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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:
2020-02-07 to 2020-05-04
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:
adopted 2006, corrected 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) No 2016/266 of December 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 3.2, 10, 32 and 100 mg/L

- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media at all test concentrations and the control at the start (without algae) and at the end of the test (with algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.

- Sample storage conditions before analysis: All samples were frozen (at -20 ± 5 °C) immediately after sampling and stored until analysis.
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 99.98 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature in the dark. The test media of the lower test item concentrations were prepared in a series of further dilution steps. The test media were prepared just before the start of the test.

- Controls: A control (test water without test item) was tested in parallel
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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: 3 days (exponentially growth)
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg/L as CaCO3
Test temperature:
22 °C
pH:
7.5 to 8.2
Dissolved oxygen:
not specified
Salinity:
not relevant
Conductivity:
not specified
Nominal and measured concentrations:
Nominal: control, 1.0, 3.2, 10, 32, 100 mg/L;
Mean Measured (geometric mean): n.a., n.a., 0.021, 0.33, 1.1, 80 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, fill volume: glass, 75 mL, 30 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: ~ 67 μE s-1 m-2

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
- Range finding study: performed
- Test concentrations: 0.1, 1.0, 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:
48 h
Dose descriptor:
EC50
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % Confidence Interval: 2.6 - 5.8
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % Confidence Interval: 0.077 - 0.17
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % Confidence Interval: 1.3 - 2.7
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.066 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % Confidence Interval: 0.043 – 0.096
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
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: The shape and size of the algal cells were obviously not affected by the test item up to at least the mean measured test concentration of 1.1 mg/L (nominal 32 mg/L)
- 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
Reported statistics and error estimates:
The 48-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear weighted regression.
For the determination of the 48-hour 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®.

Table 1: Biomass of Algae

Nominal Test

Item

Concentration (mg/L)

Mean Measured

Test Item

Concentration (mg/L)

Repl.

No.

Biomass of Algae*

24 Hours

48 Hours

72 Hours**

Control

--

1

2.11

13.4

62.4

2

1.89

13.6

57.9

3

2.52

13.0

60.6

4

2.13

14.4

63.2

5

2.08

13.6

58.4

6

2.13

13.5

60.3

Mean SD

2.14

13.6

60.5

0.204

0.447

2.09

1.0

n.a

1

2.15

16.4

64.5

2

2.54

14.2

62.1

3

2.10

16.0

63.9

Mean SD

2.26

15.5

63.5

0.238

1.15

1.28

3.2

0.021

1

1.46

13.0

59.7

2

1.57

13.5

60.6

3

2.19

13.7

61.7

Mean SD

1.74

13.4

60.7

0.397

0.358

1.00

10

0.33

1

1.77

12.4

55.1

2

1.69

15.5

53.1

3

2.04

14.1

54.5

Mean SD

1.83

14.0

54.2

0.185

1.56

1.02

32

1.1

1

1.73

6.04

20.3

2

1.37

4.00

10.8

3

1.55

6.49

19.2

Mean SD

1.55

5.51

16.8

0.181

1.33

5.18

100

80

1

0.468

0.306

0.249

2

0.468

0.634

0.121

3

0.525

0.490

0.176

Mean SD

0.487

0.477

0.182

0.0331

0.164

0.0643

n.a.: Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

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 0.696 x 104 relative fluorescence units.

**: The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Table 2: Average Growth Rates (μ)

Nominal Test Item Concentration (mg/L)

Mean Measured Concentration (mg/L)

Average Growth Rate μ [day-1] and Inhibition Ir [%]

0-24 h

0-48 h

0-72 h**

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

Control

---

1.121

0.0

1.485

0.0

1.488

0.0

1.0

n.a

1.177

-4.9

1.552

-4.5

1.505

-1.1

3.2

0.021

0.900*

19.7

1.479

0.4

1.489

-0.1

10

0.33

0.965*

14.0

1.499

-1.0

1.452

2.4

32

1.1

0.798*

28.9

1.024*

31.1

1.048*

29.6

100

80

-0.358*

131

-0.211*

114

-0.461*

131

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

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

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 3: Yield (Y)

Nominal Test Item Concentration (mg/L)

Mean Measured Concentration (mg/L)

Average Growth Rate μ [day-1] and Inhibition Ir [%]

0-24 h

0-48 h

0-72 h**

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

---

1.4

0.0

12.9

0.0

59.8

0.0

1.0

n.a

1.6

-8.4

14.8

-15.3

62.8

-5.1

3.2

0.021

1.0*

27.9

12.7

1.4

60.0

-0.3

10

0.33

1.1*

21.5

13.3

-3.4

53.5#

10.5

32

1.1

0.9*

40.8

4.8*

62.6

16.1#

73.1

100

80

-0.2*

114.4

-0.2

101.7

-0.5#

100.9

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

*:          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).

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Table 4: Section-by-Section Growth Rates

Nominal Test Item Concentration (mg/L)

Mean Measured Concentration (mg/L)

Average Growth Rate μ [day-1] and Inhibition Ir [%]

0-24 h

0-48 h

0-72 h**

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

Control

---

1.121

0.0

1.849

0.0

1.494

0.0

1.0

n.a

1.177

-4.9

1.928

-4.2

1.409

5.7

3.2

0.021

0.900

19.7

2.057

-11.3

1.511

-1.1

10

0.33

0.965

14.0

2.034

-10.0

1.357

9.2

32

1.1

0.798

28.9

1.250

32.4

1.097

26.6

100

80

-0.358

132

-0.063

103

-0.961

164

n.a.:     Not analyzed since the inhibition of growth rate and yield was below the 48 and 72-hour NOEC.

**:        The biological results after 72 hours were not used for the calculation of the ECx and NOEC/LOEC values due to the decrease of the substance after 48 hours.

Note:    Percentage inhibition values in excess of 100 % are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Table 5: Impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (results are based on mean measured concentrations of the test item)

Endpoint after 48 Hours

Growth Rate [mg/L]

Yield [mg/L]

EC10

95 % Confidence Interval

0.12

0.077 – 0.17

0.066

0.043 – 0.096

EC20

95 % Confidence Interval

0.38

027 – 0.53

0.21

0.15 – 0.29

EC50

95 % Confidence Interval

3.7

2.6 – 5.8

1.9

1.3 – 2.7

NOEC

0.33

0.33

LOEC

1.1

1.1

Analytical results

Table 6: Results for the Range-Finding Test

Sampling Day/Sample Age

[day/hours]

Nominal Concentration of Test Item Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

% from Initially Measured

[%]

0/0

(fresh)

Control

n.d.

126

< LOQ

n.a.

1

7.74

126

0.977

98

10

81.1

126

10.2

102

100

38.7

2561

103

103

1/24 (aged)

1

5.78

126

0.729

73

2/48 (aged)

1

n.d.

2.1

< LOQ

n.a.

3/72

(aged)

Control

n.d.

2.1

< LOQ

n.a.

1

n.a.

2.1

< LOQ

n.a.

10a

n.a.

51.3

< LOQ

n.a.

100

65.2

504

32.8

33

LOQ: 0.002 mg test item /L (LOQ in range-finding test)

n.d. = not detected

n.a. = not applicable

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

 

Table 7: Results for Spiked Samples

Nominal Concentration of Test Item

Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg]

Sample Preparation Factor

F

Determined Concentration of Test Item

C

[mg/L]

Recovery Rate

R

[%]

Accuracy (Average Recovery)

[%]

Precision (Relative Standard Deviation of Recovery)

[%]

0

n.d.

2

n.a.

< LOQ

n.a.

-

0.00728

4.09

2

0.00817

101

113

6.1

0.00728

3.84

2

0.00769

104

0.00728

3.97

2

0.00794

109

0.00728

4.51

2

0.00901

124

0.00728

4.13

2

0.00827

114

121

183

669

122

101

105

2.4

121

189

669

127

104

121

191

669

127

105

121

193

669

129

107

121

194

669

130

107

Acceptance Target:

80 - 120

≤ 10

LOQ: 0.00365 mg test item /L

n.d. = not detected

n.a. = not applicable

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

 

Table 8: Results for Centrifuged Spiked Samples

Nominal Concentration of Test Item

[day/hours]

Measured Concentration of Test Item in the Spiked Sample

x

[µg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

Recovery Rate

R

[%]

0.00728

3.71

2

0.00741

102

121

179

669

120

99

 

Table 9: Results for Test Samples

Sampling Day/Sample Age

[day/hours]

Nominal Concentration of Test Item Cnom

[mg/L]

Measured Concentration of Test Item

X

[µg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

[mg/L]

% of Nominal Concentration

[%]

0/0

(fresh)

Control

n.d.

2

< LOQ

n.a.

3.2

93:5

31

2:86

89

10

95:7

102

9:76

98

32

158

302

31:8

100

100

153

669

102

102

1/24

(aged)

Control

n.d.

2

< LOQ

n.a.

3.2

n.d.

2

< LOQ

n.a.

10

20.0

102

2.04

20

32

103

202

20.9

65

100

144

669

96.3

96

2/48

(fresh)

Control

n.d.

2

< LOQ

n.a.

3.2

n.d.

2

< LOQ

n.a.

10

n.d.

2

< LOQ

n.a.

32

n.d.

2

< LOQ

n.a.

100

77.1

669

51.6

52

2/72

(aged)

control

n.d.

2

< LOQ

n.a.

3.2

n.d.

2

< LOQ

n.a.

10

n.d.

2

< LOQ

n.a.

10a

n.d.

2

< LOQ

n.a.

10b

n.d.

2

< LOQ

n.a.

32

n.d.

2

< LOQ

n.a.

100

2.69

2

0.00538

0.00538

LOQ: 0.00365 mg test item/L

a: without algae

b: without algae, without light

n.d. = not detected

n.a. = not applicable

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

 

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects on Pseudokirchneriella subcapitata in a 72-hour static test performed according to OECD 201.
Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.
Therefore, the 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).
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, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The analytical measurements from test media at the start of the test show the correct preparation of the test media. As the substance is highly instable, the test item concentration decreased distinctly during the test period of 72 hours. At the end of the test, all test concentrations were below the limit of quantification (LOQ = 0.00365 mg/L), with the exception of the highest test concentration of 100 mg/L, where 0.005 % of the nominal concentration could be measured.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Photodegradation could be excluded, since a decrease of the test item was also determined in the additional samples incubated without light but it seems that the test item is not hydrolytically stable in test water.

The samples of the nominal test concentration of 1.0 mg/L were not analyzed since the concentrations were below the 48-hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations.

The 48-hour NOEC for growth rate and yield was determined to be 0.33 mg/L, since at this concentration the growth rate and yield of the algae after 48 hours were not significantly lower than in the control. The LOEC for growth rate and yield was determined to be 1.1 mg/L. The 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).

Description of key information

The test item had acute toxic effects on Pseudokirchneriella subcapitata  in a 72-hour static test performed according to OECD 201.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Therefore, the 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).

Key value for chemical safety assessment

EC50 for freshwater algae:
3.7 mg/L
EC10 or NOEC for freshwater algae:
0.12 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, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3. The nominal test item concentrations tested were 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The analytical measurements from test media at the start of the test show the correct preparation of the test media. As the substance is highly instable, the test item concentration decreased distinctly during the test period of 72 hours.

At the end of the test, all test concentrations were below the limit of quantification (LOQ = 0.00365 mg/L), with the exception of the highest test concentration of 100 mg/L, where 0.005 % of the nominal concentration could be measured.

Due to the almost complete decrease of the test item after 72 hours and since the biomass in the control already increased by a factor of 19.5 over the first 48 hours it was decided, to calculate the mean measured concentrations as the geometric mean of the concentrations measured at the sampling dates after 0, 24 and 48 hours. It was also decided to reflect the biological 48-hour endpoints based on these mean measured concentrations.

Photodegradation could be excluded, since a decrease of the test item was also determined in the additional samples incubated without light but it seems that the test item is not hydrolytically stable in test water.

The samples of the nominal test concentration of 1.0 mg/L were not analyzed since the concentrations were below the 48-hour NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Due to the decrease of the test item concentrations during the test period of 72 hours, the biological results were related to the mean measured concentrations.

The 48-hour NOEC for growth rate and yield was determined to be 0.33 mg/L, since at this concentration the growth rate and yield of the algae after 48 hours were not significantly lower than in the control. The LOEC for growth rate and yield was determined to be 1.1 mg/L. The 48-h EC50 for growth rate was calculated to be 3.7 mg/L with 95 % confidence limits of 9.0 and 12 mg/L. The 48-h EC10 for growth rate was determined to be 0.12 mg/L (95 %-CL: 0.077 - 0.17).