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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:
02/2018 to 05/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD No. 201, adopted March 2006, corrected July 28, 2011, equivalent to Commission Regulation (EC) No 761/2009, C.3., 2009.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: Blancolen® HP
Synonyms: Blancolen® HP (Disodium hydroxy(sulfonato)acetate)
Batch No.: 14102401
Active Ingredient Content: 98 %, according to certificate of analysis
Certificate of Analysis Date: October 06, 2017
Aggregate State at Room Temperature: Solid
Colour: White
Expiry Date: October 24, 2024
Storage Conditions at Test Facility: At 20 +/- 5 °C, in the dark
Analytical monitoring:
yes
Details on sampling:
Analysis of the Test Item Concentrations

Sampling:
The samples were taken from the biological phase of the study.
Collecting, storage and handing over of the samples were the Study
Director’s responsibility. The information concerning the samples
was provided by the Study Director.
Duplicate samples from the freshly prepared test media (containing
algae) of all test concentrations and from the control were taken at
the start of the test.
For the determination of the stability of the test item under the test
conditions and of the maintenance of the test item concentrations
during the test period, duplicate samples from the test media of all
test concentrations and the control (containing algae) were taken at
the end of the test (after the 72 hours test period) by pouring
together the contents of the test beakers of each treatment. In
addition, intermediate samples were taken after 24h and 48h.
The samples remained undiluted until analysis.
Additional samples of the control were taken at each sampling
without any sample treatment.

Storage:
All samples were stored in a freezer (≤ ‐ 20 °C), protected from light
until analysis was performed. Afterwards the samples were again
stored deep frozen (≤ ‐20 °C) and will be kept stored up to the date
of the final report.

Analyses:
The concentrations of the test item Blancolen® HP were analysed in
the duplicate test media samples from all test concentrations, and in
the duplicate control samples from all sampling times.


Analytical Samples

Fortified Samples:
Approximately 100 mg of the test item were dissolved (1 minute
ultrasonication) in 50 mL test water to obtain a stock solution of
approximately 2 g test item/L. Two independent stock solutions
were prepared. Appropriate amounts of these stock solutions were
diluted with test water to obtain fortified samples at a level of 8, 70,
and 1200 mg test item/L. Exact values were documented in the raw
data.


Sample Preparation Procedure

Sample Preparation: Biological treatment samples and control samples:
The samples were allowed to thaw to room temperature. They were
then shaken well and treated with ultrasound for 1 minute to obtain
homogenous samples. The samples were diluted further with test
water to match the calibration range, if necessary. The samples were
centrifuged (13,000 rpm, 3 minutes) before analysis to remove the
algae.

Sample Preparation: Fortified samples and analytical blank control samples:
The samples were shaken well. They were diluted further with test
water to match the calibration range, if necessary. The samples were
centrifuged (13,000 rpm, 3 minutes) before analysis.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Dosage of Test Item:
The test medium of the highest test concentration of nominal
1000 mg test item/L was prepared by dissolving 701 mg test item into
701 mL test water by intense stirring for 45 minutes. Adequate
volumes of this test medium were diluted with test water to prepare
the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae
(= start of the test).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
0.24 mmol/l, corresponding to 24 mg/l, as CaCO3
Test temperature:
22.7 to 23.0 °C
pH:
7.7 to 8.0 at test start
3.6 to 10.1 at test end
Salinity:
Reconstituted water was used for the test: Analytical grade salts were added to deionised water.
Nominal and measured concentrations:
Nominal 1000, 316, 100, 31.7 and 10.0 mg test item/L corresponding
to geometric mean measured concentrations of 809, 151, 40.0, 17.7 and 11.3 mg test item/L.
Details on test conditions:
Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG,
formerly known as Selenastrum capricornutum, and recently
renamed as Raphidocelis subcapitata (KORSHIKOV)

Origin: The algae were supplied by the „Sammlung von Algenkulturen,
Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität
Göttingen", 37073 Göttingen, Germany.

Breeding Conditions: The algae were cultivated in the laboratories of ibacon under
standardised conditions according to the test guidelines.

Reference Item: For the evaluation of the quality of the algae and the experimental
conditions the reference item potassium dichromate is tested at least
twice a year to demonstrate satisfactory test conditions.

Type and Size: Erlenmeyer flasks of 50 mL volume with approximately 50 mL of test
medium

Identification: Each test unit was uniquely identified with the serial number of the
study, treatment and replicate number.

Light Regime: Continuous illumination

Light Intensity: The light intensity was measured once during the test at 6 positions
distributed over the experimental area at the surface of the test media.
Mean light intensity: 6540 lux (range: 6370 to 6730 lux)

Recording: Test conditions were recorded

Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of approx.
5000 algal cells per mL test medium. These cells were taken from an
exponentially growing pre‐culture, which was set up 3 days prior to
the test start under the same conditions as in the test.

Replicates: The test was performed with three replicates per test concentration
and six replicates in the control. Volumes of approx. 50 mL of algal
suspension per replicate were continuously stirred with magnetic
stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass
dishes and incubated in a water bath. The flasks were placed in a
random order and were repositioned each day to minimize
differences in test conditions.

Blanks: Additionally, one replicate of each test concentration and of the
control was prepared without algae to provide a "blank" for the
spectrophotometric measurements. The additional replicates were
incubated under the same conditions as described above. The blank
values were subtracted from the absorption measured in the samples
containing algae in order to eliminate absorption caused by the test
item (see also "Determination of the Cell Density").

Determination of the Cell Density:
The cell density on each observation time was determined by
spectrophotometric measurement. Therefore, defined volumes of the
algal suspensions from all replicates and from the blanks were
sampled after 24, 48 and 72 hours of exposure, and were not
replaced. The algal cell densities were calculated by subtracting the
absorption of the blanks, from each of the measured absorption of
the test media (with algae).
Based on the counted cell densities and the absorption from an algal
suspension and its dilutions, a linear regression was performed for

Exposure Time: 72 hours



Reference substance (positive control):
yes
Remarks:
The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
131 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
63.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
39.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
40 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
151 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Growth Inhibition:
The 72 hour EyC50 was calculated to be 79.8 mg test item/L and
the ErC50 131 mg test item/L. The 72 hour EyC10 was calculated to be
29.2 mg test item/L and the ErC10 39.4 mg test item/L. The 72 hour
NOEyC was determined to be 40.0 mg test item/L and the associated
72 hour LOEyC of 151 mg test item/L. The 72 hour NOErC was
determined to be 40.0 mg test item/L and the associated 72 hour
LOErC is 151 mg test item/L.

Microscopic Examination:
The microscopic examination of the shape of the algal cells after 72
hours of test duration did not show any difference between the algae
that had been growing up to a geometric mean measured test
concentration of 809 mg test item/L and the algal cells in the control.
Thus, the shape of the algal cells was not obviously affected up to this
test concentration, the highest concentration tested.
Results with reference substance (positive control):
The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Reported statistics and error estimates:
Statistical Analysis:
Based on the calculated cell densities, the 72 hours ErC50 and the
72 hours EyC50 (see Definitions), the corresponding EC20 and EC10
values and where possible their 95 %-confidence limits were
calculated by Weibull analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC,
the calculated growth rates and yields at each test concentration
were tested for significant differences compared to the control values
by Williams t‐test (yield) and Bonferroni-Welch t-test (growth rate).
The software used to perform the statistical analysis was ToxRat
Professional, Version 3.2.1, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Conclusions:
The influence of Blancolen® HP on the growth of the freshwater
green algae Pseudokirchneriella subcapitata was assessed in a static
concentration-response test. The 72 hour EyC50 was calculated to be
79.8 mg test item/L, and the 72 hour ErC50 value was calculated to be
131 mg test item/L. The 72 hour NOEyC was determined to be
40.0 mg test item/L and the associated 72 hour LOEyC was 151 mg
test item/L. The 72 hour NOErC was determined to be 40.0 mg test
item/L and the associated 72 hour LOErC was 151 mg test item/L. The
initial concentrations and the maintenance of the exposure
concentrations during the test were verified in the analytical part. All
reported results refer to geometric mean concentrations, since the
test item concentrations were not within ± 20 % of the measured
initial concentrations during the test.
Executive summary:

The ErC50 value was determined to be 131 mg/l.

Since this value is above 1 mg/l, classification into the hazard class "Aquatic acute" is not applicable.

Since this value is above 100 mg/l and since the substance has shown to be readily biodegradeable, classification into a category of the hazard class "Aquatic chronic" is not applicable.

Description of key information

Substance is not acute toxic to algae.
ErC50 (72h) = 131 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
131 mg/L

Additional information