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)

Detailed information on the history of the field site from where the test soil is collected should be available. Details include exact location, vegetation cover, treatments with chemicals, treatments with organic and inorganic fertilisers, additions of biological materials or other contamination. If soils have been treated with the test substance or its structural analogues within the previous four years, these should not be used for transformation studies (10)(15).

The soil should be freshly collected from the field (from the A horizon or top 20 cm layer) with a soil water content which facilitates sieving. For soils other than those from paddy fields, sampling should be avoided during or immediately following long periods (> 30 days) of drought, freezing or flooding (14). Samples should be transported in a manner which minimises changes in soil water content and should be kept in the dark with free access of air, as much as possible. A loosely-tied polyethylene bag is generally adequate for this purpose.

The soil should be processed as soon as possible after sampling. Vegetation, larger soil fauna and stones should be removed prior to passing the soil through a 2 mm sieve which removes small stones, fauna and plant debris. Extensive drying and crushing of the soil before sieving should be avoided (15).

When sampling in the field is difficult in winter (soil frozen or covered by layers of snow), it may be taken from a batch of soil stored in the greenhouse under plant cover (e.g. grass or grass-clover mixtures). Studies with soils freshly collected from the field are strongly preferred, but if the collected and processed soil has to be stored prior to the start of the study storage conditions must be adequate and for a limited time only (4 ± 2 ^oC for a maximum of three months) to maintain microbial activity ([^E0160]). Detailed instructions on collection, handling and storage of soils to be used for biotransformation experiments can be found in (8)(10)(15)(26)(27).

Before the processed soil is used for this test, it should be pre-incubated to allow germination and removal of seeds, and to re-establish equilibrium of microbial metabolism following the change from sampling or storage conditions to incubation conditions. A pre-incubation period between two and 28 days approximating the temperature and moisture conditions of the actual test is generally adequate (15). Storage and pre-incubation time together should not exceed three months.

1.9.   PERFORMANCE OF THE TEST

1.9.1.   Test conditions

1.9.1.1.   Test temperature

During the whole test period, the soils should be incubated in the dark at a constant temperature representative of the climatic conditions where use or release will occur. A temperature of 20 ± 2 ^oC is recommended for all test substances which may reach the soil in temperate climates. The temperature should be monitored.

For chemicals applied or released in colder climates (e.g. in northern countries, during autumn/winter periods), additional soil samples should be incubated but at a lower temperature (e.g. 10 ± 2 ^oC).

1.9.1.2.   Moisture content

For transformation tests under aerobic conditions, the soil moisture content ([^E0161]) should be adjusted to and maintained at a pF between 2,0 and 2,5 (3). The soil moisture content is expressed as mass of water per mass of dry soil and should be regularly controlled (e.g. in 2 week intervals) by weighing of the incubation flasks and water losses compensated by adding water (preferably sterile-filtered tap water). Care should be given to prevent or minimise losses of test substance and/or transformation products by volatilisation and/or photodegradation (if any) during moisture addition.

For transformation tests under anaerobic and paddy conditions, the soil is water-saturated by flooding.

1.9.1.3.   Aerobic incubation conditions

In the flow-through systems, aerobic conditions will be maintained by intermittent flushing or by continuously ventilating with humidified air. In the biometer flasks, exchange of air is maintained by diffusion.

1.9.1.4.   Sterile aerobic conditions

To obtain information on the relevance of abiotic transformation of a test substance, soil samples may be sterilised (for sterilisation methods see references 16 and 29), treated with sterile test substance (e.g. addition of solution through a sterile filter) and aerated with humidified sterile air as described in Section 1.9.1.3. For paddy soils, soil and water should be sterilised and the incubation should be carried out as described in Section 1.9.1.6.

1.9.1.5.   Anaerobic incubation conditions

To establish and maintain anaerobic conditions, the soil treated with the test substance and incubated under aerobic conditions for 30 days or one half-life or DT50 (whichever is shorter) is then water-logged (1-3 cm water layer) and the incubation system flushed with an inert gas (e.g. nitrogen or argon) ([^E0162]). The test system must allow for measurements such as pH, oxygen concentration and redox potential and include trapping devices for volatile products. The biometer-type system must be closed to avoid entrance of air by diffusion.

1.9.1.6.   Paddy incubation conditions

To study transformation in paddy rice soils, the soil is flooded with a water layer of about 1-5 cm and the test substance applied to the water phase (9). A soil depth of at least 5 cm is recommended. The system is ventilated with air as under aerobic conditions. pH, oxygen concentration and redox potential of the aqueous layer should be monitored and reported. A pre-incubation period of at least two weeks is necessary before commencing transformation studies (see Section 1.8.3.2).

1.9.1.7.   Test duration

The rate and pathway studies should normally not exceed 120 days ([^E0163]) (3)(6)(8), because thereafter a decrease of the soil microbial activity with time would be expected in an artificial laboratory system isolated from natural replenishment. Where necessary to characterise the decline of the test substance and the formation and decline of major transformation products, studies can be continued for longer periods (e.g. 6 or 12 months) (8). Longer incubation periods should be justified in the test report and accompanied by biomass measurements during and at the end of these periods.

1.9.2.   Performance of the test

About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see Figures 1 and 2 in Appendix 3) and the soil treated with the test substance by one of the methods described in Section 1.8.2. When organic solvents are used for the application of the test substance, they should be removed from soil by evaporation. Then the soil is thoroughly mixed with a spatula and/or by shaking of the flask. If the study is conducted under paddy field conditions, soil and water should be thoroughly mixed after application of the test substance. Small aliquots (e.g. 1 g) of the treated soils should be analysed for the test substance to check for uniform distribution. For alternative method, see below.

The treatment rate should correspond to the highest application rate of a crop protection product recommended in the use instructions and uniform incorporation to an appropriate depth in the field (e.g. top 10 cm layer ([^E0164]) of soil). For example, for chemicals foliarly or soil applied without incorporation, the appropriate depth for computing how much chemical should be added to each flask is 2,5 cm. For soil incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions. For general chemicals, the application rate should be estimated based on the most relevant route of entry; for example, when the major route of entry in soil is through sewage sludge, the chemical should be dosed into the sludge at a concentration that reflects the expected sludge concentration and the amount of sludge added to the soil should reflect normal sludge loading to agricultural soils. If this concentration is not high enough to identify major transformation products, incubation of separate soil samples containing higher rates may be helpful, but excessive rates influencing soil microbial functions should be avoided (see Sections 1.5 and 1.8.2).

Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully mixed in an appropriate mixing machine and then transferred in small portions of 50 to 200 g into the incubation flasks (for example with the use of sample splitters). Small aliquots (e.g. 1 g) of the treated soil batch should be analysed for the test substance to check for uniform distribution. Such a procedure is preferred since it allows for more uniform distribution of the test substance into the soil.

Also untreated soil samples are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements during and at the end of the studies.

When the test substance is applied to the soil dissolved in organic solvent(s), soil samples treated with the same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples treated with the test substance. These samples are used for biomass measurements initially, during and at the end of the studies to check for effects of the solvent(s) on microbial biomass.

The flasks containing the treated soil are either attached to the flow-through system described in Figure 1 or closed with the absorption column shown in Figure 2 (see Appendix 3).

1.9.3.   Sampling and measurement

Duplicate incubation flasks are removed at appropriate time intervals and the soil samples extracted with appropriate solvents of different polarity and analysed for the test substance and/or transformation products. A well-designed study includes sufficient flasks so that two flasks are sacrificed at each sampling event. Also, absorption solutions or solid absorption materials are removed at various time intervals (7-day intervals during the first month and after one month in 17-day intervals) during and at the end of incubation of each soil sample and analysed for volatile products. Besides a soil sample taken directly after application (0-day sample) at least five additional sampling points should be included. Time intervals should be chosen in such a way that pattern of decline of the test substance and patterns of formation and decline of transformation products can be established (e.g. 0, 1, 3, 7 days; 2, 3 weeks; 1, 2, 3 months, etc.).

When using ¹⁴C-labelled test substance, non-extractable radioactivity will be quantified by combustion and a mass balance will be calculated for each sampling interval.

In the case of anaerobic and paddy incubation, the soil and water phases are analysed together for test substance and transformation products or separated by filtration or centrifugation before extraction and analysis.

1.9.4.   Optional tests

Aerobic, non-sterile studies at additional temperatures and soil moistures may be useful for the estimation of the influence of temperature and soil moisture on the rates of transformation of a test substance and/or its transformation products in soil.

A further characterisation of non-extractable radioactivity can be attempted using, for example, supercritical fluid extraction.

2. DATA

2.1.   TREATMENT OF RESULTS

The amounts of test substance, transformation products, volatile substances (in % only), and non-extractable should be given as % of applied initial concentration and, where appropriate, as mg· kg^-1 soil (based on soil dry weight) for each sampling interval. A mass balance should be given in percentage of the applied initial concentration for each sampling interval. A graphical presentation of the test substance concentrations against time will allow an estimation of its transformation half-life or DT50. Major transformation products should be identified and their concentrations should also be plotted against time to show their rates of formation and decline. A major transformation product is any product representing ≥ 10 % of applied dose at any time during the study.

The volatile products trapped give some indication of the volatility potential of a test substance and its transformation products from soil.

More accurate determinations of half-lives or DT50 values and, if appropriate, DT75 and DT90 values should be obtained by applying appropriate kinetic model calculations. The half-life and DT50 values should be reported together with the description of the model used, the order of kinetics and the determination coefficient (r²). First order kinetics is favoured unless r² < 0,7 . If appropriate, the calculations should also be applied to the major transformation products. Examples of appropriate models are described in references 31 to 35.

In the case of rate studies carried out at various temperatures, the transformation rates should be described as a function of temperature within the experimental temperature range using the Arrhenius relationship of the form:

Image omitted; see official source

or

Image omitted; see official source

,

where ln A and B are regression constants from the intercept and slope, respectively, of a best fit line generated from linearly regressing ln k against 1/T, k is the rate constant at temperature T and T is the temperature in Kelvin. Care should be given to the limited temperature range in which the Arrehenius relationship will be valid in case transformation is governed by microbial action.

2.2.   EVALUATION AND INTERPRETATION OF RESULTS

Although the studies are carried out in an artificial laboratory system, the results will allow estimation of the rate of transformation of the test substance and also of rate of formation and decline of transformation products under field conditions (36)(37).

A study of the transformation pathway of a test substance provides information on the way in which the applied substance is structurally changed in the soil by chemical and microbial reactions.

3. REPORTING

TEST REPORT

The test report must include:

Test substance:

• — common name, chemical name, CAS number, structural formula (indicating position of label(s) when radiolabelled material is used) and relevant physical-chemical properties (see Section 1.5),

• — purity (impurities) of test substance,

• — radiochemical purity of labelled chemical and specific activity (where appropriate),

Reference substances:

• — chemical name and structure of reference substances used for the characterisation and/or identification of transformation product,

Test soils:

• — details of collection site,

• — date and procedure of soil sampling,

• — properties of soils, such as pH, organic carbon content, texture (% sand, % silt, % clay), cation exchange capacity, bulk density, water retention characteristic, and microbial biomass,

• — length of soil storage and storage conditions (if stored),

Test conditions:

• — dates of the performance of the studies,

• — amount of test substance applied,

• — solvents used and method of application for the test substance,

• — weight of soil treated initially and sampled at each interval for analysis,

• — description of the incubation system used,

• — air flow rates (for flow-through systems only),

• — temperature of experimental set-up,

• — soil moisture content during incubation,

• — microbial biomass initially, during and at the end of the aerobic studies,

• — pH, oxygen concentration and redox potential initially, during and at the end of the anaerobic and paddy studies,

• — method(s) of extraction,

• — methods for quantification and identification of the test substance and major transformation products in soil and absorption materials,

• — number of replicates and number of controls.

Results:

• — result of microbial activity determination,

• — repeatability and sensitivity of the analytical methods used,

• — rates of recovery (% values for a valid study are given in Section 1.7.1),

• — tables of results expressed as % of applied initial dose and, where appropriate, as mg· kg^-1 soil (on a dry weight basis),

• — mass balance during and at the end of the studies,

• — characterisation of non-extractable (bound) radioactivity or residues in soil,

• — quantification of released CO2 and other volatile compounds,

• — plots of soil concentrations versus time for the test substance and, where appropriate, for major transformation products,

• — half-life or DT50, DT75 and DT90 for the test substance and, where appropriate, for major transformation products including confidence limits,

• — estimation of abiotic degradation rate under sterile conditions,

• — an assessment of transformation kinetics for the test substance and, where appropriate, for major transformation products,

• — proposed pathways of transformation, where appropriate,

• — discussion and interpretation of results,

• — raw data (i.e. sample chromatograms, sample calculations of transformation rates and means used to identify transformation products).

4. REFERENCES

• (1)US — Environmental Protection Agency, (1982) Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate.

• (2)Agriculture Canada, (1987) Environmental Chemistry and Fate. Guidelines for registration of pesticides in Canada.

• (3)European Union (EU), (1995) Commission Directive 95/36/EC of 14 July 1995 amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex II, Part A and Annex III, Part A: Fate and Behaviour in the Environment.

• (4)Dutch Commission for Registration of Pesticides, (1995) Application for registration of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air.

• (5)BBA, (1986) Richtlinie für die amtliche Prüfung von Pflanzenschutzmitteln, Teil IV, 4-1. Verbleib von Pflanzenschutzmitteln im Boden — Abbau, Umwandlung und Metabolismus.

• (6)ISO/DIS 11266-1, (1994) Soil Quality -Guidance on laboratory tests for biodegradation of organic chemicals in soil — Part 1: Aerobic conditions.

• (7)ISO 14239, (1997) Soil Quality — Laboratory incubation systems for measuring the mineralisation of organic chemicals in soil under aerobic conditions.

• (8)SETAC, (1995) Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed.

• (9)MAFF — Japan 2000 — Draft Guidelines for transformation studies of pesticides in soil — Aerobic metabolism study in soil under paddy field conditions (flooded).

• (10)OECD, (1995) Final Report of the OECD Workshop on Selection of Soils/Sediments. Belgirate, Italy, 18-20 January 1995.

• (11)Guth, J.A., (1980) The study of transformations. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, p. 123-157.

• (12)DFG: Pesticide Bound Residues in Soil. Wiley — VCH (1998).

• (13)T.R. Roberts: Non-extractable pesticide residue in soils and plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984).

• (14)OECD Test Guideline 304 A: Inherent Biodegradability in Soil (adopted 12 May 1981).

• (15)ISO 10381-6 (1993) Soil Quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.

• (16)Appendix V to Directive 67/548/EEC.

• (17)Guth, J.A., (1981) Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.

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

• (19)Methods of Soil Analysis (1986) Part 1, Physical and Mineralogical Methods. A. Klute, Ed.) Agronomy Series No 9, 2nd Edition.

• (20)Methods of Soil Analysis (1982) Part 2, Chemical and Microbiological Properties. A.L. Page, R.H. Miller and D.R. Kelney, Eds. Agronomy Series No 9, 2nd Edition.

• (21)ISO Standard Compendium Environment (1994) Soil Quality — General aspects; chemical and physical methods of analysis; biological methods of analysis. First Edition.

• (22)Mückenhausen, E., (1975) Die Bodenkunde und ihre geologischen, geomorphologischen, mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.

• (23)Scheffer, F., Schachtschabel, P., (1975) Lehrbuch der Bodenkunde. F. Enke Verlag, Stuttgart.

• (24)Anderson, J.P.E., Domsch, K.H., (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, p. 215-221.

• (25)ISO 14240-1 and 2 (1997) Soil Quality — Determination of soil microbial biomass — Part 1: Substrate-induced respiration method. Part 2: fumigation-extraction method.

• (26)Anderson, J.P.E., (1987) Handling and storage of soils for pesticide experiments. In Pesticide Effects on Soil Microflora. L. Somerville, M.P. Greaves, Eds. Taylor & Francis, 45-60.

• (27)Kato, Yasuhiro. (1998) Mechanism of pesticide transformation in the environment: Aerobic and bio-transformation of pesticides in aqueous environment. Proceedings of the 16^th Symposium on Environmental Science of Pesticide, p. 105-120.

• (28)Keuken O., Anderson J.P.E., (1996) Influence of storage on biochemical processes in soil. In Pesticides, Soil Microbiology and Soil Quality, 59-63 (SETAC-Europe).

• (29)Stenberg B., Johansson M., Pell M., Sjödahl-Svensson K., Stenström J., Torstensson L. (1996) Effect of freeze and cold storage of soil on microbial activities and biomass. In Pesticides, Soil Microbiology and Soil Quality, 68-69 (SETAC-Europe).

• (30)Gennari, M., Negre, M., Ambrosoli, R., (1987) Effects of ethylene oxide on soil microbial content and some chemical characteristics. Plant and Soil 102, p. 197-200.

• (31)Anderson, J.P.E. (1975) Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, p. 141-146.

• (32)Hamaker, J.W., (1976) The application of mathematical modelling to the soil persistence and accumulation of pesticides. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides, p. 181-199.

• (33)Goring, C.A.I., Laskowski, D.A., Hamaker, J.W., Meikle, R.W., (1975) Principles of pesticide degradation in soil. In ‘Environmental Dynamics of Pesticides’. R. Haque and V.H. Freed, Eds., p. 135-172.

• (34)Timme, G., Frehse, H., Laska, V., (1986) Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues. II. Pflanzenschutz — Nachrichten Bayer 39, p. 188-204.

• (35)Timme, G., Frehse, H., (1980) Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues. I. Pflanzenschutz — Nachrichten Bayer 33, p. 47-60.

• (36)Gustafson D.I., Holden L.R., (1990) Non-linear pesticide dissipation in soil; a new model based on spatial variability. Environm. Sci. Technol. 24, p. 1032-1041.

• (37)Hurle K., Walker A., (1980) Persistence and its prediction. In Interactions between Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, p. 83-122.

Appendix 1

WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC) ([^E0165])

Height of Water Column [cm]	pF [^E0166]	bar [^E0167]	Remarks
107	7	104	Dry Soil
1,6 · 104	4,2	16	Wilting point
104	4	10
103	3	1
6· 102	2,8	0,6
3,3 · 102	2,5	0,33  [^E0168]	Image omitted; see official source Range of Field capacity [^E0169]
102	2	0,1	Image omitted; see official source Range of Field capacity [^E0169]
60	1,8	0,06	Image omitted; see official source Range of Field capacity [^E0169]
33	1,5	0,033	Image omitted; see official source Range of Field capacity [^E0169]
10	1	0,01	WHC (approximation)
1	0	0,001	Water saturated soil

pF = log of cm water column.

1 bar = 10⁵ Pa.

Corresponds to an approximate water content of 10 % in sand, 35 % in loam and 45 % in clay.

Field capacity is not constant but varies with soil type between pF 1,5 and 2,5 .

Water tension is measured in cm water column or in bar. Due to the large range of suction tension it is expressed simply as pF value which is equivalent to the logarithm of cm water column.

Field capacity is defined as the amount of water which can be stored against gravity by a natural soil two days after a longer raining period or after sufficient irrigation. It is determined in undisturbed soil in situ in the field. The measurement is thus not applicable to disturbed laboratory soil samples. FC values determined in disturbed soils may show great systematic variances.

Water holding capacity (WHC) is determined in the laboratory with undisturbed and disturbed soil by saturating a soil column with water by capillary transport. It is particularly useful for disturbed soils and can be up to 30 % greater than field capacity (1). It is also experimentally easier to determine than reliable FC-values.

Notes

Appendix 2

SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM VARIOUS COUNTRIES

		Soil moisture content at	Soil moisture content at	Soil moisture content at
Soil type	Country	Soil moisture content at	Soil moisture content at	Soil moisture content at
		WHC [^E0170]	pF = 1,8	pF = 2,5
Sand	Germany	28,7	8,8	3,9
Loamy sand	Germany	50,4	17,9	12,1
Loamy sand	Switzerland	44,0	35,3	9,2
Silt loam	Switzerland	72,8	56,6	28,4
Clay loam	Brazil	69,7	38,4	27,3
Clay loam	Japan	74,4	57,8	31,4
Sandy loam	Japan	82,4	59,2	36,0
Silt loam	USA	47,2	33,2	18,8
Sandy loam	USA	40,4	25,2	13,3

Water holding capacity.

Appendix 3

Figure 1

Example of a flow-through apparatus to study transformation of chemicals in soil([^E0171])

([^E0172])

Image omitted; see official source

Text of image

1: needle valve

2: gas washing bottle containing water

3: ultramembrane (sterile conditions only), pore size 0.2 μm

4: soil metabolism flask (water-logged only for anaerobic and paddy conditions)

5: ethylene glycol trap for organic volatile compounds

6: sulphuric acid trap for alkaline volatile compounds

7, 8: sodium hydroxide trap for CO2 & other acidic volatiles

9: flow meter.

AIR or N2

Figure 2

Example of a biometer-type flask for studying the transformation of chemicals in soil([^E0173])

Image omitted; see official source

Text of image

Soda lime to absorb CO2

Oil – treated glass wool or polyurethane foam to absorb organic volatiles

Soil + test substance

C.24.   AEROBIC AND ANAEROBIC TRANSFORMATION IN AQUATIC SEDIMENT SYSTEMS

1. METHOD

This test method is a replicate of the OECD TG 308 (2002).

1.1.   INTRODUCTION

Chemicals can enter shallow or deep surface waters by such routes as direct application, spray drift, run-off, drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition. This testing method describes a laboratory method to assess aerobic and anaerobic transformation of organic chemicals in aquatic sediment systems. It is based on existing Guidelines (1)(2)(3)(4)(5)(6). An OECD Workshop on Soil/Sediment Selection, held in Belgirate, Italy in 1995 (7) agreed, in particular, on the number and type of sediments for use in this test. It also made recommendations relating to collection, handling and storage of sediment samples, based on the ISO Guidance (8). Such studies are required for chemicals which are directly applied to water or which are likely to reach the aqueous environment by the routes described above.

The conditions in natural aquatic sediment systems are often aerobic in the upper water phase. The surface layer of sediment can be either aerobic or anaerobic, whereas the deeper sediment is usually anaerobic. To encompass all of these possibilities both aerobic and anaerobic tests are described in this document. The aerobic test simulates an aerobic water column over an aerobic sediment layer that is underlain with an anaerobic gradient. The anaerobic test simulates a completely anaerobic water-sediment system. If circumstances indicate that it is necessary to deviate significantly from these recommendations, for example by using intact sediment cores or sediments that may have been exposed to the test substance, other methods are available for this purpose (9).

1.2.   DEFINITIONS

Standard International (SI) units should be used in any case.

Test substance: any substance, whether the parent or relevant transformation products.

Transformation products: all substances resulting from biotic and abiotic transformation reactions of the test substance including CO2 and bound residues.

Bound residues: ‘bound residues’ represent compounds in soil, plant or animal that persist in the matrix in the form of the parent substance or its metabolite(s) after extractions. The extraction method must not substantially change the compounds themselves or the structure of the matrix. The nature of the bond can be clarified in part by matrix-altering extraction methods and sophisticated analytical techniques. To date, for example, covalent ionic and sorptive bonds, as well as entrapments, have been identified in this way. In general, the formation of bound residues reduces the bioaccessibility and the bioavailability significantly (10) (modified from IUPAC 1984 (11)).

Aerobic transformation: (oxidising): reactions occurring in the presence of molecular oxygen (12).

Anaerobic transformation: (reducing): reactions occurring under exclusion of molecular oxygen (12).

Natural waters: are surface waters obtained from ponds, rivers, streams, etc.

Sediment: is a mixture of mineral and organic chemical constituents, the latter containing compounds of high carbon and nitrogen content and of high molecular masses. It is deposited by natural water and forms an interface with that water.

Mineralisation: is the complete degradation of an organic compound to CO2, H2O under aerobic conditions, and CH4, CO2 and H2O under anaerobic conditions. In the context of this test method, when radiolabelled compound is used, mineralisation means extensive degradation of a molecule during which a labelled carbon atom is oxidised or reduced quantitatively with release of the appropriate amount of ¹⁴CO2 or ¹⁴CH4, respectively.

Half-life, t0,5, is the time taken for 50 % transformation of a test substance when the transformation can be described by first-order kinetics; it is independent of the initial concentration.

DT 50 (Disappearance Time 50): is the time within which the initial concentration of the test substance is reduced by 50 %.

DT 75 (DISAPPEARANCE TIME 75): IS THE TIME WITHIN WHICH THE INITIAL CONCENTRATION OF THE TEST SUBSTANCE IS REDUCED BY 75 %.

DT 90 (Disappearance Time 90): is the time within which the initial concentration of the test substance is reduced by 90 %.

1.3.   REFERENCE SUBSTANCES

Reference substances should be used for the identification and quantification of transformation products by spectroscopic and chromatographic methods.

1.4.   INFORMATION ON THE TEST SUBSTANCE

Non-labelled or isotope-labelled test substance can be used to measure the rate of transformation although labelled material is preferred. Labelled material is required for studying the pathway of transformation and for establishing a mass balance. ¹⁴C-labelling is recommended, but the use of other isotopes, such as ¹³C, ¹⁵N, ³H, ³²P, may also be useful. As far as possible, the label should be positioned in the most stable part(s) of the molecule ([^E0174]). The chemical and/or radiochemical purity of the test substance should be at least 95 %.

Before carrying out a test, the following information about the test substance should be available:

• (a) solubility in water (Method A.6);

• (b) solubility in organic solvents;

• (c) vapour pressure (Method A.4) and Henry's Law constant;

• (d) n-octanol/water partition coefficient (Method A.8);

• (e) adsorption coefficient (Kd, Kf or Koc, where appropriate) (Method C.18);

• (f) hydrolysis (Method C.7);

• (g) dissociation constant (pKa) (OECD Guideline 112) (13);

• (h) chemical structure of the test substance and position of the isotope-label(s), if applicable.

Note: the temperature at which these measurements were made should be reported.

Other useful information may include data on toxicity of the test substance to microorganisms, data on ready and/or inherent biodegradability, and data on aerobic and anaerobic transformation in soil.

Analytical methods (including extraction and clean-up methods) for identification and quantification of the test substance and its transformation products in water and in sediment should be available (see Section 1.7.2).

1.5.   PRINCIPLE OF THE TEST METHOD

The method described in this test employs an aerobic and an anaerobic aquatic sediment (see Appendix 1) system which allows:

the measurement of the transformation rate of the test substance in a water-sediment system,

the measurement of the transformation rate of the test substance in the sediment,

the measurement of the mineralisation rate of the test substance and/or its transformation products (when ¹⁴C-labelled test substance is used),

the identification and quantification of transformation products in water and sediment phases including mass balance (when labelled test substance is used),

the measurement of the distribution of the test substance and its transformation products between the two phases during a period of incubation in the dark (to avoid, for example, algal blooms) at constant temperature. Half-lives, DT50, DT75 and DT90 values are determined where the data warrant, but should not be extrapolated far past the experimental period (see Section 1.2).

At least two sediments and their associated waters are required for both the aerobic and the anaerobic studies respectively (7). However, there may be cases where more than two aquatic sediments should be used, for example, for a chemical that may be present in freshwater and/or marine environments.

1.6.   APPLICABILITY OF THE TEST

The method is generally applicable to chemical substances (unlabelled or labelled) for which an analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile, non-volatile, water-soluble or poorly water-soluble compounds. The test should not be applied to chemicals which are highly volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in water and/or sediment under the experimental conditions of this test.

The method has been applied so far to study the transformation of chemicals in fresh waters and sediments, but in principle can also be applied to estuarine/marine systems. It is not suitable to simulate conditions in flowing water (e.g. rivers) or the open sea.

1.7.   QUALITY CRITERIA

1.7.1.   Recovery

Extraction and analysis of, at least, duplicate water and sediment samples immediately after the addition of the test substance gives a first indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test substance. Recoveries for later stages of the experiments are given by the respective mass balances (when labelled material is used). Recoveries should range from 90 % to 110 % for labelled chemicals (6) and from 70 % to 110 % for non-labelled chemicals.

1.7.2.   Repeatability and sensitivity of analytical method

Repeatability of the analytical method (excluding the initial extraction efficiency) to quantify test substance and transformation products can be checked by duplicate analysis of the same extract of the water or the sediment samples which were incubated sufficiently long enough for formation of transformation products.

The limit of detection (LOD) of the analytical method for the test substance and for the transformation products should be at least 0,01 mg· kg^-1 in water or sediment (as test substance) or 1 % of the initial amount applied to a test system whichever is lower. The limit of quantification (LOQ) should also be specified.

1.7.3.   Accuracy of transformation data

Regression analysis of the concentrations of the test substance as a function of time gives the appropriate information on the accuracy of the transformation curve and allows the calculation of the confidence limits for half-lives (if pseudo first-order kinetics apply) or DT50 values and, if appropriate, DT75 and DT90 values.

1.8.   DESCRIPTION OF THE METHOD

1.8.1.   Test system and apparatus

The study should be performed in glass containers (e.g. bottles, centrifuge tubes), unless preliminary information (such as n-octanol-water partition coefficient, sorption data, etc.) indicates that the test substance may adhere to glass, in which case an alternative material (such as Teflon) may have to be considered. Where the test substance is known to adhere to glass, it may be possible to alleviate this problem using one or more of the following methods:

• — determine the mass of test substance and transformation products sorbed to glass,

• — ensure a solvent wash of all glassware at the end of the test,

• — use of formulated products (see also Section 1.9.2),

• — use an increased amount of co-solvent for addition of test substance to the system; if a co-solvent is used it should be a co-solvent that does not solvolyse the test substance.

Examples of typical test apparatus, i.e. gas flow-through and biometer-type systems, are shown in Appendices 2 and 3, respectively (14). Other useful incubation systems are described in reference 15. The design of the experimental apparatus should permit the exchange of air or nitrogen and the trapping of volatile products. The dimensions of the apparatus must be such that the requirements of the test are complied with (see Section 1.9.1). Ventilation may be provided by either gentle bubbling or by passing air or nitrogen over the water surface. In the latter case gentle stirring of the water from above may be advisable for better distribution of the oxygen or nitrogen in the water. CO2-free air should not be used as this can result in increases in the pH of the water. In either case, disturbance of the sediment is undesirable and should be avoided as far as possible. Slightly volatile chemicals should be tested in a biometer-type system with gentle stirring of the water surface. Closed vessels with a headspace of either atmospheric air or nitrogen and internal vials for the trapping of volatile products can also be used (16). Regular exchange of the headspace gas is required in the aerobic test in order to compensate for the oxygen consumption by the biomass.

Suitable traps for collecting volatile transformation products include but are not restricted to 1 mol· dm^-3 solutions of potassium hydroxide or sodium hydroxide for carbon dioxide ([^E0175]) and ethylene glycol, ethanolamine or 2 % paraffin in xylene for organic compounds. Volatiles formed under anaerobic conditions, such as methane, can be collected, for example, by molecular sieves. Such volatiles can be combusted, for example, to CO2 by passing the gas through a quartz tube filled with CuO at a temperature of 900 ^oC and trapping the CO2 formed in an absorber with alkali (17).

Laboratory instrumentation for chemical analysis of test substance and transformation products is required (e.g. gas liquid chromatography (GLC), high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), mass spectroscopy (MS), gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), etc.), including detection systems for radiolabelled or non-labelled chemicals as appropriate. When radiolabelled material is used a liquid scintillation counter and combustion oxidiser (for the combustion of sediment samples prior to analysis of radioactivity) will also be required.

Other standard laboratory equipment for physical-chemical and biological determinations (see Section Table 1, Section 1.8.2.2), glassware, chemicals and reagents are required as appropriate.

1.8.2.   Selection and number of aquatic sediments

The sampling sites should be selected in accordance with the purpose of the test in any given situation. In selecting sampling sites, the history of possible agricultural, industrial or domestic inputs to the catchment and the waters upstream must be considered. Sediments should not be used if they have been contaminated with the test substance or its structural analogues within the previous four years.

1.8.2.1.   Sediment selection

Two sediments are normally used for the aerobic studies (7). The two sediments selected should differ with respect to organic carbon content and texture. One sediment should have a high organic carbon content (2,5 -7,5 %) and a fine texture, the other sediment should have a low organic carbon content (0,5 -2,5 %) and a coarse texture. The difference between the organic carbon contents should normally be at least 2 %. ‘Fine texture’ is defined as a [clay + silt] ([^E0176]) content of > 50 % and ‘coarse texture’ is defined as a [clay + silt] content of < 50 %. The difference in [clay + silt] content for the two sediments should normally be at least 20 %. In cases, where a chemical may also reach marine waters, at least one of the water-sediment systems should be of marine origin.

For the strictly anaerobic study, two sediments (including their associated waters) should be sampled from the anaerobic zones of surface water bodies (7). Both the sediment and the water phases should be handled and transported carefully under exclusion of oxygen.

Other parameters may be important in the selection of sediments and should be considered on a case-by-case basis. For example, the pH range of sediments would be important for testing chemicals for which transformation and/or sorption may be pH-dependent. pH-dependency of sorption might be reflected by the pKa of the test substance.

1.8.2.2.   Characterisation of water-sediment samples

Key parameters that must be measured and reported (with reference to the method used) for both water and sediment, and the stage of the test at which those parameters are to be determined are summarised in the Table hereafter. For information, methods for determination of these parameters are given in references (18)(19)(20)(21).

In addition, other parameters may need to be measured and reported on a case by case basis (e.g. for freshwater: particles, alkalinity, hardness, conductivity, NO3/PO4 (ratio and individual values); for sediments: cation exchange capacity, water holding capacity, carbonate, total nitrogen and phosphorus; and for marine systems: salinity). Analysis of sediments and water for nitrate, sulfate, bioavailable iron, and possibly other electron acceptors may be also useful in assessing redox conditions, especially in relation to anaerobic transformation.

Measurement of parameters for characterisation of water-sediment samples (7)(22)(23)

Parameter	Stage of test procedure	Stage of test procedure	Stage of test procedure	Stage of test procedure	Stage of test procedure	Stage of test procedure
Parameter	field sampling	post- handling	start of acclimation	start of test	during test	end of test
Parameter	Water	Water	Water	Water	Water	Water	Water
Parameter	Origin/source	x
Temperature	x
pH	x		x	x	x	x
TOC			x	x		x
O2 concentration*	x		x	x	x	x
Redox Potential*			x	x	x	x
Sediment	Sediment	Sediment	Sediment	Sediment	Sediment	Sediment
Origin/source	x
Depth of layer	x
pH		x	x	x	x	x
Particle size distribution		x
TOC		x	x	x		x
Microbial biomass [^E0177]		x		x		x
Redox potential [^E0178]	Observation (colour/smell)		x	x	x	x

Microbial respiration rate method (26), fumigation method (27) or plate count measurements (e.g. bacteria, actinomycetes, fungi and total colonies) for aerobic studies; methanogenesis rate for anaerobic studies.

Recent research results have shown that measurements of water oxygen concentrations and of redox potentials have neither a mechanistic nor a predictive value as far as growth and development of microbial populations in surface waters are concerned (24)(25). Determination of the biochemical oxygen demand (BOD, at field sampling, start and end of test) and of concentrations of micro/macro nutrients Ca, Mg and Mn (at start and end of test) in water and the measurement of total N and total P in sediments (at field sampling and end of test) may be better tools to interpret and evaluate aerobic biotransformation rates and routes.

1.8.3.   Collection, handling and storage

1.8.3.1.   Collection

The draft ISO guidance on sampling of bottom sediment (8) should be used for sampling of sediment. Sediment samples should be taken from the entire 5 to 10 cm upper layer of the sediment. Associated water should be collected from the same site or location and at the same time as the sediment. For the anaerobic study, sediment and associated water should be sampled and transported under exclusion of oxygen (28)(see Section 1.8.2.1). Some sampling devices are described in the literature (8)(23).

1.8.3.2.   Handling

The sediment is separated from the water by filtration and the sediment wet-sieved to a 2 mm-sieve using excess location water that is then discarded. Then known amounts of sediments and water are mixed at the desired ratio (see Section 1.9.1) in incubation flasks and prepared for the acclimation period (see Section 1.8.4). For the anaerobic study, all handling steps have to be done under exclusion of oxygen (29)(30)(31)(32)(33).

1.8.3.3.   Storage

Use of freshly sampled sediment and water is strongly recommended, but if storage is necessary, sediment and water should be sieved as described above and stored together, water-logged (6-10 cm water layer), in the dark, at 4 ± 2°C⁴ for a maximum of four weeks (7)(8)(23). Samples to be used for aerobic studies should be stored with free access of air (e.g. in open containers), whereas those for anaerobic studies under exclusion of oxygen. Freezing of sediment and water and drying-out of the sediment must not occur during transportation and storage.

1.8.4.   Preparation of the sediment/water samples for the test

A period of acclimation should take place prior to adding the test substance, with each sediment/water sample being placed in the incubation vessel to be used in the main test, and the acclimation to be carried out under exactly the same conditions as the test incubation (see Section 1.9.1). The acclimation period is the time needed to reach reasonable stability of the system, as reflected by pH, oxygen concentration in water, redox potential of the sediment and water, and macroscopic separation of phases. The period of acclimation should normally last between one week and two weeks and should not exceed four weeks. Results of determinations performed during this period should be reported.

1.9.   PERFORMANCE OF THE TEST

1.9.1.   Test conditions

The test should be performed in the incubation apparatus (see Section 1.8.1) with a water sediment volume ratio between 3:1 and 4:1, and a sediment layer of 2,5 cm (± 0,5 cm). ([^E0179]) A minimum amount of 50 g of sediment (dry weight basis) per incubation vessel is recommended.

The test should be performed in the dark at a constant temperature in the range of 10 to 30 °C. A temperature of (20 ± 2)°C is appropriate. Where appropriate, an additional lower temperature (e.g. 10 ^oC) may be considered on a case-by-case basis, depending on the information required from the test. Incubation temperature should be monitored and reported.

1.9.2.   Treatment and application of test substance

One test concentration of chemical is used ([^E0180]). For crop protection chemicals applied directly to water bodies, the maximum dosage on the label should be taken as, the maximum application rate calculated on the basis of the surface area of the water in the test vessel. In all other cases, the concentration to be used should be based on predictions from environmental emissions. Care must be taken to ensure that an adequate concentration of test substance is applied in order to characterise the route of transformation and the formation and decline of transformation products. It may be necessary to apply higher doses (e.g. 10 times) in situations where test substance concentrations are close to limits of detection at the start of the study and/or where major transformation products could not readily be detected when present at 10 % of the test substance application rate. However, if higher test concentrations are used they should not have a significant adverse effect on the microbial activity of the water-sediment system. In order to achieve a constant concentration of test substance in vessels of differing dimensions an adjustment to the quantity of the material applied may be considered appropriate, based on the depth of the water column in the vessel in relation to the depth of water in the field (which is assumed to be 100 cm, but other depths can be used). See Appendix 4 for an example calculation.

Ideally the test substance should be applied as an aqueous solution into the water phase of the test system. If unavoidable, the use of low amounts of water miscible solvents (such as acetone, ethanol) is permitted for application and distribution of the test substance, but this should not exceed 1 % v/v and should not have adverse effects on microbial activity of the test system. Care should be exercised in generating the aqueous solution of the test substance — use of generator columns and pre-mixing may be appropriate to ensure complete homogeneity. Following addition of the aqueous solution to the test system, gentle mixing of the water phase is recommended, disturbing the sediment as little as possible.

The use of formulated products is not routinely recommended as the formulation ingredients may affect the distribution of the test substance and/or transformation products between water and sediment phases. However, for poorly water-soluble test substances, the use of formulated material may be an appropriate alternative.

The number of incubation vessels depends on the number of sampling times (see Section 1.9.3). A sufficient number of test systems should be included so that two systems may be sacrificed at each sampling time. Where control units of each aquatic sediment system are employed, they should not be treated with the test substance. The control units can be used to determine the microbial biomass of the sediment and the total organic carbon of the water and sediment at the termination of the study. Two of the control units (i.e. one control unit for each aquatic sediment) can be used to monitor the required parameters in the sediment and water during the acclimation period (see Table in Section 1.8.2.2). Two additional control units have to be included in case the test substance is applied by means of a solvent to measure adverse effects on the microbial activity of the test system.

1.9.3.   Test duration and sampling

The duration of the experiment should normally not exceed 100 days (6), and should continue until the degradation pathway and water/sediment distribution pattern are established or when 90 % of the test substance has dissipated by transformation and/or volatilisation. The number of sampling times should be at least six (including zero time), with an optional preliminary study (see Section 1.9.4) being used to establish an appropriate sampling regime and the duration of the test, unless sufficient data is available on the test substance from previous studies. For hydrophobic test substances, additional sampling points during the initial period of the study may be necessary in order to determine the rate of distribution between water and sediment phases.

At appropriate sampling times, whole incubation vessels (in replicate) are removed for analysis. Sediment and overlying water are analysed separately ([^E0181]). The surface water should be carefully removed with minimum disturbance of the sediment. The extraction and characterisation of the test substance and transformation products should follow appropriate analytical procedures. Care should be taken to remove material that may have adsorbed to the incubation vessel or to interconnecting tubing used to trap volatiles.

1.9.4.   Optional preliminary test

If duration and sampling regime cannot be estimated from other relevant studies on the test substance, an optional preliminary test may be considered appropriate, which should be performed using the same test conditions proposed for the definitive study. Relevant experimental conditions and results from the preliminary test, if performed, should be briefly reported.

1.9.5.   Measurements and analysis

Concentration of the test substance and the transformation products at every sampling time in water and sediment should be measured and reported (as a concentration and as percentage of applied). In general, transformation products detected at ≥ 10 % of the applied radioactivity in the total water-sediment system at any sampling time should be identified unless reasonably justified otherwise. Transformation products for which concentrations are continuously increasing during the study should also be considered for identification, even if their concentrations do not exceed the limits given above, as this may indicate persistence. The latter should be considered on a case by case basis, with justifications being provided in the report.

Results from gases/volatiles trapping systems (CO2 and others, i.e. volatile organic compounds) should be reported at each sampling time. Mineralisation rates should be reported. Non-extractable (bound) residues in sediment are to be reported at each sampling point.

2. DATA

2.1.   TREATMENT OF RESULTS

Total mass balance or recovery (see Section 1.7.1) of added radioactivity is to be calculated at every sampling time. Results should be reported as a percentage of added radioactivity. Distribution of radioactivity between water and sediment should be reported as concentrations and percentages, at every sampling time.

Half-life, DT50 and, if appropriate, DT75 and DT90 of the test substance should be calculated along with their confidence limits (see Section 1.7.3). Information on the rate of dissipation of the test substance in the water and sediment can be obtained through the use of appropriate evaluation tools. These can range from application of pseudo-first order kinetics, empirical curve-fitting techniques which apply graphical or numerical solutions and more complex assessments using, for example, single- or multi-compartment models. Further details can be obtained from the relevant published literature (35)(36)(37).

All approaches have their strengths and weaknesses and vary considerably in complexity. An assumption of first-order kinetics may be an oversimplification of the degradation and distribution processes, but when possible gives a term (the rate constant or half-life) which is easily understood and of value in simulation modelling and calculations of predicted environmental concentrations. Empirical approaches or linear transformations can result in better fits of curves to data and therefore allow better estimation of half-lives, DT50 and, if appropriate, DT75 and DT90 values., The use of the derived constants, however, is limited. Compartment models can generate a number of useful constants of value in risk assessment that describe the rate of degradation in different compartments and the distribution of the chemical. They should also be used for estimation of rate constants for the formation and degradation of major transformation products. In all cases, the method chosen must be justified and the experimenter should demonstrate graphically and/or statistically the goodness of fit.

3. REPORTING

3.1.   TEST REPORT

The report must include the following information:

Test substance:

• — common name, chemical name, CAS number, structural formula (indicating position of the label(s) when radiolabelled material is used) and relevant physical-chemical properties,

• — purity (impurities) of test substance,

• — radiochemical purity of labelled chemical and molar activity (where appropriate).

Reference substances:

• — chemical name and structure of reference substances used for the characterisation and/or identification of transformation products.

Test sediments and waters:

• — location and description of aquatic sediment sampling site(s) including, if possible, contamination history,

• — all information relating to the collection, storage (if any) and acclimation of water-sediment systems,

• — characteristics of the water-sediment samples as listed in Table in section 1.8.2.2.

Test conditions:

• — test system used (e.g. flow-through, biometer, way of ventilation, method of stirring, water volume, mass of sediment, thickness of both water and sediment layer, dimension of test vessels, etc.),

• — application of test substance to test system: test concentration used, number of replicates and controls mode of application of test substance (e.g. use of solvent if any), etc.,

• — incubation temperature,

• — sampling times,

• — extraction methods and efficiencies as well as analytical methods and detection limits,

• — methods for characterisation/identification of transformation products,

• — deviations from the test protocol or test conditions during the study.

Results:

• — raw data figures of representative analyses (all raw data have to be stored in the GLP-archive),

• — repeatability and sensitivity of the analytical methods used,

• — rates of recovery (% values for a valid study are given in section 1.7.1),

• — tables of results expressed as % of the applied dose and in mg· kg^-1 in water, sediment and total system (% only) for the test substance and, if appropriate, for transformation products and non-extractable radioactivity,

• — mass balance during and at the end of the studies,

• — a graphical representation of the transformation in the water and sediment fractions and in total system (including mineralisation),

• — mineralisation rates,

• — half-life, DT50 and, if appropriate, DT75 and DT90 values for the test substance and, where appropriate, for major transformation products including confidence limits in water, sediment and in total system,

• — an assessment of the transformation kinetics of the test substance and, where appropriate, the major transformation products,

• — a proposed pathway of transformation, where appropriate,

• — discussion of results.

4. REFERENCES

• (1)BBA-Guidelines for the examination of plant protectors in the registration process., (1990) Part IV, Section 5-1: Degradability and fate of plant protectors in the water/sediment system. Germany.

• (2)Commission for registration of pesticides: Application for registration of a pesticide., (1991) Part G. Behaviour of the product and its metabolites in soil, water and air, Section G.2.1 (a). The Netherlands.

• (3)MAFF Pesticides Safety Directorate., (1992) Preliminary guideline for the conduct of biodegradability tests on pesticides in natural sediment/water systems. Ref No SC 9046. United-Kingdom.

• (4)Agriculture Canada: Environmental chemistry and fate., (1987) Guidelines for registration of pesticides in Canada. Aquatic (Laboratory) — Anaerobic and aerobic. Canada. p. 35-37.

• (5)US-EPA: Pesticide assessment guidelines, Subdivision N. Chemistry: Environmental fate (1982) Section 162-3, Anaerobic aquatic metabolism.

• (6)SETAC-Europe publication., (1995) Procedures for assessing the environmental fate and ecotoxicity of pesticides. Ed. Dr Mark R. Lynch. SETAC-Europe, Brussels.

• (7)OECD Test Guidelines Programme., (1995) Final Report of the OECD Workshop on Selection of Soils/sediments, Belgirate, Italy, 18-20 January 1995.

• (8)ISO/DIS 5667-12., (1994) Water quality — Sampling — Part 12: Guidance on sampling of bottom sediments.

• (9)US-EPA (1998a) Sediment/water microcosm biodegradation test. Harmonised Test Guidelines (OPPTS 835.3180). EPA 712-C-98-080.

• (10)DFG: Pesticide Bound Residues in Soil. Wiley-VCH (1998).

• (11)T.R. Roberts: Non-extractable pesticide residues in soils and plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984).

• (12)OECD Test Guideline 304A: Inherent Biodegradability in Soil (adopted 12 May 1981).

• (13)OECD (1993): Guidelines for Testing of Chemicals. Paris. OECD (1994-2000): Addenda 6-11 to Guidelines for the Testing of Chemicals.

• (14)Scholz, K., Fritz R., Anderson C. and Spiteller M. (1988) Degradation of pesticides in an aquatic model ecosystem. BCPC — Pests and Diseases, 3B-4, p. 149-158.

• (15)Guth, J.A., (1981) Experimental approaches to studying the fate of pesticides in soil. In Progress in Pesticide Biochemistry (D.H. Hutson, T.R. Roberts, Eds.), Vol. 1, 85-114. J. Wiley & Sons.

• (16)Madsen, T., Kristensen, P. (1997) Effects of bacterial inoculation and non-ionic surfactants on degradation of polycyclic aromatic hydrocarbons in soil. Environ. Toxicol. Chem. 16, p. 631-637.

• (17)Steber, J., Wierich, P. (1987) The anaerobic degradation of detergent range fatty alcohol ethoxylates. Studies with ¹⁴C-labelled model surfactants. Water Research 21, p. 661-667.

• (18)Black, C.A. (1965). Methods of Soil Analysis. Agronomy Monograph No 9. American Society of Agronomy, Madison.

• (19)APHA (1989) Standard Methods for Examination of Water and Wastewater (17^th edition). American Public Health Association, American Water Works Association and Water Pollution Control Federation, Washington D.C.

• (20)Rowell, D.L. (1994) Soil Science Methods and Applications. Longman.

• (21)Light, T.S., (1972). Standard solution for redox potential measurements. Anal. Chemistry 44, p. 1038-1039.

• (22)SETAC-Europe publication (1991) Guidance document on testing procedures for pesticides in freshwater mesocosms. From the Workshop ‘A Meeting of Experts on Guidelines for Static Field Mesocosms Tests’, 3-4 July 1991.

• (23)SETAC-Europe publication. (1993) Guidance document on sediment toxicity tests and bioassays for freshwater and marine environments. From the Workshop on Sediment Toxicity Assessment (WOSTA), 8-10 November 1993. Eds.: I.R. Hill, P. Matthiessen and F. Heimbach.

• (24)Vink, J.P.M., van der Zee, S.E.A.T.M. (1997) Pesticide biotransformation in surface waters: multivariate analyses of environmental factors at field sites. Water Research 31, p. 2858-2868.

• (25)Vink, J.P.M., Schraa, G., van der Zee, S.E.A.T.M. (1999) Nutrient effects on microbial transformation of pesticides in nitrifying waters. Environ. Toxicol, p. 329-338.

• (26)Anderson, T.H., Domsch, K.H. (1985) Maintenance carbon requirements of actively-metabolising microbial populations under in-situ conditions. Soil Biol. Biochem. 17, p. 197-203.

• (27)ISO-14240-2., (1997) Soil quality — Determination of soil microbial biomass — Part 2: Fumigation-extraction method.

• (28)Beelen, P. Van and F. Van Keulen., (1990), The Kinetics of the Degradation of Chloroform and Benzene in Anaerobic Sediment from the River Rhine. Hydrobiol. Bull. 24 (1), p. 13-21.

• (29)Shelton, D.R. and Tiedje, J.M. (1984) General method for determining anaerobic biodegradation potential. App. Environ. Microbiol. 47, p. 850-857.

• (30)Birch, R.R., Biver, C., Campagna, R., Gledhill, W.E., Pagga, U., Steber, J., Reust, H. and Bontinck, W.J. (1989) Screening of chemicals for anaerobic biodegradation. Chemosphere 19, p. 1527-1550.

• (31)Pagga, U. and Beimborn, D.B. (1993) Anaerobic biodegradation tests for organic compounds. Chemoshpere 27, p. 1499-1509.

• (32)Nuck, B.A. and Federle, T.W., (1986) A batch test for assessing the mineralisation of ¹⁴C-radiolabelled compounds under realistic anaerobic conditions. Environ. Sci. Technol. 30, p. 3597-3603.

• (33)US-EPA (1998b). Anaerobic biodegradability of organic chemicals. Harmonised Test Guidelines (OPPTS 835.3400). EPA 712-C-98-090.

• (34)Sijm, Haller and Schrap (1997) Influence of storage on sediment characteristics and drying sediment on sorption coefficients of organic contaminants. Bulletin Environ. Contam. Toxicol. 58, p. 961-968.

• (35)Timme, G., Frehse H. and Laska V. (1986) Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues II. Pflanzenschutz — Nachrichten Bayer, 39, p. 187-203.

• (36)Timme, G., Frehse, H. (1980) Statistical interpretation and graphic representation of the degradational behaviour of pesticide residues I. Pflanzenschutz — Nachrichten Bayer, 33, p. 47-60.

• (37)Carlton, R.R., and Allen, R., (1994) The use of a compartment model for evaluating the fate of pesticides in sediment/water systems. Brighton Crop Protection Conference — Pest and Diseases, p. 1349-1354.

Appendix 1

GUIDANCE ON THE AEROBIC AND THE ANAEROBIC TEST SYSTEMS

Aerobic test system

The aerobic test system described in this test method consists of an aerobic water layer (typical oxygen concentrations range from 7 to 10 mg·l^-1) and a sediment layer, aerobic at the surface and anaerobic below the surface (typical average redox potentials (Eh) in the anaerobic zone of the sediment range from — 80 to — 190 mV). Moistened air is passed over the surface of the water in each incubation unit to maintain sufficient oxygen in the head space.

Anaerobic test system

For the anaerobic test system, the test procedure is essentially the same as that outlined for the aerobic system with the exception that moistened nitrogen is passed above the surface of the water in each incubation unit to maintain a head space of nitrogen. The sediment and water are regarded as anaerobic once the redox potential (Eh) is lower than — 100 mV.

In the anaerobic test, assessment of mineralisation includes measurement of evolved carbon dioxide and methane.

Appendix 2

EXAMPLE OF A GAS FLOW-THROUGH APPARATUS

Image omitted; see official source

Text of image

Air

or

Nitrogen

Aquatic sediment degradation

rig design

Vacuum line

Inlet manifold

Surface water

Deionised water

Inlet traps

Safety trap

Aquarium valves outlet manifold

Sediment

Incubation unit

Safety trap

Trap 1

Trap 2

Trap 3

Trap 4

Volatile traps

Deionised water

Outlet traps

Safety trap, empty

Trap 1:

ethyleneglycol to trap organic volatiles

Trap 2:

sulphuric acid 0.1 M to trap alkaline volatiles

Appendix 3

EXAMPLE OF A BIOMETER APPARATUS

Image omitted; see official source

Text of image

System for absorption of CO2 and for adsorption of organic volatile compounds (O2 permeable)

Device for CO2 extraction

Opening for oxygen determination in H2O

Opening for gas determination (with septum closed)

Stirrer

Water

Sediment

Magnetic stirrer

Appendix 4

EXAMPLE CALCULATION FOR APPLICATION DOSE TO TEST VESSELS

Cylinder internal diameter:	= 8 cm
Water column depth not including sediment:	= 12 cm
Surface area: 3,142 × 42	= 50,3 cm2
Application rate: 500 g test substance/ha corresponds to 5 μg/cm2
Total μg: 5 × 50,3	= 251,5 μg
Adjust quantity in relation to a depth of 100 cm: 12 × 251,5 ÷ 100	= 30,18 μg
Volume of water column: 50,3 × 12	= 603 ml
Concentration in water: 30,18 ÷ 603	= 0,050 μg/ml or 50 μg/l

C.25.   AEROBIC MINERALISATION IN SURFACE WATER — SIMULATION BIODEGRADATION TEST

1. METHOD

This method is equivalent to OECD TG 309 (2004) (1).

1.1.   INTRODUCTION