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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:
Between 16 december 2011 to 29 june 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2011-08-31

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Triethyl phosphono acetate (TEPA)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.1, 1.0, 10, 100 mg/L for the pretest and 100 mg/L for the definitive test
- Sampling method: Sampling of test preparation at 0h and 72h
- Sample storage conditions before analysis: -20°C

Test solutions

Vehicle:
no
Details on test solutions:
For the range finding test:
An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solution of 10, 1.0 and 0.1 mg/L. An aliquot (450 mL) of each of the stock solution was separately inoculated with Algal suspension(6.5 mL) to give the required test concentration of 0.1, 1.0, 10 and 100 mg/L.

For the definitive test:
The test item was dissolved directly in culture medium. An amount of test item was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution. This stock solution was innoculated with algal suspension (14.6 mL) to give the required test concentration of 100 mg/L
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity
The concentration and stability of the test item in the preparations were verified by chemical analysis at 0 and 72h

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection):Culture Collection of Algae and Protozoa (CCAP) , SAMS Research services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): No data
- Method of cultivation: Culture in culture medium under constant aeration and illumination at 21°C ± 1

ACCLIMATION
- Acclimation period: No data
- Culturing media and conditions (same as test or not):Yes

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No

Test conditions

Hardness:
No data
Test temperature:
24°C ± 1 (throughout the test)
pH:
7.4 to 8.6 (measured at 0h and 72h)(See Cell densities and pH value in the definitive test)
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
nominal : 100 mg/L. the recovery after 72h was 84% of the start concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: glass conical flask
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 mL filled at 100mL
- Aeration: No
- Initial cells density: 5x10e3/mL
- Control and cells density, No. of organisms per vessel: See figure 1
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data
- Total organic carbon:No data
- Particulate matter:No data
- Metals:No data
- Pesticides:No data
- Chlorine:No data
- Alkalinity:No data
- Ca/mg ratio:No data
- Conductivity:No data
- Culture medium different from test medium:No data
- Intervals of water quality measurement:No data

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No measured using WTW PH 320 pH meter
- Photoperiod: light 24h/24h provided by warm white lighting (380-730 nm)
- Light intensity and quality:approximately 7000lux
- incubator: INFORS Multitron version 2 incubator

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter multisizer particle Counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: In the pretest, no inhibition of the growth rate occurred
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
In the valid pre-test study, four concentrations (Max 100 mg/L) of the test item were used. In this study, no inhibition on the growth of the Algal occurred.
The main study was performed likewise as a limit test using the concentration 100 mg/L. No inhibition occurred in the tested concentration.
At the beginning and the end of the test, the content of the test item in the test solutions was determined using a HPLC-MS.
The recovery after 72h was 84% of the start concentration, the determination of the biological results was based on the nominal concentration.
Results with reference substance (positive control):
- Results with reference substance were valid
ErC50(0 -72h): 1.4 mg/L, 95% confidence limits 1.2 -1.7 mg/L
EyC50(0 -72h): 0.59 mg/L, 95% confidence limits 0.53 -0.65 mg/L
Reported statistics and error estimates:
A student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and the control groups. All statistical analysis were performed using the SAS computer software package (SAS 1999-2001)

Any other information on results incl. tables

Cell densities and pH value in the definitive test

 

Nominal Concentration (mg/L)

pH

Cell densities * (Cell per mL)

pH

0h

0h

24h

48h

72h

72h

control

R1

7.4

5.45 .103

1.89. 104

8.41. 104

5.97. 105

8.6

R2

5.15 .103

2.07. 104

8.56. 104

4.14. 105

R3

5.54 .103

2.25. 104

1.13. 105

5.72. 105

R4

5.27 .103

1.94. 104

1.05. 105

4.76. 105

R5

5.76 .103

2.43. 104

1.05. 105

5.47. 105

R6

5.37 .103

2.16. 104

1.02. 105

4.92. 105

Mean

5.42 .103

2.12. 104

9.91. 104

5.16. 105

100

R1

7.4

6.33 .103

2.17. 104

1.17. 105

4.56. 105

8.5

R2

5.86 .103

2.28. 104

9.24. 104

4.64. 105

R3

5.13 .103

1.86. 104

9.69. 104

5.69. 105

R4

5.94 .103

2.49. 104

1.16. 105

6.20. 105

R5

5.66 .103

1.94. 104

1.05. 105

3.88. 105

R6

5.50 .103

2.50. 104

1.08. 105

4.75. 105

Mean

5.74 .103

2.21. 104

1.06. 105

4.95. 105

 

* = Cell densities represent the mean number of cell per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R1-R6 = Replicates 1 to 6

 

Daily specific Growth rates 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

0.055

0.062

0.082

R2

0.059

0.059

0.066

R3

0.063

0.057

0.068

R4

0.056

0.071

0.063

R5

0.066

0.061

0.069

R6

0.061

0.065

0.066

Mean

0.060

0.064

0.069

R1-R6 = Replicates 1 to 6

 

Inhibition of growth rate and yield in the definitive test

 

Nominal Concentration (mg/L)

Growth Rate

(cells/mL/hour)

Yield

(Cells/mL)

0-72h

% inhibition

0-72h

% inhibition*

control

R1

0.066

-

5.92

-

R2

0.061

4.09

R3

0.066

5.66

R4

0.063

4.70

R5

0.0652

5.42

R6

0.064

4.87

Mean

0.064

5.11

SD

0.002

6.82

100

R1

0.063

2

4.50

 

R2

0.063

2

4.58

 

R3

0.066

[3]

5.64

 

R4

0.067

[5]

6.14

 

R5

0.060

6

3.82

 

R6

0.063

2

4.70

 

Mean

0.064

1

4.90

4

SD

0.003

 

8.42

 

 

* = In accordance with the OECD test guideline only the mean value for yield is calculed

R1-R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
From the differents data, it is clear that the growth rate (r) and the yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a nominal concentration of 100 mg/L over 72-Hour exposure
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to TEPA at nominal concentrations of 100 mg/L under static conditions in accordance with the OECD 201.

The following results were determined:

NOEC = 100 mg/L

ErC50 (0 -72h) >100 mg/L

EyC50(0-72h) > 100 mg/L

In the both study (pre-test and main study), no inhibition on the growth of algae occurred.

This toxicity study is classified as acceptable and satisfies the guideline requirements for toxicity study.