Groundwater is a hidden global keystone ecosystem.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Towards evidence-based conservation of subterranean ecosystems.

Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate meta-analytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness.

Forest damage and subsequent recovery alter the water composition in mountain lake catchments.

Forest damage by insect infestation directly affects the trees themselves, but also indirectly affects water quality via soil processes. The changes in water composition may undergo different pathways depending on site-specific characteristics and forest components, especially the proportion of coniferous and deciduous trees. Here, we test whether changes in forest components and the intensity of disturbance can predict the chemical properties of water outflow from affected lake catchments. Information about forest regeneration (a phase dominated by deciduous trees) and the proportions of damaged and healthy coniferous trees and treeless areas were obtained from satellite data. The four study catchments of Prásilské, Laka, Plesné, and Certovo lakes are geographically close and located in the same mountain range (Sumava Mts., Czech Republic) at similar altitude, but they differ in extents of forest disturbances and recoveries. The water quality measured at the lake catchment outflows differed, and better reflected the development of forest components and health than did meteorological (temperature and precipitation) or hydrological (discharge) variables. Several of the outflow properties (concentrations of inorganic aluminium, protons, potassium, calcium, magnesium, alkalinity, dissolved organic carbon (DOC), nitrate, and total phosphorus), responded catchment-specifically and with different delays to forest disturbance. The most pronounced differences occurred in DOC concentrations, which started to increase in the most disturbed Plesné and Laka catchments 7 and 6 years, respectively, after the peak in tree dieback, but did not increase significantly in the Prásilské catchment, which was disturbed several times during the last 3-4 decades. This study demonstrates an importance of extents of forest disturbances, the following changes in forest composition, and catchment-specific characteristics on water composition.

Collecting eco-evolutionary data in the dark: Impediments to subterranean research and how to overcome them.

Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research.Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represents the elective habitat for the so-called "cave species." Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating laboratory experiments.We explore the advantages and disadvantages of four general experimental setups (in situ, quasi in situ, ex situ, and in silico) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.Our over-arching goal is to promote caves as model systems where one can perform standardized scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline.

Species-dependent effect of cover cropping on trace elements and nutrients in vineyard soil and Vitis.

BACKGROUND: The research focused on the evaluation of the impact of cover cropping on trace metals (Fe, Mn, Cu, Zn, Pb, Co and Cd) and nutrients in vineyard soils and Vitis vinifera L. For this purpose, two types of cover crops (Lolium perenne L. and Medicago sativa L.) and their mixture were planted between vine rows of Muscat white in the vineyard in South Crimea. Trace elements, nutrients and other parameters were analyzed in the soil layers, leaves and grapevines of control and cover cropped plots. RESULTS: The effect of cover cropping was dependent on applied plant species. Ryegrass (L. perenne L.) seems to compete with V. vinifera L. for nutrients - these were lower in the soil and vines of the treated plot. In parallel, lead (Pb) bioconcentration in grapevines was reduced. In contrast, under lucerne (M. sativa L.), nitrogen in the soil and vines, and trace metal bioconcentration (Fe, Pb and Co) were higher. CONCLUSIONS: Our results indicate that cover cropping can influence the chemical composition of soil and vines. This should be considered when selecting cover crops. © 2019 Society of Chemical Industry.

Recommendations for ecotoxicity testing with stygobiotic species in the framework of groundwater environmental risk assessment.

As a consequence of the growing global dependence on groundwater resources, environmental risk assessments (ERA) for groundwater are increasingly required and, with that, ecotoxicological studies with groundwater fauna (stygofauna). However, the literature on the ecotoxicological studies with stygobiotic species (i.e. species that complete their life cycle exclusively in groundwater) has not expanded significantly since the first paper published in this field. The limitations regarding the use of stygobiotic species for ecotoxicological testing are clear and consistent across the globe; stygobiotic species are often 1) naturally present in low numbers, 2) difficult to collect, and 3) difficult to culture under laboratory conditions. This paper reviews the methods used in ecotoxicological studies performed with stygobiotic species, and provides ten recommendations for Good Laboratory Practice (GLP) for such tests. The recommendations focused on the following 10 points: 1) the taxonomic identification, the life stage/size and gender of the test organisms; 2) collection methodology of the organisms, including collection location, conditions and methods; 3) holding and acclimation conditions in the laboratory; 4) exposure conditions such as test set up and exposure time, number of replicates and densities of organisms in tests and in test vessels; 5) range-finding test set up and schedule; 6) final test design, including details of controls and treatments, and replication options; 7) incubation conditions, specifying temperature, pH and oxygenation levels throughout the test; 8) test duration; 9) observations and endpoints; 10) test validity criteria and compliance. The recommendations were developed for the purpose of supporting future short-term ecotoxicological testing with stygofauna through providing consistency in the protocols. A discussion of the potential implications for groundwater managers and decision-makers committed to ERA for groundwater is included.

Response and recovery of a pristine groundwater ecosystem impacted by toluene contamination - A meso-scale indoor aquifer experiment.

Microbial communities are the driving force behind the degradation of contaminants like aromatic hydrocarbons in groundwater ecosystems. However, little is known about the response of native microbial communities to contamination in pristine environments as well as their potential to recover from a contamination event. Here, we used an indoor aquifer mesocosm filled with sandy quaternary calciferous sediment that was continuously fed with pristine groundwater to study the response, resistance and resilience of microbial communities to toluene contamination over a period of almost two years, comprising 132days of toluene exposure followed by nearly 600days of recovery. We observed an unexpectedly high intrinsic potential for toluene degradation, starting within the first two weeks after the first exposure. The contamination led to a shift from oxic to anoxic, primarily nitrate-reducing conditions as well as marked cell growth inside the contaminant plume. Depth-resolved community fingerprinting revealed a low resistance of the native microbial community to the perturbation induced by the exposure to toluene. Distinct populations that were dominated by a small number of operational taxonomic units (OTUs) rapidly emerged inside the plume and at the plume fringes, partially replacing the original community. During the recovery period physico-chemical conditions were restored to the pristine state within about 35days, whereas the recovery of the biological parameters was much slower and the community composition inside the former plume area had not recovered to the original state by the end of the experiment. These results demonstrate the low resilience of sediment-associated groundwater microbial communities to organic pollution and underline that recovery of groundwater ecosystems cannot be assessed solely by physico-chemical parameters.

Community barcoding reveals little effect of ocean acidification on the composition of coastal plankton communities: Evidence from a long-term mesocosm study in the Gullmar Fjord, Skagerrak.

The acidification of the oceans could potentially alter marine plankton communities with consequences for ecosystem functioning. While several studies have investigated effects of ocean acidification on communities using traditional methods, few have used genetic analyses. Here, we use community barcoding to assess the impact of ocean acidification on the composition of a coastal plankton community in a large scale, in situ, long-term mesocosm experiment. High-throughput sequencing resulted in the identification of a wide range of planktonic taxa (Alveolata, Cryptophyta, Haptophyceae, Fungi, Metazoa, Hydrozoa, Rhizaria, Straminipila, Chlorophyta). Analyses based on predicted operational taxonomical units as well as taxonomical compositions revealed no differences between communities in high CO2 mesocosms (~ 760 µatm) and those exposed to present-day CO2 conditions. Observed shifts in the planktonic community composition were mainly related to seasonal changes in temperature and nutrients. Furthermore, based on our investigations, the elevated CO2 did not affect the intraspecific diversity of the most common mesozooplankter, the calanoid copepod Pseudocalanus acuspes. Nevertheless, accompanying studies found temporary effects attributed to a raise in CO2. Differences in taxa composition between the CO2 treatments could, however, only be observed in a specific period of the experiment. Based on our genetic investigations, no compositional long-term shifts of the plankton communities exposed to elevated CO2 conditions were observed. Thus, we conclude that the compositions of planktonic communities, especially those in coastal areas, remain rather unaffected by increased CO2.

Towards an integrated understanding of how micro scale processes shape groundwater ecosystem functions.

Micro scale processes are expected to have a fundamental role in shaping groundwater ecosystems and yet they remain poorly understood and under-researched. In part, this is due to the fact that sampling is rarely carried out at the scale at which microorganisms, and their grazers and predators, function and thus we lack essential information. While set within a larger scale framework in terms of geochemical features, supply with energy and nutrients, and exchange intensity and dynamics, the micro scale adds variability, by providing heterogeneous zones at the micro scale which enable a wider range of redox reactions. Here we outline how understanding micro scale processes better may lead to improved appreciation of the range of ecosystems functions taking place at all scales. Such processes are relied upon in bioremediation and we demonstrate that ecosystem modelling as well as engineering measures have to take into account, and use, understanding at the micro scale. We discuss the importance of integrating faunal processes and computational appraisals in research, in order to continue to secure sustainable water resources from groundwater.

Biodegradation: Updating the concepts of control for microbial cleanup in contaminated aquifers.

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

Catecholamine levels in groundwater and stream amphipods and their response to temperature stress.

Temperature stress in invertebrates is known to be reflected by changes in catecholamine levels. However, the mechanisms of stress response are not fully understood. Groundwater and surface water amphipods are expected to be differently adapted to temperature elevations due to the different temperature regimes in their habitats and consequently, show a different stress response. No data have been published so far regarding the effects of stress on catecholamine patterns in groundwater invertebrates and accordingly, comparisons with surface water fauna are also missing. In this study, we compared the average catecholamine levels in two taxonomically related amphipod species: Niphargus inopinatus, living in groundwater with constant water temperatures throughout the year, and Gammarus pulex, a surface water stream amphipod frequently exposed to diurnal and seasonal temperature fluctuations. Furthermore, we tracked the immediate changes in whole-animal catecholamine levels in response to heat stress in both species. Pronounced differences in the catecholamine levels of the two species became apparent, with the average dopamine (DA) level of N. inopinatus being almost 1000 times higher than that in G. pulex. The noradrenaline (NA) concentrations in N. inopinatus were on average two orders of magnitude higher than in G. pulex, and for adrenaline (A), the difference constituted one order of magnitude. When exposed to short-term heat stress, both species showed a response in terms of catecholamine levels, but the observed patterns were different. In N. inopinatus, temperature stress was reflected by the appearance of adrenaline, while in G. pulex a significant increase in noradrenaline levels occurred in the treatment with the highest temperature elevation.

A new bioassay for the ecotoxicological testing of VOCs on groundwater invertebrates and the effects of toluene on Niphargus inopinatus.

A protocol was developed for testing the ecotoxicological effects of volatile organic compounds (VOCs) on groundwater invertebrates. Test substance volatility was addressed in a "closed from start to analysis"-design. Since manifestation of toxic effects may be delayed in 'slower metabolizing' organisms such as groundwater fauna, a time-independent (TI-) approach was adopted. Toluene was used as a model substance and its toxicity to the groundwater amphipod Niphargus inopinatus was assessed as an example. The method evaluation process considered various methodological issues such as partitioning of the toxicant between the water and the gas phase (Henry equilibrium), the possible depletion of oxygen in closed test vials, as well as microbial biodegradation of the test substance. For N. inopinatus, an LC50,14 days of 46.6mgL(-1) toluene was obtained. The ultimate LC50 value was estimated at 23.3mgL(-1) toluene. No oxygen depletion occurred in the test vials and Henry equilibrium was found to be established after 6h. The new test system proposed now awaits broad practical application.

The potential use of fauna and bacteria as ecological indicators for the assessment of groundwater quality.

The use of ecological criteria for the assessment of aquatic ecosystem status is routine for surface waters. So far no ecological parameters are considered for the assessment and monitoring of groundwater quality. It has been well known for decades that aquifers are ecosystems harbouring a vast diversity of invertebrates and microorganisms. The growing knowledge on groundwater microbial and faunal communities as well as the molecular and statistical tools available form a solid ground for the development of first ecologically sound assessment schemes. The sensitivity of groundwater communities towards impacts from land use and surface waters is exemplarily demonstrated by a data set of two geologically similar but hydrologically partially separated aquifer systems. Subgroups of the fauna in groundwater (stygobites vs. stygophiles and stygoxenes) successfully indicated elevated nitrate impacts linked to land use activities. Within the microbial communities, impacts from land use are mirrored by high bacterial biodiversity values atypical for pristine groundwater of comparable systems. The data show that there is legitimate hope for the application of ecological criteria for groundwater quality assessment in the future.

Effects of thermal energy discharge on shallow groundwater ecosystems.

The use of groundwater as a carrier of thermal energy is an important source of sustainable heating and cooling. However, the effects of thermal use on geochemical and biological aquifer characteristics are poorly understood. Here, we have assessed the impacts of heat discharge on an uncontaminated, shallow aquifer by monitoring the hydrogeochemical, bacterial and faunal parameters at an active thermal discharge facility. The observed variability between wells was considerable. Yet, no significant temperature impacts on bacterial or faunal abundance and on bacterial productivity were observed. Also, we did not observe an improved survival or growth of coliforms with temperature. In contrast, the diversity of bacterial terminal restriction fragment (T-RF) length polymorphism fingerprints and faunal populations was either positively or negatively affected by temperature, respectively, and the abundance of selected T-RFs was clearly temperature dependent. Canonical correspondence analysis indicated that both the impact of temperature and of surface water from a nearby river, were important drivers of aquifer biotic variability. These results demonstrate that aquifer thermal energy discharge can affect aquifer bacteria and fauna, while at the same time controlling only a minor part of the total seasonal and spatial variability and therefore posing no likely threat to ecosystem functioning and drinking water protection in uncontaminated, shallow aquifers.