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

Composition of the AAP-medium (US. EPA) and the OECD TG 201 medium.

Component	AAP	AAP	OECD	OECD
	mg/l	mM	mg/l	mM
NaHCO3	15,0	0,179	50,0	0,595
NaNO3	25,5	0,300
NH4Cl			15,0	0,280
MgCl2·6(H2O)	12,16	0,0598	12,0	0,0590
CaCl2·2(H2O)	4,41	0,0300	18,0	0,122
MgSO4·7(H2O)	14,6	0,0592	15,0	0,0609
K2HPO4	1,044	0,00599
KH2PO4			1,60	0,00919
FeCl3·6(H2O)	0,160	0,000591	0,0640	0,000237
Na2EDTA·2(H2O)	0,300	0,000806	0,100	0,000269*
H3BO3	0,186	0,00300	0,185	0,00299
MnCl2·4(H2O)	0,415	0,00201	0,415	0,00210
ZnCl2	0,00327	0,000024	0,00300	0,0000220
CoCl2·6(H2O)	0,00143	0,000006	0,00150	0,00000630
Na2MoO4·2(H2O)	0,00726	0,000030	0,00700	0,0000289
CuCl2·2(H2O)	0,000012	0,00000007	0,00001	0,00000006
pH	7,5	7,5	8,1	8,1

The molar ratio of EDTA to iron slightly exceeds unity. This prevents iron precipitation and at the same time, chelation of heavy metal ions is minimised.

In test with the diatom Navicula pelliculosa both media must be supplemented with Na2SiO3 ·9H20 to obtain a concentration of 1,4 mg Si/l.

The pH of the medium is obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air. An approximate relationship between pH at 25 ^oC and the molar bicarbonate concentration is:

pHeq = 11,30 + log[HCO3]

With 15 mg NaHCO3/l, pHeq = 7,5 (U.S. EPA medium) and with 50 mg NaHCO3/l, pHeq = 8,1 (OECD medium).

Element composition of test media

Element	AAP	OECD
	mg/l	mg/l
C	2,144	7,148
N	4,202	3,927
P	0,186	0,285
K	0,469	0,459
Na	11,044	13,704
Ca	1,202	4,905
Mg	2,909	2,913
Fe	0,033	0,017
Mn	0,115	0,115

Preparation of OECD medium

Nutrient	Concentration in stock solution
Stock solution 1: macro nutrients	Stock solution 1: macro nutrients
NH4Cl	1,5 g/l
MgCl2·6H2O	1,2 g/l
CaCl2·2H2O	1,8 g/l
MgSO4·7H2O	1,5 g/l
KH2PO4	0,16 g/l
Stock solution 2: iron	Stock solution 2: iron
FeCl3·6H2O	64 mg/l
Na2EDTA·2H2O	100 mg/l
Stock solution 3: trace elements	Stock solution 3: trace elements
H3BO3	185 mg/l
MnCl2·4H2O	415 mg/l
ZnCl2	3 mg/l
CoCl2·6H2O	1,5 mg/l
CuCl2·2H2O	0,01 mg/l
Na2MoO4·2H2O	7 mg/l
Stock solution 4: bicarbonate	Stock solution 4: bicarbonate
NaHCO3	50 g/l
Na2SiO3·9H20

Sterilise the stock solutions by membrane filtration (mean pore diameter 0,2 μm) or by autoclaving (120 °C, 15 min). Store the solutions in the dark at 4 °C.

Do not autoclave stock solutions 2 and 4, but sterilise them by membrane filtration.

Prepare a growth medium by adding an appropriate volume of the stock solutions 1-4 to water:

Add to 500 ml of sterilised water:

10 ml of stock solution 1

1 ml of stock solution 2

1 ml of stock solution 3

1 ml of stock solution 4

Make up to 1 000  ml with sterilised water.

Allow sufficient time for equilibrating the medium with the atmospheric CO2, if necessary by bubbling with sterile, filtered air for some hours.

Preparation of U.S. EPA medium

• 1.Add 1 ml of each stock solution in 2.1–2.7 to approximately 900 ml of deionised or distilled water and then dilute to 1 litre.

• 2.Macronutrient stock solutions are made by dissolving the following into 500 ml of deionised or distilled water. Reagents 2.1, 2.2, 2.3, and 2.4 can be combined into one stock solution.

2.1	NaNO3	12,750 g.
2.2	MgCl2·6H2O	6,082 g.
2.3	CaCl2·2H2O	2,205 g.
2.4	Micronutrient Stock Solution(see 3).	Micronutrient Stock Solution(see 3).
2.5	MgSO4·7H2O	7,350 g.
2.6	K2HPO4	0,522 g.
2.7	NaHCO3	7,500 g.
2.8	Na2SiO3·9H2O	See Note 1.

Note 1: Use for diatom test species only. May be added directly (202,4 mg) or by way of stock solution to give 20 mg/l Si final concentration in medium.

• 3.The micronutrient stock solution is made by dissolving the following into 500 ml of deionised or distilled water:

3.1	H3BO3	92,760 mg.
3.2	MnCl2·4H2O	207,690 mg.
3.3	ZnCl2	1,635 mg.
3.4	FeCl3·6H2O	79,880 mg.
3.5	CoCl2·6H2O	0,714 mg.
3.6	Na2MoO4·2H2O	3,630 mg.
3.7	CuCl2·2H2O	0,006 mg.
3.8	Na2EDTA·2H2O	150,000 mg. [Disodium (Ethylenedinitrilo) tetraacetate].
3.9	Na2SeO4·5H2O	0,005 mg See Note 2.

Note 2: Use only in medium for stock cultures of diatom species.

• 4.Adjust pH to 7,5 ± 0,1 with 0,1 N or 1,0 N NaOH or HCl.

• 5.Filter the media into a sterile container through either a 0,22 μm membrane filter if a particle counter is to be used or a 0,45 μm filter if a particle counter is not to be used.

• 6.Store medium in the dark at approximately 4 °C until use.

Appendix 4

Example of a procedure for the culturing of algae

General observations

The purpose of culturing on the basis of the following procedure is to obtain algal cultures for toxicity tests.

Use suitable methods to ensure that the algal cultures are not infected with bacteria. Axenic cultures may be desirable but unialgal cultures must be established and used.

All operations must be carried out under sterile conditions in order to avoid contamination with bacteria and other algae.

Equipment and materials

See under test method: Apparatus.

Procedures for obtaining algal cultures

Preparation of nutrient solutions (media):

All nutrient salts of the medium are prepared as concentrated stock solutions and stored dark and cold. These solutions are sterilised by filtration or by autoclaving.

The medium is prepared by adding the correct amount of stock solution to sterile distilled water, taking care that no infection occurs. For solid medium 0,8 per cent of agar is added.

Stock culture:

The stock cultures are small algal cultures that are regularly transferred to fresh medium to act as initial test material. If the cultures are not used regularly they are streaked out on sloped agar tubes. These are transferred to fresh medium at least once every two months.

The stock cultures are grown in conical flasks containing the appropriate medium (volume about 100 ml). When the algae are incubated at 20 °C with continuous illumination, a weekly transfer is required.

During transfer an amount of ‘old’ culture is transferred with sterile pipettes into a flask of fresh medium, so that with the fast-growing species the initial concentration is about 100 times smaller than in the old culture.

The growth rate of a species can be determined from the growth curve. If this is known, it is possible to estimate the density at which the culture should be transferred to new medium. This must be done before the culture reaches the death phase.

Pre-culture:

The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture is incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2 to 4 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.

Appendix 5

Data analysis by nonlinear regression

General considerations

The response in algal tests and other microbial growth tests — growth of biomass — is by nature a continuous or metric variable — a process rate if growth rate is used and its integral over time if biomass is selected. Both are referenced to the corresponding mean response of replicate non-exposed controls showing maximum response for the conditions imposed — with light and temperature as primary determining factors in the algal test. The system is distributed or homogenous and the biomass can be viewed as a continuum without consideration of individual cells. The variance distribution of the type of response for a such system relate solely to experimental factors (described typically by the log-normal or normal distributions of error). This is by contrast to typical bioassay responses with quantal data for which the tolerance (typically binomially distributed) of individual organisms are often assumed to be the dominant variance component. Control responses are here zero or background level.

In the uncomplicated situation, the normalised or relative response, r, decreases monotonically from 1 (zero inhibition) to 0 (100 per cent inhibition). Note, that all responses have an error associated and that apparent negative inhibitions can be calculated as a result of random error only.

Regression analysis

Models

A regression analysis aims at quantitatively describing the concentration response curve in the form of a mathematical regression function Y = f (C) or more frequently F(Z) where Z = log C. Used inversely C = f^{– 1} (Y) allows the calculation of, ECx figures, including the EC50, EC10 and EC20, and their 95 % confidence limits. Several simple mathematical functional forms have proved to successfully describe concentration — response relationships obtained in algal growth inhibition tests. Functions include for instance the logistic equation, the nonsymmetrical Weibul equation and the log normal distribution function, which are all sigmoid curves asymptotically approaching zero for C → 0 and one for C → infinity.

The use of continuous threshold function models (e.g. the Kooijman model ‘for inhibition of population growth’ Kooijman et al. 1996) is a recently proposed or alternative to asymptotic models. This model assumes no effects at concentrations below a certain threshold EC0+ that is estimated by extrapolation of the response concentration relationship to intercept the concentration axis using a simple continuous function that is not differentiable in the starting point.

Note that the analysis can be a simple minimisation of sums of residual squares (assuming constant variance) or weighted squares if variance heterogeneity is compensated.

Procedure

The procedure can be outlined as follows: Select an appropriate functional equation, Y = f(C), and fit it to the data by non-linear regression. Use preferably the measurements from each individual flask rather than means of replicates, in order to extract as much information from the data as possible. If the variance is high, on the other hand, practical experience suggests that means of replicates may provide a more robust mathematical estimation less influenced by systematic errors in the data, than with each individual data point retained.

Plot the fitted curve and the measured data and examine whether the curve fit is appropriate. Analysis of residuals may be a particular helpful tool for this purpose. If the chosen functional relationship to fit the concentration response does not describe well the whole curve or some essential part of it, such as the response at low concentrations, choose another curve fit option — e.g., a non-symmetrical curve like the Weibul function instead of a symmetrical one. Negative inhibitions may be a problem with for instance the log — normal distribution function likewise demanding an alternative regression function. It is not recommended to assign a zero or small positive value to such negative values because this distorts the error distribution. It may be appropriate to make separate curve fits on parts of the curve such as the low inhibition part to estimate EClowx figures. Calculate from the fitted equation (by ‘inverse estimation’, C = f^{– 1}(Y)), characteristic point estimates ECx's, and report as a minimum the EC50 and one or two EClow x estimates. Experience from practical testing has shown that the precision of the algal test normally allows a reasonably accurate estimation at the 10 % inhibition level if data points are sufficient — unless stimulation occurs at low concentrations as a confounding factor. The precision of an EC20 estimate is often considerably better than that of an EC10, because the EC20 is usually positioned on the approximately linear part of the central concentration response curve. Sometimes EC10 can be difficult to interpret because of growth stimulation. So while the EC10 is normally obtainable with a sufficient accuracy it is recommended to report always also the EC20.

Weighting factors

The experimental variance generally is not constant and typically includes a proportional component, and a weighted regression is therefore advantageously carried out routinely. Weighting factors for a such analysis are normally assumed inversely proportional to the variance:

Wi = 1/Var(ri)

Many regression programs allow the option of weighted regression analysis with weighting factors listed in a table. Conveniently weighting factors should be normalised by multiplying them by n/Σ wi (n is the number of datapoints) so their sum be one.

Normalising responses

Normalising by the mean control response gives some principle problems and gives rise to a rather complicated variance structure. Dividing the responses by the mean control response for obtaining the percentage of inhibition, one introduces an additional error caused by the error on the control mean. Unless this error is negligibly small, weighting factors in the regression and confidence limits must be corrected for the covariance with the control (Draper and Smith, 1981). Note that high precision on the estimated mean control response is important in order to minimise the overall variance for the relative response. This variance is as follows:

(Subscript i refers to concentration level i and subscript 0 to the controls)

Yi = Relative response = ri/r0 = 1 – I = f(Ci)

with a variance Var(Yi) = Var (ri/r0) ≅ (∂ Yi/∂ ri) · Var(ri) + ((∂ Yi/∂ r0)² · Var(r0)

and since (∂ Yi/∂ ri) = 1/r0 and (∂ Y I/∂ r0) = ri/r0 ²

with normally distributed data and mi and m0 replicates: Var(ri) = σ²/mi

the total variance of the relative response Yi thus becomes

Var(Yi) = σ²/(r0 ² · mi) + ri ² · σ²/r0 ⁴ · m0

The error on the control mean is inversely proportional to the square root of the number of control replicates averaged, and sometimes it can be justified to include historic data and in this way greatly reduce the error. An alternative procedure is not to normalise the data and fit the absolute responses including the control response data but introducing the control response value as an additional parameter to be fitted by non linear regression. With a usual 2 parameter regression equation, this method necessitates the fitting of 3 parameters, and therefore demands more data points than non-linear regression on data that are normalised using a pre-set control response.

Inverse confidence intervals

The calculation of non-linear regression confidence intervals by inverse estimation is rather complex and not an available standard option in ordinary statistical computer program packages. Approximate confidence limits may be obtained with standard non-linear regression programs with re-parameterisation (Bruce and Versteeg, 1992), which involves rewriting the mathematical equation with the desired point estimates, e.g. the EC10 and the EC50 as the parameters to be estimated. (Let the function be I = f (α, β, Concentration) and utilise the definition relationships f (α, β, EC10) = 0,1 and f (α, β, EC50 ) = 0,5 to substitute f (α, β, concentration ) with an equivalent function g( EC10, EC50, concentration).

A more direct calculation (Andersen et al, 1998) is performed by retaining the original equation and using a Taylor expansion around the means of ri and r0.

Recently ‘boot strap methods’ have become popular. Such methods use the measured data and a random number generator directed frequent re-sampling to estimate an empirical variance distribution.

REFERENCES

Kooijman, S.A.L.M.; Hanstveit, A.O.; Nyholm, N. (1996): No-effect concentrations in algal growth inhibition tests. Water Research, 30, 1625-1632.

Draper, N.R. and Smith, H. (1981). Applied Regression Analysis, second edition. Wiley, New York.

Bruce, R..D. and Versteeg,, D.J. (1992). A Statistical Procedure for Modelling Continuous Ecotoxicity Data. Environ. Toxicol. Chem. 11, 1485-1494.

Andersen, J.S., Holst, H., Spliid, H., Andersen, H., Baun, A. & Nyholm, N. (1998). Continuous ecotoxicological data evaluated relative to a control response. Journal of Agricultural, Biological and Environmental Statistics, 3, 405-420.

C.4.   DETERMINATION OF ‘READY’ BIODEGRADABILITY

PART I.   GENERAL CONSIDERATIONS

I.1.   INTRODUCTION

Six test methods are described that permit the screening of chemicals for ready biodegradability in an aerobic aqueous medium:

• (a) Dissolved Organic Carbon (DOC) Die-Away (Method C.4-A)

• (b) Modified OECD Screening — DOC Die-Away (Method C.4-B)

• (c) Carbon dioxide (CO2) Evolution (Modified Sturm Test) (Method C.4-C)

• (d) Manometric Respirometry (Method C.4-D)

• (e) Closed Bottle (Method C.4-E)

• (f) MITI (Ministry of International Trade and Industry — Japan) (Method C.4-F)

General and common considerations to all six tests are given in Part I of the method. Items specific for individual methods are given in Parts II to VII. The appendices contain definitions, formulas and guidance material.

An OECD inter-laboratory comparison exercise, done in 1988, has shown that the methods give consistent results. However, depending on the physical characteristics of the substance to be tested, one or other of the methods may be preferred.

I.2.   SELECTION OF THE APPROPRIATE METHOD

In order to select the most appropriate method, information on the chemical's solubility, vapour pressure and adsorption characteristics is essential. The chemical structure or formula should be known in order to calculate theoretical values and/or check measured values of parameters, e.g. ThOD, ThCO2, DOC, TOC, COD (see Appendices 1 and 2).

Test chemicals which are soluble in water to at least 100 mg/l may be assessed by all methods, provided they are non-volatile and non-adsorbing. For those chemicals which are poorly soluble in water, volatile or adsorbing, suitable methods are indicated in Table 1. The manner in which poorly water-soluble chemicals and volatile chemicals can be dealt with is described in Appendix 3. Moderately volatile chemicals may be tested by the DOC Die-Away method if there is sufficient gas space in the test vessels (which should be suitably stoppered). In this case, an abiotic control must be set up to allow for any physical loss.

Table 1:

Applicability of test methods

Test	Analytical Method	Suitability for substances which are:	Suitability for substances which are:	Suitability for substances which are:
Test	Analytical Method	poorly soluble	volatile	adsorbing
DOC Die-Away	Dissolved organic carbon	–	–	+/–
Mod. OECD Die-Away	Dissolved organic carbon	–	–	+/–
CO2 Evolution	Respirometry: CO2 evolution	+	–	+
Manometric Respirometry	Manometric respirometry: oxygen consumption	+	+/–	+
Closed Bottle	Respirometry: dissolved oxygen	+/–	+	+
MITI	Respirometry: oxygen consumption	+	+/–	+

Information on the purity or the relative proportions of major components of the test material is required to interpret the results obtained, especially when the results are low or marginal.

Information on the toxicity of the test chemical to bacteria (Appendix 4) may be very useful for selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values.

I.3.   REFERENCE SUBSTANCES

In order to check the procedure, reference chemicals which meet the criteria for ready biodegradability are tested by setting up an appropriate flask in parallel to the normal test runs.

Suitable chemicals are aniline (freshly distilled), sodium acetate and sodium benzoate. These reference chemicals all degrade in these methods even when no inoculum is deliberately added.

It was suggested that a reference chemical should be sought which was readily biodegradable but required the addition of an inoculum. Potassium hydrogen phthalate has been proposed but more evidence needs to be obtained with this substance before it can be accepted as a reference substance.

In the respirometric tests, nitrogen-containing compounds may affect the oxygen uptake because of nitrification (see Appendices 2 and 5).

I.4.   PRINCIPLE OF THE TEST METHODS

A solution, or suspension, of the test substance in a mineral medium is inoculated and incubated under aerobic conditions in the dark or in diffuse light. The amount of DOC in the test solution due to the inoculum should be kept as low as possible compared to the amount of DOC due to the test substance. Allowance is made for the endogenous activity of the inoculum by running parallel blank tests with inoculum but without test substance, although the endogenous activity of cells in the presence of the substance will not exactly match that in the endogenous control. A reference substance is run in parallel to check the operation of the procedures.

In general, degradation is followed by the determination of parameters, such as DOC, CO2 production and oxygen uptake, and measurements are taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. With automatic respirometers the measurement is continuous. DOC is sometimes measured in addition to another parameter but this is usually done only at the beginning and the end of the test. Specific chemical analysis can also be used to assess primary degradation of the test substance, and to determine the concentration of any intermediate substances formed (obligatory in the MITI test).

Normally, the test lasts for 28 days. Tests however may be ended before 28 days, i.e. as soon as the biodegradation curve has reached a plateau for at least three determinations. Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached day 28.

I.5.   QUALITY CRITERIA

I.5.1.   Reproducibility

Because of the nature of biodegradation and of the mixed bacterial populations used as inocula, determinations should be carried out at least in duplicate.

It is common experience that the larger the concentration of micro-organisms initially added to the test medium, the smaller will be the variation between replicates. Ring tests have also shown that there can be large variations between results obtained by different laboratories, but good agreement is normally obtained with easily biodegradable compounds.

I.5.2.   Validity of the test

A test is considered valid if the difference of extremes of replicate values of the removal of test chemical at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20 % and if the percentage degradation of the reference substance has reached the level for ready biodegradability by 14 days. If either of these conditions is not met, the test should be repeated. Because of the stringency of the methods, low values do not necessarily mean that the test substance is not biodegradable under environmental conditions, but indicates that more work will be necessary to establish biodegradability.

If in a toxicity test, containing both the test substance and a reference chemical, less than 35 % degradation (based on DOC) or less than 25 % (based on ThOD or ThCO2) occurred in 14 days, the test chemicals can be assumed to be inhibitory (see also Appendix 4). The test series should be repeated, if possible using a lower concentration of test chemical and/or a higher concentration of inoculum, but not greater than 30 mg solids/litre.

I.6.   GENERAL PROCEDURES AND PREPARATIONS

General conditions applying to the tests are summarised in Table 2. Apparatus and other experimental conditions pertaining specifically to an individual test are described later under the heading for that test.

Table 2

Test conditions

Test	DOC Die-Away	CO2 Evolution	Manometric Respirometry	Modified OECD Screeing	Closed Bottle	MITI (I)
Concentration of Test Substance
as mg/l			100		2-10	100
mg DOC/1	10-40	10-20		10-40
mg ThOD/1			50-100		5-10
Concentration of Inoculum (in cells/l, approximatively)	≤ 30 mg/l SS or ≤ 100 ml effluent/l (107-108)	≤ 30 mg/l SS or ≤ 100 ml effluent/l (107-108)	≤ 30 mg/l SS or ≤ 100 ml effluent/l (107-108)	0,5 ml secondary effluent/1 (105)	≤ 5 ml of effluent/l (104-106)	30 mg/l SS (107-108)
Concentration of elements in mineral medium (in mg/l):
P	116	116	116	116	11,6	29
N	1,3	1,3	1,3	1,3	0,13	1,3
Na	86	86	86	86	8,6	17,2
K	122	122	122	122	12,2	36,5
Mg	2,2	2,2	2,2	2,2	2,2	6,6
Ca	9,9	9,9	9,9	9,9	9,9	29,7
Fe	0,05 - 0,1	0,05 - 0,1	0,05 - 0,1	0,05 - 0,1	0,05 - 0,1	0,15
pH	7,4 ± 0,2	7,4 ± 0,2	7,4 ± 0,2	7,4 ± 0,2	7,4 ± 0,2	preferably 7,0
Temperature	22 ± 2 oC	22 ± 2 oC	22 ± 2 oC	22 ± 2 oC	22 ± 2 oC	25 ± 1 oC
DOC = Dissolved organic Carbon	DOC = Dissolved organic Carbon	DOC = Dissolved organic Carbon	ThoD = Theoretical Oxygen Demand	ThoD = Theoretical Oxygen Demand	SS = Suspended Solids	SS = Suspended Solids

I.6.1.   Dilution water

Deionised or distilled water, free from inhibitory concentrations of toxic substances (e.g. Cu ⁺⁺ ions) is used. It must contain no more than 10 % of the organic carbon content introduced by the test material. The high purity of the test water is necessary to eliminate high blank values. Contamination may result from inherent impurities and also from the ion-exchange resins and lysed material from bacterial and algae. For each series of tests use only one batch of water, checked beforehand by DOC analysis. Such a check is not necessary for the closed bottle test, but the oxygen consumption of the water must be low.

I.6.2.   Stock solutions of mineral components

To make up the test solutions, stock solutions of appropriate concentrations of mineral components are made up. The following stock solutions may be used (with different dilution factors) for the methods DOC Die-Away, Modified OECD Screening, CO2 Evolution, Manometric Respirometry, Closed Bottle test.

The dilution factors and, for the MITI test, the specific preparation of the mineral medium are given under the headings of the specific tests.

Stock solutions:

Prepare the following stock solutions, using analytical grade reagents.

(a)	Monopotassium dihydrogen orthophosphate, KH2PO4	8,50 g
(a)	Dipotassium monohydrogen orthophosphate, K2HPO4	21,75 g
(a)	Disodium monohydrogen orthophosphate dihydrate Na2HPO4. 2 H2O	33,40 g
(a)	Ammonium chloride, NH4Cl	0,50 g
(a)	Dissolve in water and make up to 1 litre The pH of the solution should be 7,4 .
(b)	Calcium chloride, anhydrous, CaCl2	27,50 g
(b)	or Calcium chloride dihydrate, CaCl2, 2 H2O	36,40 g
(b)	Dissolve in water and make up to 1 litre
(c)	Magnesium sulphate heptahydrate, MgSO4. 7 H2O	22,50 g
(c)	Dissolve in water and make up to 1 litre.
(d)	Iron (III) chloride hexahydrate, FeC13. 6H2O	0,25 g
(d)	Dissolve in water and make up to 1 litre.

Note: in order to avoid having to prepare this solution immediately before use add one drop of conc. HCL or 0,4 g ethylenediaminetetra-acetic acid disodium salt (EDTA) par litre.

I.6.3.   Stock solutions of chemicals

For example, dissolve 1-10 g, as appropriate, of test or reference chemical in deionised water and make up to 1 litre when the solubility exceeds 1 g/l. Otherwise, prepare stock solutions in the mineral medium or add the chemical direct to the mineral medium. For the handling of less soluble chemicals, see Appendix 3, but in the MITI test (Method C.4-F), neither solvents nor emulsifying agents are to be used.

I.6.4.   Inocula

The inoculum may be derived from a variety of sources: activated sludge, sewage effluents (unchlorinated), surface waters and soils or from a mixture of these. For the DOC Die-Away, CO2 Evolution and Manometric Respirometry tests, if activated sludge is used, it should be taken from a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. Inocula from other sources have been found to give higher scattering of results. For the Modified OECD Screening and the Closed Bottle tests a more dilute inoculum without sludge fiocs is needed and the preferred source is a secondary effluent from a domestic waste water treatment plant or laboratory-scale unit. For the MITI test the inoculum is derived from a mixture of sources and is described under the heading of this specific test.

I.6.4.1.   Inoculum from activated sludges

Collect a sample of activated sludge freshly from the aeration tank of a sewage treatment plant or laboratory-scale unit treating predominantly domestic sewage. Remove coarse particles if necessary by filtration through a fine sieve and keep the sludge aerobic thereafter.

Alternatively, settle or centrifuge (e.g. at 100 g for 10 min) after removal of any coarse particles. Discard the supernatant. The sludge may be washed in the mineral medium. Suspend the concentrated sludge in mineral medium to yield a concentration of 3-5 g suspended solids/l and aerate until required.

Sludge should be taken from a properly working conventional plant. If sludge has to be taken from a high rate treatment plant, or is thought to contain inhibitors, it should be washed. Settle or centrifuge the re-suspended sludge after thorough mixing, discard the supernatant and again re-suspend the washed sludge in a further volume of mineral medium. Repeat this procedure until the sludge is considered to be free from excess substrate or inhibitor.

After complete re-suspension is achieved, or with untreated sludge, withdraw a sample just before use for the determination of the dry weight of the suspended solids.

A further alternative is to homogenise activated sludge (3-5 g suspended solids/l). Treat the sludge in a mechanical blender for two min at medium speed. Settle the blended sludge for 30 min or longer if required and decant liquid for use as inoculum at the rate of 10 mill of mineral medium.

I.6.4.2.   Other sources of inoculum

It can be derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. Collect a fresh sample and keep it aerobic during transport. Allow to settle for 1 h. or filter through a coarse filter paper and keep the decanted effluent or filtrate aerobic until required. Up to 100 ml of this type of inoculum may be used per litre of medium.

A further source for the inoculum is surface water. In this case, collect a sample of an appropriate surface water, e.g. river, lake, and keep aerobic until required. If necessary, concentrate the inoculum by filtration or centrifugation.

I.6.5.   Pre-conditioning of inocula

Inocula may be pre-conditioned to the experimental conditions, but not pre-adapted to the test chemical. Pre-conditioning consists of aerating activated sludge in mineral medium or secondary effluent for five to seven days at the test temperature. Pre-conditioning sometimes improves the precision of the test methods by reducing blank values. It is considered unnecessary to pre-condition MITI inoculum.

I.6.6.   Abiotic controls

When required, check for the possible abiotic degradation of the test substance by determining the removal of DOC, oxygen uptake or carbon dioxide evolution in sterile controls containing no inoculum. Sterilise by filtration through a membrane (0,2 - 0,45 micrometre) or by the addition of a suitable toxic substance at an appropriate concentration. If membrane filtration is used, take samples aseptically to maintain sterility. Unless adsorption of the test chemical has been ruled out beforehand, tests which measure biodegradation as the removal of DOC, especially with activated sludge inocula, should include an abiotic control which is inoculated and poisoned.

I.6.7.   Number of flasks

The number of flasks in a typical run is described under the headings of each tests.

The following type of flask may be used:

• — test suspension: containing test substance and inoculum

• — inoculum blank: containing only inoculum

• — procedure control: containing reference substance and inoculum

• — abiotic sterile control: sterile, containing test substance (see 1.6.6)

• — adsorption control: containing test substance, inoculum and sterilising agent

• — toxicity control: containing test substance, reference substance and inoculum

It is mandatory that determination in test suspension and inoculum blank is made in parallel. It is advisable to make the determinations in the other flasks in parallel as well.

This may, however, not always be possible. Ensure that sufficient samples or readings are taken to allow the percentage removal in the 10-day window to be assessed.

I.7.   DATA AND EVALUATION

In the calculation of Dt, percentage degradation, the mean values of the duplicate measurement of the parameter in both test vessels and inoculum blank are used. The formulas are set out in the sections below on specific tests. The course of degradation is displayed graphically and the 10-day window is indicated. Calculate and report the percentage removal achieved at the end of the 10-day window and the value at the plateau or at the end of the test, whichever is appropriate.

In respirometric tests nitrogen-containing compounds may affect the oxygen uptake because of nitrification (see Appendices 2 and 5).

I.7.1.   Degradation measured by means of DOC determination

The percentage degradation Dt at each time a sample was taken should be calculated separately for the flasks containing test substance using mean values of duplicate DOC measurements in order that the validity of the test can be assessed (see 1.5.2). It is calculated using the following equation:

Image omitted; see official source

where:

Dt	=	% degradation at time t
---	---	---
Co	=	mean starting concentration of DOC in the inoculated culture medium containing the test substance (mg DOC/l)
---	---	---
Ct	=	mean concentration of DOC in the inoculated culture medium containing test substance at time t (mg DOC/l)
---	---	---
Cbo	=	mean starting concentration of DOC in blank inoculated mineral medium (mg DOC/l)
---	---	---
Cbt	=	mean concentration of DOC blank inoculated mineral medium at time t (mg DOC/l).

All concentrations are measured experimentally.

I.7.2.   Degradation measured by means of specific analysis

When specific analytical data are available, calculate primary biodegradation from:

Image omitted; see official source

where:

Dt	=	% degradation at time t, normally 28 days,
---	---	---
Sa	=	residual amount of test substance in inoculated medium at end of test (mg),
---	---	---
Sb	=	residual amount of test substance in the blank test with water/medium to which only the test substance was added (mg).

1.7.3.   Abiotic degradation

When an abiotic sterile control is used, calculate the percentage abiotic degradation using:

Image omitted; see official source

Where:

Cs(o)	=	DOC Concentration in sterile control at day 0
---	---	---
Cs(t)	=	DOC Concentration in sterile control at day t.

I.8.   REPORTING

The test report shall, if possible, contain the following:

• — test and reference chemicals, and their purity,

• — test conditions,

• — inoculum: nature and sampling site(s), concentration and any pre-conditioning treatment,

• — proportion and nature of industrial waste present in sewage if known,

• — test duration and temperature,

• — in the case of poorly soluble test chemicals, treatment given,

• — test method applied; scientific reasons and explanation should be given for any change of procedure,

• — data sheet,

• — any observed inhibition phenomena,

• — any observed abiotic degradation,

• — specific chemical analytical data, if available,

• — analytical data on intermediates, if available,

• — the graph of percentage degradation against time for the test and reference substances; the lag phase, degradation phase, 10-day window and slope should be clearly indicated (Appendix 1). If the test has complied with the validity criteria, the mean of the degradation percentages of the flasks containing test substance may be used for the graph,

• — percentage removal after 10-day window, and at plateau or at end of the test.

PART II.   DOC DIE-AWAY TEST (Method C.4-A)

II.1.   PRINCIPLE OF THE METHOD

A measured volume of inoculated mineral medium containing a known concentration of the test substance (10-40 mg DOC/l) as the nominal sole source of organic carbon is aerated in the dark or diffused light at 22 ± 2 ^oC.

Degradation is followed by DOC analysis at frequent intervals over a 28-day period. The degree of biodegradation is calculated by expressing the concentration of DOC removed (corrected for that in the blank inoculum control) as a percentage of the concentration initially present. The degree of primary biodegradation may also be calculated from supplemental chemical analysis made at the beginning and end of incubation.

II.2.   DESCRIPTION OF THE METHOD

II.2.1.   Apparatus

• (a) Conical flasks, e. g. 250 ml to 2 l, depending on the volume needed for DOC analysis;

• (b) shaking machine to accommodate the conical flasks, either with automatic temperature control or used in a constant temperature room; and of sufficient power to maintain aerobic conditions in all flasks;

• (c) filtration apparatus, with suitable membranes;

• (d) DOC analyser;

• (e) apparatus for determining dissolved oxygen;

• (f) centrifuge.

II.2.2.   Preparation of mineral medium

For the preparation of the stock solutions, see I.6.2.

Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 11 with dilution water.

II.2.3.   Preparation and pre-conditioning of inoculum

The inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters; soils or from a mixture of these.

See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.

II.2.4.   Preparation of flasks

As an example, introduce 800 ml portions of mineral medium into 2 l conical flasks and add sufficient volumes of stock solutions of the test and reference substances to separate flasks to give a concentration of chemical equivalent to 10-40 mg DOC/l. Check the pH values and adjust, if necessary, to 7,4 . Inoculate the flasks with activated sludge or other source of inocula (see I.6.4.), to give a final concentration not greater than 30 mg suspended solids/l. Also prepare inoculum controls in the mineral medium but without test or reference chemical.

If needed, use one vessel to check the possible inhibitory effect of the test chemical by inoculating a solution containing, in the mineral medium, comparable concentrations of both the test and a reference chemical.

Also, if required, set up a further, sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6.6).

Additionally, if the test chemical is suspected of being significantly adsorbed on to glass, sludge, etc., make a preliminary assessment to determine the likely extent of adsorption and thus the suitability of the test for the chemical (see Table 1). Set up a flask containing the test substance, inoculum and sterilising agent.

Make up the volumes in all flasks to 11 with mineral medium and, after mixing, take a sample from each flask to de.termine the initial concentration of DOC (see Appendix 2.4). Cover the openings of the flasks, e.g. with aluminium foil, in such a way as to allow free exchange of air between the flask and the surrounding atmosphere. Then insert the vessels into the shaking machine for starting the test.

II.2.5.   Number of flasks in typical run

Flasks 1 and 2: Test suspension

Flasks 3 and 4: Inoculum blank

Flask 5: Procedure control

preferably and when necessary:

Flask 6: Abiotic sterile control

Flask 7: Adsorption control

Flask 8: Toxicity control

See also I.6.7.

II.2.6.   Performance of the test

Throughout the test, determine the concentrations of DOC in each flask in duplicate at known time intervals, sufficiently frequently to be able to determine the start of the 10-day window and the percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each determination.

Before sampling make good evaporation losses from the flasks by adding dilution water (I.6.1) in the required amount if necessary. Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is dissolved or suspended before sampling. Membrane-filter or centrifuge (see Appendix 2..4) immediately after the sample has been taken. Analyse the filtered or centrifuged samples on the same day, otherwise store at 2-4 ^oC for a maximum of 48 h, or below - 18 ^oC for a longer period.

II.3.   DATA AND REPORTING

II.3.1.   Treatment of results

Calculate the percentage degradation at time t as given under I.7.1 (DOC determination) and, optionally, under I.7.2 (specific analysis).

Record all results on the data sheets provided.

II.3.2.   Validity of results

See I.5.2.

II.3.3.   Reporting

See I.8.

II.4.   DATA SHEET

An example of a data sheet is given hereafter.

DOC DIE-AWAY TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: … mg/1 as chemical

Initial concentration in medium, to: … mg/1 as chemical

4. INOCULUM

Source:

Treatment given:

Pre-conditioning, if any:

Concentration of suspended solids in reaction mixture: mg/1

5. CARBON DETERMINATIONS

Carbon analyser:

	Flask nr		DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)
	Flask nr		0	n1	n2	n3	nx
Test chemical plus inoculum	1	a1
Test chemical plus inoculum	1	a2
Test chemical plus inoculum	1	a, mean Ca(t)
Test chemical plus inoculum	2	b1
Test chemical plus inoculum	2	b2
Test chemical plus inoculum	2	b, men Cb(t)
Blank inoculum without test chemical	3	C1
Blank inoculum without test chemical	3	C2
Blank inoculum without test chemical	3	C, mean Cc(t)
Blank inoculum without test chemical	4	d1
Blank inoculum without test chemical	4	d2
Blank inoculum without test chemical	4	d, mean Cd(t)
Blank inoculum without test chemical	Image omitted; see official source	Image omitted; see official source

6. EVALUATION OF RAW DATA

Flask nr		% degradation afer n days	% degradation afer n days	% degradation afer n days	% degradation afer n days	% degradation afer n days
Flask nr		0	n1	n2	n3	nx
1	Image omitted; see official source	0
2	Image omitted; see official source	0
Mean [^E0064]	Image omitted; see official source	0

D1 and D2 should not be averaged if there is a considerable difference.

Note: similar formats may be used for the reference chemical and toxicity controls.

7. ABIOTIC CONTROL (optional)

	Time (days)	Time (days)
	0	t
DOC conc. (mg/l) in sterile control	Cs(o)	Cs(t)

Image omitted; see official source

8. SPECIFIC CHEMICAL ANALYSIS (optional)

	residual amount of test chemical at end of test (mg/l)	% primary degradation
Sterile control	Sb
Inoculated test medium	Sa	Image omitted; see official source

PART III.   MODIFIED OECD SCREENING TEST (Method C.4-B)

III.1.   PRINCIPLE OF THE METHOD

A measured volume of mineral medium containing a known concentration of the test substance (10-40 mg DOC/litre) as the nominal sole source of organic carbon is inoculated with 0,5 ml effluent per litre of medium. The mixture is aerated in the dark or diffused light at 22 ± 2 ^oC.

Degradation is followed by DOC analysis at frequent intervals over a 28-day period. The degree of biodegradation is calculated by expressing the concentration of DOC removed (corrected for that in the blank inoculum control) as a percentage of the concentration initially present. The degree of primary biodegradation may also be calculated from supplemental chemical analysis made at the beginning and end of incubation.

III.2.   DESCRIPTION OF THE METHOD

III.2.1.   Apparatus

• (a) Conical flasks, e.g. 250 ml to 2 litres, depending on the volume needed for DOC analysis;

• (b) shaking machine — to accommodate the conical flasks, either with automatic temperature control or used in a constant temperature room, and of sufficient power to maintain aerobic conditions in all flasks;

• (c) filtration apparatus, with suitable membranes;

• (d) DOC analyser;

• (e) apparatus for determining dissolved oxygen;

• (f) centrifuge.

III.2.2.   Preparation of mineral medium

For the preparation of the stock solutions, see I.6.2.

Mix 10 ml of solution (a) with 80 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 1 litre with dilution water.

This method uses only 0,5 ml effluent/litre as inoculum and therefore the medium may need to be fortified with trace elements and growth factors. This is done by adding 1 ml each of the following solutions per litre of final medium:

Trace element solution:

Manganese sulfate tetrahydrate, MnSO4. 4H2O	39,9 mg
Boric acid, H3BO3	57,2 mg
Zinc sulfate heptahydrate, ZnSO4. 7H2O	42,8 mg
Ammonium heptamolybdate (NH4)6 Mo7O24	34,7 mg
Fe-chelate (FeCl3 ethylenediamine-tetra-acetic acid)	100,0 mg
Dissolve in, and make up to 1 000 ml with dilution water
Vitamin solution:
Yeast extract	15,0 mg

Dissolve the yeast extract in 100 ml water. Sterilise by passage through a 0,2 micron membrane, or make up freshly.

III.2.3.   Preparation and pre-conditioning of inoculum

The inoculum is derived from the secondary effluent of a treatment plant or laboratory scale unit receiving predominantly domestic sewage. See I.6.4.2. and I.6.5.

0,5 ml per litre of mineral medium is used.

III.2.4.   Preparation of flasks

As an example, introduce 800 ml portions of mineral medium into 2-litre conical flasks and add sufficient volumes of stock solutions of the test and reference substances to separate flasks to give a concentration of chemical equivalent to 10-40 mg DOC/litre. Check the pH value and adjust, if necessary, to 7,4 . Inoculate the flasks with sewage effluent at 0,5 ml/litre (see I.6.4.2). Also prepare inoculum controls in the mineral medium but without test or reference chemical.

If needed, use one vessel to check the possible inhibitory effect of the test chemical by inoculating a solution containing, in the mineral medium, comparable concentrations of both the test and a reference chemical.

Also, if required, set up a further, sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6.6).

Additionally, if the test chemical is suspected of being significantly adsorbed on to glass, sludge, etc., make a preliminary assessment to determine the likely extent of adsorption and thus the suitability of the test for the chemical (see Table 1). Set up a flask containing the test substance, inoculum and sterilising agent.

Make up the volumes in all flasks to 1 litre with mineral medium and, after mixing, take a sample from each flask to determine the initial concentration of DOC (see Appendix 2.4). Cover the openings of the flasks, e.g. with aluminium foil, in such a way as to allow free exchange of air between the flask and the surrounding atmosphere. Then insert the vessels into the shaking machine for starting the test.

III.2.5.   Number of flasks in typical run

Flasks 1 and 2: test suspension

Flasks 3 and 4: inoculum blank

Flask 5: procedure control

and preferably and when necessary:

Flask 6: abiotic sterile control

Flask 7: adsorption control

Flask 8: toxicity control

See also 1.6.7.

III.2.6.   Performance of the test

Throughout the test, determine the concentrations of DOC in each flask in duplicate at known time intervals, sufficiently frequently to be able to determine the start of the 10-day window and the percentage removal at the end of the 10-day window. Take only the minimal volume of test suspension necessary for each determination.

Before sampling make good evaporation losses from the flasks by adding dilution water (I.6.1) in the required amount if necessary. Mix the culture medium thoroughly before withdrawing a sample and ensure that material adhering to the walls of the vessels is dissolved or suspended before sampling. Membrane-filter or centrifuge (see Appendix 2.4) immediately after the sample has been taken. Analyse the filtered or centrifuged samples on the same day, otherwise store at 2-4 ^oC for a maximum of 48 h, or below 18 ^oC for a longer period.

III.3.   DATA AND REPORTING

III.3.1.   Treatment of results

Calculate the percentage degradation at time t as given under I.7.1 (DOC determination) and, optionally, under I.7.2 (specific analysis).

Record all results on the data sheets provided.

III.3.2.   Validity of results

See I.5.2.

III.3.3.   Reporting

See I.8.

III.4.   DATA SHEET

An example of a data sheet is given hereafter

MODIFIED OECD SCREENING TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: … mg/litre as chemical

Initial concentration in medium, to: … mg/litre as chemical

4. INOCULUM

Source:

Treatment given:

Pre-conditioning, if any:

Concentration of suspended solids in reaction mixture: mg/1

5. CARBON DETERMINATIONS

Carbon analyser:

	Flask nr		DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)	DOC after n-days (mg/l)
	Flask nr		0	n1	n2	n3	nx
Test chemical plus inoculum	1	a1
Test chemical plus inoculum	1	a2
Test chemical plus inoculum	1	a, mean Ca(t)
Test chemical plus inoculum	2	b1
Test chemical plus inoculum	2	b2
Test chemical plus inoculum	2	b, men Cb(t)
Blank inoculum without test chemical	3	C1
Blank inoculum without test chemical	3	C2
Blank inoculum without test chemical	3	C, mean Cc(t)
Blank inoculum without test chemical	4	d1
Blank inoculum without test chemical	4	d2
Blank inoculum without test chemical	4	d, mean Cd(t)
Blank inoculum without test chemical	Image omitted; see official source	Image omitted; see official source

6. EVALUATION OF RAW DATA

Flask nr		% degradation afer n days	% degradation afer n days	% degradation afer n days	% degradation afer n days	% degradation afer n days
Flask nr		0	n1	n2	n3	nx
1	Image omitted; see official source	0
2	Image omitted; see official source	0
Mean [^E0065]	Image omitted; see official source	0

D1 and D2 should not be averaged if there is a considerable difference.

Note: similar formats may be used for the reference chemical and toxicity controls.

7. ABIOTIC CONTROL (optional)

	Time (days)	Time (days)
	0	t
DOC conc. (mg/l) in sterile control	Cs(o)	Cs(t)

Image omitted; see official source

8. SPECIFIC CHEMICAL ANALYSIS (optional)

	residual amount of test chemical at end of test (mg/l)	% primary degradation
Sterile control	Sb
Inoculated test medium	Sa	Image omitted; see official source

PART IV.   CO2 EVOLUTION TEST (Method C.4-C)

IV.1.   PRINCIPLE OF THE METHOD

A measured volume of inoculated mineral medium containing a known concentration of the test chemical (10-20 mg DOC or TOC/l) as the nominal sole source of organic carbon is aerated by the passage of carbon, dioxide-free air at a controlled rate in the dark or in diffuse light. Degradation is followed over 28 days by determining the carbon dioxide produced, which is trapped in barium or sodium hydroxide and which is measured by titration of the residual hydroxide or as inorganic carbon. The amount of carbon dioxide produced from the test chemical (corrected for that derived from the blank inoculum) is expressed as a percentage of ThCO2. The degree of biodegradation may also be calculated from supplemental DOC analysis made at the beginning and end of incubation.

IV.2.   DESCRIPTION OF THE METHOD

IV.2.1.   Apparatus

• (a) Flasks, 2-5 litres, each fitted with an aeration tube reaching nearly the bottom of the vessel and an outlet;

• (b) magnetic stirrers, when assessing poorly soluble chemicals;

• (c) gas-absorption bottles;

• (d) device for controlling and measuring airflow;

• (e) apparatus for carbon dioxide scrubbing, for preparation of air which is free from carbon dioxide; alternatively, a mixture of CO2-free oxygen and CO2-free nitrogen, from gas cylinders, in the correct proportions (20 % O2: 80 % N2) may be used;

• (f) device for determination of carbon dioxide, either titrimetrically or by some form of inorganic carbon analyser;

• (g) membrane filtration device (optional);

• (h) DOC analyser (optional).

IV.2.2.   Preparation of mineral medium

For the preparation of the stock solutions, see I.6.2.

Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 11 with dilution water.

IV.2.3.   Preparation and pre-conditioning of inoculum

The inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters; soils or from a mixture of these.

See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.

IV.2.4.   Preparation of flasks

As an example the following volumes and weights indicate the values for 5-litre flasks containing 3 l of suspension. If smaller volumes are used modify the values accordingly, but ensure that the carbon dioxide formed can be measured accurately.

To each 5-litre flask add 2400 ml mineral medium. Add an appropriate volume of the prepared activated sludge (see I.6.4.1 and I.6.5) to give a concentration of suspended solids of not more than 30 mg/l in the final 3 l of inoculated mixture. Alternatively first dilute the prepared sludge to give a suspension of 500-1000 mg/l in the mineral medium before adding an aliquot to the contents of the 5 litre flask to attain a concentration of 30 mg/l; this ensures greater precision. Other sources of inoculum may be used (see I.6.4.2.).

Aerate these inoculated mixtures with CO2-free air overnight to purge the system of carbon dioxide.

Add the test material and reference substance, separately, as known volume of stock solutions, to replicate flasks to yield concentrations, contributed by the added chemicals, of 10 to 20 mg DOC or TOC/l; leave some flasks without addition of chemicals as inoculum controls. Add poorly soluble test substances directly to the flasks on a weight or volume basis or handle as described in Appendix 3.

If required, use one flask to check the possible inhibitory effect of the test chemical by adding both the test and reference chemicals at the same concentrations as present in the other flasks.

Also, if required, use a sterile flask to check whether the test chemical is degraded abiotically by using an uninoculated solution of the chemical (see I.6.6). Sterilise by the addition of a toxic substance at an appropriate concentration.

Make up the volumes of suspensions in all flasks to 3 l by the addition of mineral medium previously aerated with CO2-free air. Optionally, samples may be withdrawn for analysis of DOC (see Appendix 2.4.) and/or specific analysis. Connect the absorption bottles to the air outlets of the flasks.

If barium hydroxide is used, connect three absorption bottles, each containing 100 ml of 0,0125 M barium hydroxide solution, in series to each 5-litre flask. The solution must be free of precipitated sulphate and carbonate and its strength must be determined immediately before use. If sodium hydroxide is used, connect two traps, the second acting as a control to demonstrate that all the carbon dioxide was absorbed in the first. Absorption bottles fitted with serum bottle closures are suitable. Add 200 ml 0,05 M sodium hydroxide to each bottle, which is sufficient to absorb the total quantity of carbon dioxide evolved when the test chemical is completely degraded. The sodium hydroxide solution, even when freshly prepared, will contain traces of carbonates; this is corrected by deduction of the carbonate in the blank.

IV.2.5.   Number of flasks in a typical run

Flasks 1 and 2: Test suspension

Flasks 3 and 4: Inoculum blank

Flask 5: Procedure control

and, preferably and when necessary:

Flask 6: Abiotic sterile control

Flask 7: Toxicity control

See also I.6.7.

IV.2.6.   Performance of the test

Start the test by bubbling CO2-free air through the suspensions at a rate of 30-100 ml/min. Take samples of the carbon dioxide absorbent periodically for analysis of the CO2-content. During the first ten days it is recommended that analyses should be made every second or third day and then every fifth day until the 28th day so that the 10-day window period can be identified.

On the 28th day, withdraw samples (optionally) for DOC and/or specific analysis, measure the pH of the suspensions and add 1 ml of concentrated hydrochloric acid to each flask; aerate the flasks overnight to drive off the carbon dioxide present in the test suspensions. On day 29 make the last analysis of evolved carbon dioxide.

On the days of measurement of CO2, disconnect the barium hydroxide absorber closest to the flask and titrate the hydroxide solution with HCl 0,05 M using phenolphthalein as the indicator. Move the remaining absorbers one place closer to the flask and place a new absorber containing 100 ml fresh 0,0125 M barium hydroxide at the far end of the series. Make titrations as needed, for example, when substantial precipitation is seen in the first trap and before any is evident in the second, or at least weekly. Alternatively, with NaOH as absorbent, withdraw with a syringe a small sample (depending on the characteristics of the carbon analyser used) of the sodium hydroxide solution in the absorber nearer to the flask. Inject the sample into the IC part of the carbon analyser for analysis of evolved carbon dioxide directly.

Analyse the contents of the second trap only at the end of the test to correct for any carryover of carbon dioxide.

IV.3.   DATA AND REPORTING

IV.3.1.   Treatment of results

The amount of CO2 trapped in an absorber when titrated is given by:

mgCO2 = (100 × CB - 0,5 × V × CA) × 44

where:

V	=	volume of HCl used for titration of the 100 ml in the absorber (ml)
---	---	---
CB	=	concentration of the barium hydroxide solution (M)
---	---	---
CA	=	concentration of the hydrochloric acid solution (M)

if CB is 0,0125 M and CA is 0,05 M, the titration for 100 ml barium hydroxide is 50 ml and the weight of CO2 is given by:

Image omitted; see official source

Thus, in this case, to convert volume of HCl titrated to mg CO2 produced the factor is 1,1 .

Calculate the weights of CO2 produced from the inoculum alone and from the inoculum plus test chemical using the respective titration values and the difference is the weight of CO2 produced from the test chemical alone.

For example, if the inoculum alone gives a titration of 48 ml and inoculum plus test chemical gives 45 ml,

CO2 from inoculum = 1,1 × (50-48) = 2,2 mg

CO2 from inoculum plus test chemical = 1,1 × (50-45) = 5,5 mg

and thus the weight of CO2 produced from the test chemical is 3,3 mg.

The percentage biodegradation is calculated from:

Image omitted; see official source

or,

Image omitted; see official source

3,67 being the conversion factor (44/12) for carbon to carbon dioxide.

Obtain the percentage degradation after any time interval by adding the percentage of ThCO2 values calculated for each of the days, up to that time, on which it was measured.

For sodium hydroxide absorbers, calculate the amount of carbon dioxide prodllced, expressed as IC (mg), by multiplying the concentration of IC in the absorbent by the volume of the absorbent.

Calculate the percentage degradation from:

Image omitted; see official source

Calculate DOC removals (optional) as described under I.7. Record these and all other results on the data sheets provided.

IV.3.2.   Validity of results

The IC content of the test chemical suspension in the mineral medium at the beginning of the test must be less than 5 % of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/1 medium. If values greater than 70 mg CO2/litre are obtained, the data and experimental technique should be examined critically.

See also I.5.2.

IV.3.3.   Reporting

See I.8.

IV.4.   DATA SHEET

An example of a data sheet is given hereafter.

CARBON DIOXIDE EVOLUTION TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: … mg/litre as chemical

Initial conc. in medium: … mg/litre as chemical

Total C added to flask: … mg C

ThCO2: mg CO2

4. INOCULUM

Source:

Treatment given:

Pre-conditioning if any:

Concentration of suspended solids in reaction mixture: mg/litre

Time (day)	CO2 formed Test (mg)	CO2 formed Test (mg)	CO2 formed blank (mg)	CO2 formed blank (mg)	CO2 formed cumulative (mg) (test minus blank mean)	CO2 formed cumulative (mg) (test minus blank mean)	ThCO2cumulative Image omitted; see official source	ThCO2cumulative Image omitted; see official source	ThCO2cumulative Image omitted; see official source
Time (day)	1 2	mean	3 4	mean	1	2	1	2	mean
0
n1
n2
n3
28

Note: similar formats may be used for the reference chemical and toxicity controls.

6. CARBON ANALYSIS (optional)

Carbon analyser:

Time (day)	Blank mg/l	Test chemical mg/l
0	Cb(o)	Co
28 [^E0066]	Cb(t)	Ct

Or at end of incubation

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7. ABIOTIC DEGRADATION (optional)

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PART V.   MANOMETRIC RESPIROMETRY TEST (Method C.4-D)

V.1.   PRINCIPLE OF THE METHOD

A measured volume of inoculated mineral medium, containing a known concentration of test chemical (100 mg/litre of the test substance, to give at least 50-100 mg ThOD/litre) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1^o C or closer) for up to 28 days. The consumption of oxygen is determined either by measuring the quantity of oxygen (produced electrolytically) required to maintain constant gas volume in the respirometer flask, or from the change in volume or pressure (or a combination of the two) in the apparatus. Evolved carbon dioxide is absorbed in a solution of potassium hydroxide or another suitable absorbent. The amount of oxygen taken up by the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD or COD. Optionally, primary biodegradation may also be calculated from supplemental specific analysis made at the beginning and end of incubation, and ultimate biodegradation by DOC analysis.

V.2.   DESCRIPTION OF THE METHOD

V.2.1.   Apparatus

• (a) suitable respirometer;

• (b) temperature control, maintaining ± 1 ^oC or better;

• (c) membrane-filtration assembly (optional);

• (d) carbon analyser (optional).

V.2.2.   Preparation of mineral medium

For the preparation of the stock solutions, see I.6.2.

Mix 10 ml of solution (a) with 800 ml dilution water, add 1 ml of solutions (b) to (d) and make up to 1 litre with dilution water.

V.2.3.   Preparation and pre-conditioning of inoculum

The inoculum may be derived from a variety of sources: activated sludge; sewage effluents; surface waters and soils or from a mixture of these.

See I.6.4., I.6.4.1., I.6.4.2. and I.6.5.

V.2.4.   Preparation of flasks

Prepare solutions of the test and reference chemicals, in separate batches, in mineral medium equivalent to a concentration, normally, of 100 mg chemical/litre (giving at least 50-100 mg ThOD/litre), using stock solutions.

Calculate the ThOD on the basis of formation of ammonium salts unless nitrification is anticipated, when the calculation should be based on nitrate formation (see Appendix 2.2)

Determine the pH values and if necessary adjust to 7,4 ± 0,2 .

Poorly soluble substances should be added at a later stage (see below).

If the toxicity of the test chemical is to be determined, prepare a further solution in mineral medium containing both test and reference chemicals at the same concentrations as in the individual solutions.