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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:
28 June - 17 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted: 23 March 2006; Annex 5 corrected: 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) No 761/2009.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
HPLC/UV-vis
Details on sampling:
- Concentrations: 100 % saturated solution, 50, 25, 12.5, 6.25 % v/v, resulted in measured concentrations of 80.4, 38.9, 20.1, 10.1, 5.04 mg/L, respectively.
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from pooled replicates at 72 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was prepared by stirring an excess (100 mg/L) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item. A series of dilutions was made from this saturated solution to give stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 1.8 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
- Eluate: Algal culture media
- Controls: 6 culture media only vessels prepared
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata / green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Age of inoculum (at test initiation): Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10E4 to 10E5 cells/mL. Exact timing not reported.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at
24 ±1 °C.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): Same
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
-
Test temperature:
24 ± 1 ºC
pH:
7.7 - 8.5 (control vessles did not deviate by more than 1.5 units i.e. 8.0 - 8.5)
Dissolved oxygen:
-
Salinity:
-
Conductivity:
-
Nominal and measured concentrations:
Definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution. This equated to measured concentrations of 5, 10, 20, 39 and 80 mg/L, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flask
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: Glass; 250 mL; filled to 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A - static test
- Renewal rate of test solution (frequency/flow rate): N/A - static test
- Initial cells density: 5E3
- Control end cells density: 1.19E6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: Yes OECD mediuem
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in house from salt stocks. The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux (wavelength: 380-730 nm)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- Chlorophyll measurement: Not conducted
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Spacing facotr of 2 used.
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 % v/v
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is routinely tested. The results from the previous positive control test with potassium dichromate were within the normal ranges for this reference item.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Remarks on result:
not determinable
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: N/A
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) 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 EC50: Growth rate= 1.6 mg/L; Yield= 0.77 mg/L
- 72h NOEC: Growth rate=0.25 mg/L; Yield=0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Growth rate: Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 5.0 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 5.0 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L.

Yield: Statistical analysis of the yield data was carried out as per above. All test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was less than 5.0 mg/L. Correspondingly the LOEC based on yield was 5.0 mg/L.

Table 1       Cell densities and pH values in the definitive test

0 h Measured Test Concentration (mg/L)

pH

Cell Density (cells per mL)

pH

0h

22 h

47 h

72 h

72 h

Control

R1

8.0

2.64E+04

1.62E+05

1.28E+06

8.5

R2

2.65E+04

1.58E+05

1.18E+06

R3

2.23E+04

1.58E+05

1.09E+06

R4

2.53E+04

1.61E+05

1.18E+06

R5

2.46E+04

1.67E+05

1.23E+06

R6

2.49E+04

1.59E+05

1.18E+06

Mean

2.50E+04

1.61E+05

1.19E+06

5.0

R1

7.9

2.23E+04

1.49E+05

1.04E+06

8.5

R2

2.26E+04

1.49E+05

9.58E+05

R3

2.09E+04

1.44E+05

8.91E+05

Mean

2.19E+04

1.47E+05

9.61E+05

10

R1

7.8

2.11E+04

1.19E+05

6.65E+05

8.4

R2

1.71E+04

1.07E+05

5.59E+05

R3

1.99E+04

1.11E+05

6.33E+05

Mean

1.94E+04

1.12E+05

6.19E+05

20

R1

7.7

9.68E+03

3.67E+04

1.70E+05

8.0

R2

1.13E+04

3.34E+04

1.57E+05

R3

8.62E+03

3.02E+04

1.35E+05

Mean

9.78E+03

3.34E+04

1.54E+05

39

R1

7.7

1.04E+04

2.65E+04

6.34E+04

7.8

R2

8.10E+03

2.26E+04

4.98E+04

R3

8.89E+03

2.33E+04

4.75E+04

Mean

9.14E+03

2.41E+04

5.36E+04

80

R1

7.7

9.01E+03

1.42E+04

1.66E+04

7.7

R2

9.77E+03

1.10E+04

9.06E+03

R3

1.39E+04

1.47E+04

1.13E+04

Mean

1.09E+04

1.33E+04

1.23E+04

 

Table2       Daily specific growth rates for the control cultures in the definitive test

 

Treatment

Daily specific growth rate (cells/mL/hour)

Day 0 – 1

Day 1 – 2

Day 2 - 3

Control

R1

0.076

0.073

0.082

R2

0.076

0.071

0.080

R3

0.068

0.078

0.077

R4

0.074

0.074

0.080

R5

0.072

0.077

0.080

R6

0.073

0.074

0.080

Mean

0.073

0.075

0.080

 

Table 3       Inhibition of growth rates and yield in the definitive test

 

0 h Measured Test Concentration (mg/L)

Growth rate (cells/mL/h)

Yield (cells/mL)

0-72 h

% inhibition

0-72 h

% inhibition

Control

R1

0.077

-

1.27E+06

 

R2

0.076

1.17E+06

 

R3

0.075

1.09E+06

 

R4

0.076

1.18E+06

 

R5

0.077

1.23E+06

 

R6

0.076

1.18E+06

 

Mean

0.076

1.19E+06

 

SD

0.001

6.14E+06

 

5.0

R1

0.074

3

1.03E+06

 

R2

0.073

4

9.53E+05

 

R3

0.072

5

8.86E+05

 

Mean

0.073

4

9.56E+05

19

SD

0.001

-

7.24E+04

 

10

R1

0.068

11

6.60E+05

 

R2

0.066

13

5.54E+05

 

R3

0.067

12

6.28E+05

 

Mean

0.067

12

6.14E+05

48

SD

0.001

-

5.40E+04

 

20

R1

0.049

36

1.65E+05

 

R2

0.048

37

1.52E+05

 

R3

0.046

39

1.30E+05

 

Mean

0.048

37

1.49E+05

87

SD

0.002

-

1.79E+04

 

39

R1

0.035

54

5.84E+04

 

R2

0.032

58

4.48E+04

 

R3

0.031

59

4.25E+04

 

Mean

0.033

57

4.86E+04

96

SD

0.002

-

8.63E+03

 

80

R1

0.017

78

1.16E+04

 

R2

0.008

89

4.06E+03

 

R3

0.011

86

6.29E+03

 

Mean

0.012

84

7.31E+03

99

SD

0.005

-

3.86E+03

 

 

Validity criteria fulfilled:
yes
Conclusions:
The 72 h ErC50 to green algae, under the conditions of this test was 30 mg/L. The 72 h ErC10 under the conditions of this test was 7.9 mg/L. Statistically significant reduced growth was observed in all treatment groups, with the excpetion of 5.0 mg/L, compared to the control. The 72h -NOEC for growth rate was therefore established as 5.0 mg/L.
Executive summary:

OECD 201 (2019) - In a 72 hour toxicity study, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 5.0 to 80 mg/L. There was no significant change in the measured concentrations at 72 hours (5.0 to 78 mg/L) and so the results are based on the 0-Hour measured test concentrations only; 5, 10, 20, 39 and 80 mg/L.

 

The initial cell density added to each treatment flasks was 5.0x103cells/mL at 0 h. Cell biomasses were determined, using a particle counter, at 24, 48 and 72 h.

 

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 5.0 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 5.0 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L. The 72 h ErC50 to green algae, under the conditions of this test was 30 mg/L.  The 72 h ErC10 under the conditions of this test was 7.9 mg/L.

 

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.

Description of key information

72 h ErC50: 30 mg/L; OECD 201; Vryenhoef, H. (2019)

72 h ErC10: 5.0 mg/L; OECD 201; Vryenhoef, H. (2019)

Key value for chemical safety assessment

EC50 for freshwater algae:
30 mg/L
EC10 or NOEC for freshwater algae:
7.9 mg/L

Additional information

OECD 201 (2019) - In a 72 hour toxicity study,Pseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 5.0 to 80 mg/L. There was no significant change in the measured concentrations at 72 hours (5.0 to 78 mg/L) and so the results are based on the 0-Hour measured test concentrations only;5, 10, 20, 39 and 80 mg/L.

 

The initial cell density added to each treatment flasks was 5.0x103cells/mL at 0 h. Cell biomasses were determined, using a particle counter, at 24, 48 and 72 h.

 

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 5.0 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 5.0 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L. The 72 h ErC50 to green algae, under the conditions of this test was 30 mg/L.  The 72 h ErC10 under the conditions of this test was 7.9 mg/L.

 

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.