Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

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:

08-July 2019 - 08- August 2019

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)

Version / remarks:

adopted July 17, 1992

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Treatment: Freshly obtained sludge was kept under continuous aeration until further treatment. Before use, sludge was coarsely sieved (1 mm). After treatment concentration of suspended solids (SS) was determined to be 3.0 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9 mg/L.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: 1 L mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
Stock solutions of mineral components:
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre,pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
- Test temperature: between 23 and 24°C
- pH: At t=0d:7.5 ;At t=14d: 8.0 - 8.2 (procedure and toxicity control); At t=28d:7.5 - 7.8 (blank control and test solution).
- pH adjustment: no
- Aeration: Constant aeration with a mixture of oxygen (ca. 20%) and nitrogen (ca. 80%).
- Suspendid solids concentration:suspended solids (SS) was determined to be 3.0 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 3 mL per litre of mineral medium, leading to a SS concentration of 9 mg/L.
- Illumination: Test media were excluded from light.
- Pre incubation medium: The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

TEST SYSTEM
- Culturing apparatus:2 litre brown coloured glass bottles.
- Type and number of bottles:
Test suspension: containing test item and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Procedural control: containing procedural control item and inoculum (1 bottle).
Toxicity control: containing test item, procedural control item and inoculum (1 bottle).
- Test performance in open system: yes
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sapmle frequency: Titrations were made on nominal 2, 5, 8, 12, 15, 19, 23, 29. On the penultimate day (day 14 for procedural and toxicity control an don day 28 for the blank control and testvessels), 1 mL of concentrated HCl (37%, Merck) was added to the inoculum blank and test suspension. Bottles were aerated overnight to drive off CO2. Final titration was made on Day 15 (procedural and toxicitycontrol) and on Day 29 (remaining vessels).
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.

CONTROL AND BLANK SYSTEM
- Inoclulum blank: yes
- abiotic sterile control: no
- Toxicity control: yes

PREPARATION OF TEST SOLUTION
The substance was a clear yellow liquid (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. A sample of test item was taken for determination of Total Organic Carbon (TOC) content. TOC content of the test item was determined to be 60.48%. The test item was tested in duplicate at a target concentration of 20 mg/L, corresponding to 12 mg TOC/L.
On the day of testing weighed amounts of test item were added to 2 L test bottles containing
medium with microbial organisms and mineral components (test item bottle A: 39.62 mg; test
item bottle B: 39.63 mg; toxicity control bottle: 40.16 mg). To this end, small watch glasses
were used to transfer weighed amounts of test item to respective test bottles. Test solutions
were continuously stirred during the test, to ensure optimal contact between test item and test
organisms.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

57

Sampling time:

29 d

Remarks on result:

other: Bottle A

Key result

Parameter:

% degradation (CO2 evolution)

Value:

65

Sampling time:

29 d

Remarks on result:

other: Bottle B

Details on results:

- ThCO2 of the substance was calculated to be 2.22 mg CO2/mg.
- Relative biodegradation values calculated from measurements performed during the test period revealed 57% and 65% biodegradation of the substance, for vessel A and B, respectively (based on ThCO2). Therefore, average biodegradation of the substance in vessel A and B reached ≥60%. The tenday window was not met; however, for sunstances containing homologous constituents, the 10-day window does not apply.

- Functioning of the test system was checked by testing the procedural control item sodium acetate, which showed a normal biodegradation curve.

Results with reference substance:

Procedural control item was biodegraded by at least 60% (actual result: 90%) within 14 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

the substance was readily biodegradable under the conditions of the modified Sturm test presently performed. Since the test item is a UVCB,containing homologous constitents, the 10-day window was not applicable.

Executive summary:

In a CO2 Evolution Test performed according to OECD guideline 301B an d GLP principles, the substance was evaluated for its readiy biodegradability. The Test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days. The test item was tested in duplicate at a target concentration of 20 mg/L, corresponding to 12 mg TOC/L Additionally two innoculum blanks, one procedural control and one toxicity control were tested. Relative biodegradation values calculated from measurements performed during the test period revealed 57% and 65% biodegradation of the substance, for vessel A and B, respectively. Therefore, average biodegradation of the substance in vessel A and B reached ≥60%. the 10 ady window was not met; however, for this substance, containing homologous constituents, the 10 -day window does not apply.

In the toxicity control, more than 25% biodegradation occurred within 14 days (61%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.

In conclusion, the substance was readily biodegradable under the conditions of the performed modified Sturm test. Since the test item is a UVCB containing homologous constituents, the 10-day window was not applicable.

All criteria for acceptability of the test were met, this study was considered to be valid.

Description of key information

Readily Biodegradable. Average biodegradation of the substance in vessel A and B reached ≥60%.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

In a CO2 Evolution Test performed according to OECD guideline 301B an d GLP principles, the substance was evaluated for its readiy biodegradability. The Test was performed in an aerobic aqueous medium containing activated sludge over a period of 28 days. The test item was tested in duplicate at a target concentration of 20 mg/L, corresponding to 12 mg TOC/L Additionally two innoculum blanks, one procedural control and one toxicity control were tested. Relative biodegradation values calculated from measurements performed during the test period revealed 57% and 65% biodegradation of the substance, for vessel A and B, respectively. Therefore, average biodegradation of the substance in vessel A and B reached ≥60%. The 10-day window does not apply.