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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:
02 February 2018 to 15 March 2018
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:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Dilutions of 1:1000, 1:320, 1:100, 1:32, 1:10, plus control
- Sampling method: Duplicate samples were taken at 0 (without algae) hours and 72 hours (with algae). At the end of the test, the treatment replicates were pooled.
- Sample storage conditions before analysis: All samples were frozen at a temperature of -20 ± 5 °C and protected from light.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was liquefied in a water bath at approximately 50 °C. 100 µL of the melted test item was pipetted into 1000 mL test water at 50°C. Ultrasonic treatment was applied to the test medium at 50 °C for 10 minutes, followed by intensive stirring at room temperature for 2 hours until the test media cooled to 21°C. Dilutions of this were then used for the test concentrations.
- Controls: Test water with the absence of test item was used as a negative control. Potassium dichromate was used as a positive control to test for the sensitivity of test system.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Undissolved material remained after sample preparation. A 0.45 μm membrane filter (pre-conditioned with 200 mL filtrate) was used to filter the insoluble particles from the test media. All test media were clear solutions throughout test.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Originally from the collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen /Germany).
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium.
- Method of cultivation: Algae were cultivated under standardised conditions according the OECD TG 201.

ACCLIMATION
- Acclimation period: Not specified
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No
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:
Start: 7.3
End: 7.4 - 7.8
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Dilution 1:10, 1:32, 1:100 1:320 and 1:1000 of the undiluted filtrate (loading rate 100 mg/L)
Mean measured concentrations (geometric mean): 8.7, 2.5, 0.81, 0.27 and 0.089 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 75mL Erlenmeyer flasks, covered with a glass dish
- Type: open
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 5000 algal cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Chemicals (analytical grade) were dissolved sterile purified water (Reconstituted test water: AAP Medium)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature recorded continuously, pH measured at start and end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Not reported
- Photoperiod: Continuous illumination
- Light intensity and quality: 71 to 75 μE s-1 m-2,
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED: The algal biomass in the samples was determined by fluorescence measurement (SpectraMax) I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment.
- Chlorophyll measurement: Not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Dilution factor of 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: 1:10, 1:100, 1:1000 and undiluted filtrate
- 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.:
0.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.69 - 0.75
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.15 - 0.18
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.089 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate / yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.32 - 0.36
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.16 - 0.20
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The 72-hour EC50 for growth rate was 1.1 mg/L which was within the recommended test guideline (72-hour EC50 for growth rate 0.9-1.5 mg/L)
Reported statistics and error estimates:
The 72-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 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 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

 

Dilution°

Mean Measured

Test Item Concentration

 

[mg/L]

Repl.
No.

Biomass of Algae*

 

24 Hours

48 Hours

72 Hours

Control

1

4.7

29.5

160.1

2

5.0

27.6

142.4

3

4.2

31.8

159.5

4

4.4

23.6

125.2

5

3.9

24.6

121.9

6

3.9

30.1

160.1

Mean

4.3

27.9

144.8

SD

0.4

3.2

17.9

1:1000

0.089

1

4.7

29.3

159.8

2

4.6

27.0

133.6

3

4.9

31.9

157.6

Mean

4.7

29.4

150.3

SD

0.1

2.4

14.5

1:320

0.27

1

3.2

20.8

97.0

2

3.3

18.6

97.2

3

4.6

30.7

101.6

Mean

3.7

23.4

98.6

SD

0.8

6.5

2.6

1:100

0.81

1

2.2

3.5

3.5

2

1.8

3.0

4.2

3

2.4

3.0

4.5

Mean

2.1

3.1

4.1

SD

0.3

0.3

0.5

1:32

2.5

1

2.1

1.9

2.6

2

1.6

2.6

3.5

3

1.7

2.2

3.3

Mean

1.8

2.3

3.1

SD

0.3

0.3

0.5

1:10

8.7

1

2.1

2.4

2.3

2

2.1

2.4

2.3

3

1.5

3.3

2.7

Mean

1.9

2.7

2.4

SD

0.4

0.5

0.3

°: Dilutions of the undiluted filtrateof an equilibrated test item suspension with a loading rate of 100 mg/L

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 0.92 x 104relative fluorescence units.

Table 2. Average growth rates (µ)

 

Dilution°

Mean Measured Concentration

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

0-24 h

0-48 h

0-72 h

 

[mg/L]

µ [day-1]

Ir [%]

µ [day-1]

Ir [%]

µ [day-1]

Ir [%]

Control

---

1.550

0.0

1.704

0.0

1.685

0.0

1:1000

0.089

1.645

-6.1

1.733

-1.7

1.699

-0.8

1:320

0.27

1.378

11.1

1.607

5.7

1.559*

7.5

1:100

0.81

0.831#

46.4

0.615*

63.9

0.497*

70.5

1:32

2.5

0.669#

56.8

0.445*

73.9

0.409*

75.8

1:10

8.7

0.711#

54.1

0.535*

68.6

0.324*

80.8

°: Dilutions of the undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

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

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

Table 3. Yield (Y)


Dilution°

Mean Measured Concentration

Yield Y (x 104) and Inhibition Iy [%]

0-24 h

0-48 h

0-72 h

 

[mg/L]

Y

Iy [%]

Y

Iy[%]

Y

Iy [%]

Control

---

3.42

0.0

26.94

0.0

143.93

0.0

1:1000

0.089

3.83

-12.1

28.48

-5.7

149.40

-3.8

1:320

0.27

2.77*

19.0

22.45

16.7

97.65#

32.2

1:100

0.81

1.20*

64.9

2.23#

91.7

3.18#

97.8

1:32

2.5

0.89*

74.1

1.33#

95.0

2.23#

98.4

1:10

8.7

0.97*

71.5

1.78#

93.4

1.52#

98.9

°: Dilutions of the undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L.

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

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

Table 4. Analytical results for test samples

Sampling Day/
Sample Age

Treatment and Dilutions*

Measured Concentration
 of Test Item
x

Sample Preparation Factor
F

Determined Concentration of Test Item
 c

% of Initially Measured

[day/hours]

[mg/L]

 

[mg/L]

[%]

0/0

Control

n.d.

0.1

<LOQ

-

(fresh)

Dilution 1:1000

1.04

0.1

0.104

-

 

Dilution 1:320

3.02

0.1

0.302

-

 

Dilution 1:100

9.33

0.1

0.933

-

 

Dilution 1:32

27.9

0.1

2.79

-

 

Dilution 1:10

9.42

1.0

9.42

-

3/72

Control

n.d.

0.1

<LOQ

-

(aged)

Dilution 1:1000

0.761

0.1

0.0761

73

 

Dilution 1:320

2.36

0.1

0.236

78

 

Dilution 1:100

7.06

0.1

0.706

76

 

Dilution 1:32

22.3

0.1

2.23

80

 

Dilution 1:10

7.96

1.0

7.96

85

* Dilutions of the undiluted filtrate of an equilibrated test item suspension with a loading rate of 100 mg/L

n.d. = no test item detected

LOQ: 0.322 mg/L[JB1] 

The tabulated values of the samples represent rounded results obtained by calculation using the exact Raw Data.

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 72-hour ErC50 was 0.72 mg/L and the ErC10 was 0.17 mg/L, based on growth rate.
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 OECD 201 (2006, corrected 2011) and the Commission Regulation (EU)

No. 2016/266, C3. For preparation of the highest concentrated test medium, the test item was melted at about 50 °C in a water bath before use. 100 µL of the melted test item were pipetted into 1000 mL test water at 50 °C. Then test medium was submitted to ultrasonic treatment at 50 °C for 10 minutes and intensive stirring at room temperature for two hours, until test media cooled down. Due to the undissolved particles in the test water, the suspension was filtered after stirring through a 0.45 µm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The dilutions 1:10, 1:32, 1:100, 1:320 and 1:1000 of the undiluted filtrate were used as test media. A control (test water without test item) was tested in parallel. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 73 to 85 % of the initially measured concentrations were found. Therefore, the biological results were related to the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test). In conclusion, the 72-hour ErC50 was 0.72 mg/L and the ErC10 was 0.17 mg/L, based on growth rate.

Description of key information

In conclusion, the 72-hour ErC50 was 0.72 mg/L and the ErC10 was 0.17 mg/L, based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.72 mg/L
EC10 or NOEC for freshwater algae:
0.17 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 OECD 201 (2006, corrected 2011) and the Commission Regulation (EU)

No. 2016/266, C3. For preparation of the highest concentrated test medium, the test item was melted at about 50 °C in a water bath before use. 100 µL of the melted test item were pipetted into 1000 mL test water at 50 °C. Then test medium was submitted to ultrasonic treatment at 50 °C for 10 minutes and intensive stirring at room temperature for two hours, until test media cooled down. Due to the undissolved particles in the test water, the suspension was filtered after stirring through a 0.45 µm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The dilutions 1:10, 1:32, 1:100, 1:320 and 1:1000 of the undiluted filtrate were used as test media. A control (test water without test item) was tested in parallel. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 73 to 85 % of the initially measured concentrations were found. Therefore, the biological results were related to the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test). In conclusion, the 72-hour ErC50 was 0.72 mg/L and the ErC10 was 0.17 mg/L, based on growth rate.