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)

Most hydrolysis reactions follow apparent first order reaction rates and, therefore, half-lives are independent of the concentration (see equation 4 in Appendix 2). This usually permits the application of laboratory results determined at 10^-2 to 10^-3 M to environmental conditions (≤ 10^-6 M) (10). Several examples of good agreement between rates of hydrolysis measured in both pure and natural waters for a variety of chemicals were reported by Mabey and Mill (11), provided both pH and temperature had been measured.

3. REPORTING

3.1.   TEST REPORT

The test report must include at least the following information:

Test substance:

• — common name, chemical name, CAS number, structural formula (indicating position of label when radiolabelled material is used) and relevant physical-chemical properties (see Section 1.5);

• — purity (impurities) of test substance,

• — label purity of labelled chemical and molar activity (where appropriate).

• — Buffer solutions:

• — dates and details of preparation,

• — buffers and waters used,

• — molarity and pH of buffer solutions.

Test conditions:

• — dates of the performance of the studies,

• — amount of test substance applied,

• — method and solvents (type and amount) used for application of the test substance,

• — volume of buffered test substance solutions incubated,

• — description of the incubation system used,

• — pH and temperature during the study,

• — sampling times,

• — method(s) of extraction,

• — methods for quantification and identification of the test substance and its hydrolysis products in the buffer solutions,

• — number of replicates.

Results:

• — repeatability and sensitivity of the analytical methods used,

• — recoveries (% values for a valid study are given in Section 1.7.1),

• — replicate data and means in a tabular forms,

• — mass balance during and at the end of the studies (when labelled test substance is used),

• — results of preliminary test,

• — discussion and interpretation of results,

• — all original data and figures.

The following information is only required when hydrolysis rate is determined:

• — plots of concentrations versus time for the test substances and, where appropriate, for the hydrolysis products at each pH value and temperature;

• — tables of results of Arrhenius equation for the temperature 20 ^oC/25 ^oC, with pH, rate constant [h^-1 or day^-1], half-life or DT50, temperatures [^oC] including confidence limits and the coefficients of correlation (r²) or comparable information;

• — proposed pathway of hydrolysis.

4. REFERENCES

• (1)OECD, (1981) Hydrolysis as a Function of pH. OECD Guideline for Testing of Chemicals No 111, adopted 12 May 1981.

• (2)US-Environmental Protection Agency, (1982) 40 CFR 796.3500, Hydrolysis as a Function of pH at 25 ^oC. Pesticide Assessment Guidelines, Subdivision N. Chemistry: Environmental Fate.

• (3)Agriculture Canada, (1987) Environmental Chemistry and Fate Guidelines for registration of pesticides in Canada.

• (4)European Union (EU), (1995) Commission Directive 95/36/EC amending Council Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex V: Fate and Behaviour in the Environment.

• (5)Dutch Commission for Registration of Pesticides, (1991) Application for registration of a pesticide. Section G: Behaviour of the product and its metabolites in soil, water and air.

• (6)BBA, (1980) Merkblatt No 55, Teil I und II: Prüfung des Verhaltens von Pflanzenbehandlungsmitteln im Wasser (October 1980).

• (7)SETAC, (1995) Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides. Mark R. Lynch, Ed.

• (8)OECD, (2000) Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD Environmental Health and Safety Publications Series on Testing and Assessment No 23.

• (9)OECD, (1993) Guidelines for the Testing of Chemicals. Paris. OECD (1994-2000): Addenda 6-11 to Guidelines for the Testing of Chemicals.

• (10)Nelson, H, Laskowski D, Thermes S, and Hendley P., (1997) Recommended changes in pesticide fate study guidelines for improving input to computer models. (Text version of oral presentation at the 14th Annual Meeting of the Society of Environmental Toxicology and Chemistry, Dallas TX, November 1993).

• (11)Mabey, W. and Mill, T., (1978) Critical review of hydrolysis of organic compounds in water under environmental conditions. J. Phys. Chem. Ref. Data 7, p. 383-415.

Appendix 1

Tiered hydrolysis test scheme

Image omitted; see official source

Text of image

Yes

Is the water solubility of the test substance > 2 10^-2 M ?

No

Prepare a solution in buffer of ≤ 10^-2 M

Prepare a half-saturated solution in buffer

Yes

Is the test substance known to be unstable hydrolytically ?

No

Conduct preliminary test for 5 days at pH 4, 7, 9 at 50°C

Yes

Interim Assessment: Is < 10 % of the test substance hydrolysed in 5 days* (t0.5>1y at 25°C)

No

Conduct hydrolysis tests at 3 temperatures in the range of 10-70°C and determine the hydrolysis rate for 25°C

Interim Assessment: Hydrolysis product(s) formed in significant amount (≥ 10 %) in pH ranges 4-9 at 25°C ?

No

Expert judgement of the relevance of and need for identification of hydrolysis products

Yes

Yes

Isolate and identify hydrolysis product(s)

No further hydrolysis tests

• 10 % hydrolysis of a test substance at 50°C corresponds to a half-life of approx. 30 days which corresponds to a value of approx. 1 year at 20°C.

No

Appendix 2

Definitions and units

Standard International (SI) units should be used in any case.

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

Transformation products: all substances resulting from biotic or abiotic transformation reactions of the test substance.

Hydrolysis products: all substances resulting from hydrolytic transformation reactions of the test substance.

Hydrolysis refers to a reaction of a test substance RX with water, with the net exchange of the group X with OH at the reaction centre:

RX + HOH → ROH + HX	[1]

The rate at which the concentration of RX decreases in this simplified process is given by

rate = k [H2O] [RX]	second order reaction
or	or
rate = k [RX]	first order reaction

depending on the rate determining step. Because the water is present in great excess compared to the test substance, this type of reaction is usually described as a pseudo-first order reaction in which the observed rate constant is given by the relationship

kobs = k [H2O]	[2]

and can be determined from the expression ([^E0074])

Image omitted; see official source ln Image omitted; see official source	[3]

where:

t = time

and Co, Ct = concentrations of RX at times 0 and t.

The units of this constant have the dimensions of (time)^-1 and the half-life of the reaction (time for 50 % of RX to react) is given by

Image omitted; see official source	[4]

Half-life: (t0,5) is the time taken for 50 % hydrolysis of a test substance when the reaction can be described by first order kinetics; it is independent of the concentration.

DT 50 (Disappearance Time 50): is the time within which the concentration of the test substance is reduced by 50 %; it is different from the half-life t0,5 when the reaction does not follow first order kinetics.

Estimation of k at different temperature

When the rate constants are known for two temperatures, the rate constants at other temperatures can be calculated using the Arrhenius equation:

Image omitted; see official source

or

Image omitted; see official source

A plot of ln k versus 1/T gives a straight line with a slope of - E/R

where:

k	=	rate constant, measured at different temperatures
---	---	---
E	=	activation energy [kJ/mol]
---	---	---
T	=	absolute temperature [K]
---	---	---
R	=	gas constant [8,314 J/mol.K]

The activation energy was calculated by regression analysis or the following equation:

Image omitted; see official source

where: T2 > T1.

Appendix 3

Buffer Systems

A. CLARK AND LUBS:

Buffer mixtures of CLARK and LUBS([^E0075])

Composition	pH
0,2 N HCl and 0,2 N KCl at 20 oC	0,2 N HCl and 0,2 N KCl at 20 oC
47,5 ml. HCl + 25 ml. KCl dil. to 100 ml	1,0
32,25 ml. HCl + 25 ml. KCl dil. to 100 ml	1,2
20,75 ml. HCl + 25 ml. KCl dil. to 100 ml	1,4
13,15 ml. HCl + 25 ml. KCl dil. to 100 ml	1,6
8,3 ml. HCl + 25 ml. KCl dil. to 100 ml	1,8
5,3 ml. HCl + 25 ml. KCl dil. to 100 ml	2,0
3,35 ml. HCl + 25 ml. KCl dil. to 100 ml	2,2
0,1 M potassium biphthalate + 0,1 N HCl at 20 oC	0,1 M potassium biphthalate + 0,1 N HCl at 20 oC
46,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	2,2
39,60 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	2,4
32,95 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	2,6
26,42 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	2,8
20,32 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	3,0
14,70 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	3,2
9,90 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	3,4
5,97 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	3,6
2,63 ml. 0,1 N HCl + 50 ml. biphthalate to 100 ml	3,8
0,1 M potassium biphthalate + 0,1 N NaOH at 20 oC	0,1 M potassium biphthalate + 0,1 N NaOH at 20 oC
0,40 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	4,0
3,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	4,2
7,50 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	4,4
12,15 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	4,6
17,70 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	4,8
23,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	5,0
29,95 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	5,2
35,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	5,4
39,85 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	5,6
43,00 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	5,8
45,45 ml. 0,1 N NaOH + 50 ml. biphthalate to 100 ml	6,0

Buffer mixtures of CLARK and LUBS (Continued)

0,1 M monopotassium phosphate + 0,1 N NaOH at 20 oC	0,1 M monopotassium phosphate + 0,1 N NaOH at 20 oC
5,70 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	6,0
8,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	6,2
12,60 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	6,4
17,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	6,6
23,45 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	6,8
29,63 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	7,0
35,00 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	7,2
39,50 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	7,4
42,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	7,6
45,20 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	7,8
46,80 ml. 0,1 N NaOH + 50 ml. phosphate to 100 ml	8,0
0,1 M H3B03 in 0,1 M KCl + 0,1 N NaOH at 20 oC	0,1 M H3B03 in 0,1 M KCl + 0,1 N NaOH at 20 oC
2,61 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	7,8
3,97 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	8,0
5,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	8,2
8,50 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	8,4
12,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	8,6
16,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	8,8
21,30 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	9,0
26,70 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	9,2
32,00 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	9,4
36,85 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	9,6
40,80 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	9,8
43,90 ml. 0,1 N NaOH + 50 ml. boric acid to 100 ml	10,0

B. KOLTHOFF AND VLEESCHHOUWER:

Citrate buffers of KOLTHOFF and VLEESCHHOUWER

Composition	pH
0,1 M monopotassium citrate and 0,1 N HCl at 18 oC [^E0076]	0,1 M monopotassium citrate and 0,1 N HCl at 18 oC [^E0076]
49,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	2,2
43,4 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	2,4
36,8 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	2,6
30,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	2,8
23,6 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	3,0
17,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	3,2
10,7 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	3,4
4,2 ml. 0,1 N HCl + 50 ml. citrate to 100 ml	3,6
0,1 M monopotassium citrate and 0,1 N NaOH at 18 oC [^E0076]	0,1 M monopotassium citrate and 0,1 N NaOH at 18 oC [^E0076]
2,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	3,8
9,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	4,0
16,3 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	4,2
23,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	4,4
31,5 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	4,6
39,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	4,8
46,7 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	5,0
54,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	5,2
61,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	5,4
68,0 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	5,6
74,4 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	5,8
81,2 ml. 0,1 N NaOH + 50 ml. citrate to 100 ml	6,0

Add tiny crystal of thymol or a similar substance to prevent growth of molds.

C. SÖRENSEN:

Borate mixtures of SÖRENSEN

Composition	Composition	Sörensen 18 oC	Walbum, pH at	Walbum, pH at	Walbum, pH at
ml. Borax	ml. HCl/NaOH	Sörensen 18 oC	10 oC	40 oC	70 oC
0,05 M borax + 0,1 N HCl	0,05 M borax + 0,1 N HCl	0,05 M borax + 0,1 N HCl	0,05 M borax + 0,1 N HCl	0,05 M borax + 0,1 N HCl	0,05 M borax + 0,1 N HCl
5,25	4,75	Sörensen 18 oC	7,62	7,64	7,55	7,47
5,50	4,50	Sörensen 18 oC	7,94	7,98	7,86	7,76
5,75	4,25	8,14	8,17	8,06	7,95
6,00	4,00	8,29	8,32	8,19	8,08
6,50	3,50	8,51	8,54	8,40	8,28
7,00	3,00	8,08	8,72	8,56	8,40
7,50	2,50	8,80	8,84	8,67	8,50
8,00	2,00	8,91	8,96	8,77	8,59
8,50	1,50	9,01	9,06	8,86	8,67
9,00	1,00	9,09	9,14	8,94	8,74
9,50	0,50	9,17	9,22	9,01	8,80
10,00	0,00	9,24	9,30	9,08	8,86
0,05 M borax + 0,1 N NaOH	0,05 M borax + 0,1 N NaOH	0,05 M borax + 0,1 N NaOH	0,05 M borax + 0,1 N NaOH	0,05 M borax + 0,1 N NaOH	0,05 M borax + 0,1 N NaOH
10,0	0,0	9,24	9,30	9,08	8,86
9,0	1,0	9,36	9,42	9,18	8,94
8,0	2,0	9,50	9,57	9,30	9,02
7,0	3,0	9,68	9,76	9,44	9,12
6,0	4,0	9,97	10,06	9,67	9,28

Phosphate mixtures of SÖRENSEN

Composition	pH
0,0667 M Monopotassium phosphate + 0,0667 M Disodium phosphate at 20 oC	0,0667 M Monopotassium phosphate + 0,0667 M Disodium phosphate at 20 oC
99,2 ml. KH2PO4 + 0,8 ml Na2HPO4	5,0
98,4 ml. KH2PO4 + 1,6 ml Na2HPO4	5,2
97,3 ml. KH2PO4 + 2,7 ml Na2HPO4	5,4
95,5 ml. KH2PO4 + 4,5 ml Na2HPO4	5,6
92,8 ml. KH2PO4 + 7,2 ml Na2HPO4	5,8
88,9 ml. KH2PO4 + 11,1 ml Na2HPO4	6,0
83,0 ml. KH2PO4 + 17,0 ml Na2HPO4	6,2
75,4 ml. KH2PO4 + 24,6 ml Na2HPO4	6,4
65,3 ml. KH2PO4 + 34,7 ml Na2HPO4	6,6
53,4 ml. KH2PO4 + 46,6 ml Na2HPO4	6,8
41,3 ml. KH2PO4 + 58,7 ml Na2HPO4	7,0
29,6 ml. KH2PO4 + 70,4 ml Na2HPO4	7,2
19,7 ml. KH2PO4 + 80,3 ml Na2HPO4	7,4
12,8 ml. KH2PO4 + 87,2 ml Na2HPO4	7,6
7,4 ml. KH2PO4 + 92,6 ml Na2HPO4	7,8
3,7 ml. KH2PO4 + 96,3 ml Na2HPO4	8,0

C.8.   TOXICITY FOR EARTHWORMS

ARTIFICIAL SOIL TEST

1. METHOD

1.1.   INTRODUCTION

In this laboratory test, the test substance is added to an artificial soil in which worms are placed for 14 days. After this period (and optionally after seven days) the lethal effect of the substance on the earthworms is examined. The test provides a method for relatively short-term screening of the effect of chemicals on earthworms, by dermal and alimentary uptake.

1.2.   DEFINITION AND UNIT

LC50: the concentration of a substance estimated as killing 50 % of the test animals during the test period.

1.3.   REFERENCE SUBSTANCE

A reference substance is used periodically as a means of demonstration that the sensitivity of the test system has not changed significantly.

Analytical grade chloroacetamide is recommended as the reference substance.

1.4.   PRINCIPLE OF THE TEST

Soil is a variable medium, so for this test a carefully defined artificial loam soil is used. Adult earthworms of the species Eisenia foetida (see note in Appendix) are kept in a defined artificial soil treated with different concentrations of the test substance. The content of the containers is spread on a tray 14 days (and optionally seven days) after the beginning of the test, and the earthworms surviving at each concentration counted.

1.5.   QUALITY CRITERIA

The test is designed to be as reproducible as possible with respect to the test substrate and organism. Mortality in the controls must not exceed 10 % at the end of the test, or the test is invalid.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Materials

1.6.1.1.   Test substrate

A defined artificial soil is used as a basic test substrate.

• (a) Basic substrate (percentages are in terms of dry weight)
• — 10 % sphagnum peat (as close to pH 5,5 to 6,0 as possible with no visible plant remains and finely ground),
• — 20 % kaolinite clay with preferably more than 50 % kaolinite,
• — about 69 % industrial quartz sand (dominant fine sand with more than 50 % of particle size 0,05 to 0,2 mm). If the substance is not sufficiently dispersible in water, 10 g per test container should be kept available for mixing with the test substance later on,
• — about 1 % calcium carbonate (CaCO3), pulverised, chemically pure, added to bring the pH to 6,0 ± 0,5 .

• (b) Test substrate The test substrate contains the basic substrate, the test substance and deionised water. Water content is about 25 to 42 % of the dry weight of the basic substrate. The water content of the substrate is determined by drying a sample to constant weight at 105 ^oC. The key criterion is that the artificial soil must be wetted to a point where there is no standing water. Care should be taken in mixing to obtain an even distribution of the test substance and the substrate. The way of introducing the test substance to the substrate has to be reported.

• (c) Control substrate The control substrate contains the basic substrate and water. If an additive agent is used, an additional control should contain the same quantity of the additive agent.

1.6.1.2.   Test containers

Glass containers of about one litre capacity (adequately covered with plastic lids, dishes or plastic film with ventilation holes) filled with an amount of wet test or control substrate equivalent to 500 g dry weight of substrate.

1.6.2.   Test conditions

Containers should be kept in climatic chambers at a temperature of 20 ± 2 ^oC with continuous light. Light intensity should be 400 to 800 lux.

The test period is 14 days, but mortality can be assessed optionally seven days after starting the test.

1.6.3.   Test procedure

Test concentrations

Concentrations of the test substance are expressed as weight of substance per dry weight of basic substrate (mg/kg).

Range finding test

The range of concentrations just causing mortalities of 0 to 100 % may be determined in a range-finding test to provide information on the range of concentrations to be used in the definitive test.

The substance should be tested at the following concentrations: 1 000 ; 100; 10; 1; 0,1 mg substance/kilogram test substrate (dry weight).

If a full definitive test is to be carried out, one test batch per concentration and one for the untreated control, each with 10 worms, could be sufficient for the range-finding test.

Definitive test

The results of the range-finding test are used to choose at least five concentrations in a geometric series just spanning the range 0 to 100 % mortality and differing by a constant factor not exceeding 1,8 .

Tests using these series of concentration should allow the LC50 value and its confidence limits to be estimated as precisely as possible.

In the definitive test at least four test batches per concentration and four untreated controls, each with 10 worms, are used. The results of these replicate batches are given as a mean and standard deviation.

When two consecutive concentrations, at a ratio of 1,8 , give only 0 % and 100 % mortality, these two values are sufficient to indicate the range within which the LC50 falls.

Mixture of the basic test substrate and the test substance

The test substrate should, whenever possible, be made up without any additional agents other than water. Immediately before the start of the test, an emulsion or dispersion of the test substance in deionised water or other solvent is mixed with the basic test substrate, or sprayed evenly over it with a fine chromatographic or similar spray.

If insoluble in water, the test substance can be dissolved in as small a volume as possible of suitable organic solvent (e.g. hexane, acetone or chloroform).

Only agents which volatilise readily may be used to solubilise, disperse or emulsify the test substance. The test substrate must be ventilated before use. The amount of water evaporated must be replaced. The control should contain the same quantity of any additive agent.

If the test substance is not soluble, dispersible or emulsifiable in organic solvents, 10 g of a mixture of fine ground quartz sand and a quantity of test substance necessary to treat 500 g dry weight of artificial soil are mixed with 490 g of dry weight of test substrate.

For each test batch, an amount of wet test substrate equivalent to 500 g dry weight is placed in each glass container and 10 earthworms, which have been conditioned for 24 hours in a similar wet basic substrate and then washed quickly and surplus water absorbed on filter paper before use, are placed on the test substrate surface.

The containers are covered with perforated plastic lids, dishes or film to prevent the substrate drying and they are kept under the test conditions for 14 days.

The assessments should be made 14 days (and optionally seven days) after setting up the test. The substrate is spread on a plate made of glass or stainless steel. The earthworms are examined and the numbers of surviving earthworms determined. Earthworms are considered dead if they do not respond to a gentle mechanical stimulus to the front end.

When the examination is made at seven days, the container is refilled with the substrate and the surviving earthworms are replaced on the same test substrate surface.

1.6.4.   Test organisms

Test organisms should be adult Eisenia foetida (see note in Appendix) (at least two months old with clitellum) wet weight 300 to 600 mg. (For breeding method see Appendix.)

2. DATA

2.1.   TREATMENT AND EVALUATION OF RESULTS

The concentrations of the substance tested are reported with reference to the corresponding percentages of dead earthworms.

When the data are adequate the LC50 value and the confidence limits (p = 0,05 ) should be determined using standard methods (Litchfield and Wilcoxon, 1949, for equivalent method). The LC50 should be given as mg of test substance per kilogram of the test substrate (dry weight).

In those cases where the slope of the concentration curve is too steep to permit calculation of the LC50, a graphical estimate of this value is sufficient.

When two consecutive concentrations at a ratio of 1,8 give only 0 % and 100 % mortality, the two values are sufficient to indicate the range within which the LC50 falls.

3. REPORTING

3.1.   TEST REPORT

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

• — statement that the test has been carried out in accordance with the abovementioned quality criteria,

• — test carried out (range finding test and/or definitive test),

• — exact description of the test conditions or statement that the test has been carried out in accordance with the method; any deviations have to be reported,

• — exact description of how the test substance has been mixed into the basic test substrate,

• — information about test organisms (species, age, mean and range in weight, keeping and breeding conditions, supplier),

• — method used for determination of LC50,

• — test results including all data used,

• — description of observed symptoms or changes in behaviour of test organisms,

• — mortality in the controls,

• — LC50 or highest tested concentration without mortality and lowest tested concentration with a mortality of 100 %, 14 days (and optionally seven days) after setting up the test,

• — plotting of the concentration/response curve,

• — results obtained with the reference substance, whether in association with the present test or from previous quality control exercises.

4. REFERENCES

• 1)OECD, Paris, 1981, Test Guideline 207, Decision of the Council C(81)30 final.

• 2)Edwards, C. A. and Lofty, J. R., 1977, Biology of Earthworms, Chapman and Hall, London, p. 331.

• 3)Bouche. M. B., 1972, Lombriciens de France, Ecologie et Systematique, Institut National de la Recherche Agronomique, p. 671.

• 4)Litchfield, J. T. andWilcoxon, F., A simplified method of evaluation dose effect experiments. I. Pharm. Exp. Therap., vol. 96, 1949, p. 99.

• 5)Commission of the European Communities, Development of a standardised laboratory method for assessing the toxicity of chemical substances to earthworms, Report EUR 8714 EN, 1983.

• 6)Umweltbundesamt/Biologische Bundesanstalt für land- und Forstwirtschaft, Berlin, 1984, Verfahrensvorschlag ‘Toxizitätstest am Regenwurm Eisenia foetida in künstlichem Boden’, in: Rudolph/Boje, Ökotoxikologie, ecomed, Landsberg, 1986.

Appendix

Breeding and keeping of the worms before testing

For breeding the animals, 30 to 50 adult worms, are put in a breeding box with fresh substrate and removed after 14 days. These animals maybe used for further breeding batches. The earthworms hatched from the cocoons are used for testing when mature (under the prescribed conditions after two to three months).

Keeping and breeding conditions

Climatic chamber	:	temperature 20 ± 2 oC preferably with continuous light (intensity 400 to 800 lux).
---	---	---
Breeding boxes	:	suitable shaI1ow containers of 10 to 20 l volume.
---	---	---
Substrate	:	Eisenia foetida may be bred in various animal excrements. It is recommended to use as breeding medium a mixture of 50 % by volume peat and 50 % cow or horse dung. The medium should have a pH value of about 6 to 7 (regulated with calcium carbonate) and a low ionic conductivity (less than 6 mmhos or 0,5 % salt concentration). The substrate should be moist but not too wet. Other successful procedures may be used besides the method given above.

Note: Eisenia foetida exists in two races which some taxonomists have separated into species (Bouche, 1972). These are morphologically similar but one, Eisenia foetida foetida, has typically transverse striping or banding on the segments and the other, Eisenia foetida andrei, lacks this and has a variegated reddish colour. Where possible Eisenia foetida andrei should be used. Other species may be used if the necessary methodology is available.

C.9.   BIODEGRADATION

ZAHN-WELLENS TEST

The full description of this test method has been deleted. The equivalent international test method appears in Table 3 of Part 0.

C.10.   SIMULATION TESTAEROBIC SEWAGE TREATMENT: C.10-A: ACTIVATED SLUDGE UNITS — C.10-B: BIOFILMS

C.10-A:   Activated Sludge Units

INTRODUCTION

• 1.This Test Method is equivalent to OECD Test Guideline (TG) 303 (2001). In the 1950s it was realised that the newly introduced surfactants caused excessive foaming in waste water treatment plants and in rivers. They were not fully removed in the aerobic treatment and in some cases limited the removal of other organic matter. This instigated many investigations into how surfactants could be removed from waste waters and whether new chemicals produced by industry were amenable to waste water treatment. In order to do this, model units were used representing the two main types of aerobic biological waste water treatment (activated sludge and percolating, or trickling, filtration). It would have been impractical and very costly to distribute each new chemical and to monitor large-scale treatment plants, even on a local basis.

INITIAL CONSIDERATIONS

Activated sludge units

• 2.Model activated sludge units have been described ranging in size from 300 ml up to about 2 000 ml. Some closely mimicked full-scale plants, having sludge settlement tanks with settled sludge being pumped back to the aeration tank, while others provided no settlement facilities e.g. Swisher (1). The size of the apparatus is a compromise; on the one hand, it must be large enough for successful mechanical operation and for the provision of sufficient volume of samples without affecting the operation, while on the other hand it should not be so large that it demands excessive space and materials.

• 3.Two forms of apparatus which have been extensively and satisfactorily used are the Husmann units (2) and Porous Pot units (3)(4), first used in the study of surfactants; these are described in this Test Method. Others have also been used satisfactorily, e.g. Eckenfelder (5). Because of the relatively high cost and effort of applying this simulation test, simpler and cheaper screening tests, now embodied in chapter C.4 A-F of this Annex (6) were investigated in parallel. Experience with many surfactants and other chemicals has shown that those which passed the screening tests (readily biodegradable) also degraded in the simulation test. Some of those failing the screening tests passed the inherent biodegradability tests (chapters C.12 (7) and C.19 (8) of this Annex) but only some of this latter group were degraded in the simulation test, while those chemicals which failed tests for inherent biodegradability did not degrade in the simulation tests (9)(10)(11).

• 4.For some purposes simulation tests carried out under a single set of operating conditions are sufficient; the results are expressed as a percentage removal of the test chemical or of dissolved organic carbon (DOC). A description of such a test is given in this test method. However, unlike the previous version of this chapter, which described only one type of apparatus treating synthetic sewage in the coupled mode using a relatively crude method of sludge wastage, this text offers a number of variations. Alternatives to the type of apparatus, mode of operation, sewage and sludge wastage removal are described. This text closely follows that of ISO 11733 (12), which was carefully scrutinised during its preparation, though the method has not been subject to a ring test.

• 5.For other purposes the concentration of the test chemical in the effluent is required to be known more accurately and for this a more extensive method is needed. For example, the sludge wastage rate must be more precisely controlled throughout each day and throughout the period of the test, and units have to be run at a number of wastage rates. For a fully comprehensive method, tests should also be run at two or three different temperatures: such a method is described by Birch (13)(14) and summarised in Appendix 6. However, present knowledge is insufficient to decide which of the kinetic models are applicable to the biodegradation of chemicals in waste water treatment and in the aquatic environment generally. The application of Monod kinetics, given in Appendic 6 as an example, is limited to chemicals present at 1 mg/l and above, but in the opinion of some even this remains to be substantiated. Tests at concentrations more truly reflecting those found in waste waters are indicated, in Appendix 7, but such tests, and those in Appendix 6, are included in Appendices instead of being issued as separate Test Methods.

Filters

• 6.Much less attention has been given to model percolating filters, perhaps because they are more cumbersome and less compact than activated sludge plant models. Gerike et al developed trickling filter units and operated them in the coupled mode ((15). These filters were relatively large (height 2 m; volume 60 l) and each required as much as 2 l/h of sewage. Baumann et al (16), simulated trickling filters by inserting polyester ‘fleece’ strips into 1 m tubes (14 mm int. diameter) after the strips had been immersed in concentrated activated sludge for 30 min. The test chemical as sole C source in a mineral salts solution was fed down the vertical tube and biodegradation was assessed from measurements of DOC in the effluent and CO2 in the issuing gas.

• 7.Biofilters have been simulated in another way (15); the inner surfaces of rotating tubes, inclined at a small angle to the horizontal, were fed with sewage (about 250 ml/h) with and without the test chemical, and the collected effluents analysed for DOC and/or the specific test chemical.

PRINCIPLE OF THE TEST

• 8.This method is designed to determine the elimination and the primary and/or ultimate biodegradation of water-soluble organic chemicals by aerobic micro-organisms in a continuously operated test system simulating the activated sludge process. An easily biodegradable organic medium and the organic test chemical are the sources of carbon and energy for the micro-organisms.

• 9.Two continuously operated test units (activated sludge plants or porous pots) are run in parallel under identical conditions which are chosen to suit the purpose of the test. Normally the mean hydraulic retention time is 6 h and the mean sludge age (sludge retention time) is 6 to 10 days. Sludge is wasted by one of two methods, the test chemical is normally added at a concentration of between 10 mg/l dissolved organic carbon (DOC) and 20 mg/l DOC, to the influent (organic medium) of only one of the units. The second unit is used as a control unit to determine the biodegradation of the organic medium.

• 10.In frequently taken samples of the effluents, the DOC, preferably, or chemical oxygen demand (COD) is determined, together with the concentration of the test chemical (if required) by specific analysis, in the effluent from the unit receiving the test chemical. The difference between the effluent concentrations of DOC or COD in the test and control units is assumed to be due to the test chemical or its organic metabolites. This difference is compared with the influent concentration of DOC or COD due to the added test chemical in order to determine the elimination of the test chemical.

• 11.Biodegradation may normally be distinguished from bioadsorption by careful examination of the elimination-time curve and may usually be confirmed by applying a test for ready biodegradation using an acclimatised inoculum from the unit receiving the test chemical.

INFORMATION ON THE TEST CHEMICAL

• 12.The purity, water solubility, volatility and adsorption characteristics of the test chemical should be known to enable correct interpretation of results to be made. Normally volatile and insoluble chemicals cannot be tested unless special precautions are taken (see Appendix 5). The chemical structure, or at least the empirical formula, should also be known in order to calculate theoretical values and/or to check measured values of parameters, e.g. theoretical oxygen demand (ThOD), dissolved organic carbon (DOC) and chemical oxygen demand (COD).

• 13.Information on the toxicity of the test chemical to micro-organisms (see Appendix 4) may be useful for selecting appropriate test concentrations and may be essential for the correct interpretation of low biodegradation values.

PASS LEVELS

• 14.In the original application of this simulation (confirmatory) test to the primary biodegradation of surfactants, a removal of more than 80 % of the specific chemical is required before the surfactant may be marketed. If the value of 80 % is not attained, this simulation (confirmatory) test may be applied and the surfactant may be marketed only if more than 90 % of the specific chemical is removed. With chemicals in general there is no question of pass/fail and the value of percentage removal obtained can be used in proximate calculations of the probable environmental concentration to be used in hazard assessments posed by chemicals. Results tend to follow an all or nothing pattern. In a number of studies of pure chemicals the percentage removal of DOC was found to be > 90 % in more than three quarters and > 80 % in over 90 % of chemicals which showed any significant degree of biodegradability.

• 15.Relatively few chemicals (e.g. surfactants) are present in sewage at the concentrations (about 10 mg C/l) used in this test. Some chemicals may be inhibitory at these concentrations, while the kinetics of removal of others may be different at low concentrations. A more accurate assessment of the degradation could be made by using modified methods, using realistically low concentrations of the test chemical, and the data collected could be used to calculate kinetic constants. However, the necessary experimental techniques have not yet been fully validated and neither have the kinetic models, which describe the biodegradation reactions, been established (see Appendix 7).

REFERENCE CHEMICALS

• 16.To ensure that the experimental procedure is being carried out correctly, it is useful occasionally to test chemicals whose behaviour is known simultaneously when test chemicals are investigated. Such chemicals include adipic acid, 2-phenyl phenol, 1-naphthol, diphenic acid, 1-naphthoic acid, etc. (9)(10)(11).

REPRODUCIBILITY OF TEST RESULTS

• 17.There have been far fewer reports of studies of simulation tests than of tests for ready biodegradability. Reproducibility between (simultaneous) replicates is good (within 10-15 %) for test chemicals degraded by 80 % or more but for less well degraded chemicals variability is greater. Also, with some borderline chemicals widely disparate results (e.g. 10 %, 90 %) have been recorded on different occasions within the 9 weeks allowed in the test.

• 18.Little difference has been found in results obtained with the two types of apparatus, but some chemicals have been more extensively and consistently degraded in the presence of domestic sewage than with OECD synthetic sewage.

DESCRIPTION OF THE TEST METHOD

Apparatus

Test system

• 19.The test system for one test chemical consists of a test unit and a control unit; but when only specific analyses are performed (primary biodegradation) only a test unit is required. One control unit can be used for several test units receiving either the same or different test chemicals. In the case of coupling (Appendix 3) each test unit must have its own control unit. The test system may be either an activated sludge plant model, Husmann unit (Appendx 1, Figure 1) or a porous pot (Appendix 1, Figure 2). In both cases storage vessels of sufficient size for the influents and effluents are needed, as well as pumps to dose the influent, either mixed with solution of the test chemical or separately.

• 20.Each activated sludge plant unit consists of an aeration vessel with a known capacity of about 3 litres of activated sludge and a separator (secondary clarifier) which holds about 1,5 litres; the volumes can, to some extent, be changed by adjusting the height of the separator. Vessels of different sizes are permissible if they are operated with comparable hydraulic loads. If it is not possible to keep the temperature in the test room in the desired range, the use of water-jacketed vessels with temperature controlled water is recommended. An airlift pump or a dosing pump is used to recycle the activated sludge from the separator to the aeration vessel, either continuously or intermittently at regular intervals.

• 21.The porous pot system consists of an inner, porous cylinder with a conical bottom held in a slightly larger vessel of the same shape, but made of an impervious plastic material. A suitable material for the porous vessel is porous polyethylene of maximum pore size 90 μm and 2 mm thickness. Separation of the sludge from the treated organic medium is effected by differential passage through the porous wall. Effluents collect in the annular space from where it overflows into the collecting vessel. No settlement occurs and hence there is no sludge return. The whole system may be mounted in a thermostatically controlled water-bath. Porous pots become blocked and could overflow in the initial stages. In such a case, replace the porous liner with a clean one by first siphoning the sludge from the pot into a clean bucket and removing the blocked liner. After wiping out the impervious outer cylinder insert a clean liner and return the sludge to the pot. Any sludge adhering to the sides of the blocked liner is also carefully scraped off and transferred. Clean blocked pots first by using a fine jet of water to remove remaining sludge and by soaking in dilute sodium hypochlorite solution, then in water, followed by thoroughly rinsing with water.

• 22.For aeration of the sludge in the aeration vessels of both systems, suitable techniques are required, for example sintered cubes (diffuser stones) and compressed air. The air shall be cleaned, if necessary, by passing through a suitable filter and washed. Sufficient air must pass through the system to maintain aerobic conditions and to keep sludge flocs in suspension at all times during the test.

Filtration apparatus or centrifuge

• 23.Device for filtration of samples with membrane filters of suitable porosity (nominal aperture diameter 0,45 μm) which adsorb soluble organic chemicals and release organic carbon to a minimum degree. If filters are used which release organic carbon, wash the filters carefully with hot water to remove leachable organic carbon. Alternatively, a centrifuge capable of producing 40 000 m/s² may be used.

Analytical equipment

• 24.Apparatus required to determine:

• — DOC(dissolved organic carbon) and TOC (total organic carbon), or COD (chemical oxygen demand);

• — specific chemical, if required;

• — suspended solids, pH, oxygen concentration in water;

• — temperature, acidity and alkalinity;

• — ammonium, nitrite and nitrate, if the test is performed under nitrifying conditions.

Water

• 25.Tap water, containing less than 3 mg/l DOC. Determine the alkalinity if not already known.

• 26.Deionised water, containing less than 2 mg/l DOC.

Organic medium

• 27.Synthetic sewage, domestic sewage or a mixture of both is permissible as the organic medium. It has been shown (11)(14) that the use of domestic sewage alone often gives increased percentage DOC removal and even allows the removal and biodegradation of some chemicals which are not biodegraded when OECD synthetic sewage is used. Also, the constant or intermittent addition of domestic sewage often stabilises the activated sludge, including the crucial ability to settle well. Thus, the use of domestic sewage is recommended. Measure the DOC or COD concentration in each new batch of organic medium. The acidity or alkalinity of the organic medium should be known. The organic medium may require the addition of a suitable buffer (sodium hydrogen carbonate or potassium dihydrogen phosphate) if it is of low acidity or alkalinity, to maintain a pH of about 7,5 ± 0,5 in the aeration vessel during the test. The amount of buffer to be added, and when to add it, has to be decided in each individual case. When mixtures are used either continuously or intermittently, the DOC (or COD) of the mixture must be kept at an approximately constant value, e.g. by dilution with water.

Synthetic sewage

• 28.Dissolve in each litre of tap water: peptone, 160 mg; meat extract, 110 mg; urea, 30 mg; anhydrous dipotassium hydrogen phosphate (K2HPO4), 28 mg; sodium chloride (NaCl), 7 mg; calcium chloride dihydrate (CaCl2.2H2O), 4 mg; magnesium sulphate heptahydrate (Mg2SO4.7H20), 2 mg. This OECD synthetic sewage is an example and gives a mean DOC concentration in the influent of about 100 mg/l. Alternatively, use other compositions, with about the same DOC concentration, which are closer to real sewage. If a less concentrated influent is required, dilute the synthetic sewage, for example 1:1, with tap water to obtain a concentration of about 50 mg/l. Such a weaker influent will allow better growth of nitrifying organisms and this modification should be used if the simulation of nitrifying waste water plants is to be investigated. This synthetic sewage may be made up in distilled water in a concentrated form and stored at about 1 °C for up to one week. When needed, dilute with tap water. (This medium is unsatisfactory e.g. nitrogen concentration is very high, relatively low carbon content, but nothing better has been suggested, except to add more phosphate as buffer and extra peptone).

Domestic sewage

• 29.Use fresh settled sewage collected daily from a treatment works receiving predominantly domestic sewage. It should be collected, prior to primary sedimentation, from the overflow channel of the primary sedimentation tank, or from the feed to the activated sludge plant, and be largely free from coarse particles. The sewage can be used after storage for several days (but generally should not exceed seven days) at about 4 °C, if it is proved that the DOC (or COD) has not significantly decreased (i.e. by less than 20 %) during storage. In order to limit disturbances to the system, the DOC (or COD) of each new batch should be adjusted before use to an appropriate constant value, e.g. by dilution with tap water.

Activated sludge

• 30.Collect activated sludge for inoculation from the aeration tank of a well operated waste water treatment plant or from a laboratory — scale activated sludge unit, treating predominantly domestic sewage.

Stock solutions of test chemical

• 31.For chemicals of adequate solubility, prepare stock solutions at appropriate concentrations (e.g. 1 to 5 g/l) in deionised water, or in the mineral portion of the synthetic sewage. (for insoluble and volatile chemicals, see Appendix 5). Determine the DOC and total organic carbon (TOC) of the stock solution and repeat the measurements for each new batch. If the difference between the DOC and TOC is greater than 20 %, check the water-solubility of the test chemical. Compare the DOC or the concentration of the test chemical measured by specific analysis of the stock solution with the nominal value, to ascertain whether recovery is good enough (normally > 90 % can be expected). Ascertain, especially for dispersions, whether or not DOC can be used as an analytical parameter or if only an analytical technique specific for the test chemical can be used. Centrifugation of the samples is required for dispersions. For each new batch, measure the DOC, COD or the test chemical with specific analysis.

• 32.Determine the pH of the stock solution. Extreme values indicate that the addition of the chemical may have an influence on the pH of the activated sludge in the test system. In this case neutralise the stock solution to obtain a pH of 7 ± 0,5 with small amounts of inorganic acid or base, but avoid precipitation of the test chemical.

PROCEDURE

• 33.The procedure is described for the activated sludge plant units; it has to be slightly adapted for the porous pot system.

Preparation of the inoculum

• 34.Inoculate the test system at the beginning of the test with either activated sludge or an inoculum containing a low concentration of micro-organisms. Keep the inoculum aerated at room temperature until it is used and use it within 24 h. In the first case, take a sample of activated sludge from the aeration tank of an efficiently operated biological waste water treatment plant, or a laboratory treatment plant, which receives predominantly domestic sewage. If nitrifying conditions are to be simulated, take sludge from a nitrifying waste water treatment plant. Determine the concentration of suspended solids and, if necessary, concentrate the sludge by settling so that the volume added to the test system is minimal. Ensure that the starting concentration of dry matter is about 2,5 g/l.

• 35.In the second case, use 2 ml/l to 10 ml/l of an effluent from a domestic biological waste water treatment plant as an inoculum. To get as many different species of bacteria as possible, it may be helpful to add inocula from various other sources, for example surface water. In this case, the activated sludge will develop and grow in the test system.

Dosage of organic medium

• 36.Ensure that influent and effluent containers and tubing from influent vessels and to effluent vessels are thoroughly cleaned to remove microbial growths initially and throughout the test. Assemble the test systems in a room where the temperature is controlled (normally in the range 20-25 °C) or use water-jacketed test units. Prepare a sufficient volume of the required organic medium (paragraphs 27-29). Initially fill the aeration vessel and the separator with the organic medium and add the inoculum (paragraphs 34, 35). Start the aeration such that the sludge is kept in suspension and in an aerobic state and begin dosing the influent and recycling the settled sludge. Dose organic medium out of storage vessels into the aeration vessels (paragraphs 20, 21) of the test and control units and collect the respective effluents in similar storage vessels. To get the normal hydraulic retention time of 6 h, the organic medium is pumped at 0,5 l/h. To confirm this rate, measure the daily amount of organic medium dosed by noting the reduction in volumes of the medium in the storage vessels. Other modes of dosing would be necessary for determining the effects of intermittent release and ‘shock’ loading of chemicals.

• 37.If the organic medium is prepared for use for a period longer than 1 day, cooling at about 4 °C, or other appropriate methods of conservation are necessary to prevent microbial growth and biodegradation outside the test units (paragraph 29). If synthetic sewage is used, it is possible to prepare, and store at about 4 °C, a concentrated stock solution (e.g. 10-fold the normal concentration, paragraph 28). This stock solution can be well mixed with the appropriate volume of tap water before use; alternatively, it can be pumped directly while the appropriate amount of tap water is pumped separately.

Dosage of test chemical

• 38.Add an appropriate volume of the stock solution of the test chemical (paragraph 31) to the storage vessel of the influent or dose it directly with a separate pump into the aeration vessel. The normal mean test concentration in the influent should be between 10 mg/l and 20 mg/l DOC, with an upper concentration of no more than 50 mg/l. If the water-solubility of the test chemical is low or if toxic effects are likely to occur, reduce the concentration to 5 mg/l DOC or even less, but only if a suitable specific analytical method is available and performed (dispersed test chemicals which are poorly soluble in water may be added using special dosing techniques, see Appendix 5).

• 39.Start adding the test chemical after a period in which the system has stabilised and is removing DOC of the organic medium efficiently (about 80 %). It is important to check that all units are working equally efficiently before the addition of test chemical; if they are not, it usually helps to mix the individual sludges and to re-dispense equal volumes to individual units. When an inoculum of (about) 2,5 g/l (dry weight) activated sludge is used, the test chemical may be added from the start of the test since directly adding increasing amounts from the beginning has the advantage that the activated sludge may be better able to adapt to the test chemical. In whatever manner the test chemical is added, it is recommended that the relevant flow rate and/or the volumes in the storage vessel(s) are measured at regular intervals.

Handling of activated sludge

• 40.The concentration of activated sludge solids normally stabilises between limits during the test, independent of the inoculum used, in the range 1 to 3 g/l (dry weight) depending on the quality and concentration of the organic medium, operating conditions, the nature of the micro-organisms present and the influence of the test chemical.

• 41.Either determine the suspended solids in the aeration vessels at least weekly and discard surplus sludge to maintain the concentration at 1 g/l to 3 g/l (dry weight), or control the mean sludge age at a constant value usually in the range 6 days to 10 days. If, for example, a sludge retention time of 8 days is chosen, remove daily 1/8 of the volume of the activated sludge in the aeration vessel and discard it. Carry this out on a daily basis or, preferably, by means of an automatic intermittently operating pump. Maintaining the concentration of suspended solids constant, or within narrow limits, does not maintain a constant sludge retention time (SRT), which is the operating variable that determines the value of the concentration of test chemical in the effluent.

• 42.Throughout the test, remove, at least daily, any sludge adhering to the walls of the aeration vessel and the separator so that it is resuspended. Check and clean regularly all tubes and tubing to prevent growth of biofilm. Recycle the settled sludge from the separator to the aeration vessel, preferably by intermittent pumping. No recycling takes place in the porous pot system but ensure that clean inner pots are inserted before the volume in the vessel rises significantly (paragraph 21).

• 43.Poor settlement and loss of sludge may occur in the Husmann plant units. These may be rectified by employing one or more of the actions, listed below, in parallel in test and control units:

• — fresh sludge or flocculant (for example 2 ml/vessel of 50 g/l FeCl3) could be added at regular intervals, e.g. weekly, but ascertain that no reaction or precipitation of the test chemical occurs with FeCl3;

• — the air-lift pump could be replaced by a peristaltic pump, thus enabling a sludge recirculation flow which about equals the influent flow to be used and allowing development of an anaerobic zone in the settled sludge (the geometry of the air-lift pump limits the minimum flow rate of returned sludge to be about 12-fold that of the influent); — sludge could be pumped intermittently from the separator to the aeration vessel (e.g. 5 min. every 2,5 h to recycle 1 l/h to 1,5 l/h; — a non-toxic, anti-foaming agent at minimal concentration could be used to prevent loss by foaming (e.g. silicone oil); — air could be passed through the sludge in the separator in short, shock bursts (e.g. 10 sec. every hour); — the organic medium may be dosed at intervals into the aeration vessel (e.g. 3 min. to 10 min. every hour).

Sampling and analysis

• 44.At regular intervals measure the dissolved oxygen concentration, the temperature and the pH value of the activated sludge in the aeration vessels. Ensure that sufficient oxygen is always available (> 2 mg/l) and that the temperature is kept in the required range (normally 20 °C to 25 °C). Keep the pH at 7,5 ± 0,5 by dosing small amounts of inorganic base or acid into the aeration vessel or into the influent, or by increasing the buffering capacity of the organic medium (see paragraph 27). When nitrification occurs acid is produced, the oxidation of 1 mg N producing the equivalent of about 7 mg CO3 ^–. The frequency of measuring depends on the parameter to be measured and the stability of the system, and may vary between daily and weekly measurements.

• 45.Measure the DOC or COD in the influents to the control and test vessels. Measure the test chemical concentration in the test influent by specific analysis or estimate it from the concentration in the stock solution (paragraph 31), the volume used and the amount of sewage dosed into the test unit. It is recommended that the concentration of the test chemical be calculated in order to reduce the variability of the concentration data.

• 46.Take suitable samples from the collected effluent (e.g. 24 h composites) and filter through a membrane of pore size 0,45 μm or centrifuge them at about 40,000 m/s² for about 15 min. Centrifuging should be used if filtering is difficult. Determine DOC or COD at least in duplicate to measure ultimate biodegradation and, if required, primary biodegradation by an analysis specific for the test chemical.

• 47.The use of COD may give rise to analytical problems at low concentrations and is therefore recommended only if a sufficiently high test concentration (about 30 mg/l) is used. Also, for strongly adsorbing chemicals, it is recommended that the amount of adsorbed chemical in the sludge be measured using an analytical technique specific for the test chemical.

• 48.The frequency of sampling depends on the expected duration of the test. A recommended frequency is three times per week. Once the units are operating efficiently, allow from 1 week to a maximum of 6 weeks after the test chemical has been introduced, for adaptation to reach a steady state. Preferably obtain at least 15 valid values in the plateau phase (paragraph 59), normally lasting 3 weeks, for the evaluation of the test result. The test may be completed if a sufficient degree of elimination is reached (e.g. > 90 %) and these 15 values, which represent analyses carried out each weekday over 3 weeks, are available. Normally, do not exceed a test duration of more than 12 weeks after addition of the test chemical.

• 49.If the sludge nitrifies and if the effects of the test chemical on nitrification are to be studied, analyse samples from the effluent of the test and control units at least once per week for ammonium and/or nitrite plus nitrate.

• 50.All analyses should be performed as soon as possible, especially the nitrogen determinations. If analyses have to be postponed, store the samples at about 4 °C in the dark in full, tightly stopped bottles. If samples have to be stored for more than 48 h, preserve them by deep-freezing, acidification (e.g. 10 ml/l of a 400 g/l solution of sulphuric acid) or by addition of a suitable toxic substance (e.g. 20 ml/l of a 10 g/l solution of mercury (II) chloride). Ensure that the preservation technique does not influence results of analysis.

Coupling of test units

• 51.If coupling is to be used (Appendix 3), daily exchange the same amount of activated sludge (150 ml to 1 500 ml for aeration vessels containing 3 litres of liquor) between the aeration vessels of the test unit and its control unit. If the test chemical adsorbs strongly onto the sludge, change only the supernatant of the separators. In both cases use a correction factor to calculate the test results (paragraph 55).

DATA AND REPORTING

Treatment of results

• 52.Calculate the percentage of DOC or COD elimination of the test chemical for each timed assessment, using the equation:

Image omitted; see official source

• where

Dt	=	% elimination of DOC or COD at time t
---	---	---
Cs	=	DOC or COD in the influent due to the test chemical, preferably estimated from the stock solution (mg/l)
---	---	---
E	=	measured DOC or COD value in the test effluent at time t (mg/l)
---	---	---
Eo	=	measured DOC or COD value in the control effluent at time t (mg/l)

• 53.The degree of DOC or COD elimination of the organic medium in the control unit is helpful information in assessing the biodegradative activity of the activated sludge during the test. Calculate the percentage elimination from the equation:

Image omitted; see official source

• where

DB	=	% elimination of DOC or COD of the organic medium in the control unit at time t
---	---	---
CM	=	DOC or COD of the organic medium in the control influent (mg/l)

• Optionally, calculate the percentage elimination DOC or COD due to the organic medium plus test chemical in the test unit from the equation:

Image omitted; see official source

• where

DT	=	% elimination of total test influent DOC or COD
---	---	---
CT	=	DOC or COD of total test influent or calculated from stock solutions (mg/l)

• 54.Calculate the removal of the test chemical if measured with a specific analytical method at each time assessment from equation:

Image omitted; see official source

• where

DST	=	% primary elimination of test chemical at time t
---	---	---
Si	=	measured or estimated test chemical concentration in the test influent (mg/l)
---	---	---
Se	=	measured test chemical concentration in test effluent at time t (mg/l)

• 55.If the coupling mode has been used, compensate the dilution of the test chemical in the aeration vessel by the sludge exchange using a correction factor (see Appendix 3). If a mean hydraulic retention time of 6 h and an exchange of half of the volume of the activated sludge in the aeration vessel have been used, the determined daily elimination values (Dt, paragraph 52) have to be corrected to obtain the true degree of elimination, Dtc, of the test chemical from the equation:

Image omitted; see official source

Expression of test results

• 56.Plot the percentage elimination Dt (or Dtc) and Dst, if available, versus time (see Appendic 2). From the shape of the elimination curve of the test chemical (per se or as DOC) some conclusions may be drawn about the removal process.

Adsorption

• 57.If a high DOC elimination of the test chemical is observed from the beginning of the test, the test chemical is probably eliminated by adsorption onto the activated sludge solids. It is possible to prove this by determining the adsorbed test chemical by specific analysis. It is not usual for the elimination of DOC of adsorbable chemicals to remain high throughout the test; normally, there is a high degree removal initially which gradually falls to an equilibrium value. If, however, the adsorbable test chemical was able to cause acclimation of the microbial population in some way or other, the DOC elimination of the test chemical would subsequently increase and reach a high plateau value.

Lag phase

• 58.As in static, screening tests, many test chemicals require a lag phase before full biodegradation occurs. In the lag phase, acclimation or adaptation of the degrading bacteria takes place with almost no removal of the test chemical; then the initial growth of these bacteria occurs. This phase ends and the degradation phase is taken to begin when about 10 % of the initial amount of test chemical is removed (after allowing for adsorption, if it occurs). The lag phase is often highly variable and poorly reproducible.

Plateau phase

• 59.The plateau phase of an elimination curve in a continuous test is defined as that phase in which the maximum degradation takes place. The plateau phase should be at least 3 weeks and have about 15 measured valid values.

Mean degree of elimination of test chemical

• 60.Calculate the mean value from the elimination values (Dt) of the test chemical at the plateau phase. Rounded to the nearest whole number (1 %), it is the degree of elimination of the test chemical. It is also recommended to calculate the 95 % confidence interval of the mean value.

Elimination of organic medium

• 61.Plot the percentage of elimination of the DOC or COD of the organic medium in the control unit (DB) versus time. Indicate the mean degree of elimination in the same way as for the test chemical (paragraph 60).

Indication of biodegradation

• 62.If the test chemical does not adsorb significantly on to activated sludge and the elimination curve has a typical shape of a biodegradation curve with lag, degradation and plateau phases (paragraphs 58, 59), the measured elimination can safely be attributed to biodegradation. If a high initial removal has taken place, the simulation test cannot differentiate between biological and abiotic elimination processes. In such cases, and in other cases where there is any doubt about biodegradation (e.g. if stripping takes place), analyse adsorbed test chemicals or perform additional static biodegradation tests based on parameters clearly indicating biological processes. Such tests are the oxygen uptake methods (chapter C.4 D, E and F of this Annex (6)) or a test with measurement of carbon dioxide production (chapter C.4 C of this Annex (6)) or the ISO Headspace method (18), using a pre-exposed inoculum from the simulation test. If both the DOC removal and specific chemical removal have been measured, significant differences (the former being lower than the latter) between the percentages removed indicate the presence in the effluents of intermediate organic products which may be more difficult to degrade than the parent chemical.

Validity of test results

• 63.Information on the normal biodegradation behaviour of the inoculum is achieved if the degree of elimination of the organic medium (paragraph 53) in the control unit is determined. Consider the test to be valid if the degree of DOC or COD elimination in the control unit(s) is > 80 % after two weeks and no unusual observations have been made.

• 64.If a readily biodegradable (reference) chemical has been used, the degree of biodegradation (Dt, paragraph 52) should be > 90 %.

• 65.If the test is performed under nitrifying conditions, the mean concentration in the effluents should be < 1 mg/l ammonia-N and < 2 mg/l nitrite-N.

• 66.If these criteria (paragraphs 63-65) are not met, repeat the test using an inoculum from a different source, test a reference chemical, and review all experimental procedures.

Test Report

• 67.The test report must include the following:

Test chemical:

• — identification data;

• — physical nature and, where relevant, physical-chemical properties.

Test conditions:

• — type of test system; any modifications for testing insoluble and volatile chemicals;

• — type of organic medium;

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

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

• — test chemical stock solution: DOC and TOC content; how prepared, if suspension; test concentration used; reasons if outside range of 10-20 mg/l DOC; method of addition; date first added; any changes;

• — mean sludge age and mean hydraulic retention time; method of sludge wastage; methods of overcoming bulking, loss of sludge, etc.;

• — analytical techniques employed;

• — test temperature;

• — qualities of the sludge-bulking, sludge volume index (SVI), mixed liquor suspended solids (MLSS);

• — any deviations from standard procedures and any circumstances which may have affected results.

Test results:

• — all measured data (DOC, COD, specific analyses, pH, temperature, oxygen concentration, suspended solids, N chemicals, if relevant;

• — all calculated values of Dt (or Dtc), DB, DSt obtained in tabular form and the elimination curves;

• — information on lag and plateau phases, test duration, the degree of elimination of the test chemical and that of the organic medium in the control unit, together with statistical information and statements of biodegradability and validity of the test;

• — discussion of results.

LITERATURE:

• (1) Swisher RD (1987). ‘Surfactant Biodegradation’, 2nd Edn. Marcel Dekker Inc. New York, 1085 pp.