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Ecotoxicological information

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:
2016-10-25 to 2016-10-28
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:
March 23, 2006, Annex 5 corrected: July 28, 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
March 01, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
April 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
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 obtain the concentration of 100 mg/L. The test solution was freshly prepared in the testing laboratory just before introduction of algae (start of the experiment). Untreated controls were run concurrently. 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: 61.81 SAG
- Source: SAG: Collection of Algal Cultures, 37073 Göttingen, Germany
- Pre-culturing: The pre-culture was incubated for four days in culture medium before the start of the test. Before the treatment, the cell density and the condition of the cells were checked in the pre-culture.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.2 - 22.4 °C (measured in the flasks)
22.1 - 23.8 °C (in the climate chamber)
pH:
7.85 - 8.64 in the control group
7.76 - 8.62 in the treatment group
Nominal and measured concentrations:
Nominal: up to 100 mg/L
Measure concentration: As toxic effects were not observed during the preliminary test, a single concentration of 100 mg/L (limit concentration) and an untreated concurrent control were tested in the main test. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 72 hours. The measured concentrations showed very good correlation with the nominal concentrations. The recovery rate after 72 hours was 102 %. Therefore the nominal concentration was used for the determination of the results.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- test vessel volume and fill volume: 250 mL volume, fill volume: 100 mL
- Type: closed (air-permeable stoppers)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source / preparation of growth medium: Reconstituted algal growth medium (according to OECD Guideline for Testing of Chemicals no. 201).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7649 Lux

EFFECT PARAMETERS MEASURED
The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: no effects up to 100 mg/L in preliminary tests
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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
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
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
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

Validation criteria:
The cell density in the control cultures increased by a factor of 46.33 within 72 hours. This corresponds to a specific growth rate of 1.28/day.
The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 3.41 %.
All validity criteria were met. Therefore, the study was considered as valid.

A single concentration of 100 mg/L (limit test) and a concurrent control were tested in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. In the untreated control group the test item was not detected. The measured concentration of BIS-TRIS was 100 % of the nominal at the start and 102 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 72 hours. Therefore, the biological results were based on the nominal concentration.
Results with reference substance (positive control):
- Results with reference substance valid? YES

For the evaluation of the reliability of the test system and the experimental conditions Potassium dichromate is tested at least twice a year.
The date of the last study with the reference item Potassium dichromate was: 20 – 23 September 2016 and the following results were obtained:
The 72h ErC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.77 mg/L)
The 72h EyC50: 0.46 mg/L, (95 % confidence limits: 0.42 – 0.49 mg/L)
Reported statistics and error estimates:
not applicable as no effects were observed



Table 2: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period










































Test Group



Growth Rates (μ) and % Inhibition of μ



Control



0-24 h



0–48 h



0–72 h



μ



%



μ



%



μ



%



0.0345



0.0



0.0471



0.0



0.0532



0.0



100 mg/L*



0.0373



-8.2



0.0465



1.3



0.0524



1.4





Remark: negative inhibition means growth stimulation


*Statistically not significant compared to the control group (α =0.05; p-value: 0.1115)


Table 3: Yield (y) and Percentage Inhibition of y during the Test Period





























Test Group



Yield y and % Inhibition of y



0–72 h



 



y



%



Control



45.3



0.0



100 mg/L*



42.8



5.5





*Statistically not significant compared to the control group (α=0.05; p-value: 0.1026)

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata according to OECD TG 201 showed the following results: The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L. The 72-h EC50 values based on growth rate and yield were determined to be > 100 mg/L. No abnormal appearance of the algal cells was observed during the experiment.
Executive summary:

The acute toxicity of the substance was investigated in a GLP-study according to OECD test guideline 201 using unicellular freshwater green alga Raphidocelis subcapitata. At the start and at the end of the test, the content of the test item in the test solutions was determined by HPLC. The recovery after 72 hours was 102% of the start concentration. Therefore, the determination of the results was based on the nominal concentrations. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. 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) were not statistically significantly different from the untreated concurrent control. All validity criteria were met. Therefore, the study was considered as valid. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L (limit concentration). Consequently, the 72-h EC50 values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.

Description of key information

The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata according to OECD TG 201 showed the following results: The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L. The 72-h EC50 values based on growth rate and yield were determined to be > 100 mg/L. No abnormal appearance of the algal cells was observed during the experiment (reference 6.1.5-1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The acute toxicity of the substance was investigated in a GLP-study according to OECD test guideline 201 using unicellular freshwater green alga Raphidocelis subcapitata. At the start and at the end of the test, the content of the test item in the test solutions was determined by HPLC. The recovery after 72 hours was 102% of the start concentration. Therefore, the determination of the results was based on the nominal concentrations. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth was determined over a fixed test period of 72 hours in relation to a concurrent untreated control culture. 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) were not statistically significantly different from the untreated concurrent control. All validity criteria were met. Therefore, the study was considered as valid. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be >100 mg/L (limit concentration). Consequently, the 72-h EC50 values based on growth rate and yield were also > 100 mg/L. As a limit test was performed and as no effects were observed, no further statistical analysis was performed.