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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experiment was performed during following period: 2017-06-27 to 2017-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
Version of 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
Method published 2008 in Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Information not available
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The department of health of the government of the United Kingdom (2017-11-28)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-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 26 June 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: No, inoculum was sampled at a sewage treatment plant.
- Method of cultivation: Not applicable.
- Storage conditions: No storage required. Sample was used on day of collection.
- Storage length: No storage of sample performed.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral 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 ºC and used on the day of collection.
- Pretreatment: 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 deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC 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 concentration was equal to 2.2 g/L prior to use.
- Initial cell/biomass concentration: Not reported.
- Water filtered: Yes, sample was filtered for determination of suspended solids level.
- Type and size of filter used, if any: pre-weighed GF/A filter paper (rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven)
Duration of test (contact time):
28 d
Initial conc.:
10.7 mg/L
Based on:
test mat.
Remarks:
The volume of mineral medium was adjusted to 3 liters to give a final concentration of 10.7 mg/L, equivalent to 10 mg carbon/L.
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
The biodegradation of the test item was assessed by the determination of carbon dioxide produced.
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
Solution a: KH2PO4: 8.50 g/L, K2HPO4: 21.75 g/L, Na2HPO4 x 2H2O: 33.20 g/L, NH4Cl: 0.50 g/L; pH 7.4.
Solution b: CaCl2: 27.50 g/L.
Solution c: MgSO4 x 7H2O: 22.50 g/L.
Solution d: FeCl3 x 6H2O: 0.25 g/L
To one liter (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 and 1 mL of Solution d.
- Additional substrate: No.
- Solubilising agent (type and concentration if used): Not used. The test item was dispersed directly in mineral medium.
- Test temperature: The test was carried out in a temperature controlled room at temperatures of between 22 and 25 °C.
- pH: The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.
- pH adjusted: The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution.
- CEC (meq/100 g): Information not available.
- Aeration of dilution water: No.
- Suspended solids concentration: The suspended solids concentration was equal to 2.2 g/L prior to use. Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- Continuous darkness: Yes, test was performed in darkness.

TEST SYSTEM
- Culturing apparatus: The test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution.
- Number of culture flasks/concentration: 2 vessels per concentration, 2 vessels for inoculum control, 2 vessels for reference substance and 1 vessel for toxicity control.
- Method used to create aerobic conditions: The added mineral medium had been purged overnight with CO2 free air.
- Method used to create anaerobic conditions: Not applicable.
- Measuring equipment: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer.
- 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: 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 water.

SAMPLING
- Sampling frequency: Samples of first CO2-absorber vessels were taken on day 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.
- Sampling method: Samples of 2 mL were taken from the absorber vessels.
- Sterility check if applicable: Not performed.
- Sample storage before analysis: Information not available.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.

STATISTICAL METHODS: The biodegradation parameter (percentage biodegradation, total CO2 evolution) were calculated according to the equation given in the mentioned guidelines. No further statistical proedures applied.
Reference substance:
benzoic acid, sodium salt
Remarks:
Purity of reference substance >99.5%
Preliminary study:
Information provided by the Sponsor indicated that the test item solubility in water was 0.15 mg/L. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation: From this preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when using the ultrasonication method of preparation. Refer to section above ( "Any other information on materials and methods")
Test performance:
The test item was dispersed directly in mineral medium. An amount of test item (32.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (approximately 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 10.7 mg/L, equivalent to 10 mg carbon/L. A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
Determination of IC/TC Ratio:
Samples (30 mL) were removed from the test item vessels on Day 0 prior to the addition of the test item in order to calculate the IC content in the test media. The samples were filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. Samples (30 mL) were also removed from the inoculum control vessels on Day 0 and filtered through 0.45 μm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis. IC/TC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
6 d
Parameter:
% degradation (CO2 evolution)
Value:
1
Sampling time:
10 d
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
14 d
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
21 d
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
28 d
Details on results:
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 item. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of inoculum control. Replicate 1, procedure control Replicate 1 and procedure control Replicate 2. This decrease was considered to be due to sampling/analytical variation. 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.
Results with reference substance:
Degradation of reference substance (sodium benzoate):
Sodium benzoate attained 67% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The toxicity control attained 25% biodegradation after 14 days and 33% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

Table 1: Percentage biodegradation values:

     % biodegrdation  
 Day  Procedure control Test item  Toxicity control 
 0  0  0
 2  52  0  18
 6  68  0  22
 8  70  0  28
 10  61  1  30
 14  67  0  25
 21  69  0  32
 28  84  0  29
 29  78  11  33
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)). The test item attained 11% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. The reference substance (sodium benzoate) degraded rapidly (70% after 8 days).
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)). The study was performed following GLP.

The test item, at a concentration of 10 mg carbon/L, was exposed to predominantly domestic activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 and 25°C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. samples of first CO2-absorber vessels were taken on day 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27. 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 water.

Results: The test item attained 11% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. The reference substance (sodium benzoate) degraded rapidly (70% after 8 days). The results are summarised in the following table:

Table 1: Percentage biodegradation values:

     % biodegrdation  
 Day  Procedure control Test item  Toxicity control 
 0  0  0
 2  52  0  18
 6  68  0  22
 8  70  0  28
 10  61  1  30
 14  67  0  25
 21  69  0  32
 28  84  0  29
 29  78  11  33

Description of key information

OECD 301B, GLP, domestic sewage, 28 d, CO2 evolution, not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

The test item, at a concentration of 10 mg carbon/L, was exposed to predominantly domestic activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 22 and 25°C for 28 days. The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes. samples of first CO2-absorber vessels were taken on day 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29. The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27. 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 water.

Results: The test item attained 11% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. The reference substance (sodium benzoate) degraded rapidly (70% after 8 days).

[Type of water: freshwater]