Methyl 3-aminocrotonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Methyl 3-aminocrotonate
CAS number: 14205-39-1
Description: Yellowish white crystals
Expiry date: 20 December 2019
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed at the applied test concentration levels and from the control at the beginning of the experiment and 24-hour intervals thereafter during the test in order to better define loss of the test substance during the exposure period to provide more data to allow a better interpretation.

Vehicle:

yes

Details on test solutions:

A stock solution with a nominal concentration of 100.0 mg/L was prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) using ultrasonic bath (7 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of the test lab.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

value is estimated on 96h

Hardness:

not available; standard OECD medium used

Test temperature:

The temperature was between 22.1 and 22.5°C measured in the flask and between 21.5 and 23.2 °C measured within the climate chamber.

pH:

The range of the pH was 7.38 –8.97 during the experiment.

Dissolved oxygen:

not available

Salinity:

not applicable; freshwater used

Conductivity:

not available

Nominal and measured concentrations:

Because significant inhibition was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment. The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
Due to rapid degradation of the test item in the test media the analytically measured concentrations could not be measured the whole experiment, the biological results are based on the initial measured test item concentrations. The corresponding initial measured test item concentrations were: 5.11, 10.08, 19.63, 42.56 and 88.20 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: standard
- Type (delete if not applicable): open
- Cells density: cell density in the control cultures increased by the factor of 64.00 within three days.
- No. of organisms per vessel: biomass of approximately 104 algal cells per mL test medium

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:
- Light intensity and quality: The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7502 lux (equivalent to ~101 uE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed +/- 15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 88.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

ErC50

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

84.55 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

EyC50

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

91.25 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

EbC50

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

42.56 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

NOErC

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

19.63 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks:

NOEyC

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

19.63 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

NOEbC

Details on results:

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group at the highest examined concentration of 88.20 mg/L (measured), therefore the NOEC was determined as 42.56 mg/L (measured) and LOEC was determined as 88.20 mg/L (measured). The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 42.56 – 88.20 mg/L (measured), therefore the NOEC was determined as 19.63 mg/L (measured) and LOEC was determined as 42.56 mg/L (measured). The biological results are summarised in the table mentioned above.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 19/009-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 22 – 25 January 2019.
The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)
These values are within the range of laboratory ring test data.

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (alpha = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Validity criteria fulfilled:

yes

Conclusions:

The measured EC50- and NOEC-values for aquatic green algae after 72 hours were as follows:
Growth rate: ErC50 > 88.20 mg/l / NOErC 42.56 mg/l
Yield: EyC50 >84.55 mg/l / NOEyC 19.63 mg/l
Biomass: EbC50 91.25 mg/l / NOEbC 19.63 mg/l

Executive summary:

The study was performed in 2019 following OECD method 201 and EU method C.3. The effect of test item was assessed on algal growth using the unicellular green algaPseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The nominal concentrations oftest item used in the main experimentwere:6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.Test concentrations were analytically determined at the start of the experiment andat 24-hour intervals thereafterduring thetest.Due to rapid degradation of the test item in the test media the analytically measured concentrations could not be measured the whole experiment, the biological results are based on the initial measured test item concentrations(more details are given in 4.2.).The initial measured test item concentrations were:5.11, 10.08, 19.63, 42.56 and 88.20 mg/L.

The measured EC50- and NOEC-values for aquatic green algae after 72 hours were as follows:

Growth rate: ErC50  > 88.20 mg/l / NOErC 42.56 mg/l

Yield: EyC50  >84.55 mg/l / NOEyC 19.63 mg/l

Biomass: EbC50  91.25 mg/l / NOEbC 19.63 mg/l

Description of key information

The study was performed in 2019 following OECD method 201 and EU method C.3. The effect of test item was assessed on algal growth using the unicellular green algaPseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.The nominal concentrations oftest item used in the main experimentwere:6.25, 12.5, 25.0, 50.0 and100.0 mg/L.Test concentrations were analytically determined at the start of the experiment andat 24-hour intervals thereafterduring thetest.Due to rapid degradation of the test item in the test media the analytically measured concentrations could not be measured the whole experiment, the biological results are based on the initial measured test item concentrations(more details are given in 4.2.).The initial measured test item concentrations were:5.11, 10.08, 19.63, 42.56 and 88.20 mg/L.

The measured EC50- and NOEC-values for aquatic green algae after 72 hours were as follows:

Growth rate: ErC50  > 88.20 mg/l / NOErC 42.56 mg/l

Yield: EyC50 84.55 mg/l / NOEyC 19.63 mg/l

Biomass: EbC50  91.25 mg/l / NOEbC 19.63 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

84.55 mg/L

EC10 or NOEC for freshwater algae:

19.63 mg/L