trans-cinnamic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 June 2018 and 24 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 18 July 2017 - 20 July 2017 Date of Issue: 28 November 2017

Test material

Test material information

Constituent 1

Reference substance name:

trans-cinnamic acid

EC Number:

205-398-1

EC Name:

trans-cinnamic acid

Cas Number:

140-10-3

Molecular formula:

C9H8O2

IUPAC Name:

3-phenylacrylic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20171106
- Expiration date of the lot/batch: 10 April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over silica gel

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from additional media ran alongside at 24 and 48 hours, and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. A duplicate set of samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A nominal amount of test item (200 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes, and the volume adjusted to 2 liters to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL.
- Culturing media and conditions (same as test or not): Same as test
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Any deformed or abnormal cells observed:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.0-7.8

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32, 100 mg/L
Measured: 0.69, 2.5, 9.0, 30, 87 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed (plugged with polyurethane foam bungs to reduce evaporation)
- Material, size, headspace, fill volume: 250mL glass
- Initial cells density: 1.83 x 10^6 cells per mL
- Control end cells density: 5.00 x 10^3 cells per mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture

OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: Continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Samples were taken at 21, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter
- Other: The shape and size of the algal cells was inspected microscopically and any abnormalities recorded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield values

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield values

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield values

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield values

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield values

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 0.69, 2.5 and 9.0 mg/L, however no cells were observed to be present in the test cultures at 30 and 87 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.69, 2.5 and 9.0 mg/L test cultures were observed to be light green dispersions whilst the 30 and 87 mg/L test cultures were observed to be clear colorless solutions

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L (based on growth rate)
- EC50: 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L (based on yield)

Any other information on results incl. tables

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/L)		Cell Densities * (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	7.48E+03	1.02E+06	-	-
	R2	7.16E+03	1.53E+06
	Mean	7.32E+03	1.28E+06
0.10	R1	8.65E+03	1.39E+06	1	[2]
	R2	7.42E+03	1.21E+06
	Mean	8.04E+03	1.30E+06
1.0	R1	6.60E+03	1.27E+06	[1]	[6]
	R2	7.54E+03	1.44E+06
	Mean	7.07E+03	1.35E+06
10	R1	7.60E+03	1.11E+06	6	21
	R2	7.42E+03	9.10E+05
	Mean	7.51E+03	1.01E+06
100	R1	8.30E+03	2.45E+04	86	99
	R2	7.83E+03	9.36E+03
	Mean	8.07E+03	1.69E+04

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate

- = Not applicable

[ ] = Increase in growth compared to controls

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 92% to 136% of nominal. A decline in measured test concentrations was observed at 72 hours in the range of 21% to 49% of nominal indicating that the test item was unstable under the conditions of the test.

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 80% to 101% of nominal. A marked decline in measured test concentrations was observed at 72 hours in the range of 9% to 71% of nominal indicating that the test item was unstable under the conditions of the test.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:

Nominal Test Concentrations (mg/L)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the Nominal Test Concentration (%)
1.0	0.69	69
3.2	2.5	78
10	9.0	90
32	30	94
100	87	87

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2.

Table 2 Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities * (cells per mL)			pH
		0 Hours	21 Hours	46 Hours	72 Hours	72 Hours
Control	R1	7.8	1.15E+04	5.80E+04	2.70E+05	8.1
	R2		1.14E+04	5.71E+04	3.11E+05
	R3		1.04E+04	6.36E+04	3.61E+05
	R4		1.06E+04	4.56E+04	2.63E+05
	R5		1.06E+04	5.85E+04	3.04E+05
	R6		1.19E+04	6.53E+04	3.67E+05
	Mean		1.11E+04	5.80E+04	3.13E+05
0.69	R1	7.6	1.33E+04	5.20E+04	2.83E+05	8.1
	R2		1.02E+04	5.75E+04	3.03E+05
	R3		1.26E+04	6.52E+04	3.48E+05
	Mean		1.20E+04	5.82E+04	3.11E+05
2.5	R1	7.5	1.00E+04	4.96E+04	2.46E+05	8.0
	R2		1.13E+04	5.61E+04	2.57E+05
	R3		1.26E+04	6.22E+04	2.83E+05
	Mean		1.13E+04	5.60E+04	2.62E+05
9.0	R1	7.0	1.15E+04	5.35E+04	2.76E+05	7.9
	R2		1.06E+04	5.96E+04	3.13E+05
	R3		8.07E+03	5.51E+04	2.78E+05
	Mean		1.01E+04	5.61E+04	2.89E+05
30	R1	5.3	4.99E+03	6.54E+03	1.46E+04	5.3
	R2		3.90E+03	4.08E+03	4.08E+03
	R3		3.99E+03	3.81E+03	3.49E+03
	Mean		4.29E+03	4.81E+03	7.65E+03
87	R1	4.3	2.90E+03	3.37E+03	2.82E+03	4.0
	R2		2.90E+03	3.52E+03	2.70E+03
	R3		3.64E+03	3.14E+03	3.61E+03
	Mean		3.15E+03	3.34E+03	3.04E+03

 

 Daily specific growth rates for the control cultures are given in Table 3.

Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Nominal Concentration (mg/L)		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.040	0.065	0.059
	R2	0.039	0.064	0.065
	R3	0.035	0.072	0.067
	R4	0.036	0.059	0.067
	R5	0.036	0.068	0.063
	R6	0.041	0.068	0.066
	Mean	0.038	0.066	0.065

 

Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

Table 4 Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition
Control	R1	0.055	-	2.65E+05	-
	R2	0.057		3.06E+05
	R3	0.059		3.56E+05
	R4	0.059		2.58E+05
	R5	0.057		2.99E+05
	R6	0.060		3.62E+05
	Mean	0.057		3.08E+05
	SD	0.002		4.39E+05
0.69	R1	0.056	2	2.78E+05
	R2	0.057	0	2.98E+05
	R3	0.059	[4]	3.43E+05
	Mean	0.057	[1]	3.06E+05
	SD	0.002		3.35E+04	0
2.5	R1	0.054	5	2.41E+05
	R2	0.055	4	2.52E+05
	R3	0.056	2	2.78E+05
	Mean	0.55	4	2.57E+05	17
	SD	0.001		1.91E+04
9.0	R1	0.056	2	2.71E+05
	R2	0.057	0	3.08E+05
	R3	0.056	2	2.73E+05
	Mean	0.056	1	2.84E+05	8
	SD	0.001		2.10E+04
30	R1	0.015	74	9.61E+03
	R2	0.000	100	-1.60E+02
	R3	-0.005	109	-1.51E+03
	Mean	0.003	94	2.65E+03	99
	SD	0.010		6.07E+03
87	R1	-0.008	114	-2.18E+03
	R2	-0.009	116	-2.30E+03
	R3	-0.005	109	-1.39E+03
	Mean	-0.007	113	-1.96E+03	101
	SD	0.002		4.95E+02

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

SD= Standard Deviation

- = Not applicable

[ ] = Increase in growth compared to controls

From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period

Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:

Inhibition of Growth Rate

ErC10 (0 to 72 hour) : 13 mg/L

ErC20 (0 to 72 hour) : 15 mg/L

ErC50 (0 to 72 hour) : 19 mg/L

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control, 0.69, 2.5 and 9.0 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 9.0 mg/L. Correspondingly the LOEC based on growth rate was 30 mg/L.

Inhibition of Yield

EyC10 (0 to 72 hour) : 9.4 mg/L

EyC20 (0 to 72 hour) : 11 mg/L

EyC50 (0 to 72 hour) : 14 mg/L

Where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in the above section. There were no statistically significant differences (P≥0.05), between the control, 0.69, 2.5 and 9.0 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 9.0 mg/L. Correspondingly the LOEC based on yield was 30 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 63 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours : 5.00E+03 cells per mL

Mean cell density of control at 72 hours : 3.13E+05 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.69, 2.5 and 9.0 mg/L, however no cells were observed to be present in the test cultures at 30 and 87 mg/L.

Water Quality Criteria

The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ±1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

A concentration dependent decline in pH was observed in the test preparations at 0 hours in the range of pH 7.6 at 0.69 mg/L to pH 4.3 at 87 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustments were made to the pH of the test preparations prior to the addition of algal cells at the start of the test.

Observations on Test Item Stability

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.69, 2.5 and 9.0 mg/L test cultures were observed to be light green dispersions whilst the 30 and 87 mg/L test cultures were observed to be clear colorless solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:
Based on Growth Rate: EC50 19 mg/L, LOEC 30mg/L, NOEC 9.0 mg/L
Based on Yield: EC50 14 mg/L, LOEC 30 mg/L, NOEC 9.0 mg/L

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0, 24 and 48 hours showed measured test concentrations to range from 80% to 101% of nominal. A marked decline in measured test concentrations was observed at 72 hours in the range of 9% to 71% of nominal indicating that the test item was unstable under the conditions of the test.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be 0.69, 2.5, 9.0, 30 and 87 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable	EC50 (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	19	9.0	30
Yield	14	9.0	30