Cabbage (Brassica oleracea var. capitata) as possible indicator of wartime metal and metalloid contamination in eastern Croatia (ICP-MS method)

ABSTRACT

Biomonitoring of the local population and environmental monitoring in eastern Croatia have revealed abnormalities in metal and metalloid distribution that could be related to war activities during the 1990s. The goal of this study was to determine whether there are differences in the concentrations of metals and metalloids by comparing locations of high and low-intensity combat activity; we also evaluated a possible connection between metal contamination in soil and in humans. We sampled 14 locations and measured the concentrations of 20 war related metals and metalloids (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Ni, Pb, Sb, Si, Sr, U, V and Zn). The results of principal components analysis showed two main clusters: locations Dopsin and Dalj (both characterized by high-intensity combat activity), where the concentrations of most elements (except Hg) were increased. Although the concentrations of metals and metalloids in cabbage samples collected in eastern Croatia did not exceed the maximum allowed values, the results of our study confirmed existance of environmental ‘hotspot’ with higher concentrations of war metals and metalloids. Our findings indicate that there is a possible common source and mechanism of transferring metals from the environment to the population.

KEYWORDS:

• War
• environment
• antimony
• lead
• arsenic
• soil
• vegetables
• spectrometry
• Croatia

Introduction

One of the major characteristics of war in Croatia was the destruction of civilian objects, the devastation of infrastructure and civilian casualties. The last combat activities occurred during 1999, with the destruction of all Danube bridges leading from Croatia to Serbia in a NATO bombardment campaign. The consequences on environmental and human health have not been explored to a significant extent, albeit some studies have been undertaken (Puntarić et al. 1997; Jergović et al. 2010; Vidosavljević et al. 2013; 2014). For areas with significant agricultural production and farming activities, such deadly heritage represents even a greater problem, with unforeseeable consequences (Domazet Lošo 2010). Among others, some studies warn of an increased incidence of malignant diseases in the post-war period in Croatia and neighbouring war-affected countries (Obralić et al. 2004; Ebling et al. 2005).

The possible metal and metalloid burden of eastern Croatia and the potential impact on the population’s health have only recently been systematically investigated; for example, water and soil analyses have been performed in the area (Ćurković 2010; Vidosavljević et al. 2013; Gvozdić et al. 2015). Compared with soil samples from locations of low-intensity combat activities (LICAs), soil samples from a site of high-intensity combat activities (HICAs) had high concentrations of As, Hg and Pb, which were above the values permitted by the national legislation for organic farming, as well as higher concentrations of Hg, which were even higher than the maximum allowed values for agricultural soils in general, according to the national legislation (Vidosavljević et al. 2013). First results of local population biomonitoring (serum, urine and hair) have also been published, suggesting differences depending on the level of exposure to war-related metals (Ćurković 2010; Jergović et al. 2010; Vidosavljević et al. 2014). Such studies revealed as an environmental ‘hotspot’ the area of the village Dalj and areas adjacent to the bridge across the Danube near Erdut, where higher concentrations of Sb, Pb, Al, Fe, Ni and Mg in biological (hair) and environmental samples have been measured (Vidosavljević et al. 2014).

Similar results regarding wildlife in the area have been published, especially considering older specimens of deer, which suggested contamination and long-term exposure via the environment (Lazarus et al. 2005). On the other hand, only two studies have been published on concentrations and possible impacts of metals in vegetables from the wider Zagreb area (Bošnir & Puntarić 1997; Puntarić et al. 2013). The tradition of growing vegetables in and around populated areas in Croatia, especially in eastern Croatia, is widespread, mainly due to fertile soil and the proximity to markets, but also as part of the culture. Green leafy vegetables, such as cabbage, significantly take up heavy metals and metalloids from the environment and are, therefore, of particular interest for biological monitoring. In previous studies, Brassicaceae such as spinach (Spinacea oleracea), lettuce (Lactuca sativa), endive (Cichorium endivia) and cabbage (Brassica oleracea var. Capitata) have been successfully used as biomonitors in regard to the bioaccumulation of heavy metals in vegetables growing in industrial and residential areas (Christ 1992; Voutsa et al. 1996; Uwah et al. 2009).

To date, research on metal and metalloid concentrations in food grown in war-affected areas has not been carried out. Croatia is as an area which has suffered from a five-year-long war and is still contaminated by mines. Particularly east Croatia, as a former significant food-producing area, is an important site for such research.

The main objective of this research was to measure concentrations of war-related metals in home-grown white cabbage from selected locations in eastern Croatia. We measured concentrations of selected metals and metalloids and determined whether such food is suitable for human and animal consumption.

In addition, from the perspective of ecological health, it is crucial to establish whether abnormalities in the presence of war metals in the population (civilians and soldiers) can be attributed to the intake of locally grown food, as a prolonged consequence of war, or to other sources, for example, water.

The investigation was conducted in a plain region of eastern Croatia, a part that stretches through Hungary, Serbia and Croatia. The main characteristic of the investigated area (Drava depression) is the alternation of coarse and fine-grained clastic materials. The last phases of deposition are characterized by a smaller average grain size. According to mineralogical and petrographic data, those sediments mostly represent eroded material from the Alps massif, while only smaller quantities are of Slavonian mountain provenance (Ujević et al. 2010). In a recent study, Vidosavljević et al. (2013) have measured the concentrations of 21 elements (metal and metalloids) in soil samples collected from this area. In addition, Ivezić et al. (2015) have reported the results of analysing 19 selected soil-quality indicators (bulk density, pH, soil organic carbon, dissolved organic carbon, inorganic carbon, cation exchange capacity, ammonium lactate extractable P and K, total N, Cu, Cd, Fe, Mn, Zn and water-extractable Cu, Cd, Fe, Mn and Zn).

Sampling locations were divided into two subgroups: one with high intensity of combat activities (war locations – HICAs) and one with low-intensity of combat activity or peacetime locations as control group (LICA). These results were compared with previously published results of soil analyses from the same location in order to determine the existence of so-called environmental hotspots, locations of significant metal burdening.

Materials and methods

Sampling locations

Sampling was carried during July 2013 (Figure 1) in the following settlements: Ćelije, the first destroyed village during the war in Croatia; Ernestinovo, one of the most developed agricultural villages before the war and a place with the highest electrical substation in this part of Europe, which sustained three months of heavy fighting; and Dalj, a village on the border with Serbia and in the vicinity of the Erdut–Bogojevo Bridge at the border crossing on the Danube, which was destroyed by NATO aircrafts in 1999. Other war sites (Vladislavci, Hrastin, Čepin and Dopsin) were villages on the front line, except the city of Osijek, where soil samples were also taken from army barracks. For the control group, three sampling sites, Potnjani, Draž and Našice (LICAs), were used (without registered war activities), with agricultural production and soil composition corresponding with the study group. Sampling sites were also chosen on the basis of similar geological composition so that each HICA site would have a similar LICA site, according to the geological map of eastern Croatia (CGS, Department of Geology 2009). The sites Dalj and Draž are situated on the banks of the Danube and consist of loess-like/Pleistocene sediments, while all other locations present alluvium/Holocene sediments. Apart from the city of Osijek, all other places have no significant industry. Agriculture and animal farming are the main activities.

Figure 1. Locations of the sampling sites in Croatia.

Sample collection and analysis

All samples were collected from gardens previously sampled for soil analyses (see Vidosavljević et al. 2013). External and internal cabbage leaves (white cabbage, approximately 100 g) were collected, packed into containers and sent to the laboratory for further analysis. Prior to the analyses, the leaves were rinsed with distilled water; subsequently, 1 g of cabbage was diluted with 9 ml HNO₃ and 3 ml H₂O₂. Concentrations were measured directly using inductively coupled plasma- mass spectrometry (ICP-MS) and are expressed in mg kg⁻¹.

All samples were analysed by ICP-MS (ICP-MS, ELAN DRC-e, Perkin Elmer, Waltham, MA, USA) with the following operating conditions: radio frequency generator power 1300 W; plasma gas flow 15 L/min; auxiliary gas flow 1 L/min; nebulizer gas flow 0.93–0.98 L/min; peristaltic pump speed 1 mL/min and nickel sampler/skimmer cones. Polyatomic interferences with elements such as Fe, As and Cr were eliminated with an instrumental dynamic reaction cell using the reactive gas CH₄. The instrument was calibrated after every 12th sample, using the external standard 71-Elemental Group Multi-Element Standard Solution (Inorganic Ventures, Christiansburg, VA, USA) and internal standards containing elements yttrium, indium, terbium and bismuth (Inorganic Ventures).

Although by using ICP-MS we determined the values of all 66 elements, for further data processing, we used only the 20 elements that are used in military production and have already been investigated in this particular region: Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Ni, Pb, Sb, Si, Sr, U, V and Zn (Wallace 2008; Vidosavljević et al. 2013; 2014).

Statistical analysis

To determine metal and metalloid concentrations at different sampling locations, mean and standard deviations were calculated. The data were analysed by Mann–Whitney U test and Pearson’s coefficient.

Principal components analysis (PCA) was carried out to determine the structure of the data and to observe the sources of variation in the data set. The PCA method was used to explore the relationships between the observed variables (metals and metalloids) and sampling locations (places). For statistical analyses, we used Statistica 7.0 (Statsoft Inc. Tulsa, Oklahoma, USA) (Jolliffe 2005).

Results

Except in the case of cadmium concentrations, for which we found a negative correlation between soil and cabbage (Pearson’s Cd (soil)/(cabbage) = −0.57; p = .031; p < .05), Pearson’s coefficients showed no significant correlations between the concentrations of the remaining 19 elements in the soil and cabbage samples.

The PCA provides the results given in Figure 2 for the scores (samples) and loadings (elements). Two main clusters of samples can be distinguished: a rather compact cluster (I) in the right portion of Figure 2 which contains most of the locations. It therefore defines common conditions, the normal level of contamination. A two-member cluster (II) containing locations 8 and 12 (HICA locations) in which the concentrations of most of the elements (except Hg) associated with intense military operations were slightly higher.

Figure 2. PCA of metals and metalloids in cabbage at locations of higher (HICAs, 1–12) and lower (LICAs, 13–14) intensity of combat activities in eastern Croatia.

Discussion

All measured values of analysed elements presented in Table 1 and covered by the regulations at the time of sampling were in compliance with the respective maximum permissible concentration (MAC) (Official gazette, 146/2012).

Table 1. Concentrations (mean, minimum and maximum) of metals and metalloids and maximally allowed concentrations (MAC; Puntarić et al. 2013) in cabbage samples from eastern Croatia (mg kg⁻¹ f.w.).

Element mg kg^−1	Mean (HICA)	Mean (LICA)	Min. (HICA)	Min. (LICA)	Max. (HICA)	Max. (LICA)	MAC mg kg^−1
Al	26.2 ± 20.0	19.9 ± 10.4	2.90	12.50	91.30	27.30	–
As	0.029 ± 0.018	0.017 ± 0.009	0.003	0.01	0.06	0.02	–
B	4.2 ± 2.0	4.3 ± 2.0	1.0	2.8	8.0	5.7	–
Ba	3.0 ± 2.1	2.4 ± 1.2	0.68	1.62	7.3	3.2	–
Cd	0.018 ± 0.012	0.015 ± 0.002	0.0029	0.01	0.04	0.02	0.2
Co	18.4 ± 10.3	17.3 ± 3.7	2.0	14.7	34.2	19.9	–
Cr	0.14 ± 0.09	0.07 ± 0.008	0.04	0.07	0.4	0.081	–
Cu	0.7 ± 0.6	1.2 ± 0.07	0.16	1.1	2.8	1.3	–
Fe	31.5 ± 24.0	26.0 ± 0.7	6.4	25.5	85.9	26.5	–
Hg	0.0027	ND	ND	ND	0.0027	ND	–
Li	0.09 ± 0.06	0.1 ± 0.08	0.009	0.05	0.2	0.1	–
Mg	704.86 ± 309	670.64 ± 127	70.672	580.06	1205.6	761.2	–
Ni	0.13 ± 0.08	0.14 ± 0.08	0.0015	0.0081	0.35	0.20	–
Pb	0.027 ± 0.02	0.02 ± 0.006	0.008	0.014	0.065	0.023	0.3
Sb	0.00085 ± 0.0001	ND	ND	ND	0.0022	ND	–
Si	0.03 ± 0.022	0.04 ± 0.008	0.004	0.032	0.08	0.04	–
Sr	6.0 ± 2.5	5.5 ± 1.4	1.6	4.5	1.0	6.5	–
U	0.001 ± 0.00082	0.001 ± 0.0009	ND	0,0008	0.003	0.002	–
V	0.055 ± 0.04	0.04 ± 0.002	0.011	0.038	0.15	0.04	–
Zn	4.8 ± 1.9	3.3 ± 0.5	1.8	2.9	9.0	3.6	–

Notes: Values are expressed as mg kg⁻¹ f.w. and reported as mean ± standard deviation, ND: not detected; HICA: high intensity of combat activity locations; LICA: low intensity of combat activity or peacetime locations as control group.

We have analysed zinc, cooper and iron concentrations in this study since high intake of these essential elements can result in adverse health effects. Similar low concentrations of non-toxic elements Zn (3.3–4. 8 mg kg⁻¹), Cu (0.7–1.2 mg kg⁻¹) and Fe (26–31.5 mg kg⁻¹) have been found by Vitale et al. (2007) for west Slavonia and Harmanescu et al. (2011) for Banat County, Romania. Although the results point to different concentrations in samples from HICA and LICA locations (Figure 2), those differences were not statistically significant. By comparing the measured values of arsenic presented in Table 1 with results from previous studies on green leafy vegetables from gardens in Zagreb and the north-eastern part of Croatia, we found that the measured values of As in Čepin (35.5 µg kg⁻¹), Dalj (40.63 µg kg⁻¹), Hrastin (52.90 µg kg⁻¹) and Ćelije (68 µg kg⁻¹) were twice as high compared to the ones measured in Zagreb (20 µg kg⁻¹). Interestingly, arsenic was not detected in cabbage samples in any of the investigated sites in the north-west of Croatia (Vitale et al. 2007). The elevated As concentrations in samples collected in eastern Croatia might be due to the natural presence of high amounts of arsenic in this region, mainly in water (Gvozdić et al. 2015) and soil (Ujević et al. 2010). The most likely arsenic sources are the minerals deposited in the deeper aquifers during the Middle and Upper Pleistocene; thus, the input of this element from combat activities is negligible. The mean measured arsenic values in vegetables were similar to those measured in some EU countries (Harmens 2005).

Lead and antimony concentrations in the war-affected areas such as Dopsin, Ernestinovo, Čepin and Dalj (Pb: 31, 42, 58 and 65 µg kg⁻¹; Sb: 1.6, 2.1, 2.1 and 2.2 µg kg⁻¹, respectively) significantly exceeded values measured in the remaining 10 locations (Fe: 9–19 µg kg⁻¹; Sb: <0.02 µg kg⁻¹) (Table 1). According to the research by Olive (2006), Sb and Pb are the two most important contaminants at shooting ranges, representing more than 85% of all contamination (Puntarić et al. 2013; Vidosavljević et al. 2014). The elevated Pb and Sb concentrations in soil samples and their grouping, presented in Figure 2, near the Erdut–Bogojevo Bridge in the vicinity of Dalj (Pb: 71.17 mg kg⁻¹; Sb: 3.10 mg kg⁻¹) can be considered as a result of combat activity of high intensity over a relatively small area. In 1991 this was location of fierce fighting between Croatian and Yugoslav forces, and in 1999 on the serbian side of bridge there were anti-aircraft artillery firing on NATO aircrafts bombing the bridge (Vidosavljević et al. 2014). These locations, especially the Dalj–Bogojevo bridge across the Danube, are, in theory, eligible for remediation. Phytoremediation would be most suitable in this case; however, funding is not available, especially since this bridge and the surrounding area are uninhabited and serve only as a border crossing between Croatia and Serbia.

Plants of the Brassicaceae family are considered useful for phytoremediation because of their tolerance to high levels of heavy metals; in addition, they can be used as a neutral fungicide and bacteriostatic and to improve crop yields and soil quality (Bączek-Kwinta et al. 2011; Szczyglowska et al. 2011; Kusznierewicz et al. 2012). Based on this, Antonkiewicz et al. (2016) found that due to its low tolerance to Ni, maize could be used as an indicator plant for environmental quality assessment.

Vitale et al. (2007) analysed metals in cabbage and potatoes in a number of different Croatian regions, including west Slavonia, dividing the sampled area between ‘wartime’ and ‘peacetime’ regions. The results are similar to the ones measured in our study, with a higher value of lead in samples from war zones in relation to peacetime areas. In the same study, they also calculated the approximate consumption and intake of lead, cadmium, copper and zinc by cabbage. They calculated an estimated consumption of 11.73 kg of cabbage per Croatian capital, with an average weekly intake of 0.005 mg of lead and 0.002 mg of cadmium and an average daily intake of 0.07 mg of zinc and 0.01 mg of copper per person, which are within the acceptable limits and no indicators of a significant impact of war-related activities (Vitale et al. 2007).

Cadmium and mercury are elements that are often associated with health risks (Bošnir & Puntarić 1997; Puntarić et al. 2013). Vegetables grown in urban and suburban areas are suitable indicators of environmental pollution (Voutsa et al. 1996; Hršak et al. 2003; Puntarić et al. 2013). Some of them, such as lettuce, chard and spinach, accumulate metals to a high degree, while cabbage, red cabbage, radishes and beets accumulate metals to a medium or high level (Puschenreiter & Horak 2005). Mean concentrations of cadmium presented in Table 2 (0.015–0.018 mg kg⁻¹) in cabbage samples from our study, in relation to those found in vegetables in Zagreb (Croatian capital city), Slovenia, Romania or Sweden (Jorhem et al. 2000), were lower and similar to those reported in the north-western part of Croatia (Vitale et al. 2007). The highest Hg concentration (2.7 µg kg⁻¹) was found in cabbage samples from an HICA location (Vladislavci), but was undetectable in the rest of the cabbage samples irrespective of their origin (HICA or LICA war area). Increases in soil uranium due to human activities may result in higher uranium contents in crops or vegetables, potentially entering the human food chain. Determination of uranium in a variety of foodstuffs from the USA and UK indicates that typical concentrations of uranium in fresh vegetables are around 2 µg kg⁻¹ (EFSA 2009). In contrast, uranium concentrations detected in cabbage samples from Portugal (near the Cunha Baixa uranium mine site) ranged from 14 to 255 µg kg⁻¹ (Neves et al. 2012). In our study, the mean concentration of uranium in cabbage samples was 1.35 µg kg⁻¹, suggesting that uranium exposure through the ingestion of these vegetables is insignificant.

Table 2. Overview of concentrations (mean, minimum and maximum) of metals and metalloids in cabbage samples from eastern Croatia compared to values found in other studies (Jorhem et al. 2000).

Element	Our study	Other studies (Jorhem et al. 2000)
Al	23.0 mg kg^−1 (mean)	8.8 mg kg^−1(mean)
As	0.023 mg kg^−1(mean)	0.02-0.06 mg kg^−1(min-max)
B	4.2 mg kg^−1(mean)	14 mg kg^−1(mean)
Ba	2.7 mg kg^−1(mean)	4.0 mg kg^−1(mean)
Cd	0.016 mg kg^−1(mean)	0.005-0.01 mg kg^−1
Cr	0.10 mg kg^−1(mean)	0.13 mg kg^−1(mean)
Cu	0.95 mg kg^−1(mean)	3-4 mg kg^−1 (min-max)
Li	0.09 mg kg^−1(mean)	0.5 mg kg^−1(DW); 4.9 mg kg^−1(AW) (mean)
Ni	0.135 mg kg^−1(mean)	1.03 mg kg^−1(mean)
Pb	0.02 mg kg^−1	1.7-2.4 mg kg^−1 (mean)
Sb	0.00085 mg kg^−1(mean)	0.0043 mg kg^−1(mean)
Sr	5.8 mg kg^−1(mean)	1.2-150 mg kg^−1(min-max)
U	0.001 mg kg^−1(mean)	0.0047 mg kg^−1(mean)
Zn	4.05 mg kg^−1 (mean)	27 mg kg^−1 (mean)

Published papers on metals in vegetables, in this case in cabbage, are extremely rare, especially in terms of metals and metalloids which are not considered in regulations. However, the ‘encyclopaedic’ work of Kabata-Pendias (2011) somehow fills this gap (Table 2). Comparing the author’s values with those obtained in our study, concentrations of Al, As, Cd and Cr were similar, Sr concentration was slightly lower and the values of B, Ba, Cu, Li, Ni, Pb, Sb and Zn were lower or significantly lower (Tables 1 and 2). It should be noted that no other studies have evaluated the consequences of war on metal and metalloid amounts in the environment; therefore, it is crucial to establish some sort of reference values.

The links between metals and metalloids from the soil and those found in plants depend on a number of factors, such as soil pH, presence and activity of microorganisms, length and extent of contamination and plant metabolism. In this context, this is a complex interrelationship to each other, and sometimes it is difficult to detect a connection (Bošnir & Puntarić 1997; Romić & Romić 2003). This study evaluated the relationship between soil metal concentration and metal levels in plants and only found a negative correlation for Cd; for the other studied metals, there was no significant correlation.

The results of the Mann–Whitney test showed that differences in terms of metal and metalloid contents between samples from HICA and LICA areas were statistically insignificant (p = .78, p < .05).

The PCA has depicted a compact central cluster (I) and a two-member cluster (II) for the locations Dopsin and Dalj, where, in addition to the already described higher Pb and Sb values (Dalj), levels of almost all of the elements, except Hg, were elevated (Figure 2). Here, by using cabbage as an example, we could confirm that Dalj and its surrounding area are an environmental ‘hotspot’, according to previously established positive correlations between levels of Al, Fe, Mg and Ni in the soil and in hair and plant (Taraxacum officinalis) samples and levels of B, Cu, Si, Sr and Zn in serum, urine and water, thus suggesting a common source and metal transmitting mechanisms (Vidosavljević et al. 2014; Puntarić et al. 2013). Dalj is defined as a place of large-scale destruction because, in addition to the 1991 Croatian war for independence, its bridges over the Danube have been destroyed during a NATO bombing campaign in 1999.

The source of increased uranium concentrations in samples from the wider Našice area has not been identified yet; however, there were no connections between high uranium soil values and uranium concentrations in the studied vegetables.

There is no doubt that the war in east Croatia has left residues additionally burdening the environment and the population with metals and metalloids. Despite the large-scale destruction, investigated vegetables from this area are safe for human consumption, and measured abnormalities, in general, are more of academic significance, showing the presence of the same elevated metals from soil, through vegetables to humans. Despite the confirmed ‘hotspot’ in Dalj, the use of cabbage as an indicator of environmental contamination with metals suggests that the total load of metals in eastern Croatia after the war is not significantly higher, but further research and constant monitoring of the area are needed.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Dragana Jurić is Ph.D. student at J.J. Strossmayer University Osijek.

Dinko Puntraić, MD, Ph.D., is professor of epidemiology and health ecology at Croatian Catholic University Zagreb.

Vlatka Gvozdić, Ph.D., is associate professor of chemistry at J.J. Strossmayer University in Osijek.

Domagoj Vidosavljevic, MD, Ph.D., is assistant Professor at Faculty of Medicine, J.J. Strossmayer University Osijek.

Zdenko Lončarić, Ph.D., is professor of Agroecology at Faculty for Agriculture, J.J. Strossmayer University Osijek.

Ada Puntarić is student at Faculty for Food technology and biotechnology, Zagreb University.

Eda Puntarić is researcher at Croatian environmental Agency.

Ida Puntarić, MD, is Physician at Zagreb County Community Health Center.

Marina Vidosavljević, MD, is specialist at County general Hospital Vinkovci.

Lidija Begović, Ph.D., is assistant professor of Biology, J.J. Strossmayer University Osijek.

Siniša Šijanović, MD, Ph.D., is Professor at Faculty of Medicine, J.J. Strossmayer University in Osijek.

Additional information

Funding

This work was supported by the Ministry of Science and Education, Republic of Croatia.