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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 27 July 2016 and 07 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Remarks:
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 is reflected in the GLP compliance statement.
Vehicle:
no
Details on test solutions:
Range-Finding Test

Test Item Preparation
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
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 (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 22 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).

Reference Item Preparation
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 12 minutes. The pH of this stock solution was measured to be pH 5.6 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 32, 100, 320 and 1000 mg/L

Test Item Preparation
The test item was dispersed directly in water.
Amounts of test item (5, 16, 50, 160 and 500 mg (five replicates of each)) 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 21 C and 22 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, 32, 100, 320 and 1000 mg/L (five replicates of each).

Reference Item Preparation
For the purpose of the test a reference item, 3,5-dichlorophenol was used. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 5.9 and adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
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 (10 liters) was fed synthetic sewage (500 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not reported
Test temperature:
21 - 22”C
pH:
7.1 - 7.5
Dissolved oxygen:
5.9 - 8.3 mg O2/L or 66 - 93% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Range-Finding Test
Nominal test concentrations of 10, 100 and 1000 mg/L

Definitive Test
Test concentrations were 10, 32, 100, 320 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. Two additional control vessels were then prepared prior to the test item vessels being prepared as described in "Details of Test Item" (above). 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 21 and 22 ºC
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
>= 520 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Range-Finding Test
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% 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 concentrations of 100 and 1000 mg/L.
Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain both a NOEC and an EC50 value for the test item. Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg/L were selected for the definitive test.

Definitive Test
The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 1. Oxygen consumption rates and percentage inhibition values for the control, test and reference items after 3 hours contact time are given in Table 2.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 6.8% and the specific respiration rate of the controls was 27.53 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

Inhibition of Respiration Rate
The following results were derived:
FRET 10-0367 3,5-dichlorophenol
ECx (3 Hours)(mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)

EC10 99 - 1.2 -
EC20 130 - 1.9 -
EC50 520 - 6.5 4.9 - 8.6
EC80 >1000 - 23 -
NOEC 100 - - -

It was not possible to determine an EC80 value for the test item as no concentration tested resulted in greater than 80% inhibition. It was therefore not possible to calculate 95% confidence limits for the test item as the data generated did not fit the models available for this calculation.
No statistically significantly toxic effects were shown at the test concentrations of 10, 32 and 100 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 320 and 1000 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 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.
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Results with reference substance (positive control):
See "Details on Results" (above)

T

R1 – R5 = Replicates 1 to 5

[Increase in respiration rate as compared to controls]

* Results considered to be anomalous so omitted from the calculation for the EC50 and NOEC values

able 1: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Definitive Test

Dissolved Oxygen Concentrations of the Test Preparations after

30 Minutes Contact Time in the Definitive Test

 

 Dissolved Oxygen Concentration

(mg O2/L)

 Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L

 Control

 R1

 5.8

 65

 

 R2  5.4  61
   R3  5.7  64
   R4  5.4  61
   R5  5.7  64
   R6  5.6  63
 Test Item  10 R1  5.4  61
   10 R2  5.5  62
   10 R3  5.5  61
   10 R4  5.4  61
   10 R5  5.5  62
   32 R1  8.0  90
   32 R2  6.6  74
   32 R3  5.5  62
   32 R4  5.8  62
   32 R5  5.4  61
   100 R1  5.5  62
   100 R2  6.1  69
   100 R3  6.1  69
   100 R4  5.6  63
   100 R5  5.9  66
   320 R1  6.0  67
   320 R2  5.8  65
   320 R3  5.5  62
   320 R4  5.6  63
   320 R5  6.1  69
   1000 R1  5.4  61
   1000 R2  5.5  62
   1000 R3  5.5  62
   1000 R4  6.6  74
   1000 R5  6.5  73
 3,5 -dichlorophenol  3.2  5.4  61
   10  5.6  63
   32  7.6  85

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive 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

 5.1

 4.

 2.5

 39.00

 -

 R2

 5.4

 5

 2.0

 40.80

 -

 R3

 4.9

 4

 2.1

 42.00

 -

 R4

 3.2

 2

 1.9

 39.00

 -

 R5

 4.9

 4

 1.8

 46.50

 -

 R6

 4.4

 4

 1.7

 40.50

 -

 Test Item 10 R1

 4.2

 3

 2.1

 42.00

 [2]

 10 R2

 3.3

 2

 1.9

 42.00

 [2]

 10 R3

 3.8

 3

 1.47

 42.00

 [2]

 10 R4

 3.6

 3

 1.5

 42.00

 [2]

 10 R5

 4.4

 3

 2.1

 46.00

 [11]

 32 R1

 5.1

 7

 2.3

 24.00

 42*

 32 R2

 4.2

 3

 2.0

 44.00

 [7]

 32 R3

 2.9

 1

 2.1

 48.00

 [16]

 32 R4

 3.1

 3

 1.5

 44.00

 [7]

 32 R5

 3.8

 3

 1.6

 44.00

 [7]

 100 R1  4.4  4  2.1  34.50  16
 100 R2  3.9  3  2.2  34.00  18
 100 R3  4.0  4  2.1  38.00  8
 100 R4  3.8  3  1.9  38.00  8
 100 R5  3.5  2  2.2  39.00  6
 320 R1  5.3

 8

 2.1

 23.25

 44

 320 R2

 5.1

 4

 2.1

 45.00

 [9]

 320 R3  5.2  8  2.1  23.25  44
 320 R4  4.4  4  1.5  43.50  [5]
 320 R5  5.7  10  2.1  21.60  48
 1000 R1  5.9  10  2.6  19.80  52
 1000 R2  5.8  10  3.0  18.60  51
 1000 R3  6.1  10  3.0  18.60  55
 1000 R4  5.9  10  2.4  21.00  49
 1000 R5  6.3  10  3.0  49.80  52
 3,5 -dichlorophenol 3.2  6.3  10  1.8  27.00  35
 10  6.6  10  3.6  18.00  56
 32  7.9  10  7.2  4.20  90

R1 – R5 = Replicates 1 to 5

[Increase in respiration rate as compared to controls]

* Results considered to be anomalous so omitted from the calculation for the EC50 and NOEC values

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 520 mg/L*.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for this calculation.
Executive summary:

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 a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L (5 replicates) for a period of 3 hours at measured temperatures of between 21 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 520 mg/L*.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

The reference item gave a 3-Hour EC50 value of 6.5 mg/L, 95% confidence limits 4.9 to 8.6 mg/L.

* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for this calculation.

Description of key 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)".

Methods

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L

(5 replicates) for a period of 3 hours at measured temperatures of between 21 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 520 mg/L*.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 100 mg/L.

The reference item gave a 3-Hour EC50 value of 6.5 mg/L, 95% confidence limits

4.9 to 8.6 mg/L.

* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for this calculation.

Key value for chemical safety assessment

EC50 for microorganisms:
520 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information