Ethyl 2-methyl-1,3-dioxolane-2-acetate

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:

29 April 2013 to 30 April 2013

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:

yes

Remarks:

(see below)

Principles of method if other than guideline:

The dissolved oxygen content was not above 60% to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L in several of the vessels.
This deviation was considered to have had no adverse effect on the study as all oxygen consumption values were measured over the linear portion of the traces.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTIONS
2500 mg of test material was dissolved in water and the volume adjusted to 1 litre to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 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 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
For each control, 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculums, then added to the test vessel.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of micro-organisms: a mixed population of activated sewage sludge micro-organisms was obtained on 29 April 2013 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- 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 the activated sewage sludge (10 litres) was fed synthetic sewage sludge (500 mL).
- Suspended solids concentration: The suspended solids concentration was equal to 3 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.5 - 7.7 at 0 hours; 8.1 - 8.5 at 3 hours (measured in the controls, reference substance and treatment groups)

Dissolved oxygen:

4.2 - 7.7 mg O2/L (measured after 30 minutes contact time in the controls, reference substance and treatment groups)

Nominal and measured concentrations:

0, 10, 100, 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: yes, via narrow bore glass tubes at a rate of approximately 0.5 - 1.0 litre per minute
- No. of vessels per concentration (replicates): 1 at 10 and 100 mg/L and 3 at 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 1 at each concentration

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the study was deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

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
dissolved in 1 litre of water with the aid of ultrasonication.
The pH of the synthetic sewage stock used to feed the activated sewage sludge was 6.9 and adjusted to 7.2 using 1.0 M NaOH. The pH of the synthetic sewage stock used for the study was 7.1.

OTHER TEST CONDITIONS
- Photoperiod: the test was conducted under normal laboratory lighting.

EFFECT PARAMETERS MEASURED
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 7.0 mg O2/L and 2.0 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.
Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (3.2, 10 and 32 mg/L)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
The coefficient of variation of oxygen uptake in the control vessels was 13 % and the specific respiration rate of the control was 30.08 mg oxygen per gram dry weight of sludge per hour.

Results with reference substance (positive control):

The 3-hour EC50 value for the reference substance, 3,5-dichlorophenol, was determined to be 11 mg/L (95 % confidence limits 8.7 - 14).

Reported statistics and error estimates:

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 for the test after 3 hours for the control and all test concentrations 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).

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range Finding Test

Nominal Conc. (mg/L)		Initial O2Reading (mg O2/L)	Measurement Period (min)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	4.0	2	2.3	51.00	-
	R2	4.0	3	1.7	46.00	-
	R3	5.3	4	2.2	46.50	-
	R4	5.8	6	2.1	37.00	-
Test Material	10	4.4	3	2.0	48.00	[6]
	100	5.0	4	2.0	45.00	1
	1000 R1	5.1	4	2.1	45.00	1
	1000 R2	4.9	4	2.0	43.50	4
	1000 R3	5.1	4	2.2	43.50	4
Reference Material	3.2	4.9	4	2.3	39.00	14
	10	6.5	10	2.7	22.80	49
	32	7.7	10	6.2	9.00	80

[Increase in respiration rate as compared to controls]

R1 – R4 = Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 3 hour EC50 value was determined to be in excess of 1000 mg/L and the No Observed Effect Concentration was 1000 mg/L, based on the respiration rate of the activated sludge.

Executive summary:

The effect of the test material on the respiration of activated sewage sludge was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209.

Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °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 reference item 3,5 -dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3 -hour EC50 value in excess of 1000 mg/L. The No Observed Effect Concentration after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 -hour EC50 value of 11 mg/L. All of the validity criteria of the study were satisfied.

Description of key information

3-hour EC50 > 1000 mg/L; 3-hour NOEC = 1000 mg/L, OECD 209, Roulstone (2013)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The effect of the test material on the respiration of activated sewage sludge was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 209.

Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °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 reference item 3,5 -dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3 -hour EC50 value in excess of 1000 mg/L. The No Observed Effect Concentration after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3 -hour EC50 value of 11 mg/L.

Since the study was conducted under GLP conditions and to a standardised guideline the study was assigned a reliability score of 1 in line with the criteria of Klimisch (1997).