Registration Dossier

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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Study design

Test type:
static
Total exposure duration:
72 h

Test conditions

Test temperature:
The water temperature was maintained at 23.0°C throughout the exposure period in all test vessels.
pH:
The pH in the control was 8.0 at the start and in the range of 10.1 – 10.4 at the end of the test.
Nominal and measured concentrations:
Based on the results of a range-finding test the nominal concentrations of CA 2814 B for the main test were selected as 1.88, 3.75, 7.5, 15 and30 mg/L. Additionally, a blank and control and a solvent control (0.01% DMF) without test item was included.
Details on test conditions:
TEST SYSTEM

- Test vessels: The test was performed in 100 ml Erlenmeyer flaks, each with 50 ml algae suspension, sealed with a plug
and continuously shaken with a lab shaker. .

- Light: Continuous light 79µE/m2s in the spectral range of 400-700 nm, measured at the end of the test with
Quantum LI-189 light meter.

- Cultivation: The algae were cultivated and tested in synthetic test water.

- Cell denisty: The test was started with a nominal algae cell density of 10000 cells ± 10 % per ml test solution.
These cells were taken from pre-culture, which was set up about 72 hours prior to the test at the same
concentration as in the test.

- Replicates: Three replicates were carried out for each test concentration, six for the control and three for the solvent control.
Additionally, the highest concentration was tested without algae and served for the determination of the particle
background (BG).

TEST CONCENTRATIONS

- Stock solution/Test concentrations:
- Stock solutions 1: 1999.9 mg test item was mixed with about 3 ml DMF to get a final volume of 5 ml.
The stock solution was slightly turbid, but without sediment.

- Test concentrations: The main test was performed with nominal test item concentrations of 0.88, 3.75, 7.5, 15, and 30 mg/L.
A blank control without test item and a solvent control DMF (0.01%).
To obtain the treatment concentrations, the following stock solutions were diluted from the stock solution 1.

- Stock solution 2: 3.75 ml from stock solution 1 and filled up to 5 ml with DMF, 0.1 ml from this stock solution resulted in 30 mg/L.
- Stock solution 3: 2.5 ml from stock solution 2 and 2.5 ml DMF, 0.1 ml from this stock solution resulted in 15 mg/L.
- Stock solution 4: 2.5 ml from stock solution 3 and 2.5 ml DMF, 0.1 ml from this stock solution resulted in 7.5 mg/L.
- Stock solution 5: 2.5 ml from stock solution 4 and 2.5 ml DMF, 0.1 ml from this stock solution resulted in 3.75 mg/L.
- Stock solution 6: 2.5 ml from stock solution 5 and 2.5 m DMF, 0.1 ml of this stock solution resulted in 1.88 mg/L.
- Stock solution 7: 0.1 ml DMF and filled up to 1000 ml with test medium = DMF Control (0.01%).

These solution were divided into 3 groups of 50ml.
50 ml of each test solution were placed into flasks for analytical determinations.
All test solutions were prepared as close as practically possible to the start of the test.

- Substance control: Additionally, the test item was tested in the highest concentration without algae (particle background).

EFFECT PARAMETERS MEASURED

- Cell density: The cell densities were determined after 24, 48 and 72 hours.
Additionally, at the end of the test the shape of the algae cells was examined in the highest concentration still
showing algal growth and in the control.

- pH: The pH was measured at the beginning and after 72 hours.

- Temperature: The temperature was recorded continuously in an Erlenmeyer flask filled with water and running under
the same conditions as the test flasks.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 7.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
ca. 7.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The water temperature was maintained at 23.0°C throughout the exposure period in all test vessels.
The pH in the control was 8.0 at the start and in the range of 10.1 – 10.4 at the end of the test.
Based on the results of a range-finding test the nominal concentrations of CA 2814 B for the main test were selected as 1.88, 3.75, 7.5, 15 and30 mg/L. Additionally, a blank and control and a solvent control (0.01% DMF) without test item was included.
Since the test item is instable in water it was not possible to make a stock solution of CA 2814 B in water. Hence, N,N-Dimethylformamide (DMF) was used as solubilising agent for the preparation of a stock solution.
The following nominal concentrations were chosen for the definitive test: 1.88, 3.75, 7.5, 15 and 30 mg/L in addition to a blank control and a DMF control, both without test item.
The stock solutions for the preparation of the test concentrations of 15 and 30 mg/L seemed to be not completely soluble in DMF. Neverthenless the test was carried out with these concentrations. The inhibition of biomass in the lower test concentrations of 7.5 mg/L resulted in 35.3%. In all probability the low solubility of CA 2814 B in DMF is responsible for the results found in the test concentrations of 15 and 30 mg/L. For this reason, no proper dose-response curve was found. Regardless the test concentration of 15 and 30 mg/L were above solubility limit in test water and the EC50 is clearly above 30 mg/L.
The findings mentioned above corresponded with the limit of solubility for DMF (10% in DMF) found in the daphnia test with the same test item.
Due to the instability of the test item, the actual test concentrations of CA 2814 B were not determined analytically. Hence, the results were calculated with the nominal concentrations

After 72 hours of exposure no misshaped cells or cell-debris were observed microscopically.


EC values: The Ec values bsded on nominal concentrations CA 2814 B were determined as follows:
Critical Effect Concentrations of CA 2814 B (mg/L; 0 – 72 h)

Growth rate ErC50 >30
(95 % confidence limit n.d.)

ErC10 n.d.
(95 % confidence limit n.d.)

LOEC 7.5
NOEC 3.8

Biomass rate EbC50 >30
(95 % confidence limit n.d.)

EbC10 n.d.
(95 % confidence limit n.d.)

LOEC 7.5
NOEC 3.8

n.d. = value could not be determined
Reported statistics and error estimates:
The statistical analysis of the data was performed with a statistical program (Ratte H T, 1995).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure no misshaped cells or cell-debris were observed microscopically.
Executive summary:

The water temperature was maintained at 23.0°C throughout the exposure period in all test vessels. 

The pH in the control was 8.0 at the start and in the range of 10.1 – 10.4 at the end of the test. 

Since the test item is instable in water it was not possible to make a stock solution of CA 2814 B in water. Hence, N,N-Dimethylformamide (DMF) was used as solubilising agent for the preparation of a stock solution. 

The following nominal concentrations were chosen for the definitive test: 1.88, 3.75, 7.5, 15 and 30 mg/L in addition to a blank control and a DMF control, both without test item. Due to the instability of the test item, the actual test concentrations of CA 2814 B were not determined analytically. Hence, the results were calculated with the nominal concentrations. 

 

After 72 hours of exposure no misshaped cells or cell-debris were observed microscopically.

 

Critical Effect Concentrations of CA 2814 B (mg/L; 0 – 72 h)

 

Growth rate ErC50      >30

(95 % confidence limit n.d.)

 

ErC10                              n.d.

(95 % confidence limit n.d.)

 

LOEC                              7.5

NOEC                            3.8

 

n.d. = value could not be determined