Since ancient times, plant based food has played an important role in human nutrition, being a very important source of antioxidants, vitamins and minerals [1–3]. Modern nutrition requires greater consumption of vegetables and fruits, because of their role on the quality of life . On the other hand, plant food, especially the one which is consumed without prior processing, such as raw vegetables, is the first link of the food chain through which macro and micro metals can go directly into the body. To ensure the functioning of various enzyme systems, the human body needs most micro metals, but some such as Cd, Pb, and Hg have toxic effects. Toxicology indicates that other heavy metals, such as Cu, Ni, Mn, Fe, Cr, V, Mo, can appear in the list of harmful metals, if their concentration exceeds certain limits [4, 5].
For vegetables obtained in uncontaminated areas, the levels of these metals are low, generally below the permissible limits. The situation is quite different when these crops are obtained in geogenic or anthropogenic contaminated areas, such as the mining areas where these metals are exploited. Especially after the industrial revolution, the need for metals in modern society led to the development of metal mining. Because environmental pollution problems have generally been neglected over the centuries, accumulated residues in these important areas have led to increased pollution of soil and plants (both spontaneous and cultivated). Only in recent decades has society realized the negative effects that heavy metals contamination has on the environment and on human health. Nowadays, heavy metals have been acknowledged as factors of “Global Change” scenarios. Climate change may affect HMs bioavailability in soils and hence the entire food chain .
Romania is recognized as one of the European countries with polymetallic mining (Fe, Pb, Cu, Zn, Au, Ag) since antiquity . Research conducted in Romania in recent decades has clearly shown the extent of the anthropogenic pollution of soil and plant food (both of these mining areas as well as of the large urban areas) with various heavy metals [8–13]. Such research, which calls attention to the increased contamination of soils and plants in areas contaminated by mining activities or in large conurbation areas, has been performed worldwide [14–18].
Together with soil, vegetable food constitutes the next link that can increase HMs accumulation in the food chain and that can make them a hazard for consumption. Recently, many studies on various plant foods have addressed this issue [19–32]. If Fe, Mn, Zn, Cu, Ni are essential for plant growth, other HMs (Cd, Pb, Cr, Hg, Ag) are toxic. In normal situations, plants' defences and homeostasis by complex mechanisms are processes that limit the accumulation of HMs , for example in special situations in contaminated areas where HMs accumulation in some vegetables can be high and dangerous for human health [19, 30–32].
For proper growth and development of all animals, including humans, Fe, Mn, Zn and Cu can be considered trace minerals with a central role in many metabolic processes throughout the body. They are essential as catalysts in many enzyme and hormone systems which influence growth, bone development, feathering, enzyme structure and function. On the other hand, it has been proven that, in large amounts, these metals can cause oxidative stress in the animal body, which can on the one hand be beneficial, killing tumour cells, but on the other hand it may have a negative role inducing cancer by oxidative DNA lesion . Moreover, science has proven the existence of structural interactions between heavy metals and functional peptides [35–37]. Another source of metals in animal-based food for humans is the addition of metals in forage, which can get into meat or other animal food products and hence in the human body, where it can influence human health in a positive or negative way [38–40].
The wide range of aspects regarding the presence of HMs in the food chain and their implications on human health requires further research in this field in a unitary approach on the environment which the humankind is part of. The old mining areas affected by anthropogenic contamination with heavy metals are particular areas in which the concentration of one or more heavy metals exceeds normal values in most soils and in some agricultural products used as plant food, such as vegetables and fruit, or even animal products (meat, eggs, and milk).
Given the variety and diversity of data, research in these areas involves not only modern analytical methods with sensitivity, specificity and high accuracy to obtain valid results on HMs content in soil and food but also complex statistical methods that provide the big picture in what they are concerned. Multivariate statistical techniques are the right tool for viewing and analysing some matrices of complex data . PCA and cluster analysis (CA) are two unsupervised methods that allow us to deduce how certain variables (metals concentration, other parameters of the soil or plants) that characterize objects (soil, plant) determine their association. If the CA method is used for samples grouping original variables, PCA estimates the correlation structure of the variables by finding hypothetical new variables (principal components - PC) that account for as much as possible of the variance (or correlation) in a multidimensional data set. These new variables are linear combinations of the original variables . This method helps us to identify groups of variables (i.e. heavy metals concentrations or other soil or plant parameters) based on the loadings and groups of samples (soil or vegetable species) based on the scores.
To understand the complex connection between soil or plant samples and heavy metals contents was used chemometric technique PCA. It is based on eigenanalysis of the covariance or correlation matrix. Each variable has a loading which show how well a variable is taken into account by the model components. They reflect how much each variable contributes to the meaningful variation (or correlation) in the data and to interpret variables relationship. Each sample has a score along each model component which shows the location of the sample in this model and can be used to detect sample patterns, groupings, similarities or differences [43, 44]. In practice, it will ignore higher number PC axes that explain only a small proportion of variance in the species data . The importance of a variable in a PC model is indicated by the size of its residual variance. This is useful for the variable selection; a variable with little explained variance may be removed without adding more changes to the PC model. It is not restriction in the number of variables, the rule for multiple regressions that the number of variables must be smaller than the number of objects does not apply in PCA case. The closer the similarity between the objects, the fewer terms are needed in the expansion to achieve certain approximation goodness . In order to simplify plotting, PCA may be used for reduction of the data set to only two variables (the first two components).
In recent years, chemometric evaluation has increasingly been used in food research. Most applications of chemometric methods focus on establishing correlations between different foods and their composition [41, 44, 47–49], determining geographical origin [50–52], quality of water from dew , quality of environment , modelling of heavy metals contamination of fruits and vegetables [55, 56] or authentication of organic food .
The purpose of our work is to assess the complex phenomenon of pollution of the vegetable food chain in old mining areas with heavy metals by the Principal Component Analysis. For this purpose, many PCA models were constructed and they were computed with different markers. As main markers for pollution were used two types of markers: simple markers, represented by the HMs concentrations in contaminated soils and by the HMs concentrations in the vegetables consumed by the population in these areas. It was also employed a complex marker, namely Target Hazard Quotiens (THQ). This marker connects the metals concentrations in food with their toxicity, quantity and quality of food consumption and body mass of consumers . The use of this complex parameter is more extensive in evaluating the potential health risk of HMs present in various foods [58–62].