2-butenoic anhydride

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

Study period:

2012-08-13 to 2012-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

trans-crotonic acid

EC Number:

203-533-9

EC Name:

trans-crotonic acid

Cas Number:

107-93-7

Molecular formula:

C4H6O2

IUPAC Name:

(E)-2-Butenoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling:
For determination of the test item concentration, samples were taken from the testing concentration and the control at the start and at the end of the test. Six replicate samples were taken from the test concentration (100 mg/L) at the start (before distribution into the test vessels) and one sample from each test vessel respectively (six replicates in total) at the end of the test. From the control, six replicate samples were taken at the start and at the end of the experiment.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L was first prepared by dissolving an amount of 0.1 g test item in 1000 mL dilution water (OECD medium) by mechanical dispersion. The test item is highly acidic and reduces the pH of the test solution significantly (pH 4.62; measured in the pre experiment at 100 mg/L), therefore the pH of the stock solution was adjusted to the pH of the OECD Medium using 1N NaOH. The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared immediately before introduction of algae (start of the experiment).

- Chemical name of vehicle: Algal Mineral Salts Test Medium

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): used when still exponentially growing, normally after an incubation period of about three days

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.5 – 22.1 °C

pH:

7.74 – 9.31

Nominal and measured concentrations:

The following nominal concentrations of test item were tested: 6.6, 12.5, 25.0, 50.0 and 100 mg/L. The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were: 2.6, 10.3, 18.3, 46.5 and 57.5 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Initial cells density: 10E04 cells/mL
- No. of vessels per concentration (replicates):The test was performed with three replicates at each test concentration
- No. of vessels per control (replicates): six replicates were included in the untreated control.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- in accordiance with the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, 1N NaOH
- Light intensity and quality: 7862 lux, with a spectral range of 400-700 nm


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

Reference substance (positive control):

yes

Remarks:

Toxic Reference Control: potassium dichromate; Untreated Control: Algal Mineral Salts Test Medium

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 57.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and Yield

Remarks on result:

other: 95% CL not applicable

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 57.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: 95% CL not applicable

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

57.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: 95% CL not applicable

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 57.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: 95% CL not applicable

Details on results:

TEST CONCENTRATION
The measured concentrations deviated more than 20 % from the nominal during the experiment therefore the exposure concentrations were calculated as geometric mean of the concentrations measured at the start and at the end of the test.

BIOLOGICAL RESULTS
All biological results are related to the geometric mean of the measured test item concentrations during the experiment.

GROWTH INHIBITION
The test item had no statistically significant inhibitory effect on the growth of Pseudokirchneriella subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in the whole tested concentration range.

SHAPE OF THE ALGAL CELLS
Abnormal appearance of the algal cells was not observed during the experiment.

CELL DENSITY IN CONTROL
The cell density in the control has increased from nominal N = 1 × 10E04 cells/mL at the start of the test (0 hours) to N = 59.50 × 10E4 cells/mL (mean value) after 72 hours in the in the Untreated Control. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.


BEHAVIOUR OF THE TEST ITEM
The behaviour of the test item in test medium was monitored during the test by visual (unaided eye) assessment. In the higher concentrations the test medium was observed as turbid and something yellowish compared to the green colour of the control medium at the end of the test. The turbidity and the yellowish colour of the test medium showed dose dependent relationship.

Applicant's summary and conclusion

Conclusions:

In this 72-h algal growth inhibition test with Crotonic acid, the 72-h EC50 and EC10 values based on growth rate and yield was determined to be higher than 57.5 mg/L. The 72-h NOEC related to growth rate and yield was determined to be 57.5 mg/L and the 72-h LOEC related to growth rate and yield was determined to be higher than 57.5 mg/L.