Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.

Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.

Transnasal transsphenoidal pituitary surgery in a large tertiary hospital, a retrospective study.

OBJECTIVES: Pituitary adenomas (PAs), although being small tumours, can have quite an impact on patients' lives causing hormonal and visual disturbances, for which surgery must be performed. As a large peripheral hospital with specialists in pituitary surgery, an assessment of the efficacy and safety of transnasal transsphenoidal pituitary surgery was made. METHODS: A retrospective analysis of neurosurgical reports as well as pre and postoperative imaging was made to evaluate the presenting symptoms, tumoural variables, peri-operative morbidity, and long-term outcome. RESULTS: This cohort included 105 patients who were operated for PAs over a 9-year period, with a slight male predominance. Adenomas had a mean maximum diameter of almost 25 mm, with one-third of tumours presenting with a Knosp-grade 3 or 4. As expected, most patients presented with either visual (32.4%) or hormonal (40.0%) disturbances. After surgery, 85.3% had complete resolution of visual deficits, and 97.1% had normalisation of hormonal hypersecretion. Postoperative hormonal insufficiency requiring substitution was observed in 43.1% and was significantly more frequent in males and in non-functioning pituitary adenomas (NFAs). Postoperative cerebrospinal fluid (CSF) leakage was observed in 2.9%, and merely one patient developed meningitis. Tumour recurrence was significantly more frequent in patients with partial resection as compared to complete resection (25.6 vs. 7.9%). CONCLUSIONS: This study demonstrates that transnasal transsphenoidal pituitary surgery can be performed safely and effectively in a large non-university hospital, improving visual and/or hormonal disturbances as well as providing long-term tumour control. Patients with larger adenomas are at an increased risk to develop postoperative hypopituitarism.

Three-Dimensional Reconstruction of the Tissue-Specific Multielemental Distribution within Ceriodaphnia dubia via Multimodal Registration Using Laser Ablation ICP-Mass Spectrometry and X-ray Spectroscopic Techniques.

In this work, the three-dimensional elemental distribution profile within the freshwater crustacean Ceriodaphnia dubia was constructed at a spatial resolution down to 5 µm via a data fusion approach employing state-of-the-art laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) and laboratory-based absorption microcomputed tomography (µ-CT). C. dubia was exposed to elevated Cu, Ni, and Zn concentrations, chemically fixed, dehydrated, stained, and embedded, prior to µ-CT analysis. Subsequently, the sample was cut into 5 µm thin sections that were subjected to LA-ICP-TOFMS imaging. Multimodal image registration was performed to spatially align the 2D LA-ICP-TOFMS images relative to the corresponding slices of the 3D µ-CT reconstruction. Mass channels corresponding to the isotopes of a single element were merged to improve the signal-to-noise ratios within the elemental images. In order to aid the visual interpretation of the data, LA-ICP-TOFMS data were projected onto the µ-CT voxels representing tissue. Additionally, the image resolution and elemental sensitivity were compared to those obtained with synchrotron radiation based 3D confocal µ-X-ray fluorescence imaging upon a chemically fixed and air-dried C. dubia specimen.

Systematic Evaluation of Chronic Metal-Mixture Toxicity to Three Species and Implications for Risk Assessment.

Metal contamination generally occurs as mixtures. However, it is yet unresolved how to address metal mixtures in risk assessment. Therefore, using consistent methodologies, we have set up experiments to identify which mixture model applies best at low-level effects, i.e., the independent action (IA) or concentration addition (CA) reference model. The toxicity of metal mixtures (Ni, Zn, Cu, Cd, and Pb) to Daphnia magna, Ceriodaphnia dubia, and Hordeum vulgare was investigated in different waters or soils, totaling 30 different experiments. Some mixtures of different metals, each individually causing <10% inhibition, yielded much larger inhibition (up to 66%) when dosed in combination. In general, IA was most accurate in predicting mixture toxicity, while CA was the most conservative. At low-effect levels important in risk assessments, CA overestimated mixture toxicity to daphnids and H. vulgare, on average, with a factor 1.4 to 3.6. Observed mixture interactions could be related to bioavailability or by competition interactions, either for binding sites of dissolved organic carbon or for biotic ligand sites. Our study suggests that the current metal-by-metal approach in risk evaluations may not be conservative enough for metal mixtures.

Updated Chronic Copper Bioavailability Models for Invertebrates and Algae.

Chronic copper (Cu) bioavailability models have been successfully implemented in European risk assessment frameworks and compliance evaluations. However, they were developed almost two decades ago, which calls for an update. In the study, we present updated chronic Cu bioavailability models for invertebrates and algae. They consider recent ecotoxicity data sets and use the more recent speciation model Windermere Humic Aqueous Model (WHAM) VII and an optimized model structure (i.e., a generalized bioavailability model [gBAM]). Contrary to the classic biotic ligand model, a gBAM models the effect of pH on Cu2+ toxicity via a log-linear relationship parametrized through the pH slope SpH . The recalibrated SpH parameters are -0.208 for invertebrates (Daphnia magna, two clones) and -0.975 for algae (Raphidocelis subcapitata and Chlorella vulgaris). The updated models predict 80% to 100% of the observed effect levels for eight different species within a factor of 2. The only exception was one of the two data sets considering subchronic 7-day mortality to Hyalella azteca: the prediction performance of the updated invertebrate model at pH ≥ 8.3 was poor because the effect of pH on Cu2+ toxicity appeared to be dependent on the pH itself (with a steeper pH slope compared with the updated invertebrate model at pH ≥ 8.1). The prediction performance of the updated Cu bioavailability models was similar to or better than that of the models used for regulatory application in Europe until now, with one exception (i.e., H. azteca). Together with the recently published fish bioavailability model, the models developed in the present study constitute a complete, updated, and consistent bioavailability model set. Overall, the updated chronic Cu bioavailability model set is robust and can be used in regulatory applications. The updated bioavailability model set is currently used under the European Union Registration, Evaluation, Authorisation, and Restriction of Chemicals framework regulation to guide the safe use of Cu. Environ Toxicol Chem 2024;43:450-467. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Ecotoxicity of Lead to a Phytoplankton Community: Effects of pH and Phosphorus Addition and Implications for Risk Assessment.

Ecological risk assessment and water quality criteria for lead (Pb) are increasingly making use of bioavailability-based approaches to account for the impact of toxicity-modifying factors, such as pH and dissolved organic carbon. For phytoplankton, which are among the most Pb-sensitive freshwater species, a Pb bioavailability model has previously been developed based on standard single-species exposures at a high phosphorus (P) concentration and pH range of 6.0 to 8.0. It is well known that P can affect metal toxicity to phytoplankton and that the pH of many surface waters can be above 8.0. We aimed to test whether the single-species bioavailability model for Pb could predict the influence of pH on Pb toxicity to a phytoplankton community at both low and high P supply. A 10-species phytoplankton community was exposed to Pb for 28 days at two different pH levels (7.2 and 8.4) and two different P supply levels (low and high, i.e., total P input 10 and 100 µg/L, respectively) in a full factorial 2 × 2 test design. We found that the effects of total Pb on three community-level endpoints (biodiversity, community functioning, and community structure) were highly dependent on both pH and P supply. Consistent lowest-observed-effect concentrations (LOECs) ranged between 21 and >196 µg total Pb/L and between 10 and >69 µg filtered Pb/L. Long-term LOECs were generally higher, that is, 69 µg total Pb/L (42 µg filtered Pb/L) or greater, across all endpoints and conditions, indicating recovery near the end of the exposure period, and suggesting the occurrence of acclimation to Pb and/or functional redundancy. The highest toxicity of Pb for all endpoints was observed in the pH 7.2 × low P treatment, whereas the pH 8.4 × low P and pH 8.4 × high P treatment were the least sensitive treatments. At the pH 7.2 × high P treatment, the algal community showed an intermediate Pb sensitivity. The effect of pH on the toxicity of filtered Pb could not be precisely quantified because for many endpoints no effect was observed at the highest Pb concentration tested. However, the long-term LOECs (filtered Pb) at low P supply suggest a decrease in Pb toxicity of at least 1.6-fold from pH 7.2 to 8.4, whereas the single-species algal bioavailability model predicted a 2.5-fold increase. This finding suggests that bioavailability effects of pH on Pb toxicity cannot be extrapolated as such from the single species to the community level. Overall, our data indicate that, although the single-species algal Pb bioavailability model may not capture pH effects on Pb ecotoxicity in multispecies systems, the bioavailability-based hazardous concentration for 5% of the species was protective of long-term Pb effects on the structure, function, and diversity of a phytoplankton community in a relevant range of pH and P conditions. Environ Toxicol Chem 2023;42:2684-2700. © 2023 SETAC.

European freshwater silver monitoring data do not suggest a potential European-wide risk.

European legislations frequently focus on substances that are of potential concern to human or environmental health, such as "priority substances" under the Water Framework Directive 2000/60/EC ("WFD") that are identified as substances posing a significant risk to or via the aquatic environment. The EU REACH regulation also requires the assessment of the environmental risks of chemicals put on the EU market. To properly assess the potential risk of a substance, high-quality representative monitoring data should be compared with a safe threshold concentration. The objective of this article is to evaluate different publicly available freshwater monitoring data sets for silver and investigate them for a potential European-wide risk according to the methodology used by the European Commission. Most available silver monitoring data sets contain a large proportion of undetected samples with a reported concentration below the limit of quantification (LOQ) of the analytical technique, leading to considerable uncertainty in the data set. For silver, this LOQ is often at or above the safe threshold concentration, and the method used to handle undetected samples during the data processing considerably impacts the data assessment. We demonstrate that for large data sets covering many European countries (and often a wide range of LOQs), the uncertainty in the data set does not allow us to make a general conclusion about European-wide risk. However, by examining the data sets in more detail and assessing three additional country-specific monitoring data sets, we show that silver does not pose a risk to the freshwater environment in several countries. We conclude that the available data sets need careful assessment to account for the values that are below the LOQ, and that there is currently no reliable evidence indicating a European-wide risk for silver in the aquatic environment, meaning it should not be selected as  priority substance under the WFD. Integr Environ Assess Manag 2023;19:1110-1119. © 2022 European Precious Metals Federation. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Updating the Chronic Freshwater Ecotoxicity Database and Biotic Ligand Model for Nickel for Regulatory Applications in Europe.

Bioavailability has been taken into account in the regulation of nickel in freshwater ecosystems in Europe for over a decade; during that time a significant amount of new information has become available covering both the sensitivity of aquatic organisms to nickel toxicity and bioavailability normalization. The ecotoxicity database for chronic nickel toxicity to freshwater organisms has been updated and now includes 358 individual data points covering a total of 53 different species, all of which are suitable for bioavailability normalization to different water chemistry conditions. The bioavailability normalization procedure has also been updated to include updates to the bioavailability models that enable more sensitive water chemistry conditions to be covered by the model predictions. The updated database and bioavailability normalization procedure are applicable to more than 95% of regulated European surface water conditions and have been used to calculate site-specific criteria for a variety of different water chemistry scenarios, to provide an indication of how the sensitivity to nickel varies between different water types. The hazardous concentration for 5% of a species (HC5) values for this diverse selection of water types range from 1.6 to 36 µg L-1 , clearly demonstrating the importance of accounting for nickel bioavailability in freshwaters. This updated database and bioavailability normalization procedure provide a robust basis for the derivation of regulatory thresholds for chronic nickel toxicity in freshwaters such as predicted no-effect concentrations and Environmental Quality Standards and are protective of the results of several mesocosm studies. Environ Toxicol Chem 2023;42:566-580. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Assessment of the contribution of surfactants to mixture toxicity in French surface waters.

Surfactants are widely used 'down-the-drain' chemicals with the potential to occur at high concentrations in local water bodies and to be part of unintentional environmental mixtures. Recently, increased regulatory focus has been placed on the impacts of complex mixtures in aquatic environments and the substances that are likely to drive mixture risk. This study assessed the contribution of surfactants to the total mixture pressure in freshwater ecosystems. Environmental concentrations, collated from existing French monitoring data, were combined with estimated ecotoxicological thresholds to calculate hazard quotients (HQ) for each substance, and hazard indices (HI) for each mixture. Two scenarios were investigated to correct for concentrations below the limit of quantification (LOQ) in the dataset. The first (best-case) scenario assumed all values

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Decoding the activated stem cell phenotype of the neonatally maturing pituitary.

The pituitary represents the endocrine master regulator. In mouse, the gland undergoes active maturation immediately after birth. Here, we in detail portrayed the stem cell compartment of neonatal pituitary. Single-cell RNA-sequencing pictured an active gland, revealing proliferative stem as well as hormonal (progenitor) cell populations. The stem cell pool displayed a hybrid epithelial/mesenchymal phenotype, characteristic of development-involved tissue stem cells. Organoid culturing recapitulated the stem cells' phenotype, interestingly also reproducing their paracrine activity. The pituitary stem cell-activating interleukin-6 advanced organoid growth, although the neonatal stem cell compartment was not visibly affected in Il6-/- mice, likely due to cytokine family redundancy. Further transcriptomic analysis exposed a pronounced WNT pathway in the neonatal gland, shown to be involved in stem cell activation and to overlap with the (fetal) human pituitary transcriptome. Following local damage, the neonatal gland efficiently regenerates, despite absence of additional stem cell proliferation, or upregulated IL-6 or WNT expression, all in line with the already high stem cell activation status, thereby exposing striking differences with adult pituitary. Together, our study decodes the stem cell compartment of neonatal pituitary, exposing an activated state in the maturing gland. Understanding stem cell activation is key to potential pituitary regenerative prospects.

The pituitary gland is a pea-sized structure found just below the brain that produces hormones controlling everything from growth and stress to reproduction and immunity. To perform its role, the pituitary gland needs specialised hormone-producing cells, but it also contains stem cells. These stem cells can divide to produce more cells like themselves, or differentiate into cells of different types, including hormone-producing cells. In mice, the stem cells of the pituitary gland appear to be activated in the first few weeks after birth, and later become 'quiescent' (or lazy) in the adult pituitary gland. However, it remains unclear how the activated state found in the maturing gland is established and regulated. To answer this question, Laporte et al. used single-cell RNA sequencing, a technique that allows researchers to profile which genes are active in individual cells, which can provide vital information about the state and activity of a tissue. The researchers compared the cells of the maturing pituitary gland of newborn mice to the cells in the established gland of adult mice. This analysis revealed that the maturing pituitary gland is a dynamic tissue, with populations of cells that are actively dividing (including the stem cells), which the mature pituitary gland lacks. Additionally, Laporte et al. established the molecular basis for the activated state of the stem cells in the maturing pituitary gland, which relies on the activation of a cell signalling pathway called WNT. To confirm these findings, Laporte et al. used an organoid system that allowed them to recapitulate the stem cell compartment of the maturing pituitary gland in a dish. When Laporte et al. blocked WNT signalling in these organoids, the organoids failed to form or divide. Furthermore, blocking the pathway directly in newborn mice reduced the number of dividing stem cells in the pituitary gland. Both findings support the notion that WNT signalling is required to establish the activated state of the maturing pituitary gland in newborn mice. Laporte et al. also wanted to know whether the newborn pituitary gland responded to injury differently than the adult gland. It had already been established that the adult pituitary stem cells become activated upon injury, and that the gland has some regenerative capacity. However, when Laporte et al. injured the newborn pituitary gland, the gland was able to fully regenerate, despite the stem cells not becoming more activated. This is likely because these cells are already activated (or 'primed'), and do not require further activation to divide and repair the gland with the help of other proliferating cells. With these results, Laporte et al. shed light on the activated state of the stem cells in the pituitary gland of newborn mice. This provides insight into the role of these stem cells, as well as unveiling possible routes towards regenerating pituitary tissue. This could eventually prove useful in medicine, in cases when the pituitary gland is damaged or removed.

Exploring stem cell biology in pituitary tumors and derived organoids.

Pituitary tumorigenesis is highly prevalent and causes major endocrine disorders. Hardly anything is known on the behavior of the local stem cells in this pathology. Here, we explored the stem cells' biology in mouse and human pituitary tumors using transcriptomic, immunophenotyping and organoid approaches. In the prolactinoma-growing pituitary of dopamine receptor D2 knock-out mice, the stem cell population displays an activated state in terms of proliferative activity and distinct cytokine/chemokine phenotype. Organoids derived from the tumorous glands' stem cells recapitulated these aspects of the stem cells' activation nature. Upregulated cytokines, in particular interleukin-6, stimulated the stem cell-derived organoid development and growth process. In human pituitary tumors, cells typified by expression of stemness markers, in particular SOX2 and SOX9, were found present in a wide variety of clinical tumor types, also showing a pronounced proliferative status. Organoids efficiently developed from human tumor samples, displaying a stemness phenotype as well as tumor-specific expression fingerprints. Transcriptomic analysis revealed fading of cytokine pathways at organoid development and passaging, but their reactivation did not prove capable of rescuing early organoid expansion and passageability arrest. Taken together, our study revealed and underscored an activated phenotype of the pituitary-resident stem cells in tumorigenic glands and tumors. Our findings pave the way to defining the functional position of the local stem cells in pituitary tumor pathogenesis, at present barely known. Deeper insight can lead to more efficient and targeted clinical management, currently still not satisfactorily.

Pituitary disease and recovery: How are stem cells involved?

The pituitary gland embodies our endocrine hub and rigorously regulates hormone balances in the body, thereby ruling over vital developmental and physiological processes. Pituitary dysfunction and disease strongly impact the organism's biology. Physical damage, tumour development and ageing all negatively affect pituitary state and functionality. On top of its hormone-producing cells, the pituitary contains a population of stem cells. Not only their physiological role is still largely unknown, also whether or how these stem cells are involved in pituitary disease and recovery from defective functionality remains enigmatic. Here, we summarize what is known on the phenotypical and functional behaviour of pituitary stem cells in diseased or dysfunctional gland, as particularly caused by injury, tumourigenesis and ageing.

Setting a Protective Threshold Value for Silver Toward Freshwater Organisms.

Driven by Regulation (EC) No. 1272/2008 and the European Water Framework Directive 2000/60/EC, we have re-evaluated the available chronic freshwater ecotoxicity data for ionic silver (Ag) using strict data quality criteria. In addition, we generated new chronic ecotoxicity data for species potentially sensitive to Ag (the rotifer Brachionus calyciflorus, the cyanobacteria Anabaena flos-aquae, and the aquatic plant Lemna minor) using Ag nitrate as the test substance. The 10% effect concentrations for the most sensitive endpoint per test species were 0.31 µg dissolved Ag/L for B. calyciflorus (population size), 0.41 µg dissolved Ag/L for A. flos-aquae (growth rate), and 1.40 µg dissolved Ag/L for L. minor (root length). We included these values in the set of reliable chronic freshwater data, subsequently covering a total of 12 taxonomic groups and 15 species. Finally, we applied a species sensitivity distribution approach to the data set using various models. The best-fitting model (Rayleigh distribution) resulted in a threshold value protective for 95% of the species of 0.116 µg dissolved Ag/L. This value is considered reliable and conservative in terms of species protection and can be used as a solid basis for setting thresholds for Ag in freshwater after application of an appropriate assessment factor. Furthermore, this value represents reasonable worst-case conditions for bioavailability in European Union surface waters (low hardness and low dissolved organic carbon). Environ Toxicol Chem 2021;40:1678-1693. © 2021 European Precious Metals Federation. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A Generalized Bioavailability Model (gBAM) for Predicting Chronic Copper Toxicity to Freshwater Fish.

The generalized bioavailability model (gBAM) has been proposed as an alternative to the biotic ligand model (BLM) for modeling bioavailability and chronic toxicity of copper (Cu). The gBAM combines a log-linear effect of pH on free Cu2+ ion toxicity with BLM-type parameters for describing the protective effects of major cations (calcium [Ca]2+ , magnesium [Mg]2+ , and sodium [Na]+ ). In the present study, a Windermere Humic Aqueous Model (WHAM) VII-based gBAM for fish was parametrized based on an existing chronic (30-d) dataset of juvenile rainbow trout (Oncorhynchus mykiss). The model, with defined parameters (pH slope parameter [SpH ] = 0.4449 and biotic ligand competition constants [log KCaBL = 4.0, log KMgBL = 3.4, and log KNaBL = 3.0]), was shown to accurately predict the effects of pH, dissolved organic carbon, Ca, and Mg on chronic Cu toxicity to juvenile rainbow trout at the effect levels relevant for environmental risk assessment (i.e., median prediction error of 1.3-fold for 10 and 20% lethal concentrations). The gBAM predicted the effect of pH more accurately than a previously published Cu BLM for juvenile rainbow trout, especially at pH > 8. We also evaluated the cross-species and cross-life stage applicability of the newly developed juvenile rainbow trout gBAM using existing chronic Cu toxicity data with early life stages of fathead minnow (Pimephales promelas) and rainbow trout. We did this because using a single bioavailability model for all fish species and life stages is practical from a regulatory point of view. Although the early life stage datasets exhibit considerable uncertainties, 91% of the considered toxicity values at the effect levels most relevant in European environmental regulations (10% effect on survival or growth) were predicted within a 2-fold error. Overall, the chronic Cu gBAM we developed is a valuable alternative for the existing chronic Cu BLM for rainbow trout and performs sufficiently well to be used in risk assessment according to currently accepted standards of bioavailability model performance (from the current European regulatory point of view). However, our analysis also suggests that bioavailability relations differ between different fish life stages and between endpoints (e.g., mortality vs growth), which is currently not accounted for in environmental risk assessments. Environ Toxicol Chem 2020;39:2424-2436. © 2020 SETAC.

Demonstrating the Reliability of bio-met for Determining Compliance with Environmental Quality Standards for Metals in Europe.

The importance of considering the bioavailability of metals in understanding and assessing their toxicity in freshwaters has been recognized for many years. Currently, biotic ligand models (BLMs) are being applied for the derivation and implementation of environmental quality standards (EQS) for metals under the Water Framework Directive in Europe. bio-met is a simplified tool that was developed for implementing bioavailability-based EQS for metals in European freshwaters. We demonstrate the reliability of the relationship between the full BLM predictions and the thresholds (hazardous concentration affecting 5% of species [HC5] values) predicted by bio-met in 3 stages, for the metals copper, nickel, and zinc. First, ecotoxicity data for specific species from laboratory tests in natural waters are compared with predictions by the individual species BLMs included in the full BLMs. Second, the site-specific HC5 values predicted by bio-met for the natural waters used for ecotoxicity testing are compared with those provided by the full BLMs. The reliability of both relationships is demonstrated for all 3 metals, with more than 80% of individual species BLM predictions being within a factor of 3 of the experimental results, and 99% of bio-met local HC5 predictions being within a factor of 2 of the full BLM result. Third, using a larger set of European natural waters in addition demonstrates the reliability of bio-met over a broad range of water chemistry conditions. bio-met is therefore an appropriate tool for performing compliance assessments against EQS values in Europe, due to the demonstrated consistency with the toxicity test data. Environ Toxicol Chem 2020;39:2361-2377. © 2020 SETAC.

The Effects of Nickel on the Structure and Functioning of a Freshwater Plankton Community Under High Dissolved Organic Carbon Conditions: A Microcosm Experiment.

In the present study, we aimed to test the protectiveness of the bioavailability-normalization procedure, with its associated hazardous concentrations for x% of the species (HCx), that is currently implemented to derive environmental threshold concentrations for nickel (Ni) in European environmental legislative frameworks. We exposed a natural plankton-dominated community to 3 constant Ni concentrations, that is, a control with no Ni added (background Ni of 1.2-4 µg/L) and the bioavailability-normalized HC5 and HC50 of 24 and 97 µg dissolved Ni/L, respectively, during a 56-d microcosm experiment under high dissolved organic carbon (DOC) conditions (DOC of 14 mg/L at test initiation). The effects of the bioavailability-normalized HC5 and HC50 values were evaluated at the levels of community structure (community composition and plankton group abundances), community functioning (measured as indirect physicochemical proxies for overnight respiration and carbon fluxes), and individual species abundances. The bioavailability-normalized HC50 treatment had clear effects (defined as effects occurring on at least 2 consecutive sampling days) on both the structure and functioning of the investigated aquatic community. Through its effect on community functioning (i.e., reduced pH and DOC), Ni also influenced its own bioavailability. Clear direct effects of Ni were observed for only 3 species (the Cyanobacteria Oscillatoria sp. 1 and the rotifers Asplanchna/Testidunela sp. and Trichocerca group similis). Most other effects occurring in the plankton community in the HC50 treatment were indirect and likely driven by the direct effect of Ni on the Cyanobacteria Oscillatoria sp. 1, which was the dominant phytoplankton species in the control microcosms. In contrast, the bioavailability-normalized HC5 did not induce clear effects on community structure and functioning endpoints: these were only affected on individual sampling days. Clear (direct) effects were observed for only 2 plankton species (the rotifer Trichocerca group similis and the Cyanobacteria Oscillatoria sp. 1), but their abundances recovered to control levels at the end of the study. In addition, a few species (1 phytoplankton and 3 zooplankton species) were affected in the HC5 treatment only on the last sampling day. It is uncertain whether these species would have shown clear effects over a longer exposure duration. Thus, our study shows that the bioavailability-normalized HC5 of Ni at high DOC induced clear effects on a few individual species. However, the overall conclusion is that the bioavailability-normalized HC5 of Ni as derived through the procedure that is currently implemented in European legislative frameworks protects against clear effects on community structure and function. Environ Toxicol Chem 2019;38:1923-1939. © 2019 SETAC.

Organoids from pituitary as a novel research model toward pituitary stem cell exploration.

The pituitary is the master endocrine gland, harboring stem cells of which the phenotype and role remain poorly characterized. Here, we established organoids from mouse pituitary with the aim to generate a novel research model to study pituitary stem cell biology. The organoids originated from the pituitary cells expressing the stem cell marker SOX2 were long-term expandable, displayed a stemness phenotype during expansive culture and showed specific hormonal differentiation ability, although limited, after subrenal transplantation. Application of the protocol to transgenically injured pituitary harboring an activated stem cell population, resulted in more numerous organoids. Intriguingly, these organoids presented with a cystic morphology, whereas the organoids from undamaged gland were predominantly dense and appeared more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes and showed that cystic organoids more resembled the pituitary phenotype, at least to an immature state, and displayed in vitro differentiation, although yet moderate. Organoid characterization further exposed facets of regulatory pathways of the putative stem cells of the pituitary and advanced new injury-activated markers. Taken together, we established a novel organoid research model revealing new insights into the identity and regulation of the putative pituitary stem cells. This organoid model may eventually lead to an interesting tool to decipher pituitary stem cell biology in both healthy and diseased gland.

A framework for ecological risk assessment of metal mixtures in aquatic systems.

Although metal mixture toxicity has been studied relatively intensely, there is no general consensus yet on how to incorporate metal mixture toxicity into aquatic risk assessment. We combined existing data on chronic metal mixture toxicity at the species level with species sensitivity distribution (SSD)-based in silico metal mixture risk predictions at the community level for mixtures of Ni, Zn, Cu, Cd, and Pb, to develop a tiered risk assessment scheme for metal mixtures in freshwater. Generally, independent action (IA) predicts chronic metal mixture toxicity at the species level most accurately, whereas concentration addition (CA) is the most conservative model. Mixture effects are noninteractive in 69% (IA) and 44% (CA) and antagonistic in 15% (IA) and 51% (CA) of the experiments, whereas synergisms are only observed in 15% (IA) and 5% (CA) of the experiments. At low effect sizes (∼ 10% mixture effect), CA overestimates metal mixture toxicity at the species level by 1.2-fold (i.e., the mixture interaction factor [MIF]; median). Species, metal presence, or number of metals does not significantly affect the MIF. To predict metal mixture risk at the community level, bioavailability-normalization procedures were combined with CA or IA using SSD techniques in 4 different methods, which were compared using environmental monitoring data of a European river basin (the Dommel, The Netherlands). We found that the simplest method, in which CA is directly applied to the SSD (CASSD ), is also the most conservative method. The CASSD has median margins of safety (MoS) of 1.1 and 1.2 respectively for binary mixtures compared with the theoretically more consistent methods of applying CA or IA to the dose-response curve of each species individually prior to estimating the fraction of affected species (CADRC or IADRC ). The MoS increases linearly with an increasing number of metals, up to 1.4 and 1.7 for quinary mixtures (median) compared with CADRC and IADRC , respectively. When our methods were applied to a geochemical baseline database (Forum of European Geological Surveys [FOREGS]), we found that CASSD yielded a considerable number of mixture risk predictions, even when metals were at background levels (8% of the water samples). In contrast, metal mixture risks predicted with the theoretically more consistent methods (e.g., IADRC ) were very limited under natural background metal concentrations (<1% of the water samples). Based on the combined evidence of chronic mixture toxicity predictions at the species level and evidence of in silico risk predictions at the community level, a tiered risk assessment scheme for evaluating metal mixture risks is presented, with CASSD functioning as a first, simple conservative tier. The more complex, but theoretically more consistent and most accurate method, IADRC , can be used in higher tier assessments. Alternatively, the conservatism of CASSD can be accounted for deterministically by incorporating the MoS and MIF in the scheme. Finally, specific guidance is also given related to specific issues, such as how to deal with nondetect data and complex mixtures that include so-called data-poor metals. Environ Toxicol Chem 2018;37:623-642. © 2017 SETAC.

Ecotoxicological and biochemical mixture effects of an herbicide and a metal at the marine primary producer diatom Thalassiosira weissflogii and the primary consumer copepod Acartia tonsa.

Mixture effects of chemicals and their potential synergistic interactions are of great concern to the public and regulatory authorities worldwide. Intensive agricultural activities are leading to discharges of chemical mixtures to nearby estuarine and marine waters with possible adverse effects on the aquatic communities and for the trophic food web interlinking these communities. Further information about the impacts of these stressors on aquatic organisms is needed. This study addresses ecotoxicological and biochemical effects of single and mixtures of the metal copper and the herbicide Primextra® Gold TZ on the marine diatom Thalassiosira weissflogii and on the estuarine calanoid copepod Acartia tonsa by determining growth rate and survival, respectively, and changes on fatty acid(FA) profiles in both species. Mixture effects on diatom species revealed that copper and Primextra® acted most likely additively with respect to the concentration addition (CA) and independent action (IA) models with model deviation ratios (MDR), 0.752 and 1.063, respectively. For the copepod species, copper and Primextra® were most likely non-interactive with respect to the CA model (MDR = 1.521) but acted most likely synergistically with respect to the IA model (MDR = 2.026). A significant decline in the absolute FA concentration was observed for copepod species after mixture exposure including a considerable decrease of essential FAs that cannot be synthesized de novo by these grazers. We concluded that the mixture effects are more hazardous for primary consumer than for primary producer species in terms of both abundance and biomass quality, suggesting a potential for harmful effects for higher trophic levels and thus a decrease in energy flow through the ecosystem.