Benzyl cinnamate

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 2017-06-12 to 2017-06-17

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:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Series on Testing and Assessment, No. 126 -Short Guidance on the Threshold Approach for Acute Fish Toxicity

Version / remarks:

2010

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.8 mg/L
- Sampling method: The limit concentration, control and solvent control were analytically verified from all freshly prepared and aged test media.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

yes

Remarks:

Dimethyl sulfoxide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution (nominal concentration: 8 g/L) was prepared in a measuring flask (10 mL capacity) by weighing out 80 mg of the test item and transferring it into the measuring glass flask. Thereafter, the flask was filled up with DMSO to 10 mL. The test concentration was prepared by adding 1.0 mL/10L (0.1 mL/L) of the stock solution to the dilution water.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicate.
- Solvent control: Additional a solvent control with 0.100 mL DMSO/L dilution water was prepared and tested under the same conditions as the test groups.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: Danio rerio
- Source: a single brood stock, more information on source in study report
- Average body weight: 0.20 g
- Average body length: 2.76 cm (2.7-2.8 cm)

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Sera Vipan, 4 % of the fish body weight per feeding day.
- Feeding frequency during acclimation: 3 times per week, not fed 24 h before the test start

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

52 mg/L as CaCO3

Test temperature:

22.4-24.6°C

pH:

7.35-7.98

Dissolved oxygen:

89-100%

Nominal and measured concentrations:

Nominal: 0.8 mg/L
Measured: 0.643 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: 13 L glass aquaria
- Type: closed, covered with a floating lid
- Material, size, headspace, fill volume: Glass aquaria of 13 L, filled with approximately 10 L of dilution water
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): One replicate
- No. of vessels per vehicle control (replicates): One replicate
- Biomass loading rate: 0.14 g fish per litre test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin
- Chlorine: filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Alkalinity: 0.6 mmol/L
- Intervals of water quality measurement: quarterly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 7 - 750 Lux

EFFECT PARAMETERS MEASURED :
- Biological Parameters: Observation of visible abnormalities after 2, 24, 48, 72 and 96 h. Measurement of size and weight of the control fish at the end of the exposure.
- Physical Properties: pH-value, temperature, oxygen saturation, total hardness, water temperature and light intensity were assessed throughout the trial

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Taking into account the aquatic toxicity data of the test item the test was performed without a range-finding test. The threshold concentration (TC) is based on the toxicity data of the test item of an alga toxicity test (ErC50(0-72 h) = 0.386 mg/L). Considering the loss of the substance during the exposition, a nominal concentration of 0.8 mg/L was performed in the limit test.
- Test concentrations: 0.8 mg/L were tested as a threshold concentration in a limit test.

Reference substance (positive control):

no

Remarks:

No reference item is recommended for this test according to the guideline.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.643 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.643 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 0.643 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Biological Data: No symptoms of toxicity were observed for both test and control group. No mortality occurred.
- Physicochemical Data: The environmental conditions (pH-value, temperature, O2 saturation) were within the acceptable limits. The continuous measuring of water temperature was in the valid range of 23 ± 2 °C. The measured light intensity was in the given range of 7 - 750 lux and ranged from 96 to 114 lux (mean value 110 lux).
- Analytical Data: The measured concentrations of the test item in the freshly prepared media (0, 24, 48 and 72 hours) ranged from 87% to 107% of the nominal test item concentration. In 24 hours old media the measured concentrations of the test item were between 20% and 67%.

Sublethal observations / clinical signs:

Observations in the test vessels

geometric mean conc (mg/L)	Number of fish having normal behaviour
	2h	24h	48h	72h	96h
0.643	7/7	7/7	7/7	7/7	7/7
solvent control	7/7	7/7	7/7	7/7	7/7
control	7/7	7/7	7/7	7/7	7/7

Culative mortality (%) in the test vessels

Geometric mean measured concentration (mg/L)	Cumulative mortality (%) after different exposure periods (hours)
	2	24	48	72	96
0.643	0	0	0	0	0
Solvent control	0	0	0	0	0
Control	0	0	0	0	0

Measured concentrations

geometric mean conc (mg/L)	0h		24h		48h		72h
	measured conc (mg/L)	% of nominal concentration	measured conc (mg/L)	% of nominal concentration	measured conc (mg/L)	% of nominal concentration	measured conc (mg/L)	% of nominal concentration
0.643	0.699	87	0.818	102	0.762	95	0.858	107
solvent control	not available		< LOQ		< LOQ		< LOQ
control	< LOQ		< LOQ		< LOQ		< LOQ

Validity criteria fulfilled:

yes

Conclusions:

The threshold concentration of the test item was found to have no effect on the vitality of zebrafish. In conclusion, the LC0 corresponds to the geometric mean measured concentration 0.643 mg/L. The LC50 and the LC100 correspond to the geometric mean measured concentration > 0.643 mg/L.

Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation No. 440/2008/C.1 (2008).

A semi-static limit test was performed with a threshold concentration of 0.8 mg/L. The threshold concentration selected in the fish study was based on the toxicity data of the test item in an algae toxicity test where an EC50 of 0.386 mg/L (TWA conc) was derived.

The test had a duration of 96 hours. 7 test organisms were exposed each for the test concentration and the control. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

The determination of the test concentration and the corresponding degradation product after derivatization was carried out via GC-MS after liquid-liquid extraction. Evaluation was carried out from all freshly prepared media after 0, 24, 48 and 72 h and from the corresponding 24 h aged test media after 24, 48, 72 and 96 h. The measured concentrations in freshly prepared media were in the range of 87% to 107% of the nominal values. In corresponding 24 h aged test media, the measured concentrations were in the range of 20% to 67% of the nominal value.

In conclusion, the LC0 corresponds to the geometric mean measured concentration 0.643 mg/L. The LC50 and the LC100 correspond to the geometric mean measured concentration > 0.643 mg/L.

Description of key information

LC0 = 0.643 mg/L (Limittest; Danio rerio, OECd 203)

Additional information

A semi-static limit test was performed with a threshold concentration of 0.8 mg/L. The threshold concentration selected in the fish study was based on the toxicity data of the test item in an algae toxicity test where an EC50 of 0.386 mg/L (TWA conc) was derived.

The test had a duration of 96 hours. 7 test organisms were exposed each for the test concentration and the control. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

The determination of the test concentration and the corresponding degradation product after derivatization was carried out via GC-MS after liquid-liquid extraction. Evaluation was carried out from all freshly prepared media after 0, 24, 48 and 72 h and from the corresponding 24 h aged test media after 24, 48, 72 and 96 h. The measured concentrations in freshly prepared media were in the range of 87% to 107% of the nominal values. In corresponding 24 h aged test media, the measured concentrations were in the range of 20% to 67% of the nominal value.

In conclusion, the LC0 corresponds to the geometric mean measured concentration 0.643 mg/L. The LC50 and the LC100 correspond to the geometric mean measured concentration > 0.643 mg/L.

As key value for LC50 the lowest concentration from the range was selected: 0.643 mg/L.