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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:
From July 23 to 26, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Test Item name: Dehydrocholic Acid
Batch No.: 2018110457
Appearance: White microcrystalline powder
Expiry date: 30 November 2023
Storage: Room temperature (15 - 25 ºC)

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
yes
Remarks:
water
Details on test solutions:
The test was performed with six replicates in the test concentration and six replicates in the control group. Each test unit was uniquely identified with study number, test concentration/ test group and replicate number.
The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test solution. The initial cell density was about 104 cells/mL, in each test flask.
Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks for an exposure time of 72 hours. The test flasks were covered with air-permeable stoppers.
The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined and recorded in parallel.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, Göttingen D-37073, Germany.
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.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Study design

Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
The cultures was maintained at a temperature in the range of 21 24 C controlled at  2 C, which was checked at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) in the climate chamber.
pH:
The pH was measured at the beginning and at the end of the study in the control and in the test concentration level. The pH was not adjusted throughout the test period. The pH of the control medium did not increase by more than 1.5 units during the test.
Nominal and measured concentrations:
Based on the results of the preliminary range-finding test a limit test concentration (100 mg/L nominal) and a concurrent untreated control were used in the main test.
Biological results and endpoints are based on the nominal concentration.
The test solution used in the test was prepared by mechanical dispersion without any solubilising agent. The solution was prepared by dissolving an amount of 0.0701 g test item in 701 mL dilution water (OECD medium see 5.4) resulting a nominal concentration of 100 mg/L. Thereafter this solution was placed into an ultrasonic bath for approximately 20 minutes.
After the formulation procedure algal cells were immediately introduced into the test solutions (start of the experiment).

Details on test conditions:
The test was performed with six replicates in the test concentration and six replicates in the control group. Each test unit was uniquely identified with study number, test concentration/ test group and replicate number.
The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test solution. The initial cell density was about 104 cells/mL, in each test flask.
Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks for an exposure time of 72 hours. The test flasks were covered with air-permeable stoppers.
The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined and recorded in parallel.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
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:
Abnormal appearance of the algal cells was not observed in the control and in the test item treated group during the experiment.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The purpose of the study was to determine the effect of the test item Dehydrocholic Acid on the growth of a unicellular green algal species.
Exponentially growing cultures of Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) were exposed to the limit concentration (100 mg/L) of the test item over several generations under defined conditions.
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.
Growth rate (r) Yield (y)
NOEC 72 h 100 mg/L 100 mg/L
LOEC 72 h > 100 mg/L > 100 mg/L

All biological results are based on the nominal concentration.