Long-term effects of copper exposure to agricultural soil function and microbial community structure at a controlled and experimental field site☆
Graphical abstract
Introduction
Heavy metals can persist in the environment for decades, yet relatively little is known about how soils respond to exposure over decadal timescales. Acceptable metal levels in soils are often determined by short-term, controlled exposure experiments, where impacts to microbial communities and functional endpoints are measured over weeks, days, or hours (Kuperman and Carreiro, 1997; Beare et al., 1990; Hopkins et al., 1994; Farrell et al., 2009; Chander and Joergensen, 2002; Smolders et al., 2009; Zheng et al., 2017; Pietikäinen and Fritze, 1995; EPA USA, 2007). The acclimation time in these short-term experiments may not be representative of how soil environments respond to contamination events in the long-term, as changes reflect only immediate microbial responses (Smolders et al., 2003). Previous studies have confirmed this, concluding that metal toxicity to soil microbes in freshly spiked soils bears little resemblance to the effects detected in field-contaminated soils over longer timeframes (Renella et al., 2002; Smolders et al., 2004). Often, retrospective observations from locations such as historical mining sites are used as a proxy for longer-term exposure research (Pennanen et al., 1996; Dell’Amico et al., 2008; Berg et al., 2005; Berg et al., 2012; Liu et al., 2005; Oliveira and Pampulha, 2006). However, retrospective observational studies typically only measure metal concentrations at the time of sampling, even though concentrations may have varied considerably since the contamination event began.
One of the few long-term studies that monitored and controlled exposure concentrations found that historical Cu exposure (15-year) was directly associated with tolerance to Cu exposure (Wakelin et al., 2014). However, this study only tested up to 200 mg kg−1 Cu, which is only marginally higher than Cu concentrations naturally found in agricultural soils (NSW EPA, 1997). Other microbial studies have similarly observed community resilience to functional loss (e.g. nitrification) when re-exposed to heavy metals (Smolders et al., 2003; Mertens et al., 2006; Diaz-Ravina et al., 1996; Díaz-Raviña et al., 1994; Kunito et al., 1999), demonstrating microbial ability to adapt and tolerate new, harsher environmental conditions. However, it is unknown whether there is maximum tolerance threshold after which soil microbial communities cease to adapt or build resilience and are irreversibly, functionally impacted in situ.
To address this question, we examined both soil functionality and microbial community structure in a long-term (12-year), controlled, field-based experiment, which tested a wide range of Copper (Cu) concentrations: from 0 to 3310 mg Cu kg−1. Copper was applied to agricultural soils in 22 distinct, randomised plots within a 1 km radius, and concentrations at these sites, along with two control sites, were monitored over 12 years with no amendment or human interference. We combined environmental DNA (eDNA) sequencing with a range of functionality assays (substrate induced respiration (SIR), substrate induced nitrification (SIN), proteolysis, and peptide and amino acid turnover) to analyse and compare community composition and soil function across the different Cu treatments. The functionality assays chosen, assess common soil functions, including carbon and nitrogen turnover. This long-term experimental field site presented a unique opportunity to determine the concentration at which contaminated soils can return to former functionality if contamination ceases (e.g., mining, ore extraction, or biosolid application). We also examined whether conserved functionality reflects previous (non-exposed) community structures or if novel community assemblages are created.
Section snippets
Field sites
The field site was located in an agricultural region in Menangle, NSW, Australia and soils were characterised as chromosol under the Australian soil classification system (Isbell, 2002). Treatments consisted of two control plots, where no metal was applied, and 22 plots where varying rates of copper sulphate (Cu), ranging from non-toxic (<100 mg kg−1, NSW EPA max allowable guideline; NSW EPA, 1997) to highly toxic (>1000 mg kg−1) were added. Detailed descriptions of the field trials and soil
Total and extractable Cu concentration
There was no significant difference in total Cu concentration between measurements taken at the beginning of the experiment in 2004 (data provided in Supplementary Materials: SM) and those taken upon sample collection in 2016 (Wilcoxon signed ranks test p = 0.864), demonstrating the persistence of Cu at the study site. Prior to Cu application in 2004, the background Cu levels in all plots were within normal ranges for the area and soil type (<100 mg Cu kg−1), and control plots remained within
Recovery of soils twelve years after Cu contamination event
We assessed microbial community structure and function in soils that were exposed to a range of copper concentrations (0–3310 mg kg−1 total Cu) over a decade ago. No functional differences were identified between control soils (no dose applied) and those exposed to Cu concentrations <100 mg kg−1, demonstrating that low level contamination is unlikely to negatively impact agricultural soils over decadal time frames. However, plots with treatment concentrations above 200 mg kg−1 total Cu were
Conclusions
We found that agricultural soils exposed to Cu concentrations greater than 200 mg Cu kg−1 twelve years previously, still had significantly lower functionality than control plots, and in many cases, significantly different microbial community assemblages. Soils with >200 mg Cu kg−1 were characterised by a specific microbial community assemblage, likely consisting of organisms able to tolerate medium to high metal levels. A further shift in community structure was evidenced in soils over 800 mg kg
Funding
This article was funded by the Commonwealth Scientific and Industrial Research Organisation.
CRediT authorship contribution statement
J.L.A. Shaw: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft. J.G. Ernakovich: Conceptualization, Methodology, Formal analysis, Writing - review & editing. J.D. Judy: Methodology, Formal analysis, Writing - review & editing. M. Farrell: Methodology, Writing - review & editing. M. Whatmuff: Writing - review & editing. J. Kirby: Conceptualization, Methodology, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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