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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 3 June 2009 and 4 June 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 no 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 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

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
- Date received: 26 May 2009
- Stability under test conditions: Stable
- Storage condition of test material: approximately 4°C in the dark

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Not applicable.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:
Test material Preparation
For the purpose of the test, amounts of test material (5, 16, 50, 160 and 500 mg) were each separately dispersed in approximately 250 ml of water and subjected to ultrasonication for approximately 15 minutes followed by prolonged mixing for 24 hours in order to maximise the dissolved test material concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 ml), activated sewage sludge (200 ml) and water were added to a final volume of 500 ml to give the required concentrations of 10, 32, 100, 320 and 1000 mg/l.
The control group was maintained under identical conditions but not exposed to the test material.

Reference material preparation
For the purpose of the test a reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ) was used. Two stock solutions of 50 and 160 mg/l were prepared by dissolving the reference material directly in water with the aid of ultrasonication for approximately 15 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/l. Similarly, a 100 ml aliquot of the 50 mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference material were inverted several times to ensure homogeneity of the solutions.

-Eluate
Preparation of Test System
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately
0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared according to test material preparation already described. Finally a second control was prepared.


As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 °C.

- 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): No

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.4 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/l prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

140 mg/l as CaCO3.

Test temperature:

Temperature controlled room at 21 ±1 °C.

pH:

7.5 - 8.3.

Dissolved oxygen:

In some instances, the initial dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Salinity:

Not applicable.

Nominal and measured concentrations:

Test material concentrations (nominal): 10, 32, 100, 320 and 1000 mg/l.

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

OTHER TEST CONDITIONS
- Adjustment of pH: No

- Photoperiod: 3 hours.

- Light intensity: Normal laboratory lighting.

EFFECT PARAMETERS MEASURED:
see section on any other information on materials and methods incl. tables.

TEST CONCENTRATIONS

Test Material
Test concentrations of 10, 32, 100, 320 and 1000 mg/l.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 4

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL not stated

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes.

- Relevant effect levels:
The reference material gave a 3-Hour EC50 value of 5.5 mg/l, 95% confidence limits 4.0 - 7.5 mg/l.

- Other: None.

Reported statistics and error estimates:

None.

Any other information on results incl. tables

The following results were derived:

	Reactionmass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane		3,5-dichlorophenol
	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)	ECx(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	64	-	1.4	-
EC50	160	-	5.5	4.0 – 7.5
EC80	>1000	-	22	-
NOEC	54	-	1.1	-

It was not possible to obtain an EC₈₀value for the test material as no concentration tested resulted in greater than 80% inhibition.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

Variation in respiration rates of controls 1 and 2 after 3 hours contact tiwas ± 3%.

The validation criteria for the control respiration rates have been satisfied. The EC₅₀value for the reference material after 3 hours contact time (5.5 mg/l) was within the range of 5 –30 mg/l given in the validation criteria, although the 95% confidence limits for this value ranged from 4.0 – 7.5 mg/l. The lower end of this range was slightly below the range given for the EC₅₀value for the reference material after 3 hours contact time in the validation criteria. This was considered to have had no effect on the validity of the study given that the EC₅₀value for the reference material after 3 hours contact time was within the range stated in the validation criteria.

In soinstances, the initial dissolved oxygen concentrations were below those recomnded in the test guidelines (6.5 mg O₂/l). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that all test concentrations contained clear colourless solutions.

Observations made after 30 minutes and 3 hours contact time showed that all test concentrations contained dark brown dispersions with no undissolved test material visible.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of 160 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 54 mg/l.

Executive summary:

Introduction.A study was perford to assess the effect of the test material on the respiration of activated sewage sludge. Thethod followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of 160 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 54mg/l.

The reference material gave a 3-Hour EC₅₀value of 6.7 mg/l, 95% confidence limits 5.1 – 8.8 mg/l.