Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidline Study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Identification: Aminopropyl Vinyl Ether
- Chemical name: 3-(ethenyloxy)propan-1-1amine
- Physical form: liquid
- Appearance: colourless, clear
- Water solubility: miscible
- Purity: 99.6 corrected % (treated as 100% pure)
- Density: 0.891 g/cm3 (20°C)
- Vapor pressure: 100 mbar (76°C)

Sampling and analysis

Analytical monitoring:
Details on sampling:
Sampling was performed at the start of the exposure (0 h) from the inoculated replicate 7 of each concentration and the control.
At the end of the exposure (72 h) samples were taken from the combined inoculated (+ algae) replicates of the test concentrations and of the combined inoculated control group replicates. Additionally, samples were collected from the uninoculated (-algae) replicate 0 of each test concentration and the control at the end of the exposure (72 h).

Test solutions

Details on test solutions:
The volume of the test substance required for preparation of the nominal concentration was calculated from the density (d=0.891 g/cm3). Prior to use, the test substance container was shaken to ensure homogeneity. The stock test solution (111 mg/L) was prepared by pipetting 249µL of test substance into 2L of test medium and stirring for approximately 10 min until visibly dissolved. The stock solution was prepared at 111% of the nominal concentration to compensate for the dilution by the later addition of algal inoculum. The lower test concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance.
-Test groups: 0 (control), 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L as nominal concentrations based on test substance mass without correction for purity.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata KORHIKOV
- Strain: SAG 61.81
- Source (laboratory, culture collection): Collection of algal cultures in University of Göttingen/Germany
- Age of inoculum (at test initiation): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

Test temperature:

Dissolved oxygen:
Not measured
Not measured, freshwater
Details on test conditions:
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): plugged with gas permeable silicone sponge caps
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, test volume 100 mL
- Shaking rate: continuous (appr. 85 rpm)
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 0.5x10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per test group for initial concentration control analsis only (replicates): 1
- No. of vessels per uninoculated (without algae) replicate per test group for background fluorescence correction and to determine the influence of the test design on test substance stability: 1

- Standard medium used: yes

- Source/preparation of dilution water: OECD medium prepared according to OECD Guidleine for Testing of Chemicals, No. 201 Algal growth inhibition test, Mar 2006, Annex 3
- pH-value: adjusted to 8.1 with NaOH (1M)

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily.
- Light intensity and quality: Average 6594 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Fluorescence measurement: The fluorescence of aliquots from each test vessel was measured using a Tecan Infinite 200Pro fluorometer in a 96-well flat bottom black plate with the following parameters: fluorescence top reading; excitation / emission wavelength = 430 / 670 nm; excitation / emission bandwidth = 20 / 25 nm; flashes = 5; integration time = 20 µs; shaking duration = 15s; shaking amplitude = 6 mm.
- Determination of correlation between fluorescence and cell density: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
- Algal morphology: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

- test groups: 0 (control), 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L as nominal concentrations based on test substance mass without correction for purity.
- Reason for selection of test concentrations: In the first experiment a flat dose response curve was observed for the yield and growth rate endpoints. In the lowest test concentration 1 mg/L, 49% inhibition of yield and 15% inhibition of growth rate were observed. Therefore lower test concentrations were included in the second experiment in order to estimate either an EC10 or NOEC value. The raw data of the range finding test are archived together with the raw data of this study.

Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
38.2 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 29.7-49.0
72 h
Dose descriptor:
Effect conc.:
7.93 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 4.65-13.5
Details on results:
- Observation of the following abnormalities (for algal test) were performed: no remarkable observations
Results with reference substance (positive control):
- Results with reference substance valid? Yes
In order to verify that the algal cultures are responding normally to toxic stress, tests with a reference substance (potassium dichromate) are conducted. Reference substance tests are conducted according to OECD 201 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to potassium dichromate EC50 values published in ISO test guideline 8692, which represent the typical response range for the algal species tested.

- EC50:
According to the test ISO guideline 8692 the EC50 values of the reference substance potassium dichromate should be in the range: ErC50 = 0.92 – 1.46 mg/L after 72 hours for Pseudokirchneriella subcapitata

Applicant's summary and conclusion

Validity criteria fulfilled: