1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-27 to 2010-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2006)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commision Regulation (EC) No 440/2008

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 (100 mg/L) of test item in culture medium for a period of 24 hours prior to removal of the aqueous phase by mid-depth siphon to give a saturated solution of the test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

EC Number:

700-453-0

Cas Number:

59802-05-0

Molecular formula:

C26H42O4

IUPAC Name:

1,4-Benzenedicarboxylic acid, 1,4-diisononyl ester

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: range finder test: nominal test concentrations of 0.10 %, 1.0 %, 10 % and 100 % v/v saturated solution, definitive test: nominal 100 % (v/v) saturate solution
- Sampling method: The concentration and stability of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours. Samples were taken from the control and the 100% v/v saturated solution test group ( at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored at approximately -20°C for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-study 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 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 a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (300 mg) was dispensed on to the surface of 3 litres of culture medium prior to stirring slowly via magnetic stirrer for 24 hours. After 24 hours the stirring was stopped and the aqueous phase removed by mid-depth siphoning to give a 100% v/v saturated solution of test item. An aliquot (2 litres) of the stock solution was separately inoculated with algal suspension (10.5 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.

- Differential loading: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 Desmodesmus subspicatus 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. Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution.
- Controls: Test medium without test substance. The control group was maintained under identical conditions but not exposed to the test substance.
- Vehicle: Auxiliary solvents were not used to aid dissolution.
- Evidence of undissolved material : 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 pale green dispersions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium and under constant aeration and constant illumination at 21 ± 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 10^3 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.
- Culturing media and conditions: same medium to maintain the stock culture and in the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Test temperature:

24 ± 1°C

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH values of the control cultures were observed to increase from pH 7.4 - 7.5 at 0 hours to pH 7.8 - 7.9 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.

Nominal and measured concentrations:

nominal 100 5 (v/v) saturated solution;
Analysis of the test preparations at 0 hours showed measured test concentrations range from 0.0088 to 0.0096 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.00044 mg/L. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentration is 0.0015 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 ml glass conical flasks with 100 mL of solution
- Aeration: no
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 7.63 x 10^5 cells per mL. Inoculation of 2 litres of test medium with 10.5 mL of this algal suspension gave an initial nominal cell density of 4 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- Control end cells density: 2.95x10^5 cells per mL (mean cell density after 72 h)
- No. of organisms per vessel: 4 x 10^3 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Culture medium different from test medium: same

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No. The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure.
- The flasks were plugged with polyurethane foam bungs and incubated 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.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Other: All test and control cultures were inspected microscopically at 72 hours. Observations on test item solubility at start and end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test with a saturated solution of 100 % (v/v)
- Justification for using less concentrations than requested by guideline: Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration no effect on algal growth was observed.

Range finding study
- Test concentrations: The range-finding test was conducted with a series of nominal test concentrations of 0.10 %, 1.0 %, 10 % and 100 % (v/v) saturated solution for a period of 72 hours.
- Results used to determine the conditions for the definitive study: The results of the range finding study showed no effect on growth at the test concentrations of 0.10%, 1.0%, 10% and 100% (v/v) saturated solution. Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.009 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.009 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Based on the data, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item over the 72-Hour exposure period.

- Exponential growth in the control (for algal test): yes
- Obeservation of the culture and test item solubility: ll test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours. 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 pale green dispersions.
- Any observations that might cause a difference between measured and nominal values: Exposure of Desmodesmus subspicatus to the test item gave EC50 values based on the 0-Hour mean measured test concentration of greater than 0.0092 mg/L and correspondingly the No Observed Effect Concentration was 0.0092 mg/L. Analysis of the test preparations at 0 hours showed measured test concentrations range from 0.0088 to 0.0096 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.00044 mg/L. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The EC50 values based on the geometric mean measured test concentrations were greater than 0.0015 mg/L and correspondingly the No Observed Effect Concentration was 0.0015 mg/L. This study showed that there were no toxic effects at saturation. The use of the geometric mean measured test concentrations in the calculation of the EC50 and NOEC values had no significant effect on the outcome of the study.

Results with reference substance (positive control):

- Results with reference substance valid? The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- EC50: ErC50 (0- 72 h) 0.74 mg/L; EyC50 (0- 72 h) 0.37 mg/L, 95% confidence limits 0.34 - 0.41 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 and yield data after 72 hours for the control and the 100% v/v saturated solution test concentration to determine any statistically significant differences between the test and control groups. There were no statistically significant differences (P~0.05), between the control and 0.0092 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.0092 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell concentration of the controls increased by a factor of 73 (at least 16). Variation for section by section specific growth rate for the controls: 24% (<35%). Coefficient of variation for average specific growth rate for the controls: 5% (<7%).

Conclusions:

Exposure of Desmodesmus subspicatus to the test item gave EC50 values based on the 0-Hour mean measured test concentration of greater than 0.0092 mg/L and correspondingly the No Observed Effect Concentration was 0.0092 mg/L.
Analysis of the test preparations at 0 hours showed measured test concentrations range from 0.0088 to 0.0096 mg/l. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.00044 mg/l. Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.
The EC50 values based on the geometric mean measured test concentrations were greater than 0.0015 mg/L and correspondingly the No Observed Effect Concentration was 0.0015 mg/L. This study showed that there were no toxic effects at saturation.