Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 97.04%
Impurities: Not reported

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance was directly weighed into each test flask and pure water was added.

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: Supplied by municipal sewage treatment plant (Bensheim, Germany)
- Conditioning: The activated sludge used was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge was 7.2 and therefore no adjustment was necessary.

Study design

Test type:
not specified
Total exposure duration:
3 h

Test conditions

Test temperature:
20 ± 2°C
pH:
6.3 to 8.0
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume.
- Aeration: With compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control: Six controls (pure water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the test medium.
- Nitrification inhibitor used: N-allylthiourea

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of this solution was adjusted to 7.0 with NaOH.

EFFECT PARAMETERS MEASURED: Respiration rate (oxygen consumption) was measured for 10 minutes after a 3 hour exposure period. Shorter evaluation periods were also possible and done over the most linear part of the measurement. Results were expressed as percent inhibition relative to the control.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
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 respiration due to nitrification
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Total respiration:
In comparison to the inoculum controls the respiration rates of the activated sludge up to concentrations of 320 mg/L were not inhibited. The highest test concentrations of 1000 mg/L showed a small inhibition; the inhibition was 1.6%, -13.9%, -8.7%, -2.4% and 23.0% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

Respiration without nitrification (Heterotrophic Respiration):
The inhibition of the heterotrophic respiration of the activated sludge was only moderately and not dose-related inhibited up to concentrations of 1000 mg/L; the inhibition was 25.2%, 26.7%, 21.9%, 29.5% and 32.8% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

Respiration based on nitrification:
In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification was not inhibited for all test item concentrations up to 1000 mg/L; the inhibition was -52.1%, -105.9%, -78.2%, -74.8% and 0.5% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.
Results with reference substance (positive control):
The 3-hour EC50 for total respiration was found to be 6.9 mg/L (lower cl: 4.0 mg/L, higher cl: 11.7 mg/L) thus lying in the range of 2 - 25 mg/L recommended by the test guidelines. The 3-hour EC50 for heterotrophic respiration was found to be 12.0 mg/L (lower cl: 2.6 mg/L, higher cl: 58.1 mg/L) thus lying in the range of 5 - 40 mg/L recommended by the test guidelines. For the oxygen uptake due to nitrification the 3-hour EC50 was 2.3 mg/L (lower cl: 1.0 mg/L, higher cl: 5.1 mg/L) lying in the range of 0.1 - 10 mg/L recommended by the test guidelines. The result confirms the suitability of the activated sludge used.
Reported statistics and error estimates:
For normal distribution of the data, the Shapiro-Wilk`s Test was used, the variance homogeneity was tested by the Levene`s Test. Subsequently, the Williams Multiple Sequential t-test Procedure (one-sided smaller) with Levenberg-Marquardt optimization was used to determine the 3-hour ECx and their 95 %-confidence limits and LOEC and NOEC values, if possible. For nitrification respiration of the test item, Dunnett’s multiple t-Test (one-sided smaller) with Levenberg-Marquadt optimization was used to determine the 3-hour ECx and their 95 %- confidence limits and LOEC and NOEC values if possible.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
NOEC: 320 mg/L (total respiration)
NOEC: <10 mg/L (heterotrophic respiration)
NOEC: ≥1000 mg/L (nitrification respiration)
Executive summary:

The effects of the test substance on activated sludge were determined in a 3-hour exposure laboratory study conducted under OECD Guideline 209. Activated sludge from the aeration tank of a municipal sewage treatment plant was exposed to the test substance at concentrations of 10, 32, 100, 320 and 1000 mg/L and 6 untreated controls. The same test design was repeated with the nitrification inhibitor Nallylthiourea. The test substance had no inhibiting toxic effects on the total respiration of the activated sludge up to the concentration of 320 mg/L. The inhibition was around 23% at concentrations of 1000 mg/L. Heterotrophic respiration of the activated sludge was only moderately inhibited up to concentrations of 1000 mg/L. No dose-related changes in inhibition were noticed.

The NOEC was determined to be 320 mg /L for total respiration, <10 mg /L for heterotrophic respiration and was determined to be ≥1000 mg/L for nitrification respiration.

The 3-hour EC50 could not be established for total, heterotrophic and nitrification respiration.