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Ecotoxicological information

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:
2017-05-05 to 2017-07-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
No. 2016/266
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.
Immediately after sampling, all samples were frozen (at -20 ± 5 °C). Based on pre-experiments for investigation of the storage stability (actual study), the test item was found to be stable in the test water under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium of the highest nominal concentration of 100 mg/L was prepared by completely dissolving 50.06 mg of the test item in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum: 4 days

ACCLIMATION
- Acclimation period: no, same conditions for culturing media and test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
24 °C
pH:
7.5 - 8.5
Nominal and measured concentrations:
Nominal concentrations: control, 2.2, 4.6, 10, 22, 46, 100 mg/L
Measured concentrations (0 h): Measured concentrations (72 h): n.a.: not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of biological results
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material, size, fill volume: glass, 75 mL, 30 mL
- Covered: Each test flask was covered with a glass dish
- Initial cells density: 5000 cells/mL
- Final cell density: 1660000 cells/mL
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, AAP Medium

TEST MEDIUM PARAMETERS
- Preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: 66 to 68 μE s-1 m-2
- Position: orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland), positioned randomly and repositioned daily

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study
- Test concentrations: 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CI: 77 - 96 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % CI: 85 - 93 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % CI: 32 - 39 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-hour EC50 for growth rate = 1.0 mg/L (May 2017) whithin the valid range recommended by the guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L)
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate.
Statistical analysis was performed using ToxRat Professional® [7].

Table 1: Biomass of Algae

Nominal Test Item Concentration
[mg/L]

Repl.
No.

Biomass of Algae*

24 Hours

48 Hours

72 Hours

Control

1

2.2

20.9

128.1

2

2.8

21.4

154.1

3

2.2

21.7

137.2

4

2.3

21.3

136.6

5

2.5

23.6

150.8

6

2.9

23.8

147.8

Mean

2.5

22.1

142.4

SD

0.3

1.3

10.0

2.2

1

2.5

22.1

134.6

2

2.9

20.9

138.9

3

1.9

23.3

139.0

Mean

2.4

22.1

137.5

SD

0.5

1.2

2.5

4.6

1

2.3

21.4

129.5

2

2.7

20.2

134.1

3

2.2

20.3

134.9

Mean

2.4

20.6

132.8

SD

0.3

0.6

2.9

10

1

2.4

22.1

135.5

2

2.2

21.7

137.3

3

2.6

22.0

136.8

Mean

2.4

21.9

136.5

SD

0.2

0.2

1.0

22

1

2.8

20.9

123.1

2

2.0

20.1

122.6

3

2.4

19.9

132.5

Mean

2.4

20.3

126.0

SD

0.4

0.5

5.6

46

1

2.3

18.4

125.3

2

1.8

17.3

115.2

3

2.0

17.7

128.3

Mean

2.0

17.8

122.9

SD

0.3

0.6

6.9

100

1

1.5

10.0

58.7

2

1.6

10.6

60.7

3

2.1

11.2

61.0

Mean

1.7

10.6

60.1

SD

0.3

0.6

1.3

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104 ).

At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.43 x 104 relative fluorescence units.

Table 2: Average Growth Rates (µ)

Nominal

Test Item Concentration

Average Growth Rate µ (day-1) and Inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

[mg/L]

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.747

0.0

1.970

0.0

1.934

0.0

2.2

1.719

1.6

1.969

0.0

1.923

0.6

4.6

1.706

2.4

1.936

1.7

1.911

1.2

10

1.716

1.8

1.966

0.2

1.921

0.7

22

1.723

1.4

1.928*

2.1

1.894*

2.1

46

1.550*

11.3

1.862*

5.5

1.885*

2.5

100

1.392*

20.3

1.604*

18.6

1.647*

14.8

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

Table 3: Yield (Y)

Nominal

Test Item Concentration

Yield Y (x 104) and Inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

[mg/L]

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

2.05

0.0

21.69

0.0

142.00

0.0

2.2

2.00

2.4

21.66

0.1

137.10

3.5

4.6

1.94

5.2

20.20

6.9

132.42

6.8

10

1.96

4.2

21.48

0.9

136.10

4.2

22

2.00

2.5

19.87*

8.4

125.62#

11.5

46

1.61*

21.6

17.37*

19.9

122.49#

13.7

100

1.32*

35.6

10.20*

53.0

59.69#

58.0

*: Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller, α = 0.05).

#: Mean value statistically significantly lower than in the control (according to a Welch t-test one-sided smaller, α = 0.05).

Table 4: Analytical Results for Test Samples

Sampling Day/

Sample Age

[d/h]

Nominal Concentration of
Test Item
cnom

[mg/L]

Measured Concentration
 of Test Item
x

[mg/L]

Sample Preparation Factor

F

Determined Concentration

of Test Item
c

[mg/L]

% of Nominal

Concentration

0/0

Control

n.d.

2

< LOQ

n.a.

(fresh)

10

5.05

2

10.1

101

 

22

11.2

2

22.4

102

 

46

24.2

2

48.3

105

 

100

52.8

2

105.6

106

3/72

Control

n.d.

2

< LOQ

n.a.

(aged)

10

4.97

2

9.93

99

 

22

11.1

2

22.3

101

 

46

24.0

2

47.9

104

 

100

52.6

2

105

105

n.d. = no test item detected

n.a. = not applicable

LOQ: 4.1 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity criteria fulfilled:
yes
Conclusions:
The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata is summarized as follows:
- 72-hour ErC50 (growth rate) >100 mg/L
- 72-hour ErC10 (growth rate) = 85 mg/L (95 % CI: 77 - 96 mg/L)
The results are based on nominal concentrations of the test item.
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006). The nominal test item concentrations tested were 2.2, 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The measured concentrations in the test media of the test concentrations of 10 to 100 mg/L were between 101 and 106 % of the nominal values at the start of the test and 99 and 105 % at the end of the test. Thus, the test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The biological results of the study were as follows: 72-hour ErC50 >100 mg/L; 72-hour ErC10 = 85 mg/L ( (95 % CI: 77 - 96 mg/L).

Description of key information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was determined according to OECD 201 and is summarized as follows:

- 72-hour ErC50 (growth rate) >100 mg/L

- 72-hour ErC10 (growth rate) = 85 mg/L (95 % CI: 77 - 96 mg/L)

The results are based on nominal concentrations of the test item.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
100 mg/L
EC10, LC10 or NOEC for freshwater algae:
85 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006). The nominal test item concentrations tested were 2.2, 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The measured concentrations in the test media of the test concentrations of 10 to 100 mg/L were between 101 and 106 % of the nominal values at the start of the test and 99 and 105 % at the end of the test. Thus, the test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item. The biological results of the study were as follows: 72-hour ErC50 >100 mg/L; 72-hour ErC10 = 85 mg/L ( (95 % CI: 77 - 96 mg/L).