**1. Introduction**

Berries have been collected for nutritional purposes for a long time. They can be either consumed directly as fresh or dried fruits or in various processed forms, such as jams, syrups, infusions, juices, and jellies, or as ingredient of yoghurts or ice-creams. Diet in the Western world includes vegetables and fruits, as well as other plant parts, such as roots, leaves, stems, and seeds, from more than 40 botanical families [1]. Many demonstrate a presence of phytochemicals in fruit and vegetables, which contribute to good human health by influencing metabolic processes [1], e.g., free radical scavenging, stimulating the immune system, inducing apoptosis, and detoxification. Including fruits and vegetables in the daily diet is recommended in dietary guidelines worldwide. However, the consumers' awareness of nutraceutical components is increasing. Thus, ongoing scientific interest in berries, especially wild grown ones, is needed [2]. Regarding public interest, it was estimated in the 1990s, that one family gathers approx. 20–30 kg of berries per year [3]. Even if gathering berries and fruits by oneself is nowadays still carried out, it has started to change from self-collecting in the woods to the usage of pick-your-own farms, which offer a wide range of berries, such as strawberries, raspberries, or blackberries, and processed products [4,5].

Berry fruits, in general, contain a wide range of micro- and macronutrients, such as fibres, minerals, vitamins, and folate, but their biological properties have been attributed mainly to hydrophilic phenolic-type phytochemicals. Usually not only one bioactive compound determines the positive effects of a fruit, but the presence of multiple phytochemicals exhibiting complementary, synergistic, and/or additive effects results in the wide spectrum of health promoting effects [1,2]. Thus, berry research has been focused mainly on organic composition [6–11].

Regarding inorganic analytes, investigations dealing with nutrients in different berry types, e.g., major and minor essential elements, have been published for decades [12–17]. Using old data, such as from 1944 [12] or 1974 [13], it has to be taken into account that the analytical methodology, as well as the environmental influences, have changed, reducing their significance for current evaluations. In addition to the nutritional value, the content of minerals present also influences food quality parameters, like appearance, taste, texture, stability, and even flavonoid production [18]. Conversely, ripeness affects the mineral composition, and some nutrients have been found to decrease with maturity [15], whereas the level of the harmful element arsenic increased [16]. Furthermore potentially harmful metals and metalloids have to be taken into account. Berries collected in highly contaminated areas have been analysed for this purpose [3,19,20]. Additionally, in remote areas heavy metal pollution may occur due to transmission processes. Thus, food safety is of concern [21]. Ten chemicals have been classified to be of major public health concern, including four metals/metalloids (arsenic, cadmium, lead, and mercury) [22]. In addition to the top-ten, other metals may exhibit harmful effects to humans through food intake, e.g., nickel [23,24]. Therefore, maximum allowed limits have been established for harmful elements, and food on the market should not contain certain metals in levels that might result in exceeding the respective allowable intakes. PTWI (provisional tolerable weekly intake), PTDI (provisional tolerable daily intake), or PTMI (provisional tolerable monthly intake) levels have been published by the FAO/WHO [25].

The aim of the present investigation was to determine the content of five elements: aluminium (Al), cadmium (Cd), chromium (Cr), nickel (Ni), and lead (Pb) in wild berries and fruits, since they are considered to be potentially toxic contaminants. Regarding the methodology, previously optimised digestion and measurement procedures were applied, i.e., acidic microwave-assisted digestion followed by inductively-coupled plasma atomic emission spectrometry (ICP-AES) [26]. Closed-vessel microwave digestion as a sample preparation method has been approved for the determination of 31 elements in foodstuffs [27].

#### **2. Materials and Methods**

#### *2.1. Chemicals and Glass/Plastic Ware*

Reagents for sample and CRM digestion, i.e., hydrogen peroxide (H2O2) and supra pure nitric acid (HNO3) were purchased from Sigma (Munich, Germany). The standard stock solution used for preparation of calibration standards (ICP Multielement Standard IV) was obtained from Merck (Darmstadt, Germany). Ultra-pure water, resistivity ≥ 18 MΩ·cm, was prepared by in-house equipment. Quality control measurements were based on the certified reference material strawberry leaves (CRM: LGC7162) from LGC Standards (Middlesex, UK). All glass- and plasticware used for sample storage and preparation were pre-cleaned with diluted nitric acid.

#### *2.2. Samples and Sample Preparation*

Wild berries and fruits, namely blueberries (*Vaccinium myrtillus*), lingonberries (*Vaccinium vitis-idaea*), and rosehip (*Rosa canina*), were collected during summer and fall 2010 when optimally ripe in a non-polluted rural area of Croatia (Slunj: N, 45.07◦; E, 15.36◦; A: ~280 m). The sampling site covered approx. 200 m2. For each fruit type five plants, randomly distributed in the sampling area, were sampled by collecting 5 to 15 fruits. Immediately after picking, the fruits were frozen and kept at −20 ◦C prior to further work-up. After thawing the fruits were rinsed with ultra-pure water. Rose hips were separated into flesh (mesocarp) and seeds. All fruits were dried at 105 ◦C for 24 h. Using a metal-free mortar, the samples were then ground and homogenised, resulting in a pooled sample for each plant (i.e., five subsamples for each fruit type) In order to destroy the organic matrix, all samples underwent a previously-optimised acidic microwave assisted digestion [26], whereby approx. 0.25 g to 0.5 g (weighed to the nearest 0.1 mg) were treated with 5 mL nitric acid (*c* = 7 mol/L), applying the following three-step digestion procedure: (1) 150 ◦C/10 min, (2) 160 ◦C/10 min, and (3) 190 ◦C/20 min. The obtained clear solutions were then brought to 10.0 mL with ultrapure water.

#### *2.3. Apparatus and Measurements*

Microwave-assisted digestion of the fruits, CRM, and blanks were done in a MWS-2 Microwave System Speedwave instrument (Berghof Laborprodukte GmbH, Eningen, Germany). The subsequent quantitative determination of metals and metalloids was carried out using a Prodigy High Dispersive ICP-AES spectrometer (Teledyne Leeman, Hudson, NH, USA), using a simultaneous mode, the optimal instrumental conditions are listed in Table 1. The emission lines selected along with the respective characteristics of the analytical method are given in Table 2.


**Table 1.** Operating conditions of the Prodigy High Dispersive ICP-AES.

External standards in the concentration range from 0.0500 mg/L to 5.00 mg/L were used to calibrate all analytes. The standard solutions were prepared by diluting a multi-element standard stock solution (1000 mg/L) with 1% *w*/*w* HNO3. This diluted nitric acid is also the medium of the blank solution, whose intensity was subtracted from all sample values for blank correction. In case of sample concentrations outside the calibration range, appropriate dilutions to the sample were performed with 1% *w*/*w* HNO3.
