dec-9-en-2-one

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:

From May 07, 2008 to June 18, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Date of inspection 15-16 March 2007 / Date of decision 26 April 2007

Specific details on test material used for the study:

- Water solubility: 268.1 mg/L (See Phytosafe study under 07-42-066-ES)
- ThOD = 2.91 g/g

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 5-7 days at the test temperature in the mineral medium used for testing.
The resulting solution was maintained under strong aeration and agitation for 5-7 days at 20 +/- 1°C.
The pre-conditioned inoculum was further used at a rate of 2 mL/L of medium.
The suspended solids were measured to represent 8-9 µg/L.

Duration of test (contact time):

21 d

Initial conc.:

2.18 mg/L

Based on:

test mat.

Remarks:

(nominal value)

Initial conc.:

2.11 mg/L

Based on:

test mat.

Remarks:

(measured initial value)

Initial conc.:

6.33 mg/L

Based on:

ThOD

Remarks:

(nominal value)

Parameter followed for biodegradation estimation:

O2 consumption

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

GC-FID determination

Details on study design:

Mineral medium: The mineral medium was prepared according to the Test Guideline. The mineral medium was strongly aerated for at least 20 minutes, and then allowed to stand fo approximately 20 h at the test temperature before use.

Parallel groups of 250 mL BOD bottles were prepared of sufficient number to allow assessments every 3-4 days throughout the 28-day incubation period for each of the following experimental series:
- Blank group: mineral medium alone with inoculum (duplicate determination),
- 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: In a first experiment, a stock solution was prepared using 411.1 mg of test substance in 20 mL of acetone. The stock solution was further 1/100 v/v diluted in water. Aliquots of 3 mL of the diluted stock solution served for the treatment application in each 300 mL unit. A solvent group was prepared using 3 mL aliquots of a 1/100 v/v dilution of acetone in water. After 6 days of incubation, the dissolved oxygen concentration was almost zero in the test substance treated units and in the solvent control units (data not shown), suggesting that acetone was not convenient as carrier and the test was abandoned. In a second experiment, the stock solution was prepared using 50 µL of test substance in 500 mL of water. The resulting solution was firmly shaken. Due to the low stability of the test item in water under stirring conditions, care was taken to avoid undue agitation of the test solution before the BOD bottles were stoppered. For that purpose the BOD bottles were individually prepared: 250 mL of mineral medium were added with 10 mL of the test substance stock solution and stirred for a few seconds. The volume was added with 50 mL of mineral medium and 0.6 mL of pre-conditioned medium. The pH of the resulting solution was measured at 7.21. The other groups were prepared accordingly.
Blank group: 250 mL of mineral medium were stirred for a few seconds and then added with 10 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
Reference group: A stock solution was prepared using 0.1234 g Aniline for 20 mL of water. 250 mL of mineral medium were added with 100 µL of the stock solution and stirred for a few seconds. The volume was added with 50 mL of mineral medium and 0.6 mL of pre-conditioned inoculum, resulting in a 2.10 mg/L solution.
Toxic control group: 250 mL of mineral medium were added with 10 mL of the test substance stock solution and 100 µL of the Aniline stock solution and stirred for a few seconds. The volume was added with 50 mL of mineral medium + 0.6 mL of pre-conditioned inoculum.
In every case care was taken to avoid undue bubbling of air within the aqueous volumes. The bottles were kept open for approximately 30 minutes and then stoppered.
The nominal value for the test item concentration in the test group and in the toxic control group was taken as 10*65.28/300 = 2.18 mg/L, where 10 = volume (mL) of the stock solution used for the treatment application, 65.28 = measured concentration (mg/L) of the stock solution, and 300 = volume (mL) of water in the test unit.

- 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 terminated after 21 days of incubation.
The test was considered as valid on the basis of the following fulfilled conditions:
- Mean oxygen uptake in the blank vessels < 1.5 mg/L at the end of test;
- The residual concentration of oxygen in the test vessels did not fall below 0.5 mg/L at any time;
- 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, based on total ThOD, had occurred on day 14.

Key result

Parameter:

% degradation (O2 consumption)

Value:

71.6

Sampling time:

21 d

Remarks on result:

other: within the 10-day window

Remarks:

Mean of 2 replicates

Details on results:

OXYGEN UPTAKE AND RELATED BIODEGRADATION (see tables 5.2.1/1, 2 and 3 in "Any other information on results incl. tables"):
The test was ended on day 21 as dissolved oxygen in the test vessels had reached a plateau for three successive determinations: on days 13, 17 and 21.
Mean oxygen uptake in the blank vessels was 0.76 mg/L at the end of test.
Test substance: The beginning of the 10-day window occurred within the first 3 days of incubation and the pass level was reached on day 6. The biodegradation had reached a plateau on day 13. Mean biodegradation was 71.6% on day 21. The difference between extremes values on day 21 was (72.82 - 70.46) = 2.36% biodegradation, and represented 3.3% of mean value (71.64%).
In the toxic control series, the biodegradatation based on total ThOD was 58.1% on day 13, and final value on day 21 was 58.9%.

ACTIVE INGREDIENT CONTENT (see table 5.2.1/4 in "Any other information on results incl. tables"):
The measured initial concentration represented 96-99% of the nominal value (2.18 mg/L). The treatment application was considered as valid. At the end of test, the test substance was not recovered in the test vessels nor in the toxic control vessels.

Results with reference substance:

The beginning of the 10-day window occurred between 6 and 10 days of incubation, and the pass-level (60% biodegradation) was reached on day 13. Final biodegradation on day 21 was 76.9%.

Table 5.2.1/1: Time evolution of dissolved oxygen (mg/L) in the reacting vessel

Days	Blank		Test		Toxic	Aniline
	Rep. 1	Rep. 2	Rep. 1	Rep. 2		Rep. 1	Rep. 2
0 3 6 10 13 17 21	7.69 7.25 7.13 7.05 6.92 7.02 6.92	7.70 7.16 7.05 6.97 7.05 6.86 6.95	7.69 6.47 3.07 2.89 2.82 2.35 2.47	7.69 6.33 3.24 2.85 2.69 2.23 2.32	7.66 6.50 3.56 2.94 0.41 0.35 0.26	7.69 7.27 7.14 4.16 3.47 3.03 3.32	7.70 7.32 6.96 3.94 3.64 3.23 2.94

Table 5.2.1/2: Measured oxygen up-take (mg/L) throughout the 28 -day incubation period

Days	Blank (mean)	Test		Toxic	Aniline
		Rep. 1	Rep. 2		Rep. 1	Rep. 2
3 6 10 13 17 21	0.49 0.61 0.69 0.71 0.76 0.76	1.22 4.62 4.80 4.87 5.34 5.22	1.36 4.45 4.84 5.00 5.46 5.37	1.16 4.10 4.72 7.25 7.31 7.40	0.42 0.55 3.53 4.22 4.66 4.37	0.38 0.74 3.76 4.06 4.47 4.76

Table 5.2.1/3: Measured percent biodegradation

Days	Test			Toxic	Aniline
	Rep. 1	Rep. 2	Mean		Rep. 1	Rep. 2	Mean
0 3 6 10 13 17 21	0 11.53 63.43 65.01 65.72 72.43 70.46	0 13.74 60.74 65.64 67.77 74.33 72.82	0 12.64 62.08 65.32 66.74 73.38 71.64	0 5.94 30.99 35.78 58.08 58.13 58.88	0 -1.42 -1.11 57.51 71.16 78.94 72.98	0 -2.22 2.73 62.16 67.92 75.10 80.86	0 -1.82 0.81 59.84 69.54 77.02 76.92

Table 5.2.1/4: Measured concentrations of the test substance

	Replicate samples	Test item, mg/L	% recovery
Stock solution	1	67.20 63.36	-
Test initiation	1 2 3 Mean	2.15 2.11 2.08 2.11	98.6 % 97.1 % 95.6 % 97.1 %
End of test	1 (test unit) 2 (test unit) 1 (toxic control) Mean	0.00 0.00 0.03 0.00	0.0 % 0.0 % 2.3 % 0.8 %

Validity criteria fulfilled:

yes

Remarks:

See "test performance" section.

Interpretation of results:

readily biodegradable

Conclusions:

Based on oxygen consumption, a mean of 71.6% 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 2.11 mg/L test solution (initial mean measured concentration).

In the test item treatment, the 10 -day window began within the first 3 days of testing and ended on day 6. The biodegradation reached a plateau on day 13 of testing, and the test was ended on day 21. On day 21, mean biodegradation was 71.6%.

In the toxic group, the bioidegradation was 58.1% on day 13. The test substance was thus considered as not inhibitory.

Based on analytic assessment, the removal of the test substance in the test vessels at the end of the test was total.

In conclusion, based on oxygen consumption, a mean of 71.6% 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 1:

71.6% 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, one experimental study is availabe.

This study (Phytosafe, 2008), 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 2.11 mg/L test solution (initial mean measured concentration). In the test item treatment, the 10 -day window began within the first 3 days of testing and ended on day 6. The biodegradation reached a plateau on day 13 of testing, and the test was ended on day 21. On day 21, mean biodegradation was 71.6%. In the toxic group, the bioidegradation was 58.1% on day 13. The test substance was thus considered as not inhibitory. Based on analytic assessment, the removal of the test substance in the test vessels at the end of the test was total. In conclusion, based on oxygen consumption, a mean of 71.6% biodegradation was measured after 21 days, within the 10 -day window. Therefore, the registered substance is readily biodegradable.