Diallyl 2,2'-oxydiethyl dicarbonate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2010 - February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Harlan Laboratories Ltd., UK

Analytical monitoring:

not required

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: the control group was maintained under identical conditions (see above "Method") but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable [tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener)]
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): see above "Method"
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed no evidence of undissolved material

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: no
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. Synthetic sewage served as respiratory substrate.
The composition of synthetic sewage:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCI
0.4 g CaCI2.2H20
0.2 g MgS04.7H20
2.8 g K2HP04
dissolved in 1 litre of water with the aid of ultrasonication.
- Preparation of inoculum for exposure: 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 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Pretreatment: see above "Preparation of inoculum for exposure"
- Initial biomass concentration: The suspended solids concentration was equal to 3.8 g/l prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

no

Hardness:

140 mg/l as CaC03

Test temperature:

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

pH:

The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time (please see Table 2 in "Remarks on results including tables and figures")

Dissolved oxygen:

The rate of respiration was measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe (see Table 1 in " Remarks on results including tables and figures"). The contents of the measuring vessel were stirred constantly by magnetic stirrer.

Salinity:

no data

Nominal and measured concentrations:

not applicable

Details on test conditions:

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 and second control). As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel.
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 250 ml darkened glass Biological Oxygen Demand (BOD) bottle is the measuring vessel
- Aeration: 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. The contents of the measuring vessel were stirred constantly by magnetic stirrer.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per reference material: 3
- Biomasse loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the test 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 CaC03. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 1.018 mg/l
- Particulate matter: data not available
- Metals: data not available
- Pesticides: 0.001 µg/l
- Chlorine: 0.307 mg/l
- Alkalinity: data not available
- Ca/mg ratio: data not available
- Conductivity: 386.2 µS/cm at 20°C
- Culture medium different from test medium: no
- Intervals of water quality measurement: 2008-01-01 - 2008-12-31

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not applicable
- Light intensity: normal laboratory lightening

EFFECT PARAMETERS MEASURED:
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/I and 2.5 mg O2/I). 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 ( the concentartions used 10, 32, 100, 320 and 1000 mg/l).
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 100-160 mg/L (95% CL)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations made at 0 hours, 30 minutes and 3 hours (see Table 3 in "Any other information on results incl. tables") showed that the control vessels contained a dark brown dispersion and the reference item vessels contained a dark brown dispersion with no undissolved reference item visible.
Observations of the test item vessels made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10, 32, 100 and 320 mg/l contained a clear colourless solution. The test concentration of 1000 mg/l contained a cloudy dispersion with globules of test item visible on the surface.
Observations of the test item vessels made after 30 minutes and 3 hours contact time showed that the test concentrations of 10, 32, 100 and 320 mg/l contained a dark brown dispersion with no undissolved test item visible and the test concentration of 1000 mg/l contained a dark brown dispersion with globules of test item visible on the surface.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes (see "Remarks on results including tables and figures" Table 3)
- Relevant effect levels: 3.2, 10 and 32 mg/l.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O₂/l and 2.5 mg O₂/l respectively) (Table 1). 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.

Table 1 - Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	5.7	6	2.2	0.58	-
	R2	5.8	6	2.5	0.55	-
Test Material	10	6.0	6	2.4	0.60	[6]
	32	5.7	5	2.8	0.58	[3]
	100	6.8	10	3.8	0.30	47
	320	7.7	10	6.4	0.13	77
	1000	7.8	10	6.7	0.11	81
3,5-dichlorophenol	3.2	7.1	10	2.5	0.46	19
	10	7.5	10	5.1	0.24	58
	32	8.4	10	7.4	0.10	82

[Increase in respiration rate as compared to controls]

R1, R2 replicates 1 to 2

Table 2 - pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.3	7.8
	R2	7.6	7.9
Test Material	10	7.5	7.8
	32	7.5	7.8
	100	7.5	7.9
	320	7.5	7.9
	1000	7.5	7.8
3,5-dichlorophenol	3.2	7.4	8.0
	10	7.4	8.1
	32	7.5	8.2

Table 3: Overall summary of EC values and NOEC

	Test Material (CAS 142 -22 -3)		3,5-dichlorophenol
	ECX(3 Hours) (mg/l)	95% Confidence Limits (mg/l)	ECX(3 Hours) (mg/l)	95% Confidence Limits (mg/l)
EC20	56	-	3.0	-
EC5o	130	100 -160	9.0	7.1-11
EC8o	390	-	27	-
NOEC	47	-	2.5	-

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

The validation criteria for the control respiration rates and reference item EC₅₀ values were therefore satisfied.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge gave a 3 Hour EC50 of 130 mg/l, 95% confidence limits 100 - 160 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 47 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method 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.

Activated sewage sludge was exposed to an aqueous dispersion 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.

The reference material gave a 3-Hour EC50 value of 9.0 mg/l, 95% confidence limits 7.1 -11 mg/l.

The effect of the test item on the respiration of activated sewage sludge gave a 3 Hour EC50 of 130 mg/l, 95% confidence limits 100 - 160 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 47 mg/l.

Description of key information

- EC50 (3h) = 130 mg/L for microorganisms (OECD 209)
- NOEC (3h) = 47 mg/L for microorganisms (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:

130 mg/L

EC10 or NOEC for microorganisms:

47 mg/L

Additional information

The toxicity of 'diallyl 2,2'-oxydiethyl dicarbonate' to microorganisms has been tested under GLP conditions according to OECD 209 activated sludge respiration inhibition test (Youngs, 2010). The effect of the test item on the respiration of activated sewage sludge gave a 3 hour EC50 of 130 mg/L, 95% confidence limits 100 - 160 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 47 mg/L.