盐城生物圈保护区越冬丹顶鹤受到真菌毒素威胁的研究

Potential natural exposure of endangered red-crowned crane ( Grus japonensis ) to mycotoxins aflatoxin B 1 , deoxynivalenol, zearalenone, T-2 toxin, and ochratoxin A 盐城生物圈保护区越冬丹顶鹤受到真菌毒素威胁的研究 1 2 first-author 1 3 4 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:wuwenda@njau.edu.cnluchanghu@njfu.com.cn †E-mail:wuwenda@njau.edu.cnluchanghu@njfu.com.cn wuwenda@njau.edu.cn 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:wuwenda@njau.edu.cnluchanghu@njfu.com.cn †E-mail:wuwenda@njau.edu.cnluchanghu@njfu.com.cn wuwenda@njau.edu.cn Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China Yancheng Biosphere Reserve, Yancheng 224057, China Yancheng Biosphere Reserve, Yancheng 224057, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing 210042, China Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing 210042, China A survey was conducted to determine whether mycotoxins were present in the foods consumed by red-crowned cranes ( Grus japonensis ) in the Yancheng Biosphere Reserve, China. Collected in the reserve’s core, buffer, and experimental zones during overwintering periods of 2013 to 2015, a total of 113 food samples were analyzed for aflatoxin B 1 , deoxynivalenol, zearalenone, T-2 toxin, and ochratoxin A using high performance liquid chromatography (HPLC). The contamination incidences vary among different zones and the mycotoxins levels of different food samples also presented disparity. Average mycotoxin concentration from rice grain was greater than that from other food types. Among mycotoxin-positive samples, 59.3% were simultaneously contaminated with more than one toxin. This study demonstrated for the first time that red-crowned cranes were exposed to mycotoxins in the Yancheng Biosphere Reserve and suggested that artificial wetlands could not be considered good habitats for the birds in this reserve, especially rice fields. 目的 2 调查在盐城生物圈保护区越冬的丹顶鹤食物是否受到真菌毒素污染以及评估丹顶鹤受到真菌毒素威胁的风险。 创新点 2 首次证明在盐城生物圈保护区越冬的丹顶鹤受到真菌毒素的威胁。 方法 2 2013年11月至2015年3月，两个越冬期内在盐城生物圈保护区采集丹顶鹤不同觅食生境内113份食物样品。使用高效液相色谱法对这些食物中毒素（黄曲霉毒素B 1 （AFB 1 ）、脱氧雪腐镰刀菌烯醇（DON）、玉米赤霉烯酮（ZEN）、T-2毒素（T-2）和赭曲霉毒素A（OTA））含量进行检测。 结论 2 盐城生物圈保护区内丹顶鹤食物受到真菌毒素的污染，人工湿地尤其是稻田不适宜作为鸟类的觅食地。 Food Mycotoxin Red-crowned crane Yancheng Biosphere Reserve 食物 真菌毒素 丹顶鹤 盐城生物圈保护区 Introduction 1 Mycotoxins are toxic substances produced as secondary metabolites mainly by filamentous fungi, such as Aspergillus , Penicillium , and Fusarium species (Nielsen et al., 2009 2009 ; Marin et al., 2013 2013 ). These metabolites have frequently been found worldwide in foods and feeds, and are known to cause acute and/or chronic toxicity in humans and animals, depending on the amount consumed (Scudamore et al., 1997 1997 ; Placinta et al., 1999 1999 ; Lee et al., 2010 2010 ; Li et al., 2014 2014 ). Fink-Gremmels ( 1999 1999 ) has estimated that approximately 25% of crops worldwide are contaminated with mycotoxins. To date, more than 400 mycotoxins have been identified as part of a growing interest in this field of research and availability of modern laboratory methods (Sulyok et al., 2010 2010 ). The most important mycotoxin members are aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and ochratoxin A (OTA), because of their ubiquitous nature, causing disease in humans and other domestic animals following ingestion of contaminated foods (Santos et al., 2009 2009 ; Zain, 2011 2011 ). The International Agency for Research on Cancer (IARC) has classified AFs and OTA as Groups 1 and 2B carcinogens, respectively (IARC, 1993 1993 ). AFs are fungal metabolites primarily from two species of Aspergillus , A. flavus and A. parasitius . AFs are extremely potent carcinogens in all animal species tested, including rats, mice, fish, hamsters, ducks, and humans. Among AFs, aflatoxin B 1 (AFB 1 ) is the most common and toxic form (Marin et al., 2013 2013 ). OTA, produced by many species of Aspergillus and Penicillium , is carcinogenic, nephrotoxic, teratogenic, immunotoxic, and hepatotoxic (Hussein and Brasel, 2001 2001 ). DON, ZEN, and T-2 are mainly produced by the mold genus Fusarium (Zinedine et al., 2007 2007 ; Kolf-Clauw et al., 2008 2008 ). Long-term exposure of animals to feeds contaminated with DON causes reduced live weight gain, decreased nutrient efficiency, and increased susceptibility to infectious diseases (Swamy et al., 2004 2004 ). ZEN, also known as F-2 toxin, is of relatively low toxicity. However, a ZEN property that causes estrogenic syndromes in reproductive tracts of animals, such as pigs, cattle, and poultry, has been recognized (Minervini and Dell'Aquila, 2008 2008 ). T-2 toxic effects include immunomodulation, cytotoxicity, inhibition of protein, DNA, and RNA syntheses, cell lesions in digestive tract, organs, and skin, and neural disturbances (Stafford and McLaughlin, 1973 1973 ; Sklan et al., 2003 2003 ; Parent-Massin, 2004 2004 ; Meissonnier et al., 2008 2008 ). Historically, a number of mycotoxin studies have been performed because of the economic implications of these compounds on the animal husbandry industry or public health risks. However, in recent years, studies have focused on mycotoxin impacts on wildlife populations. Lawson et al. ( 2006 2006 ) have identified hepatic AF residues in house sparrow ( Passer domesticus ) and greenfinch ( Carduelis chloris ) in Britain, and reported that the residues resulted from high dietary AF exposure, although the source(s) of dietary AFs was unknown. Some clusters of mycotoxicosis, with associated morbidity and mortality, also have occurred in cranes. About 9500 sandhill cranes ( Grus canadensis ) died in Gaines County, Texas and Roosevelt County, New Mexico, USA, between 1982 and 1987. The most probable cause of this mortality was mycotoxins produced by Fusarium spp. that grew on peanuts left in fields after harvest and found to be the predominant food of these dead cranes (Windingstad et al., 1989 1989 ). Pelleted feed contaminated by T-2 and DON sickened 80 of 300 captive whooping cranes ( G. americana ) and sandhill cranes, and caused the death of 15 of these cranes at the Patuxent Wildlife Research Center, Laurel, Maryland, USA, in 1987 (Olsen et al., 1995 1995 ). The red-crowned crane ( G. japonensis ) is one of the rarest crane species and has been classified as “endangered” on the International Union for Conservation of Nature (IUCN) Red List (IUCN, 2014 2014 ). The current population is estimated to be 2750 individuals (BirdLife International, 2015 2015 ). A large proportion of the migratory population migrates from northeastern China to Yancheng in late October and overwinters in the Yancheng Biosphere Reserve, China, itself the largest wintering area for the migratory population, until early March (Ma et al., 1999 1999 ; Wang, 2008 2008 ). Red-crowned cranes are omnivorous, whose diet includes rice, grain, wheat seedlings, shellfish, fish, shrimp, crabs, snails, seepweed seed and leaf, and reed roots (Ma et al., 2000 2000 ; Ma and Li, 2002 2002 ; Dong et al., 2005 2005 ). As Yancheng is located in a climatic zone with temperature and humidity suitable for mold growth and mycotoxin production (Fan, 2008 2008 ), it is likely that some of these foods are contaminated by mycotoxins. Such a situation might have a negative impact on the health and reproduction of these cranes, and thus potentially threatens the population. The objectives of this study are to confirm whether these five mycotoxins were present in the cranes’ food and to discuss the potential for mycotoxin impact on the health of red-crowned cranes in the Yancheng Biosphere Reserve, China. Materials and methods 1 Chemicals and instruments 2 Mycotoxin standards for AFB 1 , DON, ZEN, T-2, and OTA were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) and stored at −20 °C until use. Immunoaffinity columns (IACs) were obtained from R-Biopharm AG (Darmstadt, Germany). High performance liquid chromatography (HPLC)-grade acetonitrile, methanol, and acetic acid were obtained from Merck KGaA (Darmstadt, Germany). Ultrapure water was produced by a Millipore Milli-Q system (Millipore, Billerica, MA, USA). All other chemicals and reagents used were of analytical grade. Study area 2 This study was conducted in the Yancheng Biosphere Reserve (32°34 ' –34°28 ' N, 119°48 ' –120°56 ' E), which is located in Yancheng City, Jiangsu Province, China. The reserve was established for red-crowned cranes, other waterbirds, and coastal wetlands in 1983, and in 1992 the United Nations Educational, Scientific, and Cultural Organization (UNESCO) recognized it as an international biosphere reserve. The reserve was divided into three zones, the core, buffer and experimental zones, to integrate biodiversity conservation and economic development of local communities (Fig. 1 1 ). Red-crowned cranes in the reserve are the principal users of five major foraging habitats, including the common seepweed community ( Suaeda glauca ), a reed pond ( Phragmites communis ), farmland (wheat and rice fields), a salt pan, and an aquaculture pond. The seepweed community and reed pond were mainly located in the core zone with little human disturbance, as a type of natural wetlands, providing cranes with crabs, snails, seepweed seed and leaf, and reed root. The farmland, salt pan, and aquaculture pond comprised entirely artificial wetlands and occurred in the buffer and experimental zones, supplying cranes with rice grain, wheat seedings, shellfish, fish, and shrimp (Ma et al., 2000 2000 ; Liu et al., 2013 2013 ). Samples 2 A total of 113 samples were collected at 15–20 d intervals from November through to March 2013–2014 and 2014–2015 (Table 1 1 ). The corresponding foods were collected when cranes were observed feeding in foraging habitats. For each kind of food three sampling stations were chosen for each sampling date. The sample sites were more than 3 km from each other within the same wetland. In each site, the first available 50 g of each food was obtained at random. Then the same kind of food was combined to form a single sample. The samples were stored in an ice box in the field and transported to the laboratory within 8 h. All samples were ground using a grinder. The grinder was cleaned between samples thoroughly to avoid cross-contamination of samples. After grinding, samples were homogenized and stored in sealed plastic bags at −80 °C until subsequent HPLC analysis. NA: not available. According to the 2010–2015 winter survey, there were no cranes in salt pan, and no crane feeding was detected during the present sampling in this foraging habitat 2 Zone 2 Foraging habitat 2 Food sample 2 Number of samples Nov.–Mar. 2013–2014 Nov.–Mar. 2014–2015 Core Reed pond Crabs 7 8 Reed root 9 9 Common seepweed community Seepweed seed and leaf 9 9 Crabs and snails 6 7 Buffer and experimental Rice field Rice grain 8 7 Wheat field Wheat seedlings 9 9 Aquaculture pond Fish and shrimp 7 9 Salt pan Shellfish and fish NA a a NA AFB 1 analysis 2 A 25-g sample, prepared as above, was homogenized with 125 ml of methanol/water solution (70/30, v/v) and 5 g of NaCl for 30 min. The mixture was filtered through Whatman No. 4 paper, 15 ml of the filtrate diluted with 30 ml of ultrapure water, and then 15 ml of the resulting solution applied to an AFB 1 IAC. The column was then washed with 20 ml of ultrapure water and dried by blowing air through the column, and the target analyte was eluted three times with 1.0 ml of methanol. The eluate was evaporated to dryness at 40 °C under a stream of nitrogen. The residue was derivatized with trifluoroacetic acid according to the method of Khayoon et al. ( 2010 2010 ). Then, 1 ml of acetonitrile/water (30/70, v/v) was added. A 20-μl sample was suction-filtered through a 0.45-μm nylon membrane and injected into the HPLC system. AFB 1 analysis was performed on an HPLC system equipped with a fluorescence detector (HPLC-FLD) and separation achieved using a C 18 column (150 mm×4.6 mm, 5 μm particle size; Shimadzu Corp., Kyoto, Japan). The mobile phase was acetonitrile/water (30/70, v/v) with a flow rate of 0.8 ml/min. Fluorescence was detected at 365 and 418 nm for excitation and emission, respectively. The column temperature was 30 °C and the limit of detection and limit of quantification (LOD and LOQ, respectively) of the method were 0.03 and 0.09 μg/kg, respectively. The mean recovery was 90% with a relative standard deviation (RSD) of 4%. DON analysis 2 A 25-g sample was homogenized in 200 ml of ultrapure water for 30 min. The mixture was sequentially filtered through Whatman No. 4 and glass fiber filter papers, and then 2 ml of this extract was applied to a DON IAC. The column was then washed with 5 ml of ultrapure water and flushed with air to remove remaining water. DON bound to the column was eluted using three 1.5-ml volumes of methanol and the eluate evaporated to dryness at 40 °C under a steam of nitrogen. The residue was dissolved with 1 ml of acetonitrile/water (16/84, v/v) and a 20-μl sample was suction-filtered through a 0.45-μm nylon membrane and injected into the HPLC system. DON analysis was carried out on an HPLC system equipped with an ultraviolet detector and a C 18 column (150 mm×4.6 mm, 5 μm particle size). The mobile phase was acetonitrile/water (16/84, v/v), flow rate 0.8 ml/min, column temperature 30 °C, and detection at 218 nm. LOD and LOQ for this method were 4.2 and 13.44 μg/kg, respectively, and the mean recovery was 105% with an RSD of 5%. ZEN analysis 2 A 25-g sample was homogenized with 125 ml of acetonitrile/water (75/25, v/v) for 30 min and filtered through Whatman No. 4 paper. A 10-ml volume of the extract was then diluted with 40 ml of phosphate buffered saline (PBS) and then 25 ml of the diluted sample applied to a ZEN IAC. The column was then washed with 20 ml of PBS and dried by blowing air through the column. ZEN was finally eluted using three 1.5-ml volumes of acetonitrile. A 20-μl sample of the extract was finally suction-filtered through a 0.45-μm nylon membrane and injected into the HPLC system. Chromatographic separation was performed on a C 18 column (150 mm×4.6 mm, 5 μm particle size), with a methanol/acetonitrile/water (10/44/46, v/v/v) mobile phase and 0.8 ml/min flow rate. The excitation and emission wavelengths were 274 and 440 nm, respectively, and the column temperature was 25 °C. LOD and LOQ for this method were 2.0 and 6.2 μg/kg, respectively, and the mean recovery was 83% with an RSD of 5%. T-2 analysis 2 A 25-g sample was homogenized with 125 ml of methanol/water (90/10, v/v) for 30 min and filtered through Whatman No. 4 paper. A 7-ml volume of the extract was mixed with 28 ml of ultrapure water, passed through a glass fiber filter paper, and then 25 ml of the resulting solution was applied to a T-2 IAC. The column was washed with 20 ml of ultrapure water and T-2 eluted using three 1.5-ml volumes of methanol. The eluate was evaporated to dryness under a steam of nitrogen at 50 °C. T-2 samples were derivatized with 4-dimethylaminopyridine (4-DMAP) and 2-naphthoyl chloride (2-NC) reagents according to a previously published method (Wang et al., 2013 2013 ). The sample was then reconstituted with 1 ml of acetonitrile/water (75/25, v/v) and suction-filtered through a 0.45-μm nylon membrane, and a 20-μl volume was analyzed on the HPLC system. Chromatographic separation was performed on a C 18 column (150 mm×4.6 mm, 5 μm particle size), with an acetonitrile/water (75/25, v/v) mobile phase and 0.6 ml/min flow rate. The excitation and emission wavelengths were 381 and 470 nm, respectively, and the column temperature was 30 °C. LOD and LOQ for this method were 1.8 and 5.4 μg/kg, respectively, and the mean recovery was 96% with an RSD of 6%. OTA analysis 2 A 25-g sample was homogenized with 100 ml of acetonitrile/water (60/40, v/v) for 30 min and filtered through Whatman No. 4 paper. A 2-ml volume of the extract was diluted with 22 ml of PBS and then applied to an OTA IAC. The column was washed with 20 ml of PBS and dried by flushing the column with air. OTA was eluted using three 1.5-ml volumes of methanol and 20 μl of the eluate was suction-filtered through a 0.4-μm nylon membrane and injected into the HPLC system. Chromatographic separation was achieved on a C 18 column (150 mm×4.6 mm, 5 μm particle size), with an acetonitrile/acetic acid/water (51/2/47, v/v/v) mobile phase and 1 ml/min flow rate. Fluorescence was detected at 333 and 443 nm for excitation and emission, respectively, and the column temperature was 40 °C. LOD and LOQ were 0.09 and 0.28 μg/kg, respectively, and the mean recovery was 84% with an RSD of 3%. Statistical analysis 2 Levels of mycotoxins were analyzed with one-way analysis of variance (ANOVA). The comparisons using the chi-square test were carried out for the contamination incidence of mycotoxins in separate zones. In all the tests, the differences were considered significant at P < 0.05. Data analyses were performed using the software package SPSS 16.0. Results 1 In the core zone, only seepweed seed and leaf were contaminated with AFB 1 . The incidence and levels in the samples from buffer and experimental zones were significantly higher than those from core zone (Table 2 2 ). In the buffer and experimental zones, levels of AFB 1 and OTA were significantly higher in rice grain than in fish and shrimp, and levels of DON and ZEN were higher in rice grain than in wheat seedings (Table 3 3 ). LOD: limit of detection. The data in parentheses represent the total sample numbers. a, b Values with different superscripts are significantly different ( P < 0.05) between zones within the same mycotoxin 2 Mycotoxin 2 Zone 2 Positive samples * * 2 Contamination incidence (%) 2 Concentration (μg/kg) Mean±SD Range  AFB 1 Core 6 (64) 9.4 a 1.90±0.62 a LOD–2.43 Buffer and experimental 18 (49) 36.7 b 39.92±25.10 b 0.09–80.30  DON Core 0 (64) 0 a Buffer and experimental 18 (49) 36.7 b 778.63±400.23 LOD–1587.30  ZEN Core 0 (64) 0 a Buffer and experimental 24 (49) 49.0 b 201.01±140.97 LOD–447.30  T-2 Core 0 (64) 0 a Buffer and experimental 11 (49) 22.4 b 38.08±28.31 LOD–100.20  OTA Core 0 (64) 0 a Buffer and experimental 18 (49) 36.7 b 5.14±3.76 LOD–10.70 Data are expressed as mean±SD. a,b Values with different superscripts are significantly different ( P < 0.05) between food samples. Crabs collected from reed pond and common seepweed community 2 Zone 2 Sample type 5 Concentration (μg/kg) AFB 1 DON ZEN T-2 OTA Core Reed root Seepweed seed and leaf 1.90±0.69 a Crabs c c and snails Buffer and experimental Rice grain 47.60±19.67 b 1015.55±319.38 a 221.84±135.81 a 38.08±36.28 6.50±2.44 a Wheat seedlings 146.87±120.12 b 11.00±1.52 b Fish and shrimp 1.57±0.99 b 1.37±1.17 b The co-occurrence of mycotoxins in mycotoxin-positive samples is shown in Table 4 4 . Among the samples, 59.3% (16/27) were found to be co-contaminated with multiple mycotoxins. Among samples collected in the buffer and experimental zones, co-occurrence was 66.7% (16/24), while in the core zone, no samples were contaminated by multiple mycotoxins. In individual food-types and taking into account mycotoxin-positive samples, co-occurrence was the most frequent in rice grain (100.0%), which also showed the most frequent combination of 3 mycotoxins (40.0%), followed by 4 mycotoxins (33.3%) and 2 or 5 mycotoxins (13.3%). In fish and shrimp, the mycotoxin co-occurrence appeared in one only positive sample (6.3%). Wheat seedling samples were only contaminated with DON or ZEN. Crabs collected from reed pond and common seepweed community. Number of co-occurring mycotoxins 2 Zone 2 Sample type 4 Co-occurrence (%) 2 b b 3 b b 4 b b 5 b b Core Reed root 0 0 0 0 Seepweed seed and leaf 0 0 0 0 Crabs a a and snails 0 0 0 0 Buffer and experimental Rice grain 13.3 40.0 33.3 13.3 Wheat seedlings 0 0 0 0 Fish and shrimp 6.3 0 0 0 Discussion 1 Mycotoxins were detected in foods consumed by red-crowned cranes while overwintering in the Yancheng Biosphere Reserve. To our knowledge, this study is the first to assess mycotoxin contamination of the red-crowned crane’s food. The present results indicated that these cranes were potentially exposed to mycotoxins during the entire wintering period. Seasonal weather patterns, preharvest, harvest, and storage conditions can place agricultural crops at risk for mycotoxin contamination (Schrödter, 2004 2004 ; Georgiadou et al., 2012 2012 ; Torres et al., 2014 2014 ). Some studies have shown that rice not only contains considerable amounts of fungi but is also contaminated with mycotoxins (Park et al., 2005 2005 ; Nguyen et al., 2007 2007 ; Bansal et al., 2011 2011 ; Qiu and Shi, 2014 2014 ). The high rice contamination observed in this study probably resulted from environmental conditions, especially moisture and temperature, which are conducive to fungal growth and toxin bioproduction. Some fungi, such as Fusarium , can infect wheat at some or any stage of growth, leading to fungal diseases (Xu, 2003 2003 ; Lu et al., 2008 2008 ), and under favorable conditions, molds can produce mycotoxins (Krnjaja et al., 2015 2015 ). Fungicides are widely used to control these diseases (D'Mello et al., 1998 1998 ). However, pesticides, putting birds at risk, are not used in the reserve because of the active education program for local farmers. Therefore, pesticides are not implicated in mycotoxin production. It is most likely that time, temperature, humidity, and physical damage were primary factors interacting in complex ways to induce the toxins observed in wheat seedings. At present, all of the aquaculture production is based on the use of commercial foodstuffs. Many of the raw materials used in these feeds, including peanuts, wheat, soybean, maize, and fishmeal, have been shown to be contaminated with mycotoxin (Binder et al., 2007 2007 ; Rodrigues and Naehrer, 2012 2012 ; Streit et al., 2012 2012 ). In addition, feed production, transport, and storage are other factors that can contribute to the presence of mycotoxins in animal feed (Jouany, 2007 2007 ; Zinedine et al., 2007 2007 ). Mycotoxins in feed could carry over into animal organs or tissues (Marin et al., 2013 2013 ). AFB 1 contamination in fish and shrimp might be explained by the fact that the aquafeeds were contaminated. Among the foods collected in the core zone, only seepweed seed and leaf were contaminated by AFB 1 , and at very low concentration. To our knowledge, no study regarding mycotoxin occurrence in common seepweed has been reported. It is possible that this plant is susceptible to infection by AF-producing fungi, as are wheat, barley, and maize. Acute toxicity of mycotoxins requires intake of relatively large quantities of contaminated foods over a short period. The mean weight of adult red-crowned cranes is 6–11 kg and the average daily food consumption is 324.3 g (Ma and Li, 2002 2002 ; Dong et al., 2005 2005 ). Because of a lack of information regarding the relative sensitivity of red-crowned cranes to mycotoxins, the 50% lethal doses (LD 50 ) of other birds were referenced here to assess the possibility of acute toxicity in these cranes (Pain et al., 2004 2004 ). Toxin concentrations leading to acute toxicity are presented in Table 5 5 . If the LD 50 for cranes is similar to that for these other birds, all values of mycotoxin-positive samples were lower than these threshold concentrations and the possibility of acute toxicity in these cranes was ruled out (Tables 2 2 and 5 5 ). In spite of this, comparison of these concentrations to those used in laboratory experiments suggested that these cranes could have been subject to chronic mycotoxic effects. These negative effects include growth retardation, tumor development, and immunotoxicity, thus increasing crane susceptibility to predation and disease (Carnaghan, 1965 1965 ; Choudhury, 1971 1971 ; Huff et al., 1986 1986 ; Kamalavenkatesh et al., 2005 2005 ). Moreover, although low mycotoxin concentrations do not cause morbidity in controlled laboratory settings, it might reduce the survival probability of wild species that rely on highly efficient metabolic systems to cope with extreme environments, such as winter conditions (Oberheu and Dabbert, 2001 2001 ). In addition, the simultaneous occurrence of more than one mycotoxin was common in analyzed mycotoxin-positive samples. The implications of the interaction between different mycotoxins have been investigated. For example, when ZEN and DON occur simultaneously in the same substrate at permitted feed concentrations, the combination causes serious physiological effects in weaning piglets (Chen et al., 2001 2001 ). Specifically, ZEN and DON decrease the levels of globulin, albumin, total protein, anticlassical swine fever antibody titers, and the mRNA expression of interferon (IFN)-α, tumor necrosis factor (TNF)-γ, and interleukin (IL)-2. The serum enzyme activities of γ-glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase increase at the same time. Histopathologically, alterations of the kidney, spleen, uterus, lymph node, and liver were found. A combination of T-2 and ZEN decreases cultured renal cell viability and increases reactive oxygen species production and heat shock protein 70 expression, compared with their individual effects (Bouaziz et al., 2013 2013 ). Combined exposure to several classes of mycotoxins could lead to an additive effect, with a few minor exceptions, which indicated a synergistic interaction (Speijers and Speijers, 2004 2004 ). Therefore, multiple mycotoxin contamination might pose a serious health hazard to the endangered cranes in the Yancheng Biosphere Reserve. Although artificial wetlands offer lower food availability and increased human disturbance, cranes fly to these wetlands to feed (Wang et al., 2011 2011 ; Li et al., 2013 2013 ). This might result from the limited carrying capacity of the natural wetlands, not the presence of food types in the artificial areas. The loss and degradation of wetlands worldwide has adversely affected waterbirds, which depend on wetland habitats (Ma et al., 2010 2010 ). From 1984 to 2008, the natural wetlands in the reserve’s core zone decreased by up to 73% (Wang, 2012 2012 ). From 1980 to 2008, the area of natural wetlands was in danger, with human activities as the main driving forces of reserve wetland degradation (Gu et al., 2012 2012 ). The deterioration of natural wetland quality has led to changes in feeding habitats in many waterbird species, increasing their reliance on artificial wetlands (Zuo et al., 2004 2004 ). Before 1996, there was no record of these cranes feeding on farmlands (Li et al., 1997 1997 ). Almost all cranes select the core zone as their roost habitat, but some of them move to artificial wetlands for daytime feeding because of the limited natural wetlands (Lv, 2007 2007 ). The concentrations leading to acute toxicity in red-crowned crane are calculated based on mycotoxin LD 50 values for selected bird species, the mean weight of adult red-crowned cranes, and the average daily food consumption Mycotoxin Species LD 50 (mg/kg) Concentration * * (mg/kg)  AFB 1 Ducklings 0.335 (Lijinsky and Butler, 1966 1966 ) 6.2–11.4 Chickens 6.5–16.5 (Smith and Hamilton, 1970 1970 ) 120.3–559.6  DON Ducklings 27 (Yoshizawa and Morooka, 1974 1974 ) 499.5–915.8 Chickens 140 (Huff et al., 1981 1981 ) 2590.0–4748.3  ZEN Chickens > 15 000 (Chi et al., 1980 1980 ) >508 750.5  T-2 Chickens 4.97 (Chi et al., 1978 1978 ) 91.9–168.6  OTA Chickens 3.3 (Peckham et al., 1971 1971 ) 6.1–111.9 Artificial wetlands have been recognized as important areas for conservation of cranes and other waterbirds (Bellio et al., 2009 2009 ; Wang et al., 2011 2011 ; Dias et al., 2014 2014 ). These wetlands provide diurnal roosts and feeding grounds for birds. Lee et al. ( 2007 2007 ) found that unplowed rice fields provide more food for red-crowned cranes and white-naped cranes ( G. vipio ) than plowed fields in the Cheolwon area of the Civilian Control Zone area of South Korea, and for conservation efforts, made recommendations that fields should remain unplowed until the foraging birds leave. However, from the present study, when compared with natural wetlands in the Yancheng Biosphere Reserve, artificial wetlands, especially rice fields, might not serve as good habitats for these birds. Based on the present results, efficient and effective recommendations that conservationists could employ are needed to mitigate crane’s food safety risks associated with these toxins. Thus, the following recommendations are provided for crane conservation. First, natural wetlands should be conserved and restored, providing birds with sufficient food resources, decreasing the use of artificial areas, and reducing mycotoxin exposure. Second, fish farmers in the buffer and experimental zones should be educated regarding the harm of contaminated feeds and encouraged to use toxin-free products. Third, monitoring of rice fields for mycotoxins should be done routinely and continuously. Once mycotoxins from waste rice grain are identified as a potential cause of bird mortality, local rice growers should be persuaded to till the waste grain. Fourth, local farmers should be encouraged to plant grains that are less susceptible to the growth of mycotoxin-producing mold to minimize the birds’ exposure to toxins. In conclusion, foods potentially utilized by red-crowned cranes were contaminated with mycotoxins in the Yancheng Biosphere Reserve. The mycotoxin incidences and concentrations in samples collected in the buffer and experimental zones were higher than those from the core zone. Mycotoxin-contaminated foods might represent a serious health threat to these birds. 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BK20140691 and BK2011083), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China 2015-09-03 2016-01-14 2015-12-20 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 17