A.M.M. Gonçalves

Designing ionic liquids: the chemical structure role in the toxicity

Ionic liquids (ILs) are a novel class of solvents with interesting physicochemical properties. Many different applications have been reported for ILs as alternatives to organic solvents in chemical and bioprocesses. Despite the argued advantage of having low vapor pressure, even the most hydrophobic ILs show some degree of solubility in water, allowing their dispersion into aquatic systems and raising concerns on its pollutant potential. Moreover, nowadays most widespread notion concerning the ILs toxicity is that there is a direct relationship with their hydrophobicity/lipophilicity. This work aims at enlarging the currently limited knowledge on ILs toxicity by addressing negative impacts in aquatic ecosystems and investigating the possibility of designing hydrophobic ILs of low ecotoxicity, by the manipulation of their chemical structures. The impact of aromaticity on the toxicity of different cations (pyridinium, piperidinium, pyrrolidinium and imidazolium) and hydrophobic anions (bis(trifluoromethylsulfonyl)imide [NTf2] and hexafluorophosphate [PF6]) was analysed. Concomitantly, several imidazolium-based ILs of the type [CnCmCjim][NTf2] were also studied to evaluate the effects of the position of the alkyl chain on the ILs’ toxicity. For that purpose, standard assays were performed using organisms of different trophic levels, Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna, allowing to evaluate the consistency of the structure–activity relationships across different biological targets. The results here reported suggest the possibility of designing ILs with an enhanced hydrophobic character and lower toxicity, by elimination of their aromatic nature.

Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient

The Ecological Risk Assessment of pesticides requires data regarding their toxicity to aquatic and terrestrial non-target species. Such requirements concern active ingredient(s), generally not considering the noxious potential of commercial formulations. This work intends to contribute with novel information on the effects of short-term exposures to two herbicides, with different modes of action (Spasor®, Stam Novel Flo 480®), and an insecticide (Lannate®), as well as to corresponding active ingredients (Glyphosate, Propanil and Methomyl, respectively). The microalga Pseudokirchneriella subcapitata (growth inhibition), the cladoceran Daphnia magna (immobilisation), and the earthworm Eisenia andrei (avoidance behaviour) were used as test species. Both herbicides were innocuous to all test organisms at environmentally realistic concentrations, except for Stam and Propanil (highly toxic for Pseudokirchneriella; moderately toxic to Daphnia). Lannate and Methomyl were highly toxic to Daphnia and caused Eisenia to significantly avoid the spiked soil at realistic application rates. The toxicity of formulations either overestimated (e.g. Stam/Propanil for P. subcapitata) or underestimated (e.g. Stam/Propanil for D. magna) that of the active ingredient.

Environmental safety of cholinium-based ionic liquids: assessing structure-ecotoxicity relationships

Ionic liquids (ILs) are innovative solvents that can be tuned for their specific application through the selection, or functionalization, of the cation and the anion. Although the cation has been assumed as the main driver of toxicity, the importance of the anion must not be underestimated. This study considers a series of cholinium based ILs aiming at assessing the effects of the functionalization of the cation and the anion on their ecotoxicity. These effects were assessed using three biological models, the microalgae Raphidocelis subcapitata, the macrophyte Lemna minor and the cladoceran Daphnia magna, representing aquatic ecosystems, a major putative recipient of ILs due to their high water solubility. Since the toxicity trends fluctuated depending on the biological model, the results were integrated with previous data through a species sensitivity distribution approach in an attempt to provide a useful safety variable for the design of eco-friendlier ILs. The results reported here challenge some heuristic rules previously proposed for the design of ILs, in particular in what concerns the side-chain effect for the cholinium ILs, and the notion that cholinium-based ILs are inherently safe and less environmentally hazardous than most conventional solvents. Moreover, it was confirmed that structural changes in the ILs promote differences in toxicity highlighting the importance of the role of the anion in their toxicity. Different biological systems yielded different toxicity trends across the IL series tested, also distinct from previous data retrieved with the bacteria V. fischeri; such a novel integration effort challenges the suitability of establishing structure–ecotoxicity relationships for cholinium-based IL design. Overall, this study reinforces the need to perform complete ecotoxicological characterisation before assuming ILs as suitable, environmentally compatible, alternative solvents.

Sustainable design for environment-friendly mono and dicationic cholinium-based ionic liquids.

Cholinium-based ionic liquids are receiving crescent interest in diverse areas of application given their biological compatibility and potential for industrial application. In this work, mono and dicationic cholinium ionic liquids as well as cholinium derivatives were synthesized and their toxicity assessed using the luminescent bacteria Vibrio fischeri. A range of cholinium derivatives was synthesized, using different amines and the correspondent brominated derivatives, through the alkylation of the amine with the halide in MeCN. The results indicate that their toxicity is highly dependent on the structural modifications of the cholinium cation, mainly related to the alkyl side or linkage chain length, number of hydroxyethyl groups and insertion of carbon-carbon multiple bonds. The data indicated that it is possible to perform environmentally advantageous structural alterations, namely the addition of double bonds, which would not negatively affect V. fischeri. Moreover, the dicationic compounds revealed a significantly lower toxicity than the monocationic counterparts. The picture emerging from the results supports the idea that cholinium derivatives are promising ionic liquids with a low environmental impact, emphasizing the importance of a careful and directed design of ionic liquid structures.

The effectiveness of a biological treatment with Rhizopus oryzae and of a photo-Fenton oxidation in the mitigation of toxicity of a bleached kraft pulp mill effluent.

Huge efforts have been made both in adopting more environmental-friendly bleaching processes, and in developing advanced oxidation processes and more effective biological treatments for the reduction of deleterious impacts of paper mill effluents. Even so, the success of such treatments is frequently reported in terms of chemical parameters without a proper evaluation of the effluents toxicity mitigation. This is the first study reporting an exhaustive evaluation of the toxicity of a secondary bleached kraft pulp mill effluent, after either tertiary treatment with the soft-rot fungi Rhizopus oryzae or with a photo-Fenton oxidation, using a battery of freshwater species. As it has been reported the photo-Fenton/UV treatment has proved to be the most effective in reducing the colour and the COD (chemical oxygen demand) of the effluent. Nevertheless, extremely low EC(50) values were reported for almost all species, after this tertiary treatment. The treatment with R. oryzae was less effective in terms of colour removal and COD reduction, but proved to be the most promising in reducing toxicity.

Spatial and temporal distribution of harpacticoid copepods in Mondego estuary

Seasonal and spatial variations in the composition and relative contribution of the harpacticoid species in the Mondego estuary (western Portugal) were studied based on a monthly sampling along a salinity gradient. These benthic harpacticoids were collected in the water column by means of a 63-mm plankton net. The influence of hydrological parameters (temperature, salinity, oxygen dissolved concentration, pH, turbidity, chlorophyll-a, total suspended solids and nutrient concentrations) was analysed by means of a redundancy data analysis. In addition, this is the first study to provide a checklist of benthic harpacticoids from this estuary. In total, 13 species plus six species not yet identified but known to belong to the genus Canuella, Microsetella, Ectinosoma, Mesochra, Harpacticus and Parapseudoleptomesochra were identified. Copepodites and adults of Euterpina acutifrons and Paronychocamptus nanus were most abundant in this harpacticoid community. While P. nanus dominated in winter and spring at upstream stations, E. acutifrons dominated more downstream. Although this species occurred along the whole salinity gradient, its highest abundances were found in the north arm of the estuary, except in autumn. This distribution may be due to the adaptability of E. acutifrons to different environments and the intermediate position between marine and estuarine conditions assigned to this species. Paraleptastacus cfr. spinicauda showed a relative occurrence of 5–10% in all stations, except at the mouth of the estuary (M). Tachidius discipes was found in low densities in Mondego estuary in spite of the large numbers of copepodites of this species in the southern arm, characterized by a high level of total suspended solids. The northern arm of the estuary was characterized by a higher numerical occurrence of harpacticoid organisms throughout the study years, with the higher densities towards the freshwater part (upstream areas). Canuella sp., Ectinosoma sp. (copepodite), Ectinosoma melaniceps, Leptocaris brevicornis, Phyllognathopus viguieri, Microsetella norvegica (copepodite) and Macrosetella gracilis (copepodite) were considered rare species. Nonetheless harpacticoid species were represented by a higher number of adults in the northern arm, and juveniles in the southern arm.

Acute and chronic toxicity of Betanal(®)Expert and its active ingredients on nontarget aquatic organisms from different trophic levels.

As a way to improve the efficacy to target organisms, new pesticide generation is based on technologically advanced coformulations of two or more active ingredients. One example is Betanal®Expert, a postemergence herbicide composed of an Advanced Micro Droplet coformulation of phenmedipham, desmedipham, and ethofumesate. Although its composed formulation brings an increase in the pesticide performance, it can also enhance its toxicity to nontarget species. Therefore, the present study intends to contribute with relevant information on ecotoxicological effects of Betanal®Expert and its active ingredients on a battery of bioassays using aquatic species from different trophic levels: bacteria (Vibrio fischeri), microalgae (Pseudokirchneriella subcapitata, Chlorella vulgaris, and Chlamydomonas pseudocostata), macrophyte (Lemna minor), and cladocerans (Daphnia magna and Daphnia longispina) species. Across the organisms tested and endpoints measured, different responses concerning the toxicity of the active ingredients were found: (i) phenmedipham was the most toxic to V. fischeri and L. minor; (ii) desmedipham was the most toxic to P. subcapitata, D. magna, and D. longispina; (iii) and ethofumesate was the most toxic to C. pseudocostata and C. vulgaris. Furthermore, for C. pseudocostata and daphnids, the toxicity observed for some active ingredients was higher than the toxicity of the commercial formulation. In fact, in an attempt to evaluate the contribution of each active ingredient to the overall toxicity of Betanal®Expert, it was observed that, in general, the toxicity values obtained for desmedipham and phenmedipham were close or even lower to the values determined for Betanal®Expert, indicating that the ethofumesate can act as an antagonist in the three‐way coformulation. In spite of the most impaired species being the photosynthetic ones, this study also showed pernicious effects on nonphotosynthetic organisms with distinct target sites. Therefore, our results underline the importance of clarifying the mode of action and metabolic pathways of these compounds on nonphotosynthetic species.

Biochemical and toxicological effects of organic (herbicide Primextra(®) Gold TZ) and inorganic (copper) compounds on zooplankton and phytoplankton species.

In Europe, mainly in the Mediterranean region, an intensive usage of pesticides was recorded during the past 30 years. According to information from agricultural cooperatives of the Mondego valley (Figueira da Foz, Portugal), Primextra ® Gold TZ is the most used herbicide in corn crop fields and one of the 20 best-selling herbicides in Portugal. Copper is mainly used in pesticide formulations. This study aims to determine the ecotoxicological and biochemical (namely fatty acid profiles) effects of the herbicide Primextra® Gold TZ and the metal copper on marine plankton. The organisms used in this study are three planktonic species: the marine diatom Thalassiosira weissflogii, the estuarine copepod Acartia tonsa and nauplii of the marine brine shrimp Artemia franciscana. Fatty acids (FAs) are one of the most important molecules transferred across the plant-animal interface in aquatic food webs and can be used as good indicators of stress. The conducted lab incubations show that T. weissflogii is the most sensitive species to the herbicide followed by A. tonsa (EC50=0.0078 mg/L and EC50=0.925 mg/L, respectively), whereas the copepod was the most sensitive species to the metal followed by T. weissflogii (EC50=0.234 mg/L and EC50=0.383 mg/L, respectively). A. franciscana was the most tolerant organism both to the herbicide and to the metal (EC50=20.35 mg/L and EC50=18.93 mg/L, respectively). Changes in the FA profiles of primary producer and primary consumers were observed, with the increase of saturated FA and decrease of unsaturated FA contents, especially of highly unsaturated FAs that can be obtained mainly from food and therefore are referred to as „essential FA‟. The study suggests that discharges of Primextra ® Gold TZ or other pesticides mainly composed by copper may be a threat to plankton populations causing changes in the FA contents and thus in their nutritive value, with severe repercussions for higher trophic levels and thus the entire food web.

Ecotoxicological effects of Mikado® and Viper® on algae and daphnids

The toxicity of single and combined formulated herbicides (Mikado® and Viper®) was assessed on several endpoints in species from two trophic levels: algae growth—Pseudokirchneriella subcapitata and Chlorella vulgaris—immobilization and life‐history traits (only for single compound toxicity) of daphnids—Daphnia longispina and Daphnia magna. Viper was the most toxic formulated herbicide. It was hypothesized that the toxicity of both formulated herbicides could have been enhanced by adjuvants, especially for Viper. In most cases, the sublethal endpoints were the most sensitive and affected by both formulations, comparatively to their acute effects. Concentration addition (CA) and independent action (IA) models provided an accurate description of Mikado and Viper joint action on algae growth and immobilization of daphnids, although significant deviations were always detected. A low‐dose antagonism and high‐dose synergism were identified for P. subcapitata, whereas C. vulgaris response deviated antagonistically from CA and synergistically from IA. For both daphnids, however, synergistic effects were observed for higher mixture concentrations. Under a regulatory standpoint, CA provided the most conservative estimation either because the mixture effects were overestimated or less subestimated than IA. Overall, the great sensitivity differences observed within species did not allow the conclusion that one trophic level was more tolerant than the other. Instead, P. subcapitata was always the most sensitive species to both herbicide formulations, followed by D. longispina, while D. magna and C. vulgaris were the most tolerant species. On a whole, further studies are needed toward a comprehensive understanding of herbicides mode of action, their effects at lower biological‐level endpoints, and under different mixture designs.

Spatial and temporal distribution of microplastics in water and sediments of a freshwater system (Antuã River, Portugal)

Microplastics (particles with a size<5mm), one of the most emerging aquatic pollutants, are of particular concern since they can reach high densities and interact with biotic and abiotic environment. The occurrence of microplastics in freshwater systems is less understood than in marine environment. Hence, the present study aims to provide new insights into microplastics abundances and distribution in Antuã River (Portugal) by applying the isolation method of wet peroxide oxidation with addition of zinc chloride to water and sediment samples collected in March and October 2016, in three sampling sites. The abundance of microplastics in water ranged from 5 to 8.3mgm-3 or 58-193itemsm-3 in March and from 5.8-51.7mgm-3 or 71-1265itemsm-3 in October. In sediments, the abundance ranged from 13.5-52.7mgkg-1 or 100-629itemskg-1 in March and from 2.6-71.4mgkg-1 or 18-514itemskg-1 in October. The water and sediment samples with the greatest abundances were from São João da Madeira and Aguincheira, respectively. Spatio-temporal distribution showed different pattern according to methodological approaches, seasonal and hydrodynamic conditions and the proximity to urban/industry areas. Analysis of plastics by Fourier transform infrared spectroscopy underline polyethylene and polypropylene as the most common polymer types identified in this work. The low medium high oxidation ratio was 56:22:22 (%) in March and 61:31:8 (%) in October. Foams and fibers were the most abundant type in São João da Madeira, while fibers and fragments were the most abundant in Aguincheira and Estarreja in water and sediment samples, respectively. This study emphasizes the importance of rivers as carriage systems of microplastics. Further studies should be performed to identify point sources in order to mitigate the microplastics contamination in aquatic systems.