2-Butenedioic acid, 2-methyl-, 1,4-bis(2-ethylhexyl) ester, (2Z)-

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A guideline study (OECD 201, EC C.3) conducted under GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Media preparation trial for "Difficult Substances" (OECD 2000) was applied. No deviations from the study plan occurred.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

The study assessed the effect of the test item on the growth of green alga. The test endpoint is inhibition of growth, expressed as logarithmic increase in algal biomass (average growth rate) during the exposure period of 72 h. As the test item was insoluble in water, pre-experiments were performed to determine the solubility and stability of the test item in the test water. Preliminary range-finding tests were conducted exposing algae to saturated 100 % v/v nominal concentation. Based on the results, a limit test was conduted to confirm that no effects on algal growth was observed at the highest attainable test concentration.

The test design is static and is applied for 72 hours. Three replicates per test concentration, six replicates of the control and (if appropriate) solvent control flasks are applied. According to the study plan, at least five concentrations of the test item arranged in geometric series are tested for their toxicity.
The actual test concentrations are assigned according to preliminary range-finding tests. The concentration series should preferably cover 5 - 75 % inhibition of algal growth rate. Concentrations higher than 100 mg/L or above the solubility limit (whichever is the lower) will usually not be tested. If no effect is observed in the preliminary range-finding test at the highest concentration or the limit of solubility (whichever is the lower), a Limit Test may be conducted using only this concentration and the corresponding control(s). The control group will be exposed to the test water alone. Where auxiliary solvents/surfactants are used,, a second control will be employed.
Methods such as prolonged mixing, ultrasonication, high shear force stirring or the production of Water Accommodated Fractions (WAFs) or saturated solutions may be used. If auxiliary solvents or surfactants are used to aid in the dispersion, they will be used at maximum 100 µL/L (final volume). If the test item falls in the category of a "difficult substance", OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2009, Series on Testing and Assessment No. 23.) the recommendations are followed.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

EC Number:

807-715-4

Cas Number:

1354569-12-2

Molecular formula:

C21 H38 O4

IUPAC Name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

Test material form:

other: liquid

Details on test material:

98.09 %

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The test sample was stored in room temperature at dark.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the test media were taken at the initiation and termination of both the range-finding and definitive tests for analysis, and analysed immediately or stored frozen until analysis.

Test solutions

Vehicle:

yes

Details on test solutions:

The culture medium is described in Annex 3 of the test report (attachment 1).

Preliminary solubility work conducted indicated the test item was insoluble in water using the conventional methods of preparation such as ultrasonication and high shear mixing. A study to determine the general physico-chemical properties of the test item (Envigo Study Number YL25VY) showed the water solubility of the test item to be in the range of 1.07 x 10-5 to 1.52 x 10-5 g/L of solution at 20.0 ± 0.5 ºC. 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.

Preliminary media preparation trial (Annex 4 of the test report, attachment 2):
A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 20 mg/10 mL solvent stock solution from which a dilution was made to give a further solvent stock solution of 2.0 mg/10 mL. An aliquot (500 μL) of the 2.0 mg/10 mL solvent stock solution was dispersed in 5 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 0.020 mg/L prior to taking samples for chemical analysis after pre-treatments. 4 different pre-treatment options based on centrifugation and filtration were investigated. Based on the findings, the test item for the definitive test was prepared using a solvent spike method at an initial loading rate 0.020 mg/L, stirred for a period of 10 minutes prior to the removal of any undissolved test item by centrifugation at 40,000 g. In this way a dissolved test item concentration of approximately 0.0050 mg/L could be obtained using a solvent spike method of preparation.

Range-finding tests (Annex 5 of the Test Report as attachment 3):
First test:
The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentration of 100% v/v for a period of 72 hours.

A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 20 mg/10 mL solvent stock solution from which a dilution was made to give a further solvent stock solution of 2.0 mg/10 mL. An aliquot (100 μL) of the 2.0 mg/10 mL solvent stock solution was dispersed in 1 liter of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 0.020 mg/L stock solution. After stirring any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give the required test concentration of 100% v/v. An aliquot (500 mL) of this stock solution was inoculated with algal suspension (3.4 mL) to give the required test concentration of 100% v/v.

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 the control, solvent control and 100% v/v test concentration. The control group was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 μL/L of dimethylformamide.

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.

After the initial range-finding test had been started, the results of the preliminary media preparation trial were reassessed showing that the use of filtration was perhaps not appropriate as a means of removing any undissolved test item present. It was therefore considered appropriate to conduct a second range-finding test using centrifugation as a means for removing any undissolved test item.

Second test (Annex 5 of the test Report in Attachment 3):
The second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentration of 100% v/v for a period of 72 hours.

A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 20 mg/10 mL solvent stock solution from which a dilution was made to give a further solvent stock solution of 2.0 mg/10 mL. An aliquot (100 μL) of the 2.0 mg/10 mL solvent stock solution was dispersed in 1 liter of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 0.020 mg/L stock solution. After stirring any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give the required test concentration of 100% v/v. An aliquot (450 mL) of this stock solution was inoculated with algal suspension (3.6 mL) to give the required test concentration of 100% v/v.

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 the control, solvent control and 100% v/v test concentration.

The control group was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 μL/L of dimethylformamide.

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 the 100% v/v test concentration was taken for chemical analysis at 0 and 72 hours from both range-finding tests in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive test (Annex 5 of the test Report in Attachment 3):
Based on the results of the range-finding tests a "limit test" was conducted at a concentration of 100% v/v to confirm that at the highest attainable test concentration no effect on algal growth was observed.

Experimental Preparation : A nominal amount of test item (20 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 20 mg/10 mL solvent stock solution from which a dilution was made to give a further solvent stock solution of 2.0 mg/10 mL. An aliquot (200 μL) of the 2.0 mg/10 mL solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 0.020 mg/L stock solution. After stirring any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give the required test concentration of 100% v/v. An aliquot (1500 mL) of this stock solution was inoculated with algal suspension (13.3 mL) to give the required test concentration of 100% v/v. The stock solutions 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 (see Annex 5).

Exposure Conditions: As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control, solvent control and 100% v/v treatment group. The control group was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 μL/L of dimethylformamide.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.66 x 10E5 cells per mL. Inoculation of 1500 mL of test medium with 13.3 mL of this algal suspension gave an initial nominal cell density of 5 x 103 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 organisms

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 ± 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 1,000 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 10,000 - 100,000 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:

0.15 mmol/L (15 mg/L as CaCO3)

Test temperature:

24 +- 1 oC

pH:

7.5

Salinity:

fresh water

Nominal and measured concentrations:

A definitive water solubility test (OECD 105 under GLP) was performed before conducting this study. Therefore the substance was categorised as "difficult substance" as defined by OECD 2000., and a media preparation trial and two range-fining tests were conducted. Please see attachments 2 and 3 for details.

Details on test conditions:

INFORS Multitron Version 2 incubator under constant illumination (4440 - 8880 lux) provided a warm white lightning (380 - 730 nm). An orbital shaker was utilised (appr. 150 rpm). Temperature, light intensity and oscillation rate of the incubation chamber were recorded daily troughout the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: calculated as inhibition of average specific growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: calculated as inhibition of average specific growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: calculated as inhibition of average specific growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: based on no statistically significant difference (p>= 0.05)

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: yield

Remarks on result:

other: calculated as inhibition of yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

cell number

Remarks on result:

other: calculated as inhibition of yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

cell number

Remarks on result:

other: calculated as inhibition of yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated nominal concentration

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

cell number

Remarks on result:

other: based on no statistically significant difference (p>= 0.05)

Details on results:

Interpretation: the test item exibited no toxic effects at its limit of water solubility (100 % v/v saturated nominal concentration).

Range-fining Tests:
The cell densities and percentage inhibition of growth values of Pseudokirchneriella subcapitata from the exposure to the test item are given in Table 1 and 2 (attachment 5).

Definitive Test:
The mean cell densities for the definitive test are presented in Figure 1.

Range-finding tests:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding tests are given in Table 1 and Table 2 (attachment 5). The results showed no effect on growth at the test concentration of 100% v/v using either filtration or centrifugation as a means to remove any undissolved test item that may have been present. Based on this information a single test concentration of six replicates, of 100% v/v 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 test preparations taken from the initial and second range-finding tests at 0 and 72 hours (see Annex 5 in attachment 3) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.00090 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Verification of Test Concentrations:
Analysis of the 100% v/v test preparations at 0 and 72 hours (see Annex 5 in attachment 3) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method were obtained which was determined to be 0.00090 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Growth Data:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5 (please see attachment 5). The mean cell densities versus time for the definitive test are presented in Figure 1 (below). From the data given in Tables 3 and 5, 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 over the 72-Hour exposure period.

Results with reference substance (positive control):

The control results are included in the Test Result Tables 1 - 5 (attachment 4).

Reported statistics and error estimates:

Inhibition of the growth rate:
Statistical analysis of the growth rate data was carried out for the solvent control and 100% v/v 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 solvent control and 100% v/v test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v.

Inhibition of yield:
Statistical analysis of the yield data was carried out as for Inhibition of Growht Rate. There were no statistically significant decreases in yield (P≥0.05), between the solvent control and 100% v/v test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v.

Any other information on results incl. tables

Validation criteria:

1) The cell concentration of the control cultures must increase at least by a factor of 16 (Observed factor 171 in the control and 153 in the solvent control culture; acceptable)

2) The mean of the variation coefficients of the section by section daily growth rates must not exceed 35 % in the control cultures during the days 0 -1, 1 -2 and 2 -3. (Observed variation coefficients were 7 % for the control and 10 % for the solvent control culture; acceptable).

3) The variation coefficient of the average specific growth rate in replicate control cultures must not exceed 7 %.(Observed 1 %; acceptable).

Observations on cultures:

no abnormalities were detected in any of the control or test cultures during the microscopical inspections at 72 hours. (acceptable)

Water quality criteria:

pH values in the control cultures increased from 7.6 to 8.8 and solvent control cultures from 7.5 to 8.7 (< 1,5; acceptable)

Temperature was maintained at 24 +- 1^oC.

Observations on test item solubility:

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The result of the algal toxicity test indicates that the test substance exhibited no toxic effects at the saturation concentration.
The reported 72 h EC50 values are > 100 % (v/v) and statistically determined NOECs based on growth inhibition and yield were 100 % v/v saturated nominal concentration (p >= 0.05).

Executive summary:

The result of the algal toxicity test conducted according to OECD 201 and EC C.3 under GLP indicates that the test substance exhibited no toxic effects at the saturation concentration. The reported 72 h EC50 values are > 100 % (v/v) and statistically determined NOECs based on growth inhibition and yield were 100 % v/v saturated nominal concentration (p >= 0.05).

In addition, the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000) was followed and two preliminary studies conducted before the definitive test and concentrations were analytically determined from the test solutions at the beginning and end of the test.

As all validation criteria were met, the test result conducted under GLP is considered as reliable (Klimisch 1).