Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

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:

From December 07, 2016 to February 02, 2017

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

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test (1999).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: RE 10-7; Purity: 92.75 %; Appearance: lightly yellow paste; Solubility in water: 600 mg/L; Stability in water: stable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, 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: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 5 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (32 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.

Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic conditions.

Test duration: 28 days (last CO2 measurement on day 29). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre brown coloured glass bottles.
Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

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.

Mineral medium: 1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.

Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands) and freshly prepared 0.0125 M Ba(OH)2 solution (Merck KGaA, Darmstadt, Germany), both stored in a sealed vessel to prevent absorption of CO2 from the air.

Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20 %) and nitrogen (ca. 80 %) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in
small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).

Illumination: The test media were excluded from light.

Duration of test (contact time):

28 d

Initial conc.:

21.5 mg/L

Based on:

ThCO2

Initial conc.:

12 other: TOC/L

Based on:

ThCO2

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

PREPARATION OF BOTTLES
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 (1 % of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.

Type and number of bottles

Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).

Preparation
At the start of the test (day 0), test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
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.

Test substance
The test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. A sample of the test substance was taken for determination of the Total Organic Carbon (TOC) content. The TOC content of the test substance was determined to be 56.85 %. Based on the TOC content the ThCO2 of the test substance was calculated to be 2.08 mg CO2/mg. The test substance was tested in duplicate at a target concentration of 21.5 mg/L, corresponding to 12 mg TOC/L. No correction was made for the purity/composition of the test substance.

On the day of testing weighed amounts of the test substance were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 43.7 mg; test substance bottle B: 43.7 mg and toxicity control bottle: 43.0 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Reference substance:
A solution of sodium acetate was prepared by dissolving 400.5 mg in Milli-RO water and making this up to a total volume of 100 mL. Volumes of 20 mL from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/L).

DETERMINATION OF CO2

Experimental CO2 production:
The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).

Measurements:
- Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.

- Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1 % solution in ethanol, Merck) was used as pH-indicator.

- On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

- Theoretical CO2 production: The theoretical CO2 production was calculated from the results of the TOC-analysis.

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

10

Sampling time:

29 d

Key result

Parameter:

% degradation (CO2 evolution)

Value:

21

Sampling time:

29 d

Details on results:

Theoretical CO2 production:
- The ThCO2 of the test substance was calculated to be 2.08 mg CO2/mg.
- The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation:
- The relative biodegradation values calculated from the measurements performed during the test period revealed 10 % and 21 % biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, more than 25 % biodegradation occurred within 14 days (46 %, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity. Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

The temperature recorded in a vessel with water in the same room varied between 21.8 and 23.6 °C.
The pH values of the test substance medium:
bottle A: at the begining of experiment - 7.7 → 7.6; at the end - 7.8
bottle B: at the begining of experiment - 7.7 → 7.6; at the end - 7.7

ThCO2, expressed as mg CO2/mg test substance, was calculated from the results of carbon analysis.

The first step in calculating the amount of CO2 produced is to correct for background (endogenous) CO2 production. Thus the amount of CO2 produced by a test item is determined by the difference (in mL of titrant) between the experimental and blank Ba(OH)2 traps.

The amount of 0.05 N HCl titrated is converted into mg of CO2 produced:

mg CO₂ = (0.05 × Δ mL HCl titrated × 44) /2 = 1.1 × Δ mL HCl titrated

Relative biodegradation values were calculated from the cumulative CO2 production relative to the total expected CO2 production, based on the total carbon content of the amount of test substance present in the test bottles. A figure of more than 10 % biodegradation was considered significant.

The relative biodegradation values were plotted versus time together with the relative biodegradation of the positive control. If applicable, the number of days is calculated from the attainment of 10% biodegradation until 60% biodegradation. Should this period be ≤ 10 days (10-day window), then the test substance is designated as readily biodegradable.

Toxicity control: if less than 2 5% biodegradation (based on ThCO2 of the test and positive control items combined) occurred within 14 days, the test item was assumed to be inhibitory.

The total CO2 evolution in the inoculum blank was determined by the cumulative difference (in mL of titrant) between the blank Ba(OH)2 traps and untreated Ba(OH)2 (background).

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the study conditions, the substance was considered to be not readily biodegradable.

Executive summary:

A study was conducted to determine the ready biodegradability of the substance according to OECD Guideline 301B, EU Method C.4 - C, ISO 9439 and ISO 10634, in compliance with GLP. The test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test substance was determined to be 56.85 %. Based on the TOC content the ThCO2 of the test substance was calculated to be 2.08 mg CO2/mg. The test substance was tested in duplicate at a target concentration of 21.5 mg/L, corresponding to 12 mg TOC/L. The study consisted of six bottles: 2 inoculum blanks, 2 test bottles containing test substance, 1 positive control with sodium acetate and 1 toxicity control containing both test substance and sodium acetate. Weighed amount of the test substance were added to the 2 -litres test bottles holding medium with microbial organisms and mineral components. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and test medium. Test duration was 28 days and the last CO2 evolution measurement was on Day 29. The relative biodegradation values calculated from the measurements performed during the test period revealed 10% and 21% biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. In the toxicity control, the test substance did not inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the substance was considered to be not readily biodegradable (Desmares-Koopmans, 2017).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

A study was conducted to determine the ready biodegradability of the substance according to OECD Guideline 301B, EU Method C.4 - C, ISO 9439 and ISO 10634, in compliance with GLP. The test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test substance was determined to be 56.85 %. Based on the TOC content the ThCO2 of the test substance was calculated to be 2.08 mg CO2/mg. The test substance was tested in duplicate at a target concentration of 21.5 mg/L, corresponding to 12 mg TOC/L. The study consisted of six bottles: 2 inoculum blanks, 2 test bottles containing test substance, 1 positive control with sodium acetate and 1 toxicity control containing both test substance and sodium acetate. Weighed amount of the test substance were added to the 2 -litres test bottles holding medium with microbial organisms and mineral components. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test substance and test medium. Test duration was 28 days and the last CO2 evolution measurement was on Day 29. The relative biodegradation values calculated from the measurements performed during the test period revealed 10% and 21% biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. In the toxicity control, the test substance did not inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid. Under the study conditions, the substance was considered to be not readily biodegradable (Desmares-Koopmans, 2017)