Cinnamomum cassia, ext.

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/4/2013 - 18/4/2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guideline (OECD guideline 202) under GLP. No deviations from guideline reported. The two lowest exposures 0.10 and 0.32 mg/L loading rates were diluted from the loading rate 1 mg/L but this is of no relevance for the result. No valid verification of TOC levels.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not releavnt

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamomum cassia, ext.

EC Number:

284-635-0

EC Name:

Cinnamomum cassia, ext.

Cas Number:

84961-46-6

IUPAC Name:

(2E)-3-(2-methoxyphenyl)prop-2-enal; (2E)-3-phenylprop-2-en-1-yl acetate; (2E)-3-phenylprop-2-enal

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Cassia oil:
- Physical state: liquid

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: Duplicate samples were taken after 0 hours (fresh media) and 48 hours (old media).
- Sample storage conditions before analysis: Samples were stored frozen for further analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (20, 64 and 200 mg) were added to the surface of 20 liters of reconstituted water to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and dispersions or undissolved test item to be present. The aqueous phase or W AF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate W AFs.
Aliquots (100 and 320 mL) of the 1.0 mg/L loading rate WAF were each separately added to a final volume of 1 liter of reconstituted water to give the 0.10 and 0.32 mg/L loading rate W AFs respectively.
- Controls: Yes, blanks
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the mixing period the 1.0, 3.2 and 10 mg/L loading rates were observed to be a clear colorless water column with oily globules of test item dispersed throughout and on the surface. After 23 hours stirring and a 1-Hour standing period the 1.0, 3.2 and 10 mg/L loading rates were observed to be a clear colorless water column with oily droplets of test item on the surface. Microscopic inspection of the W AF showed no micro-dispersions or undissolved test item to be present. The 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF test preparations were observed to be clear colorless solutions throughout the duration of the test.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory strain
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Feeding during test: no

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not relevant

Test conditions

Hardness:

250 mg/L CaCO3

Test temperature:

20 - 21ºC

pH:

7.7 - 8.1

Dissolved oxygen:

8.3 - 9.0 mg/L (93 - 99% of ASV)

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal loading rates: 0.1, 0.32, 1.0, 3.2 and 10 mg/L
For measured TOC concentrations, see Section "Any other information on results incl. tables". Note that the results of the TOC analysis are considered to be invalid (see below).

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours provided highly variable results and showed no concentration dependent pattern. There was no obvious explanation as to why the TOC results were variable, however, given that there was a very high amount of carbon seen in the control samples, and large inconsistencies between replicate samples, it is reasonable to assume that contamination or instrument malfunction may have occurred during the analysis. Given that the immobilization pattern during the study was consistent between the definitive test and the range finding test, and also that there was a relationship between dosed levels in the definitive test and a previsouly conducted test but not a relationship between measured concentrations, this error is not considered to have had an effect on the outcome or ntegrity of the study.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

TEST SYSTEM
- Test vessel: Jar
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 250 mL filled with 200 mL test medium
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Culture medium different from test medium: No
- Conductivity: <5 uS/cm^-1
- Intervals of water quality measurement: 24 hours

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light (606 - 283 lux)/dark with 20 minute transition period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilisation (24 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study:
- Test concentrations: 0.001, 0.01, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

3.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 3.0 - 4.8 mg/L

Effect concentrations 3

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: At the start of the mixing period the 1.0, 3.2 and 10 mg/L loading rates were observed to be a clear colorless water column with oily globules of test item dispersed throughout and on the surface. After 23 hours stirring and a 1-Hour standing period the 1.0, 3.2 and 10 mg/L loading rates were observed to be a clear colorless water column with oily droplets of test item on the surface. Microscopic inspection of the W AF showed no micro-dispersions or undissolved test item to be present. The 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF test preparations were observed to be clear colorless solutions throughout the duration of the test.

As the results of the TOC analysis were considered to be invalid, results were based on nominal loading rates.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.45 mg/L

Reported statistics and error estimates:

The EL* so value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).

Any other information on results incl. tables

Cumulative immobility:

Nominal loading rate (mg/L)	Cumulative immobilized Daphnia (initial population: 5 per replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.10	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
3.2	0	0	0	0	0	0	2	2	2	1	7	35
10	1	0	0	2	3	15	5	5	5	5	20	100

Graphs are presented below.

Nominal and measured concentrations

Samples	Nominal loading rate (mg/L)	Concentration of TOC (mg C/L)	Concentration of TOC corrected for control (mg C/L)
0 Hours (Fresh media)	Control R1- R4	<LOQ/4.69*	-
	0.10 R1 - R4	11.72/9.35*	11.72/4.66*
	0.32 R1 - R4	19.58/13.09*	19.58/8.40*
	1.0 R1 - R4	6.26/6.95*	6.27/2.26*
	3.2 R1 - R4	9.21/9.05*	9.21/4.36*
	10 R1 - R4	11.08/12.00*	11.08/7.31*
48 Hours (old media)	Control R1- R4	5.74/3.96*	-
	0.10 R1 - R4	17.62/17.41*	11.87/3.45*
	0.32 R1 - R4	27.25/10.12*	21.51/6.16*
	1.0 R1 - R4	12.15/5.40*	6.14/1.44*
	3.2 R1 - R4	15.61/7.20*	9.87/3.24*
	10 R1 - R4	18.8*	13.06*

*) Duplicate sample frozen prior to analysis

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

No mortality in controls, physical-chemical parameters within acceptable limits

Conclusions:

The acute toxicity (48h-EL50) of Cassia oil towards Daphnia magna is 3.8 mg/L.

Executive summary:

The acute toxicity of Cassia oil towards Daphnia magna was investigated according to OECD guideline 202 under GLP. Daphnids were exposed to nominal WAF's of 0.1, 0.32, 1.0, 3.2 and 10.0 mg/L and observed for 48 hours. The 48h-EL50 was found to be 3.8 mg/L based on nominal concentrations.