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Environmental fate & pathways

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:
29 March 2017 - 28 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted July 17, 1992
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test
Version / remarks:
1999
Qualifier:
according to
Guideline:
other: ISO International Standard 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
Version / remarks:
1995
GLP compliance:
yes
Specific details on test material used for the study:
TEST ITEM INFORMATION
Test item: 207811/A
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Batch: EM97160711
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions: until 20 July 2017 (expiry date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

TEST CONCENTRATION AND PREPARATION OF TEST SOLUTIONS
On the day of testing, weighed amounts of the test item were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 33.93 mg; test item bottle B: 33.94 mg and toxicity control bottle: 34.17 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. 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 item and the test organisms.
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 used immediately. The concentration of suspended solids was determined to be 3.6 g/L in the concentrated sludge. Before use, the sludge was allowed to settle (31 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 items under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
17 mg/L
Based on:
test mat.
Remarks:
12 mg TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST PROCEDURE AND CONDITIONS

Test duration:
- 28 days for the inoculum blank and test item (last CO2 measurement on day 29).
- 14 days for the positive and toxicity control (last CO2 measurement on day 15).
- 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 and 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), 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). *Gas cylinder, Air products, Air zero, O2 20.9%±1%, H2O < 3 ppm, CO+CO2 < 1 ppm, THC (as CH4) < 0.2 ppm.
Illumination: The test media were excluded from light.
Test temperature: The temperature recorded in a vessel with water in the same room varied between 22.2 and 23.7°C.
pH: 7.4-7.8

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

Preparation: At the start of the test (day 0), test and reference item 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.

DETERMINATION OF C02

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 the penultimate day, 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 and the final titration was made.

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

MEASUREMENTS AND RECORDING:
pH: At the start of the test (day 0) and on the penultimate day (day 14 for the positive and toxicity control and day 28 for the inoculum blanks and test item), before addition of concentrated HCl.Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Reference substance:
acetic acid, sodium salt
Remarks:
Purity = 99.5%
Test performance:
The relative biodegradation values calculated from the measurements performed during the test period revealed 46% and 48% biodegradation of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol (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 (51%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity. Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
46
Sampling time:
29 d
Remarks on result:
other: Test item (Bottle A)
Remarks:
Test duration was 28 days (last CO2-measurement on day 29).
Key result
Parameter:
% degradation (CO2 evolution)
Value:
48
Sampling time:
29 d
Remarks on result:
other: Test item (Bottle B)
Remarks:
Test duration was 28 days (last CO2-measurement on day 29).
Details on results:
Acceptability of the test
1. The positive control item was biodegraded by at least 60% (85%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 2%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (47.8 mg CO2 per 2 litres of medium, corresponding to 23.9 mg CO2/L).
4. The Inorganic Carbon content (IC) of the test item (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 (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).
Results with reference substance:
The positive control item was biodegraded by at least 60% (85%) within 14 days.

CO2 production and percentage biodegradation of the control and test item (bottle A & B).

Day  

Blank

Bottle A

Bottle B

 

CO2cumulative (mg)

CO2cumulative (mg)

Degradation (%)1

CO2cumulative (mg)

Degradation (%)1

1

4

6

8

11

15

19

22

25

292

292

292

2.2

7.5

11.2

14.6

19.3

23.6

29.1

33.7

37.9

43.8

46.6

47.8

0.0

2.4

8.5

15.8

21.8

27.1

30.6

33.3

36.3

38.9

40.5

40.5

0

3

10

18

25

31

35

38

42

44

46

46

0

1.9

8.1

15.6

22.2

28.0

32.2

34.9

38.1

39.7

40.7

42.5

0

2

9

18

25

32

37

40

43

45

47

48

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test item: 87.6 mg CO2/2L.

2): CO2measured on day 29 is actually part of CO2production of day 28, since microbial activity was ended on day 28 by

addition of HCl.

Validity criteria fulfilled:
yes
Remarks:
see details on results
Interpretation of results:
not readily biodegradable
Conclusions:
Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was biodegraded significantly (46% and 48%) during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was not readily biodegradable.
Executive summary:

The ready biodegradability of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP. In this study activated sludge was exposed to 17 mg/L, corresponding to 12 mg TOC/L, of the test substance for 28 days. In the toxicity control, the test substance was found not to inhibit microbial activity. Furthermore, the validity criteria of the test were met. The relative biodegradation values calculated from the measurements performed during the test period revealed 46% and 48% 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. Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was designated as not readily biodegradable.

Description of key information

The ready biodegradability of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was investigated in a study conducted in accordance with OECDTG 301B (carbon dioxide evolution test (modified Sturm test)) and GLP. In this study activated sludge was exposed to 17 mg/L, corresponding to 12 mg TOC/L, of the test substance for 28 days. In the toxicity control, the test substance was found not to inhibit microbial activity. Furthermore, the validity criteria of the test were met. The relative biodegradation values calculated from the measurements performed during the test period revealed 46% and 48% 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. Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was designated as not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Although the substance does not meet the readily biodegradable criteria, at test end (28d), the biodegradation curve still showed ongoing biodegradation of the UVCB; the increase of % biodegradation over time was not levelling off yet and biodegradation did not seem to be completed. It is therefore likely that prolongation of the test would have shown additional biodegradation, which would indeed be expected. The expectation is based on the ready biodegradability of the source substances (epoxidized soybean oil, methanol and 1,2 -propanediol) and similar UVCB substances, like e.g. Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized (cas no. 158318-67-3); Fatty acids, C16-18 and C18-unsatd., epoxidized, Me esters, oligomeric reaction products with trimethylolpropane (cas no. 188831-96-1) and Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol (cas no. 211450-54-3), see Table below. Data originate from the ECHA website, the respective Reach dossiers were consulted 30 April 2018.

 

Soybean oil, epoxidized, Me ester, reaction products with propylene glycol

 

Fatty acids, C16-C18 and C18-unsatd., Me esters, epoxidized

 

Fatty acids, C16-18 and C18-unsatd., epoxidized, Me esters, oligomeric reaction products with trimethylolpropane

 

Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

 

Epoxidized soybean oil

 

Methanol

 

1,2-Propanediol

Cas no.

96690-51-6

158318-67-3

188831-96-1

211450-54-3

8013-07-8

67-56-1

57-55-6

TG 301B

46-48%

 

 

 

79-92%

 

 

TG 301F

 

84%

64%

78%

 

 

91.1%

Literature

 

 

 

 

 

95% (20d)

 

Conclusion

Not readily

readily

readily

readily

readily

readily

readily