α,α,α-trifluoro-m-toluidine

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

Study period:

05 November 2010 to 25 November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This endpoint was generated in accordance with OECD Guideline No. 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test). The study was performed in compliance with UK GLP standards.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2001

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:

3-trifluoromethylaniline

IUPAC Name:

3-trifluoromethylaniline

Details on test material:

Identification: 3-trifluoromethylanilie
Description: Extremely pale yellow liquid
Purity: 99.66 %
Batch Number: 101009
Date Received: 25 October 2010
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at -20 degrees celsius for further analysis if necessary.

Test solutions

Details on test solutions:

The test item was dissolved directly in the culture medium. 100 mg of test item was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. Each stock solution was inverted several times for homogeneity. Aliquots of these stock solutions were used as test solutions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 +/- 1 degrees celsius.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 +/- 1 degrees celsius throughout the test.

pH:

Control pH: 0 hours: 7.6; 72 hours: 7.8
A significant decline in pH was observed between 0 and 72 hours in the 25, 50 and 100 mg/L test cultures. This was considered to be due to the possible production of an acidic degradation product.

Nominal and measured concentrations:

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 87 % to 98 % of nominal. A marked decline in measured test concentrations was observed at 72 hours in the range of 7 % to 17 % of nominal. The substance is known to be unstable in both light and water.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for estimating EC50 values. The results were therefore based on the geometric mean of test concentrations.

Test item verification data are presented in Tables ###.

Details on test conditions:

EXPOSURE CONDITIONS
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.36 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 15 mL of this algal suspension gave an initial nominal cell density of 4 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated at 24 +/- 1 degrees celsius under continuous illumination (intensity approximately 7000 lux) provided by warm white light (380-730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 19 - 23

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 5.9 - 7.7

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

8.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours to identify any statistically significant differences (alpha = 0.05) between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table 1. Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/L)	Cell Densities (cells per mL)	Cell Densities (cells per mL)	Inhibition Values (%)	Inhibition Values (%)
	0 hours	72 Hours	Growth Rate	Yield
Control - R1	4.01E+03	2.48E+05
Control - R2	4.10E+03	2.47E+05	-	-
Control - Mean	4.05E+03	2.47E+05
0.10 mg/L - R1	4.03E+03	3.69E+05
0.10 mg/L - R2	4.02E+03	3.12E+05	9	38
0.10 mg/L - Mean	4.02E+03	3.40E+05
1.0 mg/L - R1	4.06E+-3	3.60E+05
1.0 mg/L - R2	4.06E+03	2.47E+05	5	23
1.0 mg/L - Mean	4.06E+03	3.04E+05
10 mg/L - R1	4.06E+03	3.95E+05
10 mg/L - R2	4.06E+03	4.01E+05	12	62
10 mg/L - Mean	4.06E+03	3.98E+05
100 mg/L - R1	4.02E+03	1.22E+04
100 mg/L - R2 100 mg/L - Mean	4.05E+03  4.03E+03	7.78E+03 9.98E+03	77	98

Table 2. Cell Densities and pH Values in the Definitive Test

Nominal Concentration	pH 0h	Cell Densities (cells/mL) 0 h	24h	48 h	72 h	pH 72 h
Control - R1 R2 R3 R4 R5 R6 Mean	7.6 7.6 7.6 7.6 7.6 7.6	3.80E+03 3.11E+03 3.46E+03 3.82E+03 4.65E+03 3.71E+03 3.76E+03	9.46E+03 1.21E+04 1.06E+04 9.05E+03 1.48E+04 9.40E+03 1.09E+04	4.90E+04 4.88E+04 5.05E+04 4.94E+04 5.01E+04 4.82E+04 4.93E+04	2.39E+05 2.42E+05 2.50E+05 2.30E+05 2.47E+05 2.75E+05 2.47E+05	7.8 7.8 7.8 7.8 7.8 7.8
6.25 - R1 R2 R3 Mean	7.5 7.5 7.5	4.37E+03 4.28E+03 4.09E+03 4.25E+03	1.12E+04 1.43E+04 9.70E+03 1.17E+04	7.35E+04 6.70E+04 4.71E+04 6.25E+04	4.31E+05 4.45E+05 4.34E+05 4.36E+05	7.7 7.8 7.7
12.5 - R1 R2 R3 Mean	7.5 7.5 7.5	4.50E+03 4.01E+03 4.25E+03 4.25E+03	4.63E+03 7.95E+03 9.36E+03 7.31E+03	3.52E+04 3.30E+04 2.44E+04 3.09E+04	2.22E+05 2.94E+05 2.06E+05 2.41E+05	7.5 7.5 7.5
25 - R1 R2 R3 Mean	7.4 7.4 7.4	4.42E+03 4.11E+03 4.35E+03 4.30E+03	4.10E+03 8.38E+03 4.56E+03 5.68E+03	4.55E+03 1.02E+04 9.42E+03 8.07E+03	1.84E+04 1.75E+04 1.21E+04 1.60E+04	4.0 4.1 4.0
50 - R1 R2 R3 Mean	7.4 7.4 7.4	4.11E+03 4.21E+03 4.01E+03 4.11E+03	3.46E+03 3.55E+03 3.57E+03 3.53E+03	6.72E+03 7.13E+03 8.45E+03 7.43E+03	4.18E+03 4.62E+03 2.68E+03 3.83E+03	3.5 3.6 3.5
100 - R1 R2 R3 Mean	7.4 7.4 7.4	3.99E+03 3.84E+03 3.90E+03 3.91E+03	1.43E+03 2.09E+03 3.41E+03 2.31E+03	3.70E+03 4.29E+03 3.78E+03 3.93E+03	1.66E+03 3.60E+03 4.70E+03 3.32E+03	3.3 3.3 3.3

Table 3. Daily Specific Growth Rate for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
	Day 0 -1	Day 1 -2	Day 2 -3
Control R1 R2 R3 R4 R5 R6 Mean	0.036 0.046 0.041 0.034 0.054 0.036 0.041	0.068 0.058 0.065 0.071 0.051 0.068 0.064	0.066 0.067 0.067 0.064 0.066 0.073 0.067

Table 4. Inhibition of Growth Rate amd Yield in the Definitive Test

	Growth Rate (cells/mL/hour)	Growth Rate (cells/mL/hour)	Yield (cells/mL)	Yield (cells/mL)
Nominal Concentration (mg/L)	0 -72 h	% Inhibition	0 -72 h	% Inhibition
Control R1 R2 R3 R4 R5 R6 Mean SD	0.057 0.057 0.057 0.056 0.057 0.059 0.057 0.001	-	2.35E+05 2.39E+05 2.47E+05 2.26E+05 2.43E+05 2.71E+05 2.44E+05 1.52E+04	-
6.25 R1 R2 R3 Mean SD	0.065 0.065 0.065 0.065 0.000	[14] [14] [14] [14]	4.26E+05 4.40E+05 4.30E+05 4.32E+05 7.31E+03	[77]
12.5 R1 R2 R3 Mean SD	0.060 0.056 0.055 0.057 0.003	[5] 2 4 0	2.89E+05 2.18E+05 2.02E+05 2.36E+05 4.66E+04	3
25 R1 R2 R3 Mean SD	0.021 0.020 0.015 0.019 0.003	63 65 74 67	1.39E+04 1.34E+04 7.72E+03 1.17E+04 3.44E+03	95
50 R1 R2 R3 Mean SD	0.001 0.002 -0.006 -0.001 0.004	98 96 111 102	7.20E+01 4.16E+02 -1.33E+03 -2.80E+02 9.24E+02	100
100 R1 R2 R3 Mean SD	-0.012 -0.001 0.002 -0.004 0.007	121 102 96 106	-2.34E+03 -2.40E+02 7.92E+02 -5.95E+02 1.59E+03	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Desmodesmus subspicatus gave the following results:

Nominal Concentrations:
ECr50 = 21 mg/L (95 % confidence limits: 19-23); NOEC = 12.5 mg/L; LOEC = 25 mg/L
ECY50 = 15 mg/L; NOEC = 12.5 mg/L; LOEC = 25 mg/L

Measured Concentrations (Geometric Mean):
ECr50 = 6.7 mg/L (95 % confidence limits: 5.9 - 7.7); NOEC = 3.2 mg/L; LOEC = 8.4 mg/L
ECY50 = 4.3 mg/L; NOEC = 3.2 mg/L; LOEC = 8.4 mg/L