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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:
08 February 2018 – 16 June 2018
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)
Deviations:
yes
Remarks:
Some analysed concentrations of test item were slightly outside the acceptable limit. This deviation does not have any impact on the study since test item was found stable in test medium and the EC50 is far from LOQ of analytical method
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L.
- Sampling method: All the test concentrations along with the negative control were analyzed for the test item concentration at the beginning and end of test. Test item stock (100 mg/L) was analysed at the start of test.
- Sample storage conditions before analysis: not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item stock of 0.1 mg/mL (100 mg/L) was prepared by mixing 100 mg test item and making up to 1 L in a volumetric flask using test medium. To prepare test concentrations of 0.238, 0.762, 2.44, and 7.81 mg/L, 2.38, 7.62, 24.4, and 78.1 mL test item stock was mixed and made up to 1 L in a volumetric flask using test medium separately. To prepare test concentrations of 25 and 80 mg/L, 25 and 80 mg test item was mixed and made up to 1 L in a volumetric flask using test medium separately. 4.7 mL algal cell suspension was added before final volume make up for each test concentration in the volumetric flask.
- Negative control: 4.7 mL algal cell suspension was made up to 1 L using test medium.
- Positive control: A stock solution of reference substance (Potassium dichromate- K2Cr2O7) at 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium. For preparation of reference substance at 0.9 mg/L, 0.9 mL of reference substance stock was made up to 1L using test medium. 4.7 mL algal cell suspension was added just before final volume make up.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108, USA
- Method of cultivation: not reported

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): OECD TG 201 medium was used. Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 25°C (±2°C) under a continuous illumination of 8000±1600 Lux.
To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculating Pseudokirchneriella subcapitata of continuous/mother culture to test medium 4 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions.
- Any deformed or abnormal cells observed: not reported.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
22.1 to 22.5ºC
pH:
7.78 to 7.84
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L.
Mean measured concentrations (after 72 h): 0 (control), 0.09953, 0.2004, 2.206, 8.569, 21.94 and 79.67mg/L.
Nominal concentrations were considered to calculate the EC values.
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Material, size, headspace, fill volume: 250 mL capacity, 150 mL fill volume
- Aeration: The test containers were not bubble aerated.
- Initial cells density: 10E+04 cells/mL
- Control end cells density: 69.08 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (negative) and 3 (positive)
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: he OECD medium was prepared as per the OECD guideline 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the controls and the test solutions was checked at the beginning and at the end of the test. Light intensity inside the algal chamber (at four random locations) was measured once daily using a Lux meter. Temperature inside the algal chamber was recorded once daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 6850 to 7013 Lux obtained with white fluorescent tube lights (light intensity of approximately 4000 to 10000 Lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The algal biomass in each flask was determined at 24, 48 and 72 hours after the start of the test using a haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 3.2
- Justification for using less concentrations than requested by guideline: Not applicable; 6 concentrations used for the main test.

- Range finding study
- Test concentrations: 0.0 (control), 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L there was a reduction of algal cell biomass by 7.28, 12.58, 18.54, 20.53, 30.46, 65.56, 82.45 and 91.72 %, respectively.
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
27.349 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: : (fiducial limits: 9.71335 - 77.00683 mg/L)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.204 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (fiducial limits: 2.55845 - 20.2832 mg/L)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.159 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (fiducial limits: 12.0872 - 21.6025 mg/L)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.445 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (fiducial limits: 1.82878 - 3.26844 mg/L)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.911 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (fiducial limits: 0.6814 - 1.21781 mg/L)
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Observed cells were found morphologically normal.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none.

Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: There was 22.48% and 62.30% inhibition of average growth rate and yield of alga, respectively, in the reference substance group at 0.9 mg/L when compared to negative control.
Reported statistics and error estimates:
The inhibition of growth was determined by calculating the ErC and EyC by Probit analysis of Finney (1971) using developed and validated computer program.

Table 1: Definitive Test - Cell Counts at Different Time Intervals

Group

Treatment (mg/L)

Replications

Average cell counts

(x 104cells/mL)

Yield

(x 104cells/ mL) at 72 h

0 h*

24 h

48 h

72 h

G1

Negative control

R1

1.00

5.00

26.75

67.00

66.00

R2

1.00

4.00

26.50

69.50

68.50

R3

1.00

5.75

27.00

68.00

67.00

R4

1.00

6.50

26.25

71.50

70.50

R5

1.00

5.50

28.00

69.25

68.25

R6

1.00

5.25

26.00

69.25

68.25

Average

1.00

5.33

26.75

69.08

68.08

G2

0.238

R1

1.00

4.75

24.50

69.00

68.00

R2

1.00

4.50

26.75

71.50

70.50

R3

1.00

5.50

25.00

70.25

69.25

Average

1.00

4.92

25.42

70.25

69.25

G3

0.762

R1

1.00

6.25

17.25

65.50

64.50

R2

1.00

5.50

18.75

65.00

64.00

R3

1.00

6.00

18.75

66.25

65.25

Average

1.00

5.92

18.25

65.58

64.58

G4

2.44

R1

1.00

4.00

16.75

54.25

53.25

R2

1.00

3.50

18.25

55.25

54.25

R3

1.00

5.75

18.25

54.75

53.75

Average

1.00

4.42

17.75

54.75

53.75

G5

7.81

R1

1.00

4.25

15.75

39.00

38.00

R2

1.00

4.25

15.50

38.75

37.75

R3

1.00

4.00

16.75

37.75

36.75

Average

1.00

4.17

16.00

38.50

37.50

G6

25

R1

1.00

2.75

14.25

27.50

26.50

R2

1.00

3.25

14.75

29.25

28.25

R3

1.00

2.25

13.75

29.00

28.00

Average

1.00

2.75

14.25

28.58

27.58

G7

80

R1

1.00

1.50

11.25

19.50

18.50

R2

1.00

1.25

11.00

19.25

18.25

R3

1.00

0.75

10.75

20.75

19.75

Average

1.00

1.17

11.00

19.83

18.83

G8

Positive

Control

(0.9 mg/L K2Cr2O7)

R1

1.00

4.25

6.50

29.50

28.50

R2

1.00

4.50

4.50

25.00

24.00

R3

1.00

3.50

4.00

25.50

24.50

Average

1.00

4.08

5.00

26.67

25.67

*:  0 hour observations are based on loading of algal cells.

Table 2: Definitive Test Results – Algal Cell Biomass

Group

 

Treatment

(mg/L)

Average Cell Counts (x 104cells/mL)

0 hour

24 hour

48 hour

72 hour

 

G1

Negative control

1.00

5.33

26.75

69.08

 

G2

0.238

1.00

4.92

25.42

70.25

 

G3

0.762

1.00

5.92

18.25

65.58

 

G4

2.44

1.00

4.42

17.75

54.75

 

G5

7.81

1.00

4.17

16.00

38.50

 

G6

25

1.00

2.75

14.25

28.58

 

G7

80

1.00

1.17

11.00

19.83

 

G8

 

Reference substance

(0.9 mg/L K2Cr2O7)

 

1.00

4.08

5.00

26.67

Table 3: Definitive Test Results –Percent Growth Rate Inhibition

Group

Treatment

(mg/L)

Average Cell Counts

(x 104cells/mL)

(0 hour)

Average Cell Counts

(x 104cells/mL)

(72 hour)

Natural log (average cell counts at 0 hour)

Natural log (average cell counts at 72 hour)

 

Growth Rate

(anti log values)

% Growth Rate Inhibition

 

G1

Negative control

1.00

69.08

9.21034

13.44561

0.05882

-

 

G2

0.238

1.00

70.25

9.21034

13.4624

0.05906

-0.41*

 

G3

0.762

1.00

65.58

9.21034

13.39361

0.05810

1.22

 

G4

2.44

1.00

54.75

9.21034

13.21312

0.05559

5.49

 

G5

7.81

1.00

38.50

9.21034

12.861

0.05070

13.8

 

G6

25

1.00

28.58

9.21034

12.56305

0.04657

20.83

 

G7

80

1.00

19.83

9.21034

12.19754

0.0415

29.46

 

G8

 

Reference substance

(0.9 mg/L K2Cr2O7)

1.00

26.67

9.21034

12.49388

0.0456

22.48

*: Considered as zero

Table 4: Definitive Test Results –Percent Yield Inhibition

Group

Treatment

(mg/L)

Average Cell Counts

(x 104cells/mL)

(0 hour)

Average Cell Counts

(x 104cells/mL)

(72 hour)

Yield

(x 104cells/mL) at 72 hours

% Yield

Inhibition

 

G1

Negative control

1.00

69.08

68.08

-

 

G2

0.238

1.00

70.25

69.25

-1.71*

 

G3

0.762

1.00

65.58

64.58

5.14

 

G4

2.44

1.00

54.75

53.75

21.05

 

G5

7.81

1.00

38.50

37.50

44.92

 

G6

25

1.00

28.58

27.58

59.49

 

G7

80

1.00

19.83

18.83

72.34

 

G8

 

Reference substance

(0.9 mg/L K2Cr2O7)

 

1.00

 

26.67

 

25.67

 

62.30

*: Considered as zero

Table 5: Definitive Test - Specific Growth Rate. Negative control.

Replications

Specific Growth rates

Replicate-mean specific growth rate

S.D.

% CV

24 h - 0 h

48 h - 24 h

72 h - 48 h

R1

1.61

1.68

0.92

1.40

0.42

29.93

R2

1.39

1.89

0.96

1.41

0.47

32.93

R3

1.75

1.55

0.92

1.41

0.43

30.79

R4

1.87

1.40

1.00

1.42

0.44

30.6

R5

1.71

1.62

0.91

1.41

0.44

31.01

R6

1.66

1.60

0.98

1.41

0.38

26.64

Mean ± SD (% CV)

1.67 ± 0.16 (9.68)

1.62 ± 0.16 (9.94)

0.95 ± 0.04 (3.91)

1.41 ± 0.01 (0.49)

-

30.32

Mean coefficient of variation (% CV) for section by section specific growth rates

30.32

The coefficient of variation (% CV) of average specific growth rates during the whole test period in replicate control cultures

0.49

Table 6: Results of Test Concentration Analysis at the End of Test

Claimed Con
 (mg/ L)

Sample Code

Analyzed Test Item Conc        (mg/L)

% Agreement with Claimed Conc.

 

Final Test Item

Conc.

Overall % Agreement with Claimed Conc.

Mean

SD

%RSD

Mean

SD

%RSD

0.238

G2R1-1

0.09460

39.42

0.09953

0.00807

8.1

41.47

3.36

8.1

G2R1-2

0.1089

45.35

G2R1-3

0.09515

39.65

0.762

G3R1-1

0.1825

24.01

0.2004

0.0156

7.8

26.36

2.05

7.8

G3R1-2

0.2112

27.79

G3R1-3

0.2074

27.28

2.44

G4R1-1

2.264

92.80

2.206

0.124

5.6

90.40

5.09

5.6

G4R1-2

2.290

93.84

G4R1-3

2.063

84.55

7.81

G5R1-1

8.895

113.9

8.569

0.621

7.3

109.7

7.96

7.3

G5R1-2

7.853

100.5

G5R1-3

8.960

114.7

25.00

G6R1-1

19.93

79.70

21.94

2.42

11.0

87.77

9.66

11

G6R1-2

21.28

85.14

G6R1-3

24.62

98.48

80.00

G7R1-1

87.79

109.7

79.67

8.92

11

99.59

11.1

11

G7R1-2

81.09

101.4

G7R1-3

70.13

87.66

Acceptance limits: The acceptable range is 80 to 120 % of claimed concentration with ≤ 20 % of relative standard deviation (RSD) of the concentration.

Conclusion: There was no interference in the G1 samples at the RT of the analyte. The obtained results of G2 and G3 group were out of acceptance limits (80-120%).

Validity criteria fulfilled:
yes
Remarks:
(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)
Conclusions:
In an Alga Growth Inhibition Test with Pseudokirchneriella subpicata green algae, the 72h-ErC50 of the test substance was determined to be higher than 80 mg/L and the 72h-ErC10 was calculated to be 7.2037 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the per cent reduction of algal cell biomass was 7.28, 12.58, 18.54, 20.53, 30.46, 65.56, 82.45 and 91.72 %, at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with test item concentrations of 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L (factor of 3.2). Negative control consisted of test medium alone and a solution of 0.9 mg/L potassium dichromate was used as reference substance.Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group and three replicates for the reference substance. A validated analytical method based on HPLC was used to monitor the concentration and stability of the active ingredient in the test solution. The analysis of test concentrations 2.44, 7.81, 25 and 80 mg/L showed an agreement with claimed concentrations of 87.77 to 99.59% at end of test (72 hour). Test concentrations of 0.238 and 0.762 mg/L were outside the acceptable limit (80-120% of nominal). This deviation does not have any impact on the study since stability test found that test item is stable in test medium between 1.08 and 108 mg/L and the EC50 is far from LOQ of analytical method. Thus, nominal concentrations were considered to calculate EC values. All validity criteria were fulfilled. Observed cells were found morphologically normal. The per cent inhibition of growth rate was 0, 1.22, 5.49, 13.8, 20.83 and 29.46 % and the percent yield inhibition was 0, 5.14, 21.05, 44.92, 59.49 and 72.34 % at the tested concentrations of 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L, respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 7.2037 mg/L, 72h-ErC20 = 27.3495 mg/L and 72 h-ErC50 > 80 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 = 0.9109 mg/L, 72h-EyC20 = 2.4448 mg/L and 72h-EyC50 = 16.159 mg/L.

Description of key information

Key study. Test method according to OECD 201, GLP study. The 72h-ErC50 of the test substance to Pseudokirchneriella subpicata green algae was determined to be higher than 80 mg/L and the 72h-ErC10 was calculated to be 7.2037 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
80 mg/L
EC10, LC10 or NOEC for marine water algae:
7.204 mg/L

Additional information

Key study: An Algae Growth Inhibition Test was performed with Pseudokirchneriella subpicata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the per cent reduction of algal cell biomass was 7.28, 12.58, 18.54, 20.53, 30.46, 65.56, 82.45 and 91.72 %, at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with test item concentrations of 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L (factor of 3.2). Negative control consisted of test medium alone and a solution of 0.9 mg/L potassium dichromate was used as reference substance.Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group and three replicates for the reference substance. A validated analytical method based on HPLC was used to monitor the concentration and stability of the active ingredient in the test solution. The analysis of test concentrations 2.44, 7.81, 25 and 80 mg/L showed an agreement with claimed concentrations of 87.77 to 99.59% at end of test (72 hour). Test concentrations of 0.238 and 0.762 mg/L were outside the acceptable limit (80-120% of nominal). This deviation does not have any impact on the study since stability test found that test item is stable in test medium between 1.08 and 108 mg/L and the EC50 is far from LOQ of analytical method. Thus, nominal concentrations were considered to calculate EC values. All validity criteria were fulfilled. Observed cells were found morphologically normal. The per cent inhibition of growth rate was 0, 1.22, 5.49, 13.8, 20.83 and 29.46 % and the percent yield inhibition was 0, 5.14, 21.05, 44.92, 59.49 and 72.34 % at the tested concentrations of 0.238, 0.762, 2.44, 7.81, 25 and 80 mg/L, respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 7.2037 mg/L, 72h-ErC20 = 27.3495 mg/L and 72 h-ErC50 > 80 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 = 0.9109 mg/L, 72h-EyC20 = 2.4448 mg/L and 72h-EyC50 = 16.159 mg/L.