Lead and zinc are recognized as the most widespread trace metals in nature and can, at high levels, compromise the health of wildlife and their habitat. Because of their position in a higher trophic level, wild carnivores can be valuable biological indicator species of trace-metal contamination in the environment. We assessed blood lead and zinc concentrations of four small carnivore species native to Taiwan, the small Indian civet (Viverricula indica), the masked palm civet (Paguma larvata), the ferret badger (Melogale moschata), and the crab-eating mongoose (Herpestes urva), from urban and rural areas (Yangmingshan National Park, Xiuguluan River bank, and Da-an River bank). Blood samples were acquired from the anterior vena cava under general anesthesia, and lead and zinc concentrations, hematology, and serum biochemistry results were then obtained. Blood lead levels were significantly higher in ferret badgers in the Yangmingshan area. Although lead concentrations were comparable with those in humans and cats with lead toxicosis, there was no hematological or biochemical evidence that animal health was compromised. Blood zinc levels were within an acceptable range in all four species tested. Overall, we found significant differences in blood lead and zinc levels among four species of carnivores living in areas with different levels of land development in Taiwan. Anthropogenic pollution, mining history, and volcanic activities in Yangmingshan National Park may contribute to significantly high blood lead levels in ferret badgers in this area. Our results provided information about the potential impact of land development on wildlife and may be beneficial to wildlife conservation, public health, and environmental health in Taiwan.
Microelements are crucial for maintaining normal functioning of all living organisms. An excess or lack of certain trace-metal elements in an organism often results in pathology (Rashed and Soltan 2005). With the development of modern civilization, pollutants, including trace metals from human activities, have accumulated in the air, water bodies, and soil, where they can affect living organisms (Dip et al. 2001). Among the trace metals, lead is considered one of the most toxic and widespread in the urban environment (Friberg et al. 1986; Goyer 1996), and has long been recognized as a stressor for wild animals (Bellrose 1959). Although zinc is a necessary micronutrient for humans and animals (Friberg et al. 1986), overexposure via contaminated food as well as mining activities has also been documented in wild animals, leading to zinc toxicosis (Malo et al. 1993; Sileo et al. 2003).
Free-living wild animals are common model organisms used to investigate trace-metal pollution and its potentially hazardous consequences in animals and the wider ecosystem (Hernández-Moreno et al. 2013). Predators and scavengers are higher ecological or end-of-food-chain consumers, and play an important role in ecosystems. Biomagnification of pollutants can be more apparent in these consumers, and they are therefore suitable species for biomonitoring trace-metal pollution in the environment (Hutton 1982; Kottferová et al. 1996; Dip et al. 2001). Current methods for wildlife biomonitoring of trace metals involve sampling from internal organs and tissues, which is highly invasive and often conducted postmortem (Hernández-Moreno et al. 2013). In contrast, blood samples provide an economical, less invasive, and well-established method for antemortem assessment of body zinc and lead levels. This is especially important for adhering to animal conservation laws and policies. Blood lead concentrations only indicate recent or ongoing exposure, and fluctuate because of redistribution of lead between tissue and peripheral circulation and thus do not necessarily correlate with total lead burden. Therefore, an animal with previous exposure or with long-term low-level exposure can have a high total body burden of lead despite a low blood lead level (Knight and Kumar 2003; Langlois et al. 2017).
Small Indian civet (Viverricula indica), masked palm civet (Paguma larvata), ferret badger (Melogale moschata), and crab-eating mongoose (Herpestes urva) are small carnivores native to Taiwan. Among them, small Indian civet and crab-eating mongoose are listed as protected species in the Wildlife Conservation Act of Taiwan. These four species are widespread and are often sympatric in low to medium-elevation areas in Taiwan. When living in the same habitats, they fit different ecological niches by separating their activity patterns and diets (Chuang and Lee 1997; Chiang et al. 2012). For example, the crab-eating mongoose is a diurnal animal, whereas the other three species are nocturnal (Chiang et al. 2012). Although there are certain degrees of dietary overlap between these species, the preference for and proportion of each food item differ (Chuang and Lee 1997). Whereas crab-eating mongooses predominately eat crustaceans, insects, and amphibians, a large proportion of diets of the ferret badgers is oligochaetes and insects. The diet of the small Indian civets is varied; insects, oligochaetes, plants, and small mammals are all important. Although masked palm civets share many of the same prey, they further diversify their ecological niche by supplementing their diet with fruits and small mammals (Zhou et al. 2008). In addition, all four species are relatively long-lived mammals, well adapted to developing rural and suburban environments, occupy an upper trophic level within the ecosystem, and are territorial (Chuang and Lee 1997). Therefore, biomagnification of trace metals could be detected and traced back because of the animals' stable home range and feeding habit. These factors made them suitable species to provide regional information regarding trace-metal toxicity.
Trace-metal studies in Taiwan have focused mainly on cadmium, arsenic, and mercury toxicosis in humans and invertebrates. Little attention has been paid to lead and zinc contamination in wild animals and their habitats (Hsu et al. 2006; Liu et al. 2017). Taiwan is rapidly becoming a developed country, with the impact of human activities expanding fast and beyond the region of urban areas. Although anthropogenic activities are well-known sources of exposure (Langlois et al. 2017), remarkable zinc and lead exposure had been noticed sporadically in wild animals in Taiwan. The threat of lead and zinc contamination in the environment and within native wildlife should be properly assessed. Therefore, our aim was to compare blood zinc and lead concentrations in animals from the same habitat but different ecological niches, and animals from different habitats that represent different levels and types of human activity. In addition, we examined the impact of trace-metal exposure on hematology and serum biochemistry in these wild animals. Results will be beneficial in determining the threat of trace-metal pollution in these carnivorous species and the wider ecosystem in Taiwan.
MATERIALS AND METHODS
As a national park located in the capital, Taipei City (Fig. 1), Yangmingshan National Park (YNP) is famous for its dormant volcano and floriculture, which attract citizens and tourists around Taipei City. It is a classic urban wildlife habitat, and, although it is exposed to busy traffic, approximately 10% of the land area is used for recreation (Hsu et al. 2008). Tomahawk double-door traps (Tomahawk 206, Tomahawk Live Trap, Hazelhurst, Wisconsin, USA) in YNP were set between September 2012 and April 2018 (Fig. 1). Study sites along the Xiuguluan River bank (XRB) were located in eastern Taiwan (Fig. 1). These sites represent minimally developed natural habitat without human activities and interventions. Traps along the XRB were opened between October 2014 and January 2016. Sites along the Da-an River bank (DRB) were located near the industrial area of Taichung City (Fig. 1). The industrial area was approximately 8–10 km from the study area and covered the industries involved in automobile production, nanotechnology such as optoelectronics, integrated circuit production, and production of precision machinery. The traps were set in a fruit farm, an abandoned forest farm, and a secondary forest and were opened between October 2015 and April 2018. The degree of development lies between the extremes of the sites in the YNP and XRB; however, it is the only site near an industrial area. The YNP, XRB, and DRB were located in the north, east, and west of Taiwan; each represents different degrees of land development in natural habitat.
Traps were covered with black plastic bags, and dirt and branches were on placed on top to mimic natural tunnels. Sausage, fish, and cat food were used as bait and traps were checked daily. Small carnivores were trapped opportunistically during three studies in which investigation of trace-metal concentrations was not the main purpose. Thus, blood samples were collected when the animals were under routine anesthetic protocol in the field. As a control group for the comparison among different habitats, captive small carnivores from rescue centers were also sampled following the procedures described below. All procedures were reviewed and approved by the Bureau of Forestry, the competent authority of wildlife use, and the Institutional Animal Care and Use Committee of National Taiwan University approval NTU106-EL-00165.
Anesthesia and blood sampling
Animals were transferred from each Tomahawk trap to an anesthetic induction chamber. Isoflurane (5%; Attane®, Panion & BF Biotech, Inc., Taoyuan, Taiwan) with 100% oxygen was delivered into the chamber for anesthetic induction. Body weight was measured after bringing each animal out of the chamber. Anesthetic maintenance was conducted via a mask with 2–2.5% isoflurane and 100% oxygen. Heart rate and blood oxygen saturation were monitored by blood oximeter (PureSAT® SpO2 Model 9847V, Nonin Medical Inc., Plymouth, Minnesota, USA) throughout the procedure. Animals were positioned in dorsal recumbency, and blood was collected from the cranial vena cava with a 23-gauge needle and 5-mL syringe. Blood samples were divided and placed into potassium ethylenediaminetetraacetic acid-coated tubes (BD Microtainer®, Becton Dickinson, Franklin Lakes, New Jersey, USA) for hematology, lithium heparin-coated tubes (Vacuette®, Greiner Bio-One Co., Monroe, North Carolina, USA) for lead and biochemical analyses, and nonanticoagulant-coated tubes without rubber caps for zinc analyses (Vacutainer® trace element serum, Becton Dickinson, Singapore). After the procedure, animals were removed to an oxygenized chamber and released after a full recovery.
Blood samples were placed on a rack and stored in a cooler on ice immediately after sampling and transported. All samples were processed within 6 h. A complete blood cell count was obtained using an autoanalyzer (Exigo BM800, Boule Medical AB, Spånga, Sweden) and plasma biochemistry analyses were conducted using a different autoanalyzer (VITROS® 350 chemistry system, Ortho-Clinical Diagnostics, Raritan, New Jersey, USA). Aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase, albumin, total protein, glucose, urea, and creatinine were measured. Lead and zinc concentrations were measured by atomic absorption spectrometry (GBC 932 Plus atomic absorption spectrometer, GBC Scientific Equipment Pty Ltd., Braeside, Australia). The sensitivity reported for lead and zinc were to one decimal place and to one digit respectively. When a blood sample was not sufficiently large enough to measure all analytes, blood lead and zinc levels were examined first.
All statistical analyses were performed with SPSS version 18.0 (SPSS Inc., Chicago, Illinois, USA). To appropriately represent actual field conditions, all data were included in the study. Hematology, serum biochemistry, and blood lead and zinc levels were presented as means and standard deviation. Normality was assessed with Kolmogorov-Smirnov test on the basis of species and trapping sites. Only creatinine and aspartate aminotransferase of ferret badgers from YNP, XRB, and DRB as well as blood urea nitrogen and alkaline phosphatase of ferret badgers from XRB and DRB were not normally distributed.
Because the majority of the data were normally distributed and the number of ferret badgers sampled was over 30, differences between blood lead and zinc values in the same species from different habitats (including captive populations) were analyzed by one-way analysis of variance (more than two variants) or t-tests (two variants), as were those from different species that shared the same habitat. On the basis of previous reports (Knight and Kumar 2003; Gurnee and Drobatz 2007; Langlois et al. 2017), the effect of lead or zinc exposure on animal health was monitored once blood lead and zinc concentrations were greater than 30 µg/dL or 5,500 µg/L, respectively, and animals were grouped according to whether they were below or above the selected values. Differences in hematology and serum biochemistry between groups was examined by t-tests.
During the study period, there were 118 ferret badgers (53, 43, and 22 individuals from the YNP, DRB, and XRB of Taiwan, respectively), 25 masked palm civets (nine and 16 individuals from the YNP and DRB, respectively), 17 crab-eating mongooses (five and 12 individuals from XRB and DRB, respectively), and five small Indian civets (all from the YNP) captured. In addition, four captive masked palm civets and six captive small Indian civets were included in the study; all of the captive animals had been kept in captivity for at least 6 mo. Data collection was subject to the amount of blood able to be collected (regulated by each different authority) and was also limited by the overall condition of the animal (e.g., body weight and degree of dehydration). Complete blood lead and zinc levels were able to be analyzed in the majority of trapped animals (Table 1); however, only ferret badgers had enough blood to support hematology and serum biochemistry analyses (Table 2).
Differences within each species in different areas
Ferret badgers from the YNP area had significantly higher blood lead levels than those from the DRB (P<0.001) and XRB (P<0.001), whereas they had significantly lower blood zinc levels (P<0.001). Crab-eating mongoose from the XRB had significantly higher blood lead (P=0.003) and zinc (P=0.009) concentrations than those from DRB. In contrast, masked palm civets and small Indian civets from different areas did not show any significant differences in blood lead and zinc concentrations (Table 1).
Differences among different species within the same area
In the YNP, ferret badgers had significantly higher blood lead levels (P<0.001) and significantly lower blood zinc levels than masked palm civets (P=0.021) and small Indian civets (P=0.008). In the DRB, there were no significant differences between ferret badgers, masked palm civets, or crab-eating mongoose in both blood zinc (P=0.294) and lead (P=0.151) levels. In addition, there were no significant differences between ferret badgers and crab-eating mongoose in XRB (lead: P=0.530; zinc: P=0.956) or between masked palm civets and small Indian civets (lead: P=0.237; zinc: P=0.833) in captivity (Table 1).
Effect of high blood lead and zinc concentrations on animal health
Thirteen of 46 ferret badgers in the YNP area had blood lead concentrations >30 µg/ dL. When compared with the other 89 ferret badgers in Taiwan with blood lead concentrations <30 µg/dL, the higher blood lead group had significantly higher mean hemoglobin concentration (13.90±2.50 vs. 12.07±2.03; P=0.004), mean corpuscular hemoglobin (16.85±0.74 vs. 16.27±1.59; P=0.035), and mean corpuscular hemoglobin concentration (37.78±1.77 vs. 34.95±4.29; P<0.001), and significantly lower mean corpuscular volume (44.71±2.33 vs. 46.83±3.20; P=0.024). Other indicators of animal health were not significantly different (Table 2). All animals in the study had blood zinc concentrations <5,500 µg/L and were thus considered at low risk for toxicosis.
The increasing effects of urbanization on trace-metal toxicosis have been studied in depth. For example, before the shift from leaded to unleaded gasoline in automobiles, tissue lead concentrations in three populations of pigeons (Columba livia) from London had been found to increase progressively with proximity to the city center (Hutton 1980), and lead concentrations in red foxes (Vulpes vulpes) showed a decreasing trend as distance from downtown to the suburbs increased (Dip et al. 2001). Traffic density also plays an important role in lead contamination, with studies reporting correlations between high traffic density or proximity to roadways and high lead concentrations in soil, plants, and animals (Jefferies and French 1972; Quarles et al. 1974; Goldsmith and Scanlon 1977). Overall, there is a considerable body of evidence that human activities can affect trace-metal concentrations in the tissues of wildlife that share the same territory. Anthropogenic environmental contamination such as lead-containing plumbing materials, ammunition, fishing weights, toys, and storage batteries as well as the manufacturing and recycling factories for these products are well-known sources of exposure (Langlois et al. 2017).
A study conducted in Taroko National Park in Taiwan identified two major sources of lead pollution, one of the major causes for current ecological crises for mammals (Su et al. 2013). With the first being anthropogenic contaminants from the industries, the second source stemmed from sandstorms in mainland China that were carried over by prevailing winds (Su et al. 2013). The study further concluded that long-term pollutant transmission from China and Southeast Asia and fertilizer from agricultural activities are likely the major pollution sources affecting natural habitats in Taiwan (Su et al. 2013). In addition, there is a history of mining in the YNP area, and the mining industry is known for increasing lead pollution in the environment (Ayangbenro and Babalola 2017). Finally, gas emissions from volcanic activity and geothermal springs have the potential to enrich the surrounding soil with trace metals, such as lead, which can be absorbed by surrounding vegetation and pose potential negative health effects on local communities (Varrica et al. 2000; Durowoju et al. 2016). These factors highlight the likely reasons why there is significantly higher blood lead concentrations in ferret badgers in the YNP compared with those living in XRB and DRB.
Food and water are two of the most important sources of trace-metal exposure in wild animals (Pokras and Chafel 1992). Although the four carnivorous species in the present study live in similar habitats, they experienced differences in trace-metal exposure. This may be explained by differences in their diet and food items they normally consumed (Chuang and Lee 1997; Zhou et al. 2008). Earthworms make up a significant portion of ferret badger diet, and can accumulate trace metals, including copper, zinc, lead, and cadmium, when exposed to trace-metal-contaminated soils (Hobbelen et al. 2006). Ferret badgers therefore may have elevated blood lead concentrations due to their ingestion of earthworms that accumulate these trace metals. The lower blood lead and zinc concentrations found in masked palm civets and small Indian civets in the YNP may be explained by their diet of fruit-eating birds and rodents. Plants have a relatively low trace-metal concentration despite growing in trace-metal-rich areas (Durowoju et al. 2016). A study of the transfer of trace metals from geothermal springs to surrounding soil and mango trees (Mangifera indica) in South Africa showed that, although some trace-metal concentrations were above the standard guidelines for drinking water and typical soil, concentrations in mangos were low compared with the control samples (Durowoju et al. 2016). Phytoremediation of heavy metals from the contaminated sites generally happens through any one or more of the following mechanisms or processes: phytoaccumulation, phytostabilization, phytodegradation, phytovolatilization, and hydraulic control (Muthusaravanan et al. 2018). Plants extract, degrade, and release trace metals from contaminated water and soil in a less toxic form to the atmosphere. Blood lead and zinc concentrations did not differ between captive and wild masked palm civets and small Indian civets. Captive animals were kept in controlled environments and fed using selected food and water sources.
Recently, animal hair has been used as bioindicator material for trace-metal contamination (Rashed and Soltan 2005; Hernández-Moreno et al. 2013). Hair samples can be collected by using barbed wire or glue traps set up along animal trails to collect hair from passing wild animals (Berezowska-Cnota et al. 2017). Hair trapping makes hair collection stress free for wild animals and is relatively easily done since the more laborious processes such as catching and anesthetizing the animal can be eliminated. Furthermore, hair can be stored at ambient temperature for long periods of time without degradation, making them suitable for long-term and consecutive studies (Yin et al. 2006). However, blood concentration is still considered the gold standard for the diagnosis of trace-metal toxicosis in a clinical setting (Knight and Kumar 2003). Blood tests can concurrently assess trace-metal toxicity and evaluate its impact on animal health. In addition, our study displayed the importance of selecting the appropriate study organism as an indicator species for trace-metal exposure. Theoretically, biomagnification of pollutants should be more apparent in carnivore species because of their higher trophic level. However, as our study found, bioaccumulation of trace metals is also influenced by different natural processes such as food selection and habitat. Thus, future studies should take those factors into consideration when choosing their study organism.
External signs of poisoning may be present in waterfowl with blood lead concentrations between 5 and 10 µg/dL, but susceptibility among songbird species varies (Beyer et al. 2004). Clinical signs of lead toxicosis in humans and animals historically were thought to occur at much greater blood lead concentrations, often >35 or 40 µg/dL (Langlois et al. 2017). No differences were found in blood lead concentrations of a test population of dogs grouped by the presence or absence of clinical signs (Langlois et al. 2017). However, three of four dogs with blood lead concentrations >5 µg/dL had owner-reported gastrointestinal or neurologic abnormalities at the time of screening (Langlois et al. 2017). In cats, toxic blood lead levels have been reported to be >30–35 µg/dL (Knight and Kumar 2003). The mean blood lead concentration in ferret badgers from the YNP in the current study was 25.48 µg/dL, a value at the higher end of the range of previous reports and species described above. Among these, only one ferret badger had a blood lead concentration <10 µg/dL, whereas 13 of 46 ferret badgers had concentrations >30 µg/dL. Clinical pathology features of lead poisoning include presence of nucleated red blood cells, microcytic/hypochromic anemia, basophilic stippling, and elevated liver enzymes (Knight and Kumar 2003). Although the ferret badgers with blood lead concentrations >30 µg/dL showed significantly elevated hemoglobin values instead of anemia or significantly elevated liver enzymes, the effects of lead on their health should still be monitored. This is because the classic hemograms described above in lead-poisoned animals can be a more unusual finding in other animals (Knight and Kumar 2003), and gastrointestinal or neurologic abnormalities are difficult to confirm during field studies using anesthetized animals.
Normal serum zinc concentrations in dogs and cats are 700–2,000 µg/L (Torrance and Fulton 1987) and normal blood zinc concentrations vary from 150 to 3,410 µg/L in Psittaciformes (Wismer 2016). In a retrospective study of 19 dogs with zinc toxicosis, the median blood zinc concentration was 30,600 µg/L (range: 5,500–159,000 µg/L; Gurnee and Drobatz 2007). The blood zinc concentrations of small carnivores in our study in Taiwan ranged from 471 to 4,508 µg/L, remarkably lower than in dogs with zinc toxicosis. Although the blood zinc concentrations of the crab-eating mongoose from the XRB were significantly higher than those from the DRB, values were still at the lower limits compared with dogs displaying toxicosis and therefore might be considered as normal variation between different areas rather than toxicosis.
Lead and zinc interactions are not well defined. Although research has shown experimentally that lead increases zinc excretion and decreases plasma zinc concentration (Goyer 1997; Roney and Colman 2004), others have reported that lead does not affect zinc absorption and the concentrations of zinc in plasma, liver, or kidney (Roney and Colman 2004). Meanwhile, several studies have demonstrated the protective effect of zinc on lead toxicity through lead inhibition or reactivation of lead-inhibited δ-aminolevulinic acid dehydratase activity as well as the inhibition of gastrointestinal lead absorption (Roney and Colman 2004). Toxic levels of zinc can also delay lead accumulation in the cerebrum and prevent the development of clinical signs of lead poisoning (Willoughby et al. 1972; Hietanen et al. 1982). The interactions between zinc and lead might explain the significantly lower zinc concentrations found in the ferret badgers in YNP as compared with those from the DRB or XRB, and with other carnivores in the YNP, because they had significantly greater blood lead concentrations.
In conclusion, we reported blood lead and zinc concentrations in four small carnivore species in Taiwan from different habitats. The natural habitat for small carnivores in the YNP poses a risk of lead contamination, particularly for ferret badgers. Anthropogenic pollution, mining history, and volcanic activities are unique in this area and may contribute to this finding. To discriminate these different possible sources, inductively coupled plasma mass spectrometry coupled with isotope ratio analysis might be warranted (Ferrara et al. 1995; Durowoju et al. 2016). Less invasive methods for long-term monitoring should be explored, such as the use of hair samples as a bioindicator of trace metals, and can be done so by establishing the correlation of trace metals between hair and blood levels in ferret badgers. Although other parts of Taiwan did not demonstrate a marked risk for lead and zinc toxicosis in small carnivores, studies covering the full spectrum of trace metals and that target other possible biomonitor species are still warranted in the future.
This study was supported financially by Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, project 107AS-8.7.4-BQ-B1; Bureau of Forestry, Council of Agriculture, Executive Yuan, project 106 FD-08.3-C-28; and Headquarters of Yangmingshan National Park project 1070704.
5 These authors made equal contributions to this work.