Bioassays as one of the Green Chemistry tools for assessing environmental quality: A review

Highlights

• Green chemistry principles should apply to the analysis of environmental samples.

• Toxicity as a summary assessment parameter of the quality of the environment

• Bioassays as a “green” alternative to conventional instrumental methods

Abstract

For centuries, mankind has contributed to irreversible environmental changes, but due to the modern science of recent decades, scientists are able to assess the scale of this impact. The introduction of laws and standards to ensure environmental cleanliness requires comprehensive environmental monitoring, which should also meet the requirements of Green Chemistry. The broad spectrum of Green Chemistry principle applications should also include all of the techniques and methods of pollutant analysis and environmental monitoring. The classical methods of chemical analyses do not always match the twelve principles of Green Chemistry, and they are often expensive and employ toxic and environmentally unfriendly solvents in large quantities. These solvents can generate hazardous and toxic waste while consuming large volumes of resources. Therefore, there is a need to develop reliable techniques that would not only meet the requirements of Green Analytical Chemistry, but they could also complement and sometimes provide an alternative to conventional classical analytical methods. These alternatives may be found in bioassays. Commercially available certified bioassays often come in the form of ready-to-use toxkits, and they are easy to use and relatively inexpensive in comparison with certain conventional analytical methods. The aim of this study is to provide evidence that bioassays can be a complementary alternative to classical methods of analysis and can fulfil Green Analytical Chemistry criteria. The test organisms discussed in this work include single-celled organisms, such as cell lines, fungi (yeast), and bacteria, and multicellular organisms, such as invertebrate and vertebrate animals and plants.

Introduction

The environment is a highly complex system that is split into biotic and abiotic parts, among which there is a continuous exchange of matter and energy. These processes should remain in balance, and this balance is called homeostasis. This sensitive balance may be disrupted by the release of various chemicals into the environment.

Virtually all human activities can cause environmental pollution, but some of them have important influences on the levels of anthropogenic impacts. Among these activities, the following different industrial branches can be considered: the petrochemical industry, the mining of precious metals and stones, tanneries, the lead battery industry, and industrial and/or municipal discharges. The pollution from other manifestations of human activity such as transportation, housekeeping, agriculture, sewage and municipal waste are not insignificant, either (Nadal et al., 2004, Mehlman, 1992, Kaldor et al., 1984, Cordy et al., 2011, Módenes et al., 2012, Bahadir et al., 2007, Rajaram and Das, 2008).

Environmental pollution does not respect geographic boundaries, and under favourable conditions, it may be transmitted over long distances and “travel” all over the biosphere (Oke, 2002, Walker et al., 1999, Hung et al., 2010). Pollutants may be transferred over long distances by different environmental components such as water and air (as well as particulate matter and aerosols) or by living organisms. Water and air act as a transport medium; however, transport by living organisms strongly depends on the migratory species in question (Lohmann et al., 2007).

Chemicals undergo a number of processes in the environment depending on their physicochemical properties. Hydrophilic substances remain dissolved in water, hydrophobic substances accumulate in soil or/and sediment and volatile compounds pollute the air. Chemicals may be partially bioaccumulated by living organisms (Zenker et al., 2014).

Fig. 1 shows the pathways through which xenobiotics move from the environment into the different levels of the food chain together with an indication of their bioaccumulation and biomagnification. Human beings make up the last link in the food chain, and we are particularly vulnerable to the adverse effects of accumulated xenobiotics.

To check the applicability of the analytical method, the method must be validated and optimized by determining parameters such as its accuracy, sensitivity, reproducibility, simplicity, cost effectiveness, flexibility and speed. However, none of these parameters helps to reduce the environmental burden of any specific method (Armenta et al., 2008).

At this point, it is not only “dry” validation parameters that are important but also the underlying principles, rules or guidelines are important as well. Compliance with these principles would help to reduce the burden of chemical operations on the environment, and using natural resources in a responsible and sustainable manner should be considered.

The Green Chemistry concept emerged in the 1990s and was aimed at reducing pollution by using so-called green solvents. Planning chemical processes to obtain a final product that would use the same amount of input materials (atom economy and catalysis) is essential to the Green Chemistry approach. In the late 1990s, the Green Chemistry idea began to expand slowly in Europe and across the ocean in the United States, and its first concerns were chemical synthesis and chemical engineering. In the United States, the Environmental Protection Agency played a significant role in the introduction of new “green” ideas (Anastas and Kirchhoff, 2002).

In 1998, Anastas and Warner proposed a set of twelve Green Chemistry principles that would serve as guidelines, and these guidelines would be focused on reducing the waste that was generated during chemical processes, using non-toxic solvents, applying catalysts (when possible), and designing chemical processes in accordance with the principle of atom economy (Anastas and Warner, 1998).

Over time, the concepts and principles of Green Chemistry came into effect at a smaller scale for laboratory practice. In the Handbook of Green Analytical Chemistry, de la Guardia and Garrigues (2012) state the following five Green Analytical Chemistry strategies:

• remote sensing and direct measurement of untreated samples,

• replacement of toxic reagents,

• miniaturizations of procedures and instrumentation,

• automation, and

• on-line treatment of analytical wastes De la Guardia and Garrigues, 2012).

Efforts have been made to create a list of the twelve principles and goals of Green Analytical Chemistry to apply to analytical chemistry practices. The most important aspects were adopted from Green Chemistry, which plays a key role in this approach, and they are related to the elimination or reduction of the use of chemical substances (solvents, reagents, preservatives, additives for pH adjustment and others); the minimization of energy consumption; proper management of analytical waste; and increased safety for the operator (Gałuszka et al., 2013).

Newly emerging chemicals provide a major challenge to the analytical chemist because classical analytical methods involve the use of standards or the uploading of libraries of newly emerging compounds to existing libraries. Continuously decreasing the concentration levels of chemicals does not always make sense because one has to consider environmental samples as a mixture of different chemicals, which do not remain neutral when they interact with one another. The chemicals in a given mixture may act synergistically, antagonistically or additively, resulting in a toxicity shift in the exposed organism. They are active even at ultralow concentration levels (below those set by legal regulations) and may cause adverse effects in ecosystems (Wieczerzak et al., 2015). In these cases, classical analyses (even when conducted according to Green Chemistry principles) is not sufficient and chemical quantitation should be supplemented with biological tools.

All the issues outlined above reflect the complexity of the problem of determining and removing contaminants from the environment. Green Chemistry is increasingly recognized as an overarching tool that is now included in most chemical operations and chemical analyses. In many countries, there are norms and laws that aim to protect the environment and human health during the design of chemical processes (Anastas and Warner, 1998).

Toxicology is a scientific discipline; it is the study of the toxic properties of chemical substances against living organisms. Through their behaviours, living organisms reflect both the negative and positive effects of stressors. The basis of toxicological studies is dose-response dependence. However, the observed effect is a combination of many factors such as species and individual differences, e.g., age and gender (Traczewska, 2011). The most frequently designated parameters include the ED_x/EC_x (effective dose/concentration), LD_x/LC_x (lethal dose/concentration), and the following parameters related to the threshold dose: the NOEL/NOEC (no observed effect level/concentration), LOEL/LOEC (lowest observable effect level/concentration) and LOAEL (lowest observable adverse effect level).

The pollutants that are present in the environment can affect organisms in many ways. Some compounds are characterized by high toxicity, or they are present in large enough quantities to produce immediate acute toxicity, which can ultimately lead to death.

However, most environmental stressors occur at levels below lethal concentrations or even at trace amounts, causing sub-acute, chronic or sub-chronic toxicity. These changes can be observed after a longer exposure time and within a few generations. The time of exposure to a given compound or to a mixture of compounds is also very relevant, with a prolonged exposure time increasing the chance of the appearance of a distant toxic effect such as teratogenotoxicity and mutagenic effects (see Fig. 2.) (Kuczyńska et al., 2004).

Environmental pollution is such a broad issue that a need has arisen for a new field of science that will cover all the problems connected to the environmental fate of pollutants. Ecotoxicology is the branch of science that addresses the study of xenobiotic impact on the environment and covers the entire “life cycle” of toxic substances in the biosphere. At present, ecotoxicological studies are gaining significance, and a new approach from the field of bioanalytics and biomonitoring makes it possible to assess risks and to assess environmental quality quickly; for example, to test the safety of medical products derived from bacterial toxins such as vaccines (Sesardic, 2012).

For these reasons, more and more interest is being devoted by scientists to reducing the harmful chemicals used in environmental monitoring and analyses and to replace at least some of them with biological studies. To support this approach and apply biotests and bioassays to the modern Green Analytical Chemistry field, the most significant information and parameters of both classical and novel tests are presented to facilitate the decision process by less experienced researchers. It is also necessary to account for the transformation and biotransformation of environmental pollutants within the biotic and abiotic components of the environment, e.g., the compounds that are produced over the course of wastewater treatment, e.g., during ozonation and photocatalysis. Some of these compounds belong to a group of newly emerging contaminants and may be characterized by their greater hazards to and burdens on the environment. The advantage of bioassays in this case lies in their ability to assess the toxicity of a sample as a whole, and it does not matter whether the tested sample contains compounds that are known to humankind.

Next, emphasis should be placed on the problem of sample toxicity changes that are caused during disinfection processes. The list of toxic disinfection by-products covers numerous chemicals, including the following: halogenated organics, phosphines, cyanides, polycyclic aromatic hydrocarbons, metals and organometals, biocides as well as plant and pest control chemicals. According to European regulations, reliable information on the toxicity level of water samples should be based on acute and chronic toxicity determinations that have been performed in algae, macrophytes, Daphnia, and fish (those that are characteristic of a given region) to determine the pollution standards for a given ecosystem. For these reasons, bioassays should help with or even constitute the basis for determining the legal safety regulations and procedures of environmental risk assessments for wastes or newly emerging chemicals.