Commission Directive 2004/73/ECShow full title

1. METHOD

This Test Method is a replicate of the OECD TG 307 (2002)

1.1 INTRODUCTION

This Test Method is based on existing guidelines (1)(2)(3)(4)(5)(6)(7)(8)(9). The method described in this Test Method is designed for evaluating aerobic and anaerobic transformation of chemicals in soil. The experiments are performed to determine (i) the rate of transformation of the test substance, and (ii) the nature and rates of formation and decline of transformation products to which plants and soil organisms may be exposed. Such studies are required for chemicals which are directly applied to soil or which are likely to reach the soil environment. The results of such laboratory studies can also be used to develop sampling and analysis protocols for related field studies.

Aerobic and anaerobic studies with one soil type are generally sufficient for the evaluation of transformation pathways (8)(10)(l1). Rates of transformation should be determined in at least three additional soils (8)(10).

An OECD Workshop on soil and sediment selection, held at Belgirate, Italy in 1995 (10) agreed, in particular, on the number and types of soils for use in this test. The types of soils tested should be representative of the environmental conditions where use or release will occur. For example, chemicals that may be released in subtropical to tropical climates should be tested with Ferrasols or Nitosols (FAO system). The Workshop also made recommendations relating to collection, handling and storage of soil samples, based on the ISO Guidance (15). The use of paddy (rice) soils is also considered in this method.

1.2 DEFINITIONS

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

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

Bound residues:”Bound residues” represent compounds in soil, plant or animal, which persist in the matrix in the form of the parent substance or its metabolite(s)/transformation products after extraction. 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 (12) [modified from IUPAC 1984 (13)].

Aerobic transformation: reactions occurring in the presence of molecular oxygen (14).

Anaerobic transformation: reactions occurring under exclusion of molecular oxygen (14).

Soil: is a mixture of mineral and organic chemical constituents, the latter containing compounds of high carbon and nitrogen content and of high molecular weights, animated by small (mostly micro-) organisms. Soil may be handled in two states:

(a)

undisturbed, as it has developed with time, in characteristic layers of a variety of soil types;

(b)

disturbed, as it is usually found in arable fields or as occurs when samples are taken by digging and used in this test method (14).

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

Half-life: t_0.5, 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 concentration.

DT₅₀ (Disappearance Time 50): is the time within which the concentration of the test substance is reduced by 50%; it is different from the half-life to.5 when transformation does not follow first order kinetics.

DT₇₅ (Disappearance Time 75): is the time within which the concentration of the test substance is reduced by 75%.

DT₉₀ (Disappearance Time 90): is the time within which the concentration of the test substance is reduced by 90%.

1.3 REFERENCE SUBSTANCES

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

1.4 APPLICABILITY OF THE TEST

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

1.5 INFORMATION ON THE TEST SUBSTANCE

Non-labelled or labelled test substance can be used to measure the rate of transformation. 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(1). The purity of the test substance should be at least 95 %.

Before carrying out a test on aerobic and anaerobic transformation in soil, the following information on 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)

chemical stability in dark (hydrolysis) (Method C.7);

(f)

pK_a if a molecule is liable to protonation or deprotonation [OECD Guideline 112 ] (16).

Other useful information may include data on toxicity of the test substance to soil micro-organisms [Testing Methods C.21 and C.22] (16).

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

1.6 PRINCIPLE OF THE TEST METHOD

Soil samples are treated with the test substance and incubated in the dark in biometer-type flasks or in flow-through systems under controlled laboratory conditions (at constant temperature and soil moisture). After appropriate time intervals, soil samples are extracted and analysed for the parent substance and for transformation products. Volatile products are also collected for analysis using appropriate absorption devices. Using ¹⁴C-labelled material, the various mineralisation rates of the test substance can be measured by trapping evolved ¹⁴CO₂ and a mass balance, including the formation of soil bound residues, can be esstablished.

1.7 QUALITY CRITERIA

1.7.1 Recovery

Extraction and analysis of, at least, duplicate soil 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. Recoveries should range from 90% to 110% for labelled chemicals (8) and from 70% to 110% for non-labelled chemicals (3).

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 soil, incubated 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 soil (as test substance) or 1% of applied dose 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 reliability of the transformation curve and allows the calculation of the confidence limits for half-lives (in the case of pseudo first order kinetics) or DT₅₀ values and, if appropriate, DT₇₅ and DT₉₀ values.

1.8 DESCRIPTION OF THE TEST METHOD

1.8.1 Equipment and chemical reagents

Incubation systems consist of static closed systems or suitable flow-through systems (7)(17). Examples of suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures 1 and 2, respectively. Both types of incubation systems have advantages and limitations (7)(17).

Standard laboratory equipment is required and especially the following:

• Analytical instruments such as GLC, HPLC, TLC-equipment, including the appropriate detection systems for analysing radiolabelled or non-labelled substances or inverse isotopes dilution method;

• Instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.);

• Liquid scintillation counter;

• Oxidiser for combustion of radioactive material;

• Centrifuge;

• Extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet apparatus for continuous extraction under reflux);

• Instrumentation for concentrating solutions and extracts (e.g. rotating evaporator);

• Water bath;

• Mechanical mixing device (e.g. kneading machine, rotating mixer).

Chemical reagents used include, for example:

• NaOH, analytical grade, 2 mol dm-^-3, or other appropriate base (e.g. KOH, ethanolamine);

• H₂SO₄, analytical grade, 0.05 mol dm-^-3;

• Ethylene glycol, analytical grade;

• Solid absorption materials such as soda lime and polyurethane plugs;

• Organic solvents, analytical grade, such as acetone, methanol, etc.;

• Scintillation liquid.

1.8.2 Test substance application

For addition to and distribution in soil, the test substance can be dissolved in water (deionised or distilled) or, when necessary, in minimum amounts of acetone or other organic solvents (6) in which the test substance is sufficiently soluble and stable. However, the amount of solvent selected should not have a significant influence on soil microbial activity (see sections 1.5 and 1.9.2-1.9.3 The use of solvents which inhibit microbial activity, such as chloroform, dichloromethane and other halogenated solvents, should be avoided.

The test substance can also be added as a solid, e.g. mixed in quartz sand (6) or in a small sub-sample of the test soil which has been air-dried and sterilised. If the test substance is added using a solvent the solvent should be allowed to evaporate before the spiked sub-sample is added to the original non-sterile soil sample.

For general chemicals, whose major route of entry into soil is through sewage sludge/farming application, the test substance should be first added to sludge which is then introduced into the soil sample (see sections 1.9.2 and 1.9.3)

The use of formulated products is not routinely recommended. However, e.g. for poorly soluble test substances, the use of formulated material may be an appropriate alternative.

1.8.3 Soils

1.8.3.1 Soil selection

To determine the transformation pathway, a representative soil can be used; a sandy loam or silty loam or loam or loamy sand [according to FAO and USDA classification (18)] with a pH of 5.5-8.0, an organic carbon content of 0.5-2.5% and a microbial biomass of at least 1% of total organic carbon is recommended (10).

For transformation rate studies at least three additional soils should be used representing a range of relevant soils. The soils should vary in their organic carbon content, pH, clay content and microbial biomass (10).

All soils should be characterised, at least, for texture (% sand, % silt, % clay) [according to FAO and USDA classification (18)], pH, cation exchange capacity, organic carbon, bulk density, water retention characteristic(2) and microbial biomass (for aerobic studies only). Additional information on soil properties may be useful in interpreting the results. For determination of the soil characteristics the methods recommended in references (19)(20)(21)(22)(23) can be used. Microbial biomass should be determined by using the substrate-induced respiration (SIR) method (25)(26) or alternative methods (20).

1.8.3.2 Collection, handling, and storage of soils

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(3). 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 2 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(4) 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)(5). 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(6) (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 Annex 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(7) 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 Annex 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 5 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.

(1)

For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of transformation products.

(2)

Water retention characteristic of a soil can be measured as field capacity, as water holding capacity or as water suction tension (pF). For explanations see Annex 1. It should be reported in the test report whether water retention characteristics and bulk density of soils were determined in undisturbed field samples or in disturbed (processed) samples.

(3)

Recent research results indicate that soils from temperate zones can also be stored at -20^oC for more than three months (28)(29) without significant losses of microbial activity.

(4)

The soil should neither be too wet nor too dry to maintain adequate aeration and nutrition of soil microflora. Moisture contents recommended for optimal microbial growth range from 40-60% water holding capacity (WHC) and from 0.1-0.33 bar (6). The latter range is equivalent to a pF-range of 2.0 - 2.5. Typical moisture contents of various soil types are given in Annex 2.

(5)

Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown in an EU sponsored research project [K. Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal variation in subsoil temperatures and oxygen contents. Proc. Internat. Symp. Environm. Aspects Pesticides Microbiol., 270-277, 17-21 August 1992, Sigtuna, Sweden]. Anaerobic conditions may only occur occasionally during flooding of soils after heavy rainfalls or when paddy conditions are established in rice fields.

(6)

Aerobic studies might be terminated much before 120 days provided that ultimate transformation pathway and ultimate mineralisation are clearly reached at that time. Termination of the test is possible after 120 days, or when at least 90% of the test substance is transformed, but only if at least 5% CO₂ is formed.

(7)

Calculation of the initial concentration on an area basis using the following equation:

C_soil= Initial concentration in soil [mg·kg^-1]

A = Application rate [kg·ha^-1]; l = thickness of field soil layer [m]; d = dry bulk density of soil [kg·m^-3].

As a rule of thumb, an application rate of 1 kg·ha^-1 results in a soil concentration of approximately 1 mg·kg^-1 in a 10 cm layer (assuming a bulk density of 1 g · cm^-3).