Search

Groundwater-fed springs are relatively stable freshwater environments which harbor a valuable biodiversity and represent a precious water source for human needs. However, with ongoing global warming and increasing human footprint on natural systems, spring ecosystems are more and more exposed to hydrologic and physico-chemical alterations, which would in turn threaten their biodiversity. Recently, scientists claimed that an integrated “ecohydrogeological” approach, analyzing patterns and drivers of changes in spring environments from various perspectives, would greatly help in assuring the long-term preservation of these precious ecosystems. Following this plea, we studied both the ecological and the hydrological dynamics of the Presciano karst spring system (Abruzzo, Central Italy). The Crustacea Copepoda were chosen as target invertebrate group, since they are main components of the freshwater meiofauna and occur in all the groundwater-dependent ecosystems, groundwater-fed springs included. Specifically, we analyzed temporal changes in both copepod species richness and between-site differences in assemblage composition (i.e., beta diversity). The observed patterns were also contrasted to variations in measured physico-chemical parameters. On the hydrologic side, starting from rain gauge data, we implemented the CETEMPS Hydrological Model (CHyM) to derive a rain field with hourly resolution across the Gran Sasso – Sirente hydrogeological basin, which hosts the recharge areas feeding the Presciano spring. The modelled monthly cumulative rain, averaged across the Gran Sasso – Sirente basin, was then compared to the maximum and minimum monthly flow discharge values extracted from a hydrometric station located downstream the Presciano spring outlet. Finally, future rainfall simulations for the 2081-2091 decade were performed on the same area by forcing the CHyM software under the RCP8.5 emission trajectory, based on three different Regional Climate Models. Both species richness and beta diversity of the Presciano spring noticeably varied over time but variability of hydrochemistry could not comprehensively explain such changes, except for species turnover which was tightly related to between-site variability in water oxygenation. Seasonal discharge variations may thus have a more prominent role than local water conditions in determining the overall structure of spring meiofauna assemblages. Moreover, a not-negligible signal emerged from the comparison of precipitation and discharge temporal dynamics, indicating that accurately modelling rainfall in recharge areas would permit to better estimate and possibly forecast temporal variation in spring discharge. The future projections highlighted an overall predicted drop in precipitation across the Gran Sasso − Sirente basin, warning about possible groundwater lowering in karst springs in the next future.

A group of four amphoras found in the anchialine cave of Vodeni Rat in the Croatian island of Sveti Klement, on a rocky ledge at 24 m water depth, indicates that a freshwater source was exploited at the bottom of this karstic pit by islanders and/or passing-by sailors from the late Roman Republican Period to the Early Medieval Period. In other words, prior to the 4th–7th century CE, Vodeni Rat was not an anchialine cave but a Pleistocene karstic pit with a freshwater pool at the bottom. Seawater started to infiltrate this cavity via newly opened fissures in the surrounding limestone after the 4th–7th century CE, as the archeometric age of the youngest intact amphora found down in the pit suggests. At present, the 29-m-deep water body is stratified, with a saline water layer at the bottom, a freshwater layer at the surface, and a transitional brackish water layer in the middle. Our study of the physical and biological characteristics of the cave water column revealed diversified and highly partitioned populations of stygobitic crustaceans including ostracods, copepods, amphipods, and thermosbaenaceans. Some species known to be exclusively endemic of highly saline habitats were collected from the bottom saline layer, whereas others known to be of freshwater origin were found in the upper water layer. This suggests that the freshwater dwellers were already present in this cave prior to the early medieval event that caused seepage of seawater into this karstic pit., SCOPUS: ar.j, info:eu-repo/semantics/published

1. Species of conservation concern are usually considered important elements in site prioritization for biodiversity conservation. To overcome the lack of information on species conservation status, multidimensional measures of species rarity can be used as proxies of species vulnerability. 2. Under this assumption, a two-step protocol for site prioritization of aquatic groundwater-dependent ecosystems is proposed using invertebrate vulnerability estimated from species' traits. In the first step, each species occurring in the sites of interest are scored according to their vulnerability. In the second step, sites are prioritized using species' scores. 3. Species vulnerability scores are based on five dimensions, for which various traits are scored: (i) geography, (ii) ecology, (iii) biology, (iv) population, and (v) evolutionary history. For each species, the scores of the various traits belonging to the same dimension are multiplied to obtain a synthetic score. These scores are then ranked into four classes and, for each dimension, each species receives a new score that reflects its rank. The sum of these scores represents the species' overall score. 4. Site conservation priorities are assessed by combining species scores into three indices: Sum of Species Scores, Biodiversity Conservation Concern (which relates the sum of species scores with the local species richness) and Groundwater Biodiversity Concern (which is the average of the former two). The protocol is illustrated using case studies in Italy and it is fully implemented in the software AQUALIFE which is freely available at: http://app.aqualifeproject.eu by registered users. 5. Sensitivity analyses showed that the protocol is robust against the lack of information on species biology or sampling limitations. However, trait scoring rests with the user, who must be familiar with the study group. 6. This approach can be applied at any spatial scale and to different types of aquatic groundwater-dependent 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 metaanalytical 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., This study is funded by the European Commission via the Marie Sklodowska-Curie Individual Fellowships program (H2020-MSCA-IF-2019; project number 882221), awarded to S.M. Additional support is provided by the PRIN SHOWCAVE “A multidisciplinary research project to study, classify and mitigate the environmental impact in tourist caves” (project number 2017HTXT2R; funded by the Italian Ministry of Education, University and Research). M.B.M. acknowledges support from the Kone Foundation (project number 202007611). T.D., C.F., V.Z., and M.Z. were supported by the Slovenian Research Agency, through core programme P1-0184 and P6-0119. O.T.M. was supported by a grant of the Romanian Ministry of Research, Innovation and Digitization, CNCS/CCCDI— UEFISCDI, project number 2/2019 (DARKFOOD), within PNCDI III. S.I.S. acknowledges funding by MEMOBIC (EU Operational Programme Research, Development and Education No. CZ.02.2.69/0.0/ 0.0/16_027/0008357), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant number CZ.02.1.01/0.0/0.0/16 025/0007417). E.P. is supported by the PON “Research and Innovation” Programme (Axis IV “Education and Research for recovery” – Action IV.6 “Research contracts on Green themes”).

Ammonium has detrimental effects on the survival and physiology of freshwater invertebrates, however, the effects of ammonium on the functionality of hyporheic invertebrate assemblages have never been investigated. In this study, we applied a mixed (taxonomy- and trait-based) approach to explore the taxonomic and functional trait variation along a gradient of ammonium contamination in the hyporheic zone of a Mediterranean stream. We analysed fifty-one trait modalities of thirty-eight invertebrate taxa. We tested a priori hypotheses on the functional traits that should be affected. We observed a severe erosion of taxonomic and functional diversity with the loss of 12 taxa and 11 trait modalities related to size and body form, fecundity and reproduction, resistance forms and respiration, diet, locomotion and feeding habits. The effect on taxonomic and trait modality abundances was also relevant; in particular, the decrease in detritivores seemed to have led to a low content in dissolved organic matter. Synergic detrimental effects of ammonium and the summer season were also observed. Despite some uncertainties, we found out that the mixed approach responded consistently along a gradient of ammonium contamination. Since functional traits link community organization to ecosystem goods and services, the results of our study could be prodromal to the inclusion of ammonium as indicator of functional stress in a health index of hyporheic zones in Mediterranean rivers.

Conservation planning aimed at halting biodiversity loss has seldom focused on groundwater environments due to the lack of suitable management tools and data. Using harpacticoid crustaceans as a test case, we explore the potential of implementing an approach based on Conservation-Relevant Hotspots for practical conservation of groundwater biodiversity. Conservation-Relevant Hotspots are identified by intersecting species richness, endemicity, and taxonomic distinctness with the aim to minimize the total area to protect. We show that, by targeting five Conservation-Relevant Hotspots that cover only 1.9% of the European land surface, one would protect as much as 44% of the harpacticoid crustacean richness, 93% of its endemicity, and 98% of its taxonomic distinctness. About 28% of the area occupied by these hotspots overlaps with protected areas, which calls for an increase in their protection coverage. Our framework proved a useful tool for conservation planning of environments where spatial or socio-economic constraints occur.

A new species of the genusStygepactophanesMoeschler & Rouch, 1984 (Copepoda, Harpacticoida, Canthocamptidae) is established to accommodate a small canthocamptid population collected from a spring system in the “Parc du Mercantour”, Var catchment, southern France. The population analysed in the present study is defined by a set of morphological characters of the female, namely a very large maxilliped, a rudimentary mandibular palp, P1 with 3-segmented exopod and 2-segmented endopod, a falcate terminal claw of the P1 endopod, dorsal seta of caudal rami inserted on the inner margin, and anal operculum not overreaching the insertion of the caudal rami, thus supporting its assignment into the genusStygepactophanes. The new speciesStygepactophanesoccitanusshows marked differences with the nominotypical species of the genus that was originally described by monotypy with the speciesStygepactophanesjurassicusMoeschler & Rouch, 1984. The main diagnostic traits ofS.jurassicusare the absence of the P5 and a falcate outer terminal claw of P1 endopod.Stygepactophanesjurassicusalso shows a reduced armature of the antennal exopod, bearing one seta, 1-segmented P2–P4 endopods, a reduced armature of P2–P4 exopodal segments 3 (3,4,4 armature elements, respectively), P6 bearing only one long seta, a rounded short and smooth anal operculum. Conversely the female ofS.occitanusGalassi & Fiers,sp. n.has a well-developed P5, with rudimentary intercoxal sclerite, together with a falcate outer terminal claw of P1 endopod, antennal exopod bearing two elements, P4 endopod 1-segmented versus 2-segmented in P2–P3, P2–P4 exopodal segment 3 with five armature elements, P6 with three setae of different lengths, rounded anal operculum, bearing 3–4 strong spinules.According to our present knowledge,S.occitanusGalassi & Fiers,sp. n.is assigned to the genusStygepactophanesas the most conservative solution, waiting for the male to be discovered. The genusStygepactophanesrepresents a distinct lineage within the harpacticoid family Canthocamptidae that colonised southern European groundwater, the genus being known only from the saturated karst in Switzerland and a fissured saturated aquifer in southern France. Both species of the genus are stygobites and narrow endemics, the nominotypical species being known from the type locality Source de la Doux in Délemont (Switzerland), andS.occitanusGalassi & Fiers,sp. n.described herein from a spring system of the Var catchment (France).

In this study we used the data of an extensive pesticide monitoring survey that took place in 11 alluvial aquifers of central Italy from 2010 to 2015 to explore 4 different scenarios of risk. The Scenarios 1 and 2 were used to depict the risk of failing to meet the good groundwater chemical status as defined by the Water Framework Directive. The Scenarios 3 and 4 were used to assess for the first time the ecological risk in groundwater bodies, defined as the likelihood of hazard to the groundwater communities stably residing in the 11 alluvial aquifers that may be affected by pesticide contamination. The ecological risk was assessed through a new procedure called GERAp (Groundwater Ecological Risk Assessment due to pesticides). The main results of this study highlighted that: 1) the Scenario 1 provided information of little use for risk managers; 2) more realistic information was provided by using the highest concentrations measured in the six-year monitoring period and considering the ecological risk in a combined scenario (Scenarios 2 and 4); 3) the achievement of the good chemical status by 2027 in 3 aquifers will be likely much more difficult than in the others because the ecosystem services, such as pesticide biodegradation, are likely less efficient in the 3 groundwater bodies; 4) some pesticides that were banned in Europe in 2009 should be kept monitored in the next surveys because they showed a persistent occurrence in the 11 aquifers; 5) DDT forms, Dieldrin and Heptachlor are expected to damage groundwater communities at concentrations that are lower than the present legal limits.

Groundwater invertebrate communities in karst landscapes are known to vary in response to multiple environmental factors. This study aims to explore the invertebrate assemblages’ composition of an Apennine karst system in Italy mainly described by the Rio Gamberale surface stream and the Stiffe Cave. The stream sinks into the carbonate rock and predominantly feeds the saturated karst into the cave. For a minor portion, groundwater flows from the epikarst and the perched aquifer within it. The spatial distribution of the species belonging to the selected target group of the Crustacea Copepoda between the surface stream and the groundwater habitats inside the cave highlighted a different response of surface-water species and obligate groundwater dwellers to the hydrogeological traits of the karst unit. Our results suggest that fast endorheic infiltration routes promoted the drift of epigean species from the surface to groundwater via the sinking stream while most of the obligate groundwater dwellers come from the perched aquifer in the epikarst from diffuse infiltration pathways.

Diclofenac (DCF) is one of the most widespread pharmaceutical compounds found in freshwaters as a pseudo-persistent pollutant due to its continuous release from point and diffuse sources, being its removal in Wastewater Treatment Plants incomplete. Moreover, DCF is particularly persistent in interstitial habitats and potentially toxic for the species that spend their whole life cycle among the same sediment grains. This study is aimed at offering a first contribution to the assessment of DCF effects on freshwater invertebrate species living in the interstitial habitats of springs, rivers, lakes and groundwaters. The Crustacea Copepoda are one of the main components of the freshwater interstitial communities, with the primacy taken by the worm-like and small-sized harpacticoids. A sub-lethal concentration of 50 μg L-1 DCF significantly affected six out of the eight behavior parameters of the burrower/interstitial crustacean harpacticoid Bryocamptus pygmaeus recorded by video tracking analysis. DCF exposure reduced swimming speed, swimming activity, exploration ability and thigmotaxis, and increased swimming path tortuosity. The biochemical approach revealed a reduced level of the mitochondrial superoxide dismutase 2 in individuals exposed to DCF. It could be explained by a decline in mitochondrial performance or by a reduced number of functional mitochondria. Since mitochondrial dysfunction may determine ATP reduction, it comes that less energy is produced for maintaining the cell functions of the DCF-exposed individuals. In addition, the increasing energy demand for the detoxification process further contributes to decrease the total energetic budget allocated for other physiological activities. These observations can explain the changes we have observed in the swimming behavior of the copepod B. pygmaeus.

In this study, we analyzed the structure of the stygobiotic copepod assemblages of an unconsolidated European aquifer (VO), in southern Italy, that has been subject to persistent nitrate contamination for over 15 years. To this end, we monitored 25 bores where groundwater was contaminated only by nitrate, and no other chemical pollutants were reported as being above detection limits from 2009 to 2014. We monitored these bores three times, namely in autumn 2014 and in spring and autumn 2015. We expected that the chronic exposure to high nitrate concentrations had a significant and evident impact on the stygobiotic copepod assemblages. Unexpectedly, the assemblages were highly diversified. The stygobiotic species richness (SSR) accounted 17 species, a value that exceeded the European mean value (SSR = 12 species). However, the species density was only 0.6 species/km(2), lower than the European mean value (= 1.6 species/km(2)). Moreover, the juvenile copepods were numerically less abundant than the adults and the biomass-abundance model showed signs of alteration of the structure of the copepod assemblages. This study highlighted that (i) nitrates, even at high concentrations, probably have a less severe impact on groundwater assemblages of unconsolidated aquifers than expected and (ii) the analysis of population traits and biomasses can detect signs of alteration of these assemblages that would, otherwise, not be visible from the analysis of the sole species richness and abundances.

The distribution patterns of stygobitic crustacean harpacticoids at the boundaries of three different groundwater habitat types in Europe were analysed through a GIS proximity analysis and fitted to exponential models. The results showed that the highest frequency of occurrences was recorded in aquifers in consolidated rocks, followed by the aquifers in unconsolidated sediments and, finally, by the practically non-aquiferous rocks. The majority of the stygobitic harpacticoid species were not able to disperse across the boundaries between two adjacent habitats, with 66% of the species occurring in a single habitat type. The species were not evenly distributed, and 35–69% of them occurred from 2 to 6 km to the boundaries, depending on the adjacent habitat types. The distribution patterns were shaped by features extrinsic to the species, such as the hydrogeological properties of the aquifers, and by species’ intrinsic characteristics such as the preference for a given habitat type and dispersal abilities. Most boundaries between adjacent habitat types resulted to be “breaches”, that is transmissive borders for stygobitic harpacticoids, while others were “impermeable walls”, that is absorptive borders. Our results suggest that conservation measures of groundwater harpacticoids should consider how species are distributed within the different groundwater habitat types and at their boundaries to ensure the preservation of species metapopulations within habitat patches and beyond them.

The weighted Groundwater Health Index (wGHI), introduced by Korbel and Hose (2017), is a multimetric, two-tiered framework for measuring and identifying the ecological status of groundwater ecosystems using biotic and abiotic indicators. The wGHI was conceived and tested in unconsolidated alluvial aquifers in Australia. In this study we applied and tested the index in European unconsolidated aquifers located in nitrate vulnerable zones (NVZ). A refinement of the wGHI was necessary to ensure the compliance with the requirements of the European Directives. We called the refined index wGHIN where N stands for nitrates. We tested the wGHIN in an unconsolidated aquifer (VO_EU_GWB) in the River Vomano catchment (central Italy) that was designated NVZ in 2005 and has since been subject to management measures pursuant to the Nitrate Directive. We also monitored a complex of minor confined unconsolidated aquifers (VO_CON_GWB) located in the same catchment. The wGHIN highlighted extensive nitrate contamination in both the VO_EU_GWB and VO_CON_GWB aquifers. Despite the widespread contamination, most of the monitoring sites showed only minor deviations from good ecological status. The index also highlighted the biodiversity of the aquifers which happened to be among the most diverse in Europe. The wGHIN proved to be theoretically sounding, concrete, numerical and easily understandable by the public and policy-makers. Finally, the index was economically sustainable. The wGHIN has certain limitations that have to be resolved, such as the low correlation of some indicators to the index overall score in the aquifers of the River Vomano and the fact that the index is not “operationally simple” since it requires taxonomic and ecological skills.

International audience; In this overview (introductory article to a special issue including 14 papers), we consider all main types of natural and artificial inland freshwater habitas (fwh). For each type, we identify the main biodiversity patterns and ecological features, human impacts on the system and environmental issues, and discuss ways to use this information to improve stewardship. Examples of selected key biodiversity/ecological features (habitat type): narrow endemics, sensitive (groundwater and GDEs); crenobionts, LIHRes (springs); unidirectional flow, nutrient spiraling (streams); naturally turbid, floodplains, large-bodied species (large rivers); depth-variation in benthic communities (lakes); endemism and diversity (ancient lakes); threatened, sensitive species (oxbow lakes, SWE); diverse, reduced littoral (reservoirs); cold-adapted species (Boreal and Arctic fwh); endemism, depauperate (Antarctic fwh); flood pulse, intermittent wetlands, biggest river basins (tropical fwh); variable hydrologic regime—periods of drying, flash floods (arid-climate fwh). Selected impacts: eutrophication and other pollution, hydrologic modifications, overexploitation, habitat destruction, invasive species, salinization. Climate change is a threat multiplier, and it is important to quantify resistance, resilience, and recovery to assess the strategic role of the different types of freshwater ecosystems and their value for biodiversity conservation. Effective conservation solutions are dependent on an understanding of connectivity between different freshwater ecosystems (including related terrestrial, coastal and marine systems).

Biodiversity hotspots are routinely identified by grid-based analyses, despite grids encompassing different habitats, thus hindering the potential to assess which habitat type accounts for the conservation priority assigned to a grid. In this study, we aimed at identifying the main hotspots for the conservation of the European stygobitic Crustacea Copepoda Harpacticoida at the groundwater habitat scale. A multi-metric approach was used, based on six biodiversity indicators: species richness, endemicity, evolutionary origin, phylogenetic rarity, taxonomic distinctness, habitat specificity. The Hot Spot Analysis, based on the statistics Getis-Ord Gi*, was used to compare the local to the global average values of each indicator to identify hotspots of conservation. The operational units used to perform the analyses were the groundwater habitat types, in order to gather all the possible patterns of spatial occupancy in terms of habitat variability. Eight biodiversity hotspots of stygobitic Crustacea Harpacticoida were highlighted: 1) the Pyrenees (Spain and France), 2) the Jura Massif (France), 3) the Alpine arc (France, Switzerland and Italy) embracing southward the River Po alluvial plain and the Slovenian External Dinarides, 4) the Central Apennines (Italy), 5) the Carpathian and Balkan mountains in Romania and at the boundary between western Bulgaria and north-west Macedonia, 6) the Dinaric Alps (from Croatia to Albania), 7) the Sardinia Island, 8) an area in central-northern Europe embracing Denmark, the Netherlands and Germany. The hotspots showed a clear spatial distribution in southern Europe where they were distributed predominantly south to the 45th parallel, in line to what reiteratively observed in previous studies. Many hotspots embraced more than one habitat type. The adoption of discrete groundwater habitat types as working spatial units rather than grids provided a higher resolution of where the stygobitic harpacticoid species effectively live, with the possibility of intervening more precisely to preserve them and their habitats.

We introduce a suite of software tools aimed at investigating multiple bio-ecological facets of aquatic Groundwater Dependent Ecosystems (GDEs). The suite focuses on: (1) threats posed by pollutants to GDE invertebrates (Ecological Risk, ER), (2) threats posed by hydrological and hydromorphological alterations on the subsurface zone of lotic systems and groundwater-fed springs (Hydrological-Hydromorphological Risk, HHR), and (3) the conservation priority of GDE communities (Groundwater Biodiversity Concern index, GBC). The ER is assessed by comparing tolerance limits of invertebrate species to specific pollutants with the maximum observed concentration of the same pollutants at the target site(s). Comparison is based on an original, comprehensive dataset including the most updated information on tolerance to 116 pollutants for 474 freshwater invertebrate species. The HHR is assessed by accounting for the main direct and indirect effects on both the hyporheic zone of lotic systems and groundwater-fed springs, and by scoring each impact according to the potential effect on subsurface invertebrates. Finally, the GBC index is computed on the basis of the taxonomical composition of a target community, and allows the evaluation of its conservation priority in comparison to others. The software suite is freely available at: http://app.aqualifeproject.eu by registered users.

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.

Groundwater communities residing in contaminated aquifers have been investigated mainly through taxonomy-based approaches (i.e., analyzing taxonomic richness and abundances) while ecological traits have been rarely considered. The aim of this study was to assess whether a trait analysis adds value to the traditional taxonomy-based biomonitoring in N-contaminated aquifers. To this end, we monitored 40 bores in the Vomano alluvial aquifer (VO_GWB, Italy) for two years. The aquifer is a nitrate vulnerable zone according to the Water Framework Directive. The traditional taxonomy-based approach revealed an unexpectedly high biodiversity (38 taxa and 5725 individuals), dominated by crustaceans, comparable to that of other unpolluted alluvial aquifers worldwide. This result is in contrast with previous studies and calls into question the sensitivity of stygobiotic species to N-compounds. The trait analysis provided an added value to the study, unveiling signs of impairments of the groundwater community such as low juveniles-to-adults and males-to-females ratios and a crossover of biomasses and abundances curves suggestive of an intermediate alteration of the copepod assemblages.

The ecological risk assessment (ERA) of chemical substances is based on the premise that the protection of the most sensitive taxon safeguards the overall community. Given the severe scarcity of ecotoxicological data concerning groundwater species, we felt urged to consider epigean model species’ sensitivity data to determine the safe pesticide concentrations for obligate groundwater dwelling species. To this end, we performed the ERA of pesticide mixtures occurring in eleven Mediterranean porous aquifers (Abruzzo region; central Italy). The evaluation was based on data collected between 2010 and 2015 by the environmental protection agency of Abruzzo region (ARTA Abruzzo) and included 42 pesticides and 1953 samples. We applied a step-wise procedure: we used the Measured Environmental Concentration (MEC); we estimated the Predicted No-Effect Concentration (PNEC) and we established the ecological risk as Risk Quotient (RQ) based on the ∑MECi/PNECi ratio following a concentration addition model for mixtures’ toxicity. we used the Measured Environmental Concentration (MEC); we estimated the Predicted No-Effect Concentration (PNEC) and we established the ecological risk as Risk Quotient (RQ) based on the ∑MECi/PNECi ratio following a concentration addition model for mixtures’ toxicity. The PNEC was regarded as the concentration below which a harmful effect will most likely not occur to the groundwater dwelling fauna. The toxicity data used to compute the PNEC values were obtained from the US. EPA ECOTOX database confining the search to epigean crustaceans. Missing toxicity data were estimated by ECOSAR v.1.11. PNEC values were calculated by dividing the lowest short-term L(E)C50 value (that refers to the concentration at which 50% of its maximal effect was observed in test species) by appropriate assessment factors (AF). The AF values were selected according to the difference in the sensitivities of groundwater and epigean crustaceans derived from the available studies. Groundwater crustacean species were generally less sensitive to acute exposure to chemicals than the model species Daphnia magna. However, they were more sensitive than their epigean relatives when the comparisons were made among organisms sharing the same family/order. This result suggests caution when inferring the sensitivity of groundwater species from that of epigean taxa. The ecological risk was scored using a binary ecological classification suggesting that appreciable risk is likely when RQ≥1. Pesticide mixture risks were often driven by a minimum of 2 to 11 compounds in the eleven aquifers of Abruzzo region. However, the risk-drivers (i.e., individual pesticides explaining the largest share of potential effects) differed substantially among the aquifers. The results of this study have been published by Di Lorenzo et al. (2018).

Groundwater drift collected continuously at perennial outlets of karst aquifers has been examined for its potential in assessing the spatial distribution of the resident species. To this end, we have continuously monitored the groundwater drifted at the Mazzoccolo spring (Western Aurunci karst aquifer, Central Italy) over 2 years. Concurrently to the biological monitoring, major ion geochemistry and turbidity were investigated on the same schedule. We found that the hydrochemistry did not govern the distribution pattern of the dominant taxon (Copepoda) into the Western Aurunci aquifer. In contrast, copepod drift showed clear differences in the number of individuals between the high and low water periods because of the "piston effect," which is very frequent in karst and fractured aquifers due to recharge from rainfalls. According to the results of this study, groundwater species do not inhabit all sectors of a karst aquifer. They avoid the fast-flowing conduits where groundwater rapidly flows, while colonizing the more inertial aquifer sectors, such as the slow-flowing conduits.

Species interactions between stygobites (obligate groundwater organisms) are poorly known, reflecting the difficulty in studying such organisms in their natural environments. Some insight can be gained from the study of the spatial variability in microcrustacean communities in groundwater-fed springs. Earthquakes can increase hydraulic conductivity in the recharge area of karstic aquifers and flow rates in discharge zones, thus dislodging stygobites from their original habitats to the spring outlets. Earthquakes are expected to alter species spatial niche overlap at the spring outlets, where stygobites coexist with non-stygobites living in benthic and subsurface habitats. We compared the abundance of stygobiotic and non-stygobiotic microcrustaceans in groundwater-fed springs before and after the 6.3-Mw earthquake that hit the karstic Gran Sasso Aquifer (Italy) in 2009. Pre-seismic (1997, 2005) overall niche overlaps were not different from null expectations, while post-seismic (2012) species mean niche overlaps were higher, following the redistribution of animals caused by the earthquake-triggered discharge. The reduced abundance of stygobites following their dislodgement from the aquifer and the concomitant displacement of non-stygobites led to a higher post-seismic co-occurrence of stygobites and non-stygobites. Changes in aquifer structure destroyed pre-seismic species segregation patterns by creating new or strengthening already existing interactions.

Lepidocharontidae Galassi & Bruce, fam. n. is erected, containing Lepidocharon Galassi & Bruce, gen. n. and two genera transferred from the family Microparasellidae Karaman, 1934: Microcharon Karaman, 1934 and Janinella Albuquerque, Boulanouar & Coineau, 2014. The genus Angeliera Chappuis & Delamare Deboutteville, 1952 is placed as genus incertae sedis in this family. The Lepidocharontidae is characterised by having rectangular or trapezoidal somites in dorsal view, a single free pleonite, a tendency to reduction of the coxal plates, and the unique uropodal morphology of a large and long uropodal protopod on which the slender uropodal exopod articulates separately and anteriorly to the endopod. Lepidocharon Galassi & Bruce, gen. n. has a 6-segmented antennula, a well-developed antennal scale (rudimentary exopod), long and slender pereiopods 1–7 directed outwards, coxal plates rudimentary, incorporated to the lateral side of the sternites, not discernible in dorsal view, the single pleonite narrower than pereionite 7, scale-like elements bordering the proximal part of male pleopod 1 on posterior side, and stylet-guiding grooves of male pleopod 1 which run parallel to the outer lateral margins of the same pleopod. Lepidocharon priapus Galassi & Bruce, sp. n., type species for the genus, and Lepidocharon lizardensis Galassi & Bruce, sp. n. are described from Lizard Island, northern Great Barrier Reef. The most similar genus is Microcharon, both genera sharing the same general organization of the male pleopods 1 and 2, topology and architecture of the stylet-guiding groove of male pleopod 1, morphology of female operculum, presence of 2 robust claws of different lengths on pereiopodal dactylus 1–7, not sexually dimorphic. Lepidocharon gen. n. differs from Microcharon in the shape of the pereionites, very reduced coxal plates, the presence of imbricate scale-like elements bordering the proximal postero-lateral margins of the male pleopod 1, and the topology of the pereiopods, which are ventro-laterally inserted and directed outwards in Lepidocharon gen. n. and dorso-laterally inserted and directed ventrally in Microcharon. Lepidocharon shares with the genus Janinella the morphology of the tergites and the reduced lacinia mobilis of the left mandible, but differs significantly from Janinella in having a well-developed antennal scale, very reduced coxal plates also in females bearing oostegites, the general morphology and spatial arrangement of the stylet-guiding groove of male pleopod 1 and the possession of a 6-segmented antennula. The family Microparasellidae is redefined as monotypic, the only genus being Microparasellus Karaman, 1933.

The Frasassi cave system in central Italy hosts one of the few known examples of a groundwater metazoan community that is supported by sulfur-based lithoautotrophic microbes. Despite the challenging conditions represented by high concentrations of H2S and low concentrations of O2, this cave system is home to many invertebrate species. Here, we analyzed the copepods inhabiting sulfidic lakes and non-sulfidic dripping pools in order to investigate how environmental conditions in sulfidic waters regulate the spatial distribution of the cave microcrustacean community over time. We also sampled copepod assemblages of sulfidic lakes under conditions of both high and low H2S concentration. Cluster analysis and canonical correspondence analysis separated the copepod assemblages inhabiting dripping pools from those of sulfidic lakes. H2S concentration, pH and O2 concentration were identified as the main factors regulating community structure. These results indicate that the distribution of groundwater copepods within the cave system is ecologically and spatially structured. Sulfidic lakes showed lower Simpson dominance, higher Shannon diversity and higher Pielou equitability at higher H2S concentrations. The complex community structure of the copepods of the Frasassi cave system suggests that a chemosynthetically produced food source facilitated the colonization of stygobionts in sulfidic groundwater due to their tolerance to the environmental conditions.

We investigated whether the equilibrium theory of island biogeography (ETIB) can be applied to the meiofauna of groundwater-fed springs. We tested whether copepod species richness was related with spring area, discharge, and elevation. Additionally, five hypotheses are tested based on species distribution patterns, dispersal ability, and life-history characteristics of several guilds (stygobiotic, nonstygobiotic, cold stenotherm, and noncold stenotherm species). Thirty springs in the central Apennines (Italy) were considered. A multimodel selection procedure was applied to select best-fit models using both ordinary least-squares regressions and autoregressive models. Mantel tests were used to investigate the impact of spatial autocorrelation in determining interspring similarity (ßsor), pure turnover (ßsim), intersite nestedness (ßnest = ßsor − ßsim), and matrix nestedness (measured using NODF and other metrics). Explicit consideration of spatial correlations reduced the importance of predictors of overall species richness, noncold stenotherm species (both negatively affected by elevation), cold stenotherm species, and nonstygobiotic species, but increased the importance of area for the stygobiotic species. We detected nested patterns in all cases, except for the stygobites. Interspring distances were positively correlated with ßsor and ßnest (but not with ßsim) for the entire data set and for nonstygobiotic, cold stenotherm, and noncold stenotherm species. In the case of stygobites, interspring geographical distances were marginally correlated with ßsor and no correlation was found for ßsim and ßnest. We found support for ETIB predictions about species richness, which was positively influenced by area and negatively by elevation (which expresses the size of source of immigrants). Low turnover and high nestedness are consistent with an equilibrium scenario mainly regulated by immigration and extinction. Stygobites, which include many distributional and evolutionary relicts, have a low capability to disperse through the aquifers and tend to be mainly confined to the springs where they drifted out and were trapped by springbed sediments. AQUALIFE project funded by the European Community (LIFE12 BIO/IT/000231 AQUALIFE) info:eu-repo/semantics/publishedVersion

In groundwater-fed springs, habitat characteristics are primarily determined by a complex combination of geomorphic features and physico-chemical parameters, while species assemblages are even more intricate. Springs host species either inhabiting the spring mouth, or colonizing spring habitats from the surface or from the aquifers which feed the springs. Groundwater species living in springs have been claimed as good candidates for identifying dual aquifer flowpaths or changes in groundwater pathways before reaching the spring outlets. However, the reliability of spring species as hydrological biotracers has not been widely investigated so far. Our study was aimed at analysing a large karstic spring system at three nested spatial scales in order: i) to assess, at whole spring system scale, the presence of a groundwater divide separating two aquifers feeding two spring units within a single spring system, by combining isotope analyses, physico-chemistry, and copepod distribution patterns; ii) to test, at vertical spring system scale, the effectiveness of copepods in discriminating surface and subsurface habitat patches within the complex mosaic spring environment; iii) to explore, at local spring unit level, the relative role of hydrochemistry and sediment texture as describers of copepod distribution among microhabitats. The results obtained demonstrated the presence of a hierarchical spatial structure, interestingly reflected in significant differences in assemblage compositions. Copepod assemblages differed between the two contiguous spring units, which were clearly characterized by their hydrochemistry and by significant differences in the groundwater flowpaths and recharge areas, as derived by the isotope analyses. The biological results suggested that stygobiotic species seem to be related to the origin of groundwater, suggesting their potential role as hydrological biotracers. At vertical scale, assemblage composition in surface and subsurface habitats was significantly different, both between spring units and among microhabitats, supporting strong habitat preferences of copepod species. At the smaller local scale, the response to habitat patchiness of subsurface copepod assemblages resulted in distribution patterns primarily defined by sediment texture, while the sensitivity to differences in hydrochemistry was negligible.

The family Phyllognathopodidae (Crustacea, Copepoda, Harpacticoida) is heavily affected by the floating taxonomic status of the type-genus Phyllognathopus. A revision of the different character states displayed by members of the family is presented, and new phylogenetically informative characters are described, enlarging the analysis to the remaining genera of the family, Parbatocamptus and Allophyllognathopus. Phyllognathopus viguieri (Maupas, 1892) and Parbatocamptus jochenmartensi Dumont and Maas, 1988 are redescribed in detail, and Phyllognathopus inexspectatus sp. n. is described from ground water in Italy. The new genus Neophyllognathopus is established to accommodate Phyllognathopus bassoti Rouch, 1972, originally collected from Long Island (Papua - New Guinea), and subsequently recorded also from the Bantayan Island (Philippines), and from the Indian subcontinent. The new genus is presently monotypic and is easily defined by the unique construction and morphology of leg 5 in both male and female, of male leg 6, and by the peculiar ornamentation of male third and fourth urosomites. Biogeographical and ecological considerations are presented for members of the family.

SUMMARY 1. The distribution patterns of stygobiotic invertebrates were examined with a stratified sampling design at 197 sites selected among four hydrogeographic basins in the Lessinian Massif (northern Italy). The sites were approximately evenly distributed among four hydrogeological zones: unsaturated and saturated zone of karstic aquifers, and hyporheic and saturated zone of porous aquifers. 2. Outlying Mean Index (OMI) analysis which assesses deviation of habitat conditions from reference conditions, was used to evaluate the importance of 14 selected environmental variables in shaping groundwater biodiversity patterns in the region (total of 89 stygobiotic species). The measured variables explained 80% of the variability in the data set. 3. Sampling sites were distributed along the environmental gradients defined by OMI analysis. Significant differences were detected between karstic and porous site, as well as among sites located in the four hydrogeological zones. Differences among the four hydrogeographic basins were not observed. 4. Ordination of stygobiotic species along the environmental gradients was best explained

The sensitivity of freshwater invertebrates to agricultural pollutants is supposed to increase with rising temperature due to global warming. The aim of this study was to measure the effect of temperature on the lethal toxicity of ammonia-N, the herbicide Imazamox and the mixture of the two chemicals, in the adults and the juveniles of a population of the copepod Eucyclops serrulatus. This is a widely distributed species found in surface waters, in transitional habitats between surface water and groundwater, and in genuine groundwater environments. We tested the sensitivity by short-term bioassays (96 h) at 15°C and 18°C, respectively. Our results highlighted the following: (1) increasing temperature affected the sensitivity of the adults to ammonia-N and of the juveniles to the mixture, all of which were more sensitive to its detrimental effects at 18°C; (2) the juvenile stages were more sensitive than the adults to all toxicants, and (3) for all combinations of chemicals and temperatures, the effects were synergistic and approximately one order of magnitude greater than those expected according to a concentration addition model when comparing the LC50 for each chemical in the mixture with the LC50s of chemicals individually assayed. Overall, in a context of global change, ammonia-N and mixtures of agricultural pollutants may affect the survival rate of species that spend a part or the whole life-cycle in the hyporheic habitat, with detrimental effects to biodiversity and ecosystem services provided by the hyporheic biota. Fil: di Lorenzo, Tiziana. Centre National de la Recherche Scientifique; Francia Fil: Di Marzio, Walter Dario. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cifoni, Marco. University of L’Aquila; Italia Fil: Fiasca, Barbara. University of L’Aquila; Italia Fil: Baratti, M.. Centre National de la Recherche Scientifique; Francia Fil: Sáenz, María Elena. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Galassi, Diana M. P.. University of L’Aquila; Italia

The European Union (EU) threshold values for $${\text{NH}}_{4}^{ + }$$ in groundwater range from a minimum of 0.084 mg l−1 to the maximum allowed of 5 mg l−1. The aim of our study was to determine whether these values are adequate to protect groundwater copepods in alluvial aquifers underlying intensive agriculture. To this end, we analyzed abiotic (including $${\text{NH}}_{4}^{ + }$$ concentration) and biological patterns (copepod assemblages) in an alluvial aquifer in an area of intensive agriculture. Groundwater was collected from pre-existing farmer-owned bores. Abiotic and biological patterns were not related to seasonal variation in agricultural practices, and pollutant concentrations were typically below the legal maximum threshold values. However, both abiotic and biological variables differed significantly between two groups of bores, with high and low $${\text{NH}}_{4}^{ + }$$ conditions based on separate ecotoxicological assays that set the $${\text{NH}}_{4}^{ + }$$ toxicity threshold for stygobiotic copepods at 2.5× below the current EU lowest legal threshold. In particular, phosphorus concentration was higher, and oxygen and copepod abundance and richness were lower in high $${\text{NH}}_{4}^{ + }$$ bores. Our results suggest that the present threshold value range for $${\text{NH}}_{4}^{ + }$$ adopted by EU Member States may not protect stygobiotic copepod assemblages in alluvial aquifers underlying densely cultivated areas.

International audience; Springs are amongst the most relevant Groundwater Dependent Ecosystems (GDEs) and are key research environments in freshwater ecology and biology. The strict dependency on ground water of surface spring biodiversity is widely recognized, whereas the biodiversity occurring below the spring is very poorly known. This study analyzes copepod assemblages in the subsurface habitat of a karstic rheo- limnocrenic spring in relation to seventeen environmental parameters. Subsurface copepod assemblages were sensitive to microspatial variation in habitat structure, and species distributions were mostly driven by groundwater flowpath and substratum type, resulting in biologically distinct limnocrenic and rheocrenic sectors at the spring system scale. Habitat patchiness was reflected in differences in the microdistribution of subsurface copepods, stygobiotic assemblages being more sensitive to the measured environmental gradients than non-stygobiotic ones. In spite of the apparent stability of spring environments, copepods, as a target group, performed well as de- scriptors of sediment texture and hydrodynamics, and may offer relevant information for a better understanding of the potential changes generated by anthropogenic disturbance on these ecosystems.

The Groundwater Dependent Ecosystems (GDEs) are those ecosystems which depend on groundwater quality and quantity, and are commonly ranked on the basis of the degree of dependency on ground water. This review is aimed at describing these ecosystems (with special emphasis on Subsurface Groundwater Dependent Ecosystems - SGDEs), and at assessing major threats of climate change and of synergistic factors which may alter the ecological functioning of these ecosystems. As primary productivity is missing in the SGDEs due to total darkness, communities are composed of bacteria, protists and animals (predominantly invertebrates). Climate change has detrimental effects on GDEs, along with groundwater withdrawal, excavations in the streambed, intensive agriculture with excessive use of fertilizers and pesticides, urban sewage, increasing pollution from illegal dumping of toxic substances. These activities determine loss of biodiversity and ecosystem services. Up to now the adaptation to climate change in GDEs has received poor attention, as groundwater quality and quantity need to be integrated with ecosystem ability to sustain groundwater biodiversity and related ecosystem services. Mitigation measures should consider period, duration and amount of ground water exploited in order to ensure, for alluvial aquifers, the contact between the surface water body and the underlying aquifer, for karstic aquifers, the maintenance of water saturation of the annexed capacitive subsystems.

Earthquakes are among the most destructive natural events. The 6 April 2009, 6.3-Mw earthquake in L’Aquila (Italy) markedly altered the karstic Gran Sasso Aquifer (GSA) hydrogeology and geochemistry. The GSA groundwater invertebrate community is mainly comprised of small-bodied, colourless, blind microcrustaceans. We compared abiotic and biotic data from two pre-earthquake and one post-earthquake complete but non-contiguous hydrological years to investigate the effects of the 2009 earthquake on the dominant copepod component of the obligate groundwater fauna. Our results suggest that the massive earthquake-induced aquifer strain biotriggered a flushing of groundwater fauna, with a dramatic decrease in subterranean species abundance. Population turnover rates appeared to have crashed, no longer replenishing the long-standing communities from aquifer fractures, and the aquifer became almost totally deprived of animal life. Groundwater communities are notorious for their low resilience. Therefore, any major disturbance that negatively impacts survival or reproduction may lead to local extinction of species, most of them being the only survivors of phylogenetic lineages extinct at the Earth surface. Given the ecological key role played by the subterranean fauna as decomposers of organic matter and ‘‘ecosystem engineers’’, we urge more detailed, long-term studies on the effect of major disturbances to groundwater ecosystems.

This paper examines the biodiversity patterns of natural springs across three large hydrogeological units of the central Apennines (central Italy). The biological dataset obtained was analysed by selecting the free-living Copepoda, the most species-rich group among the meiofaunal components of the springs in the study area. The analyses performed focused on distribution patterns and the prevailing factors that may determine such distributions at the scale of the spring site as well as at the wider, hydrogeological scale of the entire region. The taxonomic distinctness of the communities found was also evaluated, in order to integrate the mere species counts with additional information on the relatedness of the taxa in copepod assemblages, with the aim of obtaining a more exhaustive assessment of the aspects of the biodiversity found. Copepod diversity in the various springs did not result in an evenly distributed pattern among spring sites and the larger hydrogeological units. Low-altitude karstic springs were the most diverse in terms of species richness, and showed the highest values of taxonomic distinctness measured for the whole area. Conversely, high-altitude springs from geological formations of both sandstone and limestone were species-poor, and with relatively similar copepod assemblages.The observed differences in composition of the communities among springs and hydrogeological units are discussed under the perspective of the potential of planning freshwater conservation measures.