Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2003-02-13

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : SEPIWHITE MSH
Description : off-white powder
Batch number : 0214400005 (see Appendix 2)
Date received : 13 October 2003
Storage conditions : approximately 4ºC in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on
16 February 2004 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at
Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
Duration of test (contact time):
ca. 28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 13.8 mg/L
Based on:
test mat.
Initial conc.:
ca. 10 mg/L
Based on:
other: mg carbon /L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

Preparation of inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a
temperature of 21ºC and was used on the day of collection. Determination of the suspended
solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the
activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner
funnel. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and
allowed to cool before weighing. This process was repeated until a constant weight was attained.
The suspended solids was equal to 2.8 g/l prior to use.

Culture medium
The culture medium used in this study (see Appendix 1) was that recommended in the
OECD Guidelines.

Preparation of test system
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each
containing 3 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 concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of
10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final
concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg
suspended solids (ss)/l. The study was carried out in a temperature controlled room at 21°C, in
darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled
with 2400 ml of culture medium and 32.1 ml of inoculum and aerated overnight. On Day 0 the
test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the
addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of
approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing selfindicating
soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing
350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed
water.

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
75
Sampling time:
28 d
Details on results:
Points of degradation plot (test substance):
4 % degradation after 1 d
22 % degradation after 2 d
44 % degradation after 6 d
67 % degradation after 8 d
72 % degradation after 10 d
75 % degradation after 28 d

BOD5 / COD results

Results with reference substance:
Points of degradation plot (reference substance):
16 % degradation after 1 d
41 % degradation after 2 d
63 % degradation after 6 d
66 % degradation after 8 d
71 % degradation after 10 d
85 % degradation after 28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test material attained 75% degradation after 28 days and satisfied the 10-Day window
validation criterion, whereby 60% degradation must be attained within 10 days of the degradation
rate exceeding 10%. The test material can therefore be considered to be readily biodegradable
under the strict terms and conditions of OECD Guideline No 301B.
Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

Methods.

The test material, at a concentration of 10 mg C/l, was exposed to activated sewage sludge micro-organisms 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 carbon dioxide produced. 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 75% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.