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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 26 September 2011. Experimental Completion Date: 27 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 August 2011
Analytical monitoring:
no
Details on test solutions:
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 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). Where necessary, pH values were adjusted to between pH 7.0 to 8.0 using 1.0 M Sodium Hydroxide or 0.5 M Sulphuric acid solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and was used on the day of collection. The pH of the sample was 7.4. Determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 2.7 g/L.
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 2 °C
Nominal and measured concentrations:
10, 100 and 1000 mg/L (nominal)
Details on test conditions:
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 approximately 0.5 – 1 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.

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into a 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 approximately 7.0 mg O2/L and 2.0 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.

The test was conducted under normal laboratory lighting in a temperature controlled room at 20 ± 2 ºC.


TEST MEDIUM / WATER PARAMETERS
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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. The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

TEST CONCENTRATIONS
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
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol at concentrations of 3.2, 10 and 32 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
100 - 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
Results after 30 minutes
The dissolved oxygen concentrations in all vessels after 30 minutes contact time confirm that the dissolved oxygen concentrations in all vessels were between 65 to 99 % of the dissolved oxygen saturation level of 8.9 mg O2/L at 21 °C and thereby the sludge flocs would have been maintained in suspension. The standard deviation of the 4 replicate control dissolved oxygen concentrations was 0.28 with the standard deviation of the 3 replicate 1000 mg/L test vessel dissolved oxygen concentrations being 0.25.

Results after 3 Hours
The mean of the 4 control oxygen consumption rates was 29.80 mg O2/L/hour with a standard deviation value of 8.1.

The mean of the 1000 mg/L test vessel oxygen consumption rates was 3.20 mg O2/L/hour with a standard deviation value of 0.92.

From these results it can be seen that the effect of the test item on the respiration rate of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of between 100 and 1000 mg/L.

Other results and observations
The pH values of the test preparations at the start and end of the exposure period showed a slight increase in pH in all vessels with the exception of the 1000 mg/L test vessels over the 3 hour study period. The pH values of the four replicate control vessels at 0 hours were 7.4 for replicates R1 and R2 and 7.6 and 7.7 for replicates R3 and R4 respectively and increased to values of 7.9 for replicate R1 and 8.0 for replicates R2, R3 and R4 after 3 hours contact time. For the reference item vessels containing 3,5-dichlorophenol, the pH values at 0 hours were 7.5, 7.6 and 7.7 for the test concentrations of 3.2, 10 and 32 mg/L, which respectively increased to pH 8.1, 8.1 and 8.2 after 3 hours contact time. The pH values of the test item vessels at 0 hours were 7.6 for the test concentration of 10 mg/L, 7.7 for the test concentration of 100 mg/L and 7.6, 7.7 and 7.6 for the three replicate 1000 mg/L test vessels respectively. After 3 hours contact time these pH values were measured to be 7.9 for the test concentration of 10 mg/L, 7.8 for the test concentration of 100 mg/L and 6.5, 6.7 and 6.4 for the three replicate 1000 mg/L test vessels.

Observations made on the test preparations showed in all the test item vessels an oily layer of test item was visible at 0 hours. After 30 minutes and 3 hours contact time an increasing amount of oil was seen on the surface of a dark brown dispersion. In the reference item vessels, containing 3,5 dichlorophenol, the observations showed that no undissolved reference item was visible at 0 hours and after 30 minutes and 3 hours contact time and that the observations in all vessels remained the same after 30 minutes and 3 hours contact time. The observations of all vessels changed between 0 hours and 30 minutes contact time from pale yellow/brown dispersions to dark brown dispersions due to the addition of inoculum.

Study validation
The coefficient of variation of oxygen uptake in the control vessels was 27.0 % thereby passing the validation criterion of not being more than 30 %.

The mean specific respiration rate of the controls was 29.80 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied as the specific respiration rates of the controls should not be less than 20 mg oxygen per gram dry weight of sludge per hour.

In some instances, the initial dissolved oxygen concentrations were outside the top range of those recommended in the test guidelines (7.0 mg O2/L). 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.

Based on these results, it was considered not necessary to perform a definitive test in order to obtain a NOEC and/or an exact EC50 value for the test item.
Results with reference substance (positive control):
EC50 = 3.2 mg/L with 95% confidence limits of 2.1 - 5.0 mg/L

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)
*

Control

R1

6.7**

 

R2

6.8**

 

R3

6.2**

 

R4

6.4**

Test Item

10

7.0

 

100

6.6

 

1000 R1

6.3***

 

1000 R2

5.8***

 

1000 R3

6.1***

3,5-dichlorophenol

3.2

7.0

 

10

6.7

 

32

8.8

*Measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter

** Standard deviation= 0.28

*** Standard deviation= 0.25

R1– R4= Replicates 1 to 4

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

6.6

10

2.7

23.40*

-

 

R2

5.7

7

3.1

22.29*

-

 

R3

5.6

5

2.6

36.00*

-

 

R4

5.0

4

2.5

37.50*

-

Test Item

10

6.0

8

2.7

24.75

17

 

100

5.7

7

2.6

26.57

11

 

1000 R1

7.9

10

7.4

3.00**

90***

 

1000 R2

7.4

10

6.7

4.20**

86***

 

1000 R3

8.2

10

7.8

2.40**

92***

3,5-dichlorophenol

3.2

7.1

7

5.4

14.57

51

 

10

7.8

4

7.1

10.50

65

 

32

8.3

10

7.6

4.20

86

* Standard deviation= 8.1

** Standard deviation= 0.92

*** Statistically significant inhibition of respiration compared to control vessles (P≤0.05)

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms was assessed according to OECD Guideline 209 and gave a 3-Hour EC50 value of between 100 and 1000 mg/L.

Description of key information

Key study performed according to GLP and OECD Guideline 209:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of between 100 and 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

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

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 hr at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hr contact time and compared to data for the control and reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of between 100 and 1000 mg/L.

The reference item gave a 3-Hour EC50value of 3.2 mg/L, 95 % confidence limits 2.1- 5.0 mg/L.

Based on these results it was considered not necessary to perform a definitive test in order to obtain a NOEC and/or exact EC50value for the test item.