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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to current test guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaction Product of Saccharose, Glycerine, biodiesel
propoxylated
- Physical state: Red to brown, clear, liquid
- Analytical purity: 100%
- Lot/batch No.: T41/012/13
- Expiration date of the lot/batch: 2014-06-15
- Storage condition of test material: Ambient (room temperature)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sample storage conditions before analysis: All samples were stored at room temperature until the start of the
analysis, if necessary. Prepared samples were stored at room
temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Five Water Soluble Fractions (WSF**) with nominal loadings of the test item in the range of 10.0
to 234 mg/L were prepared, set up in a geometric series with a factor of 2.2: 10.0 - 22.0 - 48.4 -
106 - 234 mg/L. For each loading level an appropriate amount of the test item was weighed out.
The test item was applied onto glass slides and inserted in brown glass bottles with appropriate
amounts of dilution water. The test item dispersions were shaken for 24 hours with 20 rpm at
room temperature. After a separation phase of 24 hours, undissolved particles were removed by
membrane filtration (pore size 0.45 μm). During filtration, the filter was always kept
covered. The resulting Water Soluble Fractions (WSF) were used
in the test. The method of preparation of the water soluble fractions
was based on a range finding test.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriel/a subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches lnstitut der Universitat Gottingen
Nikolausberger Weg 18, D-37073 Gottingen
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used
as inoculum. For the start of the test, the preculture was directly pipetted into each test media and the control.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity
amounted to 35-70 μE ·m-2 ·s-1 for 24 hours per day.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 24 °C, controlled at± 2 °C
pH:
The pH-value were within the acceptable limits (8.14 - 8.38).
Nominal and measured concentrations:
Nominal: 10.0, 22.0, 48.4, 106, 234 mg/L
Measured: 9.13, 22.1, 50.2, 121, 263 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 ml) with cotton wool plugs
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 100 mL
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: Nominal: approximately 5 x 10³ - 10E04 cells/mL; Actual: 5.297 x 10³ cells/mL
- No. of vessels per concentration (replicates): 3 replicates per loading level
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline
- Intervals of water quality measurement: The pH-value at the start of the exposure was measured out of
one additional replicate per loading level and the control. At the
end of the exposure, it was measured from pooled replicates per
loading level and control. The room temperature was measured
continuously. Light intensity was measured prior to the start of the
exposure

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: Approximately 60 μE · m-2 · s-1, 24 hours/day light, Fluorescent tubes, OSRAM l 36 W/11-865, cool daylight


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-a-fluorescence,
excitation at 436 nm, emission at 685 nm. Dilution water was
used as background signal.
Microscopic evaluation of the cells at the start and end of the
exposure was carried out. The cells were checked for unusual cell
shapes, colour differences, differences in chloroplast morphology,
flocculation, adherence of algae to test containers and
agglutination of algae cells.

TEST CONCENTRATIONS
- Range finding study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
45.8 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, batch number MKBF6527V, purity 99.6 %, CAS RN
7778-50-9) to the unicellular freshwater green alga Pseudokirchneriel!a subcapitata was
determined over a period of 72 hours from 2013-10-22 to 2013-10-25.

Growth Rate inhibition ErC50: 0.904 [mg/L], (0-72 hours)
Yield inhibition EyC50: 0.491 [mg/L], (0-72 hours)

Applicant's summary and conclusion