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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch number: 3194/001/A17
Appearance: White to yellowish Powder
Purity: 95% (102.7% according to the Certificate of Analysis)
Expiry date: 11 July 2019
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity), protected from light and humidity (store in a tightly closed container)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed at the applied test concentration level and from the control at the beginning and at the end of the experiment.

Test solutions

Details on test solutions:
A stock solution with a nominal concentration of 100.0 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath for approximately 5 minutes. As a Limit Test was carried out, further dilution of stock solution was not performed.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
- Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.3 – 22.7 °C measured in the flask and between 21.4 and 23.1 °C measured within the climate chamber.
pH:
The pH was checked at the beginning and at the end of the test in each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.29 – 8.44 during the experiment.
Nominal and measured concentrations:
As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.
Details on test conditions:
- Replicate: The test design included six replicates at test concentration and six replicates for the untreated control.

- Light Intensity: The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7502 lux (equivalent to ~101 µE/m^2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.

- Observations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

- Data treatment: Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test ( = 0.05) by TOXSTAT software.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
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:
EC50
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:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
not specified
Details on results:
There were no any observed morphological deviations of the algal cells during the experiment.
Results with reference substance (positive control):
The date of the last study (Study Code: 19/009-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 22 – 25 January 2019.
The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Any other information on results incl. tables

* VALIDITY

The cell density in the control cultures increased by the factor of 72.17 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.64 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.97 %.

All validity criteria were met; therefore the study can be considered as valid.

* AVERAGE SPECIFIC GROWTH RATES

The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L(nominal).

Table:  Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

Test group

Growth rate µ and % inhibition of µ

0–24 h

0–48 h

0–72 h

m

%

m

%

m

%

Control

0.0573+

0.0

0.0599

0.0

0.0594

0.0

100.0 mg/L (nominal)

0.0558

2.7

0.0598

0.3

0.0597

-0.4

+: at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

* AREAS UNDER THE GROWTH CURVES

The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L(nominal).

Table:  Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group

Areas under the Growth Curves (A) and % inhibition of A

0–24 h

0–48 h

0–72 h

A

%

A

%

A

%

Control

36.0

0.0

274.0

0.0

1330.0

0.0

100.0 mg/L (nominal)

34.0

5.6

268.0

2.2

1338.0

-0.6

 

* YIELD

The results of the statistical evaluation (based on 2 Sample t-Test; alpha=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100.0 mg/L(nominal).

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

Test group

Yield (Y) and % inhibition of Y (0-72 h)

Y

%

Control

71.2

0.0

100.0 mg/L (nominal)

72.5

-1.9

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the algal growth inhibition test (OECD 201), the calculated endpoints for the effect of Dipotassium Glycyrrhizate were the following:
- The 72h EbC50 value (biomass) > 100.0 mg/L (nominal)
- The 72h ErC50 value (growth rate) > 100.0 mg/L (nominal)
- The 72h EyC50 value (yield) > 100.0 mg/L (nominal)

The 72h No-Observed Effect Concentration (NOEC) = 100.0 mg/L (nominal)
The 72h Lowest Observed Effect Concentration (LOEC) > 100.0 mg/L (nominal)