α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

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:

1999-12-23

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on test solutions:

2.50 g of the test item were diluted to 100 mL tap water to give a stock concentration of 25.0 g test item/L (5.0 g a.s./L).
The test suspensions were prepared by adding 16 mL of synthetic sewage feed together with appropriate amounts of test item stock solution were made up to 300 mL with tap water. Two hundred (200) mL of microbial inoculum was then added and the total mixture poured into the test vessel. The addition of 313 µL, 625 µL, 1.25 mL, 2.5 mL, 5 mL, and 10 mL of test item stock solution resulted in final nominal concentrations of 3.13, 6.25, 12.5, 25, 50, and 100 mg a.s./L, respectively.

Details for Reference item:
A stock solution of 500 mg/L 3,5-dichlorophenol was prepared by dissolving 100.5 mg of the reference item in 2 mL of 1 M NaOH. After addition of 4 mL of deionized water, 1 N H2S04 was added up to the point of incipient precipitation (approximately 1.6 mL). Finally the mixture was diluted to 200 mL with deionised water. The addition of 5, 15, and 30 mL of reference item stock solution resulted in final concentrations of 5, 15, and 30 mg/L, respectively.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge from a domestic sewage treatment plant form waste-water treatment Plant in Romanshorn, Switzerland was used as the microbial inoculum. After collection, the sludge is placed in a centrifuge tube, centrifuged at 1500 rpm for 10 minutes and the supernatant is decanted. More sludge is added and the tube is centrifuged again and the supernatant is decanted. The sludge is washed with tap water. The sludge solution is centrifuged once again at 1500 rpm for 10 minutes and the supernatant decanted. This procedure is repeated three times. A small aliquot (1-2 g) of the washed sludge is weighed and dried at 105°C to calculate the quantity of wet sludge required to be suspended in water to obtain an activated sludge containing a solids level of 4 g/L (±10%). This level yields a concentration of 1.6 g/L in the test medium if the procedure below is followed.

If the sludge cannot be used on the day of collection, 50 mL synthetic sewage feed is added to each liter of the activated sludge prepared as described above. This is aerated overnight at 20 ± 2°C. The sludge is then kept aerated for use during the day. Before use, the pH is measured and buffered to pH 6.0 to 8.0 using sodium bicarbonate solution.

For the definitive test, the inoculum was prepared by weighing 141.73 g of wet sludge (7.62% dry weight) and bringing it to a volume of 3 liters with tap water. The resulting activated sludge solution had a suspended solid concentration of 3.6 g/L. One hundred fifty (150) mL synthetic sewage feed were then added to 2.85 L of the activated sludge solution. The inoculum was aerated overnight before being used the following day. On the day of the definitive test, three aliquots of the inoculum were dried to determine the solid content. A suspended solid concentration of 4.97 g/L was found in the inoculum due to cell growth overnight. A volume of 2.4 L of this suspension was diluted with 0.6 L of tap water to give a solid concentration of 3.97 g/L which was in the required range of 4 g/L ± 10%. The pH of the inoculum was 7.84.

Test type:

static

Water media type:

other: Tap water

Test temperature:

Between 19.5 to 20.9°C

pH:

Between 7.0 to 8.0 pH

Nominal and measured concentrations:

3.13, 6.25, 12.5, 25, 50, and 100 mg a.s./L

Details on test conditions:

The test system of the definitive test was set up inside a temperature-controlled environmental chamber. A red light filter cut off light with wavelengths smaller than 560 nm. Each test suspension was kept in a 1000 mL glass beaker. The suspensions were constantly stirred using a magnetic stirrer to warrant homogeneous distribution and aerated at 0.5 to 1L/min using ambient air, pumped with oil free aquarium pumps (Elilte 801 and 802) via silicone tubing attached to glass frits.

After 3 hours of aeration, the suspension was transferred to a 500 mL biological oxygen demand (BOD) bottle and was positioned on a magnetic stir plate. The BOD bottle was fitted with a stopper through which an oxygen probe was placed. Immediately upon transfer, dissolved oxygen was measured and recorded every minute, over a 10 minute period. The solutions were stirred continuously with a magnetic stir bar during the measurements.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

71 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of respiration

Remarks on result:

other: The test was conducted with a contact time of 3 hours. The contact time is the time the microorganisms were in contact with the test item or reference item prior to measurement of the oxygen consumption.

Details on results:

Inhibition of respiration by the test item was -4, -27, 3, 20, 26, and 70% for 3.13, 6.25, 12.5, 25, 50 and 100 mg a.s./L (nominal), respectively.

Results with reference substance (positive control):

The calculated EC50 for the reference item was 10 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations the EC50 was determined to be 71 mg a.s./L for myclobutanil.

Executive summary:

The purpose of this study was to assess the effect of the test item on the respiration of microorganisms. Activated sewage sludge was used as the source of the microorganisms. The respiration rate of the activated sludge was measured in the presence of the test item at nominal concentrations of 3.13, 6.25, 12.5, 25, 50, and 100 mg a.s./L. Inhibition of respiration by the test item was -4, -27, 3, 20, 26, and 70% for 3.13, 6.25, 12.5, 25, 50, and 100 mg a.s./L (nominal), respectively. Based on nominal concentrations the EC50 was determined to be 71 mg a.s./L for myclobutanil.

Description of key information

In a GLP-study performed according to OECD TG 209, the EC₅₀ for microorganisms was calculated to be 71 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

71 mg/L