Methyl benzoate

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:

1993-01-18 to 1993-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were analysed at the beginning of the test and after 24 hours, samples were taken in the course of the first exchange of test medium

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: testing solutions were prepared with synthetic freshwater by using an Ultra-Turrax mixer; substance was directly weighed into testing vessels

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Zebrabärbling)
- Strain: Hamilton Buchanan
- Source: West-Aquarium (Bad Lauterberg)
- Age at study initiation (mean and range, SD): approximately 80 days
- Length at study initiation (length definition, mean, range and SD): 2.5 to 3.5 cm
- Weight at study initiation (mean and range, SD): approximately 0.4 g
- Feeding during test: no feeding

ACCLIMATION
- No details on acclimation period are reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Hardness:

13.6 ° dH

Test temperature:

Control: 21.2 to 22.5 °C
11 mg/L: 21.3 to 22.8 °C
16 mg/L: 21.3 to 22.7 °C
22 mg/L: 21.3 to 22.8 °C
31 mg/L: 21.3 to 22.8 °C
44 mg/L: 21 to 22.2 °C

pH:

Control: 7.7 to 8.1
11 mg/L: 7.6 to 8.1
16 mg/L: 7.6 to 8.1
22 mg/L: 7.6 to 8.1
31 mg/L: 7.6 to 8.1
44 mg/L: 7.6 to 8.1

Dissolved oxygen:

Control: 8.3 to 10.0 mg oxygen/L
11 mg/L: 7.7 to 10.0 mg oxygen/L
16 mg/L: 7.6 to 10.2 mg oxygen/L
22 mg/L: 7.8 to 10.0 mg oxygen/L
31 mg/L: 8.7 to 10.2 mg oxygen/L
44 mg/L: 8.9 to 9.5 mg oxygen/L

Salinity:

not reported

Nominal and measured concentrations:

Nominal: 11, 16, 22, 31, 44 mg/L
Measured: 9.4, 13.3, 18.3, 27.3, 39.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: aquarium
- Material, size, headspace, fill volume: material not reported, 5 L test medium
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): after every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.42 to 0.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater prepared according to ISO guideline
- No details on composition are reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 8 hours light/16 hours dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

13.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

39.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: The LC50 was calculated as the geometric mean of the LC0 and LC100 values

Basis for effect:

mortality (fish)

Details on results:

Lethargic swimming behaviour and immobility, uncontrolled swimming and abnormal swimming positions were noted for the two highest test concentrations of 31 and 44 mg/L

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Sublethal observations / clinical signs:

Table 1: Detailed results on mortality

Group		2 hours	24 hours	48 hours	72 hours	96 hours
0 mg/L	Absolute #	0	0	0	0	0
	Cumulative #	0	0	0	0	0
	Percentage	0	0	0	0	0
11 mg/L	Absolute #	0	0	0	0	0
	Cumulative #	0	0	0	0	0
	Percentage	0	0	0	0	0
16 mg/L	Absolute #	0	0	0	0	0
	Cumulative #	0	0	0	0	0
	Percentage	0	0	0	0	0
22 mg/L	Absolute #	0	0	0	0	2
	Cumulative #	0	0	0	0	2
	Percentage	0	0	0	0	20%
31 mg/L	Absolute #	0	4	0	1	3
	Cumulative #	0	4	4	5	8
	Percentage	0	40%	40%	50%	80%
44 mg/L	Absolute #	0	10	-	-	-
	Cumulative #	0	10	-	-	-
	Percentage	0	100%	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

The test substance methyl benzoate was acutely toxic to freshwater fish in a 96-hour semi-static test. The LC50 value was 23 mg/L.

Executive summary:

The acute toxicity of the substance methyl benzoate to freshwater fish was studied under GLP conditions in a 96-hour semi-static test in accordance with EU Method C.1 (1992). Healthy zebra fish (Brachydanio rerio, HAMILTON BUCHANAN) aged about 2.5 months, which had body lengths ranging from 2.5 to 3.5 cm were used in the test. The mortality of fish under the conditions of cultivation in the laboratory was less than 2%. Test solutions at nominal concentrations of 11, 16, 22, 31 and 44 mg/L were prepared by dissolving the test substance in synthetic freshwater prepared according to ISO. To this end, a stirrer (Ultra-Turrax) at 8000 rpm was used for 60 seconds. In addition, a control without test substance was run. The nominal concentrations were confirmed by gas chromatography analysis immediately at the start of the test and 24 hours later when the test medium was changed for the first time in the course of the semi-static operation mode (a repetition of the analysis was performed at the beginning and end of the second experimental day). The measured concentrations ranged from 94.1% to 96.4% of the nominal concentrations at the beginning of an experimental day and from 71.6% to 84.8% at the end of a day showing the relative stability of the test substance. At the beginning of the tests, ten healthy fish were placed in each vessel. No mortality occurred in the controls and at nominal test concentrations of 11 and 16 mg/L. Two fish were found dead after 96 hours at the nominal concentration of 22 mg/L. Lethargic swimming behaviour and immobility, uncontrolled swimming and abnormal swimming positions were noted for the two highest test concentrations of 31 and 44 mg/L. Eight dead fish were counted after 96 hours at the nominal concentration of 31 mg/L and ten out of ten fish died within 24 hours at the highest nominal test concentration of 44 mg/L. The LC0 of 13.3 mg/L and the LC100 of 39.9 mg/L were calculated on the basis of the arithmetic mean measured concentrations. The LC50 of 23.0 mg/L was calculated as the geometric mean of LC0 and LC100 values.

Description of key information

 The substance was moderately toxic in a 96-hour test with zebra fish resulting in a LC50 value of 23 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

23 mg/L

Additional information

A valid study on the acute toxicity of methyl benzoate to freshwater fish according to EU Method C.1 (1992) is available, in which zebra fish were exposed to the substance under semi-static conditions for a period of 96 hours. The LC50 found in the study was 23 mg/L. Information on a prolonged acute toxicity test with guppy was published (Verhaar et al. 1999). Details on the actual study were not provided in this publication. The 96-hour LC50 was 40 mg/L and the 14-day LC50 was 28.6 mg/L, which is quite similar to the value found in the study with zebra fish. Another acute toxicity study in three freshwater species - trout, bluegill sunfish and goldfish - resulted in mortality of trout and bluegill fish at a nominal test concentration of 5 ppm. However, this test cannot be considered as valid mainly due to the small number of test animals.

It can be concluded that fish appears to be the most sensitive aquatic species with regard to acute exposure to methyl benzoate when compared to other aquatic organisms.