Oxirane, 2,2'-[methylenebis[(2,6-dimethyl-4,1-phenylene)oxymethylene]]bis-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2011 to 21 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973

Version / remarks:

Amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentration of the test material in the test solutions was determined at the beginning and at the end of the study, except for the concentration of terminated limit test which was measured at the start of the test only.
- On each occasion four replicate samples were taken from the test solutions and one sample was taken from the control solution.

Vehicle:

no

Details on test solutions:

LIMIT TEST
Because the test material is poorly soluble in water, in order to achieve the maximum solubility level of the test material (limit test concentration), a test solution was prepared using a saturated solution method. A supersaturated test material stock solution (nominal loading rate of 1000 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (ISO Medium) two days before the start of the study (on day -2). This solution was shaken for about 48 hours and then the non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the saturated solution.

FULL TEST
The saturated solution prepared for the limit test was stored for about one day and then used for preparing test solutions for full test. Results of the three days stability test in ISO medium indicated that this saturated stock solution was suitable for further testing. The test solutions were prepared by appropriate dilution of the stock solution:
100% v/v saturated solution: 500 mL stock.
50% v/v saturated solution: 90 mL stock, q.s. ad 180 mL ISO medium
25% v/v saturated solution: 45 mL stock, q.s. ad 180 mL ISO medium
12.5% v/v saturated solution: 22.5 mL stock, q.s. ad 180 mL ISO medium
6.25% v/v saturated solution: 11.3 mL stock, q.s. ad 180 mL ISO medium

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Justification for species: Daphnia magna is the standard species of the acute immobilisation test.
- Source: Supplied by National Institute of Public Health. Cultured under standardised conditions at the testing laboratory.
- Age: Daphnia magna were less than 24 h old at the beginning of the test.
- Feeding during test: no

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.
- Before the test the Daphnia culture was fed with concentrated algal suspension of Pseudokirchneriella subcapitata.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

249 mg/L (as CaCO3)

Test temperature:

20.9 – 21.9 °C

pH:

7.50 – 7.70

Dissolved oxygen:

6.7 – 6.9 mg/L

Nominal and measured concentrations:

Nominal concentrations: 6.25; 12.5; 25; 50 and 100% v/v saturated solutions
Measured geometric mean concentrations: 0.06; 0.12; 0.24; 0.48 and 0.88 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Material, size, headspace, fill volume: at least 4 mL test solution/animal
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
Separate stock solutions of individual trace elements were first prepared in deionised water. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution): 25 mL of 11.76 g/L CaCl2.2H2O, 25 mL of 4.93 g/L MgSO4.7H2O, 25 mL of 0.23 g/L KCl and 25 mL of 2.59 g/L NaHCO3.
- Intervals of water quality measurement: The water temperature was measured at the start of the test and 24-hour intervals thereafter in each test vessel. The dissolved oxygen concentration and pH was measured in each test vessel at the start and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

RANGE-FINDING STUDY
- A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours.
- Test concentrations: 1, 10, 50 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: Yes, because significant immobility was not detected during the preliminary concentration range-finding test, only one test concentration (at the solubility level of the test material in the test medium) and one control group was tested in a limit test. This test was terminated after 24 hours exposure, because 80 per cent immobilisation was observed at treated group. Measured concentration of treated group at the start was 1.01 mg/L. After termination of the limit test a full test was conducted using five different concentrations in a geometric series (factor 2) plus a control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 0.65 – 0.91 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 0.59 – 0.76 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 0.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

VALIDITY
- Not more than 10 per cent of the Daphnia were immobilised in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

IMMOBILISATION
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour and the following were calculated:
- The 24h EC50 value: 0.76 mg/L (measured) (95% confidence limits: 0.65 – 0.91 mg/L)
- The 48h EC50 value: 0.67 mg/L (measured) (95% confidence limits: 0.59 – 0.76 mg/L)
- The 48h EC100 value: > 0.88 mg/L (measured)
- The 48h No-Observed Effect Concentration (NOEC): 0.48 mg/L(measured)
- The 48h Lowest Observed Effect Concentration (LOEC): 0.88 mg/L (measured)

Results with reference substance (positive control):

The 24h EC50: 1.61 mg/L, (95% confidence limits: 1.34 – 1.93 mg/L)

Reported statistics and error estimates:

The EC50 values were calculated by Probit analysis with 95% confidence limits using TOXSTAT software.
For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 1: Number and percentage of immobilised animals

Measured Concentrations (mg/L)	Number of treated animals	Immobilised Animals
		24 Hours		48 Hours
		Number	Percent	Number	Percent
Control	20	0	0	0	0
0.06	20	0	0	0	0
0.12	20	0	0	0	0
0.24	20	0	0	0	0
0.48	20	2	10	2	10
0.88	20	13*	65	17*	85

* statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 h EC50 value for the test material to the aquatic invertebrate, Daphnia magna, was determined to be 0.67 mg/L (95 % confidence limits: 0.59 – 0.76 mg/L).

Executive summary:

The acute toxicity of the test material to the aquatic invertebrate, Daphnia magna, was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2., OPPTS 850.1010 and Japanese guidelines, under GLP conditions.

Based upon the results from the preliminary experiment a limit test was started using only one concentration (100% v/v saturated solution) plus a control. This test was terminated after 24 hours exposure, because 80% immobilisation was observed in the treated group. The measured concentration of the saturated solution at study initiation was 1.01 mg/L. After termination of the limit test a full test was conducted using five concentrations of test material in a geometric series (factor 2) plus a control.

The following nominal concentrations of test material were investigated in the full experiment: 6.25; 12.5; 25; 50 and 100% v/v saturated solutions. The corresponding measured geometric mean test material concentrations were: 0.06; 0.12; 0.24; 0.48 and 0.88 mg/L.

The test design included four replicates at each test group (test material and control). Each group comprised twenty daphnia. The study was conducted for a period of 48 hours under static conditions.

The 24 h and 48 h EC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of this study, the calculated and observed endpoints for the effect of test material (measured concentrations) were: 24 h EC50 value: 0.76 mg/L (95% confidence limits: 0.65 – 0.91 mg/L), 48 h EC50 value: 0.67 mg/L (95% confidence limits: 0.59 – 0.76 mg/L), 48 h EC100 value: > 0.88 mg/L, 48 h No-Observed Effect Concentration (NOEC): 0.48 mg/L and 48 h Lowest Observed Effect Concentration (LOEC): 0.88 mg/L.

Description of key information

Under the conditions of the study, the 48 h EC50 value for the test material to the aquatic invertebrate, Daphnia magna, was determined to be 0.67 mg/L (95% confidence limits: 0.59 – 0.76 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.67 mg/L

Additional information

The acute toxicity of the test material to the aquatic invertebrate, Daphnia magna, was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2., OPPTS 850.1010 and Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Based upon the results from the preliminary experiment a limit test was started using only one concentration (100% v/v saturated solution) plus a control. This test was terminated after 24 hours exposure, because 80% immobilisation was observed in the treated group. The measured concentration of the saturated solution at study initiation was 1.01 mg/L. After termination of the limit test a full test was conducted using five concentrations of test material in a geometric series (factor 2) plus a control.

The following nominal concentrations of test material were investigated in the full experiment: 6.25; 12.5; 25; 50 and 100% v/v saturated solutions. The corresponding measured geometric mean test material concentrations were: 0.06; 0.12; 0.24; 0.48 and 0.88 mg/L.

The test design included four replicates at each test group (test material and control). Each group comprised twenty daphnia. The study was conducted for a period of 48 hours under static conditions.

The 24 h and 48 h EC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of this study, the calculated and observed endpoints for the effect of test material (measured concentrations) were: 24 h EC50 value: 0.76 mg/L (95% confidence limits: 0.65 – 0.91 mg/L), 48 h EC50 value: 0.67 mg/L (95% confidence limits: 0.59 – 0.76 mg/L), 48 h EC100 value: > 0.88 mg/L, 48 h No-Observed Effect Concentration (NOEC): 0.48 mg/L and 48 h Lowest Observed Effect Concentration (LOEC): 0.88 mg/L.