Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

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 07 March 2019 and 13 March 2019.

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: PG-RAW-90-032
Physical State/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 ºC in the dark

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
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 (three replicates for the test concentration of 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 sealed with nescofilm prior to being 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.
The exposure conditions for each flask were as described with the exception that the test was conducted at measured temperatures of approximately 20 ºC.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Based on the results of the range finding test, in order to obtain a NOEC for the test item, the following test concentrations were assigned to the definitive test: 10, 32 and 100 mg/L.

Test Item Preparation
The test item was dispersed directly in water.
Nominal amounts of test item (5.0, 16 and 50 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and sealed with nescofilm prior to being 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, 32 and 100 mg/L (five replicates of each).
The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter.
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.

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 7 March 2019 for the range finding test and on 12 March 2019 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.

Test 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
Each constituent was dissolved in a final volume of 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stocks used to feed the activated sewage sludge and used in the range finding and definitive tests was pH 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:

20 °C - 21 °C

pH:

7.2 - 7.7

Dissolved oxygen:

2.8 - 7.9 mg O2/L

Nominal and measured concentrations:

Range-finding test:
Test item: Nominal: 10, 100 and 1000 mg/L
Reference Item: Nominal: 3.2, 10 and 32 mg/L

Definitive test:
Test item: Nominal: 10, 32 and 100 mg/L
Reference item: Nominal: 3.2, 10 and 32 mg/L

Details on test conditions:

Range-Finding Test
Reference Item Preparation
A reference item, 3,5 dichlorophenol, was included in the range finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. 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 6.3 and was 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 to give the required 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.
The exposure conditions for each flask were as described.

Definitive Test
Based on the results of the range finding test, in order to obtain a NOEC for the test item, the following test concentrations were assigned to the definitive test: 10, 32 and 100 mg/L.

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 15 minutes. The pH of this stock solution was measured to be pH 6.4 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 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.

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 (9.5 liters) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.6 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.

Preparation of Test System
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 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. 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.

Assessments
Observations
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.

pH Measurements
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.

Oxygen Concentration
The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Measurement of the Respiration Rates
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 7 mg O2/L and 2 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.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

7.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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 concentration of 10 mg/L; however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 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.
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 10, 32 and 100 mg/L were selected for the definitive test.

Definitive Test
Percentage inhibition is plotted against concentration for the reference item, 3,5 dichlorophenol.

Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 7.7 mg/L.
• The specific respiration rate of the controls was 28.00 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 3.0%.
All validation criteria for the study were therefore satisfied.

Results with reference substance (positive control):

The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 7.7 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	1.5	-
EC20	2.2	-
EC50	7.7	5.9−10
EC80	26	-

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

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

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.

Dissolved Oxygen Concentrations and Saturation 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	5.80	65
	R2	6.05	68
	R3	6.04	68
	R4	5.83	66
Test Item	10	5.87	66
	100	5.70	64
	1000 R1	6.01	68
	1000 R2	5.87	66
	1000 R3	5.94	67
3,5-Dichlorophenol	3.2	5.96	67
	10	6.53	73
	32	7.60	85

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	3.9	2	2.4	45.00	-
	R2	3.6	2	2.3	39.00	-
	R3	4.3	3	2.4	38.00	-
	R4	4.2	3	2.2	40.00	-
Test Item	10	3.3	2	2.0	39.00	4
	100	2.7	1	2.2	30.00	26
	1000 R1	3.9	4	2.1	27.00	33
	1000 R2	4.0	4	2.0	30.00	26
	1000 R3	2.8	2	1.8	30.00	26
3,5-Dichlorophenol	3.2	6.4	9	2.3	27.33	33
	10	7.0	10	4.0	18.00	56
	32	7.9	10	7.1	4.80	88

 

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	7.6	7.5
	R3	7.5	7.6
	R4	7.7	7.5
Test Item	10	7.5	7.5
	100	7.3	7.3
	1000 R1	7.4	7.4
	1000 R2	7.6	7.5
	1000 R3	7.6	7.4
3,5-Dichlorophenol	3.2	7.3	7.8
	10	7.2	7.7
	32	7.4	7.6

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 slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a slight oily film of test item visible at the surface
	1000 R1	Pale yellow/brown cloudy dispersion with a layer of oily globules of test item visible at the surface and oily globules dispersed throughout	Dark brown dispersion with a layer of oily globules of test item visible at the surface	Dark brown dispersion with a layer of oily globules of test item visible at the surface
	1000 R2	Pale yellow/brown cloudy dispersion with a layer of oily globules of test item visible at the surface and oily globules dispersed throughout	Dark brown dispersion with a layer of oily globules of test item visible at the surface	Dark brown dispersion with a layer of oily globules of test item visible at the surface
	1000 R3	Pale yellow/brown cloudy dispersion with a layer of oily globules of test item visible at the surface and oily globules dispersed throughout	Dark brown dispersion with a layer of oily globules of test item visible at the surface	Dark brown dispersion with a layer of 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
Test Item	10R1	7.6
	10R2	7.4
	10R3	7.4
	10R4	7.4
	10R5	7.4
	32R1	7.5
	32R2	7.4
	32R3	7.4
	32R4	7.4
	32R5	7.2
	100R1	7.8
	100R2	7.4
	100R3	7.7
	100R4	7.5
	100R5	7.7

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	7.02	79
	R2	6.27	70
	R3	6.50	73
	R4	6.22	70
	R5	6.25	70
	R6	6.17	69
Test Item	10 R1	5.98	67
	10 R2	5.82	65
	10 R3	6.05	68
	10 R4	5.96	67
	10 R5	6.10	69
	32 R1	5.99	67
	32 R2	6.17	69
	32 R3	5.73	64
	32 R4	6.04	68
	32 R5	5.93	67
	100 R1	5.98	67
	100 R2	5.96	67
	100 R3	5.91	66
	100 R4	6.01	68
	100 R5	5.75	65

R   = Replicate

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Dissolved Oxygen Saturation Level of 8.9 mg O2/L (%)
3,5-Dichlorophenol	3.2	6.51	73
	10	6.76	76
	32	7.79	88

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	3.8	2	2.4	42.00	-
	R2	3.8	3	1.8	40.00	-
	R3	3.5	2	2.1	42.00	-
	R4	4.0	3	1.9	42.00	-
	R5	4.9	4	2.1	42.00	-
	R6	4.4	3	2.2	44.00	-
Test Item	10R1	4.2	3	2.1	42.00	0
	10R2	4.0	3	1.9	42.00	0
	10R3	3.4	2	2.0	42.00	0
	10R4	4.3	3	2.3	40.00	5
	10R5	4.4	3	2.2	44.00	[5]
	32R1	3.0	1	2.3	42.00	0
	32R2	3.3	2	2.1	36.00	14
	32R3	3.9	3	2.1	36.00	14
	32R4	2.2	1	1.6	36.00	14
	32R5	3.4	2	2.1	39.00	7

R      = Replicate

[ ]   =Increase in respiration rate as compared to controls

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 (%)
Test Item	100R1	2.4	1	1.8	36.00	14
	100R2	2.4	1	1.8	36.00	14
	100R3	3.3	2	2.2	33.00	21
	100R4	2.4	1	1.9	30.00	29
	100R5	2.8	1	2.3	30.00	29
3,5-Dichlorophenol	3.2	5.9	8	2.0	29.25	30
	10	6.3	10	3.1	19.20	54
	32	7.9	10	6.9	6.00	86

R      = Replicate

Nominal Concentration (mg/L)		pH
		0 Hours	3 Hours
Test Item	100R1	7.5	7.5
	100R2	7.9	7.6
	100R3	7.5	7.4
	100R4	7.6	7.4
	100R5	7.5	7.4
3,5-Dichlorophenol	3.2	7.6	8.0
	10	7.8	7.9
	32	7.9	7.9

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	10R1	Pale yellow/brown very slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	10R2	Pale yellow/brown very slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	10R3	Pale yellow/brown very slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	10R4	Pale yellow/brown very slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	10R5	Pale yellow/brown very slightly cloudy dispersion with a few small oily globules of test item visible at the surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible

*    Observations made prior to the addition of activated sewage sludge

R      =Replicate

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Test Item	32R1	Pale yellow/brown slightly cloudy dispersion with a layer of small oily globules of test item visible at the surface	Dark brown dispersion with a thin oily film of test item visible at the surface	Dark brown dispersion, no undissolved test item visible
	32R2	Pale yellow/brown slightly cloudy dispersion with a layer of small oily globules of test item visible at the surface	Dark brown dispersion with a thin oily film of test item visible at the surface	Dark brown dispersion, no undissolved test item visible
	32R3	Pale yellow/brown slightly cloudy dispersion with a layer of small oily globules of test item visible at the surface	Dark brown dispersion with a thin oily film of test item visible at the surface	Dark brown dispersion, no undissolved test item visible
	32R4	Pale yellow/brown slightly cloudy dispersion with a layer of small oily globules of test item visible at the surface	Dark brown dispersion with a thin oily film of test item visible at the surface	Dark brown dispersion, no undissolved test item visible
	32R5	Pale yellow/brown slightly cloudy dispersion with a layer of small oily globules of test item visible at the surface	Dark brown dispersion with a thin oily film of test item visible at the surface	Dark brown dispersion, no undissolved test item visible
	100R1	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a thin oily film of test item visible at the surface
	100R2	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a thin oily film of test item visible at the surface
	100R3	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a thin oily film of test item visible at the surface
	100R4	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a thin oily film of test item visible at the surface
	100R5	Pale yellow/brown slightly cloudy dispersion with a layer of oily globules 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 a thin oily film of test item visible at the surface

*       Observations made prior to the addition of activated sewage sludge

R      = Replicate

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 PG-RAW-90-032 is on activated sewage sludge was greater than 1000 mg/l for the 3-hour contact time.

Executive summary:

A study according to OECD 209 was performed to assess the inhibitory effect of Substance PG-RAW-90 -032 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 greater than 1000 mg/l for the 3-hour contact time. The NOEC is considered to be 10 mg/l.

Description of key information

A study according to OECD 209 was performed to assess the inhibitory effect of Substance PG-RAW-90 -032 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 greater than 1000 mg/l for the 3-hour contact time. The NOEC is considered to be 10 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L