Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-22 - 2015-01-26
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl benzene-1,2,4-tricarboxylate
EC Number:
EC Name:
Trimethyl benzene-1,2,4-tricarboxylate
Cas Number:
Molecular formula:
1,2,4-trimethyl benzene-1,2,4-tricarboxylate
Details on test material:
- Name of test material (as cited in study report): 1,2,4-Trimethylbenzenetricarboxylate
- Physical state: liquid, viscous/ amber
- Analytical purity: 98.6 area-% (233 nm); 99.15 area-% (207 nm)
- Lot/batch No.: 3723340
- Expiration date of the lot/batch: 31 Mar 2015
- Stability under test conditions: stable
- Storage condition of test material: The test substance was stored at ambient temperature

Sampling and analysis

Analytical monitoring:
Details on sampling:
-Sampling was performed at the start of the exposure (0 h) from the inoculated replicate 7 of each concentration and the control.
At the end of the exposure (72 h) samples were taken from the combined inoculated (+ algae) replicates of the test concentrations and of the combined inoculated control group replicates. Additionally, samples were taken from the uninoculated (-algae) replicate 0 of each test concentration and the control at the end of the exposure (72 h).
- Sample storage conditions before analysis: ambient temperature

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
To facilitate handling, the test substance was melted by warming the container approx. 8 min in a drying oven to 100°C before use. A stock solution (355.2 mg/L) was prepared by directly adding 710.43 mg of test substance to 2L of test medium and stirring for about one day until visibly dissolved. The lower test concentrations were prepared by diluting this stock solution. To reach the correct nominal concentrations at test start, inoculum culture was added to each 111% stock test solution at a ratio of 1:10. The stock test solution appeared colorless and clear. The pH of the stock solution was adjusted with 1 M NaOH to pH of 8.0 before preparation of the lower test concentrations.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata KORSHIKOV (SAG 61.81), formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata KORSHIKOV were obtained from the SAG (Collection of algal cultures in Göttingen, Germany).
- Age of inoculum (at test initiation): 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).

- Acclimation period: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 104 cells/mL. The inoculum culture is incubated under test conditions for 4 days prior to test initiation. The increase in biomass is verified to ensure that growth is within the normal range and algal cells are examined microscopically for normal morphology prior to use for test inoculation. Inoculum culture cell density (4 days growth) : 346 x 10^4 cells/mL (692 fold increase Inoculum culture morphology: normal and healthy

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

Test temperature:
23.4 – 23.8 °C
Nominal and measured concentrations:
Nominal: 0 (control), 1, 3.2, 10, 32, 100 and 320 mg/L
Measured: 0 (control), 0.8, 2.42, 7.43, 24.3, 80.7, 270 mg/L
Details on test conditions:
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge
- Type (delete if not applicable): open / closed
- Test volume: 100 mL
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 57 x 10^4 cells/mL
- Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 additional replicate per test group for initial concentration control analysis only.
- 1 additional uninoculated (without algae) replicate per test group for background fluorescence correction and to determine the influence of the test design on test substance stability.

- Standard medium used: yes

- Source/preparation of dilution water: media prepared according to OECD Guideline

- Adjustment of pH: no
- Light intensity and quality: Average 7006 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm)
- 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.
- 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.

Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
71.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 68.3 – 75.3
72 h
Dose descriptor:
Effect conc.:
29.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 25.9 - 33.3
Results with reference substance (positive control):
- Results with reference substance valid? yes
- The EC50 (72 h) of the control substance potassium dichromate was 0.942 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
Executive summary:

In a 72-hour algal growth study, cultures of Pseudokirchneriella subcapitata were exposed to 1,2,4-Trimethylbenzenetricarboxylate at geometric mean measured concentrations of 0 (control), 0.800, 2.42, 7.43, 24.3, 80.7, 270 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: ErC50= 71.7 mg/L (CI. 68.3 - 75.3).

The measured concentrations of the test substance in the test water were in a range of 80 - 93% of the nominal concentration at start of exposure and decreased to 62 - 80 % of the nominal concentration at the end of exposure. The geometric mean measured concentrations were in a range of 74 - 84% of the nominal concentration and are considered to be an accurate representation of exposure levels throughout the test period. 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.