(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

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:

13 Jan 2020 - 26 Feb 2020

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 (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: MEDOL-10
- Batch: 09892803
- Purity: 99.9%
- Physical State/Appearance: Clear colorless liquid
- Expiry Date: 15 February 2023
- Storage Conditions: Room temperature in the dark

Analytical monitoring:

not specified

Details on sampling:

Nominal amounts of test item (25, 80, 250, 800 and 2500 mg) were separately dissolved in water with the aid of ultrasonication for 15 minutes and the volume adjusted to 1 liter to give 25, 80, 250, 800 and 2500 mg/L stock solutions.

Vehicle:

no

Details on test solutions:

Test Item Preparation
The test item was dissolved directly in water.
Nominal amounts of test item (25, 80, 250, 800 and 2500 mg) were separately dissolved in water with the aid of ultrasonication for 15 minutes and the volume adjusted to 1 liter to give 25, 80, 250, 800 and 2500 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 80, 250, 800 and 2500 mg/L stock solutions were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/L (five replicates of each concentration). The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

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 5.9 and adjusted to pH 7.2 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 (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Date of collecition: 14 January 2020 for the range finding test and on 19 February 2020 for the definitive test.
- Preparation of inoculum for exposure: 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 4.0g/L and was adjusted to 3.0 g/L prior to the activated sewage sludge (10 liters) being fed an aliquot of synthetic sewage (500 mL).
The pH of the sample on the day of the test was 7.5 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.

Test type:

static

Water media type:

other: Deionized reverse osmosis water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19 and 21 °C

pH:

0 hour: 7.3 - 7.7
3 hour: 7.1 - 7.6

Dissolved oxygen:

30 min: 5.56 - 7.94 mg O2/L; 62 - 76 %

Nominal and measured concentrations:

Range finding test (nominal) (mg/L): 10, 100 and 1000
Definitive test (nominal) (mg/L): 10, 32, 100, 320 and 1000
Reference Item (nominal) (mg/L): 3.2, 10 and 32

Details on test conditions:

TEST SYSTEM
- Type: closed
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Weight of dry solids per volume of reaction mixture per unit of time: 3.0 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Range finding study : yes

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 Test System: Nominal amounts of test item (25, 80, 250, 800 and 2500 mg) were separately dissolved in water with the aid of ultrasonication for 15 minutes and the volume adjusted to 1 liter to give 25, 80, 250, 800 and 2500 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 80, 250, 800 and 2500 mg/L stock solutions were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/L (five replicates of each concentration). The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The pH of the test item stock solutions was measured and adjusted if necessary to between pH 7.0 and 8.0 using a Hach HQ40d Flexi handheld meter
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added (first control). The mixture was aerated with 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 as described in Section 3.4.2.1. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of between 19 °C and 21 °C.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 650 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 210 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

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

Details on results:

It was not possible to determine an EC80 value for the test item as no concentration tested resulted in greater than 80%

No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L; however statistically significant toxic effects (P < 0.05) were shown at the test concentrations of 100, 320 and 1000 mg/L. Based on these results the NOEC after 3 hours exposure was 32 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.

Results with reference substance (positive control):

The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 7.9 mg/L.

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line for the reference item.
The 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours to determine any statistically significant differences between the test and control groups.
All statistical analyses were performed using the SAS computer software package (SAS, 1999 2001).

Validity criteria fulfilled:

yes

Conclusions:

The effect of MEDOL-10 on the respiration of activated sewage sludge micro organisms gave a 3 Hour EC50 value of 650 mg/L, whereas the NOEC after 3 hours exposure was 32 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)".

 

Results

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3‑Hour EC₅₀ value of 650 mg/L*.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L.

The reference item, 3,5‑dichlorophenol, gave a 3‑Hour EC₅₀ value of 7.9 mg/L, 95% confidence limits 6.1 to 10 mg/L confirming the acceptability of the inoculum used in the test system.

* The data generated was unsuitable to calculate 95% confidence limits for the test item

 

Conclusion

The effect of the test item on the respiration of activated sewage sludge micro‑organisms gave a 3‑Hour EC₅₀ value of 650 mg/L.  The NOEC after 3 hours exposure was 32 mg/L.

Description of key information

Inhibition of Respiration Rate (3 hours)

EC₅₀ = 650 mg/L

NOEC = 32 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

650 mg/L

EC10 or NOEC for microorganisms:

120 mg/L

Additional information

An Activated Sludge Respiration Inhibition Test (ASRIT) was conducted (Covance Laboratories Limited, DR86XT, 2020) to determine the effect of MEDOL-10 on microorganisms. The test was conducted according to OECD test guideline 209, and in compliance with GLP.

A mixed population of activated sludge was taken from a sewage treatment plant predominantly treating domestic sewage. Based on range finder test results, cultures were exposed to MEDOL-10 at concentrations 10, 32, 100, 320 and 1000 mg/L for 3 hours. 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. No statistically significant toxic effects were shown at the test concentrations of 10 and 32 mg/L; however statistically significant toxic effects were shown at the test concentrations of 100, 320 and 1000 mg/L. 

The effect of the test item on the respiration of activated sewage sludge microorganisms gave a 3 Hour EC50 value of 650 mg/L. The NOEC after 3 hours exposure was 32 mg/L.