United States      Prevention, Pesticides      EPA712-C-08-018
         Environmental Protection    And Toxic Substances      EPA 712-C-08-019
         Agency        (7101)           June 2008
&EPA   Fate, Transport and
         Transformation Test
         OPPTS 835.4300
         Aerobic Aquatic

         OPPTS 835.4400
         Anaerobic Aquatic


       This guideline is one of a series of test guidelines that have been developed by the Office of
Prevention, Pesticides and Toxic Substances, 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 U. S. Environmental Protection Agency under the Toxic Substances Control
Act (15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C.  136, et seq.)
and section 408 of the Federal Food, Drug and Cosmetic Act (21 U.S.C. 346a)

       The Office of Prevention, Pesticides  and Toxic Substances (OPPTS) has developed this
guideline through a process of harmonization that blended the testing guidance and requirements that
existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I,
Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP)
which  appeared in publications of the National Technical Information Service (NTIS) and the
guidelines published by the Organization for Economic Cooperation and Development (OECD).

       The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to
minimize variations among the testing procedures.

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, etseq.) 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)(l) of this

       (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


bioavailability significantly (see paragraph (h)(3) of this guideline) (modified from IUPAC 1984 (see
paragraph (h)(4) of this guideline).

       DT so (Disappearance Time 50) is the time within which the initial concentration of the test
substance is reduced by 50%.
            (Disappearance Time 75) is the time within which the initial concentration of the test
substance is reduced by 75%.
            (Disappearance Time 90) is the time within which the initial concentration of the test
substance is reduced by 90%.

       Half-life, to.s, 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 CC>2, H2O under aerobic
conditions, and CH4, CC>2 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.

       (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

       (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)(l).

       (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.

       (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

       (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.

       (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)(l 1)  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)(l)).  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.

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.

Figure 2—Example of a Biometer Apparatus:
                                              of Qj^jar.le^ volatile
       (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 CCh by
passing the gas through a quartz tube filled with CuO at a temperature of 900 °C and trapping the CC>2
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

(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 jim.
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

       (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

        Table 1. Measurement of parameters for characterization of water-sediment samples*
O2 concentration**
Redox potential**
Depth of layer
Particle Size
Microbial biomass***
Redox potential**
Stage of Test Procedure




Start of




Start of Test



During Test




End of Test



* 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-


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)(l)). 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 °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 °C 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

       (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 °C.  A temperature of (20 ± 2) °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


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 jig/cm2

       Total |ig: 5x50.3 =251.5  |ig

       Adjust quantity in relation to a depth of 100 cm:
       12 x 251.5-f- 100 =30.18 |ig

       Volume of water column: 50.3 x 12 = 603 mL

       Concentration in water: 30.18 -r- 603  = 0.050 |ig/mL or 50 |ig/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 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.
       (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


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)(l)(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  (CC>2 and  others, i.e., volatile organic


compounds) should be reported at each sampling time. Mineralization rates should be reported. Non-
extractable (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, DTso and, if appropriate, DTys and DTgo 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

       (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

       (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.


       (ii) All information relating to the collection, storage (if any)  and acclimation of water-
sediment 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/kgin water, sediment and
total system (% only) for the test substance and, if appropriate, for transformation products and non-
extractable 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, DTso and, if appropriate, DT?s and DTgo values  for the test substance and,
where appropriate, for transformation products including confidence limits in water, sediment and in
total system.


       (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. United-

       (8) Agriculture Canada: Environmental chemistry and fate (1987). Guidelines for
registration of pesticides in Canada. Aquatic (Laboratory) - Anaerobic and aerobic. Canada, pp 35-

       (9) SET AC-Europe publication (1995). Procedures for assessing the environmental fate and
ecotoxicity of pesticides.  Ed. DrMarkR. Lynch.  SET AC-Europe, Brussels.

       (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, 38-4,149-158.

       (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.

       (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) SET AC-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) SET AC-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.

       (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-

       (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 actively-
metabolising 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-

       (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, I, Reust, H.
and Bontinck, WJ. (1989). Screening of chemicals for anaerobic biodegradation. Chemosphere 19,

       (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,

       (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.

       (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 -

       (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.