Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 5 July 2009 and 30 October 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of signature: 04/03/2009 Date of Inspection: 19/08/2008

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Constituent 2

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Organic, Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80-95%
- Lot/batch No.: 200901317-17
- Stability under test conditions: Stable
- Storage condition of test material: approximately 4°C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Definitive Test
0.56, 1.0, 1.8, 3.2 and 5.6 mg/l.

- Sampling method:
Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken.

- Sample storage conditions before analysis:
Storage conditions: approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:

Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/l. The test material was dissolved directly in dechlorinated tap water with the aid of prolonged stirring.
An amount of test material (2250 mg) was dissolved in 22.5 litres of dechlorinated tap water with the aid of prolonged stirring, using a propeller stirrer at approximately 1500 rpm at approximately 14°C for 24 hours, to give the 100 mg/l test concentration. Aliquots (200 ml and 2 litres) of the 100 mg/l test concentration were each separately diluted in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 1.0 and 10 mg/l test concentrations respectively.

Definitive Test
An amount of test material (1100 mg) was dissolved in 11 litres of dechlorinated tap water with the aid of prolonged stirring, using a propeller stirrer at approximately 1500 rpm at approximately 14°C for 24 hours, to give a 100 mg/l stock solution. Aliquots (112, 200, 360, 640 and 1120 ml) of the 100 mg/l stock solution were each separately diluted in a final volume of 20 litres of dechlorinated tap water, and stirred using a flat bladed mixer for approximately 1 minute, to give the 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l test concentrations respectively.

- Eluate: Not applicable

- Differential loading: Not applicable

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
The test preparations were observed to be clear, colourless solutions thoughout the duration of the test.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout

- Strain: Not stated

- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK

- Age at study initiation (mean and range, SD): Juvenile.

- Length at study initiation (length definition, mean, range and SD): mean standard length of 5.1 cm (sd = 0.7)

- Weight at study initiation (mean and range, SD): mean weight of 1.80 g (sd = 0.62) at the end of the definitive test

- Method of breeding: Not stated

- Feeding during test: Not applicable
- Food type:
The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Amount: Not stated

- Frequency: Not stated


ACCLIMATION
- Acclimation period: 14 October 2009 to 26 October 2009

- Acclimation conditions (same as test or not): Same as test conditions.

- Type and amount of food:
commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test

- Feeding frequency: Not stated

- Health during acclimation (any mortality observed): Zero mortality in the 7 days prior to the start of the test.


QUARANTINE (wild caught)
- Duration:
Not applicable

- Health/mortality:
Not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Any mortalities and sub-lethal effects of exposure were recorded at 4, 6, 24, 48, 72 and 96 hours after the start of exposure. The first observations were conducted after 4 hours exposure rather than 3 hours as stated in the protocol. This was considered not to have affected the test given that no effects were observed until after 24 hours exposure therefore none would be expected after 3 hours exposure.

Test conditions

Hardness:

Approximately 140 mg/l as CaCO3.

Test temperature:

Approximately 14ºC

pH:

The pH was measured using a WTW pH/Oxi 340I pH.
pH range of 7.4-7.8.

Dissolved oxygen:

The dissolved oxygen concentration was measured using a dissolved oxygen meter.

Salinity:

No Data

Nominal and measured concentrations:

Range-finding test (nominal): 1.0, 10 and 100 mg/l.
Definitive test (nominal): 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range-finding test 20 litre completely filled and sealed glass round-bottomed vessels were used for each test concentration.The test vessels received no auxiliary aeration.

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Daily

- No. of organisms per vessel: 7
-No. of vessels per concentration (replicates): 1

- No. of vessels per control (replicates): 1

- No. of vessels per vehicle control (replicates): Not applicable

- Biomass loading rate: Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener).

-Total Organic Carbon Analysis (average): 1.041 mg/l

- Particulate matter: Not measured

- Metals: Total not Stated
- Pesticides: Total value for pesticides not Stated
- Total Chlorine (average): 0.297 mg/l
- Alkalinity: Not Stated
- Ca/mg ratio: Not Stated
- Conductivity (average): 421.385 µS/cm at 20°C
- Culture medium different from test medium: Not applicable
- Intervals of water quality measurement: Not Stated


OTHER TEST CONDITIONS
The test vessels were then sealed and maintained at approximately 14ºC in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: In the range-finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study:Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits of 1.8 - 3.2 mg/l

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limit not stated

Details on results:

- Behavioural abnormalities: None recorded.

- Observations on body length and weight: None recorded

- Other biological observations: None recorded

- Mortality of control: None recorded

- Other adverse effects control: None recorded

- Abnormal responses:
Sub-lethal effects of exposure were observed at the 1.8 mg/l and above. These responses were increased pigmentation, swimming at the surface, gaping, loss of equilibrium and the presence of moribund fish (see Table 4).

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
The test preparations were observed to be clear, colourless solutions thoughout the duration of the test.

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Not applicable

Any other information on results incl. tables

Sublethal observations / clinical signs:

Range-finding Test

The results showed no mortalities at the test concentration of 1.0 mg/l. However, mortalities were observed at 10 and 100 mg/l

Based on this information test concentrations of 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l were selected for the definitive test.

Definitive Test

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be 1.8 mg/l, the lowest test concentration resulting in 100% mortality to be 3.2 mg/l and the No Observed Effect Concentration (NOEC) to be 1.0 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration (see table below).

Sub-lethal Effects

Sub-lethal effects of exposure were observed at test concentrations of 1.8 mg/l and above. These responses were increased pigmentation, swimming at the surface, gaping, loss of equilibrium and the presence of moribund fish.

After approximately 30 hours exposure five out of seven fish at 5.6 mg/l were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

After approximately 53 and 71 hours exposure three out of seven fish and two out of four fish respectively at 3.2 mg/l were observed to be moribund. Due to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the following observational time point.

Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration (mg/l)	Sub-lethal Effects	Time (Hours)
		4	6	24	48	72	96
Control	No abnormalities detected
0.56	No abnormalities detected
1.0	No abnormalities detected
1.8	Increased pigmentation Swimming at surface Gaping Loss of equilibrium				6/7	7/7 3/7 1/7	7/7   3/7 3/7
3.2	Increased pigmentation and swimming at the surface Increased pigmentation Swimming at the surface Moribund			3/7	7/7 5/7 **	A/D
5.6	Increased pigmentation Swimming at the surface Moribund			7/7 5/7 [1]	A/D

[1]After approximately 30 hours exposure five out of seven fish were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the 48-Hour time point.

** After approximately 53 and 71 hours exposure three out of seven fish and two out of four fish respectively were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the 72-Hour time point.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC50 value of 2.4 mg/l with 95% confidence limits of 1.8 - 3.2 mg/l. The No Observed Effect Concentration was 1.0 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods. Following a preliminary range-finding test, fish were exposed, in groups of seven, to an aqueous solution of the test material over a range of concentrations of 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l for a period of 96 hours at a temperature of approximately 14°C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 4 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results.The 96-Hour LC₅₀ based on nominal test concentrations was 2.4 mg/l with 95% confidence limits of 1.8 ‑ 3.2 mg/l. The No Observed Effect Concentration was 1.0 mg/l.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 87% to 105% of nominal with the exception of the 0.56 mg/l test concentration at 24 hours which showed a measured concentration of 204% of nominal. Analysis of the corresponding frozen duplicate sample showed a measured concentration of 189% of nominal. Given that the analysis of the 0.56 mg/l test concentration at 0 hours showed a measured concentration of 104% of nominal it was considered that a sampling error had occurred at 24 hours. This was considered not to affect the study as this concentration was below the NOEC and all other measured concentrations were within the 80% to 120% acceptance limits. It was therefore considered justifiable to base the results on nominal test concentrations only.

N.B.Under the test conditions employed for the ecotox studies, the test item was shown to be sufficiently stable over the period of media renewal to allow the fish test to be conducted using semi-static renewal i.e. fresh test media prepared each 24 hours.  It should be noted that the hydrolysis study indicated that the test item was more stable at higher pH values, in line with the pH of the test medium used in the ecotox studies and this, along with the different diluents used, will explain the apparent ‘conflict’ with the hydrolysis study.  It should also be noted that the test item is a mixture of components and hence toxicity cannot be attributed to any one, or more, of the components present but to the test item as a whole.  Therefore whilst the peak measured in the ecotox studies may not reflect that of the main component it does give a ‘marker’ for confirming correct preparation of the test solutions and the use of nominal test concentrations to express the results of the test is appropriate.

Conclusion. The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and gave a 96-Hour LC₅₀ value of 2.4 mg/l with 95% confidence limits of 1.8 - 3.2 mg/l. The No Observed Effect Concentration was 1.0 mg/l.