Abstract
Nowadays ecotoxicology plays the role of a theoretician – methodical unifying centre for the optimization of man – biosphere relations and sustainable existence of life on the Earth. The main basis for its development is the classical toxicology—studies of chemical compounds’ effects on man, but ecotoxicology is the original part, following it. According to the modern concept, the ecotoxicology is a science for migration, transformation and utilization of different toxic ingredients (with organic, inorganic or organic-mineral chemical nature; with natural biotic or abiotic origin and artificial, mainly anthropogenic origin) in the environment and their impact on Macro- biological systems with different levels of integration as groups of individuals, population, community, ecosystem, etc. studied in ecology. In this chapter, the types of ecotoxicological tests are discussed in detail with a set of examples about used species, advantages and disadvantages of different types of toxicity testing. The application of exposed natural ecosystems or man-made analogue systems is also commented as the environmentally more realistic approach for ecotoxicological testing. These test systems are increasingly becoming in aquatic ecotoxicology practice, but they are contemporary challenge in terrestrial testing. The development of test systems for realistic assessment of contaminant toxicity is essential for the efficient control of human influence on the environment.
Keywords
- ecotoxicology
- bio tests
- acute
- chronic
- mono-species
- multispecies
- biomarkers
- kits
1. Introduction: the contemporary meaning of ecotoxicology as a complex science
Ecotoxicology is a scientific discipline, which of the modern stage of man-biosphere relations, is developed as the theoretician – methodical unifying centre for the optimization of these relations for the sustainability of life existence on Earth. The main basis for the development of ecotoxicology is classical toxicology—the research of drugs and chemical compounds effects on man. The modern concept of ecotoxicology is that it is the original part following the classical toxicology, which studies migration, transformation, degradation and utilization of toxic ingredients (with organic, inorganic or organic-mineral chemical nature; with natural biotic or abiotic origin and artificial, mainly anthropogenic origin) in the environment and their impact on Macro- biological systems with different levels of integration as groups of individuals, population, community, ecosystem, etc. studied in ecology (MBS) [1, 2] and others.
The main objects of the ecotoxicological studies are the both: (1) toxic ingredients and their “behaviour” in the five main environments such as air, soil, terrestrial, water (incl. sediments) and biotic and (2) the responses of MBS in nature. The studied toxicants can be: (1) by the chemical nature: organic, inorganic or organic-mineral; (2) by the origin: natural (biotic or abiotic) and artificial (mainly anthropogenic origin); (3) by the toxicity: toxicants in the Black list, toxicants in the Grey list, etc. and (4) by the main environment of circulation: air, water, soil or different bio toxicants. The migration, transformation and degradation of toxic ingredients depend on the internal (endogenous) factors, that are, chemical properties of the toxicant and external (exogenous) factors or features of the environment. The studied MBS at different levels of integration can be: diverse individuals as elements of the population; homogeneous and heterogeneous populations as elements of the communities; heterogeneous communities as elements of the bio cenosis; ecosystem as a functional unity between biotope and bio cenosis; landscape, biome and biosphere formed by a corresponding set of ecosystems and their environment. The responses of MBS also depend on the endogenous factors (level of integration and features of the MBS) and exogenous factors (the characteristics of toxicants and habitats). Therefore, according to the used objects, ecotoxicology is an interdisciplinary complex science, developing on the border of chemical, biological, medical, ecological, environmental, economic, social and legal sciences. It can also be considered as applied environmental science studying the biological effects of anthropogenic ingredients. According to the main objects concerned, the main sections of ecotoxicology are
The development of analytical methods is very important in this division for solving the series of toxicological problems. For example, with the appearance of inductively coupled plasma mass spectrometry (ICP/MS) as a method of measurement, it is possible to separate and determinate the toxicity of various forms of each toxicant. Therefore, the chemical and physical measurements for assessing the toxicity of the substances and their forms are important for determining the valid toxic concentration in the bioassay and complementary test system reply.
There are few main differences between classical toxicology and ecotoxicology: (a) the usage of bio-test, including selected for this purpose; (b) the main objects for acute toxicity measurement are different –
The MBS are characterized by a complex structural and functional organization and the specificity of the set of internal regulatory mechanisms that support the system in equilibrium, which should be considered in the toxicological effect extrapolation, as well as for assessment of ecosystem health and predicting the risk. Therefore, the models, adequately reflecting the responses of MBS in nature, require the knowledge of structure, function and the mechanisms for ensuring the existence and integrity of MBS and the behaviour of toxicants in the current climate. The main functions and features of MBS have been deeply commented by Lyubenova [5].
The main aim of chapter is to comment the contemporary knowledge and established practice in the usage of bioassays to study the environmental toxicity of ingredients. The acute, chronic, mono-species and multi-species tests are discussed. Moreover, the analytical and biochemical methods for determining the initial damage at the molecular level on acute and chronic exposure are commented, too. The molecular markers (biomarkers) or indicators are very important for the early diagnosis of damages and the interests for new developments are growing steadily.
2. Ecotoxicological testing: contemporary knowledge and gaps
The ecotoxicological effects of contaminants on bio systems and MBS are developed as sub lethal and lethal responses. The earliest toxicological responses (change in biological systems) are detected at the cellular level. Some of the most important effects are changes in the structural components of the cell membrane (e.g. breach links between proteins and lipids); suppression of certain enzymes (e.g. microsomal enzymes); damage to the whole or partial metabolic changes (e.g. the synthesis of carbohydrates) [6]; changes in DNA correlation, respectively mutations and modification of cell growth [7], etc. At the macro-bio system level, effects related with their structure and functioning can be observed: efficiency of energy utilization and transmission through the food chains [8]; bio-depressant effects (inhibition of growth and reproduction) [9] and bio-stimulant effects (e.g. eutrophication) on population or community [10]; changes in the nature of biological cycle—capacity (the amount of chemical elements involved in biomass per year), intensity (of productive processes, energy transformation and destructive processes), chemistry (determined by the leading elements in the cycle), openness (e.g. balance of import and export); bioaccumulation of toxicants (higher concentration in the biomass than in the surrounding medium) [11]; bio magnification (increasing the concentrations in each higher-trophic level) [12]; bio concentration (accumulation in separate organs or elements of bio-system), etc. In most cases, plants and animals are more exposed to the combined effects of many pollutants simultaneously [13]. The interaction between them may increase or decrease the toxicity of the mixture and hence alter the response of the biological system. The effects on biosystem exposure on two or more toxicants may result in the following combining toxicological responses: supplementary response (e.g. in simultaneous action of two organophosphate compounds) [14]; synergistic (reinforced) response (e.g. response of rat to concomitant ingestion of hepatic toxins – ethanol and carbon tetrachloride) and depressed (antagonistic, reduced) response, when the antagonistic reaction between toxicants exists: for example, chemical (e.g. the toxic effect of Se and Hg) [15], competitive (e.g. the toxic effect of CO), uncompetitive (e.g. the toxic effect of atropine and organophosphorus insecticides), functional (e.g. the toxic effect of barbiturate decreased vascular pressure) and predisposing (e.g. the reduction of organophosphorus insecticides toxicity with piperonyl bioksid by blocking the activity of cytochrome P450, responsible for the metabolism of organophosphates) antagonism. The interactions between toxicants and natural chemicals in the environment result in formation of new molecules or complexes, changing their expected utilization. When toxicity is unknown, the conducting tests use a wide range of concentrations and report of “all or none” response. The dose–response relationship is the tested biological effects to 4–5 toxicant concentrations that cause from 20 to 80% mortality. This value is only representative of acute exposure, not chronic one. The LD50 and ED50 variables are influenced by many factors: behaviour of animals [16], age [17], sex [18], temperature [19], water quality (hardness) [20], pH [21], etc. Nevertheless, the 50% response rate is used, because it is the most reproducible response and can be calculated with high reliability. There are three main types of systems for the contaminants exposure of aquatic organisms:

Figure 1.
Percentage distribution of ecotoxicological studies published based on random sampling.
In the random sample of 384 published studies, 535-conducted bio-tests were considered. For the studied period (2010–2016), an exponential increase of published ecotoxicological studies to 2012 can be observed—Figure 1. The level is kept in 2013 and the percentage sharp fell in 2014, while in 2015 the published ecotoxicological studies are closed to that in 2014. We do not have compete data for 2016, but it seems that this trend will likely keep. Furthermore, the scientific community is concerned about finding new environmentally acceptable agents and technologies in industry, agriculture and households, which is gradually becoming a priority in the new solutions. No less is the role of environmental legislation, the timely testing of new toxicants and the introduction of regulations and restrictions.
Among the reviewed studies, the tests for toxicity of aquatic environment prevail – 67%, of which these for the toxicity of freshwater are 35%, the terrestrial ones are about 20% and those concerning three environments – 13% (Figure 2).

Figure 2.
Percentage distribution of ecotoxicological studies by media published in random sample.
During the years, the focus of researches has been on the toxicity of different environments, for example, in 2011, prevailed these for the aquatic environment; in 2012, for the terrestrial; in 2013, again for the aquatic; and in 2015, again for the terrestrial (Figure 3).

Figure 3.
Percentage distribution of ecotoxicological studies by media and years published in random sample.
For the research period in the toxicological testing, 25 groups of biological systems have been used as most tests have been performed with crustaceans and fishes, 22% and 20%, respectively (Figure 4). Common test objects are also insects (9%), molluscs (9%), algae (8%) and the plants (6%). In the ecotoxicological studies, 61 crustacean species, 51 fish species, 27–17 insect species, molluscs, algae and higher plants have been used (Figure 5).

Figure 4.
Percentage participation of biological groups for ecotoxicological testing in random sample (2010–2016).

Figure 5.
Participation of biological groups (number of species) for ecotoxicological testing in random sample (2010–2016).
2.1. Ecotoxicological tests for short–term (acute) and continuous (long–term,6 chronic) toxicity
The accepted types of toxicants impact on exposed biosystem are: acute (high doses and for a short time, typically 24 – 96 h); sub-acute (repeated exposure for one month or less at lower doses than those in the acute exposure); sub chronic (multiple exposure, for 1–3 months) and chronic (exposure for more than 3 months at doses representing about 1/100 to 1/1000 of the acute dose). The exposure intervals definition varies for different biological systems, media and toxicants.
For the period concerned, the studies of acute toxicity clearly prevailed over the chronic tests. In a number of studies for clarifying the actual toxicity, a series of both tests have been performed. The practical application of subacute and sub-chronic tests is low or negligible (Figure 6), but the trend of increasing the interest for these tests in 2014 and 2015 is noticeable.

Figure 6.
Percentage participation of acute and chronic tests in random sample (2010–2016).
By the
2.2. Mono–species tests and multi–species tests
For the period concerned, the studies of acute toxicity clearly prevailed over the chronic tests. In a number of studies to clarify the actual toxicity the series of both tests have also been performed. The practical application of subacute and sub-chronic tests is low or negligible (Figure 6), but the noticeable trend of increasing the interest for these tests in 2014 and 2015 is observed (Figure 7). The analyses carried show that in 2012, 2014 and 2015, the tests with two species as test objects and multi-species ones were applied in most published studies. In 2013, the focus is mainly on tests with communities and multi-species ones.

Figure 7.
Percentage participation of tests with different number of test objects used in random sample (2010–2016).
The dysfunction occurring in plant communities and its effects on the plant populations structure and functioning is very well studied, especially for systems with poor species composition and simple structure. The pioneer studies have been published [1]. The imbalance is defined as a sudden change of the resource base that led to a clearly visible response. The hierarchical series of responses to these impacts can be predicted. The responses of the plant may influence the plant populations—reproduction, density spatial structure, rate of growth, mortality, body age, genetic variations, interspecies connections, etc. The different responses of plant populations can lead to major change of plant community as species composition, species richness, distribution of included genera, succession processes, etc. At the ecosystem level, these changes affect the primary productivity, the intensity of respiration, the intensity of mineralization and other functional processes. These responses depend on the type, frequency, intensity, duration and heterogeneity of dysfunctions. In some cases, we can evaluate the different obtained effects to the intensity and combination of impacts in models of vegetation structure and dynamics changes. By the comparison between the responses of exposed plant communities and the responses of untreated ones that grow on compatible soil types under similar topography and climate, the imbalance can be evaluated. Further, it would be possible to find a correlation of the results from laboratory tests, such as root growth, the growth of algae in soils or soil extracts, with actual plant data. There are some attempts to create models and study the complex toxicity on terrestrial ecosystems, but they are mostly with cultural ecosystems. For example, a model toxicological investigation of cultural plant-soil complex treated with wastewater have been published [95, 96]. Today, the conducting ecotoxicological studies with model ecosystems are common practice in the aquatic toxicology. While it can be considered that to some degree the problems associated with the study of the toxicological effects in the aquatic toxicology are resolved, this is not the case in terrestrial toxicology.
3. About the studies of ecosystem health (ecosystem diagnosis)
The evaluation methods for ecosystems health assessment are usually based either on
The need of test systems classification leads to the publication of a set of investigations in the last century. For example, the classification [1] is based on the following criteria: environment (air, water and soil); time of exposure (long, medium, occasionally, etc.); concentration of toxicant (mg/l, mg/m3); used organisms (bacteria, fish, mammals, plants, etc.); type of exposure (through food, air, dermal, etc.); the effects on test objects (genetic, toxic, bio-accumulation, etc.); measuring methods; test type (common, standard, experienced, screening); requirements for variability, accuracy and precision of values and technical requirements for personnel and laboratory equipment. The developed protocols, however, require significant modifications depending on the type of ecosystem and environmental factors, the objectives of the study and more mentioned in the specific dynamic action analyses (SDA). To assess the possible effects on ecosystem level, the responses of dominant species are usually investigated on a set of sample plots. The mostly used indicators are grouped as indicators of plant community, plant chemistry (major cations, nitrogen, phosphorus, iron, zinc, etc.), aboveground and water insects (some populations of
For the forest ecosystem health assessment, the widespread indicator is defoliation that can do possible to calculate the ratio of damage of ecosystem: C = [∑(n·k)/NK]·100, where n is number of trees with respective scores of defoliation (first to fifth score); N—the total number of trees; K – maximum score on the scale. The forest ecosystems are considered to be damaged at C > 30% [121] Percentage of defoliation is determined by sight with guidance, where the habitus of crowns with different rates of defoliation for each tree species is given.
Nowadays,
4. Conclusion
New synthetic chemicals are recorded each year and the legislation in countries requires the immediate conduction of the both – toxicological and ecotoxicological testing. The scale of the potential ecological impacts on the environment and biota requires fast and accurate assessments of toxicological effects. The practical importance of ecotoxicology for the existence and functioning of the MBS is constantly growing. The toxicity may be different for different species in the ecosystem and for the same species in different ecosystems. Furthermore, toxicants do not only directly affect the biological system being evaluated, but may have an indirect negative effect on it, altering both abiotic and biotic parameters in the ecosystem. The various populations of the same species under different environmental conditions will respond differently to a given concentration of toxicant. In ecotoxicology practice, the number of species is used as test objects, and the results are extrapolated to all groups of organisms in the ecosystem. The variation in size, physiology, evolution, ontogeny and geographical distribution are some of the important parameters that usually do not fit exactly. However, some of the basic tests have demonstrated its great importance in the understanding of contaminants effects on the environment. The series of variables must be considered for the realistic assessment of environmental toxicity and MBS state. The reported sublethal effects often refer to changes in the structure of MBS that can lead to their degradation. A greater variation in the responses of individuals, populations and ecosystems observed in nature are compared with these reported under laboratory conditions, due to the mutual influence. This fact requires the more intensive usage of multi-testing systems—micro- and mesocosms and new developments. The analysis of situation and problems of ecotoxicological testing makes it possible to outline the directions in which to focus future efforts. They are related to the search of sensitive species for acute and risk testing, developing of new biomarkers and kits, especially for the study of terrestrial toxicity, formation of model systems (micro- and mesocosms) by key members of the ecosystem trophic network for multi-species testing and modelling the toxic effects at MBS level, which is especially true for the terrestrial ecotoxicology.
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