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Map showing the distribution of Balkan endemic nephropathy regions adapted after Tatu et al. Hypotheses that have been proposed for the etiology of BEN include: lead contamination of wheat flour Danilovic et al. Strengths and limitations of hypotheses have been reviewed elsewhere Plestina ; Stefanovic ; Tatu et al. The Pliocene lignite hypothesis posits that BEN is caused by long-term exposure to low concentrations of aromatic hydrocarbons and other toxic organic compounds leaching into well and spring water via groundwater from low-rank coals, that is, Pliocene lignites, found in the vicinity of endemic settlements Feder et al.

The rural population uses the well and spring water almost exclusively Tatu et al.

The low concentration of the toxic organic compounds may account for the slow development i. Limited information is available on the distribution of the coal deposits and the hydrology in the endemic areas Radovanovic and Peric ; Feder et al. Geologic structure differs from one village to another Craciun and Rosculescu ; Feder et al. In Romania and the former Yugoslavia, an 80—m column of Tertiary sediments would typically contain 10—15 Pliocene lignite layers Feder et al.

The mainly Pliocene age 1.

Aromatic units in coal

Therefore, the endemic villages may be located below the coal layers located in the surrounding hills , or overlying the layers, and there are instances of wells penetrating through the coal layers, for example, in the endemic villages of Poroina, Romania and Vreoci, Serbia. In Bulgaria, extensive coal deposits occur north and south of the endemic region, and the endemic region is geologically defined by Pliocene sediments Feder et al.

Furthermore, in , we discovered the presence of at least two types of coal in the Bulgarian endemic areas, one being a low-rank lignite, very similar to Romanian and Serbian endemic Pliocene lignites. The well and spring water in endemic regions is largely supplied by aquifers with a relatively fast flow rate, for example, from rain.


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However, the underground transport pathway may be highly variable Niagolova et al. The aquifers are shallow, as evidenced in part by the high nitrate levels Orem et al. Given the relatively fast flow rate and the shallowness of the aquifers, it is unlikely that the well and spring water is supplied to a significant extent by, for example, fossil aquifers and paleowater.

Inorganic geochemical analyses of well and spring water samples from Romania have shown no significant differences between endemic and nonendemic regions Orem et al. For example, a greater number of organic compounds Goldberg et al.

Organic compounds in water extracts of coal: links to Balkan endemic nephropathy | SpringerLink

Also, methanol extracts of lignite samples showed that endemic Pliocene coals possessed a complexity that was not matched by any nonendemic lignites, including a nonendemic Pliocene age lignite Orem et al. In addition, human mesenchymal stem cells exposed to a water extract of an endemic area Pliocene lignite showed increased cellular proliferation and differentiation compared to the control Suciu et al. However, a number of criticisms about the Pliocene lignite hypothesis, and many of the above previous studies have been raised Pfohl-Leszkowicz et al.


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  • For example, evaluations of the research that supports the Pliocene lignite hypothesis concluded that studies have not moved beyond a weak topographical association Voice et al. Demonstration of a cause-and-effect relationship was considered far away Radovanovic , in part because the source of the organic compounds in the well and spring water has been unconfirmed Orem et al. Some limitations of the Pliocene lignite hypothesis have been addressed in the literature, for example, regarding the topographical association between the deposits and endemic BEN areas Tatu et al.

    A comprehensive compilation of the possible limitations of the hypothesis, including whether other environmental agents may be more plausible as etiological factors than exposure to Pliocene lignites, for example, aristolochic acid from A. In addition, studies on Pliocene and other lignites from the Balkans, for example, Bulgaria Stefanova et al.

    For example, experiments using coal samples Koukouzas et al. Therefore, we have conducted aqueous leaching experiments on a variety of coal samples, including Pliocene lignites from endemic BEN areas, in order to simulate groundwater leaching of organic compounds, and to further test the role of the Pliocene lignite hypothesis in the etiology of BEN. Herein, presented for the first time, is the identification and concentration of organic compounds leached by water from endemic and nonendemic area coal samples under various experimental conditions.

    Results provide direct evidence of the water-soluble, water-leachable, and water-extractable organic compounds, and therefore, evidence for also possibly linking the organic compounds present in the well and spring drinking water from endemic locations to the proximal Pliocene lignite coal deposits.

    Preliminary results only have been published Orem et al. This study will add to the increasing body of knowledge in the re-emerging discipline known as medical geology Finkelman et al. IRC internal reference coal. Rt room temperature, Hb hot water bath, Sx Soxhlet. Ten grams of coal were used in each experiment.

    The Chemistry and Technology of Coal

    One endemic area sample i. No attempt was made to mimic the elements or compounds present in natural groundwater in the water used for experiments, in order to ensure that results could be directly linked to experimental conditions, as possible side interactions could have occurred due to the presence of introduced solutes. Furthermore, laboratory experiments that simulated groundwater transport have often not mimicked aquifer ion composition Bales et al. Finally, it is unlikely that the inorganic parameters of the water have a significant effect on the extractability of the organic compounds.

    Samples were held at maximum temperature for the entire duration of the experiments. Laboratory blanks consisted of milliQ water extracted with pesticide grade dichloromethane, subjected to the same processing conditions as samples. Average ion composition was calculated for each peak, and peak deconvolution was performed on all peaks.

    Thermal alterations of organic matter in coal wastes from Upper Silesia, Poland

    External aliphatic and aromatic standards, injected under the same conditions as samples, were used for quantification. To quantitate compounds in samples, a subset of 3 standard compounds with known retention times were chosen for each sample. Compounds in samples were quantified using standards with retention times that best corresponded to the retention time ranges i. As the majority of sample compounds were not quantified with the actual standard for that compound, the majority of sample compound concentration results are semi-quantitative.

    Inferential statistics were not employed because group sizes were too small for results, whether they would have shown statistical significant or no statistical significance, to have been meaningful. Laboratory blank concentrations of organic compounds were very low, and show that contamination associated with laboratory methods, for example, extraction and rotoevaporation, was negligible.

    Pebble-sized Husnicioara samples run under higher temperature experimental conditions i. Many samples contained phthalate esters, ubiquitous environmental contaminants of anthropogenic origin. As laboratory blank concentrations of organic compounds were very low, the phthalate esters represent contamination of the coal prior to processing in the laboratory. Greater numbers and higher concentrations of organic compounds were present in aqueous leachates of endemic area Pliocene lignites compared to the other coals examined.

    Though some organic compound information from extracts of bituminous coals have been presented before Zhao et al. Results of leaching experiments using lignite samples may be influenced by different grain sizes because of differences in surface area Izquierdo et al. The higher concentration of aromatic compounds in the higher temperature i. Similar concentrations of saturated and unsaturated aliphatic compounds and their functional derivatives were found in aqueous leachates of endemic area Pliocene lignites and the other coals examined, suggesting that these compounds do not play an etiological role in BEN.

    Many aromatic compounds, for example, PAHs and aromatic amines, are known or suspected carcinogens, or have been linked to urinary tract cancer and tubulointerstitial nephropathies Tatu et al.


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    • A BEN causative agent or agents may be among the numerous aromatic compound classes identified in the extracts phenols, PAHs, benzenes , as for example, in the experiments designed to gauge exposure to groundwater leaching of organic compounds over long periods of time i. Many organic compounds in endemic area Pliocene lignite coal extracts were also found in water supply samples Orem et al.

      In addition, phenolic compounds and aromatic derivatives present in the water supply samples were attributed to either anthropogenic pollution or a geologic source Orem et al. However, the higher concentrations of hydroxy-, methoxy-phenols, PAHs and derivatives, and hydroxy-, methoxy-benzenes present in the higher temperature extracts of endemic area Pliocene lignites compared to extracts of other coals , show that the organics detected in the endemic water supply samples are more consistent with a geologic source, that is, Pliocene lignites.

      The proposed exposure pathway of the Pliocene lignite hypothesis i. However, exposure to organic compounds leached from Pliocene lignite coal deposits into well and spring drinking water in endemic BEN areas alone cannot explain the etiology of the disease, as for example, not all individuals residing in endemic villages develop BEN. Genetic susceptibility is also involved in the emergence of the disease.

      For example, the gene-environment interaction is translated into unusual xenobiotic substance metabolism controlled by cytochrome P and other enzymes that increases the risk to develop BEN only in those individuals that bear certain gene variants that code for the detoxification enzymes Atanasova et al. Additional research on this subject is warranted for an improved understanding of the role of the Pliocene lignite hypothesis in the etiology of BEN.

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      Also, a compound-by-compound assessment for known or suspected causal links to tubulointerstitial nephropathies and urinary tract cancers appear needed, as well as a compound-by-compound comparison of endemic and nonendemic area extract yields from this study to those of water supply samples. Future experiments should be conducted on a greater number of samples, using longer time durations, and with water that better mimics the natural inorganic groundwater composition.

      Coal samples for future experiments should include Pliocene lignites from neighboring countries with no known cases of BEN, for example, Greece, Slovenia, and Turkey, in order to further narrow the comparison of coals to similar rank and flora. This study supports the role of the Pliocene lignite hypothesis as a factor in the etiology of Balkan endemic nephropathy. Water-soluble, water-leachable, and water-extractable aromatic compounds with functional groups with potential toxicity have been demonstrated to be leached from endemic area Pliocene lignite coal samples under a number of experimental conditions.

      In addition, some of these same compounds have been identified in endemic but not nonendemic area water supply samples, and therefore, indicate a link to the proximal Pliocene lignite coal deposits.