Microplastics from agricultural mulch films: Biodegradation and ecotoxicity in freshwater systems.

The application of bio-based biodegradable mulch films in agriculture has raised environmental concerns regarding their potential impacts on adjacent freshwater ecosystems. This study investigated the biodegradation of microplastics derived from a bio-based biodegradable mulch (bio-MPs) and its acute and chronic ecotoxicity considering relevant scenarios (up to 200 and 250 mg/kg of sediment, using pristine and/or UV-aged particles), using the fungus Penicillium brevicompactum and the dipteran Chironomus riparius as model organisms, respectively, due to their ecological relevance in freshwater environments. Fourier-transform infrared spectroscopy analysis suggested changes in the fungus's carbohydrate reserves and bio-MP degradation through the appearance of low molecular weight esters throughout a 28 day biodegradation test. In a short-term exposure (48 h), C. riparius larvae exposed to pristine or UV-aged bio-MPs had up to 2 particles in their gut. Exposure to pristine bio-MPs decreased larval aerobic metabolism (<20 %) and increased neurotransmission (>15 %), whereas exposure to UV-aged bio-MPs activated larval aerobic metabolism (>20 %) and increased antioxidant defences (catalase activity by >30 % and glutathione-s-transferase by >20 %) and neurotransmission (>30 %). Longer-term (28-d) exposure to UV-aged bio-MPs did not affect larval survival and growth nor the dipteran's emergence but increased male numbers (>30 %) at higher concentrations. This study suggests that the selected agricultural bio-based mulch film is prone to biodegradation by a naturally occurring fungus. However, there is a potential for endocrine disruption in the case of prolonged exposures to UV-aged microplastics. This study emphasises the importance of further research to elucidate the potential ecological effects of these plastic products, to ensure effective management practices, and to establish new regulations governing their use.

PPCPs abatement using TiO2-based catalysts by photocatalytic oxidation and ozonation: The effect of nitrogen and cerium loads on the degradation performance and toxicity impact.

Pharmaceutical and personal care products (PPCPs) have been consumed in great extension and most of these are found in water bodies, owing to the inefficiency of conventional wastewater treatments. To face against these recalcitrant contaminants, advanced oxidation processes such as photocatalysis and ozonation have been studied. Moreover, the combination of these technologies can improve the degradation of PPCPs, reducing the ozone consumption and the effluent toxicity with the presence of photocatalysts. In particular, this study aimed to evaluate the effects of different N and Ce loads in co-doping TiO2 catalysts on the efficiency of photocatalytic oxidation and photocatalytic ozonation for PPCPs abatement, as well as on the resultant toxicity to aquatic species. Different radiation sources (UVA and solar radiation) were considered for the photocatalytic oxidation. A mixture of 5 PPCPs: paracetamol, sulfamethoxazole, carbamazepine, methylparaben and propylparaben was used as a model synthetic effluent. Photocatalysis showed a low efficiency on the PPCPs removal (<20 %), which was not affected by the radiation source. In general, the tested catalysts showed no or low added-value for reducing the toxicity of the synthetic effluent. Concerning photocatalytic ozonation, the lowest N amount (2.5 % w/w) promoted the best results for PPCPs removal, achieving values up to 100 % with significant reduction of ozone dose compared to photolytic ozonation. In general, photocatalytic ozonation showed better ecotoxicological performance than single photocatalysis. Compared to single photolytic ozonation, a benefitial effect was observed for two aquatic species, using a specific catalyst. This catalyst, prepared by doping TiO2 with 2.5 % w/w N and 1.2 % w/w Ce, showed to be the most promisong one, with potential to be used in photocatalytic ozonation. Hence, this work highlights the potential role of N and Ce co-doped TiO2-based catalysts in photocatalytic ozonation for wastewater treatment.

The role of biomass elemental composition and ion-exchange in metal sorption by algae.

The use of macroalgae, microalgae and cyanobacteria for metal sorption has been widely reported. Still, there are no studies allowing a direct comparison of the performance of these biomasses, especially while evaluating metal competition. The simultaneous sorption of Co2+, Cu2+, Ni2+ and Zn2+ present in a multi-elemental solution by six macroalgae, two microalgae and three cyanobacteria was evaluated. Brown macroalgae were shown to be the most promising biosorbent, with Undaria pinnatifida having a total metal sorption capacity of 0.6 mmol g-1. Overall, macroalgae performed better than microalgae, followed by cyanobacteria. Carboxyl groups were identified as being the main functional groups involved in metal sorption, and all biomass samples were found to be selective to Cu2+. This was linked not only to its higher complexation constant value with relevant functional groups when compared to the remaining metals, but also the Irving-Williams series. The release of K+ and Ca2+ to the aqueous solution during the metal sorption was followed. The obtained results suggest they are readily exchanged with metals in the solution, indicating the occurrence of an ion-exchange mechanism in metal sorption by most biomass. Red macroalgae are an exception to the reported trends, suggesting that their metal sorption mechanism may differ from the other biomass types.

COVID-19 Lockdown: Impact on Oral Health-Related Behaviors and Practices of Portuguese and Spanish Children.

This study aimed to assess and compare the impact of COVID-19 pandemic lockdowns on the oral health attitudes, dietary habits and access to dental care of Portuguese and Spanish children. A cross-sectional observational study involving caregivers of 3−17-year-old children who had cohabited during a COVID-19 pandemic lockdown in Spain and Portugal was conducted. Caregivers completed an online anonymous questionnaire. Aiming groups comparison, chi-square test was used for qualitative variables. 770 surveys were obtained. Significant changes in the children's routine were higher in Portugal (p < 0.001). Both countries showed a large percentage of children who had between 2−3 snacks between meals (p < 0.001) and a higher consumption of snacks was particularly noticed among Spanish children with untreated dental caries during the lockdown (p = 0.003). Most caregivers reported children's oral hygiene habits did not suffer noteworthy alterations (p = 0.417), although parental supervision of toothbrushing was associated with dental attendance during the lockdown. The majority of the sample had no dental attendance during confinement. Confinement appears to have not markedly affected the oral health status and habits of the majority of these children, although an important impact of some demographic and behavioral factors upon dietary and oral care/habits was detected.

Ozonation of Selected Pharmaceutical and Personal Care Products in Secondary Effluent-Degradation Kinetics and Environmental Assessment.

The efficiency of ozonation depends on the water matrix and the reaction time. Herein, these factors were addressed by assessing the removal of five pharmaceutical and personal care products (PPCPs) by ozonation. The main aims were: (i) to assess the effects of the water matrix on the degradation kinetics of PPCPs, individually and in mixture, following ozonation; and (ii) to assess the ecotoxicological impact of the ozone reaction time on the treatment of a spiked municipal wastewater (MW) added the five PPCPs over several species. The degradation of the PPCPs was faster in ultrapure water, with all PPCPs being removed in 20 min, whereas in the MW, a 30 min ozonation period was required to achieve a removal close to 100%. Increasing the number of PPCPs in the water matrix did not affect the time required for their removal in the MW. Regarding the ecotoxicity assessment, Raphidocelis subcapitata and Daphnia magna were the least sensitive species, whereas Lemna minor was the most sensitive. The temporal variation of the observed effects corroborates the degradation of the added PPCPs and the formation of toxic degradation by-products. The removal of the parent compounds did not guarantee decreased hazardous potential to biological species.

Recovery of bacterioruberin and proteins using aqueous solutions of surface-active compounds.

Haloarchaea microorganisms are little explored marine resources that can be a promising source of valuable compounds with unique characteristics, due to their adaptation to extreme environments. In this work, the extraction of bacterioruberin and proteins from Haloferax mediterranei ATCC 33500 was investigated using aqueous solutions of ionic liquids and surfactants, which were further compared with ethanol. Despite the good performance of ethanol in the extraction of bacterioruberin, the use of aqueous solutions of surface-active compounds allowed the simultaneous release of bacterioruberin and proteins in a multi-product process, with the non-ionic surfactants being identified as the most promising. The optimum operational conditions allowed a maximum extraction yield of 0.37 ± 0.01 mgbacterioruberin gwet biomass -1 and 352 ± 9 mgprotein gwet biomass -1 with an aqueous solution of Tween® 20 (at 182.4 mM) as the extraction solvent. In addition, high purities of bacterioruberin were obtained, after performing a simple induced precipitation using ethanol as an antisolvent to recover the proteins present in the initial extract. Finally, a step for polishing the bacterioruberin was performed, to enable solvent recycling, further closing the process to maximize its circularity.

Do sediment-bound nickel and lead affect chironomids life-history? Toxicity assessment under environmentally relevant conditions.

Metal pollution in aquatic ecosystems translates into increased concentrations of sediment-bound metals, representing a risk for benthic species. This risk might be enhanced in soft and moderately hard waters, world widely distributed, due to the protective role of hardness against metal toxicity. As lead (Pb) and nickel (Ni) are amongst the more abundant metals in aquatic systems, and since their combined effects to benthic species have been overlooked, in this study we aimed to investigate the life-cycle toxicity of Pb and Ni (using spiked sediment) to the benthic species Chironomus riparius, considering both single and mixture exposures, in moderately hard water. Environmentally relevant concentrations of each metal were used (25 and 75 mg kg-1, based on a scenario of pollution by runoff waters from burnt forests), following a full factorial design. Effects of the mixture with the highest metal concentrations (Pb 75 mg kg-1 dw + Ni 75 mg kg-1 dw) were also assessed in the second generation. In the first generation, exposure to Pb increased emergence and the weight of males, and decreased time to emergence of both males and females. Conversely, exposure to Ni delayed female emergence and decreased the weight of imagoes. Summarizing, Pb affected more endpoints but showed an apparent positive effect, whereas Ni affected less endpoints but exhibited adverse effects. Reproduction was not affected by these metals. In the second generation, the mixture Pb 75 mg kg-1 + Ni 75 mg kg-1 dw delayed emergence and reduced the emerged female fraction and their weight. These results highlight that Pb and Ni can alter the structure of C. riparius populations at environmentally relevant concentrations, which signals potential repercussions in the dynamics and functioning of freshwater ecosystems under these contamination scenarios. The findings of the present study are relevant not only for metal-polluted environments, in general, but also for fire-affected ecosystems.

Cyanobacteria as Candidates to Support Mars Colonization: Growth and Biofertilization Potential Using Mars Regolith as a Resource.

Cyanobacteria are indicated as organisms that can possibly support Mars colonization, contributing to the production of oxygen and other commodities therein. In this general context, the aim of this work was to evaluate the ability of three species of cyanobacteria (Anabaena cylindrica, Nostoc muscorum, and Arthrospira platensis) and a green microalga (Chlorella vulgaris) to grow using only the resources existing in Mars, i.e., water and Martian regolith stimulant (MGS-1), under an Earth-like atmosphere. A Martian regolith extract was produced and used as a culture medium to grow these species. Their growth was assessed during a period of 25 days, using optical density and fluorometric parameters. After this period, the possible contribution of end-of-life cyanobacteria/microalga as biofertilizing agents was also assessed, using the macrophyte Lemna minor as a vegetable model. Among the three species, N. muscorum showed the best growth performance when compared to the other species, while A. platensis and C. vulgaris were not able to thrive on Mars regolith extract. Therefore, N. muscorum should be the target of future studies not only due to their role in oxygen production but also due to their possible use as a food source, as many members of the Nostoc genus. Cyanobacteria and microalgae (A. platensis and C. vulgaris) showed good abilities as biofertilizing agents, i.e., they stimulated biomass (i.e., dry weight) production at levels comparable to the plants that grew on standard synthetic medium. The highest yield was reached with A. platensis, while the lowest was achieved using the media with N. muscorum. FTIR-ATR (Fourier transform infrared with attenuated total reflectance) spectroscopy showed that the differences between the plants grown on media with or without Martian regolith seem to be related mainly to polysaccharides.

Automated Counting of Daphnid Neonates, Artemia Nauplii, and Zebrafish Eggs: A Proof of Concept.

In aquatic invertebrate (e.g., daphnids and Artemia sp.) and zebrafish cultures, in ecotoxicological bioassays, or when addressing complex population-level experimental designs, the counting of an organism's progeny is often required. This counting process is laborious, repetitive, and time-consuming, potentially posing health hazards to the operators, and necessarily entailing a higher likelihood of human error. We present an experimental evaluation of a computer-based device for counting neonates (Daphnia magna, Daphnia longispina, and Ceriodaphnia sp.), nauplii (Artemia salina and Artemia franciscana), and zebrafish (Danio rerio) eggs. Manual counts by an experienced technician were compared with the corresponding automated counts achieved by the computer-based counting device. A minimum of 55 counts/species was performed, with the number of counted organisms being up to a maximum of 150 neonates of Ceriodaphnia dubia, 200 neonates of D. magna and D. longispina, 200 nauplii of A. franciscana and A. salina, and 500 zebrafish eggs. Manual and automated counts were both performed in culture medium solutions of 50 ml of volume. Automated counts showed a mean relative acccuracy of 98.9% (97.9%-99.4%) and a relative standard deviation of 1.72%. The results demonstrate that the computer-based device can be used for accurately counting these aquatic organisms. This computer-based counting might be extended to other organisms of similar size, thus facilitating reproduction and life-cycle ecotoxicity tests. Environ Toxicol Chem 2022;41:1451-1458. © 2022 SETAC.

Bio-Based Solar Energy Harvesting for Onsite Mobile Optical Temperature Sensing in Smart Cities.

The Internet of Things (IoT) fosters the development of smart city systems for sustainable living and increases comfort for people. One of the current challenges for sustainable buildings is the optimization of energy management. Temperature monitoring in buildings is of prime importance, as heating account for a great part of the total energy consumption. Here, a solar optical temperature sensor is presented with a thermal sensitivity of up to 1.23% °C-1 based on sustainable aqueous solutions of enhanced green fluorescent protein and C-phycocyanin from biological feedstocks. These photonic sensors are presented under the configuration of luminescent solar concentrators widely proposed as a solution to integrate energy-generating devices in buildings, as windows or façades. The developed mobile sensor is inserted in IoT context through the development of a self-powered system able to measure, record, and send data to a user-friendly website.

A review on polycyclic aromatic hydrocarbons distribution in freshwater ecosystems and their toxicity to benthic fauna.

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds, found ubiquitously in all environmental compartments. PAHs are considered hazardous pollutants, being of concern to both the environmental and human health. In the aquatic environment, PAHs tend to accumulate in the sediment due to their high hydrophobicity, and thus sediments can be considered their ultimate sink. Concurrently, sediments comprise important habitats for benthic species. This raises concern over the toxic effects of PAHs to benthic communities. Despite PAHs have been the subject of several reviews, their toxicity to freshwater benthic species has not been comprehensively discussed. This review aimed to provide an overview on PAHs distribution in freshwater environments and on their toxicity to benthic fauna species. The distribution of PAHs between sediments and the overlying water column, given by the sediment-water partition coefficient, revealed that PAHs concentrations were 2 to 4 orders of magnitude higher in sediments than in water. The sediment-water partition coefficient was positively correlated to PAHs hydrophobicity. Toxicity of PAHs to benthic fauna was addressed through Species Sensitivity Distributions. The derived hazardous concentration for 5% of the species (HC5) decreased as follows: NAP (376 µg L-1) > PHE > PYR > FLT > ANT (0.854 µg L-1), varying by 3 orders of magnitude. The hazardous concentrations (HC5) to benthic species were inversely correlated to the hydrophobicity of the individual PAHs. These findings are pertinent for environmental risk assessment of these compounds. This review also identified future challenges regarding the environmental toxicity of PAHs to freshwater benthic communities, namely the need for updating the PAHs priority list and the importance of comprehensively and more realistically assess the toxicity of PAHs in combination with other stressors, both chemical and climate-related.

Synthesis of Purine-Based Ionic Liquids and Their Applications.

Bio-based ionic liquids (ILs) are being increasingly sought after, as they are more sustainable and eco-friendly. Purines are the most widely distributed, naturally occurring N-heterocycles, but their low water-solubility limits their application. In this work, four purines (theobromine, theophylline, xanthine, and uric acid) were combined with the cation tetrabutylammonium to synthesize bio-based ILs. The physico-chemical properties of the purine-based ILs were characterized, including their melting and decomposition temperatures and water-solubility. The ecotoxicity against the microalgae Raphidocelis subcapitata was also determined. The ILs show good thermal stability (>457 K) and an aqueous solubility enhancement ranging from 53- to 870-fold, in comparison to their respective purine percursors, unlocking new prospects for their application where aqueous solutions are demanded. The ecotoxicity of these ILs seems to be dominated by the cation, and it is similar to chloride-based IL, emphasizing that the use of natural anions does not necessarily translate to more benign ILs. The application of the novel ILs in the formation of aqueous biphasic systems (ABS), and as solubility enhancers, was also evaluated. The ILs were able to form ABS with sodium sulfate and tripotassium citrate salts. The development of thermoresponsive ABS, using sodium sulfate as a salting-out agent, was accomplished, with the ILs having different thermosensitivities. In addition, the purine-based ILs acted as solubility enhancers of ferulic acid in aqueous solution.

Overview of Chemotaxis Behavior Assays in Caenorhabditis elegans.

Environmental pollution related to anthropogenic pressures, and the associated repercussions on public health, represent a worldwide problem. Thus, the study of the effects that environmental contaminants can pose to natural ecosystems and human health is of vital importance. Laboratory model organisms such as Caenorhabditis elegans have played a significant role in clarifying multilevel effects of those agents. Although the evaluation of contaminant effects at the behavioral level of organisms is an emerging approach in ecotoxicology, studies assessing chemotaxis behavior in C. elegans within the ecotoxicological research context are still scarce. Chemotaxis studies in C. elegans have contributed to the understanding of both the neuronal mechanisms involved in the behavioral effects triggered by environmental cues and the impact of contaminants on natural ecosystems. Its compact and well-characterized nervous system, as well as the availability of transgenic strains and molecular tools, allows a detailed examination of behavioral, molecular, and genetic chemosensation mechanisms. This overview provides a summary and general comparison of methods used to measure chemotaxis behavior in C. elegans, with the aim of helping researchers select the most suitable approach in their chemotaxis studies. We compare methods based on the type of chemical tested, advantages and drawbacks of the different approaches, and specific experimental goals. Lastly, we hope to encourage the evaluation of C. elegans chemotaxis behavior in ecotoxicology studies, as well as its potential integration in standardized protocols assessing environmental quality. © 2021 Wiley Periodicals LLC.

Measurement of the Effects of Metals on Taxis-to-Food Behavior in Caenorhabditis elegans.

Chemosensation in nematodes is linked to processes that affect their ability to survive, such as the search for food and the avoidance of toxic substances. Since the 1970s, numerous studies have assessed chemotaxis in the nematode species Caenorhabditis elegans, focusing on a multitude of agents, including bacteria (food), ions, salts, hormones, volatile organic compounds, and, to a lesser extent, metal-contaminated medium/food. The few studies evaluating metal exposure have reported a variety of responses (neutral, attraction, avoidance), which generally appear to be contaminant and/or concentration specific. Differences in experimental designs, however, hinder appropriate comparison of the findings and attainment of firm conclusions. Therefore, we herein propose and describe a detailed protocol for the assessment of the effects of metals on taxis-to-food behavior in C. elegans. Distinct approaches are proposed in two innovative stages of testing to (1) screen metals' effects on taxis-to-food behavior and (2) classify the behavioral response as attraction/avoidance/indifference or preference. Use of such a standard protocol will allow for easy comparison across studies and direct interpretation of results. Findings using this model system can contribute to a deeper understanding of the real risks of metal contamination to nematodes and how such contaminants could impact ecosystems in general, given the key environmental roles that these organisms play. © 2021 Wiley Periodicals LLC. Basic Protocol: Assessing the effects of metal contamination on taxis-to-food behavior in Caenorhabditis elegans Support Protocol 1: Synchronization of C. elegans by hand-picking gravid worms Support Protocol 2: Synchronization of C. elegans by using a bleaching solution.

Effects of post-fire contamination in sediment-dwelling species of riverine systems.

Wildfires are an important environmental problem in forested watersheds and can significantly alter water quality. Besides the reported ecotoxicological effects on pelagic species, the accumulation of post-fire contaminants in river sediments can also impair the benthic species. In this study, three sediment-dwelling species, Chironomus riparius, Atyaephyra desmarestii and Echinogammarus meridionalis, with different sensitivities, habitats, behaviours and/or feeding strategies, were exposed to water and sediments, in in-situ and in laboratory. Four sites were selected in a partially burnt basin (Alfusqueiro river basin), within and upstream the burnt area. The sites within the burnt area showed higher metal burden in both water and sediment, as well as changes in water physico-chemistry, consistently with the typical effects of incoming post-fire runoff. Both in-situ and laboratory exposures to water and sediments affected by the wildfire induced post-exposure feeding inhibition in the three tested macroinvertebrates. In fact, laboratory and field bioassays have produced generally consistent post-exposure feeding inhibition responses, but the most impactful response could be recognised after in-situ bioassays at the river site within the burnt area, where the species respond to the physico-chemical fluctuations during the exposure period. This comparative perspective supports the importance of using in-situ bioassays as a more realistic approach when dealing with complex and intermittent natural samples such as those affected by post-fire runoff. Overall, our results reinforce the awareness about the negative effects of wildfires on benthic biota, with significant feeding depression and consequent reduction in the available energy budget to ensure successful detoxification, growth and reproduction signalling potential trophic and functional disruption at the ecosystem level. In addition, the duality conditions of sediments as a sink and source of contaminants reinforce concerns, as the exposure of benthic organisms may persist in the long term, even after runoff income ceases due to the resuspension of contaminated sediments.

Testing the response of benthic diatom assemblages to common riverine contaminants.

Benthic diatoms constitute keystone assemblages in riverine ecosystems, and their structure is used to support regulatory water quality assessment. However, no standard ecotoxicological tests exist using integrated responses of the benthic diatom assemblages. This work aimed to assess whether benthic diatom assemblages are responsive to different riverine contaminants through a previously developed rapid toxicity test, supporting future attempts towards its standardization and integration in both prospective and retrospective Environmental Risk Assessment (ERA) schemes. We selected two benthic diatoms assemblages likely responding similarly to pollution (similar IPS diatom index score), collected from two rivers in Northern-Central Portugal (sites: Palhal and Cabreia). Fresh whole diatom assemblages were exposed for 48 h to five model contaminants (glyphosate, imidacloprid, SDS, CuSO4, and Pb). At the end of the test, changes induced by the exposures in overall yield and in the yield of each diatom genus were assessed. The assemblage collected at Palhal was invariably more responsive and sensitive than that collected at Cabreia, both considering overall and genus-specific yields, regardless of the tested contaminant. Achnanthes, Fragilaria and Navicula were the most responsive genus, regardless of the tested contaminant or assemblage. The distinct response profiles observed for the two assemblages to the same contaminants at the same concentration ranges suggest that using this test method to support prospective ERA is inadequate. However, the method can be an asset supporting retrospective ERA, as the responses seem to be shaped by the interplay of resilience drivers promoted by the local conditions, e.g. adaptive changes in assemblage structure.

New insights on the effects of ionic liquid structural changes at the gene expression level: Molecular mechanisms of toxicity in Daphnia magna.

Knowledge on the molecular basis of ionic liquids' (ILs) ecotoxicity is critical for the development of these designer solvents as their structure can be engineered to simultaneously meet functionality performance and environmental safety. The molecular effects of ILs were investigated by using RNA-sequencing following Daphnia magna exposure to imidazolium- and cholinium-based ILs: 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl) and cholinium chloride ([Chol]Cl)-; the selection allowing to compare different families and cation alkyl chains. ILs shared mechanisms of toxicity focusing e.g. cellular membrane and cytoskeleton, oxidative stress, energy production, protein biosynthesis, DNA damage, disease initiation. [C2mim]Cl and [C12mim]Cl were the least and the most toxic ILs at the transcriptional level, denoting the role of the alkyl chain as a driver of ILs toxicity. Also, it was reinforced that [Chol]Cl is not devoid of environmental hazardous potential regardless of its argued biological compatibility. Unique gene expression signatures could also be identified for each IL, enlightening specific mechanisms of toxicity.

Assessment of hazardous property HP 14 using ecotoxicological tests: a case study of weathered coal fly ash.

The classification of wastes regarding hazardous property HP 14 (ecotoxicity) is essential for proper waste management. In the EU, HP 14 has been estimated based on waste chemical composition rather than using biotests, and guidelines for experimental assessment are still lacking. This study aims at evaluating the potential ecotoxicological impacts of weathered coal fly ash (CFA) from a landfill, as a case study to assess the current EU methodology used to classify wastes regarding HP 14. A large amount of CFA is still landfilled, but its valorisation would be of interest. The analysis was based on the chemical composition of CFA (in ClassifyMyWaste software), and on a battery of five biotests applied to eluates, with Lepidium sativum, Aliivibrio fischeri, Raphidocelis subcapitata, Lemna minor and Daphnia magna. Through chemical analysis, most of the simulations with data from the literature indicated "Possible Hazard", including the sample of this work. Biotests revealed low impairment for most endpoints. D. magna was the most sensitive organism, but the inhibitory effect was significantly reduced after pH adjustment of the eluate. The test with A. fischeri does not seem to be adequate to assess CFA due to the high variability observed in results. The methodology involving a simple battery of bioassays was proven to be enlightening, providing relevant results for HP 14 assessment. The chosen battery of biotests (excluding the A. fischeri test) may be a good starting point to represent the aquatic environment in this context. In short, it seems that weathered CFA can be considered non-hazardous, and therefore the material under analysis could be valorised in practical applications without significant ecotoxic effect on the environment.

Wildfire effects on two freshwater producers: Combining in-situ and laboratory bioassays.

Mediterranean forests are highly susceptible to wildfires, which can cause several impacts not only within burnt areas but also on downstream aquatic ecosystems. The ashes' washout from burnt areas by surface runoff can be a diffuse source of toxic substances, such as metals, when reaching the nearby aquatic systems, and can be noxious to aquatic organisms. The present work aimed at assessing the ecotoxicological effects of post-fire contamination on two aquatic producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor) through in-situ bioassays, validating the obtained results with the outcomes of laboratory bioassays with surface water collected simultaneously. Four distinct sites were selected in a basin partially burnt (Ceira river basin; Coimbra district, Portugal) for bioassay deployment: one site upstream the burnt area in the Ceira river (RUS); three sites located under the influence of the burnt area, one immediately downstream of the burnt area in the Ceira river (RDS) and the other two in tributary streams within the burnt area (BS1 and BS2). The in-situ bioassays lasted for 13 days and began following the first post-fire major rain events. Results showed that the microalgae growth rate was able to distinguish the three sites within and downstream of the burnt area (BS1, BS2, RDS) from the site upstream (RUS). By contrast, the macrophytes growth rate only allowed to differentiate between the sites within the burnt area (BS1 and BS2) and those up- and downstream of the burnt area (RUS and RDS). The in-situ results for both species were corroborated with the results of the laboratory experiments, supporting the use of laboratory surrogates for a screening assessment of wildfire impacts in aquatic ecosystems. Direct causal relationships between the observed ecotoxicological effects on R. subcapitata and L. minor and the physical-chemical parameters of the water samples were difficult to establish, although the results suggest (i) a role of differential major and trace metal load in explaining species growth variation; (ii) interaction between metals and/or between metals and other field parameters are likely to modulate the biological responses to the challenges deriving from wildfire runoff.