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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:
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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
See explanation in result section
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Specific details on test material used for the study:
Batch no.: 19020094
Appearance: colorless to pale yellow liquid
Purity: 99.80 % (GC)
Homogeneity: homogeneous
Production date: 20. Feb. 2019
Expiry date: 20. Feb. 2019
Storage: Room Temperature (20 ± 5 °C) store under inert gas in the dark

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
* Preparations
A stock solution will be prepared by mixing the nominal load of 100 mg/L (±10 %) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 ± 1 hours. Based on the results of validation of the analytical method, after this, complete dissolution of the test item is reached. The concentrations to be tested will be prepared by dilution of this stock solution with algal medium.

* The study was performed using 5 concentrations ranging from 0.32 to 32 mg/L (nominal).

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
* Origin and Culture
The culture of Desmodesmus subspicatus was obtained from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock cul- ture 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 will be prepared.

Study design

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

Test conditions

Test temperature:
22.0 to 24.7°C
pH:
7.7 to 7.8
Nominal and measured concentrations:
Treatments tested: 32 / 10 / 3.2 / 1 / 0.32 mg/L nominal concentration
The concentrations to be tested are based on non GLP pre-tests.

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 and after 24 hours, the correlation between nominal and measured concentration was very good (range 90 % and 107 % of the nominal concentrations).
After 72 hours the measured concentrations were in a range of 33 % - 97 % of the nominal concentration.
Because of insufficient stability under test conditions, after 72 hours only very low and strong scattering test item concentration were measurable.
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 (0.389 mL) to achieve a cell concentration of 3 *10^3 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 (0.681 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.

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 content of the test item in the test solution was analytically determined at the beginning and every 24 hours. To avoid any loss of test item by absorption onto the algae cells, analytical measurement was performed in in additional test replicates without algae.


Number of replicates: 6 replicates for the blank control; 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: within the specified range (4440 – 8880 Lux)
Replicates (Blank control): 45 ±1 mL algal medium and algae; 1 vessel filled with 430 mL test solution without algae (for analytical determination)
Replicates (Treatments): 45 ±1 mL test solution and algae; 1 vessel filled with 430 mL control solution without algae (for analytical determination)

Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) was tested as positive control in a current reference study.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 1.62 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. 1.62 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. 2.64 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. 3.64 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. 3.13 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. 1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 7.02 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. 3.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass

Any other information on results incl. tables

* Biological Results of the Reference Study (201809R301)

The results of the last study with the positive control K2Cr2O7are presented in the following table.The study was performed under GLP conditions in June 2018.

Parameter

Value

95% confidence interval

72h ErC50

0.65 mg/L

0.60 mg/L – 0.71 mg/L

72h EyC50

0.28 mg/L

0.21 mg/L – 0.38 mg/L

* Deviations

a) Deviations from the Study Plan

The following deviations were observed:

- The temperature in the experiment was in the range 22.0– 24.7°C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

- The temperature during pre-culture incubation was in the range 23.2 – 24.5°C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical. 

The deviations were assessed and signed by the study director on 27. May 2019.

b) Deviations from the Guideline         

The following deviations were observed:

- The stock solutions to be autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.       

- The temperature in the experiment was in the range 22.0– 24.7 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

- The temperature during pre-culture incubation was in the range 23.2 – 24.5 °C, and therefore not in the desired range (21.0 - 24.0 °C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.

The deviations were assessed and signed by the study director on 27. May2019

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
According to OECD 201, the study was performed using 5 concentrations ranging from 0.32 to 32 mg/L (nominal) and significant inhibition of algal growth was observed at the 2 highest concentrations 10 mg/L and 32 mg/L.
The EC50-72h for Growth Rate and Yield endpoint for the test item were 3.88 mg/L and >7.02 mg/L, respectively.