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Ecotoxicological information

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:
The study was conducted between 25 and 29 April 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For the analysis of the actual test item concentrations, the samples were taken from the biological phase of the study. 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.
All samples were extracted stand-by immediately after sampling and stored at 4 ± 4°C until analysis.

For the estimation of the algal growth, 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.
Vehicle:
no
Details on test solutions:
Before test start, a ´stock solution´ (nominal 100 mg test item/L) of Syvertal BHT in test medium was prepared by dissolving 108.3 mg test item into 1083 mL test water. The test medium was stirred with an excess of Syvertal BHT in a vessel with minimum headspace for 2 hours. Afterwards the test medium was allowed to settle for 1 hour until phase separation. This ´stock solution´ was withdrawn from a tap at the center of the vessel, after first flushing the tap and tubing with a small aliquot, and then mixed with test medium to produce the desired set of test concentrations.
An aqueous solution of the test item in test media with a concentration as close to saturation as possible (referred to as a ´stock solution´) was obtained first. This stock solution and dilutions of this “stock solution” by 1:3, 1:9, 1:27, 1:81 were prepared for testing. Additionally, a control was tested in parallel.
These dilutions correspond to following mean measured concentrations of the test item: 0.926, 2.75, 8.27, 24.2 and 77.3 mg test item/L, and a control.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
The test was carried out using Pseudokirchnerella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany. Cultures were maintained in the laboratory under standardized conditions.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/l as CaCO3
Test temperature:
Temperature was maintained at 23.2 to 23.5 °C throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. It was:
8.1 at test start and
8.6 to 10.7 at test end.
Nominal and measured concentrations:
Based on the results of the range-finding test, the following test concentrations were assigned to the definitive test:
Nominal test concentrations: 1.2, 3.7, 11.11, 33.33 and 100 mg/I.
Mean measured concentrations of the test item: 0.926, 2.75, 8.27, 24.2 and 77.3 mg test item/L, and a control.
Details on test conditions:
TEST SYSTEM
- Test vessel:
50 mL flasks were used.
The control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth.

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
The culture medium was prepared using analytical grade salts dissolved in sterile purified water.

- Photoperiod:
The flasks were incubated at 23.2 to 23.5 °C under continuous illumination at a measured light intensity of about 6888 lux- mean (range: 6280 to 7430 lux).

- Determination of cell concentrations:
Samples were taken at 24, 48 and 72 hours and the cell densities determined by spectrophotometric measurement.

- The water temperature was recorded daily. The pH was recorded in each test concentration and the control at the start and at the end of the test. The appearance of the test media was recorded daily.
Reference substance (positive control):
yes
Remarks:
potassium dichromate, tested at least once a year
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
At the start of the test, the measured levels of the test substance ranged between 73 and 79% of their nominal values, and after 72 h they ranged between 72 and 76% of their nominal values.

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 test concentration of 8.27 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. At concentrations of 24.2 and 77.3 mg test item/L, the algal cells showed deformations.
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50, the corresponding EC20 and EC10 values and where possible their 95%-confidence limits were calculated by Probit 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 Bonferroni-Welch t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Remarks:
Cell Density Increase in Control Cultures: 357.7-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 20.8 %; Coefficient of Variation of Average Growth between Control Replicates: 1.4 %
Conclusions:
Algae toxicity test (OECD TG 201): The ErC50, EC10 and NOEC were 32.0, 16.7 and 2.75 mg/l, respectively.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchnerella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchnerella subcapitata was exposed to solutions of the test material at mean measured concentrations of 77.3, 24.2, 8.27, 2.75 and 0.926 mg test item/L (3 replicate flasks per concentration), and a control for 72 hours, under constant illumination and shaking at a temperature of 23.2 - 23.5°C. At the start of the test, the measured levels of the test substance ranged between 73 and 79% of their nominal values, and after 72 h they ranged between 72 and 76% of their nominal values. The mean measured values were used for the effect assessment. The ErC50, EC10 and NOEC were 32.0, 16.7 and 2.75 mg/l, respectively.

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchnerella subcapitata. The method followed that described in the OECD TG No 201. Following a preliminary range-finding test, Pseudokirchnerella subcapitata was exposed to solutions of the test material at mean measured concentrations of 77.3, 24.2, 8.27, 2.75 and 0.926 mg test item/L(3 replicate flasks per concentration)

, and a control for 72 hours, under constant illumination and shaking at a temperature of 23.2 - 23.5°C. At the start of the test, the measured levels of the test substance ranged between 73 and 79% of their nominal values, and after 72 h they ranged between 72 and 76% of their nominal values.
The mean measured values were used for the effect assessment. The ErC50, EC10 and NOEC were
32.0, 16.7 and 2.75 mg/l, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
32 mg/L
EC10 or NOEC for freshwater algae:
16.7 mg/L

Additional information

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