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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 21 November 2017 Experimental completion date: 22 November 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 influence 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 11-0539
CAS Number: 1631962-93-0
Physical state/Appearance: clear colorless liquid
Storage Conditions: room temperature in the dark
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test Item Preparation
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 and1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 and 8.0 if necessary.
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 and Supporting Information
A mixed population of activated sewage sludge micro organisms was obtained on
21 November 2017 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 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
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 was pH 7.3 and the pH of the synthetic sewage stock used for the range finding test was pH 7.2. The pH values were measured using a Hach HQ40d Flexi handheld meter.

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 liters) was fed synthetic sewage (450 mL). The pH of the sample on the day of the test was 7.7 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 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
between 20 °C and 21 °C
pH:
The pH of the test item dispersions was measured and adjusted to between pH 7.0 and 8.0 if necessary.
Nominal and measured concentrations:
Test substance - Nominal (range-finding test): 10, 100 and1000 mg/L.
Reference substance - Nominal: 3.2, 10 and 32 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 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 20 °C and 21 °C.
Data from the control, reference item vessels and synthetic sewage was shared with similar concurrent studies.

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) with the exception of the 100 mg/L test vessel. 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 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:
EC10
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 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 concentration of 1000 mg/L. It was not possible to determine any statistically significant differences for the test concentration of 100 mg/L as the response could not be measured over the linear portion of the trace between values of between 7 mg O2/L and 2 mg O2/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 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.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 2.5% and the specific respiration rate of the controls was 31.35 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The EC50 value was 7.4 mg/L which satisfied the validation criterion for the reference item.
Results with reference substance (positive control):
3,5-dichlorophenol EC50 (3 h): 7.4 mg/I
3,5-dichlorophenol EC10 (3 h): 1.5 mg/I

The following results were derived:

 

FRET 11-0539

3,5-Dichlorophenol

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

ECx(3 Hours)
(mg/L)

95% Confidence Limits (mg/L)

EC10

<1000

-

  1.5

-

EC20

>1000

-

  2.3

-

EC50

>1000

-

  7.4

5.7 – 9.7

EC80

>1000

-

24

-

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

3.5

2

1.9

48.00

-

R2

4.8

3

2.4

48.00

-

R3

5.6

5

1.8

45.60

-

R4

4.8

4

1.7

46.50

-

Test Item

10

2.7

1

1.8

54.00

[15]

100

*

*

*

*

*

1000 R1

2.8

2

1.6

36.00

23

1000 R2

3.5

2

2.2

39.00

17

1000 R3

2.7

1

2.0

42.00

11

3,5-Dichlorophenol

3.2

6.3

7

2.4

33.43

29

10

6.8

10

3.4

20.40

57

32

8.0

10

7.0

6.00

87

 


R=   Replicate

-  =  Not applicable

*   = Oxygen consumption rate could not be calculated as the dissolved oxygen concentrations could not be measured over the linear portion of the trace as the initial and final dissolved oxygen concentrations were below 2 mg O2/L

[] =     Increase in respiration rate as compared to controls

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (respiration inhibition) values of the substance 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 11-0539 on the respiration of activated sewage sludge. Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5‑Dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50value of greater than 1000 mg/L. 

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

The reference item gave a 3‑Hour EC50value of 7.4 mg/L, 95% confidence limits 5.7 to 9.7 mg/L.

Description of key information

A study according to OECD 209 was performed to assess the inhibitory effect of SubstanceFRET 11-0539on the respiration of activated sewage sludge.Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5‑Dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50value of greater than 1000 mg/L. 

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

The reference item gave a 3‑Hour EC50value of 7.4 mg/L, 95% confidence limits 5.7 to 9.7 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information