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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:
2016-05-17 to 2016-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentration, samples were taken from each testing concentration and the control at the start of the test and 24 hours interval thereafter during the experiment. For these daily measurements an additional vessel was set in each test concentration and the control, under the same conditions as the ones used for the determination of algal cell number.
The analysis was performed according to the results of the analytical method validations (Study number: 644-100-1080 and 644-100-1399) by a HPLC method with UV detection on a HyperPrep HS C18 column.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
In order to prepare the test solutions, a stock solution (100 mg/L) was first prepared by diluting 0.2 g test item in 2 L OECD medium using 10 minutes ultrasonic dispersion. The test solutions of the chosen test concentrations were accordingly diluted from this stock solution. The test solutions were prepared just before introduction of algae (start of the experiment).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
- Controls: Potassium dichromate (K2Cr2O7)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.)
Test type:
static
Water media type:
other: OECD medium
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.6 – 22.7°C measured in the flask and between 22.4 and 23.9°C measured within the climate chamber.
pH:
The pH was checked at the beginning and at the end of the study, in the control and the test concentrations. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 6.93 – 8.49 during the study. Measured data of temperature and pH are detailed in Appendix 1; Tables 17 and 18.
Nominal and measured concentrations:
nominal concentrations: 32.8, 41.0, 51.2, 64.0, 80.0 and 100.0 mg/L
calculated geometric mean test item concentration (based on the analytical measurements): 11.01, 14.29, 17.45, 23.66, 28.46 and 40.45 mg/L (72 hours of exposure)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium
- Initial cells density: 10^4 algal cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per untreated control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algal Mineral Salts Test Medium (Dilution Water) OECD water, prepared according to OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuosly
- Light intensity and quality: 7374 lux,

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber
- morphological changes of algal cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.25
- Range finding study: yes
- Test concentrations: 32.8, 41.0, 51.2, 64.0, 80.0 and 100.0 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
35.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
64.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 40.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
14.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
17.45 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.:
13.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
21.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 40.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
14.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
17.45 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: none
- Unusual cell shape: none
- Colour differences: none
- Flocculation: none
- Adherence to test vessels: none
- Aggregation of algal cells: none
- 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
For the evaluation of the quality of the algae and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Number: 392-201-1333) with the reference item Potassium dichromate was: 01 – 04 March 2016.
The 72h ErC50: 0.70 mg/L, (95 % confidence limits: 0.64 – 0.78 mg/L)
The 72h EyC50: 0.39 mg/L, (95 % confidence limits: 0.36 – 0.43 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations were calculated for each treatment at the start, at 24 and 48 hours and at the end of the test using Excel for Windows 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 by Bonferroni t-Test (α = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test.
For determination of the 72-h ErC50 and EyC50 values with 95 % confidence limits, Probit analysis was performed using SPSS PC+ software.
Validity criteria fulfilled:
yes
Conclusions:
In the 72-h algal growth inhibition test on Raphidocelis subcapitata with the test item, the 72-h EC50 value based on growth rate was determined to be higher than 40.45 mg/L and based on yield was calculated to be higher than 40.45 mg/L. The NOEC related to growth rates was determined to be 14.29 mg/L and related to yield it was determined to be 14.29 mg/L.
The results are based on the geometric mean of the measured concentrations.
Executive summary:

The purpose of this study was to determine the effect of the test item on the growth of an unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum). A stock solution was first prepared by diluting 0.2 g test item in 2 L OECD medium. The test concentrations investigated in the study were prepared by appropriate diluting of this stock solution. The dilution water was the fully characterized (content, physico-chemical characteristics) OECD medium. Based upon the results from the preliminary experiments, nominal concentrations of 32.8, 41.0, 51.2, 64.0, 80.0 and 100.0 mg/L solutions were investigated in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 11.01, 14.29, 17.45, 23.66, 28.46 and 40.45 mg/L calculated for 72 hours of exposure. All biological results are based on the geometric mean of the measured concentrations. Untreated control group (OECD Test Medium without test item) was tested together with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated controls. In each concentration and in the control, an additional flask was maintained for analytical sampling. The alga cell concentration was 104 cells/mL in all of the test cultures at the start of the test. Glass flasks with total capacity of ~250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The test vessels were covered with air-permeable stoppers. The test item was suspected as an instabil substance and thus major decrease of the concentration was expected. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Based on the results of the preliminary tests, a full test with six test concentrations in a geometric series was performed with the separation factor of 1.25. The following nominal concentrations were tested: 32.8, 41, 51.2, 64.0, 80.0 and 100.0 mg/L.

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 by Bonferroni t-Test (α = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test. For determination of the 72-h ErC50 and EyC50 values with 95 % confidence limits, Probit analysis was performed using SPSS PC+ software.

Table 1 Summary of Biological Endpoints

Parameter

Growth rate (r)

[mg/L]

Yield (y)

[mg/L]

Calculation based on the geometric mean of the measured concentrations

72-h EC50

> 40.45 (nominal concentration: 100 mg/L)

> 40.45 (nominal concentration: 100 mg/L)

72-h EC10

35.29

13.62

72-h EC20

64.37

21.45

72-h NOEC

14.29

14.29

72-h LOEC

17.45

17.45

Description of key information

In the 72-h algal growth inhibition test on Raphidocelis subcapitata with the test item, the 72-h EC50 value based on growth rate was determined to be higher than 40.45 mg/L and based on yield was calculated to be higher than 40.45 mg/L. The NOEC related to growth rates was determined to be 14.29 mg/L and related to yield it was determined to be 14.29 mg/L. The results are based on the geometric mean of the measured concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
35.29 mg/L

Additional information

The purpose of this study was to determine the effect of the test item on the growth of an unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum). A stock solution was first prepared by diluting 0.2 g test item in 2 L OECD medium. The test concentrations investigated in the study were prepared by appropriate diluting of this stock solution. The dilution water was the fully characterized (content, physico-chemical characteristics) OECD medium. Based upon the results from the preliminary experiments, nominal concentrations of 32.8, 41.0, 51.2, 64.0, 80.0 and 100.0 mg/L solutions were investigated in the main study. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were 11.01, 14.29, 17.45, 23.66, 28.46 and 40.45 mg/L calculated for 72 hours of exposure. All biological results are based on the geometric mean of the measured concentrations. Untreated control group (OECD Test Medium without test item) was tested together with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated controls. In each concentration and in the control, an additional flask was maintained for analytical sampling. The alga cell concentration was 104 cells/mL in all of the test cultures at the start of the test. Glass flasks with total capacity of ~250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The test vessels were covered with air-permeable stoppers. The test item was suspected as an instabil substance and thus major decrease of the concentration was expected. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Based on the results of the preliminary tests, a full test with six test concentrations in a geometric series was performed with the separation factor of 1.25. The following nominal concentrations were tested: 32.8, 41, 51.2, 64.0, 80.0 and 100.0 mg/L.

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 by Bonferroni t-Test (α = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test. For determination of the 72-h ErC50 and EyC50 values with 95 % confidence limits, Probit analysis was performed using SPSS PC+ software.

Table 1 Summary of Biological Endpoints

Parameter

Growth rate (r)

[mg/L]

Yield (y)

[mg/L]

Calculation based on the geometric mean of the measured concentrations

72-h EC50

> 40.45 (nominal concentration: 100 mg/L)

> 40.45 (nominal concentration: 100 mg/L)

72-h EC10

35.29

13.62

72-h EC20

64.37

21.45

72-h NOEC

14.29

14.29

72-h LOEC

17.45

17.45