Bis[C5-(linear and branched)-alkyl] benzene-1,4-dicarboxylate

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:

supporting study

Study period:

2015-04-16 to 2016-02-09

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:

(2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control (without test item), Geometrical series with a dilution factor of 2 (6.25%, 12.5%, 25.0%, 50.0%, 100%), Test concentrations (geometric mean measured): 4.00, 5.81, 8.37, 28.3 µg test item/L
- Sampling method: All concentration levels and the control were analytically verified via LC-MS/MS after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 1 ml test item I L was prepared once with dilution water three days prior to the start of the exposure. The test item was placed by pipetting it on the surface of the dilution water. To ensure that the solubility limit of the test item in the test medium was obtained, a slow stirring procedure for 70 hours was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at approximately 25 °C. After a separation of the phases after at least 1 hour of standing, the saturated solution was taken from the homogeneous phase and was used for testing without further dilution.
Test concentrations: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25- 12.5- 25.0- 50.0- 100% of the
saturated solution.
- Controls: six replicates without test items
- Reference Item: Potassium dichromate
- Evidence of undissolved material: Since the formation of a (micro-) emulsion had to be excluded, the stock solution was checked after stirring via laser beam (Tyndall effect) for any signs of (micro-)emulsions. The test media were clear throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerie/la subcapitata
- Strain: Pseudokirchnerie/la subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Collection of Algae Cultures, Institute for Plant Physiology), University of Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 J.µE* m^-2 ·s^-1 for 24 hours per day.
Culture medium: Nutrient medium Z

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L in dilution water

Test temperature:

25 °C, nominal range 21-24 °C ±2 °C

pH:

7.98 -9.60

Nominal and measured concentrations:

The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25%, 12.5%, 25.0%, 50.0%, 100% of the saturated solution.
The measured concentrations of Di-(iso)-pentyl terephthalate (DPT) in the fresh media (0 h) were in the range of < LOQ to 69.8 µg test item/L. The measured concentrations in the old media (72 h) were in the range of < LOQ to 11.5 µg test item/L. Therefore, all effect values given are based on the geometric mean measured test item concentrations. The dilution level are corresponding to geometric mean measured test item concentrations of 4.00 -5.81 - 8.37 - 17.8 - 28.3 J.lg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile conical flasks (vol. 250 mL) with cotton wool plugs
- Material, size, headspace, fill volume: fil volume 100 mL
- Aeration: no
- Renewal rate of test solution: Static test, 72 h
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, Current: 6153 cells/mL
- Control end cells density: 1.84 *10^6 cells/mL (mean of the yield)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Fluorescent tubes (OSRAM L 36 W/11-865, cool daylight), 60 - 120 µE · m^-2 · s^-1

EFFECT PARAMETERS MEASURED: algal cell concentration determined by photometric measurement after 0, 24, 48 and 72 h
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-afluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: (non-GLP)
- Test concentrations: 100%, 10% and 1% of a saturated solution

Reference substance (positive control):

yes

Remarks:

potassium dichromate (CAS No 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

27 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 8.58µg/L - > 28.3 µg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

28 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: 8.77 µg/L- > 28.3 µg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 28.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield Inhibition

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: growth and yield were increased at concentrations of 4.00 to 17.8 µg/L (geometric mean).
- Any observations that might cause a difference between measured and nominal values: The measured concentrations in the fresh media (0 h) were in the range of< LOQ to 69.8 µg test item/L. The measured concentrations in the old media (72 h) were in the range of< LOQ to 11.5 µg test item/L. Therefore, all effect values given are based on the geometric mean measured test item concentrations of Di-(iso)-pentyl terephthalate (OPT). A separate replicate without algae was prepared and analysed after 72 h. The measured concentration of 36.4 µg/L is slightly higher than the measured concentration in samples with algae, indicating the tendency for adsorption of the test item to algae.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 0.613 mg/L (grwoth rate)

Reported statistics and error estimates:

Since only a slight effect on growth rate at the highest concentration level was observed EC10,EC20, and EC50-values were not calculated. EC10- and EC20-values with confidence intervals of yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.
The NOEC and LOEC were determined by calculation of analyses statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT's test were used for NOEC/LOEC calculations.

Validity criteria fulfilled:

yes

Remarks:

300-fold increase of cell growth in control culture (growth rate 1.90/day); Section-by section specific growth rate 12.8% (<35%); Coefficient of variation of average specific growth rate: 0.96 % (≤7 %)

Conclusions:

As the measured concentrations in the fresh media at the end of the test were < LOQ, all effect values given are based on the geometric mean measured test item concentrations. In this study a slight effect of the test item on the green alga Pseudokirchneriella subcapitata in terms of inhibition of yield was found at the geometric mean measured test item concentration of 28.3 µg/L, biologically and statistically significant. The inhibition on the growth rate (5 %) of the green alga Pseudokirchneriella subcapitata at the geometric mean measured test item concentration of 28.3 µg/L, was considered to be biologically not significant. The NOEC-values for inhibition of growth rate and yield after 72 hours were 28.3 and 17.8 µg/L. The EC50-values for inhibition of both growth rate (EC50) and yield (EC50) after 72 hours were > 28.3 µg/L (>100 % of the saturated solution). All effect values are given based on geometric mean measured test item concentrations.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-11 to 2016-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2006)

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 (see details on test solution).

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 0.080 mg/L test concentration in a limit test
- Sampling method: Concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours. Samples were taken from the control and the 0.080 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
- Sample storage conditions before analysis: The samples were analysed immediately.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (80 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give an 80 mg/10 mL solvent stock solution from which a dilution was made to give a further solvent stock solution of 8.0 mg/10 mL. An aliquot (200 μL) of the 8.0 mg/10 mL solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 30 minutes to give a 0.080 mg/L stock solution. An aliquot of this stock solution was inoculated with algal suspension to give the required test concentration of 0.080 mg/L.
Approximately 24 hours prior to the start of the test, a solution of a nominal concentration of 0.080 mg/L was prepared and dispensed to all test vessels, sample bottles and volumetric flasks to pre-condition. After 24 hours pre-conditioning the solutions were discarded.
- Controls: test medium without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Evidence of undissolved material: At the start of the test all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control and test cultures were observed to be pale green dispersions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: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 illumination at 21 ± 2 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

control cultures: pH 8.2 at 0 hours, pH 9.2 at 72 hours
solvent control: pH 8.1 at 0 hours, pH 9.2 at 72 hours

Nominal and measured concentrations:

nominal 0.080 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 to reduce evaporation
- Material, size, headspace, fill volume: 250 mL flasks containing 100 mL of test medium
- Aeration: no
- Initial cells density: 5.00 x 103 cells/mL
- No. of organisms per vessel: 10^3 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: as described in test guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control, solvent control and each test preparation was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: intensity approximately 7000 lux, provided by warm white lighting (380 – 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Multisizer Particle Counter. Three determinations were made for each sample.
- Other: To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test.The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
- Justification for using less concentrations than requested by guideline: range-finding study
- Range finding study
- Test concentrations: nominal test concentration of 0.080 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentration of 0.080 mg/L. Based on the result of the range-finding test a "limit test" was conducted at a concentration of 0.080 mg/L to confirm that at the limit of water solubility, no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

There were no statistically significant differences (P≥0.05), between the solvent control and 0.037 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.037 mg/L.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.037 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other:

Remarks:

There were no statistically significant differences (P≥0.05), between the solvent control and 0.037 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.037 mg/L.

Details on results:

Chemical analysis of the 0.080 mg/L test preparation at 0 hours (see Annex 5) showed a measured test concentration of 105% of nominal was obtained. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed which was determined to 0.033 mg/L. Analysis of an additional test sample at 72 hours which had been prepared with the omission of algal cells at 0 hours also showed a measured concentration of less than the LOQ. These results suggest that the test item was unstable under the conditions of the test and/or was adsorbing to active sites on the glassware which had not been fully pre-conditioned prior to the start of the test.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0016 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentration was determined to be 0.037 mg/L.

The growth rate and yield of Pseudokirchneriella subcapitata were not affected by the presence of the test item at a geometric mean measured test concentration of 0.037 mg/L over the 72-Hour exposure period.

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) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L; EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the solvent control and 0.037 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P≥0.05), between the solvent control and 0.037 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.037 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.037 mg/L. The No Observed Effect Concentration was 0.037 mg/L. This study showed that the test item exhibited no inhibitory effects at the limit of water solubility.

Description of key information

The test substance showed no inhibitory effects of green algae at the limit of water solubility. Based on the geometric mean the ErC50 value was greater than 0.037 mg/L.

Additional information

The acute toxicity has been tested in guideline test according to guideline OECD 201 in GLP compliance in two tests. In the key study, the exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values (72 h) based on the geometric mean measured test concentrations of greater than 0.037 mg/L. This study showed that the test item exhibited no inhibitory effects at the limit of water solubility and the No Observed Effect Concentration was 0.037 mg/L based on the geometric mean of the test substance concentration.

In a supporting study also with Pseudokirchneriella subcapitata, the ErC50 and ErC10 were found to be greater than 0.0283 mg/L and a slight reduction of the growth rate was considered to be biologically not significant. The NOErC was 0.0283 mg/l after 72 h .