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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 November 1999 through 10 January, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
UVCB
Specific details on test material used for the study:
CEDRENE
Lot No. : 9000346040
Purity : GLC profile matches standard

The test item is a multi-component complex mixture isolated from a natural source and is, consequently, defined as an UVCB.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Aïre) was used.

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110 °C for two hours and weighing the residue.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The method used is basically the one described under No. 301 F in the OECD Guidelines for Testing of Chemicals.

A measured volume of inoculated mineral medium, containing a known concentration of test substance (100 mg/l) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1 °C) for up to 28 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant the gas volume in the respirometer flask. Evolved carbon dioxide is absorbed in soda lime pellets. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide and hydrogen to water).

The respirometer used during this study is a BI-1000, made by BIOSCIENCE Inc., Bethlehem, PA, USA.

The water used during this study is deionised water containing less than 10 mg/L dissolved organic carbon.

As suggested in the OECD 301 F method, the toxicity of the test chemical for the inoculum is checked. Therefore, a pair of flasks of the volumetric respirometer are filled with mineral medium + test chemical (100 mg/l) + reference substance (100 mg/l) + inoculum and their respirations are recorded as for the other flasks. If they are lower than those of the flasks containing mineral medium + reference substance (100 mg/l) + inoculum, the test chemical can be assumed to be inhibitory to the inoculum used.

All flasks are filled with 1000 ml of mineral medium. Samples of test or reference substances, or both, are added. Then, a volume of suspended sludge corresponding to 30 mg dry weight is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. About 5 g of soda lime is placed in an attachment of the stopper, the flasks are closed and placed in the water bath of the BI-1000. After temperature and pressure equilibration, the measuring cells are started (time zero of the experiment).

The oxygen consumption of each flask is automatically recorded by the BI-1000 during the whole test duration. Correct temperature and stirring are regularly checked visually.

At the end of the test period (normally 28 days), the pH of each flask is measured again.




Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium Benzoate (Fluka, Buchs, CH, Article No. 71300), purity: minimum 99.0%

Results and discussion

% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
78
Sampling time:
28 d
Details on results:
CEDRENE undergoes 78 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 5 and reaches 52 % at the end of the 10-day window (days 5 to 15). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion for CEDRENE, which is a multi-component complex mixture isolated from a natural source and is, consequently, defined as an UVCB. Thus, CEDRENE should be regarded as readily biodegradable according to this test based on the biodegradation percentage of 78% at day 28.

Thus, CEDRENE should be regarded as Readily Biodegradable according to this test.

The curves obtained with the reference substance alone and with CEDRENE + reference substance show no toxic effect of CEDRENE to the micro-organisms at the test concentration.

BOD5 / COD results

Results with reference substance:
Sodium Benzoate underwent 84% degradation by Day 7 (minimum for validity criteria = 40%) and 90% by Day 14 (minimum for validity criteria = 65%).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
CEDRENE undergoes 78 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 5 and reaches 52 % at the end of the 10-day window (days 5 to 15). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion for CEDRENE, which is a multi-component complex mixture isolated from a natural source and is, consequently, defined as an UVCB. Thus, CEDRENE should be regarded as readily biodegradable according to this test based on the biodegradation percentage of 78% at day 28.

Thus, CEDRENE should be regarded as Readily Biodegradable according to this test.

The curves obtained with the reference substance alone and with CEDRENE + reference substance show no toxic effect of CEDRENE to the micro-organisms at the test concentration.
Executive summary:

CEDRENE undergoes 78 % biodegradation after 28 days in the test conditions. Biodegradation starts on day 5 and reaches 52 % at the end of the 10-day window (days 5 to 15). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion for CEDRENE, which is a multi-component complex mixture isolated from a natural source and is, consequently, defined as an UVCB. Thus, CEDRENE should be regarded as readily biodegradable according to this test based on the biodegradation percentage of 78% at day 28.

 

Thus, CEDRENE should be regarded as Readily Biodegradable according to this test.

 

The curves obtained with the reference substance alone and with CEDRENE + reference substance show no toxic effect of CEDRENE to the micro-organisms at the test concentration.