Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy (BEN)). In Bulgaria, much higher prevalence of ochratoxin A (OTA), exceeding 2 microg/L, was observed in the blood of affected population. OTA is found more often in the urine of people living in BEN-endemic villages. To confirm and quantify exposure to OTA in Vratza district, we followed up OTA intake for 1 month, OTA in blood and urine from healthy (20-30 years old) volunteers, from two villages with high risk for BEN disease. Food samples were collected daily, blood and urine at the beginning of each week. Relations between increasing OTA intake, blood concentration and elimination of OTA in urine have been studied in rats. Average weekly intake of OTA varies from 1.9 to 206 ng/kg body weight, twice tolerable weekly intake recommended by JECFA. OTA blood concentrations are in the same range as previously reported in this region with concentrations reaching 10 microg/L. Weekly OTA food intake is not directly correlated with blood and urine concentrations. Biomarkers of biological effects such as DNA adducts were detected in patients affected by urinary tract tumours (UTT) and in rat study. All these plead for the implication of OTA, in BEN and UTT.

Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.

Deoxynivalenol (DON) was analysed in 313 beer samples collected from the European retail market using a commercially available immunoassay kit (enzyme-linked immunosorbent assay, ELISA). The incidence rate was about 87%, while most samples (73%) had contamination levels lower than 20 ng m(-1). The contamination ranged between 4.0 and 56.7 ng ml(-1), with an average of 13.5 ng ml(-1). A statistically significant correlation between alcohol levels and DON contamination was found, as well as a significant difference between bottom, top and spontaneous fermenting beers. Twenty-seven beer samples were compared using a second ELISA kit and a good correlation was obtained between the two kits (r = 0.93). Although when compared with gas chromatography-mass spectrometry the ELISA tended to overestimate the results, a good correlation (r=0.94) between the two methods was observed. Monitoring of DON in beer is important considering that DON production is dependent on the weather and that it can contribute significantly to the tolerable daily intake of DON, especially for frequent beer consumers.

In the 1950s, a series of publications from Bulgaria, Yugoslavia, and Romania locally described a kidney disease called Balkan Endemic Nephropathy (BEN). In Bulgaria, the exposure of populations to ochratoxin A (OTA) was supported by analysis of individual food items demonstrating a higher prevalence and higher levels of OTA in food from the high-incidence areas of BEN. In this work, food consumption from a series of individuals from two villages of the BEN area during 1 month was followed using the duplicate diet method. Meals consumed by volunteers from both villages showed uneven OTA contents, spreading from below the limit of quantification (<0.07 microg/kg) to 2.6 microg/kg. The average weekly intake of OTA varies from 1.86 to 92.7 ng/kg of body weight. Some of these levels approach the provisional tolerable weekly intake (PTWI) established by the JECFA at 100 ng/kg of body weight. These results confirm previous studies performed in the same area and demonstrate the high exposure of this population to OTA, thus strengthening the hypothesis of the involvement of this mycotoxin in BEN etiology.

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 micrograms/kg (DON) and 17 micrograms/kg (ZEA), maximum concentrations were 1800 micrograms kg-1 and 120 micrograms kg-1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1% and 0.7% of the samples, at at maximum level of about 100 micrograms kg-1. Only one sample was positive for T-2 (55 micrograms/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.