2-Butenoic acid, 4,4,4-trifluoro-3-(methylamino)-, ethyl ester, (2Z)-

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

EC Number:

801-277-8

Cas Number:

507448-65-9

Molecular formula:

C7H10F3NO2

IUPAC Name:

ethyl (2Z)-4,4,4-trifluoro-3-(methylamino)but-2-enoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Specimens of each test concentration were drawn from the approximate center of the test vessels.
They were taken immediately before exposure and after 96 hours exposure.
If any of the concentrations showed 100 % mortality before the end of the test, specimens were taken when all fish had died.
The specimens were kept at -18 °C to -22 °C until analysis.

Test solutions

Details on test solutions:

100.3 – 250.7 mg CA 2245 were weighed, dissolved and made up to 100.0 ml with methanol. From these stock solutions, reference solutions in the range of the test concentrations (related to and extended to about 10 % below and above the expected concentration) were diluted with methanol/test water = 1/1 (v/v). So references with concentrations between 0.50 – 25.07 mg/L CA 2245 were used.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Source: Commercial supplier: from own culture.
- Age at study:
- Length at study initiation: (mean) 50 mm (range 46 - 56 mm) based on 7 fish of the control
- Weight at study initiation: (mean) 1.0 g (range 0.77 - 1.40 g) based on 7 fish of the control
- Feeding during test: No feeding during the test period.

ACCLIMATION
- Acclimation period: 88 days
- Acclimation conditions: In water of similar quality used in the test.
- Type and amount of food: Commercially prepared trout food supplemented by living, frozen or dried organisms until
24 hours prior to the test.
- Health during acclimation: Total mortality did not exceed 3 % during the 7 day period prior to the start of the test.

Study design

Test type:

flow-through

Total exposure duration:

96 h

Test conditions

Hardness:

Total hardness 187 mg CaCO3 /L.

Test temperature:

13.4 – 14.3°C

pH:

8.2 – 8.4

Dissolved oxygen:

Dissolved oxygen contents between 92 – 103 % of the saturation value by gentle aeration during exposure.

Nominal and measured concentrations:

The nominal concentrations were 10, 17, 31, 56 and 100 mg test item/L.
The measured concentrations were 4.1, 7.7, 14, 24 and 52 mg/L test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 20 litres filled with 15 litres of water (35/22/26 cm; depth of water: 20 cm)
- Aeration: The water was gently aerated during exposure (oil free air).
- No. of organisms per vessel: 7 per aquaria
- Biomass loading rate: Fish biomass to solution ratio was 0.073 g/L in 24 hours.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water of the town of Basel (carbon fine filtered to eliminate
chlorine and particulate material).

OTHER TEST CONDITIONS
- Photoperiod: Light/datk: 16 hours light/8 hours dark with a 30 minutes transistion period.
- Light intensity: Fluorescent light.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Test concentrations: The measured concentrations were 4.1, 7.7, 14, 24 and 52 mg/L test item/L.


OBSERVATION INTERVALS
- Water quality: The oxygen content (%), pH and temperature (°C) were measured in each test chamber at 0, 24, 48,
72 and 96 hours (as long as there remained any fish in the tank).
In addition, total hardness (as CaCO3) was measured in the blank control chamber immediately
before the start of the test.
Characteristics of the test solutions (such as precipitated materials, cloudiness, etc) were recorded in daily intervals.
- Biological: At 2, 4, 24, 48, 72 and 96 hours, observations of mortality and sublethal symptoms, such as
abnormal behavioural activity and stress were made (swimming behaviour, loss of equilibrium,
exophthalmos, respiratory function, pigmentation and other observations). Dead fish, if any,
were removed from the test solutions at least at the above mentioned intervals. At test termination,
the fish in the control were retained for measurement of weight and length.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

ca. 18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

ca. 18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

ca. 22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

WATER QUALITY DATA
- Temperature: The temperature was maintained constant at 13.4 – 14.3°C
- Oxygen: The dissolved oxygen contents between 92 – 103 % of the saturation value by gentle aeration during exposure.
- pH: The pH was determined to be 8.2 – 8.4.
- Hardness: The total hardness 187 mg CaCO3 /L.

CONCENTRATION OF TEST ITEM IN WATER
- Homogeneity: The test item appeared homogeneously distributed in the test vessels at all test concentrations over the period of
the test.
- Other observations; No precipitation was observed in the stock solutions or the testing solutions throughout the duration of the test.
- Concentrations: The nominal concentrations were 10, 17, 31, 56 and 100 mg test item/L.
The measured concentrations were 4.1, 7.7, 14, 24 and 52 mg/L test item/L.

LETHAL EFFECTS
After 96 hours exposure, mortality occurred at concentrations of 24 and 52 mg/L with 100 and 100% mortality, respectively.
A decrease of the LC50-value from 22 to 18 mg/L was observed during the period 24 to 48 h exposure.
The LC 50 emained constant until the end of the exposure.
The LC 0 (96h) was 14 mg/L and the LC 100 (96h) 24 mg/L.
The mortality in the blank control was 0 %.

SUBLETHAL EFFECTS
Sublethal effects were observed at concentrations ≥ 4.1 mg/L, such as change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal and lethal effects was < 4.1 mg/L.

Any other information on results incl. tables

Sublethal observations / clinical signs:

The following calculations and values are based on measured mean concentrations. 

 

Exposure period	LC50	95% confidence	Slope	Goodness of fit
(h)	(mg/L)	(mg/L)	(mg/L)	X^2	df	p
96 72 48 24	18 18 18 22	none none none none	25.7 25.7 25.7 15.1	0.00 0.00 0.00 0.00	3 3 3 3	1.00 1.00 1.00 1.00

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The LC 50 (96h) of CA 2455 A was determined to be 18 mg/L based on measured mean concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, CA 2455 A is “harmful to rainbow Trout” based on the inherent toxicity of CA 2455 A in this test.
The highest concentration with no sublethal and lethal effects was < 4.1 mg CA 2455 A/L.
Based on similarity of the measured concentrations of CA 2455 A at the start and at the end of the exposure, the test item was stable to the test solutions over the period of the test under Flow-Through conditions.

Executive summary:

 

Static pre-tests showed that the LC50 (96h) was between > 100 mg/L. Therefore, the following nominal concentrations were chosen for the main test: 10, 17, 31, 56 and 100 mg test item/L. 

After the preparation of the test concentrations and an equilibrium time of 4 days, 7 fish were randomly placed in each of the 15 L glass aquaria containing 15 L of test solution (beginning of exposure). Fish were exposed to CA 2455 A for 96 hours and were observed for adverse effects at 2, 24, 48, 72 and 96 hours. 

 

Findings:

The temperature was maintained constant at 13.4 – 14.3°C and the dissolved oxygen contents between 92 – 103 % of the saturation value by gentle aeration during exposure. The pH was determined to be 8.2 – 8.4 and the total hardness 187 mg CaCO₃/L. 

The test item appeared homogeneously distributed in the test vessels at all test concentrations over the period of the test. No precipitation was observed in the stock solutions or the testing solutions throughout the duration of the test. 

The nominal concentrations were 10, 17, 31, 56 and 100 mg test item/L. The measured concentrations were 4.1, 7.7, 14, 24 and 52 mg/L test item/L. 

 

The following calculations and values are based on measured meanl concentrations. 

 

Exposure period	LC50	95% confidence	Slope	Goodness of fit
(h)	(mg/L)	(mg/L)	(mg/L)	X^2	df	p
96 72 48 24	18 18 18 22	none none none none	25.7 25.7 25.7 15.1	0.00 0.00 0.00 0.00	3 3 3 3	1.00 1.00 1.00 1.00

 

 

After 96 hours exposure, mortality occurred at concentrations of 24 and 52 mg/L with 100 and 100% mortality, respectively. 

A decrease of the LC50-value from 22 to 18 mg/L was observed during the period 24 to 48 h exposure. 

The LC 0 (96h) was 14 mg/L and the LC 100 (96h) 24 mg/L. 

 

Sublethal effects were observed at concentrations ≥ 4.1 mg/L, such as change in pigmentation, loss of equilibrium, change in the swimming behaviour and in the respiratory function, hence, the highest concentration with no sublethal and lethal effects was < 4.1 mg/L. 

 

Conclusion:

The LC 50 (96h) of CA 2455 A was determined to be 18 mg/L based on measured mean concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, CA 2455 A is “harmful to rainbow Trout” based on the inherent toxicity of CA 2455 A in this test. 

The highest concentration with no sublethal and lethal effects was < 4.1 mg CA 2455 A/L. 

Based on similarity of the measured concentrations of CA 2455 A at the start and at the end of the exposure, the test item was stable to the test solutions over the period of the test under Flow-Through conditions.