Environ Sci Pollut Res DOI 10.1007/s11356-015-4098-2
Acute toxicity and sublethal effects of gallic and pelargonic acids on the zebrafish Danio rerio Didier Techer & Sylvain Milla & Pascal Fontaine & Sandrine Viot & Marielle Thomas
Received: 31 January 2014 / Accepted: 11 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Gallic and pelargonic acids are naturally found in a variety of plants and food products. Despite their extensive use in man-made applications, little is known regarding their potential risks to aquatic vertebrates. The aim of this work was to assess the acute toxicity of these polyphenolic and fatty acid compounds to the zebrafish. In order to get insights into sublethal effects, the enzyme activity of usual biomarkers related to oxidative stress and biotransformation were also assessed in fish. These latter included total superoxide dismutase, catalase as well as total glutathione peroxidase for antioxidant defence mechanisms and glutathione Stransferase for biotransformation related enzyme. Gallic acid was practically non-toxic (96-h lethal concentration (LC50) > 100 mg/L) whereas pelargonic acid was slightly toxic (96-h LC50 of 81.2 mg/L). Moreover, biomarker analyses indicated enhanced superoxide dismutase activity in fish exposed to 20, 40 and 100 mg/L of gallic acid compared to control. A dose-dependent induction of glutathione peroxidase and glutathione S-transferase was reported following gallic acid exposure at the tested concentrations of 10, 20 and 40 mg/L, with the exception of 100 mg/L of substance where basal activity levels were reported. In the case of pelargonic acid, there was no change in antioxidant enzyme activity while an inhibition of glutathione S-transferase was observed from organisms exposed to 45, 58 and 76 mg/L of test solution. The results concerning sublethal effects
Responsible editor: Philippe Garrigues D. Techer (*) : S. Milla : P. Fontaine : S. Viot : M. Thomas Université de Lorraine, UR AFPA, USC INRA 340, Campus Victor Grignard, Boulevard des aiguillettes, 54506 Vandœuvre-lès-Nancy Cedex, France e-mail: [email protected]
on biological parameters of zebrafish highlighted thereby the need for further investigations following chronic exposure to both organic acids. Keywords Zebrafish . Acute toxicity . Gallic acid . Pelargonic acid . Oxidative stress . Glutathione S-transferase Abbreviations CAT catalase CNDB 1-chloro-2,4-dinitrobenzene EDTA ethylenediaminetetraacetic acid GPx glutathione peroxidase GST glutathione transferase OECD Organisation for Economic Co-operation and Development MMWD Marin Municipal Water District NADPH nicotinamide adenine dinucleotide phosphate (reduced) SOD superoxide dismutase TOXNET TOXicology Data NETwork US EPA United States Environmental Protection Agency
Introduction Gallic acid (CAS 149-91-7) and pelargonic acid (CAS 112-05-0) are two structurally different organic acids naturally found in a wide variety of plants (Ow and Stupans 2003; US 2000). They are also present at low levels in common foodstuffs and are therefore part of human’s diet (Galati and O'Brien 2004; Ow and Stupans 2003; US EPA US 2000). Gallic acid, referring to 3,4,5-trihydroxybenzoic acid (C6H2(OH)3COOH), has been employed as a raw material in
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a number of manufacturing areas and, more recently, innovative nanomaterials, in addition to prospects for its use in pharmaceutical and health perspectives (Dena et al. 2012; Galati and O'Brien 2004; Niho et al. 2001; Ow and Stupans 2003). It is a planar polyphenolic compound that exists in free or, more frequently, in conjugated forms in hydrolysable tannins (Galati and O'Brien 2004; Ow and Stupans 2003). Due to its specific conformation with three adjacent aromatic phenoxyl groups involved in intra- and intermolecular hydrogen bonding, this secondary plant metabolite exhibits strong chelating abilities with numerous inorganic ligands and proteins (Masoud et al. 2012; Rawel et al. 2006). Despite its extensive use in man-made applications, little is known about the associated toxicological risks subsequent to passive exposure of aquatic organisms (Labieniec et al. 2007; Xie and Cui 2003). Gallic acid has been shown to possess allelopathic properties and several authors investigated its growth inhibition potential towards diatoms (Liu et al. 2012) and microalgae including the well-known toxin-producing cyanobacteria Microcystis aeruginosa (Nakai et al. 2000; Shao et al. 2013; Wang et al. 2008). In those cases, gallic acid seemed to be efficient at concentrations ranging from ~1 to 15 mg/L. Regarding aquatic invertebrates, Labienec et al. (Labieniec et al. 2003, 2007) have successively reported that water-soluble plant polyphenols such as tannic, ellagic and gallic acids can cause morphological and physiological alterations in filter-feeding organisms like bivalves. To our knowledge, no data is currently available on the short-term toxicity of gallic acid with its potential sublethal effects on aquatic vertebrates as fish and, in particular, the zebrafish (Danio rerio) as a model. The latter is considered as an alternative test species for anthropogenic chemical evaluation and is among the most used freshwater fishes in toxicological studies (Hill et al. 2005). Pelargonic acid, also known as nonanoic acid because of its nine carbon aliphatic chain (CH3(CH2)7COOH), is a fatty acid chemically produced and commercialized at a “high production volume” (TOXNET 2008). Its applications mainly focus on its herbicidal, fungicidal and sanitizing properties (TOXNET 2008; US 2000). Other industrial applications encompass organic synthesis, incorporation into lacquers, plasticizers, lubricants and related fluids (MMWD 2008). Furthermore, similarly to gallic acid, there are several prospects concerning its allelopathy potential towards cyanobacteria and aquatic weed species (Nakai et al. 2005; Shao et al. 2013; Webber et al. 2014). However, from an ecotoxicological viewpoint, there is also a few information regarding detrimental effects of pelargonic acid in the pure form on aquatic wildlife (MMWD 2008). The only available toxicological reference value (TRV) of 0.46 mg/L in the literature was derived by the “Marin Municipal Water District” (MMWD) US government agency and calculated as the 20th of the lowest lethal concentration (LC50) reported for the rainbow trout (Oncorhynchus mykiss), i.e. 9.19 mg/L, when
exposed to potassium soap salt of the fatty acid (MMWD 2008). Nevertheless, it has to be pointed out that the modelling of several accidental spill scenarios indicated that these latter may lead to uprising of water concentrations from one to two orders of magnitude of the TRV (MMWD 2008), i.e. from 42 to 151 mg/L of substance in the case of thermally stratified ponds for instance. As recently mentioned by the European Food Safety Authority (European Food Safety Authority 2013) concerning pelargonic acid use and application, there remain data gaps to address the aspects of the ecotoxicological risk assessment of this middle-chain fatty acid towards aquatic organisms, thereby highlighting the need for additional ecotoxicity experiments. Finally, it appears that the growing need for eco-compatible technologies using natural-based bioactive compounds has enlarged the scope of applications for phytochemicals like gallic and pelargonic acids (Ow and Stupans 2003; Sethiya et al. 2014; Shao et al. 2013; Sun et al. 2014). However, it has also brought up their presumably enhanced environmental exposure issues, with potential ecological impacts for aquatic organisms that remain to be clarified (European Food Safety Authority 2013; Shao et al. 2013). The main objective of this work was to evaluate the acute aquatic toxicity of gallic and pelargonic acids, both in pure form, using the standard experimental freshwater fish species D. rerio. Besides toxic potency evaluation, alteration of some biological parameters of the tested organisms was investigated at sublethal levels. For this purpose, the activities of antioxidant enzymes namely total superoxide dismutase (total SOD), catalase (CAT) and total glutathione peroxydase (total GPx) on the one hand, and glutathione S-transferase (GST) on the other, were assessed as biomarkers for oxygen-mediated toxicity and phase II detoxification pathway.
Materials and methods Chemicals Gallic acid (98 % purity) was obtained as dry powder from Acros Organics (no. 410860010). A stock solution of 8 g/L was prepared in standardized water (OECD 1992) and used for serial dilution during exposure experiments. Pelargonic acid (97 % purity) was purchased from Alfa Aesar (Lot. 10146705). It was liquid at room temperature and directly added to test solutions. Tricaine methane sulphonate (MS222) and chemicals required for SOD and CAT assays were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and were of analytical grade. Enzyme assay kits were used for glutathione peroxidase and glutathione S-transferase experiments, and were purchased from Cayman Chemical.
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Fish care All fish treatment and procedures used in this study were in accordance with the general guidelines of the Council of European Communities (European Union 2010, no. 2010/63/CEE) and the French Animal Care Guidelines. Wild-type zebrafish (D. rerio, TU strain) with similar length and age (2±1 cm, 3 months old) were provided by GIS AMAGEN (UMS 3504 CNRS/UMS 1374 INRA, Gif-surYvette, France). They were acclimatized for 2 weeks in aerated glass containers filled with artificially reconstituted water (24±2 °C; 12:12-h light/dark photoperiod) (OECD 1992). They were fed twice daily with commercial pellets (BioMar, France) and twice weekly with freshly hatched Artemia nauplii. During the acclimation period, no mortality neither abnormal behaviour were reported, thereby meeting the batch validity criteria for further experiments (Directive 2001/59/ CE; OECD 1992). Toxicity testing procedure The assays followed the Organisation for Economic Cooperation and Development (OECD) standard protocol in static conditions (OECD 1992). Briefly, a series of preliminary range-finding tests were conducted using respectively gallic and pelargonic acids. For gallic acid, given the absence of any toxicological data regarding fish mortality, animals were exposed to increasing doses arranged in a geometric series up to 1000 mg/L of test substance. Definitive assays for this compound were performed in order to confirm that the LC50 was greater than 100 mg/L, i.e. that the substance was practically non-toxic to fish according to general guidance document (US EPA 1992). Additional exposure to lower concentrations of 5, 10, 20 and 40 mg/L of gallic acid were also set up during the definitive assays so as to get insights into potential dosedependent sublethal effects of the polyphenolic compound following biomarker assessment (antioxidant enzymes and GST activities). Concerning acute toxicity of pelargonic acid, preliminary range-finding tests were performed according to previously reported LC50 from fatty acids in pure and soap salt forms (Brooke et al. 1984; US EPA 1992). Definitive assays consisted of animal exposure to five concentrations of pelargonic acid arranged in a geometric series with a factor of 1.3, i.e. 45, 58, 76, 98 and 120 mg/L of test substance. During definitive assays, a group of 10 fish was randomly distributed in each glass tank (i.e. less than 1 g fish/L of test solution according to the recommendations of OECD 1992), containing appropriate compound concentration. This procedure was repeated in triplicates for each concentration including control groups (non-exposed fish in tanks containing only freshwater). Fish were not fed 24 h before the beginning of experiments and during the 4 days of toxicity assessment. They were exposed to test substances in static conditions
without any replacement of solutions throughout the duration of the study (OECD 1992). Temperature, pH and dissolved oxygen were checked daily. Number of dead fish (immediately removed from tanks) was recorded at 24, 48, 72 and 96 h so as to determine the acute toxicity at each time-point expressed as mean lethal concentration (LC50). Fish euthanasia and morphometric measurements Following 96-h exposure period, surviving animals were euthanized by overdose of tricaine methane sulfonate (MS-222, 300 mg/L). Length (mm) and weight (mg) were determined to calculate condition factor indices according to Fulton’s formula (Nash et al. 2006): h . i K ¼ weight ðmg Þ length ðmmÞ3 100
Biochemical analysis Whole body homogenate preparation For the assessment of sublethal biological effects, i.e. biomarker measurement, five fish were randomly selected from each of the three tanks used per exposure concentration, respectively, from all test solution containing gallic acid (from 5 to 100 mg/L), and from those of 45, 58 and 76 mg/L of pelargonic acid (including control). Each individual was placed in a 1.5-mL screw-cap microcentrifuge tube and homogenized in five volumes of 100 mM Tris buffer (pH 7.8 at 4 °C) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 150 mM KCl, using a Bullet Blender (Hoyt 2009). The homogenate was centrifuged 10,000×g, 15 min at 4 °C and the resulting supernatant (S9 fraction) was kept on ice before use for total protein content and enzymatic activity measurements. Total protein determination Protein content of S9 fraction was measured following a 40fold dilution of the recovered supernatant in homogenization buffer, according to the Bradford method (1976). The Thermo Scientific Pierce Protein Assay kit (no. 23236) was used with bovine serum albumin as a standard. Absorbance was read at 595 nm after a 15-min period of incubation. Enzyme activity determination For each fish homogenate, total SOD (Cu–Zn SOD and Mn SOD), CAT, total GPx (selenium-dependent and nonselenium-dependent GPx) and GST activities were determined by spectrophotometric methods adapted to microtiter plates, and expressed as units per milligram of protein.
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Total SOD activity (EC 126.96.36.199) was measured according to the pyrogallol autoxidation method of Marklund and Marklund (1974). The assay mixture contained 50 mM Tris buffer with 1 mM EDTA (pH 8.2), and whole fish homogenate in a final volume of 275 μL per well. The reaction was initiated by the addition of 25 μL of freshly prepared 2.4 mM pyrogallol solution (in 50 mM phosphate buffer, pH 6.4) to attain a final concentration of 0.2 mM pyrogallol in the assay mixture. The inhibition of pyrogallol autoxidation by SOD was monitored at 420 nm at 10-s interval for 3 min. One unit of SOD activity was defined as the amount of enzyme required for inhibiting pyrogallol autoxidation by 50 % per min. CAT assay (EC 188.8.131.52) was performed according to the method of Beers and Sizer (1952) with 10 mM hydrogen peroxide as substrate. The absorbance drop at 240 nm following H2O2 consumption was recorded at 15-s interval for 2 min. One unit of CAT activity was defined as the micromoles of H2O2 decomposed per min (ɛ240 = 0.0394 cm2 μmol−1; Nelson and Kiesow 1972). Total GPx activity (EC 184.108.40.206) was evaluated by the indirect method of Drotar et al. (1985) using cumene hydroperoxide as substrate (Cayman Chemical Glutathione Peroxidase assay kit 703102). The decrease in nicotinamide adenine dinucleotide phosphate (reduced; (NADPH)) concentration, used as a cofactor, was monitored at 340 nm at 30-s interval for 8 min. One unit of GPx activity was defined as the amount of enzyme catalysing the oxidation of 1 nmol of NADPH per minute The activity of GST (EC 220.127.116.11) was determined according to the method of Habig and Jakoby (1981) using 1-chloro2,4-dinitrobenzene (CDNB) as substrate (Sigma-Aldrich assay kit CS0410). The increase in absorbance at 340 nm was recorded at 30-s interval for 8 min. One unit of activity was defined as the amount of enzyme required to conjugate 1 nmol of CDNB per minute. All enzymatic assays were performed at 25 °C using a LabSystems Multiskan EX microplate reader with incubator. For each fish homogenate, each enzyme activity was determined from two different aliquots of the same homogenate sample. The mean obtained from these two aliquots was then used for further calculations, so that a total of 15 enzyme activity determinations were finally obtained for each exposure concentration (corresponding to the number of fish initially sampled per exposure concentration and control). Statistical treatment Mean LC50 values (mg/L±95 % confidence intervals) were estimated after 24, 48, 72 and 96 h of fish exposure using REGTOX macro Excel (v.7.0.6) based on non-linear regression (Hill model) (Vindimian 2012). Fish condition indices and biochemical data were expressed as mean±SE (n=15). Normality and homogeneity of variance assumptions were respectively checked using Kolmogorov–Smirnov and
Levene’s test. The differences between groups were determined by ANOVAs followed by Student Newman–Keuls post-hoc tests for parametric distributions or Kruskal–Wallis and Mann–Whitney post hoc tests for non parametric ones (in the case of inequality between the treatment group size the Dunns’ test was used). All statistical analyses were carried out at a significance level of p