Pyridinium toluene-4-sulphonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

Dec 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aqueous solution with a nominal content of 80 % was prepared prior to the test. The test substance content of this working solution was analytically verified by HPLC to be 77.9 % (water-free).

A stock solution with a nominal concentration of 5 g/L was prepared by diluting 3209.53 mg of the test item working solution (corresponding to 2500 mg water-free test substance) in 500 mL deionized water and thoroughly mixed by intense stirring for 10 minutes. In this way a clear solution was obtained. The pH of the stock solution was adjusted from 4.0 to 7.3 with a 1M NaOH solution.
Adequate volumes of this stock solution (1 – 100 mL) were diluted with deionized water to a final volume of 234 mL. Then, synthetic sewage feed (16 mL) and activated sludge inoculum (250 mL) were added give a final volume of 500 mL.
- Controls: containing an equal volume of activated sludge and synthetic medium but no test or reference item

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland)
- Method of cultivation / Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in chlorine free tap water. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
Based on this determination, a calculated amount of wet sludge was suspended in chlorine free tap water to obtain a concentration equivalent to 3 g dry material per liter. The pH of the activated sludge was adjusted from 7.6 to 7.4 with a diluted sulfuric acid solution. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, on the next day, the pH of the activated sludge inoculum was measured again and found to be 7.4.
- Initial biomass concentration: 3.0 g/L (dry weight)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19 - 20 °C

pH:

test start: 7.5 - 7.9
test end: 8.0 - 8.3

Dissolved oxygen:

test start: 8.4 - 8.7 mg/L
test end: 7.1 - 8.6 mg/L

Nominal and measured concentrations:

nominal: 10, 32, 100, 320, 1000 mg/L (water free)

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L beakers, contents 500 mL
- Type (delete if not applicable): open
- Aeration: intense stirring with magnetic followers
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.5 g/L (dry weight)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water (<1 mg/L DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: test item stock solution: from pH 4.0 to pH 7.3; inoculum: from pH 7.6 to pH 7.4
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : dissolved oxygen concentration after 3 hours contact time, continuously recorded for a period of up to 10 minutes or until the oxygen concentration fell below 2 mg/L

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L (water-free)
- Results used to determine the conditions for the definitive study: oxygen consumption at the tested concentrations was inhibited by 9 %, 24 % and 16 - 30 %

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% confidence interval: 38 - 354 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: 29 - 36 mg O2/gh
- Coefficient of variation of oxygen uptake rate in control replicates:9 %

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 6 mg/L (within the guidelines-recommended range of 2–25 mg/L)

Reported statistics and error estimates:

The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using the software ToxRat® Professional (ToxRat® Solutions GmbH, Version 3.2.1).
The NOEC was derived by per-vessel statistics using the Williams t-test (one sided smaller, α = 0.05).
The 3-hour EC10, EC20, EC50 and EC80 values and their 95 % confidence limits were derived by Probit Analysis. Adverse effects of the test item compared to the control were investigated using one-tailed (smaller) hypothesis testing at p ≤ 0.05 and per treatment means.
The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis.
All test results were based on nominal concentrations.

The mean inhibitions of the respiration rates for the tested concentrations were in the range 14.5 to 27.3 % (see attached document "Table Oxygen Consumption of Activated Sludge.pdf").

No dose-response-relationship was observed at 10, 32 and 100 mg/L: mean inhibitions of the respiration rates were 14.5, 17.2 and 16.7 %, respectively. Although relatively low, these reductions in the respiration rate were found to be statistically significantly different from the control. Consequently, the 3-hour NOEC and EC10 to activated sludge microorganisms were found to be below 10 mg/L.

At 320 and 1000 mg/L, the inhibitory effect increased and reached 22.6 and 27.3 %, respectively. Concentrations exceeding 1000 mg/L were not tested in accordance with the guidelines.

Validity criteria fulfilled:

yes

Remarks:

The specific respiration rates in the controls were ≥20 mg O2/g/h and the coefficient of variation was ≤ 30 %.

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC50 > 1000 mg/L, EC20 128 mg/L, NOEC < 10 mg/L (OECD 209, EU C.11)

Additional information

EC50 for microorganisms > 1000 mg/L

EC20 for microorgansims 128 mg/L

NOEC for microorgansims < 10 mg/L