Ethyl 4-methyl-2-oxocyclohexanecarboxylate

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:

Experimental start date 15 November 2017 Experimental completion date 11 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD guidelines and in compliance with GLP without deviations that influence the result.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 13-0545
Batch: PDJ378-68
Purity: 93.2%
Physical state/Appearance: clear colorless liquid
Expiry Date: 30 June 2018
Storage Conditions: approximately 4 °C in the dark

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

Range-Finding Test
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 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).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter if necessary.

Definitive Test
The test item was dispersed directly in water.
Nominal amounts of test item (50, 160 and 500 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and 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 100, 320 and 1000 mg/L (five replicates of each).
The pH of the test item stock solutions was measured and adjusted to between pH 7.0 and 8.0 using a Hach HQ40d Flexi handheld meter

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 15 November 2018 for the range-finding test and on 10 January 2018 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.

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 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.2 measured using a Hach HQ40d Flexi handheld 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 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 use.

Dilution water:
The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 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
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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at a measured temperature of approximately 20 °C.

pH:

7.1-7.6 at start of test. 7.3-7.6 at end of test
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.

Dissolved oxygen:

3.5-6.1 mg 02/L at start of test. 1.9-2.9 mg 02/L at end of test
The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Nominal and measured concentrations:

Range finding
Test substance - Nominal: 10, 100 and 1000 mg/l.

Definitive test
Test substance - Nominal: 100, 320 and 1000 mg/l.

Reference substance - Nominal: 3.2, 10 and 32 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 via narrow bore glass tubes 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.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (C6H3CI2.OH), Purity 97%

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: EC10/NOECis based on =< x% inhibition

Details on results:

Range-Finding Test
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 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 a NOEC value for the test item. Test concentrations of 100, 320 and 1000 mg/L were selected for the definitive test.

Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 9.9 mg/L.
• The specific respiration rate of the controls was 28.61 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 2.4%.
All validation criteria for the study were therefore satisfied.

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

Results with reference substance (positive control):

3,5-dichlorophenol gave a 3-Hour EC50 value of 9.9 mg/L, 95% confidence limits 7.9 to 12 mg/L.

Inhibition of Respiration Rate

The following results were derived:

	3,5-Dichlorophenol
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	2.5	-
EC20	3.6	-
EC50	9.9	7.9 - 12
EC80	27	-

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

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	R1	4.2	3	1.6	52.00	-
	R2	4.3	3	2.0	46.00	-
	R3	5.7	5	2.4	39.60	-
	R4	4.3	3	2.3	40.00	-
Test Item	10	4.4	3	2.0	48.00	[[8]
	100	3.5	2	1.9	48.00	[8]
	1000 R1	5.5	8	2.6	21.75	51
	1000 R2	6.1	10	2.9	19.20	57
	1000 R3	6.1	10	2.6	21.00	53
3,5-Dichlorophenol	3.2	5.1	6	2.5	26.00	41
	10	6.0	10	2.4	21.60	51
	32	7.5	10	6.5	6.00	86

R   = Replicate

-       = Not applicable

[ ]   = Increase in respiration rate as compared to controls

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a NOEC after 3 hours exposure of 320 mg/L.

Executive summary:

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 13 -0545 on the respiration of activated sewage sludge. Cultures of activated sewage sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance. Aeration was interrupted after 3 hours and the rates of respiration measured electrochemically for each culture. The percentage inhibition of respiration was calculated for each culture after each contact period by comparing oxygen depletion rates for the test substance with those for the negative control culture. A positive control i.e. 3,5-dichlorophenol was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. The NOEC is considered to be 320 mg/l.

Description of key information

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 13 -0545 on the respiration of activated sewage sludge. Cultures of activated sewage sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance. Aeration was interrupted after 3 hours and the rates of respiration measured electrochemically for each culture. The percentage inhibition of respiration was calculated for each culture after each contact period by comparing oxygen depletion rates for the test substance with those for the negative control culture. A positive control i.e. 3,5-dichlorophenol was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. The NOEC is considered to be 320 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

320 mg/L