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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:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperonyl acetate
EC Number:
206-312-5
EC Name:
Piperonyl acetate
Cas Number:
326-61-4
Molecular formula:
C10H10O4
IUPAC Name:
(2H-1,3-benzodioxol-5-yl)methyl acetate
impurity 1
Chemical structure
Reference substance name:
Piperonyl alcohol
EC Number:
207-808-4
EC Name:
Piperonyl alcohol
Cas Number:
495-76-1
Molecular formula:
C8H8O3
IUPAC Name:
1,3-benzodioxol-5-ylmethanol
Specific details on test material used for the study:
Batch no.: 18040126
Appearance: colourless liquid
Purity: 99.80 % (GC)
Homogeneity: homogeneous
Production date: 24. May 2018
Expiry date: 23. May 2020
Storage: Room Temperature (20 ± 5 °C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
SAMPLE PREPARATION
* Twofold Enrichment: (10 mg/L and 32 mg/L)
2 g NaCl was dissolved in 20 mL of the test item solution; then, the clear solution was extracted two times with the solvent dichloromethane (4 mL and 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID.
* Tenfold Enrichment: (Control, 0.32 / 1 / 3.2 mg/L)
6 g NaCl was dissolved in 100 mL of the test item solution (or blank control); then, the clear solution was extracted two times with the solvent dichloromethane (4 mL and 4 mL) and the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloromethane after addition of 500 µL ISTD (internal standard) stock solution (1000 mg/L in dichloromethane) and the solution was measured via GC/FID.

Test solutions

Vehicle:
no
Details on test solutions:
* PREPARATION STOCK SOLUTION
A stock solution was prepared by mixing 80.8 µL test item (corresponding to 100 mg/L, using the density of the test item 1.237 g/cm3) with 1000 mL algal medium (demineralised water enriched with minerals but without algae) and stirring for 23 hours. The clear upper phase of this solution was used for preparation of the treatments. The treatments were prepared by dilution of the stock solution with algal medium.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
* Specification
Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

* Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
The algae are kept as stock culture on solid agar at 2 - 8°C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

* Pre-culture
For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21.2 – 23.1°C). The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Study design

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

Test conditions

Test temperature:
from 22.1 to 23.6°C
pH:
from 7.6 to 8.2
Nominal and measured concentrations:
* Treatments tested:
32 / 10 / 3.2 / 1 / 0.32 mg/L nominal concentration (based on non GLP pre-tests).

* Analytical Determination:
The content of the test item in the test solutions was determined using GC. The measured concentrations at the beginning lay between 92 % and 99 % of the nominal concentrations. For the measured concentrations at the beginning, a recovery rate calculated from the QC samples of 91 % was taken into account as sensitivity correction factor.
Because the test item is not stable under test conditions, already after 24 hours no more test item was detectable. Therefore, no further measurements were performed.
For calculation of a mean exposure concentration, the geometric mean was calculated based on the measured start concentration and the limit of quantification (LOQ) of 0.1 mg/L (lowest calibration level 1 mg/L and tenfold enrichment).
Details on test conditions:
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.304 mL) to achieve a cell concentration of 2.6E3 cells/mL.
For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.533 mL). In this mixture, the pH-value was measured.

The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.

For the analytical measurement, additional replicates without algae were prepared.

The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.

At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The concentration of the test item in the test vessels was measured at the start and at the end of the test and after 24 hours. Because no test item can be detected after 24 hours, no further measurements were performed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.56 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.:
ca. 0.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
75 h
Dose descriptor:
LOEC
Effect conc.:
ca. 0.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0.84 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.:
ca. 0.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 1.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 1.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 13.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 3.79 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 15.91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
One valid experiment was performed.
The study was performed using 5 concentrations ranging from 0.32 to 32 mg/L (nominal).
Significant inhibition of algal growth was observed at the 2 highest concentrations 10 mg/L and 32 mg/L.
The content of the test item in the test solutions was determined using GC. To avoid any loss of test item by absorption onto the algae cells, additional test replicates without algae were prepared only for analytical measurement. At the beginning of the test, the correlation between nominal and measured concentration was very good. The measured concentrations at the beginning lay between 92 % and 99 % of the nominal concentrations. Because the test item is not stable under test conditions, already after 24 hours no more test item was detectable. Therefore, no further measurements were performed.
For calculation of a mean exposure concentration, the geometric mean was calculated based on the measured start concentration and the limit of quantification (LOQ) of 0.1 mg/L (lowest calibration level 1 mg/L and tenfold enrichment).

The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.

All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.
Results with reference substance (positive control):
The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes