bis(2-methylpropyl) perylene-dicarboxylate

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,10-bis(2-methylpropyl) perylene-3,10-dicarboxylate; 3,9-bis(2-methylpropyl) perylene-3,9-dicarboxylate

EC Number:

616-749-7

Cas Number:

79869-59-3

Molecular formula:

C30H28O4

IUPAC Name:

3,10-bis(2-methylpropyl) perylene-3,10-dicarboxylate; 3,9-bis(2-methylpropyl) perylene-3,9-dicarboxylate

Details on test material:

Name of test substance: Oracet F Yellow 084
Test substance No.: 16/0121-1
Batch identification: 12-7612
CAS No.: 79869-59-3
Content: > 99.8 g/100 g
Identity: confirmed
Homogeneity: given
Storage stability: Expiry date: 26 Mar 2023
Chemical Name: Perylenedicarboxylic acid, bis(2-methylpropyl) ester
Date of production: unknown
Physical state: solid
Color: orange
Water solubility: <100 μg/L (pH at 23°C: 7.25)
Storage conditions: ambient

Sampling and analysis

Analytical monitoring:

no

Remarks:

Analyses of the test substance preparations was not conducted for technical reasons. No reliable method for analyses in the required concentration range could be developed.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance is poorly soluble in water. Therefore, the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a saturated solution of the test substance. The test solution was prepared by directly adding 200.11 mg of test substance to 2 L test media. The mixture was stirred vigorously for about 2 days to generate a saturated test solution. Undissolved test substance was removed by filtration (pore with 0.2 μm). The first 50-100 mL of filtered solution was discarded (used to condition the filter). The exposure was started after separation of the undissolved material by adding inoculum culture at a ratio of 1:100. The test solution after addition of algal inoculum was considered equivalent to the nominal concentration since no more than 1% dilution occurs. After filtration, the stock solution and the test solution were visibly clear following preparation.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test species and strain: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81), formerly known as Selenastrum capricornutum, and currently renamed as Raphidocelis subcapitata KORSHIKOV were obtained from the SAG (Collection of algal cultures in Göttingen, Germany).
Reason for selection of the test species: Recommended algae species proposed by the test guideline
Age (on study day 0): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
Supplier: Collection of algal cultures in University of Göttingen/Germany

In order to verify that the algal cultures are responding normally to toxic stress, tests with a reference substance (potassium dichromate) are conducted. Reference substance tests are conducted according to OECD 201 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to potassium dichromate EC50 values published in ISO test guideline 8692, which represent the typical response range for the algal species tested.

According to the test ISO guideline 8692 the EC50 values of the reference substance potassium dichromate should be in the range: ErC50 = 0.92 – 1.46 mg/L after 72 hours for Pseudokirchneriella subcapitata. The ErC50 (72 h) of the control substance potassium dichromate was 1.14 mg/L (Date of the last control experiment: 04 Jul 2016). These results indicate that the algae are responding normally to toxicant stress.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

23.6 – 23.8 °C

pH:

7.8-8.5

Dissolved oxygen:

n/a

Salinity:

n/a

Conductivity:

n/a

Nominal and measured concentrations:

nominal: 100 mg/L

Details on test conditions:

Test groups: 0 (control), 100 mg/L as loading rate based on test substance mass without correction for purity.
Test replicates: 6 replicates for the control; 6 replicates for each test substance concentration; 1 additional uninoculated (without algae) replicate per test group for background fluorescence correction.
Reason for selection of test concentrations: In a preliminary test a treatment prepared at a nominal (loaded) concentration of 100 mg/L and filtered to remove undissolved material caused no inhibition of algal growth relative to the control after 72 hours. According to the OECD-guideline, the highest suggested test concentration is 100 mg/L for a limit test. The raw data of the range finding test are archived together with the raw data of this study.
Test duration: 72 hours
Test vessels: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps
Test media: OECD media
Test volume: 100 mL
Initial cell density: 0.5 x 104 cells/mL
Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet.
Test temperature: 23.6 – 23.8 °C
Illumination: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily.
Light intensity: 6166 lux (within ± 15% variability) at a wave length of 400 – 700 nm
Shaking rate: Continuous (approx. 85 rpm)
Test parameter: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm)
Route of exposure: Static exposure via the test medium.

The cell density of the inoculum culture in exponential growth phase was determined and then adjusted to 50 x 104 cells/mL. To obtain the correct initial cell density in the test replicates of 0.5 x 104 cells/mL and the correct nominal concentrations of the test solutions, inoculum culture was added to each test solution at a ratio of 1:100.
Fluorescence measurement: The fluorescence of aliquots from each test vessel was measured using a Tecan Infinite 200Pro fluorometer in a 96- well flat bottom black plate with the following parameters: fluorescence top reading; excitation / emission wavelength = 430 / 670 nm; excitation / emission bandwidth = 20 / 25 nm; flashes = 5; integration time = 20 μs; shaking duration = 15s; shaking amplitude = 6 mm.
Temperature measurement: Continuous measurement during the whole test period in the climate chamber in a separate deionized water filled flask.
Determination of correlation between fluorescence and cell density: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
Algal morphology: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.
Light measurement: Light homogeneity was evaluated by measuring light intensity at 5 locations within the incubation area at the start and end of the test (Testo 545 light meter, Testo GmbH & Co, Lenzkirch, Germany). The light intensity did not vary by more than ± 15% over the incubation area.
Additional Water parameter: The TOC (Total organic carbon) values of the test concentration and the control were analyzed at the start (filtrated solution) and the end of the exposure as an additional water quality parameter from uninoculated samples (replicate 0).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria:
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
- The validity criterion for cell multiplication factor in the untreated control is > 16 in 72 hours. The cell multiplication factor in the untreated control (all replicates mixed together) was 146-fold after 72 hours
- The validity criterion for the mean coefficient of variation for section-by-section growth rates for each test day in the control cultures is ≤35%. The mean coefficient of variation for section-by-section growth rates for each test day in the control cultures was 15.3%.
- The validity criterion for the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is ≤7%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.94%.



In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to the test substance at loading rates of 0 (control), 100 mg/L under static conditions in accordance with the OECD 201 guideline. The water pH, temperature and lighting intensity were all maintained within acceptable guideline specifications. The following statistical effect concentrations were obtained after 72 hours of exposure:

ErL50 > 100 mg/L (growth rate)
NOErLR ≥ 100 mg/L (growth rate)

The given water solubility limit of the test substance is <0.1 mg/L. The test organisms were exposed to a saturated solution of the test substance in test media prepared at a loading rate of 100 mg/L in this study. The exposure was started after separation of the undissolved test substance. The test substance had no observable inhibitory effect on Pseudokirchneriella subcapitata growth up to its saturation limit in test media and under test conditions. A slight stimulatory effect on growth was observed at 100 mg/L loading concentration. Concentration control analysis was not performed since the analytical detection limit was above the water solubility of the test substance because a reliable method for analyses in the required concentration range could not be developed. However, all reasonable efforts were taken to produce a saturated solution of the test substance in test media, following the guidance in OECD 23 and the algae were exposed to the entire loaded mass of test substance for the duration of the test. According to OECD 23, for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations. The toxicity results presented here are consistent with the results from preliminary tests. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.