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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:
05 November 2019 to 08 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The following deviations were observed: • The temperature in the experiment was in the range 21.7 – 24.9 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and expo-nential growth was observed in the blank control this was stated as uncritical. • The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved. • The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
resp. EU C.3
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate is attached in the study report

Test material

Constituent 1
Chemical structure
Reference substance name:
6-methylbenzotriazole
EC Number:
205-265-8
EC Name:
6-methylbenzotriazole
Cas Number:
136-85-6
Molecular formula:
C7H7N3
IUPAC Name:
6-methylbenzotriazole
Test material form:
solid
Specific details on test material used for the study:
Batch: N6A3I
Expiry date: 07 August 2022

Sampling and analysis

Analytical monitoring:
yes
Remarks:
At the start and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS.

Test solutions

Vehicle:
not specified
Details on test solutions:
Because of insufficient solubility of the test item in test medium, a saturated solution was prepared for the test. This was done by mixing the nominal load of 100 mg/L (real load 100.25 mg/L) with the corresponding amount of algal medium (demineralised water en-riched with minerals but without algae) and shaking vigorously for 23.5 hours. After that, the solution looked clear. Nevertheless, the resulting solution was filtrered through 0.45 µm PTFE filters. The lower treatments were prepared by dilution of this solution with algal medium.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga Desmodesmus subspicatus
- SAG Strain number: 86.81
- 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 cul-ture 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 4 days under test conditions (Lighting: with-in the specified range of 4440 – 8880 lux; 21.3– 23.5 °C). The resulting culture grew ex-ponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.

Study design

Test type:
not specified
Water media type:
freshwater
Total exposure duration:
72 h

Test conditions

Test temperature:
21.7 - 24.9°C
pH:
7.6 - 8
Nominal and measured concentrations:
Nominal concentration (mg/L): 0.32; 1; 3.2; 10; 32; 100
Measured concentration (mg/L; t=0h): 0.33; 1.04;3.33; 10.51; 33.76; 104.38
Measured concentration (mg/L; t=72h): 104; 104; 104; 105; 105; 104
Details on test conditions:
TEST SYSTEM
Test vessel:open (covered with perforated plastic foil acting as a stopper).
Treatments: 3 vessels, filled with 45 ±1 mL test solution and algae
1 vessel, filled with 45 ±1 mL test solution without algae for analytical determination
Blank Control: 6 vessels, each completely filled with algal medium and algae
1 vessel filled with algal medium without algae for analytical determination


GROWTH MEDIUM:
The algal medium corresponds to the OECD TG 201 medium.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: within the specified range (4440 – 8880 Lux); Lux meter LT Lutron LX-101


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter.
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).

TEST CONCENTRATIONS:
0.32 / 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
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
Duration:
72 h
Dose descriptor:
EC50
Remarks:
Yield
Effect conc.:
ca. 15.67 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Remarks:
Growth Rate
Effect conc.:
ca. 71.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Remarks:
Yield
Effect conc.:
ca. 3.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Remarks:
Growth Rate
Effect conc.:
ca. 11.73 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Results with reference substance (positive control):
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
72h ErC50: 0.80 mg/L
72h EyC50: 0.39 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes