Synchrotron-Based X-ray Fluorescence Imaging Elucidates Uranium Toxicokinetics in Daphnia magna.

A combination of synchrotron-based elemental analysis and acute toxicity tests was used to investigate the biodistribution and adverse effects in Daphnia magna exposed to uranium nanoparticle (UNP, 3-5 nm) suspensions or to uranium reference (Uref) solutions. Speciation analysis revealed similar size distributions between exposures, and toxicity tests showed comparable acute effects (UNP LC50: 402 µg L-1 [336-484], Uref LC50: 268 µg L-1 [229-315]). However, the uranium body burden was 3- to 5-fold greater in UNP-exposed daphnids, and analysis of survival as a function of body burden revealed a ∼5-fold higher specific toxicity from the Uref exposure. High-resolution X-ray fluorescence elemental maps of intact, whole daphnids from sublethal, acute exposures of both treatments revealed high uranium accumulation onto the gills (epipodites) as well as within the hepatic ceca and the intestinal lumen. Uranium uptake into the hemolymph circulatory system was inferred from signals observed in organs such as the heart and the maxillary gland. The substantial uptake in the maxillary gland and the associated nephridium suggests that these organs play a role in uranium removal from the hemolymph and subsequent excretion. Uranium was also observed associated with the embryos and the remnants of the chorion, suggesting uptake in the offspring. The identification of target organs and tissues is of major importance to the understanding of uranium and UNP toxicity and exposure characterization that should ultimately contribute to reducing uncertainties in related environmental impact and risk assessments.

Crystal Structure of H227A Mutant of Arginine Kinase in Daphnia magna Suggests the Importance of Its Stability.

Arginine kinase (AK) plays a crucial role in the survival of Daphnia magna, a water flea and a common planktonic invertebrate sensitive to water pollution, owing to the production of bioenergy. AK from D. magna (DmAK) has four highly conserved histidine residues, namely, H90, H227, H284, and H315 in the amino acid sequence. In contrast to DmAK WT (wild type), the enzyme activity of the H227A mutant decreases by 18%. To identify the structure-function relationship of this H227A mutant enzyme, the crystal 3D X-ray structure has been determined and an unfolding assay using anilino-1-naphthalenesulfonic acid (ANS) fluorescence has been undertaken. The results revealed that when compared to the DmAK WT, the hydrogen bonding between H227 and A135 was broken in the H227A crystal structure. This suggests that H227 residue, closed to the arginine binding site, plays an important role in maintaining the structural stability and maximizing the enzyme activity through hydrogen bonding with the backbone oxygen of A135.

Grazer-Induced Chemical Defense in a Microcystin-Producing Microcystis aeruginosa (Cyanobacteria) Exposed to Daphnia gessneri Infochemicals.

Cyanobacteria are photosynthetic microorganisms that compose phytoplankton and therefore have a trophic relationship with zooplankton, which represent an important link for energy flux in aquatic food webs. Several species can form blooms and produce bioactive metabolites known as cyanotoxins. However, the ecological and adaptative role of these toxins are still under debate. Many studies have addressed the cyanotoxins' function in defense against herbivory when grazing pressure by zooplankton plays a role in phytoplankton top-down control. Thus, the present study evaluated the ecophysiological responses of the cyanobacterial strain Microcystis aeruginosa NPLJ-4 underlying the chemical induced defense against the cladoceran Daphnia gessneri. Exposure to predator infochemicals consisted of cultures established in ASM-1 medium prepared in a filtrate from a culture of adults of D. gessneri at an environmentally relevant density. Daphnia infochemicals promoted a significant increase in toxin production by M. aeruginosa. However, no differences in growth were observed, despite a significant increase in both maximum photosynthetic efficiency and electron transport rate in response to zooplankton. Additionally, there was no significant variation in the production of exopolysaccharides. Overall, although a grazer-induced defense response was demonstrated, there were no effects on M. aeruginosa fitness, which maintained its growth in the presence of Daphnia alarm cues.

Insights into the Activation of a Crustacean G Protein-Coupled Receptor: Evaluation of the Red Pigment-Concentrating Hormone Receptor of the Water Flea Daphnia pulex (Dappu-RPCH R).

The validation of a previously developed model of the interaction between the red pigment-concentrating hormone of Daphnia pulex and its cognate receptor (Jackson et al., IJBM 106, 969-978, 2018) was undertaken. Single amino acid replacements, noticeably an Ala scan, of the ligand, Dappu-RPCH, were docked to the receptor, and the binding energies calculated and compared to the one with Dappu-RPCH. As a second step, the same molecules were docked using molecular dynamics (MD) in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane. Changes in binding energy were compared to previous results on in vitro receptor activation (Marco et al., Sci. Rep. 7, 6851, 2017). Residue scanning and MD simulations both gave comparable results for binding energy. For most mutants, there was a good inverse correlation between in vitro activity and binding. There were, however, exceptions; for example: [Ala4]Dappu-RPCH bound as tightly as the cognate ligand but had little activity. This seeming discrepancy was explained when the MD data were analyzed in detail, showing that, although [Ala4]Dappu-RPCH had multiple interactions with the receptor accounting for the high binding energy, the interacting residues of the receptor were quite different to those of Dappu-RPCH. The MD calculations show clearly that the strong binding affinity of the ligand to the receptor is not sufficient for activation. Interaction of the binding of the ligand to two residues of the receptor, Ser 155 and Gln 237, is also essential. A comparison of our computational results with the experimental results of Marco et al. and comparison with the extensive data on GnRH supports the validity of our Dappu-RPCH R model.

Comparison of neutral lipid fatty acid composition in organisms from different trophic levels.

The profiles of total fatty acids (TFAs) and the neutral lipid fatty acids (NLFAs) were compared for the bacterium Rhodopirellula rubra and the alga Raphidocelis subcapitata (conventional food source for Daphnia magna). D. magna NLFAs were assessed when this crustacean was fed with bacterium and alga, individually or in combination. After NLFA extraction, the profiles of the various organisms were characterized by gas chromatography. Results evidenced the relevance of the different composition of the fatty acid (FAs) fractions in the different organisms, R. rubra and R. subcapitata. In these species, the NFLA analyses revealed high amounts of long chain FAs (C19). The FA profile of D. magna was influenced by the different diets provided although the preferred diet was the alga. D. magna showed the capacity to adapt to the available food resources as it defines its FA profile according to its needs, namely for the long chain FAs (C19).

The Influence of Structural Patterns on Acute Aquatic Toxicity of Organic Compounds.

Investigation of the influence of molecular structure of different organic compounds on acute toxicity towards Fathead minnow, Daphnia magna, and Tetrahymena pyriformis has been carried out using 2D simplex representation of molecular structure and two modelling methods: Random Forest (RF) and Gradient Boosting Machine (GBM). Suitable QSAR (Quantitative Structure - Activity Relationships) models were obtained. The study was focused on QSAR models interpretation. The aim of the study was to develop a set of structural fragments that simultaneously consistently increase toxicity toward Fathead minnow, Daphnia magna, Tetrahymena pyriformis. The interpretation allowed to gain more details about known toxicophores and to propose new fragments. The results obtained made it possible to rank the contributions of molecular fragments to various types of toxicity to aquatic organisms. This information can be used for molecular optimization of chemicals. According to the results of structural interpretation, the most significant common mechanisms of the toxic effect of organic compounds on Fathead minnow, Daphnia magna and Tetrahymena pyriformis are reactions of nucleophilic substitution and inhibition of oxidative phosphorylation in mitochondria. In addition acetylcholinesterase and voltage-gated ion channel of Fathead minnow and Daphnia magna are important targets for toxicants. The on-line version of the OCHEM expert system (https://ochem.eu) were used for a comparative QSAR investigation. The proposed QSAR models comply with the OECD principles and can be used to reliably predict acute toxicity of organic compounds towards Fathead minnow, Daphnia magna and Tetrahymena pyriformis with allowance for applicability domain estimation.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Comparison of Bayesian and numerical optimization-based diet estimation on herbivorous zooplankton.

Consumer diet estimation with biotracer-based mixing models provides valuable information about trophic interactions and the dynamics of complex ecosystems. Here, we assessed the performance of four Bayesian and three numerical optimization-based diet estimation methods for estimating the diet composition of herbivorous zooplankton using consumer fatty acid (FA) profiles and resource library consisting of the results of homogeneous diet feeding experiments. The method performance was evaluated in terms of absolute errors, central probability interval checks, the success in identifying the primary resource in the diet, and the ability to detect the absence of resources in the diet. Despite occasional large inconsistencies, all the methods were able to identify the primary resource most of the time. The numerical optimization method QFASA using χ2(QFASA-CS) or Kullback--Leibler (QFASA-KL) distance measures had the smallest absolute errors, most frequently found the primary resource, and adequately detected the absence of resources. While the Bayesian methods usually performed well, some of the methods produced ambiguous results and some had much longer computing times than QFASA. Therefore, we recommend using QFASA-CS or QFASA-KL. Our systematic tests showed that FA models can be used to accurately estimate complex dietary mixtures in herbivorous zooplankton. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

In vivo comprehensive multiphase NMR.

Traditionally, due to different hardware requirements, nuclear magnetic resonance (NMR) has developed as two separate fields: one dealing with solids, and one with solutions. Comprehensive multiphase (CMP) NMR combines all electronics and hardware (magic angle spinning [MAS], gradients, high power Radio Frequency (RF) handling, lock, susceptibility matching) into a universal probe that permits a comprehensive study of all phases (i.e., liquid, gel-like, semisolid, and solid), in intact samples. When applied in vivo, it provides unique insight into the wide array of bonds in a living system from the most mobile liquids (blood, fluids) through gels (muscle, tissues) to the most rigid (exoskeleton, shell). In this tutorial, the practical aspects of in vivo CMP NMR are discussed including: handling the organisms, rotor preparation, sample spinning, water suppression, editing experiments, and finishes with a brief look at the potential of other heteronuclei (2 H, 15 N, 19 F, 31 P) for in vivo research. The tutorial is aimed as a general resource for researchers interested in developing and applying MAS-based approaches to living organisms. Although the focus here is CMP NMR, many of the approaches can be adapted (or directly applied) using conventional high-resolution magic angle spinning, and in some cases, even standard solid-state NMR probes.

Flow-based in vivo NMR spectroscopy of small aquatic organisms.

NMR applied to living organisms is arguably the ultimate tool for understanding environmental stress responses and can provide desperately needed information on toxic mechanisms, synergistic effects, sublethal impacts, recovery, and biotransformation of xenobiotics. To perform in vivo NMR spectroscopy, a flow cell system is required to deliver oxygen and food to the organisms while maintaining optimal line shape for NMR spectroscopy. In this tutorial, two such flow cell systems and their constructions are discussed: (a) a single pump high-volume flow cell design is simple to build and ideal for organisms that do not require feeding (i.e., eggs) and (b) a more advanced low-volume double pump flow cell design that permits feeding, maintains optimal water height for water suppression, improves locking and shimming, and uses only a small recirculating volume, thus reducing the amount of xenobiotic required for testing. In addition, key experimental aspects including isotopic enrichment, water suppression, and 2D experiments for both 13 C enriched and natural abundance organisms are discussed.

Adverse effects of oxo-degradable plastic leachates in freshwater environment.

The production of biodegradable plastics is considered to be a way to reduce plastic waste issue. Among others, oxo-degradant additives enable a faster degradation of plastics in the environment. However, the introduction of these new materials could provoke the release of substances potentially toxic in the environment. This work determined and compared the toxicity of leachates from various additivated polymers (polyethylene, PE; polypropylene, PP; polystyrene, PS) upon different test organisms: plants (Sorghum saccharatum, Lepidium sativum, Sinapis alba, and Vicia faba), crustacean (Daphnia magna), and luminescent bacteria (Vibrio fischeri). Daphnia magna survival was mainly affected by PS and PP leachates (72% and 61% effect, respectively) while PS notably reduced the reproduction rate. On plants, only PP exerted a negative effect (S. saccharatum IG% 32.4), while V. fischeri always showed values around 50%. The data integration, through the Toxicity Test Battery Integrated Index (TBI) approach, allowed to rank the leachates toxicity as PE > PS > PP. This result could be mainly ascribable to the highest metals content in PE since no difference with organic compounds analysis was evidenced. In conclusion, since the polymers exerted dissimilar toxicity, the additive could not be considered the sole responsible of the measured toxicity, but its role in the enhancement of the virgin polymers leachates effects can be solidly hypothesized.

Long-term eutrophication affects vertical changes of Daphnia ephippia in the sediments of a subtropical Chinese lake.

Cladoceran remains (e.g., Daphnia ephippia) in sediments are usually able to accurately reflect the historical succession of cladoceran in lakes. However, mechanisms describing the interaction between density changes of empty ephippia and ephippia containing resting eggs in lake sediments and environmental factors remain unclear. The diversity and vertical changes of Daphnia ephippia in the 30-cm sedimentary layer (equivalent to about 90 years) were investigated in a subtropical Chinese lake, Lake Chaohu. The ephippia of D. similoides sinensis, D. pulex, and D. galeata were identified in the lake sediments. The densities and accumulation rates of both empty ephippia and ephippia containing resting eggs of three Daphnia species showed similar patterns. The values in the surface sedimentary layers (after the 1970s) were notably higher than in the bottom sedimentary layers (before the 1970s). The densities and accumulation rates of both empty ephippia and ephippia containing resting eggs of D. similoides sinensis were higher than those of D. pulex and D. galeata. Highly significant relationships (P < 0.001) between the TN contents and the ephippial densities and accumulation rates of D. similoides sinensis and D. galeata. TP contents had also significant correlations with the ephippial densities and accumulation rates of D. similoides sinensis and D. galeata, whereas it was not significant correlations with those of D. pulex (P > 0.05). Our results suggested that long-term eutrophication and cyanobacterial blooms might significantly affect the production of Daphnia ephippia and their vertical distribution in the sediments in eutrophic lakes.

Assessment of the ecotoxicity of the pharmaceuticals bisoprolol, sotalol, and ranitidine using standard and behavioral endpoints.

The pharmaceuticals bisoprolol (BIS), sotalol (SOT), and ranitidine (RAN) are among the most consumed pharmaceuticals worldwide and are frequently detected in different aquatic ecosystems. However, very few ecotoxicity data are available in the literature for them. To help fill these data gaps, toxicity tests with the algae Raphidocelis subcapitata, the macrophyte Lemna minor, the cnidarian Hydra attenuata, the crustacean Daphnia similis, and the fish Danio rerio were performed for assessing the ecotoxicity of these pharmaceuticals. Standard, as well as non-standard endpoint, was evaluated, including the locomotor behavior of D. rerio larvae. Results obtained for SOT and RAN showed that acute adverse effects are not expected to occur on aquatic organisms at the concentrations at which these pharmaceuticals are usually found in fresh surface waters. On the other hand, BIS was classified as hazardous to the environment in the acute III category. Locomotor behavior of D. rerio larvae was not affected by BIS and RAN. A disturbance on the total swimming distance at the dark cycle was observed only for larvae exposed to the highest test concentration of 500 mg L-1 of SOT. D. similis reproduction was affected by BIS with an EC10 of 3.6 (0.1-34.0) mg L-1. A risk quotient (RQ) of 0.04 was calculated for BIS in fresh surface water, considering a worst-case scenario. To the best of our knowledge, this study presents the first chronic toxicity data with BIS on non-target organisms.

Photostable Fluorescent Tracker for Imaging Mitochondria with Super Resolution.

The power factories in cells, mitochondria, play important roles in all physiological processes. It is reported that progressive mitochondrial swelling and outer mitochondrial membrane rupture could be induced by a wide variety of apoptotic and necrotic stimuli. Regrettably, although a variety of mitochondrial probes have been developed, most of them are based on the detection of active species in mitochondria. Probes that can monitor the status and distribution of mitochondria for a long time are still urgently needed. In this study, a fluorescent sensor with excellent properties, EtNBEn, is described. Outstanding performance allows it to be observed not only in cells but also in living Daphnia and zebrafish under confocal microscopy for a long time. Moreover, the swelling process of mitochondria under light stimulation is also visualized under super-resolution (SR) microscopy. All these results suggest that EtNBEn could be employed for tagging mitochondria in various physiological processes, which makes a great contribution to the cure of diseases.

Environmental DNA size sorting and degradation experiment indicates the state of Daphnia magna mitochondrial and nuclear eDNA is subcellular.

Environmental DNA analysis has emerged as a key component of biodiversity and environmental monitoring. However, the state and fate of eDNA in natural environments is still poorly understood for many ecological systems. Here we assess the state and fate of eDNA derived from the water flea, Daphnia magna, using a full factorial mesocosm experiment. We measured the quantity and degradation of eDNA over a two month period across a range of filters differing in pore size (0, 0.2, 1 and 10 µm), which spans the range of eDNA source material including subcellular, cellular and tissue. We also used two primer sets targeting mitochondrial (COI) and nuclear (18S) genomic regions. Our findings demonstrated that eDNA was most prevalent in the effluent water, but also reliably detected on the 0.2 µm filter, suggesting subcellular material is the predominate state of eDNA. Temporal eDNA quantity dynamics followed an exponential decay function over the course of 6-17 days, demonstrating a predictable decline in eDNA concentration. Nuclear eDNA was more abundant than mitochondrial eDNA, which may be a result of greater primer affinity, or indicate greater availability of nuclear eDNA gene targets in the environment. In contrast to two previous size-sorting experiments, which utilizing fish eDNA, our findings suggest that the state of invertebrate eDNA is much smaller than previously suspected. Overall, our data suggest that the detection of eDNA greatly depends on our knowledge of the state and fate of eDNA, which differ among species, and likely across environmental conditions.

Analysis of neurotransmitters in Daphnia magna affected by neuroactive pharmaceuticals using liquid chromatography-high resolution mass spectrometry.

Neurotransmission plays an essential role during the central nervous system (CNS) development. During the last years, several studies based on the changes produced in neurotransmitters of aquatic organisms caused by pharmaceuticals have been reported. Daphnia magna, the aquatic ecotoxicological model organism, shares several of the neurotransmitters targeted by antidepressant and other neuro-active drugs with vertebrates. Therefore, a method based on liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) has been applied for the first time to study the levels of 41 neurotransmitters in Daphnia magna under the effect of four different neuro-active pharmaceuticals (sertraline, venlafaxine, duloxetine and fluoxetine). In addition, the performance of LC-HRMS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method using LC-HRMS is a new tool for neurotoxicology research using the Daphnia magna model. As a result, general differences on the concentrations of those neurotransmitters exposed to the mentioned pharmaceuticals were observed.

Daphnia magna Exudates Impact Physiological and Metabolic Changes in Microcystis aeruginosa.

While the intracellular function of many toxic and bioactive cyanobacterial metabolites is not yet known, microcystins have been suggested to have a protective role in the cyanobacterial metabolism, giving advantage to toxic over nontoxic strains under stress conditions. The zooplankton grazer Daphnia reduce cyanobacterial dominance until a certain density, which may be supported by Daphnia exudates, affecting the cyanobacterial physiological state and metabolites' production. Therefore, we hypothesized that D. magna spent medium will impact the production of cyanobacterial bioactive metabolites and affect cyanobacterial photosynthetic activity in the nontoxic, but not the toxic strain. Microcystin (MC-LR and des-MC-LR) producing M. aeruginosa PCC7806 and its non-microcystin producing mutant were exposed to spent media of different D. magna densities and culture durations. D. magna spent medium of the highest density (200/L) cultivated for the shortest time (24 h) provoked the strongest effect. D.magna spent medium negatively impacted the photosynthetic activity of M. aeruginosa PCC7806, as well as the dynamics of intracellular and extracellular cyanobacterial metabolites, while its mutant was unaffected. In the presence of Daphnia medium, microcystin does not appear to have a protective role for the strain. On the contrary, extracellular cyanopeptolin A increased in M. aeruginosa PCC7806 although the potential anti-grazing role of this compound would require further studies.

Toxicological Evaluation and Quantification of Ingested Metal-Core Nanoplastic by Daphnia magna Through Fluorescence and Inductively Coupled Plasma-Mass Spectrometric Methods.

There are few studies on nanoplastic that propose quantification of the amount ingested combined with evaluation of the toxic effects on aquatic organisms. We propose 2 methods to quantify the amount of polystyrene nanoplastic (PSNP) ingested by Daphnia magna: fluorescence intensity, where a fluorescent monomer (F) is added to the PSNP and quantified through fluorescence light microscopy, and total aluminum quantification, where PSNP is synthesized with Al2 O3 metal-core nanoparticles and used for quantification of the nanoplastic ingested by the organism Daphnia magna using inductively coupled plasma-mass spectrometry. In addition, the PSNP was functionalized with palmitic acid to simulate the environmental conditions leading to biological and chemical transformations. Acute and chronic toxicity tests were performed with fluorescent PSNP (PSNP/F) and palmitic acid-functionalized PSNP/F (PSNP/F-PA). The ingestion quantified was higher by factors of 2.8 and 3.0 for PSNP/F-PA and 1.9 and 1.7 for PSNP/F applying the fluorescence intensity and total Al quantifying methods, respectively, when compared to PSNP. These results are consistent with the data obtained in the toxicity tests, which showed an approximately 3 times increase in the adverse effect of PSNP/F-PA on the mobility and reproduction of the organisms. Thus, the strong inhibition of D. magna reproduction caused by PSNP/F-PA in the chronic toxicity tests could be associated with a greater amount of this nanoplastic being ingested by the organisms. Environ Toxicol Chem 2019;38:2101-2110. © 2019 SETAC.

When Fluorescence Is not a Particle: The Tissue Translocation of Microplastics in Daphnia magna Seems an Artifact.

Previous research reported the translocation of nano- and microplastics from the gastrointestinal tract to tissues in Daphnia magna, most prominently of fluorescent polystyrene beads to lipid droplets. For particles >300 nm, such transfer is biologically implausible as the peritrophic membrane retains these in the daphnid gut. We used confocal laser scanning microscopy to study tissue transfer applying the setup from a previous study (neonates exposed to 20 and 1000 nm polystyrene beads at 2 µg L-1 for 4 and 24 h), the same setup with a fructose-based clearing, and a setup with a 1000-fold higher concentration (2 mg L-1 ). We used passive sampling to investigate whether the beads leach the fluorescent dye. Although the 1000 nm beads were visible in the gut at both exposure concentrations, the 20 nm beads were detectable at 2 mg L-1 only. At this concentration, we observed fluorescence in lipid droplets in daphnids exposed to both particle types. However, this did not colocalize with the 1000 nm beads, which remained visible in the gut. We further confirmed the leaching of the fluorescent dye using a passive sampler, a method that can also be applied in future studies. In summary, we cannot replicate the original study but demonstrate that the fluorescence in the lipid droplets of D. magna results from leaching of the dye. Thus, the use of fluorescence as a surrogate for particles can lead to artifacts in uptake and translocation studies. This highlights the need to confirm the stability of the fluorescence label or to localize particles using alternative methods. Environ Toxicol Chem 2019;38:1495-1503. © 2019 SETAC OPEN PRACTICES: The present study has earned Open Data/Materials badges for making publicly available the digitally shareable data necessary to reproduce the reported results. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

Nutritional and toxicity constraints of phytoplankton from a Brazilian reservoir to the fitness of cladoceran species.

Camorim is a small, eutrophic reservoir in Rio de Janeiro, Brazil, with a phytoplankton community dominated most of the year by the filamentous diatom Aulacoseira spp. and the toxic cyanobacterium Cylindrospermopsis raciborskii. As filamentous species can be a poor food for grazers, we hypothesize that phytoplankton from this reservoir would constrain cladoceran fitness due to nutritional limitation and/or toxicity when animals fed mixtures of cultured green algae and natural seston. Clones of different cladoceran species were exposed either to seston from Camorim reservoir sampled in different seasons or to a C. raciborskii strain (CYLCAM-2) isolated from the reservoir. In short-term assays, cladocerans were exposed to either 100% seston or mixtures of 50% seston added to green algae (200 µg C L-1), and their survivorship and somatic growth were measured for 4 days. In life table assays, neonates were exposed to the same seston treatments over 14 days and age at first reproduction, survivorship, fecundity, total offspring, and the intrinsic rate of natural increase (r) were assessed. In general, seston negatively affected cladoceran survivorship and fitness (r), but this response was seasonally and species specific. Stronger effects of CYLCAM-2 than those caused by seston on survivorship, somatic growth, and r were found for all cladoceran species, especially when the proportion of CYLCAM-2 was higher than 50% in relation to green algae in a fixed total food concentration. Our results suggest that both nutritional (C/P and morphology) and toxicity factors can act to impair cladoceran fitness and help explain the absence of cladocerans in Camorim reservoir.