1,2,4,5-Benzenetetracarboxylic acid, tetra-C6-10-alkyl esters

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:

9th - 12th November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetra-C6-10-(even and linear)-alkyl benzene-1,2,4,5-tetracarboxylate

EC Number:

700-623-4

Cas Number:

263750-17-0

Molecular formula:

C34H54O8 - C50H86O8

IUPAC Name:

Tetra-C6-10-(even and linear)-alkyl benzene-1,2,4,5-tetracarboxylate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentration of the test material in the test solutions was determined at 24 hour intervals during the 72 hours exposure period.
Each day three replicate samples were taken from the test solution. One sample was taken from both control solutions at the start of the test.

Test solutions

Vehicle:

yes

Details on test solutions:

The test item is very poorly soluble in water, furthermore due to its specific physical character there is no appropriate method for this test system to prepare a saturated test solution (limit test concentration) in aquatic media.
In order to obtain a fine homogeneous suspension which is in the analytically measurable range, acetone as organic solvent was used during the formulation procedure. An amount of 250 mg test item was dissolved in 50 mL acetone thereafter 0.1 mL from this stock solution was diluted in 1000 mL of OECD medium to give the test concentration of 0.5 mg/L (nominal).

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Initial cell number: The initial cell number in the test cultures was 10E4 cells/mL.
Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal GrowthMedium to 10E7 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

Not reported

Test temperature:

Culture temperature was checked at the beginning of the study and every 24 hours in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.7 – 23.0 °C measured in the flask and between 22.3 and 23.4 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and each concentration. The average pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.60 – 7.97 at the start and 8.90 – 9.34 at the end of the study.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

0.5 mg/L nominal
The measured concentration was 0.389 mg/L at the start of the experiment, 0.124 mg/L at day 1, below the Limit of Detection (LOD) at day 2 and below the Limit of Quantification (LOQ) at the end of the experiment (day 3).

Details on test conditions:

Dilution Water
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 109 μE/m2/s, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm) and it is checked periodically.

Equipment and Test Vessels
Normal laboratory equipment and the following were necessary for determination of the parameters of the test:
- pH meter
- thermometer
- light-meter
- microscope with counting chamber
- climate chamber
- orbital shaker
- balance
For test vessels all-glass flasks with total capacity of about 250 mL were used. The volume of the test liquid in the vessels was 100 mL.

Description of the test procedure
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 10^4 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control groups. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and control groups. The test concentration was prepared using acetone as organic solvent as described above.
The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in the following table:

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L] Untreated control Solvent control 0.125 0.25 0.5

Average of cell number at 72 hours (x 10E4cell/mL) 86.00 84.00 82.50 85.00 87.00

Concentration Levels Investigated in the Main Test
Because a significant toxic response was not observed during the preliminary concentration range-finding test, only one test concentration of 0.5 mg/L (nominal), one untreated and one solvent control were tested in a limit test.
The test results are based on the calculated test item concentrations.

Observations
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Validity
The cell density in the control cultures was increased by a factor of 70.50 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.95 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.72 %.
All validity criteria were met, therefore the study can be considered as valid.

Concentrations of the test item
The test substance was not detected in the control samples (neither normal nor solvent control).
A nominal concentration of 0.5 mg/L was tested in the experiment.
The measured concentration was 0.389 mg/L at the start of the experiment, 0.124 mg/L at day 1, below the Limit of Detection (LOD) at day 2 and below the Limit of Quantification (LOQ) at the end of the experiment (day 3). In order to calculate the geometric mean exposure concentration, the concentration was taken as LOD at day 2 and as the half of LOQ at day 3 (according to OECD 23). The calculated geometric mean concentration was 0.1 mg/L.

Average specific growth rates
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-24 h, 0-48 h and 0-72 h average specific growth rates were not statistically significantly different from the control values at the used test group.

Areas under the growth curves
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-24 h, 0-48 h and 0-72 h areas were not statistically significantly different from the control values at the used test group.

Yield
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield values were not statistically significantly different from the control values at the used test group.

Cell numbers
The cell number in each flask was determined at the 24th, 48th, 72nd hours. The shape of the algal cells growing was not affected.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate (Batch Number: 0769128) is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 10/157-022AL) with the reference item Potassium dichromate was: 22 – 25 June 2010.
The ErC 50 : 1.00 mg/L, (95 % confidence limits: 0.89 – 1.13 mg/L)
The EbC 50 : 0.61 mg/L, (95 % confidence limits: 0.54 – 0.68 mg/L)
The EyC 50 : 0.47 mg/L, (95 % confidence limits: 0.43 – 0.52 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

Statistical Analysis
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The EC50 values of the test item and their confidence limits were not calculated, due to the lack of toxic effects.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

Any other information on results incl. tables

Table 1: Yield and % inhibition.

Test Group	Yield (Y)	% Inhibition of Y at 0-72 hours
Control	69.5	0.0
Solvent control	68.8	1.0
0.5 mg/L (nominal)	69.5	0.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours at the highest achievable concentrations of test item.
Under the conditions of this algal growth inhibition test, no toxic effect was observed at the tested concentration level. As the concentration of 0.5 mg/L (nominal) represented a higher level than the limit of solubility, it can be stated that the test item had no toxic effect at saturation.
The observed endpoints for the effect of the test material are summarised in the following table:

Influence of the test material on the Growth of Pseudokirchneriella subcapitata (results below were based on the measured concentration and determined directly from the raw data)

Parameter (0-72 h) Biomass (b) Growth rate (r) Yield (y)
EC50 > 0.1 > 0.1 > 0.1
95 % conf. limits Not calculated Not calculated Not calculated
NOEC 0.1 0.1 0.1
LOEC > 0.1 > 0.1 > 0.1
All validity criteria were met during this experiment.

Executive summary:

In a 72 hour acute toxicity study (10/095-022AL), the cultures of the alga Pseudokirchnerella subcapitata were exposed to the test material at a nominal concentration of 0.5 mg/L under static conditions in accordance with EU Test Method C.3. Under the conditions of this algal growth inhibition test, no toxic effect was observed at the tested concentration level. As the concentration of 0.5 mg/L (nominal) represented a higher level than the limit of solubility, it can be stated that the test item had no toxic effect at saturation.The NOEC and EC50 values based on growth rate were 0.1 and >0.1 mg/L (actual geometic mean ceoncentration) respectively.