Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2013 - 20 March 2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Synthetic sewage
Synthetic sewage feed for activated sludge was prepared by dissolving the following in one litre of RO water:
Peptone - 16.0g/L
Meat extract - 11.0g/L
Urea - 3.0g/L
Sodium chloride - 0.7g/L
Calcium chloride dihydrate - 0.4g/L
Magnesium sulphate heptahydrate - 0.2g/L
di-Potassium hydrogen phosphate - 2.8g/L
This solution was stored in the fridge before use. The pH of this solution was 7.5 ± 0.5.

Preparation of the test substance
The test substance was poorly soluble in water. Therefore weights of the test substance (nominally 5, 50 and 500 mg) were added to glass ampoules which were then added to their respective test vessels (500 mL glass bottles). The ampoules were then carefully broken and RO water (284 mL) added.

Vehicle:

no

Details on test solutions:

In this test, a Co-ordinated Environmental Services (CES) Ltd automated respirometer and associated software was used to monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.
Mixtures were incubated in 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle. Aliquots of RO water, synthetic sewage and antifoam agent (silicone oil in water) were added to each bottle. Weights of the test substance were then added to the respective bottles to give final, nominal concentrations of 10, 100, and 1000 mg/L. Immediately prior to initiation of the instrument, the required volume of activated sludge was added to each bottle.
The mixtures were incubated at a temperature of 20 +-2°C. Mixtures were prepared to give a final volume of 500 mL

Details on inoculum:

Preparation of the microbial inoculum
A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.

On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.

On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.

Preparation of solutions of the reference substance (3,5-dichlorophenol)
A concentrated solution of 3,5-DCP (99%+) was prepared (500 mg/L). The pH of this solution was checked and adjusted to 7.5 ± 0.5 as required using 10M NaOH. Nominal concentrations of 3, 10 and 32 mg/L were prepared by dilution of this concentrated solution.

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes. The pH and temperature of the samples were measured at the start and end of the test.

Test temperature:

20 +-2°C.

pH:

7-8

Dissolved oxygen:

4-7(mgO2/L)

Nominal and measured concentrations:

10, 100, and 1000 mg/L.

Details on test conditions:

The prepared mixtures were aerated and stirred for three hours in a thermostaticallycontrolled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase.

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Details on results:

AO-26 did not significantly inhibit the respiration rates of the samples of activated sludge. At most, mean respiration rates were decreased by 3% at 1000 mg/L. Consequently, the 20%, 50% and 80% effect concentrations (EC20, EC50 and EC80) for inhibition could not be calculated but must be >1000 mg/L, the highest concentration employed in this study. As there was no significant inhibition observed in any of the test groups the study was reported as a limit test with no further testing carried out.

Results with reference substance (positive control):

The mean specific respiration rate of the control cultures was 27.8 mgO2/gh with a coefficient of variation of 9.0%. Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 8.93 mg/L (95% confidence intervals 4.89 to 16.2 mg/L). These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

Validity criteria fulfilled:

yes

Conclusions:

AO-26 did not significantly inhibit the respiration rates of the samples of activated sludge. Consequently, the 50% effect concentration (EC50) for inhibition could not be calculated but must be >1000 mg/L, the highest concentration employed in this study.
The no observed effect concentration (NOEC) for AO-26 was 1000 mg/L.

The three-hour EC50 for 3,5-DCP (8.93 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L). The validity criterion relating to the respiration rates in the control (specific respiration rate ≥20 mgO2/gh and a coefficient of variation of≤30%) was also satisfied.

Executive summary:

The study was conducted to assess the effect of AO-26 on the respiration rate of activated sludge using the methods detailed in OECD Procedure 209 of the “Guidelines for Testing of Chemicals”: Activated Sludge, Respiration Inhibition Test (carbon and ammonium oxidation), adopted 22 July 2010.

Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test substance for three hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with those controls that contained activated sludge and synthetic sewage alone.

The nominal AO-26 concentrations used in the study were 10, 100, and 1000 mg/L; five replicates were prepared for each concentration including the control. Single replicates of the

reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control.

The mean specific respiration rate (Rs) of the control cultures incubated with the test mixtures was 27.8 mgO2/gh with a coefficient of variation (CV) of 9.0% The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 8.93 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

AO-26 did not significantly inhibit the respiration rates of the samples of activated sludge. Consequently, the 50% effect concentration (EC50) for inhibition could not be calculated but must be >1000 mg/L, the highest concentration employed in this study. The no observed effect concentration (NOEC) for AO-26 was 1000 mg/L.

Description of key information

AO-26 did not significantly inhibit the respiration rates of the samples of activated sludge. Consequently, the 50% effect concentration (EC50) for inhibition could not be calculated but must be >1000 mg/L, the highest concentration employed in this study.

The no observed effect concentration (NOEC) for AO-26 was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L