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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OCSPP 850.330 Modified Activated Sludge, Respiration Inhibition Test
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Remarks:
In this test analytical concentration measurements did not follow the actual test item concentrations, because of the test item direct addition.
Vehicle:
no
Details on test solutions:
At the start of the test defined amounts of the test item (5 x 3 mg; 5 x 9.6 mg; 5 x 30 mg; 5 x 96 mg and 5 x 300 mg) were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentrations.
Additionally, 5 x 96 mg and 5 x 300 mg test item (that correspond to the highest two concentration levels of 320 and 1000 mg/L) were examined also with pH adjustment. The pH of the test mixtures at these concentration levels was checked and adjusted with 1N NaOH to pH 7-8. The neutralization step was performed just before the treatment.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, one day before the preliminary experiment.
Preparation of Activated Sludge 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 (2 L/minute) at the test temperature for about 24 hours (1 day) and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.41), pH adjustment of the inoculum was considered not necessary.
Foaming:
In this test the occurring foaming was not significant, controlling was not necessary during the incubation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no
Hardness:
Not determined.
Test temperature:
The temperature in the test mixtures during the measurements was in average: 20.0 °C, the measured minimum: 18.3 °C, maximum: 21.8 °C.
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.
pH:
The test item had a significant effect on the pH at the highest concentration levels examined (320 and 1000 mg/L). The pH in the test containers containing test item – water - synthetic sewage (320 mg/L, see Appendix I, Table 3: T4A-T4E) was pH ~ 6 for 320 mg/L, and pH ~ 5 for 1000 mg/L. After inoculation the pH was in the pH 7.2-7.3 respectively 7.3 - 7.4 range.

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.
Dissolved oxygen:
Aeration with compresed air at 0.5 L/min
Salinity:
/
Conductivity:
/
Nominal and measured concentrations:
0 - 10 - 32 - 100 - 320 - 1000 mg/L nominal. Five replicates were tested. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Additionally, 5 replicates of the highest two concentration levels of 320 and 1000 mg/L were examined also with pH adjustment. The pH of the test mixtures at these concentration levels were checked and adjusted with 1N NaOH to pH 7-8. The neutralization step was performed just before the treatment.
Details on test conditions:
The test item 7-ACA was investigated in a preliminary test (Study number: 880-209-1601). Based on the results of the preliminary experiments neither inclusion of abiotic controls nor differentiation between heterotrophic respiration and nitrification was considered as necessary.

Synthetic Sewage (the ratio of components referring to 1000 mL):
Peptone 16 g
LAB-LEMCO Powder (BEEF extract) 11 g
Urea 3 g
NaCl 0.7 g
CaCl2 x 2H2O 0.4 g
MgSO4 x 7H2O 0.2 g
K2HPO4 2.8 g
Purified, deionized water ad. 1000 mL

Blank Control (CB):
In the definite test eight controls (according to the number of the available O2 electrodes) four at the start and four at the end of the test series were investigated.
Abiotic Control (CA):
The abiotic control was not repeated because no oxygen uptake was observed in the abiotic control group in the preliminary test.
Reference Control (R):
The reference item 3,5-Dichlorophenol was tested at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions; as the test mixtures
Nitrification Control (CN):
The nitrification respiration was not significant in the preliminary test and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification was occurring. The definite test was performed in one set of test vessels, without ATU addition, but in the definite test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels.

Course of the Test:
Test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the N-allylthiourea adequate or the reference item were filled up with water (with purified, deionized water) to 150 mL before the start of the test. At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added to the test containers. The test flasks were incubated for 3 hours. The test vessels were aerated and shaken continuously such as to ensure an appropriate dissolved O2 level in the samples.
In this study no analytical measurements were performed. Because of the directly added test item the obtained results refer to the nominal test item concentration.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol. Nitrification inhibitor: N-allythiourea (ATU).
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
887 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: With pH adjustment.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
528 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: Without pH-adjustment.
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
84 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Based on the results of the preliminary experiments neither inclusion of abiotic controls nor differentiation between heterotrophic respiration and nitrification was considered as necessary.

Inhibitory effect of the test item was not observed at the lowest examined concentration of 10 mg/L. The observed-calculated oxygen consumption rates and corresponding inhibitory tendencies showed a dose-related tendency in the examined concentration range of 32-1000 mg/L. At the concentration of 32 mg/L 7 %, at 100 mg/L 10 % inhibitions were calculated. At the concentration of 320 mg/L 35 %, at 1000 mg/L 71 % inhibitions were obtained.
Based on the inhibition values, the 3-hour EC50 value of the test item was calculated as 528 mg/L (95 % confidence limits: 407-686 mg/L), without pH adjustment.

With an additional neutralization step at the concentration of 320 mg/L 21 %, at 1000 mg/L 60 % inhibitions were obtained.
Based on the inhibition values, the 3-hour EC50 value of the test item was calculated as 887 mg/L (95 % confidence limits: 613-1283 mg/L), with pH adjustment.
Results with reference substance (positive control):
Positive reference control:
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to 8 mg/L.

The test item had a significant effect on the pH at the highest concentration levels examined (320 and 1000 mg/L). The pH in the test containers containing test item – water - synthetic sewage (320 mg/L, see Appendix I, Table 3: T4A-T4E) was pH ~ 6 for 320 mg/L, and pH ~ 5 for 1000 mg/L. After inoculation the pH was in the pH 7.2-7.3 respectively 7.3 - 7.4 range.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 (with pH adjustment) is 887 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, according to the OECD guideline 209. Based on the results of the earlier performed preliminary test (significant inhibition of oxygen consumption by the test substance in the examined concentration range of 10-1000 mg/L; furthermore the strong test item effect on the pH) a definite test was performed, where pH adjusted test item groups (where needed) were investigated in parallel.

In the definite test, based on measured oxygen consumption values and calculated specific respiration rates clearly dose-related effect of the test item was established and the 3-hour EC50 value of the test item without pH adjustment is 528 mg/L (95% confidence limits: 407-686 mg/L), the 3-hour EC50 value of the test item with pH adjustment is 887 mg/L (95% confidence limits: 613-1283 mg/L). The EC10 (with pH adjustement) is 84 mg/L, the NOEC was calculated to 10 mg/L.

Description of key information

The 3-hour EC50 value of the test item was estimated to 887 mg/L with pH adjustment, and 528 mg/L without pH adjustment.

The EC10 was calculated to 84 mg/L (with pH adjustment).

Key value for chemical safety assessment

EC50 for microorganisms:
887 mg/L
EC10 or NOEC for microorganisms:
84 mg/L

Additional information

The EC10 was calculated to 84 mg/L (with pH adjustment).