Bis-O-(benzylidene)-D-glucitol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 3 July 2017. Experimental completion date: 1 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

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 culture medium 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 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Remarks:

deionised water

Details on test solutions:

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.

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 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Visual observations made on the centrifuged test 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 filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 18 mg/L.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH value of the test cultures was observed to increase from pH 7.4 at 0 hours to pH 7.8 at 72 hours.
The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
The pH was measured using a Hach HQ30d Flexi handheld meter.

Nominal and measured concentrations:

Definitive test:
Nominal concentration of 100% saturated solution.
Measured concentration of 15 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. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium 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 filter (first approximate 2 liters 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 stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.

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

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 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 the highest attainable test concentration no effect on algal growth was observed.

Experimental Preparation:
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium 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 filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. An aliquot (1 liter) of this saturated solution was inoculated with algal suspension (7.3 mL) to give the required test concentration of 100% v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Exposure Conditions:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and 100% v/v saturated solution treatment group.

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

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 6.88 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 7.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations:
Samples were taken at 26, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

0-Hour measured test concentration

Basis for effect:

other: yield

Details on results:

Range-finding Test:
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Based on this information a single test concentration of six 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 the highest attainable test concentration no effect on growth was observed.

Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 72 hours showed a measured test concentration of 15 mg/L was obtained indicating that the test item was stable under test conditions.

Definitive Test:
Verification of Test Concentrations
Chemical analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 15 mg/L was obtained. There was no significant change in the measured concentration at 72 hours and so the results are based on 0-Hour measured test concentration only.

Growth Data:
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a 0-Hour measured test concentration of 15 mg/L over the 72-Hour exposure period.

The test concentration of 15 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water.

Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h): >15 mg/L
ErC20 (0 - 72 h): >15 mg/L
ErC50 (0 - 72 h): >15 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 15 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P≥0.05), between the control and 15 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 15 mg/L.

Inhibition of Yield:
EyC10 (0 - 72 h): >15 mg/L
EyC20 (0 - 72 h): >15 mg/L
EyC50 (0 - 72 h): >15 mg/L
Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05), between the control and 15 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 15 mg/L.

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on Test Item Solubility:
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Validation Criteria.

The following data show that the cell concentration of the control cultures increased by a factor of 124 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours : 5.00 x 10³ cells per mL

Mean cell density of control at 72 hours : 6.21 x 10⁵ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Inhibition of Growth Rate and Yield in the Definitive Test

0-Hour Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.066		5.93E+05
	R2	0.065		5.34E+05
	R3	0.068		6.73E+05
	R4	0.067	-	6.10E+05	-
	R5	0.065		5.37E+05
	R6	0.070		7.45E+05
	Mean	0.067		6.16E+05
	SD	0.002		8.17E+04
15	R1	0.068	[1]	6.79E+05
	R2	0.069	[3]	6.94E+05
	R3	0.068	[1]	6.85E+05
	R4	0.066	1	5.74E+05
	R5	0.067	0	6.12E+05
	R6	0.068	[1]	6.75E+05
	Mean	0.068	[1]	6.53E+05	[6]
	SD	0.001		4.85E+04

*    In accordance with the OECD test guideline only the mean value for yield is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 15 mg/L. The No Observed Effect Concentration was 15 mg/L.
This study showed that there were no toxic effects at saturation.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

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, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium 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 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 15 mg/L was obtained. There was no significant change in the measured concentration at 72 hours and so the results are based on the 0-Hour measured test concentration only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀ values of greater than 15 mg/L. The No Observed Effect Concentration was determined to be 15 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 effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

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, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium 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 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured test concentration of 15 mg/L was obtained. There was no significant change in the measured concentration at 72 hours and so the results are based on the 0-Hour measured test concentration only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 15 mg/L. The No Observed Effect Concentration was determined to be 15 mg/L.

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

Key value for chemical safety assessment

EC50 for freshwater algae:

15 mg/L

EC10 or NOEC for freshwater algae:

15 mg/L