2-benzoylbenzoic acid

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:

25 Jan 2019 to 28 Feb 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)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals

Version / remarks:

OECD series on testing and assessment number 23, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Off-white powder
Purity/Composition: 98.8%
Test item storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

During the final test samples for possible analysis were taken from the limit concentration and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis. The method of analysis is described in the appended Analytical Report (Appendix 1).

Frequency at t=0 h, t=24 h and t=96 h
Volume 3.0 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for possible analysis for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

The batch of 2-Benzoylbenzoic acid tested was an off-white powder with a purity of 98.8% which was visually not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying approximately a 10 minute period of ultrasonic treatment followed by three days of magnetic stirring to accelerate dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: ‘De Haar Vissen’, Zodiac proefacc., Wageningen University and Research Centre, The Netherlands.
Mean length(1): Range-finding test: 3.6 ± 0.3 cm
Final test: 3.5 ± 0.5 cm
Mean weight(1): Range-finding test: 0.62 ± 0.22 g
Final test: 0.54 ± 0.15 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 23

(1) Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Holding:
Quarantine/Acclimatisation: At least 12 days after delivery
Holding Medium: Test medium (see section 4.8.2.2).
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature was continuously recorded. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence 0.5-0.8 mm, Coppens International B.V., Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

Test guideline

Post exposure observation period:

Not applicable

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

20-24°C, constant within 2°C

pH:

pH: 6.0-8.5, constant within 1 unit

Dissolved oxygen:

oxygen > 60% of air saturation

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal: 100 mg/L - remained constant throughout testing

Details on test conditions:

Test duration : 96 hours
Test type: Static
Test vessels: 20 litres, all-glass, containing 15 litres of test solution.
Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Number of fish: 7 per test group
Loading: 0.25 g fish/litre, i.e. 7 fish per 15 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: The test solutions were not aerated during the test.
Feeding: No feeding from 24 hours prior to the test and during the total exposure period
Introduction of fish: Approximately 1 hour after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, mobility

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Sublethal observations / clinical signs:

Range-Finding test

No mortality or clinical effects were observed at any of the test concentrations throughout the test.

Based on this result, samples taken from the highest test concentration were analysed. Despite the filtration step, the concentrations measured in the undiluted SS were at the level of the nominal loading rate, i.e. remained constant at 100 mg/L throughout the test (see also Table 2 of the appended Analytical Report). Hence, it can be concluded that the undissolved material removed during preparation of test solutions was not test item related.

All test conditions were maintained within the limits prescribed by the study plan.

Limit Test

Measured Concentrations

The results of analysis of the samples taken during the limit test are described in Table 3 of the appended Analytical Report.

Samples taken from the limit concentration were analysed. Despite the filtration step, the concentrations measured in the undiluted SS were at the level of the nominal loading rate, i.e. were 106-109 mg/L throughout the test. Hence, it can be concluded that the undissolved material removed during preparation of test solutions was not test item related and the effect parameters were expressed in terms of analytically confirmed nominal concentration.

It should be noted that a small response was extrapolated in the control sample at the start of the test. The reason is unknown. However, the contribution is negligible and had no effect on the results of the study.

Based on this result, effect parameters were expressed in terms of the analytically confirmed nominal concentrations.

Mortality and Clinical Effects

Table 1 shows the mortality data recorded during the final test. The obtained results were in agreement with the results of the range-finding test. No mortality or clinical effects were observed in the control and at the limit concentration throughout the test.

Table 1       

Incidence of Mortality and Total Mortality in the Limit Test

2-Benzoylbenzoic acid Nominal conc. (mg/L)	Initial Number of fish	Cumulativemortality					Total Mortality (%)
		3h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Determination of Effect Concentrations

Table 2 shows the effect parameters based on the analytically confirmed nominal concentrations.

Table 2       

Effect Parameters

Parameter	2-Benzoylbenzoic acid Nominal conc. (mg/L)
24h, 48h, 72h, 96h-LC50	>100

Experimental Conditions

The results of measurement of pH and oxygen concentrations are presented in Table 3. The temperatures measured during the study in the various test vessels are presented in Table 4. All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Table 3       

pH and Dissolved Oxygen (mg/L) in the Limit Test  

2-Benzoylbenzoic acid Nominal conc. (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^1	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	8.0	9.7	7.9	7.7	7.9	7.5	7.8	7.3	7.6	7.6
100	6.5^2	8.6	7.2	7.1	7.3	6.9	7.6	6.8	7.5	7.0

¹the pH of the holding tank medium was 7.6 upon test initiation.  

²fish were hold for approximately 30 minutes in a separate tank with test medium at pH of 7.0 (adjusted with 1 mol HCl/L)

Table 4       

Temperatures (°C) Measured in the Limit Test

2-Benzoylbenzoic acid Nominal conc. (mg/L)	Day 0^1	Day 1	Day 2	Day 3	Day 4
Control	20^2	21	21	21	21
100	23	21	21	21	21

¹temperature of culture medium was: 22°C.

²test solution was warmed up to 22°C prior to introduction of the fish.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 96h-LC50 for carp (Cyprinus carpio) exposed to 2-Benzoylbenzoic acid was beyond the regulatory limit concentration, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

The objective of the study was to evaluate 2-Benzoylbenzoic acid for its ability to generate acute toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if possible, to determine the LC₅₀ at all observation times.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2018.

The batch of 2-Benzoylbenzoic acid tested was an off-white powder with a purity of 98.8% which was visually not completely soluble in test medium at the loading rate initially prepared. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A limit test was performed based on the results of a preceding range-finding test. Seven fish per group (single tank, non-replicated) were exposed to an untreated control and to the SS prepared at a loading rate of 100 mg/L. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

No mortality or clinical effects were observed in the control and at the limit concentration throughout the test.

Samples taken from the limit concentration were analysed. Despite the filtration step, the concentrations measured in the undiluted SS were at the level of the nominal loading rate, i.e. were 106-109 mg/L throughout the test. Hence, it can be concluded that the undissolved material removed during preparation of test solutions was not test item related and the effect parameters were expressed in terms of analytically confirmed nominal concentration.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 96h-LC₅₀ for carp (Cyprinus carpio) exposed to 2-Benzoylbenzoic acid was beyond the regulatory limit concentration, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The 96h-LC₅₀ for carp (Cyprinus carpio) exposed to 2-Benzoylbenzoic acid was beyond the regulatory limit concentration, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.