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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, carried out according to recognised guidelines
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
DEVIATION FROM THE STUDY PLAN

Concerning: Preparations of the test flasks

According to the Study Plan: At the start of the test 132 mL activated sludge inoculum with a sludge concentration of 2-4 g/L (dry weight) will be added, first to first control (C1), then in time intervals of approximately 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item and the test item and finally to a second control (C2).

Deviation: At the start of the test 132 mL activated sludge inoculum with a sludge concentration of 4 g/L (dry weight) was added, first to first control (C1), then in time intervals of 15 minutes (an arbitrary but convenient interval) to the test solutions of the reference item and the test item and finally to a second control (C2). Time interval between the last test solution flask and the second control was more than 15 minutes.

Reason for this change: Technical
Presumed Effect on the Study: None
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50.

Defined amounts of test item were directly weighed into the designated test flasks to reach the planned nominal concentrations. The nominal test item concentrations were prepared by mechanical dispersion using shortly (5 min) ultrasonic bath. These test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.
Untreated Control (C1 and C2): Two controls (deionised water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
Reference Control (R1 – R3): In parallel to the study with the test item, the reference item 3,5-Dichlorophenol was tested (the nominal test concentrations of 5, 16 and 32 mg/L) under otherwise identical test conditions. A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209: 0.25 g of 3,5-Dichlorophenol was dissolved in 5 mL 1 mol/L NaOH and diluted to about 15 mL with deionised water. Excess of NaOH was neutralised with approximately 4 mL of 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to 0.5 litre with deionised water. The final pH was measured to be 7.62 and the final concentration amounted 500 mg/L.

Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL)

Peptone 16.0g
Meat extract 11.0g
Urea 3.0g
NaCl 0.7g
CaCl2 x 2H2O 0.4g
MgSO4 x 7H2O 0.2g
K2HPO4 2.8g
Deionised water add 1000.0mL

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Test organisms comprised activated sludge, microorganisms from a domestic waste water treatment plant.
The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.20. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
Room temperature (20-25 deg C)
pH:
7.21 - 7.87
Dissolved oxygen:
7.0 - 8.5
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
10, 31, 100, 313, 1000 mg/l
Details on test conditions:
Surrounding type: Climate chamber (during the incubation) and controlled environment room (during the formulation and oxygen measuring)
Temperature: 19.5 – 20.6 deg C (during the incubation) and 20.0 – 20.9 deg cC (during oxygen measurement)
Aeration: With compressed air (1 litre per minute)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (the nominal test concentrations of 5, 16 and 32 mg/L) under otherwise identical test conditions.
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:
In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration of 10 mg Reofos 65/L. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 2.1 % and 4.2 % in the examined nominal test concentration range (31 - 1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested.
Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L.
The NOEC was determined to be 1000 mg/L. The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (4.2 %) at the concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.460 (at 1000 mg/L) was not in the historical control data range (0.524 ±0.070), but the deviation from the control was within +/- 15 %, it can be considered as a biological variability of the test system.
Results with reference substance (positive control):
The following nominal concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 5, 16 and 32 mg/L. In comparison to the controls the respiration rate of the activated sludge was inhibited by 35.4 % at the lowest nominal concentration of 5 mg/L. At the nominal concentrations of 16 and 32 mg/L, the respiration rate was inhibited by 60.4 % and 81.3 %, respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 9.46 mg/L with 95 % confidence limits of 7.40 to 12.11 mg/L.

Table 2.     Influence of test item on oxygen consumption of activated sludge

Flask
No.

ID

Test group

Concentration
(mg/L)

Oxygen consumption
(mg O2/L/min)

Inhibition
(%)

pH-values

Oxygen concentration
(mg O2/L)

start *

end *

start *

end *

1

C1

Control

0.490

7.26

7.31

8.1

8.0

15

C2

Control

0.470

7.29

7.36

8.4

7.9

 

Mean

0.480

 

Deviation (%)

4.2

5

T1

Test item

10

0.480

0.0

7.28

7.82

8.4

7.3

6

T2

Test item

31

0.470

2.1

7.26

7.87

8.5

7.6

7

T3

Test item

100

0.470

2.1

7.27

7.80

8.4

7.7

8

T4

Test item

313

0.470

2.1

7.32

7.80

8.5

7.0

9

T5

Test item

1000

0.460

4.2

7.21

7.79

8.4

7.0

Validity Criteria 

-The respiration rates of the two controls did not differ by more than 15 % (4.2 %).

-The 3-hour EC50of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be in the range of 5 to 30 mg/L (9.46 mg/L).

-The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.0 mg O2/L.

Validity criteria fulfilled:
yes
Conclusions:
This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L. The substance does not inhibit the respiration rate of sewage sludge microorganisms, and is not considered to be harmful to organisms of this nature. No classification is applicable.
Executive summary:

This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%

A laboratory test was carried out with Reofos 65 to evaluate the effect of the test item on microorganisms by measuring the respiration rate. The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50.

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration of 10 mg Reofos 65/L. In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 2.1 % and 4.2 % in the examined nominal test concentration range (31 - 1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested.

In parallel to the study with the test item, the reference item 3,5-Dichlorophenol was tested (the nominal test concentrations of 5, 16 and 32 mg/L) under otherwise identical test conditions. 

The 3-hour EC50of 3,5-Dichlorophenol was calculated to be 9.46 mg/L with 95 % confidence limits of 7.40 to 12.11 mg/L.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50and EC80were higher than 1000 mg/L.

The NOEC was determined to be 1000 mg/L.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (4.2 %) at the concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.460 (at 1000 mg/L) was not in the historical control data range (0.524 ± 0.070), but the deviation from the control was within +/- 15 %, it can be considered as a biological variability of the test system.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study conducted to recognised guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
Each flask (500 ml capacity, glass, conical) containing Durad 310M was prepared by adding 16 ml synthetic sewage feed, 200 ml microbial inoculum, the appropriate weight of Durad 310M and a total volume of 284 ml deionised water. The reference material vessels were prepared with 16 ml synthetic sewage feed, 200 ml microbial inoculum, an appropriate volume of the reference material stock and sufficient deionised water to give a total volume of 500 ml. One flask was prepared at each concentration for test and reference materials. In addition, 2 control flasks, containing 16 ml synthetic sewage feed, 200 ml microbial inoculum and 284 ml deionised water were prepared. Flasks were prepared at 15 min intervals, with the control flasks being at the beginning and end of the sequence, and transferred to an aeration manifold. Air (BOC zero grade) was bubbled through each flask for 3 hours. The flow rate through the system was measured (using a flow-meter) as 5 l/min (total flow through 10 vessels i.e. 0.5 l.vessel/min).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A sample of activated sewage sludge was obtained from Haddington Municipal Sewage Works on 7 September 2000. After the sludge had been allowed to settle, the overlying, clear supernatant was removed by siphon and stored for later use. The remaining suspended solids were shaken by hand to homogenise thoroughly.

Subsamples of the homogenised sludge were dried in an oven at ca 105 °C to determine dry weight. The final concentration of solids in the sample was adjusted to be 4.0 g/l , final with the supernatant set aside earlier. Initially the concentration of solids was 6.0 g/l .
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Not conducted.
Hardness:
Not measured
Test temperature:
The test area temperature during the experiment was 19.0-19.8 °C. The flask temperatures ranged from 19.4-20.3 °C.
pH:
Not measured
Dissolved oxygen:
Inclusive within experiment; see below.
Salinity:
Not applicable.
Nominal and measured concentrations:
The test was conducted at the following nominal concentrations of Durad 310M: 1000, 316, 100, 31.6 and 10 p.p.m.
The actual weighings of test material made were: 0.5003, 0.1587, 0.0506, 0.0158, 0.0053 g
The reference material, 3,5-DCP, was tested at the following nominal concentrations: 30, 15 and 5 p.p.m.
Details on test conditions:
Reference Material
3,5-Dichlorophenol (DCP), was obtained from Aldrich Chemicals (Batch No. 00127).

For the test, a solution of 3,5-DCP was prepared at a nominal concentration of 1 g/l . 3,5-DCP (0.1003 g) was dissolved in 100 ml deionised water. The flask was placed in an ultrasonic bath for ca 5 min to aid dissolution.

Microbial Inoculum
A sample of activated sewage sludge was obtained from Haddington Municipal Sewage Works on 7 September 2000. After the sludge had been allowed to settle, the overlying, clear supernatant was removed by siphon and stored for later use. The remaining suspended solids were shaken by hand to homogenise thoroughly.
Subsamples of the homogenised sludge were dried in an oven at ca 105 °C to determine dry weight. The final concentration of solids in the sample was adjusted to be 4.0 g/l , final with the supernatant set aside earlier. Initially the concentration of solids was 6.0 g/l .

Synthetic Sewage Feed
A synthetic sewage feed was prepared by combining the following constituents and making up to a total volume of 500 ml with deionised water.

peptone (8.000 g)
meat extract (5.495 g)
urea (1.501 g)
sodium chloride (0.349 g)
calcium chloride dihydrate (0.204 g)
magnesium sulphate heptahydrate (0.1006 g)
dipotassium hydrogen phosphate (1.387 g)

Test Procedure
The test was conducted at the following nominal concentrations of Durad 310M: 1000, 316, 100, 31.6 and 10 p.p.m.
The actual weighings of test material made were: 0.5003, 0.1587, 0.0506, 0.0158, 0.0053 g
The reference material, 3,5-DCP, was tested at the following nominal concentrations: 30, 15 and 5 p.p.m.

The volumes of the reference material stock solution used to achieve these concentrations were 15, 7.5 and 2.5 ml respectively.

Each flask (500 ml capacity, glass, conical) containing Durad 310M was prepared by adding 16 ml synthetic sewage feed, 200 ml microbial inoculum, the appropriate weight of Durad 310M and a total volume of 284 ml deionised water. The reference material vessels were prepared with 16 ml synthetic sewage feed, 200 ml microbial inoculum, an appropriate volume of the reference material stock and sufficient deionised water to give a total volume of 500 ml. One flask was prepared at each concentration for test and reference materials. In addition, 2 control flasks, containing 16 ml synthetic sewage feed, 200 ml microbial inoculum and 284 ml deionised water were prepared. Flasks were prepared at 15 min intervals, with the control flasks being at the beginning and end of the sequence, and transferred to an aeration manifold. Air (BOC zero grade) was bubbled through each flask for 3 hours. The flow rate through the system was measured (using a flow-meter) as 5 l/min (total flow through 10 vessels i.e. 0.5 l.vessel/min).

After an incubation period of 3 hours, the contents of the flask were transferred to a measuring bottle and the dissolved oxygen concentration recorded at 30 s intervals for 10 min using a Yellow Springs Incorporated Limited Dissolved Oxygen Meter (Model No. 5100) and a YSl DO Probe (Model No. 5010).

Calculation Of Results
The portion of the measurement period used for calculation of respiratory rate is presented in Table 1 below. These data represented the linear portions and respiration rates were determined by linear regression. The inhibition of respiration at each concentration of the test or reference material was calculated.
A probit transformation was applied to the percentage inhibition values. The probit transformed data were subjected to a regression procedure against logarithmically transformed concentrations of the material, with a Newton-Raphson maximum likelihood iterative procedure being used to obtain parameter estimates (Finney, 1971) for the test and reference materials separately. From the fitted model, the EC20, EC50 and EC80 values were estimated (only for the reference material). This was not possible for the test material. The goodness of fit of the probit model to the data was checked via the Pearson chi-squared test statistic.

Environmental Control
The temperature range recorded in the test area (by a minimum-maximum thermometer) over the period of the test and the temperature ranges in the flasks at the beginning and end of the oxygen measurements were measured.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: ppm
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: based on ppm
Details on results:
Globules of test material were observed in vessels at 100, 316 and 1000 p.p.m. Durad 310M.

These globules were dispersed in the test medium by aeration of the flasks throughout the 3 h incubation period.

The test area temperature during the experiment was 19.0-19.8 °C. The flask temperatures ranged from 19.4-20.3 °C.

The respiration rates for the 2 control vessels were within 4% of each other at the 3 h measurement which meets the validity criteria (<15 %). The EC50 of the reference material was 7.7 p.p.m. and within the range 5 to 30 p.p.m. which also met the criteria for a valid test. The chi-squared test was not significant (p=0.26) indicating that the positive control data was a good fit to the probit model.

The test met all relevant validity criteria.
Results with reference substance (positive control):
Results for the reference substance are presented in table form - See "Any other information".
Reported statistics and error estimates:
The respiration rates for the 2 control vessels were within 4% of each other at the 3 h measurement which meets the validity criteria (<15 %). The EC50 of the reference material was 7.7 p.p.m. and within the range 5 to 30 p.p.m. which also met the criteria for a valid test. The chi-squared test was not significant (p=0.26) indicating that the positive control data was a good fit to the probit model.

The following results were obtained in this test:

 

Treatment

EC Number

Value (p.p.m.)

95% Confidence Limits (p.p.m.)

 

 

 

Lower

Upper

Durad 310M

EC20

EC50

EC80

>1000

>1000

>1000

NA

NA

NA

NA

NA

NA

3,5-DCP

EC20

EC50 EC80

2.53

7.71

23.50

1.32

5.78

18.53

3.71

9.54

33.40

 

Table 1 

Durad 310M Activated Sludge, Respiration Inhibition Test Dissolved O2Values (mg O2.t1)

 

Time (min)

Flask Identity

 

CI

T10

T31.6

T100

T316

T1000

R5

R15

R30

CM

0.5

-

-

-

-

-

-

-

-

 

-

1.0

-

-

-

-

-

-

-

-

 

-

1.5

-

-

-

-

-

-

-

-

-

-

2.0

6.41

6.08

4.97

5.29

3.70

3.34

6.47

7.72

8.03

4.87

2.5

6.02

5.68

4.54

4.90

3.29

2.90

6.21

7.65

7.99

4.51

3.0

5.67

5.30

4.12

4.48

2.89

2.47

5.97

7.55

7.91

4.15

3.5

5.32

4.89

3,72

4.08

2.49

2.03

5.74

7.45

7.84

3.74

4.0

4.93

4.52

3.32

3.64

2.10

1.60

5.52

7.37

7.80

3.37

4.5

4.58

4.12

2.91

3.25

1.70

1.17

5.26

7.24

7.73

2.98

5.0

4.21

3.72

2.50

2.85

1.31

0.75

5.02

7.16

7.64

2.60

5.5

3.84

3.32

2.12

2.44

0.92

0.37

4.78

7.04

7.58

2.23

6.0

3.48

2.93

1.69

2.04

-

-

4.53

6.95

7.53

1.85

6.5

3.11

2.53

1.32

1.64

-

-

4.31

6.84

-

1.47

7.0

2.74

2.13

0.92

1.24

-

-

4.05

6.74

-

1.11

7.5

2.37

1.74

-

0.84

-

-

3.84

6.66

-

0.74

8.0

2.01

1.35

-

-

-

-

3.58

6.55

-

-

8.5

1.65

0.96

-

-

-

-

3.33

6.45

-

-

9.0

-

-

-

-

-

-

-

-

-

-

9.5

-

-

-

-

-

-

-

-

-

-

10.0

-

-

-

-

-

-

-

-

-

-

C = Control

T = Test material

R = Reference material 3,5-DCP

- = Data not part of the linear portion

 

Table 2

 

Durad 310M Activated Sludge, Respiration Inhibition TestRespiration Rates and Percent age Inhibition

 

 

 

 

Respiration

 

Treatment

Concentration (p.p.m.)

Rate (mg O2 / l /h

%

Inhibition

Control 1

0

43.94

NA

Durad 310M

10

47.35

0

 

31.6

48.63

0

 

100

48.74

0

 

316

47.74

0

 

1000

51.86

0

3, 5-DCP

5

28.76

35.6

 

15

11.92

73.3

 

30

7.83

82.5

 

Validity criteria fulfilled:
yes
Conclusions:
This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at <5%

The EC50 value of Durad 310M was >1000 p.p.m. under the conditions of this test. Therefore, Durad 310M was determined not to inhibit respiration in this test system at concentrations up to 1000 p.p.m.
Executive summary:

Durad 310M was tested at concentrations of 1000, 316, 100, 31.6, 10 and 0 mg/l. Globules of test material were observed in vessels at 100, 316 and 1000 ppm Durad 310M. These globules were dispersed in the test medium by aeration of the flasks throughout the 3 h incubation period.

The temperature recorded in the flasks during oxygen measurements was within protocol specifications. The temperature of the test area recorded at the beginning and end of the test was also within protocol specifications.

The test met all relevant validity criteria.

In conclusion Durad 310M, at concentrations up to 1000 ppm produced no inhibitory effect on respiration in this test system.

Description of key information

Toxicity to microorganisms

Key value for chemical safety assessment

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

Additional information

Two studies are presented for Activated Sludge Respiration Inhibition with the following results:

 

Where TPP content > 5%

NOEC (3 h): 1000 mg/L test mat. (nominal) based on: respiration rate

EC50 (3 h): > 1000 mg/L test mat. (nominal) based on: respiration rate

 

Where TPP content < 5%

EC50 (3 h): > 1000 ppm dissolved (nominal) based on: respiration rate

 

The substance is not considered to pose a hazard to STP microorganisms and is not dependent on triphenyl phosphate content.