Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

21 Aug 2006 to 23 Aug 2006

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 (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

No stock solution could be prepared due to the low water solubility of the test item. Therefore, test item amounts of 4.87, 16.43, 48.42, 158.84, and 500.16 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 284 mL of tap water. The test item was mixed into the tap water by ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark, to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added. Additionally, two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the five test item concentrations under identical test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Location of sewage treatment plant where inoculum was collected: Fullinsdorf, Switzerland
- Preparation of inoculum for exposure: Aerobic activated sludge from a wastewater treatment plant treating predominantly domestic wastewater. To eliminate possible inhibitory material, the sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Pretreatment: Based on the calculated wet and dry weight ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of two days prior to use, the sludge was fed daily with 50 ml synthetic wastewater* per litre and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was adjusted from 8.3 to 7.0 with a diluted sulfuric acid solution.

* Synthetic wastewater (filled up to a final volume of 1 litre with deionized water):
- 16 g peptone
- 11 g meat extract
- 3 g urea
- 0.7 g NaCl
- 0.4 g CaCl2 · 2H2O
- 0.2 g MgSO4 · 7H2O
- 2.8 g K2HPO4

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 °C

pH:

- Test initiation: 7.7 to 7.8
- Test termination: 8.3 to 8.6

Dissolved oxygen:

- Test initiation: 7.9 to 8.2 mg O2/L
- Test termination: 8.4 to 8.9 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L glass beakers
- Aeration: Yes (continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item)
- No. of vessels per concentration: 1
- No. of vessels per control: 2
- Sludge concentration: 2.2 g/L dry weight (corresponding to about 0.9 g dry material per litre test medium).

EFFECT PARAMETERS MEASURED
For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. Then, the dissolved oxygen concentration was measured with an oxygen electrode, and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve in the range of 6.5 - 2.5 mg 0 2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above, but always within the linear part of the respiration curve. In case of low oxygen consumption, the rate was determined over a period of at least ten minutes.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol; at nominal concentrations of 5, 16 and 50 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: NOEC above the solubility of the test substance in the test medium

Details on results:

An overview of the results is provided in Table 1 in ‘Any other information on results incl. tables’.
Down to the lowest test concentration of 10 mg/L, the test item was not completely dissolved in the test media (after stirring for 24 hours). Thus, all test concentrations were clearly above the water solubility limit of the test item under the test conditions.
The test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of 3 hours up to and including the highest test item concentration of 1000 mg/L. The inhibition of the respiration rates was in the range -3 to 10%, compared to the controls. No concentration-effect relationship was determined. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at least 1000 mg/L. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 0.5% (guideline-recommended: ≤15%).

Results with reference substance (positive control):

An overview of the results is provided in Table 1 in ‘Any other information on results incl. tables’. The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was 14 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline recommended range of 5 - 30 mg/L, confirming suitability of the activated sludge used.

Table 1. Influence of the test item and 3,5-dichlorophenol on the oxygen consumption of activated sludge

Vessel no.	Nominal concentration of test chemical (mg/L)	Oxygen consumption rate (mg O2/L/min)	Inhibition (%)	pH values		Oxygen concentration (mg O2/L)
				start*	end*	start*	end*
1	0	1.122		7.7	8.4	7.9	8.5
10	0	1.128		7.8	8.4	8.0	8.7
Mean		1.125
Deviation(%)		0.5
Test item
5	10	1.080	4.0	7.8	8.5	7.9	8.4
6	32	1.153	-2.5	7.8	8.4	8.2	8.6
7	100	1.136	-1.0	7.7	8.4	8.0	8.9
8	320	1.015	9.8	7.7	8.5	7.9	8.4
9	1000	1.087	3.4	7.7	8.3	8.2	8.9
Reference item
2	5	1.028	8.6	7.8	8.5	8.0	8.3
3	16	0.387	65.6	7.7	8.6	8.2	8.5
4	50	0.109	90.3	7.8	8.6	8.3	8.4

* Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to control

Validity criteria fulfilled:

yes

Conclusions:

In an activated sludge respiratory inhibition test performed in accordance with OECD TG 209, the 3-hour NOEC value was determined to be ≥ 1000 mg/L for inhibition of the total respiration of the microorganisms in activated sludge.

Executive summary:

An activated sludge respiratory inhibition test with the test substance was performed according to OECD TG 209 and GLP principles. Activated sludge collected from sewage treatment plants was placed in test bottles to which the test substance was added (no stock could be made due to low solubility). The final nominal concentrations of the test substance were 0 (blank control), 10, 32, 100, 320 and 1000 mg/L 20 °C. Due to the low water solubility of the test item, the test item was weighed directly into the test flasks. The aqueous dispersions in tap water were treated ultrasonically and were stirred over 24 hours. 3,5-Dichlorophenole was used as positive control and tested at concentrations of 5, 16 and 50 mg/L under the same experimental condition. During the 3 hours incubation period, the test vessel was continuously aerated by intense stirring on magnetic stirrers. The dissolved oxygen concentration was continuously recorded and the respiration rates calculated. The inhibitory effect of the test item was expressed as a percentage of the mean respiration rate of the two controls.

The test item was not completely dissolved in the test media (after stirring for 24 hours) in all treatments (≥ 10 mg/L). Thus, all test concentrations were clearly above the water solubility limit of the test substance under the test conditions. After 3 hours incubation, 8.6%, 65.6% and 90.3% of inhibition on oxygen consumption were observed in the groups treated with positive control at 5, 16 and 50 mg 3,5 -Dichlorophenole/L, respectively. For the test substance treated groups, ≤ 10% of inhibition was observed in all test concentrations. Therefore, the NOEC value was determined to be above ≥ 1000 mg/L  for inhibition of the total respiration of the microorganisms in activated sludge. The EC20, EC50 and EC80 could not be calculated because of the absence of a toxic effect.

Description of key information

3-h NOEC ≥ 1000 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Seyfried 2006

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

One study is available for this endpoint, which was selected as key study. In this study performed according to OECD TG 209 and in compliance with GLP, activated sludge collected from sewage treatment plants was exposed to the test substance (transferred directly to the test vessels as no stock could be made due to low solubility) at concentrations of 0 (control), 0, 32, 100, 320 and 1000 mg/L for 3 hours at 20 °C. 3,5-Dichlorophenole was used as positive control and tested at concentrations of 5, 16 and 50 mg/L under the same experimental condition. The test item was not completely dissolved in the test media (after stirring for 24 hours) in all ≥ 10 mg/L treatments. Thus, all test concentrations were clearly above the solubility limit of the test substance under the test conditions. After 3 hours incubation, 8.6%, 65.6% and 90.3% of inhibition on oxygen consumption were observed in 5, 16 and 50 mg/L 3,5-Dichlorophenole treated groups, respectively, confirming suitability of the activated sludge used. For the test substance treated groups, ≤ 10% of inhibition was observed in all test concentrations. Therefore, the NOEC value was determined to be ≥ 1000 mg/L for inhibition of the total respiration of the microorganisms in activated sludge. The EC20, EC50 and EC80 could not be calculated because of the absence of a toxic effect.