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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition 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:

2 samples (2 times about 31 ml per concentration and controls) were taken at the start and 2 samples (2 times about 30 ml per concentration and controls) at the end of exposure. Samples were taken from the freshly prepared test solutions immediately before exposure and from two composite test vessels after 72 hours exposure. All samples were kept at -18 to -22 °C until analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS (test Item):
10.334 g of CA 2455 A were mixed and made up to 10 ml with DMF (stock solution 1). 2.5 ml of stock solution 1 were mixed and made up to 5 ml with DMF to prepare stock solution 2. 2.5, 1.25, 0.625 and 0.312 ml of stock solution 1 were mixed and made up to 10 ml with DMF to prepare stock solutions3, 4, 5 and 6 respectively. 3 µl of each stock solution were mixed and made up to 31 ml with medium to prepare the test solutions

TEST CONCENTRATIONS:
A range-finding test (test concentrations 0.1, 1.0 and 10 mg/L) was conducted to determine a concentration range of CA 2455 A to use in the definitive test. The cell concentrations were measured at the start and at the end of exposure. No inhibition of the algae growth was seen at all test concentrations.

Based on the results of the range-finding test the following nominal concentrations were selected for the definitive test:
Nominal: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L.
Control: Reconstituted water.
Replicates: Each test concentration was tested in 3 replicates, the blank control and the vehicle control in 6.

Remark: Calculated amounts of the stock solution to produce the desired test concentrations and calculated amount of DMF to achieve identical vehicle concentration were given into the water and were homogeneously distributed. The algae were then transferred to the flasks.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Common name: Green Algae
- Strain: Selenastrunm capricornutum ATCC 22662 ≡ Pseudokirch subcapiata SAG 61.81
- Source: Collection of algae cultures, Pflanzenphysiologisches Institut, University, Nikolausberger Weg 180, D-37
073 Göttingen, Germany.
- Initial cell density: 9770 cells/ml
- Pre-culture: 3 days under test conditions
- Culture: The algal culture used was demonstrated to be adequately sensitive. The ErC 50 (0 – 72 h) and the EbC
50 (0 – 72 h) of potassium dichromate were 0.97 and 0.51 mg/L.

Study design

Test type:

static

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

24 ± 1°C

pH:

.9 – 8.0 at the start and between 7.1 – 7.6 at the end of exposure

Salinity:

Conductivity was measured as 142 µS

Nominal and measured concentrations:

The nominal test concentrations were: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L.
The measured test concentrations of CA 2455 A were 1.8, 3.8, 7.5, 19.2, 37.0 and 71.8 mg/L at the start of exposure and 0.03, <0.018, <0.018, 0.018, 0.03 and 0.07 mg test item/L at the end of exposure.

Details on test conditions:

Vessels: 100 ml Erlenmeyer flasks (total volume 125 ml), cotton stoppers, on Lab shaker, 30ml test solution per flask.

Temperature: 24 ± 1°C

Lighting: Continuous illumination, cold white fluorescent light (approx. 8000 lux)

Shaking rate: 150 rpm

Duration: 72 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth inhibition

Details on results:

Cell densities were measured at 24, 48 and 72 hours exposure.
Changes in mean density indicated that exponential growth occurred in the negative control replicates.
No significance difference in mean cell density between the controls and the 3.125, 6.25, 12.5, 25, 50 and 100 mg/L treatment groups on the basis of growth rate (0 – 10 % inhibition) were observed.
On the basis of AUC a significant inhibition was observed at a concentration of 100 mg test item/L (35 % inhibition).

Calculations of EC50 and NOEC are based on actual mean concentrations of CA 2455 A – refer to Table 1

Results with reference substance (positive control):

- The algal culture used was demonstrated to be adequately sensitive. The ErC 50 (0 – 72 h) and the EbC 50 (0 – 72 h) of potassium dichromate were 0.97 and 0.51 mg/L.

Any other information on results incl. tables

 

Table 1:

	Inhibition (growth rates)	Inhibition (areas under growth curve)
EC 50 (0 – 72 h): 95 % confidence limit: Slope: NOEC (0- - 72 h): (significant on 5% level)	ErC 50 : > 100 mg/L None None NOErC : 100 mg/L	EbC 50:> 100 mg/L     NOEbC : 50 mg/L

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC 50 (0 – 72 h) of CA 2455 A was determined to be >100 mg/L based on nominal concentrations.

The NOErC growth inhibition (0 – 72 h) was 100 mg/L based on nominal concentrations of CA 2455 A.

Executive summary:

 

Range-finding tests showed that the ErC50 (72h) would be greater than 10 mg/L. Based on the range-finding test, the following nominal concentrations of CA 2455 A were chosen for the main test: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L. 10.334 g of CA 2455 A were mixed and made up to 10 ml with DMF. This stock solution was diluted with DMF to prepare further stock solutions. 

The prepared test solutions were distributed in the test vessels before the algae were placed in the test vessels. When the algae are placed in the test vessels, the exposure starts (0h). Exposure of Selenastrum capricornutum was started at an initial cell density of 9770 cells/mL after pre-culture period of three days. 

 

The test with CA 2455 A was conducted at a temperature of 24 ± 1°C. Continuous illumination with cold white fluorescent light at 112 µE/m²(approx. 8000 lux) was used. The pH of the test solutions was 7.9 – 8.0 at the start and between 7.1 – 7.6 at the end of exposure. The multiplication factor of the algae cell density in the control within 72 hours was 51 in the control. 

The nominal test concentrations were: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L. The measured test concentrations of CA 2455 A were 1.8, 3.8, 7.5, 19.2, 37.0 and 71.8 mg/L at the start of exposure and 0.03, <0.018, <0.018, 0.018, 0.03 and 0.07 mg test item/L at the end of exposure. 

Despite the fact, that the test item is not miscible with water and is not hydrolytically stable, the initial recovery directly after application was between 57.6 – 76.8 % of nominal concentration

 

Following calculations of EC50 and NOEC are based on nominal concentrations. 

 

ErC 50 (0 – 72 h):                > 100 mg/L

95% confidence limit:           none

Slope:                                       none

NOEC (0 – 72 h):                NOErC : 100 mg/L

(significant on 5% level)

 

Conclusion:

ErC 50 (0 – 72 h) of CA 2455 A was determined to be >100 mg/L based on nominal concentrations. 

 

The NOErC growth inhibition (0 – 72 h) was 100 mg/L based on nominal concentrations of CA 2455 A.