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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
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)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Federal Office for the Environment (FOEN), Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples, the test item was found to be stable under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.03 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 15 min and intense stirring for 15 min at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media with the lower test concentrations. The test media were prepared just before the start of the test.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test media were clear solutions throughout the test period.
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, Göttingen, Germany)
- Method of cultivation: Algae were cultivated at the laboratory under standardized conditions according to the guideline.

ACCLIMATION
- Acclimation period: An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg CaCO3/L
Test temperature:
23 °C
pH:
7.5 - 8.1
Nominal and measured concentrations:
nominal: control, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
measured (0 h): < LOQ, not measured, not measured, not measured, 1.02, 3.29, 10.9, 33.5, 104 mg/L
measured (72 h): < LOQ, not measured, not measured, not measured, 0.851, 2.69, 8.43, 27.7, 82.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered with a glass lid
- Material, size, headspace, fill volume: glass, 75 mL, headspace: 45 mL, fill volume: 30 mL
- Aeration: continuous shaking
- Initial cells density: 5000 cells/mL (0.4E+04 relative fluorescence)
- Control end cells density: Only the relative fluorescence units were reported (mean: 59.5E+04)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: temperature was measured continuously, pH was measured at the beginning and end of exposure

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 67 μE s-1 m-2 (range: 65 - 68 μE s-1 m-2, measured at nine places in the experimental area)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: A small volume (2 x 100 μL) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement. The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm. At the end of the test, a sample was taken from the control and from the nominal test concentration of 10 mg/L to determine a potential influence of the test item on the algal cells.
- Cell shape: The shape and size of the algal cells were visually inspected.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: control, 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Inhibition of growth rate ranged from 2.4% (0.1 mg/L) to 84% (100 mg/L).
Reference substance (positive control):
yes
Remarks:
potassium dichromate (tested twice a year)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 20 - 24 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 5.8 - 8.5 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.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 9.9 - 15 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 1.8 - 4.7 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the test concentration of 10 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, test solutions were clear and colorless
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: EC50 (72 h): 0.9 mg/L (October 2016)
- Other: The reference substance was tested twice a year in a separate study.
Reported statistics and error estimates:
The EC10, EC20 and EC50 (72 h) values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Weibull 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 the Williams t-test or Welch t-test, where appropriate. Statistical analysis was performed using ToxRat Professional®.

The measured concentrations the test media of the test concentrations of 1.0 to 100 mg/L were between 102 and 109% of the nominal values at the start of the test and between 82 and 87% at the end of the test. Thus, the effect concentrations were based on the nominal concentrations.

Table 1: Average growth rates and inhibition of growth

Nominal test item concentrations [mg/L]

Average growth rate µ [day-1] and inhibition of µ [Ir]

0-24 h

0-48 h

0-72 h

µ

Ir [%]

µ

Ir [%]

µ

Ir [%]

Control

1.529

0.0

1.701

0.0

1.670

0.0

0.032

1.610

-5.3

1.685

1.0

1.669

0.1

0.10

1.346

12.0

1.653

2.8

1.654

1.0

0.32

1.377

9.9

1.656

2.6

1.635

2.1

1.0

1.493

2.3

1.704

-0.2

1.644

1.6

3.2

1.240*

18.9

1.664

2.2

1.623

2.8

10

1.168*

23.6

1.505#

11.6

1.514

9.4

32

0.756*

50.5

0.379#

77.8

0.364#

78.2

100

0.506*

66.9

0.279#

83.6

0.313#

81.3

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

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

Table 2: Inhibition of yield (Y)

Nominal test item concentration [mg/L]

Yield Y (x E+04] and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

1.4

0.0

11.5

0.0

59.1

0.0

0.0032

1.6

-10.7

11.1

3.5

58.8

0.4

0.10

1.1*

21.5

10.4

9.5

56.2

4.9

0.32

1.2*

18.1

10.5

8.9

53.1

10.2

1.0

1.4*

4.0

11.6

-0.6

54.6

7.5

3.2

1.0*

32.2

10.7*

7.5

51.3#

13.2

10

0.9*

38.7

7.7*

33.6

36.9#

37.5

32

0.5*

66.4

0.5*

96.0

0.8#

98.6

100

0.3*

81.7

0.3*

97.4

0.6#

98.9

*: 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 3: Results from analytical measurements

Sampling day/Sample age [d/h]

Nominal concentration of test item [mg/L]

Measured concentration of test item [mg/L]

Sample preparation factor

Determined concentration of test item [mg/L]

% of nominal

0/0 (fresh)

Control

n.d.

1.05

< LOQ

n.a.

1.0

0.969

1.05

1.02

102

3.2

3.14

1.05

3.29

103

10

10.4

1.05

10.9

109

32

31.9

1.05

33.5

105

100

98.6

1.05

104

104

3/72 (aged)

Control

n.d.

1.05

< LOQ

n.a.

1.0

0.810

1.05

0.851

85

3.2

2.56

1.05

2.69

84

10

8.03

1.05

8.43

84

32

26.4

1.05

27.7

87

100

78.5

1.05

82.5

82

n.d. = no test item detected

n.a. = not applicable

LOQ: 0.27 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The substance was found to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 h of exposure. The test was conducted according to the OECD guideline 201 (GLP). ErC10- and ErC50-value were found to be 7.2 mg/L and 22.0 mg/L, respectively.

Description of key information

ErC50 (72 h): 22 mg/L (nominal, OECD 201)

ErC10 (72 h): 7.2 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
22 mg/L
EC10 or NOEC for freshwater algae:
7.2 mg/L

Additional information

One experimental study is available investigating the toxicity of the test item to aquatic algae according to OECD 201 under GLP conditions (2017). Pseudokirchneriella subcapitata was exposed to nominal concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L for 72 h. The actual test concentrations were verified by HPLC/UV measurement and indicated that the substance was stable in the test media over the test period of 72 hours (between 102 and 109 % of nominal values at test start and between 82 and 87 % at test end). Thus, the effect concentrations were based on nominal concentrations.
The growth rate of algae was affected by the test item resulting in an 72-hour ErC50 of 22 mg/L (95% CI: 20 -24 mg/L) and an 72-hour ErC10 of 7.2 mg/L (95% CI: 5.8 - 8.5 mg/L).