Toxicity Of Eugenol Research Articles

Field application of star polymer-delivered chitosan to amplify plant defense against potato late blight

• Complexation of star polymer (SPc) with chitosan leads to particle size reduction. • SPc can assemble with chitosan/eugenol through different binding forces. • Control effects of chitosan/eugenol delivered by SPc are improved. • SPc can be applied as a common adjuvant to improve control efficacy in the field. The application of environmentally-friendly fungicides, such as elicitors and botanical fungicides, to minimize the risk of exposure to the ecosystem has become a global trend. However, the bioactivity and absorbency of elicitors are critical factors that limit the large-scale field application. With the objective of exploring a sustainable method for controlling plant diseases, a star polymer (SPc) was constructed to deliver plant elicitors/botanical fungicides with a low production cost. The SPc could be complexed with chitosan through hydrogen bond and Van der Waals forces. The complexation of SPc with chitosan decreased chitosan particle size from 144.61 nm to 17.40 nm in an aqueous solution. The SPc could activate the endocytosis pathway of plants by a nano-delivery system to amplify the defense responses induced by the chitosan elicitor. The nano-sized chitosan/SPc complexes exhibited enhanced control effects against potato late blight, which is considered one of the most destructive crop pathogens threatening global food security. Similar to chitosan, the eugenol could assemble with SPc through electrostatic interactions, and the toxicity of eugenol delivered by SPc was also improved against potato late blight. As an adjuvant, SPc could be applied to increase chitosan and eugenol’s control efficacies in the field. Here we report the first application of a nano-delivery system to improve plant defenses to the best of our knowledge. The SPc adjuvant’s application appears to be suitable for delivering plant elicitors/botanical fungicides, which is beneficial for improving bioactivity and decreasing the application quantity and frequency of conventional fungicides.

Comparative cytotoxic study of silica materials functionalised with essential oil components in HepG2 cells

This work evaluated the cytotoxic effect of different EOCs-functionalised silica particle types. The in vitro toxicity of eugenol and vanillin-immobilised SAS, MCM-41 microparticles and MCM-41 nanoparticles was evaluated on HepG2 cells, and compared to free EOCs and pristine materials. The results revealed that free essential oil components and bare silica had a mild cytotoxic effect on HepG2 cells. However, the comparative study showed that free eugenol and vanillin had a milder cytotoxic effect than the equivalent concentrations of immobilised components on the different silica particles, while differences in cell viability between the bare and functionalised particles relied on the type of analysed material. The most cytotoxic materials were eugenol and vanillin-functionalised MCM-41 micro with IC50 values of 0.19 and 0.17 mg/mL, respectively, at 48 h exposure. Differences in cytotoxicity between functionalised particles may be attributed to the density of the functional components on their surface as a result of the functionalisation reaction performance for different materials. The study of the physico-chemical properties of particles demonstrated that cationic nature and increased hydrophobicity could be responsible for promoting cell-particle interactions for the eugenol and vanillin functionalised silica particles, enhancing their cytotoxic behaviour.

Effectiveness of eugenol as an anesthetic for adult spotted sea bass (Lateolabrax maculatus)

Spotted sea bass, Laterlabrax maculatus, is one of the most commercially important marine fish in China. It is very easy to suffer from stress and physical damage due to its aggressive nature and dorsal spines, which results in severe mortalities during handling and transportation. Therefore, most spotted sea bass are sold as fresh cooled products rather than live fish in domestic market, thus limiting its market value and market scale as well as the industry development. Anesthetics could be useful to reduce the physical stress during handling and transportation. However, few studies have been done to investigate the application of anesthetics in spotted sea bass. In the present study, the anesthetic efficacy of eugenol (2-methoxy-4-prop-2-enyl-phenol) in adult spotted sea bass was extensively investigated. Firstly, the acute toxicity of eugenol was measured. The estimated 10-min LC50 and 24-h LC50 were 98.13 mg•L−1 and 19.73 mg•L-1. Secondly, the needed time periods for induction and recovery from anesthesia were measured and compared to clove oil and MS-222. Based on the time criteria of ideal induction (less than 3 min) and recovery (less than 10 min), the lowest effective concentrations for spotted sea bass were 60 mg•L−1 for eugenol, 120 mg•L−1 for clove oil and 140 mg•L−1 for MS-222 at around 20 °C. In addition, the anesthetic efficacies of different concentrations of eugenol at water temperatures of 20 and 30 °C were compared. Results showed that longer induction time to deep anesthesia and shorter recovery time were observed when fish were exposed to 20 and 30 mg•L−1 eugenol at 30 °C compared to that at 20 °C. On the contrary, shorter time to induce deep anesthesia and longer time to recovery were found when fish were exposed to 40 and 50 mg•L−1 eugenol at 30 °C compared to that at 20 °C. Lastly, water quality and survival of spotted sea bass subjected to different concentrations of eugenol during simulated transportation were evaluated. Current study indicated that the addition of eugenol did not show significant improvements compared to the control group. In conclusion, eugenol was an effective anesthetic for inducing anesthesia on spotted sea bass, but the addition of it did not improve the transportation of spotted sea bass.

Fumigant toxicity of eugenol and its negative effects on biological development of Callosobruchus maculatus L

The protection of stored products from insect damages, when accomplished, is mostly relying on the application of synthetic insecticides with serious health and environmental issues in addition to risks of selection of resistant insect populations associated with such practice. The use of plants derived compounds have been presented as sound strategy for sustainable insect pest management. Eugenol is an aromatic component of natural occurrence in essential oils of numerous plants. known for its repellent and insecticidal bioactivities against different insect species. Here, we investigated the fumigant application of eugenol to control the cowpea weevil Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae) by assessing toxicity, effects on biological development (including emergence inhibition) and repellency to these weevil. Besides a good fumigant insecticidal activity, eugenol exposure resulted in a dose-dependent decrease of the growth rate of C. maculatus associated with a steady reduction in bean mass losses. Additionally, inhibition of offspring was upper 50% when adults were exposed to lethal and sublethal concentrations. significant inhibition of the offspring emergence was achieved after the exposure of parental adults to lethal and sublethal doses of eugenol. Furthermore, adult weevils were repelled away from beans exposed to high dose (LD99) of eugenol in contrast with their attraction to the beans treated with lower dose (LD50). Our findings suggest that eugenol has potential as control tool to be used in sustainable management startegies of C. maculatus.

Evaluation of eugenol toxicity in bioassays with test-organisms

ABSTRACT: Fish in both natural and farming conditions are exposed to stress of capture, handling, transport or treatment that provoke low zootechnical performance. Anesthetics like eugenol obtained from clove oil have been used strategically not only in freshwater but also in marine and estuarine fish in order to reduce the stress. Apart from the eugenol indication as anesthetic and its low toxicity for animals, its environment action is not clear. Bioassays or ecotoxicity tests with indicator organisms are used to evaluate the mode of action of the pollutants in the environment. The aim of this study was to test the acute toxicity of eugenol using the microcrustacean Daphnia magna and the bacterium Aliivibrio fischeri, and also its chronic toxicity for the algae Desmodesmus subspicatus. Eugenol in the concentrations of 50, 75 and 100mg L-1 were toxic to tested indicator organisms.

Chemosensitivity of MCF-7 cells to eugenol: release of cytochrome-c and lactate dehydrogenase

Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F0F1ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC50: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit PLUS) into culture media at eugenol concentration >EC50. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential.

Eugenol Toxicity in Human Dental Pulp Fibroblasts of Primary Teeth.

The aim of the study was to determine the eugenol concentrations at which toxicity occurs in human dental pulp fibroblasts of primary teeth. Samples of primary dental pulp tissue were taken. Tissue samples were seeded by means of explant technique and used in the 4(th)-5th pass. Single Cell Gel Electrophoresis (Comet), phenazine MeThoSulfate (MTS), LIVE/DEAD Cell Viability/Toxicity and trypan blue assays for evaluation of the cytotoxicity of increasing concentrations of eugenol (0.06 to 810 μM) were performed. The results of toxicity tests showed toxic effects on dental pulp fibroblasts, even at very low concentrations of eugenol (0.06 μM). Very low concentrations of eugenol produce high toxicity in human dental pulp fibroblasts. All of the concentrations of eugenol that we evaluated produced high toxicity in human dental pulp fibroblasts of primary teeth.

Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata

Environmental Factors and the Toxicity of Eugenol and Quercetin against Snail Lymnaea acuminata

Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.

Cch1p Mediates Ca2+ Influx to Protect Saccharomyces cerevisiae against Eugenol Toxicity

Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p, whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.

The assessment of tolerability of prolonged oral eugenol administration in rats

The potential toxicity and general tolerability of eugenol following two-week or four-week continuous p.o. administration to rats has been investigated. An experiment was performed on 72 male rats of the Wistar strain. Four groups of rats were treated with different doses of eugenol (10 mg/kg bm/day, 50 mg/kg, 200 mg/kg and 400 mg/kg bm/day), the fifth group was administered vehicle (0.5 % methylcellulose, propylene glycol and water), and the sixth group comprised absolutely untreated controls. The corresponding doses of eugenol and vehicle were applied using a gastric probe in a volume of 1 ml/100 g body mass. The general tolerability of eugenol was evaluated on the basis of the daily intake of water and food, body mass, general health condition, behaviour, and lethality in the course of the experiment. In the investigated doses, eugenol applied p.o. in the course of two or four weeks does not influence significantly the intake of food, water, or body mass of rats. The dose of 400 mg/kg/day produced undesired reactions (agitation and hyperesthesia) that were first observed on day 21 and lasted until the end of the experiment. Low subacute toxicity of eugenol was established following p.o. administration to rats. Eugenol in doses of 200 and 400 mg/kg tm/day has a low toxic potential and is safe for administration to this animal species.

Evaluation of the Toxicity of Eugenol at Anesthetic Doses in African Clawed Frogs (Xenopus laevis)

Eugenol has been shown to induce anesthesia in African clawed frogs (Xenopus laevis). The toxicity of eugenol, administered at anesthetic doses, was evaluated in Xenopus frogs with an average body weight of 28.2 ± 13.7 g. Frogs were immersed in 250 mL of an aqueous solution containing 350 µl/L of eugenol for ten minutes and received a single administration (group 1, twelve animals) or three consecutive daily administrations (group 2, twelve animals). In each group, six frogs were scheduled to be euthanized the following day (subgroup A) and the other six were scheduled to be euthanized after a one-week recovery period (subgroup B). Morphologic changes consistent with renal tubular apoptosis affecting distal tubules in the medulla were observed in all subgroup A animals, ranging from mild to moderate in group 1, and from mild to severe in group 2. In subgroup B, renal tubular regeneration was present in all but one animal examined. These findings suggest that eugenol toxicity in amphibians is first manifested by renal tubular apoptosis. Other eugenol-related lesions were massive hepatic necrosis in group 2 (n = 6), hyaline membranes in the lung (n = 5), and adipose tissue hemorrhages in group/subgroup 2B (n = 4).

Antimycotic Activity of Eugenol against Selected Water Molds

This study was conducted to investigate the antimycotic activities of eugenol, a major essential oil of clove, against some fish pathogenic species of Saprolegniaceae as well as to determine the toxicity of eugenol to selected cultured fish. Two eugenol solutions were used in this study, a 10% v/v (volume:volume) solution in dimethyl sulfoxide (DMSO) and FA 100, which contains 10% v/v eugenol. The fungal species investigated were Saprolegnia parasitica, S. diclina, S. ferax, S. salmonis, Achlya klebsiana, and Aphanomyces piscicida. The minimum inhibitory concentrations (MICs) of eugenol in DMSO against Saprolegnia spp., A. klebsiana, and A. piscicida were 500, 250, and 125 μg/mL, respectively, and the fungicidal concentrations (for complete killing) were 1,000, 500, and 250 μg/mL, respectively. In contrast, the MICs of FA 100 against S. parasitica, other Saprolegnia spp., A. klebsiana, and A. piscicida were 250, 125, 250, and 63 μg/mL, respectively, and the fungicidal concentrations were 1,000, 1,000, 1,000, and 63 μg/mL, respectively. Zoospores of the Saprolegnia spp. tested and of A. klebsiana could not germinate in the presence of a concentration of 250 μg/mL of either eugenol solution, while those of A. piscicida were killed by a concentration of 125 μg/mL of either eugenol solution. Eugenol was highly toxic to salmonids but less toxic to cyprinids.

Comparative Toxicity of Eugenol and Its Quinone Methide Metabolite in Cultured Liver Cells Using Kinetic Fluorescence Bioassays

Comparative kinetic analyses of the mechanisms of toxicity of the alkylphenol eugenol and its putative toxic metabolite (quinone methide, EQM) were carried out in cultured rat liver cells (Clone 9, ATCC) using a variety of vital fluorescence bioassays with a Meridian Ultima laser cytometer. Parameters monitored included intracellular GSH and calcium levels ([Ca2+]i), mitochondrial and plasma membrane potentials (MMP and PMP), intracellular pH, reactive oxygen species (ROS) generation, and gap junction-mediated intercellular communication (GJIC). Cells were exposed to various concentrations of test compounds (1 to 1000 μM) and all parameters monitored directly after addition at 15 s intervals for at least 10 min. Eugenol depleted intracellular GSH, inhibited GJIC and generation of ROS, and had a modest effect on MMP at concentrations of 10 to 100 μM. At high concentrations (1000 μM), eugenol also affected [Ca2+]i, PMP, and pH. Effects of EQM were seen at lower concentrations (1 to 10 μM). The earliest and most potent effects of either eugenol or EQM were seen on GSH levels and GJIC. Coadministration of glutathione ethyl ester enhanced intracellular GSH levels by almost 100% and completely protected cells from cell death caused by eugenol and EQM. These results suggest that eugenol mediates its hepatotoxic effects primarily through depletion of cytoprotective thiols and interference in thiol-dependent processes such as GJIC. Furthermore, our results support the hypothesis that the toxic effects of eugenol are mediated through its quinone methide metabolite.

Horseradish peroxidase-catalyzed conjugation of eugenol with basic amino acids.

L-Lysine is shown to yield an adduct with the quinone methide intermediate formed during the horseradish peroxidase (HRP)-catalyzed aerobic oxidation of eugenol (4-allyl-2-methoxyphenol). Adduct formation is evidenced by (i) lysine quenching of the characteristic quinone methide absorption band measured at 350 nm; arginine and gamma-aminobutyric acid, but not alanine or propionic acid showed similar behaviour (ii) lysine-promoted a 400 mV decrease of the eugenol oxidation voltammetric wave (1.00 V), concomitantly with an increase in current intensity and (iii) reverse phase HPLC isolation of the lysine eugenol adduct, followed by GC-MS analysis. The MS spectrum is consistent with a 2:1 lysine:eugenol adduct (MW = 455). If operative in vivo, binding of lysine to eugenol might lead to protein inactivation and possibly be involved in eugenol toxicity.

Eugenol cytotoxicity evaluated with continuous cell lines

The cytotoxicity of eugenol to replicating cells, as mediated by the intracellular level of glutathione and by metabolic activation, was evaluated with the neutral red (NR) assay. The cytotoxicity of eugenol to human HFF fibroblasts and human HepG2 hepatoma cells was increased somewhat in the presence of a hepatic S-9 microsomal fraction from Aroclor-induced rats or hamsters. Exposure of human HepG2 hepatoma cells to eugenol depleted the level of intracellular glutathione. Cells treated with 1-chloro-2,4-dinitrobenzene (CDNB) or buthionine sulphoximine (BSO), agents that deplete intracellular glutathione, were hypersensitive to eugenol. A 1-hr pretreatment with CDNB enhanced the cytotoxicity of eugenol, as did a 24-hr pretreatment with BSO. Intracellular glutathione levels were, apparently, significant in mediating the toxicity of eugenol.

A Simple In Vito Cytotoxicity Test Using the MTT (3-(4,5)-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide) Colorimetric Assay: Analysis of Eugenol Toxicity on Dental Pulp Cells (RPC-C2A)

A simple colorimetric assay using MTT has been developed to monitor mammalian cell survival and proliferation in vitro. In this study we used a clonal fibroblastic cell line (RPC-C2A) from rat incisal dental pulp to examine the effectiveness of the colorimetric assay to test for the toxicity of eugenol, which is frequently used to treat inflammed dental pulp. A technical problem encountered was the insolubility of MTT formazan, produced by the activity of mitochondria dehydrogenases. Dimethyl sulfoxide (DMSO) seemed to be the best solvent. Doses of eugenol causing a 50% inhibition in the colorimetric assay were calculated as 0.6 mM and 1 mM for cells in the growing phase and for cells at confluence, respectively. These values exist in the concentration range reported in the previous studies. Although the correlation between spectrophotometric absorbance and cell number was not completely linear, this method could be used effectively as a simple preliminary assay to test for the toxicity of dental drugs and materials.

A simple in vito cytotoxicity test using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay: Analysis of eugenol toxicity on dental pulp cells (RPC-C2A).

A simple colorimetric assay using MTT has been developed to monitor mammalian cell survival and proliferation in vitro. In this study we used a clonal fibroblastic cell line (RPC-C2A) from, rat incisal dental pulp to examine the effectiveness of the colorimetric assay to test for the toxicity of eugenol, which is frequently used to treat inflammed dental pulp. A technical problem encountered was the insolubility of MTT formazan, produced by the activity of mitochondria dehydrogenases. Dimethyl sulfoxide (DMSO) seemed to be the best solvent. Doses of eugenol causing a 50% inhibition in the colorimetric assay were calculated as 0.6 mM and 1 mM for cells in the growing phase and for cells at confluence, respectively. These values exist in the concentration range reported in the previous studies. Although the correlation between spectrophotometric absorbance and cell number was not completely linear, this method could be used effectively as a simple preliminary assay to test for the toxicity of dental drugs and materials.

Critique of available studies on the toxicology of kretek smoke and its constituents by routes of entry involving the respiratory tract.

Kreteks are tobacco cigarettes containing clove, imported into North America from Indonesia. Health effects, including severe pulmonary toxicity, are suspected to be associated with kretek use among adolescents and young adults. The most likely candidate for a specific toxic effect among the chemical constituents of kreteks is eugenol. Eugenol is a natural compound found in high concentrations in clove buds and is the active ingredient that distinguishes kreteks from conventional cigarettes. It is a potent and valuable topical anesthetic, as well as a pungent spice constituent. Until kretek use became a fad in North America in the early 1980s, the toxicity of eugenol by routes of exposure involving the respiratory tract had not been considered because human exposure had been predominantly oral and topical. Concern over the possible pulmonary toxicity of kretek smoke has stimulated studies on the smoke, eugenol itself, and other chemical constituents of kreteks. Two sets of studies are now available for review, one series conducted by La Voie et al. of the American Health Foundation and another conducted by Clark et al. of the Huntingdon Research Centre in the United Kingdom. Their methods and findings are very different. Common problems include low statistical power, methods of exposure that do not duplicate human exposure, and failure of the models to replicate the circumstances of the extant clinical reports, which suggest that aspiration, idiosyncratic or allergic reactions, or impaired host defense mechanisms may be involved. The available toxicologic studies must be considered preliminary.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute inhalation toxicity of eugenol in rats.

The acute toxicity of inhaled eugenol was assessed by exposure of three groups of five male and five female rats to a submicron aerosol of eugenol for 4 h followed by a 14-day observation period. A fourth group, also of five male and five female rats and exposed to air only under similar conditions, served as a control group for comparison. The three concentrations of eugenol to which the different groups were exposed were 2.58, 1.37 and 0.77 mg/l. The mass median aerodynamic diameters and geometric standard deviations of the aerosols were, respectively, 0.82 micron (sigma g 2.26), 0.88 micron (sigma g 2.05) and 0.9 micron (sigma g 1.87). Clinical signs observed during exposure consisted principally of moderately increased salivation and restlessness (indicative of irritation) and abnormal breathing patterns. The signs were graded, being less marked in animals exposed to the lower concentrations of eugenol. All three groups, exposed to high, medium and low levels of eugenol, lost weight overnight following exposure. Associated with the weight loss were marked reductions in food and water intake. The responses appeared to be largely independent of the concentration of eugenol inhaled, although there was some evidence of a graded effect on water intake. There was rapid recovery, with food and water consumption data comparable with control values throughout most of the remainder of the 14-day observation period. Also, by the end of the observation period, group mean body weights were comparable. Upon sacrifice and macroscopic examination of the animals, abnormalities were detected in the lungs only of a few animals: 3/10 control, 2/10 eugenol 2.58 mg/l, and 2/10 eugenol 0.77 mg/l.(ABSTRACT TRUNCATED AT 250 WORDS)