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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: 28 November 2017, Experimental completion date: 08 December 2017
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 (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
Please see "Principles of method if other than guideline" for details.
Principles of method if other than guideline:
The following deviation from the study plan occurred:
The incorrect terminology for the measurement for Dissolved Organic Carbon (DOC) of the deionized reverse osmosis water was given in the Study Plan. The measurement reported was Total Organic Carbon (TOC) not DOC which was a deviation to the Study Plan.
The terminology TOC rather than DOC is correct as the deionized reverse osmosis water did not need to be filtered prior to analysis therefore Total Organic Carbon was measured, not Dissolved Organic Carbon.
This deviation was considered to have had no effect on the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was dispersed directly in water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained for the initial range-finding test on 29 November 2017 and on 7 December 2017 for the second range-finding test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic 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.

In the second range-finding test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.4 and the pH of the synthetic sewage stock used for the second range-finding test was pH 7.3. 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 (9.5 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 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 two consecutive dry weights within 4% were attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not stated
Test temperature:
19 °C - 22 °C
pH:
7.3 - 7.4, The pH values were measured using a Hach HQ40d Flexi handheld meter.
Dissolved oxygen:
2 mg O2/L - 7 mg O2/L
Salinity:
n/a
Conductivity:
not stated
Nominal and measured concentrations:
nominal test concentrations: 10, 100 and 1000 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 prior to the test item vessels. 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 °C and 22 °C.
TEST CONCENTRATIONS
In the initial 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. However it was considered necessary to repeat this range-finding test due to inconsistent results shown between the replicate 1000 mg/L test vessels with two out of the three vessels showing significant inhibition. It could therefore not be concluded which results were erroneous.
In the second 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 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.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The test water used for the initial and second range-finding tests was deionized reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).
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:
growth inhibition
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Second 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 indicating that the sludge flocs were in suspension in the test.
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.
Percentage inhibition is plotted against concentration for the reference item, 3,5-Dichlorophenol.

Initial Range-Finding Test

It was considered necessary to repeat this range-finding test due to inconsistent results shown between the replicate 1000 mg/L test vessels with two out of the three vessels showing significant inhibition. It could therefore not be concluded which results were erroneous.

 Inhibition of Respiration Rate

The following results were derived:

 

Petrit T

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

-

   1.7

-

EC20

>1000

-

   2.5

-

EC50

>1000

-

   8.1

6.2 - 11

EC80

>1000

-

26

-

NOEC

  1000

-

-

-

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.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD

Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Following an initial range-finding test, in a second range-finding test activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and

1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 19 °C and 22 °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.

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 was 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 EC50 value of 8.1 mg/L, 95% confidence limits 6.2 to11 mg/L.

Description of key information

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 was 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

Additional information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD

Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

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 was 1000 mg/L.

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

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