Brittany G Perrotta
email: brittany_perrotta@baylor.edu

Marie Simonin

Jeffrey A Back

Steven M Anderson

Astrid Avellan

Christina M Bergemann

Benjamin T Castellon

Benjamin P Colman

Gregory V Lowry

Cole W Matson

Emily S Bernhardt

Ryan S King
email: ryan_s_king@baylor.edu

Ryan S King -

Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

■ INTRODUCTION

Engineered nanoparticles (NPs) are ubiquitous in many commercial products and enter the aquatic environment where they have been shown to have a wide variety of effects. Nanomaterials represent a sizeable fraction of materials used in the global economy and consequently have multiple pathways into the environment. Flow into aquatic ecosystems by runoff and wastewater is estimated to contribute up to 21,000 metric tons of NPs annually. [START_REF] Keller | Global Life Cycle Releases of Engineered Nanomaterials[END_REF] Once NPs are released into the environment, they interact with a complex aquatic ecosystem. At present, more extensive research has been conducted on NPs in laboratory settings. [START_REF] Griffitt | Exposure to Copper Nanoparticles Causes Gill Injury and Acute Lethality in Zebrafish (Danio Rerio)[END_REF][START_REF] Blaise | Ecotoxicity of Selected Nano-Materials to Aquatic Organisms[END_REF] Less research has been conducted on the ecosystem-level effects of NPs under realistic environmental conditions, such as chronic long-term exposure and NP interactions with food webs, particularly primary producers and consumers. [START_REF] Colman | Not the Dose: Comparing Chronic and Pulsed Silver Nanoparticle Exposures[END_REF][START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF] Primary producers (plants and algae) and consumers (grazers) interact extensively with NPs discharged into aquatic ecosystems. This interaction allows for the transfer of NPs from the water column or sediments into aquatic food webs, resulting in possible negative effects on ecosystems. [START_REF] Ferry | Transfer of Gold Nanoparticles from the Water Column to the Estuarine Food Web[END_REF][START_REF] Wright | Titanium Dioxide Nanoparticle Exposure Reduces Algal Biomass and Alters Algal Assemblage Composition in Wastewater Effluent-Dominated Stream Mesocosms[END_REF] Although most studies have shown relatively benign effects of NPs, some NPs have toxic effects on organisms including algae, cladocerans, snails, and fish. [START_REF] Pang | Effects of Sediment-Associated Copper to the Deposit-Feeding Snail, Potamopyrgus Antipodarum: A Comparison of Cu Added in Aqueous Form or as Nano-and Micro-CuO Particles[END_REF][START_REF] Ramskov | Bioaccumulation and Effects of Different-Shaped Copper Oxide Nanoparticles in the Deposit-Feeding Snail Potamopyrgus Antipodarum: Bioaccumulation and Effects of CuO Nanoparticles[END_REF][START_REF] Ali | Sensitivity of Freshwater Pulmonate Snail Lymnaea Luteola L., to Silver Nanoparticles[END_REF] NPs have been shown to alter organism metabolism and bind to ions such as phosphate, limiting essential nutrients from interacting with cells. [START_REF] Cedervall | Food Chain Transport of Nanoparticles Affects Behaviour and Fat Metabolism in Fish[END_REF][START_REF] Guo | Adsorption of Essential Micronutrients by Carbon Nanotubes and the Implications for Nanotoxicity Testing[END_REF][START_REF] Schultz | Aquatic Toxicity of Manufactured Nanomaterials: Challenges and Recommendations for Future Toxicity Testing[END_REF] Changes to organism metabolismhow organisms take up, transform, and expend energy and materialscan lead to indirect effects on the ecosystem. [START_REF] Galic | When Things Don't Add up: Quantifying Impacts of Multiple Stressors from Individual Metabolism to Ecosystem Processing[END_REF][START_REF] Brown | Toward a metabolic theory of ecology[END_REF] Consumers are an integral part of nutrient dynamics in ecosystems and may affect primary productivity by altering the supply and quality of resources such as nutrients, primarily nitrogen and phosphorus. [START_REF] Vanni | Nutrient Transport and Recycling by Consumers in Lake Food Webs: Implications for Algal Communities[END_REF] Consumer-mediated nutrient recycling is an ecological process, which includes the excretion of dissolved nitrogen and phosphorus by an organism into its surrounding environment over time. It is commonly used for testing the predictions of important ecological frameworks, such as the metabolic theory of ecology or ecological stoichiometry. [START_REF] Vanni | Predicting Nutrient Excretion of Aquatic Animals with Metabolic Ecology and Ecological Stoichiometry: A Global Synthesis[END_REF] Both of these frameworks use principles of physics, chemistry, and biology to link the biology or elemental composition of individual organisms to the ecology of populations, communities, and ecosystems. [START_REF] Brown | Toward a metabolic theory of ecology[END_REF][START_REF] Sterner | Ecological Stoichiometry[END_REF][START_REF] Allen | Towards an Integration of Ecological Stoichiometry and the Metabolic Theory of Ecology to Better Understand Nutrient Cycling[END_REF] Previous studies measuring consumer-mediated nutrient recycling have contributed considerably to our understanding of interactions among consumers, algae, and nutrients in aquatic ecosystems. [START_REF] Elser | The Stoichiometry of Consumer-Driven Nutrient Recycling: Theory, Observations, and Consequences[END_REF][START_REF] Hillebrand | Ecological Stoichiometry of Indirect Grazer Effects on Periphyton Nutrient Content[END_REF] Individual consumer species can directly alter ecosystem function through grazing stored forms of organic nutrients which they then excrete in simplified forms at rates comparable to major nutrient sources. These nutrients excreted by consumers are then immediately available for plant, algal, and microbial uptake and drive consumermediated nutrient recycling. [START_REF] Vanni | Nutrient Transport and Recycling by Consumers in Lake Food Webs: Implications for Algal Communities[END_REF][START_REF] Hall | Exotic Snails Dominate Nitrogen and Carbon Cycling in a Highly Productive Stream[END_REF][START_REF] Vanni | Stoichiometry of Nutrient Recycling by Vertebrates in a Tropical Stream: Linking Species Identity and Ecosystem Processes[END_REF][START_REF] Grimm | Role of Macroinvertebrates in Nitrogen Dynamics of a Desert Stream[END_REF] Further, the processing of food under varying environmental conditions, particularly contrasting background nutrient enrichment regimes, can lead to differential consumer excretion of nutrients into aquatic ecosystems. [START_REF] Evans-White | Stoichiometry of Consumer-Driven Nutrient Recycling across Nutrient Regimes in Streams[END_REF] Ecological processes have nutrient requirements, and the excess nutrients, specifically nitrogen and phosphorus, may result in altered ecosystem function. Surface waters have become nutrient-enriched, largely because of urban and agricultural practices, leading to widespread eutrophication of freshwater ecosystems. These eutrophic ecosystems have an increased capacity for algal biomass and primary production. [START_REF] Dodds | Phosphorus, and Eutrophication in Streams[END_REF][START_REF] Carpenter | Nonpoint Pollution of Surface Waters with Phosphorus and Nitrogen[END_REF][START_REF] Elser | Global Analysis of Nitrogen and Phosphorus Limitation of Primary Producers in Freshwater, Marine and Terrestrial Ecosystems[END_REF] The increase in primary productivity exacerbates diel dissolved oxygen fluctuations and contributes to an overall decline in species richness while encouraging proliferation of more pollution-tolerant species. [START_REF] Søndergaard | Role of Sediment and Internal Loading of Phosphorus in Shallow Lakes[END_REF][START_REF] Hilsenhoff | An Improved Biotic Index of Organic Stream Pollution[END_REF][START_REF] Mouthon | Compared Sensitivity of Species, Genera and Families of Molluscs to Biodegradable Pollution[END_REF] Proliferation of pollution-tolerant species that recycle nitrogen quickly may affect overall biogeochemical cycling through consumer-mediated nutrient recycling pathways. [START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF] Pollution-tolerant species, such as the aquatic snail, Physella acuta, have been shown to persist where NPs co-occur with high nutrient loads in wastewater streams and agricultural runoff. [START_REF] King | Stream Biodiversity Is Disproportionately Lost to Urbanization When Flow Permanence Declines: Evidence from Southwestern North America[END_REF] P. acuta is a freshwater pulmonate snail detritivore and herbivore that reproduces quickly, can occur at high densities, and thrives under a wide range of food and cultural conditions. [START_REF] Sturm | The Mollusks: A Guide to Their Study, Collection, and Preservation[END_REF][START_REF] Cope | Competitive Interactions between Two Successful Molluscan Invaders of Freshwaters: An Experimental Study[END_REF][START_REF] Albrecht | Invasion of Ancient Lake Titicaca by the Globally Invasive Physa Acuta (Gastropoda: Pulmonata: Hygrophila)[END_REF][START_REF] Dillon | Populations of the European Freshwater Pulmonate Physa Acuta Are Not Reproductively Isolated from American Physa Heterostropha or Physa Integra[END_REF] Previous work showed that they accumulate substantial amounts of NPs relative to other primary consumers and are a good model to study uptake of NPs. [START_REF] Ramskov | Bioaccumulation and Effects of Different-Shaped Copper Oxide Nanoparticles in the Deposit-Feeding Snail Potamopyrgus Antipodarum: Bioaccumulation and Effects of CuO Nanoparticles[END_REF][START_REF] Geitner | Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms[END_REF][START_REF] Spyra | Freshwater Alien Species Physella Acuta (Draparnaud, 1805) -A Possible Model for Bioaccumulation of Heavy Metals[END_REF][START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF][START_REF] Croteau | Bioaccumulation and Toxicity of CuO Nanoparticles by a Freshwater Invertebrate after Waterborne and Dietborne Exposures[END_REF][START_REF] Croteau | A Novel Approach Reveals That Zinc Oxide Nanoparticles Are Bioavailable and Toxic after Dietary Exposures[END_REF][START_REF] Hoang | Copper Uptake and Depuration by Juvenile and Adult Florida Apple Snails (Pomacea Paludosa)[END_REF][START_REF] Ma | Biochemical Responses to the Toxicity of the Biocide Abamectin on the Freshwater Snail Physa Acuta[END_REF][START_REF] Ramskov | Biokinetics of Different-Shaped Copper Oxide Nanoparticles in the Freshwater Gastropod, Potamopyrgus Antipodarum[END_REF][START_REF] Goncalves | Effects of Silver Nanoparticles on the Freshwater Snail Physa Acuta: The Role of Test Media and Snails' Life Cycle Stage[END_REF] We know very little of the effects of NPs and nutrients on the relationships among consumers and nutrient remineralization through consumer-mediated nutrient recycling, despite growing evidence that metal-based NPs accumulate in snails and that metals have been found to influence the physiology of snails at high concentrations. [START_REF] Bernot | Effects of Ionic Liquids on the Survival, Movement, and Feeding Behavior of the Freshwater Snail, Physa Acuta[END_REF][START_REF] Brix | The Toxicity and Physiological Effects of Copper on the Freshwater Pulmonate Snail, Lymnaea Stagnalis[END_REF] Investigations of contaminants within the metabolic theory of ecology, ecological stoichiometry, and consumer-mediated nutrient recycling framework provide an understanding of contaminant effects on aquatic food webs while also assessing the potential utility of these established ecological responses to complex mixtures of NPs and nutrient contaminants in freshwater ecosystems.

Here, we studied the interactive effects of NPs and nutrient enrichment on P. acuta with a chronic long-term, lowconcentration dosing scheme to provide a better understanding of NP effects on aquatic food webs and of how NPs may indirectly impact aquatic ecosystems by altering consumer metabolism. We investigated how chronic exposures to copper hydroxide-based (CuNP) or gold (AuNPs) (weekly additions) crossed with two levels of nutrient enrichment influenced the nutrient excretion of consumers in outdoor wetland mesocosms over 6 months. We used CuNPs found in a commercially available fungicide at a realistic dose to investigate particle and biotic interactions likely to be occurring in aquatic ecosystems. We chose AuNPs as a model NP that is easy to detect in complex environmental matrices, in part because of its low natural background, enabling us to study the fate of these particles. [START_REF] Mahapatra | Probabilistic Modelling of Prospective Environmental Concentrations of Gold Nanoparticles from Medical Applications as a Basis for Risk Assessment[END_REF] We hypothesized that consumer nitrogen (as NH 4 + -N) and phosphorus (as PO 4

3--P) excretion would be elevated in response to CuNP exposure relative to AuNPs and control treatments. We hypothesized that copper would increase excretion rates of consumers relative to gold because CuNPs have antimicrobial/antifungal properties and previous research demonstrated that the antimicrobial/antifungal agent triclosan increased snail nutrient excretion. [START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF] We hypothesized that consumers in the AuNP mesocosms would excrete similarly to the control mesocosms, as previous studies have used AuNPs as inert NP tracers because of their low dissolution rates in abiotic water and interaction with organisms as a particle. [START_REF] Glenn | Abiotic and Biotic Factors That Influence the Bioavailability of Gold Nanoparticles to Aquatic Macrophytes[END_REF][START_REF] García-Cambero | Converging Hazard Assessment of Gold Nanoparticles to Aquatic Organisms[END_REF][START_REF] Hull | Filter-Feeding Bivalves Store and Biodeposit Colloidally Stable Gold Nanoparticles[END_REF] Furthermore, we hypothesized that snails at higher background nutrient treatments would excrete at higher rates than ambient nutrient treatments because consumer food sources in these treatments are nutrient-enriched and more available. [START_REF] Cross | Consumer-Resource Stoichiometry in Detritus-Based Streams[END_REF] In our study, we saw an increased internal eutrophication in the mesocosms exposed to the combination of NPs and nutrient enrichment. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF] We wanted to investigate ecosystem level changes, which could drive increased eutrophication, other than primary producers. Consumer responses including consumer-mediated nutrient recycling might be one of the key parameters explaining this phenomenon. Consumer metabolic responses to NPs are largely unknown, but consumer metabolic responses to environmental variables have been known to alter ways in which nutrients are processed in consumers, leading to a differential excretion of nutrients to the water column. [START_REF] Devine | Spatial and Seasonal Variation in Nutrient Excretion by Benthic Invertebrates in a Eutrophic Reservoir[END_REF][START_REF] Ikeda | Metabolic Rates of Epipelagic Marine Zooplankton as a Function of Body Mass and Temperature[END_REF] ■ MATERIALS AND METHODS Wetland Mesocosm Experimental Design. This study took place in the Center for the Environmental Implications of NanoTechnology (CEINT) experimental wetland mesocosm facility located in the Duke Forest in Durham County, North Carolina, USA. We employed the same slant-board mesocosm design that has been described previously. [START_REF] Lowry | Transformations of Nanomaterials in the Environment[END_REF][START_REF] Colman | Emerging Contaminant or an Old Toxin in Disguise? Silver Nanoparticle Impacts on Ecosystems[END_REF] Mesocosms were built to dimensions (3.66 m × 1.22 m × 0.81 m), were filled with sandy-loam soil, and then with ∼250 L of ground water. This design yields an upland zone with minimal inundation, a transitional zone that is periodically inundated with water, and an aquatic zone that is permanently inundated. The detailed mesocosm setup is found in the Supporting Information, section "Mesocosm Setup", and has been previously reported. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF][START_REF] Geitner | Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms[END_REF][START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] In June 2015, we added the following organisms to the aquatic zone: rooted macrophyte (Egeria densa), aquatic snails (P. acuta and Lymnaea sp.), and eastern mosquito fish (Gambusia holbrooki).

On September 28, 2015, two levels of nutrients (ambient and enriched) were established through weekly additions. The enriched level received 1 L of mesocosm water supplemented with 88 mg of N (as KNO 3 ) and 35 mg of P (as KH 2 PO 4 ) added throughout the entire aquatic zone.

Environmental Science & Technology

On January 18, 2016, mesocosms were randomly assigned to NP treatments across the two nutrient treatments to the NP treatments (control, copper, gold). The fully factorial experiment resulted in three treatments with no nutrient additions (ambient): control, CuNP, and AuNP and three treatments with nutrient additions (enriched): control, CuNP, and AuNP. Each treatment was replicated in three independent mesocosms, resulting in a total of 18 mesocosms that were exposed for 192 days (about 6 months). NPs were dosed weekly into mesocosms by mixing freshly sonicated NPs with 1 L of mesocosm water and distributed throughout the entire aquatic zone. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF][START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] The CuNP mesocosms received a weekly dose of 35 mg of Kocide for 6 months, except for the first week of treatment when they received an initial pulse of 374 mg that resulted in a total dose of 0.9816 g of Kocide per mesocosm over the duration of the study. Kocide contains 27% Cu by dry weight, so 358 mg of Cu from CuOH 2 was added to the mesocosms. The concentration of Cu was intended to be environmentally realistic by simulating field application rates and losses. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF] This resulted in an average water column Cu ± SE concentration in CuNP mesocosms of 11 ± 1.1 and 13 ± 2.0 μg/L in the ambient and enriched mesocosms, respectively, over the course of the experiment (Table S1). The mesocosms exposed to AuNPs received a weekly dose of 19 mg Au, resulting in a total dose of 532 mg Au added over the 6 months of the experiment. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF] This resulted in an average Au ± SE concentration in the AuNP mesocosms of 6 ± 1.0 and 9 ± 1.8 μg/L in the ambient and enriched mesocosms, respectively, over the course of the experiment (Table S1).

Consumer Nutrient Excretion. The nutrient excretion study was conducted during the peak of growing season in July and after snails had been exposed to NPs for 6 months and 3 months prior to the end of the mesocosm experiment. We chose to focus on snails because nutrient excretion is wellstudied in snails, [START_REF] Hall | Exotic Snails Dominate Nitrogen and Carbon Cycling in a Highly Productive Stream[END_REF][START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF][START_REF] Moslemi | Impacts of an Invasive Snail (Tarebia Granifera) on Nutrient Cycling in Tropical Streams: The Role of Riparian Deforestation in Trinidad, West Indies[END_REF][START_REF] Vanni | A Global Database of Nitrogen and Phosphorus Excretion Rates[END_REF][START_REF] Liess | Periphyton Responds Differentially to Nutrients Recycled in Dissolved or Faecal Pellet Form by the Snail Grazer Theodoxus Fluviatilis[END_REF][START_REF] Hill | Nitrogen Processing by Grazers in a Headwater Stream: Riparian Connections[END_REF] and snails represented 68% of the nonplant biomass in our mesocosms. Ten P. acuta snails were collected from each control, Cu and Au mesocosm and placed into mesocosm-specific plastic bins with mesocosm water. We then placed single snails into 15 mL conical tubes with 7 mL of filtered ambient nutrient, control mesocosm water (0.2 μm Nalgene Filter, Thermo Scientific, USA). All snails were incubated at a water temperature of 30 °C for 95 ± 0.5 min (mean ± SE). After incubation, snails and fecal matter were removed from excretion water. Snails and excretion water were immediately frozen and shipped to Baylor University. Excretion water samples were thawed and filtered (0.45 μm Whatman Filter, Cytiva, USA) to remove any remaining particulates (e.g. feces) prior to analysis. Frozen snails were analyzed for NP uptake by quantifying body burdens of Cu and Au using inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS methods are detailed in the Supporting Information, section "ICP-MS Methods", and were previously described from this mesocosm experiment. [START_REF] Geitner | Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms[END_REF][START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Cu speciation was measured on the dried powder of soft tissues that was kept under a N 2 atmosphere. Prior to analysis, the powder was homogenized, pressed into 6 mm pellets, and sealed with Kapton tape. Pellets were analyzed at the Cu Kedge (8.98 keV) for X-ray absorption near-edge structure (XANES) spectroscopy under 77 K cryostat conditions to avoid beam damage. Additional beamline, sample information, and LCF modeling are detailed in the Supporting Information, section "Cu Speciation", and were previously described from this mesocosm experiment. [START_REF] Geitner | Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms[END_REF][START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Background water nutrient concentrations (NH 3 -N and PO 4 -P) were measured in 25 replicates of filtered, control mesocosm water that was not exposed to snails. Ammonia was determined using the phenolate method and phosphorus was determined using the molybdate colorimetric method and on a Lachat QuikChem 8500 flow-injection autoanalyzer. [START_REF] Epa | Determination of Phosphorus by Semi-Automated Colorimetry[END_REF][START_REF] Epa | Determination of Nitrate-Nitrite Nitrogen by Automated Colorimetry[END_REF] The snail excretion water was analyzed for NH 3 -N and PO 4 -P in the same manner. The mean background NH 3 -N and PO 4 -P and concentrations were subtracted from the excretion concentrations to determine the snail-excreted NH 3 -N and PO 4 -P. NH 3 -N was measured, but NH 4 -N was estimated using differences in the molecular weight. Hence, we measured ammonia, the predominant form of nitrogen excreted by aquatic mollusks, and reported ammonium, which is the standard practice for measuring nitrogen excretion in aquatic animals. [START_REF] Hall | Exotic Snails Dominate Nitrogen and Carbon Cycling in a Highly Productive Stream[END_REF][START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF][START_REF] Moslemi | Impacts of an Invasive Snail (Tarebia Granifera) on Nutrient Cycling in Tropical Streams: The Role of Riparian Deforestation in Trinidad, West Indies[END_REF][START_REF] Vanni | A Global Database of Nitrogen and Phosphorus Excretion Rates[END_REF][START_REF] Campbell | Nitrogen Metabolism in Molluscs[END_REF][START_REF] Hall | How Body Size Mediates the Role of Animals in Nutrient Cycling in Aquatic Ecosystems[END_REF][START_REF] Evans-White | Grazer Species Effects on Epilithon Nutrient Composition[END_REF] Snail and food source (floc, E. densa, periphyton from E. densa, and the sediment) were oven-dried at 60 °C, homogenized, and weighed into silver capsules. Capsules were then exposed to HCl to remove inorganic carbon, ovendried at 60 °C for 48 h, and wrapped in tin capsules for stable carbon and nitrogen analysis. [START_REF] Jaschinski | Effects of Acidification in Multiple Stable Isotope Analyses[END_REF] Analysis was conducted on a Thermo-Electron Delta V Advantage isotope ratio mass spectrometer (IRMS) with a dual inlet system.

Statistical Analysis. Snail excretion rate (NH 4 -N and PO 4 -P μg/h) and metal accumulation in snail tissues (Cu and Au μg/g DM) were modeled using linear mixed models (LMMs) to determine the effects of the NP exposure, nutrient status, snail biomass, and their interaction on both nutrient excretion and bioaccumulation. [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology[END_REF] The main effects were NP treatment (control, Au, Cu), and nutrient treatment (ambient and enriched), whereas dry mass (DM, mg) was modeled as a covariate because excretion rate was expected to be dependent on snail body mass. [START_REF] Hall | How Body Size Mediates the Role of Animals in Nutrient Cycling in Aquatic Ecosystems[END_REF][START_REF] Vanni | Nutrient Cycling by Animals in Freshwater Ecosystems[END_REF] Mesocosm number (n = 18) was a random effect, which nested individual snail measurements within mesocosms and subsequently modeled the random effect of each mesocosm on these values. [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology[END_REF] The models were fit using the lmer function of the lme4 package in R. [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF][START_REF] Bates | Fitting Linear Mixed-Effects Models Using {lme4}[END_REF] Model fits were evaluated using residual plots, which demonstrated that the model fit the data and met the assumptions of a LMM. Conditional R 2 values were calculated for the selected model. [START_REF] Nakagawa | A General and Simple Method for Obtaining R2 from Generalized Linear Mixed-Effects Models[END_REF] Significance of main effects and interactions were estimated using the analysis of variance (ANOVA) function in the car package (Table S2). [START_REF] Zuur | Mixed Effects Models and Extensions in Ecology[END_REF][START_REF] Fox | An {R} Companion to Applied Regression[END_REF] The function predict.se.MERmod in the lme4 package was used to predict NH 4 -N or PO 4

--P excretion rates using the model and different combinations of levels of significant main effects or interactions (p < 0.05). The fitted models were used to graphically illustrate the predicted mean and 95% confidence interval (CI) excretion rates by snail DM. Results were plotted in ggplot2 for both NH 4 -N (Figure 3a,b) and PO 4 -P excretion (Figure 4a,b). [START_REF] Wickham | Ggplot2: Elegant Graphics for Data Analysis[END_REF] Average snail nutrient recycling rates were determined by averaging snail NH 4 -N and PO 4 -P excretion values by treatment (Figures 3c,d and4c,d). Snail biomass was determined by averaging snail DM from the excretion experiment (Figure 1c). The lsmeans package in R was used on the full excretion models described in Table S2 to look at pairwise differences between nutrient and NP treatments. [START_REF] Lenth | Least-Squares Means: The {R} Package {lsmeans}[END_REF] Stable isotope analysis, including snail diet composition, was determined using the MixSIAR package in R. [START_REF] Moore | Incorporating Uncertainty and Prior Information into Stable Isotope Mixing Models[END_REF] The δ 13 C and δ 15 N values for the snail tissue and food sources were considered tracers, and the NP was treated as a fixed effect (Figure S2).

■ RESULTS

Metal Accumulation in Snails. Cu and Au concentrations in NP-exposed snail tissues were more than 10-fold higher than control snails (Figure 1a,b, p < 0.001). Metal concentrations for CuNP-exposed snails averaged 4622 μg Cu/g snail and 4.78 μg Au/g snail for AuNP-exposed snails, with Cu accumulating at 1000-fold higher concentrations compared to Au. Nutrient status had no effect on metal accumulation in snails (Figure 1a, p < 0.68 and Figure 1b, p < 0.11).

Average snail body DM ranged from 3.73 to 5.82 mg in the ambient AuNP mesocosms and the enriched control mesocosms, respectively. While both types of NPs accumulated in the snail tissue, CuNPs did not influence the body size of snails either in the ambient or enriched mesocosms (Figure 1c, p < 0.34 and p < 0.99). Chronic AuNP exposure in the ambient mesocosms led to a reduction of snail body DM (Figure 1c, p < 0.05), but there was no difference in DM between the enriched AuNP snails and the enriched control snails (Figure 1c, p < 0.99). Without chronic NP dosing, there was no difference in snail body size between nutrient treatments (Figure 1c, p < 0.08). Snails represented 68% of the nonplant, living organism biomass, and P. acuta represented 83% of the snail biomass (Figure 1d).

Measurements of Cu speciation in the snail tissue (Figure 2) indicate that the Cu uptake was predominately of an ionic form bound to carboxyl groups (∼70%), with the remainder represented by sulfidized Cu (∼30%). Kocide CuNPs are primarily Cu(OH) 2 , yet there was no contribution of Cu(OH) 2 in the XANES spectra, suggesting that snails are not taking up the CuNPs directly through grazing. The addition of nutrients in the system did not change the speciation of copper associated with the snails. Tissue concentrations were too low to measure Au speciation in snails.

Nitrogen Excretion. Nitrogen excretion rates increased linearly as snail DM (covariate) increased across all NP and nutrient mesocosms (Figure 3a,b). NPs had the largest effect on N excretion, but nutrient and the interaction between DM [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Results of the linear combination analysis (LCA) of the P. acuta spectra are also presented along with their quality factor (R f ) as detailed in the Methods section. More details of Cu speciation can be found in the Supporting Information, and the references used as potential additional compounds for the LCA can be found in Figure S1. and NPs also had significant effects (Table S2, p < 0.001, p < 0.05). The conditional R 2 value for the nitrogen excretion LMM is 0.57, which describes the proportion of variance explained by the fixed and random factors (Table S2). Although N excretion rates were similar for snails with low DM in all treatments, as DM increased, N excretion rates in AuNP-exposed snails had larger slopes than those observed for CuNP-exposed snails. Both AuNP and CuNP-exposed snails had larger slopes than those observed for control snails.

In ambient mesocosms, the amount of N excreted per mean individual snail was 90 and 47% higher in the CuNPs and AuNPs mesocosms, respectively, compared to the ambient control mesocosms (Figure 3c, p < 0.001, p < 0.01). However, in enriched mesocosms, the amount of N excreted per average individual snail was 38% higher in the AuNP mesocosms (Figure 3d, p < 0.05). Snails from the enriched-CuNP mesocosms experienced an 18% increase in N excretion, but this increase was not statistically different from the enriched control mesocosms. After accounting for the effect of NPs, snails from the enriched mesocosms excreted 72% more N than snails from the ambient mesocosms (Figure 3c,d,p

< 0.

001).

Phosphorus Excretion. There were significant NP and nutrient effects on P excretion rate, with NPs having a larger influence on snail P excretion than nutrients (Table S2, p < 0.001, p < 0.05). There was a significant DM and NP interaction, indicating that control, Au, and Cu NP mesocosms had different effects on snail P excretion as a function of snail DM (Table S2, p < 0.05). The conditional R 2 value for the phosphorus excretion LMM is 0.45 (Table S2). Phosphorus excretion increased significantly with increasing DM in the ambient CuNP mesocosms (Figure 4a). Conversely, ambient and enriched AuNP and enriched CuNP mesocosms showed little change in P excretion with increasing DM (Figure 4a,b). Ambient and enriched control mesocosms had a slightly negative relationship in P excretion with increasing DM.

P excretion per mean individual snail was 94 and 110% higher in the ambient CuNP and AuNP mesocosms, respectively, compared to the ambient control mesocosms (Figure 4c, p < 0.001). The mass of P excreted per mean individual snail was 52% higher in the enriched AuNP mesocosms, respectively, compared to the enriched control mesocosms (Figure 4d, p < 0.01). Snails from the enriched CuNP mesocosms had a slight increase in P excretion (11%), but it was not statistically different from the enriched control mesocosms (Figure 4d, p < 0.52). After accounting for the effect of NPs, snails excreted 71% more P per mean individual in the enriched than in the ambient mesocosms (Figure 4c,d,p < 0.001).

■ DISCUSSION

Consumer-mediated nutrient recycling balances the requirements of organisms for energy and nutrients and the resulting relationship between diet, growth rates, and excretion. [START_REF] Vanni | Predicting Nutrient Excretion of Aquatic Animals with Metabolic Ecology and Ecological Stoichiometry: A Global Synthesis[END_REF][START_REF] Sterner | Ecological Stoichiometry[END_REF][START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF] Through excretion, consumers can be important drivers of nutrient cycling and can supply nutrients at rates comparable to other major nutrient sources in aquatic ecosystems. [START_REF] Hall | Exotic Snails Dominate Nitrogen and Carbon Cycling in a Highly Productive Stream[END_REF][START_REF] Vanni | A Global Database of Nitrogen and Phosphorus Excretion Rates[END_REF][START_REF] Vanni | Nutrient Cycling by Animals in Freshwater Ecosystems[END_REF] Previous analyses have found the prediction of nutrient excretion for any animal taxon or aquatic ecosystem to be Environmental Science & Technology best described using body size, water temperature, trophic guild, and vertebrate classification. [START_REF] Vanni | Predicting Nutrient Excretion of Aquatic Animals with Metabolic Ecology and Ecological Stoichiometry: A Global Synthesis[END_REF] Here, we see that N and P excretion rates of consumers increased as much as two-fold in response to chronic NP exposure in both ambient and enriched mesocosms between snails of the same species, sizes, and at the same water temperature. The best predictors of aquatic organism excretion were all equivalent in our study, yet we see a doubling of N and P excretion, strongly indicating that NP exposure is the driving variable behind this increased excretion. This two-fold change in cycling of nutrients by consumers means that algal communities have nutrients available to them at twice the rate when consumers are exposed to chronic NPs. Thus, NP exposure to consumers may result in increased primary production rates, and in our system, it resulted in NP mesocosms spending a larger proportion of time above the nutrient threshold where blooms occur. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF][START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF][START_REF] King | Spatial Dependency of Vegetation Environment Linkages in an Anthropogenically Influenced Wetland Ecosystem[END_REF][START_REF] Singer | Anthropogenic Subsidies Alter Stream Consumer-Resource Stoichiometry, Biodiversity and Food Chains[END_REF] While body size is typically the primary driver of nutrient excretion, we saw no difference in snail body sizes between treatments, with the exception of smaller snails in the ambient AuNP mesocosms, although we saw a difference in nutrient excretion. Stable isotope analysis of snail food sources and snail body tissue across treatments showed an indistinguishable preference for periphyton, Egeria, and "floc" compared to other food sources (Figure S2). Floc is a matrix of amorphous detritus, periphyton, and animal feces (particularly snails), which is why snail isotopes aligned so closely to the flocit was likely the signature of their own waste product rather than their dominant food, which was periphyton. This indicates that there is likely a disparity in how snails are compensating for excreted phosphorus under NP treatments, whether it be compensatory feeding, increased energy demands to maintain homeostasis, increased P associated with fecal matter, or another mechanism to be determined in future work. [START_REF] Liess | Periphyton Responds Differentially to Nutrients Recycled in Dissolved or Faecal Pellet Form by the Snail Grazer Theodoxus Fluviatilis[END_REF][START_REF] Fink | Physiological Responses to Stoichiometric Constraints: Nutrient Limitation and Compensatory Feeding in a Freshwater Snail[END_REF] Particle dissolution rate is important because it determines if the organism interacts with a dissolved or a particulate metal in aquatic ecosystems. Although both NPs experienced some dissolution in different compartments of the mesocosms, CuNPs dissolve more rapidly in the water column than AuNPs. [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Measurements during this experiment indicate that CuNPs rapidly dissolve (t 1/2 = 33 h in our mesocosms), releasing Cu ions that form a complex with organic matter and sediment onto E. densa and the floc. [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF][START_REF] Vencalek | In Situ Measurement of CuO and Cu(OH)2 Nanoparticle Dissolution Rates in Quiescent Freshwater Mesocosms[END_REF] The surface of E. densa and the floc is where the snails have high grazing activity and encounter the complexed ionic Cu and some CuS species (Figures 2 andS2). While AuNPs have low dissolution rates in abiotic water and generally interact with organisms as particles, this was not true in our mesocosms containing E. densa. [START_REF] Glenn | Abiotic and Biotic Factors That Influence the Bioavailability of Gold Nanoparticles to Aquatic Macrophytes[END_REF][START_REF] García-Cambero | Converging Hazard Assessment of Gold Nanoparticles to Aquatic Organisms[END_REF][START_REF] Hull | Filter-Feeding Bivalves Store and Biodeposit Colloidally Stable Gold Nanoparticles[END_REF] The dosed AuNPs in the mesocosms associated with E. densa were transformed into oxidized species by periphyton. [START_REF] Avellan | Gold Nanoparticle Biodissolution by a Freshwater Macrophyte and Its Associated Microbiome[END_REF] At the time of the snail excretion study, most of the added AuNPs were associated with the E. densa periphyton and the floc, the primary grazing areas for the snails. All of the Au associated with E. densa and ∼10% of the Au in the floc had dissolved and become associated with new ligands. [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Therefore, snails were likely to be feeding on a combination of ionic Au species and AuNPs. While accumulation into the consumer tissue type varied by the NP type and differences in their rates of dissolution in the water column, consumer excretion rates in the mesocosms exposed to NPs are similar. This may be F because snails primarily uptake dissolved and complexed forms of the metals and not pristine NPs. [START_REF] Botha | Comparative Aquatic Toxicity of Gold Nanoparticles and Ionic Gold Using a Species Sensitivity Distribution Approach[END_REF][START_REF] Jo | Toxicity and Biokinetics of Colloidal Gold Nanoparticles[END_REF] Snails accumulated high amounts of metals in their soft tissues after 6 months of exposure, especially for CuNPs (4622 μg/g). Cu speciation in the snail tissue was mainly associated with carboxyl groups (Figure 2). Copper is a biologically relevant element to consumers; it is actively and intentionally taken up and tightly regulated by organisms. It has direct interactions with amino acid and carboxyl side chains within peptide chains and is critical to organismal biochemical reactions. [START_REF] Festa | Copper: An Essential Metal in Biology[END_REF] Conversely, gold is not a biologically relevant metal in consumers. Organisms in aquatic ecosystems are unlikely to encounter gold NPs or complexed Au ions, and therefore, it is a novel contaminant. [START_REF] Southam | The Biogeochemistry of Gold[END_REF] This could explain the lower amount of Au in snail tissues compared to Cu. However, these differences may also be the result of the faster dissolution rate of CuNPs in the water column and the resulting differences in their distribution in the mesocosms. [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] While NP body burdens were studied, they did not correlate well with consumer excretion. In future work, we intend to investigate possible mechanisms for the increase in nutrient excretion.

While we understand that nutrient recycling increases with NP exposure, we do not know the mechanism(s) behind these alterations. Previous studies have reported an increase in consumer nutrient excretion after exposure to other contaminants, including triclosan. [START_REF] Taylor | Consumer-Mediated Nutrient Recycling Is Influenced by Interactions between Nutrient Enrichment and the Antimicrobial Agent Triclosan[END_REF] Triclosan is a well-known antimicrobial and has a probabilistic mechanism to alter the gut microbiome within the consumer. In our study, CuNPs have known antimicrobial properties and, while not typically thought of as an antimicrobial, AuNPs have some effect on microbial communities. [START_REF] Avellan | Gold Nanoparticle Biodissolution by a Freshwater Macrophyte and Its Associated Microbiome[END_REF] While we could not confirm the direct uptake of AuNPs, some of the Au in snails is likely AuNPs because the floc contained primarily AuNPs. [START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] In our study, both NPs likely exhibited some antimicrobial properties. [START_REF] Avellan | Gold Nanoparticle Biodissolution by a Freshwater Macrophyte and Its Associated Microbiome[END_REF][START_REF] Reese | Microbial Nitrogen Limitation in the Mammalian Large Intestine[END_REF][START_REF] Holmes | Diet-Microbiome Interactions in Health Are Controlled by Intestinal Nitrogen Source Constraints[END_REF][START_REF] Borase | Nano-Eco Toxicity Study of Gold Nanoparticles on Aquatic Organism Moina Macrocopa: As New Versatile Ecotoxicity Testing Model[END_REF][START_REF] Elbehiry | Antibacterial Effects and Resistance Induction of Silver and Gold Nanoparticles against Staphylococcus Aureus-Induced Mastitis and the Potential Toxicity in Rats[END_REF] These properties could alter the gut microbiome in snails and alter food assimilation efficiency. [START_REF] Krajmalnik-Brown | Effects of Gut Microbes on Nutrient Absorption and Energy Regulation[END_REF][START_REF] Antunes | Effect of Antibiotic Treatment on the Intestinal Metabolome[END_REF][START_REF] Schulfer | Risks of Antibiotic Exposures Early in Life on the Developing Microbiome[END_REF] However, most Cu (and perhaps Au) accumulated by snails was as dissolved species. Both ionic Cu and Au have demonstrated toxicity in aquatic organisms and may exhibit differential toxicity to snails. [START_REF] Brix | The Toxicity and Physiological Effects of Copper on the Freshwater Pulmonate Snail, Lymnaea Stagnalis[END_REF][START_REF] Borase | Nano-Eco Toxicity Study of Gold Nanoparticles on Aquatic Organism Moina Macrocopa: As New Versatile Ecotoxicity Testing Model[END_REF][START_REF] Balfourier | Unexpected Intracellular Biodegradation and Recrystallization of Gold Nanoparticles[END_REF] Another mechanistic explanation is that metal exposure may result in increased energy demands because of metal transport and sequestration, limiting the amount of energy available for nutrient uptake processes. This could be analyzed via metabolite biomarkers and gene expression. [START_REF] Griffitt | Exposure to Copper Nanoparticles Causes Gill Injury and Acute Lethality in Zebrafish (Danio Rerio)[END_REF][START_REF] Williams | Size and Dose Dependent Effects of Silver Nanoparticle Exposure on Intestinal Permeability in an in Vitro Model of the Human Gut Epithelium[END_REF][START_REF] Poynton | Toxicogenomic Responses of Nanotoxicity in Daphnia Magna Exposed to Silver Nitrate and Coated Silver Nanoparticles[END_REF] While NPs are novel additions to consumer environments, nutrient enrichment has been well-studied and identified as a water quality issue worldwide.

Previous studies have found that nutrient enrichment increases consumer excretion and these studies concur with our results. Nutrient enrichment increases consumer excretion because consumers are not sequestering nutrients, as there is a surplus in their food. [START_REF] Dalton | The Influence of Dietary and Whole-Body Nutrient Content on the Excretion of a Vertebrate Consumer[END_REF] Independent of NP exposure, N and P excretion rates increased by 70% in the enriched mesocosms compared to ambient mesocosms. This indicates that there is an enriched versus ambient modulated effect. Previous literature has found that organismal excretion increased across agricultural gradients in mayfly species and fish assemblages. [START_REF] Wilson | Nutrient recycling by fish in streams along a gradient of agricultural land use[END_REF][START_REF] James | Land Use Controls Nutrient Excretion by Stream Invertebrates along a Gradient of Agriculture[END_REF] The excretion rates that we saw in the control snails in this study were similar to those reported in Physella sp. collected from wetland ecosystems, and our excretion pattern of increasing N excretion as size increases is consistent with those reported in previous studies. [START_REF] Grimm | Role of Macroinvertebrates in Nitrogen Dynamics of a Desert Stream[END_REF][START_REF] Vanni | A Global Database of Nitrogen and Phosphorus Excretion Rates[END_REF] Nutrient enrichment from wastewater streams are frequently mixed with other emerging contaminants, including NPs.

NPs are likely to enter aquatic ecosystems as part of waste streams that include high nutrients (e.g. agricultural fertilizers and wastewaters). This nonlethal impact of NPs on consumers has the potential to exacerbate nutrient pollution and hypoxia in aquatic ecosystems. The doubling of nutrient excretion by consumers could be an overlooked factor in the intensification of eutrophication driven by consumer exposure to contaminants such as NPs. Although our experiment focused on snails, other aquatic consumers interact with NPs and the metal species they transform into through the same exposure pathways. It is likely that this increase in nutrient excretion is applicable to other aquatic consumers who live in similar waste streams with high nutrient loading and contaminant mixtures. In our study, this resulted in an increased frequency of algal blooms in NP mesocosms. [START_REF] Simonin | Engineered Nanoparticles Interact with Nutrients to Intensify Eutrophication in a Wetland Ecosystem Experiment[END_REF] If our experimental mesocosm findings are indicative of global processes, NP and consumer interactions could contribute to widespread increases in aquatic productivity, resulting in more frequent algal blooms and more rapid degradation of aquatic ecosystems.

Figure 1 .

 1 Figure 1. Snail metal concentration ± 95% CI for Cu (a) and Au (b) in snail tissues from NP treatments. Axes and the scale are different for each plot. Black points represent replicate values. NP treatments are distinguished by bar colors. (c) Average DM (mg ± 95% CI) of snails included in excretion experiment per treatment. (d) Average total organismal DM in each mesocosm per treatment (g ± 95% CI) at the end of the mesocosm experiment.

Figure 2 .

 2 Figure 2. XANES at Cu K-edge for P. acuta sampled in either ambient or enriched mesocosms in comparison with Kocide, Cu-carboxyl, and Cu-S references, as described in (Avellan et al., 2020).[START_REF] Avellan | Differential Reactivity of Copper-and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm[END_REF] Results of the linear combination analysis (LCA) of the P. acuta spectra are also presented along with their quality factor (R f ) as detailed in the Methods section. More details of Cu speciation can be found in the Supporting Information, and the references used as potential additional compounds for the LCA can be found in FigureS1.

Figure 3 .

 3 Figure 3. Nitrogen excretion rates expressed by individual snail body DM (mg) in the (a) ambient and (b) enriched mesocosms. NPs are distinguished by shading color. Lines and shaded areas represent predicted means and 95% CIs from LMM. Mean nitrogen excretion rates by P. acuta after NP exposure in the (c) ambient and (d) enriched mesocosms. Means and 95% CIs are presented, and significant differences (***p < 0.001, **p < 0.01, *p < 0.05) are indicated by asterisks.

Figure 4 .

 4 Figure 4. Phosphorus excretion rates expressed by individual snail body DM (mg) in the (a) ambient and (b) enriched mesocosms. NPs are distinguished by shading color. Lines and shaded areas represent predicted means and 95% CIs from LMMs. Mean phosphorus excretion rate of P. acuta after NP exposure in the (c) ambient and (d) enriched mesocosms. Means and 95% confidence intervals are presented and significant differences (***p < 0.001, **p < 0.01, *p < 0.05) are indicated by asterisks.

https://dx.doi.org/10.1021/acs.est.0c02197 Environ. Sci. Technol. XXXX, XXX, XXX-XXX B

https://dx.doi.org/10.1021/acs.est.0c02197 Environ. Sci. Technol. XXXX, XXX, XXX-XXX E

https://dx.doi.org/10.1021/acs.est.0c02197 Environ. Sci. Technol. XXXX, XXX, XXX-XXX H

https://dx.doi.org/10.1021/acs.est.0c02197 Environ. Sci. Technol. XXXX, XXX, XXX-XXX I

https://dx.doi.org/10.1021/acs.est.0c02197 Environ. Sci. Technol. XXXX, XXX, XXX-XXX J

■ ACKNOWLEDGMENTS

We thank Ally Adams, Samuel Mahanes, and Jason Rodriguez for their help processing samples. Further, Mark Wiesner, Nicholas Geitner, Heileen Hsu-Kim, and Jason Unrine for their assistance in designing this mesocosm experiment. The authors acknowledge the Baylor University Mass Spectrometry Center and the Baylor University Isotope Laboratory for their support during this work. This material is based upon work supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF0830093 and DBI-1266252, Center for the Environmental Implications of NanoTechnology (CEINT). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been subjected to EPA review, and no official endorsement should be inferred. Part of this work used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Additional funding was provided by the C. Gus Glasscock, Jr. Endowed Fund for Excellence in Environmental Sciences at Baylor University.


G Nahas

B Cirée

M Quesada

T Gelain

A Loukili

S Gildas Medjigbodo

A Khelidj

M Choinska

J-P Regoin

F Benboudjema

A Darquennes

A Hilaire

R Bennacer

G Rastiello

C La Borderie

C Lawrence

J Dubreuil

M-C Barbaroux

C Gilliot

Study of the Reinforced Concrete Structures Containment

Introduction

Double-wall containment buildings for pressurized water nuclear power plants consist of a reinforced concrete outer wall and a biaxially pre-stressed concrete inner wall. The leak-tightness of the inner wall is a primary safety requirement. This structure that ensures the containment of the radionuclides may crack during its construction, or in service loading. This cracking tends to reduce the leak-tightness of the wall in the event of a serious accident (accidental pressure and temperature loads). Quantifying this risk of leakage has been the subject of several models for which the parameters were obtained from tests on the material scale. Given that it is difficult, or even impossible, to perform an experiment on the real structure, it is necessary to resort to numerical simulation. The difficulty involved is related to the numerical models used, which must be capable of simulating both the mechanical behavior of the structure (macroscopic scale) and the geometry of the cracks (mesoscopic scale). The development of a reliable tool to assess leakage through a reinforced and prestressed concrete wall under severe pressure conditions with possible thermal loads, which will allow the models and methods implemented in the calculation software to be verified, thus becomes a key objective. This is the purpose of the ECOBA project (Study of Reinforced Concrete Structure Containment) and the validation of this tool is done by means of an experimental study using a representative structure member Mock-up. This project was conducted by A. Loukili (GEM) over the period 2010-2014, as part of collaboration between the IRSN and three laboratories with complementary skills: GeM, SIAME and LMT. These teams obtained support from the French National Research

Design of the Mock-up

The design of the Mock-up is an essential step of the project, since it must allow a proper validation of the models and methods implemented in the calculation software applications used.

The important parameters that have guided this design are: the geometric representativeness of the section in relation to the current portion of a P4 type reactor inner containment wall, the representativeness of circumferential stress in accidental loads, the homogeneity of the stresses in the section in order to avoid parasitic forces localization near loads application areas and the simplicity of the boundary conditions to facilitate numerical simulations.

Five configurations with different shapes, geometries and structures were studied. These configurations were analyzed using finite element calculations with the CAST3M software, taking into account the non-linear behavior of the concrete and the reinforcement. The idea is to ensure continually relevance of each configuration with respect to the objectives of the experiment. The final configuration chosen is that of a bone-shaped Mock-up, comprising two heavily reinforced and prestressed soles and a "zone of interest" surrounded by two transition zones with brackets at 30°degrees, for connecting the "zone of interest" to the soles. This choice allows a homogeneous distribution of the deformations in the "zone of interest" for the crack aperture and leakage flow rate measurements after the area is subjected to traction. The 1.50m wide, 1.20m high and 0.90m thick "zone of interest" represents a current portion of the containment building. Thus, with the soles and transition zones, the Mock-up reaches a length of 3.90m and a height of 2.40m, with a mass of about 20 tons. The "zone of interest" is made of a type of concrete known as B11, the formulation of which is representative of the concrete used for containment buildings, and the soles are made of self-compacting concrete (Figure 1). In order to avoid the problem of loosening of the transition area and loss of mechanical force continuity during loading, pin-shaped HA8 hook connectors, with an extended length of 33 cm are distributed over the entire rear face of each steel plate (Figure 2). Two Mock-ups ("Model 1" and "Model 2") were made successively on the experimental platform to build upon the experience feedback of the first Mock-up, especially regarding special precautions to ensure the leak-tightness between the partition plates and the "zone of interest".

The geometry and construction layout of both Mock-ups are identical, but the "zone of interest" of the second Mock-up includes construction joints similar to those of containment buildings, to assess their impact on the leak-tightness of the wall.

As regards the instruments embedded in the concrete, two types of sensors are positioned in the thickness of the wall in the "zone of interest", in order to monitor internal displacements (or strains) and temperature changes:

 three dual-coil vibrating-wire sensors (CV) with 3D positioning for monitoring strains (and temperature using a thermistor) along the three axes,  four thermocouples (TC) positioned in the thickness of the wall (one of them at the same location as a vibrating-wire sensor for validation) to measure the temperature profile.

The location of the sensors is shown in Figure 3.

In addition to these sensors, Model 2 is equipped with stress gauges installed on the reinforcement and reservations are left for installing hygrothermal sensors.

Location of the hygrothermal sensors Location of the dual-coil vibrating-wire sensors

Early-age concrete behavior

This study aims to predict the state of the initial stresses in the Mock-up. Indeed, given the massiveness of the Mock-up, the exothermic hydration reaction leads to high temperatures (about 55°C, that is a temperature increase of 40°C), inducing thermal strain gradients. Moreover, given  Concrete construction joint restraint: stresses induced by the shrinkage of the lower raised edge must be added to the earlier stress state (this concern only the second Mock-up).

During the temperature increase phase, compressive stresses (weak, because the Young modulus is low at this stage) appear in the current raised edge (they are tensile stresses in the lower raised edge that may lead to cracking all the way through). Upon cooling, the stresses are reversed: the tensile stresses generated in the current raised edge can induce cracking, this time all the way through, accentuated by the autogenous shrinkage strain.

Drying can be a priori neglected in this study. Indeed, on one hand water exchanges are 1000 to 10,000 times slower than the thermal phenomena and therefore concern only a thin layer of concrete, on the surface of massive structures; on the other hand, when the formwork remains in place, the drying becomes very slow. The effects of drying are similar to the thermal effects.

Nevertheless, given the massiveness of the structure, only the water gradient (higher internal relative humidity rate than the external humidity rate) can induce a non-negligible stress state, while remaining comparable to that observed in a conventional concrete test piece (type 16X32, for example). Thus, cracking limited to the area close to the surface may occur. Then, the initial stresses in the Mock-up were calculated using the finite element software CAST3M, with a model incorporating temperature, hydration, shrinkage, creep and cracking. The parameters of this model have been identified in companion specimen tests in B11 concrete, representative of a nuclear power plant, which was used during the tests. A preliminary study has shown that drying induces only a localized surface cracking. Thus, this has not been taken into account in these calculations (there are formwork effects and the mesh must be fine enough). Supported by the soles, the jacks will put in tension the "zone of interest", thus creating cracks in this area. These correspond to cracks that may appear in a current containment wall area under ultimate pressure loads. After the creation of the cracks, the gas outputs through these cracks in the "zone of interest" are measured using an injection box (upstream side) and a recovery box (downstream side). Several sequences of measurements, at various injection pressure values upstream and at various crack apertures, are needed to cover the various flows through cracks that may appear in the containment wall during a severe accident.

Studies of the characteristics of these cracks, particularly their aperture, spacing and geometry, provide knowledge regarding the mechanical behavior of this area, particularly during the airsteam sequences. Studies of the leakage flow rates through the cracks provide knowledge about the assessment of containment building of nuclear power plants under loads of serious accidents.

The displacement measurements are performed using 3 devices (Figure 8): Three levels were achieved at 20%, 40% and 60% of the theoretical rated load respectively, estimated from a direct tensile test on companion specimen. The 60% level was maintained long enough to observe the future cracks (processing of the displacement field from a few images) and to place 3 LVDT sensors on the future cracks observed from the displacement field. After the installation of the LVDT sensors, the Mock-up was fully unloaded before resuming the initial scenario, which allowed the aperture of the cracks created to be measured by both methods (DIC and LVDT). The scenario was completed respectively at 60%, 80% and 100% of the rated load prior to a second total unloading. During this loading, three cracks appeared in the "zone of interest". First, a crack at the center of the area, followed by two other cracks on either side of the first one, which reflects the proper control of the cracking process provided by the experimental device.

On July 5 th 2013, the Mock-up was put under tension again. First, having again been subjected to a force driven load with 5 levels: (from 20% to 100% of the rated load), for the gas permeability testing. During this phase, the Digital Image Correlation (DIC) and the LVDT sensor data were not available. The downstream recovery box was dismantled during the 100% level, and the 6

LVDT sensors were installed. The Mock-up was then unloaded and reloaded with a displacement driven loading at a speed of 0.17 mm/min with several levels and a final ramp towards the estimated theoretical aperture of 500 µm. When a 4200 kN load was reached, a cracking sound was heard by the experimenters and the Mock-up was then unloaded. During this second load phase, DIC measurement was performed.

On March 12 th 2014, the Mock-up was put under tension with a displacement driven load. During this loading, up to a 2 mm displacement of the jacks, the drive proved ineffective because of the failure of a jack sensor. The Mock-up was then unloaded and reloaded with a force driven load with increasing displacement target values until a displacement of 4.2 mm per jack was achieved.

DIC measurement was performed during these two loading phases on the upstream side. LVDT sensors were placed on the downstream side in order to measure the cumulative apertures of the cracks and to easily find this displacement mark for measuring the leakage rate.

Analysis of the cracks was performed in two stages:

 Displacement measurement by digital image correlation using the CORRELIQ4 software  Crack analysis by Gaussian deconvolution of the displacement fields with a program specially developed in MATLAB software to automatically detect cracks, their aperture

Study of the containment

The leakage rates through the cracks are measured using a tracer gas dilution method. This method consists in injecting helium at a controlled flow rate into the pressurized upstream box and collecting the helium-air mixture in the measurement box next to the wall on the downstream side of the Mock-up.

The helium concentration of the mixture collected downstream is then measured by means of a mass spectrometer, which allows the leakage rate from the crack to be known by continuously recording the helium balance in an open system.

The crack aperture is obtained from the measured flow rate using a "Cracked Concrete Model" developed by IRSN. This model makes it possible to calculate the dimensional characteristics Knowledge obtained from the test results and numerical models will be used by IRSN in nuclear safety assessments, to support its expert analysis of the leak-tightness of the inner containments wall of nuclear power plants.

The full-scale Mock-ups built at the École Centrale de Nantes were achieved despite their complexity under the conditions of university laboratories. To develop them, teams had to overcome many technical and technological problems. The complementary skills of the teams involved were a great help to find accurate solutions.

These problems have led to delays in the progress of the project. Issues include the choice of the design of the Mock-up, the test control and monitoring system, the complexity of the finite element calculations, the design of the leak measurement system and the construction of the upstream injection box and downstream recovery box.

The instruments used are similar to those implemented in the containments buildings of the nuclear power plants under operation. They have been improved by additional measurements, in In the last experimental campaign, parasitic leaks appeared on the sealing surfaces placed on the upper and lower faces of the "zone of interest". This problem is currently being resolved, thus allowing the experimental program to continue.

The first test results are very interesting, especially the creation of cracks in the "zone of interest" that are similar to those found on real structures subjected to such stresses, as well as the corresponding air leakage flow rate values. All of this confirms the great interest of the project and the possibilities of assessing the sensitivity of the parameters affecting the leak-tightness of containment buildings of nuclear power plants.

2 nd

 2 Conference on Technological Innovations in Nuclear CivilEngineering TINCE 2014, Paris 1 st to 4 th September Agency (ANR) as part of a so called "White Program". The project was also labeled by the competitiveness workgroup Advancity, Sustainable City and Mobility. The construction, on the site of the Ecole Centrale de Nantes, of two Mock-ups representing a current portion of a 2.40 m high and 3.90 m wide inner containment wall was entrusted to NUVIA Travaux Spéciaux and the technical control of the test was entrusted to APAVE-Agence de Niort. The scientific issues studied are: the early-age behavior of concrete with the appearance of micro cracking, changes in its permeability and flows through localized cracks under severe accident conditions or under the effect of pressure (5 bar) and temperature (150°C), with simulations at the mesoscopic and macroscopic scales. This paper presents the experimental part of the project, the technical solutions implemented, in particular the design of Mock-ups, the devices for creating and monitoring crack apertures and leak rate measurement. An overview of the research conducted in the framework of this project is also briefly discussed.

2 nd

 2 Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2014, Paris 1 st to 4 th September

Figure 1 -

 1 Figure 1 -Schematic Mock-up

Figure 2 -

 2 Figure 2 -Reinforcement of the "zone of interest"

Figure 3 -

 3 Figure 3 -Instruments embedded in the concrete of the Mock-up

2 ndFigure 4 -Figure 5 -

 245 Figure 4 -Changes in the temperature at various measuring points in the structure over time.(Presence of soles)

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 2 Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2014, Paris 1 st to 4 th September

Figure 6 -Figure 7 -

 67 Figure 6 -Time history of maximum stresses, in the depth and the surface of the concrete structure

 6

 6 measurement sensors integrated into the hydraulic jacks  6 LVDT sensors with a stroke +/-2mm, connected to the data acquisition system  An image analysis system (DIC), for measuring displacement fields Mechanical tests on the first Mock-up took place on July 4 th and 5 th 2013 and then on March 12 th 2014. On July 4 th 2013, the Mock-up was put under tension by a force driven load using Digital Image Correlation (DIC) to measure the displacement field.

Figure 8 -

 8 Figure 8 -DIC measurement system with the LVDT sensors

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 2 Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2014, Paris 1 st to 4 th September and the total strain. The processed images are synchronized with the mechanical load data (force sensors, jack displacement sensors and LVDT sensors).

Figure 9 Figure 9 -

 99 Figure 9 shows the apertures of the residual cracks measured by LVDT sensors and calculated from the displacement field at the end of the test (July 5 th 2013)

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 2 Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2014, Paris 1 st to 4 th September (airflow opening) of a network of cracks, if the flow rate and associated pressure values are known. This model is explained in [3].From the flow rate results obtained for Mock-up (Model 1), we have determined the cracks openings compared to the expected theoretical values. (Figure12).

Figure 12 -

 12 Figure 12 -Cracks openings as a function of the total load applied by the jacks

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 2 Conference on Technological Innovations in Nuclear CivilEngineering TINCE 2014, Paris 1 st to 4 th September particular in regard to crack aperture measurements: vibrating-wire sensors, LVDT sensors, image correlation and airflow aperture method. The redundancy and diversity of the measurement methods make it possible to compare different values and validate the results more robustly.

2 nd Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2014, Paris 1 st to 4 th September that

  drying is a diffusive phenomenon, a relative humidity gradient, and therefore a drying shrinkage gradient, appears. This creates stresses on the macroscopic scale, according to several mechanisms.

	The thermal and autogenous aspects lead to two cracking mechanisms:
	 A self-stress mechanism: during heating, the temperature increases significantly deep
	inside, while remaining close to the outside temperature on the surface. The ensuing
	temperature gradient results in a thermal strain gradient (expansion) that generates tensile
	stresses on the surface and compression deep inside. These self-balanced stresses are
	weak, because the Young modulus is low at this stage, but the tensile strength is also

weak. Upon cooling, the Young modulus of the concrete increases, the strain gradient (shrinkage contraction) is reversed relative to that of the previous phase, corresponding to heating. Tensile stresses develop deep inside, while compression stresses appear on the surface. Potential cracking occurs, first on the surface and then deeper inside: it does not go all the way through.