(E)-4-Ethoxy-1,1,1-trifluoro-3-buten-2-one (stabilized with BHT)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-05 May 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. Therefore full validation applies.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD, 1984

Deviations:

no

Remarks:

The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Concentrations were analysed in all treatments: control (algal medium) and exposed to 1.0, 2.2, 5.0, 11 and 25 mg/L.
- Sampling method:
* Samples of 100 μL were taken in triplicate from the bulk solutions and algal medium at the beginning of the study,
* Samples of 1.0 mL were taken in triplicate from experimental flasks at the end of the study (on day 3).
- Sample storage conditions before analysis: The samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: On the day of test initiation, a stock solution of 100 mg/L was prepared by dissolving 50 mg ETFBO in 0.5 L algal medium. Test solutions at 0, 1.0, 2.2, 5.0, 11 and 25 mg/L were prepared from this stock solution as reported in the table "Preparation of the test solutions" (see in the field "Any other information on materials and methods incl. tables").
- Controls: Algal medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Particles of the test substance were not visible.
- Other relevant information: All test solutions were clear.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green alga.
- Strain: ATCC 22662.
- Source: A culture of Selenastrum capricornutum was received on 19 January 2004 from the Culture Collection of Algae and Protozoa, Ambleside, Cumbria, UK.
- Method of cultivation: The algae were culturedin 100-mL Erlenmeyer flasks with 50 mL filter sterilised medium. Each week a volume of 0.5 - 2 mL of this culture was transferred into fresh algal medium.
No further data.

ACCLIMATION
- Acclimation period: The preculture used for this study was transferred into fresh algal medium 4 days prior to the start of the test.
- Culturing media and conditions: The culture chamber was illuminated 24 hours a day with fluorescent lamps.
No further data.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

22 to 25 °C
This is in agreement with the criterion of 21 to 25 °C. However, it was not possible to meet the validity criterion of temperature control at +/- 2 °C as the precision of the Chessel type 346 recorder is lower than the Yokogawa temperature recoding system (see in the field "Any other information on materials and methods incl. tables").

pH:

Control: 7.8 on day 0 and 8.3 on day 3
Exposed groups (pH only available on day 3):
1.0 mg/L: 8.3
2.2 mg/L: 8.3
5.0 mg/L: 8.2
11 mg/L: 8.0
25 mg/L: 7.9

Nominal and measured concentrations:

Nominal concentrations: 0, 1.0, 2.2, 5.0, 11 and 25 mg/L.
Measured concentrations: not detected, 1.1, 2.1, 4.8, 10.5 and 24.0 mg/L (mean of concentrations measured on day 0 and day 3).
N.B. As ETFBO hydrolyses quickly (T1/2 is < 5 min at the test temperature), the concentration in the test solutions was based on the hydrolysis product, 4,4,4-trifluoro-3-oxobutanal (Eicher, 2006).

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks.
- Fill volume: 250 mL.
- Volume of solution: 100 mL.
- Shaking: The Erlenmeyer flasks were placed in a shaking incubator with a temperature setpoint of 24 +/- 1 °C. The shaking speed in the incubator was 90 - 110 rotations per minute.
- Initial cells density: 1 x 10E4 cells/mL.
- Control end cells density: 152.6 x 10E4 cells/mL (thus icrease factor > 16 which met the validity criterion).
- No. of vessels per concentration (replicates): 3.
- No. of vessels per control (replicates): 3.

GROWTH MEDIUM
- Standard medium used: Yes, the algal medium used in this study had a similar composition as OECD TG 201 medium. Algal medium was prepared in purified water (purified water was prepared according to the methods of European Pharmacopeia and the USP Purified Water).

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No.
- Intervals of water quality measurement: The pH of the algal medium was measured at test initiation. The pH of the test solutions was measured at test termination in one test vessel per concentration.

OTHER TEST CONDITIONS
- Sterile test conditions: yes.
- Adjustment of pH: no.
- Photoperiod: The Erlenmeyer flasks in the shaking incubator were illuminated continuously with fluorescent lamps.
- Light intensity and quality: The light intensity at the level of the Erlenmeyer flasks was 75 µE/m2.s, which is in agreement with the criterion of 60 - 120 µE/m2.s (ISO, 1989).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Before test initiation, the algal density was determined by using a counting chamber (Neubauer improved) and a microscope (magnification 200 times). During the test, the absorption at 750 nm was measured after 24, 48 and 72 hours in all Erlenmeyer flasks using a spectrophotometer (HP 8453) and a cuvette with a path length of 5 cm. A calibration line was used to determine the cell density of the test solutions based on measurements of the absorption (linear interpolation method). The algal preculture was used to prepare the calibration line. The cell density measurements were used to calculate the biomass integral (day 3) and growth rate (days 0-3) for each Erlenmeyer flask. These results were used to determine the mean biomass integral and mean growth rate for each test concentration. Hereafter the inhibition (%) of the biomass integral and the inhibition (%) of the growth rate were calculated for each test concentration.
- Chlorophyll measurement: No.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2.
- Range finding study: yes; not detailed except that the range-finding study allowed to select concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L during the definitive study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

BIOLOGICAL RESULTS
- In several parts of the report, it is indicated that effect concentrations could be based on nominal concentrations because the differences between the nominal and the mean measured concentrations were < 20%. In this sense, it seems the NOEC (biomass) was well expressed on a nominal basis. The EC50 (growth rate and biomass) however seem to have been expressed on a measured basis using linear interpolation with mean measured concentrations on a logarithmic scale. Anyway, the use of nominal or measured concentrations should not affect the global conclusions of the study; the difference between them being < 20 %.
- No NOEC value was available for growth rate. In the absence of details on the statistical results, it was difficult to determine the highest concentration without significant effect on growth rate. From Table 3 reported in the field "Any other information on results incl. tables", it appears that growth rate inhibitions equaled 0.9, 2.9, 14, 34 and 56 % for the nominal concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L, respectively. Considering these percentages, the fact that the other available ecotoxicological value (i.e. EC50) is significantly higher when based on growth rate than on biomass and the fact that the available 72h-NOEC value based on biomass is equal to 1 mg/L, it does not seem aberrant to conclude that the NOEC value based on growth rate should be superior to 1 mg/L. As a matter of precaution and in the absence of a precise numerical NOEC value based on growth rate, it was nevertheless decided to conclude on the long-term toxicity to algae by considering the acute EC50 value (growth rate) for algae together with environmental fate information.
- Validity criteria: In the study report, the only validity criterion cited for control algae is the factor of biomass increase which should be >= 16. This criterion was fulfilled (see in the field "Test conditions / Details on test conditions / TEST SYSTEM". The current OECD 201 guideline cites two other validity criteria which were not investigated at the time of the study report:
* The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%. It was re-calculated from raw data to be equal to 15.1% in this study.
* The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7%. It was re-calculated from raw data to be equal to 1% in this study.

ANALYTICAL RESULTS
As ETFBO hydrolyses quickly (T1/2 is < 5 min at the test temperature), the concentration in the test solutions was based on the hydrolysis product, 4,4,4-trifluoro-3-oxobutanal (Eicher, 2006):
Measured concentrations in the control group: not detected.
Measured concentrations in the exposed groups:
* 1.0 mg/L (nominal): 1.28 mg/L on day 0 and 0.83 mg/L on day 3 (mean: 1.1 mg/L).
* 2.2 mg/L (nominal): 2.47 mg/L on day 0 and 1.73 mg/L on day 3 (mean: 2.1 mg/L).
* 5.0 mg/L (nominal): 5.51 mg/L on day 0 and 4.15 mg/L on day 3 (mean: 4.8 mg/L).
* 11 mg/L (nominal): 12.1 mg/L on day 0 and 8.99 mg/L on day 3 (mean: 10.5 mg/L).
* 25 mg/L (nominal): 27.5 mg/L on day 0 and 20.4 mg/L on day 3 (mean: 24 mg/L).
The differences between the nominal and the mean measured concentrations were thus < 20%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes (72h-EC50 of potassium dichromate within the acceptance range of 0.28-1.1 mg/L for biomass integral and 0.14-1.6 for growth rate).
- 72h-EC50: 0.48 mg/L for biomass integral and 0.89 mg/L for growth rate (test conducted from 04 to 07 April 2006).

Reported statistics and error estimates:

Linear interpolation, with concentration on a logarithmic scale (base = 10), was used to determine the 72h-EC50 for biomass integral and growth rate inhibition. The 72h-NOEC, based on biomass integral, was determined with William's Test (Williams, 1972) using the statistical program SAS.

Table 1 – Mean measured algal cell densities

 

Nominal concentration (mg/L)	Mean cell density (x 10E4 cells/mL)
	Day 1	Day 2	Day 3
Control	4.315	30.26	152.6
1.0	4.315	30.83	144.8
2.2	4.468	29.75	132.0
5.0	3.432	27.92	74.41
11	3.845	8.891	28.61
25	3.332	3.506	9.159

 

Table 2 – Mean biomass integral and biomass integral inhibition (%) on day 3

 

Nominal concentration (mg/L)	Biomass integral (x 10E4)			Biomass integral inhibition on day 3 (%)
	Day 1	Day 2	Day 3
Control	1.66	17.9	108	-
1.0	1.66	18.2	106	2.6
2.2	1.73	17.8	97.7	9.8
5.0	1.22	15.9	66.1	39.0
11	1.42	6.79	24.5	77.4
25	1.17	3.58	8.92	91.8

 

Table 3 – Mean growth rate inhibition (%)

 

Nominal concentration (mg/L)	Growth rate inhibition (%)
	Day 0-1	Day 1-2	Day 2-3	Day 0-3
1.0	-0.2	-0.9	4.0	0.9
2.2	-2.3	2.5	7.9	2.9
5.0	16	-6.8	39	14
11	7.9	58	28	34
25	18	97	41	56

 

Validity criteria fulfilled:

yes

Remarks:

Factor of biomass increase in the controls > 16, mean coef. of variation for section-by-section specific growth rates in the controls =< 35% and coef. of variation of average specific growth rates during the whole test period in replicate controls =< 7%.

Conclusions:

Initial exposure to ETFBO and then to its hydrolysis product triggered a 72h-EC50 of 20 mg/L based on growth rate and 6.3 mg/L based on biomass. Considering the value based on growth rate, the preferred endpoint when assessing algal toxicity, a conclusion as harmful to Selenastrum capricornutum needs to be applied under the present experimental conditions.
No NOEC value was available for growth rate. In the absence of details on the statistical results, it was difficult to determine the highest concentration without significant effect on growth rate. Growth rate inhibitions equaled 0.9, 2.9, 14, 34 and 56 % for the nominal concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L, respectively. Considering these percentages, the fact that the other available ecotoxicological value (i.e. EC50) is significantly higher when based on growth rate than on biomass and the fact that the available 72h-NOEC value based on biomass is equal to 1 mg/L, it does not seem aberrant to conclude that the NOEC value based on growth rate should be superior to 1 mg/L. As a matter of precaution and in the absence of a precise numerical NOEC value based on growth rate, it was nevertheless decided to apply the conclusion as harmful to Selenastrum capricornutum with long-lasting effects based on the acute EC50 value (growth rate) together with environmental fate information.

Executive summary:

The acute toxicity to algae was investigated in a GPL-compliant study performed according to OECD test guideline 201.

Algal cultures (Selenastrum capricornutum) were exposed for 72 hours under static conditions to a control treatment and to treatments exposed to 1.0, 2.2, 5.0, 11 and 25 mg/L ETFBO (nominal concentrations). Algal cell densities were measured after 24, 48 and 72 hours and inhibitions of biomass integral and growth rate were calculated.

To study exposure to ETFBO, samples of test solutions were taken and analysed with HPLC at the beginning (on day 0) and at the end of the test (on day 3). As ETFBO hydrolyses quickly (T1/2 is < 5 min at the test temperature), the concentration in the test solutions was based on the hydrolysis product, 4,4,4-trifluoro-3-oxobutanal (Eicher, 2006). The means of concentrations measured on day 0 and day 3 were equal to 1.1 (1.0 nominal), 2.1 (2.2 nom.), 4.8 (5.0 nom.), 10.5 (11 nom.) and 24.0 (25 nom.) mg/L; meaning that the difference between nominal and measured concentrations was < 20 % and that effect concentrations could be thus based on nominal concentrations. NOEC (biomass) was well expressed on a nominal basis. The EC50 (growth rate and biomass) however seem to have been expressed on a measured basis. Anyway, the use of nominal or measured concentrations should not affect the global conclusions of the study; the difference between them being < 20 %.

The 72h-EC50 was set equal to 20 mg/L based on growth rate and 6.3 mg/L based on biomass. Considering the value based on growth rate, the preferred endpoint when assessing algal toxicity, a conlusion as harmful to Selenastrum capricornutum needs to be applied under the present experimental conditions. No NOEC value was available for growth rate. In the absence of details on the statistical results, it was difficult to determine the highest concentration without significant effect on growth rate. Growth rate inhibitions equaled 0.9, 2.9, 14, 34 and 56 % for the nominal concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L, respectively. Considering these percentages, the fact that the other available ecotoxicological value (i.e. EC50) is significantly higher when based on growth rate than on biomass and the fact that the available 72h-NOEC value based on biomass is equal to 1 mg/L, it does not seem aberrant to conclude that the NOEC value based on growth rate should be superior to 1 mg/L. As a matter of precaution and in the absence of a precise numerical NOEC value based on growth rate, it was nevertheless decided to apply the conclusion as harmful to Selenastrum capricornutum with long-lasting effects based on the acute EC50 value (growth rate) together with environmental fate information.

Description of key information

Initial exposure to ETFBO and then to its hydrolysis product triggered a 72h-EC50 of 20 mg/L based on growth rate and 6.3 mg/L based on biomass. Considering the value based on growth rate, the preferred endpoint when assessing algal toxicity, a conclusion as harmful to Selenastrum capricornutum needs to be applied under the present experimental conditions.

No NOEC value was available for growth rate. In the absence of details on the statistical results, it was difficult to determine the highest concentration without significant effect on growth rate. Growth rate inhibitions equaled 0.9, 2.9, 14, 34 and 56 % for the nominal concentrations of 1.0, 2.2, 5.0, 11 and 25 mg/L, respectively. Considering these percentages, the fact that the other available ecotoxicological value (i.e. EC50) is significantly higher when based on growth rate than on biomass and the fact that the available 72h-NOEC value based on biomass is equal to 1 mg/L, it does not seem aberrant to conclude that the NOEC value based on growth rate should be superior to 1 mg/L. As a matter of precaution and in the absence of a precise numerical NOEC value based on growth rate, it was nevertheless decided to apply the conclusion as harmful to Selenastrum capricornutum with long-lasting effects based on the acute EC50 value (growth rate) together with environmental fate information.

Key value for chemical safety assessment

EC50 for freshwater algae:

20 mg/L

Additional information

An experimental study, conducted according to OECD guideline and GLP, is available. It is considered as fully reliable, and the results are retained as key data.