Ethyl cinnamate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is according to OECD 202 I (1984) and GLP. However, recalculation of EC-values based on initial measured concentrations was necessary, as the recovery rate of the lowest concentration (6.3 mg/L nominal) was not within +/- 20% of the nominal concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

- Stock solution: 100 mg/L
- Test dilutions: 1:16, 1:8, 1:4, 1:2, 1:1
- Test concentrations: 6.3 — 12.5 — 25 — 50 — 100 mg/L
- Control: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Reason for the selection: Daphnia magna STRAUS belongs to the suitable Daphnia types and are bred in the test facility.
- Origin: Institut für Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Berlin, Germany
- Breeder: DR.U.NOACK-LABORATORIUM, Sarstedt, Germany
- Culture: 2-3 L glass vessels with approximatively 1.8 L culture medium, by 21 °C (temperatures of 20-25 °C were tolerated), in an incubator, 16 h illumination, illumination strength 1.5-5.0 µmol.m-2.s-1
- Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
- Feeding: 5 x weekly ad libitum with a mix of Scenedesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106cells/mL.
- Age: less than 24h old
- Acclimatization: 2h in dilution water
- Number of study organisms per concentration and control: 20 animals, divided into 4 (2*) parallel samples, each with 5 (10*) animals
- Number of animals per test vessel: 5 (10*)
- Number of parallel samples per concentration: 4 (2*)
- Test temperature: 21+/-1°C
- Illumination: Diffuse light, illumination range 1.5 - 5 µmol.me-2.se-1
- Photoperiod: 16/8 h light/dark cycle

(*) The information in brackets refer to the test of the reference item

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Total hardness of dilution water: 246 mg/L as CaCO3

Test temperature:

21 +/- 1°C

pH:

pH of dilution water: 7.65
pH at beginning of the test: 7.81 (100 mg/L), 7.79 (50 mg/L), 7.82 (25 mg/L), 7.65 (12.5 mg/L), 7.73 (6.3 mg/L), 7.77 (control)

Dissolved oxygen:

Dissolved oxygen in dilution water: 8.22 mg/L
Dissolved oxygen at beginning of the test (mg/L): 8.44 (100 mg/L), 8.61 (50 mg/L), 8.52 (25 mg/L), 8.59 (12.5 mg/L), 8.67 (6.3 mg/L), 8.53 (control)

Nominal and measured concentrations:

100, 50, 25, 12.5, 6.3 and 0 mg/L nominal concentrations
88.4, 46.5, 21.9, 10.2, 4.42 and 0 mg/L measured concentrations

Details on test conditions:

-Test vessels: Giess beakers (5 cm ID x 8 cm H), 50 mL
- Volume of the study medium: 20 mL
- Dilution water: According to EEC 92/69 L383A C.2. (Annex)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

6.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

12.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

46.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The percentage immobility was determined in all test item groups and controls after 24 h and 48 h.
The EC 10 and EC 50-values after 24 and 48h were calculated by sigmoidal dose-response regression.
The EC100-values, the NOEC and LOEC after 48 h were deduced directly from the observation data, without mathematical calculation.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100 value was determined directly from the test results. The EC10 and EC50 with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in [mg/L]:
EC10 : 1.3
EC50 : 2.0 (CI 1.8 — 2.2)
EC100 :5.8
The EC50- value of reference item potassium dichromate alter 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30.

Reported statistics and error estimates:

EC100-values were deduced directly from the dose-response- relationship after 24 and 48 h. There was no mathematical calculation.
The concentration effect relationship is shown graphically in a probability network.
EC10and EC50-values were calculated via probit analysis. Probits according to WEBER (1986).
Calculation of the confidence intervals was carried out using standard procedures according to BREITIG & TÜMPLING (1982).
Calculations were performed using software SigmaPlot rel. 2000 (2000), SPSS CORPORATION.

Immobilisation rates after 24 and 48 h of Exposure

(n= 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration (mg/L)	Measured initial concentration of test item (mg/L)	Immobilisation (%)
		24h					48h
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
100	88.4	100	100	100	100	100	100	100	100	100	100
50.0	46.5	100	100	80	100	95	100	100	100	100	100
25.0	21.9	60	40	20	40	40	80	100	80	100	90
12.5	10.2	20	20	40	20	25	40	20	60	20	35
6.3	4.42	0	20	0	0	5	0	20	0	0	5
Control		0	0	0	0	0	0	0	0	0	0

MV= mean value

DOC-analysis of new media (0h):

Nominal test item concentration (mg/L)	Dissolved Organic Carbon (DOC)		Recovery rate** (%)	Calculated test item concentration in new media ***(mg/L)
	Nominal concentration* (mg/L)	Measured concentration (mg/L)
100	74.8	53.6	88	88.4
50.0	37.4	24.9	93	46.5
25.0	18.7	12.2	88	21.9
12.5	9.4	7.19	82	10.2
6.3	4.7	4.02	70	4.42
control	/	0	/	0

* Nominal DOC concentration based on the ThTOC (0.748mg C/mg) of test item

** Percent of the nominal concentration of DOC

*** Calculation of the test item concentration at the start of the exposure (0h) based on the recovery rate of DOC.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 after 48h of exposure was 12.1mg/L.

Executive summary:

In the current study the acute immobilisation of the test item to Daphnia magna (STRAUS) was determined according to OECD TG 202/ EEC Directive 92/69/EEC Method C.2.

The study was conducted under static conditions over a duration of 48 hours. 20 test organisms were exposed to a test concentration or to a control. Five concentration were tested: 100, 50, 25, 12.5, 6.3 and 0 mg/L nominal concentrations, corresponding to 88.4, 46.5, 21.9, 10.2, 4.42 and 0 mg/L measured concentrations.

The test item solutions were clearly dissolved after filtration in all tested concentration levels throughout exposure.

 

A reference test is carried out once per month with potassium dichromate as reference item and the reference toxicity determined. The EC50-value of the reference item at 2.0 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30.

Water quality parameters of pH-value and dissolved oxygen concentration measured at 0 and 48 h were determined to be within the acceptable limits.

The validity criteria of the test guideline were fulfilled.

 

In the original test the recovery rate of the lowest concentration was not within +/- 20% of the nominal concentrations. Therefore, the sponsor requested the EC-values to be re-calculated. Re-calculations were done by sigmoidal dose- response regression based on the measured initial concentrations.

The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

 

After 24 hours the EC50 was 22.2 mg/L (CI: 18.2 – 27.6) and after 48 hours the EC50 was 12.1 mg/L (CI: 10.7 – 14.3).

Description of key information

The test item has an EC50 of 12.1 mg/L after 48 hours.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

12.1 mg/L

Additional information

For this endpoint only one test is available. In this study the acute immobilisation of the test item to Daphnia magna (STRAUS) was determined according to OECD TG 202 under static conditions over a duration of 48 hours.

The nominal test concentrations were 100, 50, 25, 12.5, 6.3 and 0 mg/L nominal concentrations, corresponding to 88.4, 46.5, 21.9, 10.2, 4.42 and 0 mg/L measured concentrations.

The reference test item had an EC50-value of 2.0 mg/L after 24 h which is within the prescribed concentration range. The water quality parameters of pH-value and dissolved oxygen concentration measured were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

The EC-values were calculated by sigmoidal dose- response regression based on the measured initial concentrations. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

 

After 24 hours the EC50 was 22.2 mg/L (CI: 18.2 – 27.6) and after 48 hours the EC50 was 12.1 mg/L (CI: 10.7 – 14.3).