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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:
from 2012-11-23 to 2013-05-23
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:
2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control) and 100 mg/L
- Sampling method: At the start of the experiment one sample was taken from the control and 12 parallel samples were taken from the test solution, while at the end of the study one sample was taken from the control and from each replicate of the 100 mg/L test item group.
- Sample storage conditions before analysis: at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solution used in the test was prepared by mechanical dispersion. 0.1055 g of test item was dissolved in 1000 mL of the dilution water (Algal Mineral Salts Test Medium, Section: 5.4) in order to give the concentration of 100 mg/L.
- Controls:
Blank control performed with the test solution without test substance
Positive control performed with the reference substance potassium dichromate
- Evidence of undissolved material: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata (Selenastrum capricornutum)
- Source: Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Untere Karspüle 2, 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 standardized conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as the test
- Any deformed or abnormal cells observed: none
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
22.0 – 22.5 °C
pH:
7.65 – 9.11 at the start of the test
8.78 – 9.98 at the end of the test
Nominal and measured concentrations:
nominal = 100 mg/L
measured (100% recovery) = 101 mg/L (start), 105 mg/L (end)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed
- Test volume: 100 mL
- Aeration: no
- Initial cells density: 10 000 cells/mL
- Control end cells density: 1 045 000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test medium according to OECD 201 (2006)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous lightening
- Light intensity and quality: approx. 8216 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED: The purpose of this aquatic toxicity study was to evaluate the inhibitory effect of the test material on the growth of the freshwater green algae. The cell count of the test organism was determined in each testing flask during the 72-hour test in 24 hours intervals. The cell morphology was examined parallel. The behavior of the test item in test medium was determined daily (at the cell counting) in all test flasks.
- Determination of cell concentrations: microscope with counting chamber
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: yes. Limit test due to results of a range finding test
- Test concentrations: 0.1, 1, 10, 50 and 100 mg test item/L
- Results used to determine the conditions for the definitive study: No significant toxic effect (cell number, growth rate, yield) was observed during the preliminary test, therefore, the main test included a concentration of 100 mg/L (limit test) and a concurrent control group.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- 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 effects observed
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: The ErC50 of potassium dichromate was determined to be 1.36 mg/L (95 % confidence limits: 1.25 – 1.49 mg/L). In addition the EyC50 of potassium dichromate was determined to be 0.88 mg/L (95 % confidence limits: 0.81 – 0.97 mg/L).
For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.
Reported statistics and error estimates:
t-Test (alpha= 0.05) using TOXSTAT software

Table 1: Algal Cell Concentrations (10 000cell/ mL) during the Test Period of 72 Hours and Yield

Concentration (mg/L)


No of Cells (x 10 000/mL)

Yield


0 h

24 h

48 h

72 h


Control


1

5

21

107

106



1

5

24

98

97



1

6

28

89

88



1

6

30

112

111



1

6

27

107

106



1

5

25

114

113


Mean

1.00

5.50

25.83

104.50

103.50


SD

0.0

0.5

3.2

9.4

9.4

100


1

6

27

116

115



1

7

28

102

101



1

5

23

92

91



1

6

24

93

92



1

6

30

108

107



1

6

24

109

108


Mean

1.00

6.00

26.00

103.33

102.33


SD

0.0

0.6

2.8

9.5

9.5


Validity criteria fulfilled:
yes
Conclusions:
The test substance was tested to Pseudokirchneriella subcapitata according to OECD 201 (2006) under GLP conditions. Under the given conditions of this study the 72 h-ErC50 value was determined to be > 100 mg/L. Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be ≥ 100 mg/L.
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours according to OECD 201 (2006) under GLP conditions. Based on the results of a preliminary test, the test item had no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed only at this concentration level of 100 mg/L. Untreated control group (Algal Mineral Salts Test Medium without test item) was tested together with the treated cultures. The test design included six replicates at the test concentration and for the untreated control. The alga cell concentration was 10 000 cells/mL in every 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 alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. In the test no morphological effects were observed in the 100 mg/L test item concentration. The test item concentration of the test solutions was measured by LC/MS method at the start and end of the test. As the performed analytical determinations confirmed that the test item concentration remained within the range of ± 20 % of the nominal concentration, the biological results are reported based on the nominal concentration of the test item.  Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The ErC50 of Potassium dichromate is determined to be 1.36 mg/L and the EyC50 of Potassium dichromate is determined to be 0.88 mg/L. The growth rate, 72h value is in accordance with the accepted 1.10 ± 0.48 mg/L range for Pseudokirchneriella subcapitata. For the determination of the LOEC and NOEC, growth rates and yield at the test concentration of 100 mg/L were tested on significant differences to the control values by 2 Sample t-Test by TOXSTAT software. A limit test was performed therefore statistical analysis to determine the EC50 value was not necessary. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates as well the 0-72 h yield values were not significantly different from that of the control group at the test item concentration of 100 mg/L. The72-hour EC50 value of the test substance was determined to be > 100 mg/L based on growth rate and yield. Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be ≥ 100 mg/L. The overall LOEC of the test item is > 100 mg/L.

Description of key information

The test substance was tested to Pseudokirchneriella subcapitata according to OECD 201 (2006) under GLP conditions. Under the given conditions of this study (reference 6.1.5-1) the 72 h-ErC50 value was determined to be > 100 mg/L and the 72 h-NOEC was determined to be ≥ 100 mg/L.

Key value for chemical safety assessment

Additional information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours according to OECD 201 (2006) under GLP conditions. Based on the results of a preliminary test, the test item had no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed only at this concentration level of 100 mg/L. Untreated control group (Algal Mineral Salts Test Medium without test item) was tested together with the treated cultures. The test design included six replicates at the test concentration and for the untreated control. The alga cell concentration was 10 000 cells/mL in every 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 alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. In the test no morphological effects were observed in the 100 mg/L test item concentration. The test item concentration of the test solutions was measured by LC/MS method at the start and end of the test. As the performed analytical determinations confirmed that the test item concentration remained within the range of ± 20 % of the nominal concentration, the biological results are reported based on the nominal concentration of the test item.  Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The ErC50 of Potassium dichromate is determined to be 1.36 mg/L and the EyC50 of potassium dichromate is determined to be 0.88 mg/L. The growth rate, 72h value is in accordance with the accepted 1.10 ± 0.48 mg/L range for Pseudokirchneriella subcapitata. For the determination of the LOEC and NOEC, growth rates and yield at the test concentration of 100 mg/L were tested on significant differences to the control values by 2 Sample t-Test by TOXSTAT software. A limit test was performed therefore statistical analysis to determine the EC50 value was not necessary. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates as well the 0-72 h yield values were not significantly different from that of the control group at the test item concentration of 100 mg/L. The72-hour EC50 value of the test substance was determined to be > 100 mg/L based on growth rate and yield. Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be ≥ 100 mg/L.

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