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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-11-08 - 1993-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP according to directive 88/320 EEC, date of inspection: 1992-03-17

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-methoxyphenyl)-3-oxobutyramide]
EC Number:
250-797-6
EC Name:
2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-methoxyphenyl)-3-oxobutyramide]
Cas Number:
31775-16-3
Molecular formula:
C34H30Cl2N6O6
IUPAC Name:
2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[N-(4-methoxyphenyl)-3-oxobutanamide]
Test material form:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Type: A mixed population of activated sludge microorganisms
Source: The aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, treating predominantly domestic sewage.
Date of collection: 8 November 1993.
Pre-Treatment: The activated sludge was homogenised for approximately 2 minutes and allowed to settle for 1/2 hour. The supernatant was centrifuged to remove coarse solids and used for testing.
Usage rate: 1% inoculum.
Duration of test (contact time):
28
Initial test substance concentration
Initial conc.:
33.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test sample:
Method of Preparation: Direct dispersion of the test substance in culture medium

Exposure
Duration: 28 days.
Vessels: 5 litre glass culture vessels containing 3 litres.
Culture medium: As specified in OECD-guideline 301
Loading: 1% inoculum per test vessel.
Test concentration: 20 mg carbon/L in duplicate.
Reference material 10 mg carbon/L sodium benzoate, in duplicate.
Test series:
1. Culture medium with inoculum in duplicate.
2. Culture medium with inoculum and sodium benzoate in duplicate
3. Culture medium with inoculum and test substance in duplicate.
4. Culture medium with inoculum and test substance, poisoned by the addition of 10 ml of a 10 g/1 mercuric chloride solution to act as an abiotic control (one vessel only).

Temperature: 21 ± 1°C
Light regime: The study was carried out in darkness.
Procedure: Approximately 24 hours prior to the start of the study the vessels were filled with 2400 mL of culture medium and 30 mL of inoculum and aerated overnight. On day 0 the test and standard materials were added and the volume in all vessels made up to 3 litres by the addition of culture medium. The CO2 absorption bottles were connected to the outlet of the vessels on day 0.

Aeration: The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 mL/min and stirred continuously by magnetic stirrers. CO2-free air was produced by sparging compressed air through the following series:
- 3 x 500 ml Dreschel bottles filled with 350 mL 10 N NaOH.
- 1 x 500 ml Dreschel bottle filled with 350 mL 0.025 N Ba(OH)2
- 1 x 500 ml empty Dreschel bottle to prevent liquid carry-over.

CO2-absorption: 2 x 500 mL Dreschel bottles filled with 350 mL 0.05 M NaOH. All CO2 absorbing solutions were prepared using purified de-gassed water.

Sampling: Samples (2 mL) were taken from the first CO2 absorber vessel on days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on days 0 and 29.
On day 28 the pH of each test vessel was measured and 1 mL of concentrated HCl added to drive off inorganic carbonate. The vessels were aerated overnight and the final samples were taken from both absorber vessels on day 29.

Apparatus: Ionics 555 TOC Anlyser. Each analysis was carried out in triplicate.

Evaluation: All calculations were performed according to the guideline (OECD 301B).
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
not applicable
Test performance:
not applicable
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
28 d
Details on results:
The pH values of the test material, standard material and control cultures an day 28 were 7.5 and 7.5 (test material), 7.5 and 7.5 (standard material), 7.5 and 7.5 (control) and 7.4 (abiotic control).
The test substance attained 1% degradation after 28 days and so, therefore, cannot be considered as readily biodegradable under the strict terms and conditions of the OECD Guidelines.
Additional investigational work was carried out using the Activated Sludge Respiration Inhibition test method (OECD Guideline No. 209) which showed that the test substance did not inhibit the respiration of sewage sludge microorganisms at the test concentration employed in the test. In the light of this the inhibition control vessel (test substance plus sodium benzoate) was omitted from the test.
Therefore from these results it is evident that the test substance is not readily biodegradable and that this result is not due to a toxic effect on the inoculum.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
A ready biodegradability test on the test substance according to OECD 301B (CO2 -evolution) and further testing for activated sludge respiration inhibition (OECD 209, results only mentioned in the study report) it is evident that the test substance is not readily biodegradable (1% biodegradation) and that this result is not due to a toxic effect on the inoculum.
Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Executive summary:

In a ready biodegradablility test (reliability categoriy 1) on the test substance according to OECD 301B (CO2 -evolution) and complient to GLP, the test substance attained 1% degradation at a concentration of 20 mg C/L. To exclude toxicity of the test item to the inoculum at the tested concentration additional investigational work was carried out using the Activated Sludge Respiration Inhibition test method (OECD Guideline No. 209, results mentioned in this report, but no study report available) which showed that the test substance did not inhibit the respiration of sewage sludge microorganisms at the test concentration employed in the test. Therefore from these results it is evident that the test substance is not readily biodegradable and that this result is not due to a toxic effect on the inoculum.

Sodium benzoate attained 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.