4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran

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 05 July 2017. Experimental completion date 10 August 2017

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 12-0492
Physical state/Appearance: very pale yellow liquid
Storage Conditions: approximately 4 ºC

Analytical monitoring:

no

Vehicle:

no

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 (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0 if necessary.

Definitive Test
Nominal amounts of test item (90, 160 and 280 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 180, 320 and 560 mg/L five replicates of each).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0.

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 05 July 2017 for the range-finding test and on 09 August 2017 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
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 (13 liters) was fed synthetic sewage (650 mL). The pH of the sample on the day of the test was 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 3 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 one 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 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 NaCI
0.4 g CaCI2. 2H20
0.2 g MgS04.7Hp
2.8 g K2HP04
dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
In the range-finding test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.2 and the pH of the synthetic sewage stock used for the range-finding test was pH 7.3. The pH values were measured using a Hach HQ40d Flexi handheld meter. In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.3 and the pH of the synthetic sewage stock used for the definitive test was pH 7.3. 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 21 ºC.

pH:

7.4-7.9

Dissolved oxygen:

4.1 - 5.6 mg O2/L

Nominal and measured concentrations:

Range-finding test: Nominal concentrations of 10, 100 and 1000 mg/L.
Definitive test: Nominal concentrations of 180, 320 and 560 mg/L.
Reference item: Nominal concentrations of of 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 being added. Two additional control vessels were then prepared prior to the test item vessels were prepared as described in Section 3.3.2.1. 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 21 ºC.

Reference substance (positive control):

yes

Remarks:

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

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.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

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. Based on this information test concentrations of 180, 320 and 560 mg/L were selected for the definitive test.

Definitive Test
No statistically significant toxic effects were shown at the test concentrations of 180 and 320 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentration of 560 mg/L. Based on these results the No Observed Effect Concentration (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.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 8.2% and the specific respiration rate of the controls was 22.28 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (3 h): 6.8 mg/L (5.1 - 9.1 95% confidence limits)
3,5-dichlorophenol EC10 (3 h): 1.2 mg/L

Results

	FRET 12-0492		3,5-dichlorophenol
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	<180	-	1.2	-
EC20	>180	-	1.8	-
EC50	>1000	-	6.8	5.1-9.1
EC80	>1000	-	26	-
NOEC	320	-	-	-

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal Concentration (mg/L)		Initial O2 Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	4.6	5	2.2	28.8	-
	R2	4.7	5	2	32.4	-
	R3	6.1	7	1.8	36.86	-
	R4	6	7	2.1	33.43	-
	R5	5.6	6	2.2	34	-
	R6	5.8	6	2.3	35	-
Test Item	180 R1	4.1	4	2	31.5	6
	180 R2	4.8	5	2.2	31.2	7
	180 R3	4.6	5	2	31.2	7
	180 R4	4.5	5	1.9	31.2	7
	180 R5	4.5	5	1.9	31.2	7
	320 R1	4.2	4	2.1	31.5	6
	320 R2	4.8	5	2.3	30	10
	320 R3	4.7	5	2	32.4	3
	320 R4	5.5	6	2.4	31	7
	320 R5	5.6	6	2.4	32	4
	560 R1	5.6	8	2	27	19
	560 R2	5.6	8	2.2	25.5	24
	560 R3	6.1	9	2.1	26.67	20
	560 R4	5.9	8	2.4	26.25	21
	560 R5	5.6	7	2.4	27.43	18
3,5-dichlorophenol	3.2	6.4	10	2.8	21.6	35
	10	7.1	10	4.6	15	55
	32	8.2	10	7.5	4.2	87

R1 – R6 = Replicates 1 to 6

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (respiration inhibition) values of the substance tested on activated sewage sludge was > 1000 mg/l after a 3-hour contact time.

Executive summary:

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 12 -0492 on the respiration of activated sewage sludge.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 180, 320 and 560 mg/L (5 replicates) for a period of 3 hours at a measured temperature of 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 item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.

The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.1 to 9.1 mg/L.

Description of key information

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 12 -0492 on the respiration of activated sewage sludge.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 180, 320 and 560 mg/L (5 replicates) for a period of 3 hours at a measured temperature of 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 item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 320 mg/L.

The reference item gave a 3-Hour EC50 value of 6.8 mg/L, 95% confidence limits 5.1 to 9.1 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

320 mg/L