Trimethyl benzene-1,2,4-tricarboxylate

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-15 - 2015-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethyl benzene-1,2,4-tricarboxylate

EC Number:

219-547-3

EC Name:

Trimethyl benzene-1,2,4-tricarboxylate

Cas Number:

2459-10-1

Molecular formula:

C12H12O6

IUPAC Name:

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:

yes

Details on sampling:

The concentration control analysis is performed with all test groups. Sampling for concentration control analysis is performed for 2 renewal intervals. For the first interval samples are collected within 30 min before insertion of the test organisms (0 h) from all test vessels and after 24 hours from all test vessels with surviving fish and the lowest concentration with 100% mortality. For the second interval, samples were collected from fresh test solutions within 30 min of test solution renewal at 72h and after 96h hours from all test vessels with surviving fish and the lowest concentration with 100% mortality. Water samples were taken from the middle of the test vessel using a glass pipette and were transported to the analytical laboratory in glass ampoules on the day of sampling.

Test solutions

Vehicle:

no

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 in a drying oven to 100°C for approx. 5 min before use. The test substance was homogenized by shaking the container. The test treatments were prepared by diluting a stock solution. The stock solution was prepared by weighing 500 mg (see preparation below) and mixed in a volumetric flask with 5 L for four intervals (adjusted to pH 6.0). Due to increasing stock solution requirements for the 1st and 2nd interval, an additional 100 mg of the test substance was weighed out and mixed in a volumetric flask with 1 L (adjusted to pH 6.0). The two stock solutions for the 1st and 2nd interval (5L and 1L) were merged before the test solution was prepared. The stock solution was stirred using a magnetic stirrer for approx. 24 hours and then checked for complete dissolution of the test substance before further use. For preparation of the test solutions, an aliquot of the stock solution was taken and mixed with the test water using a high shear mixer (Ultra Turrax). Test water (adjusted to pH 6.0) was then added as treatment was homogenized again with an ultra turrax. Fresh stock and test solutions was prepared and renewed daily. The pH of the final test solutions was not adjusted.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: The fish were hatched at the testing facility.
- Age at study initiation (mean and range, SD): Approx.9 months
- Length at study initiation (length definition, mean, range and SD): 3.8 cm (3.4 cm – 4.2 cm)
- Weight at study initiation (mean and range, SD): 0.48 g (0.28 g – 0.74 g)
- Feeding during test: no


ACCLIMATION
- Acclimation period: The fish were acclimatized to the testing conditions including test water, temperature and
light regime for at least 14 days. Fish were acclimatized to the reduced test water pH for 21
days
- Type and amount of food: Commercial fish diet ”Tetramin“ (manufacturer Tetra-Werke, Melle, Germany) daily ad libitum,
additionally generally on workdays live juvenile brine shrimp (Artemia nauplii).
No feeding from approximately 24 hours before start of exposure

- Health during acclimation (any mortality observed): Healthy, no visible abnormalities

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Approx. 1 mmol/L = 100 mg/L CaCO3

Test temperature:

22°C

pH:

6.0 - 6.6

Dissolved oxygen:

7.7 - 8.8 mg/L

Nominal and measured concentrations:

Nominal: 0 (control), 0.6, 1.3, 2.9, 6.3, 14, 30 mg/L
Mean measured concentration: (control)<0.3, 0.6, 1.2, 2.7, 5.9, 13.3, 29.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Approx. 10 Liter stainless steel aquaria (29x21x22 cm)
- Material, size, headspace, fill volume: 10 L
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Due to the instability of the test substance in the test medium, this study was conducted as a semi-static renewal exposure. The renewal period was 24 hours.
- No. of organisms per vessel: 7 Fish/ test vessel,
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.34 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: non chlorinated charcoal-filtered drinking water from the municipal water works of the city of Frankenthal mixed with deionized water, and aerated to achieve the following water characteristics. The mixed test water is sanitized by UV treatment prior to entering the aquaria.
Total hardness: Approx. 1 mmol/L = 100 mg/L CaCO3
Conductivity: Approx. 250 μS/cm (at 25°C)
Ca content: Approx. 40 mg/L
Mg content: Approx. 5 mg/L
Acid capacity: Approx. 2.5 mmol/L
pH-value: Adjusted to pH6
Oxygen content: >80% saturation
TOC: Generally <2mg/L
Temperature: 23 °C

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16h light, 8h dark
- Light intensity: 80–328 Lux (the light intensity is determined in regular intervals at the surface of the aquaria)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish were observed for mortality and toxic signs (changes appearance and abnormal behavior in comparison to the control group) within 1 hour after start of exposure and 6, 24, 48, 72 and 96 hours after start of exposure. Instantaneous temperature, dissolved oxygen and pH were measured daily (at the start and the end of each interval) in every test vessel and the appearance of the test solutions and dissolution behavior of the test substance was observed.

TEST CONCENTRATIONS
The concentrations for this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of the first experiment. In the first experiment mortality was observed at the limit. concentration. In a subsequent preliminary test using zebrafish the 96 hour LC50 was between 1 and 30 mg/L. The test is repeated using 6 test substance concentrations

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

other: Lowest 100% mortality

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

In a 96 hour acute toxicity study, zebrafish (Danio rerio) were exposed to 1,2,4 -Trimethylbenzenetricarboxylate at nominal concentrations of 0 (control), 0.6, 1.3, 2.9, 6.3, 14, 30 mg/L under static renewal conditions in accordance with the OECD test guidelines. The water pH, temperature and dissolved oxygen were within acceptable guideline specifications. The 96 hour LC50 was 9.26 mg/L based on the nominal concentration of the test substance and 8.59 mg/L based on the mean measured concentrations.

All measured concentrations were within ±20% of the mean values determined for the whole exposure period. Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained

throughout the test period.

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.