Trace metals occur naturally in Earth’s water, soil, and sediments and are incorporated into living tissue at low concentrations (Alloway, 1974; Pais and Jones, 1997). The industrial extraction and use of trace metals have led to their mobilization in the environment at unprecedented levels, increasing their availability to organisms via trophic uptake, inhalation of particulate matter, or contact with contaminated substrates (Bradl, 2005). Excessive dosages of trace metals can interfere with the basic cellular processes of organisms, and trace metal toxicity constitutes a growing concern for the health of humans, wildlife, and their shared ecosystems (Kapustka, 2004).
Industrial wastewater is a dominant source of global trace metal pollution, and the proper disposal of wastewater remains a major challenge for mitigating ecological risk (Kapustka, 2004). One strategy for remediating industrial wastewater involves harnessing the ability of natural wetlands to passively filter contaminants (Hammer and Bastian, 1989). Constructed wetlands are engineered environments designed to dilute concentrated elements and sequester them in less bioavailable forms (Vymazal, 2007). Intentional selection of wetland substrate, vegetation, and microbial inoculants can create favorable conditions for sulfate-reducing bacteria, which in turn facilitate the precipitation of mobile metal ions into insoluble mineral species (Webb et al., 1998; Wu et al., 2013). This passive biofiltration strategy is often more cost-effective than active chemical treatment and is employed widely by industrial facilities to comply with wastewater quality regulations (ITRC, 2003). In the United States, wastewater treatment wetlands are distinct from wetlands constructed to manage nonpoint sources of pollutants, such as stormwater retention ponds, in that they are subject to quality and monitoring standards imposed by the National Pollutant Discharge Elimination System (NPDES; Levy et al., 2014).
Despite their intentional and controlled design, wastewater treatment wetlands are dynamic systems that maintain complex biogeochemical exchanges with the surrounding environment. Seasonal fluctuations in temperature, rainfall, and deposition of organic material can shift chemical cycles and alter the proportion of bioavailable metal species within a wetland (Evers et al., 2007; Jacob and Otte, 2003; Xu and Mills, 2018). Resident aquatic biota can accumulate available trace metals and transport them from the treatment wetland environment upon emergence into terrestrial life stages (Fletcher, 2022) or through trophic links to terrestrial ecosystems (Zhou et al., 2019). Treatment wetlands may appear to serve as valuable habitat for wetland-dependent species, but the accumulation of trace metals in biota can impact individual fitness, with long-term consequences for population viability (Sievers et al., 2018). The NPDES standards for wastewater compliance focus on pollutant concentrations in discharge, and therefore treatment wetland effectiveness is primarily assessed by monitoring trace metals in outflow compared to inflow (ITRC, 2003). However, the evaluation of trace metal uptake by wetland biota is essential for understanding the potential for treatment wetlands to serve as sources of trace metals for surrounding ecosystems.
Passerines are abundant wetland inhabitants with trophic links to aquatic biota and can serve as bioindicators of trace metal flux in wetland ecosystems (Burger, 1993). Higher burdens of trace metals can be linked to trophic transfer from specific prey items contributing to passerine diets, including emergent aquatic insects (Beck et al., 2013) and spiders (Howie et al., 2018). The transfer of trace metal contaminants to passerines is documented in several well-studied populations inhabiting highly polluted systems, including pied flycatchers (Ficedula hypoleuca) at a closed zinc (Zn) and lead (Pb) mine in northern Sweden (Berglund, 2018; Berglund et al., 2010; Berglund et al., 2009; Lidman and Berglund, 2022), great tits (Parus major) at a Zn smelter site in Belgium (Dauwe et al., 2000; Dauwe et al., 2004; Dauwe et al., 2006), and tree swallows (Tachycineta bicolor) in the mercury (Hg) polluted South River, Virginia, USA (Brasso and Cristol, 2008; Hallinger and Cristol, 2011; Taylor and Cristol, 2015). Prior studies evaluating constructed wetlands as sources of metals for passerines are scarce and present mixed evidence. Sparling et al. (2004) found that nestling red-winged blackbirds (Agelaius phoeniceus) and their invertebrate prey inhabiting suburban stormwater retention ponds did not have higher whole-body Zn, copper (Cu), or Pb concentrations compared to birds in reference wetlands. However, adult red-winged blackbirds, marsh wrens (Cistothorus palustris), and tree swallows inhabiting restored wetlands contaminated with industrial effluent had elevated feather and blood concentrations of Pb (Tsipoura et al., 2008). The extent to which wetlands constructed to remediate point-source effluent serve as sources of trace metals to passerines remains unknown. At high enough dosages, trace metals can interfere with basic physiological processes in birds, with consequences for health, behavior, and breeding success (Dauwe et al., 2006; Gorissen et al., 2005; Janssens et al., 2003; Scheuhammer, 1987). In addition, passerines are highly mobile and can serve as biotic vectors of trace metals from contaminated environments to other ecosystems (Blais et al., 2007; Mallory et al., 2015). Passerine biomonitoring therefore allows for more informed assessments of the bioavailability, transport, and ecological impact of wastewater-derived metals in treatment wetlands.
The implementation of passerine biomonitoring in contaminated systems is complicated by the variability in trace metal concentrations across elements and tissue types (Scheuhammer, 1987). Excess trace metals are processed by the liver and kidneys and can injure these organs when the rate of uptake exceeds the rate of excretion (Gasaway and Buss, 1972; Zhuang et al., 2014). Therefore, lethal sampling of liver tissue is ideal for estimating internal accumulation and toxicity risk. However, researchers are motivated to reduce pressure on bird communities by employing non-lethal biomonitoring strategies. Feathers are sensitive indicators of exposure to some metals during the period of feather growth and are often favored due to their non-invasiveness (Burger, 1993; Markowski et al., 2013), but several studies have highlighted the pitfalls of inferences drawn from feather metal concentrations, including the lack of correlation with internal body burdens (Dauwe et al., 2000; Jaspers et al., 2019). Blood metal concentrations are promising indicators of recent dietary uptake but have their own set of limitations, such as saturation thresholds and uncertain relationships with internal tissue concentrations (Berglund, 2018). Despite the unknowns associated with feather and blood sampling, many passerine biomonitoring initiatives only employ these sampling types to investigate trace metal accumulation in contaminated environments (Cooper et al., 2017; Einoder et al., 2018; Lester et al., 2014; Zebral et al., 2022). A greater understanding of the relationships between metal concentrations in non-lethal samples and internal tissue, especially in field populations, is necessary before passerine biomonitoring can be widely applied in wastewater treatment wetlands.
Wastewater treatment wetlands are used to remediate industrial effluent containing elevated Zn, Cu, and Pb on the Savannah River Site (SRS) in South Carolina. Although the SRS wetlands have high trace metal removal efficiencies, extensive monitoring and metal speciation indicate that metals are bioavailable in pore and surface waters (Knox et al., 2021; Xu et al., 2019). Further, trace metals accumulate in dragonfly nymphs inhabiting the SRS wetlands and can remain in the bodies of emergent adults, which serve as prey for terrestrial wildlife (Fletcher, 2022; Fletcher et al., 2020). The goals of this study were to (1) conduct a multi-tissue analysis to evaluate the transfer of trace metals to terrestrial songbirds in well-characterized wastewater treatment wetlands on the SRS, and (2) compare non-lethal and lethal sampling strategies as tools for wetland biomonitoring. We hypothesized that excess trace metals sourced from wastewater accumulate in passerines inhabiting wastewater treatment wetlands. Therefore, we expected to observe higher Zn, Cu, and Pb concentrations in passerines within SRS treatment wetlands compared to reference wetlands, and we expected no difference in the concentrations of other metals. If feather and blood samples are reliable indicators of internal metal burdens, we expected that metal concentrations in these samples would correlate with concentrations in liver and muscle.