6-cyclopentylidenehexanal

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 29 april 2014 to 27 may 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals, Degradation and Accumulation. Ministry of Environmental Protection of the P. R. China .2013.

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Storage condition: Fridge, keep in full and tightly closed containers in a dark cool place
- Water solubility: 62,1 mg/L
- ThOD = 2,887 mg/mg

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The fresh activated sludge from the city sewage treatment plant was collected and then filter through a coarse filter paper and the filtrate was cultivated in the aerobic aquatic environment.

Duration of test (contact time):

28 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Temperature: 20 +/- 1°C
- Inoculum concentration: 0.05 mL/L
- Test item concentration: 2 mg/L
- Reference item concentration: 3 mg/L

PREPARATION OF TEST VESSELS
- Test vessels: 250 mL BOD bottle (with water seal unit)
- Inoculum Control Group (IC): yes
- Test Group (TS): yes
- Procedure Control Group (PC): yes
- Toxicity Control Group (TC): yes

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The ThOD of the test substance is 2.887 mg/mg.
The ThOD of the reference item is 1.667 mg/mg.

Test performance:

Oxygen depletion in the inoculum blank were 1.37 mg/L after 28 days, did not exceed 1.5 mg dissolved oxygen/L. The residual concentration of oxygen in the test bottles did not fall below 0.5 mg/L after 28 d. The difference of extremes of replicate values of the removal of the test chemical at the end of the test was less than 20%. The biodegradation rate for the toxicity control group in fourteenth days reached 27.4%, beyond 25 %, and thus the effect of the tested substance on microbial activity is thought as no inhibition under the experimental conditions. The biodegradation rate of sodium benzoate exceeded 60% after 14 days, and reached 62.9%

Key result

Parameter:

% degradation (O2 consumption)

Value:

73.5

Sampling time:

28 d

Remarks on result:

other: within the 14d window

Remarks:

Average value (n=2)

Details on results:

The test results showed that, for the samples in containing activated sludge culture medium, the average cumulative degradation rates (n=2) in 0, 1, 2, 4, 7, 10, 14, 17, 21, 24 and 28 day were 0 %, 9.52 %, 19.0 %, 39.9 %, 48.1 %, 56.3 %, 63.5%, 69.3 %, 70.2 %, 71.6 % and 73.5%, respectively. At the end of the test, the cumulative degradation rate was 73.5%. The cumulative degradation of 14-day window was 63.5%.

Results with reference substance:

The biodegradation rate of sodium benzoate exceeded 60% after 14 days, and reached 62.9%.

Table 5.2.1/1: Calculated biodegradation, %

Days	Procedure Control Group (PC)	Test item Group (TS)	Toxicity Control Group (TC)
0 1 2 4 7 10 14 17 21 24 28	0 2.00 17.0 34.5 44.7 51.5 62.9 76.6 87.6 88.0 88.8	0 9.52 19.0 40.0 48.1 56.3 63.5 69.3 70.2 71.6 73.5	0 11.1 18.5 23.0 24.1 25.8 27.4 / / / /

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

Based on oxygen consumption, a mean of 73.5% biodegradation was measured after 28 days, within the 14-day window. Therefore, the substance is considered readily biodegradable.

Executive summary:

This study tested the ready biodegradability of the test substance in the aerobic aquatic environment by the method of closed bottle test according to the OECD Guideline 301D. The test item was added at about 2 mg/L into the test culture medium. The test results showed that, for the samples with the test substance, the average cumulative degradation rates ( n=2 ) in 1, 2, 4, 7, 10, 14, 17, 21, 24 and 28 days were 9.52%, 19.0%, 39.9%, 48.1%, 56.3%, 63.5%, 69.3%, 70.2%, 71.6% and 73.5%, respectively. For the test substance in a 14 -day window its biodegradation rates and at the end of the test were more than 60%, which achieved with the level as ready biodegradability. The biodegradation rate for toxicity control reached 27.4% in fourteenth days, were more than 25%. The biodegradation rate for reference item, sodium benzoate, reached 62.9% in 14 days. These results verified that the biodegradability test for the test item was effective.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 June 2013 to 11 July 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

All validity criteria were fulfilled except the residual concentration of oxygen in the test bottles which fell below 0.5 mg/L in the test item vessel replicate 1 at day 21 and in the toxicity control vessel from day 15 to 21. Such low oxygen levels are considered acceptable as all other validity criteria were met and the test was thus terminated on day 21.

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 14 June 2012 / Signed on 10 January 2013

Specific details on test material used for the study:

- Water solubility: 62 mg a.i./L (See Phytosafe study under 13-42-005-ES)
- ThOD = 2.89 g/g
- Appearance: Colourless translucent liquid
- Storage condition: Room temperature protected from direct sun light

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic, non-adapted

Details on inoculum:

The inoculum was derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage.
The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 5 days at the test temperature in the mineral medium used for testing.
The resulting solution was continuously stirred for 6 days at 20 +/- 1°C.
The pre-conditioned inoculum was further used at a rate of 2 mL/L.

Duration of test (contact time):

21 d

Initial conc.:

3.33 mg/L

Based on:

test mat.

Remarks:

(nominal value)

Initial conc.:

3.22 mg/L

Based on:

test mat.

Remarks:

(measured initial value)

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

GC-FID determination

Details on study design:

Parallel groups of 250 mL BOD bottles were prepared of sufficient number to allow assessments every 3-4 days throughout the 28 days incubation period for each of the following experimental series:
- Blank group: mineral medium alone with inoculum (duplicate determinations),
- Test item: 2-5 mg/L test solution in mineral medium with inoculum (duplicate determinations),
- Reference item: 2-5 mg/L aniline solution in mineral medium with inoculum (duplicate determinations),
- Toxic control: solution containing 2-5 mg/L of each the test substance and aniline in mineral medium with inoculum (single determination).

The 250 mL BOD bottles were thoroughly cleaned before use using 5-10 mL of wash solution (2.5 g iodine plus 12.5 g potassium iodide per litre of 1% w/v sulphuric acid). The bottles were shaken to coat the bottle walls and left to stand for 15 min. The wash solution was poured off and the bottles were thoroughly rinsed with tap water and finally demineralised water.

- Preparation of the experimental solutions: 1 L of water were set at the saturation level using excessive amount of the test item. The volume was placed at 20 +/- 2°C and stirred for the night with a magnetic stirrer. The solution was then allowed to remain undisturbed for 2 hours so that excess of the item separated. Analytical check of the aqueous volume showed that the concentration was far higher than the limit of solubility for the test substance in water. Consequently, the aqueous volume was allowed to stand for one additional day under slow stirring. At then end of the period, the lower phase was carefully withdrawn from the bottom with a peristaltic pump. The test item concentration was checked in the resulting solution before use. The data showed that the test item concentration was approximately and evenly 40 mg/L. The mineral medium was then reconstituted using 1 mL of each the stock solutions (same as mentioned in the guidelines).
The test system was set as follows:
- Blank group: 300 mL of mineral medium + 0.6 mL of pre-conditioned inoculum,
- Test group: 25 mL of the above test solution + 275 mL of mineral medium + 0.6 mL of pre-conditioned inoculum,
- Reference group: A stock solution was prepared using 0.1217 g Aniline for 20 mL of water. 300 mL of mineral medium were added with 100 µL of the stock solution and 0.6 mL of pre-conditioned inoculum,
- Toxic control group: 25 mL of the test solution + 100 µL of the Aniline stock solution + 275 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
The nominal value for the test item concentration in the test group and in the toxic control group was taken as 40*25/300 = 3.33 mg/L.

- Maintenance of the test system: Each BOD bottle was fitted with a glass stopper and incubated at dark at 20 +/- °C

- Dissolved oxygen: Dissolved oxygen was assessed by the electrode method. The zero-time for dissolved oxygen was measured within 1h after the setting of the test system. Duplicate vessels of each the blank series, the test series and the reference series were sacrificed every 3-4 days for dissolved oxygen analysis. The toxic control series was measured out from one single vessel.

Reference substance:

aniline

Test performance:

The test was considered as valid:
- Mean oxygen uptake in the blank vessels < 1.5 mg/L on day 21;
- The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time, except for the test item vessel replicate 1 at day 21 and the toxicity control from day 15 to 21 (such low oxygen levels are acceptable as all other validity criteria were met);
- Differences 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 item (aniline) had reached the pass-level by day 14;
- In the toxic control series more than 25% biodegradation had occurred on day 14.

Key result

Parameter:

% degradation (O2 consumption)

Value:

86.56

Sampling time:

21 d

Remarks on result:

other: within the 10-day window

Remarks:

Mean of 2 replicates

Details on results:

- Oxygen uptake: The initial value for dissolved oxygen concentration was 8.57 mg/L for the control, 8.60 mg/L for the test item treatment, 8.58 mg/L for the toxic control and 8.62 mg/L for the reference item treatment. The consumption of oxygen in the controls was less than 1.5 mg/L over the test period, as required.
For the test item treatment, the oxygen concentration decrease down to approximately 0.5 mg/L on day 15 and then stabilized at 0.5-0.6 mg/L on day 18 and day 21. The test was thus terminated on day 21.
For the toxic group, the oxygen concentration decreased to less than 0.5 mg/L up from day 15. The corresponding concentrations were reported as <0.5 mg/L for days 15, 18 and 21.
- Biodegradation of the test substance: The beginning of the 10-day window was observed on day 4 of the test period. The pass-level was reached on day 11: mean value for biodegradation was 67.2%. At the end of the test period the biodegradation stabilized at approximately 85%.
- Biodegradation of the toxic control: Based on total ThOD, the biodegradation was more than 25% up from day 8 of the test period. High consumption of oxygen did not allow the calculation by more than 11 days. On this day, the percentage of biodegradation was 56.7%. Further determination proved that the biodegradation remained >55% until the end of the test.
- Active ingredient content: Measured initial concentrations ranged between 90.1 and 105.4% of the nominal value for the test group, and between 90.7% and 107.5% for the toxic group. Mean value was 3.22 mg/L. The treatment application was considered as valid. The initial concentration was thus taken as 3.22 mg a.i./L, corresponding to 9.31 ThOD/L. At the end of the 21-day test period, the test substance was not detected. In the light of the limit of detection for the test substance in the organic solvent, recovery was thus calculated to be less than 0.2% of the initial value.

Results with reference substance:

The beginning of the 10-day window was observed between day 4 and day 8 of the test period. The pass-level was reached on day 8: mean value for biodegradation was 64.9%. At the end of the test period, the biodegradation was >90%.

Table 5.2.1/1: Calculated biodegradation, %

Days	Test substance			Toxic	Aniline
	Rep. 1	Rep. 2	Mean		Rep. 1	Rep. 2	Mean
0 4 8 11 15 18 21	0 11.28 36.11 66.19 86.83 85.54 87.36	0 10.75 39.01 68.13 85.11 85.86 85.75	0 11.01 37.56 67.16 85.97 85.70 86.56	0 6.91 43.57 56.68 >55 >55 >55	0 4.71 62.83 71.84 75.53 88.04 91.21	0 5.53 66.92 76.35 73.48 82.91 92.65	0 5.12 64.87 74.10 74.51 85.47 91.93

Table 5.2.1/2: Measured concentrations of the test substance at test initiation

	Replicate unit	Test item (mg/L)	% of nominal value
Test group	1 2 3 4 5 6 7 8 9 10 Mean	3.52 3.30 3.01 3.06 3.25 3.22 3.17 3.34 3.21 3.35 3.24	105.4% 98.9% 90.1% 91.7% 97.4% 96.5% 95.0% 100.1% 96.2% 100.4% 97.2%
Toxic controls	1 2 3 4 5 Mean	3.03 3.07 3.20 3.59 3.03 3.18	90.8% 92.1% 96.0% 107.5% 90.7% 95.4%

Validity criteria fulfilled:

yes

Remarks:

Except the residual concentration of oxygen in the test bottles fell below 0.5 mg/L in the test item vessel replicate 1 at day 21 and in the toxicity control vessel from day 15 to 21. The test was thus terminated on day 21.

Interpretation of results:

readily biodegradable

Conclusions:

Based on oxygen consumption, a mean of 86.56% biodegradation was measured after 21 days, within the 10-day window. Therefore, the substance is considered readily biodegradable.

Executive summary:

This Closed-bottle test was performed according to EU Method C4.e with GLP statement. The inoculum was derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage. The test was performed using a 3.22 mg/L test solution (initial mean measured concentration).

In the test item treatment, the beginning of the 10-day window was observed on day 4 of the test period. The pass-level was reached on day 11: mean value for biodegradation was 67.2%. The oxygen up-take stabilized at approximately 0.5 mg/L after 15 days of testing, corresponding to approximately 85% biodegradation.

In the toxic group, 25% biodegradation was achieved within 8 days. The test substance was thus considered as not inhibitory. At the end of test, analytical check of the test substance in the test solutions and in the toxic control proved that the test substance was decreased to undetectable concentrations, corresponding to less than 0.2% of the mean initial value.

In conclusion, based on oxygen consumption, a mean of 86.56% biodegradation was measured after 21 days, within the 10-day window. Therefore, the substance is considered readily biodegradable.

Description of key information

EU Method C.4 -E, equivalent to OECD Guideline 301D, GLP, Key study, validity 2:

86.56% biodegradation after 21 days, within the 10-day window.

The registered substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

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

 

The first study (Phytosafe, 2013), assessed as the key study, was performed on the registered substance according to EU Method C.4-E (equivalent to OECD Guideline 301D) with GLP compliance. The inoculum was derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage. The test was performed using a 3.22 mg/L test solution (initial mean measured concentration). In the test item treatment, the beginning of the 10-day window was observed on day 4 of the test period. The pass-level was reached on day 11: mean value for biodegradation was 67.2%. The oxygen up-take stabilized at approximately 0.5 mg/L after 15 days of testing, corresponding to approximately 85% biodegradation. In the toxic group, 25% biodegradation was achieved within 8 days. The test substance was thus considered as not inhibitory. At the end of test, analytical check of the test substance in the test solutions and in the toxic control proved that the test substance was decreased to undetectable concentrations, corresponding to less than 0.2% of the mean initial value. In conclusion, based on oxygen consumption, a mean of 86.56% biodegradation was measured after 21 days, within the 10-day window. Therefore, the substance is considered readily biodegradable.

 

The second study (HOPE, 2014), assessed as the supporting study, was performed on the registered substance according to OECD Guideline 301D with GLP compliance. The test substance was added at about 2 mg/L into the test culture medium. The test results showed that, for the samples with the test substance, the average cumulative degradation rates ( n=2 ) in 1, 2, 4, 7, 10, 14, 17, 21, 24 and 28 days were 9.52%, 19.0%, 39.9%, 48.1%, 56.3%, 63.5%, 69.3%, 70.2%, 71.6% and 73.5%, respectively. For the test substance in a 14-day window its biodegradation rates and at the end of the test were more than 60%, which achieved with the level as ready biodegradability. The biodegradation rate for toxicity control reached 27.4% in fourteenth days, were more than 25%. The biodegradation rate for reference item, sodium benzoate, reached 62.9% in 14 days. These results verified that the biodegradability test for the test item was effective.

 

In conclusion, the registered substance is readily biodegradable.