Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

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)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical appearance: almost colourless liquid
- Storage: room temperature, protected from light
- Expiry date: 12 July 2002

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A sample of activated sludge was taken on September 10th, 2001 from an oxidation ditch in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic sewage. The medium was aerated with CO2-free air overnight before use. The sludge, with a measured dry weight of 3.6 g of solid substance/L was mixed and 420 mL of the sludge was used to inoculate 50 litres of medium to yield a final inoculum concentration of about 30 mg/L of solid substance.

Duration of test (contact time):

28 d

Initial conc.:

29 mg/L

Based on:

ThCO2

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST MEDIUM
A medium with a higher nitrogen content than that specified in the Guidelines was used. Its composition was derived from that specified in the NEN 6513 (Water - Screening test for ready biodegradability of organic compounds) and is given below. The modified composition was chosen to prevent nutrient limitation of degradation during the course of the study. 10 mL of solution A and 1 mL of solutions B to F were put together and made up to 1000 mL with ultrapure water.
A) Solution A contains per 1000 mL water: KH2PO4: 8.5g; K2HPO4: 21.75g; Na2HPO4 2H2O: 33.4g; NH4Cl: 1.5g
B) Solution B contains 36.4 g of CaCl2 2H2O per 1000 mL water.
C) Solution C contains 22.5 g of MgSO4 7H2O per 1000 mL water.
D) Solution D contains 0.025 g of FeCL3 6H2O per 100 mL water
E) Trace element solution (solution E). This solution contains per 1000 mL water: MnSO4 4H2O: 39.9 mg (30.2 mg MnSO4 H2O); H3BO3: 57.2 mg; ZnSO4 7H2O: 42.8 mg; (NH4)6Mo7O24: 34.7 mg (36.9 mg (NH4)6Mo7O24 4H2O); EDTA: 100 mg
F) 15 mg yeast extract dissolved in 100 mL water

TEST METHOD
- The OECD Guideline 301B 'Ready Biodegradability' C02 Evolution Test and the EEC Directive 92/69/EEG Part C.4-C have been modified to use an alternative CO2 trapping system similar to the method described in United States Code of Federal Regulations 40 CFR 796.3100 and to increase its flexibility (Painter H.A., et al., 1995). Two litre glass bottles (Scott Duran) closed with plastic screw caps were used for the test. A 20 mL glass vial containing the five mL CO2 absorbing fluid (i.e. 0.4 or 0.6 M NaOH solution) was suspended in the headspace of each bottle.
- Biodegradation test bottles: The final (nominal) test concentration of the test substance of 29.0 mg/L was prepared by dissolving 1.1607 g in 10 mL ethanol (Stock I). A volume of 250 of Stock I was applied to glass fibre filters (Whatman GF/C, Ø 47 mm). After being air-dried, the filters were added separately to one litre of inoculated medium in six separate bottles. This resulted in a final concentration of 20 mg carbon per litre. A confirmation of the amount of test substance on the filter showed that the loss of test substance due to evaporation was limited, more than 96% remained on the filter. The following controls were included: Blank control: To determine the background CO2 production of the inoculum, a total of 6 control bottles per sampling time were prepared. They contained one litre inoculated medium and glass fibre filters (treated with 250 µL ethanol and airdried). Three of these bottles were acidified after 14 days of incubation and three at the end of the test to remove the dissolved CO2 in the medium; Inoculum activity control: In order to determine the inoculum activity, 68 mg of the reference substance sodium acetate was added to four additional bottles containing one litre inoculated medium and blank glass fibre filter carriers. The final test concentration of sodium acetate was achieved by initially dissolving 6.806 g of the reference substance in a small volume of ultrapure water and adding up to 100 mL with ultrapure water. From this stock solution 1 mL volumes were added to one litre of inoculated medium; Inoculum toxicity control: In order to detect possible toxic effects of the test substance, sodium acetate (68 mg/L) was also added to further duplicate bottles containing 29.0 mg/L of test substance. Before the bottles were closed, five mL of 0.6 or 0.4 M NaOH (A 0.4 M NaOH solution was used for the 14 (acidified), 21, and 28 day determinations, and for the CO2 collected after acidification) was placed in each trapping vial. After closing the bottles, they were incubated in the dark on an orbital shaking machine in a temperature controlled room kept at 20 ± 2°C for 28 days. The CO2 absorption vials were replaced by fresh ones after 7, 14, 21 and 28 days. The amount of CO2 absorbed was determined by titration of the sodium hydroxide with 0.1 M HCI using a Metrohm 686 Titroprocessor. The pH value of the medium was also determined at each sampling time.
- Acidification: On day 14 and day 28, one mL of concentrated HCl was added to three of the blank control bottles, two of the inoculum activity bottles and three bottles of the test substance series. This was performed to remove the remaining dissolved CO2 in the medium. The CO2 evolved after acidification was trapped during a period of approximately one day and determined as described above. On day 28, concentrated HCI was also added to both toxicity controls.
- DOC and specific analysis: At the start of the test, three additional bottles containing 1 litre inoculated medium and test substance were prepared. Immediately 350 mL samples were taken, of which 250 mL was placed in polyethylene bottles, frozen and sent to the analytical laboratory for specific analysis. The remaining 100 mL was also placed in polyethylene bottles and stored frozen until DOC analysis. At the end of the test a similar sample was taken from the acidified test bottles. The content of test substance in the aqueous samples was determined by gas chromatography combined with mass spectrometry (GC/MS). The DOC determination was carried out using an O.I. Model 700 TOC analyser. The DOC was measured in the samples taken at the start of the test as described above, and in samples (100 mL) that were taken from the triplicate test bottles after acidification of the medium at the end of the test. An additional test was carried out to determine the influence of time on the DOC content of the aqueous phase. For this purpose, three additional filters were dosed with the test substance in a similar way as for the biodegradation test. These filters were added to 1 liter mineral medium (non-inoculated) and placed on the shaking machine. After 24 hours and 48 hours samples were taken for DOC analysis.

Reference substance:

acetic acid, sodium salt

Test performance:

- pH measurements: The pH of the medium in the test bottles varied between 7.10 and 7.18. The pH of the medium in the inoculum activity and toxicity control bottles with sodium acetate varied between 7.40 and 7.79 during the test. In the blank control it varied between 7.13 and 7.47. The increase of the pH of the inoculum activity control media is a consequence of the higher activity (i.e. higher CO2 production) of the bacterial cultures compared with the test bottles.
- Stability of the test substance: Three IR spectra were taken. The first series of two spectra was deliberately not carried according to Good Laboratory Practice (GLP) and it was repeated once according to GLP confirm the identity. The first two IR spectra were measured with 6 weeks time in between, and they were identical. This demonstrated that the test substance was stable during the test. The third spectrum, measured according to Good Laboratory Practice was also identical to the others and demonstrated the validity of the first spectra.
- Validity criteria: The difference of extreme replicate values of the removal of the test compound at the plateau, at the end of the test or at the end of the 10-day window, as appropriate, was less than 20%; The degradation of the reference compound has reached the pass level (i.e. > 60% degradation within 14 days); In a toxicity test, containing both the test substance and the reference compound, the degradation is > 25% (based on total ThCO2) within 14 days. The total CO2 evolution in the inoculum blank did not exceed 40 mg/L.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

73.1

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

> 79.7

Sampling time:

28 d

Details on results:

- Biodegradability: The biodegradation of the test substance (29 mg/L) was 73.1% (mean value) after 28 days of incubation at 20°C. The highest value in a single bottle was 77.0%. The 60% limit was reached after 28-days, however, it took just more than ten days to reach that level counting from the day that the 10% level was reached. The ten-day window was therefore not met. Also, a lag-phase (time to reach 10% degradation) of approximately eight days was observed. This might be an indication that the inoculum needed time to adapt their metabolism to the test substance.
- Dissolved Organic Carbon (DOC) removal: The DOC-content at the start of the test does not represent the maximum DOC value possible at equilibrium with the test medium. This was determined in a separate test where a DOC value of 16.8 mg/L was measured while a maximum value of 20 mg/L was expected. Based on these values the percentage removal of the DOC content was calculated. The percentage removal achieved based on the measured DOC values at the start of the test is low and not in line with the results of the CO2 measurements. The low DOC content in these samples was due to a slow transfer of the test substance from the filter to the aqueous phase. A more realistic value of 16.8 mg/L was obtained after 24h of equilibration with the medium. The day 28 measurements are, however, considered to be valid. Therefore, it was concluded that the DOC removal was at least 79.7%, which is a confirmation of the results based on the CO2 production.
- Primary degradation: The results of the specific analysis as measured by GC/MS and the percentage of primary degradation (metabolization of the test substance to one of its metabolites, not CO2) were calculated. The dissolved concentration of the test substance at day 0 is too low, due to the limited solubilization rate of the test substance from the carrier to the aqueous phase. As the solubility of the test substance was stated to be 91.7 mg/L it was expected that the test substance would be completely dissolved directly after dosing, but the DOC measurements demonstrated that this was not the case. The day 28 measurements are considered to be reliable. The percentage primary degradation based on dissolved and dosed concentration were both calculated. The test substance had almost disappeared from the test medium at the end of the test, with 99.7% primary degradation after 28 days. This is also a confirmation of the CO2 measurements. The mineralization was approximately 70%, which is a common value for a readily biodegradable substance. (70% of the carbon is mineralized to CO2 and 30% is used for growth of the biomass).
- Kinetic analysis: The data were fitted to a first order degradation equation of the Jandell Tablecurve library by using the software Microsoft Excel. With these models the following parameters were calculated: DT50, the time to 50% degradation, the first order rate constant (d-1) and the ten day window. The test substance reached the 60% degradation level within 28 days. However, the time between 10% and 60% degradation was longer than 10 days. The 10-day window criterion was therefore not met. The primary degradation rate was much higher than the mineralization rate, with an approximate DT50 of 3 days and a rate constant of 0.2. However, with the few measurements carried out these results only constitute a very rough estimation of the primary degradation rate.
- Stability of the test substance: Three IR spectra were taken. The first series of two spectra was deliberately not carried according to Good Laboratory Practice (GLP) and it was repeated once according to GLP confirm the identity. The first two IR spectra were measured with 6 weeks time in between, and they were identical. This demonstrated that the test substance was stable during the test. The third spectrum, measured according to Good Laboratory Practice was also identical to the others and demonstrated the validity of the first spectra.
- Inoculum activity and toxicity control: The biodegradation of sodium acetate in the control series (inoculum activity control) was used as a measure for the microbial activity of the inoculum. A reduction of the biodegradation of acetate in the presence of the test substance (toxicity control) would indicate an inhibiting effect on the inoculum activity. The results show that sodium acetate was sufficiently degraded within two weeks: at least 82.8 % was recovered in the traps as CO2 with acidification. At the end of the test sodium acetate degradation reached 86.9%. The percentage degradation of sodium acetate (68 mg/L) and test substance (29.0 mg/L) combined in the toxicity control was calculated on the basis of the sum of the theoretical CO2-production of both substances. The CO2 production was higher in the toxicity control than in the activity control. A net CO2 production of 60.4 mg CO2 could be attributed to the degradation of the test substance after 28 days. This corresponded to the mean value in the biodegradation test itself (53.7 mg). This means that the test substance was not toxic to the inoculum and was extensively degraded in the presence of acetate.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Description of key information

The substance is readily biodegradable but fails the 10-day window.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable but failing 10-day window

Type of water:

freshwater

Additional information

The biodegradability of the test substance was determined by a modified method according to OECD Guideline 301B and EU method C.4 -C, using a modified trapping system. The study was carried out in conformity with the OECD principles of Good Laboratory Practice. The CO2 -evolution biodegradability test was carried out in glass bottles containing one litre inoculated medium, and a vial suspended in the headspace of each bottle containing a CO2 absorbing fluid. The vials containing CO2 absorbing fluid were replaced at weekly intervals and the trapped CO2 was determined by titration. Activated sludge taken from an oxidation ditch used to treat domestic sewage was used as inoculum. The inoculum concentration was 30 mg (dry weight)/L. The test substance was introduced into the inoculated medium on a glass fibre filter carrier in accordance with the International Standard ISO 10634. One concentration of 29.0 mg test substance per litre medium (which corresponds to 20 mg carbon/L) was tested in six bottles. The test was completed with a control blank with inoculated medium only, a control on inoculum activity with sodium acetate as reference substance, and with a toxicity control with sodium acetate and 29.0 mg/L test substance. The inoculum activity and toxicity control tests with sodium acetate showed that the activity of the inoculum was sufficient, and that the test substance did not inhibit the degradation of the reference substance. The results demonstrated that biodegradation of the test substance took place with a maximum mean degradation of 73.1% after 28 days (degradation up to 77.0% was observed in a single bottle). The criterion given in the guidelines (i.e. > 60% degradation reached within a 10 -day period counting from the day that the level of degradation exceeded 10%) were not completely met. The test substance is therefore assessed to be readily biodegradable in this test, but not within the 10-day window. A lag-phase of approximately 8 days was observed. Specific analysis of the test substance concentration in the test medium at the start and the end of the test indicated that a slow release of the substance from the carrier to the test medium occurred. Based on the concentration measured after 28 days of incubation (0.08 mg/L) the primary degradation was 99.7% (based on dosed concentration). The DOC removal was 82.9%, based on the dosed concentration of 20 mg C/L. The DOC at the end of the test was 3.41 mg C/L. The stability of the test substance during the incubation was confirmed by IR analysis at two time points with at least 28 days in between.