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Diss Factsheets

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:
30 Aug - 02 Sep 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
adopted March 2006, corrected July 28, 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyl 4-(1,1-dimethylethyl)benzoate
EC Number:
239-510-5
EC Name:
Vinyl 4-(1,1-dimethylethyl)benzoate
Cas Number:
15484-80-7
Molecular formula:
C13H16O2
IUPAC Name:
ethenyl 4-tert-butylbenzoate

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: 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.
One sample from each freshly prepared application solution in DMF and triplicate samples from the freshly prepared test media (without algae) of all test concentrations and from the controls (control and solvent 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, triplicate samples from the test media of all test concentrations and the controls were taken after 24 hours (without algae) and 48 hours (without algae) and at the end of the test (after the 72 hours test period by pouring together the contents of the test beakers of each treatment (containing algae)).
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: A stock solution of 70 g test item/L DMF was prepared by dissolving 366.9 mg of the test item in 5.24 mL dimethylformamide. This stock solution was diluted in a series of sequential dilutions with the solvent using the same spacing factor as used to define the nominal test concentrations to allow the addition of the same volume of solvent (100 µL/L) to each test solution. The solvent additive was chosen based upon its solubility properties and its relative lack of toxicity to algae. A defined volume of the stock solution and its dilutions was mixed into test water to obtain the test media of the desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test concentration: 7.0, 2.2, 0.7, 0.2 and 0.07 mg test item/L (spacing factor 3.16)
corresponding to following geometric mean measured concentrations of the test item:
1.41, 0.298, 0.120, 0.0212 and 0.00932 mg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L), and a control

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)

The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

TEST ORGANISM
- Name/Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 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.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no

Study design

Test type:
static
Water media type:
other: reconstituted water, OECD Medium
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L)
as CaCO3
Test temperature:
Water temperature was: 21.9 to 22.6 °C
pH:
The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH value in the control at test start: 8.1,
pH value in the control at test end: 8.0
pH value in the solvent control at test start:8.0,
pH value in the solvent control at test end: 8.1
pH values in the test item treatments at test start: 8.0,
pH values in the test item treatments at test end: 7.9 to 8.0
Nominal and measured concentrations:
7.0, 2.2, 0.7, 0.2 and 0.07 mg test item/L (spacing factor 3.16)
corresponding to following geometric mean measured concentrations of the test item:
1.41, 0.298, 0.120, 0.0212 and 0.00932 mg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L), and a control.
Details on test conditions:
Test Vessels: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glasses.
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of nominal 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 controls.

Test Procedure: The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, 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 controls 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.

OTHER TEST CONDITIONS
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: 4888 lux (range: 4730 to 5040 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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) at a wavelength of 685 nm.
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Reference substance (positive control):
yes
Remarks:
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.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.377 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.195 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.139 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.298 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.889 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.:
0.401 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.:
0.265 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.:
0.12 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.:
0.298 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 0.377 mg test item/L and the ErC50 0.889 mg test item/L. The 72-hour EyC10 was calculated to be 0.139 mg test item/L and the ErC10 0.265 mg test item/L. The 72-hour NOEyC was determined to be 0.120 mg test item/L and the associated 72-hour LOEyC of 0.298 mg test item/L. The 72-hour NOErC was determined to be 0.120 mg test item/L and the associated 72-hour LOErC is 0.298 mg test item/L.

Signs of Phytotoxicity evaluated by Microscopic Observations: 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 the geometric mean measured test concentration of 1.41 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):
Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in February 2021

72-hour EC50 (Yield): 0.393 mg/L
72-hour NOEC (Yield): 0.2 mg/L
72-hour LOEC (Yield): 0.63 mg/L
72-hour EC50 (Growth rate): 0.835 mg/L
72-hour NOEC (Growth rate): 0.2 mg/L
72-hour LOEC (Growth rate): 0.63 mg/L
Reported statistics and error estimates:
Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by using Williams t-test (yield, growth rate 48 and 72 h) and Bonferroni-Welch t-test (growth rate 24h).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Any other information on results incl. tables

Validity Criteria





























Criterionvalue requiredvalue obtainedfullfilled
Cell Density Increase in control Culturesat least by factor of 16 within 72 h43.7 fold within 72 hyes
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures< 35%5.5%yes
Coefficient of Variation of Average Growth between Control Replicates< 7% 3.6%yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see any other information on results incl. tables
Conclusions:
The influence of the test item 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 0.377 mg test item/L, and the 72-hour ErC50 value was calculated to be 0.889 mg test item/L. The 72-hour NOEyC was determined to be 0.120 mg test item/L and the associated 72-hour LOEyC was 0.298 mg test item/L. The 72-hour NOErC was determined to be 0.120 mg test item/L and the associated 72-hour LOErC was 0.298 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test.
Executive summary:

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).


Test Design: This study encompassed 7 treatment groups (5 concentrations of the test item, a solvent control and a control) with three replicates per test concentration and six replicates for the controls. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement.


Endpoints: Yield and growth rate of the algae


Test Concentrations:7.0, 2.2, 0.7, 0.2 and 0.07 mg test item/L (spacing factor 3.16) corresponding to following geometric mean measured concentrations of the test item:1.41, 0.298, 0.120, 0.0212 and 0.00932 mg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L), and a control.


Test Conditions: Water temperature: 21.9 to 22.6 °C;pH value in the control at test start: 8.1, pH value in the control at test end: 8.0pH value in the solvent control at test start:8.0,pH value in the solvent control at test end: 8.1; pH values in the test item treatments at test start: 8.0,pH values in the test item treatments at test end: 7.9 to 8.0 continuous illumination; mean light intensity: 4888 lux (range: 4730 to 5040 lux)


Biological results








































ParameterYieldGrowth Rate
 mg test item/Lmg test item/L
72-hour EC50 0.3770.889
72-hour EC20 0.1950.401
72-hour EC10 0.1390.265
72-hour NOEC 0.1200.120

72-hour LOEC


0.298

0.298



Analytical results:


The quantification of the test item in the test samples was performed using gas chromatography with MS/MS detection.


At the start of the test 46 % of the nominal test concentrations were found (average of all test concentrations). After 24 hours test duration, 31 % of the nominal value was determined (average of all test concentrations). After 48 hours test duration, 14 % of the nominal value was determined (average of all test concentrations). And after 72 hours test duration, 2 % of the nominal value (average of all test concentrations) and in the two lowest test concentrations values below the limit of quantification were determined.