6,8-dimethylnon-7-enal

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:

key study

Study period:

From 03 April 2019 to 14 June 2019

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:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and river water was used as inoculum.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound), and river water was used as inoculum.

Qualifier:

according to guideline

Guideline:

ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Molecular formula: C11H20O1
- Molecular weight: 168.3 g/mol
- Water solubility: Expected close to 40 mg/L (iSafeRat v1.8 prediction)
- Vapour pressure: 68 Pa at 20°C (OECD TG 104, static method, Laus 2011)
- Log Kow: 3.87 at 20 °C (EEC A.8, shake-flask method, Phytosafe 2008)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Secondary activated sludge (28-03-2019) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge treatment plant treating predominantly domestic wastewater. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 μm cellulose nitrate filter. This filter was dried for 1.5 hour at 104.8 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume. The measured dry weight of the inoculum was 3.6 g/L.
The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end the inoculum was diluted in Closed Bottle test medium to 0.4 g Dry weight (DW)/L of activated sludge and aerated by stirring on a magnetic stirrer plate at ambient temperature (± 21 °C) for one week. The preconditioned inoculum was diluted further to a dry weight concentration of 2 mg/L in the BOD bottles (van Ginkel and Stroo, 1992).

Acitvated sludge was selected as inoculum based on the non-GLP screening test performed with both activated sludge and river water.

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 bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

- Nutrients, and stocks:
Deionized water containing <1.0 mg/L of organic carbon was prepared in a water purification system.
Deionized water used in the Closed Bottle test contained per liter of water 8.5 mg KH2PO4, 21.7 mg K2HPO4, 26.7 mg Na2HPO4, 22.5 mg MgSO4·7H2O, 36.3 mg CaCl2 2H2O, 0.25 mg FeCl3·6H2O.
Ammonium chloride was omitted from the medium to prevent nitrification. Accurate administering of the test substance was accomplished by preparing a solid stock of 3.0 mg of test substance per g of silica gel in a 50-mL serum flask. Only part of the top layer of the silica gel was brought into contact with the test substance. The serum flask was closed with a screw top and the content was mixed vigorously. Subsequently, 0.20 g of silica gel with the test substance was added to the test bottles. The resulting concentration of test substance in the bottles was 2.0 mg/L. Next the bottles were filled with nutrient medium with inoculum and closed. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.

- Test procedures:
Use was made of 10 bottles containing only inoculum, 10 bottles containing inoculum and silica gel (0.2 g silica gel / bottle), 10 bottles containing inoculum and silica gel with test substance, 6 bottles containing inoculum and sodium acetate. The concentrations of the test substance, and sodium acetate in the bottles were 2.0 mg/L and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14,
21, and 28.

- Analyses:
The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter (WTW). The pH was measured using an Eutech pH meter. The temperature was measured and recorded with a sensor connected to a data logger.

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.9%; Batch n° BCBP8197V

Preliminary study:

A non-GLP screening test was performed prior the GLP final test in order to determine the most suitable inoculum for the degradation of the test substance, in the Closed Bottle test.
Biodegradation of ≥60% was found within two to three weeks with both activated sludge and river water. The test substance should therefore be classified as readily biodegradable especially based on the results found with activated sludge. The time window criterion was only met with activated sludge as inoculum. For the final GLP test it is recommended to use activated sludge as inoculum because of the time window criterion was met.

Key result

Parameter:

% degradation (O2 consumption)

Value:

75

Sampling time:

28 d

Remarks on result:

other: Over 60% biodegradation was achieved in a period of approximately 12 days immediately following the attainment of 10% biodegradation after day 4, therefore fulfilled the 14-day time window criterion for ready biodegradable compounds.

Details on results:

- Theoretical oxygen demand (ThOD): The theoretical oxygen demand (ThOD) of the test substance used to calculate the biodegradation percentages is 2.95 g oxygen/g test substance. This ThOD was calculated with the molecular formula and based on the weighted mean calculation of the known constituents. The calculated ThOD of the know constituents is assumed to be representative for all organics present in the test substance (including the unknown fraction). The ThOD of sodium acetate is 0.78 g oxygen/g sodium acetate.

- Toxicity: Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected . Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

- Test conditions: The pH of the media for the control, control with silica gel, reference and test substance was 7.4 at the start of the test. The pH of the medium at day 28 was 7.3, 7.1, and 7.3 for the control, test substance and control with silica gel, respectively. The temperature ranged from 22.5 to 22.9 °C which is within the prescribed temperature range of 22 to 24°C.

- Biodegradability: The test substance was biodegraded by 75% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 12 days immediately following the attainment of 10% biodegradation after day 4, therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds. Hence this substance should be classified as readily biodegradable.

Results with reference substance:

The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 90 (see table 5.2.1/2 in "Any other information on results incl. tables").

Table 5.2.1/1: Dissolved oxygen concentrations (mg/L) in the closed bottles

Time (days)	Oxygen concentration (mg/L)
	Oc	Oa	Ocs	Ot
0	8.6	8.6	8.7	8.7
	8.7	8.7	8.7	8.7
Mean (M)	8.65	8.65	8.70	8.70
7	8.2	3.7	8.3	7.2
	8.2	3.7	8.3	7.2
Mean (M)	8.20	3.70	8.30	7.20
14	7.9	3.2	8.0	4.8
	7.9	3.2	8.0	4.5
Mean (M)	7.90	3.20	8.00	4.65
21	7.8		7.9	3.5
	7.8		7.9	3.6
Mean (M)	7.80		7.90	3.55
28	7.6		7.7	3.2
	7.6		7.7	3.3
Mean (M)	7.60		7.70	3.25

Oc = Mineral nutrient solution with only inoculum.

Ocs = Mineral nutrient solution with inoculum and silica gel

Ot = Mineral nutrient solution with inoculum, test substance (2.0 mg/L) and silica gel

Oa = Mineral nutrient solution with inoculum and sodium acetate (6.7 mg/L).

Table 5.2.1/2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test

Time (days)	Oxygen consumption (mg/L)		Biodegradation (%)
	Test substance	Acetate	Test substance	Acetate
0	0.00	0.00	0	0
7	1.10	4.50	19	86
14	3.35	4.70	57	90
21	4.35		74
28	4.45		75

Validity criteria fulfilled:

yes

Remarks:

Endogenous respiration of 1.05 mg/L at day 28. The differences of the replicate values at day 28 were less than 20%. The biodegradation of sodium acetate at day 14 was 90%. Oxygen concentrations >0.5 mg/L in all bottles during the test period.

Interpretation of results:

readily biodegradable

Conclusions:

The test item was biodegraded by 75% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 12 days immediately following the attainment of 10% biodegradation after day 4, therefore fulfilled the 14-day time window (10-day time window for other OECD 301 tests) criterion for ready biodegradable compounds. Hence, this substance should be classified as readily biodegradable.

Executive summary:

To assess the biotic degradation of the test substance, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item was exposed to activated sludge at an initial concentration of 2 mg/L. The closed bottles were incubated in the dark at 22-24°C for 28 days. The degradation of the test item was assessed by the measurement of oxygen consumption. According to the results of this study, the test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum.

The test item was biodegraded by 75% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 12 days immediately following the attainment of 10% biodegradation after day 4, thereby fulfilling the 14-day time window criterion considered acceptable to classify substances as readily biodegradable for this test (10 -day time window for other OECD 301 tests) . Hence, this substance should be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 1.05 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 90% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

Dec 12, 2007 - Jan 31, 2008

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study was performed according to the EU Method C.4-E with GLP but with significant methodological modifications. The test substance (adequately identified) was dissolved in a partially biodegradable solvent. Chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The incubation period was prolonged to 36 days. Noreenal is a volatile substance and 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.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15-16 March 2007 / Date of implementation: 16 March 2007 / Date of GIPC decision: 26 April 2007 / Time of validity: 18 months

Specific details on test material used for the study:

- Storage condition of test material: Stored at room temperature protected from direct sunlight

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 6 days in the mineral medium at the test temperature: 400 mL of surface water were added with 0.5 mL of the stock solutions A , B and C and 0.05 mL of the stock solution D and the volume was made to 500 mL.
The resulting solution was maintained under strong aeration and agitation at 20 ± 1°C for 6 days.
The preconditioned inoculum was further used at a rate of 0.7 Ml/L of medium .
The suspended solids were measured to represent 0.9 µg/L.

Duration of test (contact time):

36 d

Initial conc.:

2.03 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Degradation was followed by analysis of dissolved oxygen

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-day incubation period for each of the following experimental series:
- Blank group: mineral medium alone with inoculum (duplicate determination s),
- Solvent group: mineral medium with the same amount of chloroform as for the introduction of the test substance (single determinations) , with inoculum,
- Test group: 2-5 mg/L test solution in mineral medium with inoculum (duplicate determinations) ,
- Reference group : 2-5 mg/L aniline solution in mineral medium with inoculum (duplicate determinations) ,
- Toxic control group: solution containing 2-5 mg/L of each the test substance and aniline in mineral medium, with inoculum (single determinations) .

Reference substance:

aniline

Remarks:

Test reference: Aniline , C6H5NH 2 , CAS no 62-53-3, Sigma Reference A9880 (> 99.0%), batch n°034KO 126.

Test performance:

-       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 compound (aniline) had reached the pass-level by day 14,
-       In the toxic control series more than 25 % biodegradation of aniline had occurred on day 14.

Key result

Parameter:

% degradation (O2 consumption)

Value:

57.13

Sampling time:

28 d

Remarks on result:

other: More values: see details on results

Parameter:

% degradation (O2 consumption)

Value:

60.89

Sampling time:

32 d

Parameter:

% degradation (O2 consumption)

Value:

57.97

Sampling time:

36 d

Details on results:

Points of degradation plot (test substance):
10 % degradation after 4 d
19 % degradation after 8 d
24 % degradation after 12 d
37 % degradation after 16 d
44 % degradation after 20 d
63 % degradation after 24 d
57 % degradation after 28 d
61 % degradation after 32 d
58 % degradation after 36 d


Days Test

Mean
Toxic Aniline

Mean
0 0 0 0
4 10.19 6.79 -7.51
8 18.54 13.85 40.92
12 24.06 41 .65 59.99
16 37.25 42.37 63.16
20 43.94 56.59 67.60
24 63.48 57.94 69.48
28 57. 13 58.94 68.00
32 60.89 60.30 71.75
36 57.97 58.58 67.70

Results with reference substance:

Points of degradation plot (reference substance):
0 % degradation after 4 d
41 % degradation after 8 d
60 % degradation after 12 d
63 % degradation after 16 d
68 % degradation after 20 d
69 % degradation after 24 d
68 % degradation after 28 d
72 % degradation after 32 d
68 % degradation after 36 d


Days Test

Mean
Toxic Aniline

Mean
0 0 0 0
4 10.19 6.79 -7.51
8 18.54 13.85 40.92
12 24.06 41 .65 59.99
16 37.25 42.37 63.16
20 43.94 56.59 67.60
24 63.48 57.94 69.48
28 57. 13 58.94 68.00
32 60.89 60.30 71.75
36 57.97 58.58 67.70

Time evolution of dissolved oxygen (mg/L) in the reacting vessel & Measured oxygen up-take (mg/L) throughout the 28-day incubation period tables are available in the full report (attached)

Dissolved oxygen still decreased between day 20 and day 24 of the incubation period in the test group, and the observation period was prolonged to 36 days so as to ascertain that the plateau was reached.

Mean oxygen uptake in the blank vessels was 1.15 mg/L at the end of test.

The consumption of oxygen was slightly higher in the solvent vessel ( 1.27 mg/L) at the end of test.

Aniline : The beginning of the 10-day window occurred between 4 and 8 days of incubation, and the pass­level was reached on day 12 (60% biodegradation). Final biodegradation on day 28 was 68.0 %.

Test substance: The beginning of the 10-day window occurred on day 4 ( 10.2 % biodegradation), but on day 16, the percentage of biodegradation was still 37.2 %. The biodegradation still in creased on the followingdays: mean biodegradation was 57.1 % on day 28. The difference between extremes values on day 28 was (58.97-56.97) = 1.33% biodegradation, and represented 2.3% of mean value (57.13 %).

The incubation period was prolonged so as to ascertain that the plateau was reached on day 28: mean biodegradation appeared stabilized as 58.0 % was measured on day 36.

In the toxic control series, the biodegradation based on total ThOD was 41.7 % on day 12, and final value on day 28 was 58.9 %.

Actie ingredient concent:

The measured initial concentration represented 97 - 107% of the nominal value (2.03 mg/L). The treatment application was considered as valid. At the end of the test, the test substance was not recovered in the test vessels. Based on analytic assessment, the removal of the test substance in the test vessels at the end of test was total.

Validity criteria fulfilled:

yes

Remarks:

The test was terminated after 36 days of incubation. The test was considered as valid on the basis of the fulfilled conditions

Interpretation of results:

not readily biodegradable

Conclusions:

The test was performed using a 2.03 mg/L test solution .
57.1 % biodegradation occurred within 28 days and the pass-level was almost reached, but the test substance was not considered as readily biodegradable.
Based on analytic assessment, the removal of the test substance in the test vessels at the end of test was 100%.

Executive summary:

STUDY: Ready biodegradability of a substance-Closed-bottle test

GUIDELINES: EEC  C4.e

 

RESULTS

Hydrosolubility = 34.8 mg/L

Theorical COD = 2.95 g/g

Nominal test concentration: 2.03 mg/L (5.98 mg ThOD/L)

The criteria for classification of the test substance as Ready biodegradability was not achieved (mean of 57.13% removal within 28 days). After 32 days of incubation, a mean of 60.89% biodegradation was observed. Based on an analytical assessment, the removal of the test substance in the test vessels at the end of test was complete.

Some modifications to the method were made:

The test substance was dissolved in a partially biodegradable solvent. Chloroform was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid.

Description of key information

OECD Guideline 301D, EU Method C4 -E, ISO 10707, GLP, Key study, validity 1:

75% biodegradation after 28 days, within the 14-day window (activated sludge)

Readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

To assess the biodegradation potential of the registered substance, two experimental studies are available.

The first study, from Nouryon, 2019, assessed as the key study, was performed on the registered substance according to slighty modified OECD 301D, EU C.4 -E and ISO 10707 Test Guidelines and in compliance with the OECD principles of Good Laboratory Practice. The substance was exposed to activated sludge at an initial concentration of 2 mg/L. The closed bottles were incubated in the dark at 22 -24°C for 28 days, and the degradation was assessed by the measurement of oxygen consumption. According to the results of this study, the test substance did not cause a reduction in the endogenous respiration at day 7, and therefore is considered to be non-inhibitory to the inoculum.

The test substance was biodegraded by 75% at day 28 in the OECD 301D Closed Bottle test. Over 60% biodegradation was achieved in a period of approximately 12 days immediately following the attainment of 10% biodegradation after day 4, thereby fulfilling the 14 -day time window required as the criterion for classifying a substance as readily biodegradable (10-day time window for other OECD 301 tests) . Hence, this substance should be classified as readily biodegradable.

The second study, from Phytosafe, 2008, assessed as a disregarded study, was performed on the registered substance according to the EU Method C.4-E: Determination of the ready biodegradability - Closed bottle test, and in compliance with GLP. The test material was exposed to inoculum from surface waters, at a concentration of 2.03 mg/L, in culture medium, in closed bottles, in the dark, at 20 +/- 1 °C, for 36 days. The degradation of the test substance was assessed by the measurement of oxygen consumption. 57.1%, 60.9% and 58.0% biodegradation occurred within 28, 32 and 36 days of exposure. Also, based on an analytical assessment, 100% removal of the test substance in the test vessels was observed at the end of test.

The validity criteria were fulfilled. However, unacceptable test conditions were employed that are in disagreement with the principles of ready biodegradibility testing: The test substance was dissolved in a partially biodegradable solvent, chloroform, which was used to prepare a "stock solution" prior to adding an aliquot of this solution to the mineral medium. The biodegradation of the solvent in the test substance solutions was determined by substracting the degradation in a solvent control. However, it cannot be ascertained directly to what extent the chloroform degraded in the solutions containing test substance, the results of the study are not interpretable and the study is considered technically invalid. Consequently, the study should be diregarded.

In conclusion, based on the key study, the registered substance is readily biodegradable.