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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:
21 December 2018 (Study Initiation) to 13 March 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
the test guideline advises a test solution concentration of 2 to 5 mg/L, howver due to the limited water solubility of the test item, the study was performed at the maximum solubility which can be achieved for the test item.
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
yes
Remarks:
the test guideline advises a test solution concentration of 2 to 5 mg/L, howver due to the limited water solubility of the test item, the study was performed at the maximum solubility which can be achieved for the test item.
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test (release date) date: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as supplied by the Sponsor
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility of the test substance in the solvent/vehicle: Water solubility: <0.17 mg/L. Considering the low water solubility of the test item, a stock solution was prepared by mixing the required quantity of test item in the desired volume of Silicon oil AR
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary effluent from the JRF Sewage Treatment Plant treating predominantly domestic sewage was used as the inoculum.
- Laboratory culture: Yes
- Method of cultivation: The inoculum was pre-conditioned by aerating at 22 ± 2 °C for 6 days.
- Storage conditions: not specified
- Preparation of inoculum for exposure: The Secondary effluent was filtered through a Whatman No. 1 filter paper. The inoculum was pre-conditioned by aerating at 22 ± 2 °C for 6 days. The inoculum was analysed for microorganism concentration and confirmed as 3.1 x 104 CFU/mL.
- Pretreatment:
- Concentration of sludge: The inoculum blank was prepared by mixing 4.0 mL inoculum with 3996 mL of mineral medium in a conical flask of 5 L capacity.
- Initial cell/biomass concentration: The inoculum was analysed for microorganism concentration and confirmed as 3.1 x 104 CFU/mL.
- Water filtered: yes
- Type and size of filter used, if any:
Duration of test (contact time):
28 d
Initial conc.:
0.38 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
Composition of the medium:

Preparation of Mineral Medium
Mineral Medium for Stock Solution (mL) Capacity of Flask (L) Diluted With Volume of Diluents (mL)
A B C D
Inoculum Blank (IB) 4 4 4 4 5 Milli-Q water 3984
Test Solution (TS) 4 4 4 4 5 3984
Procedure Control (PC) 4 4 4 4 5 3984
Toxicity Control (TC) 2 2 2 2 2 1992
Test Blank (TB) 4 4 4 4 5 3984


- Preparation of stock solution for mineral medium: four stock solutions were used;

Stock Solution Chemicals used Quantity (g) Final volume (mL)
A Potassium dihydrogen orthophosphate 0.850 100
Dipotassium hydrogen orthophosphate 2.175
Disodium hydrogen orthophosphate dihydrate 3.340
Ammonium chloride 0.050

B Calcium chloride dihydrate 3.640 100

C Magnesium sulphate heptahydrate 2.250 100

D Iron (III) chloride hexahydrate 0.0250 100

- Additional substrate: no
- Solubilising agent: Silicon oil (Signa-Aldrich). A test item weigh (mg as supplied, and taking purity into account) was transferred into a 10 mL volumetric flask, dissolved in 7 mL silicon oil and sonicated for 30 minutes. The volume was made up with silicon oil to prepare a stock solution.
- Test temperature: 22 ± 2°C
- pH: The pH of stock solution A was 7.44.
- pH adjusted: no
- Aeration of dilution water: yes, the mineral medium was strongly aerated for 20 minutes and allowed to stand for 20 h at 22 ± 2 °C. All operations were performed in a horizontal laminar flow under aseptic conditions.
- Continuous darkness: yes, iIncubation was performed under dark conditions

TEST SYSTEM
- Culturing apparatus: Conical Flask
- Number of culture flasks/concentration: 2 (in duplicate)
- Method used to create aerobic conditions:
1) Preparation, Pre-conditioning and Analysis of inoculum: Secondary effluent from the JRF Sewage Treatment Plant treating predominantly domestic sewage was used as the inoculum. The Secondary effluent was filtered through a Whatman No. 1 filter paper. The inoculum was pre-conditioned by aerating at 22 ± 2 °C for 6 days.
2) Preparation of Mineral Medium: The mineral medium was strongly aerated for 20 minutes and allowed to stand for 20 h at 22 ± 2 °C. All operations were performed in a horizontal laminar flow under aseptic conditions.
- Measuring equipment: Dissolved oxygen was measured using a DO meter (Model: HQ-40d, Supplier: HACH – USA)
pH meter (Model: HQ-40d, Supplier: HACH – USA)
- Test performed in closed vessels due to significant volatility of test substance: No, test item non-volatile
- Test performed in open system: No, perfomed as a closed bottle test in accordance with OECD 301D

SAMPLING
- Sampling frequency: 0, 7, 14, 21 and 28 days
- Sampling method: Dissolved oxygen measured immediately within test flask using a DO meter on days 7, 14, 21, and 28
- Sample storage before analysis: The inoculum blank, test blank, test solution and procedure control were dispensed into their respective pre-labeled BOD bottles (capacity: 300 mL) in duplicate for analysis of DO on days 0, 7, 14, 21, and 28. The toxicity control was also dispensed into its respective pre-labeled bottles in duplicate for days 0, 7, and 14. The day zero samples were analysed for DO immediately after dispensing.

CONTROL AND BLANK SYSTEM
- Inoculum blank:, yes, Mineral medium with inoculum, test item and Sodium benzoate served as an inoculum blank.
- Abiotic sterile control: no
- Toxicity control: no

STATISTICAL METHODS: no
Reference substance:
benzoic acid, sodium salt
Remarks:
Final concentration in mineral medium 2.0 mg/L.
Test performance:
Treatment Criteria Results Conclusion

Inoculum Blank Change in DO over 28 days must be ≤ 1.5 mg/L 1.28 Pass
Test Blank 0.60

Procedure Control Degradation must be > 60% of ThOD within 14 days 84.33 Pass

Test Solution Residual oxygen over 28 days must be > 0.5 mg/L 5.01 Pass
Degradable, if oxygen consumption exceeds > 60% of ThOD over 28 days 243.28 Readily biodegradable

Toxicity Control Toxic, if % degradation < 25% within 14 days 138.28 Pass – Not toxic
Key result
Parameter:
% degradation (O2 consumption)
Value:
243.28
Sampling time:
28 d
Key result
Parameter:
BOD5
Value:
6.447 g O2/g test mat.
Remarks on result:
other: 28 days
Results with reference substance:
Treatment BOD (mg O2/mg ) on Day
7 14 21 28
Procedure Control 1.175 1.405 1.465 1.545
Test Solution 2.632 4.105 5.421 6.447

Treatment % Degradation (Based on ThOD)
7 14 21 28
Procedure Control 70.53 84.33 87.94 92.74
Test Solution 99.32 154.91 204.57 243.28
Toxicity Control 113.62 138.28 - -



Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The percent biodegradation data concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts is readily biodegradable. The test item showed a maximum degradation of 243.28% on day 28 of analysis, whereas the reference substance exhibited a normal pattern of degradation (up to 92.74%) within 28 days. The percent degradation by toxicity control was more than 25% after 14 days. Therefore, the test item is regarded as readily biodegradable and not toxic to microorganisms in the inoculum.

Description of key information

The percent biodegradation data concluded that Fatty acids, C16-18 (even numbered), manganese (II) salts is readily biodegradable. The test item showed a maximum degradation of 243.28% on day 28 of analysis, whereas the reference substance exhibited a normal pattern of degradation (up to 92.74%) within 28 days. The percent degradation by toxicity control was more than 25% after 14 days. Therefore, the test item is regarded as readily biodegradable and not toxic to microorganisms in the inoculum.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information