2-Propen-1-one, 3-(dimethylamino)-1-(3-pyridinyl)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 September 1999 - 11 November 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-770-8

EC Name:

-

Cas Number:

55314-16-4

Molecular formula:

C10 H12 N2 O1

IUPAC Name:

3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one

Details on test material:

Name of test material (as cited in study report): STI571 Y5a
- Substance type: Brown solid
- Physical state: Solid
- Analytical purity: 98.6%
- Lot/batch No.: 992301
- Expiration date of the lot/batch: 01 July 2001
- Stability under storage conditions: Stable
- Storage condition of test material: In refrigerator in the dark
- Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test samples were taken from three concentrations, i.e. 10, 32 and 100 mg/l and the negative control.

Frequency at t=0 h and t= 72 h
Volume 10 ml
Storage Samples were stored in a freezer until analysis.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and the end of the test period.
sample pre-treatment:
All samples were stored in a deep freeze. On the day of analysis, the frozen samples were defrosted at room temperature. The entire volume of each sample (10 ml) was transferred quantitatively into a 20 ml volumetric flask using acetonitrile. The flasks were filled up to the mark with acetonitrile. If necessary, the solutions were further diluted with 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.

Test solutions

Vehicle:

no

Details on test solutions:

STI571 Y5A was a brown solid with purity of 98.6% and completely soluble in test medium at the concentrations tested. Preparation of test solutions started with a stock solution in test medium at 100 mg/l. No special treatment other than careful mixing was necessary to dissolve the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the stock solution in test medium. All final test solutions were clear and coloured from yellow to colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

Between 22.5 and 23.0 °C.

pH:

Between 8.3 and 10.0

Nominal and measured concentrations:

Nominal concentrations:0, 10, 18, 32, 56 and 100 mg/l
Measured concentration:0, 10, 32, 100 mg/l
Analysis of samples taken at the start of the final test showed that measured concentrations were 12.5 mg/l at nominal 10 mg/l (125%), 36.1 mg/l at nominal 32 mg/l (113%) and 100 mg/l at nominal 100 mg/l with algae (100%). At the end of the 72-hour test period, the concentrations measured had not decreased and ranged from 113 to 122 % of nominal. The concentrations measured in the solution without algae (100 mg/l) ranged from 114 to 115%.

Details on test conditions:

RANGE FINDING (direct)
A range-finding test preceded the final test to provide information about the range of concentrations to be used in the final test. Algae were exposed to a range of 0.1 to 100 mg/l, increasing by a factor 10. Test procedure and conditions were comparable to those applied in the final test.

ADDITIONAL RANGE FINDING: (indirect)
An additional range finding study was performed to examine if STI571 Y5A would indirectly inhibit the growth of green algae owing to the colour of the test solutions.
TEST SYSTEM
- Type: closed petri dish
- Material, size, headspace, fill volume: 50 ml
- Aeration: no
- Replicates: two replicates for each test concentration and three replicates for the controls. Each replicate consisted of three petri dishes. Two petri-dishes were filled with the respective test solution without algae present, and one petri-dish was filled with 50 ml of algal suspension at an initial density of 20000 cell/ml and without test substance present. The petri dish with algal suspension was placed under the two petri dishes containing the test solution. These three petri dishes were placed in the incubator in such a way that the algal suspensions were exposed to the light only via the petri dishes with test solution. Three replicates were exposed to light using untreated test medium (negative controls), while two replicates were exposed via petri dishes that were completely darkened (positive controls).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: 0.1, 1, 10 and 100 mg/l

FINAL STUDY:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 950700 cells/ml
Replicates:
3 replicates of each test concentration.
6 replicates of the blank control.
1 replicate of the highest concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘GroLux’ of 30 Watt (Sylvania) yielding 3800-4300 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEbC, 72 h ErC50, 72 h EbC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: control and 10, 18, 32, 56 and 100 mg/l

Reference substance (positive control):

yes

Remarks:

POTASSIUM DICHROMATE

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

49 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 27-87 mg/l

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 16-55 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 52-70 mg/l

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

36 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 30-42 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Effect concentrations exceeding solubility of substance in test medium: No
-Results additional test( indirect): Since no decrease in the extinction values was observed at the highest test concentration, the reduction of cell growth recorded in the direct range finding was related to exposure and not caused by absorption of wave lengths necessary for algal growth due to the colour of the test solution.

Results with reference substance (positive control):

The EC50 for cell growth inhibition (EBC50:0-72h) was 0.55 mg/l with a 95 % confidence interval ranging from 0.38 to 0.81 mg/l.

The EC50 for growth rate reduction (ERC50:0-72h) was 1.5 mg/l with a 95 % confidence interval ranging from 1.1 to 2.1 mg/l.

Reported statistics and error estimates:

Inhibition of cell growth was statistically significant at test concentrations of 56 and 100 mg/l (ANOVA- Bonferroni t-Test and Tukey test of multiple comparison: P=0.05).
Reduction of the growth rate was found to be statistically significant at test concentrations of 56 and 100 mg/l (ANOVA-Tukey test of multiple comparison and the Bonferroni t-Test, P=0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, STI571 Y5A affected growth of this fresh water algae species significantly at 56 mg/l and higher

The EC50 for cell growth inhibition (EBC50:0-72h) was 49 mg/l with a 95 % confidence interval ranging from 27 to 87 mg/l.
The EC10 for cell growth inhibition (EBC10:0-72h) was 30 mg/l with a 95 % confidence interval ranging from 16 to 55 mg/l.

As growth rate is derived from the slope under the growth curve in a logarithmic plot, the measure of the specific growth rate is preferable over biomass following from the mathematical nature of exponential growth.

The EC50 for growth rate reduction (ERC50:0-72h) was 60 mg/l with a 95 % confidence interval ranging from 52 to 70 mg/l.
The EC10 for growth rate reduction (ERC10:0-72h) was 36 mg/l with a 95 % confidence interval ranging from 30 to 42 mg/l.

The NOEC was 32 mg/l for both cell growth inhibition and growth rate reduction.