Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2017 to ****

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98%; Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Yellow Liquid
- Carbon Content: 54.4%

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from one of the return lines at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic waste-water catchment. The sample was aerated using a compressed air supply. The suspended solids concentration of the activated sludge was determined to be 5.06 g/L. The activated sludge used in this study was not acclimatised or adapted to the test substance prior to exposure under the test conditions.

Duration of test (contact time):

28 d

Initial conc.:

15 other: mg C/L

Details on study design:

TEST CONDITIONS
- Composition of medium: Buffered synthetic, mineral salts medium prepared according to OECD guideline 301B
- Additional substrate: None.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ± 2°C
- pH: Measurements of pH were made in the blank control and reference substance vessels at the start of incubation and in all vessels at the end of the test prior to the addition of the hydrochloric acid.
- pH adjusted: No.
- Aeration of dilution water: Yes.
- Suspended solids concentration: The concentration of suspended solids was calculated to be 5.06 g/L. The medium was inoculated with activated sludge to give a suspended solids concentration of 30 mg/L in each control or test vessel.
- Continuous darkness: Yes.

TEST SYSTEM
- Number of culture flasks/concentration: Test substance vessels were prepared in duplicate (15 mg C/L).
- Method used to create aerobic conditions: Continuously sparged with a supply of CO2 free air (ca. 50 mL per minute).
- Measuring equipment: Titration (see details on analytical methods).
- Details of trap for CO2 and volatile organics if used: The exhaust air from each vessel was passed through a series of three traps containing a barium hydroxide solution.

SAMPLING
- Sampling frequency: Days 2, 5, 7, 8, 12, 16, 19, 23, 26, 28 and on Day 29 (following acidification and overnight aeration).
- Sampling method: Traps were removed from the series and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the remaining traps on Day 29 was performed following overnight aeration.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Two blank control vessels containing inoculated medium only were prepared to assess the validity of the test and to correct for baseline CO2 evolved.
- Procedure control: Two procedure control vessels containing a reference substance, sodium benzoate (15 mg C/L), were prepared to assess the performance of the inoculum.
- Toxicity control: An additional vessel containing a combination of the test and reference substances (15 mg C/L) served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration.
- Abiotic sterile control: Not applicable.

STATISTICAL METHODS
Not applicable.

Reference substance:

benzoic acid, sodium salt

Remarks:

15 mg C/L

Preliminary study:

A solubility trial was performed by adding the test substance to reverse osmosis (RO) water. After 5 minutes sonication and 5 minutes stirring, the test substance appeared to have gone in solution. The test substance was therefore deemed sufficiently soluble to be added as an aqueous stock solution and the test substancewas therefore added to the test vessels as an aqueous stock solution.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

9

Sampling time:

28 d

Details on results:

Measured pH values ranged from 7.42 to 7.51 on Day 0 and 7.53 to 7.71 on Day 28.

The mean total CO2 production in the blank control vessels was 33.1 mg/L at the end of the test, satisfying the validity criterion of < 70 mg/L.

Carbon dioxide evolution in the test substance vessels did not exceed 12% over the exposure period. Mean percent biodegradation of the test substance reached only 9% after 28 days. The test substance therefore cannot be considered readily biodegradable. Percent biodegradation values at each sampling interval, for the two replicates containing the test substance did not vary by more than 15%, satisfying the validity criterion of less than 20% difference.

Results with reference substance:

Biodegradation of the reference substance vessels exceeded 60% by Day 7 (68%), 89% by the end of the incubation phase on Day 28 and 89% at the end of the test on Day 29. Biodegradation of the reference substance in the presence of the test substance in the toxicity control (73% at Day 7, 93% by the end of the incubation phase on Day 28 and 94% by the end of the test on Day 29) was similar to that of the reference substance alone, indicating that the test substance did not have an inhibitory effect on the inoculum under the test conditions, and satisfying the validity criterion.

Biodegradation as a Percentage of Theoretical CO2 Yield

	Day 2	Day 5	Day 7	Day 8	Day 12	Day 16	Day 19	Day 23	Day 26	Day 28	Day 29^#
Reference Substance R1	30	56	65	68	73	78	81	84	86	87	87	87	87
Reference Substance R2	33	62	71	74	80	85	89	91	91	91	91	91	91
Mean	31	59	68	71	77	82	85	88	89	89	89	89	89
Test Substance R1	0	5	3	1	2	1	1	2	2	3	3	4	5
Test Substance R2	0	6	5	5	6	6	7	8	10	11	10	11	12
Mean	0	6	4	3	4	3	4	5	6	7	7	8	9
Toxicity Control*	28	57	73	78	81	85	88	91	93	93	94	94	94

 * Toxicity control values are corrected for mean test substance degradation.

# Day 29 refers to the day that titrations of trap content from acidified vessels were performed. Actual acidification was performed on Day 28.

Validity criteria fulfilled:

yes

Remarks:

Exception: The inorganic carbon (IC) content of the test medium was 3.81 mg C/L (or, 8.47%). There was no indication of suppression from increased IC content based on toxicity control and inoculum blanks. The results were therefore considered valid.

Interpretation of results:

not readily biodegradable

Conclusions:

The mean percent biodegradation of the test substance reached only 9% after 28 days. The test substance cannot, therefore, be considered readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

The ready biodegradability of the test substance was assessed by measurement of carbon dioxide (CO2) evolution under standard conditions according to OECD Test Guideline 301B., under GLP conditions. The test substance was administered to the test system as an aqueous stock solution. A buffered synthetic, mineral salts medium prepared according to OECD guideline 301B was added to give a test substance concentration equivalent to 15 mg C/L. The concentration of suspended solids was calculated to be5.06g/L. The medium was inoculated from a sample of non-adapted activated sludge to give a suspended solids concentration of 30 mg/L in each control or test vessel. Test vessels were incubated in darkness at 22 ± 2°C for 28 days and their contents continuously sparged with a supply of CO2 free air (ca. 50 mL per minute). The exhaust air from each vessel was passed through a series of traps containing a barium hydroxide solution to trap evolved CO2.

 

On Days 2, 5, 7, 8, 12, 16, 19, 23, 26, 28, traps were removed from the series and their contents titrated with hydrochloric acid to determine the quantity of CO2 evolved from the respective test vessels. At the end of incubation, 28 days, the test vessel contents were acidified to release any residual CO2 that may have remained in solution. Titration of the remaining traps on Day 29 was performed following overnight aeration. Measurements of pH were made in the blank control and reference substance vessels at the start of incubation and in all vessels at the end of the test prior to the addition of the hydrochloric acid.

The performance of the inoculum was checked by measuring the CO2 evolved from procedure control vessels containing a reference substance, sodium benzoate (15 mg C/L). An additional vessel containing a combination of the test and reference substances (15 mg C/L) served as a toxicity control to assess whether the test substance was inhibitory to biodegradation at the test concentration. Two blank control vessels were also prepared containing inoculated medium only to check the validity of the test and to correct for baseline CO2 evolved. Duplicate vessels were prepared for the test substance, reference substance and blank control groups. A single vessel was prepared for the toxicity control. The mean total CO2 production in the blank control vessels was 33.1mg/L at the end of the test, satisfying the validity criterion of < 70 mg/L.

 

Biodegradation of the reference substance vessels exceeded 60% by Day 7 (68%), 89% by the end of the incubation phase on Day 28 and 89% at the end of the test on Day 29. Biodegradation of the reference substance in the presence of the test substance in the toxicity control (73% at Day 7, 93% by the end of the incubation phase on Day 28 and 94% by the end of the test on Day 29) was similar to that of the reference substance alone, indicating that the test substance did not have an inhibitory effect on the inoculum under the test conditions, and satisfying the validity criterion.

 

The mean percent biodegradation of the test substance reached only 9% after 28 days. The test substance therefore cannot be considered readily biodegradable. Percent biodegradation values at each sampling interval, for the two replicates containing the test substance did not vary by more than 15%, satisfying the validity criterion of less than 20% difference.

Description of key information

The ready biodegradability of the test substance was assessed by measurement of carbon dioxide (CO2) evolution under standard conditions according to OECD Test Guideline 301B., under GLP conditions. The mean percent biodegradation of the test substance reached only 9% after 28 days. The test substance cannot, therefore, be considered readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B (Smithers Viscient (ESG) Ltd., 2018).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater