Bis-O-(benzylidene)-D-glucitol

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:

Experimental start date: 04 July 2017, Experimental completion date: 12 August 2017

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test item in test water for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Geniset D
Physical state/Appearance: White powder
Batch: 5434
Purity: 99.4%
Expiry Date: 31 October 2018
Storage Conditions: Room temperature, in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions (details below).

Saturated Solution Preparation:
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:

• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

Discussion:
Visual observations made on the centrifuged samples showed some undissolved test item remained, indicating that centrifugation was not sufficient to ensure all undissolved test item was removed.

The results obtained indicated that there was no increase in the dissolved test item concentration obtained when the preparation period was extended beyond 24 hours.

Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore (first approximate 2 litres discarded) to give a nominal test concentration of approximately 18 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test Water:
Reconstituted water (Elendt M7 medium) used for both the range-finding and definitive tests .

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

Temperature was maintained at approximately 22°C throughout the test

pH:

The pH of the prepared media was 7.9.
pH were recorded at the start and termination of the test.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test.

Nominal and measured concentrations:

Definitive Test:
Nominal: 100% v/v saturated solution
Measured: 19 mg/L

Details on test conditions:

RANGE FINDING TEST:
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 18 mg/L could be obtained using a saturated solution method of preparation.

The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore (first approximate discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10 % v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

DEFINITIVE TEST:
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore (first approximate 1 liter discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.

In the preliminary media preparation trial it was concluded that preconditioning the filters by discarding the first 2 liters gave the highest measured concentration, however, only 1 liter was discarded during media preparation in the definitive test. This was considered not to have adversely affected the outcome of the study, as measured concentrations achieved were higher than those in both the preliminary media preparation trial and the range finding test with no sub lethal effects or immobilization observed.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

EXPOSURE CONDITIONS:
As in the range-finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item. The test preparations were not renewed during the exposure period.

ASSESSMENTS
TEST ORGANISM OBSERVATIONS:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

WATER QUALITY CRITERIA:
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:
No immobilization was observed throughout the test.
No sub-lethal effects of exposure were observed throughout the test.

Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.

Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 48 hours (see Annex 4) showed measured test concentrations of 16 mg/L were obtained indicating that the test item was stable under the conditions of the test.

DEFINITIVE TEST:
VERIFICATION OF TEST CONCENTRATIONS:
Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 19 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0-Hour measured test concentration only.

IMMOBILIZATION DATA:
There was no immobilization in 20 daphnids exposed to a test concentration of 19 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:
24 h EC50: >19 mg/L
48 h EC50: >19 mg/L

SUB-LETHAL EFFECTS:
A Sub-lethal effect of exposure was observed in the 19 mg/L test concentration at 24 hours. This response was trapping at the water surface. This sub-lethal effect was not observed at the 48 hour observations.

VALIDATION CRITERIA:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA:
Temperature was maintained at approximately 22 C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 355 to 399 Lux.

Observations on Test Item Solubility:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.

Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

24 h:
EC50: 1.3 mg/L
NOEC: 1.0 mg/L
LOEC: 1.8 mg/L

48 h:
EC50: 1.2 mg/L
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Cumulative Immobilization Data and Observations in the Range-finding Test

 

Nominal Concentration (% v/v Saturated Solution)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5N	0	0	5N	5N
0.10	0	0	5N	5N	0	0	5N	5N
1.0	0	0	5N	5N	0	0	5N	5N
10	0	0	5N	5N	0	0	5N	5N
100	0	0	5N	5N	0	0	5N	5N

Cumulative Immobilization Data and Observations in the Definitive Test

 

0-Hour MeasuredTest Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
19	0	0	0	0	0	0	5 N	5N	4T 1N	5N

 

 

0-Hour MeasuredTest Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
19	0	0	0	0	0	0	5N	5N	5N	5N

 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the 0-Hour measured test concentration gave a 48-Hour EC50 value of greater than 19 mg/L. The No Observed Effect Concentration was 19 mg/L.

This study showed that there were no toxic effects at saturation.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 18 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 22°C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate l liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions of exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 19 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0-Hour measured test concentration only.

Exposure of Daphnia magna to the test item gave EC50 values based on the 0-Hour measured test concentrations of greater than 19 mg/L. The No Observed Effect Concentration was 19 mg/L.

This study showed that there were no toxic effects at saturation.

Description of key information

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 18 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 22°C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate l liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions of exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 19 mg/L. There was no significant change in the measured concentration at 48 hours and so the results are based on the 0-Hour measured test concentration only.

Exposure of Daphnia magna to the test item gave EC50 values based on the 0-Hour measured test concentrations of greater than 19 mg/L. The No Observed Effect Concentration was 19 mg/L.

This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

19 mg/L