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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 08, 2015 to October 08, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study with read across substance was conducted according to OECD Guideline 301B, EU Method C.4-C and ISO 9439, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
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:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide (P2O5)
IUPAC Name:
2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide (P2O5)
Constituent 2
Reference substance name:
1187441-10-6
Cas Number:
1187441-10-6
IUPAC Name:
1187441-10-6
Constituent 3
Reference substance name:
2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, phosphate
EC Number:
258-053-2
EC Name:
2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, phosphate
Cas Number:
52628-03-2
IUPAC Name:
2-(methacryloyloxy)ethyl phosphate
Test material form:
other: liquid

Study design

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: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 3.0 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (57 min) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium. The inoculum was not adapted to the test substance before the biodegradation test.
Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
12 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test procedure and conditions
- Test duration: 28 d (last CO2 measurement on Day 29), during the test period, the test media were aerated and stirred continuously.
- Test vessels: 2 litre glass brown coloured 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) and 1 mL of solutions (B) to (D) and Milli-RO water.
- Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), 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.

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.

Preparation of test substance:
A weighed amount of 991.2 mg of test substance was dissolved in milli-RO water and made up to 1,000 mL. Mixing (vortex) and magnetic stirring (38 min) was used to accelerate dissolution and to ensure homogeneity. The TOC concentration of the clear light brown solution was determined to be 386.8 mg/L. Test substance was tested in duplicate at 12 mg TOC/L, corresponding to 31 mL of the stock solution/L (31 mg/L). The exact amounts of the stock solution were added to the test medium, containing the microbial organisms, of test substance bottles A and B and the toxicity control (final volume: 2 L). The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

Measurements and recording:
- pH: At the start of the test (day 0) and on Day 28, before addition of concentrated HCl.
- Temperature of medium: continuously in a vessel with milli-RO water in the same room.

- 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)2with 0.05 M standardized HCl (1:20 dilution from 1 M HCl.

- Measurements:
Titrations were made every second or third day during the first 10 d, 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 d. 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) was used as pH-indicator. On Day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%) 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.

Test performance
- The positive control substance was biodegraded by at least 60% (84%) within 14 d.
- The difference of duplicate values for %-degradation of the test substance was always less than 20 (≤4%).
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (38 mg CO2 per 2 litres of medium, corresponding to 19 mg CO2/L).
- The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/L).
Reference substance
Reference substance:
acetic acid, sodium salt
Remarks:
40 mg sodium acetate per litre

Results and discussion

Test performance:
- The positive control substance was biodegraded by at least 60% (84%) within 14 d.
- The difference of duplicate values for %-degradation of the test substance was always less than 20 (≤4%).
- The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (38 mg CO2 per 2 litres of medium, corresponding to 19 mg CO2/L).
- The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water), IC was less than 5% of TC (mainly coming from the test substance, 12 mg TOC/L).
Since all criteria for acceptability of the test were met, this study was considered to be valid.
% Degradation
Parameter:
other: ThCo2
Value:
> 18 - < 22
Sampling time:
28 d
Details on results:
The TOC concentration of the 1 g/L test substance was determined to be 386.8 mg/L (39%). The ThCO2 of the test substance was calculated to be 1.42 mg CO2/mg. The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

The relative biodegradation values calculated from the measurements performed during the test period revealed 22% and 18% biodegradation of test substance (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10 d window) was not met.

In the toxicity control, more than 25% biodegradation occurred within 14 d (45%, based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Since all criteria for acceptability of the test were met, this study was considered to be valid.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The read across substance was considered as 'not readily biodegradable' in a CO2 evolution test.
Executive summary:

A study was conducted to determine the ready biodegradability of the read across substance (2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide (P2O5)) according to OECD Guideline 301B, EU Method C.4-C and ISO 9439, in compliance with GLP. The substance was tested in duplicate at the concentration of 12 mg TOC/L (corresponding to 31 mg substance/L) for a duration of 28 d. Test and reference substances (positive control: sodium acetate and toxicity control; test substance plus sodium acetate) were added to the bottles containing the microbial organisms and mineral components. The amount of CO2 produced was determined by titration. Titrations were made every second or third day during the first 10 d, and thereafter 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 14 d. All validity criteria were met and the reference substance reached the level for ready biodegradability by 14 d. No toxicity of the test substance was observed in the toxicity control. The relative biodegradation values calculated from the measurements performed during the test period revealed 22 and 18% biodegradation of the test substance (based on ThCO2) for the duplicate bottles tested. Under the study conditions, the substance was considered as 'not readily biodegradable' in a CO2 evolution test (Desmares-Koopmans MJE, 2015).

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