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
Study period:
16 March 2004 to 16 April 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
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)
Version / remarks:
Commision Directive 67/548/EEC, Annex V
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of Inspection: 2 December 2002 Date of Signature: 13 February 2003)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TIPX
- Substance type: Colourless liquid.
- Physical state: Liquid.
- Lot/batch No.: 042028.
- Storage condition of test material: Approximately 4ºC in the dark.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge : A mixed population of activated sewage sludge micro-organisms was obtained on 30 July 2007 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, United Kingdom, which treats predominantly domestic sewage.

- Condition for maintaining the laboratory culture: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21ºC.

- Preparation of inoculum for exposure: 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.

- Concentration of sludge: The suspended solids was equal to 3.4 g/l prior to use.

- Initial cell/biomass concentration: 30 mg suspended solids (ss)/l.

- Type and size of filter used, if any: Pre-weighed GF/A filter paper using a Buchner funnel.
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
10 other: mg carbon/l
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

- Preparation of test solution: The test material was prepared by adsorption onto silica gel prior to dispersion in culture medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material ex[posed to the test organisms.
An amount of test material was adsorbed onto the surface of 100 mg of granular silica gel (230 – 400 mesh Sigma Lot No 101K3700) prior to dispersal in approximately 100 ml of culture medium with the aid of high shear mixing (7500 rpm, 10 minutes). The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 15.9 mg/l, equivalent to 10 mg carbon/l.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 26.5 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.

- Composition of medium:
Solution a KH2PO4 8.50 g/l
K2HPO4 21.75 g/l
Na2HPO4.2H2O 33.40 g/l
NH4Cl 0.50 g/l

pH = 7.4

Solution b CaCl2 27.50 g/l
Solution c MgSO4.7H2O 22.50 g/l
Solution d FeCl3.6H2O 0.25 g/l

To 1 litre (final volume) of purified water(reverse osmosis purified and deionised water (Elga Optima 15+ for Elga Purelab Option R-15 BP) was added the following volumes of solutions a – d.

10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

- Additional substrate: Silica gel.

- Test temperature: 21ºC.

- pH: 7.5 - 7.6.

- pH adjusted: No.


TEST SYSTEM
- Culturing apparatus: Five litre glass culture vessels each containing 3 litres of solution. 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 self-indicating soda lime (Carbosorb®) granules.

- Number of culture flasks/concentration: 2.

- Method used to untreated conditions: 2.

- Measuring equipment: 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.

- Test performed in : closed systems.


SAMPLING
- Sampling frequency:
CO2 analysis: Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29. On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

DOC analysis: On Days 0 and 28 samples (20 ml) were removed from all culture vessels and centrifuged (3500 rpm, 15 minutes) prior to DOC analysis.

- Sample storage before analysis:
CO2 analysis: The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately - 20ºC. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.


CONTROL AND BLANK SYSTEM
- Untreated control: Prepared containing 100 mg silica gel per 3 litres of inoculated culture medium.

- Toxicity control: For the purposes of the test a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (47.7 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230 - 400 mesh Sigma Lot No 101K3700) prior to dispersal in approximately 100 ml of culture medium with the aid of high sheaker mixing (7500 rpm, 10 minutes). The test material/silica gel/culture medium dispersion was then dispersed in inoculated culture medium and an aliquot (51.4 ml) of the sodium benzoate stock solution added. The volume was adjusted to 3 litres to give a final concentration of 15.9 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

- Other: Standard Material
Sodium benzoate (Sigma Lot No 091K12451), a final concentration of 17.1 mg/l, equivalent to 10 mg carbon/l.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Not performed.
Test performance:
The total CO2 evolution in the control vessesl on Day 28 was 25.02 mg/l.
The IC/TC ratio of the test material suspension in the mineral medium at the start of the test was below 5%.
The difference between the values for CO2 production at the end of the test for the replicate vessels was < 20%.
Above satisfied the validation criterion give in the OECD Test Guidelines.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
35
Sampling time:
28 d
Details on results:
CO2 evolution and biodegradation:
See tables 1 and 2, as attached, for the result. The test material attained 35% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The results of the inorganic arbon analysis on Day 29 showed increase in all replicate vessels with the exception of control replicate R2. This was considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28. These increases coupled with the decrease within control replicate R2 resulted in an increase in the percentage degradation value for the test material from 35% on Day 28 to 45% on Day 29.

DOC analysis:
Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), gave percentage degradation values of 90% and 96% respectively for the test material Replicates R1 and R. See Table 3, as attached, for the result.

Observations made throughout the test period showed the contents of the control vessels to be slightly cloudy light brown dispersions of inoculum and silica gel and the standard material vessels were slightly cloudy light brown dispersions of inoculum and silica gel with no undissolved standard material visible. The test material vessels were cloudy light brown dispersions of inoculum, test material and silica gel and the toxicity control vessel was a cloudy light brown dispersions of inoculum, test material and silica gel with no undissolved standard material visible.


BOD5 / COD results

Results with reference substance:
See tables 1 and 2, as attached, for the results. The toxicity control attained 53% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.
Sodium benzoate attained 102% 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:
other: not readily biodegradable
Conclusions:
The test material attained 35% degradation after 28 days and therefore cannot 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; CO2Evolution 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 21ºC for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1996) and in the published literature, the test material wa absorbed onto granular silica gel prior to dispersion in the test medium in order to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

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 35% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.