Use of scanning and image recognition technology to semi-automate larval development assessment in toxicity tests with a tropical copepod.

There is a high demand for the development of reliable chronic toxicity tests using tropical marine species for subsequent use in tropical risk assessment. However, many chronic test endpoints can be laborious and time-consuming to assess, particularly if the endpoints require measurements of individuals (e.g. growth, size) or advanced taxonomic expertise (e.g. differentiating between larval development stages). In this study, we used scanning and image recognition (SIR) technology to develop and validate a chronic toxicity test with larvae of the tropical euryhaline copepod, Acartia sinjiensis. Optimisation steps are described, and included egg age, and effect of algal food type and salinity on toxicity. Comparisons were made between traditional endpoints measured using microscopy and those measured using SIR. Traditional endpoints of larval development ratio (LDR) and survival achieved using microscope examination and SIR were almost identical (R2 = 0.96-0.97). Additional endpoints made possible by SIR included larval development index (LDI; based on the number of animals at different stages of development), and a range of size measurements (e.g. surface area, perimeter and length) for individual animals and for total populations (i.e. a proxy for biomass). The SIR-derived endpoints were based on measurements that had concentration-dependant responses to tested toxicants (copper, nickel, ammonia), and were a sub-set of the full range of metrics provided by the software. Toxicity values based on SIR-measurements were similar to or more sensitive than the traditional LDR endpoint. SIR technology provides a major opportunity to improve and modernise larval development tests for a range for species, but comes at a cost of increased data size and complexity. Therefore, as a research tool, SIR has significant advantages over traditional microscope methods, but for routine toxicity testing, SIR incorporation into invertebrate toxicity testing will benefit from further improvements to the associated software and data management systems.

Application of Daphnia magna for Nanoecotoxicity Study.

Daphnia magna (D. magna), an aquatic invertebrate, is the most commonly used test organism in ecotoxicological studies on manufactured nanomaterials (MNs). Although standard protocols for undertaking acute and chronic toxicity tests of dissolved chemicals with D. magna have been endorsed by several national and international organizations, comprehensive guidance on aquatic toxicology testing of MNs with D. magna are in their infancy. Here we describe the acute and chronic toxicity methods with D. magna as test organism for nanoecotoxicity study. These methods may provide reference for the next step toward developing prescriptive aquatic toxicity standard tests for MNs with D. magna.

Assessment of the single and mixture ecotoxicity of pharmaceuticals of environmental concern using aquatic test organisms / Avaliação da ecotoxicidade individual e das misturas de fármacos de preocupação ambiental usando organismos-teste aquáticos

Pharmaceuticals are contaminants of emerging concern which have been a target of increasing attention by the scientific community. Pharmaceuticals presenting high consumption, incomplete metabolism and incomplete removal at wastewater treatment plants have been frequently detected in aquatic ecosystems worldwide. This is the case of the pharmaceuticals metformin (MET), bisoprolol (BIS), sotalol (SOT) and ranitidine (RAN). However, ecotoxicity data for these contaminants are scarce, especially regarding behavior effects and chronic toxicity. In addition, the knowledge regarding the joint toxicity of these pharmaceuticals on non-target organisms is still incipient, which makes their environment risk assessment uncertain. This study aimed to fill these knowledge gaps for these four pharmaceuticals, by carrying out toxicity tests using five test organisms from three trophic levels. Different endpoints were assessed in tests with Raphidocelis subcapitata (algae), Lemna minor (macrophyte), Daphnia similis (crustacean), Hydra attenuate (cnidarian) and Danio rerio (fish). The binary and quaternary mixture acute toxicity for these pharmaceuticals were assessed on D. similis and D. rerio embryo tests, respectively. This study also aimed to evaluate the predictive accuracy of the Concentration addition (CA) and the Independent action (IA) classic models. In addition, the nature of the possible toxicological interactions between the pharmaceuticals in binary mixtures were also evaluated, using the Combination Index-isobologram (CI) method. The modelling of the concentration-response curves and the associated statistical analyses were performed using the automated spreadsheet ToxCalcMix v.1.0 and the software OriginPro 2015. The software CompuSyn was used for performing the mixture analyses with the CI method. The experimental planning of the binary mixture tests was performed using the fractioned factorial design, in order to cover several possible ratio and level-dependent effects with a reduced number of test organisms. The results obtained in this study are shown in four articles. In article 1, we provided a critical review and discussed the misunderstandings, deficiencies and data gaps on the ecotoxicity data of pharmaceuticals and personal care products mixtures published in the literature. In the following articles, the results obtained from the single and mixture toxicity tests performed in this study were presented and discussed. The pharmaceuticals MET (article 2) and BIS (article 3) were classified as hazardous to the aquatic environment, in the acute toxicity category. However, an ecological risk is not expected for the pelagic freshwater species exposed to these two pharmaceuticals, based on the chronic data obtained. The results obtained from the mixture toxicity tests (article 4) showed that most of the observed toxicity effects from the binary mixtures were in the zone between the predicted effects by the CA and IA models. The CI model showed to be an useful tool to describe the possible toxicological interactions occurring between the pharmaceuticals in joint action. Even statistically significant non-effect concentrations of the pharmaceuticals added up to induce significant adverse effects in mixtures (something from nothing). It was concluded that ecological risk assessment based on single toxic effects can underestimate the real impact of environmental contaminants on aquatic ecosystems

A contaminação ambiental por fármacos tem sido alvo de crescente preocupação pela comunidade científica. Fármacos de elevado consumo, incompleto metabolismo e remoção incompleta em estações de tratamento de esgoto, como é o caso da metformina (MET), bisoprolol (BIS), sotalol (SOT) e ranitidina (RAN), têm sido frequentemente detectados em matrizes aquáticas do mundo todo. Apesar disso, dados ecotoxicológicos consistentes para esses contaminantes são escassos, principalmente com relação a efeitos comportamentais e oriundos de estudos crônicos. Além disso, o entendimento dos efeitos de suas ações combinadas em organismos não-alvo é ainda incipiente, o que gera incertezas na avaliação dos seus riscos ambientais. Esta pesquisa teve por objetivo preencher essas lacunas de conhecimentos para esses quatro fármacos, por meio da realização de testes com cinco diferentes organismos-teste de três diferentes níveis tróficos. Foram analisados diferentes parâmetros avaliativos em testes com os organismos aquáticos Raphidocelis subcapitata (alga), Lemna minor (macrófita), Daphnia similis (crustáceo), Hydra attenuata (cnidário) e Danio rerio (peixe). As toxicidades agudas das misturas binárias e quaternárias desses quatro fármacos também foram avaliadas em testes com D. similis e embriões de D. rerio, respectivamente. Este trabalho também teve por objetivo avaliar a acurácia preditiva dos modelos de adição de concentração (CA) e ação independente (IA) e analisar a natureza das possíveis interações toxicológicas entre os fármacos, em misturas binárias, usando o modelo do Índice de Combinação (CI). A modelagem das relações concentração-resposta e as análises estatísticas associadas foram realizadas empregando-se a planilha automatizada ToxCalcMix versão 1.0 e o software OriginPro 2015. O software CompuSyn foi utilizado para as análises envolvendo o CI. O planejamento experimental dos testes de misturas binárias foi realizado por meio do design fatorial fracionado, a fim de cobrir diversas possíveis interações em várias proporções e níveis de efeitos, com a redução do número de organismos-teste. Os resultados desta pesquisa estão apresentados em quatro artigos. No artigo 1, realizou-se uma revisão crítica com relação às lacunas de conhecimentos e deficiências identificadas a partir da análise da literatura sobre a ecotoxicologia de misturas de fármacos e de produtos de higiene pessoal. Nos artigos seguintes, foram apresentados e discutidos os resultados oriundos dos testes com os quatro fármacos avaliados neste estudo. Os fármacos MET (artigo 2) e BIS (artigo 3) foram classificados como perigosos para o ambiente aquático, na categoria de toxicidade aguda. Contudo, um risco ecológico não é esperado para as espécies pelágicas de água doce expostas a esses dois fármacos, com base nos dados de toxicidade crônica obtidos. Os resultados dos testes de misturas (artigo 4) permitiram concluir que a maior parte dos efeitos observados das misturas binárias estiveram na zona entre os efeitos preditos pelos modelos clássicos de CA e IA. O modelo do CI mostrou-se uma ferramenta útil para descrever a natureza das possíveis interações toxicológicas que ocorrem entre os fármacos em ações combinadas. Mesmo concentrações de nenhum efeito estatisticamente significativo dos fármacos causaram efeitos adversos significativos quando em misturas (something from nothing). Concluiu-se que avaliações de risco ecológicas baseadas em efeitos tóxicos individuais de contaminantes ambientais podem subestimar o real impacto desses compostos em ecossistemas aquáticos

A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing.

Liver chips have been developed to recapitulate in vivo physiological conditions to enhance hepatocyte functions for assessing acute responses to drugs. To develop liver chips that can assess repeated dosing chronic hepatotoxicity, we need to ensure that hepatocyte functions be maintained at constant values over two weeks in stable culture conditions of sterility, temperature, pH, fluidic-flow of culture media and drugs. We have designed a perfusion-incubator-liver-chip (PIC) for 3D cell culture, that assures a tangential flow of the media over the spheroids culture. Rat hepatocyte spheroids constrained between a cover glass and a porous-ultrathin Parylene C membrane experienced optimal mass transfer and limited shear stress from the flowing culture media; maintained cell viability over 24 days. Hepatocyte functions were significantly improved and maintained at constant values (urea, albumin synthesis, and CYP450 enzyme activities) for 14 days. The chip act as an incubator, having 5% CO2 pressure-driven culture-media flow, on-chip heater and active debubbler. It operates in a biosafety cabinet, thus minimizing risk of contamination. The chronic drug response to repeated dosing of Diclofenac and Acetaminophen evaluated in PIC were more sensitive than the static culture control.

Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance.

Evidence suggests that short-term inhalation studies may provide comparable prediction of respiratory tract toxicity to 90-day studies, presenting the opportunity to save time and resources in screening inhalation toxicity of test substances. The aim of this study was to develop a short-term inhalation test that could be employed to provide early evidence on respiratory tract effects which might occur from long-term exposure to aerosols of nano-materials. Male Wistar rats were exposed to aerosols of 0 (control), 2, 10 and 50 mg/m(3) nano-titanium dioxide (TiO2) by inhalation for 6 h/day for 5 days. Necropsies were performed either immediately after the last exposure or after 3 and 16 days post exposure (study days 5, 8 and 21, respectively). Treatment with nano-TiO2 resulted in morphological changes in the lung, with 50 mg/m(3) nano-TiO2 producing an increase in lung weight. Lung inflammation was associated with dose-dependent increases in bronchoalveolar lavage fluid (BALF) total cell and neutrophil counts, total protein content, enzyme activities and levels of a number of cell mediators. No indications of systemic effects could be found by measurement of appropriate clinical pathology parameters. Cell replication (determined by incorporation of 5-bromo-2'-deoxyuridine) was increased at all nano-TiO2 dose levels in large/medium bronchi and terminal bronchioles. The effects on the parameters measured were most prominent either on study day 5 or 8, with some endpoints returning to control levels by day 21. Overall, the pulmonary effects of nano-TiO2 observed in this short-term study were comparable to those previously reported in subchronic inhalation studies.

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 1: technical description.

In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints.

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 2: toxicological evaluation.

The goal of replacement, refinement and reduction of animal testing is critically dependent on the development and assessment of novel in vitro methodologies and the further development of existing methodologies. Here, we evaluated the use of a modified perfusion cell culture apparatus for application to chronic in vitro nephrotoxicity testing using DMSO, SDS, paracetamol and cyclosporine A as test compounds. Renal epithelial monolayers were cultured on microporous growth supports and exposed to test compounds under static or perfusion conditions. Alamar Blue reduction, gamma-glutamyl transpeptidase activity (GGT), lactate dehydrogenase activity (LDH) and remnant protein were used to assay cell toxicity. There was no significant difference in IC(50) values between static and perfusion cultures up to 72 hours exposure. However, the perfusion system allowed continuous real-time monitoring of plasma membrane damage, which gives important information of time, duration and scale of toxicity. The complexity of the system restrains its use to low-throughput analysis. However, the real and theoretical advantages of this and similar systems merit further investigations.