Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 08, 2019 to April 12, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: 2-(4-Phenylbenzoyl)benzoic acid
Batch/Lot Number: Sarex #7650
CAS number: 42797-18-2
EC number: 700-862-4
Description: white powder
Manufacture Date: 27 September 2016
Expiry Date: 31 December 2019
Purity: 99.6 %
Molecular weight: 302.32 g/mol
Molecular formula: C20H14O3
Storage Conditions: Controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety. Causes skin irritation and serious eye damage.

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

The dilution water (circulated and filtered) was used without of addition of the test item.

Details on test solutions:

Because the test substance is difficult to dissolve in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
A saturated test substance solution (nominal loading rate of approximately 100 mg/L) was prepared by dispersing/dissolving the amount of test substance into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 h at approximately 30°C and then equilibrated for about 24 h at approximately 20°C. The non-dissolved test substance was removed by filtration through a fine (0.22 µm) filter to give the 100 % saturated solution. As a Limit test was carried out, further dilution of stock solution was not performed.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Brachydanio rerio)
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group
Body length of animals: 2.6 – 3.1 cm
Food and Feeding: The fish were not fed during the test
Acclimatisation: more than 12 d
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test period; the fish were healthy. There was no mortality of the population for seven days before the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

147 mg/L (as CaCO3)

Test temperature:

20.7 and 21.4 °C

pH:

7.34 – 8.01

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 78 – 100% of the air saturation value at the temperature used.

Nominal and measured concentrations:

Range finding study: 0.1, 1, 10, and 100% saturated solutions
Definitive study: 100 mg/L (nominal); 100.15 mg/L (measured; geometric mean)

Details on test conditions:

TEST CONDITIONS
- The water temperature, oxygen concentration and pH were measured daily in each test aquarium.
- The test temperature was between 20.7 and 21.4 °C.
- The dissolved oxygen concentration was in the range of 78 – 100 % of the air saturation value at the temperature used.
- The hardness of the dilution water was determined as 147 mg/L (as CaCO3).
- The light-dark cycle during the test was 16 hours light and 8 hours darkness.
For further details please refer the Section ('Any other information on materials and methods incl. tables'

TEST SYSTEM
- One aquarium was used for test group and for the control group, respectively.
- Each aquarium comprised 7 fish and 5 L test solution.
- The animals were not fed during the test.
- The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST CONCENTRATIONS:

- Range-finding study: A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test substance so that appropriate test concentrations could be selected for use in the definitive test. Four fish were exposed to each 0.1, 1, 10, and 100% saturated solutions of the test substance plus a control, for 96 h under semi-static conditions (with 24-h renewal, in absence of stability data). During the formulation procedure the test substance stock solution was prepared by similar method described under 'Details on test solutions' Section. Test solutions of the lower concentrations were prepared by appropriate dilution of this stock solution.
- Definitive study: Because no toxic response was observed during the preliminary range-finding test, only one test concentration of the test substance in the test medium (approximately 100 mg/L nominal loading rate) and one control group was tested in the definitive study in a static system. The feasibility of using a static sytem was confirmed based on the analytical results.

OBSERVATIONS:
- The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 h. Mortality or any sub-lethal effects were not observed during the experiment.
- The test conditions (pH, temperature, oxygen saturation) were measured daily during the test.
- The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/litre testing liquid).

ANIMAL WELFARE
- After the experiment, all fish were humanely killed. Euthanasia for all animals was applied by an overdose of an aqueous solution of 250 mg/L Tricaine Methanesulphonate (MS 222; CAS No.: 886-86-2). This technique is considered to provide a humane euthanasia by direct depression of the brain and vital centres.

STATISTICAL EVALUATION
- No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

VALIDITY CRITERIA:
- Constant conditions should be maintained in this procedure.
- The mortality in the controls should not exceed 10 % at the end of the test (or one fish if less than ten are used). In case of limit test, no mortality should occur.
- The dissolved oxygen concentration (throughout the test) has to be at least 60% of air saturation value.
- The concentration of the test substance should be at least 80 % of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20%, results will be based on the measured concentration.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 100.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality and body weights

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality and body weights

Details on results:

Validity: The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid
Mortality: No sublethal effect was observed. For further details refer to Table 2 under 'Any other information on results incl. tables'.
Body weight: There was no considerable difference observed concerning body weights between the groups. For further details refer to Table 3 under 'Any other information on results incl. tables'.


The 24h, 48h, 72h and 96h LC50 value: > 100.15 mg/L (measured)
The 96h LC0 value: > 100.15 mg/L (measured)
The 96h No-Observed Effect Concentration (NOEC): > 100.15 mg/L (measured)
The 96h Lowest Observed Effect Concentration (LOEC): > 100.15 mg/L (measured)

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 1: Results of the PreliminaryRange-Finding Test

Nominal concentrations [% saturated solution]	Untreated control	0.1	1	10	100
Number of treated animals	4	4	4	4	4
Number of dead animals	0	0	0	0	0

Table 2: Cumulative mortality data in the Limit Test

Test Group	Cumulative Mortality (number of dead fish) [initial population = 7 fish]
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
100.15 mg/L (measured)	0	0	0	0	0	0

Table 3: Measured and calculated data of bodyweight

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	2.36	0.34	0.47
100.15 mg/L (measured)	2.37	0.34	0.47

Table 4: Measured concentrations of the test with the confidence interval (95%)

Nominal conc. mg/L	Measured concentration at the start mg/L	Measured concentration at the end mg/L
Control	Not detected	Not detected
100	97 ± 0.826	103.4 ± 0.873

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 96 h LC50 and NOEC of test substance in fish was determined to be >100.15 and 100 mg/L (measured; gemoetric mean).

Executive summary:

A study was conducted to determine the short-term toxicity of test substance, PBBA to Zebrafish (Brachydanio rerio), according to OECD Guideline 203, in compliance with GLP. As there was no toxicity observed during the preliminary range-finding test, a limit test was carried out using only one test concentration (approximately 100 mg/L nominal loading rate) and one control group in the definitive study under static conditions for 96 h. The test concentration was analytically determined at the start and at the end of the experiment. The measured concentrations at the start and end of the experiment were determined to be 97 and 103.4 mg/L, corresponding to a measured geometric mean test substance concentration of 100.15 mg/L. Therefore, the results were based on geometric mean measured concentrations. One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five liter test solution. The fish were observed at regular intervals for mortality and sub-lethal effects (body weight). The test conditions (pH, temperature, oxygen saturation) were measured daily during the test. All achievable validity criteria were met during this study. Under the study conditions, the 96-h LC50, NOEC of the test substance in fish was determined to be > 100.15 mg/L and 100.15 mg/L (measured; geometric mean) (Sipos, 2019)

Description of key information

The 96-h LC50, NOEC of the test substance in fish was determined to be > 100.15 mg/L and 100.15 mg/L (measured; geometric mean) respectively.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100.15 mg/L

Additional information

A study was conducted to determine the short-term toxicity of test substance, PBBA to Zebrafish (Brachydanio rerio), according to OECD Guideline 203, in compliance with GLP. As there was no toxicity observed during the preliminary range-finding test, a limit test was carried out using only one test concentration (approximately 100 mg/L nominal loading rate) and one control group in the definitive study under static conditions for 96 h. The test concentration was analytically determined at the start and at the end of the experiment. The measured concentrations at the start and end of the experiment were determined to be 97 and 103.4 mg/L, corresponding to a measured geometric mean test substance concentration of 100.15 mg/L. Therefore, the results were based on geometric mean measured concentrations. One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The fish were observed at regular intervals for mortality and sub-lethal effects (body weight). The test conditions (pH, temperature, oxygen saturation) were measured daily during the test. All achievable validity criteria were met during this study. Under the study conditions, the 96-h LC50, NOEC of the test substance in fish was determined to be > 100.15 mg/L and 100.15 mg/L (measured; geometric mean) respectively (Sipos, 2019)