Cinnamaldehyde

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

The stock solution (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Own breeding at University of Chemistry and Technology, Prague
- Age at study initiation (mean and range, SD): The animals used for the test shall be less than 24 h old and should not be first brood progeny
- Feeding during test: No feeding

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

± 1 hr

Test temperature:

20±1°C

pH:

Test: 7.9 (changed to 7.8 during test)
Control: 7.8 (no change during test)

Dissolved oxygen:

higher than 7.9 mg/L at the end of test both in the control and the sample

Nominal and measured concentrations:

Nominal test chemical concentration used for the study were 0, 0.5, 1, 2, 3, 4 and 8 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml glass vessel
- fill volume: 25 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as culturing and dilution water if D. magna survives in it for the duration of the culturing, acclimation and testing without showing signs of stress. Waters in the range pH 6 to pH 9, with hardness between 140 mg/l and 275 mg/l (as CaCO3) are recommended.
As an example, the preparation of dilution water meeting the requirements is described below.
Dissolve known quantities of reagents in water. The dilution water prepared shall have a pH of 7.8 ± 0.5, a hardness of (225 ± 50) mg/l (expressed as CaCO3), a molar Ca + Mg ratio close to 4 + 1 and a dissolved oxygen concentration above 7 mg/l.

Prepare the solutions specified below:
- Calcium chloride solution: Dissolve 117.6 g of calcium chloride dihydrate (CaCl2.2H2O) in water (4.2) and make up to 1 l with water (4.2).
- Magnesium sulfate solution: Dissolve 49.3 g of magnesium sulfate heptahydrate (MgSO4.7H2O) in water (4.2) and make up to 1 l with water (4.2).
- Sodium bicarbonate solution: Dissolve 25.9 g of sodium bicarbonate (NaHCO3) in water (4.2) and make up to 1 l with water (4.2).
- Potassium chloride solution: Dissolve 2.3 g of potassium chloride (KCI) in water (4.2) and make up to 1 l with water (4.2).

Mixing
Mix 2.5 ml of each of the four solutions and make up to 1 l with water.
The dilution water shall be aerated until the dissolved oxygen concentration has reached saturation and the pH has stabilized. If necessary, adjust the pH to 7.8 ± 0.5 by adding sodium hydroxide (NaOH) solution or hydrochloric acid (HCI). The dilution water prepared in this way shall not be further aerated before use.

- Sodium hydroxide solution, e.g. [NaOH] : 1 mol/l.
- Hydrochloric acid, e.g. [HCl] : 1 mol/l.

Reference substance:
Dissolve 600 mg of potassium dichromate (K2Cr2O7) in water and make up to 1 l with water (4.2).

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment done
- Photoperiod: Darkness

CALCULATION:
EC50 was calculated using non linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 2.87 to 3.51 mg/l

Details on results:

- Behavioural abnormalities:
- Mortality of control: No mortality in control
- Immobilisation of control: No immobilisation was observed in control

Results with reference substance (positive control):

- Results with reference substance valid
- EC50: 0.79 mg/L (24 hours)

Reported statistics and error estimates:

EC50 was calculated using non leniar regression equation by the software prism 4.0

Test concentrations (mg/L) Nominal	Number of surviving daphnia	Percent inhibitions
Control (0)	0	0
0.5	1	5
1.0	1	5
2.0	4	20
3.0	9	45
4.0	15	75
8.0	20	100

Validity criteria fulfilled:

yes

Remarks:

No mortality was observed in control group and dissolved oxygen is greater than 3 mg/l in control group

Conclusions:

Based on the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr EC50 value was determined to be 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration).

Executive summary:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0.5, 1, 2, 3, 4 and 8 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2016). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0.5, 1, 2, 3, 4 and 8 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.21 mg/L

Additional information

Various experimental studies of the test chemical were reviewed for the short term toxicity to aquatic invertebrate end point which are summarized as below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates (Experimental study report, 2016). The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution (20 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 0.5, 1, 2, 3, 4 and 8 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 3.21 mg/l (95 % C. I. - 2.87 to 3.51 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to aquatic invertebrates at environmental related concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrates study was carried out for 48 hrs (from peer reviewed journal, authoritative database and secondary sources, 2019). The study was performed following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of< 24 hour old (Juvenile daphnids) produced from an in-house culture of adults was used as a test organism. Prior to the 48 hr study, the daphnid culture were maintained in 100% dilution water for 48 hrs. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7 days prior to the beginning of the test. Test chemical concentrations were verified analytically by HPLC/UV detector. Test chemical concentrations used for the study were 0, 2.0, 3.3, 5.5, 9.0, 15 and 25 mg/l (nominal concentrations) and 0.00452 (control), 1.91, 3.34, 5.30, 9.57, 13.9 and 25.5 mg/l (mean measured concentrations), respectively. Renewal of test solutions was carried every 24 hours. Study was performed using 10 daphnids/replicate for 48 hrs in a semi-static system in duplicates. Test vessels were placed at a temperature of19.5 to 20.7°C, pH6.8 to 7.1, dissolved oxygen of 8.2 to 9.1 mg/lunder a photoperiod of 16: 8 hr light : dark conditions. Mortality, immobility, and sub-lethal effects were determined visually at 0, 24, and 48 hours. Statistical analysis involve the use of Probit method. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration (i.e., maintained within 93-106% of nominal concentrations) throughout the test. Therefore, the analysis of the results were based on nominal concentration. On the basis of the effect on mobility and mortality of the test organism Daphnia magna, the 48 hr NOEC, EC50 and LC50 value was determined to be 1.91 mg/l, 3.86 mg/l and 4.22 mg/l (nominal concentrations), respectively.

 

For the test chemical, short term toxicity to aquatic invertebrates study was carried out for 48 hrs (authoritative database, 2019 and secondary sources). The study was performed following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of2-24 hour old was used as a test organism. Test chemical concentrations were verified analytically by GLC. Test chemical solutions were prepared by emulsification of the test substance in water with acetone. Test chemical concentrations used for the study were 0, 3.8, 7.5, 15.0, 30.0, and 60 mg/L (nominal concentrations) and 0, 3.8, 7.5, 15, 30 and 60 mg/l (mean measured concentrations), respectively. Study was performed using 5 daphnids/vessel for 48 hrs in a static system. Test vessels (25 ml glass beaker) were placed at a temperature of20.2 to 20.5°C, pH7.75 to 8.19, dissolved oxygen of 8.4 to 8.6 mg/l under a photoperiod of 16: 8 hr light : dark conditions with light intensity of 200 lx. After a period of 24 and 48 hrs, mobility of the test organisms were determined. Daphnia which showed no reaction after 15 seconds were considered immobile. The pH, oxygen content and temperature were measured at the beginning and end of the test. Probit analysis was performed for determining the EC50 value. On the basis of the effect on mobility of the test organism Daphnia magna, the 48 hr EC50 and EC100 value was determined to be 11.5 mg/l and 30 mg/l (nominal concentrations), respectively.

 

On the basis of the above results, it can be concluded that the test chemical was consideredastoxic to aquatic invertebrates at environmental related concentrations.Since, the test chemical is readily biodegradable in water, chemical was considered to be non-toxic to aquatic invertebrate and hence, considered to be 'not classified' as per the CLP classification criteria.