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:
The study was conducted between 29 April 2009 and 27 May 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection: 19/08/2008 Date of Signature: 04/03/2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : PDE-28
Description : red powder
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : room temperature 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 (e.g. location, sampling depth, contamination history, procedure):
A mixed population of sewage sludge micro-organisms was obtained on 29 April 2009 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture:
Not stated
-
Method of cultivation:
An amount of test material (50 mg) was dispersed in culture medium and subjected to high shear mixing at approximately 7500 rpm for 15 minutes prior to the addition of inoculum (5 ml) and making up to a final volume of 500 ml with culture medium to give the test concentration of 100 mg/l.

A test concentration of 100 mg/l was selected for use in the study following the recommendations of the Test Guideline.

Analysis of the concentration, homogeneity and stability of the test material in the test preparations were not appropriate to the Test Guideline.

- Storage conditions:
Not stated

- Storage length:
Not stated

- Preparation of inoculum for exposure:
The sample of effluent was filtered through coarse filter paper (first approximate 200 ml discarded) and used immediately.

- Pretreatment:

Data supplied with the test material indicated that the test material was insoluble in water. Therefore the following pre-study solubility/dispersibility work was conducted to see how the best testable dispersion could be attained:

(i) Ultrasonication: An amount of test material (50 mg) was dispersed in 400 ml of deionised reverse osmosis purified water with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 500 ml. This formed a pale red dispersion with particles of test material on the surface and dispersed throughout.

(ii) High shear mixing: An amount of test material (50 mg) was dispersed in 400 ml of deionised reverse osmosis purified water with the aid of high shear mixing at approximately 7500 rpm for 15 minutes. The volume was then adjusted to a final volume of 500 ml. This formed a pale red dispersion with fine particles of test material on the surface and dispersed throughout.
The other methods specified in the protocol to aid dispersion of low water solubility test materials in the test system were not appropriate to this test material.

Based on the results of the pre-study solubility work it was concluded appropriate to disperse the test material into the test system with the aid of high shear mixing as this method appeared to increase the dispersibility of the test material over ultrasonication.

- Concentration of sludge:
A test concentration of 100 mg/l was selected for use in the study following the recommendations of the Test Guideline.

- Initial cell/biomass concentration:
Not stated

- Water filtered:
yes

- Type and size of filter used, if any:
coarse filter paper
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- 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* 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:
Not applicable

- Solubilising agent (type and concentration if used):
Not applicable

- Test temperature:
Sealed culture vessels in diffuse light at 21 +/- 1 deg C. Temperatures were recorded daily.

- pH:
The pH of all remaining control, test material, standard material and toxicity control vessels was measured prior to the vessels being placed in the CES Multi-channel Aerobic Respirometer.

- pH adjusted:
No

- CEC (meq/100 g):
Not stated

- Aeration of dilution water:
Not stated

- Suspended solids concentration:
Not stated

- Continuous darkness:
not stated

- Other:


TEST SYSTEM

The following test preparations were prepared and inoculated in 500 ml bottles:
a) Four replicate bottles containing inoculated culture medium to act as the control.
b) Three replicate bottles containing inoculated culture medium and the standard material, aniline, at a concentration of 100 mg/l.
c) Three replicate bottles containing inoculated culture medium and the test material at a concentration of 100 mg/l.
d) Two replicate bottles containing inoculated culture medium the standard material, aniline, at a concentration of 100 mg/l and the test
material at a concentration of 100 mg/l to act as toxicity control vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.

On Day 0 one control and one standard material were sampled for pH and DOC analysis.

The pH of all remaining control, test material, standard material and toxicity control vessels was measured prior to the vessels being placed in the CES Multi-channel Aerobic Respirometer.

The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolysing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated PC.


SAMPLING
- Sampling frequency:
Not stated

- Sampling method:
Not stated

- Sterility check if applicable:
Not stated

- Sample storage before analysis:
Not stated

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
Yes

- Abiotic sterile control:
Not stated

- Toxicity control:

For the purposes of the test, a toxicity control, containing the test material and aniline, was prepared in order to assess any toxic effect of the test material on the sewage treatment micro-organisms used in the study.
An amount of test material (50 mg) was dispersed in culture medium and subjected to high shear mixing at approximately 7500 rpm for 15 minutes prior to the addition of an aliquot (50 ml) of the aniline stock solution and inoculum (5 ml) and adjusting to a final volume of 500 ml with culture medium to give the test concentration of 100 mg test material/l and 100 mg aniline/l.

Reference substance
Reference substance:
aniline

Results and discussion

Test performance:
The mean BOD of the inoculated culture medium (control) was 39.30 mg O2/l after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The pH of the inoculated test vessels on Day 28 ranged from 7.8 to 8.0 and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate test material BOD values at the end of the test was ± 15% of the mean and therefore satisfied the validation criterion given in the OECD Test Guidelines.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The mean BOD of the inoculated culture medium (control) was 39.30 mg O2/l after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The pH of the inoculated test vessels on Day 28 ranged from 7.8 to 8.0 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate test material BOD values at the end of the test was ±15% of the mean and therefore satisfied the validation criterion given in the OECD Test Guidelines.

No degradation of the test material was observed after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.

Statistical analysis of the Day 28 BOD values for the control and test material vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test material.

The test material was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results whereby the toxicity control attained 26% degradation after 14 days and 37% degradation after 28 days.

BOD5 / COD results

BOD5 / COD
Parameter:
BOD5
Value:
>= 16.28 - <= 21.82 mg O2/g test mat.
Results with reference substance:
Aniline attained 66% degradation after 14 and 28 days thereby confirming the suitability of the inoculum and test conditions.
Aniline attained 96% degradation after 28 days, calculated from the results of the DOC analyses performed on Days 0 and 28. The degradation rate calculated from the results of the DOC analyses was higher than that calculated from oxygen consumption values. This is considered to be due to incorporation of the test material/aniline into the microbial biomass prior to degradation, and hence oxygen consumption, occurring.

Any other information on results incl. tables

The BOD values and the degree of degradation calculated from the oxygen consumption data for the control, test and standard materials and the toxicity control are given in Table 1. The daily BOD values and percentage degradation values are given in Appendices 2 and 3. The calculated Theoretical Oxygen Demand values for the test and standard materials are given in Appendix 4. BOD curves and biodegradation curves are given in Figures 2 and 3 respectively (see attached material).

The results of the DOC analyses are summarised in Table 2. Daily temperature readings are given in Table 3 and pH values of each individual vessel are given in Table 4.

The mean BOD of the inoculated culture medium (control) was 39.30 mg O2/l after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No degradation of the test material was observed after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.
Executive summary:

Introduction:

The study was perford 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 301F, “Ready Biodegradability; Manotric Respirotry Test” referenced asthod C.4-D of Commission Regulations (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (q)).

Methods:

The test material at a concentration of 100 mg/l was exposed to sewage sludge micro-organisms with culture medium in sealed culture vessels in diffuse light at 21 ± 1ºC for 28 days. 

The degradation of the test material was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the standard material, aniline, together with a toxicity control were used for validation purposes.

Results:

No degradation of the test material was observed after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.