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:
2000
Report Date:
2000

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:
yes
Details on sampling:
2 samples (2 times about 30 ml per concentration and control) were taken at the start and 2 samples (2 times about 30 ml per concentration and
control) at the end of exposure. In addition a sample of the stock solution was taken. . All samples were kept at -18 to -22 °C until analysis. :

Test solutions

Vehicle:
no
Details on test solutions:
Refer to analytical methods section.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green Algae
- Strain: Selenastrunm capricornutum ATCC 2262 ≡ Pseudokirch subcapiata SAG 61.81
- Source: Collection of algae cultures, Pflanzenphysiologisches Institut, University, Nikolausberger Weg 180, D-37073 Göttingen, Germany.
- Initial cell density: 10550 cells/ml
- Pre-culture: 3 days under test conditions

Study design

Test type:
static
Limit test:
yes
Total exposure duration:
3 d

Test conditions

Hardness:
25 mg CaCO3/L
Test temperature:
24 ± 1°C
pH:
The pH of the test solutions was 8.0 - 8.1 at the start and 8.0 at the end of exposure.
Nominal and measured concentrations:
The nominal test concentrations were: 1.0, 2.0, 4.0, 8.0, 16 and 32 mg test item/L.
The measured test concentrations of CA 2218 A were 0.73, 1.7, 3.5, 7.1, 12.0 and 27.2 mg/L at the start of exposure and 0.7, 1.5, 3.2, 6.3, 13.6 and 28.4 mg test item/L at the end of exposure. Actual mean concentrations were 0.71, 1.6, 3.35, 6.7, 12.8 and 27.8 mg/L corresponding to 71.5, 80.0, 83.8, 83.8, 80.0 and 86.9 % of nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Vessels: 100 ml Erlenmeyer flasks (total volume 125 ml), cotton stoppers, on Lab shaker, 30ml test solution per flask.
- Temperature: 24 ± 1°C
- Lighting: Continuous illumination, cold white fluorescent light (approx. 8000 lux)
- Shaking rate: 150 rpm
- Duration: 72 hours


STOCK SOLUTIONS
- Test item: 100.3 mg of CA 2218 A were mixed and made up with medium (untrsonification for 1 hour). This stock solution
(suspension) was used to prepare all test solutions.

TEST CONCENTRATIONS
- Range-fing test: A range-finding test (test concentrations 0.01, 0.1, 1.0, 10 and 100 mg/L) was conducted to determine a concentration
range of CA 2218 A to use in the definitive test. The cell concentrations were measured at the start and at the end of
exposure. An inhibition of the algae growth was seen at test concentration of 10 and 100 mg/L.

Based on the results of the range-finding test the following nominal concentrations were selected for the definitive test:

Nominal: 1.0, 2.0, 4.0, 8.0, 16.0 and 32 mg test item/L.
Control: Reconstituted water
Replicates: Each test concentration was tested in 3 replicates, the blank control in 6.
Remark: Calculated amounts of the stock solution to produce the desired test concentrations were given into the water and were
homogeneously distributed. The algae were then tranfered to the flasks.

MEASUREMENTS

Cell densities were measured at 24, 48 and 72 hours of exposure using CytoFluor II (a Fluorescence Multi-Well Plate Reader). Temperature was continuously measured in the room and daily in water in a separate test vessel.
The pH of the test solutions was measured at 0h and 72 hours of exposure.

Culture medium: see Table 2 below
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 1.64 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.715 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Changes in mean density indicated that exponential growth occurred in the negative control replicates.
No significant differences in mean cell density between the control and the 1.0 mg/L treatment group on the basis of growth rate (3.3 % inhibition) and on the basis of AUC (area under growth curve, 21.1 % inhibition) were observed. At test concentration of 1.6 mg/L 38.1 %inhibition was observed on the basis of growth rate and 82.8 % inhibition the basis of AUC was observed. At all other concentration inhibition of growth was 100 %.

Calculations of EC50 and NOEC are based on actual me concentrations – refer to Table 1

ErC50 (0 – 72 h) and EbC 50 (0 – 72h) of CA 2218 A were determined to be 1.64 mg/L and 1.03 mg/L based on actual mean concentrations, respectively.

The NOErC and NOEbC growth inhibition (0 – 72 h) were both 0.715 mg/L based on actual mean concentrations of CA 2218 A.




Results with reference substance (positive control):
Refer to Table 3 below

Any other information on results incl. tables

Table 1:

 

Test item results: 

Inhibition

(growth rates)

Inhibition

(areas under growth curve)

EC 50 (0 – 72 h):

95 % confidence limit:

Slope:

NOEC (0- - 72 h):

(significant on 5% level)

ErC 50 : 1.64 mg/L

None

22.2781 ± 0

NOErC : 0.715 mg/L

EbC 50: 1.03 mg/L

0.76 – 1.36 mg/L

3.638 ± 0.966

NOEbC : 0.715 mg/L

 

 

Table 3:

Reference item results:

 

Inhibition

(growth rates)

Inhibition

(areas under growth curve)

EC 50 (0 – 72 h):

95 % confidence limit:

Slope:

NOEC (0- - 72 h):

(significant on 5% level)

ErC 50 : 0.70 mg/L

None

5.18± 3.323

NOErC : 0.50 mg/L

EbC 50: 0.51 mg/L

None

23.453 ± 0

NOEbC : 0.25 mg/L

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:

ErC50 (0 – 72 h) of CA 2218 A was determined to be 1.64 mg/L based on actual mean concentrations.

The NOErC growth inhibition (0 – 72 h) was 0.715 mg/L based on actual mean concentrations of CA 2218 A.
Executive summary:

Range-finding tests showed that the ErC50 (72h) will be between 1.0 and 10.0 mg/L. Based on the range-finding test, the following nominal concentrations of CA 2218 A were chosen for the main test: 1.0, 2.0, 4.0, 8.0, 16 and 32 mg test item/L. 100.3 mg of CA 2218 A were mixed and made up to 1000 ml with medium. This stock solution was used to prepare all test solutions. The prepared test solutions were distributed in the vessels before the algae were placed in the test vessels, the exposure starts (0 h). Exposure of Selenastrum capricornutum was started at an initial cell density of 10550 cells/L after pre-culture period of 3 days. 

 

In the test with CA 2218 A, the temperature was maintained constant at 24 ± 1°C. Continuous illumination with cold white fluorescent light at 129 µE/m2(approx. 8000 lux) was used. The pH of the test solutions was 8.0 – 8.1 at the start and 8.0 at the end of exposure. The multiplication factor of the algae cell density in the control within 72 hours was 179 in the control. 

The nominal test concentrations were: 1.0, 2.0, 4.0, 8.0, 16 and 32 mg test item/L. 

The measured test concentrations of CA 2218 A were 0.73, 1.7, 3.5, 7.1, 12.0 and 27.2 mg/L at the start of exposure and 0.7, 1.5, 3.2, 6.3, 13.6 and 28.4 mg test item/L at the end of exposure. Actual mean concentrations were 0.71, 1.6, 3.35, 6.7, 12.8 and 27.8 mg/L corresponding to 71.5, 80.0, 83.8, 83.8, 80.0 and 86.9 % of nominal concentrations. 

 

Following calculations of EC50 and NOEC are based on nominal concentrations. 

 

ErC 50 (0 – 72 h):                1.64 mg/L

95% confidence limit:           none

Slope:                                  22.781 ± 0

NOEC (0 – 72 h):                NOErC : 0.715 mg/L

(significant on 5% level)

 

ErC50 (0 – 72 h) of CA 2218 A was determined to be 1.64 mg/L based on actual mean concentrations.