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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-04-22 to 2003-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA FIFRA 72-2

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

An 800 mg/mL primary stock solution was prepared by adding 8.3662 g of myclobutanil (7.9981 g as active ingredient) to 10 mL of acetone. This stock solution was observed to be clear and yellow in color following brief sonication. Each test solution was prepared in a 2-L Mariotte bottle containing 1.0-L of dilution water. Upon addition of the stock solution, each solution was observed to be cloudy, then clear with undissolved material present. The Mariotte bottle was placed on a magnetic stir plate and a Teflon-coated stir bar was inserted into each bottle. Following 15 minutes of mixing, the 80 mg/L exposure solution was observed to contain undissolved test substance. The clear portion of the solution was withdrawn from the stopcock at the bottom of the Mariotte bottle and used to prepare the replicate exposure solutions to avoid the transfer of any undissolved material. The remaining exposure solutions (=40 mg/L) were clear and contained no visible undissolved test substance.

Each test solution was divided into four replicate vessels for each test concentration and the controls. In addition, four dilution water control and four solvent control vessels were established and maintained under the same conditions as the treatment level solutions but contained no myclobutanil.

The test was initiated when 20 daphnids, <24 hours old, were impartially selected and distributed to each concentration and the controls (replicates A, B, C and D, five daphnids per replicate vessel). Daphnids were not fed during the exposure.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Organism: Daphnia magna
Age: < 24 hours.

Test type:

static

Water media type:

freshwater

Remarks:

Fortified well water

Hardness:

The dilution water was hard fortified well water with a hardness of 170 mg/L as CaCO3 and an alkalinity of 110 mg/L as CaCO3.

Test temperature:

20-21°C

pH:

7.5-8.2.

Dissolved oxygen:

>93% saturation.

Nominal and measured concentrations:

Nominal concentrations: 5.0, 10, 20, 40 and 80 mg/L.
Mean measured concentrations: 3.3, 8.5, 15, 18 and 31 mg a.s./L

Details on test conditions:

Test system:
Test vessels were 250-mL glass beakers containing 200 mL of dilution water. Exposure levels were monitored by measuring myclobutanil in samples of dilution water collected at 0 and 48 hours. Initial measured test concentrations were 3.2, 8.7, 15, 18 and 28 mg a.s./L representing 63, 87, 73, 44 and 35% of the nominal concentrations, respectively. Mean measured concentrations were 3.3, 8.5, 15, 18 and 31 mg a.s./L representing 66, 85, 77, 45 and 39% of the nominal concentrations, respectively.

Test dilution water:
The dilution water used during this study was from the same source as the water used in the daphnid cultures and had a total hardness and alkalinity as CaCO3 specified above , a pH of 7.9 and a specific conductivity of 500 µmhos/cm.

Test conditions:
The test vessels were impartially placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 20 ± 1°C. The test area was illuminated with fluorescent bulbs at an intensity of 70 to 90 foot-candles at the solutions' surface. The photoperiod during the test was the same as in the culture area. Sudden transitions from light to dark and vice versa were avoided. Light intensity was measured with a General Electric type 217 light meter.

The number of immobilized daphnids in each replicate test vessel was recorded at 24 and 48 hours of exposure. Immobilization was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel. Biological observations and observations of the physical characteristics of each replicate test solution were also made and recorded at 0, 24 and 48 hours. The pH, dissolved oxygen concentration and temperature were measured at 0, 24 and 48 hours in replicate A of each treatment level and the controls. Continuous temperature monitoring was performed in the water bath throughout the exposure period. The pH was measured with a Jenco model 60 pH meter or a LaMotte HA pH meter. Dissolved oxygen was measured using a Yellow Springs Instrument (YSI) model 57 dissolved oxygen meter and probe. Daily temperature was measured with a Fisher alcohol thermometer. Temperature was continuously monitored throughout this study using a Fisher Min-Max thermometer.

Evaluation of Test conditions: Water quality parameters were unaffected by the concentrations of myclobutanil tested and remained within acceptable ranges for the survival of daphnids. Daily measurement of the temperature in the test solutions and continuous temperature monitoring of the water bath was carried out.

Determination of EC50 and NOEC: Both the initial measured concentrations and the mean measured concentrations tested and the corresponding immobilization data derived from the definitive toxicity test were used to estimate the median lethal concentrations (EC50) and 95% confidence intervals. The EC50 is defined as the concentration of the test substance in dilution water which caused immobilization of 50% of the test organism population at the stated time interval. If at least one test concentration caused immobilization of ~50% of the test population, then a computer program (Toxstat, Gulley, 1996) was used to calculate the EC50 values and 95% confidence intervals.
The No-Observed-Effect Concentration (NOEC) during the 48-hour exposure period was also determined. The NOEC is defined as the highest concentration tested at and below which there were no toxicant-related immobilization or physical and behavioral abnormalities (e.g., lethargy), with respect to the control organisms. In addition the highest concentration yielding 0% immobilization and the lowest concentration yielding 100% immobilization were also determined.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Sublethal and lethal effects

Details on results:

After 48-hours, immobility/mortality was 0, 0, 0, 5, 40, 60 and 100% in the control, solvent control, 5.0, 10, 20, 40 and 80 mg a.s./L nominal treatments, respectively. All surviving daphnids exposed to the 8.5 mg a.s./L treatment level were observed to be lethargic. All surviving daphnids exposed to the 15 and 18 mg/L treatment levels were observed to be lethargic and on the bottom of the test vessel. No immobilization or adverse effects were observed among daphnids exposed to the 3.3 mg/L treatment level or the controls.

Table 1: Immobilisation data.

Nominal Concentration mg/L	Initial Measured Concentrations as mg a.s./L (% of nominal)	48-Hour Measured Concentrations as mg a.s./L (% of nominal)	Mean Measured Concentrations as mg a.s./L (% of nominal)	24 hours Cumulative % immobilised	48 hours Cumulative % immobilised
Control	<0.28	<0.28	---	0	0
Solvent Control	<0.28	<0.28	---	0	0
5.0	3.2 (63)	3.4 (68)	3.3 (66)	0	0
10	8.7 (87)	8.4 (84)	8.5 (85)	5	5^a
20	15 (73)	16 (81)	15 (77)	35^a	40^a
40	18 (44)	18 (45)	18 (45)	35^a	60^a
80	28 (35)	34 (43)	31 (39)	100	100

^a All surviving daphnids were observed to be lethargic.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations the 48-h EC50 of myclobutanil to Daphnia magna under static conditions was determined to be 17 mg a.s./L, and the NOEC was determined to be 3.3 mg a.s./L.

Executive summary:

The purpose of this study was to estimate the acute toxicity (EC₅₀) of myclobutanil to daphnids (Daphnia magna) under static test conditions for 48-hour. Based on mean measured concentrations (0- and 48-hour), the 48-hour EC₅₀ was determined to be 17 mg a.s./L (95% confidence intervals of 15 to 18 mg a.s./L). The No-Observed-Effect Concentration (NOEC) was determined to be 3.3 mg a.s./L.

Description of key information

In a GLP-study performed according to OECD TG 202, the 48h EC₅₀ of myclobutanil was revealed to be 17 mg a.s./L (static). Test organism was Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

17 mg/L