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Environmental fate & pathways

Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 16, 2003 to Septemberer 18, 2003
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)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The activated sludge was collected at the Columbia Wastewater Treatment Plant in Columbia, Missouri, which predominantly treats domestic sewage. One liter of activated sludge was collected from aeration basin #1. The sludge was collected on April 29,2003.
- Preparation of inoculum for exposure: The activated sludge was homogenized in a blender at a medium speed for two minutes. The homogenized sludge was allowed to settle for 30 to 60 minutes, then filtered through glass wool.
- Concentration of sludge: The suspended solids concentration of the prepared activated sludge was less than 100 mg/L. The total concentration of suspended solids in each reaction flask (30 mL of inoculum to 3,000 mL of test medium) was 1 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
20 other: mg carbon/L
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral medium, microbial inoculurn, reagent water, and the appropriate test or reference substance additions.
- Test temperature: The temperature of the environmental chamber ranged from 21.5 to 26.7 degrees C during the test duration. The mean and standard deviation of the temperature measurements was 22.1 ± 0.3 degrees C.
- pH: The pH of the control solutions was 7.41 and 7.37 at study initiation and 7.44 and 7.40 at termination for replicates 1 and 2, respectively. The pH of the test substance solutions was similar to the control solutions, both replicates were 7.42 at study initiation and 7.43 and 7.42 at termination for replicates 1 and 2, respectively. The pH of the reference substance system increased from 7.43 at study initiation to 7.62 at study termination.
- Suspended solids concentration: Tthe total concentration of suspended solids in each reaction flask (30 mL of inoculum to 3,000 mL of test
medium) was 1 mg/L .
- Continuous darkness: Yes, The test systems were kept in the dark (except for sampling and maintenance)
- Other: CO2-free air was introduced into each flask by positive pressure at flow rates of 50-100 ml/minute.

TEST SYSTEM
- Culturing apparatus: Each test system consisted of a 5-L glass flask (reaction flask) containing a 3-L test solution
- Number of culture flasks/concentration: 2
- Measuring equipment: Temperature of the chamber was continuously measured using a bi-metallic (aluminum-constantan) thermocouple probe and Multiscan 1200 temperature monitoring system.
- Details of trap for CO2 and volatile organics if used: The outlet from each flask was connected to three carbon dioxide absorber gas-washing traps in series, each filled with 100 mL of 0.2 N KOH solution.

SAMPLING
- Sampling frequency: Continuous
- Sampling method: Evolved gases passed through cabon dioxide traps. Traps collected an analyzed for IC content on days 2, 5, 7, 9, 14, 19, 23, 28, and 29.
- Bacterial plate counts performed on the prepared activated sludge and each reaction flask solution on Day 28.


Reference substance:
benzoic acid, sodium salt
Test performance:
Carbon Dioxide Evolution Results
Sodium benzoate exhibited a theoretical carbon dioxide value of 87.1% by day 29 of the study. The results from day 5 (70.6% theoretical carbon dioxide evolved) indicated greater than 60% theoretical carbon dioxide evolved in the first 5 days of the test. These results indicate that the inoculum was viable.

DOC Results
The DOC concentration of the sodium benzoate solution at initiation was 19.5 mg carbon/L. Correcting for the mean DOC concentration measured in the control treatment (0.35 mg CL), the DOC concentration was 19.2 mg carbon/L, corresponding to 96% of the nominal 20 mg carbon/L testing concentration. The recovery result was in the normally acceptable range (70-120%), indicating that the reference substance flask was prepared correctly. At termination, the DOC concentration of the sodium benzoate solution was 0.15 mg carbon/L. Correcting for the mean DOC concentration measured in the control treatment at termination (0.15 mg CL), the DOC concentration was 0.00 mg carbon/L. Biodegradation of the reference substance based on DOC measurements of the reaction solution at initiation (day 0) and termination (day 28) was 100%, confirming the measured biodegradation from carbon dioxide evolution.
Parameter:
% degradation (CO2 evolution)
Value:
86.9
Sampling time:
29 d
Remarks on result:
other: Replicate 1
Parameter:
% degradation (CO2 evolution)
Value:
98.5
Sampling time:
29 d
Remarks on result:
other: Replicate 2
Details on results:
Carbon Dioxide Evolution Results
Ethylene carbonate exhibited final theoretical carbon dioxide values (after correction for background carbon dioxide from the controls) of 86.9% and 98.5% for replicates 1 and 2, respectively, through day 29 of the study. The theoretical carbon dioxide values did reach 60% by day 9 (72.2% and 80.8% for replicates 1 and 2, respectively). Therefore, ethylene carbonate can be classified as readily biodegradable.

DOC Results
The DOC concentrations of the ethylene carbonate solutions at initiation were 20.5 and 20.9 mg carbon/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the control treatment (0.35 mg carbon/L), DOC concentrations were 20.2 and 20.6 mg carbon/L, corresponding to 101% and 103% of the nominal 20 mg carbon/L testing concentration, respectively. The recovery results were within the acceptable range (70-120%), indicating that the test substance flasks were dosed correctly. At termination, the DOC concentrations of the ethylene carbonate solutions were 1.90 and 2.45 mg carbon/L for replicates 1 and 2, respectively. Correcting for the mean DOC concentration measured in the control treatment at termination (0.15 mg carbon/L), DOC concentrations were 1.75 and 2.30 mg carbon/L, respectively. Biodegradation of the test substance based on DOC measurements of the reaction solutions at initiation (day 0) and termination (day 28) was 91% and 89% for replicates 1 and 2, respectively, confirming the measured biodegradation from carbon dioxide evolution.
Validity criteria fulfilled:
yes
Remarks:
The percent theoretical CO2 produced by the reference substance, sodium benzoate, was 70.6% theoretical carbon dioxide by day 5 and 87.1% theoretical carbon dioxide by day 29, demonstrating that the inoculum was viable.
Interpretation of results:
readily biodegradable
Conclusions:
The percent theoretical carbon dioxide produced by ethylene carbonate was 72.2% and 80.8% by day 9, and 86.9% and 98.5% theoretical carbon dioxide by day 29 of the study for replicates 1 and 2, respectively. Therefore, ethylene carbonate can be classified as readily biodegradable under the conditions of this test.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): STP Marl-West, Germany
- Concentration of sludge: 1,2 g dry weight/l
Duration of test (contact time):
5 d
Initial conc.:
295 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
no data
Reference substance:
not specified
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
5 d
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Executive summary:

100 % DOC removal in 5 days was observed for ethylene carbonate. Therefore, the substance was considered inherently biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 Feb 2001 - 01 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO 7827:1994 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of dissolved organic carbon (DOC)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
-Name of the test substance: Ethylencarbonat S
- Chemical name: 1,3-Dioxolan-2-on
- Batch number: from continuous production
- Substance number: 00/0884-1
- Date of production: EC-Kampagne 10/2000
- Product number: 81266
- Aggregate state: solid
- Colour: colourless to yellowish
- Homogeneity: homogenous at 50°C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Special conditions for storage: storage at room temperature under nitrogen
- Stability: the stability under storage conditions over the exposure period was guranteed by the sponsor
- Further remarks: hygroscopic

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Before sampling the test substance was melted at 50 °C
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Laboratory culture: Activated sludge from laboratory wastewater plants treating municipal sewage.
- Concentration of sludge: concentration dry substance: 30 mg/L
Duration of test (contact time):
21 d
Initial conc.:
47.7 mg/L
Based on:
test mat.
Remarks:
equivalent to 20 mg/I DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Test temperature : 22 ± 2 °C
- Test duration (days) : 21
- Duration of the adaptation phase (days) : 1
- Duration of the degradation phase (days) : 19
- pH:
* Blank control: before adding the inoculum 7.8; after correction 7.4
* Reference: before adding the inoculum 7.7; after correction 7.4
* Inhibition control: before adding the inoculum 7.8; after correction 7.4
* Abiotic control: before adding the inoculum 7.6; after correction 7.4
* Adsorption control: before adding the inoculum 7.6; after correction 7.4
* Test substance: before adding the inoculum 7.7 and 7.6; after correction 7.4

Test substance:
Stock solution : 717,4 mg/I
DOC value stock solution : 301 mg/I

Reference substance:
Stock solution : 388,5 mg/I
DOC value stock solution : 304 mg/I
Reference substance:
aniline
Preliminary study:
TOC [mg/g]: 417 (NON-GLP)
DOC [mg/g]: 420 (NON-GLP)
Test performance:
OECD criteria is reaching the threshold value of 70% within a period of 10 days during a total test period of 28 days. In this case, the percentage degradation of the test substance at the end of the 10-day window is 70-80%.

It must be noted that, if a test substance is biodegradable in this test, it can be assumed that it will be biodegraded in the environment, especially in wastewater treatment plants and surface waters.
Key result
Parameter:
% degradation (DOC removal)
Value:
70 - 80
Sampling time:
10 d
Remarks on result:
other: Degradation of the test substance at the end of the 10-day window (% DOC)
Key result
Parameter:
% degradation (DOC removal)
Value:
90 - 100
Sampling time:
19 d
Remarks on result:
other: Degradation degree of the test substance at the end of the test (% DOC)
Details on results:
- Degradation of the test substance at the end of the 10-day window (% DOC) : 70-80
- Degradation degree of the test substance at the end of the test (% DOC) : 90-100
- Physico-chemical (abiotic) elimination of the test substance (% DOC) : 10-20 at the end of the test
- Elimination of the test substance by adsorption (% DOC) : <10 after 5 days
Results with reference substance:
- Degradation degree of the reference substance after 14 days (% DOC) : 90-100
- Degradation degree in the inhibition control after 14 days (% DOC) : 80-90

Validity criteria:

-Deviation of the degradation degree of the test substance in the plateau phase <20% : yes

-Degradation degree of the reference substance >70% after 14 days : yes

-Degradation degree in the inhibition control >35 % after 14 days : yes

-The test is valid : yes

Proof of ready biodegradation according to OECD criteria:

-Degradation degree of the test substance >70% within 28 days : yes

-10-day window met : yes

-No pre-adapted inoculum used : yes

-Test substance is readily biodegradable according to OECD criteria : yes

Validity criteria fulfilled:
yes
Remarks:
Explained in the section "Any other infomation on results incl. tables"
Interpretation of results:
readily biodegradable
Conclusions:
The biodegradation degree (DOC removal) after 21 days for the test substance ethylene carbonate was 90-100%. Therefore, according to the OECD criteria, the test substance is readily biodegradable.

Description of key information

Three reliable studies were identified out of which two were guideline studies with K1 scores. Firstly, the key study Huntsman (2003) conducted according to the OECD guideline 301 B concluded that ethylene carbonate is readily biodegradable under the conditions of the test.

In addition, the study conducted at BASF (Schwarz, 2002), in accordance to EEC Method C.4-A: DOC Die-Away Test, OECD Guidelines for Testing of Chemicals Ready Biodegradability - DOC Die-Away Test 301 A and  ISO 7827:1994 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of dissolved organic carbon (DOC), also concluded the test substance to be readily biodegradable as per the OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A Klimisch 1 study of Huntsman (2003) was conducted according to GLP and OECD guideline 301B and was therefore identified as key study. The percent theoretical carbon dioxide produced by ethylene carbonate was 72.2% and 80.8% by day 9, and 86.9% and 98.5% theoretical carbon dioxide by day 29 of the study for replicates 1 and 2, respectively. Therefore, ethylene carbonate can be classified as readily biodegradable under the conditions of this test.

The study from BASF (Schwarz, 2002), showed a biodegradation (DOC removal) after 21 days for the test substance ethylene carbonate of 90-100% and also concluded the test substance to be readily biodegradable as per the OECD 301A criteria.

The Klimisch 2 supporting study of Huls (Diefenbach R, 1995) determined a 100 % DOC removal in 5 days (OECD guideline 302B) and concluded that the substance was inherently biodegradable.