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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 2012-11-09 to 2013-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: A single concentration of 100 mg/L (limit test) and a concurrent control was tested in the main test.
- Sampling method: Samples were taken from the treatment and the control groups at the start and at the end of the test.
- 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. An amount of 0.08 g test item was dissolved in 800 mL dilution water (OECD medium) in order to give the concentration of 100 mg/L.
- Evidence of undissolved material: no
- Control: The dilution water (OECD medium) without addition of test item was used as control solution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata (Selenastrum capricornutum) (Strain no.: 61.81 SAG)
- Source: Collection of Algal Cultures, 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 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:
21.4 – 22.2 °C
pH:
7.02 – 10.24
Nominal and measured concentrations:
- Nominal concentration: 100 mg/L
- Measured concentration: 95.5, 97.6, 96.9, 100.9, 101.5 and 100.4 mg/L. The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed
- Fill volume: 100 mg/L
- Aeration: no
- Initial cells density: 10 000 cellsmL
- Control end cells density (mean): 86.67 * 10 000 cells/mL
- No. of organisms per vessel: ca. 1 000 000
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

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) was used as dilution water in the experiment.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: approx. 7935 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 species. The inhibition of the growth in relation to a control was determined over a period of 72 hours.
- Determination of cell concentrations: The algae cell densities were determined with an electronic particle counter (Coulter, Z2).
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 100 mg test item/L
- Results used to determine the conditions for the definitive study: No toxic effects were observed during the preliminary test, therefore, a single concentration of 100 mg/L (limit concentration) and an untreated concurrent control was tested in the main test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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:
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:
cell number
Duration:
72 h
Dose descriptor:
NOEC
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:
ErC50 of Potassium dichromate: 1.36 mg/L, (95 % confidence limits: 1.25 – 1.49 mg/L)
EyC50 of Potassium dichromate: 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 (10 000 cells/ mL) during the Test Period of 72 Hours and Yield

Test Group


Number of Cells (× 104/mL)

Yield

0 h

24 h

48 h

72 h

Control


1

4

18

87

86


1

5

20

87

86


1

4

22

83

82


1

6

22

93

92


1

6

18

82

81


1

6

29

88

87

Mean value

1.00

5.17

21.50

86.67

85.7

SD

0.0

1.0

4.1

3.9

3.9

100 mg/L


1

3

14

88

87


1

3

20

102

101


1

3

15

78

77


1

4

14

79

78


1

3

16

79

78


1

3

12

84

83

Mean value

1.00

3.17

15.17

85.00

84.0

SD

0.0

0.4

2.7

9.2

9.2


Validity criteria fulfilled:
yes
Conclusions:
The test substance was tested to Pseudokirchneriella subcapitata (in a closed static test system 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 and the 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 in a closed static test system according to OECD 201 (2006) under GLP conditions. Based on the results of a preliminary test, the test item has no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed at 100 mg/L including a concurrent control. The measured concentration were determined to be 95.5, 97.6, 96.9, 100.9, 101.5 and 100.4 mg/L.The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentration. Exponentially-growing cultures of Pseudokirchneriella subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. The alga cell concentration was approximately 10000 cells/mL at the start of the test in all of the test cultures. 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. For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year. The 72h ErC50 of the reference substance is determined to be 1.36 mg/L. The validity criteria of the test was met. Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a = 0.05, TOXSTAT software). The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (100 mg/L) was not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L (limit concentration). As the test item had no significant effect on the growth of algae therefore statistical analysis was not performed to determine the EC values. The 72-h EC50 values based on growth rate and yield were determined to be > 100 mg/L.

Description of key information

The test substance was tested to Pseudokirchneriella subcapitata (in a closed static test system 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 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 in a closed static test system according to OECD 201 (2006) under GLP conditions. Based on the results of a preliminary test, the test item has no toxic effect on algae up to 100 mg/L (limit concentration). Therefore the main test was performed at 100 mg/L including a concurrent control. The measured concentration were determined to be 95.5, 97.6, 96.9, 100.9, 101.5 and 100.4 mg/L.The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentration. Exponentially-growing cultures of Pseudokirchneriella subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. The alga cell concentration was approximately 10000 cells/mL at the start of the test in all of the test cultures. 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. For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year. The 72h ErC50 of the reference substance is determined to be 1.36 mg/L. The validity criteria of the test was met. Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a = 0.05, TOXSTAT software). The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (100 mg/L) was not statistically significantly different from the untreated concurrent control. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L (limit concentration). As the test item had no significant effect on the growth of algae therefore statistical analysis was not performed to determine the EC values. The 72-h EC50 values based on growth rate and yield were determined to be > 100 mg/L.