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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:
December 1997 - February 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
one validity criterion was not fully met in both experiments, however this did not affect the conclusion of the results. No CoA. Temperature not in protocolled range in one experiment. Temporary breakdown of aeration on day 28 in second experiment.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands.
- Storage conditions: The sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decanted for use as inoculum at the amount of 10 mL/L mineral medum.
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
16 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: Stock solutions of mineral components:
a: 8.5 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4 * 12H2O, 0.50 g NH4Cl, dissolved in 1 L milli-Q water pH 7.4±0.2.
b. 22.50 g MgSO4 *7H2O dissolved in 1 L milli-Q water.
c: 36.40 g CaCl2 * H2O dissolved in 1 L milli-Q water
d: 0.25 g FeCl3 * 6H2O dissolved in 1 L millil-Q water
1L mineral medium contains: 10 mL of , 1 mL of solutions (b) to (d) and Milli-Q water.
- Additional substrate: Barium hydroxide, 0.0125 M: 4 g Ba(OH)2 * 8H2O per litre milli-Q water was filtered through filter paper and stored in a sealed vessel to prevent absorption of Co2 from the air.
- Test temperature: 20 - 22 °C (experiment 1) and 17 - 22 °C (experiment 2, deviation is not considered to effect the study outcome)
- pH: 7.5 - 7.8 (experiment 1) and 7.5 - 8.1 (experiment 2)
- pH adjusted: no
- Aeration of dilution water: aerated with CO2-free air overnight to purge the system of CO2
- Suspended solids concentration: The concentration of suspended solids was 4 and 2.8 g/L in the concentrated sludge in experiment 1 and 2, respectively (information obtained from the municipal sewage treatment plant).
- Continuous darkness: yes, brown coloured bottles

TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The volumes of suspensions were made up in all bottles by the addition of milli-Q water previously aerated with the CO2-free air. During the test CO2 free air was bubbling through the test solution at a rate of 1 - 2 bubbles per second (ca. 30-100 mL/min)
- Details of trap for CO2 and volatile organics if used: 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 the CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.5 M standardized HCL.

SAMPLING
- Sampling frequency: titration were made every second or third day during the first 10 days, and there after at least every fifth day until the 28th day.
- Sampling method: 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 was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 mL of concentrated HCL was added to each bottle. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, two bottles
- Toxicity control: yes, one bottle containing test substance, reference substance and inoculum
- Other: positive control containing ca. 40 mg/L sodium acetate.

Reference substance:
acetic acid, sodium salt
Remarks:
Batch 049- TA933768, TOC = 12 mg/L
Test performance:
Acceptability of the experiment 1
- The positive control substance was degraded at least 60% in ca. 12 days
- The total CO2 release in the blank reached a total value of 25 mg CO2 per 2 litres of medium
- The difference of duplicate values for %-degradation of the test substance was less than 20 throughout the test, except for day 29. At the end of the test the difference of the relative degradation values of A and B was 25%.

Acceptability of the experiment 2
- The positive control substance was degraded at least 60% in 9 days.
- The total CO2 release in the blank reached a total value of 42 mg CO2 per 2 litres of medium.
- The difference of duplicate values for %-degradation of the test substance was less than 20 until day 14; and more than 20% at day 19 until the end of the test.

As described previously the results of the two experiments performed were comparable, In both experiments a difference of the relative degradation values for A and B of more than 20% was noted. Therefore, one acceptability criterion as prescribed by the protocol was not met.

However, since the criterion for ready biodegradability (at least 60% degradation within 10 days of biodegradation exceeding 10%) was not met in these experiments, this will have no influence on the final conclusion of this study
Key result
Parameter:
% degradation (CO2 evolution)
Value:
30
Sampling time:
28 d
Remarks on result:
other: first test
Remarks:
result A, 10-day window criterion not met
Key result
Parameter:
% degradation (CO2 evolution)
Value:
55
Sampling time:
28 d
Remarks on result:
other: first test
Remarks:
result B, 10-day window criterion not met
Key result
Parameter:
% degradation (CO2 evolution)
Value:
52
Sampling time:
28 d
Remarks on result:
other: second test
Remarks:
result A, 10-day window criterion not met
Key result
Parameter:
% degradation (CO2 evolution)
Value:
22
Sampling time:
28 d
Remarks on result:
other: second test
Remarks:
result B, 10-day window criterion not met
Details on results:
Theoretical CO2 production
The Theoretical CO2 production (ThCO2) of the test substance (MF- C13H2202, MW=210.32 g) was calculated to be 2.72 mg CO2/mg.

Experiment 1:
The concentration was 31.9 (A) and 32.1 mg (B) test substance in 2 litres test medium. Hence, the theoretical CO2 production following complete degradation was 86.8 mg for A and 87.3 mg per 2 litres for B.
The positive control contained 80.6 mg sodium acetate (ThCO2= 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 86.2 mg per 2 litres.
The toxicity control contained 80.6 mg sodium acetate and 31.6 mg test substance in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of the test substance plus sodium acetate was 172.2 mg per 2 litres

Experiment 2:
The concentration was 32.4 mg (A) and 31.9 mg (B) in test substance 2 litres test medium. Hence, the theoretical CO2 production following complete degradation was 88.1 mg for A and 86.8 mg per 2 litres for B.
The positive control contained 81.9 mg sodium acetate (ThCO2= 1.07 mg CO2/mg) resulting in a theoretical C02 production following complete degradation of 87.6 mg per 2 litres.
The toxicity control contained 81.9 mg sodium acetate and 31.7 mg test substance in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of the test substance plus sodium acetate was 173.9 mg per 2 litres.

Biodegradation
In the first experiment the relative degradation values calculated from the measurements performed during the test period revealed 30 and 55% degradation of the test substance for A and B respectively. Since the difference of the relative degradation values for A and B was 25% at the end of the test a second experiment was performed.

The second experiment confirmed the findings of the first experiment. In the second experiment the relative degradation values calculated from the measurements performed during the test period revealed 52 and 22% degradation of the test substance for A and B respectively. The difference of the relative degradation values for A and B was more than 20% at day 19 until the end of the test.

In the toxicity control of both experiments more than 25% degradation occurred in 14 days (based on ThCO2). Therefore the test substance was assumed to be not inhibitory.

The test substance was degraded significantly during the test periods. Relative degradation values between 22 and 55% were noted in two experiments. However, since at least 60% biodegradation was not reached within ten days of biodegradation exceeding 10%, the criterion for ready biodegradability was not met. Thus, under the conditions of these tests, the test substance was not readily biodegradable.


Validity criteria fulfilled:
no
Remarks:
Validity criteria are not all fully met for the ready biodegradability guideline, however not meeting them did not impact the results and conclusions from the study.
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
Under the conditions of the test, relative degradation values between 22 and 55% were noted in two experiments. However, since at least 60% biodegradation was not reached within ten days of biodegradation exceeding 10%, the criterion for ready biodegradability was not met. Thus, under the conditions of these tests, the test substance was not readily biodegradable, but can be considered as inherently (primary) biodegradable.
Executive summary:

A CO2 evolution test was performed according to OECD TG 301B (GLP). The CO2 production was followed for 28-days in an aerobic system inoculated with domestic activated sludge. 16.0 mg/L (12.0 mg/L TOC) test material was added to the test system. The experiment was performed twice and in duplicate. An inoculum control, toxicity control and positive control were performed in parallel. Results of the two experiments performed were comparable. In both experiments a difference of the relative degradation of the duplicate values of more than 20% was noted. The difference of duplicate values for the %-degradation of the test substance at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, should be less than 20. Therefore, one acceptability criterion was not met. However, since the criterion for ready biodegradability (at least 60% degradation within 10 days of biodegradation exceeding 10%) was not met in these experiments, this will have no influence on the final conclusion of this study. The test substance was degraded significantly during the test periods. Relative degradation values between 22 and 55% were observed in both experiments. However, since at least 60% biodegradation was not reached within ten days of biodegradation exceeding 10%, the criterion for ready biodegradability was not met. Thus, under the conditions of these tests, the test substance was not readily biodegradable. Nevertheless, according to OECD "Principles and strategies related to testing of degradation of organic chemicals" (2006), item can be regarded as inherently (primary) biodegradable because its biodegradation is above 20%.

Description of key information

A CO2 evolution test was performed according to OECD TG 301B (GLP). The CO2 production was followed for 28-days in an aerobic system inoculated with domestic activated sludge. 16.0 mg/L (12.0 mg/L TOC) test material was added to the test system. The experiment was performed twice and in duplicate. An inoculum control, toxicity control and positive control were performed in parallel. Results of the two experiments performed were comparable. In both experiments a difference of the relative degradation of the duplicate values of more than 20% was noted. The difference of duplicate values for the %-degradation of the test substance at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, should be less than 20. Therefore, one acceptability criterion was not met. However, since the criterion for ready biodegradability (at least 60% degradation within 10 days of biodegradation exceeding 10%) was not met in these experiments, this will have no influence on the final conclusion of this study. The test substance was degraded significantly during the test periods. Relative degradation values between 22 and 55% were observed in both experiments. However, since at least 60% biodegradation was not reached within ten days of biodegradation exceeding 10%, the criterion for ready biodegradability was not met. Thus, under the conditions of these tests, the test substance was not readily biodegradable, but can be regarded as inherently (primary) biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

The biodegradation percentage was between 22 and 50%. Because these values > 20%, it can be considered as unequivocal evidence of biodegradation. According to OECD “Principles and strategies related to testing of degradation of organic chemicals” (2006), the item can be regarded as inherently (primary) biodegradable.