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Assessing the dietary bioavailability of metals associated with natural particles: Extending the use of the reverse labeling approach to zinc Marie Noele Croteau, Daniel J. Cain, and Christopher C. Fuller Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b06253 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 10, 2017
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Assessing the dietary bioavailability of metals
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associated with natural particles: Extending the use
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of the reverse labeling approach to zinc
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Marie-Noële Croteau*, Daniel J. Cain, and Christopher C. Fuller
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U.S. Geological Survey, MS 496, 345 Middlefield Road, Menlo Park, California 94025
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We extend the use of a novel tracing technique to quantify the bioavailability of zinc (Zn)
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associated with natural particles using snails enriched with a less common Zn stable isotope.
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Lymnaea stagnalis is a model species that has relatively fast Zn uptake rates from the dissolved
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phase, enabling their rapid enrichment in
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Zn during the initial phase of labeling. Isotopically
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enriched snails were subsequently exposed to algae mixed with increasing amounts of metal-rich
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particles collected from two acid mine drainage impacted rivers. Zinc bioavailability from the
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natural particles was inferred from calculations of
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Zinc assimilation efficiency (AE) varied from 28% for the Animas River particles to 45% for the
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Snake River particles, indicating that particle-bound, or sorbed Zn, was bioavailable from acid
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mine drainage wastes. The relative binding strength of Zn sorption to the natural particles was
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inversely related to Zn bioavailability; a finding that would not have been possible without using
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the reverse labeling approach. Differences in the chemical composition of the particles suggest
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that their geochemical properties may influence the extent of Zn bioavailability.
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Zn assimilation into the snail’s soft tissues.
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INTRODUCTION
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Knowledge of the bioavailability of metals bound to solid phases is central to assessing the
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risks associated with dietary metal exposure. Chemical extraction techniques have been used to
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operationally define bioavailable metals1-3. But inference of bioavailability from correlations
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does not provide information about the geochemical and biological processes at play2,4-5. These
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statistical relationships are also difficult to generalize and extrapolate to nature. Another
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approach to estimate metal bioavailability from solid phases relies on modeling. Kinetic trace
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element bioaccumulation models have proved successful at predicting metal concentration
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differences among species, metals and environments6-7. More broadly, these biokinetic (or
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biodynamic) models explain bioaccumulation using several parameters that represent
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physiological processes8. Assimilation efficiency (AE) is a key physiological parameter to these
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models and one of the most effective quantitative measures of metal bioavailability from diet9-10.
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AE represents the proportion of ingested metals that is retained into tissues after depuration. AE
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has been used to compare the bioavailability of different metals11, metal forms12-14, and exposure
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conditions15.
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The use of radioactive tracers has been central to the development of standard techniques for
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measuring metal AEs9,16. Stable isotope techniques have also been refined to estimate metal
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AEs17-19, eliminating the complicated logistics, handling, and waste issues associated with using
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radioisotopes. However the application of both tracing techniques is hampered by difficulty in
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labeling particles such that they accurately represent the assemblage of metal speciation found in
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natural particles. For example, in natural particles metals can be co-precipitated with manganese
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and/or iron-oxides, occluded, incorporated into mineral structures, or trapped under layers of
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organic matter20-23. Systematically labeling each phase to study their potential influence on
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bioavailability without altering the native chemistry of the particles is difficult. For instance,
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short labeling times are usually used to label natural particles, but this favors sorption of the
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labels onto particle surfaces24. Conclusions concerning the relationships between metal AE and
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particle geochemistry may thus be erroneous without a firm understanding of which phases are
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labeled.
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The reverse labeling approach, originally developed for Cu18, shows great potential for
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addressing questions involving the bioavailability of an element that naturally has multiple stable
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isotopes, many of which are commercially available in enriched forms. Conceptually, the
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procedure reverses the labeling from the “source” to the “receptor”, such that the particulate
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metal is not altered by the labeling and is studied in its natural state. The approach involves first
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a “labeling phase” where test organisms are artificially enriched with a rare stable metal isotope
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to alter their background metal isotopic signature. Species displaying fast accumulation kinetics
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are the best candidates for the approach because they rapidly accumulate metals, shifting the
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isotopic composition of the internalized metal. A rapid approach to isotopic steady state also
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limits the increase in metal body burden, thereby avoiding the onset of adverse effects. The
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isotopically enriched organisms are then exposed to natural particles. During this “exposure
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phase”, metal uptake from the natural particles shifts the isotopic composition of the organisms
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back toward the natural abundance of the element. The newly accumulated metal concentration
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is calculated using concentrations inferred from selected isotopes, including that used in the
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labeling phase. Croteau et al.18 used the reverse labeling approach to show assimilation of 44%
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of Cu ingested from Cu-bearing Fe-Al particles collected from an acid mine drainage impacted
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river. This demonstrated that inorganic particulate Cu can be bioavailable when ingested with
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food.
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Here we extend the use of the reverse labeling approach to characterize the bioavailability of
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metals with multiple stable isotopes, using Zn as the example. We first determine the exposure
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time needed to increase the relative abundance of 67Zn, a less abundant Zn isotope, in the soft
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tissues of a model species. Potential adverse effects elicited by the aqueous 67Zn exposure during
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the labeling phase were also assessed using growth as an indicator of potential toxicity. We
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provide equations to convert metal isotope ratios into accumulated metal concentrations as well
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as to calculate Zn assimilation efficiencies. Lastly we evaluate the influence of particle
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geochemistry on the extent of Zn bioavailability.
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METHODS
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Test species. The freshwater snail Lymnaea stagnalis was used as a model species given 1) its
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ease to culture and maintain in laboratory conditions; 2) its capability to accumulate metals from
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both dissolved and dietary routes17,25-26; 3) its trophic position (this species is an avid grazer of
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biofilms); and 4) its wide geographic distribution and ecological importance as part of the diet of
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many fish and cray fish. This species is also one of the most intensively studied gastropods in the
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scientific literature (Supporting Information). Snails were reared in the laboratory in glass tanks
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filled with moderately hard synthetic freshwater27 (MOD).
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Natural particles. Particles were harvested in September 2006 and August 2014 from two
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acid mine drainage impacted rivers in Colorado, i.e., the Animas River (AR) and the Snake River
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(SR). In 2006, particles formed in the mixing zone downstream from the confluence of Cement
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Creek (pH 4.1) with the AR (pH 7.6) (hereafter referred to as ARP) were concentrated by
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tangential flow ultrafiltration using 10 kDa filters, as described in Schemel et al.28. Likewise,
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particles formed 6.5 km downstream from the confluence of Peru Creek (pH 5.0) with the SR
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(pH 4.7) (hereafter referred to as SRP) were similarly concentrated in 2014. Inflows of acidic
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water from abandoned mines and from natural weathering of ore deposits occur along both Peru
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Creek and the SR upstream of their confluence29, with partial neutralization from neutral pH
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tributaries between the confluence and the collection site (pH 6.2). Particles were transported on
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ice to the laboratory where they were further concentrated by settling and centrifugation, and
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stored at 4 °C. Concentrations of Zn, Fe, Al and Cu in particles were determined by ICP-MS (see
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below). Total organic carbon content was determined on solids recovered from freeze drying
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aliquots of the stock suspensions.
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Desorption experiments. The relative binding strength of Zn sorption to the two natural
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particles was compared by measuring the extent of Zn desorption as a function of pH in batch
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experiments with particle suspensions diluted in MOD (pH 7.5) to attain an equal total Zn
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concentration (90 µg l-1), as described in the Supporting Information. Briefly, dissolved Zn was
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measured in diluted suspensions as pH was decreased incrementally from 7.5 to 5.5. 67
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Zn labeling and growth (Experiment 1). The rate of increase in the relative abundance of
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Zn in the snail soft tissues (p67snail) after waterborne exposure to isotopically enriched 67Zn was
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quantified over time to determine the exposure duration required to increase p67snail from ~4% to
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more than 50%. In addition to Zn accumulation, growth was monitored to examine for toxicity.
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Specifically, 50 snails of a restricted size range (i.e., soft tissue dry weight of ~ 1.5 mg) were
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transferred to an 8 L glass aquarium filled with MOD water spiked with isotopically enriched
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Snails were fed lettuce twice a week. Seven to 10 snails were sampled after 0, 14, 28, 40 and 54
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days of exposure, rinsed with both MOD and deionized (DI) water, and immediately frozen (-4
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°C). The results were compared to previously collected data18 that served as the control.
Zn (97% of which was 67Zn, Trace Sciences) to achieve a total Zn concentration of 10 µg l-1.
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Labeling and exposure phases for AE determination. For experiments 2 (ARP) and 3
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(SRP), 10 and 3 week labeling periods were used, respectively, with media replaced 2-4 times.
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The labeling phase for Experiment 2 was unintentionally extended due to circumstances beyond
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our control in October 2013. Prior to the exposure phase, particles were mixed with the benthic
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diatom Nitzschia palea to create Zn-contaminated food sources. Algae were grown axenically
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following procedures described previously17. They were harvested onto 1.2 µm Isopore
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membrane filters (Millipore) under low vacuum (
