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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:
2016-04-01 to 2016-04-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Concentrations: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution
- Sampling method: All concentration levels and the control were analytically verified via HPLC-DAD after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared with algae. For the test item analysis after 72 hours samples were taken from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis
Vehicle:
yes
Remarks:
demineralized water
Details on test solutions:
REPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal loading of 1 mL test item/L was prepared once with demineralized water 96 ± 1 hours prior to the start of the exposure. The test item was placed onto the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at 30 ± 2 °C for 72 ± 1 hours and for 24 ± 1 hours at room temperature. After a separation phase of 1 hour at room temperature the saturated solution was taken from the homogeneous phase and was used for testing. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Then the components of the dilution water were added to the saturated solution. The saturated solution was clear and colorless.
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations. Potassium dichromate was tested as a reference item.
- Concentration of vehicle in test medium: stock solution: 1 mL/L; final test solutions: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of saturated solution
- Evidence of undissolved material: No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDAK, SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE * m*exp^-2 * s*exp^-1 for 24 hours per day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 24 °C, controlled at ± 2 °C
pH:
8.1 ± 0.2
Dissolved oxygen:
No data was provided.
Salinity:
Freshwater was used.
Nominal and measured concentrations:
Nominal: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of saturated solution
Measured: 0.0695 - 0.21 - 0.673 - 2.16 - 9.01 - 29.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks, volume: 250 mL
- Type: sealed with cotton wool plugs
- Fill volume: 100 mL
- Initial cells density: 6305 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h/day
- Light intensity and quality: 68.2-79.6 µE * m^-2 * s^-1; Light homogeneity was within ± 15 % over incubation area.

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: yes, Chlorophyll-a-fluorescence daily with excitation at 436 nm, emission at 685 nm
- Microscopic evaluation: Microscopic evaluation of the cells was carried out at the start and end of the incubation. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: dilution factor of √10 (~ 3.16)
- Range finding study: yes
- Test concentrations: nominal 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.673 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.:
2.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.53-2.37 95 % Confidence Interval
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 2.37 - 3.30 95 % Confidence Interval
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 4.72 - 5.88 95 % Confidence Interval
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.673 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: < 0.0695-1.19 95 % Confidence Interval
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: 0.686-1.49 95 % Confidence Interval
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.08 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield Inhibition
Remarks on result:
other: 1.70-2.54 95 % Confidence Interval
Details on results:
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Measured concentrations: The measured concentrations of the test item during the exposure interval were 0.0763 - 0.223 - 0.713 - 2.61 - 9.03 - 29.7 mg/L (0 hours) and in the range of 69 to 100 % (72 hours) of the initially measured values of all concentrations. The geometric mean measured concentrations were determined.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: Growth rate inhibition: 0.460, Yield inhibition: 0.317
Reported statistics and error estimates:
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Geometric mean measured test item concentration

[mg/L]

 Replicate No.

Growth rate [d^-1]

Inhibition of growth rate [%]

Yield [cells/mL]

Inhibition of yield [%]

 29.5

1

< LOQ

100

< LOQ

100

2

< LOQ

100

< LOQ

100

3

< LOQ

100

< LOQ

100

mean

(+) n.a.

100

(+) n.a.

100

9.01

1

0.263

86

7572

100

2

0.432

77

1611

99

3

0.604

68

32317

98

mean

(+) 0.433

77

(+) 18867

99

2.16

1

1.64

14

847177

54

2

1.68

11

970424

47

3

1.64

13

852872

54

mean

(+) 1.65

13

(+) 890158

52

0.673

1

1.82

4

1468710

20

2

1.87

1

1725803

7

3

1.86

2

1678669

9

mean

(-) 1.85

2

(+) 1624394

12

0.210

1

1.80

5

1396544

24

2

1.93

-2

2032341

-10

3

1.88

0

189456

3

mean

(-) 1.87

1

(-) 1739447

6

0.0695

1

1.88

1

1781788

4

2

1.89

0

18202240

1

3

1.85

2

1616708

12

mean

(-) 1.87

1

(-) 1739579

6

Control

1

1.82

 

1476885

 

2

1.93

 

2025012

 

3

1.91

 

1914676

 

4

1.88

 

1748581

 

5

1.91

 

1955608

 

6

1.91

 

1959047

 

mean

1.89

 

1846635

 

< LOQ = below the limit of quantification  (2736 cells/mL)        

n.a. = data not applicable

Validity criteria fulfilled:
yes
Conclusions:
The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 5.29 (4.72 - 5.88) mg/L and 2.08 (1.70 - 2.54) mg/L, respectively. The NOEC for inhibition of growth rate and yield after 72 h was 0.673 and 0.210 mg/L, respectively.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009, C.3. under GLP. Five dilution levels of the saturated solution were tested in a geometrical series with a dilution factor of10 (approx. 3.16): 0.316 - 1.00 - 3.16 - 10.0 - 31.6 – 100 % of the saturated solution. The measured concentrations in the old media (72 h) containing algae were in the range of 69 to 100 % of the initially measured values of all concentrations, corresponding to the geometric mean measured concentrations: 0.0695 - 0.210 - 0.673 - 2.16 - 9.01 -29.5 mg/L. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 5.29 mg/L and 2.08 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 1.89 mg/L and 0.6 mg/L, respectively.

Description of key information

The test revealed an EC50 value of 5.29 mg/L, an EC10 value of 1.89 mg/L and a NOEC of 0.673 mg/L. The values are based on the growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:
5.29 mg/L
EC10 or NOEC for freshwater algae:
1.89 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009, C.3. under GLP. Five dilution levels of the saturated solution were tested in a geometrical series with a dilution factor of10 (approx. 3.16): 0.316 - 1.00 - 3.16 - 10.0 - 31.6 – 100 % of the saturated solution. The measured concentrations in the old media (72 h) containing algae were in the range of 69 to 100 % of the initially measured values of all concentrations, corresponding to the geometric mean measured concentrations: 0.0695 - 0.210 - 0.673 - 2.16 - 9.01 -29.5 mg/L. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 5.29 mg/L and 2.08 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 1.89 mg/L and 0.6 mg/L, respectively.