2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 22 October 2007 and 9 November 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal 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 materials, 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 material in cases where the test material 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 material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control (replicates R1 – R6 pooled) and the 0.40 mg/l test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Information provided indicated that the test material had a water solubility value of 1.5 x 10-3 g/L. Pre-study solubility work showed that the highest attainable test concentration (by visual inspection) that could be prepared was 0.20 mg/L using a preliminary solution in dimethylformamide. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water.
Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore media preparation trials were conducted in order to determine the solubility of the test material under test conditions.

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

An amount of test material (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 0.40 mg/L*. A dilution was made from this saturated solution to give a further stock solution of 0.040 mg/L.

Range-finding study:
An aliquot (250 mL) of each of the stock solutions was separately inoculated with algal suspension (3.4 mL) to give the required test concentrations of 0.040 and 0.40 mg/L.

Main study:
An aliquot (2 litres) of this saturated solution was inoculated with algal suspension (27.5 mL) to give the required test concentration of 0.40 mg/L.

* Concentration determined by analysis of a saturated solution prepared in an identical manner during the pre-study media preparation trial.

- Eluate: dimethylformamide

- Controls:
A positive control used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions were made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/L. An aliquot (250 mL) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L stock solutions was separately mixed with algal suspension (250 mL) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.
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. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were 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.
Samples were taken at 0, 28, 52 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


- Evidence of undissolved material (e.g. precipitate, surface film, etc): Yes, removed by filtration to give a saturated solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not stated.
- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The master cultures were maintained in the laboratory under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°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 10E3 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 10E4 - 10E5 cells/mL.

CULTURE MEDIUM:
- Culturing media and conditions (same as test or not): Same as test.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Any deformed or abnormal cells observed: None recorded.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Hardness:

No data.

Test temperature:

24 ± 1°C

pH:

pH 7.5 measured using a WTW pH 320 pH meter

Dissolved oxygen:

Not stated

Salinity:

Not applicable as freshwater used.

Nominal and measured concentrations:

Range-finding
Nominal: 0.04 and 0.40 mg/L

Main Study
Nominal: 0.40 mg/L
Measured: 0.338 - 0.345 mg/L (0 hours), 0.328 - 0.332 mg/L (72 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed (plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: 100 mL of test preparation
- Initial cells density: 10E3 (cubed) cells/mL
- Control end cells density: 72 hours: 7.79E+05 (cells per mL)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
Based on the result of the range-finding test a "limit test" was conducted. The test material solution for the definitive test was prepared by stirring an excess (50 mg/L) of the test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ELGA Purelab Option R-15 water purification
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: constant illumination
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Inhibition of growth, inhibition of yield, inhibition of biomass integral: 0, 24, 48 and 72 hours
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Nominal test concentrations, 0.40 mg/L for a period of 72 hours
- Justification for using less concentrations than requested by guideline: Material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).
- Range finding study
- Test concentrations: 0.040 and 0.40 mg/L for a period of 72 hours
- Results used to determine the conditions for the definitive study: Based on the result of the range-finding test a "limit test" was conducted. The test material solution for the definitive test was prepared by stirring an excess (50 mg/L) of the test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield, biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities observed.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: delete entry?
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes, the results obtained from the pre-study media preparation trial using both saturated solution and solvent spike methods of preparation indicated that the use of a saturated solution resulted in the highest dissolved test material concentration (0.40 mg/L following filtration with the first approximate 2 litres discarded). Furthermore the use of prolonged stirring had no significant effect on the dissolved test material concentration obtained.
Therefore, for the purposes of testing the test material was to be prepared using a saturated solution method of preparation followed by removal of any undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded) to produce a saturated solution with a dissolved test material concentration of 0.40 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
ErC50 (0 - 72 h) : 0.49 mg/L; 95% confidence limits 0.43 - 0.55 mg/L
EyC50 (0 - 72 h) : 0.22 mg/L; 95% confidence limits 0.19 - 0.24 mg/L
EbC50 (0 - 72 h) : 0.23 mg/L; 95% confidence limits 0.21 - 0.27 mg/L

NOEC based on growth rate: 0.125 mg/l
NOEC based on yield: 0.0625 mg/l
NOEC based on biomass integral: 0.0625 mg/l

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and the 0.40 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001). .

Range-finding Test

The results showed no effect on growth at the test concentrations of 0.040 and 0.40 mg/L.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 150 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.

Mean cell density of control at 0 hours                            :   4.25 x 10³cells per mL
Mean cell density of control at 72 hours                          :   6.37 x 10⁵cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% 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 that states this must not exceed 7%.

Growth data

From the data given in Tables 2 and 4 (see attached), it is clear that the growth rate (r), yield (y) and biomass (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a nominal test concentration of 0.40 mg/L over the 72-Hour exposure period.

The test concentration of 0.40 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water.

Accordingly the following results were determined from the data:

Inhibition of growth rate

E_rC₁₀(0 - 72 h)             : > 0.40 mg/L
E_rC₂₀(0 - 72 h)             : > 0.40 mg/L
E_rC₅₀(0 - 72 h)             : > 0.40 mg/L

where E_rC_x is the test concentration that reduced growth rate by x%.

Inhibition of yield

E_yC₁₀(0 - 72 h)            : > 0.40 mg/L
E_yC₂₀(0 - 72 h)            : > 0.40 mg/L
E_yC₅₀(0 - 72 h)            : > 0.40 mg/L

where E_yC_x is the test concentration that reduced yield by x%.

Inhibition of biomass integral

E_bC₁₀(0 - 72 h)            : > 0.40 mg/L
E_bC₂₀(0 - 72 h)            : > 0.40 mg/L
E_bC₅₀(0 - 72 h)            : > 0.40 mg/L

where E_bC_x is the test concentration that reduced biomass integral (area under the growth curve) by x%.

There were no statistically significant differences between the control and 0.40 mg/l test group and therefore the NOEC was 0.40 mg/l (for growth rat, yield and biomass).

Observations on test material solubility

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Physico-chemical measurements

The pH values of each test and control flask are given in Table 2 (see attached). Temperature was maintained at 24 ± 1ºC throughout the test.

The pH values of the control cultures (see Table 2 attached,) were observed to increase from pH 7.6 – 7.7 at 0 hours to pH 7.7 – 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.

Verification of test concentrations

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the EC₅₀ values in terms of the nominal test concentrations only.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 0.40 mg/L. Correspondingly the No Observed Effect Concentration was 0.40 mg/L.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Information provided indicated the test material had a water solubility value of 1.5 x 10^-3g/L. A pre-study media preparation trial showed that the use of a saturated solution method of preparation resulted in the highest dissolved test material concentration.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a measured test concentration of 0.40 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a nominal concentration of 0.40mg/L*.

*Concentration determined by analysis of a saturated solution prepared in an identical manner during the pre-study media preparation trial.

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

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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. Exposure of Desmodesmus subspicatus to the test material gave EC₅₀ values of greater than 0.40 mg/L and correspondingly the No Observed Effect Concentration was 0.40 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

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

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀ (0 - 72 h) of 0.49 mg/L; 95% confidence limits 0.43 – 0.55 mg/L, an E_yC₅₀ (0 - 72 h) of 0.22 mg/L; 95% confidence limits 0.19 ‑ 0.24 mg/L, and an E_bC₅₀ (0 - 72 h) of 0.23 mg/L; 95% confidence limits 0.21 - 0.27 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral were 0.25, 0.125 and 0.125 mg/L respectively and the No Observed Effect Concentrations were 0.125, 0.0625 and 0.0625 mg/L respectively. 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Range-finding study: 23 September 1997- 29 September 1997. Definitive study: 17 October 1997- 20 October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Physico-chemical measurements
The pH of each control, solvent control and test flask was determined at initiation of the study and after 72-h exposure. The temperature within the incubator was recorded daily.

Verification of test concentrations
Water samples were taken from the solvent control and the 0.10 mg/L test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 h for quantitative analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
For the purpose of the definitive study the test material was prepared using a preliminary solution in dimethylformamide.

An amount of test material was dispersed in auxiliary solvent and the volume adjusted to 100 mL to give a 100 mg/100 mL solvent stock solution. An aliquot (200 µl) of this solvent stock solution was dispersed in algal suspension (2 L) to give the test concentration of 0.10 mg/L.

The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 h.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Centre for Algae and Protozoa (CCAP), Institute of freshwater Ecology, Ferry House, Ambleside, Cumbria

ACCLIMATION
- Culturing media and conditions (same as test or not): the culture medium used for both the range-finding and definitive studies was the same as that used to maintain the stock culture.
- The culture was maintained in the laboratory at a temperautre of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 ± 1 °C

pH:

8.0- 9.9

Nominal and measured concentrations:

Range-finding study: nominal test concentrations of 0.010, 0.040 and 0.10 mg/L
Definitive test: nominal test concentration of 0.10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed; the flasks were covered with aluminium foil
- No. of organisms per vessel: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.54 x 107 cells per mL. This suspension was diluted to a cell density of 1.22 x 104 cells per mL prior to use. At initiation of the study the culture contained a nominal cell density of 104 cells per mL.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per solvent control (replicates): 3

OTHER TEST CONDITIONS
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux)
- Flasks were incubated at 24 ± 1 °C and constantly shaken at 100 rpm

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 h and the cell densities determined by direct counting with the aid of a Coulter® Multisizer II particle counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal test concentrations of 0.010, 0.040 and 0.10 mg/L
- Test vessel: 250 mL glass conical flask
- Type : closed; loosely covered with aluminium foil to reduce evaporation
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Results used to determine the conditions for the definitive study: The test material was dissolved in dimethylformamide. An amount of test material was dispersed in auxiliary solvent and the volume adjusted to 50 mL to give a 125 mg/50 mL solvent stock solution from which serial dilutions were made to give 1.0, 4.0 and 10 mg/10 mL solvent stock solutions. Aliquots of the solvent stock solutions were separately dispersed in algal suspension to give the test concentrations. The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µL/L of dimethylformamide. At the start of the range-finding study a sample of each test and control culture was removed and the cell density using a Coulter® Multisizer II particle counter. The flasks were then covered with aluminium foil and incubated at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 h. After 72 h the cell density of each flask was determined.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Details on results:

RANGE-FINDING STUDY
- Any stimulation of growth found in any treatment: There was no significant effect on growth at all concentrations tested.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: During the preliminary solubility work performed a precipitate of test material was observed (by visual inspection of the prepared test media) at concentrations in excess of 0.10 mg/L thereby indicating this to be the maximum limit of water solubility of the test material under these test conditions. Based on this information a single test concentration of six replicates, of 0.10 mg/L was selected for the definitive study.

DEFINITIVE STUDY
- Growth data: neither the growth or the biomass of Scenedesmus subspicatus (CCAP 276120) were affected by the presence of the test material over the 72-hexposure period.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 h. There were no abnormalities detected in any of the control or test cultures.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve data was carried out for the solvent control and 0.10 mg/L test group using a Students t-test. There were no statistically significant decreases in the area under the growth curve data (P ≥ 0.05), between the solvent control and 0.10 mg/L test group.

Growth data

The test concentration of 0.1 0 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Dimethylformamide was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

The following data show that the cell concentration of the control cultures increased by a factor of 21 and the cell concentration of the solvent control cultures increased by a factor of 18 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 h:

Mean cell density of control at 0 h: 1.08 x 10⁴cells/mL

Mean cell density of control at 72 h: 2.29 x l0⁵cells/mL

Mean cell density of solvent control at 0 h: 1.03 x l0⁴cells/mL

Mean cell density of solvent control at 72 h: 1.84 x l0⁵cells/mL

Physico-chemical measurements

The pH values of the control cultures were observed to increase from pH 8.0 at 0 h to pH 9.4 - 9.7 at 72 h. This effect is considered to be due to the large number of cells in the log phase of growth respiring oxygen and producing carbonates and bicarbonates as part of photosynthesis/ respiration which in solution give rise to alkaline conditions.

The pH deviation in the control cultures was slightly higher than that specified in the Test Guideline (1.5 pH unit deviation after 72 h). This is considered not to affect the integrity of the study given that the validation criterion for control cell multiplication was satisfied.

Verification of test concentrations

Chemical analysis of the test preparations at 0 h showed the measured test concentrations to be in excess of the required 80 % of nominal. Analysis of the test preparations at 72 h showed a marked decline in measured test concentrations to 65 % to 66 % of nominal.

Given that pre-study stability analysis showed the test material to be stable in the culture medium used over the study period, the decline in measured test concentrations is considered to be due to adsorption to the algal cells. Adsorption was not a factor in the pre-study stability analyses as no algal cells were present.

In order to give a worst case analysis of the data, it was considered justifiable to base the results on the time-weighted mean measured test concentration.

Cell densities and pH values in the definitive study

Nominal Concentration (mg/L)		pH	Cell densities * (cells/ mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.0	9.59 x 10^3	1.36 x 10^4	3.64 x 10^4	1.81 x 10^5	9.4
	R2	8.0	1.01 x 10^4	1.35 x 10^4	3.71 x 10^4	2.21 x 10^5	9.5
	R3	8.0	1.28 x 10^4	1.25 x 10^4	5.25 x 10^4	2.85 x 10^5	9.6
	Mean	8.0	1.08 x 10^4	1.32 x 10^4	4.20 x 10^4	2.29 x 10^5
Solvent Control	R1	8.0	1.02 x 10^4	9.51 x 10^3	3.16 x 10^4	2.60 x 10^5	9.6
	R2	8.0	1.27 x 10^4	1.25 x 10^4	4.07 x 10^4	1.72 x 10^5	9.5
	R3	8.0	8.11 x 10^3	1.77 x 10^4	4.38 x 10^4	1.19 x 10^5	9.7
	Mean	8.0	1.03 x 10^4	1.32 x 10^4	3.87 x 10^4	1.84 x 10^5
0.10	R1	8.0	9.60 x 10^3	1.32 x 10^4	5.20 x 10^4	2.63 x 10^5	9.3
	R2	8.0	1.10 x 10^4	9.91 x 10^4	4.05 x 10^4	2.24 x 10^5	9.4
	R3	8.0	1.01 x 10^4	1.26 x 10^4	3.60 x 10^4	2.82 x 10^5	9.5
	R4	8.0	1.02 x 10^4	8.86 x 10^3	4.14 x 10^4	2.34 x 10^5	9.9
	R5	8.0	1.09 x 10^4	1.11 x 10^4	4.90 x 10^4	2.48 x 10^5	9.5
	R6	8.0	9.93 x 10^3	1.08 x 10^4	4.36 x 10^4	2.16 x 10^5	8.7
	Mean	8.0	1.03 x 10^4	1.11 x 10^4	4.38 x 10^4	2.44 x 10^5

R₁- R₆= Replicates 1-6; *cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Inhibition of growth

Nominal Concentration (mg/L)	Area under curve at 72 h	% inhibition	Growth rate (0- 72 h)	% inhibition
Control	3.42 x 10^6	-	0.042	-
Solvent	2.83 x 10^6	-	0.040	-
0.10	3.63 x 10^6	[28]	0.044	[10]

[increase in growth as compared to solvent control]

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 0.10 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The EC50 values based on the time-weighted mean measured test concentration were greater than 0.080 mg/l, and correspondingly the No Observed Effect Concentration was greater than or equal to 0.080 mg/l.

Executive summary:

Methods

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Results

Exposure of Scenedesmus subspicatus to the test material gave EC₅₀ values of greater than 0.1 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test solutions at 0 and 72 h showed a marked decline in test concentrations and so it was considered justifiable to base the results on the time-weighted mean measured test concentration also. The EC₅₀, values, based on the time-weighted mean measured test concentration were > 0.080 mg/l and correspondingly the No Observed Effect Concentration was ≥ 0.080 mg/l.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5, CAS 87826-41-3)

Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield, biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield, biomass

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 0.40 mg/L. Correspondingly the No Observed Effect Concentration was 0.40 mg/L.

Executive summary:

Introduction. A study was performed to assess the effect of the test material (EC 402-950-5) on the growth of the green alga Desmodesmus subspicatus. 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Information provided indicated the test material had a water solubility value of 1.5 x 10^-3g/L. A pre-study media preparation trial showed that the use of a saturated solution method of preparation resulted in the highest dissolved test material concentration.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a measured test concentration of 0.40 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a nominal concentration of 0.40mg/L*.

*Concentration determined by analysis of a saturated solution prepared in an identical manner during the pre-study media preparation trial.

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

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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. Exposure of Desmodesmus subspicatus to the test material gave EC₅₀ values of greater than 0.40 mg/L and correspondingly the No Observed Effect Concentration was 0.40 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

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

It is proposed that this result can be used in the assessment of the target substance (EC 406-176-9).

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical:
1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC 413-110-2, CAS 135861-56-2)

Taget chemical:
2,6-bis(4-ethylphenyl)perhydro-1,3,5,7-tetraoxanaphth-4-ylethane-1,2-diol (EC 406-176-9, CAS 79072-96-1)

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see 'Read-across justification to support the REACH registration of EC 406-176-9' document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.08 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the time-weighted mean measured test concentration

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 0.10 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The EC50 values based on the time-weighted mean measured test concentration were greater than 0.080 mg/l, and correspondingly the No Observed Effect Concentration was greater than or equal to 0.080 mg/l.

Executive summary:

Methods

A study was performed to assess the effect of the test material (EC 413 -110-2) on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Results

Exposure of Scenedesmus subspicatus to the test material gave EC₅₀ values of greater than 0.1 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test solutions at 0 and 72 h showed a marked decline in test concentrations and so it was considered justifiable to base the results on the time-weighted mean measured test concentration also. The EC₅₀, values, based on the time-weighted mean measured test concentration were > 0.080 mg/l and correspondingly the No Observed Effect Concentration was ≥ 0.080 mg/l.

It is proposed that this result can be used in the assessment of the target substance (EC 406-176-9).

Description of key information

The toxicity to algae (algal inhibition of the substance (EC 406-176-9) has been assessed by reading across the results of studies conducted on two structurally similar analogue substances.

It is proposed that these results can be used in the assessment of the target substance (EC 406-176-9).

Source Substance;1,3:2,4-bis-O-(3,4-dimethylbenzylidene)-D-glucitol (EC-413 -110 -2):

Methods

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Procedure

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Results

Exposure of Scenedesmus subspicatus to the test material gave EC₅₀ values of greater than 0.1 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 0.10 mg/l.

The test concentration of 0.10 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test solutions at 0 and 72 h showed a marked decline in test concentrations and so it was considered justifiable to base the results on the time-weighted mean measured test concentration also. The EC₅₀, values, based on the time-weighted mean measured test concentration were > 0.080 mg/l and correspondingly the No Observed Effect Concentration was ≥ 0.080 mg/l.

Source Substance;1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol (EC 402-950-5):

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. Information provided indicated the test material had a water solubility value of 1.5 x 10^-3g/L. A pre-study media preparation trial showed that the use of a saturated solution method of preparation resulted in the highest dissolved test material concentration.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test material at a measured test concentration of 0.40 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a nominal concentration of 0.40mg/L*.

*Concentration determined by analysis of a saturated solution prepared in an identical manner during the pre-study media preparation trial.

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. Exposure of Desmodesmus subspicatusto the test material gave EC₅₀ values of greater than 0.40 mg/L and correspondingly the No Observed Effect Concentration was 0.40 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

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

Key value for chemical safety assessment

EC50 for freshwater algae:

0.1 mg/L

EC10 or NOEC for freshwater algae:

0.1 mg/L

Additional information

It is proposed that the results on the structurally similar analogue substance can be used in the assessment of the target substance (EC 406-176-9) and it is proposed that the target substance would not show toxicity up to its limit of solubility in algal inhibition testing.