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.

Some like it hot: Thermal preference of the groundwater amphipod Niphargus longicaudatus (Costa, 1851) and climate change implications.

Groundwater is a crucial resource for humans and the environment, but its global human demand currently exceeds available volumes by 3.5 times. Climate change is expected to exacerbate this situation by increasing the frequency of droughts along with human impacts on groundwater ecosystems. Despite prior research on the quantitative effects of climate change on groundwater, the direct impacts on groundwater biodiversity, especially obligate groundwater species, remain largely unexplored. Therefore, investigating the potential impacts of climate change, including groundwater temperature changes, is crucial for the survival of obligate groundwater species. This study aimed to determine the thermal niche breadth of the crustacean amphipod species Niphargus longicaudatus by using the chronic method. We found that N. longicaudatus has a wide thermal niche with a natural performance range of 7-9 °C, which corresponds to the thermal regime this species experiences within its distribution range in Italy. The observed range of preferred temperature (PT) was different from the mean annual temperature of the sites from which the species has been collected, challenging the idea that groundwater species are only adapted to narrow temperature ranges. Considering the significant threats of climate change to groundwater ecosystems, these findings provide crucial information for the conservation of obligate groundwater species, suggesting that some of them may be more resilient to temperature changes than previously thought. Understanding the fundamental thermal niche of these species can inform conservation efforts and management strategies to protect groundwater ecosystems and their communities.

Effects of pH, temperature and salinity on P3HB synthesis culturing the marine Rhodovulum sulfidophilum DSM-1374.

Rhodovulum sulfidophilum DSM-1374 is a potential producer of polyester when growing in phototrophic conditions. The present study investigated on a polyester product (P3HB) by culturing Rhodovulum sulfidophilum DSM-1374 in two different photobioreactors (PBR-1 and PBR-2) both with 4-L working volumes. PBR-1 is equipped with an internal rotor having 4 paddles to mix the bacterial culture while PBR-2 has an internal coil-shaped rotor. After selecting PBR-1, which best performed in the preliminary experiment, the effect of different stressing growth conditions as pH (7.0, 8.0, and 9.0), temperature (25, 30, and 35 °C), and medium salinity (1.5, 2.5, 3.5, and 4.5%) were tested. When the pH of the culture was set to 8.0, the capability of the bacterium to synthetize the polyester increased significantly reaching a concentration of 412 mg (P3HB)/L; the increase of the pH at 9.0 caused a reduction of the P3HB concentration in the culture. The medium salinity of 4.5% was the best stress-growth condition to reach the highest concentration of polyester in the culture (820 ± 50 mg (P3HB)/L) with a P3HB mass fraction in the dry biomass of 33 ± 1.5%. Stresses caused by culture temperature are another potential parameter that could increase the synthesis of P3HB.

The ecotoxicity of binary mixtures of Imazamox and ionized ammonia on freshwater copepods: Implications for environmental risk assessment in groundwater bodies.

Groundwater bodies are impacted by substances such as pesticides and N-fertilizers, which usually occur in the environment as complex mixtures rather than isolated pollutants. The threat that these mixtures pose to groundwater-dwelling organisms is still poorly understood. The aims of the present study were to test the acute effect of a binary mixture of a herbicide (Imazamox) and NH4+ on epigean (Eucyclops serrulatus) and hypogean (Diacyclops belgicus) freshwater copepod species. In addition, to evaluate if the effect of the mixture can be explained by referencing non-interaction models or by more complex interaction models; and the implications for groundwater risk assessment. Compared with the action of the compounds evaluated separately, the effects of Imazamox and NH4+ in the binary mixture were more than additive or synergistic for both species. MixTox models evidenced a dose ratio and dose level deviations from concentration addition and independent action traditional models. The hypogean species was three times more sensitive to NH4+ that the epigean species when assayed as a single chemical. However, D. belgicus was only 1.13 times more sensitive than E. serrulatus when NH4+ was assayed in the mixture. The use of an integrated approach for substances that are known to interact in groundwater, should include copepods species as test organisms.

Environmental risk of diclofenac in European groundwaters and implications for environmental quality standards.

Groundwater harbours unique species adapted to perpetual darkness. Groundwater fauna plays a crucial role in global ecosystem services, but contamination poses a threat to this keystone ecosystem. Diclofenac is a common non-steroidal anti-inflammatory drug of particular concern, due to its presence in both surface and groundwater. We assess the environmental risk of diclofenac in European groundwaters using different scenarios, analyzing Measured Environmental Concentrations (MECs) of diclofenac and estimating the Predicted No Effect Concentration (PNECs) through two approaches: considering the sensitivity of the groundwater crustacean Proasellus lusitanicus (Isopoda: Asellidae), and using surface water species as proxies. Our results show that scenarios based on surrogate species predict that groundwater ecosystems are at risk due to diclofenac contamination. On the other hand, the MECs of diclofenac were consistently lower than the PNEC of P. lusitanicus, suggesting that the current MECs do not pose a significant threat to this groundwater-adapted species. However, risk scenarios differ considering the sensitivity of other groundwater species, emphasizing the importance of considering multiple species' sensitivities in risk assessment. Therefore, we recommend establishing an environmental quality standard for diclofenac in groundwater at 5 ng/L, a value that accounts the need for precautionary measures to safeguard groundwater ecosystems, essential for preserving their unique biota and services.

Subchronic Effects of Tetrachloroethylene on Two Freshwater Copepod Species: Implications for Groundwater Risk Assessment.

Aliphatic chlorinated hydrocarbons, notably tetrachloroethylene (also known as perchloroethylene [PCE]), are persistent, mobile, and toxic (PMT) and/or very persistent, mobile, and toxic (vPMT) groundwater pollutants, often exceeding safe drinking water thresholds. The present study delves into the groundwater risk assessment of PCE with a novel focus on the sensitivity of stygobitic species-organisms uniquely adapted to groundwater environments. Through a comparative analysis of the subchronic effects of PCE on the locomotion behavior of two copepod species, the stygobitic Moraria sp. and the nonstygobitic Bryocamptus zschokkei, we highlighted the inadequacy of the current European predicted-no-effect concentration of PCE for groundwater ecosystems. Our findings indicate significant behavioral impairments in both species at a concentration (32 ng/L PCE) well below the threshold deemed safe, suggesting that the current European guidelines for groundwater risk assessment may not adequately protect the unique biodiversity of groundwater habitats. Importantly, B. zschokkei demonstrated sensitivity to PCE comparable to or greater than that of the target stygobitic species, suggesting its utility as a substitute species in groundwater risk assessment. The present study adds to the limited research on the ecotoxicological sensitivity of groundwater species to PMT/vPMT chemicals and highlights the need for refined groundwater risk-assessment methodologies that consider the susceptibilities of stygobitic species. Environ Toxicol Chem 2024;00:1-13. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Fire up Biosensor Technology to Assess the Vitality of Trees after Wildfires.

The development of tools to quickly identify the fate of damaged trees after a stress event such as a wildfire is of great importance. In this context, an innovative approach to assess irreversible physiological damage in trees could help to support the planning of management decisions for disturbed sites to restore biodiversity, protect the environment and understand the adaptations of ecosystem functionality. The vitality of trees can be estimated by several physiological indicators, such as cambium activity and the amount of starch and soluble sugars, while the accumulation of ethanol in the cambial cells and phloem is considered an alarm sign of cell death. However, their determination requires time-consuming laboratory protocols, making the approach impractical in the field. Biosensors hold considerable promise for substantially advancing this field. The general objective of this review is to define a system for quantifying the plant vitality in forest areas exposed to fire. This review describes recent electrochemical biosensors that can detect plant molecules, focusing on biosensors for glucose, fructose, and ethanol as indicators of tree vitality.

Perspectives and pitfalls in preserving subterranean biodiversity through protected areas.

Subterranean ecosystems (comprising terrestrial, semi-aquatic, and aquatic components) are increasingly threatened by human activities; however, the current network of surface-protected areas is inadequate to safeguard subterranean biodiversity. Establishing protected areas for subterranean ecosystems is challenging. First, there are technical obstacles in mapping three-dimensional ecosystems with uncertain boundaries. Second, the rarity and endemism of subterranean organisms, combined with a scarcity of taxonomists, delays the accumulation of essential biodiversity knowledge. Third, establishing agreements to preserve subterranean ecosystems requires collaboration among multiple actors with often competing interests. This perspective addresses the challenges of preserving subterranean biodiversity through protected areas. Even in the face of uncertainties, we suggest it is both timely and critical to assess general criteria for subterranean biodiversity protection and implement them based on precautionary principles. To this end, we examine the current status of European protected areas and discuss solutions to improve their coverage of subterranean ecosystems.

Acetaminophen induced antioxidant and detoxification responses in a stygobitic crustacean.

A variety of veterinary and human medicinal products (VHMPs) are found in groundwater, an often-neglected habitat inhabited by species with unique traits, stygobitic species. It is crucial to understand the effect of VHMPs on stygobitic species because they may respond differently to stressors than surface species. Our hypothesis is that groundwater species may be more susceptible to environmental contaminants due to less plasticity in their detoxification response and acquisition of energy because subterranean habitats are more stable and isolated from anthropogenic activities. We performed a battery of biomarkers associated with important physiological functions on the stygobitic asellid crustacean Proasellus lusitanicus, after a 14-day exposure to acetaminophen, a commonly used pharmaceutical and pollutant of groundwaters. Our results show a decrease in total glutathione levels and an increase in glutathione S-transferase activity, suggesting a successful detoxification response. This helps explaining why acetaminophen did not cause oxidative damage, as well as had no effect on cholinesterase activity nor in aerobic production of energy. This study shows the remarkable capacity of P. lusitanicus to tolerate sublethal concentrations of VHMP acetaminophen. Most ecotoxicological studies on stygobitic species focused on the lethal effects of these compounds. The present study focuses on consequences at sublethal concentrations. Future studies should assess the stress levels induced to better predict and estimate the impacts of contaminants on groundwater ecosystems.

Ingestion of microplastics and textile cellulose particles by some meiofaunal taxa of an urban stream.

Microplastics (MPs) and textile cellulose are globally pervasive pollutants in freshwater. In-situ studies assessing the ingestion of MPs by freshwater meiofauna are few. Here, we evaluated MP and textile cellulose ingestion by some meiofaunal taxa and functional guilds of a first-order stream in the city of Florence (Italy) by using a tandem microscopy approach (fluorescence microscopy and µFTIR). The study targeted five taxa (nematodes, oligochaetes, copepods, ephemeropterans and chironomids), three feeding (scrapers, deposit-feeders, and predators), and three locomotion (crawlers, burrowers, and swimmers) guilds. Fluorescent particles related to both MPs and textile cellulose resulted in high numbers in all taxa and functional guilds. We found the highest number of particles in nematodes (5200 particles/ind.) and deposit-feeders (1693 particles/ind.). Oligochaetes and chironomids (burrowers) ingested the largest particles (medium length: 28 and 48 µm, respectively), whereas deposit-feeders ingested larger particles (medium length: 26 µm) than scrapers and predators. Pellets were abundant in all taxa, except for Chironomidae. Textile cellulose fibers were present in all taxa and functional guilds, while MP polymers (EVA, PET, PA, PE, PE-PP) differed among taxa and functional guilds. In detail: EVA and PET particles were found only in chironomids, PE particles occurred in chironomids, copepods and ephemeropterans, PA particles were found in all taxa except in nematodes, whereas particles made of PE-PP blend occurred in oligochaetes and copepods. Burrowers and deposit-feeders ingested EVA, PET, PA, PE and PE-PP, while crawlers and scrapers ingested PE and PA. Swimmers and predators ingested PE, PA and PE-PP. Our findings suggest a pervasive level of plastic and textile cellulose pollution consistent with an urban stream which propagates in the meiofaunal assemblage of the stream ecosystem.

Thermal acclimation and metabolic scaling of a groundwater asellid in the climate change scenario.

Metabolic rate has long been used in animal adaptation and performance studies, and individual oxygen consumption is used as proxy of metabolic rate. Stygofauna are organisms adapted to groundwater with presumably lower metabolic rates than their surface relatives. How stygofauna will cope with global temperature increase remains unpredictable. We studied the thermal acclimation and metabolic scaling with body mass of a stygobitic crustacean, Proasellus lusitanicus, in the climate change scenario. We measured oxygen consumption rates in a thermal ramp-up experiment over four assay temperatures and tested two hypotheses: (i) P. lusitanicus exhibits narrow thermal plasticity, inadequate for coping with a fast-increasing thermal regime; and (ii) oxygen consumption rates scale with the body mass by a factor close to 0.75, as commonly observed in other animals. Our results show that P. lusitanicus has low thermal plasticity in a fast-increasing thermal regime. Our data also suggest that oxygen consumption rates of this species do not follow mass-dependent scaling, potentially representing a new trait of metabolic optimization in groundwater habitats, which are often limited in food and oxygen. Species with limited dispersal capacities and rigid metabolic guilds face extinction risk due to climate change and omitting groundwater ecosystems from climate change agendas emphasizes the unprotected status of stygofauna.

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.

The impact of nitrate on the groundwater assemblages of European unconsolidated aquifers is likely less severe than expected.

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/km2, lower than the European mean value (= 1.6 species/km2). 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.

Effects of diclofenac on the swimming behavior and antioxidant enzyme activities of the freshwater interstitial crustacean Bryocamptus pygmaeus (Crustacea, Harpacticoida).

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.

Spatial distribution of stygobitic crustacean harpacticoids at the boundaries of groundwater habitat types in Europe.

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.

Poly-3-hydroxybutyrate and H2 production by Rhodopseudomonas sp. S16-VOGS3 grown in a new generation photobioreactor under single or combined nutrient deficiency.

The main goal of this investigation was setting up a growth strategy to separate H2 evolution from P3HB synthesis in order to increase cumulative P3HB in Rhodopseudomonas cells. The accumulation of poly-3-hydroxybutyrate (P3HB) was investigated culturing Rhodopseudomonas sp. S16-VOGS3 with three carbon substrates either as acetate, butyrate or lactate and with two nitrogen sources either as ammonium or glutamate. The investigation was carried out under several stress conditions caused by single or double nutrient deficiency. The content of P3HB in cell dry weight (CDW) was 21.8% with lactate; 24.6% with acetate and 27.6% with butyrate under sulfur deficient conditions. The P3HB content increased significantly culturing Rhodopseudomonas sp. S16-VOGS3 with butyrate following three phases of growth: phase-1, nutrient sufficient conditions; phase-2, nitrogen-deficiency and phase-3, sulfur-deficient conditions. Under this last phase, the highest P3HB content was achieved (34.4% of CDW). A combined production of P3HB and molecular H2 was obtained when Rhodopseudomonas sp. S16-VOGS3 was cultured with either acetate or butyrate under nitrogen sufficiency (glutamate) or nitrogen deficiency.

Whey and molasses as inexpensive raw materials for parallel production of biohydrogen and polyesters via a two-stage bioprocess: New routes towards a circular bioeconomy.

Consecutive dark-fermentation and photo-fermentation stages were investigated for a profitable circular bio-economy. H2 photo-production versus poly(3-hydroxybutyrate) (P3HB) accumulation is a modern biotechnological approach to use agro-food industrial byproducts as no-cost rich-nutrient medium in eco-sustainable biological processes. Whey and molasses are very important byproducts rich in nutrients that lactic acid bacteria can convert, by dark-fermentation, in dark fermented effluents of whey (DFEW) and molasses (DFEM). These effluents are proper media for culturing purple non-sulfur bacteria, which are profitable producers of P3HB and H2. The results of the present study show that Lactobacillus sp. and Rhodopseudomonas sp. S16-VOGS3 are two representative genera for mitigation of environmental impact. The highest productivity of P3HB (4.445 mg/(L·h)) was achieved culturing Rhodopseudomonas sp. S16-VOGS3, when feeding the bacterium with 20% of DFEM; the highest H2 production rate of 4.46 mL/(L·h) was achieved when feeding the bacterium with 30% of DFEM.

Environmental risk assessment of propranolol in the groundwater bodies of Europe.

A growing concern for contamination due to pharmaceutical compounds in groundwater is expanding globally. The ß-blocker propranolol is a ß-adrenoceptors antagonist commonly detected in European groundwater bodies. The effect of propranolol on stygobiotic species (obligate groundwater dweller species) is compelling in the framework of environmental risk assessment (ERA) of groundwater ecosystems. In fact, in Europe, ERA procedures for pharmaceuticals in groundwater are based on data obtained with surrogate surface water species. The use of surrogates has aroused some concern in the scientific arena since the first ERA guideline for groundwater was issued. We performed an ecotoxicological and a behavioural experiment with the stygobiotic crustacean species Diacyclops belgicus (Copepopda) to estimate a realistic value of the Predicted No Effect Concentration (PNEC) of propranolol for groundwater ecosystems and we compared this value with the PNEC estimated based on EU ERA procedures. The results of this study showed that i) presently, propranolol does not pose a risk to groundwater bodies in Europe at the concentrations shown in this study and ii) the PNEC of propranolol estimated through the EU ERA procedures is very conservative and allows to adequately protect these delicate ecosystems and their dwelling fauna. The methodological approach and the results of this study represent a first contribution to the improvement of ERA of groundwater ecosystems.

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.

The role of freshwater copepods in the environmental risk assessment of caffeine and propranolol mixtures in the surface water bodies of Spain.

In this study we aimed at assessing: (i) the environmental risk posed by mixtures of caffeine and propranolol to the freshwater ecosystems of Spain; (ii) the sensitivity of freshwater copepod species to the two compounds; (iii) if the toxicity of caffeine and propranolol to freshwater copepods contributes to the environmental risk posed by the two compounds in the freshwater bodies of Spain. The environmental risk was computed as the ratio of MECs (i.e. the measured environmental concentrations) to PNECs (i.e. the respective predicted no-effect concentrations). The effects of caffeine and propranolol on the freshwater cyclopoid Diacyclops crassicaudis crassicaudis were tested both individually and in binary mixtures. Propranolol posed an environmental risk in some but not in all the surface water ecosystems of Spain investigated in this study, while caffeine posed an environmental risk to all the investigated freshwater bodies, both as single compound and in the mixture with propranolol. Propranolol was the most toxic compound to D. crassicaudis crassicaudis, while caffeine was non-toxic to this species. The CA model predicted the toxicity of the propranolol and caffeine mixture for this species. D. crassicaudis crassicaudis was much less sensitive than several other aquatic species to both compounds. The sensitivity of D. crassicaudis crassicaudis does not increase the environmental risk posed by the two compounds in the freshwater bodies of Spain, however, further testing is recommended since the effect of toxicants on freshwater copepods can be more pronounced under multiple stressors and temperature increasing due to climate change.