Cyclooctane

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

Study period:

2021-11-11 to 2021-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Version / remarks:

September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant, 31137 Hildesheim, Germany
- Storage length: Sludge was obtained and pretreatment started without delay.
- Pretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours. Thereafter, the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and was maintained in an aerobic condition by aeration with CO2-free air for 3 days until test start. 8.89 mL/L were used to initiate inoculation (4 mg/L dry weight). pH-adjustment was not necessary.
- Concentration of sludge:
- Initial cell/biomass concentration: 10^5 – 10^8 CFU/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

12 mg/L

Based on:

test mat.

Remarks:

corresponds to 10.2 mg C/L in vessel

Parameter followed for biodegradation estimation:

other: total inorganic carbon (TIC)

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 310
- Additional substrate: no
- Test temperature: Nominal 20 ± 1 °C, actually measured 20.0 – 21.5 °C
- pH: not specified
- pH adjusted: no
- CEC (meq/100 g): not specified
- Aeration of dilution water: not specified
- Continuous darkness: no, low light conditions

TEST SYSTEM
- Culturing apparatus: Headspace flasks (120 mL volume), headspace to liquid ratio 1:2
- Number of culture flasks/concentration: 5
- Method used to create aerobic conditions: Agitation Shaker (150 - 200 rpm)
- Measuring equipment: Carbon analyser, Multi N/C 3100, ANALYTIK JENA
- Test performed in closed vessels: yes
- Test performed in open system: no

SAMPLING
- Sampling frequency: Analysis was carried out at test start, and on 7 further sampling times. The inoculum control and test item replicates were sampled at test start and on day 1, 4, 7, 11, 14, 21 and 28. The functional control and toxicity control were sampled at test start and on day 7, 14 and 28.
- Sampling method: The CO2 production in the test vessels was determined by measuring the increase of the inorganic carbon (IC) concentration during the incubation. Sodium hydroxide solution (0.74 mL of 7 mol/L solution to 80 mL medium) was injected to each vessel sampled. The vessels were shaken on an overhead shaker (approximately 100 rpm) for at least 1 hour at test temperature and analysed.

CONTROL AND BLANK SYSTEM
- Inoculum blank: The inoculated test medium, consisting of the required volumes of mineral medium stock solutions and inoculum in ultrapure water, was prepared in a measuring flask. 80 mL inoculated test medium was placed in each test vessel of the inoculum control.
- Functional control: The reference item was weighed out and transferred into a measuring flask with ultrapure water. The required volumes of mineral medium stock solutions and inoculum were added and the flask was filled up with ultrapure water. 80 mL of this solution was placed in each test vessel of the reference item replicates.
- Toxicity control: 1.15 μL test item was pipetted directly at the wall of the test vessels of the toxicity control replicates. 80 mL reference item medium was added and the test vessels were immediately closed.

STATISTICAL METHODS:
The 95 % confidence interval for the mean percentage biodegradation after 28 days was calculated for the test item, the functional controls and the toxicity control, using software SigmaPlot (Windows) rel. 8.02, 2002, SPSS Corporation.

Reference substance:

benzoic acid, sodium salt

Remarks:

test concentration: 30 mg/L

Key result

Parameter:

% degradation (inorg. C analysis)

Value:

0

Sampling time:

28 d

Details on results:

- Carbon Content of the Test Item
The test item was tested at a concentration of 12 mg/L, corresponding to a carbon content of 10.2 mg C/L.
- TIC Production in the Inoculum Controls
The mean amount of TIC present in the inoculum controls (C1 - C5) on day 28 is 0.45 mg C/L. The validity criterion for the inoculum controls at the end of the test according to the guidelines (< 3 mg C/L) was met.
Colony Forming Units
Colony forming units (CFU) of the inoculum at test start were determined by standard dilution plate count: 0.7 x10^9 CFU/L, corresponding to 5.6 x10^7 CFU/L in the test vessel.
- CO2-Production and Biodegradation
The total amount of CO2 produced in 28 days was analysed by TIC measurements at 8 sampling times.
In the toxicity control, containing test and reference item, a biodegradation of 58 % was determined after 14 days and 59 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The 95 % confidence interval on day 28 was 59-60 %.
The test item replicates did not reach the 10 % level (beginning of biodegradation) within 28 days. The biodegradation on day 28 was 0 %. The 95 % confidence interval on day 28 was 0-1 %.

Results with reference substance:

The adaptation phase of the functional control changed within 7 days to the degradation phase (degradation ≥ 10 %). The course of the degradation phase was rapid and the pass level of 60 % was reached within 7 days. The biodegradation came to a maximum of 93 % after 14 days. The validity criterion degradation > 60 % after 14 d was fulfilled. The 95 % confidence interval on day 28 was 91 – 94 %.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item is classified as not readily biodegradable within the 28 day period of the study.

Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Headspace Test. The study was conducted according to OECD 310. The test item was tested at a concentration of 12 mg/L in triplicates (test end: 5 replicates), corresponding to a carbon content of 10.2 mg C/L. The biodegradation of the test item was followed by TIC analyses of the quantity of CO2 produced by the respiration of bacteria. The amount of TIC produced was calculated by correcting the results of the test and reference item for endogenous TIC production of the control groups and the blind value of the sodium hydroxide solution. The biodegradation was calculated for each sampling time. To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 7 days and came to a maximum of 93 % on day 14. The 95 % confidence interval on day 28 was 91 - 94 %. In the toxicity control, containing test and reference item, a biodegradation of 58 % was determined after 14 days and 59 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The 95 % confidence interval on day 28 was 59 – 60 %. The test item replicates did not reach the 10 % level (beginning of biodegradation) within 28 days. The biodegradation on day 28 was 0 %. The 95 % confidence interval on day 28 was 0–1 %. The validity criteria according to the guideline are fulfilled. The test item is classified as not readily biodegradable within the 28 day period of the study.

Description of key information

The test item is classified as not readily biodegradable within the 28 day period of the study (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days in the Headspace Test. The study was conducted according to OECD 310. The test item was tested at a concentration of 12 mg/L in triplicates (test end: 5 replicates), corresponding to a carbon content of 10.2 mg C/L. The biodegradation of the test item was followed by TIC analyses of the quantity of CO2 produced by the respiration of bacteria. The amount of TIC produced was calculated by correcting the results of the test and reference item for endogenous TIC production of the control groups and the blind value of the sodium hydroxide solution. The biodegradation was calculated for each sampling time. To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 7 days and came to a maximum of 93 % on day 14. The 95 % confidence interval on day 28 was 91 - 94 %. In the toxicity control, containing test and reference item, a biodegradation of 58 % was determined after 14 days and 59 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control. The 95 % confidence interval on day 28 was 59 – 60 %. The test item replicates did not reach the 10 % level (beginning of biodegradation) within 28 days. The biodegradation on day 28 was 0 %. The 95 % confidence interval on day 28 was 0–1 %. The validity criteria according to the guideline are fulfilled. The test item is classified as not readily biodegradable within the 28 day period of the study.