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Dietary uptake of Cu sorbed to hydrous iron oxide is linked to cellular toxicity and feeding inhibition in a benthic grazer Daniel Cain, Marie Noele Croteau, Christopher C. Fuller, and Amy H. Ringwood Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04755 • Publication Date (Web): 23 Dec 2015 Downloaded from http://pubs.acs.org on January 11, 2016

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Copper in prepared food (nmol/g)

ACS Paragon Plus Environment

Cu influx rate (nmol/g/d)

Environmental Science & Technology

Relative ingestion rate Lysosomal destabilization (%)

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Dietary Uptake of Cu Sorbed to Hydrous Iron Oxide

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is Linked to Cellular Toxicity and Feeding Inhibition

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in a Benthic Grazer

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Daniel J. Cain*†, Marie-Noële Croteau†, Christopher C. Fuller†, and Amy H. Ringwood‡

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†U.S. Geological Survey, Menlo Park, CA, USA

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‡University of North Carolina

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Keywords: dietary metal exposure, Cu, feeding inhibition, lysosomal destabilization, Lymnaea

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stagnalis

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* Corresponding Author: [email protected]

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Whereas feeding inhibition caused by exposure to contaminants has been extensively

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documented, the underlying mechanism(s) are less well understood. For this study, the behavior

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of several key feeding processes, including ingestion rate and assimilation efficiency, that affect

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the dietary uptake of Cu were evaluated in the benthic grazer Lymnaea stagnalis following 4 – 5

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hour exposures to Cu adsorbed to synthetic hydrous ferric oxide (Cu-HFO). The particles were

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mixed with a cultured alga to create algal mats with Cu exposures spanning nearly three orders

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of magnitude at variable or constant Fe concentrations, thereby allowing first order and

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interactive effects of Cu and Fe to be evaluated. Results showed that Cu influx rates and


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ingestion rates decreased as Cu exposures of the algal mat mixture exceeded 104 nmol/g.

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Ingestion rate appeared to exert primary control on the Cu influx rate. Lysosomal destabilization

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rates increased directly with Cu influx rates. At the highest Cu exposure where the incidence of

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lysosomal membrane damage was greatest (51%), ingestion rate was suppressed 80%. The

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findings suggested that feeding inhibition was a stress response emanating from excessive uptake

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of dietary Cu and cellular toxicity.

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INTRODUCTION

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Feeding inhibition caused by the exposure to dietborne toxicants is a general response having

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potentially severe ecological implications.1,2 Exposure to toxicants that would impair normal

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feeding and growth rates could reduce fitness and threaten population persistence.3,3-5 For

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example, fecundity is highly correlated with female body size in invertebrates.6,7 Allen et al.2

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proposed a general mechanism of feeding inhibition that emphasized the importance of

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particulate-bound contaminants and dietary exposure. Differences in experimental objectives and

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designs have complicated interpretations of dietary metal toxicity, however. For example,

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uncertainties arise if the contaminant is not measured in the ingested matter and in the consumer,

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concurrently. Furthermore, feeding inhibition studies have focused on the development of the

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response as a sensitive, sublethal endpoint for invertebrate models, such as Daphnia magna8,

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Gammarus pulex9, and snails10,11 based on aqueous metal exposures. Consequently, more

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attention has been placed on deriving exposure-response relationships than on feeding processes

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and underlying mechanisms resulting in feeding inhibition from dietary metal exposure.

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Feeding processes affecting metal uptake, including food ingestion rate, can be studied within

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the framework of a biokinetic metal bioaccumulation model12-14. The model predicts dietary

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metal influx rates from three terms as shown below:


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= × × (1) 43

where InfluxM is the metal influx rate expressed as concentration/unit time (e.g., nmol/g/d where

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‘g’ is gram weight and ‘d’ is day), AE is assimilation efficiency, a measure of metal

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bioavailability (unitless); IR is the daily ingestion rate (e.g., g/g/d); and [M]food is the metal

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concentration of the ingested food (e.g., nmol/g). Differences in AE among different solid phases

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and the rate and total quantity of metal ingested at various exposures can be quantified from

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short-term, pulse-chase experiments.15 Correspondence between terms, such as ingestion rate and

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dietary metal exposure can be examined for exposure-response relationships from which

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mechanisms mediating toxicity may be inferred.16 For example, the absence of feeding could

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indicate a behavioral response (avoidance) to the contaminated medium17,18 while the slowing of

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feeding rates with increasing dietary metal exposure could indicate the perturbation of metabolic

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pathways by absorbed metal.19,20

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The inclusion of toxicological endpoints in bioaccumulation studies reduces uncertainties

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concerning relationships between dietary metal uptake and toxicity. Cellular biomarkers are used

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to assess whether exposure to a contaminant alters functions at lower levels of biological

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organization (at cellular and subcellular levels), and to gain insights to mechanistic causes of

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higher level responses. Lysosomal membrane stability assays have been used worldwide in a

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variety of marine and freshwater fish and molluscan species as a valuable, sensitive indicator of

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pollutant and nanoparticle stress.21-29 Lysosomes are membrane-bound organelles that contain an

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assortment of acid hydrolases that are essential for the intracellular digestion of macromolecules

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by snails and other invertebrates (especially those incorporated through endosomal pathways), as

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well as autophagy in all species.30 Contaminants, including metals, can destabilize lysosomal

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membranes, thus releasing lysosomal contents into the cytosol, resulting in cell injury and

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death.31


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Previous studies of the gastropod Lymnaea stagnalis have shown a depression in feeding rates

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when snails were exposed to a diatom (Nitzschia palea) enriched in Cu.16 The authors suggested

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that feeding inhibition manifested as a response to Cu influx across the gut mucosa rather than

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impairment of digestive processes because Cu assimilation efficiency (AE, i.e., the portion of

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ingested Cu that is assimilated into the soft tissues of the animal) did not vary with exposure

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(mean AE ranged from 0.72 – 0.82). Because Cu influx varies with the Cu bioavailability of the

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food source, the authors suggested that feeding inhibition would correspond more closely to

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influx rates of Cu than to total dietary Cu exposure. That is, to the extent that metals bound to

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ingested particles are bioavailable they too may provoke feeding inhibition.

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In their natural environment, benthic grazers encounter complex biofilms that include metals

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sorbed to inorganic particles.32-34 Another study showed that most of the Cu sorbed to a synthetic

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hydrous ferric oxide (HFO) was assimilated (AE > 0.70) by L. stagnalis.15 This particle was

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intended to represent a type of common inorganic particle formed by the neutralization of acid

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mine drainage waters and infiltrates benthic biofilms. In that experiment, concentrations of

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dietary Cu (2 – 812 nmol/g) were established to avoid potential toxicological effects on

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measured parameters, including ingestion rate.

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Given the similarly high Cu bioavailabilities of Cu-HFO and Cu-enriched diatoms shown in

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the previous studies, we hypothesized that Cu-HFO would elicit feeding inhibition and the effect

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level would be similar to that of the Cu-enriched diatom. This hypothesis is tested in the present

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study. In a series of four experiments, L. stagnalis were exposed to Cu-HFO. Copper loadings of

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the particles and particle concentrations were manipulated to create Cu concentrations ranging

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from 200 to 85,000 nmol/g at variable or constant iron (Fe) concentrations. The design allowed

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first order and interactive effects of Cu and Fe to be evaluated. As in the previous studies15,16,


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mechanisms regulating Cu influx focused on those parameterized by the biokinetic model. In

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addition, measurements of lysosomal destabilization were performed on digestive gland cells to

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determine whether dietary Cu uptake impaired digestive processes.

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EXPERIMENTAL SECTION

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Test Species. Experiments were conducted with juvenile L. stagnalis reared from eggs in the

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laboratory16,35. Snails were held in synthetic freshwater formulated for moderate hardness (SFW)

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(ASTM 1998), at room temperature (≈ 20⁰ C), and constantly fed lettuce. When the snails were

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11 – 15 weeks old, 40 – 50 individuals were selected for experimentation based on mass

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uniformity and randomly distributed among treatments. Dry weights of the soft tissues did not

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differ among treatments within an experiment (p > 0.05, ANOVA), but did differ among

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experiments (p < 0.05, ANOVA).

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Cu-HFO preparation. Synthetic colloidal hydrous ferric hydride (HFO) was prepared,

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based on the methods described in36, labeled with

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before it was used. Coagulation of the 2 to 6 nm HFO particles occurs over the first 4 days

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following precipitation to form micron-sized highly porous aggregates.37 The quantity of

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was varied relative to Fe to create

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0.001 to 0.28 (Table S1). Additional information on the preparation

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Supporting Information.

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Cu, and then aged for at least two weeks

Cu-HFO stocks with

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Cu

Cu/Fe molar ratios ranging from 65

Cu-HFO is provided in

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Dietary exposures (experiments 1 – 3). Cu-enriched food was prepared in the form of diatom

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mats following procedures previously described.15 Briefly, batch cultures of N. palea were

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concentrated onto polycarbonate filters (1.2 µm pore size), and then resuspended in SFW.

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Because the Fe content of ingested matter might influence feeding behavior independently of Cu,

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for example by rendering the food unpalatable34, experiments were designed to analyze primary


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and interactive effects of Cu and Fe. Aliquots of

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subsamples of algal suspensions to achieve the desired dietary 65Cu and Fe concentrations (Table

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1). Details of the food preparations are provided in Supporting Information. Untreated (no 65Cu-

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HFO addition) and 65Cu-HFO spiked algal suspensions were collected by filtration onto 47 mm

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filters (1.2 µm). Each filter was subsampled (n = 5) for Cu and Fe analysis (Table 1). A previous

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study showed that mass transfer of 65Cu from HFO to the algae was less than 1% and had little

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effect on estimates of AE.15 The remaining portion was presented to L. stagnalis in 250 mL

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polypropylene containers placed into a tray partially filled with SFW. Two openings were made

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to the sides of the exposure chambers, and these were covered with nylon mesh small enough to

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contain feces and allow for exchange of water. All experiments were conducted in SFW at 13 ± 1

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⁰C in darkness. Desorption of

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assessed from water samples collected after the exposure phase of the experiment (see below).

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Samples were immediately filtered (0.45 µm) and acidified (1% nitric acid) for metals analyses.

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Cu-HFO stocks were dispensed into

Cu and the potential for aqueous uptake by the snails was

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To quantify Cu influx rate, snails were exposed to the contaminated food ([M]food in eq. 1; n =

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8 – 10 snails/treatment) for 4 – 5 hours during which their feeding behavior was visually

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monitored. At the end of the exposure, the snails were removed from the containers, carefully

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rinsed in SFW, and then transferred to individual containers for two days to allow depuration of

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unassimilated

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which was circulated through granulated carbon to trap any

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depuration period. Animals were fed ad libitum (lettuce) during depuration. After depuration,

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each animal and its feces were separately collected for metals analyses from which assimilation

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efficiency (AE) and ingestion rate (IR) were determined by mass balance (see below). The

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Cu.35 These containers were held in a 40 L aquarium filled with 30 L SFW 65

Cu excreted during the 2-day


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depuration water from each experiment was sampled (n = 1 – 3) at the end of the depuration

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period.

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Lysosomal destabilization assay (experiment 4). Duplicate sets of three

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Cu-HFO

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treatments were prepared (Table 1) to assess lysosomal function as well as Cu-assimilation

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kinetics. Copper concentrations between the duplicate preparations did not differ (ANOVA

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performed for each of three treatments: F(1,8) = 0.98 to 2.6 among treatments, p > 0. 1). Ten

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snails were assigned to each preparation. After a 4-hour exposure period, five snails were

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removed from each duplicate preparation of each treatment, placed into a single container (n = 5

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snails per replicate x 2 replicates per treatment = 10) with lettuce and depurated for 2 days as

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described above, and the tissues were then used for metals analyses. The remaining five snails

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from each treatment were processed for the lysosomal destabilization assay. Additionally, a

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group of snails (n = 10) not exposed to 65Cu-HFO was assayed to establish the basal lysosomal

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destabilization rate. Water samples were collected before and after the exposure and depuration

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phases for metals analyses.

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The lysosomal destabilization assay was performed on hepatopancreas (also known as

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digestive gland) cells. Sample preparation and analysis generally followed the procedure for

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oyster lysosomal destabilization assay described in Ringwood et al.39 Briefly, hepatopancreas

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tissues were carefully dissected and primary cell preparations were generated using calcium-

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magnesium free saline (CMFS, ionic strength 20 psu, pH 7.4) and trypsin, and incubated in

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neutral red for 1 hour. Cells with neutral red confined to intact lysosomes were scored as stable

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and cells with neutral red leaking out of impaired lysosomes were scored as destabilized (Figure

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1S). At least 50 cells were counted from each individual snail preparation (n = 10

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snails/treatment), and the results were expressed as % destabilized lysosomes.


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Metal analyses. Copper and Fe in all samples were determined by inductively coupled plasma

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mass spectrometry (ICP-MS, Perkin Elmer NexION 300Q). Subsamples of the diatom

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preparations, soft tissues and feces were prepared for metals analyses by methods described

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previously.16,32,35 Dry weights for diatoms and soft tissues were determined to the nearest 0.001

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mg (Sartorius Model M2P microbalance). Samples were digested in sealed PTFE cups with

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addition of 16N HNO3 at room temperature for 7 days followed by 30% H2O2 for 1 day.

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Volumes used for individual samples are given in Supporting Information. Deionized water (18

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MΩ-cm) was added to bring the acid concentration to ≈ 0.8 – 1.6N. Samples were spiked with an

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internal standard (germanium) to control for signal drift, and then filtered (0.45 µm) prior to

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analyses.

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Estimates of dietary uptake parameters. The 65Cu that was present in the soft tissue of each

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snail before the exposure (i.e., the background 65Cu) was accounted for and subtracted from the

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total

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Δ ) in eq. 2 and 3. Similarly, feces were corrected (Δ ) to account for

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background 65Cu. Assimilation efficiency (AE) and ingestion rate (IR, g/g/d) for individual snails

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were estimated by mass balance of ∆65Cu in fecal and tissue samples (nmol), the

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concentration of the contaminated diatom preparations (

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the snail’s soft tissue (g), and the exposure time, d (day), as shown in equations 2 and 3.

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Cu to yield only the

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Cu acquired from the contaminated food35 (symbolized as

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Cu

!, nmol/g), the dry weight of

= Δ ÷ #Σ Δ + Δ & (2) =

#Σ Δ + & #

! × ( × &

(3)

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The contribution of aqueous 65Cu to 65Cu body burdens (eq. 2 and 3) during the exposures was estimated from equation 4 and the Michaelis-Menton equation:


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Cu!tissue = (

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Cu ]aq × *+ × (4)

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where [65Cu]aq is the dissolved 65Cu concentration in the exposure media (nmol/L), ku is the

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published aqueous Cu uptake rate constant for L. stagnalis derived for the test water at Cu

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concentrations less than 140 nM40, and d is the exposure time (day). Tissue 65Cu concentrations

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estimated from eq. 4 were multiplied by the tissue weight of the snail (g) and this value was

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subtracted from the 65Cu tissue burden, providing the ∆65Cu tissue burden (eq. 2 and 3) acquired

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from 65Cu-HFO. For concentrations greater than 140 nM, dissolved uptake was estimated with

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the Michaelis-Menton equation using values for Vmax and KM of 171 nmol/g/d and 110 nM,

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respectively.40 Dissolved 65Cu concentrations reflect desorption of Cu from HFO and elimination

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of Cu by the snail. Elimination of Cu from L. stagnalis is approximately 0.026/d (≈ 3% /d).16

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The small loss of Cu from the snail was not considered in the estimates of aqueous Cu

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accumulation.

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Quality assurance. Glass and plastic materials used for the experiments and for sample

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preparation were cleaned to minimize metal contamination following procedures previously

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described.15,35 Ultra-pure reagents (nitric acid and hydrogen peroxide) and high purity water (18

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MΩ-cm,

Dietary Uptake of Cu Sorbed to Hydrous Iron ... - ACS Publications

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