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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09 - 2018-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
Adopted July 22 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2015-03-09

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyl hydrogen phosphate
EC Number:
221-485-7
EC Name:
Dioctyl hydrogen phosphate
Cas Number:
3115-39-7
Molecular formula:
C16H35O4P
IUPAC Name:
Dioctyl hydrogen phosphate
Constituent 2
Chemical structure
Reference substance name:
Octyl dihydrogen phosphate
EC Number:
223-638-3
EC Name:
Octyl dihydrogen phosphate
Cas Number:
3991-73-9
Molecular formula:
C8H19O4P
IUPAC Name:
octyl dihydrogen phosphate
impurity 1
Chemical structure
Reference substance name:
Trioctyl phosphate
EC Number:
217-305-1
EC Name:
Trioctyl phosphate
Cas Number:
1806-54-8
Molecular formula:
C24H51O4P
IUPAC Name:
trioctyl phosphate
impurity 2
Chemical structure
Reference substance name:
Octanol
EC Number:
249-405-6
EC Name:
Octanol
Cas Number:
29063-28-3
Molecular formula:
C8H18O
IUPAC Name:
octan-1-ol
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The activated sludge was obtained from the sewage treatment works in Flörsheim/Main (Germany) treating predominantly domestic sewage.
- Method of cultivation:
Washing medium for activated sludge: reconstituted water (OECD 1992)
Frequency of washing: three times; between washings the activated sludge was centrifuged for 2 minutes at 1000 g
Storage time before test start: 2 days
Storage temperature: 20 ± 2 °C
During storage 50 mL (nominal) of synthetic sewage feed were added per litre activated sludge and per day. The activated sludge was stirred and aerated during storage

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
20.2–20.7 °C (n = 34)
pH:
pH-value of the activated sludge: 6.8
pH value in the test solutions (before addition of the inoculum): 7.0–7.6 (adjusted with NaOH)
Dissolved oxygen:
Aeration of test vessels: 0.5–1 L of air per hour (ambient air, oil-free air-compressor)
Nominal and measured concentrations:
0, 62.5, 125, 250, 500, 1000 mg/L of test item
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass beakers (sterilised in a drying oven at 150 °C for 3 hours)
- Material, size, headspace, fill volume: Volume of test mixture per test vessel: 500 mL; Volume of microbial inoculum per test mixture: 200 mL; Volume of synthetic sewage feed per test mixture: 16 mL
- Aeration: 0.5–1 L of air per hour (ambient air, oil-free air-compressor)
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- Sludge concentration (weight of dry solids per volume): 1.56 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage = 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; filled up to 1 litre with deionised water.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted with NaOH

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The inhibition of the bacterial respiration activity, the 3-hour EC10 EC20 and EC50 including the 95% confidence limits were determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
The non-GLP range-finding test including a nitrification inhibition control showed less than 50% of control respiration at 1000 mg test item/L. Inhibition of nitrification was 70% of the control respiration (corresponding to 9.7% of control total respiration).
Since even at 1000 mg/L (highest level to be tested according to the guideline) the overall inhibition of total respiration was less than 50%, a separate evaluation of nitrification was not considered relevant.
There was less than 10% inhibition of total respiration at 100 or 10 mg/L.
A relevant specific inhibition of nitrification by the test item was not observed in the rangefinder. The definitive test was therefore performed without ATU.

- Test concentrations: 0, 62.5, 125, 250, 500, 1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
356 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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
Details on results:
The definitive test showed a concentration-response relationship with an inhibition of total respiration rate of up to 36.7% of control respiration at 1000 mg test item/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: the 3-hour EC50 was found to be 6.7 mg/L thus laying in the range of 2-25 mg/L recommended by the guidelines. The result confirms the suitability of the activated sludge used.

Any other information on results incl. tables

Decrease of total respiration rate (inhibition in % of control) as caused by the test item after 3 h.

Treatment

[mg test item/L]

mean respiration rate [mg O2/(l*h)]

standard deviation

number of replicates

inhibition of

respiration rate [% of control value]

Control

69.9

5.29

6

0.0

62.5

69.4

6.26

3

0.7

125

68.2

4.85

3

2.4

250

61.2

6.20

3

12.5

500

62.7

5.63

3

10.3

1000

44.3

2.46

3

36.7

Selected effective concentrations (ECx) of the test item, and their 95%- confidence limits (according to Fieller's theorem).

Parameter

EC10

EC20

EC50

EC80

Value [mg/L]

356

613

1397

n.d.

lower 95%-cl

1.76

80.8

968

n.d.

upper 95%-cl

566

862

43783

n.d.

cl: confidence limit; n.d.: not determined.

Results of the non-GLP range-finding test

Code

Test conc. [mg test item/L]

Mean respiration rate[mg O2/(L*h)]

sd

Inhibition [% of control]

C0

0.0

53.3

2.66

-

C1

10.0

54.1

-

-1.6

C2

100

54.1

-

-1.6

C3

1000

28.5

-

46.5

 

 

C0ATU

0.0

34.9

-

-

C1ATU

10.0

35.9

-

-3.0

C2ATU

100

31.7

-

9.2

C3ATU

1000

23.3

-

33.1

 

 

C0nitrif

0.0

18.4

-

-

C1nitrif

10.0

18.2

-

0.9

C2nitrif

100

22.4

-

-22.1

C3nitrif

1000

5.17

-

71.9

 

Legend: Blank controls (C0), blank controls + ATU (C0ATU), treatments (C1: 10 mg test item/L; C2: 100 mg test item/L; C3: 1000 mg test item/L) with and without ATU. Nitrification-related respiration (C0nitrif, C1nitrif, C2nitrif, C3nitrif) calculated as difference between total and and ATU-inhibited respiration

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The CV% of O2 consumption rate in controls was ≤30%: 7.6%; The O2 consumption rate in the controls was ≥20: 44.9 mg O2/(g dw × h); The 3h-EC50 of 3,5-dichlorophenol was in the accepted range of 2–25 mg/L: 6.7 mg/L
Conclusions:
The 3 -hour EC10, EC20 and EC50 for Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate are 356, 613 and > 1000 mg/L, respectively.
Executive summary:

The influence of the test item Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate on the activated sludge was evaluated by measuring the respiration rate under defined conditions, according to the OECD 209 Guideline. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item (0, 62.5, 125, 250, 500 and 1000 mg/L) after an incubation period of 3 hours.

The inhibition of the bacterial respiration activity was in the range of 0.7 to 36.7 % for the test concentrations of 62.5 to 1000 mg/L. The maximum inhibition was 36.7% at a test concentration of 1000 mg/L.

Based on measured inhibition rates, the 3 -hour EC10, EC20 and EC50 are 356, 613 and > 1000 mg/L, respectively. The definitive test showed a concentration-response relationship with an inhibition of total respiration rate of up to 36.7% of control respiration at 1000 mg test item/L.

The positive control 3,5 -Dichlorophenol was tested in the same way as the test item with nominal concentrations of 1.8, 5.9, 18.8 and 60.0 mg/L. The 3 -hour EC50 was found to be 6.7 mg/L thus laying in the range of 2 -25 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.

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