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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-02-2013 to 27-09-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Reason / purpose:
read-across: supporting information
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:
inspected: July 2012 ; signature: November 2012
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h and t=72 h
- Sampling method: 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. All 0-Hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: Stored in a freezer until analysis.
Vehicle:
no
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, Argyll, Scotland.
- Age of inoculum (at test initiation): Not reported.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and constant illumination at a temperature of 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Yes, until algal cell density was approximately 10^4 - 10^5 cells/mL
- Culturing media and conditions (same as test or not): Yes.
Medium : NaNO3 25.5 mg/l; MgCl2.6H2O 12.16 mg/l; CaCl2.2H2O 4.41 mg/l; MgSO4.7H2O 14.6 mg/l; KH2PO 1.044 mg/l; FeCl3.6H2O 0.16 mg/l; Na2EDTA.2H2O 0.3 mg/l; H3BO3 0.186 mg/l; MnCl2.4H2O 0.415 mg/l; ZnCl2 0.00327 mg/l; CoCl2.6HO 0.00143 mg/l; CuCl2.2H2O 0.000012 mg/l; Na2MoO4.2H2O 0.00726 mg/l; NaHCO3 15 mg/l; pH 7.5 ± 0.1
- Any deformed or abnormal cells observed: None
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours.
Test temperature:
Temperature within the incubator was recorded daily. 24 ± 1 °C
pH:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. pH = 7.2 ± 0.2
Nominal and measured concentrations:
Combined limit/range-finder test: 0.10, 1.0, 10 and 100 % (v/v) saturated solution.
Final test: See table 3 for nominal and measured concentrations at intial exposure and during the course of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 250 ml, containing 100 ml of test solution
- Aeration: Vessel shaken continuously.
- Initial cells density: (Mean cell density of control at 0 hours): 5.41 x 10^3 cells/ml ; RF and definitive tests: ca. 5 x 10^3 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 6.16 x10^5 cells/ml.
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control.
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: No.
- Detailed composition if non-standard medium was used: NaNO3 25.5 mg/l; MgCl2.6H2O 12.16 mg/l; CaCl2.2H2O 4.41 mg/l; MgSO4.7H2O 14.6 mg/l; KH2PO 1.044 mg/l; FeCl3.6H2O 0.16 mg/l; Na2EDTA.2H2O 0.3 mg/l; H3BO3 0.186 mg/l; MnCl2.4H2O 0.415 mg/l; ZnCl2 0.00327 mg/l; CoCl2.6HO 0.00143 mg/l; CuCl2.2H2O 0.000012 mg/l; Na2MoO4.2H2O 0.00726 mg/l; NaHCO3 15 mg/l; pH 7.5 ± 0.1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elga Optima 15+ or Elga Purelab Option R-15 BP reverse osmosis purified deionised water
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 7000 lux at 380-730 nm
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- Other: Cell densities were determined by Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Two replicates per concentration were exposed to dilutions representing nominal concentrations 0 (control), 0.1, 1.0, 10 and 100% (v/v) saturated solution. Definitive test: six replicates for control and three replicates at each test concentration.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10 and 14 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
14 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.:
14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.3 and 4.4 mg/L.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Other: Cell debris was observed to be present in the 14 mg/L test cultures whilst cell debris and transparent cells were observed to be present in the 43 and 138 mg/L test cultures.
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: During the exposure period the measured concentrations decreased in the range finder and definitive test from nominal values. No observations were made to account for the difference.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/l with a 95% confidence interval ranging from 1.0 to 1.3 mg/l. The EC50 for yield inhibition (EYC50: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 0.45 to 0.62 mg/l. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- Other: Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Reported statistics and error estimates:
Statistical analysis of the growth rate and yield data was carried out for the control and all test concentrations using 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).
Growth rate and Yield: There were no statistically significant differences between the control, 1.3 and 4.4 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 4.4 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 14 mg/L.

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

 

 

Nominal Concentration

(% (v/v) Saturated Solution)

Cell Densities* (cells per mL)

Inhibition Values (%)

 

0 Hours

 

72 Hours

 

Growth Rate

 

Yield

 

 

Control

R1

6.90E+03

7.48E+05

 

 

-

 

 

-

R2

4.85E+03

6.97E+05

Mean

5.87E+03

7.23E+05

 

 

0.10

R1

5.46E+03

6.98E+05

 

 

4

 

 

26

R2

5.20E+03

3.80E+05

Mean

5.33E+03

5.39E+05

 

 

1.0

R1

5.15E+03

8.75E+05

 

 

[3]

 

 

[3]

R2

5.33E+03

6.06E+05

Mean

5.24E+03

7.41E+05

 

 

10

R1

5.60E+03

1.72E+04

 

 

78

 

 

99

R2

4.72E+03

1.25E+04

Mean

5.16E+03

1.49E+04

 

 

100

R1

6.61E+03

7.07E+03

 

 

99

 

 

100

R2

5.63E+03

5.86E+03

Mean

6.12E+03

6.46E+03

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

R1 and R2 = Replicates 1 and 2

[Increase in growth compared to controls]

 

Table 2 Measured concentrations versus nominal concentrations: Definitive test

 

Nominal Test Concentration

(% (v/v) Saturated Solution)

 

Geometric Mean Measured Test

Concentration (mg/L)

 

Expressed as a % of the 0-Hour

Measured Test Concentration

1.0

1.3

69

3.2

4.4

72

10

14

71

32

43

69

100

138

81

 

Table 3 Cell Densities and pH Values in the Definitive Test

 

Nominal Concentration

(% (v/v) Saturated Solution)

pH

Cell Densities* (cells per mL)

pH

 

0 h

 

0 h

 

24 h

 

48 h

 

72 h

 

72 h

 

 

 

 

 

Control

R1

 

 

 

 

 

7.4

5.90E+03

2.00E+04

1.13E+05

6.12E+05

 

 

 

 

 

8.4

R2

5.20E+03

2.21E+04

1.12E+05

7.27E+05

R3

4.78E+03

2.08E+04

1.21E+05

6.86E+05

R4

5.79E+03

2.21E+04

1.16E+05

5.38E+05

R5

5.71E+03

2.18E+04

1.04E+05

5.83E+05

R6

5.04E+03

1.99E+04

1.06E+05

5.53E+05

Mean

5.41E+03

2.11E+04

1.12E+05

6.16E+05

 

 

 

1.0

R1

 

 

 

7.3

5.23E+03

2.61E+04

1.20E+05

5.73E+05

 

 

 

8.4

R2

4.66E+03

2.25E+04

1.04E+05

4.83E+05

R3

4.90E+03

2.06E+04

9.72E+04

5.94E+05

Mean

4.93E+03

2.31E+04

1.07E+05

5.50E+05

 

 

 

3.2

R1

 

 

 

7.3

4.91E+03

2.04E+04

1.04E+05

6.28E+05

 

 

 

8.4

R2

4.82E+03

2.42E+04

1.13E+05

5.69E+05

R3

4.85E+03

2.31E+04

1.07E+05

6.08E+05

Mean

4.86E+03

2.26E+04

1.08E+05

6.02E+05

 

 

 

10

R1

 

 

 

7.2

4.78E+03

1.22E+04

1.38E+04

3.42E+04

 

 

 

8.2

R2

4.93E+03

1.26E+04

1.35E+04

2.78E+04

R3

5.17E+03

1.23E+04

1.42E+04

2.04E+04

Mean

4.96E+03

1.24E+04

1.38E+04

2.75E+04

 

 

 

32

R1

 

 

 

7.2

5.26E+03

5.57E+03

5.26E+03

1.06E+04

 

 

 

7.9

R2

4.86E+03

6.67E+03

5.57E+03

9.30E+03

R3

5.26E+03

6.02E+03

5.86E+03

1.00E+04

Mean

5.13E+03

6.09E+03

5.56E+03

9.97E+03

 

 

 

100

R1

 

 

 

7.1

5.90E+03

5.68E+03

5.31E+03

9.39E+03

 

 

 

7.7

R2

6.02E+03

5.25E+03

5.42E+03

8.83E+03

R3

5.01E+03

5.06E+03

5.46E+03

7.74E+03

Mean

5.64E+03

5.33E+03

5.40E+03

8.66E+03

* Cell densities represent the mean number of cells 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

 

Table 4 Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration

(% (v/v) Saturated Solution)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

 

 

 

 

 

Control

R1

0.067

 

6.06E+05

 

R2

0.069

 

7.22E+05

 

R3

0.068

 

6.81E+05

 

R4

0.065

-

5.32E+05

-

R5

0.066

 

5.77E+05

 

R6

0.065

 

5.48E+05

 

Mean

0.067

 

6.11E+05

 

SD

0.002

 

7.57E+04

 

 

 

 

1.0

R1

0.066

1

5.68E+05

 

R2

0.063

6

4.78E+05

 

R3

0.066

1

5.89E+05

 

Mean

0.065

3

5.45E+05

11

SD

0.002

 

5.88E+04

 

 

 

 

3.2

R1

0.067

0

6.23E+05

 

R2

0.066

1

5.64E+05

 

R3

0.067

0

6.03E+05

 

Mean

0.067

0

5.97E+05

2

SD

0.001

 

2.98E+04

 

 

 

 

10

R1

0.027

60

2.95E+04

 

R2

0.024

64

2.29E+04

 

R3

0.020

70

1.53E+04

 

Mean

0.024

65

2.25E+04

96

SD

0.004

 

7.10E+03

 

 

 

 

32

R1

0.010

85

5.33E+03

 

R2

0.009

87

4.43E+03

 

R3

0.010

85

4.77E+03

 

Mean

0.010

86

4.84E+03

99

SD

0.001

 

4.53E+02

 

 

 

 

100

R1

0.009

87

3.49E+03

 

R2

0.008

88

2.82E+03

 

R3

0.006

91

2.74E+03

 

Mean

0.008

89

3.01E+03

100

SD

0.002

 

4.13E+02

 

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

Validity criteria fulfilled:
yes
Conclusions:
The test item 72h-EC50 (growth rate reduction) was 12 (95% CL: 10 – 14) mg/L based on geometric mean measured concentrations. The equivalent EC10 was 5.4 mg/L. The NOEC was 4.4 mg/L.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. Following a preliminary range-finding test, P.subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 % (v/v) saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (500 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % (v/v) saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.9 to 170 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.89 and 112 mg/L (47 % to 66 % of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The study met the acceptability criteria prescribed by the guideline. The test item 72h-EC50 (growth rate reduction) was 12 (95% CL: 10 – 14) mg/L based on geometric mean measured concentrations. The equivalent EC10 was 5.4 mg/L. The NOEC was 4.4 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
25-02-2013 to 27-09-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have common structural features in the same relative positions. The source and target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a chemically similar substance with common metabolism and common or similar degradants of the target substance. Further information is included in attachment to IUCLID section 13

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
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:
inspected: July 2012 ; signature: November 2012
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h and t=72 h
- Sampling method: 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. All 0-Hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: Stored in a freezer until analysis.
Vehicle:
no
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, Argyll, Scotland.
- Age of inoculum (at test initiation): Not reported.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and constant illumination at a temperature of 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Yes, until algal cell density was approximately 10^4 - 10^5 cells/mL
- Culturing media and conditions (same as test or not): Yes.
Medium : NaNO3 25.5 mg/l; MgCl2.6H2O 12.16 mg/l; CaCl2.2H2O 4.41 mg/l; MgSO4.7H2O 14.6 mg/l; KH2PO 1.044 mg/l; FeCl3.6H2O 0.16 mg/l; Na2EDTA.2H2O 0.3 mg/l; H3BO3 0.186 mg/l; MnCl2.4H2O 0.415 mg/l; ZnCl2 0.00327 mg/l; CoCl2.6HO 0.00143 mg/l; CuCl2.2H2O 0.000012 mg/l; Na2MoO4.2H2O 0.00726 mg/l; NaHCO3 15 mg/l; pH 7.5 ± 0.1
- Any deformed or abnormal cells observed: None
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours.
Test temperature:
Temperature within the incubator was recorded daily. 24 ± 1 °C
pH:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. pH = 7.2 ± 0.2
Nominal and measured concentrations:
Combined limit/range-finder test: 0.10, 1.0, 10 and 100 % (v/v) saturated solution.
Final test: See table 3 for nominal and measured concentrations at intial exposure and during the course of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 250 ml, containing 100 ml of test solution
- Aeration: Vessel shaken continuously.
- Initial cells density: (Mean cell density of control at 0 hours): 5.41 x 10^3 cells/ml ; RF and definitive tests: ca. 5 x 10^3 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 6.16 x10^5 cells/ml.
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control.
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: No.
- Detailed composition if non-standard medium was used: NaNO3 25.5 mg/l; MgCl2.6H2O 12.16 mg/l; CaCl2.2H2O 4.41 mg/l; MgSO4.7H2O 14.6 mg/l; KH2PO 1.044 mg/l; FeCl3.6H2O 0.16 mg/l; Na2EDTA.2H2O 0.3 mg/l; H3BO3 0.186 mg/l; MnCl2.4H2O 0.415 mg/l; ZnCl2 0.00327 mg/l; CoCl2.6HO 0.00143 mg/l; CuCl2.2H2O 0.000012 mg/l; Na2MoO4.2H2O 0.00726 mg/l; NaHCO3 15 mg/l; pH 7.5 ± 0.1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elga Optima 15+ or Elga Purelab Option R-15 BP reverse osmosis purified deionised water
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 7000 lux at 380-730 nm
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- Other: Cell densities were determined by Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Two replicates per concentration were exposed to dilutions representing nominal concentrations 0 (control), 0.1, 1.0, 10 and 100% (v/v) saturated solution. Definitive test: six replicates for control and three replicates at each test concentration.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 10 and 14 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
14 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.:
14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.3 and 4.4 mg/L.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Other: Cell debris was observed to be present in the 14 mg/L test cultures whilst cell debris and transparent cells were observed to be present in the 43 and 138 mg/L test cultures.
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: During the exposure period the measured concentrations decreased in the range finder and definitive test from nominal values. No observations were made to account for the difference.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.2 mg/l with a 95% confidence interval ranging from 1.0 to 1.3 mg/l. The EC50 for yield inhibition (EYC50: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 0.45 to 0.62 mg/l. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- Other: Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Reported statistics and error estimates:
Statistical analysis of the growth rate and yield data was carried out for the control and all test concentrations using 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).
Growth rate and Yield: There were no statistically significant differences between the control, 1.3 and 4.4 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 4.4 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 14 mg/L.

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

 

 

Nominal Concentration

(% (v/v) Saturated Solution)

Cell Densities* (cells per mL)

Inhibition Values (%)

 

0 Hours

 

72 Hours

 

Growth Rate

 

Yield

 

 

Control

R1

6.90E+03

7.48E+05

 

 

-

 

 

-

R2

4.85E+03

6.97E+05

Mean

5.87E+03

7.23E+05

 

 

0.10

R1

5.46E+03

6.98E+05

 

 

4

 

 

26

R2

5.20E+03

3.80E+05

Mean

5.33E+03

5.39E+05

 

 

1.0

R1

5.15E+03

8.75E+05

 

 

[3]

 

 

[3]

R2

5.33E+03

6.06E+05

Mean

5.24E+03

7.41E+05

 

 

10

R1

5.60E+03

1.72E+04

 

 

78

 

 

99

R2

4.72E+03

1.25E+04

Mean

5.16E+03

1.49E+04

 

 

100

R1

6.61E+03

7.07E+03

 

 

99

 

 

100

R2

5.63E+03

5.86E+03

Mean

6.12E+03

6.46E+03

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

R1 and R2 = Replicates 1 and 2

[Increase in growth compared to controls]

 

Table 2 Measured concentrations versus nominal concentrations: Definitive test

 

Nominal Test Concentration

(% (v/v) Saturated Solution)

 

Geometric Mean Measured Test

Concentration (mg/L)

 

Expressed as a % of the 0-Hour

Measured Test Concentration

1.0

1.3

69

3.2

4.4

72

10

14

71

32

43

69

100

138

81

 

Table 3 Cell Densities and pH Values in the Definitive Test

 

Nominal Concentration

(% (v/v) Saturated Solution)

pH

Cell Densities* (cells per mL)

pH

 

0 h

 

0 h

 

24 h

 

48 h

 

72 h

 

72 h

 

 

 

 

 

Control

R1

 

 

 

 

 

7.4

5.90E+03

2.00E+04

1.13E+05

6.12E+05

 

 

 

 

 

8.4

R2

5.20E+03

2.21E+04

1.12E+05

7.27E+05

R3

4.78E+03

2.08E+04

1.21E+05

6.86E+05

R4

5.79E+03

2.21E+04

1.16E+05

5.38E+05

R5

5.71E+03

2.18E+04

1.04E+05

5.83E+05

R6

5.04E+03

1.99E+04

1.06E+05

5.53E+05

Mean

5.41E+03

2.11E+04

1.12E+05

6.16E+05

 

 

 

1.0

R1

 

 

 

7.3

5.23E+03

2.61E+04

1.20E+05

5.73E+05

 

 

 

8.4

R2

4.66E+03

2.25E+04

1.04E+05

4.83E+05

R3

4.90E+03

2.06E+04

9.72E+04

5.94E+05

Mean

4.93E+03

2.31E+04

1.07E+05

5.50E+05

 

 

 

3.2

R1

 

 

 

7.3

4.91E+03

2.04E+04

1.04E+05

6.28E+05

 

 

 

8.4

R2

4.82E+03

2.42E+04

1.13E+05

5.69E+05

R3

4.85E+03

2.31E+04

1.07E+05

6.08E+05

Mean

4.86E+03

2.26E+04

1.08E+05

6.02E+05

 

 

 

10

R1

 

 

 

7.2

4.78E+03

1.22E+04

1.38E+04

3.42E+04

 

 

 

8.2

R2

4.93E+03

1.26E+04

1.35E+04

2.78E+04

R3

5.17E+03

1.23E+04

1.42E+04

2.04E+04

Mean

4.96E+03

1.24E+04

1.38E+04

2.75E+04

 

 

 

32

R1

 

 

 

7.2

5.26E+03

5.57E+03

5.26E+03

1.06E+04

 

 

 

7.9

R2

4.86E+03

6.67E+03

5.57E+03

9.30E+03

R3

5.26E+03

6.02E+03

5.86E+03

1.00E+04

Mean

5.13E+03

6.09E+03

5.56E+03

9.97E+03

 

 

 

100

R1

 

 

 

7.1

5.90E+03

5.68E+03

5.31E+03

9.39E+03

 

 

 

7.7

R2

6.02E+03

5.25E+03

5.42E+03

8.83E+03

R3

5.01E+03

5.06E+03

5.46E+03

7.74E+03

Mean

5.64E+03

5.33E+03

5.40E+03

8.66E+03

* Cell densities represent the mean number of cells 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

 

Table 4 Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration

(% (v/v) Saturated Solution)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

 

 

 

 

 

Control

R1

0.067

 

6.06E+05

 

R2

0.069

 

7.22E+05

 

R3

0.068

 

6.81E+05

 

R4

0.065

-

5.32E+05

-

R5

0.066

 

5.77E+05

 

R6

0.065

 

5.48E+05

 

Mean

0.067

 

6.11E+05

 

SD

0.002

 

7.57E+04

 

 

 

 

1.0

R1

0.066

1

5.68E+05

 

R2

0.063

6

4.78E+05

 

R3

0.066

1

5.89E+05

 

Mean

0.065

3

5.45E+05

11

SD

0.002

 

5.88E+04

 

 

 

 

3.2

R1

0.067

0

6.23E+05

 

R2

0.066

1

5.64E+05

 

R3

0.067

0

6.03E+05

 

Mean

0.067

0

5.97E+05

2

SD

0.001

 

2.98E+04

 

 

 

 

10

R1

0.027

60

2.95E+04

 

R2

0.024

64

2.29E+04

 

R3

0.020

70

1.53E+04

 

Mean

0.024

65

2.25E+04

96

SD

0.004

 

7.10E+03

 

 

 

 

32

R1

0.010

85

5.33E+03

 

R2

0.009

87

4.43E+03

 

R3

0.010

85

4.77E+03

 

Mean

0.010

86

4.84E+03

99

SD

0.001

 

4.53E+02

 

 

 

 

100

R1

0.009

87

3.49E+03

 

R2

0.008

88

2.82E+03

 

R3

0.006

91

2.74E+03

 

Mean

0.008

89

3.01E+03

100

SD

0.002

 

4.13E+02

 

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

Validity criteria fulfilled:
yes
Conclusions:
The target substance 72h-EC50 (growth rate reduction) is expected to be 12 (95% CL: 10 – 14) mg/L based on geometric mean measured concentrations. The equivalent EC10 is expected to be 5.4 mg/L and the NOEC 4.4 mg/L.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out on a source substance according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. Following a preliminary range-finding test, P.subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 % (v/v) saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (500 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % (v/v) saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.9 to 170 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.89 and 112 mg/L (47 % to 66 % of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The study met the acceptability criteria prescribed by the guideline. The test item 72h-EC50 (growth rate reduction) was 12 (95% CL: 10 – 14) mg/L based on geometric mean measured concentrations. The equivalent EC10 was 5.4 mg/L. The NOEC was 4.4 mg/L.

Description of key information

72-h EC50 (Read-Across: 4-methyl-2-phenyltetrahydro-2H-pyran ; aquatic algae; growth rate) = 12 mg/L (C.I. 10 - 14 mg/L), 72 hour, freshwater, OECD TG 201, 2013

72-h EC10 (Read-Across: 4-methyl-2-phenyltetrahydro-2H-pyran ; aquatic algae; growth rate) = 5.4 mg/L, 72 hour, freshwater, OECD TG 201, 2013

NOEC (Read-across: 4-methyl-2-phenyltetrahydro-2H-pyran ; aquatic algae; growth rate) = 4.4 mg/L, 72 hour, freshwater, OECD TG 201, 2013

Key value for chemical safety assessment

EC50 for freshwater algae:
12 mg/L
EC10 or NOEC for freshwater algae:
4.4 mg/L

Additional information

Key study : OECD TG 201, 2013 : Read-Across - SOURCE (4-methyl-2-phenyltetrahydro-2H-pyran): The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. Following a preliminary range-finding test, P.subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 % (v/v) saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (500 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100 % (v/v) saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 1.9 to 170 mg/L. A decline in measured test concentration was observed at 72 hours to between 0.89 and 112 mg/L (47 % to 66 % of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The study met the acceptability criteria prescribed by the guideline. The test item 72h-EC50 (growth rate reduction) was 12 (95% CL: 10 – 14) mg/L based on geometric mean measured concentrations. The equivalent EC10 was 5.4 mg/L. The NOEC was 4.4 mg/L.