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:
17/09/2008 - 24/10/2008
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:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A mixed population of activated sewage sludge micro-organisms was obtained on 22 September 2008 from the aeration stage of the
Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

- Laboratory culture:
Not applicable

- Method of cultivation:
The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive
amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 deg C and 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 washed activated sewage sludge by suction
through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the
filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately
105 deg C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The
suspended solids concentration was equal to 2.4 g/l prior to use.
*Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

- Preparation of inoculum for exposure:
As above.

- Pretreatment:
Not stated.

- Concentration of sludge:
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l.

- Initial cell/biomass concentration:
Not stated.

- Water filtered: yes/no
No

- Type and size of filter used, if any:
None
Duration of test (contact time):
29 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
The culture medium used in this study was that recommended in the OECD Guidelines (see below).

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
* Reverse osmosis purified and deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

- Additional substrate:
None.

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

- Test temperature:
21 deg C

- pH:
See remarks section.

- pH adjusted: yes/no
No.

- CEC (meq/100 g):
Not stated.

- Aeration of dilution water:
Not stated.

- Suspended solids concentration:
30 g/l prior to use.

- Continuous darkness:
yes

TEST SYSTEM
- Culturing apparatus:
5 litre glass culture vessels

- Number of culture flasks/concentration:
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).

- Method used to create aerobic conditions:
The culture vessels were sealed and CO3-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.

- Method used to create anaerobic conditions:
Not applicable.

- Measuring equipment:
Tekmar-Dohrmann apollo 9000 TOC analyses and a Shimadzu TOC-Vcsh TOC analyser

- Test performed in closed vessels due to significant volatility of test substance:
No.

- Test performed in open system:
Yes.

- Details of trap for CO2 and volatile organics if used:
Not applicable.

SAMPLING
- Sampling frequency:
-CO2
Samples (2 ml) were taken from the first CO2 absorber vessel on 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.
-DOC
Daily (0-28)

- Sampling method:
-CO2
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. Each analysis was carried out in triplicate.
-DOC
On Days 0 and 28 samples (20 ml) were removed from all culture vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis

- Sterility check if applicable:
Not applicable.

- Sample storage before analysis:
-CO2
The samples taken on Days 12 and 18 were stored at approximately 20 deg C. However, these samples were not analysed for CO2 as the results obtained from previous and
subsequent analyses showed that no significant change in degradation occurred during this time and therefore additional analyses were
considered to be unnecessary.
-DOC
Samples were analysed immediately.


CONTROL AND BLANK SYSTEM
- Inoculum blank:
Not applicable.
- Abiotic sterile control:
Not stated.
- 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 aliquot (177 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 17.7 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

- Other:
- Standard material:
For the purposes of the test, a standard material, sodium benzoate (C6H5COONa) (Sigma Aldrich Lot No 095K0681), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the standard material directly in culture medium with the aid of ultrasonication for approximately 3 minutes. An aliquot (51.4 ml) of this stock solution was added to the test vessel and the volume adjusted to 3 litres to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the standard material was inverted several times to ensure homogeneity of the solution.


STATISTICAL METHODS:
See 'Any other information on materials and methods section including tables' section below.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium Benzoate

Results and discussion

Preliminary study:
During the study, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-5050A analyser (see Appendix 1).
An amount of test material (100 mg) was dissolved in culture medium (1000 ml) with the aid of ultrasonication for approximately 15 minutes to give a 100 mg/l stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a Gelman 0.45 µm Acrocap filter (discarding the initial 5 ml to pre-condition the filter). A further amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 minutes and inoculated at a concentration of 30 mg suspended solids (ss)/l prior to adjusting to a final volume of 1000 ml. Two samples were taken for DOC analysis; one after filtration through a Gelman 0.45 µm Acrocap filter (discarding the initial 5 ml to pre-condition the filter) and the other after centrifugation at 3500 rpm for 15 minutes. Control samples were prepared by inoculating culture medium (1000 ml) at a suspended solids level of 30 mg ss/l and then filtering or centrifuging as per the test material samples.
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge (see Appendix 1). Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test material
Test performance:
The test system is considered valid as the standard reference substance (sodium benzoate) attained 80% after 14 days and 82% degradation after 28 days.
The toxicity control attained 66% degradation after 14 days and 67% degradation after 14days and 67% degradation after 28 days, therefore confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
29
Sampling time:
28 d
Details on results:
Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.
The total CO2 evolution in the control vessels on Day 28 was 32.19 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test material attained 29% 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 toxicity control attained 66% degradation after 14 days and 67% 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 80% degradation after 14 days and 82% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 100% for both the test material Replicates R1 and R2 and for the toxicity control. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to adsorption of the test material to the glassware and/or activated sewage sludge and the subsequent removal by filtration prior to DOC analysis of the test samples. The results obtained from the preliminary investigational work indicated that the test material did not absorb to the activated sewage sludge (see Appendix 1). However, the samples taken from this work were sampled immediately after the test material stock solution had been inoculated and therefore, it was considered that over the test period of 28 days the test material could have adsorbed to the activated sewage sludge and/or the glassware.
Sodium benzoate attained 100% degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.
Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved test material or standard material visible.


Percentage Degradation values table is included in the Overall Remarks on Results section below.

BOD5 / COD results

Results with reference substance:
Not applicable.

Any other information on results incl. tables

Table 2 Percentage Biodegradation Values

 Day % Degradation Sodium Benzoate % Degradation Test Material  % Degradation Test Materials plus Sodum Bensoate Toxicity Control   
0
21  24 
40  37 
36  10  50 
33  24  58 
58  30  70 
10  65  23  65 
14  80  25  66 
16  87  30  70 
20 82  25  68 
22  75  19  66 
24  75  19  69 
27  80  19  62 
28  79  23  65 
29  82  29  67 

Table3               Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon*

(mg/l)

Inorganic Carbon*

(mg/l)

IC Content (% of TC)

Sodium Benzoate

10 mg C/lR1

8.27

-0.74

0

Sodium Benzoate

10 mg C/l R2

8.76

-0.41

0

Test Material

10 mg C/l R1

10.36

0.18

2

Test Material

10 mg C/l R2

8.61

-0.54

0

Test Material plus Sodium Benzoate Toxicity Control

20 mg C/l

16.79

-0.41

0


R1– R2= Replicates 1 and 2

*Corrected for control values. Negative values are due toasured concentrations being less than control values

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The reference substance achieved 82% degradation at 28 days therefore the test was validated according to the OECD guideline.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material attained 29% 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 Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 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 29% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.