Open access peer-reviewed chapter
Standardized toxicity tests using earthworms as test organisms.
Abstract
Terrestrial ecotoxicology is used to evaluate the effects of substances that, whenever added to the soil, have impact on organisms and help measuring the responses from changes in the lethality, reproduction, development, and behavior of standardized soil organisms. Terrestrial ecotoxicology is a new tool that has been introduced in many countries, including Brazil, and yet little used. However, it is already widely used in Europe, besides being mandatory in research to indicate the toxicity of waste discharged in the soil. The aim of this chapter is to emphasize the importance and need of developing studies focused on the use of terrestrial ecotoxicology as a tool to assess fast and reliable responses from the toxicity of substances incorporated to the soil.
Keywords
- contamination
- bioindicators
- biomarkers
- pesticides
- soil fauna
1. Introduction
The environment has been exposed to contamination caused by many toxic agents due to human action. Environmental impact, such as soil, surface water, and groundwater contamination, is mainly caused by deactivated industries, contaminant leakage, pesticide use, and inadequate solid and liquid waste disposal. Soil protection and the protection of its inherent communities are acknowledged as the primary aim of environmental policies worldwide [1].
Although the most obvious use of soil lies on plant cultivation for food production, it is also responsible for helping sustainability maintenance. Soil retains water, regulates water resources, and filters and conducts rain water to underground aquifers. It also plays the role of recycling raw materials and nutrients, besides being the habitat of a wide variety of organisms such as bacteria, fungi, viruses, nematodes, insects, and worms, among others. The food consumed by living organisms is produced in the soil [2].
The soil, besides being the substance for most man-made food, holds a large amount of pollutants. Chemical and organic fertilizers, pesticides, and other materials applied to the soil often contribute to water and air pollution. Soil is a key component of environmental chemical cycles and an important part of natural resources on Earth. The quality of the soil and climate, which enables productivity, is the most valuable asset of the society [3, 4].
Ecotoxicological tests are internationally acknowledged as complementary tools to chemically analyze soil contamination. However, countries, such as Brazil, do not require the conduction of ecotoxicological tests with soil organisms to evaluate contamination, since it is only based on chemical indicators [5]. The behavior and toxicity of soil elements, or compounds, should not be only assessed based on chemical parameters. Actually, it should be of the utmost importance to include biological parameters in these investigations, since chemical analyses separately applied to the compounds may not show their behavior in the environment [6].
Terrestrial ecotoxicology seeks knowledge about the consequences of chemical-substance discharge in the environment on the organisms living in it. Therefore, it is essential understanding, to which extent, how hazardous the use of chemicals, alone or in mixtures, is, as well as where its effects are observed by monitoring the lethal, morphological, behavioral, physiological, cytogenetic, and biochemical effects on organisms exposed to these pollutants [6, 7].
Among many organisms in soil fauna, which is divided into macro-, meso-, and microfauna, one finds bacteria and fungi and species known as bioindicators. These organisms indicate environmental changes at their early stages and identify several modification types before these changes become worse, besides determining the pollution types capable of affecting a given ecosystem. They also have the ability to help monitoring and making more accurate and less impacting decisions on soil management. There are standardized bioindicator species around the world, such as
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2. Soil quality
The quality of the soil is defined by “its ability to function within an ecosystem in a way that it sustains biological productivity, maintains environmental quality, and promotes plant and animal health” [8]. Overall, knowing the soil characteristics involved in the development and sustainability of ecosystems is a useful tool.
Soil quality is assessed through the characteristic of indicators that measure or reflect environmental status or sustainability conditions of ecosystems. Soil quality indicators can be classified as physical, chemical, and biological.
2.1. Physical indicators
The main physical indicators in place are texture, thickness (horizons), soil density, porosity, water, and structure.
Texture refers to particle proportions or to sand, silt, and clay fractions in the soil mass. It is closely related to water retention and transport, soil structure, nutrient content, and organic matter, besides its strong influence on soil erosion processes [9, 10].
Thickness, mainly that of the surface horizon, is essential, since it is the place where the greatest biota activity takes pace. Consequently, the surface horizon is a suitable place for organic matter and nutrient cycling. Soil depth plays a key role given its water storage ability and influence on nutrient supply for plants [10].
The diversity of mineral and organic components, as well as their proportion, determines the density of soil materials. This density changes depending on texture and on soil structural conditions [9, 11].
Porosity is determined by porous space. Part of the soil after the arrangement of solid components is occupied by water and air under natural conditions. Sand retains little water, because its large porous space allows free water drainage from the soil. Clay adsorbs relatively large amounts of water, and its smaller porous space retains water, even with gravity forces [11, 12].
Water amount in the soil depends on climatic factors and on soil texture, structure, and porosity. Water retention capacity reveals the amount of water the soil can store. Hydraulic conductivity is defined by the speed water reaches when it moves in the soil and by its infiltration rates [11, 12].
Soil structure is determined by the geometric arrangement of primary (sand, silt, and clay) and secondary particles (aggregates maintained by cementing agents such as iron, silica, and organic matter) [13].
2.2. Chemical indicators
Many soil functions such as nutrient reservoir, filtration of substances dissolved in water, chemical reaction accelerator, and pollutant immobilizer would not be possible without the presence of chemical components. Organic matter (OM), cation exchange capacity (CEC), hydrogen ionic potential (pH), and chemical elements are often used as chemical indicators of the quality of the soil [14].
Organic matter (OM) is the set of all organic materials in the soil. It is correlated to most soil properties, besides being a key indicator of the quality of the soil [15].
CEC stands out as one of the basic functions of the soil that lies on providing nutrients to plants. This indicator is found on the surface of solid particles in the soil due to electrical charges (Al, Ca, K, Mg, Mn, and Na), although it can be changed by pH and OM [14, 15].
pH is the indicator used to measure soil acidity, and changes in it can affect nutrient availability, microbial populations, and the availability of chemical elements in the soil.
Chemical elements play many roles, but it is possible roughly framing them in nutrients or toxic elements.
Soil nutrients can be divided into two groups:
Macronutrients (Ca, K, Mg, N, P, S), which are mostly needed by plants.
Toxic elements, which are considered heavy metals.
Have in mind that the same chemical element, in a certain concentration, can be a nutrient but toxic in another one.
2.3. Biological indicators
Biological indicators, such as microbial biomass, mineralizable nitrogen, microbial respiration, enzyme activity, and metabolic quotient, are fundamental for nutrient cycling and for estimates on soil ability to influence plant growth. In addition, microorganisms provide rapid responses to changes in the environment due to the abundance of metabolic and biochemical activities; therefore, they have great potential to be used as a tool to assess the quality of the soil [16].
The microbial biomass is represented by living components in soil organic matter [17]. This variable controls functions such as the decomposition and accumulation of organic matter or transformations involving mineral nutrients. Biomass provides information about changes in the organic properties of the soil, about changes caused by crops or by vegetation removal, about regeneration after topsoil removal, and about the effects from pollutants such as heavy metals and pesticides [16, 17].
The nitrogen (N) in the soil is the organic compound indirectly available to plants. The N content conversion from its organic form to its mineral one (mineralization) is mainly carried out by bacteria belonging to genus
Soil respiration reflects the microbial activity and is defined as the biological oxidation of organic matter into carbon dioxide (CO2), which is conducted by aerobic microorganisms [18]. This indicator occupies a key position in carbon cycles observed in terrestrial ecosystems due to oxidation [19].
Enzymes rule the biological catabolism of organic and mineral components in the soil. This process is closely related to organic matter, physical properties, and microbial activity and biomass; therefore, it is widely used to assess the quality of the soil, since it indicates changes in microbial activity and the presence of pollutants in the soil [14, 20, 21].
The respiratory coefficient represents the relation between carbon in the microbial biomass and total organic carbon (TOC). This coefficient indicates microbial biomass efficiency in using the available carbon for biosynthesis [21, 22].
Besides the aforementioned biological indicators, soil fauna can be used as quality indicator. The biological diversity of the soil is fundamental to maintain soil productive ability; therefore, it is of great importance for the decomposition and mineralization of organic residues, since it favors nutrient availability for plants and even for other individuals. In addition, the soil fauna is sensitive to environmental changes, biological, physical, or chemical. Earthworms (
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3. Contamination of soils
Soil contamination is one of the main environmental issues worldwide [1]. The unconscious use of the soil for agricultural activities, for the disposal of waste, chemicals, and industrial waste has been the cause of concern for centuries. Besides huge damages to the environment, a large amount of contaminated soils unviable for agriculture or for construction sites is the consequence of such inappropriate discharge [24]. Agricultural production has led to the increased use of pesticides to control pests and weeds. In addition to active toxic ingredients, many agricultural products contain potentially polluting elements or compounds, such as trace elements and emulsifying surfactants, among others [25, 26]. There has been simultaneous increase in the application of sewage sludge, industrial waste—composed of urban waste—and of agricultural waste for disposal or recycling purposes. These residues have high contents of organic matter and mineral elements capable of improving the chemical, physical, and biological properties of the soil. However, these residues may contain trace elements, pathogens, and many other substances that cause environmental damages [26, 27].
The term “soil contamination” refers to the presence of toxic substances belonging to chemical classes such as inorganic ions (metals), organic solvents, radioactive substances, pharmaceuticals, polycyclic aromatic hydrocarbons (PAHs), and pesticides (herbicide, insecticide, and fungicide) [28].
According to the International Union of Pure and Applied Chemistry (IUPAC), inorganic pollutants are described as “toxic elements.” They comprise metal and metalloid elements formerly called “heavy metals.” This nomenclature includes elements that, at low concentration, are biologically essential to living organisms. However, when these essential elements are observed at concentrations higher than the required ones or above the recommended limits, they also cause imbalance and exhibit some toxicity.
The displacement of water, soil, and air contaminants, as well as the interface between different compartments, is determined by processes related to the chemical properties of substances in the soil and to environmental compartments. Soil is one of the most complex matrices in the environment due to its heterogeneity. Different soil types present a wide variety of particle sizes and ecophysiological characteristics [14].
Contaminants that reach the lithosphere move by diffusion, that is, water moves through gaps between soil particles. Contaminant displacement speed depends on molecular weight and on the concentration of the contaminant gradient, as well as on soil characteristics such as humidity, clay type, specific area, cation exchange capacity, pH, redox potential, temperature, porosity, and permeability. Soil contamination presents intrinsic characteristics such as cumulative characters and low mobility of pollutants. However, this mobility becomes greater in soil recording relatively low pH or large amounts of sand to the detriment of clay, since these factors make the solubility of toxic elements easier in the environment. Consequently, the soil becomes susceptible to water leach to underground sheets and to other water bodies. Therefore, interactions among the chemical structure of contaminants, soil properties, and entry mode in the environment determine whether a specific substance is persistent and whether it is potentially hazardous to soil compartment [12, 13, 14, 15].
The soil must be understood as a living being due to its characteristics, since its biota plays a fundamental role in physical-chemical equilibrium. The soil gets polluted by negative changes in the existing equilibria, which lead to conditions that impair, or makes unfeasible, the life in the assessed area. They also cause environmental damages that can take millennia to be fixed.
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4.3.
4.3.1. Ecotoxicological studies already carried out with earthworms belonging to genus
4. Terrestrial ecotoxicology
Terrestrial ecotoxicology aims at evaluating the effect of toxic substances on organisms representative of terrestrial ecosystems. This area involves the transportation, distribution, transformation, and final destination given to contaminants found in the terrestrial environment.
Biological processes may be more sensitive to soil changes based on physical and chemical properties, since they suggest that biological indicators could potentially provide early warnings about risks posed to ecosystems [7].
The soil is a heterogeneous mixture of biotic and abiotic factors, besides being inhabited by a wide community of organisms. The fundamental functions of the system depend on its structural and functional integrity. This functionality faces direct impact from changes, and it requires many parameters in order to accomplish a better evaluation [4, 8, 10].
Terrestrial ecotoxicology uses ecotoxicological tests—internationally standardized by the International Organization for Standardization (ISO) and by the Organization for Economic Cooperation and Development (OECD)—which expose bioindicators (animals or plants) to soils contaminated with products or residues of interest in order to qualify and quantify the aforementioned negative effects. Different contamination levels are used in studies in this field [5, 29, 30]; therefore, Inferring the toxicity of the assessed substance and contributing to establish the limits of its use or disposal is possible.
Invertebrate populations in the soil are an appropriate tool to assess the degree of soil disturbance or land use intensification [5] due to human activities [30].
4.1. Ecotoxicological tests
Ecotoxicology is applied through tests that measure the toxicity of substances found in the environment by exposing standard living organisms to them [31].
Ecotoxicological tests allow assessing environmental contamination caused by different pollution sources. These tests have the advantage of covering a wide variety of biological substances available in a sample. They have the inherent ability to detect deleterious effects produced by a toxic agent, or mixture, on living organisms. Thus, these tests allow evaluating how hazardous these substances are.
A large number of ecotoxicological trials have been developed, or improved, due to a wide variety of studied species and ecosystems. Aquatic, sedimentary, and terrestrial systems can be used in tests to verify the degree of contaminations caused by toxic agents and their possible ecological implications [32, 33].
4.1.1. Avoidance test
Among the ecotoxicological tests available, the behavioral test (avoidance test) is a rapid method applied to determine the bioavailability of chemicals or the contaminants in the soil. Although this test is a simple and rapid assay, it has high ecological significance, since avoidance indicates site rejection and population decrease caused by stressor agents such as contamination [34].
The behavioral response results from the level of the organism, which can be defined as the action, reaction, or activation of a given system. This system is subjected to a set of specific conditions that represent the integration of biochemical and physiological processes [35].
Avoidance tests also substantiate the application of other ecotoxicological tests (acute and chronic), since they indicate whether a substance is influencing the physiological and metabolic functions of test organisms.
4.1.2. Acute toxicity test
Acute toxicity tests (mortality test) assess contamination after short-term exposure to high contaminant doses (from 24 h to 14 days). Overall, acute toxicity test results showed that lethality is the main effect of contamination; however, other manifestations such as decreased mobility can be observed [36].
Acute toxicity tests are relatively simple, are inexpensive, and can be applied to a wide variety of organisms. However, they have the disadvantages of not indicating the contaminants responsible for the observed toxicity and the effects of contamination on the dynamics of assessed populations [37].
Results of acute toxicity tests include LC50/LD50 values (concentration/dose causing mortality to 50% of organisms tested) or EC50 (effective concentration or concentration causing an effect, other than mortality, that is, immobility to exposed organisms) [38].
4.1.3. Chronic toxicity test
Chronic toxicity (reproduction test) tests are closely linked to results of acute toxicity tests, since sublethal concentrations are calculated based on LC50. These tests evaluate the effects of lower contaminant concentrations for long exposure time. The observed effects are sublethal and emerge when the toxic agent concentration the organisms are exposed to allows the survival of these organisms. However, these concentrations affect one, or more, biological function of these organisms, and it influences reproduction and egg development and growth [40].
Chronic toxicity tests may be long and laborious; sometimes they range from 4 to 7 weeks, and these factors are the main disadvantage of applying this test type [37]. However, they are the most sensitive tests, which are considered the most relevant to predict the impacts on ecosystems, since they demonstrate the dynamics of populations over time [41].
Results of chronic toxicity tests can be expressed in CENO (concentration of observed effect, the highest concentration of the tested sample, which does not cause deleterious effect) or in CEO (concentration of observed effect, the effect of the lowest concentration on the body) [39].
The use of ecotoxicological tests aims at integrating different information to plan, to make decisions about public health, to take environmental control measures, to define remediation techniques, and to hierarchize the areas to be prioritized by environmental recovery programs.
4.2. Biomarkers and bioindicators
Biomarkers are tools used to measure the effects of the exposure to toxic compounds and its potential impact on living organisms, including humans. Bioindicators are changes resulting from the presence of xenobiotics in components and in cell biochemical processes, structures, or functions measurable in a system or sample [42].
Indicator organisms are used in the testes due to their characteristics, since they present very short ecological tolerance limit. Therefore, they present some physiological, morphological, and/or behavioral changes when they are exposed to certain contaminants [32].
The low cost of bioindicators is their advantage in comparison to conventional methods used to evaluate environmental quality. They can also be used in the cumulative evaluation of events observed within a given period of time to recover an environmental history that cannot be detected through other methods [23, 31, 32].
The soil hosts the greatest diversity of organisms on the planet; however, these organisms can be affected by the substances deposited in it [16, 34]. Hundreds of thousands of invertebrate species contribute to decomposition processes by crushing organic matter, improving the mineralization process and, consequently, the nutrient cycling and energy flow in ecosystems [16, 43].
Ecotoxicological tests have been widely used to evaluate the environmental impact of several pollution sources such as hydrocarbons [34, 36], toxic sludge from the ceramic [44] and textile industries [45], domestic effluent [46, 47], persistent organic pollutants (POPs) [48], and bovine manure [49].
However, ecotoxicological studies conducted in terrestrial environments remain relatively incipient in comparison to the ones involving aquatic environments. Most studies in this field are concentrated in Northern countries, and this evidence highlights the need of verifying the possible impacts of this situation on the soil of different regions of the world [27, 30].
Edaphic organisms are among the indicators used to measure the quality of the soil, given their importance to the decomposition, cycling, and regularization of nutrients in biological systems [23].
Earthworms (
4.3. Eisenia sp. (Oligochaeta , Lumbricidae )
Species belonging to genus
Earthworms are exposed to contaminants through skin absorption and intake. A yellow fetid substance acts in defense of the earthworms when they are threatened by pores in the upper surface of the body—this substance keeps predators away from them [51, 52, 53].
According to [58], earthworms are widely used given their suitability for the bioavailability evaluation of many soil chemicals due to the following factors:
Earthworms live on the ground and are in constant contact with the soil.
They live in contaminated sites, and it allows the validation of chemical availability in the field.
They are found in a wide variety of soil horizons.
The epidermis of the worms is vascularized, but it has no cuticle, and it allows absorbing the contaminants straight from the soil.
Earthworms ingest specific fractions of soil, and it provides the means for contaminant uptake.
They present high body mass, and this factor helps determining the concentration of contaminants in the assessed individuals.
Their physiology and the metabolism of metals in their organisms are well known.
Availability of standard test protocols.
Some species, such as
E. fetida , are easy to cultivate, can be kept in laboratory under controlled conditions, and are tolerant to different soil types.
Pollutants in soils have direct contact with clay and with organic materials highly capable of binding to chemical compounds and substances [14, 15, 19]. Earthworms get in contact with pollutants when they excavate and ingest contaminated soil or litter; they also absorb contaminants from the soil solution that passes through the cuticle. So earthworms can poison themselves with these pollutants; they can die or survive by incorporating or even bioaccumulating these pollutants in their tissues. This ability can be a threat to their predators, since earthworms are an important link in the terrestrial trophic chain; they are on the diet of several animal species [59, 60, 61, 62, 63].
Procedures followed in ecotoxicological tests conducted with earthworms are established by national and international standards. There are internationally acknowledged standards proposed by International Organization for Standardization (ISO) and Organization for Economic Cooperation and Development (OECD). However, some countries have their own standards. For example, in Brazil, the NBR 15537 (2014) and NBR ISO 17512-1 (2012) regulate the application of test with earthworm conducted to evaluate acute toxicity and behavior, respectively.
Table 1 shows a selection of standardized toxicity test protocols developed by acknowledged institutions involving worms currently available for researchers.
Many studies focus on artificially contaminated soil; they use to add a single substance to the assessed soil. In addition, these studies are conducted under conditions that do not properly reflect the reality in the field.
4.3.1. Ecotoxicological studies already carried out with earthworms belonging to genus Eisenia sp.
Table 2 shows a summary of results recorded by some authors in studies conducted with
| Test | Duration (days) | Species | Standard |
|---|---|---|---|
| Lethality | 14 | OECD 2017 (1984) ISO 11268 (2012) | |
| Reproduction | 28 + 281 | ISO 11268-2 (2012) OECD 222 (2004) | |
| Avoidance | 2 | ISO 17512-1 (2012) | |
| Bioaccumulation | Until 21 + 212 | OECD 317 (2010) | |
| 7 to 28 | ASTM E 1676-12 (2010) |
Table 1.
28 days for cocoon production + 28 days for juvenile hatching.
First stage, up to 21 days for the absorption of the test substance; second stage, 21 days for elimination.
| Author | Results |
|---|---|
| [37] | They exposed Results: avoidance—copper sulfate, 320 mg/kg, earthworms in the assessed mine showed no preference between the contaminated soil and the control |
| [36] | They evaluated the behavior of Results: 96% of individuals fled the section containing the contaminated sample |
| [64] | They evaluated the behavior of Results: Organisms presented greater attraction to sludge from the sewage treatment plant and treated canning industry effluent due to the higher organic matter concentrations in them. There was avoidance reaction at low concentrations of sludge from the galvanic industry, mainly due to the high content of chromium in it |
| [65] | The authors analyzed soil and sludge mixtures from three sewage treatment plants in Germany. Results: The sludge was toxic to the test organisms ( |
| [66] | Assessed the effects of herbicides Diuron and fluazifop-p-butyl on Results: The avoidance behavior in the soil evidenced that both herbicides caused significant avoidance response |
| [67] | Studied the avoidance behavior to silver forms (nanomaterials (NMs)) at four time points (24, 48, 72, and 96 h) Results: AgNO3 and NM300K induced avoidance response in |
| [34] | The authors investigated the toxicity of a binary petroleum hydrocarbon (PHC) mixture to the avoidance response of five soil invertebrate species ( Results: The avoidance of invertebrates in PHC-contaminated soil was similar in growth measurements applied to plant species sensitive to PHC-contaminated soil |
Table 2.
List of results recorded by some authors in a study focused behavioral tests conducted with earthworms.
Table 3 summarizes the results recorded by some authors in studies with
| Author | Results |
|---|---|
| [63] | Eight soil samples were collected from seven abandoned mines in the United Kingdom Results: Cocoon production and hatching rates showed that they were more sensitive to survival conditions or to weight changes. The most toxic soil presented low organic carbon, and sandy soil presented intermediate metal concentrations (7150–13,100 mg Pb/kg, 2970–53,400 mg Zn/kg) |
| [64] | Evaluated the behavior of Results: Mixtures containing domestic sewage and sludge from the olive industry increased the production of juvenile organisms and stimulated the growth of earthworms. However, the mixture with sludge from the galvanic industry caused significant decrease in the production of juveniles |
| [68] | The authors carried out an ecotoxicological evaluation with oligochaetes ( Results: The applied doses did not present significant mortality under acute exposure, but they caused lethal effects under chronic exposure. In addition, the test organisms bioaccumulated metals (Cu, Hg, Pb, Zn) because they are bioavailable and due to the role played by Cu and Zn in the metabolism and physiology of oligochaetes |
| [48] | Evaluated the ability of Results: Annelids accumulated hexachlorobenzene in their tissues, and this process may result in biomagnification along food chains |
| [49] | Evaluated the toxicity of bovine clearance residues on the survival rate of Results: They found 100% lethality in individuals subjected to the treatment with waste at concentrations 84.12 mg/kg Zn and 323.11 mg/kg Cu. The increased Cu and Zn concentrations reduced the number of cocoons |
| [47] | The authors carried out acute and chronic toxicity tests with Results: The sewage sludge showed no acute toxicity effect. The reproduction test presented the deleterious effect of it on the reproduction of worms, since there was the absence of cocoons and of young organisms after the 14th day of testing |
| [69] | Evaluated dredged sediments in Guabanara Bay, Rio de Janeiro, Brazil, through toxicity tests. The bay has been highly impacted by the disposal of liquid domestic and industrial waste Results: |
| [29] | The toxicity of five pesticides (trichlorfon, dimethoate, carbendazim, tebuconazole, and prochloraz) typically used in rice farming was evaluated through the mortality and body weight of Results: The insecticide dimethoate showed moderate acute toxicity, whereas the other tested pesticides showed low toxicity potential. However, weight loss was identified as a sensitive endpoint with the use of these pesticides |
| [70] | Evaluated the chronic toxicity of the abovementioned six neonicotinoids (NEOs) to Results: Cocoon production and hatchability were more sensitive than cocoon weight and adult weight. The reproduction of earthworms was significantly reduced at 56 days half-maximal effective hatchability concentrations (EC50) 0.37, 0.74, 1.30, 3.57, 1.20, and 0.70 mg/kg (acetamiprid, dinotefuran, clothianidin, thiacloprid, nitenpyram, imidacloprid), respectively. Most tested NEOs were highly toxic to |
Table 3.
List of results recorded by some authors in a study focused on lethality and reproduction conducted with earthworms.
Laboratory soil toxicity tests have advanced in recent years given the introduction of soil invertebrates in them; consequently, the adoption of avoidance toxicity tests has increased [34].
There is lack of data about the effects of herbicides on earthworms because they are often seen as low or nontoxic. [40] investigated whether the widely used commercial formulations of glyphosate (GLF), tembotrione (TBT), and nicosulfuron (NCS), which are applied at three environmentally relevant concentrations, have adverse effects on different biomarkers and on the reproduction of the epigeic earthworm species
Ref. [68] evaluated bioaccumulation in organisms of oligochaetes exposed to soil treated with sewage sludge and found that earthworms absorbed Zn and Cu metals in their tissues. In addition, the chronic assay caused lethal effects, as well as the absence of cocoons, at the end of the bioaccumulation stage. This outcome evidences that the toxics found in sewage sludge were able to affect the reproduction of oligochaetes.
There was significant copper (Cu) uptake increase by earthworm (
Ref. [71] verified the toxicity rates in
Ref. [72] evaluated the toxicity of different aluminum concentrations to
Some factors influence test results, and they should be carefully evaluated based on their interference.
According to [73], soil characteristics in extremely sandy soils influence the results of ecotoxicological tests. Earthworms tolerate a wide range of pH (from 4 to 9), but they prefer neutral to slightly acidic pH conditions (from 5 to 7). Ref. [74] studied the reproduction of
Earthworms prefer soil with high levels of organic matter. According to studies, earthworms strongly tend to avoid soil with low organic matter content [75]. In addition, several authors [76, 77, 78] indicate that organic matter forms stable complexes with metals by reducing their bioavailability and geochemical mobility.
Ref. [64, 69] point out that oligochaetes recognize the organic matter in domestic sewage and in sediments as real food sources.
Ref. [71] sought to analyze the reproduction of earthworms and the production of cocoons of organisms exposed to different Al concentrations and pH. Based on the recorded results, low Al concentrations and higher pH values reduced the production of cocoons. These authors suggest that increased oligochaete biomass may have implications in the reproduction of these animals, since organisms use much of their metabolism energy to increase their biomass; thus they reduce their reproduction and cocoon production rates.
Ref. [69] found biomass loss in test organisms subjected to the treatment with 20% dredged sediment from a pond receptor of domestic and industrial effluent. Weight loss likely resulted from the fact that energy reserves are mobilized to allow detoxification processes that reduce the energy allocated for growth purposes.
The greater attraction of organisms to soil with high organic matter content does not exclude toxicity likelihood, and this observation evidences the need of carrying out a test at chronic level.
Thus, it is essential knowing soil characteristics, since they can influence stress factors in organisms, other than the ones related to the contaminant that influence test results.
Recently, lethality and reproduction are not only analyzed through toxicity trials. Biochemical responses and DNA damage are complementary approaches to standard toxicity tests, since they provide more information about body responses to stress in mixtures [79].
Ref. [79] evaluated the activity of superoxide dismutase, acetylcholinesterase, cellulase, and DNA damages in
Ref. [80] evaluated changes in superoxide dismutase, catalase, peroxidase, cellulase, and malondialdehyde in
The biochemical and genetic toxicity of dinotefuran on
Research such as those conducted by [79, 80] are promising, but further research is needed in order to explain certain mechanisms, given the complexity of some enzymes, as well as DNA damage and its possible consequences on organisms.
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5. Conclusions
Natural soil resources must be interpreted and studied by taking into account all their compartments, since they act together in different soil functions.
Contaminated soils have been largely monitored through physical-chemical analyses. However, soil quality assessments and synergistic effects caused by contaminants are not taken into account. Ecotoxicological tests are critical for the evaluation of potential environmental risks posed by these contaminants.
It is important carrying out tests by using organisms that represent terrestrial ecosystems in toxicological evaluations. Toxicity tests are tools that help the management of contaminated areas. They indicate the viability of natural environmental recovery, since organisms in the field degrade and mineralize substances in contaminated environments.
It is recommended to extend the present study to other organisms, such as microcrustaceans, living in different habitat types (sediments and water) in order to cover different trophic levels and to assess whether there is, or not, toxicity transfer between food chain levels.
References
- 1.
Filser A, Koehler H, Ruf A, Römbke J, Prinzing A, Schaefer M. Ecological theory meets soil ecotoxicology: Challenge and chance. Basic and Applied Ecology. 2008; 9 :346-355. DOI: 10.1016/j.baae.2007.08.010 - 2.
Jules Pretty J, Bharucha ZP. Sustainable intensification in agricultural systems. Annals of Botany. 2014; 114 :1571-1596. DOI: 10.1093/aob/mcu205 - 3.
Bruulsema T. Managing nutrients to mitigate soil pollution. Environmental Pollution. 2018; 243 (Pt B):1602-1605. DOI: 10.1016/j.envpol.2018.09.132 - 4.
Drobnik T, Greiner L, Keller A, Grêt-Regamey A. Soil quality indicators—From soil functions to ecosystem services. Ecological Indicators. 2018; 94 :151-169. DOI: 10.1016/j.ecolind.2018.06.052 - 5.
Bianchi MO. Importance of ecotoxicological studies with invertebrates from soil. Seropédia: Rio de Janeiro; 2010. 32 p. ISSN: 1980-3075 - 6.
Wang QY, Sun JY, Xu XJ, Yu HW. Integration of chemical and toxicological tools to assess the bioavailability of copper derived from different copper-based fungicides in soil. Ecotoxicology and Environmental Safety. 2018; 161 :662-668. DOI: 10.1016/j.ecoenv.2018.06.041 - 7.
Souza TS, Christofoletti CA, Bozzatto V, Fontanetti CS. The use of diplopods in soil ecotoxicology—A review. Ecotoxicology and Environmental Safety. 2014; 103 :68-73. DOI: 10.1016/j.ecoenv.2013.10.025 - 8.
Doran JW, Parkin TB. Defining and assessing soil quality. In: Doran JW, editor. Defining Soil Quality for a Sustainable Environment. Madison: SSSA and ASA; 1994. pp. 3-21. DOI: 10.2136/sssaspecpub35.c1 - 9.
Vezzani FM, Mielniczuk JM. An overview of soil quality. Brazilian Journal of Soil Science. 2009; 33 :743-755. DOI: 10.1590/S0100-06832009000400001 - 10.
Muñoz-Rojas M. Soil quality indicators: A critical tool in ecosystem restoration. Current Opinion in Environmental Science & Health. 2018; 5 :47-52. DOI: 10.1016/j.coesh.2018.04.007 - 11.
Ball BC, Campbell DJ, Douglas JT, Henshall JK, O'Sullivan MF. Soil structural quality, compaction and land management. European Journal of Soil Science. 1997; 48 :593-601. DOI: 10.1111/j.1365-2389.1997.tb00559.x - 12.
Baver LD, Gardner WH, Gardner WR. Soil Physics. 4th ed. New York: John Wiley & Sons; 1972. 498 p. ISBN: 0-471-05974-9 - 13.
Brady NC, Weil RR. The Nature and Properties of Soils. 15th ed. New Jersey: Prentice Hall; 2008. ISBN: 978-0133254488 - 14.
Sparks DL. Environmental Soil Chemistry. San Diego: Academic Press; 1995. 267 p. ISBN: 9780080494807 - 15.
Sposito G. The Chemistry of Soils. New York: Oxford Univ. Press; 1989. 277 p. ISBN: 0-19-504615-3 - 16.
Coleman DC, Crossley DA Jr. Fundamentals of Soil Ecology. San Diego: Academic Press; 1995. 205 p. ISBN: 9780080472812 - 17.
Jenkinson DS, Ladd JN. Microbial biomass in soil: Measurement and turnover. In: Paul EA, Ladd JN, editors. Soil Biochemistry. New York: Marcel Dekker; 1981. pp. 415-471 - 18.
Anderson JPE. Soil respiration. In: Page AL, Miller RH, Keeney DR, editors. Method of Analysis, Part 2. 2nd ed. Madison: American Society of Agronomy: Soil Science Society of America; 1982. pp. 831-871 - 19.
Manahan ES. Environmental Chemistry. 9th ed. Florida: CRC Press; 2010. 783 p. ISBN: 978-1-4200-5920-5 - 20.
Chen X, Xu Y, Gao H, Mao J, Chu W, Thompson ML. Biochemical stabilization of soil organic matter in straw-amended, anaerobic and aerobic soils. Science of the Total Environment. 2018; 625 :1065-1073. DOI: 10.1016/j.scitotenv.2017.12.293 - 21.
Shen D, Ye C, Hu Z, Chen X, Guo H, Li J, et al. Increased chemical stability but decreased physical protection of soil organic carbon in response to nutrient amendment in a Tibetan alpine meadow. Soil Biology and Biochemistry. 2018; 126 :11-21. DOI: 10.1016/j.soilbio.2018.08.008 - 22.
Ali RS, Kandeler E, Marhan S, Demyan MS, Ingwersen J, Mirzaeitalarposhti R, et al. Controls on microbially regulated soil organic carbon decomposition at the regional scale. Soil Biology and Biochemistry. 2018; 118 :59-68. DOI: 10.1016/j.soilbio.2017.12.007 - 23.
Baretta D, JCP JCPS, Segat JC, Geremia EV, LCI OF, Alves MV. Edafic fauna and soil quality. In: Topics in Soil Science. 2011. pp. 119-170. ISSN: 1519-3934 - 24.
Petrik A, Thiombane M, Lima A, Albanese S, Buscherb JT, De Vivo B. Soil contamination compositional index: A new approach to quantify contamination demonstrated by assessing compositional source patterns of potentially toxic elements in the Campania region (Italy). Applied Geochemistry. 2018; 96 :264-276. DOI: 10.1016/j.apgeochem.2018.07.014 - 25.
Liu X, Lonappan L, Brar SK, Yang S. Impact of biochar amendment in agricultural soils on the sorption, desorption, and degradation of pesticides: A review. Science of the Total Environment. 2018; 645 :60-70. DOI: 10.1016/j.scitotenv.2018.07.099 - 26.
Xing Y, Meng XM, Wang L, Zhang J, Wu Z, Gong X, et al. Effects of benzotriazole on copper accumulation and toxicity in earthworm ( Eisenia fetida ). Journal of Hazardous Materials. 2018;351 :330-336. DOI: 10.1016/j.jhazmat.2018.03.019 - 27.
Melo W, Delarica D, Guedes A, Lavezzo L, Donha R, Araújo A, et al. Ten years of application of sewage sludge on tropical soil. A balance sheet on agricultural crops and environmental quality. Science of the Total Environment. 2018; 643 :1493-1501. DOI: 10.1016/j.scitotenv.2018.06.254 - 28.
Oga S, Camargo MM, Batistuzzo JA. Toxicology Fundamentals. 4th ed. São Paulo: Atheneu; 2014. 685 p. ISBN: 9788574541075 - 29.
Rico A, Sabater C, Castillo MA. Lethal and sub-lethal effects of five pesticides used in rice farming on the earthworm Eisenia fetida . Ecotoxicology and Environmental Safety. 2016;127 :222-229. DOI: 10.1016/j.ecoenv.2016.02.004 - 30.
Gunnarsson JS, Castillo LE. Ecotoxicology in tropical regions. Environmental Science and Pollution Research. 2018;(14):13203-13206. DOI: 10.1007/s11356-018-1887-4 - 31.
Chasin AAM, Pedrozo MFMO. The toxicology study. In: Azevedo FA, AAM C, editors. The Toxicological Bases of Ecotoxicology. São Carlos: Inter Tox; 2003. pp. 1-25 ISBN: 8589843017 - 32.
O’Brien AL. What are the roadblocks to using population models in ecotoxicology studies? Marine Pollution Bulletin. 2017; 124 :5-8. DOI: 10.1016/j.marpolbul.2017.08.038 - 33.
Fowles J, Lewis C, Rushton E. Studies on the environmental fate, ecotoxicology and toxicology of 2-methyl1,3-propanediol. Regulatory Toxicology and Pharmacology. 2017; 91 :240-248. DOI: 10.1016/j.yrtph.2017.10.031 - 34.
Gainer A, Hogan N, Siciliao SD. Soil invertebrate avoidance behavior identifies petroleum hydrocarbon contaminated soils toxic to sensitive plant species. Journal of Hazardous Materials. 2019; 361 :338-347. DOI: 10.1016/j.jhazmat.2018.08.086 - 35.
Delgadillo V, Verdejo J, Mondaca P, Verdugo G, Gaete H, Hodson ME, et al. Proposed modification to avoidance test with Eisenia fetida to assess metal toxicity in agricultural soils affected by mining activities. Ecotoxicology and Environmental Safety. 2017;140 :230-234. DOI: 10.1016/j.ecoenv.2017.02.038 - 36.
Sissino CLS, Bulus MRM, Rizzo AC, Moreira AC. Avoidance test using earthworms ( Eisenia fetida ) for contaminated sites evaluation: Preliminary results for hydrocarbon contamination. Journal of the Brazilian Society of Ecotoxicology. 2006;1 :41-44. DOI: 10.5132/jbse.2006.02.009 - 37.
Loureiro S, Soares AMVM, Nogueira AJA. Terrestrial avoidance behaviour tests as screening tool to assess soil contamination. Environmental Pollution. 2005; 138 :121-131. DOI: 10.1016/j.envpol.2005.02.013 - 38.
OECD. Guidelines for the Testing of Chemicals no 207: Earthworm Acute Toxcity Test. Paris: Organisation for Economic Co-operation and Development; 1984. 9 p - 39.
OECD. Guidelines for the Testing of chemicals no 222: Earthworm Reproduction Test. Paris: Organisation for Economic Co-operation and Development; 2004. 18 p - 40.
Hackenberger DK, Stjepanovi N, Loncaric Z, Hackenberger BK. Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta . Chemosphere. 2018;208 :722-730. DOI: 10.1016/j.chemosphere.2018.06.047 - 41.
Žaltauskaitė J, Sodienė I. Effects of total cadmium and lead concentrations in soil on the growth, reproduction and survival of earthworm Eisenia fetida . Ekologija. 2010;56 :10-16. DOI: 10.2478/v10055-010-0002-z - 42.
Huggett RJ, Kimerle RA, Mehrle PM, Bergman HC. Biomarkers: Biochemical, Physiological and Histological Markers of Anthropogenic Stress. 1st ed. Boca Raton: Lewis Publishers; 1992. ISBN: 978-0873715058 - 43.
Eijsackers H. Ecotoxicology of soil organisms: Seeking the way through a pitch-dark labyrinth. In: Donker MH, Eijsackers H, Heimbach F, editors. Ecotoxicology of Soil Organisms. Boca Raton: Lewis Publishers; 1994. pp. 3-32. ISBN: 0873715306 - 44.
Taiwo AM, Gbadebo AM, Oyedepo JA, Ojekunle ZO, Alo OM, Oyeniran AA, et al. Bioremediation of industrially contaminated soil using compost and plant technology. Journal of Hazardous Materials. 2016; 304 :166-172 - 45.
Liang J, Ning X, Sun J, Song J, Lu J, Cai H, et al. Toxicity evaluation of textile dyeing effluent and its possible relationship with chemical oxygen demand. Ecotoxicology and Environmental Safety. 2018; 166 :56-62. DOI: 10.1016/j.ecoenv.2018.08.106 - 46.
Natal da Luz T, Ojeda G, Pratas J, Van Gestel CAM, Sousa JP. Toxicity to Eisenia andrei andFolsomia candida of a metal mixture applied to soil directly or via an organic matrix. Ecotoxicology and Environmental Safety. 2011;74 :1715-1720 - 47.
Dores Silva PRD, Landgraf MD, Rezende MOO. Use of bioassays to evaluate the effect of acute toxicity, reproduction and increase of biomass of earthworms Eisenia fetida in acclimated domestic domestic sewage sludge. Ecotoxicology and Environmental Contamination. 2013;3 :143-146 - 48.
Vampré TM, Fuccillo R, Andréia MM. Eisenia andrei (Oligochaeta ) as bioindicator of soil contamination by hexachlorobenzene (HCB). Brazilian Journal of Soil Science. 2010;34 :59-66 - 49.
Domínguez-Crespo MA, Sánchez-Hernández E, Torres-Huerta AM, egrete-Rodríguez MLX, Conde-Barajas E, Flores-Vela A. Effects of the heavy metals Cu, Ni, Cd and Zn on the growth and reproduction of epigeic earthworms ( E. fetida ) during the vermistabilization of municipal sewage sludge. Water, Air, and Soil Pollution. 2012;223 :915-931. DOI: 10.1007/s11270-011-0913-7 - 50.
Lavelle P, Decaëns T, Aubert M, Barot S, Blouin M, Bureau F, et al. Soil invertebrates and ecosystem services. European Journal of Biology. 2006; 42 :3-15. DOI: 10.1016/j.ejsobi.2006.10.002 - 51.
Roubalová R, Procházková P, Dvořák J, Škanta F, Bilej M. The role of earthworm defense mechanisms in ecotoxicity studies. Invertebrate Survival Journal. 2015; 12 :203-213. ISSN: 1824-307X - 52.
Liu S, Zhou Q, Wang Y. Ecotoxicological responses of the earthworm Eisenia fetida exposed to soil contaminated with HHCB. Chemosphere. 2011;83 :1080-1086. DOI: 10.1016/j.chemosphere.2011.01.046 - 53.
Saint-Denis M, Narbonne JF, Arnaud C, Thybaud C, Ribera D. Biochemical responses of the earthworm Eisenia fetida ,Eisenia andrei exposed to contaminated artificial soil: Effects of benzo(a)pyrene. Soil Biology and Biochemistry. 1999;31 :1837-1846. DOI: S0038-0717(99)00106-6 - 54.
Smídova K, Sera J, Bielska L, Hofman J. Influence of feeding and earthworm density on compound bioaccumulation in earthworms Eisenia andrei . Environmental Pollution. 2015;207 :168-175. DOI: 10.1016/j.envpol.2015.09.025 - 55.
Xiao K, Song M, Liu J, Chen H, Li D, Wang K. Differences in the bioaccumulation of selenium by two earthworm species ( Pheretima guillemi andEisenia fetida ). Chemosphere. 2018;202 :560-566. DOI: 10.1016/j.chemosphere.2018.03.094 - 56.
Miao J, Chen X, Xu T, Yin D, Hu X, Sheng GD. Bioaccumulation, distribution and elimination of lindane in Eisenia foetida : The aging effect. Chemosphere. 2018;190 :350-357. DOI: 10.1016/j.chemosphere.2017.09.138 - 57.
Piron D, Boizard H, Heddadj H, Pérès G, Hallaire V, Cluzeau D. Indicators of earthworm bioturbation to improve visual assessment of soil structure. Soil and Tillage Research. 2017; 173 :53-63. DOI: 10.1016/j.still.2016.10.013 - 58.
Lanno R, Wells J, Conder J, Bradham K, Basta N. The bioavailability of chemicals in soil for earthworms. Ecotoxicology and Environmental Safety. 2004; 57 :39-47. DOI: 10.1016/j.ecoenv.2003.08.014 - 59.
Viswanathan R. Earthworms and assessment of ecological impact of soil xenobiotics. Chemosphere. 1994; 28 :413-420. DOI: 10.1016/0045-6535(94)90137-6 - 60.
Hirano T, Tamae K. Earthworms and soil pollutants. Sensors. 2011; 11 :11157-11167. DOI: 10.3390/s111211157 - 61.
Cortet J, Vauflery AGD, Poinsot-Balaguer N, Gomot L, Texier C, Cluzeau D. The use of invertebrate soil fauna in monitoring pollutant effects. European Journal of Soil Biology. 1999; 35 :115-134 - 62.
Burger J. Bioindicators: Types, development, and use in ecological assessment and research. Environmental Bioindicators. 2006; 1 :22-39. DOI: 10.1080/15555270590966483 - 63.
Nahmani J, Hodson ME, Black S. A review of studies performed to assess metal uptake by earthworms. Environmental Pollution. 2007; 145 :402-424. DOI: 10.1016/j.envpol.2006.04.009 - 64.
Natal da Luz T, Tidona S, Jesus B, Morais PV, Sousa JP. The use of sewage sludge as soil amendment. The need for an ecotoxicological evaluation. Journal of Soils and Sediments. 2009; 9 :246-260. DOI: 10.1007/s11368-009-0077-x - 65.
Rastetter N, Gerhardt A. Toxic potential of different types of sewage sludge as fertilizer in agriculture: Ecotoxicological effects on aquatic and soil indicator species. Journal of Soils and Sediments. 2015; 15 :565-577. DOI: 10.1007/s11368-016-1468-4 - 66.
Lackmann C, Velki M, Seiler TB, Hollert H. Herbicides diuron and fluazifop-p-butyl affect avoidance response and multixenobiotic resistance activity in earthworm Eisenia Andrei . Chemosphere. 2018;210 :110-119. DOI: 10.1016/j.chemosphere.2018.07.008 - 67.
Mariyadas J, Amorim MJB, Jensen J, Scott-Fordsmand JJ. Earthworm avoidance of silver nanomaterials over time. Environmental Pollution. 2018; 239 :751-756. DOI: 10.1016/j.envpol.2018.04.059 - 68.
Cesar R, Alvaro T, Silva M, Colonese J, Polivanov H, et al. Bioavailability of contaminants in Brazilian soils treated with sewage sludge: An ecotoxicological approach using bioassays with organisms aquatic and edaphic. Geochimica Brasiliensis. 2010; 24 :41-49. DOI: 10.21715/gb.v24i1.319 - 69.
Cesar R, Natal-da-Luz T, Silva F, Bidone E, Castilhos Z, Polivanov H, et al. Ecotoxicological assessment of a dredged sediment using bioassays with three species of soil invertebrates. Ecotoxicology. 2015; 24 :414-423. DOI: 10.1007/s10646-014-1390-8 - 70.
Ge J, Xiao Y, Chai Y, Yan H, Wu H, Xin X, et al. Sub-lethal effects of six neonicotinoids on avoidance behavior and reproduction of earthworms ( Eisenia fetida ). Ecotoxicology and Environmental Safety. 2018;162 :423-429. DOI: 10.1016/j.ecoenv.2018.06.064 - 71.
Van Gestel CAM, Hoogerwerf G. Influence of soil pH on the toxicity of aluminium for Eisenia andrei (Oligochaeta :Lumbricidae ) in an artificial soil substrate. Pedobiologia. 2001;45 :385-395. DOI: 10.1078/0031-4056-00094 - 72.
Kuperman RG, Checkai RT, Simini M, Phillips CT. Manganese toxicity in soil for Eisenia fetida ,Enchytraeus crypticus (Oligochaeta ), andFolsomia candida (Collembola ). Ecotoxicology and Environmental Safety. 2004;57 :48-53. DOI: 10.1016/j.ecoenv.2003.08.010 - 73.
Chelinho S, Domene X, Campana P, Natal-da-Luz T, Römbke ASJ, Andrés P, et al. Improving ecological risk assessment in the Mediterranean area: Selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two Oligochaeta species. Environmental Toxicology and Chemistry. 2011;30 :1050-1058. DOI: 10.1002/etc.480 - 74.
Van Gestel CAM, Van Breemen EM, Baerselman R. Influence of environmental conditions on the growth and reproduction of the earthworm Eisenia andrei in an artificial soil substrate. Pedobiologia. 1993;36 :109-120 - 75.
Jänsch S, Amorim M, Römbke J. Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environmental Reviews. 2005; 13 :51-83. DOI: 10.1139/a05-007 - 76.
Ritschie JD, Perdue EM. Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter. Geochimica et Cosmochimica Acta. 2003; 67 :85-96. DOI: 10.1016/S0016-7037(02)01044-X - 77.
Vijver MG, Van Gestel CAM, Roman RP, Van Straalen NM, Peijnenburg WJGM. Internal metal sequestration and its ecotoxicological relevance—A review. Environmental Science & Technology. 2004; 38 :4705-4712. DOI: 10.1021/es040354g - 78.
Wang K, Qiao Y, Li H, Zhang H, Yue S, Ji X, et al. Structural equation model of the relationship between metals in contaminated soil and in earthworm ( Metaphire californica ) in Hunan Province, subtropical China. Ecotoxicology and Environmental Safety. 2018;156 :443-451. DOI: 10.1016/j.ecoenv.2018.03.038 - 79.
Zheng K, Liu Z, Li Y, Cui Y, Li M. Toxicological responses of earthworm ( Eisenia fetida ) exposed to metal-contaminated soils. Environmental Science and Pollution Research. 2013;20 :8382-8390. DOI: 10.1007/s11356-013-1689-7 - 80.
Zhang Q, Zhang B, Wang C. Ecotoxicological effects on the earthworm Eisenia fetida following exposure to soil contaminated with imidacloprid. Environmental Science and Pollution Research. 2014;21 :12345-12353. DOI: 10.1007/s11356-014-3178-z - 81.
Liu T, wang X, Xu J, You X, Chen D, Wang F, et al. Biochemical and genetic toxicity of dinotefuran on earthworms ( Eisenia fetida ). Chemosphere. 2017;176 :156-164. DOI: 10.1016/j.chemosphere.2017.02.113