2-[(2S,3S)-3-[[(1,1dimethylethoxy)carbonyl]amino]-2-hydroxy-4-phenylbutyl]-2-[[4-(2pyridinyl)phenyl]methyl]-,1,1-dimethylethyl ester

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:

2000-05-30 to 2000-06-01

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)

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and R90076
- Expiration date of the lot/batch: 2000-06-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of the test substance in DMF was prepared at a concentration of 1.0 mg/ml
- Final dilution of a dissolved solid, stock liquid or gel: a 0.1 ml was taken and diluted with 1 L of dilution water to create the test solution.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Dissolved in DMF and then diluted to a concentration of 100 ug/L

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 ug/L
- Sampling method: Not specified
- Sample storage conditions before analysis: Not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test substance was dissolved in dimethylformamide (DMF) to give a primary stock solution with a nominal concentration of 10 mg/mL This solution was then further diluted with DMF to give a working stock solution of 1.0 mg/mL. An 0.1 mL aliquot of the working stock solution was added to diluent water to give a nominal test concentration of 100 µg/L. This provided an excess of test material in diluent water, and was stirred with a magnetic stirrer for approximately 30 minutes to give a saturated solution.
- Controls: There was a true negative control and a solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 ml of DMF/1 L dilution water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Aquatic daphnia
- Strain/clone: Daphnia magna
- Source: Cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Applique (IRChA), France.
- Age of parental stock (mean and range, SD): <24 hours
- Feeding during test: No

ACCLIMATION
- Acclimation period: 5 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Unicellular green algae
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): All mortalities were removed

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

Not specified

Test temperature:

21 C

pH:

7.7

Dissolved oxygen:

7.9

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal Concentration: 0 and 100 ug/L for the test and control respectively
Measured Concentration:

Details on test conditions:

TEST SYSTEM
- Test vessel: Jars
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass , 250 ml, 150 ml, 100 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Elendt M4 medium prepared using analytical grade reagents and deionized water.
NAME mg/L
1. Trace elements:
H3BO3 2.86
MnCl2.4H2O 0.36
LiCl 0.31
RbCI 0.071
SrCl2.6H2O 0.152
NaBr 0.016
Na2MoO4.2H2O 0.063
CuCl2.2H2O 0.017
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2.SeO3 0.0022
NH4VO3 0.00058
Fe-EDTA solution 3.50
2. Macro nutrients:
CaCl,.2H2O 294
MgSO4.7H2O 123
KCl 5.80
NaHCO3 64.8
3. Buffer nutrients:
Na2.SiO3.9H2O 10.0
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
4. Vitamins:
Thiamine hydrochloride 0.075
Cyanocobalamine (Vitamin Bl2) 0.0010
Biotin (Vitamin H) 0.00075

The above analytical grade reagents are dissolved in reverse osmosis deionized water.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Immediately prior to testing

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was not adjusted
- Photoperiod: 16 horus light/8 hours dark
- Light intensity: Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnia mobility at 24 and 48 hours into the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: Not specified
- Results used to determine the conditions for the definitive study: A preliminary study was conducted to determine the solubilty of the test substance in water. The study found that the solubility of the test substance in water was maximized by using a cofactor solvent. The maximum solubility that was able to be achieved with this method was ~61 ug/L, so a nominal concentration of 100 ug/L was chosen to ensure the solution was saturated.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 98 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 98 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 98 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No immobilisation was noted in a group of twenty, first instar Daphnia, less than 24 hours old, exposed to a saturated solution of BMS 233101-01 for a period of 48 hours.
- Effect concentrations exceeding solubility of substance in test medium: The nominal concentration of I 00 µg/L was greater than the estimated limit of solubility under the conditions of this study; 75 µg/L. Mean measured concentrations at O and 48 hours remained above the estimated limit of solubility of
BMS 233101-01 in Elendt M4; 75 µg/L.

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

The protocol states that a range finding test will be conducted before the definitive test. An initial range finding test was performed, using a different preparation method to that subsequently used in the definitive test. Following the decision to perform the study at a concentration greater than the limit of solubility, the range finding was not repeated, because the test compound was expected to have little, if any, toxicity. Therefore the definitive test was conducted immediately as a limit test.
These deviations from protocol are not believed to have affected the validity of this study.

Validity criteria fulfilled:

yes

Conclusions:

This study validly assessed the test substance's potential short-term toxicity to aquatic invertebrates according to the specified guidelines.

Description of key information

In a 48h acute aquatic toxicity test in Daphnia magna with the test substance BMS 233101,resulted in a EC50 value for, of > 98 µg/l based on the mean measured test concentrations. The NOEC was 98 µg/l. There were no toxic effects at the limit of solubility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

98 µg/L