N,N-Dimethyl-9-dodecenamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February - 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions at the start (t=0h) and every 24 hours thereafter in old and new solutions.

Vehicle:

no

Details on test solutions:

The test was carried out using test solutions prepared individually. The test solutions (for fresh media at t=0 h, t=24 h, t=48 h and t=72 h) were prepared under closed conditions and by slow-stirring. The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the test solutions. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each vessel and test water was added. Then an appropriate amount of test item was weighed on a weighing boat (for each test solution preparation) that afterwards was placed above each mixing vessel and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water to obtain 5 L of solution and closed. Mixing was initiated with the vortex in the centre extending to around 10% of the vessel depth from the top to the bottom of the vessel. After approx. 65 hours (test solutions for t =0 h were stirred throughout the weekend) or 24 ± 2 hours (for fresh media at t=24 h, t=48 h and t=72 h) of gentle stirring in the dark at room temperature, the contents of the vessels were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the test solutions were directly added into test vessels that were closed immediately after introduction of fish.
- Test concentration separation factor: Approx. 1.78
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. The test solutions were observed to be clear and colourless at all concentrations (Tyndall effect, checked via laser beam, was negative in all treatments).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: UAR TEFOR Paris-Saclay - CNRS UMS2010 / INRAE UMS1451 - Université Paris-Saclay - Bat 151, 151 route de la Rotonde - 91400 SACLAY
- Age at study initiation: Juveniles

ACCLIMATION
- Acclimation period: At least 9 days after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: flaked, powdered or live food (such as brine shrimp nauplii)
- Feeding frequency during acclimation: Daily until 24 - 48 hours before the start of the test.
- Health during acclimation (any mortality observed): no mortality during the seven days prior to the start of the test was observed

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

See Table 4 below

pH:

See Table 2 below

Dissolved oxygen:

See Table 3 below

Nominal and measured concentrations:

Nominal: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Geometric mean measured concentration: 0.86, 1.36, 2.39, 4.24 and 9.90 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Type: closed ( by placing a glass plate on the aquarium and sealing with Vaseline).
- Material, size, headspace, fill volume: All-glass, of approx. 3.5 L capacity and filled to minimum headspace.
- Aeration: no
- Renewal rate of test solution: Daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: not exceeding 0.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted water, as prescribed by the OECD Guideline 203.
Stock solutions:
a) CaCl2.2H2O 11.76 g/L
b) MgSO4.7H2O 4.93 g/L
c) NaHCO3 2.59 g/L
d) KCl 0.23 g/L
An aliquot of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 μS/cm).
The pH of this solution was in the range of 6 to 8.5 and the water hardness was between 40 and 250 mg/L (as CaCO3).
The Total Organic Carbon (TOC) in the test water was < 0.3 mg/L.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8 h dark
- Light intensity: 1140 lux

EFFECT PARAMETERS MEASURED: Mortality and abnormal behaviour - Fish were inspected 3 and 6 hours after the start of exposure, and every day thereafter (twice a day) until the end of the test. Dead fish recorded were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 1.78
- Range finding study
- Test concentrations: 0.32, 1.0, 3.2 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: See Any other information on results

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.94 mg/L

95% CI:

>= 2.53 - <= 3.41

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities:
- Observations on body length and weight: Ten fish from the test batch were measured at the end of the test. Their average length (0.81 cm) was slightly below the recommended length range given in the test guideline (1-2 cm). This was probably due, according to the fish supplier, to their time spent in polyculture being shorter than usual (5 to 10 days post-fertilisation (dpf), compared to 5 to 15 dpf in normal time), due to a crash in the rotifer culture, which is the food given to the fry especially during their polyculture. This was considered not to affect the results of the test as no impact on fish/abnormal behaviour was observed in the controls throughout the test. In any case, the fish used in the test were juveniles, as recommended by the test guideline and stated in the study plan.
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: See Table 5 below
- 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

Reported statistics and error estimates:

See Appendix VI attached

Sublethal observations / clinical signs:

2nd Range-finding test results

This 2nd range-finding test was carried out using test solutions prepared individually (and not by serial dilution of a single stock solution as was the case in the first range-finder).

Incidence of mortality and total mortality during the 2nd range-finding test.

	Nominal concentration (mg test item/L)
Nominal	Control		0.32		1.00		3.20		10.00
Cumulative mortality	M	S	M	S	M	S	M	S	M	S
t=3 & 6 h	0	0	0	0	0	0	0	3a	3c	0
3 & 6h Total mortality (%)	0		0		0		0		100
t=24 h	0	0	0	0	0	0	0	3a	3	0
24h Total mortality (%)	0		0		0		0		100
t=48 h	0	0	0	0	0	0	0	3a	3	0
48h Total mortality (%)	0		0		0		0		100
t=72 h	0	0	0	0	0	0	0	3b	3	0
72h Total mortality (%)	0		0		0		0		100
t=96 h	0	0	0	0	0	0	0	3b	3	0
96h Total mortality (%)	0		0		0		0		100

M: Number of dead fish; S: Number of surviving fish but with sublethal effects / visible abnormalities.

The initial number of fish in each test vessel was 3.

^aFish with abnormal swimming behaviour (hypoactivity, abnormal surface distribution, under-reactive to stimulus) and hypo or irregular ventilation.

^bSame as above, except that all the fish were distributed to the bottom of the vessel (critical state).

^cFish were fish distributed to the bottom of the test vessel directly after introduction.

Table 2. pH-values during the final test

		Nominal concentration and corresponding analytically confirmed concentration (geometric mean) (mg test item/L) pH
		Control (0)	1.0 (0.86)	1.8 (1.36)	3.2 (2.39)	5.6 (4.24)	10 (9.90)
Start t=0 h		6.81	6.97	7.04	7.11	7.48	7.40
t=24 h	Old	7.07	6.91	6.78	6.90	6.92	6.80
	Fresh	7.22	6.96	6.79	7.07
t=48 h	Old	7.06	7.15	7.11	7.19
	Fresh	7.19	7.26	7.16	7.29
t=72 h	Old	6.91	7.12	7.25	7.35
	Fresh	7.11	7.11	7.31	7.13
End t=96 h		7.40	7.41	7.44	7.44

no further analysis was performed for concentrations in which all fish were dead

Table 3. Dissolved oxygen concentrations (mg/L) and corresponding air saturation values (%) during the final test

		Nominal concentration and corresponding analytically confirmed concentration (geometric mean) (mg test item/L) O2
		Control (0)	1.0 (0.86)	1.8 (1.36)	3.2 (2.39)	5.6 (4.24)	10 (9.90)
Start t=0 h		8.99 [94.3%]	9.29 [97.1%]	9.22 [96.6%]	9.20 [96.3%]	9.32 [97.5%]	9.26 [96.7%]
t=24 h	Old	9.50 [98.1%]	9.45 [97.0%]	9.42 [96.9%]	9.47 [96.2%]	9.53 [97.3%]	9.53 [96.4%]
	Fresh	9.56 [98.2%]	9.50 [97.1%]	9.57 [97.1%]	9.63 [97.6%]
t=48 h	Old	9.02 [95.2%]	9.23 [96.9%]	9.13 [96.0%]	9.08 [95.6%]
	Fresh	9.40 [98.9%]	8.70 [91.6%]	9.39 [98.9%]	9.32 [97.9%]
t=72 h	Old	9.04 [97.2%]	8.56 [91.9%]	9.02 [97.3%]	9.03 [96.9%]
	Fresh	8.72 [94.0%]	9.31 [99.0%]	9.32 [98.9%]	9.31 [98.9%]
End t=96 h		8.59 [93.5%]	8.82 [95.3%]	8.94 [96.6%]	8.84 [95.0%]

no further analysis was performed for concentrations in which all fish were dead

Table 4. Temperatures (°C) measured during the final test

		Nominal concentration and corresponding analytically confirmed concentration (geometric mean) (mg test item/L)
		Control (0)	1.0 (0.86)	1.8 (1.36)	3.2 (2.39)	5.6 (4.24)	10 (9.90)
Start t=0 h		23.2	23.1	23.0	23.1	23.2	23.5
t=24 h	Old	23.0 Min 23.0 Max 23.4	23.0	23.0	23.0	23.1	23.4
	Fresh	23.1	23.2	23.0	23.1
t=48 h	Old	23.0 Min 23.0 Max 23.4	23.0	23.0	23.6
	Fresh	23.0	23.1	23.0	23.2
t=72 h	Old	23.4 Min 23.0 Max 23.4	23.6	23.3	23.1
	Fresh	23.2	23.3	23.2	23.2
End t=96 h		23.4 Min 23.2 Max 23.6	23.4	23.0	23.9

no further analysis was performed for concentrations in which all fish were dead.

Temperature was measured continuously in the control in order to obtain the values of temperature fluctuations throughout the test; Min: lowest temperature recorded; Max: highest temperature recorded.

Table 5. Incidence of mortality and total mortality during the final test

	Nominal concentration and corresponding analytically confirmed concentration (geometric mean) (mg test item/L)
Cumulative mortality	Control (0)		1.0 (0.86)		1.8 (1.36)		3.2 (2.39)		5.6 (4.24)		10 (9.90)
	M	S	M	S	M	S	M	S	M	S	M	S
t=3 h	0	0	0	0	0	0	0	7b	7	0	7	0
t=6 h	0	0	0	0	0	0	0	7b	7	0	7	0
t=24 h	0	0	0	0	0	7a	0	7b	7	0	7	0
24 h Total mortality (%)	0		0		0		0		100		100
t=48 h	0	0	0	0	0	7a	0	7b	7	0	7	0
48 h Total mortality (%)	0		0		0		0		100		100
t=72 h	0	0	0	0	0	7a	0	7b, c	7	0	7	0
72 h Total mortality (%)	0		0		0		0		100		100
t=96 h	0	0	0	0	0	7a	1	6b	7	0	7	0
96 h Total mortality (%)	0		0		0		14.3		100		100

M: Number of dead fish S: Number of surviving fish but with sublethal effects / visible abnormalities The initial number of fish in each test vessel was 7 ^aAll fish (7/7) with abnormal swimming behaviour (hypoactivity). ^bAll fish (7/7) with abnormal swimming behaviour (hypoactivity and abnormal distribution) and loss of equilibrium (abnormal horizontal orientation and loss of buoyancy control). ^cSame as b for one fish + under-reaction to stimulus. 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 IV). Samples taken from all treatments were analysed at the start (t=0h) and every 24 hours thereafter in old and new solutions with surviving fish in order to determine maintenance of actual concentrations of the test item. The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were reproducible. Analyses of samples taken from all test concentrations during the test revealed that test item levels were maintained within or close to 80% (75 to 99%) of the nominal concentrations. The evaluation of the effects on Danio rerio was based on the measured concentrations (geometric means of the analytically confirmed concentrations), as recommended by OECD Guideline 203.

Validity criteria of the study Controls: No mortality was observed in the control at the end of the test. Dissolved [O2]: The dissolved oxygen concentration was higher than 60% of the air saturation value throughout the test in all test vessels (see Table 3). Test substance: Concentrations of the test item were overall stable during the test; results were based on the measured concentrations (geometric means of the analytically confirmed concentrations), as recommended by the test guideline. Therefore, the present study can be considered valid.

 

Validity criteria fulfilled:

yes

Remarks:

See Any other information on results

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item N,N-dimethyldodec-9-enamide to the zebrafish Danio rerio. The method followed was designed to be compliant with OECD TG 203, "Fish Acute Toxicity Test", referenced as Method C.1 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 measured criterion was the LC50 (Median Lethal Concentration), a statistically derived concentration which is expected to cause death in 50% of test animals within a period of 96 hours.

Methods:

Following a preliminary range-finding test, groups of seven fish were exposed to test solutions prepared individually over 96 hours at the required nominal concentrations 1.0, 1.8, 3.2, 5.6 and 10 mg/L in aquaria closed over the study period with a minimum headspace. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 3, 6, 24, 48, 72 and 96 hours. Samples taken from the control and all test solutions were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

Results:

The analytical results of this test showed that test item levels were overall satisfactory maintained within or close to 80% (75% to 99%) of the nominal concentrations throughout the duration of the test. The evaluation of the effects on Danio rerio was based on the measured concentrations (geometric means of the analytically confirmed concentrations), as recommended by OECD TG 203: 0.86, 1.36, 2.39, 4.24 and 9.90 mg/L. After the 96-hours exposure, mortality was 0% at 0.86 and 1.36 mg/L, 14.3% at 2.39 mg/L and 100% at 4.24 and 9.90 mg/L. Therefore, the highest concentration without effect (= NOEC) was 1.36 mg/L and the lowest concentration with 100% mortality was 4.24 mg/L.

The 24, 48, 72 and 96-hour LC50 values were as follows:

Time (h)	LC50 (mg test item/L)	95% Confidence limits (mg test item/L)
24 & 48 & 72	3.18*	2.39 – 4.24**
96	2.94	2.53 – 3.41

*This value was determined by ToxRat but can also be determined using the geometric mean of the highest concentration causing no mortality and the lowest concentration producing 100% mortality.

**The highest concentration causing no mortality and the lowest concentration producing 100% mortality were used as confidence limits.

Conclusion. The toxic effect of N,N-dimethyldodec-9-enamide to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 2.94 mg/L. The NOEC was 1.36 mg/L.

Description of key information

The toxic effect of N,N-dimethyldodec-9-enamide to the zebrafish (Danio rerio) was investigated in a closed semi-static test performed according to OECD TG 203. Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 2.94 mg/L. The NOEC was 1.36 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

2.94 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item N,N-dimethyldodec-9-enamide to the zebrafish Danio rerio. The method followed was designed to be compliant with OECD TG 203, "Fish Acute Toxicity Test", referenced as Method C.1 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 measured criterion was the LC50 (Median Lethal Concentration), a statistically derived concentration which is expected to cause death in 50% of test animals within a period of 96 hours.

Following a preliminary range-finding test, groups of seven fish were exposed to test solutions prepared individually over 96 hours at the required nominal concentrations 1.0, 1.8, 3.2, 5.6 and 10 mg/L in aquaria closed over the study period with a minimum headspace. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 3, 6, 24, 48, 72 and 96 hours. Samples taken from the control and all test solutions were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

The analytical results of this test showed that test item levels were overall satisfactory maintained within or close to 80% (75% to 99%) of the nominal concentrations throughout the duration of the test. The evaluation of the effects on Danio rerio was based on the measured concentrations (geometric means of the analytically confirmed concentrations), as recommended by OECD TG 203: 0.86, 1.36, 2.39, 4.24 and 9.90 mg/L. After the 96-hours exposure, mortality was 0% at 0.86 and 1.36 mg/L, 14.3% at 2.39 mg/L and 100% at 4.24 and 9.90 mg/L. Therefore, the highest concentration without effect (= NOEC) was 1.36 mg/L and the lowest concentration with 100% mortality was 4.24 mg/L.

The 24, 48, 72 and 96-hour LC50 values were as follows:

Time (h)	LC50 (mg test item/L)	95% Confidence limits (mg test item/L)
24 & 48 & 72	3.18*	2.39 – 4.24**
96	2.94	2.53 – 3.41

*This value was determined by ToxRat but can also be determined using the geometric mean of the highest concentration causing no mortality and the lowest concentration producing 100% mortality.

**The highest concentration causing no mortality and the lowest concentration producing 100% mortality were used as confidence limits.