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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:
12 June - 11 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Stability under test conditions: Biodegradation of tes item in the test system was monitored.
- Solubility and stability of the test substance in the solvent/vehicle: solvent vehicle not used.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: no
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : An amount of test item (40.5 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 13.5 mg/L, equivalent to 10 mg carbon/L.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A
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): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: N/A
- Storage length: N/A
- 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 °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 oC 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 2.3 g/L prior to use.
- Pretreatment: N/A
- Concentration of sludge: See above.
- Initial cell/biomass concentration: N/A
- Water filtered: No
- Type and size of filter used, if any: N/A
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
TOC
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 dihydrate (36.40 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 - 24 ºC
- pH: 7.4 - 7.7
- pH adjusted: Yes
- CEC (meq/100 g): N/A
- Aeration of dilution water: Yes, CO2-free air
- Suspended solids concentration: 2.3 g/L
- Continuous darkness: Yes
- Other: The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

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 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 used in the calculation.
- Test performed in closed vessels due to significant volatility of test substance: Yes
- 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: An amount of test item (44.4 mg) was weighed onto a filter paper* prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 14.8 mg/L, equivalent to 10 mg carbon/L.

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 used in the calculation.
- 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 - 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.
An amount of test item (44.4 mg) was weighed onto a filter paper* prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 14.8 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.
- Other: reference item - A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

STATISTICAL METHODS: N/A
Reference substance:
other: sodium benzoate
Test performance:
The toxicity control attained 64% 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 88 % biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 96 % biodegradation was attained. 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:
80
Sampling time:
28 d
Details on results:
The test item attained 80 % biodegradation after 28 days.
Results with reference substance:
Sodium benzoate attained 88% biodegradation after 14 days with greater than 60 % degradation being attained in a 10-Day window. After 28 days 96 % biodegradation was attained. 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

55

6

31

6

73

28

51

8

84

39

57

10

85

46

77

14

88

56

64

21

71

56

74

28

85

70

71

29*

96

80

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, but failing 10-day window
Conclusions:
The test item was readily biodegradable under the conditions of this test. Under the conditions of OECD Guideline No. 301B, the test item can be considered to be readily biodegradable. Please note that the 10-d window criteria does not apply to UVCB substances "where the test method can lead to misleading results" (UN/SCEGHS/16/INF.17). However, as this study will be applied to a mono-constituent form in this registration, the 10-day window should apply. Therefore, the substance is considered to be readily biodegradable, but failing the 10-day window.
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 88 % biodegradation at Day 14, confirming the suitability of the test system. The toxicity control achieved 64 % 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 in mixtures containing the test item was 80 % after 28 days. As the test item is a UVCB, the 10-day window criteria does not apply. The test item can therefore be classified as readily biodegradable under the conditions of this test.

However, as this study will be applied to a mono-constituent form in this registration, the 10-day window should apply.  Therefore, the substance is considered to be readily biodegradable, but failing the 10-day window.

Description of key information

28 day biodegradation = 80 %, readily biodegradable but failing the 10-day; OECD 301B; Best, N. (2018)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
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 88 % biodegradation at Day 14, confirming the suitability of the test system. The toxicity control achieved 64 % 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 in mixtures containing the test item was 80 % after 28 days. As the test item is a UVCB, the 10-day window criteria does not apply. The test item can therefore be classified as readily biodegradable under the conditions of this test.

However, as this study will be applied to a mono-constituent form in this registration, the 10-day window should apply.  Therefore, the substance is considered to be readily biodegradable, but failing the 10-day window.