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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 25, 2012 to Jan. 31, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to OECD principles of GLP
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of the test substance was prepared at a concentration of 2.5 mg/mL in 1,000 mL reagent water. Stock solution volumes of 0.78, 3.2, 12.6, 50, and 200 mL were added to the 3.9, 16, 63, 250, and 1,000 mg/L contact flasks, respectively.
Further details are not provided in the study report.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of inoculum: Approximately 15 liters of activated sludge were collected on 26 June 2012 from Aeration Basin #1 at the Columbia Wastewater Treatment Plant in Columbia, Missouri.
- Preparation of inoculum for exposure: Twelve 250 mL centrifuge bottles were filled with activated sludge and concentrated by centrifugation at 3,000 rpm for approximately 5 minutes. The supernatant was decanted, and the bottles were refilled with activated sludge and centrifuged to concentrate the activated sludge. After the final concentration run, the supernatant was decanted, and the microbial inoculum pellet was rinsed by adding ABC well water, shaking, and centrifuging as above. The supernatant was then decanted. The microbial inoculum pellet in each bottle was washed two more times in the same manner. The final supernatant was decanted, and a suspension of the sludge pellet was prepared by adding 200 mL of well water to each bottle.
-Storage condition: After preparation, the carboy containing microbial inoculum were placed on a stir plate in an environmental chamber maintained at 20 ± 2 ˚C, and aerated while stirring using compressed air overnight. A synthetic sewage feed solution composed was used to sustain the inoculum overnight.
- pH of the prepared microbial inoculum: 8.40.
-Suspended solid determination: The suspended solid concentration at the time of collection is not reported. The microbial inoculum solution was diluted with ABC well water as needed to obtain a suspended solids concentration between 2 and 4 g/L

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not reported
Test temperature:
Temperature of the environmental chamber ranged from 19.8 to 20.1°C during the test and storage of the microbial inoculum prior to test initiation.
pH:
The pH values for the control, reference toxicant, and test substance contact flasks ranged from 8.45 to 8.65 during the test.
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
Nominal concentration: 0, 3.9, 16, 63, 250 and 1000 mg/L.
No measurement of test concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: Closed
- Material: 1L Erlenmeyer flasks containing 500 mL test solution. Each test vessel contained 250 mL inoculum, 16 mL synthetic sewage, test material (either as stock solution) and well water to make a total volume of 500 mL.
- Aeration: Yes, the contents were stirred and aerated at 500 to 1,000 mL/minute.
- Temperature: 19.8 ± 20.1 °C
- No. of vessels per concentration: One per test concentration, one per reference substance concentration and 3 biotic control flasks.
- Light/dark conditions: Ambient lighting

CONTROL
- Blank control: Yes (inoculum without test material)
- Positive control: Yes (containing reference material and inoculum)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water (non-chlorinated) was used for all inoculum preparation procedures, synthetic sewage feed preparation, and test flask preparations.
ABC reagent water was well water that was purified, deionized, and filtered using a Millipore Water System. Reagent water was used to prepare the test and reference substance stock solutions.
- Synthetic waste water composition: A prepared synthetic sewage feed from Liquid Analytic Resources was used during the study. The composition of test medium is provided in the study report.
- Intervals of water quality measurement: Samples of ABC well water were chemically characterized periodically to verify that they are free of contaminants that might interfere with test results.

EFFECT PARAMETERS MEASURED: The respiration rate was measured for reference and test material after 3 hours incubation.

RESPIRATION RATE MEASUREMENT: Monitoring of oxygen consumption was performed with a Strathkelvin Instruments Activated Sludge Respirometer (ASR).

TEST CONCENTRATIONS
- Range finding study: Yes (A first range-finding test was conducted on to estimate the nominal exposure concentration range for the definitive test. A second portion of the range-finding test investigated the potential effect of nitrifying bacteria on heterotrophic respiration of activated sludge in the presence of the test substance and a nitrification inhibitor).
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes. 0, 67 and 58% inhibition was observed at 10, 100 and 1000 mg/L test concentration for the 3 hour incubation. The abiotic control respiration rates were ≤3% of their respective biotic controls. This small amount of respiration is likely due to the use of nonchlorinated well water in the contact flask preparations and indicates the test substance does not have strong reducing properties to cause abiotic respiration.
In the second portion of the range finding study, the percent inhibition of ATU-treated test material at 10, 100, and 1,000 mg /L were 12, 58, and 24%for the 3-hour incubation.
While there was variation among the test treatments, the trend observed was similar for inhibited or uninhibited samples. The control values did not vary by more than 5% among the inhibited or uninhibited values, ranging from 61.6 to 63.8 mg O2/L/h (CV=1%). This indicates there was no effect to potential nitrifying microorganisms in the microbial inoculum and no further nitrifying work with this batch of inoculum was necessary.
Details of range finding study were provided in the study report.
Based on the results of range finding study, a definitive test was performed at nominal concentrations of 3.9, 16, 63, 250, and 1,000 mg /L

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol at 1, 10 and 30 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
228 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: No
- Inhibition of respiration: The percent inhibitions for the test substance flasks during the 3 hour exposure were 0, 0, 0, 79, and 73% at the nominal test concentrations of 3.9, 16, 63, 250, and 1,000 mg/L.
- Respiration rates of control flask: During the 3-hour exposure, respiration rates were 61.8 and 61.5 mg O2/ L/h (mean: 61.7 mg O2 /L/h), and 61.5 and 62.4 mg O2/ L/h (mean: 62.0 mg O2/L/h). The mean respiration rates of all control flasks were >20 mg oxygen per one gram of activated sludge in an hour which also satisfies the acceptability criterion outlined by OECD Method 209.
For details on inhibition of respiration rate of activated sludge by test material refer to 'Table 1' under 'Any other information on results incl. tables' section.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
-% inhibition: During the 3-hour exposure, the inhibition values were 0, 17, and 61% at 3,5-dichlorophenol concentrations of 1.0, 10, and 30 mg/L, respectively
- Relevant effect levels: The 3 h EC50 = 23.5 mg/L (95% CI = 0 to 66.9 mg/L). The EC50 value was within the 5 to 30 mg/L acceptability range as outlined in OECD Method 209.
For details on results, please refer to 'Table 1' under ‘Any other information on results incl. tables’.
Reported statistics and error estimates:
None

Table 1: Inhibition of respiration rate of activated sludge by Colipa A050 after 3 hours (Study # 68341)

Concentration (mg/L)

Respiration rate [mgO2/(L·h)]

Inhibition (%)

Control 1

-

61.8

-

Control 2

-

61.5

-

Mean

-

61.7

-

Control 3

-

62.4

-

Meana

-

62.0

-

Test material (Colipa A050)

1000

16.8

73

250

12.8

79

63

66.2

0

16

69.6

0

3.9

66.3

0

Reference material (3,5-dichlorophenol)

30

24.0

61

10

51.0

17

1

66.1

0

aCalculated using Control 2 and Control 3.

Validity criteria fulfilled:
yes
Conclusions:
The 3 hour EC50 of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate for inhibition of respiration rate of activated sludge was 228 mg/L, based on nominal concentration of test material.
Executive summary:

An activated sludge respiration inhibition test, following OECD 209 guideline, was conducted on N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate according to GLP. The inoculum was activated sludge collected from Aeration Basin #1 at the Columbia Wastewater Treatment Plant in Columbia, Missouri. The respiration inhibition of sludge microorganisms was determined after 3 hour incubation time under static conditions.

The nominal test concentrations were 0, 3.9, 16, 63, 250 and 1000 mg/L.

The percent inhibitions for the test substance flasks during the 3 hour exposure were 0, 0, 0, 79, and 73% at the nominal test concentrations of 3.9, 16, 63, 250, and 1,000 mg/L.The 3 hour EC50 of the test substance for inhibition of respiration rate of activated sludge was 228 mg/L, based on the nominal concentration of the test material.

The 3 hour EC50 value for the reference material, 3,5-dichlorophenol, was 23.5 mg/L. The EC50 value was within the 5 to 30 mg/L acceptability range as outlined in OECD Method 209.

The mean respiration rates of all control flasks were >20 mg oxygen per one gram of activated sludge in an hour which also satisfies the acceptability criterion outlined by OECD Method 209.

This activated sludge, respiration inhibition test is classified as acceptable, and satisfies the guideline requirements for the OECD 209.

Description of key information

The 3 hour EC50 of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate for inhibition of respiration rate of activated sludge was 228 mg/L, based on nominal concentration of test material.

Key value for chemical safety assessment

EC50 for microorganisms:
228 mg/L

Additional information

An activated sludge respiration inhibition test, following OECD 209 guideline, was conducted on N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate according to GLP. The inoculum was activated sludge collected from Aeration Basin of of a predominantly domestic sewage Treatment Plant. The respiration inhibition of sludge microorganisms was determined after 3 hour incubation time under static conditions.

The percent inhibitions for the test substance flasks during the 3 hour exposure were 0, 0, 0, 79, and 73% at the nominal test concentrations of 3.9, 16, 63, 250, and 1,000 mg/L.The 3 hour EC50 of the test substance for inhibition of respiration rate of activated sludge was 228 mg/L, based on the nominal concentration of the test material. All the validity criteria were met.