2,2-bis({[3-(4-ethylpiperazin-1-yl)propanoyl]oxy}methyl)butyl 3-(4-ethylpiperazin-1-yl)propanoate

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:

2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The difference of the pH in the controls was 1.8 units and therefore higher than stated in the guideline. Because the controls showed a normal exponential growth, this can be stated as uncritical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The difference of the pH in the controls was 1.8 units and therefore higher than stated in the guideline. Because the controls showed a normal exponential growth, this can be stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis({[3-(4-ethylpiperazin-1-yl)propanoyl]oxy}methyl)butyl 3-(4-ethylpiperazin-1-yl)propanoate

EC Number:

700-404-3

Cas Number:

127929-37-7

Molecular formula:

C33H62N6O6

IUPAC Name:

2,2-bis({[3-(4-ethylpiperazin-1-yl)propanoyl]oxy}methyl)butyl 3-(4-ethylpiperazin-1-yl)propanoate

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Details on test solutions:

As solubility surpasses 100 mg/L, a stock solution containing 125 mg/L in deionised water was prepared. The solutions of the amounts needed were prepared by dilution of this stock solution with deionised water. As the test item isn't stable in aqueous solutions, the stock solution was prepared directly before usage.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellar freshwater green alga.
Genus, Species: Desmodesmus subspicatus
Strain: CHODAT
Family: Chlorophyceae
Order: Chlorococcales
Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
The alga are kept as stock culture on solid agar at 8°C. From an aliquot of this stock culture the pre-culture was prepared.

Study design

Test type:

static

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

At the end of the test, the treatments were observed microscopially in order to identify changes in the appearance of the alga (e.g. caused by the exposure of the test item).

Test conditions

Test temperature:

21 - 24 °C

Details on test conditions:

Experimental conditions:
Lighting: 4440 - 8880 Lux
Control: deionised water with nutrient medium and alga
Treatments: test solution with nutrient medium and alga

Main study:
Test solutions: A stock solution containing 125 mg/L test item in deionised water was prepared. The concentrations to be tested were
prepared by diluting of this stock solution with deionised water.
Study Date: 13. - 16. April 2010
Vessels: glass flasks, nominal volume 50 mL (main study)
Treatments tested: 100 / 46 / 22 / 10 / 4.6 / 2.2 / 1.0 / 0.46 mg/L
Number of replicates: six replicates for the control
three replicates for each treatment

Reference study:
Test solutions: A stock solution containing 250 mg/L positive control in deionised water was prepared.
Study Date: 23. - 26. Februar 2010
Vessels: round cuvettes d = 10 mm (reference study)
Treatments tested: 0.04 / 0.063 / 0.1 / 0.16 / 0.25 / 0.4 / 0.63 / 1.0 / 1.6 mg/L
Number of replicates: six replicates for the control
three replicates for each treatment

Reference substance (positive control):

yes

Remarks:

potassium dicromat K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 72 h: 1.8 mg/L
- NOEC 72 h: 0.04 mg/L
- LOEC 72 h: 0.063 mg/L

Any other information on results incl. tables

The following results for the test item B 1061 were determined:

Endpoint               NOEC              LOEC               EC50

Growth Rate        0.55 mg/L        0.67 mg/L        > 5.1 mg/L

AUC                   0.55 mg/L        0.67 mg/L       5.0 mg/L

Yield               0.55 mg/L        0.67 mg/L        2.7 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria except for the difference in pH values at the start and at the end of the experiment were met. For the estimation of the EC50s of the test and reference item, the fits showed sufficient statistical correspondence of the data with the

Executive summary:

The study was performed in order to evaluate the toxic potential of B 1061 towards Desmodesmus subspicatus CHODAT, an unicellar freshwater green alga, which belongs to the order of chlorococcales. The incubation period of 72 hours corresponds to a subchronic test, as during this period, about five cell divisions take place.

One valid main study was performed.

The study was performed using eight concentrations ranging from 0.46 to 100 mg/L. The treatments were used to incubate the unicellar freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate, the area under the growth curve (AUC) and the Yield were determined.

As the test item isn't stable in aqueous solutions, the content of test item was determined using APCI-MS (Atmospheric Pressure Chemical Ionisation-Mass Spectrometry) after 0, 24 and 48 hours.

The determination of the results was based on the time-weighted mean of the measured concentrations.

The EC50s of potassium dichromate was tested in a current reference test. The values were within the normal range of the laboratory.

The following results for the test item B 1061 were determined:

Endpoint                     NOEC                   LOEC                     EC50

Growth Rate              0.55 mg/L              0.67 mg/L              > 5.1 mg/L

AUC                         0.55 mg/L              0.67 mg/L               5.0 mg/L

Yield                      0.55 mg/L              0.67 mg/L               2.7 mg/L