4-acetyl-2-methylbenzoic acid

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:

Study initiation date: November 06, 2019; Experimental start date: December 03, 2019; Experimental completion date: January 31, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of decision 2018-01-10; Swiss Ordinance relating to GLP, adopted May 18, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted November 26, 1997 by decision of the OECD Council [C(97)186/Final].

Test material

Test material information

Constituent 1

Reference substance name:

4-acetyl-2-methylbenzoic acid

EC Number:

856-079-4

Cas Number:

55860-35-0

Molecular formula:

C10H10O3

IUPAC Name:

4-acetyl-2-methylbenzoic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Storage condition of test material: : < 30 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L treatment
- Sampling method: For the determination of the test item concentrations, duplicate samples were taken from the test media of the test concentration and the control at the start and at the end of the test.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicate test medium samples of the single test concentration and the control, taken at the start and end of the test.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, a stock solution with a nominal test item concentration of 500 mg/L was prepared by mixing 1.500 g test item into 3000 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. This intensively mixed stock solution was added to test water to make up a volume of 15 L test medium, resulting in a test item nominal concentration of 100 mg/L. The test medium was freshly prepared just before introduction of the fish (at the start of the exposure). For the treatment and the control 15 liters of test medium were used for exposure of the fish.
- Controls: A control (test water without test item) was tested in parallel.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test medium. Test medium was clear throughout the entire test duration.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

•TEST ORGANISM
- Common name: Rainbow trout
- Source: The test fish were obtained from fish breeding farm Störk, 88348 Bad Saulgau, Germany.
- Age at study initiation: The study was performed with juvenile fish. The fish were obtained from a commercial breeder at least 20 days before start of the test.
- Length at study initiation: From the acclimatized test fish batch, a representative sub-batch of 10 fish was measured at the start of the test. The mean total body length (measured from the mouth to the end of the tail fin) of the fish was 4.9 ± 0.40 cm (Mean ± SD). The measured fish were not used for the test.
- Weight at study initiation: From the acclimatized test fish batch, a representative sub-batch of 10 fish was measured at the start of the test. The mean body wet weight was 1.1 ± 0.30 g (Mean ± SD). The measured fish were not used for the test.

•ACCLIMATION
- Acclimation period: The fish were acclimatized to the test water and test temperature for seven days prior to test start.
- Acclimation conditions: Same as test
- Type and amount of food during acclimation: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Hokovit, Hofmann Nutrition AG, CH-4922, Bützberg, Switzerland).
- Health during acclimation (any mortality observed): During the 20 days prior to the main test, no significant mortality was observed in the test fish batch and all fish were healthy. No medication was applied during holding and acclimatization of the fish.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Test conditions

Hardness:

The total hardness of the reconstituted test water was measured to be 125 mg/L (as CaCO3), i.e. in the recommended range of 40-250 (preferably <180) mg/L as CaCO3 (in the reconstituted test water before the initiation of the test).

Test temperature:

The water temperature in the test vessels was maintained at 13 °C during the test period.

pH:

The pH throughout the test ranged from 6.5 to 7.1.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 10.1 to 10.4 mg/L at the start of the test and in the range of 7.7 to 9.7 mg/L (i.e. 73-92 % saturation) from day 1 to the end of the test.

Conductivity:

The conductivity was 354 μS/cm (measured in the reconstituted test water before the initiation of the test)

Nominal and measured concentrations:

Nominal 100 mg/L.
- The measured concentration of the test item in the test medium was 108 and 106 % of the nominal value at the start and the end of the test, respectively.

Details on test conditions:

•TEST SYSTEM
- Test vessel: Mono block aquarium
- Material, size, headspace, fill volume: Glass, 20 L, 5 L, 15 L
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached. The test medium and the control were slightly aerated from Day 1 to Day 4, to keep the air saturation high enough to fulfill test guideline validity requirements.
- No. of organisms per vessel: At the start of the test, seven fish were introduced into each aquarium in a random order.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading rate of the fish was 0.50 g fish wet weight per liter medium.

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:
- Ingredient / Concentration mmol/L / Concentration mg/L
• CaCl2 × 2H2O / 1.0 / 147
• MgSO4 × 7H2O / 0.25 / 61.5
• NaHCO3 / 0.38 / 32.5
• KCl / 0.038 / 2.9
- Alkalinity: The alkalinity was measured to be 0.4 mmol/L (in the reconstituted test water before the initiation of the test)
- Ca/Mg ratio (molar): 1.0÷0.25 = 4

•OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test water after equilibration was 7.1. Thus no pH adjustment was required.
- Photoperiod: A 16-hour light to 8-hour dark photoperiod with a 30-minute transition period was used.
- Light intensity: The light intensity during the light period was approximately 16 to 17 microeinstein [μE/(m²·s)]

•EFFECT PARAMETERS MEASURED
The test fish were observed for mortality and visible abnormalities approximately 2 and 5 hours after the start of the exposure and further on each exposure day in the morning and the afternoon. The fish were checked for the mandatory clinical signs presented below, including the corresponding sub-categories (as presented in Annex 4 of the test guideline):
•E: Loss of equilibrium
•S: Abnormal swimming behavior
•V: Ventilatory function
•P: Abnormal skin pigmentation
•O: Other visible abnormalities

•TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable in a limit test

•RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No effects observed

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

EC50

Remarks:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

In the control and the test item nominal concentration of 100 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish.
- Behavioural abnormalities: None.
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None.
- Any observations that might cause a difference between measured and nominal values: None. No remarkable observations were made concerning the appearance of the test medium. Test medium was clear throughout the entire test duration.
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

No statistics needed due to the absence of toxicity of the test item. The 24 and 48-hour EC50 of the test item could not be calculated.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

No fish in the control died during test period (mortality should be ≤ 10 % or 1 fish if seven used). Oxygen concentration did not drop below actual minimum 73 % (should be 60 % ). Analytical dose verification confirmed the exposureto the test item.

Conclusions:

The test item was found not acute toxic to fish, represented by the rainbow trout (Oncorhynchus mykiss) in a 96-hour static limit test up to the test item nominal concentration of 100 mg/L.

Executive summary:

The acute toxicity of the test item to juvenile fish of the species rainbow trout (Oncorhynchus mykiss) was determined in a 96 hour static test according to the OECD Guideline for the Testing of Chemicals, No. 203, adopted 2019. The validity criteria were met.
On the basis of the pre-test results, a limit test was performed in accordance with the test guideline to demonstrate that the test item has no toxic effect on the test organisms up to and including the nominal concentration of 100 mg/L during an exposure period of 96 h. Thus, the only concentration tested was 100 mg/L. Additionally a control was tested in parallel. A static test design, without medium renewal, was applied. Seven fish were used at the treatment and the control. The fish were observed for visible abnormalities and mortality at approximately 2 and 5 hours after the start and twice every 24 hours period thereafter (in the morning and the afternoon).
No remarkable observations were made concerning the appearance of the test medium. Test medium was clear throughout the entire test duration. The measured concentration of the test item in the test medium was 108 and 106 % of the nominal value at the start and the end of the test, respectively. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period. Therefore, the endpoint values were reported on the nominal concentrations of the test item. The test item showed no toxicity under the conditions of the test. Accordingly the 96-hour LC50 and the LOEC were derived to be >100 mg/L and the NOEC is ≥100 mg/L.
In conclusion, the test item was found not acute toxic to fish, represented by the rainbow trout (Oncorhynchus mykiss) in a 96-hour static limit test up to the test item nominal concentration of 100 mg/L.