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Ecotoxicological information

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:
other: 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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 concentrationsopen allclose all
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
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
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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
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
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