N,N-Dimethyl-9-dodecenamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2021 - 31 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test solutions at the start (t=0 h) and at the end of the test (t=48 h).

Vehicle:

no

Details on test solutions:

- Preparation of stock and test solutions
The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water was added. Then 100.6 mg test item were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1 L of stock solution and closed. Mixing was initiated with the vortex in the centre extending to maximally around 10% of the vessel depth from the top to the bottom of the vessel. After approx. 22 hours of gentle stirring in the dark at room temperature, the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port and samples were taken from the stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary to obtain the required test concentrations into 500-mL volumetric flasks based on the measured concentration of the stock solution (around 65 mg/L). Each flask was inverted several times before filling the test vessels (without headspace) to ensure adequate mixing and homogeneity of test solutions. After filling and introduction of daphnids the vessels were sealed immediately.
- Test concentration separation factor: Approx 2.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test solutions in test vessels were observed to be clear and colourless. The Tyndall effect (checked via laser beam) was negative in all treatments.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus (Cladocera, Crustacea)
- Strain/clone: clone 5
- Sex: female
- Age at study initiation: < 24 hours old at the test initiation and were not first brood progeny.
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Method of breeding: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C/Daphnia/day. The water was changed twice a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured daphnids.
- Feeding during the test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

approximately 250 mg/L (as CaCO3)

Test temperature:

20.1 - 20.7°C

pH:

7.85 - 8.23 (Table 2)

Dissolved oxygen:

8.43 - 9.03 mg/L (Table 3)

Nominal and measured concentrations:

Nominal concentration: 0.32, 0.70, 1.5, 3.4 and 7.5 mg/L
Geometric mean measured concentration: 0.35, 0.53, 1.61, 3.56 and 8.55 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Type: closed
- Material, size, headspace, fill volume: no headspace
- Volume of solution: 60 mL
- Aeration: No aeration of the test solutions occurred throughout the test.
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates
- Biomass loading rate: 5 daphnids per vessel each completely filled with test solution and without headspace

TEST MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: 0.32, 1.0, 3.2, 10.0, 32.0 and 70 mg test item/L
- Results used to determine the conditions for the definitive study: see Table below

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.24 mg/L

95% CI:

>= 0.97 - <= 1.58

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none noted
- Other biological observations: None noted
- Other adverse effects control: none reported
- Immobilisation of control: No immobilistation in controls
- Abnormal responses: none noted
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

On October 12, 2021 (KD21-002; most recent test), the 24h-EC50 was 1.495 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L) at this time.

Reported statistics and error estimates:

See Appendix VII attached

Range-finding test results

Acute immobilisation of daphnids after 24 and 48 hours in the range-finding test (static test). 

Nominal concentration (mg test item/L)	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1 2	5 5	0 0	0	0 0	0
0.32	1 2	5 5	0 0	0	0 0	0
1.0	1 2	5 5	5 0	50	5 0	50
3.2	1 2	5 5	3 3	60	5 4	90
10.0	1 2	5 5	5 5	100	5 5	100
32.0	1 2	5 5	5 5	100	5 5	100
70.0	1 2	5 5	5 5	100	5 5	100

 

Table 2. pH-values during the final test

	Analytically confirmed concentration (geometric mean) (mg/L)
	Control	0.35	0.53	1.61		3.56	8.55
Start t=0 h	8.04	8.10	7.96	7.91	8.15		8.23
End t=48 h	7.85	7.92	7.86	7.87	7.92		7.99

 

Table 3. Dissolved oxygen concentrations (mg/L) during the final test.

	Analytically confirmed concentration (geometric mean) (mg/L)
	Control	0.35	0.53	1.61		3.56	8.55
Start t=0 h	8.59	8.62	8.45	8.43	8.44		8.51
End t=48 h	8.93	9.03	8.45	8.85	8.74		8.98

All these dissolved-oxygen concentrations correspond to values ≥ 60% of the air-saturation value.

Table 4. Acute immobilisation of daphnids after 24 and 48 hours in the final test.

Analytically confirmed concentration (mg/L)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.35	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.53	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 1 0	5
1.61	1 2 3 4	5 5 5 5	0 1 3 4	40	2 4 5 4	75
3.56	1 2 3 4	5 5 5 5	4 2 2 4	60	5 5 4 5	95
8.55	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

Analytical results

The results of analysis of the samples taken during the final test are described in the Table 6 of the attached Analytical Report (Appendix V). Samples taken from the control and all test concentrations were analysed at the start and the end of the test in order to determine maintenance of actual concentrations of the test item. Chemical analysis revealed that test item levels found were relatively stable at the three highest concentrations, with test item losses < 20%. However, concentrations measured at the two lowest concentrations showed slight test item losses between fresh and old solutions (around 25%; see Table 6 of Appendix V for details). For this reason, since the deviation of the exposure concentrations of the test item was sometimes greater than 20% of the initial concentrations, the evaluation of the effects on Daphnia magna was based on the geometric means of the analytically confirmed concentrations (as recommended by OECD Guideline 202).

Validity criteria fulfilled:

yes

Remarks:

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 3 mg/L in controls and test vessels (see Table 3).

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of test item N,N-dimethyldodec-9-enamide to Daphnia magna. The method followed was designed to be compliant with OECD TG 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (2) and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Methods.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 0.32, 0.70, 1.50, 3.40 and 7.50 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

Results.

The test item levels were found to be relatively stable at the three highest concentrations throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). However, concentrations measured at the two lowest concentrations showed slight test item losses (around 25%) between fresh and old solutions. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric means of the analytically confirmed concentrations: 0.35, 0.53, 1.61, 3.56 and 8.55 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.35 mg/L, 5% at 0.53 mg/L, 75% at 1.61 mg/L, 95% at 3.56 mg/L and 100% at 8.55 mg/L.

The 48 -h EC50 value at the end of the test was 1.24 mg test item/L; 95% confidence limits 0.97 - 1.58 mg test item/L

Statistical analyses were performed by the computer program ToxRat.

Conclusion.

The toxic effect of N,N-dimethyldodec-9-enamide to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under experimental conditions, the 48-hour EC50 value was estimated to be 1.24 mg/L, based upon analytically confirmed concentrations.

Description of key information

The toxic effect of N,N-dimethyldodec-9-enamide to the freshwater invertebrate Daphnia magna was investigated in a closed static test performed according to OECD TG 202. Under experimental conditions, the 48-hour EC50 value was estimated to be 1.24 mg/L, based upon analytically confirmed concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

1.24 mg/L

Additional information

A study was performed to assess the acute toxicity of test item N,N-dimethyldodec-9-enamide to Daphnia magna. The method followed was designed to be compliant with OECD TG 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (2) and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over 48 hours at the required nominal test concentrations 0.32, 0.70, 1.50, 3.40 and 7.50 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. Samples taken from the control and all test concentrations were analysed at the start and the end of the test, in order to determine if concentrations of the test item were maintained.

The test item levels were found to be relatively stable at the three highest concentrations throughout the test (within ± 20% of the initial and nominal concentrations throughout the test). However, concentrations measured at the two lowest concentrations showed slight test item losses (around 25%) between fresh and old solutions. Therefore, the evaluation of the effects on Daphnia magna was based on the geometric means of the analytically confirmed concentrations: 0.35, 0.53, 1.61, 3.56 and 8.55 mg/L. After 48 hours of exposure, immobilisation rates were 0% at 0.35 mg/L, 5% at 0.53 mg/L, 75% at 1.61 mg/L, 95% at 3.56 mg/L and 100% at 8.55 mg/L.

The 48 -h EC50 value at the end of the test was 1.24 mg test item/L; 95% confidence limits 0.97 - 1.58 mg test item/L

Statistical analyses were performed by the computer program ToxRat.