2-methylenepropane-1,3-diyl diacetate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 04 February 2019. Experimental completion date:
08 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methylenepropane-1,3-diyl diacetate

EC Number:

223-225-8

EC Name:

2-methylenepropane-1,3-diyl diacetate

Cas Number:

3775-29-9

Molecular formula:

C8H12O4

IUPAC Name:

2-[(acetyloxy)methyl]prop-2-en-1-yl acetate

Specific details on test material used for the study:

Identification: 2-methylene-1, 3-propanediol diacetate
CAS Number: 3775-29-9
Batch: 20160308-88
Purity: 99.7%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 31 March 2020
Storage Conditions: Room temperature in the dark

Test solutions

Details on test solutions:

Range-Finding Tests
Test Item Preparation
The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with Nescofilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.
The exposure conditions for each flask were as described in Section 3.4.2.4 with the exception that the test was conducted at measured temperatures of between 20 ºC and 21 ºC.
The control group was maintained under identical conditions but not exposed to the test item.
Reference Item Preparation
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 5.4 and was adjusted to pH 7.3 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.

Definitive Test
The test item was dispersed directly in water.
Nominal amounts of test item (5, 16, 50, 160 and 500 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with parafilm prior to being subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 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 (five replicates of each).
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
The control group was maintained under identical conditions but not exposed to the test item.
Reference Item Preparation
For the purpose of the definitive test a reference item, 3,5-dichlorophenol was used. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. The pH of this stock solution was measured to be pH 5.7 and adjusted to pH 7.4 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 20 February 2019 for the range-finding test and on 07 January 2020 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Test Water
The test water used for the range- tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).
The test water used for the definitive test was deionized reverse osmosis water containing 1.19 mg/L Total Organic Carbon (TOC).

Synthetic Sewage
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g: Peptone
11 g: Meat extract
3 g: Urea
0.7 g: NaCl
0.4 g: CaCl2.2H2O
0.2 g: MgSO4.7H2O
2.8 g: K2HPO4
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge and used in the range-finding and definitive tests was between pH 7.2 and 7.4. The pH values were measured using a Hach HQ40d Flexi handheld meter.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection, determination of the suspended solids level of the activated sewage sludge was carried out. A sample (100 mL) of the activated sewage sludge was filtered by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed three times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC 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 3.0 g/L prior to the activated sewage sludge (14 liters) being fed an aliquot of synthetic sewage (700 mL).
The pH of the sample on the day of the test was 7.4 measured using a Hach HQ40d Flexi handheld meter. The suspended solids level of the activated sewage sludge was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L.
* Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven

Study design

Test type:

static

Water media type:

other: deionized reverse osmosis water

Limit test:

no

Total exposure duration:

3 h

Test conditions

pH:

The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
7.3 -7.5 at 0 hours
7.2 -7.5 at 3 hours

Dissolved oxygen:

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.

Nominal and measured concentrations:

Range-Finding Tests
An initial range-finding test was conducted which gave inconclusive results for the percentage inhibition values for the test concentrations of 10 and 100 mg/L. Therefore it was considered necessary to perform a further range-finding test.
In the second range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.


Definitive Test
Based on the results of the range-finding test, in order to obtain a NOEC and a 3-Hour EC50 value for the test item, the following test concentrations were assigned to the definitive test: 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item. Two additional control vessels were then prepared prior to the test item vessels being prepared. Finally, two further control vessels were prepared. The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of approximately 20 °C.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

300 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

Second Range-Finding Test
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L; however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain both a NOEC and an EC50 value for the test item. Test concentrations of 10, 32, 100, 320 and 1000 mg/L were selected for the definitive test.

Inhibition of Respiration Rate
No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L and 100 mg/L; however statistically significant toxic effects (P < 0.05) were shown at the test concentrations of 320 and 1000 mg/L. Based on these results the NOEC after 3 hours exposure was 100 mg/L.
In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). 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.

Any other information on results incl. tables

The specific respiration rate of the controls was 25.47 mg oxygen per gram dry weight of sludge per hour.
The coefficient of variation of oxygen uptake in the control vessels was 3.4%.
All validation criteria for the study were therefore satisfied.

 

Inhibition of Respiration Rate

	2-methylene-1, 3-propanediol diacetate		3,5-dichlorophenol
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	92	-	1.1	-
EC20	150	-	1.6	-
EC50	300	250 - 350	6.2	4.6 - 8.3
EC90	530	-	23	-
NOEC	100	-	-	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 300 mg/L, 95% confidence limits 250 to 350 mg/L. The NOEC after 3 hours exposure was 100 mg/L.

Executive summary:

Introduction
A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for the Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods
Following a preliminary range-finding test, at the request of the Sponsor, a test to determine the No Observed Effect Concentration (NOEC) and the 3-Hour EC50 value for the test item was performed. Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L (five replicates each) for a period of 3 hours at measured temperatures of approximately 20 °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 item, 3,5-dichlorophenol.

Results
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 300 mg/L, 95% confidence limits 250 to 350 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.
The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 6.2 mg/L, 95% confidence limits 4.6 to 8.3 mg/L confirming the acceptability of the inoculum used in the test system.