α,α,α-trifluorotoluene

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:

from 2005-05-10 to 2005-07-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Methods of Testing New Chemical Substances (Algae growth inhibition test, Daphnia acute immobilization test, and Fish acute toxicity test) (Yakushokuhatsu No. 1121002, Heisei 15・11・ 13 Seikyoku No. 2, Kampo Kihatsu No. 031121002, 2003)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Stock solution of concentration 57 mg/L was prepared by dissolving the test chemical in test medium and stirring with ultrasonic agitation for 5 minutes. Then this solution was diluted with test medium to give each test concentration. The test chemical was stored at room temperature, darkness and light-interception. IR absorption spectra of the chemical were taken at the start and the end of the test, and both spectra were not contradictory to each other.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source: Obtained from American Type Culture Collection and cultivated aseptically using Gorham medium in laboratory.
- Method of cultivation: sterile

ACCLIMATION
- Pretreatment: Test algae were pre-incubated for 4 days under the same methods of the test.
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: none

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.9 - 22.2 °C

pH:

7.8 - 10.5

Nominal and measured concentrations:

Nominal Concentrations: control, 3.00, 6.30, 13.0, 27.0 and 57.0 mg/L
Measured Concentrations (TWA): control, 0.576, 1.49, 3.03, 5.63 and 12.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed system, stoppered flasks
- Material, size, headspace, fill volume: glass, 500 mL, 100 mL medium
- Aeration: no
- Initial cells density: 5000 cells/mL.
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD Medium

OTHER TEST CONDITIONS
- Sterile test conditions: none
- Adjustment of pH: none
- Photoperiod: continuously
- Light intensity and quality: 65 µE/m2/s
- Shaking: 100 rpm

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.4 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.96 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

other: area under growth curve

Remarks on result:

other: 95 % CI: 1.96 - 4.46 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.49 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Remarks:

and area under growth curve

Details on results:

- Analytical results
The ratio of measured concentration to nominal concentration was 81 - 103 % at the start of the exposure, and 5 - 11 % at the end of exposure. The main cause of the decrease was assumed to be due to the volatilization of test substance. Mean measured concentration (time weighted mean) was used to calculate the inhibition concentration.

- Morphological observation of algae
The result of the observation of cell morphology at the end of the exposure revealed that there were no changes in cell appearances (contraction, expansion, rupture, etc.) or no cell aggregation in all test groups, and that there was no difference compared with the control.

Results with reference substance (positive control):

EbC50 (0 - 72 h) for the reference substance was 0.431 mg/L.

Preliminary test

Concentrations 0.057, 0.57, 5.7 and 57 mg/L were tested for 72 hours. Inhibition above 50 % was observed at the highest tested concentration.

Main Test

The results of test substance concentration measurements showed that the ratio of measured concentration to nominal concentration at the start of the exposure was 81 - 103 %, and 5 - 11 % at the end of the exposure. The main cause of the decrease was assumed to be due to the volatilization of test substance.

Table 1. Concentration of the Test Substance in test medium

Nominal Conc. (mg/L)	Measured Concentration (mg/L) (Percent of Nominal, %)				Time-weighted mean* Conc. (mg/L) (Percent of Nominal, %)
	0 h	24 h	48 h	72 h
Control	-	-	-	-	-
3.00	3.08 (103)	0.331 (11)	0.253 (8)	0.163 (5)	0.576 (19)
6.30	5.93 (94)	1.00 (16)	0.862 (14)	0.708 (11)	1.49 (24)
13.0	11.9 (92)	2.03 (16)	1.77 (14)	1.47 (11)	3.03 (23)
27.0	21.8 (81)	3.88 (14)	3.23 (12)	2.71 (10)	5.63 (21)
57.0	49.3 (86)	7.98 (14)	7.04 (12)	5.74 (10)	12.2 (21)

* Time-weighted mean (as defined in OECD TG 211).

Growth curve

The cell concentration in the control increased by an average of 90 times during 72 hours of cultivation. In addition, the coefficient of variation of growth rate in replicate culture and section-by- section did not

exceed 15 % and 35 %, respectively, thus showing normal growth under the test condition (closed system). The cell concentration of each test group decreased (dependent on concentration – response relationship) with increasing concentration.

Table 2. Growth Rate, Inhibition and Cell density of Pseudokirchneriella subcapitata

Measured Mean Conc.^a (mg/L)	Growth rate (0 - 72 h)		Area under the growth curves (0 - 72 h)
	Rate µ (Mean)	Inhibition* (%)	Area A (Mean)	Inhibition* (%)
Control	0.0624	-	9143000	-
0.576	0.0649	-4.0	10182000	-11.4
1.49	0.0640	-2.6	8758000	4.2
3.03	0.0534	14.4**	4157000	54.5**
5.63	0.0296	52.6**	906000	90.1**
12.2	0.0079	87.3**	206000	97.7**

^a Time-weighted measured mean concentration (TWA)

* Values are the growth inhibition (%) relative to the control.

** Indicates a significant difference (α=0.01) from the control.

Observations

As the result of macroscopic color observation of test solution, there was an increasing trend in the extent of green color with time, in the control and test groups below 27.0 mg/L. For test group 57.0 mg/L, the test solution did not green until the end of the exposure (72 hours). As the result of microscopic observation of cell morphology at the end of the exposure, there was no changes in cell appearances (contraction, expansion, rupture, etc.) or no cell aggregation in all test groups, and that there was no difference compared with the control.

Validity

The cell concentration in the control group increased by 90 times on average in the 72-hour incubation period. The coefficient of variation of the growth rate for section-by-section and the coefficient of variation of the daily growth rate were 15 % and 35 %, respectively. It did not exceed and showed normal growth under the test conditions (closed system conditions). Cell concentration in each concentration range tended to decrease with increasing concentration (dose-dependently).

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure, the growth inhibition effects of the test item on Raphidocelis subcapitata were determined. Based on the growth rate the EC50 was determined to be 5.4 mg/L and the NOEC was 1.49 mg/L.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201. The study was conducted under static conditions with an initial cell density of 5000 cells/mL for a period of 72 hours. Nominal concentrations 3.00, 6.30, 13.0, 27.0 and 57.0 mg/L, and control was tested. The concentration in the test solutions was measured every 24 hours. At the beginning of the test the test concentrations were in the range of 81 to 103 % and at the end of the exposure period the concentration was reduced to 5 - 11 % of the nominal concentrations. The main cause of the decrease in concentration is the volatilization of the test item from the medium. The average value of the measured concentrations (time-weighted average, TWA) was used to calculate the inhibitory concentrations. The EC50-value for inhibition of growth rate (ErC50) was determined to be 5.4 mg/L. The NOEC was determined to be 1.49 mg/L. The EC50-value for the area under growth curve was determined to be 2.96 mg/L. The test item showed inhibitory effects on the green algae under the test conditions.

Description of key information

After 72 hours of exposure, the growth inhibition effects of the test item on Raphidocelis subcapitata were determined. Based on the growth rate the EC50 was determined to be 5.4 mg/L and the NOEC was 1.49 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.4 mg/L

EC10 or NOEC for freshwater algae:

1.49 mg/L

Additional information

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201 (reference 6.1.5-1). The study was conducted under static conditions with an initial cell density of 5000 cells/mL for a period of 72 hours. Nominal concentrations 3.00, 6.30, 13.0, 27.0 and 57.0 mg/L, and control was tested. The concentration in the test solutions was measured every 24 hours. At the beginning of the test the test concentrations were in the range of 81 to 103 % and at the end of the exposure period the concentration was reduced to 5 - 11 % of the nominal concentrations. The main cause of the decrease in concentration is the volatilization of the test item from the medium. The average value of the measured concentrations (time-weighted average, TWA) was used to calculate the inhibitory concentrations. The EC50-value for inhibition of growth rate (ErC50) was determined to be 5.4 mg/L. The NOEC was determined to be 1.49 mg/L. The EC50-value for the area under growth curve was determined to be 2.96 mg/L. The test item showed inhibitory effects on the green algae under the test conditions.