2,2'-[ethylenebis(oxymethylene)]bisoxirane

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted: 23 March 2006, Annex 5 corrected: 28 July 2011

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The required amount of the test substance was weighed and mixed with the culture medium. It was used as stock solution for the dilution of each test solution. For the range finding study, a stock solution of 100 mg/L was prepared and the test solutions of 0.1, 1, 10 and 20 mg/L were prepared by mixing the stock solution with the culture medium. For the definitive study, a stock solution of 100 mg/L was prepared and the test solutions of 30.1, 40.6, 54.9 and 74.1 mg/L were prepared by mixing the stock solution with the culture medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): American Type Culture Collection (ATCC, U.S.A.)
- Method of cultivation:
- Sub-culture: The cells were thawed and inoculated in the culture medium under aseptic conditions. Then, the cells were incubated at a temperature of 23±2°C under continuous illumination (intensity 4,440–8,880 Lux) and constantly shaken for 7 days. After incubation, a biomass (the number of cells per volume) was counted and the suspension was diluted with the culture medium to produce a biomass of 10000 cells/mL. Cells were sub-cultured continuously under the same conditions at intervals of 3 days. Exponentially growing cells, which were sub-cultured for 3 passages, were observed for atypical cell morphological abnormalities such as flocculation, discoloration, swelling, atrophy and rupture, and cells without abnormalities were inoculated in agar medium for storage. They were then incubated at 23±2°C under continuous illumination (intensity 4,440–8,880 Lux). These stock culture cells were maintained under refrigeration (2–8°C) until use.
- Pre-culture: A colony of algal cells in agar medium for long-term storage was collected and inoculated in culture medium 4 days prior to the treatment of the test substance. Biomass was counted using a particle counter and/or hemocytometer and diluted with the culture medium to produce a biomass of 5000 cells/mL. Counted cells were incubated by shaking at 23±2°C under continuous illumination (intensity 4,440–8,880 Lux).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

range finding study: 23.0 - 23.8 (measured)
main study: 22.5 - 23.8 (measured)

pH:

range finding study: control: 7.4 - 7.5, treatment: 7.3 - 8.1 (measured)
main study: control: 7.4 - 7.6, treatment: 7.4 - 7.6 (measured)

Nominal and measured concentrations:

Range-finding study
Nominal: 0, 0.1, 1, 10, 20, 100 mg/l
Main study
Nominal: 0, 30.1, 40.6, 54.9, 74.1, 100 mg/l
Measured (corrected for recovery) at 0 h: N.D., 28.78, 38.02, 52.64, 71.42, 96.69 mg/l
Measured (corrected for recovery) at 72 h: N.D., 30.61, 41.75, 57.13, 76.28, 103.7 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: glass erlenmeyer flask (300 mL)
- Initial cells density: 10000 cells / mL
- Control end cells density: 1930000 cells / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 5040 - 5460 lux (measured)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: particle counter (Vi-Cell XR, Beckman Coulter, USA)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.35
- Results used to determine the conditions for the definitive study: results range-finding study

Reference substance (positive control):

yes

Remarks:

but not concurrent

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

40.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no flocculation, decoloration, swelling, atrophy or rupture observed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 1.39 mg/l (within the permissible range of historical data (mean±2SD: 0.84–1.48 mg/L) of Biotoxtech Co., Ltd)

Validity criteria fulfilled:

yes

Remarks:

The biomass of the control was increased by 193 x. The mean COV for section-by-section specific growth rates in the control was 19.8%. At the end of exposure, the COV of average specific growth rates in the replicate control group was 0.53%

Executive summary:

This study was performed to assess the effect of the test substance, 2,2’-[ethylenebis (oxymethylene)]bisoxirane, on the growth of the fresh water alga, Pseudokirchneriella subcapitata, when exposed to the test substance for 72 h by the shaking culture method and to determine the median effect concentration (EC50) from the average specific growth rate of each treatment group. Based on the result of the range finding study, the selected treatment levels of the main study were 30.1, 40.6, 54.9, 74.1 and 100 mg/L and a control group.

The biomass in the control group was increased by 16 times or more at 72 h after exposure compared to the biomass at the start of exposure, and each coefficient of variation was confirmed to be acceptable in this study.

As a result of the concentration analysis of the test substance, the measured values in the 30.1, 40.6, 54.9, 74.1 and 100 mg/L treatment groups during the exposure period were from 80 to 120% when compared to the nominal concentrations. Therefore, all test results were determined based on the nominal concentrations.

Based on the result of this study, the ErC50 value obtained under the test conditions with the test substance, 2,2’-[ethylenebis(oxymethylene)]bisoxirane, was determined to be >100 mg/L and the EyC50 value was determined to be 86.1 mg/L (95% confidence limits: 82.7–90.2 mg/L) at 72 h after exposure.

The No Observed Effect Concentration (NOEC) for the results of the average specific growth rate and the yield was determined to be 40.6 mg/L at 72 h after exposure. The Lowest Observed Effect Concentration (LOEC) for the results of the average specific growth rate and the yield was determined to be 54.9 mg/L at 72 h after exposure.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

40.6 mg/L