HPSA

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April - 28 May 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-B (Determination of the "Ready" Biodegradability - Modified OECD Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Source :
A mixed population of sewage treatment micro-organisms was obtained on 30 April 1996 from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
Pre-Treatment :
The sample of effluent was filtered through Postlip filter paper (approximately first 200 ml discarded) and the filtrate maintained on continuous aeration at a temperature of 21°C prior to use.

Duration of test (contact time):

28 d

Initial conc.:

40 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Experimental Preparation
For the purpose of the definitive study the test material was prepared by a direct dispersion in culture medium.
An amount of test material (2000 mg) was dispersed in water and the volume adjusted to 1 litre to give a 2000 mg/I stock solution. An aliquot (200 ml) of this stock solution was dispersed in 2 litres of culture medium to give the final test concentration of 200 mg/I, equivalent to 40 mg carbon/l.
Analysis of the concentration, homogeneity and stability of the test material in the test solutions were not appropriate to the Test Guideline.

Standard Material
For the purposes of the study a standard material, sodium benzoate (Fisons Analar Lot No 9401096045), was used. An initial stock solution of 1000 mg/I was prepared by direct dispersion in water and a 68.6 ml aliquot added to the test vessel to give a final test concentration of 34.3 mg/I, equivalent to 20 mg carbon/I.

The culture medium used in this study was that recommended in the OECD Guidelines.

Preparation of test system
The following test solutions were prepared and inoculated in 3 litre glass culture vessels each containing 2 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final test concentration of 20 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final test concentration of 40 mg carbon/l.
Each test vessel was inoculated with sewage treatment micro-organisms at a rate of 0.5 ml/l. The study was carried out at a temperature of 24°C in darkness.
The culture vessels were covered with a polypropylene top to reduce evaporation and constantly aerated with compressed air via a narrow bore glass tube. Each culture vessel was also stirred constantly by magnetic stirrers.

Sampling and Dissolved Organic Carbon (DOC) Analysis
Samples (approximately 20 ml) were withdrawn by plastic disposable syringe and filtered through 0.45 µm Gelman Acrocap disposable filters. The first approximate 5 ml of filtrate was discarded.
Samples were taken for analysis at 0 hours and on days 1, 2, 3, 6, 8, 10, 14, 16, 21, 23, 27 and 28.
Prior to sampling for analysis any losses due to evaporation were corrected by the addition of deionised water and any material adhering to the culture vessel resuspended. Immediately prior to sampling the temperature and dissolved oxygen concentration (by means of a Yellow Springs dissolved oxygen meter) of each vessel were recorded. The pH of each vessel was also determined and adjusted, to pH 7.4, where necessary with sodium hydroxide or sulphuric acid solutions.

Reference substance:

benzoic acid, sodium salt

Remarks:

34.3 mg/l

Parameter:

% degradation (DOC removal)

Value:

89

Sampling time:

28 d

Details on results:

•The test item can be considered as "readily biodegradable".
•The degree of biodegradation was 89 % after 28 days.
•Degradation exceeded 10% on day 2 of the study and attained 82% on day 6 of the study and therefore satisfied the 10-day window validation criterion given in the OECD Guidelines.
•Preliminary investigational work conducted showed that no significant adsorption of the test material to glassware occurred and therefore the abiotic control vessel was omitted from the study.
•The standard material, sodium benzoate, attained 97% degradation after 28 days, thereby confirming the suitability of the inoculum and culture conditions.
•The pH values did not fall below 6.0 in any culture vessel during the study and were readjusted to pH 7.4 on sampling days. Dissolved oxygen concentrations remained at approximately 5.2 mg 02/1 or above in all culture vessels.

Results with reference substance:

Degradation of the reference item was 97% after 28 days

DISSOLVED ORGANIC CARBON (DOC) VALUES ON EACH SAMPLING OCCASION

Vessel		DOC (mg C/I)
		Day
		0 hours	1	2	3	6	8	10	14	16	21	23	27	28
Control	R1	2,19	2,21	2,13	2,07	1,63	2,98	3,99	3,56	3,41	4,07	2,91	2,88	2,62
	R2	2,19	2,30	1,74	1,60	3,90	2,45	3,23	2,90	3,35	2,83	2,29	2,26	1,84
	Mean	2,19	2,26	1,94	1,84	2,77	2,72	3,61	3,23	3,38	3,45	2,60	2,57	2,23
Sodium benzoate	R1	21,39	2,57	1,73	2,62	1,69	3,52	4,33	3,73	3,28	3,67	2,62	2,62	2,93
	R2	22,56	4,36	3,19	4,50	2,78	3,99	5,06	3,69	3,87	3,11	2,37	2,45	2,63
	Mean	21,98	3,47	2,46	3,56	2,24	3,76	4,70	3,71	3,58	3,39	2,50	2,54	2,78
Test item	R1	40,40	38,20	33,17	28,39	6,51	9,32	9,61	8,14	8,61	8,02	6,21	6,64	6,67
	R2	45,48	45,50	36,66	45,78	13,97	10,16	9,35	11,42	10,39	8,14	6,52	6,39	6,73
	Mean	42,94	41,85	34,92	37,09	10,24	9,74	9,48	9,78	9,50	8,08	6,37	6,52	6,70

R1 - R2 = Replicate vessels

CALCULATION OF PERCENTAGE DEGRADATION

The percentage biodegradation was calculated from:

D_t (%) = [1 - (C_t - C_B)/(C_A - CBA)] * 100

D_t = percentage degradation at time t

C_t = mean concentration (mg C/I) of DOC in the test or standard material suspensions at time t.

C_B = mean concentration (mg C/I) of DOC in the control suspensions at time t.

C_A = mean concentration (mg C/I) of DOC in the test or standard material suspensions at 0 hours.

C_BA = mean concentration (mg C/I) of DOC in the control suspensions at 0 hours.

PERCENTAGE DEGRADATION VALUES

Day			% Degradation*
			Sodium benzoate					Test item
1			94					3
2			97					19
3			91					13
6			103					82
8			95					83
10			94					86
14			98					84
16			99					85
21			100					89
23			101					91
27			100					90
28			97					89

*Corrected for control levels of DOC

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the guideline were fulfilled.

Interpretation of results:

readily biodegradable

Conclusions:

The test item was tested using a concentration of 40 mg organic carbon/L in test medium following OECD 301E/EU-Guideline C.4-B. The study was conducted under GLP compliance and a well documented study report is available. The test was left running for 28 days. To measure the degradation of test and reference item, the content of dissolved organic carbon (DOC) in the test vessels was measured twelve times during the test. All validity criteria were met. Degradation of the reference item was 97% after four days.
The following data could be determined for the test item :
•The degree of biodegradation was 89 % after 28 days.
•Degradation exceeded 10% on day 2 of the study and attained 82% on day 6 of the study and therefore satisfied the 10-day window validation criterion given in the OECD Guidelines.
Therefore the test item is readily biodegradable following OECD 301 E/EU C.4-B.

Executive summary:

The biodegradability of the test item was investigated according to OECD guideline 301E and EU method C.4 –B. The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). The test material was exposed to activated sewage sludge micro-organisms at a concentration of 40 mg C/I with culture medium in the dark at 24°C for 28 days. The degradation of the test material was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the standard material, sodium benzoate were used for validation purposes. All validity criteria were met. Degradation of the reference item was 97% after 28 days. The following data could be determined for the test item :

•The degree of biodegradation was 89 % after 28 days.

•Degradation exceeded 10% on day 2 of the study and attained 82% on day 6 of the study and therefore satisfied the 10-day window validation criterion given in the OECD Guidelines.

Therefore the test item is readily biodegradable following OECD 301 E/EU C.4-B.

Description of key information

- OECD 301E (Experimental investigation, GLP guideline study): 89 % degradation after 28 days - readily biodegradable, fulfilling the 10-d window 
- OECD 302B (Experimental investigation, no information about GLP): 98,5 % degradation after 28 days - inherently biodegradable

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

Regarding biodegradation in water (screening tests) 2 experimental results (one key study, Klimisch score 1 as well as one supporting study, Klimisch score 2) are available for the test substance.

The key study was conducted according to OECD Guideline 301E (Mead,OECD301E,1996). The study was conducted in compliance with the Principles of Good Laboratory Practice (GLP). The test material was exposed to activated sewage sludge micro-organisms at a concentration of 40 mg C/I with culture medium in the dark at 24°C for 28 days. The degradation of the test material was assessed by the determination of dissolved organic carbon removal. Control cultures with inoculum and the standard material, sodium benzoate were used for validation purposes. All validity criteria were met. Degradation of the reference item was 97% after 28 days. The following data could be determined for the test substance :

•The degree of biodegradation was 89 % after 28 days.

•Degradation exceeded 10% on day 2 of the study and attained 82% on day 6 of the study and therefore satisfied the 10-day window validation criterion given in the OECD Guidelines.

Therefore the test substance is readily biodegradable following OECD 301 E/EU C.4-B.

 

The supporting study was conducted according to OECD Guideline 302B (Simec,OECD302B,1996). The degradation parameter was the DOC removal in the samples. After 28 days incubation 98,5 % degradation had occured. Due to this result the test substance can be considered as inherently biodegradable. Ethylene glycol was used as reference substance. After 14 days incubation the reference substance is degraded at least 70% within the 14d-window, confirming the inoculum and the test set-up to be appropriate .