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)

If measurement of the physico-chemical uptake of oxygen is required, prepare a solution of the test chemical at, normally, 100 mg ThOD/litre which has been sterilised by the addition of a suitable toxic substance (see I.6.6).

Introduce the requisite volume of solutions of test and reference chemicals, respectively, into at least duplicate flasks. Add to further flasks mineral medium only (for inoculum controls) and, if required, the mixed test/reference chemical solution and the sterile solution.

If the test chemical is poorly soluble, add it directly at this stage on a weight or volume basis or handle it as described in Appendix 3. Add potassium hydroxide, soda lime pellets or other absorbent to the CO2-absorber compartments.

V.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

preferably, and when necessary:

Flask 6: sterile control

Flask 7: toxicity control

See also I.6.7.

V.2.6.   Performance of the test

Allow the vessels to reach the desired temperature and inoculate appropriate vessels with prepared activated sludge or other source of inoculum to give a concentration of suspended solids not greater than 30 mg/litre. Assemble the equipment, start the stirrer and check for air-tightness, and start the measurement of oxygen uptake. Usually no further attention is required other than taking the necessary readings and making daily checks to see that the correct temperature and adequate stirring are maintained.

Calculate the oxygen uptake from the readings taken at regular and frequent intervals, using the methods given by the manufacturer of the equipment. At the end of incubation, normally 28 days, measure the pH of the contents of the flasks, especially if oxygen uptakes are low or greater than ThODNH4 (for nitrogen-containing compounds).

If required, withdraw samples from the respirometer flasks, initially and finally, for analysis of DOC or specific chemical (see Appendix 2.4). At the initial withdrawal, ensure that the volume of test suspension remaining in the flask is known. 'When oxygen is taken up by N-containing test substance, determine the increase in concentration of nitrite and nitrate over 28 days and calculate the correction for the oxygen consumed by nitrification (Appendix 5).

V.3.   DATA AND REPORTING

V.3.1.   Treatment of results

Divide the oxygen uptake (mg) of the test chemical after a given time (corrected for that by the blank inoculum control after the same time) by the weight of the test chemical used. This yields the BOD expressed as mg oxygen/mg test chemical, that is:

Image omitted; see official source

= mg O2 per mg test chemical

calculate the percentage biodegradation either from:

Image omitted; see official source

or form

Image omitted; see official source

It should be noted that these two methods do not necessarily give the same value; it is preferable to use the former method.

For test substances containing nitrogen, use the appropriate ThOD (NH4 or NO3) according to what is known or expected about the occurrence of nitrification (Appendix 2.2). If nitrification occurs but is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate (Appendix 5).

When optional determinations of organic carbon and/or specific chemical are made, calculate the percentage degradation, as described under I.7.

Record all results on the data sheets attached.

V.3.2.   Validity of results

The oxygen uptake of the inoculum blank is normally 20-30 mg O2/litre and should not be greater than 60 mg/litre in 28 days. Values higher than 60 mg/litre require critical examination of the data and experimental techniques. If the pH value is outside the range 6-8,5 and the oxygen consumption by the test chemical is less than 60 %, the test should be repeated with a lower concentration of test chemical.

See also I.5.2.

V.3.3.   Reporting

See I.8.

V.4.   DATA SHEET

An example of a data sheet is given hereafter.

MANOMETRIC RESPIROMETRY TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: … mg/litre

Initial concentration in medium, Co: … mg/litre

Volume in test flask (V): … ml

ThOD or COD: … mg O2/mg test substance (NH4 or NO3)

4. INOCULUM

Source:

Treatment given:

Pre-conditioning, if any:

Concentration of suspended solids in reaction mixture: … mg/1

5. OXYGEN UPTAKE: BIODEGRADABILITY

		Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)
		0		7		14			21			28
O2 upt. (mg) test chemical	1
O2 upt. (mg) test chemical	2
O2 upt. (mg) test chemical	a, mean
O2 upt. (mg) blank	3
O2 upt. (mg) blank	4
O2 upt. (mg) blank	b, mean
Corrected BOD (mg)	(a1 - bm)
Corrected BOD (mg)	(a2 - bm)
BOD per mg test chemical	Image omitted; see official source
BOD per mg test chemical	Image omitted; see official source
% degradation Image omitted; see official source	D1 (a1)
% degradation Image omitted; see official source	D2 (a2)
% degradation Image omitted; see official source	Mean [^E0067]
V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask	V = volume of medium in test flask

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

N.B.: Similar formats may be used for the reference chemical and the toxicity controls.

6. CORRECTION FOR NITRIFICATION (see Annex V)

Day	0	28	Difference
(i) Concentration of nitrate (mg N/litre)			(N)
(ii) Oxygen equivalent (4,57 × N × V) (mg)	—	—
(iii) Concentration of nitrite (mg N/litre)			(N)
(iv) Oxygen equivalent (3,43 × N × V) (mg)	—	—
(ii + iv)  Total oxygen equivalent	—	—

7. CARBON ANALYSIS (optional)

Carbon analyser:

Time (day)	Blank mg/litre	Test chemical mg/litre
0	(Cblo)	(Co)
28 [^E0068]	(Cblt)	(Ct)

Or at end of incubation

Image omitted; see official source

8. SPECIFIC CHEMICAL (optional)

Sb	=	concentration in physico-chemical (sterile) control at 28 days
---	---	---
Sa	=	concentration in inoculated flask at 28 days,

Image omitted; see official source

9. ABIOTIC DEGRADATION (optional)

a	=	oxygen consumption in sterile flasks after 28 days, (mg)

Image omitted; see official source

(see Sections 1 and 3)

Image omitted; see official source

PART VI.   CLOSED BOTTLE TEST (Method C.4-E)

VI.1   PRINCIPLE OF THE TEST METHOD

The solution of the test chemical in mineral medium, usually at 2-5 mg/litre, is inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at constant temperature. Degradation is followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD or COD.

VI.2   DESCRIPTION OF THE METHOD

VI.2.1.   Apparatus

• a) BOD bottles, with glass stoppers, e.g. 250-300 ml;

• b) water bath or incubator, for keeping bottles at constant temperature (± 1 ^oC or better) with the exclusion of light;

• c) large glass bottles (2-5 litres) for the preparation of media and for filling the BOD bottles;

• d) oxygen electrode and meter, or equipment and reagents for Winkler titration.

VI.2.2.   Preparation of mineral medium

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

Mix 1 (one) ml of solution (a) to (d) and make up to 1 litre with dilution water.

VI.2.3.   Preparation of the inoculum

The inoculum is normally derived from the secondary effluent of a treatment plant or laboratory-scale unit receiving predominantly domestic sewage. An alternative source for the inoculum is surface water. Normally use from one drop (0,05 ml) to 5 ml of filtrate per litre of medium; trials may be needed to discover the optimum volume for a given effluent (See I.6.4.2 and I.6.5).

VI.2.4.   Preparation of flasks

Strongly aerate mineral medium for at least 20 min. Carry out each test series with mineral medium derived from the same batch. Generally, the medium is ready for use after standing for 20 h, at the test temperature. Determine the concentration of dissolved oxygen for control purposes; the value should be about 9 mg/litre at 20 ^oC. Conduct all transfer and filling operations of the air-saturated medium bubble-free, for example, by the use of siphons.

Prepare parallel groups of BOD bottles for the determination of the test and reference chemicals in simultaneous experimental series. Assemble a sufficient number of BOD bottles, including inoculum blanks, to allow at least duplicate measurements of oxygen consumption to be made at the desired test intervals, for example, after 0, 7, 14, 21 and 28 days. To ensure being able to identify the 10-day window, more bottles may be required.

Add fully aerated mineral medium to large bottles so that they are about one-third full. Then add sufficient of the stock solutions of the test chemical and reference chemical to separate large bottles so that the final concentration of the chemicals is normally not greater than 10 mg/litre. Add no chemicals to the blank control medium contained in a further large bottle.

In order to ensure that the inoculum activity is not limited, the concentration of dissolved oxygen must not fall below 0,5 mg/litre in the BOD bottles. This limits the concentration of test chemical to about 2 mg/litre. However, for poorly degradable compounds and those with a low ThOD, 5-10 mg/litre can be used. In some cases, it would be advisable to run parallel series of test chemical at two different concentrations, for example, 2 and 5 mg/litre. Normally, calculate the ThOD on the basis of formation of ammonium salts but, if nitrification is expected or known to occur, calculate on the basis of the formation of nitrate (ThODNO3: see Appendix 2.2). However, if nitrification is not complete but does occur, correct for the changes in concentration of nitrite and nitrate, determined by analysis, (see Appendix 5).

If the toxicity of the test chemical is to be investigated (in the case, for example, of a previous low biodegradability value having been found), another series of bottles is necessary.

Prepare another large bottle to contain aerated mineral medium (to about one-third of its volume) plus test chemical and reference chemical at final concentrations normally the same as those in the other large bottles.

Inoculate the solutions in the large bottles with secondary effluent (one drop or about 0,05 ml, to 5 ml/litre) or with another source such as river water (see I.6.4.2.). Finally, make up the solutions to volume with aerated mineral medium using a hose which reaches down to the bottom of the bottle to achieve adequate mixing.

VI.2.5.   Number of flasks in a typical run

In a typical run the following bottles are used:

• — at least 10 containing test chemical and inoculum (test suspension),

• — at least 10 containing only inoculum (inoculum blank),

• — at least 10 containing reference chemical and inoculum (procedure control),

• — and, when necessary, six bottles containing test chemical, reference chemical and inoculum (toxicity control). However, to ensure being able to identify the 10-day window, about twice as many bottles would be necessary.

VI.2.6.   Performance of the test

Dispense each prepared solution immediately into the respective group of BOD bottles by hose from the lower quarter (not the bottom) of the appropriate large bottle, so that all the BOD bottles are completely filled. Tap gently to remove any air bubbles. Analyse the zero-time bottles immediately for dissolved oxygen by the Winkler or electrode methods. The contents of the bottles can be preserved for later analysis by the Winkler method by adding manganese (II) sulfate and sodium hydroxide (the first Winkler reagent). Store the carefully stoppered bottles, containing the oxygen fixed as brown manganese (III) hydrated oxide, in the dark at 10-20 ^oC for no longer than 24 hours before proceeding with the remaining steps of the Winkler method. Stopper the remaining replicate bottles ensuring that no air bubbles are enclosed, and incubate at 20 ^oC in the dark. Each series must be accompanied by a complete parallel series for the determination of the inoculated blank medium. Withdraw at least duplicate bottles of all series for dissolved oxygen analysis at time intervals (at least weekly) over the 28 days incubation.

Weekly samples should allow the assessment of percentage removal in a 14-day window, whereas sampling every 3-4 days should allow the 10-day window to be identified, which would require about twice as many bottles.

For N-containing test substances, corrections for uptake of oxygen by any nitrification occurring should be made. To do this, use the O2-electrode method for determining the concentration of dissolved oxygen and then withdraw a sample from the BOD bottle for analysis for nitrite and nitrate. From the increase in concentration of nitrite and nitrate, calculate the oxygen used (see Annex V).

VI.3.   DATA AND REPORTING

VI.3.1.   Treatment of results

First calculate the BOD exerted after each time period by subtracting the oxygen depletion (mg O2/litre) of the inoculum blank from that exhibited by the test chemical. Divide this corrected depletion by the concentration (mg/litre) of the test chemical, to obtain the specific BOD as mg oxygen per mg test chemical. Calculate the percentage biodegradability by dividing the specific BOD by the specific ThOD (calculated according to Appendix 2.2) or COD (determined by analysis, see Appendix 2.3), thus:

Image omitted; see official source

= mg O2 per mg test chemical

Image omitted; see official source

Or

Image omitted; see official source

It should be noted that these two methods do not necessarily give same value; it is preferable to use the former method.

For test substances containing nitrogen, use the appropriate ThOD (NH4 or NO3) according to what is known or expected about the occurrence of nitrification (Appendix 2.2). If nitrification occurs but is not complete, calculate a correction for the oxygen consumed by nitrification from the changes in concentration of nitrite and nitrate (Appendix 5).

VI.3.2.   Validity of results

Oxygen depletion in the inoculum blank should not exceed 1,5 mg dissolved oxygen/litre after 28 days. Values higher than this require investigation of the experimental techniques. The residual concentration of oxygen in the test bottles should not fall below 0,5 mg/litre at any time. Such low oxygen levels are valid only if the method of determining dissolved oxygen used is capable of measuring such levels accurately.

See also I.5.2.

VI.3.3.   Reporting

See I.8.

VI.4.   DATA SHEET

An example of a data sheet is given hereafter.

CLOSED BOTTLE TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: … mg/litre

Initial concentration in bottle: … mg/litre

ThOD or COD: … mg O2/mg test substance

4. INOCULUM

Source:

Treatment given:

Pre-conditioning if any:

Concentration in the reaction mixture: … mg/litre

5. DO DETERMINATION

Method: Winkler/electrode

Flask analyses

Time of incubation (d)	Time of incubation (d)	Time of incubation (d)	DO (mg/l)	DO (mg/l)	DO (mg/l)	DO (mg/l)
Time of incubation (d)	Time of incubation (d)	Time of incubation (d)	0	n1	n2
Blank (without chemical)	1	C1
Blank (without chemical)	2	C2
Mean	Image omitted; see official source	Image omitted; see official source
Test chemical	1	a1
	2	a2
Mean	Image omitted; see official source	Image omitted; see official source

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

6. CORRECTION FOR NITRIFICATION (see Annex V)

Time of incubation (d)	0	n1	n2	n3
(i) Concentration of nitrate (mg N/litre)
(ii) Change in nitrate concentration (mg N/litre)	—
(iii) Oxygen equivalent (mg/litre)	—
(iv) Concentration of nitrite (mg N/litre)
(v) Change in nitrite concentration (mg N/litre)	—
(vi) Oxygen equivalent (mg/litre)	—
(iii + vi)  Total oxygen equivalent (mg/litre)	—

7. DO DEPLETION: % DEGRADATION

	Depletion after n days (mg/litre)	Depletion after n days (mg/litre)	Depletion after n days (mg/litre)	Depletion after n days (mg/litre)
	n1	n2	n3
FLASK 1: (mto - mtx) - (mbo - mbx)
FLASK 2: (mto - mtx) - (mbo - mbx)
FLASK 1: Image omitted; see official source
FLASK 2: Image omitted; see official source
% D mean [^E0069] = Image omitted; see official source

Do not take mean if there is considerable difference between duplicates.

mto	=	value in the flask at time 0
---	---	---
mtx	=	value in the flask at time x
---	---	---
mbo	=	mean blank value at time 0
---	---	---
mbx	=	mean blank value at time x

Apply also correction for nitrification from iii + vi in section 6.

8. BLANK DO DEPLETIONS

Oxygen consumption by blank: (mbo - mb28) mg/litre. This consumption is important for the validity of the test. It should be less than 1,5 mg/litre.

PART VII.   M.I.T.I. TEST (Method C.4-F)

VII.1.   PRINCIPLE OF THE METHOD

The oxygen uptake by a stirred solution, or suspension, of the test chemical in a mineral medium, inoculated with specially grown, unadapted micro-organisms, is measured automatically over a period of 28 days in a darkened, enclosed respirometer at 25 ± 1 ^oC. Evolved carbon dioxide is absorbed by soda lime. Biodegradability is expressed as the percentage oxygen uptake (corrected for blank uptake) of the theoretical uptake (ThOD). The percentage of primary biodegradability is also calculated from supplemental specific chemical analysis made at the beginning and end of incubation and, optionally, by DOC analysis.

VII.2.   DESCRIPTION OF THE METHOD

VII.2.1.   Apparatus

• (a) Automatic electrolytic BOD meter or respirometer normally equipped with six bottles, 300 ml each and equipped with cups to contain CO2 absorbent;

• (b) constant temperature room and/or water-bath at 25 ^oC ± 1 ^oC or better;

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

• (d) carbon analyser (optional).

VII.2.2.   Preparation of mineral medium

Prepare the following stock solutions, using analytical grade reagents and water (I.6.1.):

(a)	Monopotassium dihydrogen ortho phosphate, KH2PO4	8,50 g
(a)	Dipotassium monohydrogen ortho phosphate, K2HPO4	21,75 g
(a)	Disodium monohydrogen ortho phosphate dodecahydrate Na2HPO4 12 H2O	44,60 g
(a)	Ammonium chloride, NH4Cl	1,70 g
(a)	Dissolve in water and make up to 1 litre
(a)	The pH value of the solution should be 7,2
(b)	Magnesium sulphate heptahydrate, MgSO4 7 H2O	22,50 g
(b)	Dissolve in water and make up to 1 litre
(c)	Calcium chloride anhydrous, CaCl2	27,50 g
(c)	Dissolve in water and make up to 1 litre
(d)	Iron (III) chloride hexahydrate, FeCl3 6 H2O	0,25 g
(d)	Dissolve in water and make up to 1 litre

Take 3 ml of each solution (a), (b), (c) and (d) and make up to 1 litre.

VII.2.3.   Preparation of inoculum

Collect fresh samples from no fewer than ten sites, mainly in areas where a variety of chemicals are used and discharged. From sites such as sewage treatment works, industrial waste-water treatment, rivers, lakes, seas, collect 11 samples of sludge, surface soil, water, etc. and mix thoroughly together. After removing floating matter and allowing to stand, adjust the supernatant to pH 7 ± 1 with sodium hydroxide or phosphoric acid.

Use an appropriate volume of the filtered supernatant to fill a fill-and-draw activated sludge vessel and aerate the liquid for about 23 1/2 h. 30 minutes after stopping aeration, discard about one third of the whole volume of supernatant and add an equal volume of a solution (pH 7) containing 0,1 % each of glucose, peptone and monopotassium ortho phosphate, to the settled material and recommence aeration. Repeat this procedure once per day. The sludge unit must be operated according to good practice: effluents should be clear, temperature should be kept at 25 ± 2^o C, pH should be 7 ± 1, sludge should settle well, sufficient aeration to keep the mixture aerobic at all times, protozoa should be present and the activity of the sludge should be tested against a reference substance at least every three months. Do not use sludge as inoculum until after at least one month's operation, but not after more than four months. Thereafter, sample from at least 10 sites arregular intervals, once every three months.

In order to maintain fresh and old sludge at the same activity, mix the filtered supernatant of an activated sludge in use with an equal volume of the filtered supernatant of a freshly collected ten-source mixture and culture the combined liquor as above. Take sludge for use as inoculum 18-24 h after the unit has been fed.

VII.2.4.   Preparation of flasks

Prepare the following six flasks:

No 1: test chemical in dilution water at 100 mg/1

No 2, 3 and 4: test chemical in mineral medium at 100 mg/1

No 5: reference chemical (e.g. aniline) in mineral medium at 100 mg/1

No 6: mineral medium only

Add poorly soluble test chemicals directly on a weight or volume basis or handle as described in Appendix 3, except that neither solvents nor emulsifying agents should be used. Add the CO2 absorbent to all flasks in the special cups provided. Adjust the pH in flasks No 2, 3 and 4 to 7,0 .

VII.2.5.   Performance of the test

Inoculate flasks No 2, 3 and 4 (test suspensions), No 5 (activity control) and No 6 (inoculum blank) with a small volume of the inoculum to give a concentration of 30 mg/1 suspended solids. No inoculum is added to flask No 1 which serves as an abiotic control. Assemble the equipment, check for air-tightness, start the stirrers, and start the measurement of oxygen uptake under conditions of darkness. Daily check the temperature, stirrer and coulometric oxygen uptake recorder, and note any changes in colour of the contents of the flasks. Read the oxygen uptakes for the six flasks directly by an appropriate method, for example, from the six-point chart recorder, which produces a BOD curve. At the end of incubation, normally 28 days, measure the pH of the contents of the flasks and determine the concentration of the residual test chemical and any intermediate and, in the case of water soluble substance, the concentration o f DOC (Appendix 2.4). Take special care in the case of volatile chemicals. If nitrification is anticipated, determine nitrate and nitrite concentration, if possible.

VII.3.   DATA AND REPORTING

VII.3.1.   Treatment of results

Divide the oxygen uptake (mg) by the test chemical after a given time, corrected for that taken up by the blank inoculum conttol after the same time, by the weight of the test chemical used. This yields the BOD expressed as mg oxygen/mg test chemical, that is:

Image omitted; see official source

= mg O2 per mg test chemical

The percentage biodegradation is then obtained from:

Image omitted; see official source

For mixtures, calculate the ThOD from the elemental analysis, as for simple compound. Use the appropriate ThOD (ThODNH4 or ThODNO3) according to whether nitrification is absent or complete (Appendix 2.2). If however, nitrification occurs but is incomplete, make a correction for the oxygen consumed by nitrification calculated from the changes in concentrations of nitrite and nitrate (Appendix 5).

Calculate the percentage primary biodegradation from loss of specific (parent) chemical (see 1.7.2).

Image omitted; see official source

If there has been a loss of test chemical in the flask No 1 measuring physico-chemical removal, report this and use the concentration of test chemical (Sb) after 28 days in this flask to calculate the percentage biodegradation.

When determinations of DOC are made (optional), calculate the percentage ultimate biodegradation from:

Image omitted; see official source

as described under point I.7.1. If there has been a loss of DOC in the flask No 1, measuring physico-chemical removal, use the DOC concentration in this flask to calculate the percentage biodegradation.

Record all results on the data sheets attached.

VII.3.2.   Validity of results

The oxygen uptake of the inoculum blank is normally 20-30 mg O2/1 and should not be greater than 60 mgl/l in 28 days. Values higher than 60 mgl/l require critical examination of the data and experimental techniques. If the pH value is outside the range 6-8,5 and the oxygen consumption by the test chemical is less than 60 %, the test should be repeated with a lower concentration of test chemical.

See also I.5.2.

If the percentage degradation of aniline calculated from the oxygen consumption does not exceed 40 % after seven days and 65 % after 14 days, the test is regarded as invalid.

VII.3.3.   Reporting

See I.8.

VII.4.   DATA SHEET

An example of a data sheet is given below.

MITI (I) TEST

• 1. LABORATORY

• 1. DATE AT START OF TEST

3. TEST SUBSTANCE

Name:

Stock solution concentration: mg/1 as chemical

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

Volume of reaction mixture, V: … ml

ThOD: … mg O2/1

4. INOCULUM

Sludge sampling sites:

(1) …	(6) …
(2) …	(7) …
(3) …	(8) …
(4) …	(9) …
(5) …	(10) …

Concentration of suspended solids in activated sludge after acclimatisation with synthetic sewage = ... mg/1

Volume of activated sludge per litre of final medium = ... ml

Concentration of sludge in final medium = ... mg/1

5. OXYGEN UPTAKE: BIODEGRADABILITY

Type of respirometer used:

			Time (Days)	Time (Days)	Time (Days)	Time (Days)	Time (Days)
			0	7	14	21	28
O2 upt. (mg) test chemical	a1	a1
O2 upt. (mg) test chemical	a2	a2
O2 upt. (mg) test chemical	a3	a3
O2 upt. (mg) blank	b	b
Corrected BOD (mg)	(a1 - b1) (a1 - b1) (a1 - b1)	(a1 - b1) (a1 - b1) (a1 - b1)
BOD per mg test chemical	Image omitted; see official source	Flask 1
BOD per mg test chemical	Image omitted; see official source	Flask 2
BOD per mg test chemical	Image omitted; see official source	Flask 3
% degradation Image omitted; see official source		1
% degradation Image omitted; see official source		2
% degradation Image omitted; see official source		3
% degradation Image omitted; see official source		Mean [^E0070]

Do not take a mean if there are considerable differences between replicates.

N.B.: Similar formats may be used for the reference compound.

6. CARBON ANALYSIS (optional)

Carbon analyser:

Flask	DOC	DOC	DOC	DOC	% DOC removed	Mean
Flask	Measured	Measured	Corrected	Corrected	% DOC removed	Mean
Water + test substance	a				—	—
Sludge + test substance	b1		b1 - c
Sludge + test substance	b2		b2 - c
Sludge + test substance	b3		b3 - c
Control blank	c		—		—	—

Image omitted; see official source

7. SPECIFIC CHEMICAL ANALYTICAL DATA

	Residual amount of test chemical at end of test	% degradation
blank test with water	Sb
inoculated medium	Sa1
inoculated medium	Sa2
inoculated medium	Sa3

Image omitted; see official source

Calculate % degradation for flasks a1, and a3 respectively

8. REMARKS

BOD curve against time, if available, should be attached.

Appendix 1

ABBREVIATIONS AND DEFINITIONS

DO	:	Dissolved oxygen (mg/l) is the concentration of oxygen dissolved in an aqueous sample.
---	---	---
BOD	:	Biochemical oxygen demand (g) is the amount of oxygen consumed by micro-organisms when metabolising a test compound; also expressed as g oxygen uptake per g test compound. (See method C.5).
---	---	---
COD	:	Chemical oxygen demand (g) is the amount of oxygen consumed during oxidation of a test compound with hot, acidic dichromate; it provides a measure of the amount of oxidisable matter present; also expressed as 9 oxygen consumed per g test compound. (See method C.6).
---	---	---
DOC	:	Dissolved organic carbon is the organic carbon present in solution or that which passes through a 0,45 micrometre filter or remains in the supernatant after centrifuging at 40 000 m.s-2 (± 4 000 g) for 15 min.
---	---	---
ThOD	:	Theoretical oxygen demand (mg) is the total amount of oxygen required to oxidise a chemical completely; it is calculated from the molecular formula (see Appendix 2.2) and is also expressed as mg oxygen required per mg test compound.
---	---	---
ThCO2	:	Theoretical carbon dioxide (mg) is the quantity of carbon dioxide calculated to be produced from the known or measured carbon content of the test compound when fully mineralised; also expressed as mg carbon dioxide evolved per mg test compound.
---	---	---
TOC	:	Total organic carbon of a sample is the sum of the organic carbon in solution and in suspension.
---	---	---
IC	:	Inorganic carbon
---	---	---
TC	:	Total carbon, is the sum of the organic and inorganic carbon present in a sample.

Primary biodegradation:

is the alteration in the chemical structure of a substance, brought about by biological action, resulting in the loss of specific property of that substance.

Ultimate biodegradation (aerobic):

is the level of degradation achieved when the test compound is totally utilised by micro-organisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass).

Readily biodegradable:

an arbitrary classification of chemicals which have passed certain specified screening tests for ultimate biodegradability; these tests are so stringent that it is assumed that such compounds will rapidly and completely biodegrade in aquatic environments under aerobic conditions.

Inherently biodegradable:

a classification of chemicals for which there is unequivocal evidence of biodegradation (primary or ultimate) in any recognized test of biodegradability.

Treatability:

is the amenability of compounds to removal during biological wastewater treatment without adversely affecting the normal operation of the treatment processes. Generally, readily biodegradable compounds are treatable but not all inherently biodegradable compounds are. Abiotic processes may also operate.

Lag time

is the time from inoculation, in a die-away test, until the degradation percentage has increased to at least 10 %. The lag time is often highly variable and poorly reproducible.

Degradation time

is the time from the end of the lag time till the time that 90 % of maximum level of degradation has been reached.

10-day window

is the 10 days immediately following the attainment of 10 % degradation.

Appendix 2

CALCULATION AND DETERMINATION OF SUITABLE SUMMARY PARAMETERS

Depending on the method chosen, certain summary parameters will be required. The following section describes the derivation of these values. The use of these parameters is described in the individual methods.

1. Carbon content

The carbon content is calculated from the known elemental composition or determined by elemental analysis of the test substance.

2. Theoretical oxygen demand (ThOD)

The theoretical oxygen demand (ThOD) may be calculated if the elemental composition is known or derermined by elemental analysis. It is for the compound:

CcHhClclNnNanaOoPpSs

without nitrification,

Image omitted; see official source

mg/mg

or with nitrification

Image omitted; see official source

mg/mg

3. Chemical Oxygen Demand (COD)

The chemical oxygen demand (COD) is determined according to method C.6.

4. Dissolved organic carbon (DOC)

Dissolved organic carbon (DOC) is by definition the organic carbon of any chemical or mixture in water passing through a 0,45 micrometre filter.

Samples from the test vessels are withdrawn and filtered immediately in the filtration apparatus using an appropriate membrane filter. The first 20 ml (amount can be reduced when using small filters) of the filtrate are discarded. Volumes of 10-20 ml or lower, if injected (volume depending on the amount required for carbon analyser) are retained for carbon analysis. The DOC concentration is determined by means of an organic carbon analyser which is capable of accurately measuring a carbon concentration equivalent or lower than 10 % of the initial DOC concentration used in the test.

Filtered samples which cannot be analysed on the same working day can be preserved by storage in a refrigerator at 2-4 ^oC for 48 h, or below - 18 ^oC for longer periods.

Remarks:

Membrane filters are often impregnated with surfactants for hydrophilisation. Thus the filter may contain up to several mg of soluble organic carbon which would interfere in the biodegradability determinations. Surfactants and other soluble organic compounds are removed from the filters by boiling them in deionised water for three periods each of one hour. The filters may then be stored in water for one week. If disposable filter cartridges are used each lot must be checked to confirm that it does not release soluble organic carbon.

Depending on the type of membrane filter the test chemical may be retained by adsorption. It may therefore be advisable to ensure that the test chemical is not retained by the filter.

Centrifugation at 40 000 m.sec^-2 (4 000 g) for 15 min may be used for differentiation of TOC versus DOC instead of filtration. The method is not reliable at initial concentration of < 10 mg DOC/l since either not all bacteria are removed or carbon as part of the bacterial plasma is redissolved.

BIBLIOGRAPHY

• — Standard Methods for the Examination of Water and Wastewater, 12th ed, Am. Pub. Hlth. Ass., Am. Wat. Poll. Control Fed., Oxygen Demand, 1965, P 65.

• — Wagner, R., Von Wasser, 1976, vol. 46, 139.

• — DIN-Entwurf 38 409 Teil 41 Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung, Summarische Wirkungs- und Stoffkenngrößen (Gruppe H). Bestimmung des Chemischen Sauerstoffbedarfs (CSB) (H 41), Normenausschuß Wasserwesen (NAW) in DIN Deutsches Institut für Normung e. V.

• — Gerike, P., The biodegradability testing of poorly water soluble compounds. Chemosphere, 1984, vol 13 (1), 169.

Appendix 3

EVALUATION OF THE BIODEGRADABILITY OF POORLY SOLUBLE SUBSTANCES

In biodegradability tests with poorly soluble substances the following aspects should receive special attention.

While homogeneous liquids will seldom present sampling problems, it is recommended that solid materials be homogenised by appropriate means to avoid errors due to non-homogeneity. Special care must be taken when representative samples of a few milligrams are required from mixtures of chemicals or substances with large amounts of impurities.

Various forms of agitation during the tests may be used. Care should be taken to use only sufficient agitation to keep the chemical dispersed, and to avoid overheating, excessive foaming and excessive shear forces.

An emulsifier which gives a stable dispersion of the chemical may be used. It should not be toxic to bacteria and must not be biodegraded or cause foaming under test conditions.

The same criteria apply to solvents as to the emulsifiers.

It is not recommended that solid carriers be used for solid test substances but they may be suitable for only substances.

When auxiliary substances such as emulsifiers, solvents and carriers are used, a blank run containing the auxiliary substance should be performed.

Any of the three respirometric tests CO2, BOD, MITI can be used to study the biodegradability of poorly soluble compounds.

BIBLIOGRAPHY

• — de Morsier, A. et al., Biodegradation tests for poorly soluble compounds. Chemosphere, 1987, vol. 16, 833.

• — Gerike, P, The Biodegradability testing of poorly water soluble compounds. Chemosphere, 1984, vol. 13, 169.

Appendix 4

EVALUATION OF THE BIODEGRADABILITY OF CHEMICALS SUSPECTED TO BE TOXIC TO THE INOCULUM

When a chemical is subjected to ready biodegradability testing and appears to be non-biodegradable, the following procedure is recommended if a distinction between inhibition and inertness is desired (Reynolds et al., 1987).

Similar or identical inocula should be used for the toxicity and biodegradation tests.

To assess the toxicity of chemicals studied in ready biodegradability tests, the application of one or a combination of the inhibition of Sludge Respiration rate (activated sludge respiration inhibition test — Directive 87/302/EEC), BOD and/or Growth Inhibition methods would seem appropriate.

If inhibition due to toxicity is to be avoided, it is suggested that the test substance concentrations used in ready biodegradability testing should be less than 1/10 of the EC50 values (or less than EC20 values) obtained in toxicity testing. Compounds with an EC50 value of greater than 300 mg/1 are not likely to have toxic effects in ready biodegradability testing.

EC50 values of less than 20 mg/1 are likely to pose serious problems for the subsequent testing. Low test concentrations should be employed, necessitating the use of the stringent and sensitive Closed Bottle test or the use of ¹⁴C-labelled material. Alternatively, an acclimatised inoculum may permit higher test substance concentrations to be used. In the latter case, however, the specific criterion of the ready biodegradability test is lost.

BIBLIOGRAPHY

Reynolds, L. et al., Evaluation of the toxicity of substances to be assessed for biodegradability. Chemosphere, 1987, vol. 16, 2259.

Appendix 5

CORRECTION FOR OXYGEN UPTAKE FOR INTERFERENCE BY NITRIFICATION

Errors due to not considering nitrification in the assessment by oxygen uptake of the biodegradability of test substances not containing N are marginal (not greater than 5 %), even if oxidation of the ammonium-N in the medium occurs erratically as between test and blank vessels. However, for test substances containing N, serious errors can arise.

If nitrification has occurred but is not complete the observed oxygen uptake by the reaction mixture may be corrected for the amount of oxygen used in oxidising ammonium to nitrite and nitrate, if the changes in concentration during incubation of nitrite and nitrate are determined by consideration of the following equations:

2 NH4Cl + 3 O2 = 2 HNO2 + 2 HCl + 2 H2O	(1)
2 HNO2 + O2 = 2 HNO3	(2)
Overall:
2 NH4Cl + 4 O2 = 2 HNO3 + 2 HCl + 2 H2O	(3)

From equation (1), the oxygen uptake by 28 g of nitrogen contained in ammonium chloride (NH4Cl) in being oxidised to nitrite is 96 g, i.e. a factor of 3,43 (96/28). In the same way, from equation (3) the oxygen uptake by 28 g of nitrogen in being oxidised to nitrate is 128 g, i.e. a factor of 4,57 (128/28).

Since the reactions are sequential, being carried out by distinct and different bacterial species, it is possible for the concentration of nitrite to increase or decrease; in the latter case an equivalent concentration of nitrate would be formed. Thus, the oxygen consumed in the formation of nitrate is 4,57 multiplied by the increase in concentration of nitrate, whereas the oxygen associated with the formation of nitrite is 3,43 multiplied by the increase in the concentration of nitrite or with the decrease in its concentration the oxygen loss is - 3,43 multiplied by the decrease in concentration.

That is:

O2 consumed in nitrate formation = 4,57 × increase in nitrate concentration	(4)
and
O2 consumed in nitrite formation = 3,43 × increase in nitrite concentration	(5)
and
O2 lost in nitrite disappearance = - 3,43 × decrease in nitrate concentration	(6)
So that
O2 uptake due to nitrification = ± 3,43 × change in nitrite conc. + 4,57 × increase in nitrate conc.	(7)
and thus
O2 uptake due to C oxidation = total observed uptake uptake due to nitrification	(8).

Alternatively, if only total oxidised N is determined, the oxygen uptake due to nitrification may be taken to be, as a first approximation, 4,57 × increase in oxidised N

The corrected value for oxygen consumption due to C oxidation is then compared with ThOD NH3, as calculated in Appendix 2.

C.5   DEGRADATION — BIOCHEMICAL OXYGEN DEMAND

1. METHOD

1.1.   INTRODUCTION

The purpose of the method is the measurement of the biochemical oxygen demand (BOD) of solid or liquid organic substances.

Data elaborated with this test pertain to water-soluble compounds; however, volatile compounds and those of low water solubility may also, at least in principle, be tested.

The method is applicable only to those organic test materials which are not inhibitory to bacteria at the concentration used in the test. If the test material is not soluble at the test concentration, special measures, such as the use of ultrasonic dispersion, may have to be employed to achieve good dispersion of test material.

Information on the toxicity of the chemical may be useful to the interpretation of low results and in the selection of appropriate test concentrations.

1.2.   DEFINITION AND UNITS

The BOD is defined as the mass of dissolved oxygen required by a specified volume of solution of the substance for the process of biochemical oxidation under prescribed conditions.

The results are expressed as grams of BOD per gram of tested substance.

1.3.   REFERENCE SUBSTANCES

The use of a suitable reference substance to check the activity of the inoculum is desirable.

1.4.   PRINCIPLE OF THE TEST METHOD

A predetermined amount of the substance, dissolved or dispersed in a well-aerated suitable medium, is inoculated with micro-organisms and incubated at a constant defined ambient temperature in the dark.

The BOD is determined by the difference in dissolved oxygen content at the beginning and at the end of the test. The duration of the test must be at least five days and not more than 28 days.

A blank must be determined in a parallel assay containing no test substance.

1.5.   QUALITY CRITERIA

The BOD determination cannot be considered as a valid determination of the biodegradability of a substance. This test may only be regarded as a screening test.

1.6.   DESCRIPTION OF THE TEST METHOD

A preliminary solution or dispersion of the substance is prepared to obtain a BOD concentration compatible with the method used. The BOD is then determined following any suitable national or international standardised method.

2. DATA AND EVALUATION

The BOD contained in the preliminary solution is calculated according to the selected normalised method, and converted into grams of BOD per gram of tested substance.

3. REPORTING

The method used shall be stated.

The biochemical oxygen demand should be a mean of at least three valid measurements.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities, physical state, toxic effects and inherent composition of the substance which would affect the results.

The use of an additive to inhibit biological nitrification must be reported.

4. REFERENCES

List of standardised methods, for example:

NF T 90-103: Determination of the biochemical oxygen demand.

NBN 407: Biochemical oxygen demand.

NEN 32355.4: Bepaling van het biochemish zuurstofverbruik (BZV).

The determination of biochemical oxygen demand, Methods for the examination of water and associated materials, HMSO, London.

ISO 5815: Determination of biochemical oxygen demand after n days.

C.6.   DEGRADATION — CHEMICAL OXYGEN DEMAND

1. METHOD

1.1.   INTRODUCTION

The purpose of the method is the measurement of the chemical oxygen demand (COD) of solid or liquid organic substances in a standard, arbitrary manner, under fixed laboratory conditions.

Information on the formula of the substance will be useful to conduct this test and interpret the result obtained (e.g. halogen salts, ferrous salts of organic compounds, organochlorine compounds).

1.2.   DEFINITIONS AND UNITS

The chemical oxygen demand is a measure of the oxidisability of a substance, expressed as the equivalent amount in oxygen of an oxidising reagent consumed by the substance under fixed laboratory conditions.

The result is expressed in grams of COD per gram of tested substance.

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should serve primarily to calibrate the method from time to time and to allow comparison of results when another method is applied.

1.4.   PRINCIPLE OF THE TEST METHOD

A predetermined amount of the substance, dissolved or dispersed in water, is oxidised by potassium dichromate in a strong sulphuric acid medium with silver sulphate as a catalyst, under reflux for two hours. The residual dichromate is determined by titration with standardised ferrous ammonium sulphate.

In case of chlorine-containing substances, mercuric sulphate ([^E0071]) is added to reduce chloride interference.

1.5.   QUALITY CRITERIA

Because of the arbitrary manner of determination, COD is an ‘oxidisability indicator’ and as such is used as a practical method to measure organic matter.

Chloride can interfere in this test; inorganic reducing or oxidising agents may also interfere with the COD determination.

Some cyclic compounds and many volatile substances (e.g. lower fatty acids) are not fully oxidised by this test.

1.6.   DESCRIPTION OF THE TEST METHOD

A preliminary solution or dispersion of the substance is prepared to obtain a COD between 250 and 600 mg per litre.

Remarks:

In the case of poorly soluble and non-dispersible substances, an amount of finely powdered substance or liquid substance corresponding to about 5 mg of COD can be weighed and put in the experimental apparatus with water.

The chemical oxygen demand (COD) is often and especially in case of poorly soluble substances determined advantageously in a variant of the method, i.e., in a closed system with a pressure equaliser (H. Kelkenberg, 1975). In this modification compounds which are only with difficulty determined by the conventional method — e.g. acetic acid — may often be successfully quantified. The method also fails, however, in the case of pyridine. If the potassium dichromate concentration, as prescribed in ref.(1), is raised to 0,25 N (0,0416 M), the direct weighing-in of 5-10 mg of substance is facilitated which is essential for the COD determination of poorly water soluble substances (ref. (2)).

Otherwise, the COD is then determined following any suitable national or international standardised method.

2. DATA AND EVALUATION

The COD contained in the experimental flask is calculated following the selected normalised method, and converted to grams of COD per gram of tested substance.

3. REPORTING

The reference method used should be stated.

The chemical oxygen demand should be a mean of at least three measurements. All information and remarks relevant to the interpretation of the results have to be reported, especially with regard to impurities, physical state and inherent properties of the substance (if known) which would affect the results.

The use of mercuric sulphate to minimise the chloride interference must be reported.

4. REFERENCES

• (1)Kelkenberg, H.,Z. von Wasser und Abwasserforschung, 1975, vol. 8, 146.

• (2)Gerike, P. The biodegradability testing of poorly water soluble compounds. Chemosphere, 1984, vol. 13,169.

• List of standardised methods, for example:

NBN T 91-201 Determination of the chemical oxygen demand.

ISBN O 11 7512494 Chemical oxygen demand (dichromate value) of polluted and waste waters.

NF T 90-101 Determination of the chemical oxygen demand.

DS 217 = water analysis Determination of the chemical oxygen demand.

DIN 38409-H-41 Determination of the chemical oxygen demand (COD) within the range above 15 mg per litre.

NEN 3235 5.3 Bepaling van het chemisch zuurstofverbruik.

ISO 6060 Water quality: chemical oxygen demand dichromate methods.

C.7.   DEGRADATION — ABIOTIC DEGRADATION: HYDROLYSIS AS A FUNCTION OF PH

1. METHOD

This testing method is equivalent to the OECD TG 111 (2004).

1.1.   INTRODUCTION

Chemicals can enter surface waters by such routes as direct application, spray drift, run-off, drainage, waste disposal, industrial, domestic or agricultural effluent and atmospheric deposition and may be transformed in those waters by chemical (e.g. hydrolysis, oxidation), photochemical and/or microbial processes. This Guideline describes a laboratory test method to assess abiotic hydrolytic transformations of chemicals in aquatic systems at pH values normally found in the environment (pH 4-9) and is based on existing Guidelines (1)(2)(3)(4)(5)(6)(7).

The experiments are performed to determine (i) the rate of hydrolysis of the test substance as a function of pH and (ii) the identity or nature and rates of formation and decline of hydrolysis products to which organisms may be exposed. Such studies may be required for chemicals which are directly applied to water or that are likely to reach the environment by the other routes described above.

1.2.   DEFINITIONS AND UNITS

See Appendix 2

1.3.   APPLICABILITY OF THE METHOD

The method is generally applicable to chemical substances (unlabelled or labelled) for which an analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile and non-volatile compounds of sufficient solubility in water. The test should not be applied to chemicals that are highly volatile from water (e.g. fumigants, organic solvents) and thus cannot be kept in solution under the experimental conditions of this test. The test may be difficult to conduct with substances of minimal solubility in water (8).

1.4.   PRINCIPLE OF THE TEST METHOD

Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) are treated with the test substance and incubated in the dark under controlled laboratory conditions (at constant temperatures). After appropriate time intervals, buffer solutions are analysed for the test substance and for hydrolysis products. With labelled test substance (e.g. ¹⁴C), a mass balance can be more easily established.

This testing method is designed as a tiered approach which is shown and explained in Appendix 1. Each tier is triggered by the results of the previous tier.

1.5.   INFORMATION ON THE TEST SUBSTANCE

Non-labelled or labelled test substance can be used to measure the rate of hydrolysis. Labelled material is generally preferred for studying the pathway of hydrolysis and for establishing mass balance; however, in special cases, labelling may not be absolutely necessary. ¹⁴C-labelling is recommended but the use of other isotopes, such as ¹³C, ¹⁵N, ³H, may also be useful. As far as possible, the label should be positioned in the most stable part(s) of the molecule. For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable information on formation of hydrolysis products. The purity of the test substance should be at least 95 %.

Before carrying out a hydrolysis test, the following information on the test substance should be available:

• (a) solubility in water [Testing Method A.6];

• (b) solubility in organic solvents;

• (c) vapour pressure [Testing Method A.4] and/or Henry's Law constant;

• (d) n-octanol/water partition coefficient [Testing Method A.8];

• (e) dissociation constant (pKa) [OECD Guideline 112] (9);

• (f) direct and indirect phototransformation rate in water where appropriate.

Analytical methods for quantification of the test substance and, if it is relevant, for identification and quantification of hydrolysis products in aqueous solutions should be available (see also Section 1.7.2).

1.6.   REFERENCE SUBSTANCES

Where possible, reference substances should be used for the identification and quantification of hydrolysis products by spectroscopic and chromatographic methods or other suitably sensitive methods.

1.7.   QUALITY CRITERIA

1.7.1.   Recovery

Analysis of, at least, duplicate buffer solutions or of their extracts immediately after the addition of the test substance gives a first indication of the repeatability of the analytical method and of the uniformity of the application procedure for the test substance. Recoveries for later stages of the experiments are given by the respective mass balances (when labelled material is used). Recoveries should range from 90 % to 110 % for labelled and non labelled chemicals (7). In case it is technically difficult to reach this range, a recovery of 70 % for non labelled chemicals is acceptable, but justification should be given.

1.7.2.   Repeatability and sensitivity of analytical method

Repeatability of the analytical method(s) used to quantify the test substance and hydrolysis products at later times can be checked by duplicate analysis of the same buffer solutions (or of their extracts) after sufficient quantities of hydrolysis products have formed for quantification

The analytical method should be sufficiently sensitive to quantify test substance concentrations down to 10 % or less of the initial concentration. If relevant, analytical methods should also be sufficiently sensitive to quantify any hydrolysis product representing 10 % or more of applied (at any time during the study) down to 25 % or less of its peak concentration.

1.7.3.   Confidence intervals for hydrolysis kinetic data

Confidence intervals should be computed and presented for all regression coefficients, rate constants, half-lives, and any other kinetic parameters (e.g. DT50).

1.8.   DESCRIPTION OF THE TEST METHOD

1.8.1.   Equipment and apparatus

The study should be performed in glass containers (e.g. test tubes, small flasks) under dark and sterile conditions, if necessary, unless preliminary information (such as the n-octanol-water partition coefficient) indicates that the test substance may adhere to glass. In such cases, alternative materials (such as Teflon) may have to be considered. It may also be possible to alleviate the problem of adhere to glass by using one or more of the following methods:

• — determine the mass of test substance and hydrolysis products sorbed to the test vessel,

• — use of an ultrasonic bath,

• — ensure a solvent wash of all glassware at each sampling interval,

• — use of formulated products,

• — use an increased amount of co-solvent for addition of test substance to the system; if a co-solvent is used it should be a co-solvent that does not hydrolyse the test substance.

Temperature-controlled water bath shakers or thermostatically controlled incubators for incubation of the various test solutions are normally required.

Standard laboratory equipment is required, including, in particular, the following:

• — pH meter,

• — analytical instruments such as GC, HPLC, TLC equipment, including the appropriate detection systems for analysing radiolabelled and non-labelled substances or inverse isotopes dilution method,

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

• — liquid scintillation counter,

• — separating funnels for liquid-liquid extraction,

• — instrumentation for concentrating solutions and extracts (e.g. rotating evaporator),

• — temperature control devise (e.g. water bath).

Chemical reagents include, for example:

• — organic solvents, analytical grade, such as hexane, dichloromethane, etc.,

• — scintillation liquid,

• — buffer solutions (for details see Section 1.8.3).

All glassware, reagent-grade water and buffer solutions used in the hydrolysis tests should be sterilised.

1.8.2.   Test substance application

The test substance should be applied as aqueous solution into the different buffer solutions (see Appendix 3). If it is necessary for adequate dissolution, the use of low amounts of water miscible solvents (such as acetonitrile, acetone, ethanol) is permitted for application and distribution of the test substance but this should not normally exceed 1 % v/v. In case a higher concentration of solvents is considered (e.g. in the case of poorly soluble test substances), this could only be allowed when it can be shown that the solvent has no effect on the hydrolysis of the test substances.

The use of formulated product is not routinely recommended, as it cannot be excluded that the formulation ingredients may influence the hydrolysis process. However, for poorly water-soluble test substances or for substances that adhere to glass (see Section 1.8.1), the use of formulated material may be an appropriate alternative.

One concentration of the test substance should be used; it should not exceed 0,01 M or half of the saturation concentration (see Appendix 1).

1.8.3.   Buffer solutions

The hydrolysis test should be performed at pH values of 4, 7 and 9. For this purpose, buffer solutions should be prepared using reagent grade chemicals and water. Some useful buffer systems are presented in Appendix 3. It should be noted that the buffer system used may influence the rate of hydrolysis and where this is observed an alternate buffer system should be employed ([^E0072]).

The pH of each buffer solution should be checked with a calibrated pH meter to a precision of at least 0,1 at the required temperature.

1.8.4.   Test conditions

1.8.4.1.   Test temperature

The hydrolysis experiments should be carried out at constant temperatures. For extrapolation purposes, it is important to maintain the temperature to at least ± 0,5 ^oC.

A preliminary test (Tier 1) should be conducted at a temperature of 50 ^oC if the hydrolytic behaviour of the test substance is unknown. Higher Tier kinetic tests should be carried out with a minimum of three temperatures (including the test at 50 ^oC) unless the test substance is stable to hydrolysis as determined by the Tier 1 testing. A suggested temperature range is 10-70 ^oC (preferably with at least one temperature below 25 ^oC utilised), which will encompass the reporting temperature of 25 ^oC and most of the temperatures encountered in the field.

1.8.4.2.   Light and oxygen

All of the hydrolysis tests should be carried out using any suitable method to avoid photolytic effects. All suitable measures should be taken to avoid oxygen (e.g. by bubbling helium, nitrogen or argon for five minutes before preparation of the solution).

1.8.4.3.   Test duration

The preliminary test should be carried out for 5 days whereas the higher Tier tests should be conducted until 90 % hydrolysis of the test substance or for 30 days whichever comes first.

1.8.5.   Performance of the test

1.8.5.1.   Preliminary test (Tier 1)

The preliminary test is performed at 50 ± 0,5 ^oC and pH 4,0 , 7,0 and 9,0 . If less than 10 % of hydrolysis is observed after 5 days (t0,5 25 ^oC > 1 year), the test substance is considered hydrolytically stable and, normally, no additional testing is required. If the substance is known to be unstable at environmentally relevant temperatures ([^E0073]), the preliminary test is not required. The analytical method must be sufficiently precise and sensitive to detect a reduction of 10 % in the initial concentration.

1.8.5.2.   Hydrolysis of unstable substances (Tier 2)

The higher Tier (advanced) test should be performed at the pH values at which the test substance was found unstable as defined by the preliminary test above. The buffered solutions of the test substance should be thermostated at the selected temperatures. To test for first-order behaviour, each reaction solution should be analysed in time intervals which provide a minimum of six spaced data points normally between 10 % and 90 % hydrolysis of the test substance. Individual replicate test samples (a minimum of duplicate samples contained in separate reaction vessels) should be removed and the contents analysed at each of at least six sampling times (for a minimum of twelve replicate data points). The use of a single bulk sample from which individual aliquots of the test solution are removed at each sampling interval is considered to be inadequate, as it does not allow for the analysis of data variability and it may lead to problems with contamination of the test solution. Sterility confirmation tests should be conducted at the end of the higher Tier test (i.e. at 90 % hydrolysis or 30 days). However, if no degradation (i.e. transformation) is observed, sterility tests are not considered necessary.

1.8.5.3.   Identification of hydrolysis products (Tier 3)

Any major hydrolysis products at least those representing > 10 % of the applied dose should be identified by appropriate analytical methods.

1.8.5.4.   Optional tests

Additional tests at pH values other than 4, 7 and 9 may be required for a hydrolytically unstable test substance. For example, for physiological purposes a test under more acidic conditions (e.g. pH 1,2 ) may be required employing a single physiologically relevant temperature (37 ^oC).

2. DATA

The amounts of test substance and of hydrolysis products, if relevant, should be given as % of applied initial concentration and, where appropriate, as mg/L for each sampling interval and for each pH and test temperature. In addition, a mass balance should be given in percentage of the applied initial concentration when labelled test substance has been used.

A graphical presentation of the log-transformed data of the test substance concentrations against time should be reported. Any major hydrolysis products at least those representing ≥ 10 % of the applied dose should be identified and their log-transformed concentrations should also be plotted in the same manner as the parent substance to show their rates of formation and decline.

2.1.   TREATMENT OF RESULTS

More accurate determinations of half-lives or DT50 values should be obtained by applying appropriate kinetic model calculations. The half-life and/or DT50 values (including confidence limits) should be reported for each pH and temperature together with a description of the model used the order of kinetics and the coefficient of determination (r²). If appropriate, the calculations should also be applied to the hydrolysis products.

In the case of rate studies carried out at different temperatures, the pseudo first-order hydrolysis rate constants (kobs) should be described as a function of temperature. The calculation should be based on both the separation of kobs into rate constants for acid catalysed, neutral, and base catalysed hydrolysis (kH, kneutral, and kOH respectively) and the Arrhenius equation:

Image omitted; see official source

where Ai and Bi are regression constants from the intercept and slope, respectively, of the best fit lines generated from linearly regressing ln ki against the reciprocal of the absolute temperature in Kelvin (T). Through the use of the Arrhenius relationships for acid, neutral and base catalysed hydrolysis, pseudo first-order rate constants, and thus half-lives can be calculated for other temperatures for which the direct experimental determination of a rate constant is not practicable (10).

2.2.   EVALUATION AND INTERPRETATION OF RESULTS