Jesús Víctor

García-Morales

Meredith Root-Bernste
email: meredith.root-bernstein@mnhn.fr

Pierre Du Plessis

Matías Guerrero-Gatica

Trupthi Narayan

Samuel Roturier

Helen C Wheeler

What Are ILK in Relation to Science? Using the 'Ethic of Equivocation' to Co-Produce New Knowledge for Conservation

Keywords: indigenous and local knowledges, conservation, ecology, equivocation, dialogue, integration, co-production

Attention to epistemological relationships between Indigenous and local knowledges (ILK) and conservation science is increasing. Some approaches for doing so have been developed, but in general, serious engagement between ILK and science still feels experimental and does not have broad uptake. Here we address some of the main issues that arise when considering the epistemological relationships between ILK and conservation or ecological science. A key issue is the relationship between ILK and science that sets the expectations for how they may form dialogues, collaborations, or co-production. After reviewing several perspectives on this relationship, and their limitations, we suggest that a focus on shared meanings within an 'ethic of equivocation' is a productive path forward. In an ethic of equivocation, neither ILK nor science validates the other, yet meaning can be created. We explain and develop the concepts of meanings and equivocation in the ILK-science context. We, thus, argue for a broad and rich understanding of the joint roles of ILK and science, which goes beyond treating ILK as a data source. We argue that diverse styles of reasoning exist in science, and scientists may already treat observations, models, and collaborations in the ways that we discuss, providing ready-made analogies for thinking about ILK. We also discuss how to avoid abuses of power while engaging with ILK to co-produce new knowledge.

Introduction

The recent Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) reports on the state of biodiversity are part of an important call for decision-makers to stop the deterioration of ecosystems. They also mark a milestone in the use of Indigenous and local knowledges (ILK) in high-level international science policy documents, the result of almost 30 years of research triggered by Article 8j of the Convention on Biological Diversity (CBD). IPBES set up an ILK task force with a clearly defined mandate, which was implemented by the Technical Support Unit (TSU) hosted at UNESCO. The TSU facilitated a series of dialogue workshops involving ILK holders and academics, and put together an "ILK library of materials as a resource for IPBES authors" [START_REF]Indigenous and Local Knowledge in IPEBS[END_REF][START_REF] Hill | Working with indigenous, local and scientific knowledge in assessments of nature and nature's linkages with people[END_REF]. This effort highlights the increasing prominence of science and ILK working together and the rapidly evolving discussion on the synthesis and use of science and ILK in decision-making.

The epistemological relationships between ILK, science, management, and policy currently have many forms. We use the term "epistemological relationship" (see Box 1 for definitions of terms in bold) as a broad formulation that points to how different forms of knowledge are formed and used ("epistemology") without specifying a model for their interaction ("relationship"). This is because some existing terms, such as "integration", have come to be associated with methods that imply an unbalanced relationship of power in which ILK may be subsumed by the scientific framework, detached from the producers of that knowledge. Not only can this be derogatory, but it also risks further marginalizing and disempowering ILK holders. The methodological pluralism resulting from the range of epistemological relationships reflects the many different socioecological contexts in which ILK meets Western conceptions of conservation and management, and the many questions ILK may address. Undoubtedly, there is not one right way to engage with ILK, due to the diversity of these knowledge systems and their multiple relationships to varying social, political, and ecological contexts, and different scientific endeavors [START_REF] Olazabal | Subaltern forms of knowledge are required to boost local adaptation[END_REF]. At the same time, different methods and philosophies for engaging ILK create different kinds of epistemological relationships, different forms of value, and different ways in which knowledge can be used in the world.

Box 1. Definitions of some key terms.

Cosmology: a culture's claims about the kinds of things in the world (e.g., species, materials, spirits, gods, etc.), their natures or properties and capabilities, and their distribution. Epistemology: A system of reasoning about what constitutes knowledge, the desirable properties of knowledge, and how knowledge is formed. This is usually associated with a particular cosmology and ontology. For example, the epistemology of science can be characterized by claims that knowledge is formed through the experimental method, and should strive to be universal, objective, reductionist, falsifiable, etc. Equivocation: a partial, contextual alignment between two concepts or practices that are not the same, and which actors may be aware are not the same, but which form a functional basis for interaction and exchange. Holistic-mechanistic distinction: Holistic thinking attempts to understand various aspects of a complex whole, simultaneously. Mechanistic thinking attempts to break the whole into parts and to understand how each part behaves in a causal process. Ontology: a description of a culture's understanding of the origins and structure of the world, and the kinds of interconversions and relationships between the things defined by cosmology. In short, "how the world works." For example, a simplified scientific ontology claims matter originated with the Big Bang and subsequently developed into two kinds, abiotic and biotic, which are recycled back and forth through biogeochemical processes.

We direct this paper towards ecologists and conservation biologists (students, academics, and professionals) in an effort to encourage more meaningful, in-depth approaches for engaging with ILK in their research. We are a diverse group of non-Indigenous researchers speaking to the scientific research community. We should not be understood to represent, or to speak in place of, Indigenous scientists and non-scientist knowledge holders. We speak to scientists not because the engagement with ILK by scientists is a privileged or standard form of epistemological relationship, but because ecologists and conservationists are increasingly encouraged to engage with ILK, yet for many, this may still feel experimental and beyond a science skill set (e.g., [START_REF] Molnár | Inviting ecologists to delve deeper into traditional ecological knowledge[END_REF]). Discussions of epistemological relationships with ILK are common within the literature on socioecological, biocultural, ethnobiological, or environmental anthropology interdisciplinary approaches (e.g., [START_REF] Berkes | Rediscovery of traditional ecological knowledge as adaptive management[END_REF][START_REF] Green | Indigenous knowledge' and 'science': Reframing the debate on knowledge diversity[END_REF][START_REF] Verran | Engagements between disparate knowledge traditions: Toward doing difference generatively and in good faith[END_REF][START_REF] Gómez-Baggethun | Reinterpreting change in traditional ecological knowledge[END_REF]). While interdisciplinary interaction with anthropologists is valuable, our goal in this paper is to suggest that ecological and conservation science can engage directly with ILK and that conservationists who are willing to be self-reflective about their methods and their thinking processes can develop rich epistemological relationships with ILK holders. These relationships should go beyond a vision where ILK serves science, to see science as also serving ILK, and together serving better knowledge of and action in the environment [START_REF] Brondízio | Locally based, regionally manifested, and globally relevant: Indigenous and local knowledge, values, and practices for nature[END_REF].

We start this paper with a short review aimed at ecologists and conservation biologists who do not have a background in anthropology and may not feel very confident about what ILK consist of. We address the simple but difficult question, "what are ILK?" By this, we do not mean a list of all the things that may qualify as ILK, but rather a general description. We attempt to bring some clarity to the set of debates over what a general description of ILK would be. The question must be resolved in some way in order to ask the corollary, "How are ILK and science related, or how can they be brought into relation?" If we were going to express this in a simplistic and reductive way, we would ask whether ILK and science are the same or different-but as we hope to illustrate, this question cannot be satisfactorily answered. The question of the relation between science and ILK may sound philosophical, and perhaps irrelevant to the action-oriented "crisis discipline" of conservation. However, the relationship between ILK and science sets the expectations for how they may form dialogues and collaborations, co-productions, or other methods that put epistemological relationships into action (see e.g., [START_REF] Reyes-García | Recognizing Indigenous peoples' and local communities' rights and agency in the post-2020 Biodiversity Agenda[END_REF][START_REF] Reyes-García | Response to "Practice what you preach: Ensuring scientific spheres integrate Indigenous Peoples' and Local Communities' rights and agency too" by Lopez-Maldonado[END_REF][START_REF] Lopez-Maldonado | Practice what you preach: Ensuring scientific spheres integrate Indigenous Peoples' and Local Communities' rights and agency too[END_REF][START_REF] Gillette | Inevitable epistemological conflict: Reflections on a disagreement over the relationship between science and indigenous and local knowledge[END_REF]). Throughout the paper, we use analogies and metaphors to try to help ecologists and conservation biologists think about ILK in ways they may not have before. After reviewing several perspectives on what ILK are and how they are similar or different to science, we suggest that the limitations of most existing answers to these questions can be overcome with the 'ethic of equivocation', an approach that comes out of South American anthropological theory, as we describe below. While there may be a perspective that the ethic of equivocation may be specifically South American in its concerns and framing of the issues, similar ideas have been developed in other contexts, and we believe it can be successfully adapted outside of South America.

How Are ILK Different from Science, and So What?

What is knowledge? Although there is no standard definition, even in Western cultures many people would agree that knowledge takes many forms, including as art, as manual skills, or as feelings. Nevertheless, in academic and formal Western settings, knowledge is valued when it is transmitted in particular formats of words and numbers, such as scientific papers. This affects how scientists often think about ILK. Knowledge can also be thought of as necessarily including a continuous process of learning and engaging with the unknown, or simply as discrete outcomes of that process, such as facts, for example, should you separate scientific facts from the scientific method when teaching science? As we will see, this distinction between knowledge processes and outcomes is not always clarified and can lead to misunderstandings.

The varieties of ILK are widely believed to be different from science (see [START_REF] Whyte | On the Role of Traditional Ecological Knowledge as a Collaborative Concept: A Philosophical Study[END_REF]). In the past, ILK has been characterized as non-analytic, dogmatic, complacent about contradictions, non-innovative, and closed to other worldviews-a view that was subsequently critiqued as not inherently or generally the case [START_REF] Agrawal | Dismantling the divide between indigenous and scientific knowledge[END_REF]. Rather, any form of knowledge can become dogmatic, complacent, and closed, and the history of science is full of such examples (e.g., [START_REF] Kuhn | The Structure of Scientific Revolutions[END_REF]). Similarly, in early literature, ILK were often characterized as an artifact-a sacred body of knowledge completed in the ancient past [START_REF] Davidson-Hunt | Researchers, indigenous peoples, and place-based learning communities[END_REF]. This view, solidified by anthropologists in the mid-20th century, has since been thoroughly critiqued and discredited by more recent anthropological research (e.g., [START_REF] Brosius | Representing Communities: Histories and Politics of Community-Based Natural Resource Management[END_REF][START_REF] Dove | Indigenous People and Environmental Politics[END_REF]). Today, ILK are much more likely to be characterized as dynamic, adaptive, and open to incorporating modern technologies and science ideas [START_REF] Berkes | Rediscovery of traditional ecological knowledge as adaptive management[END_REF][START_REF] Gómez-Baggethun | Reinterpreting change in traditional ecological knowledge[END_REF][START_REF] Raffles | Intimate knowledge[END_REF][START_REF] Dove | Hybrid histories and indigenous knowledge among Asian rubber smallholders[END_REF].

This shift toward de-emphasizing an opposition between ILK and Western science in favor of emphasizing ILK's dynamic and adaptive qualities is not total, however. For example, Whyte et al. [START_REF] Whyte | Weaving Indigenous science, protocols and sustainability science[END_REF] envision ILK as relational, contextual, anecdotal, and embedded in a worldview of morality and animism, which they contrast with a universal Western scientific knowledge system predicated on emotional distance and exploitation of an inanimate, mechanistic world. However, in practice, neither scientists nor science are unemotional, uninfluenced, uninterested, or detached (e.g., [START_REF] Myers | Rendering Life Molecular: Models, Modelers, and Excitable Matter[END_REF][START_REF] Fletcher | Science, Ideology, and the Media: The Cyril Burt Scandal[END_REF]). Both ILK and science are dynamic, engaged, and adaptive because both have social and intellectual histories and are embedded in diverse societies (e.g., [START_REF] Titz | Uncovering 'community': Challenging an elusive concept in development and disaster related work[END_REF]).

Several other objections to a dualist view of ILK vs. science have also been advanced in the literature. Ludwig and Poliseli [START_REF] Ludwig | Relating traditional and academic ecological knowledge: Mechanistic and holistic epistemologies across cultures[END_REF] argue that this kind of contrast rests on a holistic-mechanistic distinction, which they argue is a false distinction. Contrary to this holistic-mechanistic distinction, they argue that scientists can be holistic thinkers too, and ILK can embed mechanistic causal analyses inherently in the form of management practices, values, and worldviews (e.g., [START_REF] El-Hani | Beyond the divide between indigenous and academic knowledge: Causal and mechanistic explanations in a Brazilian fishing community[END_REF]). However, it is because causality may be embedded in cultural forms that, according to Ludwig and Poliseli [START_REF] Ludwig | Relating traditional and academic ecological knowledge: Mechanistic and holistic epistemologies across cultures[END_REF], ILK lose meaning when extracted from their cultural contexts (see also [START_REF] Raffles | Intimate knowledge[END_REF][START_REF] Löfmarck | Bumping against the boundary: IPBES and the knowledge divide[END_REF]). An analogous loss of context in science would be extracting the data points from papers in order to do a meta-analysis, without reading the methods sections to see if the studies are really comparable to one another. While making this argument, Ludwig and Poliseli [START_REF] Ludwig | Relating traditional and academic ecological knowledge: Mechanistic and holistic epistemologies across cultures[END_REF] accept that science is universal and general. Green [START_REF] Green | Indigenous knowledge' and 'science': Reframing the debate on knowledge diversity[END_REF] (see also [START_REF] Agrawal | Dismantling the divide between indigenous and scientific knowledge[END_REF]) explicitly challenges this universality vs. contextuality as a valid distinction between science and ILK, arguing that models and data are also contextual, being true only in particular scales or frames. Quantum physics does not apply to stoichiometry; stoichiometry does not explain convergent evolution; etc.

In light of these debates, in which a series of proposed ILK-science distinctions are sequentially cast in doubt, perhaps it is not surprising that some researchers take the view that any differences within ILK and between ILK and science have limited relevance, and universal commonalities should be emphasized (e.g., [START_REF] Horsthemke | The idea of indigenous knowledge[END_REF]; see [START_REF] Singleton | Toward productive complicity: Applying 'traditional ecological knowledge' in environmental science[END_REF]). Löffmarck and Liedskog [START_REF] Löfmarck | Bumping against the boundary: IPBES and the knowledge divide[END_REF] argue that the IPBES framework also takes this perspective with its claims that all knowledge systems represent a common universal reality and that ILK is just science that is complicated to validate (see [START_REF] Díaz | The IPBES Conceptual Framework-Connecting nature and people[END_REF][START_REF] Díaz | A Rosetta Stone for nature's benefits to people[END_REF]). The IPBES resolve this complicatedness by claiming that each knowledge system is self-validating, creating equally valid strands of evidence that can be synthesized [START_REF] Tengö | Connecting diverse knowledge systems for enhanced ecosystem governance: The multiple evidence base approach[END_REF][START_REF] Tengö | Weaving knowledge systems in IPBES, CBD and beyond-Lessons learned for sustainability[END_REF]. However, there are challenges related to this act of synthesis that have not yet been fully resolved [START_REF] Löfmarck | Bumping against the boundary: IPBES and the knowledge divide[END_REF]. Synthesis of knowledge from multiple worldviews will not, by definition, be intellectually neutral. Synthesis of evidence is an analytical, epistemological process: it requires knowledge-as-process. Knowledge cannot exist without a worldview. The worldview provides the implicit and explicit means to interpret and act on observations, methods, and analyses. Documented cross-cultural differences in how we understand the world [START_REF] Descola | Beyond Nature and Culture[END_REF] are the fundamental underpinnings of knowledge. It is increasingly understood that these differences in worldviews are meaningful and necessary for knowledge interpretation and action. We fail to take into account the interpretive role of the worldview if we imagine that a neutral power balance across multiple worldviews engenders a neutral form of knowledge with a basis in nobody's worldview. There is no position outside of all worldviews from which to make a neutral form of knowledge. We, and others [START_REF] Green | Indigenous knowledge' and 'science': Reframing the debate on knowledge diversity[END_REF][START_REF] Löfmarck | Bumping against the boundary: IPBES and the knowledge divide[END_REF], consider that any claim of universal commonality across ILK and science amounts to a claim that the distinctive worldviews of ILK are not important for interpreting their observations of the world, a claim that makes no sense. We suggest that many approaches to synthesis that use multiple strands of evidence from ILK and science are either implicitly synthesizing that knowledge under a single (science) worldview, or implicitly using the ethic of equivocation without perhaps having a name for it (see below).

To think about the issues in the preceding paragraph through an analogy, a claim of universal commonality across ILK and science is akin to saying that data on plant richness produced using 1 m square quadrats placed at fixed distances from termite nests can legitimately be reanalyzed as if it had been collected using remote sensing on systematically placed transects of 1 km × 50 m. Obviously, such a reanalysis makes no sense, and neither does the proposition that ILK can be understood as if it had no significant differences from science. Ecological data are only interpretable with reference to the sampling strategy and methods with which they were collected, just as ILK and science are only interpretable within the worldviews from which they emerged. In both cases, means of comparison do exist: to compare the results of both studies of termite nest effects on plants, one could do a meta-analysis. In this analogy, the ethic of equivocation, which we will introduce in a moment, has the same role as meta-analysis.

To reiterate, the critical differences and similarities between ILK and science are not found in qualities, such as contextuality, dynamism, morality, etc. Rather, the critical differences and similarities are found among epistemologies, ontologies, and cosmologiesthe fundamental underpinnings of how people reason and construct understandings about the world. This is analogous to how bilby species and rabbit species have phenotypic traits that are both similar and different, yet we do not understand their relationships according to convergence or divergence in ear and leg morphology alone (similar to how an 18th-or early 19th-century natural historian might have done [START_REF] Burkhardt | The Spirit of System: Lamarck and Evolutionary Biology[END_REF]). What allows us to understand the relationship between bilbies and rabbits is the fundamental underpinnings of biogeography, phylogeny, and evo-devo (evolution of development). What allows us to understand the relationships between ILK and science are the fundamental underpinnings of epistemologies, ontologies, and cosmologies.

Further, the problem with looking for universal commonalities is the focus on comparing ILK as a unitary whole to science as a unitary whole [START_REF] Green | Indigenous knowledge' and 'science': Reframing the debate on knowledge diversity[END_REF]. By analogy, behavioral ecology, ethology, and cognitive science are all "about" animal behavior but are different fields. While some of their predictions can be made coherent, each of these subdisciplines has rich, non-overlapping research lines. A better approach to answering questions about the comparability of ILK and science may be to find particular parts of knowledge systems that coincide or complement one another [START_REF] Verran | Engagements between disparate knowledge traditions: Toward doing difference generatively and in good faith[END_REF][START_REF] Ludwig | Philosophy of ethnobiology: Understanding knowledge integration and its limitations[END_REF]. A problem-led or question-led approach can avoid bias in the choice of which parts to focus on (see section "How to do equivocation in practice"). In summary, if there is no agreement that ILK and science are truly different, or that they have any fundamental commonality, we can come to understand that this is because 'are they the same or different' is fundamentally the wrong question.

The Ethic of Equivocation Described

The ethic of equivocation helps us work with ILK by identifying partial overlaps of knowledge systems, but without having to resolve issues of sameness and difference ( [START_REF] Viveiros De Castro | Perspectival anthropology and the method of controlled equivocation[END_REF][START_REF] Viveiros De Castro | Métaphysiques Cannibales; Métaphysiques[END_REF][START_REF] Ludwig | Overlapping ontologies and Indigenous knowledge. From integration to ontological self-determination[END_REF][START_REF] Furlan | Ethnobiological equivocation' and other misunderstandings in the interpretation of natures[END_REF], for related ideas see also [START_REF] Singleton | Toward productive complicity: Applying 'traditional ecological knowledge' in environmental science[END_REF][START_REF] Rottenburg | Experimental Engagements and Metacodes[END_REF]). Viveiros de Castro [START_REF] Viveiros De Castro | Perspectival anthropology and the method of controlled equivocation[END_REF] argues for equivocation as the basis of all communication, translation, and cross-cultural interaction. Equivocation is the idea that ILK and science can find translations that preserve differences and non-equivalencies while producing similar meanings for both parties. An equivocation is a form of mistake, a "second-level" mistake. A "first-level" kind of mistake is to assume that there is a universal, correct, or superior set of perceptual categories-something that ethnobiology has disproven. An example is Dupré's study [START_REF] Dupré | Are whales fish[END_REF] showing how a particular folk taxonomy category of "fish" includes whales. "Correcting" fishermen to inform them that whales are not fish would miss that "fish" in this context is being used as a functional category akin to "omnivore" or "shrub". The ethic of equivocation operates at a "second level" in which two different forms of categorization or conceptualization are, in some context, treated by their respective users as equivalent, even if both parties know that each concept has other, non-equivalent aspects or ramifications. This is similar to the question of which appendage you shake when "shaking hands" with a lobster. The fact that one of its appendages will work perfectly well for a handshake does not mean that anyone actually believes that it is anatomically a hand.

Fitzgerald et al. [START_REF] Fitzgerald | equivocation and the politics of experimental knowledge: A transdisciplinary neuroscience encounter[END_REF] also describe equivocation as a working method for interdisciplinary research. They describe the dynamics of the collaboration between a group of social scientists and a group of neuroscientists at the same university. They were able to work together, exchange knowledge, and complete research, for the purposes of the project, without claiming to form a universal or stable set of equivalencies between their two knowledge systems. As this example illustrates, the ethic of equivocation is not limited to or predicated on researcher-informant relationships but works in any situation where there is dialogue, including interdisciplinary collaborations, applied actions, and co-generation of knowledge. The outcome of this interaction is multiple lines of evidence embedded in different epistemologies, which generate different interpretations according to peoples' worldviews. What makes equivocation unique is the focus on an interactive process that allows the coexistence of different understandings of key areas of overlap, without reducing, or subsuming, those differences into a unitary or global framework.

Achieving science and conservation goals through processes of dialogue, collaboration, or co-production does not require establishing points of direct and absolute equivalency, or cross-validation. The ethic of equivocation allows space for the creative and generative potential of second-level mistakes that work, such as a genetic mutation that makes a flower look similar to a female bee and leads to better cross-pollination. The mutation obviously does not make the flower turn into a bee, but both the bee and the flower benefit from a male bee treating the flower as a female bee, and from the flower treating the mate-seeking bee as a potential pollinator.

Standard Methods and How These Might Be Done Differently in an Ethic of Equivocation

Standard methods for working with ILK may be divided into documentation, merging with science, and co-production of new knowledge [START_REF] Roué | Indigenous and local knowledge and science: From validation to knowledge coproduction[END_REF]. Self-validating multiple evidence bases may be woven into any of these methods. Many studies see ILK as an information resource to be documented. They are thus concerned with issues of comparability or homogeneity of data standards, asking whether the information from ILK is precise and accurate and whether this can be validated scientifically (e.g., [START_REF] Danielsen | Local participation in natural resource monitoring: A characterization of approaches[END_REF][START_REF] Paniagua-Zambrana | The influence of socioeconomic factors on traditional knowledge: A cross scale comparison of palm use in northwestern South America[END_REF][START_REF] Fernández-Llamazares | Rapid ecosystem change challenges the adaptive capacity of local environmental knowledge[END_REF][START_REF] Cámara-Leret | Indigenous knowledge networks in the face of global change[END_REF]). Following this reasoning, some researchers still feel they cannot consider ILK that have not been scientifically validated, or directly generated through a standards-based protocol [START_REF] Roué | Knowledge and foresight: The predictive capacity of traditional knowledge applied to environmental assessment[END_REF]. In their research, scientists exclude their personal observations that they perceive as unvalidatable and may apply the same reasoning to ILK. Merging-with-science approaches typically also share the reasoning and approaches of the documentation approach, but go beyond documentation to use validated ILK to complement or constitute scientific datasets within projects designed (primarily) by scientists for scientific (e.g., statistical) analyses (e.g., [START_REF] Gagnon | Integrating traditional ecological knowledge and ecological science: A question of scale[END_REF][START_REF] Gagnon | Merging indigenous and scientific knowledge links climate with the growth of a large migratory caribou population[END_REF]).

From an ethic of equivocation perspective, when considering data validation and dataset archiving, we see two issues for further consideration. Ecologists and conservationists might consider whether ILK has the same meaning to ILK holders when integrated into datasets and archives. Meaning is about function-not in the sense of a teleological or ultimate purpose, but in the ecological sense of the effects of a practice or outcomes of a process. Just as a detritivore has the function of decomposition, and plants have the function of fixing carbon, meanings have social functions. Within science, examples of meaning-making include the analysis of data, the framing and interpretation given in the Introduction and Discussion sections of papers, and the claims made about the importance of findings. The function of those meanings is to allow further research, understanding, and applications. So if we ask whether a piece of ILK-as-data; that is, a given dataset derived from ILK, has the same meaning in a given social context as that piece of ILK had (or continues to have in parallel) as not-data, we are asking whether ILK-as-data can do the same things e.g., to inform people's resource use practices or adaptive capacities, as in its original non-data form. For example, if a hypothetical society uses personal narratives to incorporate knowledge of weather into crop planting decisions, do quantitative data about past weather do the same things in that society? Are they used to make similar arguments? Do they lead to similar decisions? This leads to the second issue for further consideration, which is, of what use to conservation and ecology are aspects or sections of ILK that are not reformatted as data?

Aspects of ILK that are not converted into data that the ecologist or conservationist encounters for example being used by ILK-holders in the field, can be treated in several ways. In some cases, an established equivocation is available for a specific discrete outcome of ILK. For example, when an Indigenous tracker identifies an animal track whose location and ID are then saved into a GPS, the particular observation of the track is transformed into scientific data. In other cases, an equivocation can allow a scientist an opportunity to make novel scientific data directly, through overlaps in the processes of knowledge-making. For example, while a tracker and a wildlife biologist may have different explanations or uses for animal movements, the tracker's superior tracking skills help the wildlife biologist identify sites where certain samples can be obtained.

Finally, a third possibility is that ecologists and conservationists can treat pieces of ILK that are not data in the same way that fieldworkers treat other observations of study phenomena not carried out in the framework of a scientific protocol. Things that don't immediately fit one's disciplinary knowledge, or that are akin to what scientists generally consider to be their unvalidatable personal observations, can be signs of interesting new research areas. They do not immediately yield data, but rather questions or hypotheses. The examples of Charles Darwin and Alfred Wallace (among others) remind us that detailed observation and comparison can lead to theory development [START_REF] Bertrand | Le Détail du Monde[END_REF]. The increasing number of natural history formats in scientific journals also reflects the growing recognition that exploratory research is a necessary complement to confirmatory research.

The ethic of equivocation approach leads us to argue that both documentation and merging approaches should constantly be on the look-out for opportunities to do the third approach, 'co-production of knowledge' [START_REF] Roué | Indigenous and local knowledge and science: From validation to knowledge coproduction[END_REF][START_REF] Norström | Principles for knowledge co-production in sustainability research[END_REF], which can be defined as "a process that brings together diverse groups to iteratively create new knowledge and practices" [START_REF] Jagannathan | Great expectations? Reconciling the aspiration, outcome, and possibility of co-production[END_REF] that are "actionable . . . credible, legitimate and salient to decision-makers" [START_REF] Latulippe | Making room and moving over: Knowledge co-production, Indigenous knowledge sovereignty and the politics of global environmental change decision making[END_REF]. Work on sustainable development and community conservation are subfields that have embraced this approach (e.g., [START_REF] Jagannathan | Great expectations? Reconciling the aspiration, outcome, and possibility of co-production[END_REF][START_REF] Berkes | Rethinking community-based conservation[END_REF][START_REF] Wyborn | Co-Producing Sustainability: Reordering the Governance of Science, Policy, and Practice[END_REF][START_REF] González | The potential of Amazon indigenous agroforestry practices and ontologies for rethinking global forest governance[END_REF][START_REF] Mach | Actionable knowledge and the art of engagement[END_REF]). Co-production is often aimed at applied management contexts and strives to produce concrete benefits for all parties; it is, thus, about situated knowledge. Documentation-focused and merging-with-science work with ILK should remain attentive to the large mass of situated knowledge in forms other than data that may be more effective for certain kinds of problems, and which may inspire novel hypotheses, analytical categories, and research questions (see e.g., [START_REF] Riseth | Sámi traditional ecological knowledge as a guide to science: Snow, ice and reindeer pasture facing climate change[END_REF][START_REF] Cogos | Forest fire and indigenous Sami land use: Place names, fire dynamics, and ecosystem change in Northern Scandinavia[END_REF][START_REF] Molnár | Knowledge co-production with traditional herders on cattle grazing behaviour for better management of species-rich grasslands[END_REF][START_REF] Root-Bernstein | Silvopastoralism, local ecological knowledge and woodland trajectories in a category V-type management area[END_REF][START_REF] Root-Bernstein | Does shrub encroachment reduce foraging grass abundance through plant-plant competition in Lesotho mountain rangelands?[END_REF]). However, since all of the goals of ILK holders and scientists will not be common goals, even after the development of an epistemological relationship predicated on an ethic of equivocation, either party may also continue in parallel to produce knowledge for "internal consumption" within their discipline or tradition.

In summary, the ethic of equivocation approach insists that ILK is always richer, and often more motivating and more generative of novel hypotheses than is captured by its reduction to data. It invites the conservationist or ecologist to consider that all conversions to data are first-level mistakes with the potential to become second-level mistakes.

How to Do Equivocation in Practice

When conservationists and ecologists look "behind" or "beyond" the data they can validate from ILK, they may find things they do not know exactly what to do with. How can an ecologist or conservation biologist react when their informants tell them, for example, about taboos, spirits, magic spells, or myths? These are concepts, units, or materials that many scientists are not used to including in their research. Some aspects of ILK seem impossible to validate-what a scientist might identify as a belief rather than a fact supported by evidence. Of course, facts should also be doubted: in science, theories and facts are never proven, only disproven. However, scientists also think with and about ideas that are not facts (nor beliefs), such as classic predator-prey models that do not accurately predict dynamics, the intermediate disturbance hypothesis, or metaphors, such as the selfish gene or the tree of life [START_REF] Green | Indigenous knowledge' and 'science': Reframing the debate on knowledge diversity[END_REF]. The scientific issue is whether such models, concepts, and metaphors are useful for thinking about a particular problem or topic [START_REF] Hejnol | Trees, Complexity, And Other Metaphors in Evolutionary Thinking[END_REF]. Faced with (what appear to be) others' models, concepts, metaphors, or beliefs lacking (convincing) evidence, one can usefully ask, what do they mean? To answer such questions, many anthropologists think about the meanings of beliefs/facts/knowledge by attending to their effects on how people act in the world.

An ethic of equivocation approach to thinking about how ILK affect how people act in the world is to find a scientific approach, format, or formalism, which allows (in practice) or represents (in analysis) a similar effect, and to use that as the basis for dialogue and knowledge co-production.

Concretely, an equivocation can be a concept, a formality, a practice, or an institution. Furlan et al. [START_REF] Furlan | Ethnobiological equivocation' and other misunderstandings in the interpretation of natures[END_REF] provide several examples of equivocations, mainly around concepts and practices that arose during their ethnobiological research in South America. Here, we highlight several other examples. For example, in the Andean Kichwa culture, there is a concept called Sumak Kawsay. While this has been widely translated and popularized with the Spanish term buen vivir ('living well') [START_REF] Gudynas | Vivir: Today's tomorrow[END_REF], the original concept is much broader, with a richer set of implications, than suggested by this translation. Although conservationists arguably misunderstand this when they equate buen vivir to "sustainable development" [START_REF] Virtanen | Introduction: Toward more inclusive definitions of sustainability[END_REF], this misunderstanding may be a productive starting point for working towards common goals.

Congretel and Pinton [START_REF] Congretel | Local knowledge, know-how and knowledge mobilized in a globalized world: A new approach of indigenous local ecological knowledge[END_REF] provide an example of equivocation as a formality. Although the meaning(s) of guaraná (Paullinia cupana) is/are radically different between Indigenous cultivators (as a sacred food source) and industrial producers for export (as a coffee-like stimulant), the co-development of a formal document describing how to produce guaraná gave legitimacy to both understandings while allowing conservation cooperation. The formality has not reduced the number of understandings of guaraná circulating in the world, but it has allowed different parties to work together towards the common goals that it enshrines and facilitates. Legal rulings about the personhood of geographical entities or biocultural rights, and other protocols and agreements protecting communities' knowledge and rights provide similar examples [START_REF] Nemogá | Protecting Indigenous and Local Knowledge through a Biocultural Diversity Framework[END_REF].

At the intersection of practices and formalities, one well-described example of equivocation is how early colonial settlers in New England understood property rights, and how they interpreted whether local Indigenous groups had property rights [START_REF] Cronon | Changes in the Land: Indians, Colonists and the Ecology of New England[END_REF]. Colonists were often explicitly aware that their ideas about rights, usufruct, and ownership of land did not correspond to local Indigenous concepts and practices. Although some arrangements regarding sale/land sharing may initially have seemed satisfactory to both parties, this equivocation about land rights and usufructs over time came to be deliberately misrepresented by the colonists to their political advantage. These deliberate misrepresentations, which can be contrasted with honest equivocations, had direct ecological impacts due to land-use change on lands taken over by colonists, as well as due to Indigenous groups' being forced to find alternative land-use practices in reduced spaces impacted by surrounding land-use changes. Ultimately, satisfactory and productive equivocations were abandoned as colonists promulgated deliberate misunderstandings to justify the mass displacement and genocide of Indigenous peoples. This example thus also highlights the difference between an equivocation, which all parties accept, and a deliberate misunderstanding which is dishonest, exploitative, and not mutually accepted.

Finally, an entire institution can be the focus of an equivocation. Protected areas, for example, may be institutional arrangements of which different stakeholders have widely divergent perceptions and expectations, e.g., they should prevent industrial projects, bring economic revenue, or increase certain species' populations. Some protected areas achieve outcomes that are satisfactory equivocations for multiple parties, others do not (e.g., [START_REF] Zinda | Hazards of collaboration: Local state co-optation of a new protected-area model in southwest China[END_REF][START_REF] Narayan | A culturally appropriate redesign of the roles of protected areas and community conservation: Understanding the features of the Wangchuck Centennial National Park (WCNP), Bhutan[END_REF]).

Learning How to Do Equivocation Well

The reader will note from the preceding section that equivocations require honest engagement from both parties. A mutually honest and respectful equivocation is not immune to being abandoned and replaced by a deliberate and exploitative misrepresentation. This is why the ethic of equivocation may be referred to as "controlled equivocation", since if not done attentively it may also escape control and be exploited by malicious actors [START_REF] Viveiros De Castro | Perspectival anthropology and the method of controlled equivocation[END_REF][START_REF] Furlan | Ethnobiological equivocation' and other misunderstandings in the interpretation of natures[END_REF]. The importance of attention and care is also why it is an "ethic" and not just a method or procedure. Although we cannot force people to be or remain ethical, we argue that without practicing the ethic of equivocation scientists may (unintentionally) incur ethical problems in their relations to ILK holders. As we explain above, asserting that a piece of scientific knowledge and a part ofILK are exactly equivalent or radically incomparable are both untenable political positions with suboptimal outcomes for knowledge creation. Accepting that we will make mistakes, and trying to make good, productive mistakes via the ethic of equivocation, is, we suggest, the best course of action. As we discuss below (Section 7: Issues of power relations in epistemological relationships) the worst outcomes can be avoided through respect, attention, taking seriously the claims of one's collaborative partner, and through appropriate political representation within decision-making contexts. Experience over time, training in transdisciplinarity, and personal engagement with the culture of ILK holders may also help [START_REF] Manuel-Navarrete | Fostering horizontal knowledge co-production with Indigenous people by leveraging researchers' transdisciplinary intentions[END_REF]. It can also help to learn from successful, comparable situations.

Collaborations are a perhaps more-familiar place for ecologists and conservation biologists to start looking for situations from which to learn. Collaborations in general, and interdisciplinary collaborations in particular, pose some similar issues to the ethic of equivocation in an ILK context, and advice and reflections on researchers' struggles to collaborate may provide some inspiration (e.g., [START_REF] Fitzgerald | equivocation and the politics of experimental knowledge: A transdisciplinary neuroscience encounter[END_REF]). However, not only is collaboration generally messy and less than ideal, but working across (sub)disciplines even within science/academia cannot be reduced to a list of best-practice steps to follow.

Another (and even more pertinent) source of inspiration for working with ILK and ILKholders comes from ILK-holders working with and/or as scientists. Many cross-cultural complexities have been described from the perspective of ILK-holders engaging science to develop Indigenous research methodologies (e.g., [START_REF] Botha | Mixing methods as a process towards indigenous methodologies[END_REF][START_REF] Chalmers | The transformation of academic knowledges: Understanding the relationship between decolonising and indigenous research methodologies[END_REF][START_REF] Gone | Considering Indigenous research methodologies: Critical reflections by an Indigenous knower[END_REF]).

ILK-holders are experts in the merging of science with ILK and may provide concrete examples that we interpret as equivocation in practice. For example, Berenice Sanchez reports, "Indigenous Peoples have always depended on the full understanding of the seasons to survive and have accumulated knowledge through the centuries based on observations. Everything, absolutely all the factors, has been observed and interpreted . . . What we offer is science because we have systematized and interpreted these observations" [START_REF] Anonymous | Voices from Ixtlán de Juárez. Excerpts of interviews conducting at Respecting Our Traditional Science and Ways of Knowing event. Berenice Sanchez[END_REF] (p. 22). Eddie Haikau Huitarau writes, "Although the Ahetaha community [of the Soloman Islands] has been going through lots of changes in adopting a more Western lifestyle, our traditions remain part of us, like our traditions of governance, shifting cultivation, bridal dowry, mourning rituals, and the conservation method known in our dialect as Asiia. . . . We encourage our children to attain education and become specialists in areas of conservation already in place." [START_REF] Huitarau | We live by the river, we live by the sea: Preserving natural resources of the Ahetaha Peoples in the Solomon Islands[END_REF] (pp. [START_REF] Raffles | Intimate knowledge[END_REF][START_REF] Dove | Hybrid histories and indigenous knowledge among Asian rubber smallholders[END_REF]. Hindou Oumarou Ibrahim has stated, "Where our traditional knowledge and science meet, we say that climate science uses modern knowledge . . . But for us, we are just using our information and other factors we observe . . . When we break [a fruit], we see the liquid inside. If the liquid is abundant, we know [what] the tree is predicting for the next year." [START_REF] Portalewska | A champion for Indigenous peoples against climate change[END_REF] (p. 11). These ILK holders have already integrated scientific concepts into their knowledge systems. They are holding multiple representations in mind simultaneously, and in some cases claiming these as points of what we describe as equivocation, e.g., claiming that what they are doing is science; or that they become 'Western' scientists in order to keep doing what they were already doing. Within an ethic of equivocation, they may simultaneously self-validate their knowledge (from their perspective), and validate their knowledge from a science perspective; but no cross-validation occurs.

Issues of Power Relations in Epistemological Relationships

There are many relevant issues around the balance of power that deserve attention when developing an epistemological relationship [START_REF] Singleton | Toward productive complicity: Applying 'traditional ecological knowledge' in environmental science[END_REF]. The historical case of colonists' interpretations of land rights in New England also illustrates how a political power imbalance, in which both parties are not mutually considered and treated as equals, can allow an initially valid equivocation to be replaced by a justification for exploitation and even genocide [START_REF] Cronon | Changes in the Land: Indians, Colonists and the Ecology of New England[END_REF]. However, equal political representation by itself does not automatically translate into satisfactory outcomes. As we discussed above, the idea that the synthesis of ILK and science constructs common knowledge through a neutral synthesis process (in which no one's ontology or cosmology is implicated) is a false and unrealistic pretense. However, as in the example of the formal document governing guaraná production [START_REF] Congretel | Local knowledge, know-how and knowledge mobilized in a globalized world: A new approach of indigenous local ecological knowledge[END_REF], it can be possible, through a sustained process, to develop joint understandings that can be simultaneously interpreted in different ways and represented in different forms within multiple ontologies and cosmologies in ways that lead to coordinated actions that are satisfactory to all parties.

The development of satisfactory equivocations takes time, and scientists should not imagine that they will succeed rapidly and immediately. Developing new equivocations for novel purposes is unlikely to work within short-term engagements with a field site. Indeed, the search for "efficient" processes for arriving at satisfactory equivocations may lead to poor outcomes and abuses of power. Abuses of power in the development of such an epistemological relationship can consist of, for example:

•

Insisting that two things are the same and refusing to acknowledge that any claim of equivalence must be to some degree a mistake. The acknowledgment of a latent degree of mistakenness allows the process to continue to progress towards more satisfactory, or differently satisfactory, equivocations. The insistence that two things are the same can be interpreted as an attitude, whether intended or not, of reductionistic closed-mindedness that may impede trust, cooperation, and the development of new knowledge.

•

Insisting that two things are satisfactory equivocations when the other party does not agree; imposing the conditions for agreement about satisfactory equivocations on the other party.

•

Taking a satisfactory equivocation and using this as a justification to impose a scientific worldview on related subjects because "a point of equivocation has been established"-in fact, each subject requires its own equivocation. You might think of this as similar to the problem of triangulating two satellite images in GIS in order to overlay them-but since ontologies are much more complex than two-dimensional planes, you need many more than three points to make this alignment. Certainly, it will take time to extend the field of satisfactory equivocations to a wide range of subjects.

Acknowledging that it remains unestablished how other ILK and science concepts, practices, formalisms, or institutions relate to one another during this process is a good starting point.

•

Taking an initial equivocation and failing to engage in some form of ongoing dialogue, collaboration, or other mechanism bringing parties together and allowing correction and adjustment of the equivocation over time and across contexts. Without such a process, the possibilities increase over time for obfuscation, mystification, dishonesty, abuse, and so on.

There are also numerous issues related to methods, acknowledgment and credit, authorial representation, benefit sharing, and many other issues of politics and collaborative ethics, which have been treated by other authors in great depth (e.g., [START_REF] Molnár | Inviting ecologists to delve deeper into traditional ecological knowledge[END_REF][START_REF] Nadasdy | The Politics of Tek: Power and the "Integration" of Knowledge[END_REF][START_REF] Craig | Ethical relationships for biodiversity research and benefit-sharing with indigenous peoples[END_REF][START_REF] Carothers | Introduction: Conceptual, methodological, practical, and ethical challenges in studying and applying indigenous knowledge[END_REF][START_REF] Golan | Intellectual Property Rights and Ethnobiology: An Update on Posey's Call to Action[END_REF][START_REF] Trisos | Decoloniality and anti-oppressive practices for a more ethical ecology[END_REF]), and by research guidelines issued by Indigenous communities themselves. Many of these issues need to be dealt with in ways specific to particular communities.

Our general advice is that if, as a scientist, you want to work with ILK but you have the feeling that the local community does not want you to be there doing that, you should stop doing it. One should not fool oneself that any ecological question or conservation problem absolutely requires one's personal scientifically-informed intervention to solve it. Some communities do not want outside ecologists and conservation biologists to collaborate with them; others welcome them. Our second piece of general advice is that scientists choosing field sites and working methods should do background research on the societies where they want to work, in order to determine whether there are current tensions around post-colonialism, neo-colonialism, or other political issues that might put a scientist who either engages (in specific ways) or does not engage (in specific ways) with ILK holders in a politically tense or complex situation [START_REF] Trisos | Decoloniality and anti-oppressive practices for a more ethical ecology[END_REF]. Individuals who feel strongly that they have adequate social and intercultural skills to deal with tense political situations may thrive and do good work in such contexts; other scientists who judge that they lack these skills may find it more productive to work with "local ecological knowledge" in their societies of origin, or in areas where such tensions are more resolved or less developed. Even there, ethical concerns about one's position as a scientist relative to society do not disappear. As a third and final general piece of advice, the ethic of equivocation is an approach to epistemological relationships that embraces ambiguity and uncertainty. No one should feel obliged to develop epistemological relationships that make them, or the people they want to interact with, uncomfortable. In this case, however, if one chooses to avoid the ethic of equivocation in one's own work, one should be doubly attentive to not fall into the epistemological abuses of power described above.

Conclusions

Although collaborative approaches combining ecologists, conservationists, anthropologists, and ILK holders are very valuable, here, we demonstrate to ecologists and conservationists that they may themselves have more tools than they are aware of with which to directly engage in sophisticated ways with ILK holders. Throughout this paper, we point to these intellectual tools by using several analogies and metaphors that may help to clarify what ILK are and how the ethic of equivocation suggests methods for engaging with ILK. Pluralism in styles of reasoning and working already exists in science (e.g., [START_REF] Hacking | Style'for historians and philosophers[END_REF]), and a first step to engaging with ILK is to become self-aware of one's own style, and to acknowledge and potentially mobilize different styles. Most reasonable scientists recognize that styles of argument and method, subdisciplines, or disciplines cannot be simply reduced to correct or incorrect, but have points of disagreement, contact, comparison, divergence, and convergence. Engaging with ILK can be seen as a profound and radically challenging extension of interdisciplinarity or transdisciplinarity, given the differences in underlying worldviews that provide basic interpretive schema. There is no perfect correspondence between any ILK and any academic discipline, no Rosetta Stone, and no perfect way to engage across them, which is why an ethic of equivocation based on second-order mistakes forming contextually functional equivalencies can be a respectful and productive way forward.

We conclude with a final analogy. We suggest that a productive way to imagine a science-ILK collaboration is along the lines of a collaboration between two artists from different genres. As an example, consider the performance of "The Dying Swan" by Lil Buck and Yo-Yo Ma (https://www.youtube.com/watch?v=C9jghLeYufQ, accessed 23 October 2020). In this example, neither Lil Buck nor Yo-Yo Ma is a perfect analogy for either a scientist or an ILK holder. Lil Buck is a dancer in the Jookin style, a popular dance from Memphis, USA. Yo-Yo Ma is a classical cellist, part of an elite European tradition. The rigorously trained cellist's prestige within the classical music world was not lessened, but rather increased, by his association with the self-taught and, at the time, relatively unknown dancer, and vice versa. The performance brought new dimensions to the work of each artist, creating a novel whole that was startling and exciting to millions of observers. An ethic of equivocation across ILK and science can result in equally pathbreaking advances in knowledge of and care for our natural world.

Acknowledgments: M.R.-B. considers this as a contribution to the Jefferson Fund grant no. TJF18_067. S.R. and M.R.-B. consider this as a contribution to the ECOS-Sud grant C18B0.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement:

No data were produced or used in this paper.

Author Contributions: M.R.-B. conceived the presented idea. All authors contributed equally to the writing and editing of the paper. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.


Christina M Bergemann

Astrid Avellan

Brittany G Perrotta

Emily S Bernhardt

Marie Simonin

Seasonal Differences and Grazing Pressure Alter the Fate of Gold Nanoparticles in a Microcosm Experiment

Keywords: Snails, nanomaterials, emerging contaminants, macrophytes, microcosms

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

drove the fate of AuNPs within aquatic ecosystems and that seasonality was an important abiotic factor in the fate of AuNPs. Therefore, the present work aims to study if grazers, by feeding on these interfaces, modify the AuNP fate and if this is altered by seasonal fluctuations. Microcosms were dosed with 44.8 μg/L of AuNP weekly for 4 weeks and maintained in environmental chambers simulating Spring and Fall light and temperature conditions. We discovered that seasonal changes and the presence of grazers significantly altered the fate of Au. Higher temperatures in the warmer season increased dissolved organic carbon (DOC) content in the water column, leading to stabilization of Au in the water column. Additionally, snail grazing on biofilm growing on the Egeria densa surface led to a transfer of Au from macrophytes to the organic matter above the sediments. These results demonstrate that climate and grazers significantly impacted the fate of Au from AuNPs, highlighting the role that grazers might have in a large and biologically more complex ecosystem.

Graphical abstract: Introduction:

Engineered nanomaterial use is increasing in a wide variety of applications ranging from drug delivery to agriculture use [START_REF] Ghosh | Gold nanoparticles in delivery applications[END_REF][START_REF] Lowry | Opportunities and challenges for nanotechnology in the agri-tech revolution[END_REF][START_REF] Li | Nanopesticides: A Comprehensive Assessment of Environmental Risk Is Needed before Widespread Agricultural Application[END_REF] . While many studies have shown that nanomaterials can cause toxicity [START_REF] Miralles | Uptake, and Translocation of Engineered Nanomaterials in Vascular plants[END_REF][START_REF] Garner | Emerging patterns for engineered nanomaterials in the environment: A review of fate and toxicity studies[END_REF] , further research is needed to fully understand how these chemicals interact within the environment and in the presence of other stressors. Gold nanoparticles (AuNPs) are frequently used as a nanomaterial model [START_REF] Ferry | Transfer of gold nanoparticles from the water column to the estuarine food web[END_REF][START_REF] Wray | Modeling the influence of physicochemical properties on gold nanoparticle uptake and elimination by Daphnia magna[END_REF][START_REF] Surette | Effects of surface coating character and interactions with natural organic matter on the colloidal stability of gold nanoparticles[END_REF][START_REF] Smith | Quantification of Heteroaggregation between Citrate-Stabilized Gold Nanoparticles and Hematite Colloids[END_REF] but use is also growing in the fields of biomedicine, technology, and pharmaceuticals [START_REF] Santhoshkumar | Phyto-assisted synthesis, characterization and applications of gold nanoparticles -A review[END_REF][START_REF] Elahi | Recent biomedical applications of gold nanoparticles: A review[END_REF][START_REF] Farzin | AuNP-based biosensors for the diagnosis of pathogenic human coronaviruses: COVID-19 pandemic developments[END_REF] . Previous research has shown that citrate capped AuNPs have high chemical stability in abiotic, surface waters and overall low toxicity in aquatic environments, suggesting low environmental consequences if AuNPs enter aquatic ecosystems [START_REF] Avellan | Gold nanoparticle biodissolution by a freshwater macrophyte and its associated microbiome[END_REF][START_REF] Alkilany | Toxicity and cellular uptake of gold nanoparticles: What we have learned so far?[END_REF] . Though inert in sterile conditions, AuNPs can undergo oxidation in the presence of biofilms [START_REF] Avellan | Gold nanoparticle biodissolution by a freshwater macrophyte and its associated microbiome[END_REF] where they can interact with other biological systems [START_REF] Alkilany | Toxicity and cellular uptake of gold nanoparticles: What we have learned so far?[END_REF][START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[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] Perrotta | Copper and gold nanoparticles increase nutrient excretion rates of primary consumers[END_REF] . Therefore, AuNPs are used as a model for understanding how biological responses can impact the chemical and physical processes and influence the fate and transport of Au within systems of varying complexity.

Our previous work demonstrated that low-level, chronic AuNP enrichment led to significant increases in algal blooms and hypoxia [START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] in large-scale freshwater mesocosm setups. We hypothesize that this interaction was largely modulated by primary consumers and a macrophyte Egeria densa, but the large-scale experimental design limited our ability to test this interaction implicitly. Other work using E. densa has shown that AuNPs are not taken up through shoot or root uptake and adsorption of AuNPs on plant surfaces was influenced by dissolved organic carbon present in the water column [START_REF] Glenn | Abiotic and Biotic Factors That Influence the Bioavailability of Gold Nanoparticles to Aquatic Macrophytes[END_REF] . Yet, we found that the dominant fate of AuNPs was deposition on, and/or absorption into the macrophyte Egeria densa resulting in suppression of growth and productivity [START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] . Due to this suppression, we concluded that algae within the water column were able to outcompete rooted macrophytes for nutrients, shifting the mesocosms from clear water to an algal bloom state [START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] . It has also been shown that an increase in algae and epiphyton is capable of suppressing macrophyte growth leading to algal blooms [START_REF] Yang | Glutaminolysis: A Hallmark of Cancer Metabolism[END_REF] . Furthermore, it was found that Au from supposedly inert AuNPs present at the macrophyte interface had largely undergone oxidation due to microbial activity within the biofilms attached to macrophyte leaves leading to Au dissolution [START_REF] Avellan | Gold nanoparticle biodissolution by a freshwater macrophyte and its associated microbiome[END_REF][START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] . The Au fate and transformation at the macrophyte interface seem to be one of the major drivers for the ecosystem responses [START_REF] Avellan | Gold nanoparticle biodissolution by a freshwater macrophyte and its associated microbiome[END_REF] . To add to this complexity, biofilm grazers are also present at this interface and will feed on the macrophyte leaves and biofilm, accumulating Au within their tissues [START_REF] Perrotta | Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers[END_REF] . Snail grazing has also been shown to significantly impact the water column quality through nitrogen, phosphorus, and carbon excretions [START_REF] Perrotta | Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers[END_REF][START_REF] Hall | Exotic snails dominate nitrogen and carbon cycling in a highly productive stream[END_REF] . Grazing on the surface of macrophytes can significantly reduce algal assemblages and improve macrophyte growth by grazing on epiphyton and increasing light attenuation [START_REF] Nifong | Experimental effects of grazers on autotrophic species assemblages across a nitrate gradient in Florida springs[END_REF][START_REF] Yang | Mesocosm experiment reveals a strong positive effect of snail presence on macrophyte growth, resulting from control of epiphyton and nuisance filamentous algae: Implications for shallow lake management[END_REF] . Therefore, grazing activity at this interface may thus modulate AuNP fate and transformation, and potentially impact their overall effect at the ecosystem level.

In this present work, we aimed at understanding the extent to which biofilm grazers (snails) could alter the fate of Au in simplified systems regarding its association with the different compartments of the microcosms (Egeria densa, biofilm, sediment floc, or water column). We hypothesized that grazers may reduce the amount of AuNP associated with macrophytes by reducing biofilm growth. If true, grazing may alter AuNPs fate, transport, and form of Au within the microcosm. We used the pulmonate, Physa gyrina, which is an air-breathing freshwater snail that primarily consumes filamentous algae, detritus, and senescent plant tissue [START_REF] Newman | Watercress Allelochemical Defends High-Nitrogen Foliage Against Consumption: Effects on Freshwater Invertebrate Herbivores[END_REF] . P. gyrina was chosen as a grazer for this experiment due to their ability to consume large amounts of filamentous algae while not directly consuming live plant tissue since we wanted to test Au toxicity and concentration in aquatic macrophytes. Furthermore, aquatic snails are readily found in freshwater habitats that are susceptible to runoff and chemical exposures and have shown to accumulate nanoparticles in their tissues [START_REF] Oliver | Does water chemistry affect the dietary uptake and toxicity of silver nanoparticles by the freshwater snail Lymnaea stagnalis?[END_REF][START_REF] Kulacki | How do stream organisms respond to, and influence, the concentration of titanium dioxide nanoparticles? A mesocosm study with algae and herbivores[END_REF] .

Additionally, we wanted to understand how grazer activity and seasonal differences will influence the impact and fate of AuNP in a simplified aquatic ecosystem. Previous research has shown that gold nanoparticles impacted E. densa growth rates and ecosystem metabolism rates more substantially in the spring months than in the fall [START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] . In order to manipulate both biotic and abiotic conditions while controlling the exposure and growth conditions parameters, we used microcosms to test our ecological questions with a high replication, while simplifying the chemical and biological complexity of the system to isolate the grazers contribution in the observation. Our simplified food web treatments included three biotic factors: phytoplankton, the aquatic macrophyte Egeria densa, and the aquatic snail Physa gyrina. The microcosms were randomly sorted into two treatment groups: control and AuNP addition. The AuNP treatments were dosed each week for four weeks to investigate the effects of chronic AuNP exposure. Both treatment groups received two seasonal conditions, fall (12.5°C average) and spring (25°C average), and with and without grazing pressure. We hypothesized that spring would have higher biological metabolism causing stress between organisms through competition. Higher competition coupled with AuNP stress could result in less E. densa growth and productivity.

Materials and Methods:

Nanoparticle characterization and concentration

Citrate-coated gold nanoparticles were synthesized by the Center for the Environmental Implication of Nanotechnology (CEINT). In filtered (0.45µm), control mesocosms water (pH 7.5, conductivity 80 µS/cm), AuNPs have a hydrodynamic diameter of 10.86 ± 1.44 nm and zeta potential in ultrapure water of -13 ± 6.24 mV. Full characterization of gold nanoparticles used in this experiment has previously been reported [START_REF] Surette | Effects of surface coating character and interactions with natural organic matter on the colloidal stability of gold nanoparticles[END_REF][START_REF] Nifong | Experimental effects of grazers on autotrophic species assemblages across a nitrate gradient in Florida springs[END_REF] . Each week 31.36 µg of AuNP were added to each microcosm for a total of 125.44 µg added over the four weeks of the experiment. To dose, each microcosm received 196 µL of 160 ppm AuNP stock solution pipetted across the water surface for even dispersal of nanoparticles.

Experimental Design and microcosm setup

Microcosms were built into acid-washed one-quart Ball® glass mason jars (16.36 x 8.59 cm). Water and E. densa were collected in July 2017 from a control mesocosm setup from the previous experiment in Duke Forest. Each microcosm was inoculated with phytoplankton communities collected from a local wetland and filtered through a 0.25 mm sieve to remove large organisms and debris. Physa gyrina were purchased from Carolina Biological Supply. We used two seasonal conditions (spring vs fall), two AuNP conditions (AuNP vs Control), and two food web conditions. The two food web conditions created were Phytoplankton + Macrophyte, and Phytoplankton + Macrophyte + Snails. This full factorial design resulted in 8 treatments with 48 microcosms in total (2 food webs x 2 seasons x 2 AuNP x 6 technical replicates). Macrophyte shoots were added to a bin with DI water and loaded onto a shaker for one hour at medium speed to ensure E. densa did not contain any phytoplankton or organisms. Shoots were examined again before placing them into the microcosms. Each microcosm was assembled using 100 g of Quikrete pool filter sand thoroughly rinsed with DI water and dried at 100°C, 700 mL of filtered (0.25mm) mesocosm water, 1 mL of phytoplankton communities, and five shoots of rinsed E. densa with a total wet biomass of 6g.

Microcosms were then capped with Parafilm® to allow the exchange of gases as well as light infiltration and placed in their respective controlled environmental growth chamber. Macrophytes and phytoplankton were acclimated to the microcosms for one week. During the acclimation period, microcosms were dosed with 0.1433 mg of Nitrogen (N) as KNO3 and 0.05697 mg of phosphorus (P) as KH2PO4. Nutrient addition continued weekly for the duration of the experiment for a total of 0.7165 mg of N and 0.2849 mg of P over five weeks. After the one-week acclimation period, snails and AuNP treatments were added.

For the snail treatments, five P. gyrina were added to the microcosms. After receiving snails from Carolina Biological, snails were kept in holding aquariums filled with the same water, sand, phytoplankton, and E. densa as the microcosms, allowing them to clear their intestinal tract for 48 hours and acclimate to the experimental conditions [START_REF] Oliver | Does water chemistry affect the dietary uptake and toxicity of silver nanoparticles by the freshwater snail Lymnaea stagnalis?[END_REF] . For each microcosm that received snails, five snails were removed from the holding aquariums, lengths and weights were recorded, and unique enamel paint markings were applied to each snail before placing the snails in their respective microcosms. The size variation for individual snails was maintained for each microcosm replicate (Table S1). To control for any effects on the systems from the addition of the enamel paint, glass beads were tagged in the same way as the snails and placed in all microcosms that did not contain snails. During the experiment, we replaced dead snails and removed any snail egg masses to keep the number of snails and snail biomass consistent across all treatments. Microcosms were monitored twice a week for egg clutches and snail death. Egg clutches were removed with a disposable transfer pipette with the tip cut. When snail death occurred, they were removed and replaced with a snail similar in size and weight, and the timing of exposure in the microcosms for each snail was tracked if they were introduced later in the experiment.

Incubation chambers

Our seasonality treatments were maintained by running the experiment in two controlled environmental growth chambers that were set to mimic the average seasonal conditions recorded during our previous field mesocosm experiment. In the spring treatment, the chamber oscillated between a 15-hour day at 30°C and a 9-hour night at 20°C. In the fall treatment, the chamber cycled between a 12-hour day at 15°C and a 12-hour night at 10°C. All work was performed inside of the incubators, and microcosms were maintained on wire shelving units equipped with fluorescent lights. Jars were randomized within these shelving units, with replicates rotated twice a week to eliminate any shading effects.

Sample collection and analysis

After 28 days of AuNP exposure, microcosms were disassembled. Water samples were collected and dissolved organic matter and total nitrogen (TN) were measured using TOC-VCPH Analyzer with a TNM-1 module (Shimadzu, Kyoto, Japan), and ammonium and phosphate were analyzed using Lachat QuikChem 8500 autoanalyzer. Au concentration was determined using ICP-MS after acidification of the samples with HNO3.

We determined the biomass and tissue Au concentrations of macrophytes, macrophyte associated biofilm, and snails. To remove the biofilm from E. densa shoots, shoots from each microcosm were placed into 100 mL of ultrapure water in a 250 mL Nalgene bottle and vigorously shaken by hand for 30 seconds. The cleaned shoots were then placed in aluminum tins and dried at 60˚C for 48 hours to determine total dry biomass. The biofilm that was removed from the E. densa shoots was transferred to a weigh boat and dried at 60˚C for 48 hours and weighed. Snails were removed from their microcosms and placed into a 15 mL conical tube and stored in the freezer until mass and Au content were measured. After thawing, snails were removed from their shell and dried at 60°C for 24 hours. After weighing all dried biomass samples were ground to a fine powder and weighed before digestion for metal analysis.

We expected unconsolidated organic matter, or floc, which accumulated on top of the sand throughout the experiment to be an important reservoir of Au. The constituents of the floc that accumulated varied by treatment. The Phytoplankton + Macrophyte mainly consisted of decaying E. densa debris, and Phytoplankton+Macrophyte+Snails was a combination of E. densa debris and snail excrement. At the end of the experiment, transfer pipettes were used to collect all the floc from each replicate. Floc samples were then homogenized, and subsamples were concentrated onto a 0.7 µm pore glass fiber filter attached to a filtering apparatus. All filters were dried at 60˚C for 48 hours and weighed to determine dry floc mass. After weighing, each filter was cut in half and re-weighed. Half of the filter was subsequently combusted at 500˚C for 1 hour in a muffle furnace to determine the ash-free dry mass (AFDM). The second half was then processed to determine AuNP content.

All dried solid samples were subsequently digested and analyzed for their Au content. The glass fiber filters used to determine AFDM were cut into small pieces and transferred into Teflon vials with 1 mL of 15 N hydrofluoric acid and 2 mL of 15 N nitric acid. Samples were heated for 30 min and allowed to air dry under a fume hood until all acid was removed. Rehydrated snails, biofilm, pre-digested filters, and E. densa powders were then digested in 70% HNO3 overnight and heated for 30 minutes at 95°C. HCl 30% was added to match a 3:1 HNO3: HCl volume ratio and heated additionally at 95°C for 30 minutes. All digested samples were diluted to 2% HNO3, filtered at 0.45µm, and analyzed for Au concentration by ICP-MS (Agilent 7700 for plant and floc samples and Agilent 7900 for snail and biofilm samples). Spike and certified reference material recoveries were 95.6 ± 4%, and detection limits were determined as (mean of 3 different reagent blanks) + (3 × SD of 10 replicate analyses of the reagent blank solution).

Statistical analyses

Endpoints were analyzed using either a one-way ANOVA to test for AuNP and snail treatment effects or a t-test when testing for snail effects or AuNP effects separately. One-way ANOVAs were followed by a Tukey posthoc test. All statistical analyses were performed in GraphPad Prism 8.0.1 on panels C and G graphs. Letters represent a significant difference (P < 0.05) from Tukey's post hoc test. Asterisks indicate statistical significance from a Student's t-test, * P< 0.05, ** P< 0.001, *** P< 0.001.

To assess how seasonal changes, AuNP, and snail presence influenced biotic growth after 28 days of AuNP exposure, total dry biomass was measured for each compartment in the microcosms (Fig 2).

Limited effects of AuNP on biomass pools or water chemistry

Overall, AuNP exposure had limited effects on biomass pools or water chemistry in either the fall or spring microcosms or with snail grazing presence. However, in the fall microcosms, the amount of biofilm significantly decreased with the addition of AuNP compared to control without snails (-52%, P= 0.003) (Fig. 2B). While no effects were seen on the snail, E. densa, or floc biomass in the fall microcosms (Fig. 2 C-D). Additionally, no AuNP effects were seen in the spring microcosms on biomass in any of the compartments measured. Additionally, AuNP did not alter water chemistry parameters in either season (Fig. 3). Furthermore, we did not observe a difference in E. densa photosynthetic and respiration rates with Au present compared to control (Fig. S6).

Strong seasonal and grazer effects on biomass pools and water chemistry

Seasonal differences had a substantial impact on the amount of biomass for all compartments. Snail and E. densa biomass decreased in the spring conditions compared to the fall (-63%, P<0.001 and -4.1%, P=<0.001, respectively) (Fig. S1) while biofilm and floc organic matter (OM) increased by 3.7-fold and 4.3-fold in the spring conditions compared to the fall (P< 0.001 and P=<0.001, respectively) (Fig. S1). Overall, the spring season had an increase in DOC, total nitrogen (TN), ammonium nitrogen (NH4-N), and phosphorus (PO4-P) in the water column compared to the fall (P<0.001, Fig. S1-S2). Additionally, dissolved oxygen (DO) and specific conductance also increased in the spring compared to the fall by 32% and 70%, respectively (P<0.001, Fig. S3), while pH remained similar between the two seasonal conditions.

In the fall conditions, snail grazing reduced the amount of biofilm by 66% in the controls and 25% in the AuNP treatments (P<0.001 and P= 0.77, respectively) (Fig. 2B). Snail presence did not influence the biomass of E. densa or floc; however, DOC within the water column increased in the control treatments by 73% (P<0.001) and AuNP treatments by 55% (P<0.001) with the addition of snails (Fig. 3A). Further, snail presence increased the amount of total nitrogen and phosphate in both control and AuNP treatments (Fig. S4).

In the spring conditions, snail grazing reduced the amount of biofilm by 97% in the control and 89% in the AuNP treatments (P<0.001 and P<0.001, respectively) (Fig. 2F). Snail presence also increased the amount of floc (OM) in the control treatments by 1.3-fold (P=0.009, Fig. 2H).

Snail presence also increased the amount of DOC in the control only treatment by 59% (P=0.03, Fig. 3E) and had no significant impact on TN or phosphorus amounts (Fig. S2.

Seasonal differences and grazing altered AuNP fate

Seasonal changes and snail grazing also played a significant role in the AuNP concentration found in each compartment in the microcosms. In the fall microcosms, the presence of snails significantly increased the Au concentration in the water column by 1.23 fold (Fig. 4A, P=0.005), while decreasing the concentration found in the biofilm by 41% (Fig. 4C,P=0.001) and E. densa shoots by 69% (Fig. 4D, P= 0.02).

In the spring microcosms, snail presence decreased the Au concentration in the E.densa shoots by 86% (Fig. 4I, P=0.001). There was also a trend for higher Au concentrations in the floc in the fall and spring conditions with the presence of snails (P= 0.63 and P=0.09).

Au mass balance and recovery

Au mass balance was calculated in the microcosms with almost complete recovery in the treatments without snails for both fall and spring conditions. While 96-104% of Au was recovered in the microcosms without snails, 50-60% of Au was recovered in the treatments with snails (SI Fig. 5).

In the fall microcosms, snail presence altered the fate of Au compared to the treatments without snails. The majority of Au added to the treatment without snails was found associated with E. densa (shoots and roots combined), followed by biofilm and floc. In the treatment with snails, most of the Au recovered was found in the floc and E. densa with a substantial portion not being recovered.

In the spring microcosms without snails, similar amounts of Au were recovered in the different compartments, with values ranging on average from 20% in the water column to 31% in the biofilm. In the treatments with snails, Au was mainly accumulated in the floc and water, with a large portion not being recovered. In the spring season, the Au concentration in the water column was higher than in the fall.

Discussion

After 28 days of chronic AuNP exposure, the different seasonal conditions did not alter AuNP effects but they did have a significant impact on the biomass, nutrients, and competitive interactions between the E. densa and their biofilm. In the spring conditions without snails, biofilm growth was abundant, which could cause stress to the macrophyte by increasing competition for nutrients and light [START_REF] Yang | Mesocosm experiment reveals a strong positive effect of snail presence on macrophyte growth, resulting from control of epiphyton and nuisance filamentous algae: Implications for shallow lake management[END_REF] . Higher biofilm growth and macrophyte stress with the warmer season could result in higher DOC concentrations in the water column that was observed [START_REF] Zhang | Effects of Rising Temperature on the Growth, Stoichiometry, and Palatability of Aquatic Plants[END_REF][START_REF] Reitsema | The future of freshwater macrophytes in a changing world: Dissolved organic carbon quantity and quality and its interactions with macrophytes[END_REF] . Although the spring temperature range was well within the tolerant range for both the macrophyte E. densa and the snail P. gyrina, both species showed visual stress from these conditions. The E. densa had a slight overall decrease in biomass in the spring conditions compared to the fall, while P. gyrina saw a higher death rate and lower biomass.

Concerning the impact of the AuNP chronic addition, Simonin et al have shown that an association of AuNP with the macrophyte tissues could reduce the photosynthetic and respiratory rates of E. densa [START_REF] Simonin | Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment HHS Public Access[END_REF] . In this study, we found that E. densa was a large sink of Au, similar to what was described in more complex mesocosm systems [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] . However, we did not observe a decrease in photosynthetic or respiratory rates. This could have been due to the total duration of the microcosm experiment, which only lasted five weeks, compared to the mesocosm experiment which lasted nine months. Also, we did not observe a difference in biomass from the E. densa stems that were placed in any microcosms meaning they did not undergo substantial growth. These factors could result in similar photosynthetic and respiratory rates despite having the Au addition.

Little impact was thus observed on the primary producer's and consumer's biomasses. However, while the E. densa biomass and photosynthetic activity were not altered by any of the treatments, we did see a reduction in biofilm growth in the fall microcosms exposed to AuNP without the presence of snails. Since biofilm growth decreased in the colder season and narrower window of light, the addition of AuNP could have posed as an additional stressor, further reducing the amount of biofilm growth. Although a reduction of biofilm was not observed in the spring microcosms, this could have been due to the abundant growth of biofilm in ideal conditions as well as the higher amount of DOC in the water column leading to the stabilization of Au in the water column, thus reducing deposition on the biofilm, as discussed below.

Although the impacts of AuNP were minimal on biomass, the fate of AuNP was dramatically altered with the addition of the aquatic snail, P. gyrina. The primary mechanisms by which P. gyrina altered Au fate in the microcosms were likely by the consumption of biofilm that accumulated on the leaves of the aquatic macrophyte, E. densa. In our study, P. gyrina reduced the biofilm amount by 89-97% in the spring season and 25-66% in the fall. Macrophyte associated biofilm growth has been shown to transform AuNP into oxidized, dissolved Au 13 , which could be more readily available to other aquatic organisms, but also increase its stability among the water column. With the addition of P. gyrina, biofilm growth in the spring conditions resulted on average a 20% reduction of Au associated with the E. densa tissue and an accumulation in the floc at the bottom of the microcosms. In the fall conditions, the addition of snails also decreased the amount of biofilm growth and accumulation in E. densa; however, the reduction of biofilm was smaller due to less growth overall. Remarkably, the addition of snails had the most dramatic reduction of Au associated with E. densa in the fall conditions with an average reduction of 34%.

The addition of snails also increased the amount of DOC found in the water column possibly due to snail excretions. This accumulation of DOC in the water column likely contributed to the stabilization of Au seen in the water column, as suggested by the strong correlation observed between these two traits. The presence of DOC in the water column has been shown to stabilize AuNP and reduce adsorption into macrophyte tissues [START_REF] Glenn | Abiotic and Biotic Factors That Influence the Bioavailability of Gold Nanoparticles to Aquatic Macrophytes[END_REF][START_REF] Oliver | Does water chemistry affect the dietary uptake and toxicity of silver nanoparticles by the freshwater snail Lymnaea stagnalis?[END_REF] . Similarly, organic matter released by E. densa in mesocosms has been shown to stabilize AgNP in the water column [START_REF] Unrine | Biotic and Abiotic Interactions in Aquatic Microcosms Determine Fate and Toxicity of Ag Nanoparticles[END_REF] .

While Au in both seasons without snails had full recovery (96-104%, Fig. S5), microcosms with snails had lower recovery (50-60%, Fig. 5S). There are several possible scenarios why lower Au recovery was seen with snails, such as a result of Au attaching to the shells of the snails, which were not measured for Au concentration. Similar to biofilm growth on aquatic macrophytes, biofilm growth can also occur on hard substrates such as the shell of mollusks 30 . Since snail death was observed in both seasons, the removal of dead snails could also be a sink for Au both within tissues and shells. Further, aquatic snails can secrete extracellular polysaccharides, which can also be a sink for metals [START_REF] Ballance | Influence of sediment biofilm on the behaviour of aluminum and its bioavailability to the snail Lymnaea stagnalis in neutral freshwater[END_REF][START_REF] Jugdaohsingh | Mucus Secretion by the Freshwater Snail Lymnaea stagnalis Limits Aluminum Concentrations of the Aqueous Environment[END_REF] . Alternatively, it could be due to the heterogeneity of Au associated with floc. While the majority of the floc was recovered from the microcosms complete recovery was not possible, which could account for the remaining Au.

This study highlights how critical climate and ecosystem complexity can alter the fate and effects of emerging chemicals. Using simplified systems allowed us to highlight the significant role of aquatic grazers in altering the fate of Au in freshwater environments. The addition of only five snails grazing on epiphyton significantly shifted the fate of Au from the macrophyte tissues to other compartments within the microcosms. This shift in fate can have significant impacts on water quality by increasing macrophyte growth, reducing periphyton buildup, and possibly reducing the frequency of algal blooms. Although the use of microcosms allowed for higher replication and ease of testing different climate conditions, this setup is likely far from any natural ecosystem. Therefore, future studies should focus on using large scale mesocosms or field sites.

ASSOCIATED CONTENT

Supporting Information

Additional information on overall differences in biomass and water chemistry between the fall and spring microcosms, as well as overall Au recovery and photosynthesis measurements in E. densa.

(PDF)

Figure 1 :

 1 Figure 1: Experimental design presenting food web, seasons, and AuNP treatments.

Figure 3 :

 3 Figure 3: Dissolved organic carbon (DOC), Dissolved oxygen (DO), pH, and Specific

Figure 4 :

 4 Figure 4: Au concentration in each compartment in the microcosms. Data shown are the averages

Figure 5 :

 5 Figure 5: Au recovery in microcosm compartments. Microcosms without snails showed a higher

Figure 6 :

 6 Figure 6: Correlation of Au concentration vs. DOC in the water column. P-value based on a

ACKNOWLEDGEMENTS

We would like to thank Dr. Ryan King, Eva May, Nia Bartolucci, Benjamin Wernette, Gary Dwyer, Jeffrey A. Back, and Jenny Rocca for their help in the experimental setup, sample collection, and overall feedback. Funding was supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093 and DBI-1266252, Center for the Environmental Implications of Nanotechnology (CEINT).

Results: