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Phthalates, such as di-n-butyl phthalate (DBP) and di-isopentyl phthalate (DiPeP), are pollutants with a high potential for endocrine disruption. This study aimed to evaluate parameters of endocrine disruption in specimens of the Neotropical fish Rhamdia quelen exposed to DBP and DiPeP through their food. After 30 days of exposure, the fish were anesthetized and then euthanized, and blood, hypothalamus, liver, and gonads were collected. DBP caused statistically significant alterations in the serotoninergic system of males (5 and 25 ng/g) and females (5 ng/g) of R. quelen and it increased testosterone levels in females (25 ng/g). DiPeP significantly altered the dopaminergic system in females, reduced plasma estradiol levels (125 ng/g) and hepatic vitellogenin expression (25 ng/g), and changed the antioxidant system in gonads (125 ng/g). The results suggest that DBP and DiPeP may have different response patterns in females, with the former being androgenic and the latter being anti-estrogenic. These findings provide additional evidence regarding the molecular events involving DBP and DiPeP in the endocrine disruption potential in juvenile specimens of Rhamdia quelen.

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Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.

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The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

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Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73-0.90; R2FET48h = 0.79-0.90; R2FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70-0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68-0.74 and R2FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

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Diisopentyl phthalate (DiPeP) is a plasticizer with significant offer and application in Brazilian industries. This is attributed to its origin, which is closely linked to the refining process of sugarcane for ethanol production in the country. In this work, we developed a model for trophic exposure to environmentally relevant doses (5, 25, and 125 ng/g of DiPeP) to identify possible target tissues and toxic effects promoted by subchronic exposure to DiPeP in a Neotropical catfish species (Rhamdia quelen). After thirty days of exposure, blood, liver, kidney, brain, and muscle were collected and studied regarding DNA damage in blood cells and biochemical analyses. The kidney was the most affected organ, as in the head kidney, genotoxicity was evidenced in all groups exposed to DiPeP. Besides, the caudal kidney showed a reduction in the superoxide dismutase and glutathione peroxidase activities as well as a reduced glutathione concentration. In the liver, exposure to 125 ng/g of DiPeP increased glutathione S-transferase activity and reduced glutathione levels. In muscle, acetylcholinesterase (AChE) was reduced. However, in the brain, an increase in AChE activity was observed after the exposure to lowest doses. In contrast, a significant reduction of brain AChE activity after exposure to the highest dose was detected. The pronounced genotoxicity observed in head kidney cells is of concern, as it may compromise different functions performed by this organ (e.g., hematopoiesis, immune and endocrine functions). In our study, DiPeP proved to be a compound of environmental concern since we have evidenced its nephrotoxic and neurotoxic potential even in low doses.

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One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.

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Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

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Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

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Alkylphenols ethoxylates are industrial surfactants, and the release in the environmental matrices produces degraded products, of which nonylphenol (NP) and octylphenol (OP) were the most common. They can be classified as endocrine disruptors since the estrogenic potential is widely recognized, but some others toxic aspects are in discussion. This study aimed to evaluate the toxicity of NP, OP, and mixtures of both through cellular, biochemical and genetic biomarkers in fish gonadal cell line RTG-2 exposed to nominal concentrations of 0.05; 0.5; 5; 50, and 100 µg mL-1 of each chemical and their mixtures of 0.05, 0.5; 5 µg mL-1 concentrations. After 24 h, the cells were collected for cytotoxic (neutral red - NR; crystal violet - CV, resazurin assay - RA and lactate-dehydrogenase - LDH), antioxidant system (glutathione-s-transferase - GST; superoxide-dismutase - SOD; glutathione-peroxidase - GPx and malondialdehyde - MDA) and genotoxic assays (alkaline comet assay and Fpg-modified alkaline comet assay). The chemicals and their mixtures were cytotoxic at 50 and 100 µg mL-1, in general aspect, but LDH showed cytotoxicity since 0.05 µg mL-1. The GST and SOD showed an activity increase trend in most tested groups, while GPx decreased at 5 µg mL-1 of the mixture. The MDA increase in all groups resulted in lipid peroxidation. The reactive oxygen species caused DNA damage for all groups. The tested chemicals and concentrations have been found in the freshwater systems. They can induce cell toxicity in several parameters that could impair the gonadal tissues considering the RTG-2 responses.

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Aluminum chlorohydrate (ACH) is a major aerosol component frequently used as the active ingredient in antiperspirants, and in vivo studies have raised a concern about its inhalation toxicity. Still, few studies have addressed its effects on the human respiratory tract. Therefore, we developed a study on ACH inhalation toxicity using an in vitro human alveolar cell model (A549 cells) with molecular and cellular markers of oxidative stress, immunotoxicity, and epigenetic changes. The chemical characterization of ACH suspensions indicated particle instability and aggregation; however, side-scatter analysis demonstrated significant particle uptake in cells exposed to ACH. Exposure of A549 cells to non-cytotoxic concentrations of ACH (0.25, 0.5, and 1 mg/ml) showed that ACH induced reactive oxygen species. Moreover, ACH upregulated TNF, IL6, IL8, and IL1A genes, but not the lncRNAs NEAT1 and MALAT1. Finally, no alterations on the global DNA methylation pattern (5-methylcytosine and 5-hydroxymethylcytosine) or the phosphorylation of histone H2AX (γ-H2AX) were observed. Our data suggest that ACH may induce oxidative stress and inflammation on alveolar cells, and A549 cells may be useful to identify cellular and molecular events that may be associated with adverse effects on the lungs. Still, further research is needed to ensure the inhalation safety of ACH.

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Sulfated polysaccharides (SPs) from seaweeds are potential bioactive natural compounds, but their DNA protective activity is poorly explored. This article aimed to evaluate the genotoxic/antigenotoxic potentials of a sulfated heterofucan from brown seaweed Spatoglossum schröederi (Fucan A - FA) and a sulfated galactan from green seaweed Codium isthomocladum (3G4S) using in vitro Comet assay (alkaline and oxidative versions) with HepG2 cells. The antioxidant activity of these SPs was evaluated by total antioxidant capacity, radical scavenging, metal chelating, and antioxidant enzyme activity assays. Both SPs were not genotoxic. FA and 3G4S displayed strong antigenotoxic activity against oxidizing chemical (H2O2) but not against alkylating chemical (MMS). The DNA damage reduction after a pre-treatment of 72 h with these SPs was 81.42% to FA and 81.38% to 3G4S. In simultaneous exposure to FA or 3G4S with H2O2, HepG2 cells presented 48.04% and 55.41% of DNA damage reduction compared with the control, respectively. The antigenotoxicity of these SPs relates to direct antioxidant activity by blockage of the initiation step of the oxidative chain reaction. Therefore, we conclude that FA and 3G4S could be explored as functional natural compounds with antigenotoxic activity due to their great protection against oxidative DNA damage.

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Melanoma is a type of tumor that originates from melanocytes. Irradiation of melanin with UVA and visible light can produce reactive oxygen species (ROS) such as singlet molecular oxygen (1 O2 ). The objective of this study was to examine DNA damage in melanoma cells (B16-F10) with different melanin contents, subjected to 1 O2 generation. To this end, we used the photosensitizer Rose Bengal acetate (RBAc) and irradiation with visible light (526 nm) (RBAc-PDT). We used the modified comet assay with the repair enzymes hOGG1 and T4 endonuclease V to detect the DNA damage associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers lesions, respectively. We observed increased formation of hOGG1- and T4endoV-sensitive DNA lesions after light exposure (with or without RBAc). Furthermore, 18 h after irradiation, hOGG1-sensitive DNA lesions increased compared to that at the initial time point (0 h), which shows that a high melanin content contributes to post-irradiation formation of them, mainly via sustained oxidative stress, as confirmed by the measurement of ROS levels and activity of antioxidant enzymes. Contrastingly, the number of T4endoV-sensitive DNA lesions decreased over time (18 h). Our data indicate that in melanoma cells, a higher amount of melanin may affect DNA damage levels when subjected to RBAc-PDT.

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Since organic flame retardants (FRs) have several industrial applications, they have been largely detected in environmental and biological samples, and humans have been highly exposed to them. Although the effects of oral and inhaled FRs have been well studied, dermal exposure to them has only recently been pointed out as a potential route of human exposure. Consequently, the effects of FRs on the skin and secondary target organs have been poorly investigated. This review article summarizes the main findings regarding dermal exposure to FRs, points the limitation of the published studies, and suggests future perspectives for better understanding of how dermal exposure to FRs impacts the human health. This review lists some gaps that must be filled in future studies, including characterization of the bioavailable fraction and assessment of exposure for new FRs, to establish their physiological significance and to improve the development of 3D dermal tissue for more reliable results to be obtained.

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Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

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Endocrine-disrupting chemicals (EDCs) are primarily studied regarding endocrine-mediated effects in mammals and fish. However, EDCs can cause toxicity by mechanisms outside the endocrine system, and, as they are released continuously into soils, they may pose risks to terrestrial organisms. In this work, the plant Allium cepa and the earthworm Eisenia foetida were used as test systems to evaluate the toxicity and cyto-/geno-toxicity of three environmental phenols known as EDCs (Bisphenol A - BPA, Octylphenol - OP, Nonylphenol - NP). The tested phenols were evaluated in environmentally relevant concentrations (µg/L) and in single forms and mixture. BPA, OP, and NP did not inhibit the seed germination and root development in A. cepa in their single forms and mixture. However, all single forms of the tested phenols caused cellular and DNA damages in A. cepa, and although these effects persist in the mixtures, the effects were verified at lower levels. These phenols caused acute toxicity to E. foetida after 48 h of exposure and at both conditions evaluated (single forms and mixture); however, unlike A. cepa, in earthworms, mixtures and single forms presented the same level of effects, indicating that interspecies physiological different might influence the mixture toxicity. In summary, our results suggest that BPA, OP, and NP are toxicants to earthworm and cyto-/geno-toxicants to monocotyledonous plants at low concentrations. However, interaction among these phenols reduces the magnitude of their individual effects (antagonistic effect) in the plant test system. Therefore, this study draws attention to the need to raise knowledge about the ecotoxicity of phenolic compounds to help predict their ecological risks and protect non-target terrestrial species.

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Dermal contact is the main route of exposure for most cosmetics; however, inhalation exposure could be significant for some formulations (e.g., aerosols, powders). Current cosmetic regulations do not require specific tests addressing respiratory irritation and sensitisation, and despite the prohibition of animal testing for cosmetics, no alternative methods have been validated to assess these endpoints to date. Inhalation hazard is mainly determined based on existing human and animal evidence, read-across, and extrapolation of data from different target organs or tissues, such as the skin. However, because of mechanistic differences, effects on the skin cannot predict effects on the respiratory tract, which indicates a substantial need for the development of new approach methodologies addressing respiratory endpoints for inhalable chemicals in general. Cosmetics might present a particularly significant need for risk assessments of inhalation exposure to provide a more accurate toxicological evaluation and ensure consumer safety. This review describes the differences in the mechanisms of irritation and sensitisation between the skin and the respiratory tract, the progress that has already been made, and what still needs to be done to fill the gap in the inhalation risk assessment of cosmetic ingredients.

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Lignin is a complex phenolic biopolymer present in plant cell walls and a by-product of the cellulose pulping industry. Lignin has functional properties, such as antioxidant activity, that make it a potential natural active ingredient for health-care products. However, not all safety aspects of lignin fractions have been adequately investigated. Herein, we evaluated the antioxidant and genotoxic potential of two hardwood kraft lignins (F3 and F5). The chemical characterization of F3 and F5 demonstrated their thermal stability and the presence of different phenolic units, while the DPPH assay confirmed the antioxidant activity of these lignin fractions. Despite being antioxidants in the DPPH assay, F3 and F5 were capable of generating intracellular reactive oxygen species (ROS) and subsequently causing oxidative DNA damage (Comet assay) in HepG2 cells. The biological relevance of the DPPH assay might be uncertain in some cases; therefore, we suggest combining in chemico tests with biological system-based tests to determine efficacy and safety levels of lignins and define appropriate applications of lignins for consumer products. Moreover, kraft lignins obtained by acid precipitation may pose risks to human health; however, as genotoxicity is not the sole endpoint of toxicity required in hazard assessments, additional toxicological evaluations are needed.

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Thymol, a monoterpene phenol, is used as a natural biocide. To circumvent its chemical instability, we propose use of thymol-loaded biogenic silica nanoparticles (BSiO2 #THY NPs); however, the toxicity of this system for aquatic organisms is unknown. Thus, the present study aimed to evaluate the toxicogenetic effects induced by thymol, BSiO2 NP, and BSiO2 #THY on Artemia salina and zebrafish (Danio rerio) early life stages. We also investigated the impact of BSiO2 aggregation in different exposure media (saline and freshwater). Based on the median lethal concentration at 48 h (LC5048h ), BSiO2 #THY (LC5048h = 1.06 mg/L) presented similar toxic potential as thymol (LC5048h = 1.03 mg/L) for A. salina, showing that BSiO2 had no influence on BSiO2 #THY toxicity. Because BSiO2 aggregated and sedimented faster in A. salina aqueous medium than in the other medium, this NP had lower interaction with this microcrustacean. Thus, BSiO2 #THY toxicity for A. salina is probably due to the intrinsic toxicity of thymol. For zebrafish early life stages, BSiO2 #THY (LC5096h = 13.13 mg/L) was more toxic than free thymol (LC5096h = 25.60 mg/L); however, BSiO2 NP has no toxicity for zebrafish early life stages. The lower aggregation of BSiO2 in the freshwater medium compared to the saline medium may have enhanced thymol's availability for this aquatic organism. Also, BSiO2 #THY significantly induced sublethal effects as thymol, and both were genotoxic for zebrafish. In conclusion, although BSiO2 #THY still needs improvements to ensure its safety for freshwater ecosystems, BSiO2 NP seems to be a safe nanocarrier for agriculture. Environ Toxicol Chem 2021;40:333-341. © 2020 SETAC.

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Titanium dioxide nanoparticles (TiO2NPs) are widely used and may impact the environment. Thus, this study used a high concentration of TiO2NP (1000 mg/L) to verify the defense mechanisms triggered by a plant system - an indicator of toxicity. Furthermore, this study aimed at completely characterizing TiO2NP suspensions to elucidate their toxic behavior. TiO2NPs were taken up by meristematic cells of Allium cepa, leading to slight inhibition of seed germination and root growth. However, severe cellular and DNA damages were observed in a concentration-dependent manner (10, 100, and 1000 mg/L). For this reason, we used the highest tested concentration (1000 mg/L) to verify if the plant cells developed defense mechanisms against the TiO2NPs and evaluated other evidences of TiO2NP genotoxicity. Nucleolar alterations and plant defense responses (i.e., increased lytic vacuoles, oil bodies and NP phase change) were observed in meristematic cells exposed to TiO2NP at 1000 mg/L. In summary, TiO2NPs can damage the genetic material of plants; however, plants displayed defense mechanisms against the deleterious effects of these NPs. In addition, A. cepa was found to be a suitable test system to evaluate the cyto- and genotoxicity of NPs.

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Pyroligneous acid (PA) is a by-product of bio-oil, which is obtained by pyrolysis of the wood. This product has been tested for use in several areas, such as agriculture, as a promising green herbicide; however, there are few scientific data regarding its environmental impacts. For this study, an ecotoxicity testing battery, composed of Daphnia magna acute toxicity test, Allium cepa test and in vitro Comet assay with the rainbow trout gonad-2 cell fish line (RTG-2) were used to evaluate the acute toxicity and genotoxicity of PA obtained from fast pyrolysis of eucalyptus wood fines. The PA presented acute toxicity to D. magna (microcrustacea) with EC50 of 26.12 mg/L, and inhibited the seed germination (EC50 5.556 g/L) and root development (EC50 3.436 g/L) of A. cepa (higher plant). No signs of genotoxicity (chromosomal aberrations and micronuclei in A. cepa and primary DNA lesions in RTG-2 cells) were detected to this product. The acute toxicity and absence of genotoxicity may relate to the molecules found in the PA, being the phenolic fraction the key chemical candidate responsible for the toxicity observed. In addition, daphnids seem to be more sensitivity to the toxicity of PA than higher plants based on their EC50 values. This first ecotoxicological evaluation of PA from fast pyrolysis pointed out the need of determining environmental exposure limits to promote the safer agriculture use of this product, avoiding impacts to living organisms.

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