Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
A newly developed method was created and tested alongside the OECD Guideline for Ready Biodegradability (301B). The results of which were directly comparable to the accepted OECD guideline.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: New Biodegradability Test developed. The Sturm method was used in conjuntion to validate the results of the new method
Principles of method if other than guideline:
The test substance in a dilute mineral salts solution is incubated in sealed vessels with appropriate micro-organisms for a period of up to 28 days. Only two thirds of the volume of the vessel is filled with liquid. At the test concentrations used only about 15% of the available oxygen in the headspace gas is required for the complete oxidation of all test compound carbon to carbon dioxide. Any carbon dioxide produced by the breakdown of the test material is distributed between the liquid and gaseous phases. Periodically a vessel is taken, a sample of the headspace gas withdrawn using a gas syringe and the concentration of carbon dioxide in the headspace gas determined. The seal is then broken and the concentration of dissolved inorganic carbon (DIC) in the solution is measured. Similar determinations are made for a control vessel which does not contain the test substance. The difference in the total inorganic carbon found in the test and control vessels allows the quantity of carbon dioxide produced from the test compound to be ascertained. From a knowledge of the quantity of test material added and its carbon content the extent of mineralisation can be calculated.
GLP compliance:
no
Remarks:
Published data
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
The inoculum used in the ring test was 30 mg/L of activated sludge. Assuming the carbon content of activated sludge to be about 50% then 15 mg/L of organic carbon, i.e. more carbon than is present as test compund, is introduced with the inoculum. If some of this carbon is metabolised during the course of the test then high levels of carbon dioxide will be produced in the controls thereby reducing the precision of the technique.

Since only about 1 % of cells in activated sludge are active it was considered that a 'cleaner' inoculum of similar activity could be obtained using secondary effluent from an activated sludge plant. The level of organic carbon introduced when using 10 % by volume of secondary effluent to inoculate the test is only about 1-2 mg/L.

The tests described were inoculated with secondary effluent from an activated sludge plant treating domestic sewage. The collected effluent was first passed through a coarse filter, Whatman No. 1, to remove any gross particulate matter. The level of inorganic carbon in the inoculum was reduced before use by sparging with carbon dioxide-free air for about one hour while maintaining the pH at 6.5.

In the tests reported here the CO2 produced in the control vessels, using the maximum inoculum concentration of 10 % secondary effluent, was in the range 0.4 - 1.3 mg/L. hence, in cases where positive results were obtained less than 10 % of the carbon dioxide produced was derived from the control. Finally, while any source of bacteria can be employed it should be stressed that if the procedure is used as a test of 'ready biodegradability' then micro-organisms which have been pre-exposed to the test compound must not be used. If pre-adapted inocula are used then the classification of those materials giving a positive result is lowered from 'readily' to 'inherently' biodegradable.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium used was adopted from that recommended in the 1988 OECD Ring Test of Ready Biodegradability.

Stock solutions (a)-(d) were prepared by dissolving the following salts in 1 litre of deionised water:
(a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.40 g Na2HPO4.2H2O, 0.5 g NH4Cl
(b) 36.4 g CaCl2.2H2O
(c) 22.50 g MgSO4.7H2O
(d) 0.2 g FeCl3.6H2O, 0.4 g EDTA disodium salt.

The test medium was prepared by diluting 10 ml of solution (a) and 1 ml of solutions (b)-(d) in 1 L aliquots of water.

- pH: 6.5

TEST SYSTEM
Using suitable volumetric apparatus 100 ± 1 mL of the mineral salts media is dispensed into '125 mL' Hypo-Vial. The media is prepared so as to contain 0.5 to 10 % by volume of inoculum and 2 to 10 mg/L of test substance as organic carbon. Controls containing the same inoculum concentration but no test compound are also prepared. The vials are sealed with butyl rubber septa and aluminium crimp seals and placed on an orbital shaker in a temperature controlled environment. To follow the course of biodegradation and to statistically evaluate the extent of biodegradation on the final day of the test a minimum of 12 vessels is required per test substance. This provides for a data point every fourth day and 6 replicated for the assessment of the final extent of biodegradation on the 28th day of the test. A vessel is removed from the shaker as required, a sample of the headspace gas is withdrawn using a gas syringe and the concentration of carbon dioxide determined. The seal is then broken and the concentration of dssolved inorganic carbon (DIC) in the solution is measured immediately. Similar determinations are made for a control vessel which does not contain the test substance. The difference in the total inorganic carbon found in the test and control vessels allows the quantity of carbon dioxide produced from the test compound to be ascertained.
Preliminary study:
A preliminary study was conducted using 2 mg/L which indicated equally reliable data.
Test performance:
The ring test results (Sturm test) were used as comparison. The data suggest that there are no significant differences in the overall results and that the same conclusions with regard to the ultimate biodegradability of the test materials would be reached in each case.
Parameter:
% degradation (CO2 evolution)
Value:
100.9
St. dev.:
2.5
Sampling time:
28 d
Remarks on result:
other: 96.9 - 104.9 % confidence interval
Details on results:
Four substances were tested for Ready Biodegradability using the new method (Monoethanolamine, 1,6 Hexanediol, 2,4,6 Trichlorophenol, Pentaerythritol) these were shown to be readily and ultimately biodegradable and the inherent precision of the method is clearly very high. The ring test results (Sturm test) were used as comparison. The data suggest that there are no significant differences in the overall results and that the same conclusions with regard to the ultimate biodegradability of the test materials would be reached in each case. Comparison of the results obtained by the two methods suggests that the biodegradation potential achieved by the use of secondary effluent as a source of micro-organisms is at least comparable to activated sludge. In all cases the rate of degradation in the sealed vessel test is noticeably higher than that observed in the ring test even though the weight of biomass used is only about 4 % of that introduced with the activated sludge inoculum.
Results with reference substance:
No details supplied

No further information

Validity criteria fulfilled:
yes
Remarks:
Four substances were tested for Ready Biodegradability using the new method (Monoethanolamine, 1,6 Hexanediol, 2,4,6 Trichlorophenol, Pentaerythritol) these were shown to be readily and ultimately biodegradable and the inherent precision of the method is
Interpretation of results:
readily biodegradable
Conclusions:
Although the test described is essentially the same as the Sturm CO2 Production Test as described in the OECD Guidelines the greater simplicity of the technique and the high precision of the data obtained greatly increases its range of application. The ease with which results can be replicated facilitates statistical evalutaion of the data and makes the test particularly useful for comparative purposes. The effects on biodegradability of changes in chemical structure or temperature, for example, should be readily detected. In the studies reported a test compund concentration of 10 mg/L as DOC was employed but preliminary tests at 2 mg/L indicate that equally reliable data can be obtained at the lower concentration. Testing at these lower concentrations reduces the number of instances when toxic or inhibitory effects of the test chemical rather than its poor biodegradability limit the extent of microbial attack. Since the system remains sealed throughout the incubation period volatile materials can also be examined and no additional difficulties were observed when the compounds were also sparingly soluble. As benzyl acetate is sparingly soluble this test was perfecting suited to te test substance.

benzyl acetate was found to be readily biodegradable as it degraded by 100.9 % in 28 days.
Executive summary:

As Benzyl acetate is a sparingly soluble compound, the CO2 production method is deemed beneficial. A new method based on OECD 301B was developed. The method used infra-red analysis to precisely determine carbon dioxide in both aqueous and gaseous samples thus allowing the test system to be scaled down considerably. In addition a 'cleaner' inoculum of similar activity to that recommended by the OECD guideline was obtained by using the secondary effluent from an activated sludge plant. This resulted in very much lowered CO2 production in the controls. The method was proven to be accurate and reliable by testing the four compounds used in the 1988 OECD ring testing together with a further eight reference compounds.

Benzyl acetate in a dilute mineral salts solution is incubated in sealed vessels with appropriate micro-organisms for 28 days. Only two thirds of the volume of the vessel is filled with liquid. At the test concentrations used only about 15% of the available oxygen in the headspace gas is required for the complete oxidation of all test compound carbon to carbon dioxide. Any carbon dioxide produced by the breakdown of the test material is distributed between the liquid and gaseous phases. Periodically a vessel is taken, a sample of the headspace gas withdrawn using a gas syringe and the concentration of carbon dioxide in the headspace gas determined. The seal is then broken and the concentration of dissolved inorganic carbon (DIC) in the solution is measured. Similar determinations are made for a control vessel which does not contain the test substance. The difference in the total inorganic carbon found in the test and control vessels allows the quantity of carbon dioxide produced from the test compound to be ascertained. From a knowledge of the quantity of test material added and its carbon content the extent of mineralisation was calculated.

Benzyl acetate was found to biodegrade 100.9% over 28 days proving that the test substance is readily biodegradable.

Description of key information

As Benzyl acetate is a sparingly soluble compound, the CO2 production method is deemed beneficial. A new method based on OECD 301B was developed. The method used infra-red analysis to precisely determine carbon dioxide in both aqueous and gaseous samples thus allowing the test system to be scaled down considerably. In addition a 'cleaner' inoculum of similar activity to that recommended by the OECD guideline was obtained by using the secondary effluent from an activated sludge plant. This resulted in very much lowered CO2 production in the controls. The method was proven to be accurate and reliable by testing the four compounds used in the 1988 OECD ring testing together with a further eight reference compounds.
Benzyl acetate in a dilute mineral salts solution is incubated in sealed vessels with appropriate micro-organisms for 28 days. Only two thirds of the volume of the vessel is filled with liquid. At the test concentrations used only about 15% of the available oxygen in the headspace gas is required for the complete oxidation of all test compound carbon to carbon dioxide. Any carbon dioxide produced by the breakdown of the test material is distributed between the liquid and gaseous phases. Periodically a vessel is taken, a sample of the headspace gas withdrawn using a gas syringe and the concentration of carbon dioxide in the headspace gas determined. The seal is then broken and the concentration of dissolved inorganic carbon (DIC) in the solution is measured. Similar determinations are made for a control vessel which does not contain the test substance. The difference in the total inorganic carbon found in the test and control vessels allows the quantity of carbon dioxide produced from the test compound to be ascertained. From knowledge of the quantity of test material added and its carbon content the extent of mineralisation was calculated.
Benzyl acetate was found to biodegrade 100.9% over 28 days proving that the test substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

As Benzyl acetate is a sparingly soluble compound, the CO2 production method is deemed beneficial. A new method based on OECD 301B was developed. The method used infra-red analysis to precisely determine carbon dioxide in both aqueous and gaseous samples thus allowing the test system to be scaled down considerably. In addition a 'cleaner' inoculum of similar activity to that recommended by the OECD guideline was obtained by using the secondary effluent from an activated sludge plant. This resulted in very much lowered CO2 production in the controls. The method was proven to be accurate and reliable by testing the four compounds used in the 1988 OECD ring testing together with a further eight reference compounds.

Benzyl acetate in a dilute mineral salts solution is incubated in sealed vessels with appropriate micro-organisms for 28 days. Only two thirds of the volume of the vessel is filled with liquid. At the test concentrations used only about 15% of the available oxygen in the headspace gas is required for the complete oxidation of all test compound carbon to carbon dioxide. Any carbon dioxide produced by the breakdown of the test material is distributed between the liquid and gaseous phases. Periodically a vessel is taken, a sample of the headspace gas withdrawn using a gas syringe and the concentration of carbon dioxide in the headspace gas determined. The seal is then broken and the concentration of dissolved inorganic carbon (DIC) in the solution is measured. Similar determinations are made for a control vessel which does not contain the test substance. The difference in the total inorganic carbon found in the test and control vessels allows the quantity of carbon dioxide produced from the test compound to be ascertained. From knowledge of the quantity of test material added and its carbon content the extent of mineralisation was calculated.

Benzyl acetate was found to biodegrade 100.9% over 28 days proving that the test substance is readily biodegradable.