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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:
2012-06-22 to 2012-07-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study was performed according to OECD Guideline 301 F. All validity criteria were fulfilled. However, this study is considered reliable with restrictions because the results of biodegradation are different for all three tests.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on February 22 and 23, 2011 / signed on September 19, 2011
Oxygen conditions:
aerobic
Inoculum or test system:
other: mixed population of activated sewage sludge micro-organisms
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms (Test System) was obtained on June 27, 2012 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thonex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.
- Preparation of inoculum: The sample of activated sewage sludge was maintained on continuous aeration upon receipt, A Sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30 mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
99.75 - 100.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: culture medium used in this study was that recommended in the OECD Guideline No 301 F.
- Test temperature: 22 ± 1 °C
- pH: On day 28, all vessels were sampled for pH
- Suspended solids concentration: 30 mg/L dry weight activated sludge
- Continuous darkness: No

TEST SYSTEM
- Culturing apparatus: 1000 mL glass culture vessels
- Number of culture flasks/concentration: A control, in duplicate, consisting of inoculated culture medium; test material (item no 967406), in triplicate, in inoculated culture medium.
- Measuring equipment: WTW oxitopC calculate automatically the consumption of oxygen.
- Tbe system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer. As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into NaOH causing a net reduction in gas pressure within the sample flask.

SAMPLING
- Sampling frequency: The daily Biochemical Oxygen Demand (BOD) values for the control, test and reference materials and the toxicity control were performed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: Yes; test material and the reference material in inoculated culture medium as a toxicity control.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None
Test performance:
No data
Parameter:
% degradation (O2 consumption)
Value:
59
Sampling time:
28 d
Remarks on result:
other: Test 1: 99.90 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
28 d
Remarks on result:
other: Test 2: 100.60 mg/L; 10-day window not fulfilled
Parameter:
% degradation (O2 consumption)
Value:
47
Sampling time:
28 d
Remarks on result:
other: Test 3: 99.75 mg/L
Details on results:
- The test material (test 1) attained 59 % degradation after 28 days. The test material (test 2) attained 65 % degradation after 28 days but it didn't satisfy the 10-day window validation criteria. The test material (test 3) attained 47 % degradation after 28 days.
- The toxicity test attained 47 % degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the study.
Results with reference substance:
Sodium Benzoate attained 91 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

None

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test material (test 1 and 3) attained respectively 59 % and 47 % degradation after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.
The test material (test 2) attained 65 % degradation after 28 days but it didn't satisfy the 10-day window validation criteria. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301F.
Executive summary:

The study was performed according to OECD Guideline 301 F and in compliance with GLP to assess the ready biodegradability of the test material in an aerobic aqueous media.

 

The test material, at a concentration of 99.90 mg/L in test 1, at a concentration of 100.60 mg/L in test 2 and at a concentration of 99.75 mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22 ± 1 °C for 28 days. The degradation of the test material was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test material (test 1 and 3) attained respectively 59 % and 47 % degradation after 28 days. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F. The test material (test 2) attained 65% degradation after 28 days but it didn't satisfy the 10-day window validation criteria. Therefore the test material cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
October 17th 2000 to November 16th 2000
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This study was performed according to a method very similar to the ISO Guideline 14593 and the OECD Guideline 310 without GLP statement. This study is well documented but a reference material was not tested. The results from the reference test are part of the validity criteria for OECD 310 so the study cannot be considered as valid with or without restrictions in the absence of this information.
Qualifier:
according to guideline
Guideline:
other: The application of dissolved inorganic carbon measurements to the study of aerobic biodegradability, R R Birch and R J Fletcher, Chemosphere, 1991, 23(4), pp507-524.
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
other: ISO Guideline N° 14593: "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test) (adopted 1999)".
Deviations:
yes
Remarks:
non-inclusion of a reference substance, replacing the air in the headspace by pure oxygen to ensure no oxygen transfer limitation and CO2 was measured in the headspace and in the liquid phase without acidification
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
not specified
Principles of method if other than guideline:
To avoid distribution during the test (low solubility), the test occurs in 5L bottle.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on October 17th, 2000 from the secondary treatment stage of the sewage treatment plant at Aïre (STEP Aïre, ch. de la verseuse, 1219 Aïre (Geneva, Switzerland)), which treats predominantly domestic sewage.
Upon reception, the sample of activated sewage sludge was filtered through filter paper 1 (100 μm). The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and aerated with pure oxygen at room temperature. Determination of sludge dry weight was performed to inoculate the final solution with 30 mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
27.15 mg/L
Based on:
test mat.
Initial conc.:
18.09 mg/L
Based on:
other: C
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The culture medium used in this study was that recommended in the OECD Guideline N° 310.

On Day 0, the following solutions were inoculated in 5 L glass culture vessels (total volume when full 5.5 L) each containing 2 L of solution:
- A control, in duplicate, consisting of inoculated culture medium, 2 glass culture vessels.
- The test material, in duplicate, in inoculated culture medium, 2 glass culture vessels to give a final concentration of 27.15 mg/L.
All vessels were sealed using Teflon lined silicon septa and incubated at 28°C ± 2°C with constant shaking at approximately 150 rpm (Kuhner ISF-1-W) in diffused light for 28 days.

Carbon dioxide production in the vessels was determined by measuring the increase of IC (Inorganic Carbon) in the headspace and the liquid phase of each vessel. Headspace samples were collected using a gas-tight syringe and needle through the Teflon septum (10 ml) and direct injection on the TOC (Total Organic Carbon) analyser. The sampling of the liquid phase was done through the bottom of the vessel and the samples were analysed using a Shimadzu 5000 TOC analyser. The IC concentration was measured using a non-dispersive infrared detector utilizing CO2-free synthetic air as carrier gas. Each analysis was carried out in triplicate. Calibration of the TOC analyser for IC analysis was done using standard solutions of sodium carbonate and sodium bicarbonate in ultra-pure water.
Reference substance:
not specified
Remarks:
not tested
Preliminary study:
Not applicable
Test performance:
In this study, a standard material was not tested. Therefore, the validity criterion cannot be assessed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
83
Sampling time:
28 d
Remarks on result:
other: satisfied the 10-day window validation criterion
Details on results:
-1% degradation after 5 and 8 days, 49% degradation after 12 days, 65% degradation after 15 days, 75% degradation after 19 days, 81% degradation after 22 days and 83% degradation after 29 days. See graph in "Illustration".
Results with reference substance:
Not applicable

None

Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
The test material attained 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the test material can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline.
Executive summary:

This study was performed to investigate the ready biodegradability of the test material when exposed to sewage sludge micro-organisms under aerobic conditions during 28 days. The method followed the protocol as described by Birch and Fletcher (Chemosphere, vol. 23, pp. 507-524, 1991). This method is very similar to the ISO Guideline N° 14593 (CO2 headspace test, adopted 1999) and of the OECD Guideline 310 (adopted March 23rd, 2006).

The test material, at a concentration of 18.09 mg C/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 28°C ± 2°C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced on Days 0, 5, 8, 12, 15, 19, 22 and 29.

The test material attained 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore the test material can be considered to be readily biodegradable. However, this result can only be considered as a weight of evidence study as no reference substance was tested alongside the study.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances (stereoisomers) and comparable properties related to the target endpoints.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
See attached document in Iuclid section 13

4. DATA MATRIX
See attached document in Iuclid section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Remarks:
Read-Across justification document
GLP compliance:
no
Key result
Parameter:
% degradation (CO2 evolution)
Value:
83
Sampling time:
28 d
Remarks on result:
other: satisfied the 10-day window validation criterion
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Conclusions:
According to the experimental study performed on the source substance, the substance attained 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore, the target substance can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline.
Executive summary:

This study was performed on a source substance to investigate the ready biodegradability of the test material when exposed to sewage sludge micro-organisms under aerobic conditions during 28 days. The method followed the protocol as described by Birch and Fletcher (Chemosphere, vol. 23, pp. 507-524, 1991). This method is very similar to the ISO Guideline N° 14593 (CO2 headspace test, adopted 1999) and of the OECD Guideline 310 (adopted March 23rd, 2006).

The test material, at a concentration of 18.09 mg C/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 28°C ± 2°C for 28 days. The degradation of the test material was assessed by the determination of carbon dioxide produced on Days 0, 5, 8, 12, 15, 19, 22 and 29.

The test material attained 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore the test material can be considered to be readily biodegradable. However, this result can only be considered as a supporting study as no reference substance was tested alongside the study.

Description of key information

OECD Guideline 301F, GLP, key study, validity 2:

65% degradation after 28 days without fulfilled the 10-day window validation criteria.

According to the OECD Guidelines, a mixture containing isomers that are expected to degrade sequentially is not required to meet the 10 -day window obligations and therefore, as the degradation was greater than 60% after 28 days, the test substance can be considered to be readily biodegradable.  

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

To assess the biodegradation of the registered substance, two studies are available.


 


The first study (Firmenich, 2012), assessed as the key study, was performed on the registered substance according to OECD Guideline 301F with GLP compliance, in an aerobic aqueous media. The test substance, at a concentration of 99.90 mg/L in test 1, 100.60 mg/L in test 2 and 99.75 mg/L in test 3, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22 ± 1 °C for 28 days. The degradation of the test substance was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.


The test substance (test 1 and 3) attained respectively 59 % and 47 % degradation after 28 days. Therefore the test substance cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F. The test substance (test 2) attained 65% degradation after 28 days but it didn't satisfy the 10 -day window validation criteria, therefore it cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 F. However, this study was performed on a mixture of stereoisomers. According to the OECD Guidelines, a mixture containing isomers that are expected to degrade sequentially is not required to meet the 10 -day window obligations and therefore, as the degradation was greater than 60% after 28 days, the test substance can be considered to be readily biodegradable. A positive biodegradability test is sometimes difficult to obtain because it's dependent on the quality of the inoculum used. Therefore, a positive result is sufficient to conclude on the readily biodegradation potential of the substance. An additional study is available in the dossier to support this conclusion.


 


The second study (Firmenich, 2000), assessed as a supporting study, was performed on a source substance, Reaction mass of (3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and (3S*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol (EC# 942-425-2). The target and the source substances are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. The two trans 1R,6S diasteoisomers, with both 3R and 3S hydroxyl group, of the registered/target substance are constituents of the source substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantiomers). This non-GLP study followed the protocol as described by Birch and Fletcher (Chemosphere, vol. 23, pp. 507-524, 1991). This method is very similar to the ISO Guideline N° 14593 (CO2 headspace test, adopted 1999) and of the OECD Guideline 310 (adopted March 23rd, 2006). The substance, at a concentration of 18.09 mg C/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 28°C ± 2°C for 28 days. The degradation of the test substance was assessed by the determination of carbon dioxide produced on Days 0, 5, 8, 12, 15, 19, 22 and 29. The test substance attained 83% degradation after 28 days and satisfied the 10-day window validation criterion. Therefore the test substance can be considered to be readily biodegradable.


In conclusion, based on the key and supporting studies, we can assume that the registered substance is readily biodegradable. Based on structural similarity (stereoisomers) and on similar basic physico-chemical properties, the target and source substances are not expected to have a different environmental profile.