*2.4. Calculations*

All solutions, i.e., fruit and berry digests, blanks, and CRM digest were measured thrice. The blank-corrected values (mass concentrations) were converted into contents in mg/kg dried plant material considering dilution factor, final volume, and mass of dry matter. Finally, means and standard deviations were calculated for all samples. In order to see statistically significant differences between the three fruits analysed, ANOVA test was carried out, based on *p* < 0.05. All calculations were performed using Microsoft Excel 2010 and 2013.

#### *2.5. Characterisation of the Analytical Method*

In order validate the analysis, parameters of trueness, precision, and day-to-day repeatability were determined from strawberry leaves which were chosen as a certified reference material for plant matrix. The samples were analysed after calibration on two different days. The recoveries were obtained using the following formula:

$$\text{recovery}\_{\text{x}} \text{in } \%= \frac{\text{content}\_{\text{x}, \text{found}} \text{ in mg/kg}}{\text{content}\_{\text{x}, \text{cerified in mg/kg}}} \times 100$$

Based on Bouman's procedure [28], the limits of detection (LOD; 3σ) and quantification (LOQ; 10σ) were calculated.

## **3. Results**

#### *3.1. Analytical Method*

The limits of detection (LOD) for the dried fruits, based on a digested mass of 0.25 g and a final volume of 10.0 mL, are below 0.3 mg/kg for all analytes except for Al, whose LOD is 2.9 mg/kg. In addition to the LOD values, the recoveries obtained by analysing the CRM (ranging from 85% to 113%) are given in Table 2. These figures of merit are in the range of reported ones for similar studies [26,29]. The coefficients of determination ( *R*2) of the calibration curves are all beyond 0.9995. The precision presented as relative standard deviation (RSD) for triplicate measurements is below 1.2% for all analytes. The RSD obtained for the day-to-day-repeatability is not higher than 2.5%.



#### *3.2. Elemental Content of Wild Berries and Fruits*

The results obtained for the three kinds of berries and fruits for the five metals analysed are summarised in Table 3. In all samples the Cd, as well as Cr, levels were below the respective LODs. For the other analytes (Al, Ni, Pb) the minimum, mean, and maximum values are given. Statistically significant differences in the element content were found for Al and Ni, the *p*-values are 2.6 × 10−<sup>14</sup> and 0.0023, respectively. Conversely, the Pb contents do not differ statistically significantly between the three sample types analysed (*p* = 0.24)



1 d.w. = dry weight.

## **4. Discussion**

#### *4.1. Analytical Method*

The above described figures of merit for the analytical method applied are in the range for the determination of trace elements in biological samples. LODs in a similar range were also obtained by other working groups [17,29]. Limit values for Cd and Pb in fruits and vegetables are stipulated (see Table 4). For both metals the calculated LOD is lower than the maximum allowed content. The latter are given for fresh weight. Since the water content of fruits and berries is around 90% [14], the elemental content referred to fresh weight is 10 times lower than referred to dry weight. Based on the characteristic data found, the analytical method chosen has been proven to be appropriate for the given analytical task.


1 f.w. = fresh weight; bw = body weight; PTWI = Provisional tolerable weekly intake; PTMI = Provisional tolerable monthly intake; Rfd = Reference oral dose; Trv = Toxicity reference value.

#### *4.2. Elemental Content of Wild Berries and Fruits*

Based on the five elements determined, all three fruit types differ statistically significantly from each other regarding Al and Ni content. In the following all elements analysed are discussed in detail.

Aluminium has no known physiological role in the human body. Until the 1970s it was considered to be innocuous, but starting in the 1980s toxic effects to animals, plants, and humans have been reported [36]. Al rarely shows acute toxicity, but chronic intoxications have to be considered, especially due to the fact that Al accumulates with age [37,38]. Diet is regarded as being a significant contributor to the body burden of aluminium [39], and its weekly intake is limited by the FAO/WHO [25]. The Al content found in the fruits and berries analysed was highest in rose hips, followed by blueberries and, finally, lingonberries, the mean values being 8242 mg/kg, 1248 mg/kg, and 42.5 mg/kg, respectively. The data differ from each other in a wide range, even since all samples were collected in the same area having a similar environmental background. Thus, it can be clearly seen that the uptake differs between the plants. Nile and Park do not give any value for Al in berries in their review on berry composition, they just mention that berries are rich in Al [10]. Rose hips used for the preparation of infusions collected in Turkey show a mean Al content of 157 mg/kg [40], whereby it is not reported if the entire fruit or only the flesh (as in the present study) was analysed. However, comparable Al contents were found in strawberries from Pakistan (Lahore region), whereby the values decreased during ripeness from 740 mg/kg to 230 mg/kg d.w. [15]. The PTWI given by the FAO/WHO limits the weekly Al dietary intake with 1 mg/kg bw. Thus, an 80 kg-person should not consume more than 100 g of rose hips per week. For blueberries and lingonberries more than 600 g, and even 2 kg, are to

be eaten resp. in order to reach the maximum, considering no other Al sources. Chinese blueberries contained 41 mg/kg [41], much lower than the results from this study, but in the same order of magnitude than the data obtained for lingonberries. This fact can be explained by the impact of bedrock composition on Al content in plants. A potential health threat might only be caused by rose hip intake. This is a drawback, since rose hips have recently been defined as a functional food based on bioactive ingredients, as well as due to their content of essential elements, such as Ca, Mg, K, S, Si, Se, Mn, and Fe [42].

Cadmium, being a highly toxic metal, is one of ten chemicals classified by the WHO to be a major public health concern [22]. It occurs naturally in soil, but it is also a pollutant in the environment due to anthropogenic impact. Since it is easily taken up and accumulated by plants and crops through the root systems, it may easily enter food [43], especially berries and fruits. In all samples, including blueberries, lingonberries, as well as rose-hips, no Cd was detected, meaning that the contents are below the LOD of 0.028 mg/kg d.w. Commission Regulation (EU) No. 2015/1005 limits Cd in fruits and vegetables to 0.050 mg/kg f.w., corresponding to 0.50 mg/kg d.w. [30]. Thus, all fruits analysed are supposed to be safe for humans regarding this element. The PTMI is 0.025 mg/kg bw, which would be 2 mg per month for an 80 kg person. Considering Cd present in the berries at the LOD level, this corresponds to more than 700 kg of fresh blueberries in one month. An investigation of blackberries, elderberries, autumn olives, and candleberry-myrtles revealed that they also contained Cd only below LOD [17]. Whereas no Cd was found in Azorean blueberry, levels between LOD and limit of quantitation (LOQ) were registered for Madeiran blueberry [17]. A Russian study reports Cd in blue- and cranberries in the range from 0.03 mg/kg to 0.06 mg/kg d.w. [3]. Strawberries collected in Serbia did not contain Cd at detectable concentrations either [29]. Blueberries collected in China were reported to contain 0.034 mg/kg Cd [41], slightly above the LOD of the current study. Rose hips from Turkey have been reported to contain quite a high amount of Cd, namely 0.81 mg/kg d.w. [44]. A working group from Bangladesh found Cd contents ranging from 0.012 mg/kg and 0.216 mg/kg in leafy and non-leafy vegetables [43].

Chromium, as Cr(III), plays a crucial role in human health and is, thus, defined as an essential trace element [45]. Special attention has to be drawn to its interaction in glucose tolerance and Cr supplementation in persons with diabetes, hypoglycaemia, and obesity [46]. In addition to the positive effects of Cr(III), Cr(VI) is a toxic, carcinogenic substance, this fact being reflected in the 500-times lower reference oral dose for the latter species [32,33] compared to the former species. No limit for Cr in foods is given in the Codex Alimentarius Commission, or by Australia, New Zealand, Japan, the United States, and Taiwan [41]. Even if Cr(III) is reported to be mainly found in fruits, vegetables, and grain products [47,48], no Cr could be detected in all samples of this investigation (<0.018 mg/kg). Data for Cr in blueberries are given by Hua et al., who found 0.77 mg/kg [41]. Additionally, in fresh strawberries the Cr content is quite low, ranging from 0.01 mg/kg up to 0.03 mg/kg [29]. Wild berries from Portugal, analysed by Llorent-Martínez and colleagues, found Cr in blackberries, elderberries, autumn olives, and candleberry-myrtles up to 0.6 mg/kg fresh weight [17]. Cranberries from Russia have been reported to contain Cr from 0.02 mg/kg up to 1.5 mg/kg, and blueberries 0.03 to 0.06 mg/kg d.w. [3]. Literature data for rose hips are given by Duran and colleagues, who found 0.80 mg/kg d.w. Cr [44].

Nickel is naturally occurring in soils ranging from 4 to 80 mg/kg in the USA [49] and, for agricultural soil in Velika Gorica, Croatia (N 45.7173◦–E 16.0571◦; A 107 m), a mean content of 55 mg/kg was found [50]. The main exposure route to nickel is via food intake, approx. 0.100 to 0.300 mg/day are taken up by per adult [23,49]. Apart from contact dermatitis, genotoxicity, haematotoxicity, teratogenicity, immunotoxicity, and carcinogenicity have been identified as harmful effects of Ni [24]. Thus, its intake is limited to 0.020 mg/kg bw per day [23,34]. In the present study the highest Ni content was found in blueberries (25 mg/kg), followed by rose hips (11 mg/kg) and lingonberries (2.5 mg/kg). The daily consumption of 100 g fresh blueberries would lead to an intake of 0.250 mg, being approx. 16% of the allowed limit for an 80 kg person. Thus, no harmful effects are to be expected by the berries analysed. Cranberries from polluted areas (close to a Ni-Cu-smelter) showed elevated levels (up to 97 mg/kg d.w.), whereas the background contents were in the same range as the results from the current study, i.e., 2 mg/kg to 9 mg/kg [3]. No high Ni accumulation in lingonberries collected in the vicinity of a chromium mine was reported by a Finnish research group [51]. Blueberries from the same contaminated area did not accumulate Ni that much, with contents being in the range from 4 mg/kg to 11 mg/kg for all sampling sites [3]. A Chinese working group found 2.2 mg/kg d.w. Ni in blueberries [41]. Wild berries from Portugal contain Ni in the same order of magnitude, namely from 0.6 mg/kg to 1.6 mg/kg [17], as our results for lingonberries. Additionally, results for strawberries are in the same range [29]. Regarding rose hips, the literature data are available for Turkish fruits, with the mean content being 6.5 mg/kg d.w. [44].

Lead is well known as a toxic element, exhibiting developmental neurotoxicity in young children and cardiovascular effects and nephrotoxicity in adults [35]. Thus, the Pb content in fruits and berries is limited to 0.10 mg/kg f.w. and 0.20 mg/kg f.w., respectively [35], whereas the PTI value was withdrawn in 2010 [22]. The highest maximum and mean level for Pb were found in rose hips (15 mg/kg; 3.3 mg/kg, resp.), exceeding the limit value in foods, which would correspond to approx. 1 mg/kg d.w. This result is comparable to that form Turkish rose hips with 10 mg/kg d.w. [44]. Blueberries' Pb content showed the smallest range, compared to rose hips and lingonberries. Only one sample exceeds the limit in berries (2.4 mg/kg > 2 mg/kg). The mean value of Pb in blueberries (1.66 mg/kg) is similar to the data from Barcan et al., who reported contents ranging from 0.7 mg/kg up to 1.5 mg/kg [3]. Conversely, the Chinese working group found less, their average being 0.135 mg/kg d.w. [41]. Lingonberries contained the least Pb of all fruits analysed, except for one sample (9.3 mg/kg) all results are below 1 mg/kg, thus being within the allowed range for berries. Even less Pb was found in lingonberries from Finland, whose contents ranged from 0.0006 mg/kg to 0.0011 mg/kg f.w. [51], corresponding to approx. 0.006 mg/kg to 0.011 mg/kg dried berries. Wild berries gathered in Portugal all had Pb levels below the LOD (0.012 mg/kg f.w.) [17].

## **5. Conclusions**

Wild berries and fruits collected in a remote and, thus, considered unpolluted area in Croatia have been analysed for potentially toxic elements, revealing that no health threat is to be expected by eating these fruits and berries regarding Cd, Cr, and Ni. Due to high Al and Pb contents in rose hips, their intake should not exceed 100 g per week for an adult, especially in the case of chronic renal failure. Only in cases of high consumption of blueberries are harmful effects by Al and Pb to be expected. Lingonberries were found to have the lowest contents of all metals investigated.

**Author Contributions:** M.Z. and I.J.C. conceived and designed the experiments; I.J.C. carried out the digestions and measurements; M.Z. and I.J.C. analysed the data; M.Z. wrote the paper; and M.Z. revised the paper.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
