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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
2007-11-02 to 2008-01-31
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study was performed according to the EU Method C.4-E : Determination of the ready biodegradability - Closed bottle test with significant methodological modifications. The test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Remarks:
Inspection on March 15th and16th 2007
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material:Test material provided by the sponsor, batch n° 713060STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: stored at room temparature and protected from direct sunlightTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Chloroform was used as solvent for the preparation of the test solution : a stock solution was prepared in chloroform.
Oxygen conditions:
aerobic
Inoculum or test system:
natural water
Remarks:
The inoculum was derived from surface waters.
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The inoculum was derived from surface waters collected in the near area of Pau, France. The surface waters receive predominantly domestic sewage. - Preparation of inoculum for exposure: The freshly collected sample of surface water was previously pre-conditionned to the experimental conditions under aerobic conditions for 6 days in the mineral medium at the test temperature : 400 mL of surface water were added with 0.5 mL of stock solutions (a mixture of chloroform and test substance). - Storage conditions: The resulting solution was maintained under strong aeration and agitation at 20 +/- 1 °C for 6 days. - Concentration of inoculum : The pre-conditionned inoculum wa further used at a rate of 2 mL/L of medium (for Test 1) or 0.7 mL/L of medium (Test 2).- Suspended solids : were measured to represent 4.4 µg/L (Test 1) or 0.9 µg/L (Test 2).
Duration of test (contact time):
>= 28 - < 36 d
Initial conc.:
5.02 mg/L
Based on:
test mat.
Initial conc.:
2.04 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS - Composition of medium: 8.5 g/L of KH2PO4, 21.75 g/L K2HPO4, 33.40 g/L Na2HPO4 - 2H2O, 0.50 g/L NH4Cl, 27.50 g/L CaCl2, 22.50 g/L MgSO4 - 7H2O, 2.5 g/L FeCl3 - 6H2O - Test temperature: 20 +/- 1 °C - pH : Test 1 : 7.50 ; Test 2 : 7.40 - Suspended solids concentration: 4.4 µg/L (Test1), 0.9 µg/L (Test 2). - bottles were incubated in dark TEST SYSTEM - Culturing apparatus: vessels - Number of culture flasks/concentration: test 1 : 2 blank replicates, 2 test replicates, 1 solvent replicate, 1 toxic replicate, 2 aniline replicates ; test 2 : 2 blank replicates, 2 test replicates, 1 solvent replicate, 1 toxic replicate, 2 aniline replicates - Measuring equipment: Dissolved oxygen was assessed by the electrode method : Oxi38 equipped with StirrOx G probe, WTW SAMPLING - Sampling frequency: blank series, test series and reference series were withdrawn every 3-4 days CONTROL AND BLANK SYSTEM - Inoculum blank: Yes, 300 mL mineral medium + 50 mL pre-conditionned inoculum - Abiotic sterile control: Not performed - Toxicity control: Yes, 300 mL mineral medium + 100 µL aniline + 20 µL Figolide + 50 mL pre-conditionned inoculum
Reference substance:
aniline
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
50
Sampling time:
36 d
Remarks on result:
other:
Remarks:
The 10-day window criteria was not fulfilled.
Details on results:
50.0 % biodegradation occurred after 36 days of incubation, in Test 2. The pass-level was almost reached but it did not occur within the 10-day window and the tested substance was not considered as easily biodegradable.
Results with reference substance:
In the toxic control series more than 25 % biodegradation of aniline had occurred on day 14.

The validity criteria were fulfilled for Test 2, at 2.04 mg/L :

-Mean oxygen uptake in the blank vessels < 1.5 mg/L at the end of the test,

-the residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time,

-differencies of extremes of replicate values of the removal of the test item was less than 20 % of mean value at the end of test,

percentage biodegradation of the reference compound (aniline) had reached the pass-level by day 14,

-in the toxic control series more than 25 % biodegradation of aniline had occurred on day 14.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
50 % biodegradation occurred within 36 days, the pass-level was almost reached but this did not occur within the 10-day window. Consequently, the test substance was not considered as easily biodegradable.
Executive summary:

This study was performed according to the EU Method C.4-E : Determination of the ready biodegradability - Closed bottle test.

The test material was exposed to inoculum from surface waters, at a concentration of 2.04 mg/L in Test 2, in culture medium, in closed bottles, in the dark, at 20 +/- 1 °C, for 36 days.

The degradation of the test material was assessed by the measurement of oxygen consumption.

50 % biodegradation occurred within 36 days and the 10-day window criteria was not fulfilled, even if the pass-level was almost reached.

However, in both tests, the test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.

Consequently, the test material should be regarded as not readily biodegradable according to this test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-02 to 2012-08-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The guidelines that were followed in this study are : 1) Guideline for the testing of chemicals, 301F Ready Biodegradability: Manometric Respirometry test. Beijing: China Environmental Science Press. 2004: 363-369. 2) OECD guidelines for the testing of chemicals, 301F Manometric Respirometry test. Paris: OECD, Adopted 17 July 1992. 3) GB/T 21801 - 2008 Chemicals - Ready Biodegradability: Manometric Respirometry Test.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to
Guideline:
other: Guideline for testing of chemicals, 301F Ready Biodegradability: Manometric Respirometry test. First edition. Beijing:China Environmental Science Press. 2004: 363-369.
GLP compliance:
yes (incl. certificate)
Remarks:
GLP Compliance Statement performed by China National Accreditation Service for Conformity Assessment, datinf from 2010-11-24, valid until 2013-11-23
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Store in a cool place in original container and protect from sunlight and moistureTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: the test substance was weighted and added directly to the bottles. After adding 100 mL of test medium, an ultrasonic treatment of 15 min was performed.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
No data
Duration of test (contact time):
28 d
Initial conc.:
21.4 mg/L
Based on:
not specified
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS - Composition of medium: 8.5 g KH2PO4, 28.5 g K2HPO4-3H2O, 67.15 g Na2HPO4-12H2O, 0.5 g NH4Cl ; 13.75 g CaCl2 ; 11.25 g MgSO4-7H2O ; 0.125 g FeCl3 6H2O - Test temperature: 22 +/- 11°C - pH: 7.4 +/- 0.2 - pH adjusted: yes - Continuous darkness: yes TEST SYSTEM - Culturing apparatus: - Number of culture flasks/concentration: 3 bottles used as "test suspensions containing test substance inoculum and test medium : initial concentration of 21.4 mg/L 3 bottles used as "inoculum blank" containing inoculum and test medium : do not contain test substance1 bottle used as "procedure control" containing reference substance, inoculum and test medium : initial concentration of 30.1 mg/L1 bottle used as "toxicity control" containing test substance, reference substance, inoculum and test medium : initial concentration of 30.4 mg/L. - Method used to create aerobic conditions: The test bottles were aerated on OxiTop Control BOD at 22°C +/- 1°C - Measuring equipment: The consumption of BOD of each test bottle was determined and recorded automatically by the respirometer OxiTop Control BOD - Test performed in closed vessels as volatility of the test substance was unknown. - Details of trap for CO2 and volatile organics if used: No details SAMPLING - Sampling frequency: At the start of the test and at the end of the test. - Sampling method: The consumption of BOD of each test bottle was determined and recorded automatically by the respirometer OxiTop Control BOD. pH values of 2 specific bottles were determined and the pH adjusted in each test bottle at the start of the test. pH values of each test bottle were also determined at the end of the test. CONTROL AND BLANK SYSTEM - Inoculum blank: 3 bottles containing inoculum and test medium - Abiotic sterile control: No abiotic sterile control performed - Toxicity control: 1 bottle containing test substance, reference substance, inoculum and test medium
Reference substance:
other: sodium benzoate
Preliminary study:
None
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
67.6
Sampling time:
28 d
Remarks on result:
other: At the end of the test, the percentage biodegradation of the 2 test suspensions were 62.5% and 72.7 %. The average was 67.6%. During the 10-day window the percentage biodegradation of the test substance did not reach 60% under the test conditions.
Details on results:
The consumption of BOD of each test bottle was determined and recordred automatically by the respirometer OxiTop Control BOD.At the end of the test , the percentage biodegradation of the two test supsensions were 62.5 % and 72.7 %. The average was 67.6 %. During the 10-day window, the percentage biodegradation of the test substance did not reach 60 % under the conditions of the test.
Results with reference substance:
On the 14th day of the test, the percentage biodegradation (based on ThOD) of the reference substance was 83.8%. It passed the level of 60 %, thereby passing the validity criteria.

Table 1 : Biochemical oxygen demend (BOD) of each test bottle during the test

Time (d) Test suspensions Inoculum blank Procedure
control
Toxicity
control 
bottle 1
(mg/L)
bottle 2
(mg/L)
bottle 3
(mg/L)
bottle 4
(mg/L)
Mean (mg/L) bottle 5
(mg/L)
bottle 6
(mg/L)
1 0,0 0,0 0,0 0,0 0,0 0,0 0,0
4 6,2 7,9 9,0 8,4 8,7 42,4 39,5
7 13,0 17,0 10,9 10,1 10,5 49,2 53,5
10 27,7 31,7 12,6 12,1 12,4 53,7 66,2
14 36,2 40,1 14,3 14,3 14,3 56,5 77,5
18 43,0 47,5 15,4 16,0 16,0 59,4 86,0
20 43,0 47,5 16,3 16,3 16,3 59,9 84,5
22 43,5 48,0 16,6 16,5 16,5 61,0 86,0
25 46,9 51,4 17,1 17,3 17,3 62,2 78,9
28 49,2 54,3 17,7 18,0 18,0 62,7 94,4

Table 2 : The percentage degradation calculated by BOD

Group 7d 14d 21d 28d
BOD (mg) degradation (%) BOD (mg) degradation (%) BOD (mg) degradation (%) BOD (mg) degradation (%)
test
suspension 1
13,0 5,0 36,2 43,8 43,0 53,4 49,2 62,5
test
suspension 2
17,0 13,0 40,1 51,6 47,5 62,4 54,3 72,7
Procedure
control 
49,2 76,9 56,5 83,8 59,9 86,6 62,7 88,9
Toxicity
control
53,5 43,2 77,5 63,0 84,5 67,9 94,4 75,9
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
At the end of the test , the percentage biodegradation of the two test supsensions were 62.5 % and 72.7 %. The average was 67.6 %. The 10-day window criterion was not fulfilled.
Executive summary:

The ready biodegradability of the test material has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301 F and the Chinese guideline for the testing of chemicals : 301 F ready biodegradability : Manometric Respirometry Test.

A nominal concentration of test material of 21.4 mg/L was introduced in the system.

The percentage biodegradation of the two test suspensions, based on ThOD, were 62.5 % and 72.7 %. The average was 67.6 %. The 10-day window criteria was not fulfilled.

On the 14th day of the test, the percentage biodegradation (based on ThOD) of the reference substance was 83.8% surpassing the level of 60 % and thereby passing the validity criteria.

The test material can consequently be considered as ready biodegradable failing the 10-day window.

Description of key information

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

67.6% biodegradation after 28d.

Readily biodegradable but failling the 10-day window criteria.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

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

The first study, from Guangdong Detection Center of Microbiology, 2012, assessed as the key study, was performed on the registered susbstance according to OECD Guideline 301F with GLP compliance, and according to the Chinese Guideline 301F Ready Biodegradability: Manometric Respirometry test. Activated sludge was exposed to the test substance at a concentration of 21.4 mg/L, with culture medium, for 28 days, in aerobic conditions. According to this test, the degree of degradation, calculated from the O2 consumption of two replicates was 62.5 % and 72.7 % respectively, mean of 67.6 %, after 28 days. The percentage biodegradation of the toxicity control was 63.0 % after 28 days. No abiotic control was performed. The percentage biodegradation of sodium benzoate (reference substance) was 83.8 % on the 14th day of the test. The activity of the inoculum is thus verified and the test is considered as valid. Thus, the test substance should be regarded as readily biodegradable but failling the 10-day window criteria.

It was evaluated that this study has a reliability of 2 (Klimisch rating) as :

-no sterile abiotic control was performed

-the concentration of tested substance was 21,4 mg/L instead of 100 mg/L like the guideline demands

-more measurements could have been made during the 10-day window, allowing a better evaluation of the 10-day window criteria

-the unknown volatility of the substance was not discussed in the results, whereas it could have helped in the assessmejnt of the amount of remaining substance.

The second study, from Phytosafe, 2008, assessed as a supporting study, was performed on the registered substance according to the EU Method C.4-E: Determination of the ready biodegradability - Closed bottle test. The test material was exposed to inoculum from surface waters, at a concentration of 2.04 mg/L in Test 2, in culture medium, in closed bottles, in the dark, at 20 +/- 1 °C, for 36 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. The validity criteria were fulfilled for Test 2, for a concentration of test substance of 2.04 mg/L. 50 % biodegradation occurred within 36 days and the 10-day window criteria was not fulfilled, even if the pass-level was almost reached. However, in both tests, the test substance was dissolved in a partially biodegradable solvent, chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid. Consequently, the test cannot be considered valid. In conclusion, based on the first study, the registered substance is readily biodegradable but the 10-day window criteria is not fulfilled.