Piperidine-4-carbothioamide

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted: 22 July 2010 and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-treatment of test item: Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 10.5 – 10.6. The pH was adjusted to pH 7.3 – 7.4 with HCl.
Pre-treatment of test item with ATU: Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 10.5 – 10.6. The pH was adjusted to pH 7.2 – 7.4 with HCl.
For the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Date of collection : 2017-10-09
- Method of cultivation: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Preparation of inoculum for exposure: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- pH of the suspension before application: 7.6

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

19.4 - 20.5 °C

pH:

7.4 - 8.4

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

- 100 mg/L
- Test concentrations are given as nominal concentrations as they were not confirmed by analytical methods.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Type (delete if not applicable): closed
- Aeration: permanent aeration
- No. of vessels per concentration (replicates): 3 replicates without ATU, 2 replicates with ATU
- No. of vessels per control (replicates): 6 replicates without ATU, 4 replicates with ATU
- Sludge concentration (weight of dry solids per volume): 1200 mg/L suspended solids
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)
- Stirring period of the test item before start of incubation time: 17 hours

OTHER TEST CONDITIONS
-To determine the heterotrophic oxidation a second set of control vessels, test item vessels, reference substance vessels and vessels to determine the physic-chemical oxygen consumption was prepared. Before they were filled up with deonised water to 250 mL end volume, to each vessel 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L was added. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
- Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally for both sets. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption, temperature and ph were measured after an aeration time of 3 hours

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol , concentration (2.5, 5, 10, 20, 40 mg/L)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid
- Relevant effect levels: EC50 = 13.281 mg/L

As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50.

The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

Validity criteria fulfilled:

yes

Remarks:

(oxygen uptake rate blank controls differ not less than 20 mg oxygen -coefficient of variation of oxygen uptake in control not more than 30 % at end of test - EC50 reference compound in the range of 2–25 mg/L total respiration, 5-40 mg/L heterotrophic)

Conclusions:

After an incubation period of 3 hours, analysis of the respiration rates gave the following values: EC 50: > 100 mg/L, NOEC: >= 100 mg/L. Piperidine-4-carbothioamide showed 1.5 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L.

Executive summary:

The study was conducted in accordance with OECD Guideline 209 and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02). The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated. The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration.To measure the oxygen consumption, 250 mL of sludge with Piperidine-4-carbothioamide (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode).

Six controls without test substance were included in the test design, three at the start and the others at the end of the test series. A limit test was performed with 3 replicates with a test item concentration of 100 mg/L. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound. Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8. 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C. Control vessels (inoculated sample without test item) were prepared the same way. Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge. To determine the heterotrophic oxidation four additional controls and two replicates with the test item concentration 100 mg/L, all containing 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L, were prepared. Oxygen consumption, temperature and pH were measured and recorded after an aeration time of 3 hours.

After an incubation period of 3 hours, analysis of the respiration rates gave the following values: EC 50: > 100 mg/L, NOEC: >= 100 mg/L. The test substance showed 1.5 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The test substance showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L.The effect value relates to a nominal concentration, since no analytical monitoring was performed.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms

Description of key information

After an incubation period of 3 hours, analysis of the respiration rates gave the following values:

EC50: > 100 mg/L, NOEC: >= 100 mg/L.

The test substance showed 1.5 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

should read: EC 50: > 100 mg/L, NOEC: >= 100 mg/L