2-Ethylhexyl trans-4-methoxycinnamate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- CAS no.: 83834-59-7
- Molecular formula: C18 H26 O3
- Molecular weight: 290.40 g/mole

Analytical monitoring:

yes

Details on sampling:

- Analytical chemistry sample schedule: study day (SD) 0, 3, and 5 fresh (post-renewal) solution and SD 3, 5, and 7 (pre-renewal solution composite [waste])

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: recirculation through a saturator column
- Controls: negative (blank) control
- Evidence of undissolved material: no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duck weed
- Clone: unknown
- Source: The L. minor culture originated from hydroponic cultures at Pond Megastore (Canton, OH USA) and has been cultured at the laboratory
- Age of inoculum: 3 weeks
- Method of cultivation: in SIS for 3 weeks
- Health status of test organisms: free from contamination of other organisms, healthy plant colonies (2-5 fronds) with high incidence of plants with >2 fronds, functioned as young rapidly growing plants without visible lesions or discoloration; no indications of algal contamination or infestation by other organisms

ACCLIMATION
- Acclimation period: 10 days
- Culturing media and conditions: same as test, sterile SIS medium
- Any deformed or abnormal plants observed: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

24º +/- 2 ºC (±2 ºC variation during in-life)

pH:

6.5 - 9 (variability < 1.5)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal: 0.0 (control), 0.0637 mg/L; measured:

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes, temperature was maintained at 24 ± 2°C, with intra-replicate and treatment and inter-replicate and treatment variation not exceeding 2°C at any point during the in-life phase
- Test vessel: beakers
- Material, size: glass, 250 mL
- Aeration: no active aeration
- Agitation: no
- Renewal rate of test solution: renewal on study day 3 and 5
- No. of fronds per colony: 2 - 4
- Density on day 0: 10 fronds per replicate
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes, SIS (modified per OECD 221)


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 16 h Light : 8 h dark
- Light intensity and quality: 6500 to 10000 lux (Measured at Water Surface)

EFFECT PARAMETERS MEASURED:
- Determination of frond number: manual counting
- Frond dry weight: On study day (SD) 0, the baseline frond dry weight was determined in six randomly selected samples containing 10 fronds each. On SD 7, all colonies were collected from each of the test vessels and rinsed with deionized water. The colonies were then blotted to remove excess water and then dried at 60°C overnight. The dry weight was expressed to an accuracy of 0.1 mg.
- Determination of frond area: Computer-aided digitization of digital photographs (Sigma Scan Pro, SPSS, Chicago, IL USA)
- Other: Changes in plant development, including frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, and in root length and appearance, were monitored during the in-life phase. Significant features of the test medium (e.g. presence of undissolved material, growth of algae in the test vessel) were also monitored.

RANGE-FINDING STUDY
- Test concentrations: ca. 0.050 mg/L, 0.013, 0.0032 and 0.0 (contrl) mg/L
- Results used to determine the conditions for the definitive study: yes, based on the range-finding, a limit test based on an estimated practical water solubility of 0.05 mg/L OMC was designed for definitive study.

Reference substance (positive control):

no

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

dry weight

yield

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

dry weight

yield

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

dry weight

growth rate

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

dry weight

growth rate

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

frond area

growth rate

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

frond area

yield

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

frond area

growth rate

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

frond area

yield

Remarks on result:

other: no effect detected up to and including the water solubility limit of the substance

Details on results:

I. Fronds
- Frond number: The mean frond number at study day (SD) 7 in the control (0.0 mg/L) and 0.05 mg/L OMC treatment were 130 ± 2 (SEM) and 130 ±2 (SEM), respectively. The mean total frond count at SD 7 in the 0.05 mg/L OMC treatment was not significantly different from the control (t-test, p = 0.846).
- Doubling Time : The mean and median doubling time in the control (0.0 mg/L) and 0.05 mg/L OMC treatment were both 1.90±0.01 (SEM) days, respectively. The doubling time through SD 7 in the 0.05 mg/L OMC treatment was not significantly different from the control (Mann-Whitney Rank Sum test, p=1.000).
- Total Frond Area: Mean total frond area were 871 ± 18 (SEM) and 916 ± 18 (SEM) mm2 in the control (0.0 mg/L) and 0.05 mg/L OMC treatment on SD 7, respectively. Frond area measured in the 0.05 mg/L OMC on SD 7 was not significantly different than the control (t-test, p = 0.105).
- Frond Weight: Mean frond dry weight were 0.0220± 0.0019 and 0.0203 ±0.0029 g in the 0.00 mg/L (control) and the 0.05 mg/L OMC treatment on SD 7, respectively. Mean frond dry weight measured in the 0.05 mg/L OMC treatment on SD 7 was not significantly different than the control (t-test, p = 0.646).

II. Clinical Signs of Toxicity: Clinical signs of toxicity were not observed during the conduct of the present study.

III. Average Specific Growth Rates
- Frond Area: The average specific growth rate based on frond area for the control (0.0 mg/L) and 0.05 mg/L OMC treatment were 0.3 ± 0.0 and 0.3±0.0, respectively. The CVs for the average specific growth rate based on frond area for the control (0.0 mg/L) and 0.05 mg/L OMC treatment were 0.0 % and 0.0 %, respectively. The mean average specific growth rate based on frond area for the 0.05 mg/L OMC treatment was not significant different from the control (Mann-Whitney Rank Sum test, p = 1.000). The percent inhibition in growth (Ir) was 0.0 %.
- Frond Dry Weight: The average specific growth rate based on frond dry weight for the control (0.0 mg/L) and 0.05 mg/L OMC treatment were 0.3320 ± 0.0129 and 0.3160 ± 0.0223, respectively. The CVs for the average specific growth rate based on frond dry weight for the control (0.0 mg/L) and 0.05 mg/L OMC treatment were 9.4900 % and 17.2989 %, respectively. The mean average specific growth rate based on frond dry weight for the 0.05 mg/L OMC treatment was not significant different from the control (t-test, p = 0.543). The percent inhibition in growth (Ir) was 4.9 %. However, since the study design was based on a limit test and no significant difference in average specific growth weight based on dry weight was found between the 0.05 mg/L OMC treatment and the control (0.0 mg/L), the minimal percent inhibition calculated was deemed insignificant and inconsequential to the interpretation of the study results.

IV. Yield
- Frond Area: The normalized biomass (bc) for the control and 0.05 mg/L OMC treatment were 785 and 814 mm^2, respectively. The mean yield based on frond area for the 0.05 mg/L OMC treatment was not significant different from the control (t-test, p = 0.279). The percent reduction in yield was -4% (calculated value, or theoretically 0.0).
- Frond Dry Weight: The normalized biomass (bc) for the control and 0.05 mg/L OMC treatment were 0.0198 and 0.0182 g, respectively. The mean yield based on frond dry weight for the 0.05 mg/L OMC treatment was not significant different from the control (t-test, p =0.646). The reduction in yield was 8.1%. However, since the study design was based on a limit test and no significant difference in average specific growth weight based on dry weight was found between the 0.05 mg/L OMC treatment and the control (0.0 mg/L), the minimal percent inhibition calculated was deemed insignificant and inconsequential to the interpretation of the study results.


- Any visual signs of phytotoxicity (abnormalities): no
- Decrease in frond size: no
- Necrosis / chlorosis: no
- Sinking of fronds: no
- Sterility: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

positive control not performed

Reported statistics and error estimates:

Formal statistical analysis was based on hypothesis tests comparing the average specific growth rate and yield (biomass) in the control to the OMC treatment. The data were evaluated for normality (Shapiro-Wilk’s test) and homogeneity (Levene’s test). For non-normally distributed data or data with a heterogeneous distribution of variance; the Mann-Whitney Rank Sum test was used. For normally distributed data sets with homogeneous variance, a Student’s t-test was used to evaluate the data. The statistical significance of all tests indicated was assessed at p = 0.05 and the replicate was considered the experimental unit.

Table 1: Summary of Results

Endpoints		0.0 OMC (mg/L)	0.06 OMC (mg/L)
Day 7	Fronds (no.)	---	NS
	Doubling Time (days)	---	NS
	Frond Area (mm)	---	NS
	Frond Weight (g)	---	NS
	ASGR (area)	---	NS
	ASGR (weight)	---	NS
	Yield (bt[1], area)	---	NS
	Yield (bt1, weight)	---	NS
	Growth Inhibition (%, area)	---	6.1
	Growth Inhibition (%, weight)	---	4.9
	Yield Inhibition (%, area)	---	0.0
	Yield Inhibition (%, weight)	---	8.1
	Clinical	NF	NF
NS – not signficant compared to the control. NF – No clinical findings.

 

 

[1] Treatment biomass.

 

Table 1: Summary of Performance Criteria

Criterion	Acceptable Limits	Criteria Passed
Control doubling time	The doubling time of frond number in the control must be less than 2.5 days (60 hours), corresponding to approximately a seven-fold increase in seven days.	Yes
Control average specific growth rate	An average specific growth rate of 0.275/d.	Yes
pH	Not increase by more than 1.5 s.u.	Yes
Light Intensity	6500-10000 not exceeding ±15% within test area.	Yes
Water temperature	24 ± 2°C with inter-replicate variability ≤ 2°C	Yes

 

Table 3: Summary of OMC Concentrations in the Lemna minor Growth Inhibition Test

Study Day	Treatment Target Concentration (mg/L)	Replicate	Sample ID[1]	Measured Concentration (mg/L)[2]	24-h Stability (%)
0	0.00	Fresh	009	<LOD	---
	0.050	Fresh	010	0.0574	---
3	0.00	Fresh	016	<LOD	---
		A (Waste)	012	<LOD	100.0
		B-G (Waste)	013	<LOD	100.0
	0.050	Fresh	017	0.0548	---
		A (Waste)	014	0.0547	99.8
		B-G (Waste)	015	0.0550	100.4
5	0.00	Fresh	023	<LOD	---
		A (Waste)	019	<LOD	100.0
		B-G (Waste)	020	<LOD	100.0
	0.050	Fresh	024	0.0630	---
		A (Waste)	021	0.0598	94.9
		B-G (Waste)	022	0.0600	95.2
7	0.00	Fresh	030	<LOD^3	---
		A (Waste)	026	<LOD	100.0
		B-G (Waste)	027	<LOD	100.0
	0.050	Fresh	031	0.0687[3]	---
		A (Waste)	028	0.0659	95.9
		B-G (Waste)	029	0.0648	94.3

 

[1] Pre-renewal (waste) samples were collected from replicate A and a composite of replicates B-G of the control and the OMC treatment.

[2] Limit of Detection (LOD) = 0.000972 mg/L.

[3] Tested for verification, but not used in in-life phase.

 

Table 5: Mean Measured and Time-Weight Mean OMC Concentrations in the Lemna minor Growth Inhibition Test

Treatment Target Concentration (mg/L)	Replicate	Mean Measured Concentration (mg/L)[1]	Time-Weighted Mean (mg/L)[2]	Intra-Replicate CV[3] (%)	24-h Stability[4] (%)
0.00	Fresh	<LOD[5]	<LOD	0.0	---
	A (Waste)	<LOD	<LOD	0.0	100.0
	B-G (Waste)	<LOD	<LOD	0.0	100.0
0.050	Fresh	0.0584	---	10.1	---
	A (Waste)	0.0601	0.0579	9.3	105.5
	B-G (Waste)	0.0599	0.0578	8.2	105.1

 

[1] The fresh solution mean measured concentration represent the arithmetic mean of each data point from SD 0, 3, and 5. The pre-renewal (waste) mean measured concentration represents the arithmetic mean of each data point from SD 3, 5, and 7 for replicate A and composite of replicates B-G of the control and OMC treatment.

[2] Determined in accordance with OECD 211, Annex 6.

[3] Coefficient of variation = (Standard deviation / mean)*100.

[4] Stability = (Mean Measured Waste / Mean Measured Fresh)*100.

[5] Limit of Detection (LOD) = 0.000972 mg/L.

 

Table 6: Effect of OMC Exposure on Lemna minor Frond Number – SD 7

Treatment Target Concentration [Time Weighted Mean] (mg/L)	ID	Count	Frond Number	N (Replicates)	Mean	Median	Standard Deviation	Standard Error
0.00 [<LOD]	B	1	130	6	130	129	4	2
	C	1	134
	D	1	128
	E	1	135
	F	1	126
	G	1	124
0.050 [0.0578][1]	B	1	132	6	130	130	4	2
	C	1	126
	D	1	128
	E	1	136
	F	1	125
	G	1	133

 

[1] Not significantly different from control (t-test, p=0.846).

 

Table 7: Effect of OMC Exposure on Lemna minor Average Specific Growth Rate (Weight) – SD 7

Treatment Target Concentration [Time Weighted Mean] (mg/L)	ID	Count	ASGR[1]	N (Replicates)	Mean	Median	Standard Deviation	Standard Error
0.00 [<LOD]	B	130	0.3527	6	0.3320	0.3420	0.0316	0.0129
	C	134	0.3045
	D	128	0.3438
	E	135	0.3685
	F	126	0.3407
	G	124	0.2846
0.050 [0.0578][2]	B	132	0.3527	6	0.3160	0.3360	0.0547	0.0223
	C	126	0.2604
	D	128	0.3556
	E	136	0.3711
	F	125	0.3184
	G	133	0.2391

 

[1] ASGR = Average Specific Growth Rate

[2] Not significantly different from control (t-test, p=0.543).

Validity criteria fulfilled:

yes

Conclusions:

Results from the present study indicated that L. minor growth was not significantly inhibited by exposure to the practical soluble level of OMC (0.06 mg/L OMC, measured time weighted average) in the present study. Therefore, the 7-day EC50 and 7-day NOEC based on growth and yield, respectively is considered to be 0.06 mg/L OMC (measured time weighted average).

Executive summary:

To assess the potential adverse effect of the test item on growth of Lemna minor in hydroponic culture, a Lemna sp. Growth Inhibition Test according to OECD TG 221 was carried out.  Actively growing L. minor cultures were inoculated as monocultures in one test item concentration of 0.0637 mg/L representing the limit of practical solubility in SIS media over a period of seven days (limit test). For this assay 6 replicates with 10 fronds per replicate were prepared using a saturator column. The nominal test concentration was measured using LC-MS/MS to be 0.0578 mg/L OMC (time weighted mean). The stability over the course of the in-life phase ranged from 105.5 % to 105.1 % and the CVs of the intra-replicate was 12.1 %for the fresh solution and ranged between 9.3 and 8.2 % for the old media, which is in line with the criteria given in the guideline. Frond number was the primary measurement endpoint, whereas total frond area and dry weight were also measured.  A semi-static study design with renewal on days 3 and 5 was used. Control doubling time was <2.5 days.  The range of pH measured in the control and treatments was between 6.5 and 9 and ≤1.5 throughout this study, the temperature in the study was maintained at 20±2ºC, and the inter-replicate range in temperature was maintained at ≤2 ºC.  In summary, the present study met all performance criteria established for the OECD Test Guideline 221. As a result, the mean frond number at SD 7 in the control (0.0 mg/L) and 0.05 mg/L OMC treatment were both 130±2 (SEM).  The total frond count at SD 7 in the 0.05 mg/L OMC treatment was not significantly different from the control (t-test, p=0.846). The mean average specific growth rate based on frond area for the 0.05 mg/L OMC treatment was not significantly different from the control (Mann-Whitney Rank Sum test, p=1.000).  The percent inhibition in growth (Ir) was 0.0%. Clinical signs of toxicity were not observed during the conduct of the present study. In conclusion, results from the present study indicated that L. minor growth was not significantly inhibited by exposure to the practical soluble level of OMC (0.06 mg/L OMC) in the present study. The 7-day EC50 and NOEC for growth rate and yield were both determined to be > 0.06 and >=  0.06 mg/L (measured, TWA), respectively, i.e. higher than the water solubility level of the substance.

Description of key information

Results from the study indicated that L. minor growth was not significantly inhibited by exposure to the practical soluble level of OMC (0.06 mg/L OMC, measured time weighted average). Therefore, the 7-day EC50 and 7-day NOEC based on growth and yield, respectively is considered to be > 0.06 mg/L and >= 0.06 mg/L (measured time weighted average), respectively.

Additional information

To assess the potential adverse effect of the test item on growth of Lemna minor in hydroponic culture, a Lemna sp. Growth Inhibition Test according to OECD TG 221 was carried out.  Actively growing L. minor cultures were inoculated as monocultures in one test item concentration of 0.0637 mg/L representing the limit of practical solubility in SIS media over a period of seven days (limit test). For this assay 6 replicates with 10 fronds per replicate were prepared using a saturator column. The nominal test concentration was measured using LC-MS/MS to be 0.0578 mg/L OMC (time weighted mean). The stability over the course of the in-life phase ranged from 105.5 % to 105.1 % and the CVs of the intra-replicate was 12.1 % for the fresh solution and ranged between 9.3 and 8.2 % for the old media, which is in line with the criteria given in the guideline. Frond number was the primary measurement endpoint, whereas total frond area and dry weight were also measured.  A semi-static study design with renewal on days 3 and 5 was used. Control doubling time was <2.5 days.  The range of pH measured in the control and treatments was between 6.5 and 9 and ≤1.5 throughout this study, the temperature in the study was maintained at 20 ± 2 ºC, and the inter-replicate range in temperature was maintained at ≤2 ºC.  In summary, the present study met all performance criteria established for the OECD Test Guideline 221. As a result, the mean frond number at SD 7 in the control (0.0 mg/L) and 0.05 mg/L OMC treatment were both 130±2 (SEM).  The total frond count at SD 7 in the 0.05 mg/L OMC treatment was not significantly different from the control (t-test, p=0.846). The mean average specific growth rate based on frond area for the 0.05 mg/L OMC treatment was not significantly different from the control (Mann-Whitney Rank Sum test, p=1.000).  The percent inhibition in growth (Ir) was 0.0%. Clinical signs of toxicity were not observed during the conduct of the present study. In conclusion, results from the present study indicated that L. minor growth was not significantly inhibited by exposure to the practical soluble level of OMC (0.06 mg/L OMC) in the present study. The 7-day EC50 and NOEC for growth rate and yield were both determined to be > 0.06 and >=  0.06 mg/L (measured, TWA), respectively, i.e. higher than the water solubility level of the substance.