Cinnamaldehyde

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

The stock solution (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5000 cells /ml
- Method of cultivation: No data available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Test temperature:

23±2°C

pH:

Test: 7.9 (no change during tests)
Control: 8.0 (changed to 8.8 during test)

Nominal and measured concentrations:

Nominal test chemical conc. used for the study were 0, 3, 6, 12, 24 and 50 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Glass vessel
- Type (delete if not applicable): closed (with air permeable stopper)
- Sample volume: 15 ml
- Initial cells density: 5000 cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.
- Other: ErC50 was calculated using non-linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 25.9 - 38.6 mg/l

Results with reference substance (positive control):

Results with reference substance valid
- EC50: 0.65 mg/L

Reported statistics and error estimates:

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

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical, the 72hr median effective concentration (ErC50) value was determined to be 31.6 mg/l (95% CL: 25.9 to 38.6 mg/l) (nominal concentration).

Executive summary:

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 3, 6, 12, 24 and 50 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 100 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 31.6 mg/l (95 % CI 25.9 - 38.6 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2016). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 3, 6, 12, 24 and 50 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 100 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 31.6 mg/l (95 % CI 25.9 - 38.6 mg/l) (nominal concentration). Thus, based on the EC50 value, test chemical was considered as toxic to aquatic algae. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

31.6 mg/L

Additional information

Various experimental studies of the test chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2016),toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution (200 g/L) was prepared by dissolving test chemical in DMSO. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical conc. used for the study were 0, 3, 6, 12, 24 and 50 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 100 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 31.6 mg/l (95 % CI 25.9 - 38.6 mg/l) (nominal concentration).

 

Another algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2015) Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 1 х 10E4 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 50 mg of test chemical in 100 ml of BBM to get the final concentration of 500 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. Green algae were exposed to nominal concentration of test chemical (1.8 mg/L,3.6 mg/L, 7.2 mg/L, 14.4 mg/L,28.8 mg/L and 57.6 mg/L) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 1500 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (EC50) value was determined to be 16.09 mg/l.

 

For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (from A.M. Api et. al. (2019), authoritative database and secondary sources, 2019). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Selenastrum capricornutum (green algae) of strain UTEX 1648 with a control cell density of 366000 cells/ml was used as a test organism. Green algae, U. of Texas was maintained at test conditions for 14 days prior to the test. The culture was growing in at least 2 subcultures prior to the initiation of the test. Test chemical concentrations were verified analytically by HPLC/UV detector. Range finding study was performed. Test chemical concentrations used during the range finding study were 0.1, 1.0, and 100 mg/l. In a range finding test, the number of cells/mL was >100 % of controls at 0.10 mg/L, 80% at 1.0 mg/L, and <1% at 100 mg/L after three days. Test chemical concentrations used for the definitive study were 0, 0.50, 1.0, 2.0, 4.0 and 8.0 mg/l (nominal concentrations) and 0, 0.523, 1.04, 2.00, 3.80, and 7.03 mg/l (initial mean measured concentrations), respectively. Test organisms were exposed to test chemical for a period of 72 hrs at 23.2 to 24.0°C temperature, adjusted pH of 7.5 under a photoperiod of 16: 24 light: dark with light intensity provided using 400-410 candles. All test experiments were carried out in triplicates and controls were setup in 6 replicate. After an exposure period of 72 hrs, the number of algal cells/ml, relative size, cell shapes, color, adherence and aggregation of cells was determined. At 24, 48, and 72 hours treatment and control vessels were sacrificed to determine the number of algal cells/ml.EC50 values determined by weighted least squares non-linear regression. NOEC was determined using a one-way analysis of variance (ANOVA) and Bonferroni's test. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration (i.e., maintained within 88-105% of nominal concentrations) throughout the test. Therefore, the analysis of the results were based on nominal concentration. On the basis of the effect on number of cells/ml of the test organism Selenastrum capricornutum, the 72 hrs NOEC was determined to be 2.0 mg/l and based on the effect on growth rate, number of cells/ml & area under the growth curve, theEC50 value was determined to be 6.87 mg/l, 4.56 mg/l and 4.07 mg/l, respectively.

 

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