Natural geochemical risk in the Pollino Massif: a case-study of chromium
-
1
Inail, Italian Workers' Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Rome, Italy , Italy
-
2
CNR-IMAA, – Laboratorio di Geologia Medica e Ambientale,, Italy
The geochemical risk linked to heavy metal contamination of soils and waters, commonly associated with anthropogenic causes, may, however, be linked to the presence of rocks and soils that naturally contain these metals. Chromium and nickel are particularly abundant in the ultramafic rocks and ophiolitic and in some crystalline rocks, as well as in soils resulting from these pedogenetic processes (Kierczak et al., 2007). The phenomenon of alteration in charge of such rocks can also determine the release in ionic form, in the circulating waters, of high amounts of such metals. In this regard, the north-central sector of the Pollino massif is an important pilot case, characterized by the presence of ophiolite and crystalline rocks of continental origin which are natural reservoirs of chromium and nickel. In the Pollino Massif outcrop ultramafic rocks, such as serpentinites and metabasites, and lower-crustal continental rocks, such as garnet-rich gneiss exhibit high concentrations of Mg, V, Cr, Co, and Ni, more than one or two orders of magnitude higher than the average composition of the upper continental crust. In general, the weathering of these rocks may cause groundwater pollution (Kierczak et al., 2007) that, in the case of the Pollino Massif, is mostly related to the release of Cr and Ni in solution and, to a lesser extent, also to the release of Mg and Pb (Margiotta et al., 2012). The principal geochemical process able to release Cr (VI) starting from the rock is the dissolution of chromite and, to a lesser extent, magnetite and diopside, all phases containing Cr (III). Very high concentrations of Cr (VI) were also detected in water circulating in Pleistocene conglomerates, due to the presence of a significant serpentine component. Within the project " Geochemical vulnerability monitoring aimed at a correct management of the soil use in some risk areas of the Pollino National Park (Basilicata)" (MoGeSPol), funded by the Region of Basilicata in continuity with other research projects, a detailed geochemical study aimed at defining mobilization or immobilization of contaminants related to the different phases of minerals and organic matter in soils and debris, has been carried out. Attention has been focused on a sample area where there are several sources, some of which are used for drinking purposes and/or irrigation, which present high levels of Cr (VI) (Figure 1). In this area, there is also quite developed horticulture on soils that have elevated concentrations of nickel and chromium, resulting, mainly, from the alteration of serpentinite rocks (Figure 2). Within the project MoGeSPol, laboratory tests are in progress aimed at identifying biomarkers and possible pathways of migration of contaminants from soils and waters to products intended for human consumption. Toxicity tests in vivo are performed, aimed at assessing the phytotoxicity and genotoxicity of contaminants in the soil or in the waters of model plants (Vicia faba) allowed to grow directly on the solid matrix and/or irrigated with contaminated aqueous matrix. The study of the toxic effects of contaminants on plants is very important because of the ecological role they play: being at the base of the food chain, a potential accumulation of toxic substances (e.g. heavy metals) in their tissues can become a potential danger to animal and human health. Generally, the toxicity of a chemical substance consists in causing changes at different levels (biochemical, molecular, cellular, tissue or population level) that cause the alteration or impairment of one or more functions such as survival, growth, reproduction, motility, photosynthesis, behaviour (Maffiotti et al., 1997). The identification and quantification of different alterations can be used as biomarkers in organisms exposed to contaminated environments. In our case, the phytotoxicity of Cr (VI), contained in the irrigation water, is evaluated in terms of reducing the length of the primary roots of Vicia faba plants grown in contaminated soil compared to the negative control (plants grown on non-serpentine soil) (Sturchio et al., 2011). Moreover, the genotoxicity expresses the ability of a substance to interact with DNA causing structural alterations which, if not repaired, can lead to the fixing of abnormalities in the genetic material. In this context, the genotoxicity of chromium is assessed through the test of micronucleus test performed on the primary root of Vicia faba (De Marco et al., 1990). In our study we hypothesized that Cr(VI) contained in irrigation water could be transformed by the action of natural processes in Cr (III) that could be accumulated in edible plants and interfere with nutrition of populations exposed chronically to trivalent chromium. Then, the project aims to study the genotoxic effect of Cr (VI) in cell lines of human liver carcinoma HepG2. For this purpose, the execution of the comet assay as suggested by many reports, represents one of the more effective tests for the evaluation of the damage to the DNA of human cells following exposure to Cr (VI) as it is able to detect damage to DNA, such as Cr-DNA adducts alkaline-sensitive, DNA cross-links, and incomplete excision repair events (Gedik, 1992).
Materials and Methods
In vivo testing
Sampling
Firstly, an inspection was carried out at the area of San Severino Lucano and three types of soil were identified to be tested and for which sampling has been carried out:
Soil (1) = agricultural serpentine soil in the San Severino Lucano area
Soil (2) = nonfarm serpentine soil sampled in the San Severino Lucano area
Soil (3) = non serpentine and nonfarm soil outside the area of San Severino Lucano area
Local water used by farmers to irrigate fields was also sampled; it contained hexavalent Cr.
Micronucleus test
Tests have been performed in aluminum trays each containing an aliquot of one of the soils sampled (250 gr) on which were allowed to germinate 25 seeds of Vicia faba. Each type of soil was irrigated with 50 ml of local water (containing Cr (VI)), and distilled water was used as negative control test.
Similarly, inert quartz sand was used to evaluate only the effect of the local water containing Cr(VI). Each test was performed in triplicate. The genotoxic effects were evaluated by detecting the frequency of micronucleated cells in root meristems of Vicia faba. The root tips were fixed in ethyl alcohol and acetic acid solution 3:1 (v/v). The micronucleated cells frequency was scored from 15,000 cells (15 root tips, 1,000 cells for tip). Each experimental data set was tested by ANOVA with Dunnett test for comparing the difference among the treated groups. The statistical software package SPSS (Chicago, IL) was used.
In vitro testing
Cell line
In this study, we used human hepatoma HepG2 cells, which have been used in a number of genotoxicity tests, as a model to study oxidative stress and genotoxicity associated with exposure to potassium chromate at concentration 0.5,1,2,4 µM at 24h. The cells obtained from the Riken Cell Bank (Tsukuba, Japan) were cultured in Eagle’s minimal essential medium (MEM) (Nissui, Tokyo, Japan) supplemented with 1% nonessential amino acid (Invitrogen, Carlsbad, CA), 10% fetal bovine serum, and 60 mg/mL kanamycin at 37°C and 5% CO2.
Comet assay
After incubation with potassium chromate, the cells were centrifuged, washed with phosphate buffered saline (PBS) free calcium and magnesium, and re-suspended in 100mL PBS. In a 2 mL tube, 50 mL of the cells suspension and 500 mL of melted LM Agarose were mixed and 75 mL pipetted onto a pre-warmed comet slide. The slides were placed flat in the dark at 4°C for 10 minutes to allow the mixture to solidify and then immersed in prechilled lysis solution at 4°C for 40 minutes. Slides were removed from lysis solution, tapped, and immersed in alkaline solution for 40 minutes at room temperature in the dark. Slides were washed twice for 5 min with Tris-HCl . Slides were electrophoresed at low voltage (300 mA, 25V, 4°C) for 30 minutes. Slides were placed in 70% ethanol for 5 min, removed, tapped, and air dried overnight. Slides were stained and comet slides were viewed with a Nikon fluorescence microscope
Results and discussion
In vivo testing
The preliminary results of the phytotoxicity and genotoxicity tests on Vicia faba seeds do not seem to evidence adverse significant effects detectable when exposed with soil and/or water contaminated with hexavalent chromium coming from Pollino Massif. We focused our attention on a sample area with developed horticulture where there are several contaminated sources which present high levels of Cr (VI). Our results demonstrate how natural processes (microbial activity, geochemical composition and biochemical processes in plants) could influence toxicity of Chromium (VI).
In vitro testing
In the present study we have analyzed the genotoxic potential of Cr (VI) in HepG2 cell line. The results demonstrate that Cr (VI) induces significant cytotoxic effect at the higher concentrations (2 and 4 µM) and DNA damage at early stages of exposure at concentration of 1 μM in cell line. It is interesting to study the correlation of oxidative stress to the cellular damaging potentials of Cr (VI).
We will focus our experimental proposal on the idea that DNA damage can be used as a biomarker of exposure in the domestic population exposed chronically to trivalent chromium as the population of San Severino Lucano. In fact, many studies suggest that exposure to trivalent chromium can lead to DNA damage detectable in human lymphocytes and that the absorption of Cr (III) in the exposed population may be slow but effective if the exposure is continued for prolonged periods.
References
Kierczak J., Neel C., Bril H., Puziewicz J. (2007) – Effect of mineralogy and pedoclimatic variations on Ni and Cr distribution in serpentine soils under temperate climate. Geoderma 142 (2007), 165–177.
Margiotta S., Mongelli G., Summa V., Paternoster M., Fiore S. (2012) – Trace element distribution and Cr(VI) speciation in Ca-HCO3 and Mg-HCO3 spring waters from the northern sector of the Pollino massif, southern Italy. Journal of Geochemical Exploration 115, 1-12.
Maffiotti A., Bona F. and Volterra L. (1997) - Introduzione all’ecotossicologia. Analisi e recupero dei sedimenti marini. Quaderni di Tecniche di Protezione Ambientale. Pitagora (Ed.), Bologna, pp. 139.
Sturchio E., Boccia P., Meconi C., Zanellato M., Marconi S., Beni C., Aromolo R., Ciampa A., Diana G. e Valentini M. (2011) - Effects of arsenic on soil–plant systems. Chemistry and Ecology. 27 (1), 67-78.
De Marco A., Boccardi P., De Simone C., Piccolo A., Raglione M., Testa A. and Trinca S. (1990) - Induction of micronuclei in Vicia faba root tips treated in different soils with the herbicide alachlor. Mutat. Res., 24, 6-11.
Gedik CM, Ewen SWB and Collins AR., (1992) Single cel gel electrophoresis applied to analysis of UV-C damage and its repair in human cells. Int J Radiat Biol; 62:313-20.
Keywords:
Cr (VI),
Vicia faba,
HepG2 cells,
Pollino Massif,
Comet Assay
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Oral Presentation
Topic:
Ecogenotoxicology
Citation:
Boccia
P,
Meconi
C,
Sturchio
E,
Margiotta
S,
Ragone
P and
Summa
V
(2015). Natural geochemical risk in the Pollino Massif: a case-study of chromium.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00023
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
02 Jun 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
Dr. Priscilla Boccia, Inail, Italian Workers' Compensation Authority (INAIL), Department of Technological Innovation and Safety of Plants, Product and Anthropic Settlements (DIT), Rome, Italy, ROME, 00198, Italy, pri31@libero.it