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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
other: OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
Convenient amounts of TETRAMETHYLTHIURAM MONOSULFIDE are directly pour into flasks: 100 mg/L, i.e. 25 mg, corresponding to a theoretical O2 mass of 46 mg per flask (ThOD 1.843 mg O2/mg). A parent solution of sodium benzoate in the dilution water to 2.5 mg/mL, was used in a proportion of 10 mL for 250 mL of dilution water, i.e. 25 mg, corresponding to a theoretical O2 mass of 41.65 mg per flask (ThOD 1.67 mg O2/mg). In the inhibition monitoring flask, equivalent quantities of test and reference items are introduced. For this flask, the corresponding theoretical mass of O2 is 87.7 mg per flask. The flasks were installed in the respirometer where the stirring system was started. After a waiting period of approximately 2 hours, which is necessary to reach the thermal equilibrium of the solutions at a temperature of 22 ± 1°C, the aerated inoculum, prec onditioned if required, is added to all the flasks that are then plugged.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
The inoculum came from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64). The effluent, obtained one day before seeding the flasks, was centrifuged at approximately 20°C for 10 min at 1000 g; the base was re-suspend ed in dilution water in order to keep the concentration unchanged with respect to the sample. The inoculum was preconditioned (aeration) until the flasks were inoculated. The amount of dry material in the inoculum, determined by desiccation at 105 °C until a constant weight is obtained, was 7.17 g/L.
Test type:
Water media type:
Limit test:
Total exposure duration:
28 d
Test temperature:
22 +/- 1°C
the pH value in the test suspension was 7.55 at day 28
Nominal and measured concentrations:
100 mg/L (nominal concentration)
Reference substance (positive control):
benzoic acid, sodium salt
28 d
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
The percentage degradation of the reference item reached a level of 88 % by 14 days which is above the requested level of 60 %
Validity criteria fulfilled:
not applicable
The ready biodegradabilyt test performed with TETRAMETHYLTHIURAM MONOSULFIDE allows to determine a NOEC for micro-organisms: NOEC = 100 mg/L
Executive summary:

In the toxicity control flasks of the ready biodegradability test, containing both the reference item and TETRAMETHYLTHIURAM MONOSULFIDE the percentage biodegradation reached 40% after 14 days. Since this value is higher than 25%, the test item is not inhibitory for inoculum, that's why the initial test item concentration (100 mg/L) can be considered as a NOEC.

Description of key information

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The effects of tetramethylthiuram monosulphide on the nitrification were assessed through two Klimish 2 studies which the followings data were determined:

  • LOEC-3h = 32 mg/L

  • EC75-2h = 16 mg/L

These data are not useful.

Indeed, according to the Integrated Testing Strategy described in the paragraph R.7.8.21 on ECHA's guidance R.7b (version 4.0 June 2017) "Stage 3.1. IF the chemical is readily biodegradable, or if there is evidence of good degradation of a positive control in the presence of the test substance, THEN derive PNECstp. IF PEC/PNEC <1 THEN stop"

Per the validity criteria established in the OECD 301 technical guideline. " If in a toxicity test, containing both the test substance and a reference compound, less than 35% degradation (based on total DOC) or less than 25% (based on total ThOD or ThCO2) occurred within 14 days, the test substance can be assumed to be inhibitory"

Based on the result from the ready biodegradation test performed on Tetramethylthiuram monosulfide the biodegradation measured in the toxicity control flask was 40% after 14 days thus higher than the cut-off indicated in the OECD 301 technical guideline. 

Therefore, the initial test item concentrationof 100 mg/L used in the ready biodegradation test can be considered as the NOEC.