S-ethyl N-cyclohexylthiocarbamate

Toxicity to microorganisms

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cycloate

IUPAC Name:

Cycloate

Details on test material:

Cycloate

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

not specified

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the inoculum plant for domestic sewage in Balatonfüred, Hungary, on 19 November 2018 (two days before the test).
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.367 g wet weight), dried and the ratio of wet sludge to dry weight (0.4496 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (15 g dry weight that was equivalent to 179 g wet sludge) was suspended in isotonic saline solution (ad. 5 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 48 hours (2 days) and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.58), additional pH adjustment of the inoculum was considered not necessary.
Foaming:
In this test the occurring foaming was not significant, controlling was not necessary during the incubation.

Study design

Test type:

not specified

Water media type:

not specified

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen concentration measuring) at a temperature of 20  2 C according to the guideline. The recorded temperatures in the environmental room varied between: 20.0-20.7 oC.

The test flasks were aerated with compressed air (0.5 L/minute).
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.
The test conditions were measured with suitable instruments and documented in the raw data.

pH:

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures (controls, test item treatments) etc. were in the acceptable pH 7-8 range. The measured pH values, the pH changes are summarized in Appendix I, Table: 3.
The mixtures were aerated at 0.5 L/min in the temperature range of 20±2oC. The temperature in the test mixtures during the measurements in average: 20.8oC, the measured minimum: 19.5 oC, maximum: 22.0 oC.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

>= 1 000 mg/L

Conc. based on:

test mat.

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

>= 1 000 mg/L

Conc. based on:

test mat.

Effect concentrations 3

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Conc. based on:

test mat.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
In conclusion this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, in line with OECD 209, a definite test is not required.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test itemCycloate technicalon the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours.

This preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption.

The test itemCycloate technicalwas investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels.

Triplicate vessels were prepared and investigated at the highest examined test item concentration.

In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated.

Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/L, synthetics sewage feed, but no inoculum. In this test no abiotic oxygen consumption was noticed.

This experiment was performed without pH adjustment. The test item did not have any effect on the pHwithin the test system and additional neutralization step of test item containing mixtures before inoculum addition was not necessary.

All validity criteria of the study were met. The average specific respiration rate of the blank was 43.63 mg O₂/ g activated sludge (based on dry weight) in an hour with a coefficient of variation of 4.58 %. The 3-hour EC₅₀of the reference item 3,5-Dichlorophenol was 14.70 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study thedifferentiation between heterotrophic respiration and nitrification was considered as not necessary).

The observed oxygen consumption rates and consequently the specific respiration rates in the test item concentrations of 10 and 100 mg/L remained in the range of the blank controls, and slightly lower (but statistically not significantly different from that of the control) oxygen consumption rates were noticed at 1000 mg/L. The average specific respiration rate at 1000 mg/L: 41.03 mg O₂/gh, that corresponds to about 6 % inhibition.

Based on measured oxygen consumption values and calculated specific respiration rates it can be statedthat the 3-hour EC₁₀and EC₅₀values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

The specific respiration rates at the examined highest test item concentration of 1000 mg/L were compared with the blank controlvalues using 2-Sample T-test (a=0.05). No statistically significant differences were observed, compared tothe blank control values.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L, therefore in line with OECD 209 a definite test is not required.