Biodiversity is declining at an alarming rate. Multiple stressors are driving many of these declines with freshwater (FW) ecosystems particularly impacted. Ephemeral FWs (e.g. marshes, ponds) are exceptionally biodiverse and highly exposed to varied environmental stressors but are generally overlooked within academia and regulation. Amphibians have been a major faunal component of these habitats for at least 350 million years, being highly evolved to these ecosystems. Amphibians and wetlands are some of the most highly threatened Phyla/ecosystems globally, with wetland health key to the climate crisis, due to the high methane levels emitted from human impacted systems. Using both field and laboratory approaches, here we will investigate the environmental stressor combinations driving negative impacts in amphibians (common frog, Rana temporaria) and seek to develop a biomonitoring approach to assess the health of these vital ecosystems. As amphibians are the most highly threatened vertebrate Phyla, this project is highly relevant to conservation priorities. General health, disease status, stress markers and global gene expression in wild and caged tadpoles will be measured. The use of toxicogenomics and alterations to physiology to assess impacts on tadpoles allows both the anchoring of molecular initiating events to downstream physiological endpoints and resulting adversity, as well as mapping these responses to stressor combinations. This mapping presents a highly novel approach, allowing the identification of specific stressors and their combinations that are driving negative impacts, and is widely applicable across biota. Catchment-scale eco-epidemiological studies between wild taxa and the presence/severity of stressors often rank pollution as amongst the most important variables driving negative effects in FWs. However, studies on effects of pollution at environmentally relevant levels and mixture combinations are scarce, particularly in the context of multiple stressors. Here pollutant mixture formulations will be based directly on measured levels in ephemeral FWs and combined with other ubiquitous stressors (salinity, heat wave and/or invasive crayfish - Pacifasticus leniusculus cue), all at environmentally relevant levels and combinations. These laboratory exposures will be highly novel and of vital importance to understand the true impacts of multiple stressors on iconic amphibian biota that inhabit vital ephemeral FWs. It will be tested how best to utilise data from single stressor exposures, to predict effects using theoretical models. For this, we will apply novel theoretical paradigms to the data - dominance (few stressors contribute disproportionately to observed effects) and burden (total stressor load determines effects) - which have huge potential for wide applicability for multi-stressor science. In contrast to the single-endpoint approach, here we propose to use ecological modelling to investigate effects on whole organisms and their populations in order to drastically improve the utility of these data for conservation. Finally, by transplanting spawn and sampling both caged and native tadpoles, the utility of naïve/locally adapted tadpoles as a biomonitoring tool to assess the health of FW wetlands will be assessed. This work will address an important gap in the literature between field-based catchment-level evidence demonstrating the importance of multiple stressors and the current limited laboratory-based evidence/understanding; as well as developing a new testing paradigm with practical application for conservation. The research team combines excellence in FW ecotoxicology, multiple stressors/mixture effect biology, FW ecology, ecological modelling, bioinformatics and chemistry needed for this project. In addition, the project partners and supporting organisations comprise a range of stakeholders that are focused on the health of FW ecosystems and reducing the impacts of pollution.

The project aims to unlock two significant but untapped resources for citizen science: the use of environmental DNA to detect species and the capacity of the National Trust (NT) as a major landowning and volunteering organisation. We propose a scoping study and co-designed pilot to bring these together. Environmental DNA (eDNA) are traces of DNA released into the environment by species. Sources of eDNA include secreted faeces, mucous, gametes, shed skin, hair and carcasses and these can be detected in samples of water or soil. The potential for eDNA to transform species recording and open it up to people with few or no traditional species identification skills is huge. So far, the most developed application is for fish and amphibians in aquatic environments where a simple water sample can be analysed to produce a list of species present and even provide some indication of relative proportions of different species. Initial work suggests non-specialists (anglers and schoolchildren) can collect adequate (uncontaminated) water samples that provide robust data of which species are present in a waterbody. There is an opportunity to get interested communities collecting information on what lives in their local river, lakes or pond in a way that was hitherto impossible (due to complicated and expensive survey methods or lack of species identification skills). We believe that being able to collect these data will enthuse people to find out more about the health of their local waters leading to further action either in getting more involved in data collection or in tackling issues to improve freshwaters. The use of an exciting and novel scientific tool could open up a pathway of engagement and direct action. By involving volunteers in the decision-making process and supporting them to identify local need, we believe participants will develop the confidence to share their skills with others. The National Trust is public-facing IRO and charity as well as large landowner (250,000ha) with ambitions to transform our land to be better for wildlife and to provide a wide range benefits of benefits to society. With 5.5million members and more than 60,000 volunteers the potential to reach and engage a large community with this work is considerable. Partnership working, and public engagement are embedded strongly across the NT's overall research programme. One of the NT's challenges is to monitor the effectiveness of the changes we are making across our estate but also to ensure that our monitoring effort plays a part in a wider UK network tracking the health of our landscapes. We strongly believe that citizen science, capitalising on our member, volunteer and visitor assets, could play an important role in this respect. Through this project we will bring together NT researchers, leading UK freshwater and eDNA scientists, citizen science specialists and a group of NT volunteers to explore the state of the art in terms of eDNA monitoring for freshwaters. Through a workshop and co-design process we will: Generate a series of recommendations for the development of a UK-wide citizen science project based on eDNA in freshwaters Develop and implement a co-designed pilot to test citizen science collection of eDNA species data (fish and amphibians) in a catchment where NT are leading a partnership project to improve and restore the freshwater environment (Upper Bure, Norfolk) Review lessons learned from the pilot to inform further development of eDNA based citizen science The project will lead directly to the development of a future funding bid for a national scale eDNA based citizen science research project.

In recent years, three major innovations have occurred in ecology. (1) The emergence of new statistical methods for analysing community data; (2) the rapid detection of species and whole communities from environmental DNA (eDNA) and bulk-sample DNA; and (3) the wide availability of remotely sensed environmental covariates. The efficiency gains are such that hundreds or even thousands of species can now be detected and, to an extent, quantified in hundreds or even thousands of samples. Collectively, these three innovations have the potential to relieve the problems of data limitation and analysis that environmental management has been struggling with, opening the way to near-real-time tracking of state and change in biodiversity and its functions and services over whole landscapes. The aim of our project is to develop an integrated statistical framework for DNA-based surveys of biodiversity. The framework will allow the estimation of community compositions and the identification of the landscape characteristics that drive them. We will develop a Bayesian hierarchical model accounting for the probabilistic nature of DNA-based data due to observation error and taxonomic uncertainty and for model uncertainty due to the unknown strength and direction of landscape effects on the system. We will build sophisticated and efficient algorithms within a Bayesian framework for identifying the important landscape covariates that predict community structure and provide guidelines on optimal allocation of resources in DNA-based surveys for achieving the required power to infer species distributions and to link them to landscape covariates. The huge potential contribution of DNA-based data to landscape decision-making is demonstrated by how Natural England, Local Planning Authorities, and the NatureSpace Partnership use eDNA to create a biodiversity-offset market ('District Licensing') for the protected Great Crested Newt (GCN). Water samples from 500 ponds across the South Midlands (spanning ~3320 sq km) were tested for GCN and used to create a distribution map, which was then zoned into four 'impact risk' levels. Builders pay a known, sliding-scale fee, and a portion of the fee is used to build and manage new habitat. District Licensing is only feasible with eDNA's greater efficiency. GCN District Licensing expands to at least 16 LPAs in 2020, aiming to go nationwide, which would make it the largest biodiversity-focused, land-use decision scheme in the UK, if not the world. The natural-and highly desirable-extension to the GCN scheme would be to map 'all biodiversity' and to make land-use decisions (e.g. impact risk maps, offset markets, habitat creation) on this broader basis. In fact, samples originally collected for GCN can be repurposed for this larger goal by using 'metabarcoding,' meaning that the eDNA is PCR-amplified for a larger range of taxa. Given the District-Licensing expansion plans, pond eDNA metabarcoding alone could provide an efficient way to map biodiversity across much of the UK. This is far from the only such programme. Ecologists in industry and academia around the world are plunging ahead with large-scale DNA-sampling campaigns, and there is, as yet, no comprehensive set of statistical methods for modelling the individual steps of the new observation processes, quantifying the resulting uncertainty, and assessing how it affects decision-making at the landscape level. Our proposed modelling framework will provide such tools by explicitly capturing measurement bias within biodiversity models as a set of observation processes, and not merely as error. Improving sampling designs and workflows as a result of our proposed models will profoundly increase the efficiency and credibility of inference and therefore reduce the risk of biodiversity loss during the political process of allocating land to different uses.

In our changing world there is an increasing urgency to understand interactions among multiple environmental stressors, such as pollution and warming. Much of the concern surrounding multiple stressors is due to their potential to interact, creating more severe impacts than they would do independently. Freshwater ecosystems are particularly vulnerable and freshwater biodiversity is the most threatened across the globe: a recent report estimated average population declines of >80% among freshwater vertebrate species compared to <40% in terrestrial and marine species (since 1970; WWF Living Planet Report, 2018). Although the combined impacts of multiple stressors has started to receive more attention, our knowledge on their interactive effects still remains almost non-existent. In reality, stressors are unlikely to occur in the same space at exactly the same time, yet studies that measure the combined effects of multiple stressors often assume this to be the case. In other words, they lack temporal realism. Most of these studies also lack biological realism by quantifying the effects of stressors on model species at lower levels of organisation (e.g. range shifts, survival, abundance) and ignoring feeding interactions. Here, we will consider how the order, or sequence, of stressor events alters individual-to-ecosystem responses of freshwaters, with a focus on food web interactions. Ecosystems will have multiple responses to the multiple stressors they face, including changes in diversity, abundance, body size and feeding behaviour. Even minor alterations to any of these can shift food web structure, with implications for the effects of future stressors, yet these critically important interactions have been largely ignored to date. This leaves us with little or no predictive ability about the consequences of future change in natural systems. Therefore, here we will use mesocosm experiments to quantify the combined effects of staggered nutrient pollution and warming events (i.e. previous exposure) on freshwater ecosystems, and scale our results up to the catchment level by adapting a suite of dynamic water quality models. Our experimental results will be used to parameterize temperature and nutrient controlled population sizes and growth rates, and to simulate how these changed rates will alter food web structure at the larger river system scale. This interdisciplinary study will generate an unprecedented breadth and depth of data: from individual changes in fitness and population shifts in size structure to food web complexity. We will show how the order of multiple stressor events (i.e. previous exposure) affects community resistance and resilience to change. These unique data sets will allow us to ask numerous novel questions in pure and applied ecology, and to characterise the little known multiple impacts of multiple stressors on freshwater food webs. Such a comprehensive coverage of responses has never been attempted before and this study will address this glaring gap in our knowledge of stressor impacts.

Freshwater ecosystems provide critical ecosystem services that underpin human societies and wellbeing: including water purification, carbon capture, and the maintenance of sustainable fisheries. However, these ecosystems are under an increasing array of threats, both in the UK and worldwide, especially from a wide range of new and emerging chemical stressors (e.g. novel antibiotics and pesticides). Freshwater biosciences and applied ecology are under-equipped for dealing with these new threats: the evidence base is lacking, there is often little or no mechanistic understanding, or predictive capacity for anticipating how these novel chemicals will operate in the real world. This is particularly true for the ecosystems of the future that are being reshaped and constructed by climate and other environmental changes. Our project will address all these shortcomings by taken a radically different approach from the classical biomonitoring and ecotoxicology tools that have dominated for many decades. We aim to unearth the general rules by which emerging chemical stressors operate through, and alter, networks of interacting species - from microbes at the base of the food web, through to apex predators in the fish community at the top. This will involve the development of indicators of both proximate pollution, as the chemical first enters the biological system (commonly as a new food source for microbes), and also of its indirect effects as its impact propagates through the food web. For instance, we will be able to answer questions such as: if a new insecticide wipes out the invertebrates in the middle of the food web, does this trigger blooms of nuisance algae as they are no longer kept in check? To achieve this, we will develop a new suite of methods at the ecosystem level that combine lab and field experiments to detect the causal mechanisms that we currently do not understand. The experiments will be combined with mathematical modelling to predict ecosystem-level impacts. We will address both, contemporary ecosystems that could be under imminent threat from new chemical stressors, and ecosystems of the future that will emerge under different scenarios of land-use and climate change. This will provide a completely new paradigm in chemical stressor monitoring, based on using first principles to derive a novel means of predicting "ecological surprises" that commonly arise due to the inadequacies of our current simplistic approaches when dealing with the true biocomplexity of natural systems. Our scope is for our approach to serve as a diagnostic tool for management, with research findings, for example, supporting the selection of mitigation options that deliver reduction of ecological effects. This paradigm shift will allow far more robust predictions and therefore more informed management decisions about the freshwaters of the future. The work will bring together the field of pure and applied ecological science, to the mutual benefit of both sets of disciplines. Our proposal represents the first steps along this path to the more multidisciplinary perspective that is going to be critical for dealing with future threats to our ecosystems - from emerging chemical stressors in freshwaters to the growing list of other environmental threats looming on the horizon. Because the approach is general, it will not only pave the way for the next generation of ecological assessment in freshwaters, but it can also be adapted for applications in marine and terrestrial ecosystems.