N,N-Dimethyl-9-dodecenamide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 December 2021 - 2 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test vessels were 1 L cylindrical glass bottles closed with PTFE screw caps. A magnetic stirring bar was placed in each test vessel. Blank controls were prepared with 16 mL synthetic sewage feed and 234 mL test water. Test mixtures were prepared by addition of the required amounts of test item in pre-filled test vessels (with synthetic sewage feed and test water). The 3,5-DCP stock solution was diluted with test water as necessary to obtain the required test concentrations into reference mixtures after filling with synthetic sewage and water. Then, 250 mL of sludge suspension were sequentially added in all test vessels. After addition of the inoculum, the test vessels were immediately closed and the mixtures were stirred under aeration for 3 hours.
See Table 2 for information on the contents of the test vessels.
The test was carried out without adjustment of the pH.
- Test concentration separation factor: Approx 3.16
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Origin: Activated sludge was collected (13 Dec. 2021) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.
- Pre-treatment: Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve), washed 3 times with chlorine-free tap water and re-suspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (50 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined, then the sludge was diluted in chlorine-free tap water to obtain the required sludge solids concentration of approximately 3.0 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Temperatures recorded continuously were situated between 18.4 and 21.6°C throughout the test (average value: 20.2°C)

pH:

The pH of the reaction mixtures at the end of the test period ranged from 7.44 to 8.11 (see Table 3)

Nominal and measured concentrations:

nominal: 10, 32, 100, 316 and 1000 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass bottles of approximately 1 L capacity closed with PTFE screw caps, containing 500 mL of reaction mixture. Each test vessel was uniquely identified with study code, replicate number, start time of the test and type of series.
- Test water: Deionised water, containing less than 1 mg/L DOC (Dissolved Organic Carbon); determined value: < 0.3 mg/L DOC; DOC analysis was performed in accordance with ISO 17025.
- Inoculum: The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g/L of suspended solids.
- 6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls)
- 3 replicates bottles per test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)
- 1 replicate per reference substance concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference mixtures)
- No. of vessels per abiotic control (replicates): The test item was not known to have strong reducing properties, therefore it was judged not necessary to perform abiotic controls.

The content of the test vessels is given in table 2 below.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Aeration: All suspensions were aerated and stirred during the exposure period.
- Details on termination of incubation: At the end of incubation, samples were withdrawn to measure the rate of decrease of dissolved O2 in completely filled all-glass BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Total oxygen uptake inhibition was determined (carbon and ammonium oxidation not separated)
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 3.16
- Range finding study
- Test concentrations: 10, 100, 320 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
After 3 hours of exposure, no significant effect on the total respiration rate was observed at 100 mg/L, but significant effects were found at 320 and 1000 mg/L (see Table below); all validity criteria were met (CV of oxygen total respiration rate in control replicates: 2.4%; mean total oxygen uptake rate in the blank controls: 29.9 mg O2.g-1 dry weight activated sludge.h-1; EC50 of 3,5-DCP for total respiration: 15.1 mg/L).
The test item had not a greater effect on the oxidation of ammonium (RN=RT- RH) than on general heterotrophic oxidation; this is why it was decided to perform the measurement of total oxygen uptake inhibition only in the final test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

17 mg/L

95% CI:

>= 11.8 - <= 22.5

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

116 mg/L

95% CI:

>= 99.3 - <= 135

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: No
- Blank controls oxygen uptake rate: Mean oxygen uptake rate was 29.5 mg O2/g dry weight activated sludge/h, and therefore was well above the (minimum) required value of 20 mg O2/g/h.
- Coefficient of variation of oxygen uptake rate in control replicates:7.0% at the end of the definitive test (below the recommended upper limit of 30%).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of 3,5-DCP was 8.2 mg/L for total respiration and thus remained within the prescribed range of 2 to 25 mg/L.

Reported statistics and error estimates:

The statistical outputs from ToxRat are reported in Appendix IV (attached)

Range-finding test results (respiration rate and inhibition at the end (3h) of the test)

		Total respiration     (RT) without ATU		Heterotrophic respiration (RH)        with ATU
Content	Concentration (mg/L)	Oxygen uptake rate (mg O2.g-1.h-1)	Inhibition (%)	Oxygen uptake rate (mg O2.g-1.h-1)	Inhibition (%)
Control (1)	0	29.546	-	-	-
Control (2)	0	29.969	-	31.122	-
Ref (1)	5.00	19.072	2.896
Ref (2)	8.40	29.664	0.917
Ref (3)	14.21	13.904	53.557
Ref (4)	23.81	6.253	79.115
Ref (5)	40.02	4.548	84.809
Test item	10.0	35.587	-18.866	25.129	13.807
Test item	10.0	34.401	-14.904	26.812	8.034
Test item	100.0	29.412	1.761	20.378	30.102
Test item	100.0	29.459	1.602	22.333	23.397
Test item	320.0	20.702	30.853	13.087	55.112
Test item	1000.0	9.395	68.619	8.203	71.865
Test item	1000.0	8.753	70.763	8.059	72.359
Control (3)	0	29.288	-	-	-
Control (4)	0	30.952	-	27.187	-

Activated sludge respiration inhibition test

Table 3 and Figure 1 (attached) show the responses recorded during the final test.

Table 3. Test results (oxygen uptake rate, inhibition and pH at the end of the test).

Content	Concentration (mg/L)	Oxygen uptake rate (mg O2/g/h)	Inhibition (%)	pH
Control (1)	0	27.243	-	7.88
Control (2)	0	28.001	-	7.80
Ref (1)	5.03	20.125	31.851	7.89
Ref (2)	8.44	14.804	49.871	7.82
Ref (3)	14.27	8.303	71.884	7.94
Ref (4)	23.92	4.505	84.744	7.93
Ref (5)	40.20	3.119	89.437	7.97
Control (3)	0	29.120	-	7.44
Control (4)	0	28.710	-	7.81
Test item	11.40	28.183	4.563	7.72
Test item	11.42	28.399	3.833	7.76
Test item	10.92	29.231	1.017	7.74
Test item	32.68	24.673	16.450	7.74
Test item	32.42	24.721	16.290	7.74
Test item	33.18	25.508	13.622	7.76
Test item	100.66	13.189	55.338	7.86
Test item	101.92	15.254	48.346	7.82
Test item	100.02	14.874	49.633	7.85
Test item	316.36	7.994	72.931	8.00
Test item	316.34	6.964	76.418	8.00
Test item	315.98	6.609	77.622	8.02
Test item	1001.16	4.401	85.097	8.09
Test item	1000.32	4.282	85.499	8.09
Test item	998.86	3.458	88.289	8.11
Control (5)	0	31.786	-	7.74
Control (6)	0	32.325	-	7.74

Validity criteria fulfilled:

yes

Executive summary:

Introduction

A study was performed to assess the effects of test item N,N-dimethyldodec-9-enamide on microorganisms from activated sludge by performing a respiration inhibition test. The method followed was designed to be compliant with OECD TG 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", referenced as Method C.11 of Commission Regulation No. 440/2008.

Methods

A definitive test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item at nominal concentrations of 10, 32, 100, 316 and 1000 mg test item/L (three replicates for each concentration) and to six blank controls. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol (3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids.

After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

Results

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O2/g/h, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC50 of 8.2 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration).

The ECx and NOEC values at the end of the test were as follows:

Parameter	Respiration rate - Critical effect and threshold concentration (mg/L)	95% confidence limits (mg/L)
3-hour EC10	17.0	11.8 – 22.5
3-hour EC20	32.8	25.1 – 40.7
3-hour EC50	116	99.3 – 135
3-hour NOEC	10.0	-

Statistical analyses were performed by the computer program ToxRat.

Conclusion

The effect of test item N,N-dimethyldodec-9-enamide on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions and based upon nominal concentrations, the 3-hour EC50 value was determined to be 116 mg/L.

Description of key information

The effect of test item N,N-dimethyldodec-9-enamide on microorganisms from activated sludge was investigated in a respiration inhibition test performed according to OECD TG 209. Under the experimental conditions and based upon nominal concentrations, the 3-hour EC50 value was determined to be 116 mg/L. The 3 -hour EC10 and NOEC were 17.0 and 10.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

116 mg/L

EC10 or NOEC for microorganisms:

17 mg/L

Additional information

A study was performed to assess the effects of test item N,N-dimethyldodec-9-enamide on microorganisms from activated sludge by performing a respiration inhibition test. The method followed was designed to be compliant with OECD TG 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", referenced as Method C.11 of Commission Regulation No. 440/2008.

A definitive test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item at nominal concentrations of 10, 32, 100, 316 and 1000 mg test item/L (three replicates for each concentration) and to six blank controls. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol (3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids.

After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O2/g/h, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC50 of 8.2 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration).

The ECx and NOEC values at the end of the test were as follows:

Parameter	Respiration rate - Critical effect and threshold concentration (mg/L)	95% confidence limits (mg/L)
3-hour EC10	17.0	11.8 – 22.5
3-hour EC20	32.8	25.1 – 40.7
3-hour EC50	116	99.3 – 135
3-hour NOEC	10.0	-

Statistical analyses were performed by the computer program ToxRat.