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EC number: 949-790-7
CAS number: -
The table below shows the pH recorded at
the beginning and the end of the test. The pH was within the limits
prescribed by the study plan (6-9, preferably not varying by more than
During the exposure period the temperature
measured in the incubator was maintained between 22 and 23°C.
Temperature remained within the limits prescribed by the study plan (21
24°C, constant within ±1°C).
The objective of the study was to evaluate
Potassium dichromate for its ability to generate toxic effects in
Raphidocelis subcapitata (formerly known as Pseudokirchneriella
subcapitata) (strain: NIVA CHL-1) during an exposure period of 72 hours
and, if possible, to determine the EC50 for inhibition of growth rate
(Test Facility Study No. 20196511).
Start of first exposure: 29 Apr 2019
Completion last exposure: 02 May 2019
The study procedures described in this
report were based on the OECD guideline No. 201, Adopted March 23, 2006;
Annex 5 corrected 28 July 2011 and ISO Standard 8692, Third edition, 15
This reference test was carried out to
check the sensitivity of the test system used by the Test Facility to
potassium dichromate (Merck KGaA, Art. 1.04864, Batch K50664264).
Algae were exposed for a period of 72
hours to nominal K2Cr2O7 (potassium dichromate) concentrations of 0.18,
0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell
density was 1.0 x 104 cells/ml.
Overview of % inhibition of growth rate in
the reference test:
The study met the acceptability criteria
prescribed by the study plan and was considered valid.
Potassium dichromate inhibited growth rate
of this fresh water algae species at nominal concentrations of 0.18 mg/L
The 72h-EC50 for growth rate inhibition
(ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10
to 1.13 mg/L.
The 72h-ERC50 was within the expected
range of 0.92 and 1.46 mg/L as specified in ISO International Standard
8692, February 2012, and the historical range of 0.86 and 2.3 mg/L,
which is based on reference tests performed at the Test Facility during
the last ten years.
In conclusion, the sensitivity of this
culture of Raphidocelis subcapitata was in agreement with ISO
International Standard 8692, February 2012 and the historical data
collected at the Test Facility.
The study plan, raw data and report of
this study are kept in the Test Facility’s archives.
The test described above was performed
under GLP conditions.
COMPARISON OF AVERAGE GROWTH RATES
The average specific growth rate for a
specific period is calculated as the logarithmic increase in the biomass
from the equation for each single vessel of controls and treatments:
µi-j = (ln Xj -
ln X) / (tj - ti) (day-1)
= the average specific growth rate from time i to j
= the biomass at time i
= the biomass at time j
The average growth rate at each test item
concentration is then compared with the control value and the percentage
inhibition in growth rate is calculated:
%Ir = ((µC
- µT) / µC) x 100
= percent inhibition in average specific growth rate
= mean value for average specific growth rate in the control group
= average specific growth rate for the treatment replicate
The percent inhibition in yield is
calculated for each treatment replicate as follows:
%Iy = ((YC - YT)
/ YC) x 100
= percent inhibition of yield
= mean value for yield in the control group
= value for yield for the treatment replicate
DETERMINATION OF AVERAGE EXPOSURE
Average exposure concentrations were
24 x √(Ct=0 x Ct=24)
+ 48 x √(Ct=24 x Ct=72) , being the Time
Weighted Average (TWA) of the concentrations of Esacure 3644 measured in
the samples taken at the start (Ct=0), after 24 hours (Ct=24)
and the end of the test (Ct=72).
In case concentrations measured were below
the concentration of the lowest calibration solution, the final exposure
concentration(s) were taken as a factor of 2 below this limit. This
procedure is based on the OECD “Guidance document on the use of the
harmonised system for the classification of chemicals which are
hazardous for the aquatic environment”.
DETERMINATION OF THE EFFECT PARAMETERS
A student t-Test (parametric, α=0.05,
two-sided) was performed to test for presence of statistically
significant differences in algal growth between the blank and solvent
For determination of the effect parameters
(i.e. NOELR, NOEC, ELx, and ECx-values) the approaches recommended in
the OECD guideline 201 were used. An effect was considered to be
significant if statistical analysis of the data obtained for the test
concentrations compared with those obtained in the controls (pooled
data) revealed significant inhibition of growth rate or inhibition of
yield (Multiple Sequentially-rejective U-test after Bonferroni-Holm
correction, α=0.05, one-sided, smaller).
The ELx and ECx-values could not be
determined since the observed effects were below 10%.
ToxRat Professional v 3.2.1 (ToxRat
Solutions® GmbH, Germany) was used to perform the analysis.
Critical computerized systems used in the
study are listed below or presented in the appropriate Phase Report.
All computerized systems used in the conduct of this study have been
validated; when a particular system has not satisfied all requirements,
appropriate administrative and procedural controls were implemented to
assure the quality and integrity of data.
The objective of the study was to evaluate
Esacure 3644 for its ability to generate toxic effects in Raphidocelis
subcapitata during an exposure period of 72 hours and, if possible, to
determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth
rate and inhibition of yield.
The study procedures described in this
report were based on the OECD guideline No. 201, 2006; Annex 5 corrected
28 July 2011. In addition, procedures were based on the test methods
described in the OECD series on testing and assessment number 23, 2019.
The batch of Esacure 3644 tested was a
light yellow solid UVCB and not completely soluble in test medium at the
loading rates initially prepared. Water Accommodated Fractions (WAFs)
were individually prepared at loading rates ranging between 0.010 and
1.0 mg/L and used as test concentrations. Test solutions were prepared
by using stock solutions in methanol (MeOH). Since the test item is a
UVCB, the solvent was completely evaporated addition of test medium to
avoid modification of the WAF-composition due to presence of a
water-miscible solvent. Additionally, the preparation of test solutions
was performed with minimized exposure to light.
A final test was performed based on the
results of a preceding range-finding test. Six replicates of
exponentially growing algal cultures per group were exposed to a blank
control and a solvent control. Three replicates per test concentration
were exposed to WAFs individually prepared at loading rates of 0.010,
0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 104
cells/mL. The total exposure period was 72 hours and samples for
analytical confirmation of exposure concentrations were taken at the
start, after 24 and 72 hours of exposure.
Samples taken from the nominal
concentration solutions of Esacure 3644 in methanol, used in the
preparation of test solutions, were analysed. The analysed
concentrations were at 94 – 110% relative to the nominal concentrations,
indicating proper preparation of the methanol solutions.
In addition, samples taken from all test
concentrations and the controls were analysed. Small responses were
observed at the retention time of the test item in the control and the
lowest test concentration at the start and at the end of the test. These
responses were below the response of the lowest calibration standard and
probably caused by carry-over in the analytical system. At the start of
the test, the measured concentrations in WAFs prepared at 0.032, 0.10,
0.32 and 1.0 mg/L were 0.98, 9.0, 65 and 134 µg/L, respectively. These
concentrations decreased during the test. The two highest test
concentrations decreased to 3% of the initial concentrations at the end
of the test, while the lower test concentrations had decreased below the
level of the lowest calibration standard used in the analysis of the
Since the test item is a UVCB, the effect
parameters were expressed in terms of loading rates.
Stimulation instead of inhibition of
growth rate and yield was recorded at all test concentrations. No
statistically significant differences were found between test item
treatments and the pooled controls. The study met the acceptability
criteria prescribed by the study plan and was considered valid.
The effect parameters (based on loading
rates) obtained in this study are summarized in the tables below.
Study performed to recognised OECD
guidlines with GLP certification
EC50 = > 1.0 mg/mL (loading rate) / > 33 µg/mL
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