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Administrative data

activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
1. The oxygen content measurement time was lower and higher than 8 minutes 2. The addition of the sludge to the test assay with reference substance RS6 was done accidentally 15 minutes before the scheduled time
GLP compliance:
yes (incl. certificate)

Test material

Specific details on test material used for the study:
Name of test substance: Dodecatrienal
Test substance No.: 18/0072-2
Batch identification: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8.
CAS no.: 1000399-21-2
Purity: 90.1 area-% (GC, RTX-1701 capillary); 92.7 area-% (GC, DB-35 MS capillary)
Homogeneity: Homogeneous
Storage stability: Expiry date: May 2020
Date of production: May 2018
Physical state/
liquid / colorless, clear
Water solubility: 43 mg/L +/- 4 mg/L at T = 20.0°C +/- 0.5 °C
Storage conditions: Ambient at room temperature; under light exclusion

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The temperature was measured for seven times in a separate vessel filled with aerated deionized water during incubation phase.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Test system: municipal Activated sludge
Reason for the selection of the test system: required by the test guidelines
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 04 Sep 2018
Arrival in the test facility: 04 Sep 2018
After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L Dw.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h

Test conditions

Test temperature:
20.3 – 20.6°C
7.2 - 8.2
Dissolved oxygen:
7.0 - 9.0 (start of exposure)
Nominal and measured concentrations:
1000, 500, 250, 125 and 62.5 mg/L as nominal concentration based on test substance
Details on test conditions:
Test replicates: 6 replicates for the control, 3 replicates for each test substance concentration, 2 replicates for each reference substance concentration.
Incubation time: 3 hours
Test vessels: Glas-beakers (nominal volume 1L)
Test volume: 500 mL
Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L
Duration of the measurement of oxygen consumption: About 8 - 10 min (two deviations as described above)
Sludge concentration in the test vessel: 1.5 g/L Dw

Results and discussion

Effect concentrationsopen allclose all
Key result
3 h
Dose descriptor:
Effect conc.:
150 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
3 h
Dose descriptor:
Effect conc.:
49 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
EC10 = 49 mg/L, EC20 = 72 mg/L, EC50 = 150 mg/L, EC80 = 320 mg/L were found in the activated sludge respiration inhibition test with 3 hour residence time for the test substance Dodecatrienal.

Validity criteria:
The coefficient of variation of the six replicates of blank control was 10.6 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 4.3 mg/L.
The mean oxygen uptake of the blank controls was 21 mg/g*h.

In conclusion, the test conformed to all validity criteria and is valid.

Results with reference substance (positive control):
In order to verify that the microorganisms are responding normally to toxic stress, tests with a reference substance are conducted according to OECD 209 guideline. The results from the reference substance test are compared to EC50 values published in OECD 209 guideline, which represent the typical response range.
According to the test guideline OECD 209 the EC50 values of the reference substance 3,5-dichlorophenol should be in the range of 2-25 mg/L after 3 hours of incubation. The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3
hours. These results indicate that the microorganisms are responding normally to toxicant stress.
Reported statistics and error estimates:
The consumption rates were used for the determination of the ECx by the probit method based on Finney with the software TOXRAT Professional 2.10. The effect concentrations were given with an accuracy of 2 significant digits.

Applicant's summary and conclusion

Validity criteria fulfilled:
In conclusion the 3 hours effect concentrations for Dodecatrienal in the Activated Sludge
Respiration Inhibition Test are:

Toxicity Response Variable Based on nominal effect concentration [mg/L] Confidence Limits (95%) lower/upper
EC10 49 9.9/80.6
EC20 72 23.3/106.8
EC50 150 99.1/222.8
EC80 320 215.6/908.5

The value of effect concentrations of EC10 EC20 EC50 EC80 were given with an accuracy of 2
significant digits. The degree of inhibition was evaluated by Probit analysis according to Finney.
The results in this study are consistent with all validity criteria and the test is valid according to
the guidelines used for this study.