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 aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 30 June 2009 and 4 September 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 affect 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of Signature: 04/03/2009

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:
Range-finding Test: 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l.

Definitive Test: 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

- Sampling method:
Water samples were taken from the control (replicates R1 – R2 pooled) and each test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Samples used immediately.Duplicate samples were taken and stored at 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 material was dissolved directly in reconstituted water to give nominal concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l.
An amount of test material (100 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 20 minutes to give the 100 mg/l test concentration. Serial dilutions were prepared in reconstituted water from the 100 mg/l test concentration to give the remainder of the test series of 10, 1.0, 0.10, 0.010 and 0.0010 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

An amount of test material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (200 ml) of this stock solution was diluted in a final volume of 2 litres of reconstituted water to give the 10 mg/l test concentration. Aliquots (5.0, 9.0, 16, 28, 50, 90, 160 and 280 ml) of the 10 mg/l test concentration were each separately diluted in a final volume of 500 ml of reconstituted water to give the 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l test concentrations respectively.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.

- Controls:
A positive control conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure conditions for the positive control were similar to those used in the definitive test.

- Chemical name of vehicle : Not applicable

- Concentration of vehicle in test medium: Not applicable

- Evidence of undissolved material :
The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea

- Source: Derived from in-house laboratory cultures.

- Age at study initiation: Less than 24 hours old.

- Feeding during test: Received no food during exposure

ACCLIMATION
- Acclimation period: Not stated

-Test Water: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

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:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 21 °C to 22 °C throughout the test. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.

Salinity:

freshwater used.

Nominal and measured concentrations:

Range-finding test: 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l.
Definitive test: 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass jars

- Type (delete if not applicable): closed

- Material, size, headspace, fill volume: 250 ml glasses containing 200 ml solution. The test vessels were then sealed, with minimal headspace to prevent losses of the test material due to its volatile nature,

- Aeration: None.

- Type of flow-through (e.g. peristaltic or proportional diluter): None.

- Renewal rate of test solution (frequency/flow rate): None.

- No. of organisms per vessel: 10 daphnids

- No. of vessels per concentration (replicates): Duplicate (2)

- No. of vessels per control (replicates): Duplicate (2)
- No. of vessels per vehicle control (replicates): Duplicate (2)

- Biomass loading rate: Not recorded.


- Source/preparation of dilution water:

i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.



- Total organic carbon: Not recorded

- Particulate matter: Not recorded

- Metals: Not recorded

- Pesticides: Not recorded

- Chlorine: Not recorded

- Alkalinity: Not recorded

- Ca/mg ratio: Not recorded

- Conductivity: Not recorded

- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Intervals of water quality measurement: Not recorded

OTHER TEST CONDITIONS

- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Photoperiod: A photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity: Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.

- Justification for using less concentrations than requested by guideline: Not applicable

- Range finding study
Test concentrations: 0.0010, 0.010, 0.10, 1.0, 10 & 100 mg/l.

- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 0.96 - 1.5 mg/l.

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL Not stated

Details on results:

- Behavioural abnormalities: Not recorded
- Other biological observations: None recorded

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- Mortality: No mortalities recorded.

- EC50/LC50: not stated

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material during the positive control are given in Table 3.

Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EL50 (mg/l) 95% Confidence limits (mg/l)
3 >3.2 -
24 1.0 0.90 - 1.2
48 0.78 0.68 - 0.88

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 7.8 (SE = 1.7) and 12 (SE = 2.4) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.20).

- Other: None.

Reported statistics and error estimates:

An estimate of the EC50 value at 3 hours was given by inspection of the immobilisation data.
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curves and standard errors were calculated by the maximum-likelihood probit nethod (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.

Any other information on results incl. tables

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1.

No immobilisation was observed at the test concentrations of 0.0010, 0.010 and 0.10 mg/l. However, immobilisation was observed at 1.0, 10 and 100 mg/l.

Based on this information test concentrations of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l were selected for the definitive test.

DefinitiveTest

 

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.

Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50(mg/l)	95% Confidence limits (mg/l)
24	2.1	1.7 - 2.6
48	1.2	0.96 - 1.5

The No Observed Effect Concentrations after 24 and 48 hours exposure were 0.56 and 0.18 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 3.3 (SE = 0.46) and 2.9 (SE = 0.37) respectively. 

*EL = Effective Loading Rate

Physico-chemical Measurements

Temperature was maintained at 21°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The majority of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

Verification of Test Concentrations

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 94% to 117% of nominal valuewith the exception of the 0.10 mg/l test concentration at 0 hours which showed a measured concentration of 129% of nominal.  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.  Given thisthe results are based on nominal test concentrations only.

Table 1.  Cumulative Immobilisation Data in the Range-findingTest

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.0010	0	0
0.010	0	0
0.10	0	0
1.0	2	3
10	10	10
100	10	10

Table 2. Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.10	0	0	0	0	0	0	0	0
0.18	0	0	0	0	0	0	0	0
0.32	0	0	0	0	2	0	2	10
0.56	0	0	0	0	2	2	4	20
1.0	2	5	7	35	2	6	8	40
1.8	3	2	5	25	4	6	10	50
3.2	6	6	12	60	10	9	19	95
5.6	10	10	20	100	10	10	20	100
10	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Table 3 Cummulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	0	0	0	0	1	0	1	5
1.0	0	0	0	0	7	3	10	50	10	8	18	90
1.8	0	0	0	0	10	9	19	95	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Validation

A range of standard solutions covering 0 to 20 mg/l (exceeding the range of the working sample concentrations) was analysed.

Linearity was confird (R²=0.9998) in the range 0 to 20 mg/l.

The results are presented graphically in Attachment 3, Appendix 2 (page 26).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 1.2 mg/l with 95% confidence limits of 0.96 - 1.5 mg/l. The No Observed Effect Concentration at 48 hours was 0.18 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material toDaphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods. Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l for 48 hours at a temperature of approximately 21°C to 22°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of 21°C to 22°C understatic test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results.The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was 1.2 mg/l with 95% confidence limits of 0.96 - 1.5 mg/l. The No Observed Effect Concentration was 0.18 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 94% to 117% of nominal value with the exception of the 0.10 mg/l test concentration at 0 hours which showed a measured concentration of 129% of nominal.  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.  Given this the results are based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.68 – 0.88 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Conclusion. The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 1.2 mg/l with 95% confidence limits of 0.96 - 1.5 mg/l. The No Observed Effect Concentration at 48 hours was 0.18 mg/l.