Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

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 as conducted between 01 November 2015 and 18 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 209 using a Carbon and Ammonium Oxidation method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Range-finding test
Test Item Preparation
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. The test item was dispersed directly in water.

Nominal amounts of test item (5, 50 and 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 temperatures of between 20 °C and 21 °C, 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 a Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Test Item Preparation
The test item was dispersed directly in water.

Nominal amounts of test item (50, 90 and 160 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 temperatures of between 20 °C and 21 °C, 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 and 320 mg/L (five replicates of each).

The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 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:

Test System
A mixed population of activated sewage sludge micro-organisms was obtained on 2 November 2015 for the range-finding test and on 17 November 2015 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.

Definitive test
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 7.6 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 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.
* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Between 19 and 22 °C

pH:

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:

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

Nominal and measured concentrations:

Range-Finding Test: Nominal Concentrations of 10, 100 and 1000 mg/L.
Definitive Test: Nominal Concentrations of 100, 180 and 320 mg/L.

Details on test conditions:

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 via narrow bore glass tubes at a rate of 0.5 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 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 19 and 22 ºC.

Evaluations
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.

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.

Data Evaluation
Calculation of the Oxygen Uptake Rates
The respiration rate, R, expressed in milligrams oxygen per liter per hour (mg O2/L/h), was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:

R = [(Q1 - Q2) / Δt] x 60

Where:
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L);
Δt = the time interval between the beginning and end of the selected section of the linear phase (min).

The specific respiration rate, RS, expressed as the amount of oxygen consumed per gram dry weight of sludge per hour (mg O2/g/h) was deduced according to the following equation:

RS = R / SS

Where:
SS = the concentration of suspended solids in the test mixture (g dry weight/L).


Calculation of Percentage of Inhibition
The percentage inhibition was calculated according to the following equation:

% inhibition = [1 – (R/Rbc)] x 100

Where:
Rbc = the mean respiration rate of the blank controls.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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.:

180 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 to 70% 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 100, 180 and 320 mg/L were selected for the definitive test.

The results from the range-finding test indicated that the effect of the test item on the respiration of activated sewage sludge gave a 3 Hour EC50 value of greater than 1000 mg/L.


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

Inhibition of Respiration Rate
No statistically significantly toxic effects were shown at the test concentrations of 100 and 180 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentration of 320 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 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 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.

Results with reference substance (positive control):

The 3 h EC10 was 2.5 mg/L.
The 3 h EC20 was 3.5 mg/L.
The 3 h EC50 was 9.5 with 95 % confidence limits of 7.6 - 12 mg/L.
The 3 h EC80 was 26 mg/L.

Reported statistics and error estimates:

ECX and NOEC
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.

95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

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 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).

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 4.06% and the specific respiration rate of the controls was 27.12 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

 

The validation criterion for the reference item EC₅₀value was also satisfied.

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	6.7	7.1
	100	5.9	7.1
	1000 R1	6.0	7.2
	1000 R2	6.2	7.1
	1000 R3	6.1	7.1

 R₁– R₃= Replicates 1 to 3

  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)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	6.2	70
	R2	6.3	71
	R3	6.1	69
	R4	7.2	81
Test Item	10	6.7	75
	100	6.8	76
	1000 R1	6.0	67
	1000 R2	8.0	90
	1000 R3	5.9	66
3,5-dichlorophenol	3.2	7.5	84
	10	7.9	89
	32	8.8	99

R₁– R₄= Replicates 1 to 4

Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Timein 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	5.4	6	1.9	35.00	-
	R2	5.6	6	2.1	35.00	-
	R3	5.0	5	2.0	36.00	-
	R4	4.8	5	2.0	33.60	-
Test Item	10	4.6	4	2.1	37.50	[7]
	100	5.0	5	2.1	34.80	0
	1000 R1	5.8	10	2.1	22.20	36
	1000 R2	7.2	10	3.9	19.80	43
	1000 R3	5.3	9	2.0	22.00	37
3,5-dichlorophenol	3.2	6.2	9	2.1	27.33	22
	10	7.3	10	5.0	13.80	60
	32	8.3	10	7.5	4.80	86

[Increase in respiration rate as compared to controls]

R₁– R₄= Replicates 1 to 4

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	7.6	7.9
	R2	7.6	7.8
	R3	7.7	7.8
	R4	7.7	7.8
Test Item	10	7.7	7.8
	100	7.7	7.8
	1000 R1	7.7	7.7
	1000 R2	7.7	7.9
	1000 R3	7.7	7.7
3,5-dichlorophenol	3.2	7.6	8.0
	10	7.6	8.1
	32	7.7	8.2

R₁– R₄= Replicates 1 to 4

Observations on the Test Preparations throughout the Test Periodin 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 dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R1	Pale yellow/brown dispersion, globules of test item visible on surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R2	Pale yellow/brown dispersion, globules of test item visible on surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R3	Pale yellow/brown dispersion, globules of test item visible on surface	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
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

R₁– R₄= Replicates 1 to 4

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	100 R1	6.3	7.4
	100 R2	6.6	7.2
	100 R3	6.7	7.4
	100 R4	6.6	7.4
	100 R5	6.6	7.4
	180 R1	6.5	7.3
	180 R2	6.8	7.9
	180 R3	6.6	7.1
	180 R4	6.8	7.3
	180 R5	6.6	7.2
	320 R1	6.6	7.1
	320 R2	6.9	7.1
	320 R3	6.8	7.4
	320 R4	6.9	7.7
	320 R5	6.8	7.5

 R₁– R₅= Replicates 1 to 5

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

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	5.5	62
	R2	5.4	61
	R3	5.5	62
	R4	5.6	63
	R5	6.6	74
	R6	6.3	71
Test Item	100 R1	6.0	67
	100 R2	6.4	72
	100 R3	5.8	65
	100 R4	5.9	66
	100 R5	6.5	73
	180 R1	5.6	63
	180 R2	6.1	69
	180 R3	6.3	71
	180 R4	6.5	73
	180 R5	6.4	72

R₁– R₆= Replicates 1 to 6

DissolvedOxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test Continued 

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Test Item	320 R1	6.3	71
	320 R2	6.5	73
	320 R3	6.3	71
	320 R4	5.8	65
	320 R5	6.9	78
3,5-dichlorophenol	3.2	6.2	70
	10	6.4	72
	32	8.0	90

 R₁– R₅= Replicates 1 to 5

Validity criteria fulfilled:

yes

Conclusions:

Based on the range-finding results the effect of the test item on the respiration of activated sewage sludge gave a 3 Hour EC50 value of greater than 1000 mg/L.
The No Observed Effect Concentration (NOEC) after 3 hours exposure in the definitive test was 180 mg/L.

Executive summary:

The activate sludge respiration inhibition of the test substance, IFF 215 (Floriane) was assessed according to OECD 209 using an Carbon and Ammonium Oxidation method. The 3-Hour EC₅₀value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L.

Description of key information

The activate sludge respiration inhibition of the test substance, IFF 215 (Floriane) was assessed according to OECD 209 using an Carbon and Ammonium Oxidation method. The 3-Hour EC50 value was greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 180 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

180 mg/L