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)

• 9.In the EC ring-test where seawater samples were taken at different locations and at different times of the year, the lag phase (tL) and the time to achieve 50 per cent degradation (t50), not including the lag phase, were 0 to 2 days and 1 to 4 days respectively for sodium benzoate. For aniline the tL and t50 values were 0 to 7 and 2 to 12 days respectively.

REPRODUCIBILITY

• 10.The reproducibility of the methods was established in the EC ring test (3).

DESCRIPTION OF THE METHOD

Apparatus

• 11.Normal laboratory equipment and:

• (a) 250-300 ml BOD bottles with glass stoppers or narrow neck 250 ml bottle with glass stoppers may be used;

• (b) Several 2-, 3- and 4- litre bottles with litre marks for the preparation of the experiment and for the filling of the BOD bottles;

• (c) Waterbath or constant temperature room for keeping the bottles at constant temperature (± 1 °C) with the exclusion of light.

• (d) Equipment for analysis of dissolved oxygen;

• (e) Membrane filters, 0,2-0,45 μm (optional);

• (f) Equipment for specific analysis (optional).

Seawater

• 12.Collect a seawater sample in a thoroughly cleansed container and transport to the laboratory, preferably within one or two days of collection. During transport do not allow the temperature of the sample to exceed significantly the temperature to be used in the test.

• 13.Identify the sampling location precisely and describe it in terms of its pollutional and nutritional status. Especially for coastal or polluted waters, include in this characterisation a heterotrophic microbial colony count and the determination of concentrations of dissolved nitrate, ammonium and phosphate.

• 14.Provide the following information for the seawater sample itself:

• — date of collection;

• — depth of collection;

• — appearance of sample — turbid etc.;

• — temperature at the time of collection;

• — salinity;

• — dissolved organic carbon (DOC);

• — delay between collection and use in the test.

• 15.If the DOC content of the sample is found to be high or if it is thought that the blank BOD after 28 days would be more than 30 per cent of that of the reference substances, it is recommended that the seawater be aged for about a week prior to use.

• 16.Age the sample by storing it under aerobic conditions at the test temperature and in the dark or in diffuse light. If necessary, maintain aerobic conditions by gentle aeration. During ageing, the content of easily degradable organic material is reduced. In the ring-test (3), no difference was revealed between the degradation potential of aged and freshly collected seawater samples.

• 17.Prior to use, pretreat the seawater to remove coarse particles e.g. by filtration through a nylon filter or a coarse paper filter (not membrane or GF-C filters), or by sedimentation and decanting. Report the procedure used. Pretreat after ageing, if used.

Stock solutions for mineral nutrients

• 18.Prepare the following stock solutions using analytical grade reagents:

(a)	Potassium dihydrogen orthophosphate, KH2PO4	8,50 g
(a)	Dipotassium hydrogen orthophosphate, K2HPO4	21,75 g
(a)	Disodium hydrogen orthophosphate dihydrate, Na2HPO4·2H2O	33,30 g
(a)	Ammonium chloride, NH4Cl	0,50 g
(a)	Dissolve and make up to 1 litre with distilled water.
(b)	Calcium chloride, CaCl2	27,50 g
(b)	Dissolve and make up to 1 litre with distilled water.
(c)	Magnesium sulphate heptahydrate, MgSO4·7H2O	22,50 g
(c)	Dissolve and make up to 1 litre with distilled water.
(d)	Iron (III) chloride hexahydrate, FeCl3·6H2O	0,25 g
(d)	Dissolve and make up to 1 litre with distilled water.

• Precipitation in solution (d) may be prevented by adding one drop of concentrated HCl or 0,4 g ethylenediaminetetra-acetic acid (EDTA, disodium salt) per litre. If a precipitate forms in a stock solution, replace it with freshly made solution.

Preparation of test medium

• 19.Add per litre of pre-treated seawater 1 ml of each of the above stock solutions. Saturate the test medium with air at the test temperature by aerating with clean compressed air for about 20 minutes. Determine the concentration of dissolved oxygen for control purposes. The saturated concentration of dissolved oxygen as a function of salinity and temperature may be read from the nomogram enclosed with this test method (Appendix 4).

Inoculum

• 20.Do not add a specific inoculum in addition to the micro-organisms already present in the seawater. Determine (optionally) the number of colony-forming heterotrophs in the seawater test medium (and preferably also in the original seawater sample), e.g. by plate count using a marine agar. This is particularly desirable for samples from coastal or polluted sites. Check the heterotrophic microbial activity in the seawater by performing a test with a reference substance.

Preparation of test bottles

• 21.Perform all necessary manipulations including ageing and pre-treatment of the seawater at the chosen test temperature between 15 to 20 °C, ensuring cleanliness, but not sterility of all glassware.

• 22.Prepare groups of BOD bottles for the determination of the BOD of the test and reference substances in simultaneous experimental series. Perform all analyses on duplicate bottles (blanks, reference and test substances), i.e. prepare two bottles for each determination. Perform analyses at least on days 0, 5, 15 and 28 (four determinations). For oxygen analyses, four determinations require a total of 3 × 2 × 4 = 24 bottles (blank, reference and test substance), and thus about 8 litres of test medium (for one concentration of test substance).

• 23.Prepare separate solutions of test and reference substances in large bottles of sufficient volume (paragraph 11) by first adding test and reference substances either directly or by using a concentrated stock solution to the partly filled large bottles. Add further test medium to give the final desired concentrations. If stock solutions of test and/or reference substances are used, ensure that the salinity of the seawater medium is not significantly altered.

• 24.Select concentrations of test and reference substances by taking into account:

• (a) the solubility of dissolved oxygen in seawater at the prevailing test temperature and salinity (see the enclosed nomogram — Appendix 4);

• (b) the blank BOD of the seawater; and

• (c) the expected biodegradability of the test substance.

• 25.At 15 °C and 20 °C and 32 parts per thousand salinity (ocean water), the solubility of dissolved oxygen is about 8,1 and 7,4 mg/l respectively. The oxygen consumption of the seawater itself (blank respiration) may be 2 mg O2/l or more, if the seawater is not aged. Therefore in order to ensure a significant oxygen concentration remaining after oxidation of the test substance, use a starting concentration of test substance of about 2-3 mg/l (depending on the ThOD) for the substances that are expected to become completely degraded under the conditions of the test (such as reference substances). Test less degradable substances at higher concentrations, up to about 10 mg/l, provided that toxic effects do not occur. It can be advantageous to run parallel tests with a low (about 2 mg/l) and a high (about 10 mg/l) concentration of test substance.

• 26.An oxygen blank must be determined in parallel in bottles containing neither test or reference substance.

• 27.If inhibitory effects are to be determined, prepare the following series of solutions in separate large bottles (paragraph 13):

• (a) 2 mg per litre of an easily-degradable substance, e.g. any of the reference substances mentioned;

• (b) x mg per litre of test substance (x is usually 2);

• (c) 2 mg per litre of the easily-degradable substance plus x mg per litre of test substance.

Physical-chemical control test (optional)

• 28.If the option of using specific analyses is used, a physical-chemical experiment may be performed in order to check whether the test substance is removed by abiotic mechanisms, such as hydrolysis or adsorption. A physical-chemical control test may be performed by adding mercury (II) chloride (HgCl2) ([^E0211]) (50-100 mg/l) to duplicate flasks with test substance in order to stop microbial activity. A significant decrease in specific substance concentration in the course of the test indicates abiotic removal mechanisms.

Number of BOD bottles in a typical run

• 29.In a typical run the following bottles are used:

• — at least 8 containing test substance;

• — at least 8 containing nutrient-fortified seawater only;

• — at least 8 containing reference substance, and when necessary

• — 6 bottles containing test and reference substances (toxicity control).

PROCEDURE

• 30.After preparation, immediately siphon each solution, from the lower quarter (not from the bottom) of the appropriate large bottle, to fill the respective group of BOD bottles. Immediately analyse the zero controls (time zero) for dissolved oxygen (paragraph 33) or preserve them for later chemical analysis by precipitation with MnCl2 (manganese (II) chloride) and NaOH (sodium hydroxide).

• 31.Incubate the remaining parallel BOD bottles at the test temperature (15-20 °C), keep in the dark, and remove from the incubation area at appropriate time intervals, (e.g. after 5, 15 and 28 days as a minimum) and analyse for dissolved oxygen (paragraph 33).

• 32.Membrane filter (0,2-0,45 μm) or centrifuge, for 15 minutes, samples for specific analyses (optional). Store for up to 48 hours at 2-4 °C, or for longer periods at – 18 °C, if not analysed immediately (if it is known that the test substance will remain unaffected, acidify to pH 2 before storing).

Dissolved oxygen determination

• 33.Determine the concentration of dissolved oxygen using a chemical or electrochemical method which is recognised nationally or internationally.

DATA AND REPORTING

Treatment of Results

• 34.Record analytical results on the attached data sheets (Appendix 5).

• 35.Calculate the BOD as the difference of the oxygen depletion between a blank and a solution of test substance under the conditions of the test. Divide the net oxygen depletion by the concentration (w/v) of the substance in order to express the BOD as mg BOD/mg test substance. The degradation is defined as the ratio of the biochemical oxygen demand to either, preferably, the theoretical oxygen demand (ThOD) or the chemical oxygen demand (COD) and expressed as a percentage (see paragraph 36).

• 36.Calculate the biodegradation values for each sampling time for both test and reference substances using one or other of the equations:

Image omitted; see official source

Image omitted; see official source

• where:

ThOD	=	theoretical oxygen demand (calculation, Appendix 3)
---	---	---
COD	=	chemical oxygen demand, determined experimentally.

Note: Sometimes the two ways of calculation (percentage of the ThOD or percentage of the COD) do not give the same results; it is preferable to use ThOD, since some substances are not fully oxidised in the COD test.

• 37.Illustrate the course of the degradation test graphically in a diagram (see example in section on ‘Validity and interpretation of results’. If there are sufficient data, calculate the lag phase (tL) and the time (t50) to reach 50 per cent removal from the end of the lag phase from the biodegradation curve.

• 38.If specific analysis is used (optional), state the percentage of primary degradation as the percentage of specific substance removal within the test period (corrected for analytical blanks).

Test Report

• 39.The test report must contain the following information:

Test substance:

• — physical nature and, where relevant, physicochemical properties;

• — identification data.

Test conditions:

• — location and description of the sampling site: pollutional and nutrient status (colony count, nitrate, ammonium, phosphate if appropriate);

• — characteristics of the sample (date of sampling, depth, appearance, temperature, salinity, DOC (optional), delay between collection and use in the test);

• — method used (if any) for ageing of the seawater;

• — method used for pre-treatment (filtration/sedimentation) of the seawater;

• — method used for the COD determination (if performed);

• — method used for the oxygen measurements;

• — dispersion procedure for substances which are poorly soluble under the test conditions;

• — method used for determining the number of heterotrophs in the seawater (plate count method or alternative procedure);

• — method used for determining DOC in seawater (optional);

• — method used for specific analysis (optional);

• — other optional methods used to characterise the seawater (ATP measurements, etc.).

Results:

• — analytical data reported on a data sheet (as attached, Appendix 5);

• — the course of the degradation test represented graphically in a diagram showing the lag phase, (tL), slope and time (starting from the end of the lag phase) to reach 50 per cent of the final oxygen uptake caused by oxidation of the test substance (t50). The lag phase may be estimated graphically as shown in the attached figure, or conveniently taken as the time needed for 10 per cent degradation;

• — per cent degradation measured after 28 days.

Discussion of results.

Validity and interpretation of results

• 40.The blank respiration should not exceed 30 per cent of the oxygen in the test bottle. If it is not possible to meet this criterion using freshly collected seawater, the seawater must be aged (stabilized) before use.

• 41.The possibility that nitrogen-containing substances may affect the results should be considered.

• 42.Results obtained with the reference substances sodium benzoate and aniline should be comparable to the results obtained in the ring-test (3) (paragraph 9). If results obtained with reference substances are atypical, the test should be repeated using another seawater sample.

• 43.The test substance can be considered to be inhibitory to bacteria (at the concentration used) if the BOD of the mixture of reference and test substances is less than the sum of the BOD of the separate solutions of the two substances.

• 44.Owing to the relatively high test concentrations as compared with most natural systems, and consequently an unfavourable ratio between the concentrations of test substance and other carbon sources, the method is to be regarded as a preliminary test which can be used to indicate whether or not a substance is easily biodegradable. Accordingly, a low result does not necessarily mean that the test substance is not biodegradable in marine environments, but indicates that more work will be necessary in order for this to be established.

• An example of a theoretical degradation experiment illustrating a feasible way of estimating the values of tL (length of ‘lag phase’) and t50, time interval (starting at tL), needed to reach 50 % of the final oxygen uptake caused by oxidation of the test substance, is given below:

Image omitted; see official source

Text of image

Exclude control without solvent

Are both controls equal? U-Test

Are both controls equal? t-Test

Bonferroni — U-Test

Jonckheere-Terpstra Test

Shirley Test

Dunn's Test

Both controls might be pooled

Additional solvent control?

Additional solvent control?

Statistical testing not recommended

At least four replicates per treatment?

No success

Transform data

Data:

Variation homogenous?

Distribution normal?

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

No

Dunnett's Test

William Test

Exclude control without solvent

Both controls might be pooled

Start

Non-parametric Tests

Parametric Tests

LITERATURE

• (1)de Kreuk J.F. and Hanstveit A.O. (1981). Determination of the biodegradability of the organic fraction of chemical wastes. Chemosphere, 10 (6); 561-573.

• (2)Chapter C.4-B of this Annex: Determination of ‘Ready’ Biodegradability Part III Modified OECD Screening Test

• (3)Nyholm N. and Kristensen P. (1987). Screening Test Methods for Assessment of Biodegradability of Chemical Substances in Seawater. Final Report of the ring test programme 1984-1985, March 1987, Commission of the European Communities.

• (4)Chapter C.11 of this Annex: Biodegradation — Activated Sludge, Respiration Inhibition Test.

• (5)Chapter C.4-E of this Annex: Determination of ‘Ready’ Biodegradability, Part VI. Closed Bottle Test.

Appendix 1

Determination of organic carbon in seawater

SHAKE FLASK METHOD

For the determination of organic carbon of a water sample, the organic compounds in the sample are oxidized to carbon dioxide using generally one of the following three techniques:

• — wet-oxidation by persulphate/UV-irradiation;

• — wet-oxidation by persulfate/elevated temperature (116-130 °C);

• — combustion.

Evolved CO2 is quantified employing infra-red spectrometry or titrimetry. Alternatively, CO2 is reduced to methane, which is quantified on a flame ionization detector (FID).

The persulfate/UV-method is commonly used for the analysis of ‘clean’ water with low content of particulate matter. The latter two methods can be applied to most kinds of water samples, the persulfate/elevated temperature-oxidation being most suitable for low-level samples, and the combustion technique being applicable for samples with non-volatile organic carbon (NVOC) content well above 1 mg C/l.

Interferences

All three methods are dependent on eliminating or compensating for inorganic carbon (IC) present in the sample. Purging of CO2 from the acidified sample is the most frequently used method to eliminate the IC, although this also results in a loss of volatile organic compounds (1). The complete elimination or compensation of IC must be ensured for each sample matrix, and volatile organic carbon (VOC) must be determined in addition to NVOC dependent on the sample type.

High chloride concentrations result in decreased oxidation efficiency using the persulfate/UV-method (2). Application of an oxidation reagent modified by the addition of mercury (II) nitrate may, however, remove this interference. It is recommended that the maximum tolerable sample volume be used to evaluate each type of chloride-containing sample. High salt concentrations in sample analysed using the combustion method can cause salt coating of the catalyst and excessive corrosion of the combustion tube. Precautions should be taken according to the manufacturer's manual.

Highly turbid samples as well as samples containing particulate matter may be incompletely oxidized when employing the persulfate/UV-method.

An example of a suitable method

Non-volatile organic carbon is determined by oxidation with persulfate/UV-irradiation and subsequent quantification of evolved CO2 employing non-dispersive infra-red spectrometry.

The oxidation reagent is modified in accordance with the suggestions given in (2) as described in the manufacturer's manual:

• a) 8,2 g HgCl2 and 9,6 g Hg(NO3)2·H2O are dissolved in several hundred millilitres of low carbon concentration reagent water.

• b) 20 g K2S2O8 are dissolved in the mercuric salt solution.

• c) 5 ml HNO3 (conc.) are added to the mixture.

• d) the reagent is diluted to 1 000 ml.

The interference from chloride is removed using a 40 μl sample volume for 10 per cent chloride and 200 μl sample volume for 1,9 per cent chloride. Samples of high chloride concentrations and/or larger sample volumes can be analysed according to this method provided that build-up of chloride in the oxidation vessel is prevented. Determination of volatile organic carbon can subsequently be performed, if relevant, for the sample type in question.

LITERATURE

• (1)ISO, Water quality — determination of total organic carbon. Draft International Standard ISO/DIS 8245, January 16, 1986.

• (2)American Public Health Association, Standard Methods for the Estimation of Water and Wastewater. American Water Works Association & Water Pollution Control Federation, 16th edition, 1985.

• Also of interest (gives a description of an autoanalysis system):

• (3)Schreurs W. (1978). An automated colorimetric method for the determination of dissolved organic carbon in seawater by UV destruction. Hydrobiological Bulletin 12, 137-142.

Appendix 2

Biodegradation in seawater

SHAKE FLASK METHOD

DATA SHEET

• 1. LABORATORY:

• 1. DATE AT START OF TEST:

• 1. TEST SUBSTANCE:

• Name:

Stock solution concentration:	mg/l as substance
Initial concentration in medium, to:	mg/l as substance
:	mg DOC/l

• 1. SEAWATER:

• Source:

• Date of collection:

• Depth of collection:

• Appearance at time of collection (e.g. turbid, etc.):

Salinity at collection:	‰
Temperature at collection:	°C
DOC ‘x’ hours after collection:	mg/l

• Pretreatment prior to testing (e.g. filtration, sedimentation, ageing, etc.):

Microbial colony count	—  original sample:	colonies/ml
	—  at start of test:	colonies/ml
Other characteristics:

• 1. CARBON DETERMINATIONS:

• Carbon analyser:

	Flask no.		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 no.		0	n1	n2	n3	nx
Test: nutrient-fortified seawater with test substance	1	a1
Test: nutrient-fortified seawater with test substance	1	a2
Test: nutrient-fortified seawater with test substance	1	mean, Ca(t)
Test: nutrient-fortified seawater with test substance	2	b1
Test: nutrient-fortified seawater with test substance	2	b2
Test: nutrient-fortified seawater with test substance	2	mean, Cb(t)
Blank: nutrient-fortified seawater without test substance	1	c1
Blank: nutrient-fortified seawater without test substance	1	c2
Blank: nutrient-fortified seawater without test substance	1	mean, Cc(t)
Blank: nutrient-fortified seawater without test substance	2	d1
Blank: nutrient-fortified seawater without test substance	2	d2
Blank: nutrient-fortified seawater without test substance	2	mean, Cd(t)
Blank: nutrient-fortified seawater without test substance	mean,Image omitted; see official source	mean,Image omitted; see official source
Blank: nutrient-fortified seawater without test substance	mean,Image omitted; see official source	mean,Image omitted; see official source

• 1. EVALUATION OF RAW DATA:

Flask No.	Calculation of results	% Degradation after n days	% Degradation after n days	% Degradation after n days	% Degradation after n days	% Degradation after n days
Flask No.	Calculation of results	0	n1	n2	n3	nx
1	Image omitted; see official source	0
2	Image omitted; see official source	0
Mean [^E0212]	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 when degradation is followed by specific analysis and for the reference substance and toxicity controls.

• 1. ABIOTIC DEGRADATION (optional)

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

Image omitted; see official source

Appendix 3

Calculation of the theoretical biochemical oxygen demand

CLOSED BOTTLE METHOD

The ThOD of the substance CcHhClclNnNanaOoPpSs of the molecular weight MW is calculated according to:

Image omitted; see official source

This calculation implies that C is mineralised to CO2, H to H2O, P to P2O5 and Na to Na2O. Halogen is eliminated as hydrogen halide and nitrogen as ammonia.

Example:

Glucose C6H12O6, MW = 180

Image omitted; see official source

Molecular weights of salts other than those of the alkali metals are calculated on the assumption that the salts have been hydrolysed.

Sulphur is assumed to be oxidised to the state of + 6.

Example:

Sodium n-dodecylbenzenesulphonate C18H29SO3Na, MW = 348

Image omitted; see official source

In the case of nitrogen-containing substances the nitrogen may be eliminated as ammonia, nitrite, or nitrate corresponding to different theoretical biochemical oxygen demands.

Image omitted; see official source

Image omitted; see official source

Suppose full nitrate formation had been observed by analysis in the case of a secondary amine:

(C12H25)2 NH, MW = 353

Image omitted; see official source

Appendix 4

Image omitted; see official source

Text of image

temperature (°C)

salinity

in: ‰

Nomogram giving:

Saturation concentration of oxygen of various temperatures and salinities

mg O2/l

Appendix 5

Biodegradation in seawater

CLOSED BOTTLE METHOD

DATA SHEET

• 1. LABORATORY:

• 1. DATE AT START OF TEST:

• 1. TEST SUBSTANCE:

• Name:

Stock solution concentration:	mg/l
Initial conc. in seawater medium:	mg/l
ThOD or COD:	mg O2/mg test substance

• 1. SEAWATER:

• Source:

• Date of collection:

• Depth of collection:

• Appearance at time of collection (e.g. turbid, etc.):

Salinity at collection:	‰
Temperature at collection:	°C
DOC ‘x’ hours after collection:	mg/l

• Pre-treatment prior to testing (e.g. filtration, sedimentation, ageing, etc.):

Microbial colony count	—  original sample:	colonies/ml
	—  at start of test:	colonies/ml
Other characteristics:

• 1. TEST MEDIUM:

Temperature after aeration:	°C
O2 concentration after aeration and standing before start of test:	mg O2/l

• 1. DO DETERMINATION:

• Method: Winkler/electrode

	Flask no.		mg O2/l after n days	mg O2/l after n days	mg O2/l after n days	mg O2/l after n days
	Flask no.		0	5	15	28
Test: nutrient — fortified seawater with test substance	1	a1
Test: nutrient — fortified seawater with test substance	2	a2
Test: nutrient — fortified seawater with test substance	Mean test	Image omitted; see official source
Blank: nutrient — fortified seawater, but without test substance	1	c1
Blank: nutrient — fortified seawater, but without test substance	2	c2
Blank: nutrient — fortified seawater, but without test substance	Mean blank	Image omitted; see official source

Note: Similar format may be used for reference substance and toxicity controls.

• 1. DO DEPLETION: % DEGRADATION (%D):

	DO depletion after n days	DO depletion after n days	DO depletion after n days
	5	15	28
(mb – mt) [^E0213]
Image omitted; see official source

This assumes that mb(o) = mt(o), where mb(o) = blank value at day 0, mt(o) = test substance value at day 0.

If mb(o) does not equal mt(o), use (mt(o) – mt(x)) – (mb(o) – mb(x)), where

mb(x) = blank value at day x, mt(x) = test substance value at day x.

C.43.   ANAEROBIC BIODEGRADABILITY OF ORGANIC SUBSTANCES IN DIGESTED SLUDGE: BY MEASUREMENT OF GAS PRODUCTION

INTRODUCTION

• 1.This test method is equivalent to OECD Test Guideline (TG) 311 (2006). There are a number of screening tests for assessing aerobic biodegradability of organic substances (Test methods C.4, C.9, C.10, and C.11 (1) and OECD TG 302C (2)) and the results of applying these have been successfully used to predict the fate of substances in the aerobic environment, particularly in the aerobic stages of waste water treatment. Various proportions of water-insoluble substances, as well as of those which adsorb on to sewage solids, are also dealt with aerobically, since they are present in settled sewage. However, the larger fractions of these substances are bound to the primary settled sludge, which is separated from raw sewage in settlement tanks before the settled, or supernatant, sewage is treated aerobically. The sludge, containing some of the soluble substances in the interstitial liquid, is then passed to heated digesters for anaerobic treatment. As yet there are no tests in this series for assessing anaerobic biodegradability in anaerobic digesters and this test is targeted to fill this gap; it is not necessarily applicable to other anoxic environmental compartments.

• 2.Respirometric techniques that measure the amounts of gas produced, mainly methane (CH4) and carbon dioxide (CO2), under anaerobic conditions have been used successfully for assessing anaerobic biodegradability. Birch et al (3) reviewed these procedures and concluded that the work of Shelton and Tiedje (4), based on earlier studies (5)(6)(7), was the most comprehensive. The method (4), which was further developed by others (8) and has become the American standards (9)(10), did not resolve problems related to the differing solubilities of CO2 and CH4 in the test medium and to the calculation of the theoretical gas production of a test substance. The ECETOC report (3) recommended the additional measurement of the dissolved inorganic carbon (DIC) content of the supernatant liquid, which made the technique more widely applicable. The ECETOC method was subjected to an international calibration exercise (or ring test) and became the ISO Standard, ISO 11734 (11).

- 3.This test method, which is based on ISO 11734 (11), describes a screening method for the evaluation of potential anaerobic biodegradability of organic substances under a specific condition (i.e. in an anaerobic digester at a given time and range of concentration of micro-organisms). Because a diluted sludge is used with a relatively high concentration of test substance and the duration of the test typically is longer than the retention time in anaerobic digesters, the conditions of the test do not necessarily correspond to the conditions in anaerobic digesters, nor is it applicable for the assessment of anaerobic biodegradability of organic substances under different environmental conditions. Sludge is exposed to the test substance for up to 60 days, which is longer than the normal sludge retention time (25 to 30 days) in anaerobic digesters, though at industrial sites retention times may be much longer. Predictions from the results of this test cannot be made as convincingly as they can be made in the case of aerobic biodegradation, since the evidence accrued on the behaviour of test substances in ‘ready’ aerobic tests and in simulation tests and the aerobic environment is sufficient to be confident that there is a connection; little similar evidence exists for the anaerobic environment. Complete anaerobic biodegradation can be assumed to occur if 75 %-80 % of theoretical gas production is achieved. The high ratios of substance to biomass used in these tests mean that a substance which passes is more likely to be degraded in an anaerobic digester. Additionally, substances which fail to be converted to gas in the test may not necessarily persist at more environmentally realistic substance-to-biomass ratios. Also, other anaerobic reactions occur by which substances may be at least partially degraded, e.g. by dechlorination, but this test does not detect such reactions. However, by applying specific analytical methods for determining the test substance, its disappearance may be monitored (see paragraphs 6, 30, 44 and 53).

PRINCIPLE OF THE TEST

• 4.Washed digested sludge ([^E0214]), containing low (< 10 mg/l) concentrations of inorganic carbon (IC), is diluted about ten-fold to a total solids concentration of 1 g/l to 3 g/l and incubated at 35 °C ± 2 °C in sealed vessels with the test substance at 20 to 100 mg C/l for up to 60 days. Allowance is made for measuring the activity of the sludge by running parallel blank controls with sludge inoculum in the medium but without test substance.

• 5.The increase in headspace pressure in the vessels resulting from the production of carbon dioxide and methane is measured. Much of the CO2 produced will be dissolved in the liquid phase or transformed into carbonate or hydrogen carbonate under the conditions of the test. This inorganic carbon is measured at the end of the test.

- 6.The amount of carbon (inorganic plus methane) resulting from the biodegradation of the test substance is calculated from the net gas production and net IC formation in the liquid phase in excess of blank control values. The extent of biodegradation is calculated from total IC and methane-C produced as a percentage of the measured or calculated amount of carbon added as test substance. The course of biodegradation can be followed by taking intermediate measurements of gas production only. Additionally the primary biodegradation can be determined by specific analyses at the beginning and end of the test.

INFORMATION ON THE TEST SUBSTANCE

- 7.The purity, water solubility, volatility and adsorption characteristics of the test substance should be known to enable correct interpretation of results to be made. The organic carbon content (% w/w) of the test substance needs to be known either from its chemical structure or by measurement. For volatile test substances, a measured or calculated Henry's law constant is helpful in deciding whether the test is applicable. Information on the toxicity of the test substance for anaerobic bacteria is useful in selecting an appropriate test concentration, and for interpreting results showing poor biodegradability. It is recommended to include the inhibition control unless it is known that the test substance is not inhibitory to anaerobic microbial activities (see paragraph 21 and ISO 13641-1 (12)).

APPLICABILITY OF THE TEST METHOD

- 8.The test method may be applied to water-soluble substances; it may also be applied to poorly soluble and insoluble substances, provided that a method of exact dosing is used e.g. see ISO 10634 (13). In general, a case by case decision is necessary for volatile substances. Special steps may have to be taken, for example, not releasing gas during the test.

REFERENCE SUBSTANCES

- 9.To check the procedure, a reference substance is tested by setting up appropriate vessels in parallel as part of normal test runs. Phenol, sodium benzoate and polyethylene glycol 400 are examples and would be expected to be degraded by more than 60 % theoretical gas production (i.e. methane and inorganic carbon) within 60 days (3)(14).

REPRODUCIBILITY OF TEST RESULTS

• 10.In an international ring test (14) there was good reproducibility in gas pressure measurements between triplicate vessels. The relative standard deviation (coefficient of variation, COV) was mainly below 20 %, although this value often increased to > 20 % in the presence of toxic substances or towards the end of the 60-d incubation period. Higher deviations were also found in vessels of volume < 150 ml. Final pH values of the test media were in the range 6,5-7,0.

• 11.The following results were obtained in the ring test.

Test substance	Total data n1	Mean degradation (of total data) (%)	Relative Standard deviation (of total data) (%)	Valid data n2	Mean degradation (of valid data) (%)	Relative Standard deviation (of valid data) (%)	Data > 60 % degradation in valid tests n3
Palmitic acid	36	68,7 ± 30,7	45	27	72,2 ± 18,8	26	19 = 70 % [^E0215]
Polyethylene Glycol 400	38	79,8 ± 28,0	35	29	77,7 ± 17,8	23	24 = 83 % [^E0215]

Proportion of n2

- 12.The coefficients of variation of the mean for all values obtained with palmitic acid and polyethylene glycol 400 were as high as 45 % (n = 36) and 35 % (n = 38) respectively. When values of < 40 % and > 100 % were omitted (the former being assumed to be due to sub-optimal conditions, the latter due to unknown reasons), the COVs were reduced to 26 % and 23 %, respectively. The proportions of ‘valid’ values attaining at least 60 % degradation were 70 % for palmitic acid and 83 % for polyethylene glycol 400. The proportions of the percentage biodegradation derived from DIC measurements were relatively low but variable. For palmitic acid the range was 0-35 %, mean 12 %, with COV of 92 % and for polyethyleneglycol 400 0-40 %, mean 24 %, with COV of 54 %.

DESCRIPTION OF THE TEST METHOD

Apparatus

• 13.Usual laboratory equipment and the following are required:

• (a) Incubator — spark-proof and controlled at 35 °C ± 2 °C;

• (b) Pressure-resistant glass test vessels of an appropriate nominal size ([^E0216]), each fitted with a gas-tight septum, capable of withstanding about 2 bar. The headspace volume should be about 10 % to 30 % of the total volume. If biogas is released regularly, about 10 % headspace volume is appropriate, but if the gas release is made only at the end of the test 30 % is appropriate. Glass serum bottles, of nominal volume 125 ml, total volume around 160 ml, sealed with serum septa ([^E0217]) and crimped aluminium rings are recommended when the pressure is released at each sampling time;

• (c) Pressure-measuring device ([^E0218]) adapted to enable measurement and venting of the gas produced, for example, a hand-held precision pressure meter connected to a suitable syringe needle; a 3-way gas-tight valve facilitates the release of excess pressure (Appendix 1). It is necessary to keep the internal volume of the pressure transducer tubing and valve as low as possible, so that errors introduced by neglecting the volume of the equipment are insignificant; Note — The pressure readings are used directly to calculate the amount of carbon produced in the headspace (paragraphs 42 to 44). Alternatively, the pressure readings may be converted to volumes (at 35 °C, atmospheric pressure) of gas produced using a conversion graph. This graph is constructed from data obtained by injecting known volumes of nitrogen gas into a series of test vessels (e.g. serum bottles) at 35° +/– 2 °C and recording the resulting stabilised pressure readings (See Appendix 2). The calculation is shown in the Note in paragraph 44. Warning — Take care to avoid needle-stick injuries when using micro-syringes.

• (d) Carbon analyser, suitable for the direct determination of inorganic carbon in the range of 1 mg/l to 200 mg/l;

• (e) Syringes of high precision for gaseous and liquid samples;

• (f) Magnetic stirrers and followers (optional);

- (g) Glove box (recommended).

Reagents

- 14.Use analytical grade reagents throughout.

Water

- 15.Distilled or deionised water (de-oxygenated by sparging with nitrogen gas containing less than 5 μl/l oxygen), containing less than 2 mg/l dissolved organic carbon (DOC).

Test medium

• 16.Prepare the dilution medium to contain the following constituents at the stated amounts;

Anhydrous potassium dihydrogen phosphate (KH2PO4)	0,27 g
Disodium hydrogen phosphate dodecahydrate (Na2HPO4 · 12H2O))	1,12 g
Ammonium chloride (NH4Cl)	0,53 g
Calcium chloride dihydrate (CaCl2·2H2O)	0,075 g
Magnesium chloride hexahydrate (MgCl2·6H2O)	0,10 g
Iron (II) chloride tetrahydrate (FeCl2·4H2O)	0,02 g
Resazurin (oxygen indicator)	0,001 g
Sodium sulphide nonahydrate (Na2S·9H2O)	0,10 g
Stock solution of trace elements (optional, paragraph 18)	10 ml
Add de-oxygenated water (paragraph 15)	to 1 litre

Note: Freshly supplied sodium sulphide should be used or it should be washed and dried before use, to ensure sufficient reductive capacity. The test may be performed without using a glove box (see paragraph 26). In this case, the final concentration of sodium sulphide in the medium should be increased to 0,20 g of Na2S · 9H2O per litre. Sodium sulphide may also be added from an appropriate anaerobic stock solution through the septum of the closed test vessels as this procedure will decrease the risk of oxidation. Sodium sulphide may be replaced by titanium (III) citrate, which is added through the septum of closed test vessels at a final concentration of 0,8 to 1,0 mmol/l. Titanium (III) citrate is a highly effective and low-toxicity reducing agent, which is prepared as follows: Dissolve 2,94 g of trisodium citrate dihydrate in 50 ml of de-oxygenated water (to result in a solution of 200 mmol/l) and add 5 ml of a 15 % (w/v) titanium (III) chloride solution. Neutralise to pH 7 ± 0,2 with mineral alkali and dispense to an appropriate vessel under a stream of nitrogen. The concentration of titanium (III) citrate in this stock solution is 164 mmol/l.

- 17.Mix the components of the test medium except the reducing agent (sodium sulphide titanium citrate) and sparge the solution with nitrogen gas for about 20 min immediately before use to remove oxygen. Then add the appropriate volume of freshly prepared solution of the reducing agent (prepared in de-oxygenated water) just before use of the medium. Adjust the pH of the medium, if necessary, with dilute mineral acid or alkali to 7 ± 0,2.

Stock solution of trace elements (optional)

• 18.It is recommended that the test medium should contain the following trace elements to improve anaerobic degradation processes, especially if low concentrations (e.g. 1g/l) of inoculum are used (11).

| | |

| --- | --- | | Manganese chloride tetrahydrate (MnCl2 · 4H2O) | 50 mg | | Boric acid (H3BO3) | 5 mg | | Zinc chloride (ZnCl2) | 5 mg | | Copper (II) chloride (CuCl2) | 3 mg | | Disodium molybdate dihydrate (Na2MoO4 · 2H2O) | 1 mg | | Cobalt chloride hexahydrate (CoCl2 · 6H2O) | 100 mg | | Nickel chloride hexahydrate (NiCl2 · 6H2O) | 10 mg | | Disodium selenite (Na2SeO3) | 5 mg | | Add de-oxygenated water (paragraph 15) | to 1 litre |

Test substance

• 19.Add the test substance as a stock solution, suspension, emulsion, or directly as solid or liquid, or as absorbed on to glass-fibre filter to give a concentration of no more than 100 mg/l organic carbon. If stock solutions are used, prepare a suitable solution with water (paragraph 15) (previously de-oxygenated by sparging with nitrogen gas) of such a strength that the volume added is less than 5 % of the total volume of reaction mixture. Adjust the pH of the stock solution to pH 7 ± 0,2 if necessary. For test substances which are insufficiently soluble in water, consult ISO 10634 (13). If a solvent is used, prepare an additional control, with the solvent only added to the inoculated medium. Organic solvents which are known to inhibit methane production, such as chloroform and carbon tetrachloride, should be avoided.

Warning — Handle with care toxic test substances, and those whose properties are not known.

Reference substances

- 20.Reference substances such as sodium benzoate, phenol and polyethylene glycol 400 have been used successfully to check the procedure, being biodegraded by more than 60 % within 60 days. Prepare a stock solution (in de-oxygenated water) of the chosen reference substance in the same way as for the test substance and adjust to pH 7 ± 0,2 if necessary.

Inhibition control (conditional)

- 21.In order to obtain information on the toxicity of the test substance to anaerobic micro-organisms to find the most appropriate test concentration, add the test substance and reference substance to a vessel containing the test medium (see paragraph 16), each at the same concentrations as added, respectively (see paragraphs 19 and 20 and see also ISO 13641-1 (12)).

Digested sludge

• 22.Collect digested sludge from a digester at a waste water treatment plant which treats predominantly domestic sewage. The sludge should be fully characterised and its background information should be reported (see paragraph 54). If use of adapted inoculum is intended, digested sludge from an industrial sewage treatment plant may be considered. Use wide-necked bottles constructed from high-density polyethylene or a similar material, which can expand, for the collection of the digested sludge. Add sludge to within about 1cm of the top of the bottles and seal tightly, preferably with a safety valve. After transport to the laboratory, the collected sludge may be used directly or placed in a laboratory-scale digester. Release excess biogas by opening bottles of sludge carefully. Alternatively, laboratory-grown anaerobic sludge may be used as a source of inoculum but its spectrum of activity may have been impaired.

Warning — Digested sludge produces flammable gases which present fire and explosion risks: it also contains potentially pathogenic organisms, so take appropriate precautions when handling sludge. For safety reasons, do not use glass vessels for collecting sludge.

• 23.In order to reduce background gas production and to decrease the influence of the blank controls, pre-digestion of the sludge may be considered. If pre-digestion is required, the sludge should be allowed to digest without the addition of any nutrients or substrates at 35 °C ± 2 °C for up to 7 days. It has been found that pre-digestion for about 5 days usually gives an optimal decrease in gas production of the blank without unacceptable increases in either lag or incubation periods during the test phase or loss of activity towards a small number of substances tested.

- 24.For test substances which are, or are expected to be, poorly biodegradable, consider pre-exposure of the sludge to the test substance to obtain an inoculum which is better adapted. In such a case, add the test substance at an organic carbon concentration of 5 mg/l to 20 mg/l to the digested sludge and incubated for up to 2 weeks. Wash the pre-exposed sludge carefully before use (see paragraph 25) and indicate in the test report the conditions of the pre-exposure.

Inoculum

- 25.Wash the sludge (see paragraphs 22 to 24) just prior to use, to reduce the IC concentration to less than 10 mg/l in the final test suspension. Centrifuge the sludge in sealed tubes (e.g. 3 000 g during 5 min) and discharge the supernatant. Suspend the resulting pellet in de-oxygenated medium (paragraphs 16 and 17), re-centrifuge the suspension and discharge the supernatant liquid. If the IC has not been sufficiently lowered, the washing procedure of the sludge could be repeated twice as a maximum. This does not appear to affect the micro-organisms adversely. Finally, suspend the pellet in the requisite volume of test medium and determine the concentration of total solids [e.g. ISO 11923 (15)]. The final concentration of total solids in the test vessels should be in the range of 1 g/l to 3 g/l (or about 10 % of that in undiluted digested sludge). Conduct the above operations in such a way that the sludge has minimal contact with oxygen (e.g. use a nitrogen atmosphere).

TEST PROCEDURE

- 26.Perform the following initial procedures using techniques to keep the contact between digested sludge and oxygen as low as practicable, for example, it may be necessary to work within a glove box in an atmosphere of nitrogen and/or purge the bottles with nitrogen (4).

Preparation of test and control assays

• 27.Prepare at least triplicate test vessels (see paragraph 13-b) for the test substance, blank controls, reference substance, inhibition controls (conditional) and pressure control chambers (optional procedure) (see paragraphs 7, 19 to 21). Additional vessels for the purpose of evaluating primary biodegradation using test substance specific analyses may also be prepared. The same set of blank controls may be used for several test substances in the same test as long as the headspace volumes are consistent.

• 28.Prepare the diluted inoculum before adding it to the vessels e.g. by the means of a wide-mouthed pipette. Add aliquots of well-mixed inoculum (paragraph 25) so that the concentration of total solids is the same in all vessels (between 1 g/l and 3 g/l). Add stock solutions of the test and reference substance after adjustment to pH 7 ± 0,2, if necessary. The test substance and the reference substance should be added using the most appropriate route of administration (paragraph 19).

• 29.The test concentration of organic carbon should normally be between 20 and 100 mg/l (paragraph 4). If the test substance is toxic, the test concentration should be reduced to 20 mg C/l, or even less if only primary biodegradation with specific analyses is to be measured. It should be noted that the variability of the test results increases at lower test concentrations.

• 30.For blank vessels, add an equivalent amount of the carrier used to dose the test substance instead of a stock solution, suspension or emulsion. If the test substance was administered using glass fibre filters or organic solvents, add to the blanks a filter or an equivalent volume solvent that has been evaporated. Prepare an extra replicate with test substance for the measurement of the pH value. Adjust the pH to 7 ± 0,2, if necessary, with small amounts of dilute mineral acid or alkali. The same amounts of neutralising agents should be added to all the test vessels. These additions should not have to be made since the pH value of the stock solutions of the test substance and reference substance have already been adjusted (see paragraphs 19 and 20). If primary biodegradation is to be measured, an appropriate sample should be taken from the pH-control vessel, or from an additional test vessel, and the test substance concentration should be measured using specific analyses. Covered magnets may be added to all the vessels if the reaction mixtures are to be stirred (optional).

- 31.Ensure that the total volume of liquid V1 and the volume of headspace Vh are the same in all vessels; note and record the values of V1 and Vh. Each vessel should be sealed with a gas septum and transferred from the glove box (see paragraph 26) into the incubator (see paragraph 13-a).

Insoluble test substances

- 32.Add weighed amounts of substances, which are poorly soluble in water, directly to the prepared vessels. When the use of a solvent is necessary (see paragraph 19), transfer the test substance solution or suspension into the empty vessels. Where possible, evaporate the solvent by passing nitrogen gas through the vessels and then add the other ingredients, namely, diluted sludge (paragraph 25), and de-oxygenated water as required. An additional solvent control should also be prepared (see paragraph 19). For other methods of adding insoluble substances, ISO 10634 (13) can be consulted. Liquid test substances may be dosed with a syringe into the completely prepared sealed vessels, if it is expected that the initial pH will not exceed 7 ± 1, otherwise dose as described above (see paragraph 19).

Incubation and gas pressure measurements

• 33.Incubate the prepared vessels at 35 °C ± 2 °C for about 1h to allow equilibration and release excess gas to the atmosphere, for example, by shaking each vessel in turn, inserting the needle of the pressure meter (paragraph 13-c) through the seal and opening the valve until the pressure meter reads zero. If at this stage, or when making intermediate measurements, the headspace pressure is less than atmospheric, nitrogen gas should be introduced to re-establish atmospheric pressure. Close the valve (see paragraph 13-c) and continue to incubate in the dark, ensuring that all parts of the vessels are maintained at the digestion temperature. Observe the vessels after incubation for 24 to 48 h. Reject vessels if the contents of the vessels show a distinct pink coloration in the supernatant liquid, i.e. if Resazurin (see paragraph 16) has changed colour indicating the presence of oxygen (see paragraph 50). While small amounts of oxygen may be tolerated by the system, higher concentrations can seriously inhibit the course of anaerobic biodegradation. The rejection of the occasional single vessel of a set of triplicates may be accepted, but the incidence of more failures than this must lead to an investigation of the experimental procedures as well as the repeating of the test.

• 34.Carefully mix the contents of each vessel by stirring or by shaking for a few minutes at least 2 or 3 times per week and soon before each pressure measurement. Shaking re-suspends the inoculum and ensures gaseous equilibrium. All pressure measurements should be taken quickly, since the test vessels could be subject to lowering of temperature, leading to false readings. While measuring pressure the whole test vessel including the headspace should be maintained at the digestion temperature. Measure the gas pressure, for example, by inserting through the septum the syringe needle (paragraph 13-c) connected to the pressure-monitoring meter. Care should be taken to prevent entry of water into the syringe needle; if this occurs the wet parts should be dried and a new needle fitted. The pressure should be measured in millibars (see paragraph 42). The gas pressure in the vessels may be measured periodically e.g. weekly, and optionally the excess gas is released to the atmosphere. Alternatively the pressure is measured only at the end of the test to determine the amount of biogas produced.

• 35.It is recommended that intermediate readings of gas pressure be made, since pressure increase provides guidance as to when the test may be terminated and allows the kinetics to be followed (see paragraph 6).

- 36.Normally end the test after an incubation period of 60 days unless the biodegradation curve obtained from the pressure measurements has reached the plateau phase before then; that is the phase in which the maximal degradation has been reached and the biodegradation curve has levelled out. If the plateau value is less than 60 % interpretation is problematic because it indicates that only part of the molecule has been mineralised or that an error has been made. If at the end of the normal incubation period, gas is being produced but a plateau phase is obviously not reached, then it should be considered to prolong the test to check whether the plateau (> 60 %) will be reached.

Measurement of inorganic carbon

• 37.At the end of the test after the last measurement of gas pressure, allow the sludge to settle. Open each vessel in turn and immediately take a sample for the determination of the concentration (mg/l) of inorganic carbon (IC) in the supernatant liquor. Neither centrifugation nor filtration should be applied to the supernatant liquor, since there would be an unacceptable loss of dissolved carbon dioxide. If the liquor cannot be analysed on being sampled, store it in a sealed vial, without headspace and cooled to about 4 °C for up to 2 days. After the IC measurement, measure and record the pH value.

• 38.Alternatively, the IC in the supernatant may be determined indirectly by release of the dissolved IC as carbon dioxide that can be measured in the headspace. Following the last measurement of gas pressure, adjust the pressure in each of the test vessels to atmospheric pressure. Acidify the contents of each vessel to approximately pH 1 by adding of concentrated mineral acid (e.g. H2SO4) through the septum of the sealed vessels. Incubate the shaken vessels at 35 °C ± 2 °C for approximately 24 hours and measure the gas pressure resulting from the evolved carbon dioxide by using the pressure meter.

• 39.Make similar readings for the corresponding blank, reference substance and, if included, inhibition control vessels (see paragraph 21).

• 40.In some cases, especially if the same control vessels are used for several test substances, measurements of intermediate IC concentrations in test and control vessels should be considered, as appropriate. In this case, a sufficient number of vessels should be prepared for all the intermediate measurements. This proceeding is preferred to taking all samples from one vessel only. The latter can only be done if the required volume for DIC analysis is not deemed to be too high. The DIC measurement should be made after measuring the gas pressure without release of excess gas as described below:

• — take as small a volume as possible of supernatant samples with a syringe through the septum without opening the vessels and IC in the sample is determined;

• — after having taken the sample the excess gas is released, or not;

• — it should be taken into account that even a small decrease in the supernatant volume (e.g. about 1 %) can yield a significant increase in the headspace gas volume (Vh);

- — the equations (see paragraph 44) are corrected by increasing Vh in equation 3, as necessary.

Specific analyses

- 41.If primary anaerobic degradation (see paragraph 30) is to be determined, take an appropriate volume of sample for specific analyses at the beginning and at the end of the test from the vessels containing the test substance. If this is done, note the volumes of headspace (Vh) and of the liquid (Vl) will be changed and take this into account when calculating the results of gas production. Alternatively samples may be taken for specific analyses from additional mixtures previously set up for the purpose (paragraph 30).

DATA AND REPORTING

Treatment of results

- 42.For practical reasons, the pressure of the gas is measured in millibars (1 mbar = 1h Pa = 10² Pa; 1 Pa = 1 N/m²), the volume in litres and temperature in degrees Celsius.

Carbon in the headspace

• 43.Since 1 mol of methane and 1 mol carbon dioxide each contain 12 g of carbon, the mass of carbon in a given volume of evolved gas may be expressed as:

m = 12 × 103 × n	Equation [1]

• where:

m	=	mass of carbon (mg) in a given volume of evolved gas;
---	---	---
12	=	relative atomic mass of carbon;
---	---	---
n	=	number of moles of gas in the given volume.

• If a gas other than methane or carbon dioxide (e.g. N2O) is generated in considerable amounts, the formula [1] should be amended in order to describe the possibility of effects by gases generated.

• 44.From the gas laws n may be expressed as:

Image omitted; see official source	Equation [2]

• where:

p	=	pressure of the gas (Pascals);
---	---	---
V	=	volume of the gas (m3);
---	---	---
R	=	molar gas constant [8,314 J/(mol K)];
---	---	---
T	=	incubation temperature (Kelvins).

• By combination of equations [1] and [2] and rationalising to allow for blank control production of gas:

Image omitted; see official source	Equation [3]

• where:

mh	=	mass of net carbon produced as gas in the headspace (mg);
---	---	---
Δp	=	mean of the difference between initial and final pressures in the test vessels minus the corresponding mean in the blank vessels (millibars);
---	---	---
Vh	=	volume of headspace in the vessel (l);
---	---	---
0,1	=	conversion for both newtons/m2 to millibars and m3 to litres.

• Equation [4] should be used for the normal incubation temperature of 35 °C (308 K):

mh = 0,468(Δp · Vh)	Equation [4]

Note: Alternative volume calculation. Pressure meter readings are converted to ml of gas produced using the standard curve generated by plotting volume (ml) injected versus meter reading (Appendix 2). The number of moles (n) of gas in the headspace of each vessel is calculated by dividing the cumulative gas production (ml) by 25 286 ml/mole, which is the volume occupied by one mole of gas at 35 °C and standard atmospheric pressure. Since 1 mole of CH4 and 1 mole of CO2 each contain 12 g of carbon, the amount of carbon (mg) in the headspace (mh) is given by Equation [5]:

mh = 12 × 103 × n	Equation [5]

• Rationalising to allow for blank control production of gas:

Image omitted; see official source	Equation [6]

• where:

mh	=	mass of net carbon produced as gas in the headspace (mg);
---	---	---
ΔV	=	mean of the difference between volume of gas produced in headspace in the test vessels and blank control vessels;
---	---	---
25 286	=	volume occupied by 1 mole gas at 35 °C, 1 atmosphere.

- 45.The course of biodegradation can be followed by plotting the cumulated pressure increase Δp (millibars) against time, if appropriate. From this curve, identify and record the lag phase (days). The lag phase is the time from the start of the test until significant degradation starts (for example see Appendix 3). If intermediate samples of supernatant were taken and analysed (see paragraphs 40, 46 and 47), then the total C produced (in gas plus that in liquid) may be plotted instead of only the cumulative pressure.

Carbon in the liquid

• 46.The amount of methane in the liquid is ignored since its solubility in water is known to be very low. Calculate the mass of inorganic carbon in the liquid of the test vessels using equation [7]:

ml = Cnet × Vl	Equation [7]

• where:

ml	=	mass of inorganic carbon in the liquid (mg);
---	---	---
Cnet	=	concentration of inorganic carbon in the test vessels minus that in the control vessels at the end of the test (mg/l);

| | | |

| --- | --- | --- | | Vl | = | volume of liquid in the vessel (l). |

Total gasified carbon

• 47.Calculate the total mass of gasified carbon in the vessel using equation [8]:

mt = mh + ml	Equation [8]

• where:

mt = total mass of gasified carbon (mg);

mh and ml are as defined above.

Carbon of test substance

• 48.Calculate the mass of carbon in the test vessels derived from the added test substance using equation [9]:

mv = Cc × Vl	Equation [9]

• where:

mv	=	mass of test substance carbon (mg);
---	---	---
Cc	=	concentration of test substance carbon in the test vessel (mg/l)

| | | |

| --- | --- | --- | | Vl | = | volume of liquid in the test vessel (l). |

Extent of biodegradation

• 49.Calculate the percentage biodegradation from headspace gas using equation [10] and the total percentage biodegradation using equation [11]:

Dh = (mh/mv) × 100	Equation [10]
Dt = (mt/mv) × 100	Equation [11]

• where:

Dh = biodegradation from headspace gas (%);

Dt = total biodegradation (%);

mh, mv and mt are as defined above.

• The degree of primary biodegradation is calculated from the (optional) measurements of the concentration of the test substance at the beginning and end of incubation, using equation [12]:

Dp = (1 – Se/Si) × 100	Equation [12]

• where:

Dp	=	primary degradation of test substance (%);
---	---	---
Si	=	initial concentration of test substance (mg/l);
---	---	---
Se	=	concentration of test substance at end (mg/l).

• If the method of analysis indicates significant concentrations of the test substance in the unamended anaerobic sludge inoculum, use equation [13]:

Dp 1 = [1 – (Se – Seb)/(Si – Sib)] × 100	Equation [13]

• where:

Dp 1	=	corrected primary degradation of test substance (%);
---	---	---
Sib	=	initial ‘apparent’ concentration of test substance in blank controls (mg/l);

| | | |

| --- | --- | --- | | Seb | = | ‘apparent’ concentration of test substance in blank controls at end (mg/l). |

Validity of results

• 50.Pressure readings should be used only from vessels that do not show pink coloration (see paragraph 33). Contamination by oxygen is minimised by the use of proper anaerobic handling techniques.

• 51.It should be considered that the test is valid if the reference substance reaches a plateau that represents more than 60 % biodegradation ([^E0219]).

- 52.If the pH at the end of the test has exceeded the range 7 ± 1 and insufficient biodegradation has taken place, repeat the test with increased buffer capacity of the medium.

Inhibition of degradation

- 53.Gas production in vessels containing both the test substance and reference substance should be at least equal to that in the vessels containing only reference substance; otherwise, inhibition of gas production is indicated. In some cases gas production in vessels containing test substance without reference substance will be lower than that in the blank controls, indicating that the test substance is inhibitory.

Test report

• 54.The test report must include the following information:

Test substance:

• — common name, chemical name, CAS number, structural formula and relevant physical-chemical properties;

• — purity (impurities) of test substance.

Test conditions:

• — volumes of diluted digester liquor (Vl) and of the headspace (Vh) in the vessel;

• — description of the test vessels, the main characteristics of biogas measurement (e.g. type of pressure meter) and of the IC analyser;

• — application of test substance and reference substance to test system: test concentration used and any use of solvents;

• — details of the inoculum used: name of sewage treatment plant, description of the source of waste water treated (e.g. operating temperature, sludge retention time, predominantly domestic, etc.), concentration, any information necessary to substantiate this and information on any pre-treatment of the inoculum (e.g. pre-digestion, pre-exposure);

• — incubation temperature;

• — number of replicates.

Results:

• — pH and IC values at the end of the test;

• — concentration of test substance at the beginning and end of the test, if a specific measurement has been performed;

• — all the measured data collected in the test, blank, reference substance and inhibition control vessels, as appropriate (e.g. pressure in millibars, concentration of inorganic carbon (mg/l)) in tabular form (measured data for headspace and liquid should be reported separately);

• — statistical treatment of data, test duration and a diagram of the biodegradation of test substance, reference substance and inhibition control;

• — percentage biodegradation of test substance and reference substance;

• — reasons for any rejection of the test results;

- — discussion of results.

LITERATURE

• (1)The following chapters of this Annex:

C.4, Determination of Ready Biodegradability;

C.9, Biodegradation — Zahn-Wellens Test;

C.10, Simulation Test — Aerobic Sewage Treatment:

A: Activated Sludge Units, B: Biofilms

C.11, Biodegradation — Activated sludge respiration inhibition

• (2)OECD (2009) Inherent Biodegradability: Modified MITI Test (II), OECD Guideline for Testing of Chemicals, No. 302C, OECD, Paris