CHAPTER 1
Introduction to bioanalytical tools in water quality assessment
BACKGROUND
Chemical monitoring provides a quantitative assessment of single organic contaminant concentrations in a water sample but does not account for the presence of unknown compounds such as transformation products, untargeted chemicals (i.e., not previously known to be present) or for interactions among chemicals. Biological-based monitoring is complementary to chemical analysis and provides information on all bioactive micropollutants in a sample ranked according to potency, i.e., more toxic chemicals are weighted higher than less toxic chemicals.
Classical aquatic toxicity tests used in water quality assessment include in vivo assays with fish and aquatic invertebrates that measure endpoints such as mortality, growth, reproduction and feeding responses. Ecosystem level assessment is based on functional or structural measures of whole ecosystems such as species diversity or genetic structure. The sensitivity of in vivo tests has been greatly improved with the development of early life-stage tests (e.g., the zebrafish embryo toxicity test), which is considered to be ethically more acceptable than adult fish tests. In vitro molecular and cell-based assays are sensitive, cost- and time-efficient alternatives to whole animal testing. Implementation of human and other mammalian cell lines in water testing has facilitated evaluation of toxicological endpoints relevant for assessing the potential for deleterious effects on human health.
For the purpose of this book, we define 'bioanalytical tools' as cell-based and low-complexity in vitro bioassays indicative of specified endpoints relevant for human and/or environmental health. These tools include whole cell assays and assays with genetically modified cells, where natural features have been over-expressed to enable more sensitive detection and/or where foreign features have been added for visualisation of effects. Further, we include assays with unicellular organisms, such as algae, yeast or bacteria, and some enzymes but exclude other cell-free assays (immunoassays and direct receptor binding assays).
A major advantage of bioanalytical tools is the ability to detect the toxicity of mixtures of known and unknown compounds, while chemical analysis can only quantify the concentration of known, targeted chemicals irrespective of toxicity. By measuring the mixture toxicity of a water sample, the bioassay approach is weighted by risk as it explicitly accounts for the differential in toxicity across different chemicals and for interactions among chemicals in a mixture. Many bioassays yield specific information on a given mode of toxic action rather than merely answering whether or not the cells are dead or alive after exposure to the sample. This mechanistic information can be exploited by running a series of bioassays indicative of a range of different modes of toxic action in parallel. In this way, a comprehensive bioanalytical test battery provides an integrated measure of the toxicity of the biologically active substances in a water sample. A bioassay can also be selected to target a specific protection goal such as maintenance of hormonal balance.
This book focuses exclusively on organic chemicals. Although many of the bioassays discussed here are also applicable for metals and inorganic pollutants, sample treatment and data interpretation differs between organics and inorganics. In addition, while there are millions of organic chemicals, many of which may never be identified by chemical analysis, the limited number of elements allows comprehensive chemical analysis of metals and inorganics, reducing the need for effect-based analysis.
1.2 ORGANIC MICROPOLLUTANTS
1.2.1 Defining the issue
Organic micropollutants are a group of man-made chemicals such as pesticides, industrial chemicals, consumer products and pharmaceuticals but also include natural compounds such as hormones (Schwarzenbach et al., 2006) (Table 1.1). As the name implies, micropollutants occur in water and the environment in the microgram per litre concentration range (1 µg/L = 10-6 g/L = 0.000001 g/L) or even lower, in the nanogram to picogram range (1 ng/L = 0.000000001 g/L; 1 pg/L = 0.000000000001 g/L).
In contrast to micropollutants, macropollutants are naturally occurring compounds that exist locally in excess concentration, e.g., phosphate and nitrogen compounds, which can lead to eutrophication of surface waters (Schwarzenbach et al., 2006). Macropollutants were the big environmental problem of the 1960s and 1970s in the developed world. Nowadays macropollutants are managed quite well with the introduction of source controls and additional wastewater treatments. As a result, attention has shifted to micropollutants, including inorganic and organic chemicals.
The widespread distribution of organic micropollutants in our waterways presents a hazard to aquatic life. Risks to humans can occur through the consumption of food and drinking water and through other exposure routes such as inhalation and dermal contact. Micropollutants enter the aquatic environment via direct sources, such as industry and sewage effluent discharge, and via non-point sources such as urban runoff and agricultural applications. Due to the complex nature of the chemical mixtures occurring in domestic wastewater used for water recycling schemes, conventional treatment is not always sufficient to remove the entire contaminant load. Disinfection steps such as chlorination have been introduced to wastewater and recycled water treatment to control human pathogens (microorganisms that cause diseases). While conventional biological treatment and advanced treatment processes are very effective in eliminating most unwanted pathogens and many micropollutants, they may also introduce other potentially harmful substances such as disinfection by-products and transformation products. Membrane processes such as reverse osmosis and adsorptive processes such as activated carbon filtration reduce a wide range of micropollutants to safe levels. Removal efficiency is, however, related to the chemical structure and properties, and some compounds may not be fully removed by these processes alone. Further remains the problem of contaminated waste generated during filtration and adsorption processes.
1.2.2 Transformation products
Transformation products are micropollutants that have undergone chemical reactions. It is unknown, which and how many transformation products are formed, in what quantities and what level of harm they may cause. Transformation products can arise from a variety of sources and can be formed in the environment as well as in engineered systems (Table 1.2).
Pharmaceuticals are extensively metabolised in humans and animals and, hence, are typically not excreted in the same form as they were ingested but in a variety of metabolites (Lienert et al., 2007). Many pharmaceutical drugs are activated inside the body to the pharmacologically active form, which may also be more potent than the precursor with respect to its adverse effect. Most pesticides and other micropollutants undergo biotic and abiotic transformation reactions in the environment. In surface water, for example, exposure to sunlight can cause direct photodegradation or indirect oxidation of micropollutants via formation of reactive oxygen species (Table 1.2).
Biodegradation is particularly extensive during biological wastewater treatment, yet mineralisation (complete degradation to carbon dioxide and water) is incomplete for many chemicals allowing biotransformation products to be formed. Hydrophobic micropollutants are also removed from water by mere adsorption to the sewage sludge without any transformation. Existing micropollutants in water can be transformed during advanced oxidation and disinfection processes to more persistent and/or toxic disinfection by-products (Table 1.2).
Disinfection by-products are particularly important as they can also be formed from natural organic matter present even in the purest water used as source for drinking water (Table 1.2). During chlorination of drinking water, a wide range of chlorinated chemicals are formed, e.g., trihalomethanes and haloacetic acids. Nitrosamines are further formed during chloramination. As some disinfection by-products are known to cause cancer and other adverse effects (Richardson et al., 2007), their regulation and management is important while keeping in mind that protection from pathogens is of high priority to safeguard health in the short term.
While most transformation products are less persistent, bioaccumulative and toxic than the original compounds (Boxall et al., 2004), there are a number of prominent exceptions. Some transformation products are more persistent than their compounds of origin and thus accumulate in higher concentrations in the environment. Other transformation products are more toxic than the original chemicals (Escher and Fenner, 2011). An example is nonylphenol, which is a degradation product of the industrial surfactant nonylphenol polyethoxylate (NPE). Nonylphenol is highly persistent, bioaccumulative and in addition to being more toxic than NPE in terms of acute toxicity, it exhibits weak estrogenic effects (Fenner et al., 2002).
1.2.3 Low concentrations and mixtures
Regulators are faced with vast numbers of largely unknown micropollutants and transformation products in water. Individual contaminants may be present in very low concentrations, most far below any concentration expected to cause adverse effects on their own, but acting together in mixtures their biological activity may lead to detectable effects.
All chemical analysis is limited to the lowest level of resolution of each analytical method. In most analytical laboratories today, routine analysis is limited to the microgram per litre range, while specialised methods may resolve individual chemicals down to the nanogram or picogram per litre level. Chemical analysis can only identify the tip of the iceberg with no quantitative measure of the fraction that remains unaccounted for. Although bioanalytical tools do not identify the individual components in the submerged part of the iceberg, they contribute a more complete picture of its total size, thereby improving our ability to predict the possible health significance of micropollutants. Bioassays indicative of specific modes of toxic action, such as estrogenicity and genotoxicity, may help refine this picture further by pointing to specific groups of micropollutants with common modes of toxic action, which often, although not always, comprise structurally similar chemicals.
1.3 ENVIRONMENTAL TOXICOLOGY
Environmental toxicology has evolved over the last few decades from an amalgamation of various scientific disciplines including biology, toxicology, environmental chemistry, biochemistry, pharmacology, medicine and ecology. The overall objective of environmental toxicology is to understand the impact of environmental pollutants on humans and ecosystems, encompassing all levels of biological organisation. This ranges from biochemical interactions within organisms to whole animals, populations and ecosystems. In human toxicology, this sequence is paralleled starting from the toxicity pathways occurring at the cellular level to failure or malfunction at the organ level and ultimately, to population effects and epidemiological studies (e.g., cancer clusters).
Traditionally, environmental toxicology has been divided into ecological toxicology (or ecotoxicology) and human health toxicology. While the former discipline was generally associated with environmental sciences and biology, the latter is rooted in pharmacology and medicine. As these disciplines have become more focused at the molecular level, it has been recognised that mechanistic toxicity pathways have many common pathways and modes of action in all biota, and the fields have again grown closer.
1.4 ENVIRONMENTAL RISK ASSESSMENT
Environmental toxicology, as a science, plays a central role in the development of robust methods for environmental risk assessment of chemicals. A chemical poses a risk to the environment and to human health if (a) it is likely that environmental organisms and humans are exposed to the chemical and (b) if the chemical poses a hazard, i.e., if it has an inherent potential to cause toxic effects.
Formally, environmental risk assessment consists of four steps (Figure 1.1). Hazard identification includes the collection and evaluation of all available information for the given chemical to assess its potential adverse effect and is followed by parallel exposure and effect assessment. Effect assessment includes the classification of the potential of a chemical to be categorised as persistent, bioaccumulative and toxic (PBT) as well as dose-response characterisation, which yield predicted "no effect levels" (NEL) for humans and "predicted no effect concentrations" (PNEC) for the environment. The exposure assessment involves evaluation of the expected exposure levels relevant for a given situation. For each exposure scenario, the risk is then characterised by comparing the expected exposure level with the NEL or PNEC, which should correspond to a safe dose over the entire lifetime of a human or environmental organism.
In vitro assays are used in an early screening stage of hazard assessment as part of an integrated test strategy but if there is any indication that a chemical is of concern, risk assessment needs to be based on in vivo information.
1.5 BIOANALYTICAL TOOLS
Bioanalytical tools are defined as in vitro cell-based and low complexity in vivo bioassays indicative of modes of toxic action that are relevant for human and/or ecosystem health. These assays include whole cell and reporter gene assays, tests with unicellular organisms as well as some enzyme assays. Previous reviews had wider or narrower definition. Behnisch et al. (2001) included, for example, biomarkers and enzyme immunoassays, while Eggen and Segner (2003) only included assays describing a defined chemical-biological interaction excluding general cytotoxicity assays. In the following sections, we provide an overview of where bioanalytical tools fit within the field of toxicity testing, followed by a more detailed description of the scope of bioanalytical tools.
1.5.1 In vivo and in vitro bioassays
Toxicity testing can be performed at all levels of biological organisation. Epidemiological studies attempt to link observed clusters of disease with human exposure to chemicals. These studies do provide the ultimate answers, however, only in hindsight, long after the harm was done. Most toxicological studies to date have therefore been performed at the level of single organisms (Figure 1.2). In vivo studies on individuals utilise historic case studies of human poisoning or perform animal tests (e.g., using rodents) in order to obtain toxicological information at the whole organism or organ level (Figure 1.2). As with human toxicology, the in vivo scope of ecotoxicology may range across organisms, populations, ecosystems and model ecosystems (more details in Chapter 3).
All organisms are made up of cells. Testing the responses to a chemical at the cellular level in an in vitro system in the laboratory thus provides a tool to screen a large number of chemicals and environmental samples in a much shorter time and with lower costs than with in vivo assays (Figure 1.2). As a trade-off the relevance is lower as intercellular communication and systemic effects cannot be assessed in an in vitro assay.
Additional very promising tools for hazard assessment of chemicals arise from the emerging field of toxicogenomics. Toxicogenomics is the science of applying genomic technologies to elucidate the toxicity pathways and modes of toxic action triggered by a micropollutant (Nuwaysir et al., 1999). Technologies applied in toxicogenomics include profiling at the gene (transcriptomics) and protein (proteomics) expression levels as well as profiling of the metabolic products arising from biological reactions (metabolomics) (Figure 1.2). Ecotoxicogenomics takes this approach one step further by linking these cellular-level effects with adverse outcomes for whole organisms, populations and ecosystems (Ankley et al., 2010; Fedorenkova et al., 2010).
In vitro assays and toxicogenomic techniques share the advantages of being highly sensitive and reproducible, to lend themselves to automation and of being of lower ethical concern (if cell lines are used to extract the material used for the assays) although their relevance is less well established (Figure 1.2).
Toxicogenomic techniques are yet to be validated for use in regulatory risk assessment of chemicals and, to date, their application for quantitative assessment and identification of modes of toxic action remains limited. Furthermore, ecotoxicogenomic techniques are not sufficiently advanced for application with mixtures and environmental samples because causal relationships are still hard to derive.
