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)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition 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): Biphenyl DCHA Salt
- 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=72 h
Volume 2 ml
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start and at the end of the test period.

Additionally, singular reserve samples of 2 ml were taken from all test solutions for possible analysis. If not already 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 1 hour (final test) or 3 hours (combined limit/range-finding test) 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.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol(24 mg CaCO3/L)

Test temperature:

between 21.3 and 22.3°C

pH:

between 7.8 and 8.0

Dissolved oxygen:

Not applicable

Nominal and measured concentrations:

Nominal: Control and 10, 18, 32, 56 and 100 mg/l
Measured: Analysis of the samples taken at the start of the final test showed that measured concentrations based on the cation ranged between 103 and 112% relative to nominal. For the anion the measured concentrations ranged between 88 and 94% relative to nominal. During the exposure period the measured concentrations remained constant, i.e. did not decrease by 20% or more. All results were therefore based on the nominal test concentrations.

Details on test conditions:

FINAL STUDY:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 2100000 cells/ml
Replicates:
6 replicates of the control
3 replicates of each test concentration
1 replicate of the highest concentration without algae

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 68 to 76 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: control and 10, 18, 32, 56, 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield inhibition

Remarks on result:

other: 95 % confidence interval ranging from 33 to 84 mg/l

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

both growth rate inhibition and yield inhibition

Details on results:

Biphenyl DCHA Salt reduced growth rate and inhibited the yield of the fresh water algae species Pseudokirchneriella subcapitata significantly at 18 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) was 53 mg/l based on analytically confirmed nominal concentrations with a 95 % confidence interval ranging from 33 to 84 mg/l.

The NOEC for both growth rate reduction and yield inhibition was 10 mg/l.

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.8 mg/l with a 95% confidence interval ranging from 1.4 to 2.4 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.69 mg/l with a 95% confidence interval ranging from 0.50 to 0.95 mg/l.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
No EC50-value for growth rate could be calculated (EC50 > maximum concentration tested).

Any other information on results incl. tables

Table 1        Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Concentration Biphenyl DCHA Salt (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
Control	0.07790		272.42
10	0.07799	-0.1	273.89	-0.5
18	0.07513	3.6	222.93	18.2
32	0.07367	5.4	200.99	26.2
56	0.06855	12.0	138.18	49.3
100	0.05825	25.2	65.29	76.0

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Biphenyl DCHA Salt reduced growth rate and inhibited the yield of the fresh water algae species Pseudokirchneriella subcapitata significantly at 18 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) was 53 mg/l based on analytically confirmed nominal concentrations with a 95 % confidence interval ranging from 33 to 84 mg/l.

The NOEC for both growth rate reduction and yield inhibition was 10 mg/l.