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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The defintive study was conducted between 13 March 1998 and 10 April 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Sponsor's identification: BHES-100X
Description: off white solid block
Batch number: 1A0110
Date received: 20 January 1998
Storage conditions: ambient temperature in darkness
Oxygen conditions:
aerobic
Inoculum or test system:
other: mixed population of sewage treatment micro-organisms
Details on inoculum:
A mixed population of sewage treatment micro-organisms was obtained from the secondary treatment stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
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 (100 mg) was dispersed in culture medium and the volume adjusted to 100 ml to give a 1000 mg/I stock solution. An aliquot (24 ml) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 6 litres to give a final test concentration of 4 mg/I.
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 (C6H5COONa) (Sigma Lot No 125H0060), was used. An initial stock solution of 1000 mg/I was
prepared by direct dispersion in culture medium and an aliquot (18 ml) dispersed in inoculated culture medium and the volume adjusted to 6 litres to give a final test
concentration of 3 mg/I.

Procedure
Preparation of inoculum:
The sample of effluent was filtered (Postlip Filter paper approximately 200 ml discarded) and the filtrate maintained on continuous aeration in a temperature control led room at 21 °C prior to use.

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

Preparation of the test system
The following test solutions were prepared and inoculated in 250-300 ml Biological Oxygen Demand (BOO) bottles (darkened glass) with ground glass stoppers:
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 3 mg/I.
c) The test material, in duplicate, in inoculated culture medium to give a final test concentration of 4 mg/I.
d) The test material (4 mg/I) plus the standard material (1.5 mg/I), in duplicate, in inoculated culture medium to act as a toxicity control.
Test media a-d were inoculated with sewage treatment micro-organisms at a rate of 1 drop of inoculum per litre.

The test media were transferred by siphon to BOD bottles, which were firmly stoppered to exclude all air bubbles. Sufficient bottles were prepared to allow a single oxygen determination per bottle with duplicate bottles for each test media at each sampling occasion.
The BOD bottles were incubated in a temperature controlled water bath at 21 °C.

Sampling and oxygen analysis
Dissolved oxygen concentrations for each test media were determined, in duplicate on days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28 by means of a Yellow Springs BOD Probe and oxygen meter.
Reference substance:
other: sodium benzoate
Parameter:
% degradation (O2 consumption)
Value:
14
Sampling time:
28 d
Remarks on result:
other: Substance cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301 D.
Details on results:
The variation in percentage degradation values obtained between sampling days is considered to be due to biological variation between the
respiration rates of the micro-organisms in the control and test vessels.
The toxicity control attained 80% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Results with reference substance:
The standard material, sodium benzoate, attained 100% degradation after 28 days thereby confirming the suitability of the test method and culture conditions.

Oxygen depletion and mean percentage biodegradation values are presented in the table below

Test series

Day

3

6

9

12

15

18

21

24

28

(a) Culture medium with inoculum

Mean O2depletion (mgO2/l)

0.325

0.350

0.450

0.375

0.300

1.000

0.325

0.425

0.475

(b) Standard material, sodium benzoate (3 mg/l) with inoculum

O2depletion (mgO2/l) R1

R2

% degredation (mean)

2.975

3.350

3.950

4.425

4.650

4.050

4.525

4.975

4.925

3.075

3.450

4.100

4.925

4.800

4.300

4.775

5.875

5.075

60

68

81

93

95

84

93

108

100

(c) Test material, BHES-100X (4 mg/l) with inoculum

O2depletion (mgO2/l) R1

R2

% degredation (mean)

0.275

0.550

0.350

0.525

0.800

0.250

0.475

0.625

0.625

0.275

0.450

0.400

0.625

0.700

0.500

0.775

0.575

0.675

5

10

8

12

15

8

13

13

14

(d) Test material, BHES-100X (4 mg/l) plus sodium benzoate with inoculum

O2depletion (mgO2/l) R1

R2

% degredation (mean)

3.525

3.550

4.450

4.425

5.100

4.500

5.875

6.025

6.025

3.575

3.550

4.200

5.025

5.500

4.900

5.325

6.125

6.025

48

47

58

67

71

63

76

81

80

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test material attained 14% degradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.
Executive summary:

Methods

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301 D, "Ready Biodegradability; Closed Bottle Test" referenced as Method C.4-E of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedures

The test material was exposed to sewage treatment micro-organisms at a concentration of 4 mg/l with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test material attained 14 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 D.

Description of key information

Methods

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous media. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301 D, "Ready Biodegradability; Closed Bottle Test" referenced as Method C.4-E of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedures

The test material was exposed to sewage treatment micro-organisms at a concentration of 4 mg/l with culture medium in sealed culture vessels in the dark at 21 °C for 28 days. The degradation of the test material was assessed by the determination of the amount of oxygen consumed. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test material attained 14 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 D.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information