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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-01-2012 to 23-02-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2011; signature: August 2011
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 6.25; 12.5, 25, 50 and 100 mg/L. Samples from control replicates and test group replicates of equal nominal concentration were pooled for analysis. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Sampling method: Samples were taken at 0 h and 72 h for quantitative analysis. Samples were analysed following addition of formic acid (0.1 mL per 100 mL of sample). If required, samples were diluted to 10 mg/L with 0.1% formic acid in test medium.
- Sample storage conditions before analysis: stored at approximately -20ºC.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium under constant aeration and illumination (light intensity not reported) at 21 ± 1 ºC temperature.

ACCLIMATION
- Acclimation period: No. However, prior to the start of the test exposure sufficient master culture was added to approximately 100 ml culture medium in flasks at 10^3 cells/mL and stirring at 100 – 150 rpm under constant conditions until algal density 10^4 – 10^5 cells/mL.
- Culturing media and conditions (same as test or not): Yes.
Culture medium: (mg/L) - equivalent or similar to AAP medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.
- Any deformed or abnormal cells observed: None reported.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1ºC
pH:
0-hours pH: 7.6 - 8.0
72-hours pH: 7.9 - 8.2.
Nominal and measured concentrations:
Nominal concentrations were analytically confirmed in the presence of algae at 0 and 72 hours and absence of algae at 72 hours.
0-hour - Nominal Concentrations were: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L
72-hour – Measured Concentrations with algae: 4.38, 9.08, 20.2, 43.9, 92.6 mg/L
Percentage of nominal concentrations: 70 to 93%
72-hour - Measured Concentrations without algae: 5.74, 11.3, 23.4, 47.3, 94.0 mg/L
Percentage of nominal concentrations: 91 to 95%
For detailed results, refer to Table 1.
Note: 91-95% recoveries measured in samples without algae. Concentration-dependent decline in measured test concentrations attributed to adsorption to algal cells. On this basis nominal concentrations were considered appropriate for effect level calculations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): Closed – Static. vessels plugged with PU foam bungs.
- Headspace, fill volume: 100 mL fill volume, approximately 150 mL headspace
- Aeration: Vessel shaken continuously.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: nominal: 5.12x10^3 cells/mL
- Control end cells density: 8.87x10^5 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate without algae for sampling purposes
- 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: Yes. Equivalent or similar to AAP medium, according to OECD 201, Annex 3
NaNO3 25.5, MgCI2.6H2O 12.164, CaCI2.2H2O 4.41, MgSO4.7H20 14.7, K2HPO4 1.044, NaHC03 15.0, H3BO3 0.1855, MnCI2.4H2O 0.415, ZnCI2 0.00327, CoCI2.6H2O 0.00143, CuCI2.2H2O 0.000012, NaMoO4.2H2O 0.00726, FeCl3.6H2O 0.159, Na2-EDTA 0.3, . pH 7.5 +/- 0.1.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not applicable.

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter(R) Multisizer particle counter
- Other: Microscopic evaluation of the cells was carried out at the end of the exposure for abnormalities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0, in definitive test justified from the results of the range finding study.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to 0.10, 1.0, 10 and 100 mg/L and control based on nominal concentrations.
- Results used to determine the conditions for the definitive study: Yes. Growth observed to be reduced at 100 mg/L level.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 57 - 68 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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:
EC50
Effect conc.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits: 29 - 38 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
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): Microscopic evaluation of the cells at the end of the exposure period revealed no abnormalities in the test item concentrations or controls.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Low dose stimulation was observed in the range finder at 0.1 mg/L. In the definitive test no low dose stimulation was observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Differences in nominal versus measured between with and without algae ascribed to substance adsorption to algal cells.
- 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 1.4 mg/L with a 95% confidence interval ranging from 1.2 to 1.7 mg/L. The EC50 for yield inhibition (EYC50: 0-72h) was 0.59 mg/L with a 95% confidence interval ranging from 0.53 to 0.65 mg/L. ; NOEC = 0.25 mg/L
The results were within the test facility (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.
Reported statistics and error estimates:
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by dose-response regression. Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
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 for the control and all test concentrations to determine any statistically significant differences between the test and control groups.

Table 2: Effect of test substance on growth of P. subcapitata

Nominal Concentration (mg/L)

pH

Cell Densities (cells per mL)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.0

5.12E+03

3.05E+04

1.94E+05

7.56E+05

8.2

R2

5.16E+03

3.60E+04

1.96E+05

5.06E+05

R3

5.06E+03

2.84E+04

1.51E+05

9.46E+05

R4

5.21E+03

3.64E+04

1.63E+05

7.93E+05

R5

5.17E+03

3.13E+04

1.63E+05

9.33E+05

R6

5.01E+03

3.72E+04

2.05E+05

1.09E+06

Mean

5.12E+03

3.33E+04

1.84E+05

8.87E+05

6.25

R1

7.9

5.08E+03

2.53E+04

1.12E+05

6.90E+05

8.2

R2

5.10E+03

3.17E+04

1.55E+05

7.90E+05

R3

5.09E+03

3.74E+04

1.62E+05

7.59E+05

Mean

5.09E+03

3.15E+04

1.43E+05

7.46E+05

12.5

R1

7.8

5.19E+03

3.34E+04

2.07E+05

7.99E+05

8.1

R2

5.13E+03

3.47E+04

1.53E+05

1.04E+06

R3

5.11E+03

2.42E+04

1.15E+05

6.58E+05

Mean

5.14E+03

3.05E+04

1.69E+05

5.33E+05

25

R1

7.8

5.07E+03

2.75E+04

1.47E+05

5.63E+05

8.1

R2

5.10E+03

2.57E+04

1.12E+05

5.77E+05

R3

5.02E+03

2.62E+04

1.23E+05

6.12E+05

Mean

5.06E+03

2.65E+04

1.27E+05

5.84E+05

50

R1

7.7

5.16E+03

2.03E+04

6.35E+04

3.17E+05

8.0

R2

5.06E+03

2.11E+04

6.79E+04

2.62E+05

R3

5.14E+03

1.77E+04

5.96E+04

2.13E+05

Mean

5.12E+03

1.97E+04

6.38E+04

2.64E+05

100

R1

7.6

5.26E+03

1.16E+04

1.00E+04

9.93E+03

7.9

R2

5.06E+03

1.18E+04

6.19E+03

6.22E+03

R3

5.12E+03

1.25E+04

4.05E+03

5.84E+03

Mean

5.14E+03

1.20E+04

6.76E+03

7.33E+03

Table 3: Inhibition of growth rate and yield in the definitive test

Nominal Concentration (mg/L)

Growth rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% inhibition

0 – 72 h

% inhibition

Control

R1

0.070

-

7.51E+05

-

R2

0.071

8.01E+05

R3

0.073

9.41E+05

R4

0.070

7.88E+05

R5

0.073

9.28E+05

R6

0.075

1.08E+06

Mean

0.072

8.81E+05

SD

0.002

1.25E+05

6.25

R1

0.068

6

6.85E+05

16

R2

0.070

3

7.85E+05

R3

0.070

3

7.54E+05

Mean

0.069

4

7.41E+05

SD

0.001

 

5.16E+04

12.5

R1

0.070

3

7.94E+05

6

R2

0.074

[3]

1.04E+06

R3

0.068

6

6.52E+05

Mean

0.071

2

8.28E+05

SD

0.003

 

1.94E+05

25

R1

0.066

8

5.58E+05

34

R2

0.066

8

5.72E+05

R3

0.067

7

6.07E+05

Mean

0.066

8

5.79E+05

SD

0.001

 

2.54E+04

50

R1

0.058

19

3.12E+05

71

R2

0.055

24

2.57E+05

R3

0.052

28

2.08E+05

Mean

0.055

24

2.59E+05

SD

0.003

 

5.19E+04

100

R1

0.010

86

4.67E+03

100

R2

0.003

96

1.17E+03

R3

0.002

97

7.20E+02

Mean

0.005

93

2.19E+03

SD

0.004

 

2.16E+03

 

 

Validity criteria fulfilled:
yes
Conclusions:
The test item 48h-EC50 was 62 mg/L (C.I: 57 – 68 mg/L) based on analytically confirmed nominal concentrations. The corresponding EC10 was 41 mg/L and the NOEC was 12.5 mg/L.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0.1, 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density nominal of 5.12x10^3 cells/mL. Five test item solutions were employed with a dilution factor of 2 (nominal) and concentrations of 0 (control), 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 24 ± 1 °C. Environmental conditions were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The concentrations of the test item were analytically verified via HPLC-DAD at test start (0 hours), and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 93 - 97 %. After 72 hours with algae, the recovery ranges were 70 - 93 % and < Limit of Quantification (LOQ), for test groups and control respectively. After 72 hours without algae, the recovery ranges were 91- 95 %, for the test groups compared with nominal. The decline of test item in the presence of algae was considered as being related to algal cell adsorption. On this basis it was considered that the algal cells had been exposed to nominal concentrations throughout the test period. All effect values were based on the nominal test item concentration after 72 hours. The EC50 for growth rate reduction (72h-ErC50) based on nominal concentrations was 62 (C.I. 57 – 68) mg/L. The corresponding EC10 was 41 mg/L and the NOEC was 12.5 mg/L.

Description of key information

ErC50 (algae; growth rate) = 62 (C.I. 57 - 68) mg/L based on analytically confirmed nominal concentrations, 72hour-freshwater, OECD TG 201, 2012

ErC10 (algae; growth rate) = 41 (C.I. - ) mg/L based on analytically confirmed nominal concentrations, 72hour-freshwater, OECD TG 201, 2012

NOEC (algae) = 12.5 mg/L based on analytically confirmed nominal concentrations, 72hour-freshwater, OECD TG 201, 2012

Key value for chemical safety assessment

EC50 for freshwater algae:
62 mg/L
EC10 or NOEC for freshwater algae:
41 mg/L

Additional information

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0.1, 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density nominal of 5.12x10^3 cells/mL. Five test item solutions were employed with a dilution factor of 2 (nominal) and concentrations of 0 (control), 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 24 ± 1 °C. Environmental conditions were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The concentrations of the test item were analytically verified via HPLC-DAD at test start (0 hours), and at the end (72 hours) of the exposure. The recovery rates of the test item at the start were in the range of 93 - 97 %. After 72 hours with algae, the recovery ranges were 70 - 93 % and < Limit of Quantification (LOQ), for test groups and control respectively. After 72 hours, the recovery ranges were 91- 95 %, for the test groups not inoculated with algae compared with nominal. The decline of test item in the presence of algae was considered as being related to algal cell adsorption. On this basis it was considered that the algal cells had been exposed to nominal concentrations throughout the test period. All effect values were based on the nominal test item concentration after 72 hours. The EC50 for growth rate reduction (72h-ErC50) based on nominal concentrations was 62 (C.I. 57 – 68) mg/L. The corresponding EC10 was 41 mg/L and the NOEC was 12.5 mg/L.

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