Ph.D. Thesis
Use of microalgae to report on the environmental impact of metallic
nanoparticles
Jara Hurtado Gallego
Universidad autónoma de Madrid, December 2019
Doctoral program in: Microbiology
Use of microalgae to report on the environmental impact of metallic nanoparticles
Doctoral thesis presented by:
Jara Hurtado Gallego
Dpto. De Biología, Facultad de ciencias UNIVERSIDAD AUTÓNOMA DE MADRID
Supervisors:
Dr. Francisca Fernández Piñas Dpto. de Biología
Facultad de Ciencias
Universidad Autónoma de Madrid
Dr. Roberto Rosal García Dpto. Química Analítica,
Química Física e Ingeniería Química Universidad de Alcalá
A mis padres, a Zoe y por supuesto, a ti
Llegados a este punto, no es fácil escribir unos agradecimientos en los que estén incluidas todas las personas que a su manera han ayudado a que esta tesis, esta experiencia, salga adelante. En primer lugar, me gustaría agradecer a mis directores de tesis Francisca Fernández Piñas y Roberto Rosal, la oportunidad de haber podido realizar mi tesis en su grupo de investigación. Ambos habéis aportado a esta tesis los conocimientos y la experiencia necesarios para que salga adelante y de vosotros he aprendido a “hacer ciencia”. Gracias también a Susana Cristóbal que, desde la Universidad de Linköping, Suecia, fue un gran apoyo y ejemplo de mujer investigadora. Gracias además a mis evaluadoras internacionales por sacar un huequito para evaluar mi tesis, que se que sois unas mujeres muy ocupadas.
Gracias Paco y Paqui porque gracias a vuestros conocimientos y formas de ser (que, aunque muy diferentes, consiguen formar un ying-yang de libro), habéis formado un grupo de investigación que no sólo es eficiente a la hora de trabajar, sino que somos un grupo de amigos, dispuestos a ayudarnos en lo que haga falta. Gracias a todo el pasillo de Fisio Vegetal por muchos momentos divertidos incluidas fiestas de cumpleaños, fiestas de navidad, fiestas de despedida, fiestas de bienvenida….. muchas fiestas y buen ambiente. Gracias en especial a mis compañeras de prácticas (Cris, Pili, María, Mabel) de las que he aprendido como ser una buena profesora. Gracias a tod@s mis compañer@s de la sala de becari@s donde tantos momentos hemos vivido, alegría, tristeza, nervios pero sobre todo mucho apoyo entre nosotr@s.
Gracias a mi familia por el apoyo, los ánimos y la comprensión en todos los momentos de mi vida académica. Especialmente gracias a mis padres por
incondicionalmente en todos los momentos de mi vida, en esta tesis va un trocito de vosotros.
Gracias a mi pequeña Zoe por enseñarme lo realmente importante.
Finalmente, pero no por ello menos importante, gracias, Ángel por todo tu apoyo y por compartir conmigo todas estas aventuras, sin ti nada de esto hubiera sido posible.
This Thesis was supported by a pre-doctoral FPI contract (FPI 2014 program) from Spanish Ministry of Economy and European Union (Ref.: BES-2014- 070093). Financial support for this Thesis was provided by the research projects:
CTM2013-45775-C2-1-R/2-R and CTM2016-74927-C2-1-R/2-R from Spanish Ministry of Economy.
TABLE OF CONTENTS
SUMMARY ... 1
RESUMEN ... 5
CHAPTER 1: General introduction ... 13
1.1 Emerging pollutants in the aquatic environment... 13
1.1.1 Nanoparticles in the aquatic environment ... 15
1.2 Bioassays in ecotoxicology ... 27
1.3 Biosensors: further evolution of bioassays ... 33
1.3.1 Optical biosensors ... 35
1.3.1.1 Naturally bioluminescent microorganisms used for environmental toxicity ... 35
1.3.1.2 Recombinant microbial bioreporters ... 46
1.3.1.2.1 Oxidative stress bioreporters ... 69
1.4Cyanobacteria and their applications in environmental monitoring ... 74
1.4.1 Cyanobacterial bioreporters ... 77
1.5 References ... 83
OBJECTIVES ... 113
CHAPTER: 2 Construction of cyanobacterial ROS-detecting bioreporters .... 123
2.1: Two novel cyanobacterial bioluminescent whole-cell bioreporters based on superoxide dismutases MnSod and FeSod to detect superoxide anion .. 119
2.1.1 Abstract ... 119
2.1.2 Introduction ... 120
2.1.3 Materials & Methods... 123
2.1.4 Results ... 127
2.1.5 Discussion ... 132
2.1.6 Conclusions ... 139
2.1.7 References ... 140
2.1.8 Supplementary Material ... 144
environment ... 147
2.2.1 Abstract ... 147
2.2.2 Introduction ... 148
2.2.3 Materials and Methods ... 150
2.2.4 Results ... 157
2.2.5 Discussion... 166
2.2.6 Conclusions ... 172
2.2.7 References ... 173
2.2.8 Supplementary Material ... 177
2.2.9 Supplementary references ... 178
CHAPTER 3: Use of cyanobacterial luminescent bioreporters to report on the environmental impact of metallic nanoparticles ... 181
3.1 Abstract ... 181
3.2 Introduction ... 182
3.3 Materials and Methods ... 185
3.4 Results ... 190
3.5 Discussion ... 201
3.6. Conclusions ... 205
3.7 References ... 206
3.8 Supplementary Material ... 210
CHAPTER 4: Toxicity of superparamagnetic iron oxide nanoparticles to the microalga Chlamydomonas reinhardtii ... 215
4.1 Abstract ... 215
4.2 Introduction ... 216
4.3 Materials and Methods ... 218
4.4 Results and Discussion ... 224
4.5 Conclusions ... 240
4.6 References ... 241
4.7 Supplementary Material ... 247
5.1 References ... 262
GENERAL CONCLUSIONS ... 267
CONCLUSIONES GENERALES ... 269
ABBREVIATIONS ... 271
APPENDIX I ... 275
APPENDIX II ... 279
APPENDIX III ... 283
1
SUMMARY
Nanotechnology has become one of the most successful technology nowadays.
Many nanoparticles (NPs) have been synthesized and used in numerous applications including medicine, industry and remediation processes. Due to this increasing production and widespread use of NPs, huge amounts of them are discharged in the aquatic environment daily. Their release in aquatic environment constitutes a potential harmful effect to aquatic organisms.
Among that toxic effect, reactive oxygen species (ROS) production stands out.
Within aquatic organisms the primary producers play an important role since any deleterious effect on them will affect the rest of the trophic chain.
Therefore, the study of the mechanisms of toxic action of NPs in these aquatic organisms is necessary to evaluate the potential ecotoxicological effects of these NPs not only for these organisms but for the rest of the aquatic life.
Several of these ecotoxicological studies are performed by using bioassays with different organisms which give information about the toxicity of a substance (such as NPs). A further evolution of bioassays is the use of bioreporters which allow to know the toxic information with a less laborious, cost-effective and more rapid toxicity bioassays. Furthermore, the biological effect of NPs will change once they are in the aquatic environment considering their possible physical and chemical interactions with co-occurring pollutants and organic matter in the environment; so that, experiments in real matrices are necessary to understand in a real manner NPs toxicity. The overall aim of this Thesis was to address the mechanisms of toxic action of different metallic NPs by focusing on their ROS production and the construction and use of cyanobacterial bioreporters as a useful tool in ecotoxicology.
Chapter 1 is a general introduction which includes some basic concepts about emerging pollutants (EPs) and NPs and their role in aquatic environments. The main characteristics of NPs and their potential mechanisms of action to aquatic
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organisms are described in this section. Furthermore, the use of bioassays and biosensors in ecotoxicity is reviewed, with special focus on ROS-detecting- bioreporters. it also includes a section dedicated to cyanobacteria as environmentally relevant organisms and the cyanobacterial bioreporters constructed to date.
Chapter 2 describes the construction of four luminescent cyanobacterial ROS- detecting bioreporters. In Chapter 2.1 the promoter regions of sodA and sodB genes were fused to luxCDABE genes to construct the ROS-detecting bioreporters Nostoc sp. PCC7120 pBG2154 and Nostoc sp. PCC7120 pBG2165 respectively. In Chapter 2.2 the promoter regions of 2-cys-prx and katA genes were fused to luxCDABE genes to construct the ROS-detecting bioreporters Nostoc sp. PCC7120 pBG2172 and Nostoc sp. PCC7120 pBG2173 respectively. All the bioreporters were characterized both in growth medium and in environmental water samples. The results showed a specific character of Nostoc sp. PCC7120 pBG2154, Nostoc sp. PCC7120 pBG2165 and Nostoc sp. PCC7120 pBG2173 detecting only superoxide anion, while Nostoc sp. PCC7120 pBG2172 was induced by both superoxide anion and hydrogen peroxide. The cyanobacterial bioreporters were able to detect these ROS in pollutants-spiked real water samples although they were less sensitive in polluted waters where the pollutants may suffer a complexation process with the organic matter or other present substances in the water. Their low limits of detection make them the most sensitive ROS-detecting bioreporters constructed to date.
In the last decade whole-cell bioreporters have been used in ecotoxicity studies, giving information not only about the toxicity of different pollutants but furthermore about their bioavailability. In this Thesis (Chapter 3) a battery of cyanobacterial bioreporters has been used to report the toxicity of AgNPs, TiNPs, ZnNPs and CuNPs in aquatic environment. The toxicity of these NPs was measured by using the global toxicity bioreporter Nostoc sp. CPB4337 and
3 calculating their EC50. As one of the mechanisms of toxic action of metallic NPs is the toxicity associated to the release of free-ions, the strain Synechococcus sp.
PCC7942 pBG2120 (capable to detect bioavailable metal ions) was used. The results confirm that the presence of rereleased free-ions was negligible.
However, this strain was capable to detect the metallic NPs per se so giving a new application to this cyanobacterial bioreporter. As oxidative stress is a primary toxic mechanism of metallic NPs, the ROS-detecting bioreporters constructed in Chapter 2 were also used in this study in order to evaluate the ROS formation by the metallic NPs. Firstly, the results confirmed that ROS production was due to the NPs per se and not by the released free-ions. The cyanobacterial bioreporters showed that ROS production varied depending on the growth medium or environmental matrices conditions and on the metallic NPs type. This work demonstrated the different levels of ROS production induced by metallic NPs and the importance of nanotoxicology studies in real environmental matrices.
Superparamagnetic iron nanoparticles (SPION) have become one of the most used NPs due to their magnetic properties. They are used in biomedical fields and remediation processes and they may become widespread in the environment. Several studies have been performed to elucidate SPION toxicity in animal cells but only a few of them have been done to study their ecotoxicity so more studies in relevant organisms of the aquatic environment are necessary.
In Chapter 4 the toxicity and potential mechanisms of toxic action of two SPION to the microalgae Chlamydomonas reinhardtii were evaluated. The results showed a dose-dependent toxicity. The potential mechanisms of action were measured mostly by flow cytometry indicating that both SPION produced ROS generating an oxidative stress which triggered a series of physiological changes as a decrease in metabolic activity and changes in their plasma membrane potential and in their mitochondrial membrane potential. Furthermore, and for
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the first time in this study a shading effect and a clear internalization via endocytosis were observed. These results confirm a potential risk for the environment as the potential concentrations of SPION in the environments may surpass those that cause toxicity.
Finally, Chapter 5 consists of a general discussion highlighting the main findings of the Thesis in the context of a global discussion. The Thesis ends with general conclusions both in English and Spanish.
Overall, this Thesis aims to understand the mechanisms of toxic action of metallic NPs focusing on ROS production which may trigger different alterations in the organisms. Furthermore, novel cyanobacterial ROS-detecting bioreporters have been constructed and characterized as a tool in the study of the ecotoxicity of EPs and therefore NPs in aquatic environments. These new tools contribute to increase the knowledge about the toxicity of pollutants in these environments and aim to solve the surface water contamination problems and a responsible use of nanotechnology.
5
RESUMEN
Hoy en día, la nanotecnología ha llegado a ser una de las tecnologías con más éxito. Muchas nanopartículas (NPs) se han sintetizado y utilizado en numerosas aplicaciones, incluida la medicina, la industria y los procesos de remediación.
Debido a esta creciente producción y al uso generalizado de NPs, se descargan enormes cantidades de ellas al medio ambiente acuático a diario. Esta liberación en el medio ambiente acuático constituye efectos nocivos para los organismos acuáticos. Entre estos efectos tóxicos, se destaca la producción de especies reactivas de oxígeno (EROS). Dentro de los organismos acuáticos, los productores primarios juegan un papel importante ya que cualquier efecto perjudicial sobre ellos afectará al resto de la cadena trófica. Por lo tanto, el estudio de los mecanismos de acción tóxica de las NPs en estos organismos acuáticos es necesario para evaluar los posibles efectos ecotoxicológicos de estos NP no solo para estos organismos sino también para el resto de la vida acuática. Varios de estos estudios ecotoxicológicos se realizan mediante el uso de bioensayos con diferentes organismos que brindan información sobre la toxicidad de un contaminante (como las NPs). Un tipo de bioensayos más evolucionados es el uso de cepas biorreporteras que permiten estudiar la toxicidad de los contaminantes de una manera más sencilla, barata y rápida.
Además, una vez en el medio ambiente, las NPs sufren cambios tanto físicos como químicos debido a las posibles interacciones con los contaminantes o la materia orgánica presentes en él. Estos cambios hacen que varíe su toxicidad para los organismos, por ello son necesarios los experimentos en muestras ambientales de agua para entender de una manera más precisa y real la toxicidad de las NPs en ambientes acuáticos. En general, el objetivo de esta Tesis ha sido abordar los mecanismos de acción tóxica producidos por diferentes NPs metálicas centrándose en la producción de EROS y la construcción y uso de
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cepas biorreporteras basadas en cianobacterias como una útil herramienta en ecotoxicología.
El Capítulo 1 es una introducción general que incluye algunos conceptos básicos sobre contaminantes emergentes y NPs y su papel en ambientes acuáticos. Las características principales de las NPs y sus potenciales mecanismos de acción tóxica se detallan en esta sección. Además, se hace una revisión acerca del uso de bioensayos y biosensores en el mundo de la ecotoxicología haciendo hincapié en las cepas biorreporteras capaces de detectar EROS. También se incluye un apartado dedicado a las cianobacterias como organismos relevantes en el medio ambiente acuático, así como las cepas biorreporteras basadas en cianobacterias construidas hasta la fecha.
El Capítulo 2 describe la construcción de cuatro cepas biorreporteras luminiscentes capaces de detectar EROS basadas en Nostoc sp. PCC7120. En el Capítulo 2.1 las regiones promotoras de los genes sodA y sodB se han fusionado a los genes de luminiscencia luxCDABE para construir las cepas biorreporteras que detectan EROS Nostoc sp. PCC7120 pBG2154 y Nostoc sp. PCC7120 pBG2165 respectivamente. En el Capítulo 2.2 las regiones promotoras de los genes 2-cys-prx y katA se han fusionado a los genes luxCDABE para construir las cepas biorreporteras que detectan EROS Nostoc sp. PCC7120 pBG2172 y Nostoc sp. PCC7120 pBG2173 respectivamente. Todas las cepas biorreporteras han sido caracterizadas tanto en medio de cultivo como en aguas naturales. Los resultados mostraron que las cepas Nostoc sp. PCC7120 pBG2154, Nostoc sp.
PCC7120 pBG2165 y Nostoc sp. PCC7120 pBG2173 detectaron específicamente anión superóxido mientras que la cepa y Nostoc sp. PCC7120 pBG2172 se indujo en presencia tanto de anión superóxido como de peróxido de hidrógeno. Las cepas biorreporteras fueron capaces de detecta las EROS producidas por contaminantes en aguas naturales dopadas con dichos contaminantes, aunque en aguas más contaminadas fueron menos sensibles debido probablemente al
7 acomplejamiento de los contaminantes con la materia orgánica o con otras sustancias presentes en el agua. Los bajos límites de detección de estas cepas biorreporteras hacen de ellas las cepas biorreporteras capaces de detectar EROS más sensibles construidas hasta la fecha.
En la última década, numerosas cepas biorreporteras han sido utilizadas en ecotoxicología, aportando información no solo a cerca de la toxicidad de diferentes contaminantes sino también a cerca de su biodisponibilidad. En esta tesis (capítulo 3) se ha usado una batería de cepas biorreporteras cianobacterianas para evaluar distintos aspectos de la toxicidad de AgNPs, TiNPs, ZnNPs y CuNPs en ambientes acuáticos. La toxicidad de las NPs se midió utilizando la cepa biorreportera Nostoc sp. CPB4337 que mide toxicidad global y a partir de esos resultados se calcularon las EC50 para cada NP. Debido a que uno de los mecanismos de acción tóxica de las NPs metálicas es la liberación de iones metálicos, se utilizó la cepa biorreportera Synechococcus sp. PCC7942 pBG2120 (capaz de detectar iones metálicos). Los resultados confirmaron que la presencia de iones metálicos liberados por las NPs era insignificante. Sin embargo, la cepa fue capaz de detectar a las NPs per se añadiendo una nueva aplicación a esta cepa biorreportera. Como el estrés oxidativo es un mecanismo de acción tóxica primario producido por las NPs metálicas, las cepas biorreporteras capaces de detectar EROS construidas en el Capítulo 2 fueron también utilizadas en este trabajo con el objetivo de evaluar la formación de EROS por las NPs seleccionadas. Por un lado, los resultados confirmaron que la producción de EROS era desencadenada por las NPs per se y no por la presencia de sus iones liberados. Las cepas biorreporteras mostraron que la producción de EROS depende tanto de el tipo de NP como del medio en el que se encuentren dichas NPs. Este estudio demostró las diferentes EROS que son producidas por las diferentes NPs y la importancia en el estudio de la nanotoxicología de hacer experimentos en muestras ambientales reales.
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Superparamagnetic iron nanoparticles (SPION) han llegado a ser una de las NPs más utilizadas debido principalmente a sus propiedades magnéticas que hacen que se utilicen en campos como biomedicina o biorremediación con lo cual están ampliamente distribuidas en el medio ambiente. En la actualidad, muchos estudios se han llevado a cabo para saber más acerca de la toxicidad de las SPION en células animales, en cambio no son tantos los que se han hecho para saber su repercusión en organismos relevantes en el medio ambiente acuático.
en el Capítulo 4 la toxicidad y los potenciales mecanismos de acción tóxica de dos SPION en la microalga Chlamydomonas reinhardtii fueron evaluados mostrando una toxicidad dosis-respuesta. La mayoría de los mecanismos de acción tóxica fueron medidos mediante citometría de flujo indicando que ambas SPION produjeron EROS generando estrés oxidativo que desencadena una serie de cambios fisiológicos como una disminución en la actividad metabólica y cambios en el potencial de membrana plasmática y en el potencial de membrana mitocondrial. Además, y por primera vez, en este estudió se vio un efecto “shading” o de sombreado y una internalización vía endocitosis de las SPION en Chlamydomonas reinhardtii. Estos resultados confirman el riesgo de las SPION en el medio ambiente ya que las concentraciones potenciales de ellas que pueden ser encontradas en el medio ambiente acuático son mayores a las que causan toxicidad en organismos relevantes.
Finalmente, el Capítulo 5 consiste en una discusión general resaltando los resultados de la Tesis incluyendo unas conclusiones generales tanto en inglés como en español.
En general, esta Tesis trata de entender los mecanismos de acción tóxica de las NPs metálicas incidiendo en la producción de EROS que puede desencadenar diferentes alteraciones en los organismos. Además, se han construido y caracterizado unas nuevas cepas biorreporteras basadas en cianobacterias capaces de detectar EROS como nuevas herramientas para estudios
9 ecotoxicológicos de contaminantes emergentes y por tanto de NPs en el medio ambiente acuático. Estas nuevas herramientas contribuyen a un mayor conocimiento acerca de la toxicidad de los contaminantes es estos ambientes y ayuda a solventar los problemas de contaminación de agua y a hacer un uso responsable de la nanotecnología.
CHAPTER: 1
General Introduction
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CHAPTER 1: General introduction
1.1 Emerging pollutants in the aquatic environment
The environment is experiencing the adverse consequences of unbound development of multiple human activities such as industry, transport, agriculture, or urbanisation. Water pollution is a stern worldwide problem that requires the enhancement of monitoring practices and the implementation of treatment and remediation solutions [1]. Every day, 2 million of tonnes of sewage, industrial and agricultural waste are discharged into the world’s water [2]. In the year 2000, the European Union (EU) created the Water Framework Directive (WFD) [3]. This regulation establishes a legislative framework for the protection of n surface waters including rivers, lakes, transitional waters and coastal waters and groundwater throughout the EU. This regulation follows three strategies: a) prevention of further deterioration of aquatic ecosystems, b) promotion of the sustainable water utilization based protection of available water resources and c) to enhance protection and improvement of the aquatic environment through the progressive reduction of discharges of priority substances and the cessation of emissions of priority hazardous substances. The Environmental Quality Standards (EQs) and Maximum Allowance Concentrations (MACs) are a very important factor to improve the water quality.
Despite this regulation, the last decades, increasing emerging pollutants (EPs) have been found in waterbodies [4-6]. EPs can be defined as synthetic or naturally occurring substances, which are not currently included in routine environmental monitoring programs and may be candidates for future legislation due to their adverse effects and/or persistency in aqueous compartments [7]. EPs include a wide range of ubiquitous substances which are indispensable for modern society due to their use in many applications [8]. They
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are categorized into more than 20 classes related to their origin (www.norman- network.net). The principal groups are: pharmaceuticals (urban, stock farming), pesticides (agriculture), disinfection by-products (urban, industry), wood preservation and industrial chemicals (industry) [1].
The aquatic environment receives huge amounts of EPs through different sources ranging from agricultural or industrial discharges [9, 10] to the treated effluents from wastewater treatment plants (WWTPs) [11, 12]. The discharge of WWTPs has been recognized as a major way of entry of EPs in aquatic environments [13]. Furthermore, conventional WWTPs are not specifically designed to remove EPs, so they are continuously released to the environment making them pseudo-persistent pollutants that spread into different compartments even reaching drinking water sources as trace pollutants [14-17].
The term trace pollutant indicates low concentrations of an environmental contaminant normally in the nanogram (ng) or microgram (μg) per litre range.
Some trace pollutants are referred to as EPs because they have recently been detected and are believed to adversely affect human health or the environment such as antibiotics [18] or emerging organic contaminants such as bisphenol A or their degradation by-products [19]. It is challenging to develop a comprehensive list of compounds labelled as EPs because such a list must be dynamic, as new chemicals are continuously developed and produced. Limited data are available regarding the occurrence of EPs in the freshwater environment because of the difficulty and expense involved in analysis.
Sampling and analysis protocols have focused on regulated compounds [20].
The detection, identification and quantification of EPs and their transformation products in the environment is essential for determining their risk. However, because EPs exist at trace levels in the environment, difficulties associated to sampling (grab or passive sample), the interference due to matrix effects, and the need for complex analytical processes are important obstacles to study the
15 environmental occurrence, fate, and effects of EPs [21]. Furthermore, studies about the ecotoxicity of EPs are necessary for evaluating the environmental quality of water bodies. In this sense, ecotoxicity bioassays should be designed to give information about the environmental effects of EPs in organisms potentially exposed to them [22-24]. Toxicity bioassays for EPs are normally carried out using standard tests with freshwater model organisms such as microalgae or fish following toxicity endpoints such as growth inhibition, immobilization, feeding rate, mortality or reproduction, as well as biochemical markers such as pigment content, enzyme activities and many others [25, 26].
The most used toxicity parameters are the lethal concentration (LCx), the effective concentration (ECx) and the inhibitory concentration (ICx) defined as the concentration of a chemical which induce an x-percent of the measured effect on the studied endpoint parameter with respect to the control [27]. The ecotoxicity bioassays make the quality criteria for the protection of aquatic life, through regulatory measures and environmental quality standards [24].
1.1.1 Nanoparticles in the aquatic environment
The rapid growth of nanotechnology converted nanomaterials (NMs) into an important new group of EPs whose potential toxic effects in the environment are still unknown [28, 29]. According to the International Organization for Standardization (ISO), NMs are defined as materials with any external dimension in the nanoscale (size range from approximately 1 to 100 nm) or having internal structure or surface structure in the nanoscale [30]. Nanomaterials include nanoparticles (NPs) which are particles with all external dimensions in the nanoscale where the lengths of the longest and the shortest axes of the nano- object do not differ significantly [31].
The NPs which can be found in the environment may originate by natural, incidental, or industrial processes. Natural NPs include particles from forest fires
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or volcanic emissions as well as those made of humic materials and biological particles such as bacteria and viruses [32]. The anthropogenic sources of NPs are linked to the fabrication of a wide range of products including electronic components, cosmetics, cigarette filters, antimicrobial and stain-resistant fabrics more specifically as batteries, skin-care products, paints, wound dressings, food additives or toothpastes, among many other [33, 34]. Incidental NPs are produced as a side product of anthropogenic processes. Likewise, engineered NPs are intentionally manufactured in order to serve as an ingredient in some product, whereas incidental NPs are unintentionally produced as a by-product of some other activity. For example, driving a gasoline-powered car produces incidental nanoparticles from combustion of the fuel.
Due to their small size, NPs have special physicochemical properties. Firstly, they have considerable larger surface area than similar masses of larger-scale objects and specific charge and shape, which result in larger surface reactivity and higher mobility, as well as different electronic, magnetic, optical and mechanical properties than their bulk counterparts (Figure 1.1).
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Figure 1.1. Different shapes, sizes and surface area of nanoparticles. A) Various gold nanostructures with different potential applications. B) Changes In the colour due to the aspect ratio, shell thickness and/or galvanic displacement and sizes of these gold nanoparticles. C) Changes on surface area as a function of cube size from cm to nanoscale. Adapted from [35]
and [36].
Within a solution (as the aquatic environment), NPs do not form a real solution, but they form colloidal suspensions where they can be uniformly dispersed. This capability of dispersion depends on their electric charge and aggregation. Both electric charge and aggregation of the NPs strongly depend on the medium where they are suspended, influenced therefore by the pH, the ionic strength and temperature of the surrounding medium and the NPs concentration. The electric charge of the NPs has a structure of the electrical double layer (Figure
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1.2). This structure mediates colloidal interactions, regulates surface structure, controls reactivity, sets capacitance, and represents the central element of electrochemical interactions [37]. The first layer around the particle surface is the stern layer: rigid layer of ions tightly bound to the particle. The particle attracts opposite ions which will move closer to the particle. The next layer is the slipping layer: boundary of the stern layer. The ions beyond this layer will not move with the particle as an entity but the ions within this boundary will move with the particle. Finally, the diffuse layer englobes free ions around the particle [38]. The Zeta potential (ζ- potential), is the electric potential of the slipping layer that is, is the potential difference between the mobile dispersion medium and the rigid stern layer of the dispersion medium attached to the dispersed particle. The measure of ζ- potential is related to the suspension stability; a high ζ- potential (both negative and positive) indicates more stability [39]. So that, high ζ- potential produces electrostatic repulsion between adjacent particles. If the charge is high enough, the colloids will remain discrete, disperse and in suspension (stable). In contrast, when the charge is reduced, the particles tend to form aggregates (instable suspension), modifying their properties.
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Figure 1.2. Schematic graphic of the electrical double layer on the surface of a nanoparticle.
The energy potential curve is depicted below where the surface potential is the electrical potential surrounding the particle while the stern potential is the electrical potential at the stern layer. The zeta potential is the electrical potential at the slipping plane. Adapted from [38].
NPs are classically divided into various categories depending on their morphology, size and chemical properties [40]:
- Carbon-based NPs, which englobe the highly used materials like carbon nanotubes and fullerenes. Carbon nanotubes are based in graphene sheets rolled into a tube conferring them a tensile strength about 100 times that of steel for the same weight. Fullerenes are allotropes of carbon having a structure of hollow cage of sixty or more carbon atoms
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having a pentagonal and hexagonal arrangement that confer them a high electrical conductivity and electron affinity.
- Ceramic NPs are inorganic solids made up of oxides, carbides, carbonates and phosphates with a high heat resistance and chemical inertness which allow their use in photocatalysis, photodegradation of dyes, drug delivery, and imaging.
- Metal and metal oxide NPs are synthesised from metal precursors and they have the ability to adsorb small molecules and have high surface reactivity and specific electromagnetic properties such as Surface Plasmon Resonance [41]. These NPs have applications in research areas, detection and imaging of biomolecules, environmental and biomedical applications and even they are present in cosmetics products. For example, due to their magnetic properties, iron-based NPs have been used to remove pollutants from the different environments [42-46] as well as in the biomedical field where they are highly used in contrast imaging or cancer therapy [44]. Silver NPs for example, are widely used in medical applications [47].
- Semiconductor NPs are based in elements which are found in the periodic table mainly in group IV or combinations of group III and group V (called III-V semiconductors), or group II and group VI (called II-VI semiconductors). Semiconductors share properties of both metals and non-metals and find applications in photocatalysis (such as water splitting), and photo-electronics devices [48, 49]. Titanium or Zinc NPs for example are present in many cosmetic products such as sunscreen lotions because they are capable to absorb UV light [50].
- Polymeric NPs are organic based nanoparticles, which, depending on their method of preparation, present different structures including nanocapsules or nanospheres with many different applications. For
21 example, protection of drug molecules, ability to combine therapy and imaging or specific targeting [48].
- Lipidic NPs consist of a solid core made of lipid and a matrix containing soluble lipophilic molecules stabilized by surfactants and emulsifiers [40, 51]. These nanoparticles find application in the biomedical field as a drug carrier [52] or in cancer therapy [53].
NPs have become one of the most studied groups of emerging pollutants due to their widespread use in different applications, which leads to their release to the aquatic environment though different ways. The main routes of entry of these NPs to the aquatic environment correspond to the production and manufacturing of nanomaterial-containing products, the use of nanomaterial- containing products and their fate after disposal in sewage treatment facilities or landfilling [54, 55]. Nevertheless, the main route of entry of NPs to the aquatic environment is the discharge of wastewater effluents due to the fact that WWTPs are generally unable to achieve their complete removal [56].
One persistent problem regarding NPs in the aquatic environment is that it is very difficult to detect them and accurately measure their concentrations.
Various techniques have been developed and have been applied to cope with this problem, such as field-flow fractionation (FFF) combined with inductively coupled plasma mass spectrometry and electron microscopy in transmission or scattering mode [57, 58]. Nonetheless, actual measurements of NPs in an aquatic system are rare. Furthermore, the concentration of a new substance in the environment is not known at the time of its initial risk assessment.
Therefore, expected concentrations have to be estimated with the help of extrapolations and analogies [59]. A variety of studies have created different models to determine the predicted environmental concentrations (PECs) of NPs and understand the NPs transformations and fate in the environment. PEC for
22
several important NPs are summarized in Table 1.1 (adapted from Maurer-Jones et al., 2013 [60]).
Table 1.1 Predicted Environmental Concentrations (PECs) of highly produced and used Ag, TiO2, ZnO and carbon-based NPs in three of the main pathways in the aquatic environments (adapted from Maurer-Jones et al., 2013 [60]).
NPs Environment PECs References
Ag Surface water 0.088–10,000 ng/L [61-64]
WWTP effluent 0.0164–17 μg/L [62, 64]
WWTP sludge 1.29–39 mg/kg [62, 64]
TiO2 Surface water 21–10 000 ng/L [54, 61, 63, 65, 66]
WWTP effluent 1–100 μg/L [62, 67, 68]
WWTP sludge 100–2,000 mg/kg [62, 67]
ZnO Surface water 1–10,000 ng/L [62]
WWTP effluent 0.22–1.42 μg/L [62]
WWTP sludge 13.6–64.7 mg/kg [62]
Carbon-based Surface water 0.001–0.8 ng/L [61]
WWTP effluent 3.69–32.66 ng/L [62]
WWTP sludge 0.0093–0.147 mg/kg [62]
Once in the aquatic environment, these NPs interact with the matrices of receiving bodies (Figure 1.3). For example, they can interact with natural organic matter (NOM), suffer aggregation (both homo- and heteroaggregation) or deposition processes. Furthermore, some NPs such as metallic NPs may release dissolved metallic ions, which in turn interact with environmental matrices.
These interactions are known to change NPs properties such as surface transformation, which is one of the most important factors that govern their stability as colloidal suspensions or their aggregation into larger particles and deposition in aquatic systems [57]. These interactions depend on environmental conditions such as natural organic matter, pH, and ionic strength, and influence their behaviour and bioavailability and, therefore, their fate and toxicity for the organisms in aquatic environments [69, 70].
23
Figure 1.3. Representation of the potential interactions of NPs in the aquatic environment.
Adapted from [71].
The increasing occurrence of NPs in the aquatic environment is expected to negatively affect aquatic organisms whose physiological parameters could be altered after the exposure to these NPs [72-74]. Even at relatively low concentrations, the potential toxicity of these pollutants should be evaluated [75, 76]. The recent studies about NPs toxicity are based in organisms at different trophic levels such as bacteria, microalgae, and microcrustaceans. NPs result toxic to different kind of microorganisms but bacteria are comparatively less affected because of the protective effect of their matrix of extracellular polymeric substances that act as barrier against the damage produced by NPs [77]. Besides, bacteria exhibit mechanisms that involve rapid adaptation to toxic effects and quick recovery after exposure to stress factors and several studies have used them as a model to study NPs toxic effects [78-81]. In the microcrustacean case, the invertebrate Daphnia magna is an excellent candidate for ecotoxicological studies because of its presence in almost all
24
aquatic habitats, high reproduction rate, short life, and relatively high sensitivity to contaminants and environmental stressors. Besides, their position in the trophic chain makes them useful for studying the transport, bioconcentration and bioaccumulation of NPs and they have also been used in NPs ecotoxicological studies [74, 82-88]. This thesis will focus, however, on the study of microalgae as NPs ecotoxicity organism model.
Microalgae (described below), constituting the basis for the aquatic trophic chain and involved in nutrient cycling of aquatic ecosystems, have been shown as a sensitive receptor with low EC50 of NPs [89]. Figure 1.4 summarizes the potential mechanisms of toxic action of NPs towards microalgae. The toxicity of NPs in microalgae may be due to: I) direct (including adsorption and internalization) or indirect physical effects of the NPs and II) production of reactive oxygen species (ROS). The indirect physical effects caused by NPs are those that imply not a direct contact with a biological target. For example, the inhibition on growth due to changes in the surrounding medium such as pH, nutrient bioavailability, ions dissolution in the case of metallic NPs or shading effect (decrease in light available for the organisms due to the presence of NPs) [90-93].
25
Figure 1.4. Mechanisms of NP toxicity to the algal cell membrane and organelles. Physical constraints and oxidative stress contribute to the toxicity of NPs. NPs exposure induces ROS production, and formation of ROS results in membrane lipid peroxidation and activation of antioxidative enzymes (SOD and POD). Over-accumulation of ROS leads to impairment of algal photosynthesis as well as damage on the mitochondrial membrane and DNA. At the “omics”
level, NP exposure suppresses genes encoding the reaction center protein of PSII (D1), light- harvesting proteins of the photosystem (LHC), electron transport chain (cox3, nad5, atpA, psaB, petF, and psbD) and RuBisCo of carbon fixation (rbcL); besides, proteins (e.g., cytochrome b6- f complex) involved in the photosynthesis are down-regulated upon exposure. The lowered synthesis of NADPH and ATP thus inhibits the assimilation of CO2 followed by a decrease in sugar production in the Calvin cycle. Abbreviations: Pq: plastoquinone; Pc: plastocyanin; Fd:
ferrodoxin; SOD: superoxide dismutase; POD: peroxidase; CAT: catalase. Taken from [94].
As previously described, NPs surface area is a key factor for their intrinsic toxicity because of the interaction of their surfaces with biological systems. Physical contact occurs for example when the NPs are attached to the algal surface [90].
Other example of physical contact is NPs internalization into the microalgae.
This internalization begins with the NPs passing through the cell wall and then through the plasmatic membrane where the NPs are involved by the lipidic bilayer, they are transported across the membrane via endocytosis or passive diffusion and they are pulled into the cell [95, 96]. Once in the cytoplasm the
26
NPs may interfere with the metabolic processes probably as a result of the production of ROS [57]. NPs internalization in microalgae has been reported by several authors. For example, Wang et al., (2016) [97] and Zhao et al., (2016) [89] observed the internalization of Ag and CuO NPs in Chlamydomonas reinhardtii and Chlorella pyrenoidosa, respectively. More recently Pulido-Reyes et al., (2019) [98] demonstrated the internalization of cerium oxide NPs in Chlamydomonas reinhardtii.
As can be seen in Figure 1.4 and as a large number of studies indicate, ROS (O2.-
, H2O2, .OH) production is the main mechanism of toxic action produced by NPs [99]. The accumulation of ROS into the algal cells may cause oxidative stress. In response to this oxidative stress, the cells increase the activity of their antioxidant system mainly formed by antioxidant enzymes. Superoxide dismutases (SOD), peroxidases (POD) and catalases (CAT) are the major antioxidant enzymes to scavenge ROS produced by NPs [94]. This increment of ROS in the cells, triggers a cascade of cellular processes such as lipid peroxidation, membrane permeability alterations, genetic damage or mitochondrial membrane potential alterations [73, 100] (Figure 1.4).
Furthermore, ROS production has been related with the decrease in chlorophyll content via altering the lipid–protein ratio of the pigment–protein complexes [101-103]. The decrease in chlorophyll content implies the impairment of photosynthesis, the decrease of algal productivity and ultimately the decrease in cell growth. Such as microalgae are part of the phytoplankton, base of the trophic chain, their growth inhibition suppresses the growth of higher trophic consumers in aquatic ecosystems. Therefore, further studies about the toxicity of NPs towards microalgae are necessary to help prevent the damage and maintain cell growth.
27
1.2 Bioassays in ecotoxicology
Worldwide contamination of natural resources, water, soil an even air poses a threat for human health and the environment. There is a need for real-time, in situ or online monitoring of pollutants. Chemical analysis techniques using chromatographic and spectroscopic methods are highly accurate and sensitive, but also costly as they require complex laboratory equipment, skilled personnel and, quite often, pretreatment of samples or extensive sample extraction before measurements can be done [104, 105]. Bioassays are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter and whose principal goal is the establishment of causality relationships [106]. Biological toxicity assays should also be performed as they complement chemical analyses and report on a simple question that chemical analysis cannot answer: is the sample toxic? In order to response to this question and since the first work made in 1944 with Daphnia magna [107], many bioassays have been used to determine the detrimental effects on organisms and reveal toxicity mechanisms [108]. Furthermore, toxicity bioassays report on the bioavailability of pollutants, which is a parameter closely related to toxicity, and on potential interactions of pollutants towards the tested organism among them and with other stressors, whether synergistically or antagonistically.
The information given by toxicity bioassays is very valuable for risk assessment strategies as they give information at a global scale. Representative bacteria, yeast, algae, plants and animals have been used in toxicity assays. Although there exist different endpoints of bioassays the mostly used are survival, growth, oxidative stress and reproduction. In general, regardless of the endpoint measured the bioassays can be divided in quantal and graded assays.
A quantal assay involves an "all or none response" while graded assays are based
28
on the observation that there is a proportionate increase (or decrease) in the observed response following an increase in the concentration or dose of the pollutants. The graded bioassays allow to establish an experimental dose- response relationship and to calculate relevant parameters related with the threshold level of the tested substance. These parameters (some of them previously described) are No Observed Effect Concentrations (NOECs) which is the highest concentration of a substance at which no statistically significant effects with respect to the control are observed; or parameters related with the potency of the substance as, Effective Concentrations (ECx) or Lethal Concentrations (LCx) which, as already described, are the concentration of a substance which produced an x percent of the measured effect on the studied parameter with respect to the control (the LC corresponds to pollutant concentration causing death of a percentage of the tested population). The most used Effective Concentrations are those producing 50% of effect, named as EC50 and LC50 [27, 106, 109] (Figure 1.5).
29
Figure 1.5. A typical plot depicting effect (% inhibition) vs substance concentration.
In order to evaluate and manage the risks for the aquatic environment, a variety of toxicity bioassays have been performed. These bioassays englobe a wide range of microorganisms, algae, invertebrates and vertebrates [110-112]. As previously described, microalgae are widely used in toxicity bioassays principally because they are primary producers and any deleterious effect on them will affect to the rest of the trophic chain. Furthermore, they are very sensitive to toxic compounds, these organisms respond more quickly than larger organisms, have a short generation time, and are easy to handle. The microalgae are small and only separated from the surrounding toxic medium by membranes or a cell wall, which enables rapid uptake of toxic compounds [113].
One of the most used microalgae in toxicity bioassays belongs to the genus Chlamydomonas. The genus Chlamydomonas is widely distributed on the Earth, including the freshwater ecosystems [114]. It belongs to the division
30
Chlorophyta, class Chlorophyceae, order Volvocales, family Chlamydomonadacea and genus Chlamydomonas [115, 116]. The species Chlamydomonas reinhardtii is the most used in ecotoxicology bioassays [115]. It was isolated in Amherst, Massachusetts, in 1945 by Gilbert M. Smith [114, 117].
Figure 1.6 shows the schematic diagram of a typical cell of Chlamydomonas reinhardtii. The cells possess a wall cells mainly composed of hydroxyproline- rich glycoproteins [117]. Chlamydomonas reinhardtii cells feature a single chloroplast (chlorophyll a and b), two contractile vacuoles are located at the anterior end of the cell; the cells also contain mitochondria, an eye-spot (to perceive light information), a pyrenoid and two apical flagella (Figure 1.6) [114, 117]. Furthermore, Chlamydomonas reinhardtii can grow in chemoheterotrophic (in dark, using acetate as the organic carbon source) and in photoautotrophic (using light, H2O and CO2) conditions [115].
Figure 1.6. Schematic diagram of a Chlamydomonas reinhardtii cell.
31 Chlamydomonas reinhardtii is one of the most studied microalgae due to its easy growth and genetic manipulation and is a well-known organism useful also for ecotoxicology studies in response to several pollutants [114, 115, 117-124]. To date, there exist a variety of toxicological studies of pollutants in Chlamydomonas reinhardtii which use techniques such as flow cytometry using different fluorochromes to measure cellular parameters such as ROS formation, cytoplasmatic and mitochondrial membrane potential, membrane integrity, apoptosis, intracellular pH, intracellular free calcium, metabolic activity or cellular cycle [125-128].
Specifically, ecotoxicological studies in response to exposure to NPs have been made with Chlamydomonas reinhardtii. Table 1.2 summarizes the most recently works carried out with NPs and Chlamydomonas reinhardtii. There exist multiple known mechanisms of toxic action in Chlamydomonas reinhardtii after the exposure to NPs, but two of them are common in almost all the cases: ROS production and alterations in the photosynthetic system. Furthermore, in several studies, the internalization of NPs has been demonstrated implying direct damage of the NPs in the interior of the cell.
32
Table 1.2. Summary of recent studies of NPs effect towards Chlamydomonas reinhardtii. Nanoparticle Toxic trigger Mechanisms of action References
Silver (Ag)
- Contact with the surface cell - Release of Ag+ - Internalization
- Photosynthetic system damaged - Inhibition in cell
growth
[97, 129]
Copper oxide
(CuO) - Internalization
- DNA damage - ROS production - Photosynthesis
alterations - Decrease in
metabolism activity - Lipid
peroxidation
[130]
Titanium oxide
(TiO2) - Internalization
- Accumulation
- ROS production - Membrane
damage
[131, 132]
Quantum dots
(QDs) -
- Cell aggregation - ROS production - Lipid
peroxidation - Alterations in
photosynthesis
[132]
Carbon Nanotubes (Ag and Pt) (CNT)
- Internalization in vesicles - Ag+ dissolution
- Distortion of cell wall
- Damage in membrane integrity - Photosynthetic
system affected
[133]
Cerium oxide (CeO2)
- Internalization via endocytosis
- ROS production - Damage in
plasma membrane - Alterations in
photosynthetic machinery
[98, 134, 135]
Graphene oxide (GO)
- Contact with cell wall
- ROS production - Decrease in
metabolic activity - Plasma and
mitochondrial membrane damaged - Alterations in
intracellular free Ca2+
[136]
33
poly(isobutyl‐
cyanoacrylate) (iBCA‐NPs)
- Internalization - Contact with
cell wall
- ROS production - Abnormal
swimming behaviour - Damage in
plasma membrane - Cell wall
damaged
[137]
1.3 Biosensors: further evolution of bioassays
Over the years, less laborious, cost-effective and more rapid toxicity bioassays have been developed: i.e, biosensors. Biosensors are defined as devices which detect, transmit and record information regarding a physiological or biochemical change [138, 139]. This device integrates a biological recognition element with a physical transducer to generate a measurable signal proportional to the concentration of the analytes [138, 140, 141]. A typical biosensor is represented in Figure 1.7 and it consists of the following components:
- Bioreceptor; a molecule that recognize the analyte. Usually, the biosensors utilize whole cells, antibodies, DNA, RNA, enzymes or receptor proteins as the recognition elements [142-146].
- Transducer; element that converts the bio-recognition event into a measurable signal. Most transducers produce either optical or electrical signals that are usually proportional to the amount of analyte–
bioreceptor interactions [147].
- Electronics; this is the part of a biosensor that processes the transduced signal and prepares it for display.
- Display; interpretation system such as the liquid crystal display of a computer or a direct printer that generates data understandable by the user [147]
34
Figure 1.7. Schematic representation of a biosensor. Taken from Bhalla et al., (2016) [147].
Biosensors can be grouped according to their biological element or their transduction element. Biological elements include enzymes, antibodies, micro- organisms, biological tissue, and organelles. Among these biological recognition elements, enzymes are the most widely used because they are highly selective.
However, the need for costly and difficult purification is a limitation in the construction of enzyme-based biosensors [139]. Whole cells are a good alternative to enzymes since they have the benefit of low cost, improved stability comparing to enzymes or other proteins and give information about the bioavailability of the pollutant [145, 148]. Among them, those based on microorganisms such as bacteria, yeast and algae are very useful due to their ease of cultivation and use, and the possibility of using them in high-throughput techniques. Due to their rapid growth rates, low cost, easy maintenance/preservation and the possibility of genetic manipulation, microorganism have been used to asses toxicity in the environment [140, 149- 151].
35 Depending on their transduction element, biosensors can be divided in optical, electrochemical, thermometric, piezoelectric or magnetic biosensors. Optical are the most used biosensors and their detection is based in the interaction of the optical field with a biorecognition element. Optical whole cell microbial biosensors, both natural and transgenic, respond to the presence of chemicals or physiological stresses through the synthesis of a reporter protein, such as luciferase ( bacterial lux or firefly or click beetle luc genes), β-galactosidase (lacZ gene from Escherichia coli), or green fluorescent protein (Aequorea victoria gfp gene). Thus, the reporter protein exhibits specific luminescence, fluorescence, or color development as the detectable signal [152-154].
This section of the thesis will, then, focus on optical whole-cell biosensors.
1.3.1 Optical biosensors
1.3.1.1 Naturally bioluminescent microorganisms used for environmental toxicity
Between optical biosensors, bioluminescent organisms are the most used in environmental studies. Since the first research works during the 60’s decade in which the effect of air pollutants on luminescent bacteria was determined [155, 156], the use of naturally bioluminescent microorganisms has been widely expanded as a sensitive test for the quick assessment of environmental toxicity.
The first organism used for this purpose was the aquatic bacterium Photobacterium phosphoreum (P. phosphoreum) [156], however other prokaryotic and eukaryotic organisms have been also used in bioluminescent assays. Table 1.3 provides an extensive summary of all the naturally luminescent microorganisms which have been used during last decades, featuring the type of organism, species, main applications and commercial devices if available. All these bioassays are based on the inhibition of light emitted by a nonpathogenic luminescent organism upon exposure to a toxic sample. Essentially, as bioluminescence mainly depends on cell metabolism, any toxic compound that
36
may compromise the metabolic status of cells will cause a reduction in light emission, generally, proportional to the substrate concentration.
The robustness and ease of this assessment strategy was quickly implemented in other studies [251, 252] and more bioluminescent bacteria such as Aliivibrio fischeri (A. fischeri, formerly known as Vibrio fischeri), Aliivibrio logei (A. logei), Vibrio harveyi (V. harveyi), Photobacterium leiognathi (P. leiognathi), P.
phosphoreum, Photorhabdus asymbiotica, (P. aymbiotica), Photorhabdus luminescens (P. luminescens, previously called Xenorhabdus luminescens) or Vibrio qinghaiensis sp. Q67 (V. qinghaiensis sp. Q67) were also included in this kind of works (Table 1.3; please, see Figure 1.8 with examples of naturally bioluminescent microorganisms). One of the best known microbial bioassay is based on the marine bioluminescent bacterial strain A. fischeri NRRL B-11177 which was used to develop a commercial bioluminescence test in the late 70’s under the name of Microtox™ (Microbics Corporation; Carlsbad, USA), as a rapid screening substitute to toxicity tests with animals such as fish or small invertebrates [252]. A wide revision on the toxicity of a large number of chemicals that have been obtained using the Microtox system was completed by Kaiser and Palabrica (1991) [251], more recently, just based on A. fischeri bioluminescence inhibition assay by Abbas, et al., (2018) [253].
37
Table 1.3. Naturally bioluminescent microorganisms which have been used for environmental toxicity evaluation organized by type, specie, tested pollutants and main applications (most reports before 2014 already reviewed in Fernández-Piñas et al., (2014) [148].
Type Microorganism Tested pollutants Environmental
applications Commercial test References
Prokaryotic
Aliivibrio fischeri -several strains
NPs, heavy metals, organotin compounds, PAH,
PCB, Personal care products, pesticides,
pharmaceuticals, plasticizers, Surfactants, allelopathic compounds, antibiotics, hydrophobic organic substances, azo-
dyes, herbicide.
Freshwater, groundwater, mixtures, seawater sediment, soil, wastewater, agriculture, industrial wastewater, domestic wastewater, marine sediments.
Microtox M500 Microtox ToxAlert® 10 ToxAlert 100 LUMISTox 300
Inorganic pollutants: [157-
182].
Organic pollutants:
[183-203].
Water : [148, 193, 204-218]
Soil: [219].
Sediments: [181, 189, 220, 221].
Aliivibrio logei Heavy metals, PAH, pesticides.
Industrial wastewater,
seawater. [148].
Vibrio harveyi Heavy metals, NPs. LumiMARA [171, 222].
Photobacterium leiognathi Fuel traces, heavy metals,
NPs, PAH, PCBs, pesticides. Freshwater.
LumiMARA Microtox ToxScreen®
[148, 171].
Photobacterium phosphoreum -several strains-
Heavy metals, NPs, PAH, pesticides.
Mixtures, sediments, freshwater, drinking
water.
Microtox LumiMARA LUMISTox 300 Microbiosensor B17-677F
Inorganic pollutants: [148,
171, 223-226].
Organic pollutants:
[227, 228].
Water: [229].
Sediments: [230].
Photorhabdus asymbiotica NPs. LumiMARA [171].
