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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Analytical monitoring:
no
Details on sampling:
The test flasks were incubated for 3 hours. The test vessels were aerated continuously such as to ensure that the dissolved O2 did not fall below 2.5 mg/L and that, immediately before the measurement of the respiration rate, the O2 concentration was higher than 6.5 mg/L. The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time. The treatment sample was not further aerated. The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method] under stirred conditions and was recorded for 4-10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason several O2 electrodes were used. Simultaneous (four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in ~6 cycles using four O2 electrodes. The oxygen consumption rate (in mg O2 L-1 hour-1) was determined from the linear part of the respiration curve, (in the range between approximately of 2.5 – 6.5 mg O2/L)
Vehicle:
no
Details on test solutions:
Defined amounts of the test item were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentration. Concentrations in excess of nominal 1000 mg test item/L were not tested
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 01 October 2014 (one day before the test.).

PREPARATION OF INOCULUM:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking 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 amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2L/minute) at the test temperature for about 24 hours (1 day) and fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.48) and before use (pH: 7.69). pH adjustment of the inoculum was considered not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time.
Post exposure observation period:
None
Hardness:
not measured
Test temperature:
The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen measuring) at a temperature of 20 ± 2 °C according to the guideline. The noticed temperatures in the environmental room varied between: 20.8-22.0°C.
pH:
The pH-value was in the range of 7.28 - 8.34 during the study. Adjustement of pH was not necessary.
Dissolved oxygen:
Dissolved oxygen concentration was 5.62 - 8.51 mg O2/l.
Salinity:
Freshwater used
Conductivity:
No data
Nominal and measured concentrations:
Just before the start of the test defined amounts (3 x 3; 3 x 30; 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations, furthermore 3 x 300 mg for abiotic controls) of the test item were administered directly.
Details on test conditions:
ENVIRONMENTAL CONDITIONS:
The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen measuring) at a temperature of 20 ± 2 °C according to the guideline. The noticed temperatures in the environmental room varied between: 20.8-22.0 oC.
The test flasks were aerated with compressed air (0.5 L/minute).
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.
The test conditions were measured with suitable instruments and documented in the raw data.

TEST UNITS:
- Type and Size: Erlenmeyer glass bottles of approximately 300 mL volume.
- Identification: Each test flask was uniquely identified with study number, treatment and replicate codes.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
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
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 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.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
In conclusion this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, a definite test was considered as unnecessary.
Results with reference substance (positive control):
The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 8.99 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 19.08 % and 65.83 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 16.63 mg/L, (95 % confidence limits: 12.91-21.43 mg/L).
Reported statistics and error estimates:
The EC50 value of the reference item and its 95 % confidence limits were calculated using Probit analysis. The analysis was done using the TOXSTAT statistical software program.
For the determination of the NOEC the calculated specific respiration rates were tested on significant differences to the control values by Bonferroni t-Test and Dunnett’s Test (α=0.05) by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test. In view of the variability often observed in the results, it may in many cases be sufficient to express the results additionally in order of magnitude.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10; 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the examined test item concentrations. The test was performed without pH adjustment. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L. In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L, therefore a definite test was considered unnecessary.

Description of key information

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Key value for chemical safety assessment

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

Additional information

Source: GLP-report