D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-, 2,3,4,6-tetraacetate, (1S)-

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 2016 - 22 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of diffiult items and mixtures, OECD series on testing and assessment number 23.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

EC Number:

930-010-9

Cas Number:

461432-25-7

Molecular formula:

C29H33ClO10

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}oxan-2-yl]methyl acetate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystalline solid
Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

- Solubility and stability of the test substance in the solvent/vehicle: < 0.0001 g/l @ 19.5 - 20.5 °C (insoluble); stability not indicated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method:during the final test single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: 1. At the start of the test and after 72 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.
Volume: 1.5 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

Additionally, the filter containing the undissolved test item residue was kept and single reserve samples of 1.5 mL were taken from all test solutions for possible analysis.

- Sample storage conditions before analysis: stored in a freezer (≤ -15°C) and protected from light.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with an aqueous mixture prepared at 100 mg/L. The mixture was magnetically stirred for 3 days to ensure maximum dissolution in test medium. The resulting dispersion was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved test item. The lower test concentrations were prepared by subsequent dilutions of the filtered Saturated Solution (SS) in test medium. Note that weighing and formulation were performed under dimmed light conditions.
- Evidence of undissolved material: no

Test organisms

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Strain: Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation: 2.4 ± 0.2 cm
- Weight at study initiation: 0.22 ± 0.04 g
- Method of breeding: bred in UV-treated water

ACCLIMATION
- Acclimation period: 12 days after delivery
- Acclimation conditions: same as test
- Type and amount of food during acclimation: pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International BV, Helmond, The Netherlands.
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): mortality of less than 5% during 7 days prior to the test

FEEDING DURING TEST
No feeding from 24 hours prior to the test and during the total test period.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

180 mg CaCO3 per litre

Test temperature:

Between 20.8 °C and 21.5°C throughout the test

pH:

Between 7.3 and 7.8 throughout the test

Dissolved oxygen:

Between 6.2 mg/L and 9.2 mg O2/L throughout the test

Nominal and measured concentrations:

Nominal concentration: 10, 18, 32, 56 and 100 % of a SS prepared at a loading rate of 100 mg/L
Measured concentration: 0.013 mg/L at t=0 and t=72 (fresh solutions) and 0.0074 and 0.010 mg/L at t=24 and t=48 (old solutions) respectively.
The average exposure concentration was calculated to correspond with 0.011 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 3.5 litres, all-glass, open, fill volume: 3 litres
- Aeration: no
- Renewal rate of test solution: every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 0
- Biomass loading rate: 0.51 g fish/litre

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- Intervals of water quality measurement: once a week

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily
- Light intensity: 5 lux (dimmed light conditions)

EFFECT PARAMETERS MEASURED: mortality after 96 hours; conductivity, pH, nitrate, nitrite and ammonia concentration (once a week); temperature (continuous); In addition, pH and temperature were measured before transferring the fish to the test system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study concentrations: 1.0, 10 and 100% of SS at 100 mg/L (measured concentrations at 0.017, 0.015 and 0.0087 mg/L respectively at highest test concentrations)
- Results used to determine the conditions for the definitive study: based on these results, it was decided to renew test solutions on a daily basis in the final test as to achieve better stability of concentrations over the test period.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (January 2016)

Results and discussion

Effect concentrations

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.011 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

No behavioural abnormalities, observations on body length and weight, other biological observations or abnormal responses were observed.
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: yes, >0.011 mg/L
- Temperature, dissolved oxygen levels and pH remained within the ranges prescribed by the study plan.

Results with reference substance (positive control):

Results with reference substance:
- Mortality: 100% at 0.46 mg/L, 20% at 0.22 mg/L and 0% at 0.10 mg/L
- LC50: 0.24 mg/L
- Other: effect was already reached withing 48 hours of exposure.

Reported statistics and error estimates:

No observations performed for dead or severely distressed fish in the range-finding test.

The following system was used for data acquisition: REES Centron Environmental Monitoring system version SQL 2.0

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1          Concentrations of the test item in test medium - final test

Time of sampling [days]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0 (fresh)	0	n.d.
	100	0.013^3
1 (old)	0	n.d.	n.a.
	100	0.0074^3	57
3 (fresh)	0	n.d.
	100	0.013^3
4 (old)	0	n.d.	n.a.
	100	0.010^3	81

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

³      Estimated value, calculated by extrapolation of the calibration curve.

n.d.Not detected.

n.a.Not applicable.

Table 2          Incidence of mortality and total mortality during the final test

Test group BMS-587172-01 (% SS)	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3¼h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
10	7	0	0	0	0	0	0
18	7	0	0	0	0	0	0
32	7	0	0	0	0	0	0
56	7	0	0	0	0	0	0
100 (0.011 mg/L*)	7	0	0	0	0	0	0
* Between brackets; average measured concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1) No mortality was observed in the control. 2) Test conditions were maintained constant 3) Dissolved oxygen concentration has been at least 60% of the air saturation (> 5mg/L at 22°C). 4) Results were based on the average concentrations

Conclusions:

No effects were observed in any of the concentrations tested up to and including the limit of solubility. The 96h-LC50 exceeded 0.011 mg/L, being the average measured concentration in a filtered saturated solution initially prepared at a loading rate of 100 mg/L. This concentration was considered the limit of solubility in test medium. Due to the low solubility of the test item in test medium, concentrations toxic for carp could not be reached.

Executive summary:

This study was performed to assess the effect of the test substance on the mortality of carp (Cyprinus carpio) after 96 hours of exposure. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 203 and GLP, under semi-static conditions.

A static range-finding test was performed to determine nominal concentrations for the final test. Three fish per concentration were exposed to 1.0, 10 and 100% of a SS prepared at a loading rate of 100 mg/L. Based on the results of the range-finding test, the following nominal concentrations were tested in the definitive test: 10, 18, 32, 56 and 100% of a SS prepared at a loading rate of 100 mg/L.

For each concentration and control group, 7 carps were exposed to the test substance and an untreated control for 96 hours in all-glass, open vessels. Test solutions were renewed every 24 hours. Mortality was recorded for each test and control group at 3¼ h, 24 h, 48 h, 72 h and 96 h. Samples taken from fresh and 24h spent solutions, for the 0% and 100% SS concentrations, were analysed to determine the test concentrations. Measured concentrations of the freshly prepared 100% SS solutions were 0.013 mg/L for both the first and last renewal interval. Measured concentrations of the 24 -hour spent 100% SS solutions were 0.0074 and 0.010 mg/L for the first and last renewal period respectively. The average exposure concentration was calculated to correspond with 0.011 mg/L.

The 96h-LC50 exceeded 0.011 mg/L, being the average measured concentration in a filtered saturated solution initially prepared at a loading rate of 100 mg/L. This concentration was considered the limit of solubility in test medium. Due to the low solubility of the test item in test medium, concentrations toxic for carp could not be reached.