(2E,2'E)-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(2-methylpropane-2,1-diyl) bis(2-cyano-3-(3,4-dimethoxyphenyl)acrylate)

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2E,2'E)-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(2-methylpropane-2,1-diyl) bis(2-cyano-3-(3,4-dimethoxyphenyl)acrylate)

Cas Number:

2233585-18-5

Molecular formula:

C39H46N2O12

IUPAC Name:

(2E,2'E)-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diylbis(2-methylpropane-2,1-diyl) bis(2-cyano-3-(3,4-dimethoxyphenyl)acrylate)

Test material form:

solid

Remarks:

Light yellow solid (powder)

Test organisms

Test organisms (species):

other: Pseudokirchneriella subcapitata

Details on test organisms:

The unicellular green alga species Pseudokirchneriella subcapitata used is part of the Chloroficeae group, Chlorococcales order.

Study design

Test type:

static

Water media type:

other: deionized water

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0 to 22.1oC

Details on test conditions:

A range-finding test was carried out using the following concentrations of test item: control; 0.1; 1; 10 and 100 mg.L-1 of test Item, with two replicates per concentration. The algae inoculum was transferred aseptically to each test flask, with calculated volume to yield an initial cell concentration of 5 x 103 cells.mL-1.

After 72 hours, samples of each test flask were taken in order to count the cells and to determine the concentrations, using a Neubauer chamber and a microscope, that caused algal growth reduction.

Based on the results obtained in the range-finding test, the definitive test was carried out with the following concentrations of test item: control; 1; 3.2; 10; 32 and 100 mg.L-1. The test was performed with three replicates per each concentration and control. One more replicate per concentration was prepared for determinations of pH and chemical analysis. The algae inoculum was transferred aseptically to each test flask, with calculated volume to yield an initial cell concentration of 5 x 103 cells.mL-1.

All test flasks were randomly positioned in an incubating chamber with controlled temperature and kept under continuous shaking and lighting conditions. Every day the flasks were relocated to different places inside the incubating chamber.

Tables 3 and 4 show the stock solution and medium volumes used to obtain the concentrations of the range-finding and definitive tests.

The definitive test was carried out under the conditions described below:

Temperature: 22.0 to 22.1oC
Light: 5987 lux (average)
Photoperiod: continuous light
Shaker velocity: 130.8 rpm (average)
Test duration: 72 hours

Temperature was measured daily using a digital thermometer. The pH was measured at the beginning of the test and at 72 hours (Table 8).

One aliquot was taken from each test flask in order to determine the algal growth over the period of 24, 48 and 72 hours after the start of the test. A Neubauer chamber and a microscope were used to determine the cell number (cells.mL-1).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 13.68 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 3.2 mg/L

Results with reference substance (positive control):

The 72-hours EyC50 value of the reference item potassium dichromate was 0.66 mg.L-1 (confidence limits at 95% = 0.60 to 0.72 mg.L-1), demonstrating that the toxic sensitivity of the algae was within the accepted range (ISO, 1989), which report toxicity ranged between 0.60 to 1.03 mg.L-1.

Any other information on results incl. tables

In order to check the validity of the test, the biomass in the control cultures should increase exponentially by a factor of at least 16 within 72 hours. The mean coefficient of variation (CV) for section-by-section specific growth rates in the control cultures must not exceed 35% and the CV of average specific growth rate during the entire test period in replicate control cultures must not exceed 7%. In fact, in the present study, the biomass in the control cultures increased 901 times at end of the test (Table 8). The mean coefficient of variation (CV) of the growth rate in the control group was 34.5% (Table 9) and the CV of average specific growth rate during the entire test period in replicate control cultures was 0.1% (Table 10), showing therefore, the validity of this study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72-hours EyC50 value for alga Pseudokirchneriella subcapitata exposed to Chiguard GA403 was 13.68 mg.L-1 and the 72-hours ErC50 value was not determined, because the higher tested concentration of the 100 mg.L-1 only caused 22% of the inhibition of growth rate. The highest test item concentration with no observed significant adverse effect (NOEC) on algal growth was 1 mg.L-1 and the lowest test item concentration at which was observed significant reducing effect on growth (LOEC) was 3.2 mg.L-1.