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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:
05 July 2018 to 15 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 301B guideline without deviation. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
07-July-1992
Deviations:
yes
Remarks:
Guidleline recommended temperature exceeded on day 19, this was marginal and did not impact the study.
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
Guidleline recommended temperature exceeded on day 19, this was marginal and did not impact the study.
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
yes
Remarks:
Guidleline recommended temperature exceeded on day 19, this was marginal and did not impact the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Confidential
- Expiration date of the lot/batch: Confidential

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not relevant
- Specific activity: not relevant
- Locations of the label: not relevant
- Expiration date of radiochemical substance: not relevant

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Ready biodegradability test.
- Solubility and stability of the test substance in the solvent/vehicle: not relevant
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not relevant

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was weighed onto a filter paper prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A

OTHER SPECIFICS: not relevant
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK - predominantly domestic sewage.
- Laboratory culture: No
- Method of cultivation: Not relevant (sludge collected from Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK)
- Storage conditions: Not relevant
- Storage length: Not relevant
- Preparation of inoculum for exposure: Sludge was used on used on same day as collection. 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 °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 deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 °C for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Pretreatment: full process as above.
- Concentration of sludge: The suspended solids concentration was equal to 2.9 g/L prior to use.
- Initial cell/biomass concentration: no data
- Water filtered: deionized reverse osmosis water
- Type and size of filter used, if any: no data
Duration of test (contact time):
28 d
Initial conc.:
14.8 mg/L
Based on:
test mat.
Initial conc.:
10 other: mg/ carbon/L
Based on:
TOC
Remarks:
data supplied by Registrant indicates the test item contains 67.39 % carbon
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral salts medium (MSM) for the test was prepared by establishing 10 mL of stock solution 1 and 1 mL of solutions 2, 3 and 4 in each litre of water required for the test.
Stock 1 - Potassium dihydrogen phosphate (8.50 g/L); di-Potassium hydrogen phosphate (21.75 g/L); di-Sodium monohydrogen phosphate dihydrate (33.40 g/L); Ammonium chloride (0.5 g/L)
Stock 2 - Magnesium sulphate heptahydrate (22.50 g/L)
Stock 3 - Calcium chloride (27.50 g/L)
Stock 4 - Iron (III) chloride hexahydrate (0.25 g/L)
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22 - 25 ºC
- pH: 7.4 - 7.5
- pH adjusted: Yes
- CEC (meq/100 g): N/A
- Aeration of dilution water: Yes, CO2-free air
- Suspended solids concentration: 2.9 g/L (30 mg/L in test vessels)
- Continuous darkness: Yes
- Other: N/A

TEST SYSTEM
- Culturing apparatus: 5 L test culture vessels
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: CO2-free air drawn through test system.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer. Samples (50 μL) were injected into the TC and IC channel of the TOC analyzer. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas. IC analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionized water. Each analysis was carried out in at least triplicate with three replicates being reported.
- Test performed in closed vessels due to significant volatility of test substance: Yes, but substance not assumed to be greatly volatile.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Duplicate 350 mL 0.05 M NaOH traps.
- Other: N/A

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Sampling method: The samples were analyzed for IC using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in at least triplicate with three replicates being reported. TC analysis is carried out at 680 °C using a platinum based catalyst and zero grade air as the carrier gas, resulting CO2 was measured by an IR detector.
- Sterility check if applicable: N/A
- Sample storage before analysis: Samples analysed immediately.
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No
- Toxicity control: Yes
- Other: Reference control

STATISTICAL METHODS: N/A
Reference substance:
benzoic acid, sodium salt
Test performance:
The toxicity control attained 56 % biodegradation after 14 days and 83 % 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.

Sodium benzoate attained 93 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 104 % biodegradation was attained. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
66
Sampling time:
28 d
Details on results:
The test item attained 66 % biodegradation after 28 days. The test item did not meet the 10-day window criterion as prescribed within the Guideline, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%.
Results with reference substance:
Sodium benzoate attained 93 % biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 104 % biodegradation was attained. Biodegradation values in excess of 100% were considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Table 1 Biodegradation results

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

57

2

27

6

83

19

42

8

84

26

52

10

85

34

61

14

93

50

56

21

91

54

66

28

94

62

74

29*

104

66

83

*Day 29 values are corrected to account for any carry-over of CO2 detected in absorber trap 2

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The reference substance flask achieved 93 % biodegradation at Day 14, confirming the suitability of the test system. The toxicity control achieved 56 % biodegradation after 14 days and 83 % after 28 days, confirming that the test item did not have an inhibitory effect on the micro-organisms in the test system.  The test item degraded by 66 % in the test system.

Executive summary:

OECD 301B (2018) - The ready biodegradability of the test item was assessed in accordance with OECD Procedure 301B ‘Ready Biodegradability, CO2 Evolution Test’, adopted 17 July 1992.

 

Test item was added to two bottles containing mineral salts medium inoculated with activated to give a nominal test concentration of 10 mg C/L. Two control cultures contained inoculated mineral salts medium alone (inoculum blank). Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mg C/L) (procedural control) of which one also contained the test item (total 20 mg C/L) (toxicity control) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. The test system comprised of the test vessels connected to two CO2traps containing 0.05 M NaOH. CO2-free air was drawn through the system.

 

The reference substance flask achieved 93 % biodegradation at Day 14, confirming the suitability of the test system. The toxicity control achieved 56 % biodegradation after 14 days and 83 % after 28 days, confirming that the test item did not have an inhibitory effect on the micro-organisms in the test system. 

 

Mean biodegradation of the the test item was 66 % after 28 days.

Description of key information

28 day biodegradation = 66 %, readily biodegradable; OECD 301B; Best, N. (2018)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

OECD 301B (2018) - The ready biodegradability of the test item was assessed in accordance with OECD Procedure 301B ‘Ready Biodegradability, CO2Evolution Test’, adopted 17 July 1992.

 

Test item was added to two bottles containing mineral salts medium inoculated with activated to give a nominal test concentration of 10 mg C/L. Two control cultures contained inoculated mineral salts medium alone (inoculum blank). Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mg C/L) (procedural control) of which one also contained the test item (total 20 mg C/L) (toxicity control) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. The test system comprised of the test vessels connected to two CO2traps containing 0.05 M NaOH. CO2-free air was drawn through the system.

 

The reference substance flask achieved 93 % biodegradation at Day 14, confirming the suitability of the test system. The toxicity control achieved 56 % biodegradation after 14 days and 83 % after 28 days, confirming that the test item did not have an inhibitory effect on the micro-organisms in the test system. 

 

Mean biodegradation of the the test item was 66 % after 28 days. The test item was considered by the laboratory report to not be readily biodegradable under the conditions of this test. This was due to a failure of the 10-day window.  However, please note that the 10-d window criteria does not apply to mixtures or UVCB substances (UN/SCEGHS/16/INF.17 and Regulation (EC) 1272/2008). Under the conditions of OECD Guideline No. 301B, the test item can be considered to be readily biodegradable.