.beta.-D-Ribofuranoside, methyl 2,3-O-(1-methylethylidene)-, 5-(4-methylbenzenesulfonate)

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:

May 17, 2005 to July 08, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute Immobilisation Test, 2004. EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

and OECD No.202

Deviations:

no

Test guideline 2

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:

[(3aS,4S,6S)-6-methoxy-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl 4-methylbenzene-1-sulfonate

EC Number:

609-920-2

Cas Number:

4137-56-8

Molecular formula:

C16H22O7S

IUPAC Name:

[(3aS,4S,6S)-6-methoxy-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl 4-methylbenzene-1-sulfonate

Details on test material:

Identity: Tosylfuranosid
Expiration date: July 31, 2005
Solubility in water: Not miscible
Aggregate state/physical form at room temperature: Solid
Color: White
Storage conditions: At room temperature at about 20 °C, away from direct sunlight

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE TEST ITEM CONCENTRATIONS
For the analysis of the actual test item concentrations, the following samples were taken:
Just before the start of the test: - duplicate samples from each test medium (without daphnids)
- duplicate samples from the control (without daphnids)
After 48 hours: - duplicate samples from each test medium
(stability samples) - duplicate samples from the control
For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (non-GLP), the test item is sufficiently stable in the test water under the storage conditions.
The concentrations of the test item Tosylfuranosid were analyzed in the test medium samples of the dilutions 1:4, 1:2 and of the undiluted filtrate from both sampling times (0 and 48 hours). The samples of the dilutions 1:8 and 1:16 were not analyzed, since the test concentrations were below the 48-hour NOEC, determined in this test. From the control samples only one of the duplicate samples was analyzed from each of the sampling times.

Test solutions

Vehicle:

yes

Details on test solutions:

DOSAGE AND CONCENTRATIONS
According to the results of a pre-experiment (non-GLP), the test item was not completely soluble at the concentration of 100 mg/L in the test water.
Therefore, a filtrate of a supersaturated dispersion of the test item and dilutions of the filtrate were prepared. The undiluted filtrate and the dilutions 1:2, 1:4, 1:8 and 1:16 were tested. Additionally, a control was tested in parallel (test water without test item).
The supersaturated dispersion with the loading rate of 100 mg/L was prepared by weighing 80.9 mg of the test item into 800 mL of test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
The stirring period of 24 hours was chosen according to the results of a pre-test (non-GLP) which showed that the solution equilibrium was reached after this time. In this pre-test similar test item concentrations were analytically measured in filtrates of dispersions stirred for 3, 24 and 96 hours.
Then, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) just before the preparation of the test media. The undiluted filtrate of the dispersion with the maximum concentration of dissolved test item was used as the highest concentrated test medium. Additionally, aliquots of the filtrate were diluted with test water to prepare the test media with lower test item concentrations. The test media were prepared just before introduction of the daphnids (i.e., start of the test).
The actual concentrations of the test item in the test media were analytically determined.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of a clone of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since that time, the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
At the start of the test, the daphnids used for the test were 6–24 hours old and were not first brood progeny.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Does not apply

Test conditions

Hardness:

250 mg/L as CaCO3

Test temperature:

20 °C

pH:

between 7.9 and 8.0

Dissolved oxygen:

at least 8.4 mg/L

Salinity:

Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
CaCl2 × 2H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 × 7H2O : 0.5 mmol/L (= 123 mg/L)
NaHCO3 : 0.75 mmol/L (= 65 mg/L)
KCl : 0.075 mmol/L (= 5.8 mg/L)
Water Hardness : 2.5 mmol/L (= 250 mg/L as CaCO3)
Alkalinity : 0.8 mmol/L

Nominal and measured concentrations:

7.6, 14, and 25 mg/L

Details on test conditions:

EXPERIMENTAL CONDITIONS
The test was performed in 100 mL glass beakers filled with 50 mL of test medium. The beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.
At each test concentration and for the control 20 daphnids were used divided into four replicates of five daphnids each. The daphnids were randomly distributed to the test vessels at initiation of the test. The loading rate was less than one daphnia per 5 mL of test solution.
A static test without test medium renewal was chosen since in a pre-experiment (non-GLP) the test item concentration in the test medium was constant over 72 hours.
The test was performed under the following conditions:
Water temperature: 20 °C during the test period. The test was performed in a temperature-controlled room (room temperature continuously monitored).
Light conditions: A 16-hour light to 8-hour dark photoperiod with a 30 minute transition period (light intensity during the light period was between approximately 570 and 740 Lux).
Test duration: 48 hours
Test water: Reconstituted test water: analytical grade salts were dissolved in purified water to obtain the following nominal concentrations:
CaCl2 × 2H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 × 7H2O : 0.5 mmol/L (= 123 mg/L)
NaHCO3 : 0.75 mmol/L (= 65 mg/L)
KCl : 0.075 mmol/L (= 5.8 mg/L)
Water Hardness : 2.5 mmol/L (= 250 mg/L as CaCO3)
Alkalinity : 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Na : K = 10 : 1 (based on molarity)
The test water was aerated until oxygen saturation was reached. During the test period the test water was not aerated.
Feeding: None
Positive Control: For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in February 2005 (48-hour EC50: 0.53 mg/L, RCC Study No. 859199) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 48-hour EC50: 0.53 - 1.1 mg/L).

Reference substance (positive control):

yes

Remarks:

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in February 2005 (48-hour EC50: 0.53 mg/L, RCC St

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 25 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

14 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 25 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 25 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

7.6 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

7.6 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Effect concentrations 7

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 25 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

mobility

Details on results:

The analytically measured test item concentrations in the analyzed test media samples (dilutions 1:4 and 1:2, and the undiluted filtrate) amounted to 7.6, 13, and 25 mg/L at the start of the test. At the end of the test, the concentrations found in the test media ranged from 98 to 104% of the initially measured values. Under the conditions of the test the test item was stable over the test period of 48 hours.
The biological results are related to the mean measured test item concentrations (calculated as the average over all measurements per test concentration) which were 7.6 mg/L (dilution 1:4), 14 mg/L (dilution 1:2), and 25 mg/L (undiluted filtrate).
After the test period of 24 hours, no immobilized test organisms were observed in the control and in all dilutions of the filtrate up to and including the mean measured test item concentration of 14 mg/L. Only in the undiluted filtrate with the mean measured concentration of 25 mg/L, two of the daphnids were immobile, corresponding to an immobilization rate of 10%.
The 24-hour EC0 was 14 mg/L. The 24-hour EC50 and the 24-hour EC100 of the test item were determined to be above the highest test concentration of 25 mg/L.
After 48 hours of exposure, no significant immobilization was determined in the dilution 1:16, 1:8, and 1:4 up to and including the mean measured concentration of 7.6 mg/L. At the test concentrations of 14 and 25 mg/L (dilution 1:2 and undiluted filtrate), the immobilization rates were 15 and 25%, respectively.
At the test concentration of 7.6 mg/L (dilution 1:4), one test organism was immobile at the observation after 48 hours. However this immobilization rate was not estimated as a significant toxic effect, because according to the test guidelines this immobilization rate is also tolerated in the control.
In addition to the immobilization of the daphnids, symptoms of toxicity were observed at the two highest test concentrations of 14 and 25 mg/L. At these test concentrations, 40 and 50% of the daphnids, respectively, showed a slow reaction after agitation of the test beakers, the antennae stuck together, and an abnormal swimming behavior was observed. However, these test organisms were capable of swimming and were therefore counted among the mobile daphnids.
The 48-hour EC0 and also the 48-hour NOEC (highest concentration tested without toxic effects after 48 hours) of Tosylfuranosid were 7.6 mg/L since no significant immobilization was observed in the test organisms up to and including this test concentration. The 48-hour EC50 and the 48-hour EC100 could not be determined due to the low immobilization rates of the test item up to the highest test concentration of 25 mg/L. These values were above the highest test concentration of 25 mg/L or higher than the solubility limit of the test item in the test water.
No remarkable observations were made concerning the appearance of the test media prepared by dilution of the filtrate. These test media were clear solutions throughout the entire test duration. The undiluted filtrate was also a clear solution at the start of the test and after 24 hours test duration. After 48 hours test duration, a part of the test item was precipitated and was lying at the bottom of the test beakers.
At the beginning and the end of the test period, the dissolved oxygen concentrations in the test media and the control were at least 8.4 mg/L. The pH values were between 7.9 to 8.0 and the water temperature was 20 °C at the start and the end of the test.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in February 2005 (48-hour EC50: 0.53 mg/L, RCC Study No. 859199) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2005: 48-hour EC50: 0.53 - 1.1 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of the test item Tosylfuranosid to Daphnia magna was determined in a 48-hour static test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

Since the test item was not completely soluble in the test water at the concentration of 100 mg/L, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 24 hours. Then, the dispersion was filtered and the undiluted filtrate with the maximum concentration of dissolved test item was used as the highest test medium concentration and as stock solution for dilutions in a geometric series differing by a constant factor of 2. The undiluted filtrate of the dispersion and the dilutions 1:2, 1:4, 1:8, and 1:16 were used as test media. Additionally, a control was tested in parallel.

The analytically measured test item concentrations in the analyzed test media samples (dilutions 1:4 and 1:2, and the undiluted filtrate) amounted to 7.6, 13, and 25 mg/L at the start of the test. At the end of the test, the concentrations found in the test media ranged from 98 to 104% of the initially measured values. Under the conditions of the test, the test item was stable over the test period of 48 hours.

The biological results are related to the mean measured test item concentrations (calculated as the average over all measurements per test concentration) which were 7.6 mg/L (dilution 1:4), 14 mg/L (dilution 1:2), and 25 mg/L (undiluted filtrate).

The biological test results are (on basis of mean measured test item concentrations):

- 24-hour EC50: > 25 mg/L

- 24-hour EC0 : 14 mg/L

- 24-hour EC100: > 25 mg/L

- 48-hour EC50: > 25 mg/L

- 48-hour EC0 and 48-hour NOEC: 7.6 mg/L

- 48-hour EC100: > 25 mg/L