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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:
22 February to 9 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Kyoeisha Chemical Co., Ltd
- Lot/batch No.of test material: 6033175
- Expiration date of the lot/batch: 17 March 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 20-25 0C (Ambient), in original container as supplied by the Sponsor (tightly closed and away from heat or sunlight).
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Dissolved in DMSO and sonicated, no solubility of the test item in the selected solvent performed.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dissolved in DMSO and sonicated before addition to mineral medium.
- Final dilution of a dissolved solid, stock liquid: 2.01 mg/L bulk solution of test item
-

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Solution at 2.01 mg/mL
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
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. The Secondary effluent was filtered through a Whatman No. 1 filter paper. The inoculum was pre-conditioned by aerating at 22 ± 2 °C for 7 days.

The Inoculum was analysed for microorganism concentration and confirmed as 1.99 x 104 CFU/mL.


- Pretreatment: Aeration at 22 ± 2 °C for 7 days
- Concentration of sludge: 1 mL/L
- Initial cell/biomass concentration: The Inoculum was analysed for microorganism concentration and confirmed as 1.99 x 104 CFU/mL.
- Water filtered: yes
- Type and size of filter used, if any: Whatman No. 1 filter paper
Duration of test (contact time):
28 d
Initial conc.:
2.01 mg/L
Based on:
test mat.
Remarks:
in mineral medium
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
other: ThOD
Details on study design:
TEST CONDITIONS
- Composition of medium:
Preparation of Stock Solution for Mineral Medium: The stock solutions A, B, C and D for mineral medium were prepared in Milli-Q water as described below:
Stock Solutions 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

The pH of stock solution A was 7.44.

Preparation of Stock Solution for Mineral Medium: The stock solutions A, B, C and D for mineral medium were prepared in Milli-Q water as described below:
Stock Solutions 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

The pH of stock solution A was 7.44.

- Additional substrate: None
- Solubilising agent (type and concentration if used): Dimethyl Sulfoxide 4021.0 mg/L
- Test temperature: 22 +/- 2°C
- pH: Stock solution: pH of 7.44
- pH adjusted: no

- Aeration of dilution water: Diluted mineral medium was strongly aerated for 20 mins and allowed to stand for 20 h at 22 +/-2°C. All operations were performed in a horizontal laminar flow under aseptic conditions.
- Suspended solids concentration: None
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: 300 mL (capasity) flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: 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, Make/Supplier: HACH – USA)
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No

SAMPLING
- Sampling frequency: Dissolved Oxygen (DO) Day 0, 7, 12, 21, 28 for IB, TB, TS and PC. For TC, day 0,7 and 14.
- Sampling method: All samples were dispensed into pre-labeled BOD bottles on Day 0 for analysis on day 0,7,14, 21 and 28
- Sterility check if applicable: Not applicable
- Sample storage before analysis: All test bottles were incubated at 22 ± 2 ºC in a BOD incubator.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Bulk Solution of Inoculum Blank: 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. The solution was mixed thoroughly. This mean values of DO in the inoculum were measured on day 0, 7, 14 , 21 and 28.

Bulk Solution for Procedure Control
- The procedure control was prepared by mixing 4.0 mL stock solution of Potassium hydrogen phthalate and 4.0 mL of inoculum to 3992 mL of mineral medium in a conical flask of 5 L capacity. The solution was mixed thoroughly. The final concentration of reference substance in mineral medium was 2.01 mg/L.

- Toxicity control: The toxicity control was prepared by mixing 2.0 mL stock solution of Potassium hydrogen phthalate 1.0 mL stock solution of test item and 2.0 mL of inoculum to 1995 mL of mineral medium in a conical flask of 2 L capacity. The solution was mixed thoroughly. The final concentration was 2.01 mg/L of reference substance and 2.01 mg/L of test item in mineral medium. This was dispensed into pre-labeled BOD bottles (capacity: 300mL) in duplicate for analysis of DO on day 0, 7 and 14.

STATISTICAL METHODS: None
Reference substance:
other: Potassium hydrogen phthalate
Test performance:
Test performed in accordence with OECD guidence
Key result
Parameter:
% degradation (DOC removal)
Remarks:
Test solution
Value:
5.99
Sampling time:
28 d
Remarks on result:
other: PASS
Key result
Parameter:
other: % degradation based on ThOD
Value:
18.52
Sampling time:
28 d
Remarks on result:
other: Not readily biodegradable
Details on results:
Mean Value of DO
The mean value of DO in the inoculum blank on day 0, 7, 14, 21, and 28 was 8.17, 7.89, 7.63, 7.25, and 7.01 mg/L, respectively. The mean value of DO in the test blank on day 0, 7, 14, 21, and 28 was 8.26. 8.00, 7.69, 7.16 and 6.87 mg/L, respectively. The mean value of DO in test solutions on day 0, 7, 14, 21, and 28 was 8.29, 7.71, 7.09, 6.47 and 5.99 mg/L, respectively. The mean value of DO in procedure controls on day 0, 7, 14, 21, and 28 was 8.14, 6.53, 5.99, 5.26 and 4.94 mg/L, respectively. The mean DO value for the toxicity controls on day 0, 7, and 14 was 8.21, 6.63 and 6.15 mg/L, respectively.

The percent degradation for the reference substance (Potassium hydrogen phthalate) on day 7, 14, 21, and 28 was 56.34, 68.17, 82.98, and 86.38%, respectively. The percent degradation of the test item on day 7, 14, 21, and 28 was 6.50, 12.80, 14.64 and 18.52 %, respectively. The percent degradation in the toxicity control on day 7 and 14 was 17.86 and 20.88%, respectively.

Results with reference substance:
The BOD value for the reference substance in the procedure control on day 7, 14, 21, and 28 was 0.662, 0.801, 0.975 and 1.015 mg O2/mg, respectively.

Treatment BOD (mg O2/mg ) on Day
7 14 21 28
Procedure Control 0.662 0.801 0.975 1.015
Test Solution 0.159 0.313 0.358 0.453

Treatment

Criteria

Results

Conclusion

Inoculum Blank

Change in DO over 28 day must be ≤ 1.5 mg/L

1.16

Pass

Test Blank

1.39

Procedure Control

Degradation must be > 60% of ThOD within 14 days

68.17

Pass

Test Solution

Residual oxygen over 28 day must be > 0.5 mg/L

5.99

Pass

Degradable, if oxygen consumption exceeds > 60% of ThOD over 28 day

18.52

Not readily biodegradable

Toxicity Control

Toxic, if % degradation < 25% within 14 days

20.88

Toxic

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The percent biodegradation data concluded that N, N’-bis{6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide is not readily biodegradable. The test item showed a maximum degradation of 18.52% on day 28 (i.e. outside guideline pass levels of 70% removal of DOC and 60% of ThOD), whereas the reference substance exhibited a normal pattern of degradation (up to 86.38%) within 28 days. The percent degradation by the toxicity control was less than 25% after 14 days. Therefore, the test item is regarded as not readily biodegradable and toxic to microorganisms in the inoculum.
Executive summary:

The ready biodegradability of N, N’-bis {6-[12-(hydroxy) octadecanoy-lamino] hexyl} decanediamide(as supplied) was determined in a closed bottle test in accordance with OECD test guideline 301D and EU method C.4-E at a concentration of 2.01 mg/L in mineral medium inoculated with secondary effluent from a treatment plant (1 mL/L). Potassium hydrogen phthalate was used as a reference substance to check the validity of the method. Mineral medium with inoculum (1 mL/L) without test item and Potassium hydrogen phthalate served as an inoculum blank. Separate test solutions were maintained in parallel (in duplicate) for test, reference and control samples for observation on day 0, 7, 14, 21, and 28.

During the experiment, the residual concentration of oxygen in test bottles on day 28 was 5.99 mg/L (guideline limit >0.5 mg/L at all times).The percent biodegradation data revealed that N, N’-bis {6-[12-(hydroxy) octadecanoylamino] hexyl} decanediamide was not readily biodegradable. The maximum degradation was 18.52% on day 28.

The ThOD value for the test and reference item was 2.446 mg O2/mg and 1.175 mg O2/mg, respectively. Based on the results of this test, the test item was not readily biodegradable and toxic to microorganisms in the inoculum.

Description of key information

The percent degradation by the toxicity control was less than 25% after 14 days. Therefore, the test item is regarded as not readily biodegradable and toxic to microorganisms in the inoculum.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information