e.g., 25 % or 50 % reduction in seedling emergence, shoot weight, final number of plants present, or increase in visual injury would constitute an EC25/ER25 or EC50/ER50 respectively).

Emergence is the appearance of the coleoptile or cotyledon above the soil surface.

Formulation is the commercial formulated product containing the active substance (active ingredient), also known as final preparation ([^E0186]) or typical end-use product (TEP).

LOEC (Lowest Observed Effect Concentration) is the lowest concentration of the test chemical at which effect was observed. In this test, the concentration corresponding to the LOEC, has a statistically significant effect (p < 0,05) within a given exposure period when compared to the control, and is higher than the NOEC value.

Non-target plants: Those plants that are outside the target plant area. For crop protection products, this usually refers to plants outside the treatment area.

NOEC (No Observed Effect Concentration) is the highest concentration of the test chemical at which no effect was observed. In this test, the concentration corresponding to the NOEC, has no statistically significant effect (p < 0,05) within a given exposure period when compared with the control.

Phytotoxicity: Detrimental deviations (by measured and visual assessments) from the normal pattern of appearance and growth of plants in response to a given chemical.

Replicate is the experimental unit which represents the control group and/or treatment group. In these studies, the pot is defined as the replicate.

Visual assessment: Rating of visual damage based on observations of plant stand, vigour, malformation, chlorosis, necrosis, and overall appearance compared with a control.

Test Chemical: Any substance or mixture tested using this test method.

Appendix 2

List of species historically used in plant testing

Family	Species	Common names
DICOTYLEDONAE	DICOTYLEDONAE	DICOTYLEDONAE
Apiaceae (Umbelliferae)	Daucus carota	Carrot
Asteraceae (Compositae)	Helianthus annuus	Sunflower
Asteraceae (Compositae)	Lactuca sativa	Lettuce
Brassicaceae (Cruciferae)	Sinapis alba	White Mustard
Brassicaceae (Cruciferae)	Brassica campestris var. chinensis	Chinese cabbage
Brassicaceae (Cruciferae)	Brassica napus	Oilseed rape
Brassicaceae (Cruciferae)	Brassica oleracea var. capitata	Cabbage
Brassicaceae (Cruciferae)	Brassica rapa	Turnip
Brassicaceae (Cruciferae)	Lepidium sativum	Garden cress
Brassicaceae (Cruciferae)	Raphanus sativus	Radish
Chenopodiaceae	Beta vulgaris	Sugar beet
Cucurbitaceae	Cucumis sativus	Cucumber
Fabaceae (Leguminosae)	Glycine max (G. soja)	Soybean
Fabaceae (Leguminosae)	Phaseolus aureus	Mung bean
Fabaceae (Leguminosae)	Phaseolus vulgaris	Dwarf bean, French bean, Garden bean
Fabaceae (Leguminosae)	Pisum sativum	Pea
Fabaceae (Leguminosae)	Trigonella foenum-graecum	Fenugreek
Fabaceae (Leguminosae)	Lotus corniculatus	Birdsfoot trefoil
Fabaceae (Leguminosae)	Trifolium pratense	Red Clover
Fabaceae (Leguminosae)	Vicia sativa	Vetch
Linaceae	Linum usitatissimum	Flax
Polygonaceae	Fagopyrum esculentum	Buckwheat
Solanaceae	Solanum lycopersicon	Tomato
MONOCOTYLEDONAE	MONOCOTYLEDONAE	MONOCOTYLEDONAE
Liliaceae (Amarylladaceae)	Allium cepa	Onion
Poaceae (Gramineae)	Avena sativa	Oats
Poaceae (Gramineae)	Hordeum vulgare	Barley
Poaceae (Gramineae)	Lolium perenne	Perennial ryegrass
Poaceae (Gramineae)	Oryza sativa	Rice
Poaceae (Gramineae)	Secale cereale	Rye
Poaceae (Gramineae)	Sorghum bicolor	Grain sorghum, Shattercane
Poaceae (Gramineae)	Triticum aestivum	Wheat
Poaceae (Gramineae)	Zea mays	Corn

Appendix 3

List of potenatial non-crop species

OECD Potential Species for Plant Toxicity Testing

Note: The following table provides information for 52 non-crop species (references are given in brackets for each entry). Emergence rates provided are from published literature and are for general guidance only. Individual experience may vary depending upon seed source and other factors.

FAMILY Species Botanical Name (English Common Name)	Lifespan [^E0187] & Habitat	Seed Weight (mg)	Photoperiod for germination or growth [^E0188]	Planting Depth (mm) [^E0189]	Time to Germinate (days) [^E0190]	Special Treatments [^E0191]	Toxicity Test [^E0192]	Seed Suppliers [^E0193]	Other References [^E0194]
APIACEAE Torilis japónica (Japanese Hedge-parsley)	А, В disturbed areas, hedgerows, pastures (16, 19)	1,7-1,9 (14, 19)	L = D (14)	0 (1, 19)	5 (50 %) (19)	cold stratification (7, 14, 18, 19) maturation may be necessary (19) germination inhibited by darkness (1, 19) no special treatments (5)	POST (5)
ASTERACEAE Bellis perennis (English Daisy)	Ρ grassland, arable fields, turf (16, 19)	0,09-0,17 (4, 19)	L = D (14)	0 (4)	3 (50 %) (19) 11 (100 %) (18)	germination not affected by irradiance (18, 19) no special treatments (4, 14)	POST (4)	A, D, F	7
Centaurea cyanus (Cornflower)	A fields, roadsides, open habitats (16)	4,1 -4,9 (4, 14)	L = D (14)	0-3 (2, 4, 14)	14-21 (100 %) (14)	no special treatments (2, 4)	POST (2,4)	A, D, E, F	7
Centaurea nigra (Black Knapweed)	Ρ fields, roadsides, open habitats (16, 19)	2,4-2,6 (14, 19)	L = D (14)	0 (19)	3 (50 %) (19) 4 (97 %) (18)	maturation may be necessary (18, 19) germination inhibited by darkness (19) no special treatments (5, 14, 26)	POST (5, 22, 26)	A
Inula helenium Elecampane	Ρ moist, disturbed sites (16)	1-1,3 (4, 14, 29)		0 (4, 29)		no special treatments (4)	POST (4)	A, F
Leontodon hispidus (Big Hawkbit)	Ρ fields, roadsides, disturbed areas (16, 19)	0,85 -1,2 (14, 19)	L = D (14)	0 (19)	4 (50 %) (19) 7 (80 %) (18)	germination inhibited by darkness (17, 18, 19) no special treatments (5, 23)	POST (5, 22, 23)
Rudbeckia hirta (Black-eyed Susan)	Β, Ρ disturbed (16)	0,3 (4, 14)	L = D (14)	0 (4, 33)	< 10 (100 %) (33)	no special treatments (4, 14, 33)	POST (4, 33)	C, D, E, F
Solidago canadensis Canada Goldenrod	Ρ pasture, open areas (16)	0,06-0,08 (4, 14)	L = D (11)	0 (4)	14-21 (11)	mix with equal part sand and soak in 500 ppm GA for 24 hrs (11) no special treatments (4)	POST (4)	E, F
Xanthium pensylvanicum (Common Cocklebur)	A fields, open habitats (16)	25-61 (14, 29)		0(1) 5(29)		germination may be inhibited by darkness (1) soak in warm water for 12 hrs (29)	PRE & POST (31)	A
Xanthium spinosum (Spiny Cocklebur)	A open habitats (16)	200 (14)	L = D (14) L > D (6)	10 (6)		scarification (14) no special treatments (6)	PRE & POST (6)	A
Xanthium strumarium (Italian Cocklebur)	A fields, open habitats (16)	67,4 (14)	L = D (14)	10-20 (6, 21)		no special treatments (6, 14, 21)	PRE & POST (6, 21, 28, 31)	A
BRASSICACEAE Cardamine pratensis (Cuckoo Flower)	Ρ fields, roadsides, turf (16, 19)	0,6 (14, 19)	L = D (14)	0 (19)	5 (50 %) (19) 15 (98 %) (18)	germination inhibited by darkness (18, 19) no special treatments (5, 14, 22)	POST (5, 22)	F
CARYOPHYLLACEAE Lychnis flos-cuculi (Ragged Robin)	Ρ (16)	0,21 (14)	L = D (14)		< 14 (100 %) (14, 25)	maturation may be necessary (18) no special treatments (5, 14, 15, 22-26)	POST (5, 15, 22-26)	F
CHENOPODIACEAE Chenopodium album (Lamb's Quarters)	A field margins, disturbed areas (16, 19)	0,7- 1,5 (14, 19, 34)	L = D (14)	0 (1, 19)	2 (50 %) (19)	treatment differs depending on seed colour (19) dry storage dormancy (19) germination inhibited by darkness (1, 18, 19) cold stratification (18) no special treatments (14, 34)	PRE & POST (28, 31, 34)	A	32
CLUSIACEAE Hypericum perforatum (Common St. John's Wort)	Ρ fields, arable land, open habitats (16, 19)	0,1 -0,23 (14, 19)	L = D (14)	0 (1, 19)	3 (19) 11 (90 %) (18)	germination inhibited by darkness (1, 18, 19) no special treatments (5, 14, 15, 25, 27)	POST (5, 15, 25, 27)	A, E, F
CONVOLVULACEAE Ipomoea hederacea (Purple Morning Glory)	A roadsides, open habitats, cornfields (16)	28,2 (14)	L > D (6, 10)	10-20 (6, 10, 21)	4 (100 %) (10)	germination not affected by irradiance (1) no special treatments (6, 21)	PRE & POST (6, 12, 21, 28)	A
CYPERACEAE Cyperus rotundus (Purple Nutsedge)	Ρ arable land, pastures, roadsides (16, 30)	0,2 (14)	L = D (14)	0 (1) 10-20 (6, 10)	12 (91 %) (10)	germination inhibited by darkness (1) no special treatments (6, 10, 14)	PRE & POST (6, 28, 31)	B	7
FABACEAE Lotus corniculatus (Bird's-foot Trefoil)	Ρ grassy areas, roadsides, open habitats (16, 19)	1-1,67 (14, 19)	L = D (14)		1 (50 %) (19)	scarification (14, 19) germination not affected by irradiance (18, 19) no special treatments (23, 25)	POST (5, 23, 25)	A, D, E, F
Senna obtusifolia (Cassia, Sicklepod)	A moist woods (16)	23-28 (9)	L = D (14) L > D (9)	10-20 (6,9)		soak seeds in water for 24 hours (9) scarification (14) seed viability differs depending on colour (1) no special treatments (6)	POST (6,9)	A
Sesbania exaltata (Hemp)	A alluvial soil (16)	11- 13 (9, 14)	L > D (9)	10-20 (9, 21)		soak seeds in water for 24 hours (9) germination not affected by irradiance (1) no special treatments (21)	PRE & POST (9, 21, 28, 31)	A
Trifolium pratense (Red Clover)	Ρ fields, roadsides, arable land (16, 19)	1,4- 1,7 (14, 19)	L = D (14)		1 (50 %) (19)	scarification (14, 18) may need maturation (19) germination not affected by irradiance (1, 19) no special treatments (5)	POST (5)	A, E, F
LAM IAC E AE Leonurus cardiaca (Motherwort)	Ρ open areas (16)	0,75 -1,0 (4, 14)	L = D (14)	0 (4)		no special treatments (4, 14)	POST (4)	F
Mentha spicata (Spearmint)	Ρ moist areas (16)	2,21 (4)		0 (4)		no special treatments (4)	POST (4)	F
Nepeta cataria (Catnip)	Ρ disturbed areas (16)	0,54 (4, 14)	L = D (14)	0 (4)		no special treatments (2, 4, 14)	POST (2,4)	F
Prunella vulgaris (Self-heal)	Ρ arable fields, grassy areas, disturbed sites (16, 19)	0,58 -1,2 (4, 14, 19)	L = D (14)	0 (4, 19)	5 (50 %) (19) 7 (91 %) (18)	germination inhibited by darkness (18, 19) greater germination with larger seeds (1) no special treatments (4, 14, 22)	POST (4, 22)	A, F
Stachys officinalis (Hedge-nettle)	Ρ grasslands, field margins (19)	14-18 (14, 19)	L = D (14)		7 (50 %) (19)	no special treatments (5, 14, 22)	POST (5, 22)	F
MALVACEAE Abutilón theophrasti (Velvetleaf)	A fields, open habitats (16)	8,8 (14)	L = D (14)	10-20 (6, 10, 21)	4 (84 %) (10)	scarification (14) no special treatments (5, 10, 21)	PRE & POST (6, 22, 28, 31)	A, F
Sida spinosa (Prickly Sida)	A fields, roadsides (16)	3,8 (14)	L = D (14)	10-20 (6, 21)		scarification (14) germination not affected by irradiance (1) no special treatments (6, 21)	PRE & POST (6, 21, 28, 31)	A, F
PAPAVERACEAE Papaver rhoeas (Poppy)	A fields, arable land, disturbed sites (16, 19)	0,1 -0,3 (4, 14, 19, 29)	L = D (14)	0 (4, 29)	4 (50 %) (19)	cold stratification & scarification (1, 19, 32) no special treatments (4, 14, 29)	POST (4)	A, D, E, F, G
POACEAE Agrostis tenuis (Common Bentgrass)	lawns, pastures (16)	0,07 (14)	L > D (Ю)	20 (10)	10 (62 %) (10)	germination inhibited by darkness (1, 17-19) no special treatments (10)	POST (10)	A, E
Alopecurus myosuroides (Foxtail)	A fields, open habitats (16)	0,9-1,6 (29, 34)	L = D (14)	2 (29)	< 24 (30 %) (34)	scarification (14) treat with 101 mg/L KNO3 (14) warm stratification (1) germination inhibited by darkness (1) no special treatments (34)	PRE & POST (28, 34)	A	32
Avena fatua (Wild Oats)	A cultivated areas, open habitats (16)	7-37,5 (14, 30)	L = D (14) L > D (6)	10-20 (6, 10)	3 (70 %) (18)	scarification (7, 32) darkness inhibits germination (1) cold stratification (1, 18) no special treatments (6, 10, 14)	PRE & POST (6, 10, 28, 31)	A
Bromus tectorum (Downy Brome)	A fields, roadsides, arable land (16)	0,45-2,28 (14, 29)	L = D (14)	3 (29)		maturation period (1, 7, 32) germination inhibited by light (1) no special treatments (14)	PRE & POST (28, 31)	A
Cynosurus cristatus (Dog's-tail Grass)	P fields, roadsides, open habitats (16, 19)	0,5-0,7 (14, 19, 29)	L = D (14)	0 (29)	3 (50 %) (19)	germination not affected by irradiance (19) no special treatments (14, 29)	POST (5)	A
Digitaria sanguinalis (Crabgrass)	A fields, turf, open habitats (16)	0,52-0,6 (14, 30)	L = D (14)	10-20 (21)	7 (75 %) 14 (94 %) (7)	scarification, cold stratification, & maturation (1, 7, 14, 32) treat with 101 mg/L KNO3 (14) germination inhibited by darkness (1) no special treatments (21)	PRE & POST (18, 25, 31)	A
Echinochloa crusgalli (Barnyard Grass)	A (16)	1,5 (14)	L = D (14) L > D (3)	10-20 (7, 21)		scarification (7, 32) germination not affected by irradiance (1) no special treatments (3, 14, 21)	PRE & POST (3, 21, 28, 31)	A
Elymus canadensis (Canada Wild Rye)	P riparian, disturbed sites (16)	4-5 (14, 30)	L = D (11)	1 (11)	14-28 (11)	no special treatments (2, 11)	POST (2)	C, D, E
Festuca pratensis (Fescue)	P fields, moist areas (16, 19)	1,53-2,2 (16, 19)	L = D (14) L > D (10)	20 (10)	9 (74 %) (10) 2 (50 %) (19)	no special treatments (10, 19)	POST (10)	A	7
Hordeum pusillum (Little Barley)	A pastures, roadsides, open habitats (16)	3,28 (14)				warm stratification (1) germination not affected by irradiance (1)	PRE (31)		7
Phieum pratense (Timothy)	P pastures, arable fields, disturbed sites (16, 19)	0,45 (14, 19)	L > D (10, 14)	0-10 (10, 19)	2 (74 %) (10) 8 (50 %) (19)	germination inhibited by darkness (19) germination not affected by irradiance (17) no special treatments (10, 14, 17, 19)	POST (10)	A, E
POLYGONACEAE Polygonum convolvulus (Black Bindweed)	A open habitats, roadsides (16)	5-8 (4, 14, 29)	L = D (20)	0-2 (4, 29)		cold stratification for 4 — 8 weeks (1, 2, 4, 20, 29) germination not affected by irradiance (1)	PRE & POST 1, 2, 20, 28, 31	A	32
Polygonum lapathifolium (Pale Persicaria)	A moist soil (16)	1,8-2,5 (14)	L > D (6)		5 (94 %) (18)	germination not affected by irradiance (1) germination inhibited by darkness (18) cold stratification (1) no special treatments (5)	PRE & POST (6)	A, E
Polygonum pennsylvanicum (Pennsylvania Smartweed)	A fields, open habitats (16)	3,6-7 (14, 29)		2 (29)		cold stratification for 4 wks at 0 — 5oC (1, 29) germination inhibited by darkness (1)	PRE (31)	A, E
Polygonum periscaria (Smartweed)	A disturbed areas, arable land (16, 19)	2,1 -2,3 (14, 19)	L > D (13)	0 (19)	< 14 (13) 2 (50 %) (19)	scarification, cold stratification, GA treatment (14) cold stratification, maturation (17-19) germination inhibited by darkness (19) no special treatments (13)	POST (13)	A	32
Rumex crispus (Curly Dock)	P arable fields, roadsides open areas (16, 19)	1,3-1,5 (4, 14, 19)	L = D (14, 33)	0 (4, 19, 33)	3 (50 %) (19) 6 (100 %) (33)	germination inhibited by darkness (18, 19) maturation may be necessary (18) no special treatments (4, 14, 33)	POST (4, 33)	A, E	32
PRIMULACEAE Anagallis arvensis (Scarlett Pimpernel)	A arable fields, open areas, disturbed sites (16, 19)	0,4-0,5 (4, 14, 19)	L = D (14)		1 (50 %) (19)	cold stratification, GA treatment (1,14, 18, 19, 32) light required for germination (1) no special treatments (2, 4)	POST (2,4)	A, F
RANUNCULACEAE Ranunculus acris (Common Buttercup)	Ρ arable fields, roadsides, open areas (16, 19)	1,5-2 (14, 19, 29)	L = D (14)	1 (29)	41 -56 (19, 29)	no special treatments (5, 14, 22, 24 -26)	POST (5, 22, 24-26)		32
ROSACEAE Geum urbanum (Yellow Avens)	Ρ hedgerows, moist areas (16, 19)	0,8 — 1,5 (14, 19)	L = D (14)	0 (19)	5 (50 %) (19) 16 (79 %) (18)	germination inhibited by darkness (18, 19) warm stratification (1) no special treatments (5, 14, 22, 25, 26)	POST (5, 22, 25, 26)	A
RUBIACEAE Galium aparine (Cleavers)	A arable fields, moist areas, disturbed sites (16, 19)	7-9 (14, 19)	L = D (14)		5 (50 %) (19) 6 (100 %) (18)	cold stratification (1, 18, 19) germination not affected by irradiance (18, 19) light inhibits germination (1) no special treatments (6, 14)	PRE & POST (6, 28)	A	32
Galium mollugo (Hedge Bedstraw)	Ρ hedgebanks, open areas (8)	7 (29)	L = D (14)	2 (29)		no special treatments (5, 14, 22, 24, 26, 29)	POST (5, 22, 24, 26)	A
SCROPHULARIACEAE Digitalis purpurea (Foxglove)	Β, Ρ hedgerows, open areas (16, 19)	0,1 -0,6 (4, 14, 19)	L = D (14)	0 (4, 19)	6 (50 %) (19) 8 (99 %) (18)	germination inhibited by darkness (1, 17-19) no special treatments (4, 22-26)	POST (4, 22 — 26)	D, G, F
Veronica persica (Speedwell)	A arable fields, open areas, disturbed sites (16, 19)	0,5-0,6 (14, 19)	L = D (14)	0 (19)	3(19) 5 (96 %) (18)	germination inhibited by darkness (18, 19) cold stratification (18) no special treatments (14)	PRE & POST (28)	A	32

A = Annuals, В = Biennials, Ρ = Perennials.

References 11,14 and 33 referto proportion of light (L) and darkness (D) required to induce seed germination. References 3, 6, 9, 10, 13, 20 referto growing conditions in greenhouses.

0 mm indicates seeds were sown on the soil surface or that seeds need light to germinate.

The numbers provided represent the number of days in which a percent of seeds germinated according to provided reference, e.g., 3 days (50 %) germination (reference 19).

Duration of maturation and or stratification not always available. Except for cold treatment requirements, temperature conditions are not specified since in greenhouse testing there is limited temperature control. Most seeds will germinate under normal fluctuation of temperatures found in greenhouses.

Indicates species was utilized in either a pre-emergence (PRE) and/or post-emergence (POST) plant toxicity test involving herbicides.

Provides example(s) of commercial seed suppliers.

Provides two alternative reference(s) that were consulted.

Seed Suppliers Cited

Supplier ID	Supplier Information
A	Herbiseed New Farm, Mire Lane, West End, Twyford RG10 0NJ ENGLAND +44 (0) 1189 349 464 www. herbiseed.com
B	Tropilab Inc. 8240 Ulmerton Road, Largo, FL 33771-3948 USA (727) 344 - 4050 www.tropilab.com
C	Pterophylla — Native Plants & Seeds #316 Regional Road 60, RR#1, Walsingham, ON N0E 1X0 CANADA (519) 586 - 3985
D	Applewood Seed Co. 5380 Vivian St., Arvada, CO 80002 USA (303) 431 - 7333 www.applewoodseed.com
E	Ernst Conservation Seeds 9006 Mercer Pike, Meadville, PA 16335 USA (800) 873 - 3321 www.ernstseed.com
F	Chiltern Seeds Bortree Stile, Ulverston, Cumbria LA12 7PB ENGLAND +44 1229 581137 www.chiltemseeds.co.uk
G	Thompson & Morgan P.O. Box 1051, Fort Erie, ON L2A 6C7 CANADA (800) 274 - 7333 www.thompson-morgan.com

REFERENCES CITED

• (1)Baskin, C.C. & Baskin, J.M. 1998. Seeds. Academic Press, Toronto

• (2)Blackburn, L.G. & Boutin, C. 2003. Subtle effects of herbicide use in the context of genetically modified crops: a case study with glyphosate (Round-Up®). Ecotoxicology, 12:271-285.

• (3)Boutin, C., Lee, H-B., Peart, T., Batchelor, P.S., & Maguire, R.J. 2000. Effects of the sulfonylurea herbicide metsulfuron methyl on growth and reproduction of five wetland and terrestrial plant species. Environmental Toxicology & Chemistry, 19(10):2532-2541.

• (4)Boutin, C., Elmegaard, N., & Kjaer, C. 2004. Toxicity testing of fifteen non-crop plant species with six herbicides in a greenhouse experiment: implications for risk assessment. Ecotoxicology, 13:349-369.

• (5)Breeze, V., Thomas, G., & Butler, R. 1992. Use of a model and toxicity data to predict the risks to some wild plant species from drift of four herbicides. Annals of Applied Biology, 121:669-677.

• (6)Brown, R.A., & Farmer, D. 1991. Track-sprayer and glasshouse techniques for terrestrial plant bioassays with pesticides. In: Plants for toxicity assessment: 2nd volume. ASTM STP 1115, J.W. Gorsuch, W.R. Lower, W. Wang, & M.A. Lewis, eds. American Society for Testing & Materials, Philadelphia. pp 197 - 208.

• (7)Buhler, D.D. & Hoffman, M.L. 1999. Anderson's guide to practical methods of propagating weeds and other plants. Weed Science Society of America, Lawrence, K.

• (8)Clapham, A.R., Tutin, T.G., & Warburg, E.F. 1981. Excursion flora of the British Isles, 3rd ed. Cambridge University Press, Cambridge

• (9)Clay, P.A. & Griffin, J.L. 2000. Weed seed production and seedling emergence response to late-season glyphosate applications. Weed Science, 48:481-486.

• (10)Cole, J.F.H. & Canning, L. 1993. Rationale for the choice of species in the regulatory testing of the effects of pesticides on terrestrial non-target plants. BCPC — Weeds. pp. 151 - 156.

• (11)Fiely, M. (Ernst Conservation Seeds). 2004. Personal communication. (www.ernstseed.com)

• (12)Fletcher, J.S., Johnson, F.L., & McFarlane, J.C. 1990. Influence of greenhouse versus field testing and taxonomic differences on plant sensitivity to chemical treatment. Environmental Toxicology & Chemistry, 9:769-776.

• (13)Fletcher, J.S., Pfleeger, T.G., Ratsch, H.C., & Hayes, R. 1996. Potential impact of low levels of chlorsulfuron and other herbicides on growth and yield of nontarget plants. Environmental Toxicology & Chemistry, 15(7):1189-1196.

• (14)Flynn, S., Turner, R.M., and Dickie, J.B. 2004. Seed Information Database (release 6.0, Oct 2004) Royal Botanic Gardens, Kew (www.rbgkew.org.uk/data/sid)

• (15)Franzaring, J., Kempenaar, C., & van der Eerden, L.J.M. 2001. Effects of vapours of chlorpropham and ethofumesate on wild plant species. Environmental Pollution, 114:21-28.

• (16)Gleason, H.A. & Cronquist, A. 1991. Manual of vascular plants of northeastern United States and adjacent Canada, 2nd ed. New York Botanical Garden, Bronx, NY

• (17)Grime, J.P. 1981. The role of seed dormancy in vegetation dynamics. Annals of Applied Biology, 98:555-558.

• (18)Grime, J.P., Mason, G., Curtis, A.V., Rodman, J., Band, S.R., Mowforth, M.A.G., Neal, A.M., & Shaw, S. 1981. A comparative study of germination characteristics in a local flora. Journal of Ecology, 69:1017-1059.

• (19)Grime, J.P., Hodgson, J.G., & Hunt, R. 1988. Comparative plant ecology: a functional approach to common British species. Unwin Hyman Ltd., London

• (20)Kjaer, C. 1994. Sublethal effects of chlorsulfuron on black bindweed (Polygonum convolvulus L.). Weed Research, 34:453-459.

• (21)Klingaman, T.E., King, C.A., & Oliver, L.R. 1992. Effect of application rate, weed species, and weed stage of growth on imazethapyr activity. Weed Science, 40:227-232.

• (22)Marrs, R.H., Williams, C.T., Frost, A.J., & Plant, R.A. 1989. Assessment of the effects of herbicide spray drift on a range of plant species of conservation interest. Environmental Pollution, 59:71-86.

• (23)Marrs, R.H., Frost, A.J., & Plant, R.A. 1991. Effects of herbicide spray drift on selected species of nature conservation interest: the effects of plant age and surrounding vegetation structure. Environmental Pollution, 69:223-235.

• (24)Marrs, R.H., Frost, A.J., & Plant, R.A. 1991. Effects of mecoprop drift on some plant species of conservation interest when grown in standardized mixtures in microcosms. Environmental Pollution, 73:25-42.

• (25)Marrs, R.H., Frost, A.J., Plant, R.A., & Lunnis, P. 1993. Determination of buffer zones to protect seedlings of non-target plants from the effects of glyphosate spray drift. Agriculture, Ecosystems, & Environment, 45:283-293.

• (26)Marrs, R.H. & Frost, A.J. 1997. A microcosm approach to detection of the effects of herbicide spray drift in plant communities. Journal of Environmental Management, 50:369-388.

• (27)Marshall, E.J.P. & Bernie, J.E. 1985. Herbicide effects on field margin flora. BCPC — Weeds. pp. 1021-1028.

• (28)McKelvey, R.A., Wright, J.P., & Honegger, J.L. 2002. A comparison of crop and non-crop plants as sensitive species for regulatory testing. Pest Management Science, 58:1161-1174.

• (29)Morton, S. (Herbiseed). 2004. Personal communication. (http://www.herbiseed.com)

• (30)USDA, NRCS. 2004. The Plants Database, version 3.5. (http://plants.usda.gov). National Plant Data Centre, Baton Rouge, LA 70874-4490 USA

• (31)USEPA. 1999. One-Liner Database. [U.S. E.P.A./Office of Pesticide Programs/Environmental Fate and Effects Division/Environmental Epidemiology Branch].

• (32)Webster, R.H. 1979. Technical Report No. 56: Growing weeds from seeds and other propagules for experimental purposes. Agricultural Research Council Weed Research Organization, Oxford.

• (33)White, A. L. & Boutin, C. (National Wildlife Research Centre, Environment Canada). 2004. Personal communication.

• (34)Zwerger, P. & Pestemer, W. 2000. Testing the phytotoxic effects of herbicides on higher terrestrial non-target plants using a plant life-cycle test. Z. PflKrankh. PflSchutz, Sonderh., 17:711-718.

Appendix 4

Examples for appropriate growth conditions for certain crop species

The following conditions have been found suitable for 10 crop species, and can be used as a guidance for tests in growth chambers with certain other species as well:

Carbon dioxide concentration: 350 ± 50 ppm;

Relative humidity: 70 ± 5 % during light periods and 90 ± 5 % during dark periods;

Temperature: 25 ± 3 °C during the day, 20 ± 3 °C during the night;

Photoperiod: 16 hour light/8 hour darkness, assuming an average wavelength of 400 to 700 nm;

Light: luminance of 350 ± 50 μE/m²/s, measured at the top of the canopy.

The crop species are:

• — tomato (Solanum lycopersicon);

• — cucumber (Cucumis sativus);

• — lettuce (Lactuca sativa);

• — soybean (Glycine max);

• — cabbage (Brassica oleracea var. capitata);

• — carrot (Daucus carota);

• — oats (Avena sativa);

• — perennial ryegrass (Lolium perenne);

• — corn (Zea mays);

• — onion (Allium cepa).

C.32.   ENCHYTRAEID REPRODUCTION TEST

The full description of this test method has been deleted. The equivalent international test method appears in Table 3 of Part 0.

C.33.   EARTHWORM REPRODUCTION TEST (EISENIA FETIDA/ EISENIA ANDREI )

The full description of this test method has been deleted. The equivalent international test method appears in Table 3 of Part 0.

C.34.   DETERMINATION OF THE INHIBITION OF THE ACTIVITY OF ANAEROBIC BACTERIA — REDUCTION OF GAS PRODUCTION FROM ANAEROBICALLY DIGESTING (SEWAGE) SLUDGE

INTRODUCTION

• 1.This test method is equivalent to the OECD test guideline (TG) 224 (2007). Chemicals discharged to the aquatic environment pass through both aerobic and anaerobic zones, where they may be degraded and/or can inhibit bacterial activity; in some cases they can remain in anaerobic zones undisturbed for decades or longer. In waste water treatment the first stage, primary settlement, is aerobic in the supernatant liquid and anaerobic in the subnatant sludge. This is followed in the secondary stage by an aerobic zone in the activated sludge aeration tank and an anaerobic zone in the subnatant sludge in the secondary settlement tank. Sludge from both of these stages is usually subjected to anaerobic treatment, producing methane and carbon dioxide which are normally used to produce electricity. In the wider environment, chemicals reaching sediments in bays, estuaries and the sea are likely to remain in these anaerobic zones indefinitely if they are not biodegradable. Larger proportions of some chemicals will preferably reach these zones because of their physical properties, such as low solubility in water, high adsorption to suspended solids, as well as inability to be biodegraded aerobically.

• 2.While it is desirable that chemicals discharged to the environment should be biodegradable under both aerobic and anaerobic conditions, it is essential that such chemicals do not inhibit the activity of microorganisms in either zone. In the UK there have been a few cases of complete inhibition of methane production caused by, for example, pentachlorophenol in industrial discharges, leading to very costly transportation of inhibited sludge from the digesters to ‘safe’ sites and importation of healthy digesting sludge from neighbouring installations. But there have been many cases of less severe disruption of digestion by several other chemicals, including aliphatic halohydrocarbons (dry-cleaning) and detergents, leading to significant impairment of digester efficiency.

• 3.Only one test method, C.11 (1), deals with inhibition of bacterial activity (Respiration of activated sludge), which assesses the effect of test chemicals on the rate of oxygen uptake in the presence of substrate. The method has been widely used to give early warning of possible harmful effects of chemicals on the aerobic treatment of wastewaters, as well as indicating non-inhibitory concentrations of test chemicals to be used in the various tests for biodegradability. Test method C.43 (2) offers a limited opportunity for determining the toxicity of a test chemical to gas production by anaerobic sludge, diluted to one tenth of its normal concentration of solids to allow the required precision in the assessment of percentage biodegradation. Because diluted sludge could be more sensitive to inhibitory chemicals, the ISO group decided to prepare a method using undiluted sludge. At least three texts were examined (from Denmark, Germany and the UK) and finally two ISO standards were prepared, one using undiluted sludge, ISO 13 641-1 (3) and the other using one hundredth diluted sludge, ISO 13 641-2 (4), to represent muds and sediments having low bacterial populations. Both methods were subjected to a ring-test (5); part 1 was confirmed as an acceptable standard but there was disagreement over part 2. The UK considered that, because a significant proportion of participants reported very little or no gas production, partly because the percentage gas space was too high (at 75 %) for optimal sensitivity, the method requires further investigation.

• 4.Earlier work in the UK (6)(7) described a manometric method using undiluted digesting sludge, plus raw sewage sludge as the substrate, in 500 ml flasks; the apparatus was cumbersome and the stench of the raw sludge was offensive. Later the more compact and convenient apparatus of Shelton and Tiedje (8) as developed by Battersby and Wilson (9) was successfully applied by Wilson et al. (10). Kawahara et al (11) successfully prepared more standard sludges in the laboratory for use in tests for anaerobic biodegradability and inhibition on a number of chemicals. Also, raw sludge as the substrate was replaced to carry out a test either with one hundredth diluted anaerobic sludge or with muds, sediments etc. of low bacterial activity.

• 5.This method can provide information that is useful in predicting the likely effect of a test chemical on gas production in anaerobic digesters. However, only longer tests simulating working digesters more closely can indicate whether adaptation of the microorganisms to the test chemical can occur or whether chemicals likely to be absorbed and adsorbed onto sludge can build up to a toxic concentration over a longer period than allowed in this test.

PRINCIPLE OF THE TEST

• 6.Aliquots of a mixture of anaerobically digesting sludge (20 g/l to 40 g/l total solids) and a degradable substrate solution are incubated alone and simultaneously with a range of concentrations of the test chemical.in sealed vessels for up to 3 days. The amount of gas (methane plus carbon dioxide) produced is measured by the increase in pressure (Pa) in the bottles. The percentage inhibition of gas production brought about by the various concentrations of the test chemical is calculated from the amounts produced in the respective test and control bottles. The EC50 and other effective concentrations are calculated from plots of percentage inhibition against the concentration of the test chemicals or, more usually, its logarithm.

INFORMATION ON THE TEST CHEMICAL

• 7.Test chemicals should normally be used in the purest form readily available, since impurities in some chemicals, e.g. chlorophenols, can be much more toxic than the test chemical itself. However, the needs to test chemicals in the form in which they are produced/made commercially available should be considered. The use of formulated products is not routinely recommended, but for poorly soluble test chemicals the use of formulated material may be appropriate. Properties of the test chemical which should be available include solubility in water and some organic solvents, vapour pressure, adsorption coefficient, hydrolysis and biodegradability under anaerobic conditions.

APPLICABILITY OF THE METHOD

• 8.The test is applicable to chemicals which are soluble or insoluble in water, including volatile chemicals. But special care is necessary with materials of low water-solubility (see ref. (12)) and of high volatility. Also, inocula from other anaerobic sites, e.g. muds, saturated soils, sediments, may be used. Anaerobic bacterial systems that have previously been exposed to toxic chemicals may be adapted to maintaining their activity in the presence of xenobiotic chemicals. Inocula from adapted bacterial systems may show a higher tolerance to the test chemicals compared to inocula obtained from non-adapted systems.

REFERENCE CHEMICALS

• 9.To check the procedure, a reference chemical is tested by setting up appropriate vessels in parallel as part of normal test runs; 3, 5-dichlorophenol has been shown to be a consistent inhibitor of anaerobic gas production, as well as of oxygen consumption by activated sludge and other biochemical reactions. Two other chemicals have been shown to be more inhibitory to methane production than 3, 5-dichlorophenol, namely methylene bis-thiocyanate and pentachlorophenol but results with them have not been validated. Pentachlorophenol is not recommended since it is not readily available in a pure form.

REPRODUCIBILITY OF THE RESULTS

• 10.In an international ring test (5) there was only fair reproducibility in EC50 values between the 10 participating laboratories for 3, 5-dichlorophenol and 2-bromo-ethane sulphonic acid. (The range for the former was 32 mg/l to 502 mg/l and for the latter 220-2 190 mg/l.)

Number of laboratories	As mg/l	As mg/l	As mg/l	As mg/g sludge	As mg/g sludge	As mg/g sludge
Number of laboratories	mean	s.d.	cv(%)	mean	s.d.	cv(%)
	3, 5-Dichlorophenol	3, 5-Dichlorophenol	3, 5-Dichlorophenol	3, 5-Dichlorophenol	3, 5-Dichlorophenol	3, 5-Dichlorophenol
10	153	158	103	5	4,6	92
	2-Bromo-ethane sulphonic acid	2-Bromo-ethane sulphonic acid	2-Bromo-ethane sulphonic acid	2-Bromo-ethane sulphonic acid	2-Bromo-ethane sulphonic acid	2-Bromo-ethane sulphonic acid
10	1 058	896	85	34	26	76

EC50 data from ring test — undiluted sludge

• 11.The high coefficients of variation between laboratories to a large extent reflect differences in the sensitivity of the sludge microorganisms due to either pre-exposure or no pre-exposure to the test chemical or other chemically related chemicals. The precision with which the EC50 value based on the sludge concentration was determined was barely better than the ‘volumetric’ value (mg/l). The three laboratories which reported the precision of their EC50 values for 3,5-dichlorophenol showed much lower coefficients of variation (22, 9, and 18 % respectively for EC50 mg/g) than those of the means of all ten laboratories. The individual means for the three laboratories were 3,1, 3,2 and 2,8 mg/g, respectively. The lower, acceptable coefficients of variation within laboratories compared with the much higher coefficients between laboratory values, namely 9-22 % cf. 92 %, indicate that there are significant differences in the properties of the individual sludges.

DESCRIPTION OF THE METHOD

Apparatus

• 12.Usual laboratory equipment and the following are required:

• (a) Incubator — spark-proof and controlled at 35 °C ± 2 °C;

• (b) Pressure-resistant glass test vessels of an appropriate nominal size ([^E0195]), each fitted with a gas-tight coated septum, capable of withstanding about 2 bar or 2 × 10⁵ Pa (for coating use e.g. PTFE = polytetrafluorethene). Glass serum bottles of nominal volume 125 ml, with an actual volume of around 160 ml, sealed with serum septa ([^E0196]) and crimped aluminium rings are recommended; but bottles of total volume between 0,1 and 1 litre may be used successfully;

• (c) Precision pressure-meter ([^E0197]) and needle attachment Total gas production (methane plus carbon dioxide) measured by means of a pressure-meter adapted to enable measurement and venting of the gas produced. An example of a suitable instrument is a hand-held precision pressure-meter connected to a syringe needle; a three-way gas-tight valve facilities the release of excess pressure (Appendix 1). It is necessary to keep the internal volume of the pressure transducer tubing and valve as low as possible, so that errors introduced by neglecting the volume of the equipment are insignificant;

• (d) Insulated containers, for transport of digesting sludge;

• (e) Three-way pressure valves;

• (f) Sieve, having a 1 mm square mesh;

• (g) Reservoir, for digesting sludge, a glass or high-density polyethylene bottle, capacity about 5 litre, fitted with a stirrer and facilities for passing a stream of nitrogen gas (see paragraph 13) through the headspace;

• (h) membrane filters (0,2 μm) for sterilising the substrate;

• (i) micro syringes, for the gas-tight connection of the pressure transducer (see paragraph 12(c)) to the headspace in the bottles (see paragraph 12(b)); also for adding insoluble liquid test materials into the bottles;

• (j) glove box, optional but recommended, with a slight positive pressure of nitrogen.

Reagents

• 13.Use analytical grade reagents throughout. Nitrogen gas, of high purity with a content of less than 5 μl/l oxygen, should be used throughout.

Water

• 14.If dilution is necessary at any stage, use deionised water previously de-aerated. Analytical controls on this water are not necessary, but ensure that the deionising apparatus is regularly maintained. Use deionised water also for the preparation of stock solutions. Prior to the addition of the anaerobic inoculum to any solution or dilution of test material, make sure that these are oxygen-free. This is done either by blowing nitrogen gas through the dilution water (or through the dilutions) for 1 hour before adding the inoculum, or alternatively by heating the dilution water to the boiling point and cooling to room temperature in an oxygen-free atmosphere.

Digested Sludge

• 15.Collect actively digesting sludge from a digester at a wastewater treatment plant, or alternatively, from a laboratory digester, treating sludge from predominantly domestic sewage. Practical information regarding sludge from a laboratory digester can be found elsewhere (11). If use of an adapted inoculum is intended, digesting sludge from an industrial sewage treatment plant may be considered. Use wide-necked bottles constructed from high-density polyethylene or a similar material, which can expand, for sludge collection. Add sludge to the sample bottles to within about 1 cm from the top of the bottles, seal them tightly, preferably with a safety valve (paragraph 12(e)), and place in insulated containers (paragraph 12(d)) to minimise temperature shock, until being transferred to an incubator maintained at 35 °C ± 2 °C. When opening the bottles, take care to release excess gas pressure either by cautiously loosening the seal, or by means of the three-way pressure-release valve (paragraph 12(e)). It is preferable to use the sludge within a few hours of collection, otherwise store at 35 °C ± 2 °C under a headspace of nitrogen for up to 3 days, when little loss of activity normally occurs.

Warning — Digesting sludge produces flammable gases which present fire and explosion risks: it also contains potentially pathogenic organisms, so take appropriate precautions when handling sludge. For safety reasons, do not use glass vessels for collecting sludge.

Inoculum

• 16.Immediately prior to use, mix the sludge by gentle stirring and pass it through a 1 mm² mesh sieve (paragraph 12(f)) into a suitable bottle (paragraph 12(g)) through the headspace of which a stream of nitrogen is passed. Set aside a sample for measurement of the concentration of total dry solids (see e.g. ISO 11 923 (13) or equivalent EU standard). In general, use the sludge without dilution. The solids concentration is usually between 2 % and 4 % (w/v). Check the pH value of the sludge and, if necessary, adjust to 7 ± 0,5.

Test substrate

• 17.Dissolve 10 g nutrient broth (e.g. Oxoid), 10 g of yeast extract and 10 g of D-glucose in deionised water and dilute to 100 ml. Sterilise by filtration through a 0,2 μm membrane filter (paragraph 12(h)) and use immediately or store at 4 °C for not longer than 1 day.

Test chemical

• 18.Prepare a separate stock solution for each water-soluble test chemical to contain, for example, 10 g/l of the chemical in oxygen-free dilution water (paragraph 14). Use appropriate volumes of these stock solutions to prepare the reaction mixtures containing graded concentrations. Alternatively, prepare a dilution series of each stock solution so that the volume added to the test bottles is the same for each required final concentration. The pH of the stock solutions should be adjusted to 7 ± 0,2 if necessary.

• 19.For test chemicals which are insufficiently soluble in water, consult ISO 10 634 (12) or equivalent EU standard. If an organic solvent is needed to be used, avoid solvents such as chloroform and carbon tetrachloride, which are known strongly to inhibit methane production. Prepare a solution of an appropriate concentration of water-insoluble chemical in a suitable volatile solvent, for example, acetone, di-ethylether. Add the required volumes of solvent solution to the empty test bottles (paragraph 12(b)) and evaporate the solvent before the addition of sludge. For other treatments use ISO 10 634 (12) or equivalent EU standard, but be aware that any surfactants used to produce emulsions may be inhibitory to anaerobic gas production. If it is thought that the presence of organic solvents and emulsifying agents causes artefacts, the test chemical could be added directly to the test mixture as a powder or liquid. Volatile chemicals and water-insoluble liquid test chemicals may be injected into inoculated serum bottles, using micro-syringes (paragraph 12(i)).

• 20.Add test chemicals to the bottles to give a geometric series of concentrations, for example, 500 mg/l, 250 mg/l, 125 mg/l, 62,5 mg/l, 31,2 mg/l and 15,6 mg/l. If the range of toxicity is not known from similar chemicals, first carry out a preliminary range-finding test with concentration of 1 000 mg/l, 100 mg/l and 10 mg/l to ascertain the appropriate range.

Reference chemical

• 21.Prepare an aqueous solution of 3,5-dichlorophenol (10 g/l) by gradually adding the minimum amount of 5 mol/l of sodium hydroxide solution to the solid, while shaking, until it has dissolved. Then add de-oxygenated dilution water (paragraph 14) to the required volume; sonication may aid dissolution. Other reference chemicals may be used when the average range of the EC50 has been obtained in at least three tests with different inocula (different sources or different times of collection).

INTERFERENCE/ERRORS

• 22.Some constituents of sludge presumably could react with potential inhibitors making them unavailable to micro-organisms so giving lower, or no, inhibition. Also, if the sludge already contains a chemical which is inhibitory, erroneous results would be obtained when that chemical was subjected to the test. Apart from these possibilities, there are a number of identified factors which can lead to false results. These are listed in Appendix 3, together with methods of eliminating or at least reducing errors.

TEST PROCEDURE

• 23.The number of necessary replicates depends on the degree of precision required for the inhibition indices. If the bottle seals are sufficiently gas-tight over the duration of the test, set up just one batch (at least triplicates) of test bottles at each concentration required. Similarly, set up one batch of bottles with reference chemical and one set of controls. However, if the seals of the bottles are reliable for only one or a few piercings, set up a batch (e.g. triplicates) of the test bottles for each interval (t) for which results are required for all concentrations of a test chemical to be tested. Similarly, set up ‘t’ batches of bottles for the reference chemical and for the controls.

• 24.The use of a glove box (paragraph 12(j)) is recommended. At least 30 minutes before starting the test, start a flow of nitrogen gas through the glove box containing all the necessary equipment. Ensure that the temperature of the sludge is within 35 °C ± 2 °C during handling and sealing of the bottles.

Preliminary Test

• 25.If the activity of the sludge is unknown, it is recommended to carry out a preliminary test. Set up controls to give, for example, concentrations of solids of 10 g/l, 20 g/l and 40 g/l plus substrate but use no test chemical. Also, use different volumes of reaction mixture in order to have three or four ratios of volume of headspace to volume of liquid. From the results of gas volumes produced at various time intervals, the most suitable conditions which allow two daily measurements yielding significant volumes of gas and release of pressure per day at optimal sensitivity ([^E0198]) without fear of explosions.

Addition of test chemicals

• 26.Add water-soluble test chemicals to empty test bottles (paragraph 12(b)) as aqueous solutions (paragraph 18). Use at least triplicate sets of bottles for each of a range of concentrations (paragraph 20). In the case of insoluble and poorly soluble test chemical, inject solutions of these in organic solvents using a micro-syringe into empty bottles to give replicate sets of each five concentrations of test chemical. Evaporate the solvent by passing a jet of nitrogen gas over the surface of the solutions in the test bottles. Alternatively, add insoluble solid chemicals as weighed amounts of the solid directly to the test bottles.

• 27.If insoluble and poorly water-soluble liquid test chemicals are not added using a solvent, add them directly by micro-syringe to the test bottles after addition of inoculum and test substrate (see paragraph 30). Volatile test chemicals may be added in the same way.

Addition of inoculum and substrate

• 28.Stir an appropriate volume of sieved digesting sludge (see paragraph 16) in a 5 litre bottle (paragraph 12(g)), while passing a stream of nitrogen gas through the headspace. Flush test bottles, containing aqueous solutions or evaporated solvent solutions of test chemicals, with a stream of nitrogen gas, for about two minutes to remove air. Dispense aliquots, e.g. 100 ml, of the well-mixed sludge into the test bottles using a large-tipped pipette or a measuring cylinder. It is essential to fill the pipette in one step to the exact volume of sludge required because of the ease of settlement of sludge solids. If more is taken up, empty the pipette and start again.

• 29.Then add sufficient substrate solution (paragraph 17) to give a concentration of 2 g/l of each of the nutrient broth, yeast extract and D-glucose in the mixture, while nitrogen is still flushing through. The following is an example for test batches.

Final mass concentration of test chemical in test bottles (mg/l)	Volume of test chemical (ml)	Volume of test chemical (ml)	Reagents and media (ml)	Reagents and media (ml)	Reagents and media (ml)
Final mass concentration of test chemical in test bottles (mg/l)	Stock solution (a) 10 g/l para. 18	Stock solution (b) 1 g/l para. 18	Dilution water para. 14	Inoculum para. 16	Substrate para. 17
0	—	0	1,0	100	2
1	—	0,1	0,9	100	2
3,3	—	0,33	0,67	100	2
10	0,1	—	0,9	100	2
33	0,33	—	0,67	100	2
100	1,0	—	0	100	2
Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.	Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.	Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.	Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.	Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.	Total volume of bottle = 160 ml. Volume of liquid = 103 ml Gas volume = 57 ml, or 35,6 % of total volume.

• 30.Similarly flush out with nitrogen gas sufficient empty test bottles to deal with any volatile and insoluble liquid test chemical (see paragraph 27).

Controls and reference chemical

• 31.Set up at least triplicate sets of bottles, containing sludge and substrate only, to act as controls. Set up further replicate bottles containing sludge and substrate plus sufficient stock solution of the reference chemical, 3,5-dichlorophenol (paragraph 21) to result in a final concentration of 150 mg/l. This concentration should inhibit gas production by about 50 %. Alternatively, set up a range of concentrations of the reference chemical. In addition, set up four extra bottles for pH measurement which contain sludge, de-oxygenated water and substrate. Add the test chemical to two bottles at the highest concentration being tested and add de-oxygenated water to the remaining two bottles.

• 32.Ensure that all bottles — test and reference chemicals, and controls — contain the same volume (VR) of liquid; where necessary, add de-oxygenated deionised water (paragraph 14) to make up the volume. The headspace should be between 10 % and 40 % of the bottle volume, the actual value being selected from the data obtained from the preliminary test. After adding all constituents to the bottles, remove the needle supplying the gas and seal each bottle with a rubber stopper and an aluminium cap (Paragraph 12(b)) moistening the stopper with a drop of deionised water to aid insertion. Mix the contents of each bottle by shaking.

Incubation of bottles

• 33.Transfer the bottles to the thermostatically controlled incubator, preferably equipped with a shaking device, and maintained at 35 °C ± 2 °C. The bottles are incubated in the dark. After about 1 hour, equalise the pressure in the bottles to atmosphere by inserting the syringe needle, attached to the pressure-meter (paragraph 12(c)), through the seal of each bottle in turn, open the valve until the pressure-meter reads zero and finally close the valve. The needle should be inserted at an angle of about 45° to prevent gas leaking from the bottles. If the bottles are incubated without shaking facility, shake manually twice each day during the total incubation period to equilibrate the system. Incubate the bottles and invert them to prevent any loss of gas through the septum. Inversion is, however, not appropriate in cases in which insoluble test chemicals may adhere to the bottom of the flask.

Pressure measurement

• 34.When the bottles have reached 35 °C ± 2 °C, measure and record the pH of the contents of two of the four bottles set up for the purpose and discard the contents; continue incubating remaining bottles in the dark. Measure and record the pressure in the bottles twice a day over the following 48 hours to 72 hours by inserting the needle of the pressure-meter through the seal of each bottle, in turn, drying the needle between measurements. Keep all parts of the bottle at the incubation temperature during the measurement, which should be carried out as quickly as possible. Allow the pressure reading to stabilise and record it. Then open the valve for ventilation and close it when the pressure reads zero. Continue the test usually for 48 hours from the time of first equalising the pressure, designated ‘time 0’. The number of readings and ventilations should be limited for volatile chemicals to one (at the end of incubation) or two to minimise loss of test chemical (10).