L-Leucine, 4-fluoro-N-[(1S)-2,2,2-trifluoro-1-[4'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]ethyl]-, compd. with N-cyclohexylcyclohexanamine (1:1)

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:

Jan-Feb 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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

EC Number:

700-778-8

Cas Number:

1064076-86-3

Molecular formula:

C21H23F4NO4S.C12H23N

IUPAC Name:

N-cyclohexylcyclohexanaminium (2S)-4-fluoro-4-methyl-2-{[(1S)-2,2,2-trifluoro-1-{4'-methanesulfonyl-[1,1'-biphenyl]-4-yl}ethyl]amino}pentanoate

Details on test material:

Name of test material (as cited in study report): Wheat Solubles, Hydrolyzed
- Substance type: white powder
- Physical state: powder
- Analytical purity: 98.3% w/w (by HPLC)
- Lot/batch No.: 3141100578
- Expiration date of the lot/batch: 28 November 2012 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature protected from light desiccated
-Stability in water: Unknown
-Solubility in water: Not indicated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h and t=48 h
Volume 2 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.

Additionally, reserve samples of 2 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with the highest test concentration of 100 mg/l. Magnetic stirring and treatment with ultrasonic waves were applied for a period of approximately 2 hours to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age at study initiation: < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
- Feeding during test: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

between 18.3 and 20.6°C

pH:

between 7.7 and 8.0

Dissolved oxygen:

between 8.9 and 9.8 mg/l

Nominal and measured concentrations:

Nominal: control and 10, 18, 32, 56 and 100 mg/l
Measured concentrations: Analysis of the samples taken at the start of the final test showed that measured concentrations based on the cation ranged between 111 and 118% relative to nominal. For the anion the measured concentrations ranged between 94 and 103% relative to nominal. During the exposure period the measured concentrations remained constant. All results were therefore based on the nominal test concentrations.

Details on test conditions:

- Vessel type: open
- Material, size, headspace, fill volume: glass, 100 ml, normal headspace, 80 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:
CaCl2.2H2O : 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO3 : 46.7 mg/l
KCl : 4.2 mg/l
The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3 after aeration.

- Culture medium different from test medium: yes, for culturing M7 medium is used
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50 t=24, 48 h and NOEC
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations::control and 10, 18, 32, 56 and 100mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval between 77 and 91 mg/l

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Behavioural abnormalities: no
- Immobility of control: no immobility was observed.
- Other adverse effects control: no

Results with reference substance (positive control):

The 24h-EC50 was 0.53 mg/l with a 95% confidence interval between 0.47 and 0.62 mg/l.

The 48h-EC50 was 0.28 mg/l with a 95% confidence interval between 0.26 and 0.32 mg/l.

Reported statistics and error estimates:

The EC50-value was calculated at 48 hours of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

Table 1           Acute immobilisation of daphnids after 24 and 48 hours in the final test

Concentration Biphenyl DCHA Salt (mg/l)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
10	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
18	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
32	A  B C D	5 5 5 5	0 1 0 0	5	0 1 0 0	5
56	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100	A  B C D	5 5 5 5	0 0 0 0	0	5 5 3 4	85

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Biphenyl DCHA Salt did not induce acute immobilisation of Daphnia magna at 56 mg/l after 48 hours of exposure (NOEC).

The 48h-EC50 was 82 mg/l based on analytically confirmed nominal concentrations (95% confidence interval between 77 and 91 mg/l).