STI571 F8

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 022401

Sampling and analysis

Analytical monitoring:

yes

Remarks:

At the start and at the end of the incubation period of each experiment, the concentrations of the test substance were determined by HPLC.

Details on sampling:

At the start and at the end of the incubation period of each experiment, the concentrations of the test substance were determined in aliquots
• of the blank containing test substance, but no algae
• of one replicate of each of the test cultures (after filtering through a 0.20 µm filter Acrodisc Premium, Fa. Pall, USA).
The test substance solutions were stored in a deep freezer at ca. -18 °C until analysis.

Test solutions

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: The test substance was dissolved in test medium.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Selenastrum capricornutum ATCC (American Type Culture Collection) 22662, obtained directly from ATCC, 12301 Parklawn Drive, Rockville, M 3.ryland 20852, USA.

Study design

Test type:

static

Water media type:

other: For each experiment, one stock solution was prepared by dissolving appropriate amounts of the test substance in nutrient medium, consisting of sterile double distilled water and sterile concentrated nutrient medium 9+1 .

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

The temperature of the incubation chamber was protocolled at the begining of incubation and each time when cell counts were performed. All cultures and the blank were incubated at 22±1 °C.

pH:

The pH was determined using a pH meter (Piccolo ATC. HANNA Instruments, Germany) in all test cultures, in the control and in the blank at the start of the exposure and 72 hours thereafter.

Conductivity:

The conductivity of the double distilled water used was below 5 µ Scm-1.

Nominal and measured concentrations:

First experiment (nominal concentrations):
6.3 mg/l, 12.5 mg/l, 25.0 mg/l, 50.0 mg/l and 100.0 mg /L.

Second experiment (nominal concentrations):
0.63 mg/l, 1.25 mg/l, 2.50 mg/l, 5.00 mg/l and 10.00 mg/L.

At the start of the incubation, all actual test substance concentrations but the lowest one were between 93.6 % and 101.2 % of the nominal concemtrations. The actual concentration of the lowest tested concentration (0.63 mg/L) was 119.0 % of the nominal one. This was accepted, as the test substance concentration of 0.63 mg/L is slightly below the quantification limit of the analytical method used (0.80 mg/L).
After 72 hours of incubation, the actual test substance concentrations were in the test cultures between 96.4 % and 103.3 % of the actual starting concentrations. In the blanks without algae the actual test substance concentrations v,ere 98.1 % and 100.6 % of the actual starting concentrations. These results give no indication for an uptake of the test substance by the algae or for adherence of the test substance to them.

Details on test conditions:

At the onset of each experiment the cell concentration in the preculture was determined and the inoculum was prepared by diluting an appropriate volume of the preculture with nutrient medium for precultivation to give a calculated cell concentration of 10 to the power of 5 cells/ml.
All test cultures and the blank were set up in 250 ml conical flasks (Erlenmeyer). The total culture volume was 100 ml in each case.


The test substance did not markedly alter the pH of the test media. The pH was between 8.2 and 8.8 at the start of the incubation in the test cultures and it was 8.1 or 8.6 in the control cultures.
After 72 hours of incubation there was no obvious differrence in the pH values of the control and test cultures in either experiment. The maximum change in pH in the control cultures during the 72 hours of incubation was 1.1,
i.e. within the limit of 1.5 stated by the guideline.

For each experiment, one stock solution was prepared by dissolving appropriate amounts of the test substance in nutrient medium, consisting of sterile double distilled water and sterile concentrated nutrient medium 9+1 (v/v, see also Appendix 1). The conductivity of the double distilled water used was below 5 µ Scm-1. Aliquots of this stock solution were further diluted with sterile nutrient medium to obtain the concentrations needed.

In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the betIin and at the end of the incubation period.
Possible test substance effects were determined by comparison of the areas under the growth curves and by comparison of the growth rates.
The actual concentrations of the test substance were determined in filtered samples of the cultures exposed to the test substance and also in one blank with the highest concentration of the test substance, but no algae, at the start of each experiment and after 72 hours of incubation.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.3 mg/L

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Basis for effect:

biomass

Details on results:

Algal growth was inhibited by more than 100 % at the highest tested concentration of 100.0 mg per L medium. The lowest tested concentrations of 0.63 and 1.25 mg/L resulted in a slight enhancement of algal growth compared to the negative controls.

Reported statistics and error estimates:

The nominal test substance concentrations were used for the calculations.

Two "no observed effect concentrations" (NOECs) were derived - based on the area under the growth curves and based on the average growth rates.
The nominal test substance concentrations were used for the calculations. Two EC5O(O-72h)-values were derived:
The EC50 (0-72 h) was based on the areas under the growth curves. The EC50 (0-72 h) was based on the average growth rates.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Two "no observed effect concentrations" (NOECs) and two EC50 values were derived - based on the area under the growth curves and based en the average growth rates. The nominal test substance concentrations were used for the calculations.