United States 

Environmental Protection 

Agency 

Prevention, Pesticides 

And Toxic Substances 

(7101) 

 EPA 712-C-08-018 

 EPA 712-C-08-019 

October 2008 

Fate, Transport and 

Transformation Test 

Guidelines 

 OPPTS 835.4300 

Aerobic Aquatic 

Metabolism 

OPPTS 835.4400 

Anaerobic Aquatic 

Metabolism 

 
INTRODUCTION 

 This guideline is one of a series of test guidelines that have been 

developed by the Office of Prevention, Pesticides and Toxic Substances 

(OPPTS), United States Environmental Protection Agency for use in the testing 

of pesticides and toxic substances, and the development of test data to meet the 

data requirements of the Agency under the Toxic Substances Control Act (TSCA) 

(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act 

(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and 

Cosmetic (FFDCA) (21 U.S.C. 346a). 

 OPPTS developed this guideline through a process of harmonization of 

the testing guidance and requirements that existed for the Office of Pollution 

Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code 

of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in 

publications of the National Technical Information Service (NTIS) and in the 

guidelines published by the Organization for Economic Cooperation and 

Development (OECD). 

 For additional information about OPPTS harmonized guidelines and to 

access this and other guidelines, please go to http://www.epa.gov/oppts and 

select “Test Methods & Guidelines” on the left side menu.
1

OPPTS 835.4300 Aerobic aquatic metabolism 

OPPTS 835.4400 Anaerobic aquatic metabolism. 

(a) Scope—(1) Applicability. This guideline is intended for use in meeting testing 

requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.136, et 

seq.) and for testing pursuant to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.) It 

describes procedures that, if followed, would result in data that would generally be of scientific merit 

for the purposes described in paragraph (b) of this guideline. 

 (2) Background. The source materials used in developing this OPPTS test guideline are OPP 

162-3 Anaerobic aquatic metabolism studies and OPP 162-4 Aerobic aquatic metabolism studies 

(Pesticide Assessment Guidelines Subdivision N - Chemistry: Environmental Fate, EPA report 540/9-

82-021, October 1982); and OECD Guideline for the Testing of Chemicals 308 Aerobic and Anaerobic 

Transformation in Aquatic Sediment Systems (OECD, adopted April 2002). 

(b) Purpose. (1) 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. 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. This guideline describes a laboratory test method to assess 

aerobic and anaerobic transformation of organic chemicals in aquatic sediment systems. An aerobic 

test simulates an aerobic water column over an aerobic sediment layer that is underlain with an 

anaerobic gradient. An anaerobic test simulates a completely anaerobic water-sediment system. Such 

studies are used for chemicals which are directly applied to water or which are likely to reach the 

aqueous environment by the routes described above. 

(2) 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 (see paragraph (h)(1) of this 

guideline). 

(c) Definitions. 

Aerobic transformation (oxidising) reactions are reactions occurring in the presence of 

molecular oxygen (see paragraph (h)(2) of this guideline). 

Anaerobic transformation (reducing) reactions are reactions occurring under exclusion of 

molecular oxygen (see paragraph (h)(2) of this guideline). 

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 should 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 
2

bioavailability significantly (see paragraph (h)(3) of this guideline) (modified from IUPAC 1984 (see 

paragraph (h)(4) of this guideline). 

 

DT 50 (Disappearance Time 50) is the time within which the initial concentration of the test 

substance is reduced by 50%. 

DT75 (Disappearance Time 75) is the time within which the initial concentration of the test 

substance is reduced by 75%. 

DT90 (Disappearance Time 90) is the time within which the initial concentration of the test 

substance is reduced by 90%. 

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. 

Mineralization 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 guideline, when 

radiolabeled compound is used, mineralization means extensive degradation of a molecule during 

which a labeled carbon atom is oxidized or reduced quantitatively with release of the appropriate 

amount of 14CO2 or 

14CH4, respectively. 

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. 

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

Transformation products are all substances resulting from biotic and abiotic transformation 

reactions of the test substance including CO2 and bound residues. 

(d) General considerations—(1) Principle of the test—(i) Method. The method described in 

this guideline employs an aerobic and an anaerobic aquatic sediment 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 

mineralization rate of the test substance and/or its transformation products (when 14C-labeled test 

substance is used); the identification and quantification of transformation products in water and 

sediment phases including mass balance (when labeled test substance is used); and 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. 

Existing guidelines are cited at (h)(5) through (h)(9) in this guideline. 
3

(ii) Half-lives determinations. Half-lives, DT50, DT75 and DT90 values are determined where 

the data warrant, but should not be extrapolated far past the experimental period. 

(iii) Number of sediments. At least two sediments and their associated waters are used for 

both the aerobic and the anaerobic studies respectively (see paragraph (h)(10) of this guideline). 

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. Sediments should be 

representative of intended use sites. 

(iv) Aerobic test system. The aerobic test system described in this test guideline consists of an 

aerobic water layer (typical oxygen concentrations range from 7 to 10 mg/L) 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. 

(v) 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 mineralization includes measurement of evolved carbon dioxide and 

methane. 

(vi) Conditions. 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. 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 (h)(1). 

(vii) 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. 

(2) Applicability of the test (i) The method is generally applicable to chemical substances 

(unlabeled or labeled) 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. 
4

(ii) 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. 

(e) Test procedure—(1) Test substance (i) Non-labeled or isotope-labeled test substance can 

be used to measure the rate of transformation although labeled material is preferred. Labeled material 

is used for studying the pathway of transformation and for establishing a mass balance. 14C-labeling 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. For example, if the 

substance contains one ring, this ring should be labeled; if the test substance contains two or more 

rings, separate studies may be needed to evaluate the fate of each labeled ring and to obtain suitable 

information on formation of transformational products. The chemical and/or radiochemical purity of 

the test substance should be at least 95%. 

(ii) Before carrying out a test, the following information about the test substance should be 

available: solubility in water; solubility in organic solvents; vapor pressure and Henry’s Law constant; 

n-octanol/water partition coefficient; adsorption coefficient (Kd, Kf or Koc, where appropriate); 

hydrolysis; dissociation constant (pKa); and chemical structure of the test substance and position of the 

isotope-label(s), if applicable. The temperature at which these measurements were made should be 

reported. 

(iii) 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. 

(2) Analytical methods (i) 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. 

 (ii) Reference substances should be used for the identification and quantification of 

transformation products by spectroscopic and chromatographic methods. 

(iii) Recovery. Extraction and analysis of, at least, duplicate water and sediment samples 

immediately after the addition of the test substance give 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 labeled material is 

used). Recoveries should range from 90% to 110% for labeled chemicals (see paragraph (h)(9) of this 

guideline) and from 70% to 110% for non-labeled chemicals. 

(iv) Repeatability and sensitivity of analytical method. (A) 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 long enough for formation of transformation products. 
5

(B) 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.

(v) 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. 

(3) Test system and apparatus (i) 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 paragraph (f)(3)(iii) of 

this guideline); and/or 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 solvolyze the test substance. 

(ii) Examples of typical test apparatus, i.e., gas flow-through and biometer-type systems, are 

shown in Figures 1 and 2, respectively (see also paragraph (h)(11) of this guideline). Other useful 

incubation systems are described in paragraph (h)(12) of this guideline. 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 should be such that the requirements of the test are 

complied with (see paragraph (f)(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 (see paragraph (h)(13)of this guideline). Regular exchange of the headspace gas is 

important in the aerobic test in order to compensate for the oxygen consumption by the biomass. 
6

Figure 1—Example of a Gas Flow-through Apparatus. 

Safety trap, empty 

Trap 1: 

Ethyleneglycol to trap organic volatiles 

Trap 2: 

Sulphuric acid 0.1 M to trap alkaline volatiles. 
7

Figure 2—Example of a Biometer Apparatus: 

(iii) 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 and ethylene 

glycol, ethanolamine or 2% paraffin in xylene for organic compounds. As these alkaline absorption 

solutions also absorb the carbon dioxide from the ventilation air and that formed by respiration in 

aerobic experiments, they have to be exchanged at regular intervals to avoid their saturation and thus 

loss of their absorption capacity. 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 °C and trapping the CO2

formed in an absorber with alkali (see paragraph(h)(14) of this guideline). 

(iv) Laboratory instrumentation for chemical analysis of test substance and transformation 

products is used (e.g., gas liquid chromatography (GLC), high performance liquid chromatography 
8

(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 radiolabeled or non-labeled chemicals as 

appropriate. When radiolabeled material is used, a liquid scintillation counter and combustion oxidiser 

(for the combustion of sediment samples prior to analysis of radioactivity) should be used. 

(4) Sediments. Selection and number of aquatic sediments—(i) Sampling sites. 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 should be considered. Sediments should not be used if they have 

been contaminated with the test substance or its structural analogues within the previous 4 years. The 

types of sediments selected should be representative of areas where the pesticide will be used or 

released into the aquatic environment. 

(ii) Sediment selection. (A) Two sediments are normally used for the aerobic studies (see 

paragraph (h)(10) of this guideline. 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) content of >50% and "coarse texture" is defined as a (clay + silt) 

content of <50%. (Clay + silt) is the mineral fraction of the sediment with particle size of < 50 μm. 

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. 

(B) For the strictly anaerobic study, two sediments (including their associated waters) should 

be sampled from the anaerobic zones of surface water bodies (see paragraph (h)(10)of this guideline). 

Both the sediment and the water phases should be handled and transported carefully under exclusion of 

oxygen. 

(C) 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. 

(iii) Characterization of water-sediment samples. (A) Key parameters that should 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 Table 1. For information, 

methods for determination of these parameters are given in paragraphs (h)(15) through (h)(20) of this 

guideline. 
9

Table 1. Measurement of parameters for characterization of water-sediment samples* 

Parameter Stage of Test Procedure 

Field 

Sampling 

PostHandling Start of 

Acclimation 

Start of Test During Test End of Test 

Water

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 

Origin/source x 

Depth of layer x 

pH x x x x x 

Particle Size 

distribution 

x 

TOC x x x x 

Microbial biomass*** x x x 

Redox potential** Observation 

(color/smell) 

x x x x 

* See paragraphs (h)(10), (h)(21) and (h)(22). 

** 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 water are 

concerned (see paragraphs (h)(23) and (h)(24) of this guideline). 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. 

*** Microbial respiration rate method (see paragraph (h)(25) of this guideline), fumigation method (see paragraph (h)(26) 

of this guideline) or plate count measurements (e.g., bacteria, actinomycetes, fungi and total colonies) for aerobic studies; 

methanogenesis rate for anaerobic studies. 

(B) In addition, other parameters may have 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. 

(5) Collection. The draft ISO guidance on sampling of bottom sediment (see paragraph (h)(27) 

of this guideline) 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 (see paragraph (h)(28) of this 

guideline) (see also paragraph (e)(4)(i)(B). Some sampling devices are described in the literature (see 

paragraphs (h)(22), (h)(27) of this guideline). 

(6) Handling. The sediment is separated from the water by filtration and the sediment wet-
10

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 paragraph (f)(2)(i)) in incubation flasks and 

prepared for the acclimation period (see paragraph (f)(1)). For the anaerobic study, all handling steps 

have to be done under exclusion of oxygen (see paragraphs (h)(29) through (h)(33) of this guideline). 

(7) 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 o

C for a maximum of 4 weeks (see paragraphs 

(h)(10), (h)(22), (h)(27) of this guideline). Recent studies have shown that storage at 4 oC can lead to a 

decrease of the organic carbon content of the sediment which may possibly result in a decrease of 

microbial activity (see paragraph (h)(34) of this guideline). 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 should not 

occur during transportation and storage. 

(f) Test conduct—(1) 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 paragraphs (f)(2)(i), (f)(2)(ii)). 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. In the aerobic test, the sediment should have a surface layer that is 

aerobic at the time of treatment. Results of determinations performed during this period should be 

reported. 

(2) Test conditions. (i) The test should be performed in the incubation apparatus (see 

paragraphs (e)(3)(i) through (e)(3)(iii)) with a water:sediment volume ratio between 3:1 and 4:1, and a 

sediment layer of 2.5 cm (± 0.5 cm). A minimum amount of 50 g of sediment (dry weight basis) per 

incubation vessel is recommended. 

(ii) The test should be performed in the dark at a constant temperature in the range of 10 to 

30 oC. A temperature of (20 ± 2) o

C is appropriate. Where appropriate, an additional lower 

temperature (e.g., 10 °C) may be considered on a case-by-case basis, depending on the information 

called for from the test. Incubation temperature should be monitored and reported. 

(3) Treatment and application of test substance. (i) One test concentration of chemical is 

used. Test with a second concentration can be useful for chemicals that reach surface waters by 

different entry routes resulting in significantly different concentrations, as long as the lower 

concentration can be analyzed with sufficient accuracy. 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. For chemicals applied on 

land, the study should be conducted at the aqueous concentration expected when the maximum label 

rate is applied to a 1-hectare pond, 2-meters deep. In all other cases, the concentration to be used 
11

should be based on predictions from environmental emissions. Care should 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. Data from exaggerated dose studies should only be used for degradate identification purposes. 

 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 the following example calculation. 

Example Calculation for Application Dose to Test. 

Cylinder internal diameter: =8 cm 

Water column depth not including sediment: =12 cm 

Surface area: 3.142 x 42 =50.3 cm2

Application rate: 500 g test substance/ha corresponds to 5 μg/cm2

Total μg: 5 x 50.3 = 251.5 μg 

Adjust quantity in relation to a depth of 100 cm: 

12 x 251.5 ÷ 100 = 30.18 μg 

Volume of water column: 50.3 x 12 = 603 mL 

Concentration in water: 30.18 ÷ 603 = 0.050 μg/mL or 50 μg/L 

(ii) 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 premixing 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. 

(iii) 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 
12

water and sediment phases. Studies should be conducted with the active ingredient (technical grade or 

better), not with a formulated product. 

(iv) The number of incubation vessels depends on the number of sampling times (see paragraph 

(f)(4)(i)). 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 of each aquatic sediment) can 

be used to monitor the required parameters in the sediment and water during the acclimation period 

(see Table 1). Two additional control units have to be included if the test substance is applied by 

means of a solvent to measure adverse effects on the microbial activity of the test system. 

(4) Test duration and sampling. (i) The duration of the experiment should normally not 

exceed 100 days (see paragraph (h)(9) of this guideline), and should continue until the degradation 

pathway and water/sediment distribution pattern are established or when 90 % of the test substance has 

been removed by transformation and/or volatilisation. The study should be conducted until the decline 

of parent and the formation and decline of the degradates are established. The number of sampling 

times should be at least six (including zero time), with an optional preliminary study (see paragraph 

(d)(1)(vii)) being used to establish an appropriate sampling regime and the duration of the test, unless 

sufficient data are 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. 

(ii) At appropriate sampling times, whole incubation vessels (in replicate) are removed for 

analysis. Sediment and overlying water are analyzed separately. In cases where rapid re-oxidation of 

anaerobic transformation products may readily occur, anaerobic conditions should be maintained 

during sampling and analysis. The surface water should be carefully removed with minimum 

disturbance of the sediment. The extraction and characterization 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. 

(5) Measurements and analysis. 

(i) 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. 

(ii) Results from gases/volatiles trapping systems (CO2 and others, i.e., volatile organic 
13

compounds) should be reported at each sampling time. Mineralization rates should be reported. Nonextractable (bound) residues in sediment are to be reported at each sampling point. 

(6) Calculations. (i) Total mass balance or recovery (see paragraph (e)(2)(iii)) 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. 

(ii) Half-life, DT50 and, if appropriate, DT75 and DT90 of the test substance should be calculated 

along with their confidence limits (see paragraph (e)(2)(v)). 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 (see paragraphs (h)(35) through (h)(37) of this guideline). 

(iii) 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 should 

be justified and the experimenter should demonstrate graphically and/or statistically the goodness of 

fit. 

(g) Test report—The report should include the following information: 

(1) Test substance. (i) Common name, chemical name, CAS number, structural formula 

(indicating position of the label(s) when radiolabeled material is used) and relevant physical-chemical 

properties. 

(ii) Purity (impurities) of test substance. 

(iii) Radiochemical purity of labeled chemical and molar activity (where appropriate). 

(2) Reference substances. Chemical name and structure of reference substances used for the 

characterization and/or identification of transformation products. 

(3) Test sediments and waters: (i) Location and description of aquatic sediment sampling 

site(s) including, if possible, contamination history. 
14

(ii) All information relating to the collection, storage (if any) and acclimation of watersediment systems. 

(iii) Characteristics of the water-sediment samples as listed in Table 1. 

(4) Test conditions. (i) Test system used (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.). 

(ii) 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. 

(iii) Incubation temperature. 

(iv) Sampling times. 

(v) Extraction methods and efficiencies as well as analytical methods and detection limits. 

(vi) Methods for characterization/identification of transformation products. 

(vii) Deviations from the test protocol or test conditions during the study. 

(5) Results. (i) Raw data figures of representative analyses (all raw data have to be stored in 

the GLP-archive). 

(ii) Repeatability and sensitivity of the analytical methods used. 

(iii) Rates of recovery (% values for a valid study are given in paragraph (e)(2)(iii)). 

(iv) Tables of results expressed as % of the applied dose and in mg/kg in water, sediment and 

total system (% only) for the test substance and, if appropriate, for transformation products and nonextractable radioactivity. 

(v) Mass balance during and at the end of the studies. 

(vi) A graphical representation of the transformation in the water and sediment fractions and in 

total system (including mineralization). 

(vii) Mineralization rates. 

(viii) Half-life, DT50 and, if appropriate, DT75 and DT90 values for the test substance and, 

where appropriate, for transformation products including confidence limits in water, sediment and in 

total system. 
15

(ix) An assessment of the transformation kinetics of the test substance and, where appropriate, 

the transformation products. 

(x) A proposed pathway of transformation, where appropriate. 

(xi) Discussion of results. 

(h) References. The following references should be consulted for additional background 

information on this guideline: 

(1) Environmental Protection Agency (1998). Sediment/water microcosm biodegradation 

test. Harmonised Test Guidelines (OPPTS 835.3180). EPA 712-C-98-080. 

(2) Organization for Economic Cooperation and Development (1981). Test Guideline 

304A: Inherent Biodegradability in Soil. 

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

(4) Roberts, T.R. (1984): Non-extractable pesticide residues in soils and plants. Pure 

Appl. Chem. 56, 945-956 (IUPAC 1984). 

(5) 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. 

(6) 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. 

(7) MAFF Pesticides Safety Directorate (1992). Preliminary guideline for the conduct of 

biodegradability tests on pesticides in natural sediment/water systems. Ref No SC 9046. UnitedKingdom. 

(8) Agriculture Canada: Environmental chemistry and fate (1987). Guidelines for 

registration of pesticides in Canada. Aquatic (Laboratory) - Anaerobic and aerobic. Canada. pp 35-

37. 

(9) SETAC-Europe publication (1995). Procedures for assessing the environmental fate and 

ecotoxicity of pesticides. Ed. Dr Mark R. Lynch. SETAC-Europe, Brussels. 
16

(10) Organization for Economic Cooperation and Development. OECD Test Guidelines 

Programme (1995). Final Report of the OECD Workshop on Selection of Soils/sediments, 

Belgirate, Italy, 18-20 January 1995. 

(11) Scholz, K., Fritz R., Anderson C. and Spiteller M. (1988) Degradation of pesticides in 

an aquatic model ecosystem. BCPC - Pests and Diseases, 3B-4, 149-158. 

(12) 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. 

(13) 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, 631-637. 

(14) Steber, J., Wierich, P. (1987). The anaerobic degradation of detergent range fatty 

alcohol ethoxylates. Studies with 14C-labelled model surfactants. Water Research 21, 661-667. 

(15) Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods (A. 

Klute, Ed.). Soil Science Society of America Book Series 5. 

(16) Methods of Soil Analysis (1994). Part 2. Microbiological and Biochemical Properties 

(R.W. Weaver, S. Angle and P. Bottomley, Eds.). Soil Science Society of America Book Series 5. 

(17) Methods of Soil Analysis (1996). Part 3. Chemical Methods (D.L. Sparks, Ed.). Soil 

Science Society of America Book Series 5. 

(18) American Public Health Association (1989). Standard Methods for Examination of 

Water and Wastewater (17th edition). American Public Health Association, American Water 

Works Association and Water Pollution Control Federation, Washington D.C. 

(19) Rowell, D.L. (1994). Soil Science: Methods and Applications. Longman Scientific & 

Technical, Longman Group UK Ltd, Harlow, Essex, UK (co-published in the USA with John 

Wiley & Sons Inc. New York). 

(20) Light, T.S. (1972). Standard solution for redox potential measurements. Anal. 

Chemistry 44, 1038-1039. 

(21) 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. 

(22) SETAC-Europe publication (1993). Guidance document on sediment toxicity tests and 

bioassays for freshwater and marine environments. From the Workshop On Sediment Toxicity 
17

Assessment (WOSTA), 8-10 November 1993. Eds.: I.R. Hill, P. Matthiessen and F. Heimbach. 

(23) 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, 2858-

2868. 

(24) 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., 329-338. 

(25) Anderson, T.H., Domsch, K.H. (1985). Maintenance carbon requirements of activelymetabolising microbial populations under in-situ conditions. Soil Biol. Biochem. 17, 197-203. 

(26) ISO-14240-2. (1997). Soil quality - Determination of soil microbial biomass - Part 2: 

Fumigation-extraction method. 

(27) ISO/DIS 5667-12(1994) Water quality - Sampling - Part 12: Guidance on sampling of 

bottom sediments. 

(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), 13-

21. 

(29) Shelton, D.R. and Tiedje, J.M. (1984). General method for determining anaerobic 

biodegradation potential. App. Environ. Microbiol. 47, 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, 

1527-1550. 

(31) Pagga, U. and Beimborn, D.B. (1993). Anaerobic biodegradation tests for organic 

compounds. Chemoshpere 27, 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, 

3597-3603. 

(33) Environmental Protection Agency (1998). Anaerobic biodegradability of organic 

chemicals. OPPTS Harmonised Test Guideline 835.3400. EPA 712-C-98-090. 

(34) Sijm, R.T.H.M., Haller, M., and Schrap, S.M. (1997). Influence of storage on 

sediment characteristics and drying sediment on sorption coefficients of organic contaminants. 

Bulletin Environ. Contam. Toxicol. 58, 961-968. 
18

(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, 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, 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, pp 1349-1354.