[No public or meaningful name is available]

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 26 June 2020
Experimental completion date 27 October 2020

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 influenc the result.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 18-0091
Chemical Name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1yl)oxy]pentan‐2‐ol and 1 [(1,4,4trimethyltricyclo[6.3.1.02,5]dodecan‐8yl)oxy]pentan‐2‐ol
Batch: RDEA571-42
Purity: 92%
Physical state / Appearance: Light yellow viscous liquid
Expiry date: 01 December 2021
Storage conditions: Room temperature 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 (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.
An initial test was conducted however 3 replicate readings at the 1000 mg/L could not be achieved therefore the test was repeated and the results of the initial test not reported. 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 parafilm 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 Hach HQ30d 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:

Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on 26 October 2020 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, 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, determination of the suspended solids level of the activated sewage sludge was carried out. A sample (100 mL) of the activated sewage sludge was filtered by suction through a pre-weighed GF/A filter paper (rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven) 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 2.3 g/L and was adjusted to 3.0 g/L prior to the activated sewage sludge (7 L) being fed an aliquot of synthetic sewage (350 mL).
The pH of the sample on the day of the test was 7.4 measured using a Hach HQ30d Flexi handheld meter. The suspended solids level of the activated sewage sludge was checked prior to use to ensure the suspended solids concentration was equal to 3.0 g/L.

Dilution water:
The test water used for the test 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 L 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 test was between pH 7.2 and 7.3. The pH values were measured using a Hach HQ30d Flexi handheld meter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 °C

pH:

7.4 to 7.5 at 0 hours
7.5 to 7.6 at 3 hours

Dissolved oxygen:

2.8 (mg O2/L) to 4.1 (mg O2/L) at 0 hours
1.7 (mg O2/L) to 2.1 (mg O2/L) at 3 hours

Nominal and measured concentrations:

Test substance - Nominal: 10 mg/l 100 mg/l 1000 mg/l
Reference substance - Nominal: 3.2 mg/l 10mg/l 32mg/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 at a rate of 0.5 to 1.0 L 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, 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 a measured temperature of 20 °C.

Reference substance (positive control):

yes

Remarks:

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

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

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 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 any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validation Criteria

• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 6.3 mg/L.
• The specific respiration rate of the controls was 27.67 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 2.41%. 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 10, 100 and 1000 mg/L. Based on these results the NOEC after 3 hours exposure was 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.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (3 h): 6.3 mg/I 95% confidence limits 2.3 - 17 mg/l

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	7.3
	100	6.5	7.1
	1000 R1	6.6	7.5
	1000 R2	6.3	7.6
	1000 R3	6.5	7.7

 

R    = Replicate

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	1	6.02	68
	2	6.08	68
	3	6.05	68
	4	6.19	70
Test Item	10	5.89	66
	100	5.92	67
	1000 R1	6.10	69
	1000 R2	6.04	68
	1000 R3	6.11	69
3,5-dichlorophenol	3.2	6.11	69
	10	6.27	70
	32	8.10	91

 

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 O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	Inhibition (%)
Control	1	3.1	1	2.4	42.00	-
	2	3.5	2	2.1	42.00	-
	3	4.6	4	1.8	42.00	-
	4	4.0	3	2.0	40.00	-
Test Item	10	3.9	3	1.8	42.00	[1]
	100	3.2	2	1.8	42.00	[1]
	1000 R1	2.8	1	2.1	42.00	[1]
	1000 R2	3.0	2	1.7	39.00	6
	1000 R3	4.1	3	2.1	40.00	4
3,5-dichlorophenol	3.2	5.8	9	1.9	26.00	37
	10	6.4	10	3.3	18.60	55
	32	7.6	10	6.8	4.80	88

 

-       = Not applicable

R    = Replicate

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	1	7.4	7.3
	2	7.2	7.4
	3	7.4	7.6
	4	7.3	7.6
Test Item	10	7.5	7.6
	100	7.4	7.6
	1000 R1	7.5	7.5
	1000 R2	7.5	7.5
	1000 R3	7.4	7.6
3,5-dichlorophenol	3.2	7.3	7.9
	10	7.4	7.7
	32	7.3	7.7

 

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	1	Pale yellow/brown dispersion	Very dark brown/black dispersion	Very dark brown/black dispersion
	2	Pale yellow/brown dispersion	Very dark brown/black dispersion	Very dark brown/black dispersion
	3	Pale yellow/brown dispersion	Very dark brown/black dispersion	Very dark brown/black dispersion
	4	Pale yellow/brown dispersion	Very dark brown/black dispersion	Very dark brown/black dispersion
Test Item	10	Pale yellow/brown dispersion, with some small oily globules of test item visible at the surface	Very dark brown/black dispersion, with a few small oily globules of test item at the surface	Very dark brown/black dispersion, with a few small oily globules of test item at the surface
	100	Pale yellow/brown slightly hazy dispersion, with oily globules of test item visible at the surface	Very dark brown/black dispersion, with some oily globules of test item visible at the surface	Very dark brown/black dispersion, with some oily globules of test item visible at the surface
	1000 R1	Pale yellow/brown cloudy dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface
	1000 R2	Pale yellow/brown cloudy dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface
	1000 R3	Pale yellow/brown cloudy dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface	Very dark brown/black dispersion, with many oily globules of test item visible at the surface

 

*          Observations made prior to the addition of activated sewage sludge

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
3,5-dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Very dark brown/black dispersion, no undissolved reference item visible	Very dark brown/black dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Very dark brown/black dispersion, no undissolved reference item visible	Very dark brown/black dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Very dark brown/black dispersion, no undissolved reference item visible	Very dark brown/black 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 18-0091 is on activated sewage sludge was >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 FRET 18-0091 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  >1000 mg/l for the 3-hour contact time. The NOEC is considered to be 1000 mg/l.

Description of key information

A study according to OECD 209 was performed to assess the inhibitory effect of Substance FRET 18-0091 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  >1000 mg/l for the 3-hour contact time. The NOEC is considered to be 1000 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

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