Methyl 2-benzoylbenzoate

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:

February - August 2015

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:

Identification: RCX 14-672
Chemical name: methyl-2-benzoylbenzoate
Batch no.: N14003
CAS no.: 606-28-0
EC no.: 210-112-3
Molecular formula: C15H12O3
Molecular mass: 240.3 g/mol
Description: white to light yellowish powder
Purity: >99% (gas chromatography)
Water solubility: 117.7 mg/l
Test item storage: at room temperature, protected from light
Stability: stable under storage conditions
Expiry date: 30 November 2015

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Since RCX 14-672 has a limited solubility, the test concentrations were prepared by dilutions of a saturated solution in which the test item’s concentration was first determined by HPLC. Semi-static conditions were applied. Fresh test solutions were prepared after 24, 48 and 72 h, into which the fish were transferred after each 24 h exposure period. Fresh test solutions were prepared as follows: the day before starting the test, and before each exchange of test media, about 100 mg/l of the test item was directly applied to empty glass vessels, natural water was added, and moderately stirred overnight, filtered (MILLIPORE AP15 glass fiber filter), and then measured by HPLC. Based on the result of this HPLC measurement, the obtained saturated solution was diluted to the desired concentrations. A blank consisting of natural water only without any addition of test item or other additives served as a control. At least 1 h before the start of the exposure, the test vessels were filled with the test solutions to allow equilibration of pH, oxygen and temperature. The pH was adjusted to pH 6.5–7.0.

The exposure began after an equilibration time of at least 1 h, when all the fish had randomly been placed in the test vessels. The transfer was performed in an impartial manner to eliminate bias. A fine-mesh dip net was used to transfer fish, taking care to minimise possible stress due to handling. Fish which were damaged or dropped during the transfer were not used. Transfer by fine-mesh dip net to the fresh media was performed every 24 h.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Test organism: Danio rerio (Zebrafish)
Culturing: Population of parental and young fish are held in either 200 l or 40 l glass aquaria. The bottom of the aquaria is covered with gravel (≤1.5 cm).
Illumination: 16 h a day
Temperature: 21–25 °C, controlled by a thermostat.
Medium: Continuously aerated natural water of the town of Schlieren

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness: 206-242 mg/l
CO3 hardness: 186-217 mg/l

Test temperature:

Measurements were made prior to the addition of the fish to the test solutions and after 2, 24, 48, 72 and 96 h of exposure. The water temperature was 22 °C at all exposure times.

pH:

Measurements were made prior to the addition of the fish to the test solutions and after 2, 24, 48, 72 and 96 h of exposure and have been recorded in the report.

Dissolved oxygen:

Measurements were made prior to the addition of the fish to the test solutions and after 2, 24, 48, 72 and 96 h of exposure. The dissolved oxygen concentration throughout the test (≥6.5 mg O2/l) was above the required 5.23 mg O2/l (i.e. above 60% of the air saturation value at 22°C; guideline OECD 203).

Salinity:

Chloride: 16.1 mg/l

Nominal and measured concentrations:

In the definitive test, the nominal concentrations were: 1.60, 2.73, 4.64, 7.88 and 13.4 mg/l. The concentrations of RCX 14-672 in the test solutions were measured by HPLC analysis at the beginning, and 2, 24, 48, 72 and 96 h of exposure, in the old and the freshly prepared test solutions, respectively. Since the test item hydrolyses, the effective concentrations were based on the arithmetic mean of the respective geometric means of all four 24 periods of exposure. These arithmetic means ranged between 79% and 90% of the nominal concentration (see Table 8 and 9 in the report).

Details on test conditions:

Test vessel: Glass aquaria of 2 l (14.5 x 14.0 x 9.5 cm) filled with 1.0 liter of water.
Test medium: Continuously aerated natural water of the town of Schlieren
Oxygen concentration: The total dissolved oxygen content is kept at >60% of the saturation value with aeration via Pasteur pipettes.
Number of fish: 7 fish per test concentration and control (total length of 1–3 cm)
Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes, no transition period.
Temperature (target): 21–25 °C, controlled by a thermostat.
Test type: Static exposure conditions
Test duration: 96 h

Handling:
The exposure started after an equilibration time of at least 1 h, after all the fish were randomly placed in the test vessels. The transfer was performed in an impartial manner to eliminate bias. A fine-mesh dip net was used to transfer fish, taking care to minimise possible stress due to handling. Fish which are damaged or dropped during the transfer were not used.

Observations and measurements:
Oxygen, pH, temperature measurement were made prior to the addition of the fish to the test solutions and after 2, 24, 48, 72 and 96 h of exposure

Observation of the fish were made after 2, 24, 48, 72 and 96 h of exposure. Mortality of the fish was recorded at the same time intervals. Dead fish were removed immediately from the test solution and weight and length were recorded.

The test concentrations were determined by HPLC prior to the addition of the fish to the test solutions and after 2 h of exposure, as well as in the old and fresh test solutions after each 24 h period until the end of the test

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 6.82-12.5 mg/l

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

4.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Mortality and other symptoms
Incidences of mortality in the 3rd definitive test are presented in Table 2 of the report, while specification of symptoms observed on affected fish and data of dead and surviving fish are listed in Table 3 and 4 of the report, respectively. At the nominal concentration of 13.4 mg/l a total of 6 out of 7 fish were dead after 72 h of exposure. At 7.88 mg/l, one fish died after 72 h of exposure. At all other concentrations and in the blank control no mortality was observed within 96 h of exposure.

Estimation of LC50 and NOEC
The lethal concentration (LC50) of RCX 14-672 to Danio rerio (Zebrafish) was calculated to be 9.16 mg/l (95% confidence limits: 6.82–12.5 mg/l) with respect to the measured concentrations. Since hypoactivity was observed at 7.88 mg/l between 24 and 96 h of exposure, this was regarded as a significant effect. Consequently, the no-observed-effect concentration (NOEC) was 4.64 mg/l. 100% mortality was not observed in the semi-static test.

Validity criteria fulfilled:

yes

Conclusions:

The lethal concentration (LC50) of RCX 14-672 to Danio rerio (Zebrafish) was calculated to be 9.16 mg/l (95% confidence limits: 6.82–12.5 mg/l) with respect to the measured concentrations. Since hypoactivity of the fish was observed at 7.88 mg/l between 24 and 96 h of exposure, this was regarded as a significant effect. Consequently, the no-observed-effect concentration (NOEC) was 4.64 mg/l. 100% mortality was not observed in the semi-static test.

Executive summary:

The acute lethal toxicity of RCX 14-672 (CAS No. 606-28-0) to Danio rerio (Zebrafish) was investigated under semi-static exposure conditions over a period of 96 h. The test concentrations were 1.60, 2.70, 4.64, 7.88 and 13.4 mg/l. These concentrations were chosen based on the results of a preceding limit test where 100% mortality was observed at 26.8 mg/l; as well as on the results of a full test under static exposure conditions where 100% mortality was already observed at 13.4 mg/l.

Semi-static exposure conditions were used since the full test performed under static exposure conditions suggested that hydrolysis of the test item occurred, what could be confirmed in a parallel Tier 1 hydrolysis study according to OECD 111. Since RCX 14-672 has a limited solubility, the test concentrations were prepared by dilutions of a saturated solution in which the test item’s concentration was first determined by HPLC. Fresh test solutions were prepared at the beginning (0 h) and after 24, 48 and 72 h of exposure.

The concentrations of RCX 14-672 in the test solutions were measured by HPLC analysis at the beginning and after 2, 24, 48, 72 and 96 h of exposure in the old and the freshly prepared test solutions, respectively. Since the test item hydrolyses, the effective concentrations were based on the arithmetic mean of the respective geometric means of all four 24 h periods of exposure. These arithmetic means ranged between 79% and 90% of the nominal concentration.

At the nominal concentration of 13.4 mg/l a total of 6 out of 7 fish were dead after 72 h of exposure. At 7.88 mg/, one fish died after 72 h of exposure. At all other concentrations and in the blank control no mortality was observed within 96 h of exposure. Toxic effects other than mortality, such as hypoactivity, were observed at 13.4 and 7.88 mg/l, respectively.

Based on these data, the lethal concentration (LC₅₀) of RCX 14-672 to Danio rerio (Zebrafish) was calculated to be 9.16 mg/l (95% confidence limits: 6.82–12.5 mg/l) with respect to the measured concentrations. Since hypoactivity of the fish was observed at 7.88 mg/l between 24 and 96 h of exposure, this was regarded as a significant effect. Consequently, the no-observed-effect concentration (NOEC) was 4.64 mg/l. 100% mortality was not observed in the semi-static test.

Description of key information

The 96-hour LC50 was determined to be 9.16 mg/l (95% confidence limits: 6.82–12.5 mg/l) with respect to the measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

9.16 mg/L

Additional information

The acute lethal toxicity of the substance to Danio rerio (Zebrafish) was investigated under semi-static exposure conditions over a period of 96 h. The LC50 was determined to be 9.16 mg/l (95% confidence limits: 6.82–12.5 mg/l) with respect to the measured concentrations. Since hypoactivity of the fish was observed at 7.88 mg/l between 24 and 96 h of exposure, this was regarded as a significant effect. Consequently, the no-observed effect concentration (NOEC) was 4.64 mg/l.