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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:
28 day
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):MEA Polyborate 1:3
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:reaction product
- Physical state:liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:68% in water
- Isomers composition:
- Purity test date:
- Lot/batch No.:EU-SMG -1696
- Expiration date of the lot/batch:02 APRIL 2011
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:STABLE
- Storage condition of test material:room temerature in the dark
- Other:

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch,The Netherlands, receiving predominantly domestic sewage.
- Laboratory culture:
- Method of cultivation:
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further treatment.
- Storage length:fresh
- Preparation of inoculum for exposure:
- Pretreatment:
- Concentration of sludge: 4.0 g/L
- Initial cell/biomass concentration:
- Water filtered: yes
- Type and size of filter used, if any: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ionexchange
cartridges (Milli-Q) (Millipore Corp., Bedford, Mass., USA).
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 4 g/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: test material in
- Additional substrate:
- Solubilising agent (type and concentration if used):
- Test temperature:22+/-.2 deg C
- pH:7+/-1.5
- pH adjusted: no
- CEC (meq/100 g):
- Aeration of dilution water:
- Suspended solids concentration:
- Continuous darkness: yes/no
- Other:

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration:two
- Method used to create aerobic conditions:synthetic air
- Method used to create anaerobic conditions:
- Measuring equipment:Shimadzu TOC-Control V version 2.00.00 (Shimadzu, Kyoto, Japan): carbon analysis.
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific,
Trenton, NJ, USA): Temperature.
- Test performed in closed vessels due to significant volatility of test substance:
- Test performed in open system:
- Details of trap for CO2 and volatile organics if used:
- Other:

SAMPLING
- Sampling frequency:
- Sampling method:
- Sterility check if applicable:
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- Toxicity control:
- Other:

STATISTICAL METHODS:
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
not carried out
Test performance:
Acceptability of the test
1. The positive control substance was biodegraded by at least 60% (70%) within 14 days.
2. The difference of duplicate values for %-degradation of the test substance was always less
than 20.
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/l
(44.8 mg CO2 per 2 litres of medium, corresponding to 22.4 mg CO2/l).
4. The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral
medium at the beginning of the test was less than 5% of the Total Carbon content (TC).
Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO
water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than
5% of TC (mainly coming from the test substance, 12 mg TOC/l).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
77
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
28 d
Details on results:
Theoretical CO2 production
The TOC content of MEA Polyborate 1:3 was determined to be 8.818%. Based on the TOC
content the ThCO2 of MEA Polyborate 1:3 was calculated to be 0.32 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
The ThCO2 per test bottle are given in Tables 5-8 (APPENDIX I).
7.2. Biodegradation
All data are presented in APPENDIX I. The results of CO2 production and biodegradation in
blank bottles, back ground bottles and each test bottle are listed in Table 2 to 8. Table 9
contains the comparison of biodegradation of MEA Polyborate 1:3 in bottles A and B.
Figure 1 shows the curves for biodegradation of the two bottles with MEA Polyborate 1:3, the
positive control and the toxicity control.
0
20
40
60
80
100
0 10 20 30
Course of biodegradation in time (days)
% Biodegradation
Positive
control
Bottle A
Bottle B
Toxicity
control
Figure 1 Biodegradation of MEA Polyborate 1:3 and sodium acetate in the modified
Sturm test
The relative biodegradation values calculated from the measurements performed during the test
period revealed 77 and 73% biodegradation of MEA Polyborate 1:3, for the duplicate bottles
tested. Furthermore, in test bottle A biodegradation of MEA Polyborate 1:3 of 60% was reached
within a 10-day window (10% biodegradation was reached on day 4 and 60% biodegradation
was reached on day 13 (nominal days round to integer), the criterion for Ready
biodegradability).

BOD5 / COD results

Results with reference substance:
In the toxicity control more than 25% biodegradation occurred within 14 days (63%, based on
ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Any other information on results incl. tables

ThCO2, expressed as mg CO2/mg test substance, was calculated from the results of carbon analysis. The first step in calculating the amount of CO2 produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2 produced by a test substance is determined by the difference (in ml of titrant) between the experimental and blank Ba(OH)2 traps. The amount of 0.05 N HCl titrated is converted into mg of CO2 produced: 44 1.1 ml HCl titrated 2 0.05 ml HCl titrated mg CO2 Relative biodegradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production based on the total carbon content of the amount of test substance present in the test bottles. A figure of more than 10% biodegradation was considered significant. The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. If applicable, the number of days is calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10-day window), then the test substance is designated as readily biodegradable. Toxicity control: if less than 25% biodegradation (based on ThCO2) occurred within 14 days, the test substance was assumed to be inhibitory. The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in ml of titrant) between the blank Ba(OH)2 traps and fresh Ba(OH)2.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
MEA Polyborate 1:3 was readily biodegradable under the conditions of the modified Sturm test conducted under GLP conditions.
Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with MEA Polyborate 1:3. The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C and the ISO International Standard 9439, 1999. The batch of MEA Polyborate 1:3 was a clear colourless liquid. MEA Polyborate 1:3 was tested as delivered by the sponsor. The organic carbon content of MEA Polyborate 1:3 could not be calculated and therefore the Total Organic Carbon (TOC) content was determined. The TOC content of MEA Polyborate 1:3 was determined to be 8.818%. MEA Polyborate 1:3 was tested in duplicate at 136 mg/l, corresponding to 12 mg TOC/l. Based on the TOC content the ThCO2 of MEA Polyborate 1:3 was calculated to be 0.32 mg CO2/mg. The study consisted of six bottles: - 2 inoculum blanks (no test substance), - 2 test bottles (MEA Polyborate 1:3), - 1 positive control (sodium acetate) and - 1 toxicity control (MEA Polyborate 1:3 plus sodium acetate). Weighed amounts of MEA Polyborate 1:3 were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on the 29th day). The relative biodegradation values calculated from the measurements performed during the test period revealed 77 and 73% biodegradation of MEA Polyborate 1:3, for the duplicate bottles tested. Furthermore, in test bottle A biodegradation of MEA Polyborate 1:3 of 60% was reached within a 10-day window (10% biodegradation was reached on day 4 and 60% biodegradation was reached on day 13 (nominal days round to integer), the criterion for Ready biodegradability). In the toxicity control, MEA Polyborate 1:3 was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. In conclusion, MEA Polyborate 1:3 is designated as readily biodegradable.