**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*

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

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.

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 warn-

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

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

Invertebrate populations in the soil are an appropriate tool to assess the degree of soil distur-

Ecotoxicology is applied through tests that measure the toxicity of substances found in the

Ecotoxicological tests allow assessing environmental contamination caused by different pollution sources. These tests have the advantage of covering a wide variety of biological

requires many parameters in order to accomplish a better evaluation [4, 8, 10].

whether it is potentially hazardous to soil compartment [12–15].

18 Soil Contamination and Alternatives for Sustainable Development

**4. Terrestrial ecotoxicology**

ings about risks posed to ecosystems [7].

establish the limits of its use or disposal is possible.

**4.1. Ecotoxicological tests**

bance or land use intensification [5] due to human activities [30].

environment by exposing standard living organisms to them [31].

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].

Earthworms (*Oligochaeta*) are among the most studied edaphic organisms to be used as bioindicators. These organisms are bigger than 2 mm (soil macrofauna) and have direct influence

Ecotoxicological Tests as a Tool to Assess the Quality of the Soil

http://dx.doi.org/10.5772/intechopen.82192

21

Species belonging to genus *Eisenia* sp. (*Oligochaeta*, *Lumbricidae*), commonly known as Californian or red worms of California [16, 23], stand out among terrestrial fauna species used in toxicity tests. *Eisenia fetida* and *Eisenia andrei* naturally live in the topsoil, animal manure, and compost materials. They are soil organisms that participate in soil aggregation processes and in the decomposition of plant residues, thus maintaining soil fertility and the quality of

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

*Oligochaetes* are often used to evaluate soil contamination because they ingest a large amount of soil and demonstrate the ability of accumulating pollutants [54–56]. They crush organic matter and produce excellent-quality humus, fact that facilitates water and air entry into the

According to [58], earthworms are widely used given their suitability for the bioavailability

• They live in contaminated sites, and it allows the validation of chemical availability in the field.

• The epidermis of the worms is vascularized, but it has no cuticle, and it allows absorbing

• Earthworms ingest specific fractions of soil, and it provides the means for contaminant

• They present high body mass, and this factor helps determining the concentration of con-

• Some species, such as *E. fetida*, are easy to cultivate, can be kept in laboratory under con-

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

• Their physiology and the metabolism of metals in their organisms are well known.

surface of the body—this substance keeps predators away from them [51–53].

soil and helps combating erosion and recovering degraded soils [57].

• Earthworms live on the ground and are in constant contact with the soil.

evaluation of many soil chemicals due to the following factors:

trolled conditions, and are tolerant to different soil types.

• They are found in a wide variety of soil horizons.

the contaminants straight from the soil.

taminants in the assessed individuals.

• Availability of standard test protocols.

uptake.

on soil functioning [50].

**4.3.** *Eisenia* **sp. (***Oligochaeta***,** *Lumbricidae***)**

agricultural and natural ecosystems [40].

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 (*Oligochaeta*) are among the most studied edaphic organisms to be used as bioindicators. These organisms are bigger than 2 mm (soil macrofauna) and have direct influence on soil functioning [50].

## **4.3.** *Eisenia* **sp. (***Oligochaeta***,** *Lumbricidae***)**

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

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.

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,

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,

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

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

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

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

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

possible impacts of this situation on the soil of different regions of the world [27, 30].

and/or behavioral changes when they are exposed to certain contaminants [32].

ecosystems, since they demonstrate the dynamics of populations over time [41].

**4.2. Biomarkers and bioindicators**

or functions measurable in a system or sample [42].

20 Soil Contamination and Alternatives for Sustainable Development

be detected through other methods [23, 31, 32].

[16, 43].

bovine manure [49].

systems [23].

Species belonging to genus *Eisenia* sp. (*Oligochaeta*, *Lumbricidae*), commonly known as Californian or red worms of California [16, 23], stand out among terrestrial fauna species used in toxicity tests. *Eisenia fetida* and *Eisenia andrei* naturally live in the topsoil, animal manure, and compost materials. They are soil organisms that participate in soil aggregation processes and in the decomposition of plant residues, thus maintaining soil fertility and the quality of agricultural and natural ecosystems [40].

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 [51–53].

*Oligochaetes* are often used to evaluate soil contamination because they ingest a large amount of soil and demonstrate the ability of accumulating pollutants [54–56]. They crush organic matter and produce excellent-quality humus, fact that facilitates water and air entry into the soil and helps combating erosion and recovering degraded soils [57].

According to [58], earthworms are widely used given their suitability for the bioavailability evaluation of many soil chemicals due to the following factors:


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 [59–63].

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 *Eisenia* sp., based on behavioral tests (avoidance).

**Table 3** summarizes the results recorded by some authors in studies with *Eisenia* sp. based on ecotoxicological trials conducted to measure lethality (acute test) and reproduction (chronic test).

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 *Dendrobaena veneta.* The tested herbicides did not have significant effect on reproduction success. GLF induced the acetylcholinesterase (AChE) activity after 7 days and NCS, after 28 days, whereas TBT caused up to 47% inhibition after 7 days. Only TBT caused significant change in catalase (CAT) after 7 days of exposure. Malondialdehyde concentrations (MDA) increased after NCS exposure (at any exposure period), but it only happened in GLF and in TBT after 7 and 28 days, respectively. The activity of measured biomarkers changed depending on the applied herbicide and on exposure time; it also suggested that oxidative stress plays an important role in the toxicity of

**Table 2.** List of results recorded by some authors in a study focused behavioral tests conducted with earthworms.

[37] They exposed *E. andrei* specimens to different substances (copper sulfate, pesticides, dimethoate,

[36] They evaluated the behavior of *E. fetida* specimens in samples contaminated with hydrocarbons Results: 96% of individuals fled the section containing the contaminated sample

treated canning industry effluent, and the sludge from a galvanic treatment plant

[65] The authors analyzed soil and sludge mixtures from three sewage treatment plants in Germany.

13.4 g sludge/kg soil). About 100% contaminated soil evasion was observed at 45 g/kg

[66] Assessed the effects of herbicides Diuron and fluazifop-p-butyl on *E. andrei*

applied to plant species sensitive to PHC-contaminated soil

Results: avoidance—copper sulfate, 320 mg/kg, earthworms in the assessed mine showed no preference

Ecotoxicological Tests as a Tool to Assess the Quality of the Soil

http://dx.doi.org/10.5772/intechopen.82192

23

[64] They evaluated the behavior of *E. andrei* specimens exposed to three sludge types: treated domestic effluent,

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

Results: The sludge was toxic to the test organisms (*E. fetida*) at concentration 9 g sludge/kg soil (EC50,

Results: The avoidance behavior in the soil evidenced that both herbicides caused significant avoidance

and NM300K induced avoidance response in *E. fetida* at concentrations 10 and 100 times

[67] Studied the avoidance behavior to silver forms (nanomaterials (NMs)) at four time points (24, 48, 72, and

lower than the concentrations required for AgNM-PVP coated and AgNM-non-coated nanomaterials [34] The authors investigated the toxicity of a binary petroleum hydrocarbon (PHC) mixture to the avoidance

response of five soil invertebrate species (*E. fetida, Enchytraeus crypticus, Folsomia candida, Oppia nitens*, and

Results: The avoidance of invertebrates in PHC-contaminated soil was similar in growth measurements

carbendazim, benomyl) in soil collected in a deactivated mine

between the contaminated soil and the control

the tested herbicides.

**Author Results**

chromium in it

response

Results: AgNO<sup>3</sup>

*Hypoaspis aculeifer*)

96 h)


1 28 days for cocoon production + 28 days for juvenile hatching.

2 First stage, up to 21 days for the absorption of the test substance; second stage, 21 days for elimination.

**Table 1.** Standardized toxicity tests using earthworms as test organisms.


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

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,

**Table 1** shows a selection of standardized toxicity test protocols developed by acknowledged

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

**Table 2** shows a summary of results recorded by some authors in studies conducted with

**Table 3** summarizes the results recorded by some authors in studies with *Eisenia* sp. based on ecotoxicological trials conducted to measure lethality (acute test) and reproduction (chronic test). 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].

7 to 28 *E. fetida* ASTM E 1676-12 (2010)

ISO 11268 (2012)

OECD 222 (2004)

*4.3.1. Ecotoxicological studies already carried out with earthworms belonging to genus* 

institutions involving worms currently available for researchers.

**Test Duration (days) Species Standard**

Lethality 14 *E. fetida/E. andrei* OECD 2017 (1984)

Reproduction 28 + 281 *E. fetida/E. andrei* ISO 11268-2 (2012)

Avoidance 2 *E. fetida/E. andrei* ISO 17512-1 (2012) Bioaccumulation Until 21 + 21<sup>2</sup> *E. fetida/E. andrei* OECD 317 (2010)

First stage, up to 21 days for the absorption of the test substance; second stage, 21 days for elimination.

*Eisenia* sp., based on behavioral tests (avoidance).

28 days for cocoon production + 28 days for juvenile hatching.

**Table 1.** Standardized toxicity tests using earthworms as test organisms.

several animal species [59–63].

22 Soil Contamination and Alternatives for Sustainable Development

reflect the reality in the field.

respectively.

*Eisenia sp.*

1

2

**Table 2.** List of results recorded by some authors in a study focused behavioral tests conducted with earthworms.

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 *Dendrobaena veneta.* The tested herbicides did not have significant effect on reproduction success. GLF induced the acetylcholinesterase (AChE) activity after 7 days and NCS, after 28 days, whereas TBT caused up to 47% inhibition after 7 days. Only TBT caused significant change in catalase (CAT) after 7 days of exposure. Malondialdehyde concentrations (MDA) increased after NCS exposure (at any exposure period), but it only happened in GLF and in TBT after 7 and 28 days, respectively. The activity of measured biomarkers changed depending on the applied herbicide and on exposure time; it also suggested that oxidative stress plays an important role in the toxicity of the tested herbicides.


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

Ecotoxicological Tests as a Tool to Assess the Quality of the Soil

http://dx.doi.org/10.5772/intechopen.82192

25

There was significant copper (Cu) uptake increase by earthworm (*Eisenia fetida*) when there was combined benzotriazole (BTR) pollution in the soil [26]. According these authors, water and soil environments contaminated with triazole can form complexes of metal ions and,

Ref. [71] verified the toxicity rates in *Eisenia andrei* species exposed to different aluminum concentrations and found that the metal was toxic (457 mg Al/kg) at lower pH values (3.24).

Ref. [72] evaluated the toxicity of different aluminum concentrations to *E. fetida*. Their results indicated that its survival was not affected until 1444 mg Al/kg, but it reduced by 85% at

Some factors influence test results, and they should be carefully evaluated based on their

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 *E. andrei* in artificial soil and found reduced number of juveniles at pH values 4, 6.5, 7.5, and 8, as well as

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 [76–78] indicate that organic matter forms stable complexes with metals by reducing their

Ref. [64, 69] point out that oligochaetes recognize the organic matter in domestic sewage and

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 bio-

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

mass; thus they reduce their reproduction and cocoon production rates.

were able to affect the reproduction of oligochaetes.

therefore, affect the bioavailability and toxicity of some heavy metals.

The growth and production of cocoons reduced at this pH.

optimum cocoon production at pH between 5 and 6.

bioavailability and geochemical mobility.

the energy allocated for growth purposes.

in sediments as real food sources.

concentration 2222 mg Al/kg.

interference.

**Table 3.** List of results recorded by some authors in a study focused on lethality and reproduction conducted with earthworms.

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.

**Author Results**

with sewage

pollutant (POP)

physiology of oligochaetes

biomagnification along food chains

24 Soil Contamination and Alternatives for Sustainable Development

84.12 mg/kg Zn and 323.11 mg/kg Cu.

and 14 days after the beginning of the experiment.

after the 14th day of testing

these pesticides

earthworms.

[63] Eight soil samples were collected from seven abandoned mines in the United Kingdom

effluent from an olive plant, and sludge from a galvanic treatment plant

[49] Evaluated the toxicity of bovine clearance residues on the survival rate of *E. fetida*

The increased Cu and Zn concentrations reduced the number of cocoons

been highly impacted by the disposal of liquid domestic and industrial waste

intermediate metal concentrations (7150–13,100 mg Pb/kg, 2970–53,400 mg Zn/kg)

the galvanic industry caused significant decrease in the production of juveniles

[64] Evaluated the behavior of *Eisenia andrei* specimens exposed to three sludge types: treated sewage, treated

[68] The authors carried out an ecotoxicological evaluation with oligochaetes (*E. andrei*) living in soil treated

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

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

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

[48] Evaluated the ability of *E. andrei* species in bioaccumulate hexachlorobenzene, which is a persistent organic

Results: Annelids accumulated hexachlorobenzene in their tissues, and this process may result in

[47] The authors carried out acute and chronic toxicity tests with *E. fetida* specimens in sanitary effluent sludge.

[69] Evaluated dredged sediments in Guabanara Bay, Rio de Janeiro, Brazil, through toxicity tests. The bay has

[29] The toxicity of five pesticides (trichlorfon, dimethoate, carbendazim, tebuconazole, and prochloraz)

[70] Evaluated the chronic toxicity of the abovementioned six neonicotinoids (NEOs) to *E. fetida*—its cocoon

production, hatchability, cocoon weight, and adult weight were affected in the test

Results: *E. andrei* reproduction was sensitive to dilute sediment samples presenting EC 20 = 1.26%, EC 50 = 2.94%. In addition, surviving earthworms showed visible morphological damage in their epidermis

typically used in rice farming was evaluated through the mortality and body weight of *E. fetida* specimens 7

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

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 *E. fetida*

**Table 3.** List of results recorded by some authors in a study focused on lethality and reproduction conducted with

Results: They found 100% lethality in individuals subjected to the treatment with waste at concentrations

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

There was significant copper (Cu) uptake increase by earthworm (*Eisenia fetida*) when there was combined benzotriazole (BTR) pollution in the soil [26]. According these authors, water and soil environments contaminated with triazole can form complexes of metal ions and, therefore, affect the bioavailability and toxicity of some heavy metals.

Ref. [71] verified the toxicity rates in *Eisenia andrei* species exposed to different aluminum concentrations and found that the metal was toxic (457 mg Al/kg) at lower pH values (3.24). The growth and production of cocoons reduced at this pH.

Ref. [72] evaluated the toxicity of different aluminum concentrations to *E. fetida*. Their results indicated that its survival was not affected until 1444 mg Al/kg, but it reduced by 85% at concentration 2222 mg Al/kg.

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 *E. andrei* in artificial soil and found reduced number of juveniles at pH values 4, 6.5, 7.5, and 8, as well as optimum cocoon production at pH between 5 and 6.

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 [76–78] 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.

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 lev-

Ecotoxicological Tests as a Tool to Assess the Quality of the Soil

http://dx.doi.org/10.5772/intechopen.82192

27

[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.

[2] Jules Pretty J, Bharucha ZP. Sustainable intensification in agricultural systems. Annals of

[3] Bruulsema T. Managing nutrients to mitigate soil pollution. Environmental Pollution.

[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.

[5] Bianchi MO. Importance of ecotoxicological studies with invertebrates from soil.

[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.

[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:

[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.

els and to assess whether there is, or not, toxicity transfer between food chain levels.

\* and Roberta de Moura Lisbôa<sup>2</sup>

2 Federal University of Santa Maria - UFSM, Santa Maria, Brazil

Botany. 2014;**114**:1571-1596. DOI: 10.1093/aob/mcu205

Seropédia: Rio de Janeiro; 2010. 32 p. ISSN: 1980-3075

2018;**243**(Pt B):1602-1605. DOI: 10.1016/j.envpol.2018.09.132

DOI: 10.1016/j.baae.2007.08.010

ecolind.2018.06.052

ecoenv.2018.06.041

10.1016/j.ecoenv.2013.10.025

DOI: 10.2136/sssaspecpub35.c1

1 State University of Rio Grande do Sul - UERGS, Três Passos, Brazil

\*Address all correspondence to: barbara-clasen@uergs.edu.br

**Author details**

Barbara Clasen<sup>1</sup>

**References**

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 *E. fetida* living in soil contaminated with heavy metals. Based on their results, there was lower sensitivity to superoxide dismutase enzyme activity, whereas dismutase, acetylcholinesterase, and DNA damage were more sensitive.

Ref. [80] evaluated changes in superoxide dismutase, catalase, peroxidase, cellulase, and malondialdehyde in *E. fetida* exposed to the insecticide imidacloprid, which is widely used in agriculture. Results showed that all evaluated enzymes recorded changes in their activity as the imidacloprid concentration increases (>0.66 mg/kg).

The biochemical and genetic toxicity of dinotefuran on *Eisenia fetida* were evaluated at 0, 0.1, 0.5, 1.0, and 2.0 mg/kg in a study conducted by [81]. Dinotefuran induced excessive reactive oxygen species (ROS) generation at 1.0 and 2.0 mg/kg, and it resulted in significant changes in the activity of antioxidant enzymes and on the functional gene expression. Moreover, lipids, proteins, and nucleic acids were oxidized and damaged by the excess of ROS induced by dinotefuran—this process results in serious destruction of cell structure and function.

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.
