Phytochelatin Synthesis in Response to Hg Uptake in Aquatic Plants Near A
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Chemosphere 176 (2017) 74e80 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Phytochelatin synthesis in response to Hg uptake in aquatic plants near a chlor-alkali factory * Marta Turull b, Gabriela Grmanova a, Angela Dago a, Cristina Arino~ a, , Sergi Díez b, Jose Manuel Díaz-Cruz a, Miquel Esteban a a Departament de Química Analítica, Facultat de Química, Universitat de Barcelona, Martí i Franques, 1-11, 08028, Barcelona, Spain b Environmental Chemistry Department, Institute of Environmental Assessment and Water Research, IDÆA-CSIC, E-08034, Barcelona, Spain highlights graphical abstract The effect of a Hg-rich industrial waste in Ebro River macrophytes was studied. Total Hg was analyzed in plants, sediments and suspended matter. The use of DGT allowed the estima- tion of the bioavailable Hg. PCs were detected at low levels with decreasing content downstream from the hot spot. PCs production by macrophytes is an indicator of the bioavailable Hg in the river. article info abstract Article history: The effects of mercury (Hg) released from a chlor-alkali factory in aquatic plants along the Ebro River Received 14 October 2016 basin (NE Spain) were analysed considering the phytochelatins (PCn) and their isoforms content in these Received in revised form plants. These compounds were analyzed using HPLC with amperometric detection, and the macrophytes 16 February 2017 species Ceratophyllum demersum and Myriopyllum spicatum were collected in two sampling campaigns, Accepted 17 February 2017 autumn and spring, respectively. To correlate the PC content in macrophytes with the Hg contamination, Available online 20 February 2017 n analysis of total Hg (THg) content in plants and suspended particulate matter, as well as the dissolved- Handling Editor: Martine Leermakers bioavailable fraction of Hg in water measured by the diffusive gradient in thin film (DGT) technique were done. The results confirm the presence of PC2-Ala in extracts of C. demersum and PC2-desGly in Keywords: M. spicatum, and the concentration of these thiol compounds depends clearly on the distance between Phytochelatins the hot spot and the downstream sites: the higher the levels are, the closer the hot spot is. Since most of Mercury the Hg is hypothesized to be associated with SPM and transported downstream, our results of the DGT Ebro River suggest that trace amounts of Hg in water can be released as free metal ions yielding a certain accu- Ceratophyllum demersum mulation in plants (reaching the ppb level) that are enough for activation of induction of PCs. A few PCs Myriophyllum spicatum species have been determined, at different seasons, indicating that they can be used as good indicators of the presence of bioavailable Hg in aquatic media throughout the year. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction * Corresponding author. E-mail address: [email protected] (C. Arino).~ The effect that trace metal contamination has on terrestrial and http://dx.doi.org/10.1016/j.chemosphere.2017.02.092 0045-6535/© 2017 Elsevier Ltd. All rights reserved. M. Turull et al. / Chemosphere 176 (2017) 74e80 75 aquatic ecosystems is a matter of major concern. Some trace toxic organisms of the Ebro River could be through the analysis of PCn metals, like mercury (Hg), are capable of increasing in concentra- and related compounds synthetized by macrophytes growing in the tion upward through the food chain, reaching high levels in top river, since they are good biomarkers for heavy metal stress. It is predators (Morel et al., 1998). Then, an interesting point to be interesting to remark that most of the environmental studies considered in contamination studies is the negative effect that related with this factory were devoted to the effects of Hg exposure pollutants have on living organisms. It is well known that some in the aquatic environment (e.g. fish species, crayfish, molluscs) mammalians, plants, algae and some fungi have the capability to (Carrasco et al., 2008, 2011a; 2011b; Navarro et al., 2009), or synthetize molecules to prevent the harmful effects produced by focused on atmospheric Hg and its incorporation in soils and li- metals stress. In the case of plants, algae and some fungi, phy- chens (Esbrí et al., 2015), however, determination of PCn and related tochelatin (PCn) synthesis is considered necessary to tightly regu- compounds in aquatic plants has never been reported. late the distribution of metal and to minimize damage under excess Recently, there is a growing interest in the use of aquatic mac- metal supply conditions. Phytochelatins are small cysteine-rich rophytes in the abatement of heavy metal pollution and as sentinel peptides, which play an essential role in heavy metal detoxifica- organisms of pollution in aquatic ecosystems (Rezania et al., 2016). tion by chelating metals through thiol groups in the cytosol and Among them, the submerged species are particularly useful in the transporting the complexes formed to the vacuoles. The general monitoring of heavy metals (Rai, 2009). In this sense, since Cera- structure of PCn is (g-Glu-Cys)n-Gly (n ¼ 2 to 5). Glutathione (GSH) tophyllum demersum and Myriophyllum spicatum are invasive sub- serves as the substrate of PCn biosynthesis through the trans- merged aquatic plants and the dominant species growing in the peptidation of the g-Glu-Cys moiety of GSH onto a second GSH to Ebro River basin, they were selected for investigation in this work. form PC2 or onto a PCn molecule to produce an nþ1 oligomer Both have a large capacity to adsorb metal ions and by this reason (Stillman et al., 1992; Bordin, 2000; Cobbett, 2000; Cobbett and both can potentially be used to remove metals from the aquatic Goldsbrough, 2002; Riordan and Vallee, 1991; Suzuki et al., 1993). media (Abdallah, 2012; Keskinkan et al., 2007; Milojkovic et al., By other hand, PCn synthesis depends not only on the plant species 2014). However, as far as we know, studies of PC synthesis by but also of the metal stressor, being Cd the best activator followed these plants are scarce, only Mishra et al. (2006) determine the PCn by Ag, Bi, Pb, Zn, Cu, Hg and Au cations (Bundy et al., 2014; Dago induced in C. demersum plants exposed to different levels of lead, et al., 2014b). This relationship of PCn with trace metal contami- and the presence of PC2 and PC3 was reported. nation allows us to use PCn content as an indicator of metal Thus, the aim of this work is to evaluate the toxic effect that pollution. In the analysis of PCn and their Hg complexes different upstream Hg contamination by the industrial waste and the wall methodologies have been applied. Among them the use of MS- built have on downstream macrophytes of this basin. In order to HPLC is the most common, although detection by fluorescence or accomplish this, we carried out a field study in the low Ebro River absorption spectroscopy is also considered (Serrano et al., 2015). In basin, where two submerged macrophytes were collected and a previous work of our research group, a method to determine phytochelatin production was reported as a measure of the metal glutathione, PCn and their Hg complexes using amperometric stress response. In order to correlate the PCn synthetized with Hg detection in a glassy carbon electrode has been developed (Dago concentration in the media, total Hg in aquatic plants, in suspended et al., 2009, 2011). This method has been applied to study the ef- particulate matter and in sediment samples, as well as the fect that Hg and other metal ions have in the synthesis of PCs in dissolved-bioavailable fraction of Hg have also been reported. Hordeum vulgare plants cultured in controlled conditions in the lab in the presence of these toxic substances (Dago et al., 2014a, 2014b) 2. Materials and methods or in Asparagus acutifolius plants growing naturally in the mining district of Almaden (Dago et al., 2014c). These studies determined 2.1. Chemicals PCn and their Hg-PC complexes in plants and established a corre- lation between the metal content in the growing media and the Glutathione (GSH) was obtained from Merck (Darmstadt, Ger- level of synthesized PCn. On the other hand, some PC related many). Phytochelatins (g-Glu-Cys)n-Gly (n ¼ 3e5), as tri- compounds can also be synthesized simultaneously by plants; fluoroacetate salts, were provided by DiverDrugs S.L. (Barcelona, these compounds, also called iso-phytochelatins, are structural Spain) with a purity ranging from 86.2% to 99.0%. Phytochelatin 2 variants of PCn that differ from them in one of their constituent (PC2,(g-Glu-Cys)2-Gly) and its isoforms (PC2desGly ((g-Glu-Cys)2), aminoacids. Four families have been described, (g-Glu-Cys)n-b-Ala, PC2Ala ((g-Glu-Cys)2-Ala), PC2Glu ((g-Glu-Cys)2-Glu) and CysPC2 (g-Glu-Cys)n-Ser, (g-Glu-Cys)n or (g-Glu-Cys)n-Glu, that together (Cys-(g-Glu-Cys)2-Gly)) were provided by Genosphere Bio- with the phytochelatins described above form the metallothioneins technologies (Paris, France) with a purity of 95%. class III (Grill et al., 1987; Rauser, 1995; Zenk, 1996). Another For preparing the mobile phase, acetonitrile from Panreac methodology, also based in HPLC with amperometric detection, has (Barcelona, Spain), NaCl, formic acid and KOH from Merck and been proposed in a previous work of our research group for the trifluoroacetic acid (TFA) from Sigma-Aldrich (St. Louis, MO, USA) determination of the isoforms of PC2, which, in principle, are the were used. Methanol from Merck was used for cleaning the column, most abundant in natural samples (Dago et al., 2015). and ethanol (96% purity) from Panreac was used for cleaning the In the present work, a case of residue disposal from a chlor-alkali glassy carbon electrode.