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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:
2020-06-02 to 2020-06-05
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:
Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
01 March 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For determination of the test item concentrations in the test solutions, all three test vessels were analysed at the start and at the end of the study. In case of control sample, all six test vessels were analysed at the start and at the end of the study. Extra replicate sample from each testing concentration and the control sample were analysed on the first and second day of the study on three replicate dilution.
- Sample storage conditions before analysis: no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test was prepared by mechanical dispersion without using of any solubilising agent.
For preparation of test solutions a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.1003 g test item in 1003 mL OECD medium. The test solutions of subsequent concentrations were prepared by appropriate dilution of this stock solutions
- Controls: negative (blank) control, positive control
- Evidence of undissolved material: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany.
- Age of inoculum: 4 days (exponentially growth)
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
not specified
Test temperature:
22.7 – 23.1°C
pH:
7.86 – 9.31
Dissolved oxygen:
not specified
Salinity:
not relevant
Conductivity:
not specified
Nominal and measured concentrations:
nominal: 0 (blank control), 6.25, 12.5, 25, 50 and 100 mg/L
measured: 4.5, 9.4, 21.0, 45.7 and 95.9 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed with permeable stoppers
- Material, size, fill volume: glass, ~250 mL nominal volume,100 mL fill volume
- Aeration: agitated by using an orbital shaker
- Initial cells density: 10^4 cells/mL in each test flask
- Control end cells density: 10^4 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (OECD medium, according to OECD 201) prepared with deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: 6720 lux, wavelength range: 400 – 700 nm

DETERMINATION OF CELL CONCENTRATION
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: performed
- Test concentrations: 0 (control), 0.01, 0.1, 1, 10 and 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.:
34.9 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.:
12.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
17.5 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.:
9.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
7.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
9.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: No morphological abnormalities were observed in the control and in the concentrations of 4.5 and 9.4 mg/L (measured) during the test. Swollen cells were observed in the concentrations of 21.0, 45.7and 95.9 mg/L (measured).
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: none
- 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):
72h ErC50: 0.52 mg/L (95 % confidence limits: 0.46 – 0.58 mg/L)
72h EyC50: 0.31 mg/L (95 % confidence limits: 0.28 – 0.35 mg/L)
Reported statistics and error estimates:
For determination of the ECx values Probit analysis was performed. For the determination of the NOEC and LOEC, data were first checked for normality by Kolmogorov-Smirnov and Shapiro-Wilk tests. Based ont he results of normality test, NOEC and LOEC for growth rates and yield were determined using Jonckheere-Tersptra Test.

Table 1: Algal cell number (× 104cell/mL) during the test period of 72 hours and Yield

Concentration

 

Number of Cells (× 104/mL)

Yield

nominal
[mg/L]

measured
[mg/L]

0 h

24 h

48 h

72 h

Control

Not detected

 

1

2

9

52

51

 

1

3

7

46

45

 

1

2

9

45

44

 

1

2

8

46

45

 

1

2

9

37

36

 

1

3

8

39

38

Mean

1.00

2.33

8.33

44.17

43.2

SD

0.00

0.52

0.82

5.42

5.42

6.25

4.5

 

1

2

9

45

44

 

1

3

7

35

34

 

1

3

9

38

37

Mean

1.00

2.67

8.33

39.33

38.3

SD

0.00

0.58

1.15

5.13

5.13

12.5

9.4

 

1

2

7

33

32

 

1

3

8

35

34

 

1

3

7

34

33

Mean

1.00

2.67

7.33

34.00

33.0

SD

0.00

0.58

0.58

1.00

1.00

25

21.0

 

1

2

6

20

19

 

1

3

6

21

20

 

1

2

8

17

16

Mean

1.00

2.33

6.67

19.33

18.3

SD

0.00

0.58

1.15

2.08

2.08

50

45.7

 

1

2

2

7

6

 

1

1

2

8

7

 

1

2

2

6

5

Mean

1.00

1.67

2.00

7.00

6.0

SD

0.00

0.58

0.00

1.00

1.00

100

95.9

 

1

1

1

1

0

 

1

1

1

1

0

 

1

1

1

1

0

Mean

1.00

1.00

1.00

1.00

0.0

SD

0.00

0.00

0.00

0.00

0.00

Table 2: The specific Growth Ratesduring the test period of 72 hours

Concentration

 

Growth Rates (µ)

nominal
[mg/L]

measured
[mg/L]

0 – 24 h

0 – 48 h

0 – 72 h

Control

Not detected

 

0.0289

0.0458

0.0549

 

0.0458

0.0405

0.0532

 

0.0289

0.0458

0.0529

 

0.0289

0.0433

0.0532

 

0.0289

0.0458

0.0502

 

0.0458

0.0433

0.0509

Mean

0.0345

0.0441

0.0525

SD

0.0087

0.0021

0.0017

6.25

4.5

 

0.0289

0.0458

0.0529

 

0.0458

0.0405

0.0494

 

0.0458

0.0458

0.0505

Mean

0.0401

0.0440

0.0509

SD

0.0098

0.0030

0.0018

12.5

9.4

 

0.0289

0.0405

0.0486

 

0.0458

0.0433

0.0494

 

0.0458

0.0405

0.0490

Mean

0.0401

0.0415

0.0490

SD

0.0098

0.0016

0.0004

25

21.0

 

0.0289

0.0373

0.0416

 

0.0458

0.0373

0.0423

 

0.0289

0.0433

0.0394

Mean

0.0345

0.0393

0.0411

SD

0.0098

0.0035

0.0015

50

45.7

 

0.0289

0.0144

0.0270

 

0.0000

0.0144

0.0289

 

0.0289

0.0144

0.0249

Mean

0.0193

0.0144

0.0269

SD

0.0167

0.0000

0.0020

100

95.9

 

0.0000

0.0000

0.0000

 

0.0000

0.0000

0.0000

 

0.0000

0.0000

0.0000

Mean

0.0000

0.0000

0.0000

SD

0.0000

0.0000

0.0000

Table 3: Growth Rates (µ) and percentage inhibition of µ during the test period

Concentration

Growth Rates (µ)and % Inhibition of µ

nominal
[mg/L]

measured
[mg/L]

0–24 h

0–48 h

0–72 h

µ

%

µ

%

µ

%

Control

Not detected

0.0345

0.0441

0.0525

6.25

4.5

0.0401

-16.3

0.0440

0.1

0.0509

3.0

12.5

9.4

0.0401

-16.3

0.0415

5.9

0.0490

6.8

25

21.0

0.0345

0.0

0.0393

10.8

0.0411

21.8

50

45.7

0.0193

44.2

0.0144

67.2

0.0269

48.7

100

95.9

0.0000

100.0

0.0000

100.0

0.0000

100.0

Table 4: Morphological changes of algal cells observed after 24, 48 and 72 hours of exposure

Concentration

Morphological changes

nominal
[mg/L]

measured
[mg/L]

24 h

48 h

72 h

Control

Not detected

- normal cells

- normal cells

- normal cells

6.25

4.5

- normal cells

- normal cells

- normal cells

12.5

9.4

- normal cells

- normal cells

- normal cells

25

21.0

- normal cells

- normal cells

- normal cells
- some swollen cells

50

45.7

- normal cells
- some swollen cells

- normal cells
- swollen cells

- normal cells
- swollen cells

100

95.9

- normal cells
- swollen cells

- normal cells
- swollen cells

- swollen cells

Table 5: Concentration of Test Item measured in the Test Solutions at the beginning of the test

Nominal concentration mg/L

Mean of the measured concentrations

Start (June 02, 2020)

1stday (June 03, 2020)

Concentration (mg/L) 

% of the nominal

Concentration (mg/L) 

% of the nominal

Control

not detected

-

not detected

-

6.25

4.295

69

4.425

71

12.5

9.343

75

9.651

77

25

21.283

85

21.233

85

50

46.410

93

46.814

94

100

96.644

97

96.666

97

Table 6: Concentration of Test Item measured in the Test Solutions at the end of the test

Nominal concentration mg/L

Mean of the measured concentrations

2ndday (June 04, 2020)

End (June 05, 2020)

Concentration (mg/L) 

% of the nominal

Concentration (mg/L) 

% of the nominal

Control

not detected

-

not detected

-

6.25

4.606

74

4.546

73

12.5

9.529

76

9.146

73

25

21.561

86

19.981

80

50

47.555

95

42.082

84

100

103.054

103

88.015

88

Validity criteria fulfilled:
yes
Conclusions:
In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72-hour EC50 and EC10 for the growth rate was determined to be 34.9 and 12.2 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 16.3 and 5.1 mg/L, respectively. The 72-hour NOEC was 9.4 mg/L both for the growth rate and biomass.
Executive summary:

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 6.25, 12.5, 25, 50 and 100 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 4.5, 9.4, 21.0, 45.7 and 95.9 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72 EC50 and EC10 for the growth rate was determined to be 34.9 and 12.2 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 16.3 and 5.1 mg/L, respectively. The 72-hour NOEC was 9.4 mg/L both for the growth rate and biomass.

Description of key information

In an algae growth inhibition test according to OECD TG 201 with Raphidocelis subcapitata the 72 EC50 and EC10 for the growth rate was determined to be 34.9 and 12.2 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 16.3 and 5.1 mg/L, respectively. The 72-hour NOEC was 9.4 mg/L both for the growth rate and biomass.

Key value for chemical safety assessment

EC50 for freshwater algae:
34.9 mg/L
EC10 or NOEC for freshwater algae:
12.2 mg/L

Additional information

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata were exposed for 72 hours to nominal test item concentration of 6.25, 12.5, 25, 50 and 100 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 4.5, 9.4, 21.0, 45.7 and 95.9 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72 EC50 and EC10 for the growth rate was determined to be 34.9 and 12.2 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 16.3 and 5.1 mg/L, respectively. The 72-hour NOEC was 9.4 mg/L both for the growth rate and biomass.