Olivine avoidance behaviour by marine gastropods (Littorina littorea L.) and amphipods (Gammarus locusta L.) within the context of ocean alkalinity enhancement.

Gigaton scale atmospheric carbon dioxide (CO2) removal (CDR) is needed to keep global warming below 1.5 °C. Coastal enhanced olivine weathering is a CDR technique that could be implemented in coastal management programmes, but its CO2 sequestration potential and environmental safety remain uncertain. Large scale olivine spreading would change the surficial sediment characteristics, which could potentially reduce habitat suitability and ultimately result in community composition changes. To test this hypothesis, we investigated the avoidance response of the marine gastropod Littorina littorea (Linnaeus, 1758) and marine amphipod Gammarus locusta (Linnaeus, 1758) to relatively coarse (83 - 332 µm) olivine and olivine-sediment mixtures during short-term choice experiments. Pure olivine was significantly avoided by both species, while no significant avoidance was observed for sediment with 3% or 30% w/w olivine. For L. littorea, aversion of the light green colour of pure olivine (i.e. positive scototaxis) was the main reason for avoidance. Moreover, olivine was not significantly avoided when it was 7.5 cm (45%) closer to a food source/darker microhabitat (Ulva sp.) compared to natural sediment. It is inferred that the amphipod G. locusta avoided pure olivine to reduce Ni and Cr exposure. Yet, a significant increase in whole body Ni concentrations was observed after 79 h of exposure in the 30% and 100% w/w olivine treatments compared to the sediment control, likely as a result of waterborne Ni uptake. Overall, our results are significant for ecological risk assessment of coastal enhanced olivine weathering as they show that L. littorea and G. locusta will not avoid sediments with up to 30% w/w relatively coarse olivine added and that the degree of olivine avoidance is dependent on local environmental factors (e.g. food or shelter availability).

Challenges and Recommendations in Assessing Potential Endocrine-Disrupting Properties of Metals in Aquatic Organisms.

New tools and refined frameworks for identifying and regulating endocrine-disrupting chemicals (EDCs) are being developed as our scientific understanding of how they work advances. Although focus has largely been on organic chemicals, the potential for metals to act as EDCs in aquatic systems is receiving increasing attention. Metal interactions with the endocrine system are complicated because some metals are essential to physiological systems, including the endocrine system, and nonessential metals can have similar physiochemical attributes that allow substitution into or interference with these systems. Consequently, elevated metal exposure could potentially cause endocrine disruption (ED) but can also cause indirect effects on the endocrine system via multiple pathways or elicit physiologically appropriate compensatory endocrine-mediated responses (endocrine modulation). These latter two effects can be confused with, but are clearly not, ED. In the present study, we provide several case studies that exemplify the challenges encountered in evaluating the endocrine-disrupting (ED) potential of metals, followed by recommendations on how to meet them. Given that metals have multiple modes of action (MOAs), we recommend that assessments use metal-specific adverse outcome pathway networks to ensure that accurate causal links are made between MOAs and effects on the endocrine system. We recommend more focus on establishing molecular initiating events for chronic metal toxicity because these are poorly understood and would reduce uncertainty regarding the potential for metals to be EDCs. Finally, more generalized MOAs such as oxidative stress could be involved in metal interactions with the endocrine system, and we suggest it may be experimentally efficient to evaluate these MOAs when ED is inferred. These experiments, however, must provide explicit linkage to the ED endpoints of interest. Environ Toxicol Chem 2023;42:2564-2579. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Acute bioaccumulation and chronic toxicity of olivine in the marine amphipod Gammarus locusta.

Active atmospheric carbon dioxide removal (CDR) is needed at a gigaton scale in the next decades to keep global warming below 1.5 °C. Coastal enhanced silicate weathering (CESW) aims to increase natural ocean carbon sequestration via chemical weathering of finely ground olivine (MgxFe(1-x)SiO4) rich rock dispersed in dynamic coastal environments. However, the environmental safety of the technique remains in question due to the high Ni and Cr content of olivine. Therefore, we investigated the short term bioaccumulation and chronic toxicity of olivine in the marine amphipod Gammarus locusta. Acute 24-h olivine exposure resulted in significant grain size dependent olivine ingestion and subsequent Ni and Cr accumulation in tissues. Thousands of small (mainly ≤ 10  µm) olivine grains were ingested by G. locusta, but their importance for trace metal bioaccumulation requires additional research. Most olivine grains were egested within 24 h. Chronic 35-day olivine (3-99 µm) exposure reduced amphipod survival, growth, and reproduction, likely as a result of metal induced oxidative stress and disturbance of major cation homeostasis. Amphipod reproduction was significantly reduced at olivine concentrations of 10% w/w and higher. In the context of ecological risk assessment, application of an arbitrary assessment factor of 100 to the highest no observed effect concentration of 1% w/w olivine yields a very low predicted no-effect concentration (PNEC) of 0.01% w/w olivine. This low PNEC value highlights the urgent need for additional marine olivine toxicity data to accurately assess the environmentally safe scale of coastal enhanced weathering for climate change mitigation.

Accumulation and release of organic pollutants by conventional and biodegradable microplastics in the marine environment.

The issue of microplastic (MP) litter in the aquatic environment and its capability of accumulating and/or releasing pollutants has been brought to light in recent years. Biodegradable plastics have been proposed as one of the different solutions to decrease environmental input of discarded plastics; however, their ability to accumulate and release pollutants once in the marine environment has not been assessed yet. In this study, we compare the accumulation and the release of a wide range of compounds by biodegradable (polyhydroxyalkanoates (PHA) and polybutylene succinate (PBS)) and conventional (polyethylene (PE)) MPs following exposure to natural seawater for 64 days. We quantified polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organophosphorus flame retardants (PFRs), phthalates, and alternative plasticizers in MPs, before and after exposure. Results indicated that PBS- and PHA-MPs accumulated the largest amount of PAHs and PFRs, respectively. Leaching of PFRs and plasticizers was observed for all polymers and was approximately twofold greater for PE- when compared to PBS- and PHA-MPs. Overall, our study suggests that biodegradable MPs may release less additives and accumulate a larger amount of contaminants from seawater compared to conventional ones: these findings may have implications on the risk assessment of biodegradable polymers for marine biota; and on potential widespread adoption of these types of plastics.

Effect of incubation time of three single extraction procedures on trace element extraction from sediment and soil.

The use of standard single-extraction procedures to evaluate the mobility and availability of trace elements is a common practice in most laboratories dealing with soil or sediment analysis. Most standard single-extraction procedures describe incubations last for 2 h. However, these were tested and validated for soil analysis. Applying them for sediment analysis without further investigation might be misleading and should be reviewed with care. This paper investigates the effect of incubation time on the extraction efficiency of three standard single-extracting reagents (0.01 M CaCl2, 1 M NH4NO3, and 0.05 M EDTA). Incubation experiments with sediment and soil samples lasting for 2 h, 10 h, and 10 d were performed. The results indicated that 2 h appears sufficient to reach equilibrium using CaCl2 or NH4NO3 for soil analysis; but when analyzing sediments, incubation for 10 d resulted in higher concentrations. Incubation experiments with 0.05 M EDTA showed that incubation for 2 h was enough to extract Cd from the soil sample, Mn and to a lesser extent Cd from the sediment samples; while for the other elements, incubation for 10 d yielded higher concentrations for both sample types compared to that obtained after 2 h and 10 h separately. Relative to the pseudo-total metal contents, more than 55% of all studied elements were extracted by using 0.05 M EDTA, indicating high bioavailable metal fraction.

Evaluation of effects-based methods as monitoring tools for assessing ecological impacts of metals in aquatic ecosystems.

Effects-based methods (EBMs) are considered part of a more integrative strategy for regulating substances of concern under the European Union Water Framework Directive. In general, EBMs have been demonstrated as useful indicators of effects on biota, although links to population and community-level effects are sometimes uncertain. When EBMs are sufficiently specific and sensitive, and links between measured endpoints and apical or higher level effects are established, they can be a useful tool in assessing effects from a specific toxicant or class of toxicants. This is particularly valuable for toxicants that are difficult to measure and for assessing the effects of toxicant mixtures. This paper evaluates 12 EBMs that have been proposed for potential use in the assessment of metals. Each EBM was evaluated with respect to metal specificity and sensitivity, sensitivity to other classes of toxicants, and the strength of the relationship between EBM endpoints and effects observed at the whole organism or population levels of biological organization. The evaluation concluded that none of the EBMs evaluated meet all three criteria of being sensitive to metals, insensitive to other classes of toxicants, and a strong indicator of effects at the whole organism or population level. Given the lack of suitable EBMs for metals, we recommended that the continued development of mixture biotic ligand models (mBLMs) may be the most effective way to achieve the goal of a more holistic approach to regulating metals in aquatic ecosystems. Given the need to further develop and validate mBLMs, we suggest an interim weight-of-evidence approach that includes mBLMs, macroinvertebrate community bioassessment, and measurement of metals in key macroinvertebrate species. This approach provides a near-term solution and simultaneously generates data needed for the refinement and validation of mBLMs. Once validated, it should be possible to rely primarily on mBLMs as an alternative to EBMs for metals. Integr Environ Assess Manag 2023;19:24-31.  © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Interactive toxicity of copper and cadmium in regenerating and adult planarians.

In a polluted environment, metals are present as complex mixtures. As a result, organisms are exposed to different metals at the same time, which affects both metal-specific as well as overall toxicity. Detailed information about the molecular mechanisms underlying the adverse effects of combined exposures remains limited in terms of different life stages. In this study, the freshwater planarian Schmidtea mediterranea was used to investigate developmental and physiological responses associated with a combined exposure to Cu and Cd. In addition, the cellular and molecular mechanisms underlying the provoked adverse effects were studied in different exposure scenarios. Mixed exposure resulted in a decline in survival, diverse non-lethal morphological changes, neuroregenerative impairments, altered behaviour and a limited repair capacity. Underlying to these effects, the cellular redox state was altered in all exposure conditions. In adult animals, this led to DNA damage and corresponding transcriptional changes in cell cycle and DNA repair genes. In regenerating animals, changes in hydrogen peroxide and glutathione contents led to regenerative defects. Overall, our results demonstrate that (1) developing organisms are more susceptible to metal exposures, and (2) the toxicity of an individual metal increases significantly in a mixed exposure scenario. These aspects have to be included in current risk assessment strategies.

The relevance of European Biota Quality Standards on the ecological water quality as determined by the multimetric macro-invertebrate index: A Flemish case study.

European Biota Quality Standards (EQSbiota), for compounds with low water solubility and high biomagnification, were created to sustain water quality and protect top predators and humans from secondary poisoning. In reality, for multiple compounds, an exceedance of these standards is often reported in literature without a decrease in ecological water quality determined by biotic indices. In the present study, threshold concentrations were defined in biota (from 44 sampling locations throughout Flanders (Belgium)), above which a good ecological water quality, assessed by the Multimetric Macroinvertebrate Index Flanders (MMIF), was never reached. Threshold values were compared to current EQSbiota. Accumulated perfluoroctane sulfonate (PFOS), mercury (Hg), hexabromocyclododecane (HBCD), polybrominated diphenyl ethers (PBDEs), dioxins and polychlorinated biphenyls (PCBs) concentrations were measured in muscle tissue of European yellow eel (Anguilla anguilla) and perch (Perca fluviatilis). Fluoranthene and benzo(a)pyrene were also analyzed in translocated mussels (Dreissena bugensis, D. polymorpha and Corbicula fluminea). Threshold values could only be calculated using a 90th quantile regression model for PFOS (in perch; 12 µg/kg ww), PCBs (in eel; 328 µg/kg ww) and benzo(a)pyrene (in mussels: 4.35 µg/kg ww). The lack of a significant regression model for the other compounds indicated an effective threshold value higher than the concentrations measured in the present study. Alternatively, the 95th percentile of concentrations measured in locations with a good ecological quality (MMIF≥0.7), was calculated for all compounds as an additional threshold value. Finally, fish concentrations were standardized for 5% lipid content (or 26% dry weight content for PFOS and Hg). Threshold values for PFOS and benzo(a)pyrene and the 95th percentiles for dioxins and fluoranthene were comparable to the existing standards. For all other compounds, the 95th percentile was higher than the current EQSbiota, while for HBCD, it was lower. These results strongly advise revising and fine-tuning the current EQSbiota, especially for ∑PBDE and HBCD.

Arabidopsis root growth and development under metal exposure presented in an adverse outcome pathway framework.

Due to human activities, soils become more and more polluted with metals, which imposes risks for human health and wildlife welfare. As most of the metals end up in the food chain through accumulation in plants, we need to establish science-based environmental criteria and risk management policies. To meet these necessities, a thorough understanding is required of how these metals accumulate in and affect plants. Many studies have been conducted towards this aim, but strikingly, only a few entries can be found in ecotoxicological databases, especially on Arabidopsis thaliana, which serves as a model species for plant (cell) physiology and genetic studies. As experimental conditions seem to vary considerably throughout literature, extrapolation or comparison of data is rather difficult or should be approached with caution. Furthermore, metal-polluted soils often contain more than one metal, yet limited studies investigated the impact of metal mixtures on plants. This review aims to compile all data concerning root system architecture under Cu, Cd and Zn stress, in single or multi-metal exposure in A. thaliana, and link it to metal-induced responses at different biological levels. Global incorporation into an adverse outcome pathway framework is presented.

Mercury accumulation in muscle and liver tissue and human health risk assessment of two resident freshwater fish species in Flanders (Belgium): a multilocation approach.

Detrimental effects of chemical pollution-primarily caused by human activities-on aquatic ecosystems have increasingly gained attention. Because of its hydrophobic qualities, mercury is prone to easily bioaccumulate and biomagnify through the food chain, decreasing biodiversity and eventually also affecting humans. In the present study, accumulated mercury concentrations were measured in muscle and liver tissue of perch (Perca fluviatilis) and European eel (Anguilla anguilla) collected at 26 sampling locations in Flemish (Belgian) waterbodies, allowing a comparison of these species within a variety of environmental situations. Furthermore, effects of size and weight have been assessed, expected to influence accumulation and storage of pollutants. Mercury concentrations in perch ranged up to 1.7 µg g-1 dw (median: 0.29 µg g-1 dw) in muscle and from 0.02 to 0.77 µg g-1 dw (median: 0.11 µg g-1 dw) in liver tissue. For eel, these concentrations were between 0.07 and 1.3 µg g-1 dw (median: 0.39 µg g-1 dw) and between 0.08 and 1.4 µg g-1 dw (median: 0.55 µg g-1 dw) respectively. We found a correlation of accumulated mercury with length in perch, independent of location. Furthermore, a significant difference in accumulated mercury concentrations between the targeted species was measured, with the highest mean concentrations per dry weight in eel liver and muscle tissue. In perch, higher concentrations were found in muscle compared to liver tissue, while in eel, liver tissue showed the highest concentrations. These findings were further considered with concentrations corrected for lipid content, excluding the fat compartment, which is known to a hold negligible portion of the total and methyl mercury concentrations. This confirmed our previous conclusions, except for mercury concentrations in eel. Here there was no longer a significant difference between muscle and liver concentrations. Finally, health risk analyses revealed that only frequent consumption of local eel (> 71 g day-1) could pose risks to humans.

Effect of abiotic factors and environmental concentrations on the bioaccumulation of persistent organic and inorganic compounds to freshwater fish and mussels.

Many aquatic ecosystems are under persistent stress due to influxes of anthropogenic chemical pollutants. High concentrations can harm entire ecosystems and be toxic to humans. However, in case of highly hydrophobic compounds, their low water solubility precludes direct measurement in water, and thus alternative monitoring strategies are needed. In the present study, we investigated the extent to which bioaccumulated concentrations of persistent compounds can be predicted by concentrations in environmental compartments (water and sediment). Due to their high biomagnification potential, Hg and PFOS were included in this analysis as well. At 44 field locations in Flanders (Belgium), we monitored the concentrations of 11 priority compounds and their derivatives, included in the Water Framework Directive, in both sediment and water (where feasible) and biota (European perch, European eel and freshwater mussels). Besides, some sediment (i.e. total organic carbon (TOC) and clay content) and water characteristics were measured (i.e. pH, oxygen level, conductivity, nitrate, nitrite and dissolved organic carbon (DOC)). Measurements of HCB, HCBD, cis-heptachlorepoxide, HBCD and PFOS in sediment and ∑PCB in water showed a lower detection frequency than in fish samples. While PCB profiles were comparable between all matrices, for PBDE clear differences were detected between sediment and fish profiles, with BDE99 contributing the most for sediment (34%) and BDE47 for fish (≥44%), followed by BDE99 for perch (28%) and BDE100 for eel (25%). Water concentrations for PFOS and benzo(a)pyrene were predictive of respective bioaccumulated concentrations. HCB, ∑PCB and ∑PBDE, concentrations in fish were dependent on sediment concentrations and negatively related to organic compound levels (p < 0.05). Furthermore, pH and nitrite were negatively associated with accumulated concentrations in eel for HCB and PFOS, respectively (p < 0.05). Strong relationships between bioaccumulation and sediment and/or water concentrations strengthened the basis for surrogate monitoring methods. Finally, the extrapolation potential of Hg, ∑PBDE, PFOS, HBCD and ∑PCB between both fish species offered new opportunities in extrapolating different European monitoring frameworks.

Deriving Nickel (Ni(II)) and Chromium (Cr(III)) Based Environmentally Safe Olivine Guidelines for Coastal Enhanced Silicate Weathering.

Enhanced silicate weathering (ESW) by spreading finely ground silicate rock along the coastal zone to remove atmospheric carbon dioxide (CO2) is a proposed climate change mitigation technique. The abundant and fast-dissolving mineral olivine has received the most attention for this application. However, olivine contains nickel (Ni) and chromium (Cr), which may pose a risk to marine biota during a gigaton-scale ESW application. Herein we derive a first guideline for coastal olivine dispersal based on existing marine environmental quality standards (EQS) for Ni and Cr. Results show that benthic biota are at the highest risk when olivine and its associated trace metals are mixed in the surface sediment. Specifically, depending on local sedimentary Ni concentrations, 0.059-1.4 kg of olivine m-2 of seabed could be supplied without posing risks for benthic biota. Accordingly, globally coastal ESW could safely sequester only 0.51-37 Gt of CO2 in the 21st century. On the basis of current EQS, we conclude that adverse environmental impacts from Ni and Cr release could reduce the applicability of olivine in coastal ESW. Our findings call for more in-depth studies on the potential toxicity of olivine toward benthic marine biota, especially in regard to bioavailability and metal mixture toxicity.

Towards harmonization of water quality management: A comparison of chemical drinking water and surface water quality standards around the globe.

Water quality standards (WQS) set the legal definition for safe and desirable water. WQS impose regulatory concentration limits to act as a jurisdiction-specific legislative risk-management tool. Despite its importance in shaping a universal definition of safe, clean water, little information exists with respect to (dis)similarity of chemical WQS worldwide. Therefore, this paper compares chemical WQS for drinking and surface water matrices in eight jurisdictions representing a global geographic distribution: Australia, Brazil, Canada, China, the European Union, the region of Flanders in Belgium, the United States of America, and South Africa. The World Health Organization's list is used as a reference for drinking water standards. Sørensen-Dice indices (SDI) showed little qualitative similarity in the compounds that are regulated in drinking water (median SDI = 40%) and surface water (median SDI = 33%), indicating that the heterogeneity within a matrix is substantial at the level of the standard. Quantitative similarity for matching standards was higher than the qualitative per Kendall correlation (median = 0.73 and 0.58 for drinking water and surface water respectively), yet variance observed within standards remained inexplicably high for organic compounds. Variations in WQS were more pronounced for organic compounds. Most differences cannot be easily explained from a toxicological or risk-based point-of-view. Historical development, ease of measurement, and (toxicological) knowledge gaps on the risk of a vast number of organic compounds are theorized to be the drivers. Therefore, this study argues for a more tailored, risk-based approach in which standards incorporated into water safety plans are dynamically set for compounds that are persistent and could pose a risk for human health and/or aquatic ecosystems. Global variations in WQS should therefore not necessarily be avoided but rather globally harmonized with enough flexibility to ensure a global, up-to-date definition of safe and desirable water everywhere.

Sublethal Effect Concentrations for Nonpolar Narcosis in the Zebrafish Embryo.

Nonpolar narcosis, also known as baseline toxicity, has been described as the minimal toxicity that an organic chemical may elicit based on its lipophilicity. Although lethal effects of narcosis-inducing chemicals (NICs) have been thoroughly investigated, knowledge of sublethal effects is still very limited. We investigated the effects of 3 well-known NICs (phenanthrene, 1,3,5-trichlorobenzene, and pentachlorobenzene) on a variety of organismal endpoints (malformations, swim bladder inflation, respiration, heart rate, swimming activity, and turning angles), which can be plausibly linked to narcosis in zebrafish embryos. Baseline toxicity recorded as mortality is typically observed in similar exposure ranges in a wide variety of species including fish, corresponding to a chemical activity range between 0.01 and 0.1. In the present study, we found that sublethal effects occurred at concentrations approximately 5 times below lethal concentrations. Altered swimming activity and impaired swim bladder inflation were the most sensitive endpoints occurring at exposure levels below the generally accepted threshold for baseline toxicity for 2 out of 3 compounds. Overall, most effective exposure levels across the sublethal endpoints and compounds did fall within the range typically associated with baseline toxicity, and deviations were generally limited to a factor 10. Although there could be benefit in adding sublethal endpoints to toxicity tests, such as the fish embryo acute toxicity (FET) test, based on the present sublethal endpoints and available evidence from our and other studies, the underestimation of toxicity as a result of the sole assessment of mortality as an endpoint in an FET test may be limited for narcosis. Environ Toxicol Chem 2021;40:2802-2812. © 2021 SETAC.

Temperature Effects During a Sublethal Chronic Metal Mixture Exposure on Common Carp (Cyprinus carpio).

The aquatic environment is the final sink of various pollutants including metals, which can pose a threat for aquatic organisms. Waterborne metal mixture toxicity might be influenced by environmental parameters such as the temperature. In the present study, common carp were exposed for 27 days to a ternary metal mixture of Cu, Zn, and Cd at two different temperatures, 10 and 20°C. The exposure concentrations represent 10% of the 96 h-LC50 (concentration lethal for the 50% of the population in 96 h) for each metal (nominal metal concentrations of Cu: 0.08 µM; Cd: 0.02 µM and Zn: 3 µM). Metal bioaccumulation and toxicity as well as changes in the gene expression of enzymes responsible for ionoregulation and induction of defensive responses were investigated. Furthermore the hepatosomatic index and condition factor were measured as crude indication of overall health and energy reserves. The obtained results showed a rapid Cu and Cd increase in the gills at both temperatures. Cadmium accumulation was higher at 20°C compared to 10°C, whereas Cu and Zn accumulation was not, suggesting that at 20°C, fish had more efficient depuration processes for Cu and Zn. Electrolyte (Ca, Mg, Na, and K) levels were analyzed in different tissues (gills, liver, brain, muscle) and in the remaining carcasses. However, no major electrolyte losses were observed. The toxic effect of the trace metal ion mixture on major ion uptake mechanisms may have been compensated by ion uptake from the food. Finally, the metal exposure triggered the upregulation of the metallothionein gene in the gills as defensive response for the organism. These results, show the ability of common carp to cope with these metal levels, at least under the condition used in this experiment.

Field application of a novel active-passive sampling technique for the simultaneous measurement of a wide range of contaminants in water.

A first test of the field capabilities of a novel in situ sampling technique combining active and passive sampling (APS) was conducted in the sea. The proof-of-concept device uses a pump to draw water into a diffusion cell where dissolved target substances are accumulated onto sorbents which are selective for different classes of contaminants (i.e., metal cations, polar and non-polar organic compounds), simultaneously. A controlled laminar flow established in the diffusion cell enables measurements of contaminant concentrations that are fully independent from the hydrodynamic conditions in the bulk solution. APS measurements were consistent with those obtained using conventional passive sampling techniques such as organic diffusive gradients in thin films (o-DGT) and silicone rubber (SR) samplers (generally < 40% difference), taking into account the prevailing hydrodynamic conditions. The use of performance reference compounds (PRC) for hydrophobic contaminants provided additional information. Field measurements of metal ions in seawater showed large variability due to issues related to the device configuration. An improved field set-up deployed in supplementary freshwater mesocosm experiments provided metal speciation data that was consistent with passive sampling measurements (DGT), taking into account the hydrodynamic conditions. Overall, the results indicate that the APS technique provides a promising approach for the determination of a wide range of contaminants simultaneously, and independently from the hydrodynamic conditions in the bulk solution.

A comparative study on the effects of three different metals (Cu, Zn and Cd) at similar toxicity levels in common carp, Cyprinus carpio.

To improve our understanding of underlying toxic mechanisms, it is important to evaluate differences in effects that a variety of metals exert at concentrations representing the same toxic level to the organism. Therefore, the main goal of the present study was to compare the effects of waterborne copper (Cu(II)), zinc (Zn(II)) and cadmium (Cd (II)) on a freshwater fish, the common carp (Cyprinus carpio), at concentrations being 0%, 25%, 50% and 100% of the 96 h LC50 (the concentration which is lethal to 50% of the population in 96 h). All the exposures were performed for a period of 1 week at 20°C. Our results show a rapid increase in the amount of copper and cadmium accumulated in the gills, while zinc only started to increase by the end of the experiment. All three metal ions increased metallothionein gene expression in both gills and liver. However, clear adverse effects were mainly observed for the Cu exposed group. Cu caused a decrease in Na level in gill tissue; it altered the expression of genes involved in ionoregulation such as Na+ /K+ -ATPase and H+ -ATPase as well as the expression of oxidative stress-related genes, such as catalase, glutathione reductase and glutathione S-transferase. Zinc and cadmium exposure did not alter the ion levels in the gills. In addition, no obvious effect of oxidative stress was observed, except for a transient increase in glutathione reductase at the highest cadmium concentration.

Do Aptamers Always Bind? The Need for a Multifaceted Analytical Approach When Demonstrating Binding Affinity between Aptamer and Low Molecular Weight Compounds.

In this manuscript, we compare different analytical methodologies to validate or disprove the binding capabilities of aptamer sequences. This was prompted by the lack of a universally accepted and robust quality control protocol for the characterization of aptamer performances coupled with the observation of independent yet inconsistent data sets in the literature. As an example, we chose three aptamers with a reported affinity in the nanomolar range for ampicillin, a ß-lactam antibiotic, used as biorecognition elements in several detection strategies described in the literature. Application of a well-known colorimetric assay based on aggregation of gold nanoparticles (AuNPs) yielded conflicting results with respect to the original report. Therefore, ampicillin binding was evaluated in solution using isothermal titration calorimetry (ITC), native nano-electrospray ionization mass spectrometry (native nESI-MS), and 1H-nuclear magnetic resonance spectroscopy (1H NMR). By coupling the thermodynamic data obtained with ITC with the structural information on the binding event given by native nESI-MS and 1H NMR we could verify that none of the ampicillin aptamers show any specific binding with their intended target. The effect of AuNPs on the binding event was studied by both ITC and 1H NMR, again without providing positive evidence of ampicillin binding. To validate the performance of our analytical approach, we investigated two well-characterized aptamers for cocaine/quinine (MN4), chosen for its nanomolar range affinity, and l-argininamide (1OLD) to show the versatility of our approach. The results clearly indicate the need for a multifaceted analytical approach, to unequivocally establish the actual detection potential and performance of aptamers aimed at small organic molecules.

Toxicity and bioaccumulation of Cadmium, Copper and Zinc in a direct comparison at equitoxic concentrations in common carp (Cyprinus carpio) juveniles.

The individual toxicity and bioaccumulation of cadmium, copper and zinc for common carp juveniles was evaluated in a direct comparison in two experimental setups. First, fish were exposed for 10 days to different metal concentrations in order to link metal bioaccumulation to LC50 values (concentration lethal to 50% of the animals) and incipient lethal levels (ILL, concentration where 50% survives indefinitely). Accumulated metals showed a positive dose dependent uptake for cadmium and copper, but not for zinc. Toxicity was in the order cadmium>copper>zinc with 96h LC50 values for cadmium at 0.20±0.16 µM, for copper at 0.77±0.03 µM, and for zinc at 29.89±9.03 µM respectively. For copper, the 96h exposure was sufficient to calculate the incipient lethal level and therefore 96h LC50 and ILL levels were the same, while for cadmium and zinc 5 to 6 days were needed to reach ILL resulting in slightly lower values at 0.16 µM and 28.33 µM respectively. Subsequently, a subacute exposure experiment was conducted, where carp juveniles were exposed to 2 equitoxic concentrations (10% and 50% of LC50 96 h) of the three metals for 1, 3 and 7 days. Again a significant dose-dependent increase in gill cadmium and copper, but not in zinc, was observed during the 7-day exposure. Copper clearly affected sodium levels in gill tissue, while zinc and cadmium did not significantly alter any of the gill electrolytes. The overall histopathological effects (e.g. hyperemia and hypertrophy) of the metal exposures were mild for most of the alterations. Our study showed that copper an cadmium (but not zinc) showed dose dependent metal accumulation, however this bioaccumulation was only correlated with mortality for cadmium. Metal specific alterations were reduced gill sodium levels in copper exposed fish and oedema of the primary epithelium which typically occurred in both levels of zinc exposure.

The synergistic toxicity of Cd(II) and Cu(II) to zebrafish (Danio rerio): Effect of water hardness.

We have evaluated the interactive toxicity of Cu(II) and Cd(II) in water with different hardness levels using adult zebrafish (Danio rerio). Zebrafish were exposed to Cd(II) (0.2-22 µM) or Cu(II) (0.1-8 µM) in single or binary exposures in very soft, moderately hard or very hard water. The whole body burdens of Cd(II) and Cu(II) reflect the net effect of biouptake and elimination, mortality was the indicator of toxicity, and whole body major ion content was measured to assess ion regulatory functions. Cu(II) was found to be more toxic than Cd(II) for zebrafish, and Cu(II) and Cd(II) exhibited a significant synergistic effect. The toxicity of metal ions increased upon decreasing the ionic strength of the exposure medium, probably due to elevated competition between metal ions with other cations in hard water and increased activity of Ca2+ pathways in soft water treatments. Whole body metal accumulation and the accumulation rate of both Cu and Cd increased as the metal ion concentration in the exposure medium increased. Nevertheless, neither parameter explained the observed synergistic effect on mortality. Finally, we observed a significant loss of whole body Na+ in fish which died during the metal exposure compared to surviving fish, irrespective of exposure conditions. Such an effect was not observed for other major cations (K+, Ca2+ and Mg2+). This observation suggests that, under the applied exposure conditions, survival was correlated to the capacity of the organism to maintain Na+ homeostasis.