2-Ethylhexyl trans-4-methoxycinnamate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-01 to 2021-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Common name: 2-ethylhexyl trans-4-methoxy cinnamate
- IUPAC Name: 2-Ethylhexyl trans-4-methoxycinnamate
- CAS no.: 83834-59-7

Analytical monitoring:

yes

Details on sampling:

- Sampling frequency: pre-renewal (waste) samples were collected on SD 1, 8, 15 and 21 and post-renewal (fresh) were collected on SD 0, 7, 14, and 20 from the control and OMC limit treatment and submitted for analysis. Since the fresh solution was prepared and distributed to each of the replicate vessels, the fresh solution collected on SD 0, 7, 14, and 20 was comprised of one sample of the OMC treatment and control. The waste samples collected on SD 1, 8, 15, and 21 were comprised of an equal volume composite from each of the 10 replicates of the control and OMC treatment.
- Storage of samples: samples were preserved by dilution with acetonitrile (20 mL sample + 20 mL acetonitrile), with the exception of blanks, which were not preserved

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: saturator columns
- Controls: negative control
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: < 24 hours old
- Source: housed brood stock
- Feeding during test: yes
- Food type: suspension of Pseudokirchneriella subcapitata (3 x 107 cells/mL)
- Amount: 0.2 mg C/organism/day which equated to 0.1 mL/day
- Frequency: fed daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

218 mg/L as CaCO3 (mean)

Test temperature:

20º ±2 ºC

pH:

6.5-9 (variability <1.5)

Dissolved oxygen:

>3.0 mg/L

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal: 0.06 mg/L; measured: 0.06 mg/L (fresh test solutions, time weighted mean), 0.0582 mg/L (old test solution; arithmetic mean). The 24-hour stability of the test item concentration ranged from 90.3% to 98.4% .

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size: glass, 100 mL
- Aeration: no
- Renewal rate of test solution: 24 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h Light:8 h dark
- Light intensity: 1000 to 1500 lux (Measured at Water Surface)

EFFECT PARAMETERS MEASURED: Mortality (daily), reproduction (daily), growth (length; day 21), time to first brood (daily), male neonates (gaily)

TEST CONCENTRATIONS
- Range finding study: performed
- Results used to determine the conditions for the definitive study: yes, based on results of the preliminary study a limit test was performed

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 60 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 60 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 60 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality: No mortality was observed during the study
- Reproduction: The mean and median number of neonates produced in the control (0.00 mg/L) and 0.06 mg/L OMC treatment were 91 and 91, and 90 and 92, respectively. The CV% for the control (0.00 mg/L) and 0.06 mg/L OMC treatment were 5 and 4%, respectively. The total of neonates produced at SD 21 in the 0.06 mg/L OMC treatment was not significantly different from the control (t-test, p=0.755). No signs of asynchronous reproduction were noted.
- Body Length: mean and median body length ranged from 3 mm in the 0.0 mg/L (control) to 4 mm in the 0.06 mg/L OMC treatment on SD 21. Body length measured in the OMC treatments on SD 21 was not significantly different from the control (Mann-Whitney Rank Sum test, p=0.168).
- Clinical signs of toxicity: not observed
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

CONSIDERATIONS ON VALIDITY OF TEST
The mean measured test concentration in the 0.06 mg/L OMC treatment was consistently within 20% of target and 24-hour stability was 94.8%. Control mortality was ≤20% in the control. Control reproductive performance was >60 neonates/female. The range of pH measured in the control and treatments was between 6.5 and 9 ( and the overall variability during the study ≤1.5), 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 211.

Results with reference substance (positive control):

not available

Reported statistics and error estimates:

A statistical analysis of the results was not necessary as no toxic effects were detected.

Table 1: Measured concentrations of OMC in test solutions

Study Day^1	Sample Description	Treatment Target Concentration (mg/L)	Replicate	Sample ID^2	Measured Concentration (mg/L)^3	24-h Stability (%)
0	Fresh	0.00	A-J	009	<LOD	---
		0.060	A-J	010	0.0611	---
1	Waste	0.00	A-J	011	<LOD	---
		0.060	A-J	012	0.0552	90.3
7	Fresh	0.00	A-J	014	<LOD	---
		0.060	A-J	015	0.0633	---
8	Waste	0.00	A-J	016	<LOD	---
		0.060	A-J	017	0.0595	94.0
14	Fresh	0.00	A-J	019	<LOD	---
		0.060	A-J	020	0.0597	---
15	Waste	0.00	A-J	021	<LOD	---
		0.060	A-J	022	0.0577	96.6
20	Fresh	0.00	A-J	024	<LOD	---
		0.060	A-J	025	0.0615	---
21	Waste	0.00	A-J	026	<LOD	---
		0.060	A-J	027	0.0605	98.4

 

¹ SD 0, 7, 14, and 20 are post-renewal (fresh) samples; SD 1, 8, 15, and 21 are pre-renewal (waste) samples.

² Samples are comprised of an equal volume composite from each of the 10 replicates of the control and OMC treatment.

³ Limit of Detection (LOD) = 0.000972 mg/L.

 

Table 2: Effect of OMC Exposure on Daphnia magna Mortality – SD 21

Treatment [Mean Measured Conc.] (mg/L)	ID	N	Mortality	Mean	Median	Standard Deviation	Standard Error	CV (%)
0.00 [<LOD]	A	1	0.00	0.00	0.00	0.00	0.00	0.00
	B	1	0.00
	C	1	0.00
	D	1	0.00
	E	1	0.00
	F	1	0.00
	G	1	0.00
	H	1	0.00
	I	1	0.00
	J	1	0.00
0.060 [0.0598]^1	A	1	0.00	0.00	0.00	0.00	0.00	0.00
	B	1	0.00
	C	1	0.00
	D	1	0.00
	E	1	0.00
	F	1	0.00
	G	1	0.00
	H	1	0.00
	I	1	0.00
	J	1	0.00

¹ Formal statistical analyses not performed.

 

Table 3: Table 9 Effect of OMC Exposure on Daphnia magna Reproduction (Neonates/Adult) – SD 21

Treatment [Mean Measured Conc.] (mg/L)	ID	N	Neonates Produced	Mean	Median	Standard Deviation	Standard Error	CV (%)
0.00 [<LOD]	A	1	96	91	90	5	1	5
	B	1	90
	C	1	89
	D	1	84
	E	1	99
	F	1	86
	G	1	93
	H	1	87
	I	1	92
	J	1	89
0.060 [0.0598]	A	1	95	91	92	4	1	4
	B	1	90
	C	1	91
	D	1	88
	E	1	94
	F	1	95
	G	1	87
	H	1	95
	I	1	84
	J	1	92

¹ Not significantly different from control (t-test, p=0.755).

Table 4: Table 10 Effect of OMC Exposure on Daphnia magna Body Length (mm) – SD 21

Treatment [Mean Measured Conc.] (mg/L)	ID	N	Length (mm)	Mean	Median	Standard Deviation	Standard Error	CV (%)
0.00 [<LOD]	A	1	4	3	3	1	0	20
	B	1	3
	C	1	4
	D	1	3
	E	1	3
	F	1	2
	G	1	4
	H	1	3
	I	1	4
	J	1	3
0.060 [0.0598]^1	A	1	3	4	4	0	0	13
	B	1	4
	C	1	4
	D	1	4
	E	1	4
	F	1	4
	G	1	4
	H	1	3
	I	1	4
	J	1	3

¹ Not significantly different from control (Mann-Whitney Rank Sum Test, p=0.168)

 

Table 5: General Test Performance Criteria

Criterion	Acceptable Limits	Criteria Passed
Control mortality	≤ 20 % of parental (female) daphnids at in-life conclusion	Yes
Control reproduction	Mean number of living neonates produced per parental organism survival at the conclusion of exposure is ≥ 60%	Yes
DO	> 3 mg/L	Yes
pH	6.5 – 9 within ≤ 1.5	Yes
Water temperature	20 ± 2°C with inter-replicate variability ≤ 2°C	Yes

Validity criteria fulfilled:

yes

Conclusions:

In a Daphnia magna Reproduction Test according to OECD TG 211 the 21-day NOELR of the test item was determined to be >= 60 µg/L (nominal).

Executive summary:

To assess the potential adverse effect of OMC on the reproductive output of Daphnia magna a study according to OECD TG 211 was carried out. The study design did entail exposure of < 24-hour old females to the test substance and a negative control for 21 days in a semi-static exposure format. At the conclusion, the total number of living offspring produced were assessed.  The semi-static study design with renewal of the test solutions in 24-hour intervals allowed counting of the number of living offspring produced by each individual parental organism.  The practical solubility limit under test conditions was determined as a part of the study by recirculation of test medium through saturation columns until a constant concentration was reached. The test solutions used for exposure of the test organisms were prepared in the same way.  Based on the range-finding study results, no adverse effects were observed at the practical limit of water solubility determined prior to the range-finding test.  Therefore, a limit test design comparing a control with the limit of practical water solubility determined in the present study was used for the definitive in-life phase. The target OMC treatment concentration was 0.06 mg/L.  The test item concentration was monitored using a LC-MS/MS method. Dilution water control samples collected during the study contained < LOD (LOD=0.000972 mg/L).  The arithmetic mean measured concentration of the fresh test solution used on SD 0, 7, 14, and 20 of the control and treatment were <LOD and 0.0614 mg/L, respectively.  The arithmetic mean measured concentration of the waste test solution used on SD 1, 8, 15, and 21 of the control and treatment were <LOD and 0.0582 mg/L.  The time weighted mean concentrations were <LOD and 0.0598 mg/L OMC for the 0.0 (control) and 0.06 mg/L OMC treatment, respectively. No mortality was observed during the study.

The mean neonates produced per surviving female in the control and 0.06 mg/L OMC treatment were 91±2 (SEM) and 91±1 (SEM), respectively.  The total neonates produced at SD 21 in the 0.06 mg/L OMC treatment was not significantly different from the control (t-test, p=0.755).  No signs of asynchronous reproduction were noted. No signs of asynchronous development were noted. The mean body length of surviving daphnids in the control and 0.06 mg/L OMC treatment were 3±0 (SEM) and 4±0 (SEM), respectively.  Body length measured in the OMC treatments on SD 21 was not significantly different from the control (Mann-Whitney Rank Sum test, p=0.168).  No significant effects on behavior or signs of overt toxicity were noted.

In conclusion, results from the present study indicated that survival, reproduction and growth in Daphnia magna exposed to the practical soluble level of OMC (0.06 mg/L OMC) were not adversely affected. The NOEC under the test conditions was >= 0.06 mg/L. All validity criteria of the guideline were fulfilled.

Description of key information

In a Daphnia magna Reproduction Test according to OECD TG 211 the 21-day NOELR of the test item was determined to be >= 60 µg/L (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 60 µg/L

Additional information

To assess the potential adverse effect of OMC on the reproductive output of Daphnia magna a study according to OECD TG 211 was carried out (FEL, 2021). The study design did entail exposure of < 24-hour old females to the test substance and a negative control for 21 days in a semi-static exposure format. At the conclusion, the total number of living offspring produced were assessed.  The semi-static study design with renewal of the test solutions in 24-hour intervals allowed counting of the number of living offspring produced by each individual parental organism.  The practical solubility limit under test conditions was determined as a part of the study by recirculation of test medium through saturation columns until a constant concentration was reached. The test solutions used for exposure of the test organisms were prepared in the same way.  Based on the range-finding study results, no adverse effects were observed at the practical limit of water solubility determined prior to the range-finding test.  Therefore, a limit test design comparing a control with the limit of practical water solubility determined in the present study was used for the definitive in-life phase. The target OMC treatment concentration was 0.06 mg/L.  The test item concentration was monitored using a LC-MS/MS method. Dilution water control samples collected during the study contained < LOD (LOD=0.000972 mg/L).  The arithmetic mean measured concentration of the fresh test solution used on SD 0, 7, 14, and 20 of the control and treatment were <LOD and 0.0614 mg/L, respectively.  The arithmetic mean measured concentration of the waste test solution used on SD 1, 8, 15, and 21 of the control and treatment were <LOD and 0.0582 mg/L.  The time weighted mean concentrations were <LOD and 0.0598 mg/L OMC for the 0.0 (control) and 0.06 mg/L OMC treatment, respectively. No mortality was observed during the study.

The mean neonates produced per surviving female in the control and 0.06 mg/L OMC treatment were 91±2 (SEM) and 91±1 (SEM), respectively.  The total neonates produced at SD 21 in the 0.06 mg/L OMC treatment was not significantly different from the control (t-test, p=0.755).  No signs of asynchronous reproduction were noted. No signs of asynchronous development were noted. The mean body length of surviving daphnids in the control and 0.06 mg/L OMC treatment were 3±0 (SEM) and 4±0 (SEM), respectively.  Body length measured in the OMC treatments on SD 21 was not significantly different from the control (Mann-Whitney Rank Sum test, p=0.168).  No significant effects on behavior or signs of overt toxicity were noted. In conclusion, results from the present study indicated that survival, reproduction and growth in Daphnia magna exposed to the practical soluble level of OMC (0.06 mg/L OMC) were not adversely affected. The NOEC under the test conditions was >= 0.06 mg/L.