Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

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:

The study was conducted between 25 January 2018 and 04 April 2018.

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:

The measurement reported was Total Organic Carbon (TOC) not DOC which was a deviation to the Study Plan.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 14-0383
Physical state/Appearance: colorless liquid
Storage Conditions: approximately 4 ºC in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

In the range finding test, activated sewage sludge micro organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. 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 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.


Based on the results of the range finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (50, 90, 160, 280 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, 180, 320, 560 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.

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 25 January 2018 for the range finding test and on 03 April 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.

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 7.3 and 7.4. The pH values were measured using a Hach HQ40d Flexi handheld meter.

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 (14 liters) was fed synthetic sewage (700 mL). The pH of the sample on the day of the test was 7.5 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).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Between 20 °C and 21 °C

pH:

7.3-7.8

Dissolved oxygen:

2.9-5.0 mg O2/L

Nominal and measured concentrations:

Range-Finding Test
Test item: 10, 100 and 1000 mg/L
Reference item: 3.2, 10 and 32 mg/L

Definitive test
Test item: 100, 180, 320, 560 and 1000 mg/L
Reference item: 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. Finally, two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 °C and 21 °C.

Reference substance (positive control):

yes

Remarks:

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

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

380 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 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 both a NOEC and an EC50 value for the test item. Test concentrations of 100, 180, 320, 560 and 1000 mg/L were selected for the definitive test.

Definitive test:
The effect of the test item on the respiration of activated sewage sludge micro organisms gave a 3 Hour EC50 value of 380 mg/L, 95% confidence limits 320 to 450 mg/L. The NOEC after 3 hours exposure was 100 mg/L.

No statistically significant toxic effects were shown at the test concentration of 100 mg/L, however statistically significant toxic effects (P < 0.05) were shown at the test concentrations of 180, 320, 560 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.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (3 h): 8.1 mg/I

Validation Criteria

·       The EC₅₀ value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.1 mg/L.

·       The specific respiration rate of the controls was 27.33 mg oxygen per gram dry weight of sludge per hour.

·       The coefficient of variation of oxygen uptake in the control vessels was 8.0%.

All validation criteria for the study were therefore satisfied.

 Inhibition of Respiration Rate

The following results were derived:

	FRET 14-0383		3,5-Dichlorophenol
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC10	110	-	1.5	-
EC20	140	-	2.3	-
EC50	380	320 - 450	8.1	6.4 - 10
EC80	1300*	-	28	-
NOEC	100	-	-	-

 -  =   Not appliciable

*Value obtained by extrapolation of the concentration response curve

pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	10	6.8	7.5
	100	6.5	7.4
	1000R1	4.1	7.3
	1000 R2	4.1	7.1
	1000 R3	4.2	7.9

R = Replicate

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	R1	6.86	77
	R2	6.70	75
	R3	7.21	81
	R4	5.63	63
Test Item	10	5.73	64
	100	5.62	63
	1000 R1	5.61	63
	1000 R2	5.59	63
	1000 R3	5.71	64
3,5-Dichlorophenol	3.2	6.59	74
	10	7.86	88
	32	7.12	80

R = Replicate

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	4.9	4	2.0	43.50	-
	R2	4.5	3	2.3	44.00	-
	R3	4.7	4	1.9	42.00	-
	R4	5.7	5	2.1	43.20	-
Test Item	10	4.1	3	1.9	44.00	[2]
	100	4.5	3	2.2	46.00	[7]
	1000 R1	6.9	10	5.9	6.00	86
	1000 R2	6.5	10	4.8	10.20	76
	1000 R3	6.9	10	5.4	9.00	79
3,5-Dichlorophenol	3.2	5.6	6	2.4	32.00	26
	10	6.8	10	3.3	21.00	51
	32	8.0	10	6.9	6.60	85

R = Replicate

-  = Not applicable

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.5	7.4
	R2	7.5	7.3
	R3	7.5	7.5
	R4	7.3	7.4
Test Item	10	7.5	7.4
	100	7.6	7.6
	1000 R1	7.8	7.6
	1000 R2	7.5	7.6
	1000 R3	7.5	7.6
3,5-Dichlorophenol	3.2	7.7	7.8
	10	7.6	7.9
	32	7.8	7.9

R = Replicate

Observations on the Test Preparations throughout the Test Period in the Range-Finding Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/brown slightly hazy dispersion with an oily layer of test item visible at the surface	Dark brown dispersion with an oily layer of test item visible at the surface	Dark brown dispersion with an oily layer of test item visible at the surface
	1000 R1	Pale yellow/brown hazy dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface
	1000 R2	Pale yellow/brown hazy dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface
	1000 R3	Pale yellow/brown hazy dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface	Dark brown dispersion with an oily layer and oily globules of test item visible at the surface
3,5-Dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

*Observations made prior to the addition of activated sewage sludge

R = Replicate

pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Definitive Test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	100R1	6.2	7.5
	100R2	5.7	7.7
	100R3	5.9	7.4
	100R4	6.1	7.7
	100R5	5.8	7.4
	180R1	5.3	7.9
	180R2	5.1	7.1
	180R3	5.3	7.2
	180R4	5.0	7.9
	180R5	5.1	7.1
	320R1	5.0	7.2
	320R2	4.9	7.6
	320R3	4.9	7.2
	320R4	4.8	7.9
	320R5	4.8	7.2

R  = Replicate

pH Values of the Test Item Preparations after Stirring and Prior to the Addition of Inoculum in the Definitive Test

Nominal Concentration (mg/L)		pH
		Prior to Adjustment	After Adjustment
Test Item	560R1	4.4	7.4
	560 R2	4.4	7.6
	560 R3	4.6	7.3
	560 R4	4.1	7.7
	560 R5	4.3	7.2
	1000 R1	4.3	7.1
	1000R2	4.2	7.2
	1000 R3	3.9	7.2
	1000 R4	4.2	7.6
	1000 R5	4.3	7.3

R = Replicate

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Control	R1	6.78	76
	R2	8.22	92
	R3	7.71	87
	R4	7.81	88
	R5	7.23	81
	R6	6.61	74
Test Item	100R1	6.10	69
	100R2	6.99	79
	100R3	7.25	81
	100 R4	6.70	75
	100 R5	5.96	67
	180R1	6.11	69
	180R2	6.26	70
	180R3	7.32	82
	180 R4	5.88	66
	180 R5	6.94	78
	320R1	6.76	76
	320R2	7.01	79
	320R3	5.79	65
	320R4	5.66	64
	320R5	6.25	70
	560R1	6.02	68
	560R2	5.80	65
	560R3	6.47	73
	560R4	6.59	74
	560R5	6.72	76

R = Replicate

(continued) Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
Test Item	1000R1	5.69	64
	1000R2	6.30	71
	1000R3	6.72	76
	1000R4	7.07	79
	1000R5	6.44	72
3,5-Dichlorophenol	3.2	8.52	96
	10	8.49	95
	32	8.49	95

R   = Replicate

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 O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	R1	3.3	2	1.9	42.00	-
	R2	2.9	2	1.7	36.00	-
	R3	3.8	3	1.6	44.00	-
	R4	3.8	3	1.6	44.00	-
	R5	3.8	3	1.9	38.00	-
	R6	3.9	3	1.8	42.00	-
Test Item	100R1	3.4	2	2.0	42.00	[2]
	100R2	4.5	4	1.9	39.00	5
	100R3	3.8	3	2.0	36.00	12
	100R4	5.0	5	2.0	36.00	12
	100R5	3.5	3	1.7	36.00	12
	180R1	4.4	5	2.0	28.80	30
	180R2	4.5	5	1.9	31.20	24
	180R3	4.6	5	2.3	27.60	33
	180R4	3.8	3	2.3	30.00	27
	180R5	4.0	4	1.9	31.50	23

R = Replicate

(continued)    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)	O2Consumption Rates (mg O2/L/hour)	Inhibition (%)
Test Item	320R1	5.4	9	2.1	22.00	46
	320R2	4.1	6	2.0	21.00	49
	320R3	4.9	8	2.0	21.75	47
	320R4	5.0	8	2.1	21.75	47
	320R5	4.7	7	2.0	23.14	44
	560R1	5.4	10	2.7	16.20	60
	560R2	4.8	7	2.5	19.71	52
	560R3	5.7	10	2.9	16.80	59
	560R4	6.1	10	3.3	16.80	59
	560R5	6.4	10	3.7	16.20	60
	1000R1	7.3	10	5.7	9.60	77
	1000R2	7.1	10	5.5	9.60	77
	1000R3	7.3	10	5.8	9.00	78
	1000R4	6.6	10	4.7	11.40	72
	1000R5	6.9	10	4.9	12.00	71
3,5-Dichlorophenol	3.2	5.5	7	2.1	29.14	29
	10	5.7	10	2.5	19.20	53
	32	7.1	10	6.0	6.60	84

R = Replicate

pH Values of the Test Preparations at the Start and End of the Exposure Period in the Definitive Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Control	R1	7.6	7.4
	R2	7.5	7.5
	R3	7.4	7.4
	R4	7.6	7.5
	R5	7.5	7.4
	R6	7.4	7.5
Test Item	100R1	7.6	7.4
	100R2	7.7	7.5
	100R3	7.5	7.4
	100R4	7.4	7.5
	100R5	7.6	7.4
	180R1	7.5	7.5
	180R2	7.4	7.5
	180R3	7.4	7.4
	180R4	7.3	7.5
	180R5	7.3	7.4

R = Replicate

(continued) pH Values of the Test Preparations at the Start and End of the Exposure Period in the Definitive Test

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Test Item	320R1	7.4	7.3
	320R2	7.3	7.2
	320R3	7.6	7.3
	320R4	7.3	7.2
	320R5	7.5	7.3
	560R1	7.5	7.3
	560R2	7.3	7.3
	560R3	7.4	7.2
	560R4	7.4	7.4
	560R5	7.4	7.3
	1000R1	7.4	7.4
	1000R2	7.4	7.4
	1000R3	7.9	7.6
	1000R4	7.6	7.4
	1000R5	7.5	7.4
3,5-Dichlorophenol	3.2	7.6	7.9
	10	7.5	7.8
	32	7.4	7.7

R  = Replicate

Observations on the Test Preparations throughout the Test Period in the Definitive Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R5	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R6	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	100R1	Pale yellow/brown slightly hazy dispersion with a layer of oily specks of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	100R2	Pale yellow/brown slightly hazy dispersion with a layer of oily specks of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	100R3	Pale yellow/brown slightly hazy dispersion with a layer of oily specks of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	100R4	Pale yellow/brown slightly hazy dispersion with a layer of oily specks of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	100R5	Pale yellow/brown slightly hazy dispersion with a layer of oily specks of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface

*Observations made prior to the addition of activated sewage sludge

R  = Replicate

(continued) Observations on the Test Preparations throughout the Test Period in the Definitive Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Test Item	180R1	Pale yellow/brown hazy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	180R2	Pale yellow/brown hazy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	180R3	Pale yellow/brown hazy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	180R4	Pale yellow/brown hazy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	180R5	Pale yellow/brown hazy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface	Dark brown dispersion with oily globules of test item on the surface
	320R1	Pale yellow/brown cloudy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface
	320R2	Pale yellow/brown cloudy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface
	320R3	Pale yellow/brown cloudy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface
	320R4	Pale yellow/brown cloudy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface
	320R5	Pale yellow/brown cloudy dispersion with a layer of oily specks and globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface	Dark brown dispersion with a layer of oily globules of test item on the surface

*Observations made prior to the addition of activated sewage sludge

R = Replicate

(continued)  Observations on the Test Preparations throughout the Test Period in the Definitive Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Test Item	560R1	Pale yellow/brown cloudy dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface
	560R2	Pale yellow/brown cloudy dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface
	560R3	Pale yellow/brown cloudy dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface
	560R4	Pale yellow/brown cloudy dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface
	560R5	Pale yellow/brown cloudy dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface	Dark brown dispersion with an oily layer of test item on the surface
	1000R1	Pale yellow/brown cloudy dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface
	1000R2	Pale yellow/brown cloudy dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface
	1000R3	Pale yellow/brown cloudy dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface
	1000R4	Pale yellow/brown cloudy dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface
	1000R5	Pale yellow/brown cloudy dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface	Dark brown dispersion with an oily layer and oily globules of test item on the surface

*Observations made prior to the addition of activated sewage sludge

R  = Replicate

(continued) Observations on the Test Preparations throughout the Test Period in the Definitive Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours^*	30 Minutes Contact Time	3 Hours Contact Time
3,5-Dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

*Observations made prior to the addition of activated sewage sludge

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (respiration inhibition) values of the substance FRET 14-0383 on activated sewage sludge was 380 mg/l for after 3-hour contact time.

Executive summary:

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 14 -0383 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 30 minutes and again 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 EC50 (respiration inhibition) values for the substance FRET 14 -0383 was 380 mg/l after 3-hour contact time. The NOEC is considered to be 100 mg/l.

Description of key information

 A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 14 -0383 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 30 minutes and again 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 EC50 (respiration inhibition) values for the substance was 380 mg/l after 3-hour contact time. The NOEC is considered to be 100 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

380 mg/L

EC10 or NOEC for microorganisms:

100 mg/L