[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: 30 May 2011. Experimental completion date: 31 May 2017

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 13-0460
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 ºC, in the dark
Intended use/Application: fragrance ingredient for use in cleaners, detergents, fabric softeners and air fresheners

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Preparation
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).

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 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 (4.5 liters) was fed synthetic sewage (225 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 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 test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

SYNTHETIC SEWAGE
A synthetic sewage was prepared by dissolving the following ingredients per litre of dilution water:
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

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 measured temperatures of between 21 and 22 ºC.

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:

Test substance - Nominal: 10, 100, 1000 mg/l.
Reference substance - Nominal: 3.2, 10, 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. Finally two further control vessels were prepared.

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.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (C6H3CI2.OH), Purity 99.6%

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

Details on results:

The EC50 values for 3 hours contact time, derived by interpolation, are given below:
EC10: >1000 mg/L
EC20: > 1000 mg/L
EC50: > 1000 mg/L
EC80: > 1000 mg/L
NOEC: 1000 mg/L

The coefficient of variation of oxygen uptake in the control vessels was 4.26% and the specific respiration rate of the controls was 28.55 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.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Results with reference substance (positive control):

3,5-dichlorophenol EC50 (3 h): 7.5 mg/I

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	R1	4.8	4	1.8	45	-
	R2	5.1	4	2.2	43.5	-
	R3	5.5	5	2.1	40.8	-
	R4	4.3	3	2.2	42	-
Test Item	10	5	4	2	45	[5]
	100	3.9	3	1.8	42	2
	1000 R1	4.2	3	2.2	40	7
	1000 R2	5.2	5	1.9	39.6	8
	1000 R3	5.3	5	2	39.6	8
3,5- dichlorophenol	3.2	6.5	9	1.9	30.67	28
	10	7.3	10	4.3	18	58
	32	7.9	10	6.9	6	86

R1 – R4 = Replicates 1 to 4

[Increase in respiration rate as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (respiration inhibition) values of the substance 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 FRET 13-0460 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 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 FRET 13-0460

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 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

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L