N,N-Dimethyl-9-dodecenamide

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:

23 November 2021 - 8 July 2022

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: see details on inoculum below

Details on inoculum:

- Source of inoculum/activated sludge: The sample of inoculum was collected from the secondary effluent of a treatment plant receiving predominantly domestic sewage (Aureilhan, France).
- Pre-conditioning: The sample was aerated for about 6 days before use as inoculum at the test temperature to reduce endogenous respiration.
- Initial cell/biomass concentration: A concentration of inoculum of 3.1 E05 cells/L was used.
- Water filtered: No

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:

Mineral medium:
The mineral medium was prepared by adding one mL of each of the following stock solutions (a to d) to each liter of ultrapure water:
Stock solutions - Concentration in stock solution
(a)
KH2PO4 - 8.50 g/L
K2HPO4 - 21.75 g/L
NA2HPO4. 2H2O - 33.40 g/L
NH4Cl - 0.50 g/L
The pH of this solution was approximately 7.5.
(b) CaCl2. 2H2O - 36.40 g/L
(c) MgSO4. 7H2O - 22.50 g/L
(d) FeCl3. 6H2O - 0.25 g/L
This reconstituted water was aerated for at least 1 hour and thereafter set aside at the test temperature for about 20 h before use.
Inoculum:
The sample of inoculum was collected from the secondary effluent of a treatment plant receiving predominantly domestic sewage (Aureilhan, France).
Pre-conditioning:
The sample was aerated for about 6 days before use as inoculum at the test temperature to reduce endogenous respiration.
Administration of the test and reference substances:
Due to its properties, administration of the test item was accomplished with the help of silica gel. For this purpose, approximately 30 mg of test item (actual measured amount: 30.70 mg) were added to 10 g of silica gel present in a 100-mL glass flask. The flask was closed with a screw cap and the contents were mixed vigorously by hand shaking (for about 5 min). Then approx. 0.2 g of silica gel dosed with test item was added to the respective test vessels in order to obtain a nominal concentration of 2 mg test item/L that were thereafter completely filled with the inoculated mineral medium (300 mL of test medium) and closed. No small bubble was observed in the test vessels. The test was carried out without adjustment of the pH.
The reference substance sodium benzoate was used at a nominal concentration of 2 mg/L. A concentrated stock solution (1000 mg/L) was prepared in mineral medium. Then it was diluted with mineral medium and a fixed amount of inoculum into volumetric flasks to obtain the required reference item concentration to be filled into test vessels.
Test procedures and conditions:
Test duration - 28 days
Test vessels - All-glass sealed with ground glass stoppers BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of the start of the biodegradability test (=J0) and type of series.
Test water - Mineral medium, Reconstituted water, as prescribed by OECD Guideline No. 301 (1) (see above)
Inoculum - A concentration of inoculum of 3.1×105 cells/L was used.
Study design
- 10 bottles containing inoculum, mineral medium and silica gel (inoculum blank with silica gel)
- 10 bottles containing inoculum and mineral medium (inoculum blank without silica gel)
- 10 bottles containing test item, inoculum, mineral medium and silica gel (test suspension)
- 6 bottles containing reference substance, inoculum and mineral medium (procedure control)
- 6 bottles containing test item (and silica gel), reference substance, inoculum and mineral medium (toxicity control)
Test environment - Controlled environment cabinet (22°C ± 2°C); the test was performed in the dark.
Measurements and recordings:
Dissolved [O2] (DO) - Determinations of the concentration of dissolved oxygen using an oxygen electrode in duplicate vessels taken from each group (except the procedure and toxicity control) were made on day 0, 7, 14, 21 and 28, and only at the start of the test and after 7 and 14 days of incubation for the procedure and toxicity control.
Temperature - Measured continuously in the growth chamber.
Nitrate/nitrite analysis - Since the test item is a N-containing substance, samples from the test vessels (test suspension and inoculum blank) were prepared for analysis of nitrite and nitrate concentrations. These analyses were not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.
Measurements were recorded with suitable instruments and documented in the raw data.
Interpretation
Validity criteria of the test
Inoculum blank - Oxygen depletion in the inoculum blank should not exceed 1.5 mg dissolved oxygen/L after 28 days.
Residual [O2] - The residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L at any time.
Replicate values - The differences of the replicate values with the test item at day 28 should be less than 20%.
Reference substance - The test is valid if the level of biodegradation of the reference substance achieves 60% or more within 14 days of the start of the test.
Calculation of the results:
Calculation of endogenous respiration:
The endogenous respiration (oxygen depletion in the control) was calculated as follows:
Oxygen depletion (endogenous respiration) (mg/L) = mb (day 0) - mb (day 28)
mb is the mean oxygen level in the inoculum blank
Calculation of the theoretical oxygen demand (ThOD):
The ThODs of the test chemicals (test item / reference substance) were calculated from their molecular formulae (CcHhClclNnNanaOoPpSs) and molecular weights (MW) as follows:
ThODNH3 = (16[2c + 1/2 (h-cl-3n) + 3s + 5/2p + 1/2na - o])/MW mgO2/mg
Calculation of the biodegradation percentages:
The biodegradation after n days of the test was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD) as follows:
% degradation = BOD (mg O2/mg test chemical) x 100/ ThOD (mg O2/mg test chemical)
with BOD = (a(o) - a(n)) - (mb(o) - mb(n))/mg test chemical/L in vessel
and where:
n = number of days after the start of the test
a(0) = test chemical value at day 0
a(n) = test chemical value at day n
mb(0) = mean inoculum blank value at day 0
mb(n) = mean inoculum blank value at day n.
Inoculum blank with silica gel was used for the calculation of the biodegradation percentage of the test item; inoculum blank without silica gel was used for the calculation of the biodegradation percentage of the reference substance.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

70

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

90

Sampling time:

28 d

Results with reference substance:

The biodegradation percentage of the reference substance, sodium benzoate, was 78% at day 14, thus confirming the suitability of the inoculum used.
Moreover, in the toxicity control containing both the test item and reference item sodium benzoate, 70% biodegradation was noted on day 14. According to the test guideline, the test item can be assumed to be not inhibitory to the microbial inoculum because degradation was greater than 25% within 14 days (based on ThOD).

Validity criteria of the test:

Inoculum blank - Oxygen depletion in the inoculum blank (with and without silica gel) did not exceed 1.5 mg dissolved oxygen/L after 28 days.

Residual [O2] - Oxygen concentrations were > 0.5 mg/L in all bottles during the test period.

Replicate values - The differences of the replicate (test suspension) values at day 28 were less than 20%.

Reference substance - The biodegradation percentage of the reference substance, sodium benzoate, was 78% at day 14, thus confirming the suitability of the inoculum used.

Moreover, in the toxicity control containing both the test item and reference item sodium benzoate, 70% biodegradation was noted on day 14. According to the test guideline, the test item can be assumed to be not inhibitory to the microbial inoculum because degradation was greater than 25% within 14 days (based on ThOD).

Thus all validity criteria of the test have been fulfilled in the present study (see Tables 2 and 3 below and Figure 1, attached, for details).

Test measurements and conditions:

The results of measurement of oxygen concentrations (mg/L) are presented in Table 2 below.

Temperatures were situated between 21.7 and 22.4°C throughout the test (average value: 22.1°C), and complied with the requirements as laid down in the study plan (22°C ± 2°C, constant within 1°C).

Besides, analysis of nitrite and nitrate concentrations in the inoculum blank and the test suspension at the start and the end of the test (see Table 4 below) revealed there was no nitrification process due to nitrogen from the test item. Indeed, the values obtained with inoculum blanks and test suspensions were in the same order of magnitude (close to or below the LOQ of the analytical methods). Thus, a correction for the oxygen consumed by nitrification was not relevant for the present study.

Calculation of Theoretical oxygen demand (ThOD):

The calculated ThOD of the test item and reference substance sodium benzoate was calculated from their empirical formulae to be:

Test item: C14H27NO, MW = 225.37 g/mol ThOD = 2.77 mgO2/mg

Reference substance: C7H5O2Na, MW = 144 g/mol ThOD = 1.67 mgO2/mg

Biodegradability of the test item:

According to the OECD 301 guidelines, substances are considered to be readily biodegradable in the closed bottle test if the degradation of the test item is equal to or greater than 60% (based on ThOD) in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).

In this study, test item N,N-dimethyldodec-9-enamide was biodegraded by 90% (mean) at day 28 (see Table 3 below and Figure 1 attached). The pass level of 60% was reached after less than 7 days of incubation and the 10-day window criterion was successful.

Therefore, under the test conditions and according to the requirements of the test guideline, the test item can be considered as readily biodegradable.

Table 2. Dissolved oxygen concentrations (mg/L) throughout the test.

 

	Time (days)
	0	7	14	21	28
Inoculum Blank + silica gel	8.73	8.05	7.89	7.47	7.37
	7.69	8.04	7.90	7.24	7.22
Mean	8.21	8.05	7.90	7.36	7.30
Test Suspension (2 mg test item/L)	8.61	4.00	3.32	2.83	2.34
	8.66	4.21	3.31	2.97	2.17
Mean	8.64	4.11	3.32	2.90	2.26
Inoculum Blank (without silica gel)	8.60	8.01	7.70	7.46	7.22
	8.59	7.94	7.77	7.38	7.22
Mean	8.60	7.98	7.74	7.42	7.22
Procedure Control(2 mg reference substance/L)	8.65	5.32	5.21	-	-
	8.53	5.44	5.06	-	-
Mean	8.59	5.38	5.14	-	-
Toxicity Control (2 mg reference substance/L + 2 mg test item/L)	8.65	1.93	0.83	-	-
	8.58	1.84	1.08	-	-
Mean	8.62	1.89	0.96	-	-

Table 3.% biodegradation of the test item, the reference substance and the toxicity control throughout the test.

 

 

		Time (days)
		0	7	14	21	28
Test Suspension (2 mg test item/L)	Replicate 1	0.00	80.23	89.80	88.90	96.66
	Replicate 2	0.00	77.35	90.88	87.27	100.63
	Mean	0	79	90	88	99
Procedure Control (2 mg reference substance/L)	Replicate 1	0.00	81.14	77.25	-	-
	Replicate 2	0.00	73.95	78.14	-	-
	Mean	0	78	78	-	-
Toxicity Control (2 mg reference substance/L + 2 mg test item/L)*	Mean	0	66	70	-	-

  * % biodegradationtox. Control=      (BOD tox. cont./ ThODref. subst.) x 100

 with BOD tox. cont = [(mTox(o) - mTox(n)) - (ma(o) - ma(n))]/ mg test ref subst/L in vessel

and where:

n = number of days after the start of the test

mTox(0) = mean toxicity control value at day 0

mTox (n) = mean toxicity control value at day n

ma(0) = mean test suspension value at day 0

ma(n) = mean test suspension value at day n.

  

Table 4.Analytical monitoring of nitrate & nitrite concentrations in inoculum blanks and test suspensions at the start and the end of the test.

 

			Start (Day 0)	End (Day 28)
Inoculum blank	Concentration of Nitrate (mg N/L)	Rep. 1	<0.1	0.463
		Rep. 2	<0.1	0.298
	Concentration of Nitrite (mg N/L)	Rep. 1	<0.02	<0.02
		Rep. 2	<0.02	<0.02
Test suspension (100 mg test item/L)	Concentration of Nitrate (mg N/L)	Rep. 1	<0.1	0.586
		Rep. 2	<0.1	0.793
	Concentration of Nitrate (mg N/L)	Rep. 1	<0.02	0.0506
		Rep. 2	<0.02	<0.02

LOQ_Nitrate: 0.1 mg NO₃/L; LOQ_Nitrite: 0.02 mg NO₂/L

 

                                                

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Executive summary:

A study was performed to assess the biotic degradation of the test item N,N-dimethyldodec-9-enamide by performing a ready biodegradability test. The method followed was designed to be compliant with OECD TG 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008 and with ISO 10634.

BOD bottles were filled with mineral medium inoculated with a secondary effluent of a treatment plant and test item added at a nominal concentration of 2 mg/L with the help of silica gel. In the meantime, two series of blanks (with and without silica gel) were filled with inoculated mineral medium. Furthermore, bottles containing reference substance sodium benzoate (2 mg/L) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum. The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test (except for the procedure and toxicity control, where measurements were made until day 14).

The validity of the test was demonstrated by an endogenous respiration < 1.5 mgO2/L and by a biodegradation of the reference substance of 78% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg/L in all bottles during the test period. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

According to the OECD 301 guidelines, substances are considered to be readily biodegradable in the closed bottle test if the degradation of the test item is equal to or greater than 60% (based on ThOD) in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).

In this study, the test item was biodegraded by 99% (mean) at day 28. The pass level of 60% was reached after less than 7 days of incubation and the 10-day window criterion was successful, even though this criterion is not relevant for surface active agents.

Under the test conditions and according to the requirements of the test guideline, the test item can be considered as readily biodegradable.

Description of key information

The substance is readily biodegradable based on the results of a study performed according to OECD TG 301D.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

A study was performed to assess the biotic degradation of the test item N,N-dimethyldodec-9-enamide by performing a ready biodegradability test. The method followed was designed to be compliant with OECD TG 301 D, "Ready biodegradability – Closed Bottle Test", referenced as Method C.4-E of Commission Regulation No. 440/2008 and with ISO 10634.

BOD bottles were filled with mineral medium inoculated with a secondary effluent of a treatment plant and test item added at a nominal concentration of 2 mg/L with the help of silica gel. In the meantime, two series of blanks (with and without silica gel) were filled with inoculated mineral medium. Furthermore, bottles containing reference substance sodium benzoate (2 mg/L) were tested in order to check the procedure. A toxicity control, containing both the test item and the reference substance, was also performed in order to check the absence of test item effect on the microbial inoculum. The concentrations of dissolved oxygen in duplicate bottles from each group incubated at 22°C ± 2°C in darkness were measured at the start of the test and thereafter once a week until the end of the test (except for the procedure and toxicity control, where measurements were made until day 14).

The validity of the test was demonstrated by an endogenous respiration < 1.5 mgO2/L and by a biodegradation of the reference substance of 78% of its ThOD after 14 days of incubation. Moreover, the differences of the replicate values at day 28 were less than 20% and oxygen concentrations were > 0.5 mg/L in all bottles during the test period. Furthermore, the toxicity control showed that the test item had no inhibitory effect on the activity of the microbial inoculum.

According to the OECD 301 guidelines, substances are considered to be readily biodegradable in the closed bottle test if the degradation of the test item is equal to or greater than 60% (based on ThOD) in the ten-day window within the 28-day period of the test (the 10-day window begins when the degree of biodegradation has reached 10% of the ThOD).

In this study, the test item was biodegraded by 99% (mean) at day 28. The pass level of 60% was reached after less than 7 days of incubation and the 10-day window criterion was successful, even though this criterion is not relevant for surface active agents.

Under the test conditions and according to the requirements of the test guideline, the test item can be considered as readily biodegradable.