4-[4-[4-[(3R, 5R)[5-(2,4-difluorophenyl) tetrahydro-5-(1H-1,2,4-triazol-1-yl-methyl)-furan-3-yl]methyloxy]phenyl]-1-piperazinyl]phenyl-2-[(1S,2S)(1-ethyl-2-phenylmethoxypropyl)]-2,4-dihydro-3Htriazol-3-one

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2012 - 12 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

(1996)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Paz-E

IUPAC Name:

Paz-E

Details on test material:

Identification: Paz-E
Molecular formula: C44H48O4N8F2
Molecular weight: 790.9
Description: White powder

Purity: 99.7% w/w
Test substance storage: At room temperature protected from light in desiccator
Stability under storage conditions: Stable
Expiry date: 25 November 2012 (allocated by NOTOX, 1 year after receipt of the test substance)

Hygroscopic: Yes, in desiccator
Volatile: No
Test substance handling: Use amber-coloured glassware or wrap container in tin-foil.
Stability in water: Unknown
Solubility in water: <10 mg/mL

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
-

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter used forpreparation of the highest test concentration was retained for possible analysis of the residue.
Frequency: at t=0 h and t=48 h
Volume: 2.4 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period the replicates were pooled at each concentration before sampling, except for the replicates of the highest test concentration. Due to the presence of a floating layer, a sample was taken from one replicate only.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Paz-E tested was a white powder with a purity of 99.7% w/w and not completely soluble in test medium at a loading rate of 100 mg/l. Weighing of test substance and preparation of test solutions was performed under dimmed light conditions.

Preparation of test solutions started with a loading rate of 100 mg/l applying two days of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 µm membrane filter (Whatman, RC 55) where after the clear and colourless filtrate was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

-

Post exposure observation period:

-

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

between 19.6 and 20.6°C

pH:

between 7.8 and 7.9 units

Dissolved oxygen:

9.0 mg/l

Salinity:

-

Nominal and measured concentrations:

Nominal concentrations: 0.10, 1.0, 10 and 100% of a filtered solution prepared at a loading rate of 100 mg/l.
Measured concentrations: Analysis of the sample taken from the highest test concentration at the start of the test showed a measured concentration of 133 µg/l. During the exposure period the measured concentration decreased to 8.0 µg/l (6% of initial). Based on these results, the average exposure concentration was calculated to be 33 µg/l.

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 100 ml, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 each for the control and the highest test concentration; 10 for each lower test concentration
Loading: 5 per vessel containing 80 ml of test solution
Light: No illumination
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 20 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

33 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 33 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: -

Details on results:

No immobility of daphnids was observed at any of the test concentrations and the control during the exposure period. At the highest test concentration a floating layer was observed from 24 hours onwards, indicating that the test substance concentration was above the solubility limit of Paz-E in test medium.

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 were just outside (below) the ranges of the expected historical responses at the different concentrations, i.e. a 48h-EC50 between 0.3 and 1.0 mg/l. Hence, the sensitivity of this batch of D. magna was slightly higher when compared to the historical data collected at NOTOX.
The 24h-EC50 was 0.49 mg/l with a 95% confidence interval between 0.45 and 0.55 mg/l.
The 48h-EC50 was 0.28 mg/l with a 95% confidence interval between 0.14 and 0.65 mg/l.

Reported statistics and error estimates:

No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration tested).

Any other information on results incl. tables

-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Paz-E did not induce acute immobilisation of Daphnia magna at an average exposure concentration of 33 µg/l after 48 hours of exposure (NOEC).
The 48h-EC50 was >33 µg/l and was considered to be above the solubility limit of Paz-E in test medium.