cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

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:

6 Jun 2002 to 14 Jun 2002

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:

1993

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

During the definitive test 0.07295 g of the test substance were added to a total volume of 500 mL aerated dechlorinated water, synthetic sewage and activated sludge. The sample was stored in the dark, at ambient temperature, in the container in which it was received until required for testing, when an appropriate subsample was provided for the test operator.

Test organisms (species):

activated sludge

Details on inoculum:

- Activated sludge: Activated sludge was obtained from Buckland Sewage Treatment Works, Newton Abbot, Devon, UK, 1 day prior to the study. This plant treats sewage of predominantly domestic origin. On return to the laboratory, the activated sludge was settled and the concentrated sludge washed with aerated dechlorinated water. The washed settled sludge was fed with 50 mL of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature, until it was used in the test. The total filterable solids concentration was determined on the day of the test and was found to be 6085 mg/L. The pH was measured as 7.10, and required no subsequent adjustment.

- Synthetic sewage: A synthetic sewage mixture described by the OECD guideline was prepared containing the following constituents per litre of deionised water: 15.2 g of peptone, 10.5 g of meat extract, 2.9 g of urea, 0.7 g of NaCl, 0.4 g of CaCl2.2H2O, 0.2 g of MgSO4.7H2O and 2.8 g of K2HPO4.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.10

Nominal and measured concentrations:

- Nominal concentration: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
An overview of the experimental design is provided in Table 1 in 'Any other information on materials and methods incl. tables'.
- No. of vessels per test substance concentration: 1
- No. of vessels per control : 2
- No. of vessels per reference substance concentration: 1
- Volume of active sludge: 160 mL
Each flask contained an excess of the synthetic sewage, sufficient activated sludge to give final solids concentrations of 1600 mg/L, an appropriate quantity of either the test substance or 3,5-dichlorophenol stock solution and aerated dechlorinated water to give a final flask contents volume of 500 mL.

EFFECT PARAMETERS MEASURED
The respiration rate of each culture was measured after 3 hours and compared with the mean respiration rate of the two control cultures. The rate of oxygen uptake was measured in a confined perspex cell into which a polarographic oxygen electrode was inserted. The electrode was connected to a meter whose output was recorded on a potentiometric chart recorder. The dissolved oxygen concentration after the 3 hour aeration period was at least 6.5 mg O2/L. The respiration rate was calculated between approximately 6.5 and 2.5 mg O2/L, or over approximately a 10 minute period where respiration was low. The rates of oxygen uptake were expressed as mg O2/L/h.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (at least 97% w/w)

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

An overview of the results is provided in Table 2 in ‘Any other information on results incl. tables’.

In test flasks dosed with the test substance, <10% to 15% inhibition was observed in the tested concentrations. Therefore, 3-hour EC20, EC50 and EC80 were determined to be above 100 mg/L. The EC10 was determined to be 32 mg/L.

Results with reference substance (positive control):

The reference substance, 3,5-dichlorophenol, caused substantial inhibition of the respiration rate of the activated sludge. From the results obtained the 3 hour EC50 value was estimated to be 9 mg/L. This is within the expected normal range of 5 to 30 mg/L indicating the sludge was responding normally and confirming the viability of the sludge organisms. The respiration rates in the two control flasks were within 15% of each other. Therefore, the mean control respiration rate was used in calculation of the percentage inhibition. The temperatures of the flask contents after the 3 hours aeration were all within the range 20 ± 2°C.

Table 2 Inhibition results

Test substance	Nominal Test concentration (mg/L)	pH		Respiration rate (mg O2/L/h)	Percentage mean control respiration rate^a	Percentage inhibition
		Start	End
Control	Control	7.40	8.16	29.3	-	-
3,5-DCP^b	100	7.50	7.92	2.7	9	91
3,5-DCP	32	7.40	8.09	6.0	20	80
3,5-DCP	10	7.40	8.12	13.7	45	55
3,5-DCP	3.2	7.39	8.24	27.4	90	10
3,5-DCP	1.0	7.40	8.21	28.5	93	<10
test substance	100	7.42	8.14	26.0	85	15
test substance	32	7.40	8.13	29.5	97	<10
test substance	10	7.39	8.09	27.4	90	10
test substance	3.2	7.39	8.10	28.5	93	<10
test substance	1.0	7.40	8.11	29.3	96	<10
Control	Control	7.39	8.14	31.7	-	-

a Mean control respiration rate= 30.5 mg O2/L/h

b 3,5-dichlorophenol

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the EC50 was determined to be > 100 mg ai/L and NOEC was 32 mg a.i./L.

Executive summary:

The effect of the test substance on bacterial activity in sewage sludge was investigated by exposing activated sludge from a working treating predominantly domestic sewage to the test substance at nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L. The study was conducted in accordance with OECD TG 209 and in compliance with GLP criteria. The test incorporated a dose response to a reference standard (3,5-dichlorophenol) at five nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L, and 2 replicates of an untreated control. After continuous aeration for 3 hours at 20 ± 2 ˚C and feeding on a synthetic sewage, the respiration rate of each culture was determined by measuring the oxygen consumption. The respiration rates of treated cultures were expressed as a percentage of the mean rates seen in control cultures.

After 3 hours incubation, 10% or less inhibition was observed in the 1, 3.2, 10 and 32 mg/L treatments. The 100 mg/L treatment resulted in 15% inhibition after 3 hours. Percentage inhibition less than or equal to 10% was within the expected experimental variability of the test and was considered not to be an effect of the test substance. Therefore, NOEC was equivalent to the EC10 in this test. The 3-h NOEC/EC10 was thus derived to be 32 mg/L. The 3-h EC50 was derived to be >100 mg/L

Description of key information

3 -h NOEC = 32 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Wallace 2002

3-h EC50 > 100 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Wallace 2002

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

32 mg/L

Additional information

The effect of the test substance on bacterial activity in sewage sludge was investigated by exposing activated sludge from a working treating predominantly domestic sewage to the test substance at nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L. The study was conducted in accordance with OECD TG 209 and in compliance with GLP criteria. The test incorporated a dose response to a reference standard (3,5-dichlorophenol) at five nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L, and 2 replicates of an untreated control. After continuous aeration for 3 hours at 20 ± 2 ˚C and feeding on a synthetic sewage, the respiration rate of each culture was determined by measuring the oxygen consumption. The respiration rates of treated cultures were expressed as a percentage of the mean rates seen in control cultures.

After 3 hours incubation, 10% or less inhibition was observed in the 1, 3.2, 10 and 32 mg/L treatments. The 100 mg/L treatment resulted in 15% inhibition after 3 hours. Percentage inhibition less than or equal to 10% was within the expected experimental variability of the test and was considered not to be an effect of the test substance. Therefore, NOEC was equivalent to the EC10 in this test. The 3-h NOEC/EC10 was thus derived to be 32 mg/L. The 3-h EC50 was derived to be >100 mg/L