4-hydroxy-3-methoxycinnamic acid

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effect of stock-culture period on the sensitivity of the Lemna bioassay to four different phenolic substances was evaluated in this study.

GLP compliance:

no

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Remarks on result:

other: corresponding to 0.05mM

After a seven-day exposure period, the number of fronds from L. minor was significantly reduced by all four phenolic substances at 0.05 mM when the stock-culture period was 14 days.

Validity criteria fulfilled:

not applicable

Conclusions:

After a seven-day test ferulic acid (FA) with a concentration of 0.05 mM (9.7 mg/L) significantly reduced the number of fronds and the dry weight compared with the untreated control when the stock-culture period had been exactly 14 days.

Executive summary:

The effect of stock-culture period on the sensitivity of the Lemna bioassay to four different phenolic substances was evaluated in this study. The sensitivity of the bioassay interacted with the stock-culture period of either 11, 14, or 18 days. After a seven-day test p-hydroxybenzoic acid (HBA), vanillic acid (VA), trans-cinnamic acid (CA), and ferulic acid (FA) with a concentration of 0.05 mM (9.7 mg/L) significantly reduced the number of fronds and the dry weight compared with the untreated control when the stock-culture period had been exactly 14 days. The sensitivity after the shorter ( 1 l days) or longer (18 days) stock-culture period was reduced, and the differences in the dry weight to the untreated control were not significant after a stock culture period of 18 days. The two higher concentrations (0. l and 0.25 mM) showed stronger inhibition. A comparison of the inhibition at 0.05 mM revealed that the stock culture period affected the relative toxicity of the four phenolic substances. Since the pH increased in the stock-culture flasks during the 18-day period from 6.25 to 7.9, we hypothesize that differences in the Lemna assay can be at least partly attributed to a pH effect, possibly in combination with a relative nutrient deficiency.

Reference 2

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Lemna gibba L. G3, (duckweed) was used as a bioassay organism to test the allelochemical effects of salicylic acid (SA), ferulic acid (FA), and umbelliferone (UM). Growth rate (K), dry weight (DW) and total chlorophyll (CHL) production were measured after seven days of growth. The bioassay procedure used 50 ml of E medium with and without sucrose in 125-ml Erlenmeyer flasks plus the selected concentration of allelochemical.
The objective of this research was to study L. gibba G3 as a bioassay for allelochemicals and to consider any impact on chlorophyll production as a potential indicator of interference.

GLP compliance:

no

Specific details on test material used for the study:

salicylic acid (SA; 2-hydroxybenzoic acid), ferulic acid (FA; 4 hydroxy-3-methoxycinnamic acid), and umbelliferone (UM; 7-hydroxycoumarin),

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Lemna gibba

Details on test organisms:

Lemna gibba L. strain G3
The aquatic duckweed, Lemna gibba L. strain G3 maintained aseptically, was used exclusively in this research. All cultures were grown in cotton-stoppered 125-ml Erlenmeyer flasks with 50 ml of E medium (Cleland, 1979), prepared at a pH of 4.6, and sterilized by autoclaving (15-20 min) Clones containing a total of 12 fronds were transferred to each flask using a culture loop. The cultures were allowed to grow for seven days in an environmental chamber at 28°C under constant (236 µE/sec/m 2) fluorescent and incandescent light.

Test type:

static

Water media type:

freshwater

Total exposure duration:

7 d

Nominal and measured concentrations:

100 µM to 1000µM

Details on test conditions:

The allelochemicals, salicylic acid (SA; 2-hydroxybenzoic acid), ferulic acid (FA; 4 hydroxy-3-methoxycinnamic acid), and umbelliferone (UM; 7-
hydroxycoumarin), were added as the dry chemical to the medium in the first experiments prior to autoclaving and in later experiments after autoclaving. Four replications in a completely randomized design were prepared for each treatment. To determine the effects of organic amendments on the response of L. gibba G3 to allelochemicals, E medium with and without sucrose and tartaric acid was prepared with known concentrations of the allelochemicals and autoclaved.
Cultures were prepared using plants grown in complete E medium. In another experiment, E medium with and without sucrose was prepared and autoclaved, then the allelochemicals were added to the medium after autoclaving. In order to test the effects of prior conditioning of the plants, half the cultures were prepared using L. gibba G3 that had been grown for a minimum of two weeks in complete E medium; the other half of the cultures utilized plants previously grown without sucrose and tartaric acid. Controls were cultured according to each experimental protocol but without the allelochemical.

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: Corresponding to 500 µM

Details on results:

Thie Cultures were inspected daily and, on day 7, fronds were removed from the flasks, counted, each placed in a test tube, and the total chlorophyll (CHL) extracted.
The fronds were allowed to air-dry in a hood at room temperature for one week, then weighed (DW).
The growth rate (K) was calculated according to Hillman (1961)

The threshold for Ferulic acid-induced chlorophyll reduction was lower in cultures with organic amendments (sucrose and tartaric acid); 100µM vs 750 µM without amendments.
At 500 µM Ferulic acid, bleaching was apparent, but was not severe. The fronds were smaller than in controls, not well developed, and clumped (daughter fronds did not separate from mother fronds). Fronds were less gibbous and had shorter (0.5 cm or less) or no roots. At 1000 µM FA the pattern of injury was similar but much more severe than at 500 µM.

in medium without amendment (sucrose and tartaric acid), the NOEL for the growth rate (K), dry weight and total chlorophyll is 500 µM.

Validity criteria fulfilled:

not applicable

Conclusions:

In medium without amendment (sucrose and tartaric acid),  the 7d-NOEL for the growth rate, dry weight and total chlorophyll is 97 mg/L (i.e.500 µM).

Executive summary:

Lemna gibba L. G3, (duckweed) was used as a bioassay organism to test the allelochemical effects of salicylic acid (SA), ferulic acid (FA), and umbelliferone (UM). Growth rate (K), dry weight (DW) and total chlorophyll (CHL) production were measured after seven days of growth.  The bioassay procedure used 50 ml of E medium with and without sucrose in 125-ml Erlenmeyer flasks plus the selected concentration of allelochemical.

The objective of this research was to study L. gibba G3 as a bioassay for allelochemicals and to consider any impact on chlorophyll production as a potential indicator of interference. Thus, even though the main objective of this research was not the hazard assessment of the ferulic acid, it gives useful data for hazard assessment of the ferulic acid.

Therefore as a results, in medium without amendment (sucrose and tartaric acid),  the 7d-NOEL for the growth rate, dry weight and total chlorophyll was 97 mg/L (i.e. 500µM).

Description of key information

Two studies on Lemna species were found.

In the first study (Ramirez et al 1987), Lemna gibba L. G3, (duckweed) was used as a bioassay organism to test the allelochemical effects of salicylic acid, ferulic acid, and umbelliferone. Growth rate, dry weight and total chlorophyll production were measured after seven days of growth.  The bioassay procedure used E medium with and without sucrose in Erlenmeyer flasks plus the selected concentration of allelochemical (100 up to 1000 µM). Even though the main objective of this research was to study L. gibba G3 as a bioassay for allelochemicals and to consider any impact on chlorophyll production as a potential indicator of interference, this study gives useful data for hazard assessment of Ferulic acid.

Therefore as a results, in medium without amendment (sucrose and tartaric acid),  the NOEL for the growth rate, dry weight and total chlorophyll was 97 mg/L (i.e.500 µM)

 

In the second study (Christen et al 1996), the effect of stock-culture period on the sensitivity of the Lemna bioassay to four different phenolic substances was evaluated. The sensitivity of the bioassay interacted with the stock-culture period of 11, 14, or 18 days. After a seven-day test ferulic acid with a concentration of 9.7 mg/L (0.05 mM) significantly reduced the number of fronds and the dry weight compared with the untreated control when the stock-culture period had been exactly 14 days. The two higher concentrations (0. l and 0.25 mM) showed stronger inhibition.

There is a discrepancy between these both studies indeed a LOEC of 0.05 mM was found in the study of christen but In contrast Ramirez Toro et al. (1988), using L, gibba, established a LOEC for ferulic acid of 0.75 mM (and Einhellig et at. (1985) reported a threshold of 0.25 mM for ferulic acid in L. minor). The authors explained that the results of their experiment demonstrate that the reason for such a difference in the sensitivity of the Lemna bioassay might be differences in the stock-culture period.

Therefore as the standard conditions of Ramirez study are closer to the standard conditions (OECD guideline) for REACH regulation, it is used a Key study.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

97 mg/L