We found significant differences in metals/metalloid concentrations in biological samples between adult populations in eastern Croatia that had experienced heavy and those that had experienced moderate fighting during the 1991-1995 war. Currently, there are no internationally recommended reference values for exposure to the metals analyzed in this work. Nevertheless, the concentrations of most elements investigated in the present study exceeded the reference ranges recommended by particular authors (17
). To the best of our knowledge, this is the first study relating elevated concentrations of metals and metalloids in human samples to previous armed activities and war actions.
The strengths of this study lie in the fact that three different biological samples were analyzed from each participant, and the analysis was performed using ICP-MS, a remarkably powerful method for (ultra)trace element determinations (29
). Superior to other methods, ICP-MS has extremely low limits of detection for various elements, wide multielement capability, and high sample throughput. The method allows simultaneous detection of a large number of elements in small samples and differentiates between different isotopes of the same element (18
Serum concentrations of Al, As, Ba, and V were higher in participants from areas of heavy fighting than in participants from areas of moderate fighting. These results are in accordance with the previously established association between these elements and heavy artillery projectiles (7
), and they indicate extensive and indiscriminate use of heavy artillery during the war in Croatia, especially by the Yugoslav Army. The fact that the elevated serum concentrations of these elements in our participants have persisted to the present, almost two decades after the outbreak of war, suggests that the populations living in areas of heavy fighting may still be exposed to residual metal contamination from heavy artillery ammunition.
As and Cd were the only two elements that showed higher urine concentrations in participants from areas of heavy fighting than in participants from areas of moderate fighting. As urine concentrations are indicators of acute exposure, these findings suggest that the level of contamination with most of the metals and the metalloid under study has decreased with time.
Hair concentrations of Al, As, Cd, Fe, Pb, and V, all of which are associated with bombs and heavy artillery ammunition, were higher in participants from areas of heavy fighting than in participants from areas of moderate fighting. These findings confirm hair as a useful indicator of long-term metal exposure, making it a valuable matrix for use in biomonitoring studies, as reported by other authors (29
). Low U concentration in the hair of participants from areas of heavy fighting confirmed the assumptions that depleted-uranium weapons were rarely used or not used at all in the 1991-1995 war in Croatia (23
Unexpectedly, participants from areas of moderate fighting showed higher serum concentrations of Cr and Ni and higher U concentrations in hair. Cr and U exposure may be related to cement industry, which is present in the studied areas (17
). Non-warfare sources of Ni exposure are unclear, since there are no mining, steel, or electroplating production industries in this area (17
Since some authors have demonstrated an effect of Co, Ni, and U from embedded shrapnel on genotoxic and tumorigenic pathways (14
), we considered wounding as a possible confounding factor in our study. When we compared levels of metals in wounded participants from areas of heavy fighting with non-wounded participants from the same areas, only one element (Al) in one matrix (serum) was found to be significantly higher in wounded participants. Therefore, we believe that different proportions of wounded participants from areas of heavy and moderate fighting can be excluded as a confounding factor.
A larger proportion of participants from areas of heavy fighting than of participants from areas of moderate fighting was at risk for occupational exposure. Although we did not find higher concentrations of any studied element among farmers compared with non-farmers, we cannot exclude pesticide as a confounding factor due to the small number of farmers in our subgroup of participants with occupational exposure. The differences in serum As, Cd, and Cu concentrations and hair As concentrations between participants with and without occupational exposure may be caused by confounding factors.
We also investigated correlations between the elements usually connected with occupational exposure and smoking. It is unlikely that As, Cd, and Pb had a significant confounding influence on our results: the coefficients for these metals were similar between participants with and without occupational exposure, as well as between smokers and non-smokers. One exception is As, for which significantly stronger correlations between sample matrices were found in smokers than in non-smokers, as expected. Nevertheless, since the percentage of smokers is similar in the groups from areas of moderate and heavy fighting, it seems less likely that smoking affected the overall results.
A limitation of the study is the small sample size. This made it impossible to estimate age, sex, and occupational exposure differences between the groups. Participants from areas of heavy fighting were older and more often male than participants from areas of moderate fighting. This makes sense given the predominance of men among war veterans and the war wounded. Another limitation is that this study did not consider all possible contributions from non-combat factors, such as variations in the concentrations of metals and metalloids in drinking water, local hydrogeologic characteristics, and exposure from local industry (17
Future scale up of this small-scale study, including a larger number of participants, may help us to verify the results and possibly detect additional important differences, to establish national and international reference values, comprehensively assess the risk of metal and metalloid exposure in populations living in former conflict zones in Croatia, identify other mechanisms of metal exposure in these and other populations of Croatia, and lead to the development and implementation of preventive and corrective measures.