Ethylene dinitrate

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:

2010-07-19 to 2010-08-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene dinitrate

EC Number:

211-063-0

EC Name:

Ethylene dinitrate

Cas Number:

628-96-6

Molecular formula:

C2H4N2O6

IUPAC Name:

ethane-1,2-diyl dinitrate

Specific details on test material used for the study:

Test substance was produced on site in a micro-reactor and used on the same day it was produced. Purity was reported as >99% using HPLC; batch number was EGDN-LN-20100727. Stability of the test solutions was checked at the start and end of the exposure period (72 h), and measured concentrations were within 20% of nominal concentrations for most treatments and final concentrations were within 10% of initial concentrations for all treatments.

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

The test substance is volatile; therefore, closed test vessels were used. No other physical/chemical properties were expected to be critical for study design. Measured concentrations were close to nominal concentrations indicating stability of EGDN in the test medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test species was Desmodesmus subspicatus CHODAT. Culture organisms were obtained in June 2010 from MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The stock culture was kept on solid agar at 8°C. Four days prior to the test, an aliquot of stock culture was put into pre-culture medium and incubated for 72 h. After this incubation period, the pre-culture was checked to confirm that it was in exponential growth phase. Its cell concentration was adjusted to 60,000 cells/mL and then mixed with an equal volume of 10-fold nutrient solution for use in the test. To prepare each control and test solution, 200 mL of this test culture was added to a total volume of 1000 mL. All media were sterilized prior to use. Media were prepared as specified in the OECD 201 test method.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 - 26°C

Nominal and measured concentrations:

Biomass was measured as number of cells per mL of test solution (i.e., cell number). Cell number was determined indirectly from measuring light absorbance at 440 nm (absorbance was converted to cell number using an equation). Absorbance measurements were made at 0, 24, 48, and 72 h.
At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The recovery after 72 hours was in the range of 98 and 106% of the start concentration, the correlation between nominal and measured was very good. Therefore, the determination of the biological results was based on the nominal concentration.
The nominal concentrations: 1.0, 3.2, 10, 32, 100 mg/L

Details on test conditions:

Static system without renewal.
Acute test.
Negative control with 6 replicates was run concurrently.
Three replicates per test concentration.
Exposure period was 72 h.
Exposure pathway was aqueous.
Five concentrations arranged in a geometric series with a dilution factor of 3.1 to 3.2 were tested:
Nominal concentrations: 1.0, 3.2, 10, 32, 100 mg/L
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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 17 - < 39 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf intervals

Details on results:

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 µ, the area under the growth rate (AUC) and the Yield were determined.

Results with reference substance (positive control):

The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory and were thus considered valid.

Reported statistics and error estimates:

Nominal concentrations were used to derive the toxicity estimates. EC50s were determined graphically (linear fit) by plotting percent inhibition on a probit scale against concentration on a logarithmic scale. The software Origin™ was used for these analyses.
NOECs and LOECs were derived using either t-test (if equality of variance was verified) or the WEIR test. In these tests, the three highest concentrations were compared to the control (i.e., 10 mg/L vs control; 32 mg/L vs control; 100 mg/L vs control).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Due to the volatility of the test item, the test was performed in a closed system. The study was performed using five concentrations ranging from 1.0 to 100 mg/L. The treatments were used to incubate the unicellular freshwater green algae Desmodesmus subspicatus for a period of 72 hours. The determination of the biological results was based on the nominal concentration (analytical verification). After 72 hours a NOErC = 10 mg/L, a LOErC = 32 mg/L and a ErC50 > 100 mg/L were determined for the test item ethylene dinitrate.