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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:
from 2018-04-10 to 2018-05-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 23rd March 2006, corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
01 March 2016
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
April 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sample preparation: Three parallel samples were taken from each test concentration and six samples from the control solutions on each day of the analytical occasions. Formulation samples were diluted with ultrapure water before the analysis or directly injected.
- Sample storage conditions before analysis: No storage
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 any solubilising agents. A stock solution was first prepared by dissolving an amount of 0.08 g test item in 800 mL dilution water (OECD Medium) to obtain the nominal concentration of 100 mg/L. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution. After the formulation procedure algal cells were immediately introduced into test solution. Untreated control ran parallel in the test.
- Controls: Untreated control, toxic reference control (with the reference substance potassium dichromate )
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 (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum: 4 days

ACCLIMATION
- Acclimation period: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.4 – 22.8 °C
pH:
8.09 – 9.51
Nominal and measured concentrations:
Nominal concentration: 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L
Calculated geometric mean measured concentrations: 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, fill volume: Glass, ~250 mL volume, 100 mL test medium
- Aeration: No
- Initial cells density: N = 1 × 10^4 cells/mL
- Control end cells density: N = 79.33× 10^4 cells/mL (mean value)
- No. of vessels per concentration: 3
- No. of vessels per untreated control: 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD Medium

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water. Separate stock solutions were prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE Direct-Q 5 water purification system). Then, the growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 7971 lux, fluorescent lamps (400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and, therefore, provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 24, 48 and 72 h manual cell counting using microscope with counting chamber

RANGE FINDING TEST
- Test concentrations: 0.1 , 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes

STATISTICAL ANALYSIS
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h and 48 h, and at the end of the test (72 hours after the start of the test) using Excel for Windows software. Percentage inhibition of growth rate (µ) and yield (y) were calculated using Excel for Windows software. The ECx values and their confidence limits for growth rate and yield were calculated using Probit analysis by SPSS software. For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (α = 0.05; 2-sided) by SPSS software.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 2.11 - 2.78
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 3.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 2.71 - 3.83
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.02 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conf. limits: 3.81 - 8.10
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.9 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.:
3.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.71 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 1.50 - 1.87
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 1.84 - 2.17
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.78 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% conf. limits: 2.58 - 3.04
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.02 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: Some swollen cells were noticed in one replicate of the 1.18 mg/L concentration (measured). Thin cells were observed in the concentrations of 1.18, 1.90 and 3.02 mg/L (measured) together with the normal cells during the test.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: 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
- 72h ErC50: 1.08 mg/L (95 % confidence limits: 0.79 – 1.51 mg/L)
- 72h EyC50: 0.61 mg/L (95 % confidence limits: 00.49 – 0.76 mg/L)
Reported statistics and error estimates:
Statistical procedure for ErCx (growth rate) and for EyCx (yield) estimation for Growth rates: Probit analysis, Pearson Goodness-of-Fit Test.
Statistical procedure for NOEC estimation: Dunnett´s test

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

Algal cell number (× 104 cell/mL) during the test period of 72 hours and YieldConcentration

 

Number of Cells (× 104/mL)

Yield

Nominal
[mg/L]

measured
[mg/L]

0 h

24 h

48 h

72 h

Control

Not detected

 

1

4

27

71

70

 

1

4

26

103

102

 

1

4

21

62

61

 

1

4

25

83

82

 

1

4

23

102

101

 

1

3

21

55

54

Mean

1.00

3.83

23.83

79.33

78.33

SD

0.00

0.41

2.56

20.25

20.25

2.6

0.47

 

1

4

27

90

89

 

1

2

23

79

78

 

1

4

29

71

70

Mean

1.00

3.33

26.33

80.00

79.00

SD

0.00

1.15

3.06

9.54

9.54

6.4

0.70

 

1

5

25

103

102

 

1

4

23

82

81

 

1

3

15

90

89

Mean

1.00

4.00

21.00

91.67

90.67

SD

0.00

1.00

5.29

10.60

10.60

16.0

1.18

 

1

3

25

98

97

 

1

4

12

85

84

 

1

3

21

78

77

Mean

1.00

3.33

19.33

87.00

86.00

SD

0.00

0.58

6.66

10.15

10.15

40.0

1.90

 

1

4

17

48

47

 

1

4

24

80

79

 

1

3

19

64

63

Mean

1.00

3.67

20.00

64.00

63.00

SD

0.00

0.58

3.61

16.00

16.00

100.0

3.02

 

1

2

13

36

35

 

1

4

13

40

39

 

1

3

15

29

28

Mean

1.00

3.00

13.67

35.00

34.00

SD

0.00

1.00

1.15

5.57

5.57

Table 2: The specific Growth Rates during 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

 

0.0578

0.0687

0.0592

 

0.0578

0.0679

0.0644

 

0.0578

0.0634

0.0573

 

0.0578

0.0671

0.0614

 

0.0578

0.0653

0.0642

 

0.0458

0.0634

0.0557

Mean

0.0558

0.0660

0.0604

SD

0.0049

0.0023

0.0036

2.6

0.47

 

0.0578

0.0687

0.0625

 

0.0289

0.0653

0.0607

 

0.0578

0.0702

0.0592

Mean

0.0481

0.0680

0.0608

SD

0.0167

0.0025

0.0016

6.4

0.70

 

0.0671

0.0671

0.0644

 

0.0578

0.0653

0.0612

 

0.0458

0.0564

0.0625

Mean

0.0569

0.0629

0.0627

SD

0.0107

0.0057

0.0016

16.0

1.18

 

0.0458

0.0671

0.0637

 

0.0578

0.0518

0.0617

 

0.0458

0.0634

0.0605

Mean

0.0498

0.0608

0.0620

SD

0.0069

0.0080

0.0016

40.0

1.90

 

0.0578

0.0590

0.0538

 

0.0578

0.0662

0.0609

 

0.0458

0.0613

0.0578

Mean

0.0538

0.0622

0.0575

SD

0.0069

0.0037

0.0036

100.0

3.02

 

0.0289

0.0534

0.0498

 

0.0578

0.0534

0.0512

 

0.0458

0.0564

0.0468

Mean

0.0441

0.0544

0.0493

SD

0.0145

0.0017

0.0023

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

Concentration

Growth Rates (µ)and % Inhibition ofµ

Nominal
[mg/L]

measured
[mg/L]

0–24 h

0–48 h

0–72 h

µ

%

µ

%

µ

%

Control

Not detected

0.0558

0.0660

0.0604

2.6

0.47

0.0481

13.7

0.0680

-3.2

0.0608

-0.7

6.4

0.70

0.0569

-2.0

0.0629

4.6

0.0627

-3.9

16.0

1.18

0.0498

10.7

0.0608

7.9

0.0620

-2.7

40.0

1.90

0.0538

3.6

0.0622

5.7

0.0575

4.8

100.0

3.02

0.0441

20.8

0.0544

17.5

0.0493

18.4

Table 4: Yield (y) and percentage inhibition of y during the test period       

Concentration

Yield y and % Inhibition of y

Nominal
[mg/L]

measured
[mg/L]

0–72 h

y

%

Control

Not detected

78.33

2.6

0.47

79.00

-0.9

6.4

0.70

90.67

-15.7

16.0

1.18

86.00

-9.8

40.0

1.90

63.00

19.6

100.0

3.02

34.00

56.6

Table 5: Sodium pyruvate concentration measured on 10 April 2018 (at the start of the test)       

Nominal concentration
(mg/L)

Mean of the measured concentrations (mg/L)

Measured concentration in % of the nominal

2.6

2.45

 

94

6.4

 

6.10

 

95

16

 

14.9

 

93

40

 

37.1

 

93

100

 

92.3

 

92

Table 6: Sodium pyruvate concentration measured on 11 April 2018

Nominal concentration
(mg/L)

Mean of the measured concentrations (mg/L)

Measured concentration in % of the nominal

2.6

2.01

77

6.4

3.98

62

16

12.8

80

40

35.0

87

100

90.7

91

Table 7: Sodium pyruvate concentration measured on 12 April 2018

Nominal concentration
(mg/L)

Mean of the measured concentrations (mg/L)

Measured concentration in % of the nominal

2.6

0

-

6.4

0

-

16

0

-

40

0

-

100

below the quantitation limit

-

On 13 April 2018 (at the end of the test) concentrations of all the samples were zero. The test item was not detected in the control samples.

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 3.02 mg/L and 2.78 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L and 1.71 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out at nominal concentrations of 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L because of the results of the pretest. The corresponsing measured concentrations were 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L. In this study, the test item was found to inhibit the growth of the freshwater green alga Raphidocelis subcapitata after 72 hours with the following effect values (based on geometric means of measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >3.02 mg/L (95 % conf. limits: 3.81 - 8.10) and 2.78 mg/L (95 % conf. limits: 2.58 - 3.04), respectively. The EC-10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L (95 % conf. limits: 2.11 – 2.78) and 1.71 (95% conf. limits: 1.50 - 1.87), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met, therefore, the study is considered as valid.

Description of key information

The EC50-values of the substance for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours are > 3.02 mg/L and 2.78 mg/L (geometric mean measured), respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L and 1.71 mg/L (geometric mean measured), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L (geometric mean measured).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
2.45 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) was determined according to the principles of OECD 201, Council Regulation (EC) No. 761/2009 C.3 and EPA OPPTS 850.5400. The study was conducted in compliance with GLP criteria and under static conditions. The definitive test was carried out at nominal concentrations of 2.6, 6.4, 16.0, 40.0 and 100.0 mg/L because of the results of the pretest. The corresponsing measured concentrations were 0.47, 0.70, 1.18, 1.90 and 3.02 mg/L. In this study, the test item was found to inhibit the growth of the freshwater green alga Raphidocelis subcapitata after 72 hours with the following effect values (based on geometric means of measured test item concentrations): The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were >3.02 mg/L (95 % conf. limits: 3.81 - 8.10) and 2.78 mg/L (95 % conf. limits: 2.58 - 3.04), respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.45 mg/L (95 % conf. limits: 2.11 – 2.78) and 1.71 (95 % conf. limits: 1.50 - 1.87), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.90 mg/L. In conclusion, the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met, therefore, the study is considered as valid (reference 6.1.5-1).