3,4,5-trimethoxycinnamic acid

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted April 13, 2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,4,5-trimethoxycinnamic acid

EC Number:

201-999-8

EC Name:

3,4,5-trimethoxycinnamic acid

Cas Number:

90-50-6

Molecular formula:

C12H14O5

IUPAC Name:

3-(3,4,5-trimethoxyphenyl)acrylic acid

Test material form:

solid: crystalline

Remarks:

light yellow

Details on test material:

CH03216 / E1A

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sampling method:
Frequency at t=0 h and t=48 h
Volume 3.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:

yes

Remarks:

water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of 3,4,5-TRIMETHOXYCINNAMIC ACID tested was a light yellow crystalline solid with a purity of approximately 99% and completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Weighing and preparation of test solutions was performed under dimmed light.
Preparation of test solutions started with the highest concentration of 100 mg/L applying a 11- minute period of ultrasonic waves followed by a 24 minutes of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: In-house laboratory culture with a known history.
- Justification for species other than prescribed by test guideline: This system has been selected as an internationally accepted invertebrate species.
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): <4 weeks
- Feeding during test: no
- Food type: /
- Amount: /
- Frequency: /

ACCLIMATION
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Daily, a suspension of fresh water algae

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

18-22°C

pH:

78-8.1

Dissolved oxygen:

8.7-9.2 mg/L

Nominal and measured concentrations:

0.10, 1.00, 10 and 100 mg/L.
The measured concentrations were at the level of nominal throughout the test duration (94-96%).

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Type (delete if not applicable): static
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5 per vessel containing 50 mL of test solution
- No. of vessels per concentration (replicates): 4 for the control and the highest test concentration.
2 per intermediate test concentration.
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 0 hours photoperiod daily
- Light intensity: 0

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality) At 24 hours and at 48 hours.
pH and Dissolved oxygen At the beginning and at the end of the test, for the highest concentration and the control.
Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test.


VEHICLE CONTROL PERFORMED: yes (medium)

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.00, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: combined limit/range-finding study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

In conclusion, the actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.

The 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to 3,4,5-TRIMETHOXYCINNAMIC ACID exceeded an analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

The objective of the study was to evaluate 3,4,5-TRIMETHOXYCINNAMIC ACID for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC₅₀at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.

The batch of 3,4,5-TRIMETHOXYCINNAMIC ACID tested was a light yellow crystalline solid with a purity of approximately 99% and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest concentration of 100 mg/L applying a 10-minute period of ultrasonic waves followed by a 20-minute period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

A combined limit/range-finding test was performed. wenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 100 mg/L, in a limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed to 0.10, 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No daphnids became immobilized in the control and in the three lowest test concentrations. After 24 hours 25% of the daphnids were immobilized at the highest test concentration (100 mg/l). This increased to 35% immobilization after 48 hours.

The study met the acceptability criteria prescribed by the study plan and was considered valid. Samples taken from the highest test concentration were analysed. The measured concentrations were at the level of nominal throughout the test duration (94-96%).

In conclusion, the 48h-EC₅₀for Daphnia magna exposed to 3,4,5-TRIMETHOXYCINNAMIC ACID exceeded an analytically confirmed nominal concentration of 100 mg/L.