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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:
2017-05-05 - 2017-06-06
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:
adopted 23 March 2006, Annex 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg: Statement of GLP Compliance according to § 19b Chemikaliengesetz and Directive 2004/9/EC, Date of Inspection: 10.10.2013
Analytical monitoring:
yes
Details on sampling:

Analytical data are required by the guidelines for verification of test item loading rates as well as the stability of the test item over the entire test period. Analytical samples were taken at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions from all test item loading rates and control. For each sampling also a retain sample was taken.

All samples were stored deep frozen until they were transferred to the analytical laboratory.

The maximum storage period from sampling to analysis was 18 days within this study. Residues are regarded as stable if the samples are stored deep-frozen for up to 30 days between sampling and analysis (EU COM 7032/VI/95). Therefore, the storage stability of COSMACOL ETI was not verified.


Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

The test organism was exposed to different loading rates of the test item under defined conditions in a synthetic growth medium for a time period of 72 h. Initial target cell density was 0.5 × 10exp4 cells/mL. Three replicates were employed for each of the test item loading rates and six replicates for the control. By comparing the cell division under test conditions with and without the influence of the test item, an inhibition of the cell multiplication can be calculated. This inhibition is a value for toxicity.
At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was determined by fluorescence detection and used to calculate effect test item loading rates which resulted in 10, 20 and 50 % inhibition of the cell growth rate (ErL10, 20, 50), and yield (EyL10, 20, 50). The test item loading rate which did not cause any significant inhibition (NOELR) and the lowest observed effect loading rate (LOELR) were also determined where possible.
Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. The summary of the most recently performed reference test was appended to Appendix C. The EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.

The following nominal test item loading rates were tested (spaced by a factor of 3.1): 100, 32.3, 10.4, 3.36, 1.08 mg/L and control. Stock solutions (S1, S2 and S3) were prepared by directly weighing 100 mg, 32.3 mg and 10.4 mg in 1000 mL test medium, respectively. These stock solutions were stirred in the dark at room temperature for 48 h (based on OECD Series on Testing and Assessment No. 23). The solutions were clear and transparent. Subsequently the undissolved test item was allowed to sediment and/or float for a period of 10 minutes until the phases had separated. After the settling the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. The test item solutions V1 and V2 were made by diluting the stock solution S2 with test medium without algae to give the required test item loading rates. Algae were added to each solution individually resulting in nominal cell densities of 0.5 × 10exp4 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel. The preparation of the test solutions is shown below.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): commercial supplier, e.g. MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.

Culture conditions are as follows:
• Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1)
• Temperature: 21 - 24 °C
• Culture flasks: 100 mL Erlenmeyer flasks
• CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Cells from this semi-continuous liquid stock culture were used for the test.
3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not menitioned
Test temperature:
22.8 - 23.2 °C
pH:
7.63- 7.69
Dissolved oxygen:
no data
Salinity:
freshwater test according to guideline
Nominal and measured concentrations:
Nominal: 1.08 - 3.36 - 10.4 - 32.3 - 100 mg/L (nominal test item loading rates)
1.08 mg/L nominal; measured :< LOQ
3.36 mg/L nominal; measured: 0.0476 (0 h fresh) and 0.00690 (72 h aged)
10.4 mg/L nominal; meausured: 0.195 (0 h fresh) and 0.0825 mg/L (72 h aged)
32.3 mg/L nominal; measured: 0.335 (0 h fresh) and 0.0603 mg/L (72 h aged)
100 mg/L nominal; meaused: 0.351 (0 h fesh) and 0.063 mg/L (72 h aged)

Details on test conditions:
TEST SYSTEM
- Initial cells density: approximately 0.5 × 10exp4 cells/mL
- Control end cells density: 21.3 x 10exp4 cells/ml (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM
- Dilution water: according to OECD test guideline 201

OTHER TEST CONDITIONS
- Photoperiod: 24 h/d light
- Light intensity and quality: 88.8 µEm-2s-1 at cell culture level

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell density: fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control.
- Measurement intervall: at 0, 24, 48 and 72 hours

The number of cells in each replicate was determined in duplicate. By the means of a calibration curve, where fluorescence signals (y axis) were plotted versus cell numbers (x axis), the cell numbers were derived from the fluorescence signals. To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture.

Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.


TEST CONCENTRATIONS
Nominal: 1.08 - 3.36 - 10.4 - 32.3 - 100 mg/L

Stock solutions (S1, S2 and S3) were prepared by directly weighing 100 mg, 32.3 mg and 10.4 mg in 1000 mL test medium, respectively. These stock solutions were stirred in the dark at room temperature for 48 h (based on OECD Series on Testing and Assessment No. 23). The solutions were clear and transparent. Subsequently the undissolved test item was allowed to sediment and/or float for a period of 10 minutes until the phases had separated. After the settling the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. The test item solutions V1 and V2 were made by diluting the stock solution S2 with test medium without algae to give the required test item loading rates. Algae were added to each solution individually resulting in nominal cell densities of 0.5 × 104 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.



- Range finding study
A non-GLP range-finding test was performed with the following test item loading rates: Control, 100, 10.0 and 1.00 mg/L.
After 72 h at termination of the test no concentration response relation was observed for the inhibition of growth rate and yield. The inhibition of growth rate peaked in 2.6 % at a nominal test item loading rate of 10.0 mg/L and the inhibition of yield peaked in 0.7 % at a nominal test item loading rate of 10.0 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (experimental date 2016-11-15 to 2016-11-1)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield inhibition
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no (slight increase of growth rate in lower concentrations, but within coefficient of variation of average growth in replicate control cultures)
- 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: n.a.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 1.64 mg/l/72h
- EyC50: 0.976 mg/l/72h
Reported statistics and error estimates:
EL10, 20, 50- and NOELR-values of the test organism exposed to the test item evaluated using nominal test item loading rates. No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 100 mg/L (nominal) at test end.

Table 1: Cell densities

 Test item loading rate [mg/L]  Cell density x 104[cells/mL]          

 0 h

24 h 

 48 h

 72 h

 control

0.5

1.24

4.29

21.3

1.08

0.5

1.64

5.61

28.77

3.36

0.5

1.54

6.79

31.07

10.4

0.5

1.80

7.16

 27.38

32.3

0.5

2.33

12.52

70.92

100

0.5

2.26

12.74

70.94

Table 2: Evaluation after 72 h

 Test item loading rate

[mg/L)

 % Inhibition of growth rate

 % Inhibition of yield

 control

 0

 0

1.08

-8.9

-35.9

3.36

-10.8 

-47

10.4

-7.2

-29.2

 32.3

-33.1

-238.6

100

-33.2

-238.7

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 100 mg/L (nominal) at test end. Thus the overall LOELR was not determinable and the overall NOELR was observed to be at 100 mg/L (nominal). The EL10-, EL20- and EL50-value for growth rate (ErL10, 20, 50) and the EL10-, EL20- and EL50-value for yield (EyL10, 20, 50) were > 100 mg/L (nominal).
Executive summary:

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 100 mg/L (nominal) at test end. Thus the overall LOELR was not determinable and the overall NOELR was observed to be at 100 mg/L (nominal).

The EL10-, EL20- and EL50-value for growth rate (ErL10, 20, 50) and the EL10-, EL20- and EL50-value for yield (EyL10, 20, 50) were > 100 mg/L (nominal).

Description of key information

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item loading rates, including the highest test item loading rate of 100 mg/L (nominal) at test end. Thus the overall LOELR was not determinable and the overall NOELR was observed to be at 100 mg/L (nominal).

The EL10-, EL20- and EL50-value for growth rate (ErL10, 20, 50) and the EL10-, EL20- and EL50-value for yield (EyL10, 20, 50) were > 100 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

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