Ecotoxicology and Environmental Safety 103 (2014) 68–73
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Review
The use of diplopods in soil ecotoxicology – A review Tatiana da Silva Souza a,n, Cintya Aparecida Christofoletti b, Vlamir Bozzatto b, Carmem Silvia Fontanetti b a Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, UFES, Alto Universitário, s/número Guararema, 29500-000 Alegre, ES, Brasil b Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, Bela Vista, 13506-900 Rio Claro, SP, Brasil
art ic l e i nf o
a b s t r a c t
Article history: Received 28 June 2013 Received in revised form 9 October 2013 Accepted 16 October 2013 Available online 8 November 2013
Diplopods play an important role in the dynamics of terrestrial ecosystems, actively participating in the decomposition of organic matter and soil aeration. They have gained increased attention from ecotoxicology research because they are continuously exposed to soil contaminants and biological effects of chemical stressors can be measurable at various levels of biological organization. This paper is the first review on the use of diplopods as soil bioindicators and compiles the effects of the different toxic chemical agents on these animals. Special emphasis is given on the interpretation of the effects of heavy metals and complex mixtures in target organs of diplopods. & 2013 Elsevier Inc. All rights reserved.
Keywords: Millipedes Terrestrial ecotoxicology Soil toxicity Biomarkers
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effects of environmental contaminants on diplopods and main biomarkers used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Radioactive elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Aromatic hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Complex mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Sensivity of diplopods: comparison with others soil bioindicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bioindicators are species, groups of species or biological communities whose presence, abundance and biological conditions, in real ecosystems or through the use of laboratory toxicity tests, to make inferences about the quality of the environment. The use of bioindicators in monitoring programs may be helpful to detect
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Corresponding author. E-mail addresses: [email protected] (T. da Silva Souza), [email protected] (C.A. Christofoletti), [email protected] (V. Bozzatto), [email protected] (C.S. Fontanetti). 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.025
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environmental changes in early stage or the efficacy of measures taken to improve environmental quality (Van Straalen, 1998). Diplopoda includes ca. 12,000 described species distributed worldwide, with highest diversity in the tropics. With over an estimated global fauna of more than 80,000 species, the Diplopoda (millipedes) is the third largest class of terrestrial Arthropoda following Insecta and Arachnida (Golovatch and Kime, 2009). They are generally nocturnal, living in humid environments and being commonly found underneath fallen trunks and leaves. They occupy the decomposer trophic level, eating organic matter, detritus, fruits, and some material of mineral origin (Schubart, 1942; Hoffmann et al., 2002; Ruppert and Barnes, 2005). Diplopods play an important role in the dynamics of terrestrial ecosystems,
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actively participating in soil aeration and facilitating the decomposition of organic matter by fungi and bacteria. Moreover, they excrete ammonia and uric acid, important sources of soil nitrates. In this way, they stimulate microbial metabolism, essential in nutrient recycling (Schubart, 1942; Hopkin and Read, 1992). Diplopods have gained increased attention from ecotoxicology research in last 20 years due to a number reasons: (a) they are components of the edaphic fauna and they are continuously exposed to soil contaminants; (b) there are large number of representatives distributed worldwide; (c) most species of diplopods are relatively large and therefore easy to handle; thus, they can be used both under laboratory and field conditions for biomonitoring; and (d) biological effects of environmental contaminants can be measurable at various levels of biological organization. This manuscript is the first review on the use of diplopods as soil bioindicators and compiles the effects of the different environmental contaminants on these animals.
2. The effects of environmental contaminants on diplopods and main biomarkers used 2.1. Heavy metals One way to evaluate how pollution affects an area is to conduct a species survey and ascertain which species are present and which species are not (Read and Martin, 1990). Hopkin et al. (1985) showed that most diplopod species present in non-contaminated areas are absent from contaminated areas with heavy metals. In contrast with the results mentioned above, Read and Martin (1990) and Grelle et al. (2000) failed to find a relationship between the number of species or individuals collected and the concentration of metals in the soil. Grelle et al. (2000) suggesting that there is an upper threshold for metal concentration above which diplopods are not found and concluded that availability of organic matter, type of vegetation, and soil structure can be very important factors determining species abundance and the presence of dominant species. Results of Hobbelen et al. (2006) showed that metal pollution is not a dominating factor determining the species richness and densities of the selected detritivore group that consisted of isopods, millipedes and earthworms. According to authors, possible explanations for the lack of negative effects of heavy metals were a combination of low metal bioavailability in the soil, development of resistance to metal pollution and the presence of confounding factors, such as variation in organic matter content and flooding. Even though the number of diplopod species may be affected by high amounts of metal contamination, some species will develop strategies that minimize the effects of these substances on their survival. These strategies may include a decrease in food intake, a decrease in nutrient assimilation, or both. For example, individuals of Glomeris marginata significantly decreased their intake of litter contaminated with Cd, Cu, Pb and Zn, which resulted in a decrease in biomass (Hopkin et al., 1985). Furthermore, a decrease in nutrient assimilation was observed in individuals of Allaiulus nitidus, Glomeris conspersa, Julus scandinavius and Polydesmus denticulatus (Köhler et al., 1992a) fed with Pb contaminated litter. A similar phenomenon was observed in individuals of Rossiulus kesseleri given feed contaminated with Cd and Hg (Zhulidov and Dubova, 1988). Köhler et al. (1992a) reported that individuals of G. conspersa were able to compensate for decreased nutrient assimilation by eating more litter. The others of species of Julidae investigated in that study, however, did not compensate for reduced assimilation, but nevertheless survived.
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The Polysdemidae P. denticulatus also did not compensate but died in the end of the experiment. The same was observed for R. kesseleri, fed with Cd and Hg contaminated feed (Zhulidov and Dubova, 1988). The authors concluded that a decrease in nutrient assimilation is a defense mechanism against the effects of toxic compounds in the substrate. In addition, Köhler et al. (1992a) concluded that the efficacy of this strategy depends on the species nutritional needs. Large species such as those in Julidae consume large amounts of food and absorb only small quantities of nutrients, whereas smaller species are more efficient in nutrient assimilation. The latter was the case with the individuals of P. denticulatus (see above) who were not able to obtain enough energy for survival. The total accumulation of metals in terrestrial saprophagous invertebrates based on the total concentration of these elements in the soil and in their bodies can be used as a key parameter in terrestrial ecotoxicology (Heikens et al., 2001). Nakamura and Taira (2005) and Nakamura et al. (2005) suggested that diplopods can give information on dangerous metal concentrations and metal regulation mechanisms in the soil. Hubert (1977, 1978a, 1978b, 1979a, 1979b) was the first to identify where metals accumulate within the bodies of diplopods. In general, granules containing metals are associated with organs that perform digestive, storage and excretion, as for example the midgut, hepatic cells, fat body and Malpighian tubules. Köhler and Alberti (1992) identified several granules containing calcium phosphate in the midgut of the diplopod Mycogona germanica. Studies on the diplopod G. marginata (Hopkin et al., 1985; Köhler et al., 1995) and Leptoiulus belgicus (Köhler et al., 1995) revealed that Cd (60–68%) and Pb (47–64%) are preferentially stored in the epithelial cells of the midgut. By contrast, only a small percentage of Zn (3–6.5%) is stored in the midgut, the cuticle being the favored storage place for this metal. Cells of the fat body also store metals in insoluble granules. Their composition, however, has not been studied (Hopkin and Read, 1992; Fontanetti et al., 2006). Read and Martin (1990) found high concentrations of metals in specimens of G. marginata from polluted locations, and low concentrations of them in the bodies of specimens from unpolluted places. In laboratory, juveniles accumulated Cd and Zn fast when fed with contaminated litter, and developed at a significantly slower rate when compared with juveniles reared in uncontaminated litter. Likewise, juveniles of Tachypodoiulus niger accumulated metals quickly, and presented low growth and survival rates when compared with the control group. An increase in the production of the stress protein hsp70 by organisms exposed to proteotoxic environmental stress (adversely affecting the integrity of intracellular proteins) has been considered as a universal stress marker by many researchers. A few studies focusing on this protein have been conducted on diplopods, particularly J. scandinavius, exposed to different concentrations of metals (Zanger and Köhler, 1996; Zanger et al., 1996, Köhler et al., 1996). For instance, Zanger et al. (1996) showed that adults of J. scandinavius fed with litter contaminated with different concentrations of Cd (10, 30, 50 and 60 mg/Kg CdCl2) increased their hsp70 expression. In another study (Zanger and Köhler, 1996), high levels of hsp70 were reported in diplopods exposed to high concentrations of Zn (2518, 4705 and 22,203 mg/Kg ZnCl2) and Cd (51, 133, 216 and 257 mg/Kg CdCl2); however, extremely high concentrations of Cd (422 mg/Kg CdCl2) were associated with decreased hsp70 production. The authors suggested that the latter might be a consequence of severe histopathologic damage of the midgut. Also, the expression of hsp70 after heat shock or exposure to heavy metals/molluscicides was investigated in three diplopods species (G. marginata, Cylindroiulus punctatus, and T. niger), two slugs (Deroceras reticulatum and Arion ater), and one isopod (Oniscus asellus). The comparison of laboratory and field experiments
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demonstrated the suitability of O. asellus for monitoring tests. In contrast, the mentioned diplopods and slugs showed no differences compared to the respective control. It is known from previous studies that ultrastructural alterations in the midgut of diplopods occur under heavy metal stress in laboratory as well as in the field. The preferentially affected cell components (plasma membrane and microtubules) are assigned to be binding sites for heat shock proteins. Therefore, a relation between the influence of hsp on these components and the observed ultrastructural alterations in certain cells seems to be possible group (Köhler et al. 1992b). Köhler et al. (2000) studied 38 populations of diplopods (J. scandinavius) from areas chronically contaminated with metals (particularly Cd, Pb and Zn) and found that their average levels of hsp70 were low. The authors then concluded that these populations might not be very sensitive to those contaminants. Using mathematical models and statistical analyses, they proposed that this lack of vulnerability could be a result of the selection of resistant phenotypes that do not display the stress response (increased production of hsp70). The alternative, i.e. selection for high levels of hsp70 to compensate for the adverse effects of chronic contamination, was not observed in their data. The selection of less vulnerable phenotypes would favor response mechanisms that would include avoidance of stressor agents, with little cost to the survival and reproduction of individuals. This would leave more energy available for detoxification and food selection. During the last decades, histopathological biomarkers have been applied to invertebrate species for the identification of cell and tissue damage resulting from environmental contamination (Triebskorn and Künast, 1990; Triebskorn et al., 1991, 1999; Fontanetti et al., 2010, 2011). In those studies, organs involved in the absorption and assimilation of essential nutrients such as lipids, carbohydrates and proteins have been preferentially investigated (Hopkin, 1989; Pigino et al., 2005). In diplopods, the midgut has been used because it plays an important role in detoxification processes and excretion of xenobiotics (Hopkin et al., 1985; Triebskorn et al., 1991; Köhler and Triebskorn, 1998; Godoy and Fontanetti, 2010; Nogarol and Fontanetti 2010, 2011; Souza and Fontanetti, 2011). Triebskorn et al. (1991) reported ultrastructural alterations in C. punctatus after ingestion of sublethal concentrations of Pb: cytoplasmic condensation, reduction in cell volume, dilatation of the intercellular spaces and disorganization of the ER-cisternae and microtubules. The mitochondriae, normally located apically, became distributed throughout the cell. According to the authors, Pb induces the changes described above by interfering with the cell cytoskeleton and osmotic balance. The ultrastructure of the midgut of C. alemannicum, Cylindroiulus silvarum, G. marginata, J. scandinavius, M. germanica, Ommatoiulus rutilans, Polydesmus angustus and T. niger, collected from an area contaminated with Cd, Pb and Zn, revealed alterations not found in the control group. The main alteration observed was condensation of the cytoplasm of the main cell types. As a consequence of this reduction in volume, the absorptive cells and the regenerative cells became disconnected from one another in their basal part, resulting in extensive intercellular spaces. Furthermore, the spherocrystals appeared almost completely electron-dense. In addition, mitochondria were distributed evenly throughout the cell and the ER appeared smaller. The basal lamina appeared thin and condensed. The hepatic cells showed a higher amount of lipids and glycogen, as well as the elongation of the microvilli (Köhler and Alberti, 1992). In the same paper, Köhler and Alberti (1992) show that diplopods fed with Pb-contaminated litter in the laboratory display alterations similar to those described above, but less strong, indicating that the histopathological impacts can influence the ecological role of these animals in the ecosystem.
Köhler and Triebskorn (1998) developed an impact index that combines quantitative and qualitative analyses to evaluate ultrastructural changes after metal exposure on soil invertebrates, including the diplopod (J. scandinavius). The authors selected four cell organelles (microvilli, mitochondria, ER and nucleus) and classified the observed alterations into three categories: (1) not altered (control), (2) compensation (symptomatic of a cell reaction to stress) and (3) non-compensation (symptomatic of cell destruction). 2.2. Radioactive elements In 1982, Krivolutzky et al. (1982) concluded that soil arthropods present a convenient subject for elucidating the ways and quantitative patterns of migration of radionucleides in ecosystems. Krivoluckij et al. (1972) reported a decrease in the number of diplopods in places contaminated with Sr90. Bioaccumulation of radioactive materials has been evaluated in diplopods. Rantala (1990) investigated the accumulation of radioactive Cs in Cylindroiulus britannicus after the Chernobyl accident of 1986. The diplopods from contaminated locations had high levels of radioactive compost and juveniles were the most affected age-group. Makimova (2002) also investigated the impact of a radioactive contamination caused by the Chernobyl nuclear accident on Diplopoda communities and concluded that is a correlation between the viability of millipedes and the background gammaradiation intensity. It has been found that at an early stage of radiological lesion, the amount of hemocytes involved in phagocytosis reactions increases. After, has been found a drastic increase of dead cells. With radiological lesion, the restoration function of hemocytes appears disturbed, causing a decrease in the amount of juvenile cells. Morphological changes are as follows: lysis, fission shift of the nucleus to the cell periphery, disturbance of circular shape in some cells, elongation, cytoplasm budding and vacuolization. Cytoplasm of some cells loses its granular texture and becomes homogeneous. 2.3. Pesticides Pesticides are potentially toxic for non-target species. However, diplopods exposed to either one of the following organic contaminants, γ-hexaclorociclohexan (lindane) or 2,2,5,5-tetraclorobifenil (PCB 52) did not show elevated hsp70 (Zanger and Köhler, 1996). According to the authors, the low proteotoxicity of the organic contaminants suggests the induction/presence of biotransformation enzymes in diplopods. In conclusion, diplopods would be able to detoxify low amounts of organic substances before intracellular protein degradation, and consequently, before the hsp70 response. Topical application of the pesticide methiocarb carbamate on Gymnostreptus olivaceus and Plusiorus setiger caused low toxicity, with high DL50 values for the two species: 515, 216, 137 and 93 mg/g for G. olivaceus and 489, 222, 108 and 71 mg/g for P. setiger, respectively, 24, 48, 72 and 96 h after application of the pesticide (Boccardo and Fernandes, 2000). Boccardo and Fernandes (2001) also found low toxicity of the pyretroid deltamethrin to diplopods of the same species. These responses were attributed to the protection of a calcified exoskeleton, which might have interfered with pesticides penetration. The effects of the fungicide dithane M-45 (mancozeb) on several arthropods were studied by Adamski et al. (2007), who reported high mortality rates for diplopods. The short period of exposure to the fungicide (24 h) revealed a low value for LC (LC50 ¼0.351 and LC95 ¼6.13). According to the authors, these results are likely to represent an underestimation of the effects of the fungicide in the field, where entire diplopod populations could be wiped out.
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Weber and Nentwig (2006) did not find higher mortality rates for individuals of Allajulus latestriatus fed with N4640Bt corn, which expresses the Bt Cry1Ab protein, used as an herbicide. The authors however found that the diplopods excreted considerable amounts of the active protein, making it available for other soil organisms. Regarding food intake and biomass, the authors did not find a significant difference between specimens of A. latestriatus fed with transgenic N4640Bt corn, and the control group. Merlini et al. (2012) evaluated different concentrations of trifluralin herbicide in the midgut of Rhinocricus padbergi and reported the disruption or total loss of the epithelium and others histological alterations describe in Section 2.5. 2.4. Aromatic hydrocarbons Induction of the cytochrome P450 has been accepted as a wellestablished sublethal stress biomarker that results from exposure to xenobiotics. Studies on the organization and distribution of the cytochrome P450 of invertebrates are restricted to only a few species (Livingstone, 1990). Zanger et al. (1997) reported the induction of cytochrome P450 compounds in J. scandinavius (Diplopoda) fed with β-naphthoflavone contaminated litter. However, a larger sample is required to substantiate the results of authors, because their enzymatic analyses were performed on a statistically small pool of specimens (10 diplopods). Moreover, the authors noted that individual analyses would be necessary, as well as the identification of the enzymes produced, in order to reveal similarities and differences with the P450 system of vertebrates. 2.5. Complex mixtures Fontanetti et al. (2012) analyzed diplopods behavior and the survival rate by log-rank test (p ¼0.05). The analysis showed that the animals exposed to pure sewage sludge presented higher mortality index than the specimens exposed to different concentrations of the sludge mixed with soil. Ultrastructural alterations in midgut of R. padbergi showed that nucleus and other cellular compartiments were damaged after exposure to sewage sludge sample (Nogarol and Fontanetti, 2011). Tissue alterations in the midgut of the diplopod R. padbergi were studied after exposure to soil contaminated with different proportions of residues of a complex nature, such as sewage sludge (Godoy and Fontanetti, 2010; Nogarol and Fontanetti, 2010, 2011; Perez and Fontanetti, 2010; Christofoletti et al., 2012; Bozzato and Fontanetti, 2012; Souza and Fontanetti, 2012) and soil from a deactivated bioremediation system from a petroleum refinery, contaminated with metals and polycyclic aromatic hydrocarbons (Souza and Fontanetti, 2011). Collectively, the results obtained by the above research indicated the presence of toxic substances in the samples and imply that the exposed animals display defense mechanisms that attempt to neutralize or eliminate the effects of toxic residues. In summary, the main tissue biomarkers observed in those papers were: (a) Death of the cell: Vacuolization of the cytoplasm of epithelial and hepatic cells, nuclear chromatin condensation and dilatation of the intracellular space. According to Nogarol and Fontanetti (2010), cell death could represent a physiological response that facilitates the discharge of the altered tissues, being important in the maintenance of the organ structure. However, cell death is a direct result of the environmental contaminants on the tissue and extensive cell death can lead to total or partial organ failure (Souza and Fontanetti, 2011). (b) Increase in the renewal rate of the epithelium: The process of epithelium renewal involves the removal of damaged cells to the lumen of the intestine, where they are digested. An
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increase in the renewal rate indicates that at least a portion of the toxic compounds are being eliminated through epithelium renewal. (c) Increase in the number of secretory vesicles: Apocrine secretory vesicles (Fantazzini et al., 2002), which discharge their contents together with a portion of their cytoplasms, were observed all over the epithelium of R. padbergi. The glycoproteic nature of these vesicles was interpreted as evidence that they correspond to a defense against the entrance of potentially toxic substances. However, in some treatments, the secretion vesicles display vacuolization, indicating that the toxic compounds present in the soil reach the cytoplasm of the main epithelial gut. Also, an increase in the amount of secretion vesicles released toward the lumen could represent a response of the organism to the effects of the pollutants, since the excretion of toxic compounds stored in the epithelium of the gut of diplopods would be accelerated. (d) Increase in the number of hemocytes between the hepatic cells layer: Hemocytes are cells of the arthropod immune system that are able to detect, encapsulate, or perform phagocytosis of pathogens and alien substances (Xylander, 2009). The total number of circulating hemocytes can be affected by changes in environmental conditions. Different stressors can activate the proliferation and migration of hemocytes to the affected tissues. A large number of isolated and lumped hemocytes have been observed between the hepatic cells of R. padbergi, indicating tissue lesion and possible inflammation. Therefore, they represent a defense mechanism aimed to destroy toxins and also participate in tissue renewal, through absorption of the damaged tissue. (e) Increased number of cytoplasmatic granules in the layer of hepatic cells: In diplopods, as in other invertebrates, the accumulation of potentially toxic materials as insoluble and inactive substances is an important detoxification mechanism that ensures homeostasis (Hopkin and Read, 1992). The biological origin of these cytoplasmic granules is not yet completely understood. It has been suggested that they come directly from the Golgi complex or associated cisterns; or that they are produced in the rER. Granules that appear in vacuoles or vesicles of undetermined origin are a third possibility. A fourth possible place of origin of these granules is the internal portion of the mitochondria (Köhler, 2002). In some species, the cytoplasmic granules may remain in the cell for long periods of time before being excreted. After precipitating, they can be transported to the intestinal lumen and subsequently freed (Vandenbulcke et al., 1998). Molting is also an opportunity for detoxification, once the intestinal epithelium disrupts and the contents of the main cells are eliminated together with the feces (Hubert, 1979b). An increase in the amount of intracellular granules in the tissues of aquatic and terrestrial invertebrates as a function of high concentrations of metal in the substrate has been used as a biomarker for environmental contamination (Köhler et al., 1996; Barka, 2007). This seems to be true for the diplopod R. padbergi. The mobilization of detoxification processes and survival in stressful situations require utilization of energy reserves (Köhler et al., 1996). It is possible to measure the usage of these reserves to detect substances such as glycogen, proteins and lipids, and to use these results to complement morphological analyses. Lately, the depletion of these substances has been used in ecotoxicologic evaluations when diplopods are used as bioindicators (Souza and Fontanetti, 2011). The perivisceral fat body of diplopods presents intense metabolic activity and functions in lipid, glycogen, protein and uric acid storage, as well as in the neutralization and storage of substances
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that are neither needed nor useful (Hubert, 1979a, 1979b; Hopkin and Read, 1992; Fontanetti et al., 2006). Histological and histochemical analyses have been conducted in the perivisceral fat body of R. padbergi exposed to landfarming derived from refinery oil by Souza et al. (2011). The loss of integrity of the cell boundary, the cytoplasmic disorganization, and depletion of neutral polysaccharides, in these diplopods, can be used as environmental contamination markers. Souza and Fontanetti (2012) analyzed the parietal and perivisceral fat body of R. padbergi exposed to substrate containing sewage sludge. The parietal fat body showed no alterations after variations of time exposure and concentrations. Addition to the changes reported by Souza et al. (2011) the perivisceral fat body showed increase in the quantity of spherocrystals and nuclei with altered morphology.
3. Sensivity of diplopods: comparison with others soil bioindicators Isopods (Köhler et al., 1996; Lemos et al., 2010), nematodes (Sochová et al., 2006), earthworms (Natal Da Luz et al., 2004; Sissino et al., 2006; Pérès et al., 2011) and collembolans (Crouau et al., 2002; Natal Da Luz et al., 2004; Eom et al., 2007) are established models of soil ecotoxicology. Despite their ecological importance, however, diplopods have seldom been used as bioindicators, when compared with other saprophagous terrestrial invertebrates. Due to a growing number of contamination sources, however, the potential utility of this group has gained increased attention in recent years. Metal bioaccumulation in various arthropod groups was investigated by Heikens et al. (2001). Diplopoda and Isopoda bioaccumulated copper and zinc significantly. Lead concentration was extremely high in Isopoda and Collembola, intermediate in Diplopoda, low in Chilopoda and Coleoptera. As in diplopods, heavy metals have been promoted ultrastructural and histological alterations in earthworms (Eudrillus eugeniae) (Sharma and Satyanarayan, 2011), mites (Nothrus silvestris) (Triebskorn et al., 1991), centipedes (Lithobius forficatus) (Vandenbulcke et al., 1998) and isopods (Porcellio scaber) (Köhler et al., 1996). Also, Khöler and Triebskorn (1998) found similar patterns of cellular responses to cadmium and lead in the tissues of isopods (P. scaber) and diplopods (J. scandinavius). The diplopod was more sensitive to cadmium than slugs (D. reticulatum). From these comparisons, it would appear that sensitivity of diplopods to heavy metals is comparable with other soil bioindicators when bioaccumulation and morphological alterations are employed as biomarkers. Already, Zanger et al. (1997) provides evidence of the induction of the cytochome P450 system in diplopods (J. scandinavius) and isopods (O. asellus) fed with β-naphthoflavone contaminated litter.
4. Conclusions and future prospects The response of diplopods to environmental contaminants can be evaluated in several ways such as at the community level (e.g. abundance, biomass species and functional structures) as well as through the individual response of selected species (e.g. measuring the biochemical biomarkers and histopathology). In diplopods the effects of heavy metals have been largely studied and sensitivity to these compounds has been reported. Even though the number of diplopod species may be affected by high amounts of metal contamination, some species will develop strategies that minimize the effects of these substances on their survival such as decrease in food intake, decrease in nutrient assimilation, or both. This review also clearly indicates that diplopods have efficient potential for bioaccumulation heavy metals in their tissues which
can be used as an ecological indicator of soil pollution. The assessment of structural alterations on the cell and tissue is the early stage of a reaction to cell injury, before an integrated cellular response would manifest at level of whole animal physiological processes, and long before there were any changes at the population level. Diplopods also good bioindicators of soils contaminated with radioactive elements and complex mixtures. Pesticides are potentially toxic for non-target species however there has been little progress in understanding of the effects those organic pollutants in diplopods. Most of the studies related in this review, conducted in laboratory, showed that the pesticides caused low toxicity in diplopods. But, considering that these compounds are widely used in agricultural soils, future research requires attention to understanding the biological effects of pesticides. Also, the effects of other persistent organic pollutant need to be clarified.
Acknowledgments We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Process nos. 06/52383-7 and 09/50578-3, the Fundação para o Desenvolvimento da UNESP (FUNDUNESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support.
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Review
The use of diplopods in soil ecotoxicology – A review Tatiana da Silva Souza a,n, Cintya Aparecida Christofoletti b, Vlamir Bozzatto b, Carmem Silvia Fontanetti b a Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo, UFES, Alto Universitário, s/número Guararema, 29500-000 Alegre, ES, Brasil b Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, Bela Vista, 13506-900 Rio Claro, SP, Brasil
art ic l e i nf o
a b s t r a c t
Article history: Received 28 June 2013 Received in revised form 9 October 2013 Accepted 16 October 2013 Available online 8 November 2013
Diplopods play an important role in the dynamics of terrestrial ecosystems, actively participating in the decomposition of organic matter and soil aeration. They have gained increased attention from ecotoxicology research because they are continuously exposed to soil contaminants and biological effects of chemical stressors can be measurable at various levels of biological organization. This paper is the first review on the use of diplopods as soil bioindicators and compiles the effects of the different toxic chemical agents on these animals. Special emphasis is given on the interpretation of the effects of heavy metals and complex mixtures in target organs of diplopods. & 2013 Elsevier Inc. All rights reserved.
Keywords: Millipedes Terrestrial ecotoxicology Soil toxicity Biomarkers
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effects of environmental contaminants on diplopods and main biomarkers used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Radioactive elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Aromatic hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Complex mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Sensivity of diplopods: comparison with others soil bioindicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bioindicators are species, groups of species or biological communities whose presence, abundance and biological conditions, in real ecosystems or through the use of laboratory toxicity tests, to make inferences about the quality of the environment. The use of bioindicators in monitoring programs may be helpful to detect
n
Corresponding author. E-mail addresses: [email protected] (T. da Silva Souza), [email protected] (C.A. Christofoletti), [email protected] (V. Bozzatto), [email protected] (C.S. Fontanetti). 0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.10.025
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environmental changes in early stage or the efficacy of measures taken to improve environmental quality (Van Straalen, 1998). Diplopoda includes ca. 12,000 described species distributed worldwide, with highest diversity in the tropics. With over an estimated global fauna of more than 80,000 species, the Diplopoda (millipedes) is the third largest class of terrestrial Arthropoda following Insecta and Arachnida (Golovatch and Kime, 2009). They are generally nocturnal, living in humid environments and being commonly found underneath fallen trunks and leaves. They occupy the decomposer trophic level, eating organic matter, detritus, fruits, and some material of mineral origin (Schubart, 1942; Hoffmann et al., 2002; Ruppert and Barnes, 2005). Diplopods play an important role in the dynamics of terrestrial ecosystems,
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actively participating in soil aeration and facilitating the decomposition of organic matter by fungi and bacteria. Moreover, they excrete ammonia and uric acid, important sources of soil nitrates. In this way, they stimulate microbial metabolism, essential in nutrient recycling (Schubart, 1942; Hopkin and Read, 1992). Diplopods have gained increased attention from ecotoxicology research in last 20 years due to a number reasons: (a) they are components of the edaphic fauna and they are continuously exposed to soil contaminants; (b) there are large number of representatives distributed worldwide; (c) most species of diplopods are relatively large and therefore easy to handle; thus, they can be used both under laboratory and field conditions for biomonitoring; and (d) biological effects of environmental contaminants can be measurable at various levels of biological organization. This manuscript is the first review on the use of diplopods as soil bioindicators and compiles the effects of the different environmental contaminants on these animals.
2. The effects of environmental contaminants on diplopods and main biomarkers used 2.1. Heavy metals One way to evaluate how pollution affects an area is to conduct a species survey and ascertain which species are present and which species are not (Read and Martin, 1990). Hopkin et al. (1985) showed that most diplopod species present in non-contaminated areas are absent from contaminated areas with heavy metals. In contrast with the results mentioned above, Read and Martin (1990) and Grelle et al. (2000) failed to find a relationship between the number of species or individuals collected and the concentration of metals in the soil. Grelle et al. (2000) suggesting that there is an upper threshold for metal concentration above which diplopods are not found and concluded that availability of organic matter, type of vegetation, and soil structure can be very important factors determining species abundance and the presence of dominant species. Results of Hobbelen et al. (2006) showed that metal pollution is not a dominating factor determining the species richness and densities of the selected detritivore group that consisted of isopods, millipedes and earthworms. According to authors, possible explanations for the lack of negative effects of heavy metals were a combination of low metal bioavailability in the soil, development of resistance to metal pollution and the presence of confounding factors, such as variation in organic matter content and flooding. Even though the number of diplopod species may be affected by high amounts of metal contamination, some species will develop strategies that minimize the effects of these substances on their survival. These strategies may include a decrease in food intake, a decrease in nutrient assimilation, or both. For example, individuals of Glomeris marginata significantly decreased their intake of litter contaminated with Cd, Cu, Pb and Zn, which resulted in a decrease in biomass (Hopkin et al., 1985). Furthermore, a decrease in nutrient assimilation was observed in individuals of Allaiulus nitidus, Glomeris conspersa, Julus scandinavius and Polydesmus denticulatus (Köhler et al., 1992a) fed with Pb contaminated litter. A similar phenomenon was observed in individuals of Rossiulus kesseleri given feed contaminated with Cd and Hg (Zhulidov and Dubova, 1988). Köhler et al. (1992a) reported that individuals of G. conspersa were able to compensate for decreased nutrient assimilation by eating more litter. The others of species of Julidae investigated in that study, however, did not compensate for reduced assimilation, but nevertheless survived.
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The Polysdemidae P. denticulatus also did not compensate but died in the end of the experiment. The same was observed for R. kesseleri, fed with Cd and Hg contaminated feed (Zhulidov and Dubova, 1988). The authors concluded that a decrease in nutrient assimilation is a defense mechanism against the effects of toxic compounds in the substrate. In addition, Köhler et al. (1992a) concluded that the efficacy of this strategy depends on the species nutritional needs. Large species such as those in Julidae consume large amounts of food and absorb only small quantities of nutrients, whereas smaller species are more efficient in nutrient assimilation. The latter was the case with the individuals of P. denticulatus (see above) who were not able to obtain enough energy for survival. The total accumulation of metals in terrestrial saprophagous invertebrates based on the total concentration of these elements in the soil and in their bodies can be used as a key parameter in terrestrial ecotoxicology (Heikens et al., 2001). Nakamura and Taira (2005) and Nakamura et al. (2005) suggested that diplopods can give information on dangerous metal concentrations and metal regulation mechanisms in the soil. Hubert (1977, 1978a, 1978b, 1979a, 1979b) was the first to identify where metals accumulate within the bodies of diplopods. In general, granules containing metals are associated with organs that perform digestive, storage and excretion, as for example the midgut, hepatic cells, fat body and Malpighian tubules. Köhler and Alberti (1992) identified several granules containing calcium phosphate in the midgut of the diplopod Mycogona germanica. Studies on the diplopod G. marginata (Hopkin et al., 1985; Köhler et al., 1995) and Leptoiulus belgicus (Köhler et al., 1995) revealed that Cd (60–68%) and Pb (47–64%) are preferentially stored in the epithelial cells of the midgut. By contrast, only a small percentage of Zn (3–6.5%) is stored in the midgut, the cuticle being the favored storage place for this metal. Cells of the fat body also store metals in insoluble granules. Their composition, however, has not been studied (Hopkin and Read, 1992; Fontanetti et al., 2006). Read and Martin (1990) found high concentrations of metals in specimens of G. marginata from polluted locations, and low concentrations of them in the bodies of specimens from unpolluted places. In laboratory, juveniles accumulated Cd and Zn fast when fed with contaminated litter, and developed at a significantly slower rate when compared with juveniles reared in uncontaminated litter. Likewise, juveniles of Tachypodoiulus niger accumulated metals quickly, and presented low growth and survival rates when compared with the control group. An increase in the production of the stress protein hsp70 by organisms exposed to proteotoxic environmental stress (adversely affecting the integrity of intracellular proteins) has been considered as a universal stress marker by many researchers. A few studies focusing on this protein have been conducted on diplopods, particularly J. scandinavius, exposed to different concentrations of metals (Zanger and Köhler, 1996; Zanger et al., 1996, Köhler et al., 1996). For instance, Zanger et al. (1996) showed that adults of J. scandinavius fed with litter contaminated with different concentrations of Cd (10, 30, 50 and 60 mg/Kg CdCl2) increased their hsp70 expression. In another study (Zanger and Köhler, 1996), high levels of hsp70 were reported in diplopods exposed to high concentrations of Zn (2518, 4705 and 22,203 mg/Kg ZnCl2) and Cd (51, 133, 216 and 257 mg/Kg CdCl2); however, extremely high concentrations of Cd (422 mg/Kg CdCl2) were associated with decreased hsp70 production. The authors suggested that the latter might be a consequence of severe histopathologic damage of the midgut. Also, the expression of hsp70 after heat shock or exposure to heavy metals/molluscicides was investigated in three diplopods species (G. marginata, Cylindroiulus punctatus, and T. niger), two slugs (Deroceras reticulatum and Arion ater), and one isopod (Oniscus asellus). The comparison of laboratory and field experiments
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demonstrated the suitability of O. asellus for monitoring tests. In contrast, the mentioned diplopods and slugs showed no differences compared to the respective control. It is known from previous studies that ultrastructural alterations in the midgut of diplopods occur under heavy metal stress in laboratory as well as in the field. The preferentially affected cell components (plasma membrane and microtubules) are assigned to be binding sites for heat shock proteins. Therefore, a relation between the influence of hsp on these components and the observed ultrastructural alterations in certain cells seems to be possible group (Köhler et al. 1992b). Köhler et al. (2000) studied 38 populations of diplopods (J. scandinavius) from areas chronically contaminated with metals (particularly Cd, Pb and Zn) and found that their average levels of hsp70 were low. The authors then concluded that these populations might not be very sensitive to those contaminants. Using mathematical models and statistical analyses, they proposed that this lack of vulnerability could be a result of the selection of resistant phenotypes that do not display the stress response (increased production of hsp70). The alternative, i.e. selection for high levels of hsp70 to compensate for the adverse effects of chronic contamination, was not observed in their data. The selection of less vulnerable phenotypes would favor response mechanisms that would include avoidance of stressor agents, with little cost to the survival and reproduction of individuals. This would leave more energy available for detoxification and food selection. During the last decades, histopathological biomarkers have been applied to invertebrate species for the identification of cell and tissue damage resulting from environmental contamination (Triebskorn and Künast, 1990; Triebskorn et al., 1991, 1999; Fontanetti et al., 2010, 2011). In those studies, organs involved in the absorption and assimilation of essential nutrients such as lipids, carbohydrates and proteins have been preferentially investigated (Hopkin, 1989; Pigino et al., 2005). In diplopods, the midgut has been used because it plays an important role in detoxification processes and excretion of xenobiotics (Hopkin et al., 1985; Triebskorn et al., 1991; Köhler and Triebskorn, 1998; Godoy and Fontanetti, 2010; Nogarol and Fontanetti 2010, 2011; Souza and Fontanetti, 2011). Triebskorn et al. (1991) reported ultrastructural alterations in C. punctatus after ingestion of sublethal concentrations of Pb: cytoplasmic condensation, reduction in cell volume, dilatation of the intercellular spaces and disorganization of the ER-cisternae and microtubules. The mitochondriae, normally located apically, became distributed throughout the cell. According to the authors, Pb induces the changes described above by interfering with the cell cytoskeleton and osmotic balance. The ultrastructure of the midgut of C. alemannicum, Cylindroiulus silvarum, G. marginata, J. scandinavius, M. germanica, Ommatoiulus rutilans, Polydesmus angustus and T. niger, collected from an area contaminated with Cd, Pb and Zn, revealed alterations not found in the control group. The main alteration observed was condensation of the cytoplasm of the main cell types. As a consequence of this reduction in volume, the absorptive cells and the regenerative cells became disconnected from one another in their basal part, resulting in extensive intercellular spaces. Furthermore, the spherocrystals appeared almost completely electron-dense. In addition, mitochondria were distributed evenly throughout the cell and the ER appeared smaller. The basal lamina appeared thin and condensed. The hepatic cells showed a higher amount of lipids and glycogen, as well as the elongation of the microvilli (Köhler and Alberti, 1992). In the same paper, Köhler and Alberti (1992) show that diplopods fed with Pb-contaminated litter in the laboratory display alterations similar to those described above, but less strong, indicating that the histopathological impacts can influence the ecological role of these animals in the ecosystem.
Köhler and Triebskorn (1998) developed an impact index that combines quantitative and qualitative analyses to evaluate ultrastructural changes after metal exposure on soil invertebrates, including the diplopod (J. scandinavius). The authors selected four cell organelles (microvilli, mitochondria, ER and nucleus) and classified the observed alterations into three categories: (1) not altered (control), (2) compensation (symptomatic of a cell reaction to stress) and (3) non-compensation (symptomatic of cell destruction). 2.2. Radioactive elements In 1982, Krivolutzky et al. (1982) concluded that soil arthropods present a convenient subject for elucidating the ways and quantitative patterns of migration of radionucleides in ecosystems. Krivoluckij et al. (1972) reported a decrease in the number of diplopods in places contaminated with Sr90. Bioaccumulation of radioactive materials has been evaluated in diplopods. Rantala (1990) investigated the accumulation of radioactive Cs in Cylindroiulus britannicus after the Chernobyl accident of 1986. The diplopods from contaminated locations had high levels of radioactive compost and juveniles were the most affected age-group. Makimova (2002) also investigated the impact of a radioactive contamination caused by the Chernobyl nuclear accident on Diplopoda communities and concluded that is a correlation between the viability of millipedes and the background gammaradiation intensity. It has been found that at an early stage of radiological lesion, the amount of hemocytes involved in phagocytosis reactions increases. After, has been found a drastic increase of dead cells. With radiological lesion, the restoration function of hemocytes appears disturbed, causing a decrease in the amount of juvenile cells. Morphological changes are as follows: lysis, fission shift of the nucleus to the cell periphery, disturbance of circular shape in some cells, elongation, cytoplasm budding and vacuolization. Cytoplasm of some cells loses its granular texture and becomes homogeneous. 2.3. Pesticides Pesticides are potentially toxic for non-target species. However, diplopods exposed to either one of the following organic contaminants, γ-hexaclorociclohexan (lindane) or 2,2,5,5-tetraclorobifenil (PCB 52) did not show elevated hsp70 (Zanger and Köhler, 1996). According to the authors, the low proteotoxicity of the organic contaminants suggests the induction/presence of biotransformation enzymes in diplopods. In conclusion, diplopods would be able to detoxify low amounts of organic substances before intracellular protein degradation, and consequently, before the hsp70 response. Topical application of the pesticide methiocarb carbamate on Gymnostreptus olivaceus and Plusiorus setiger caused low toxicity, with high DL50 values for the two species: 515, 216, 137 and 93 mg/g for G. olivaceus and 489, 222, 108 and 71 mg/g for P. setiger, respectively, 24, 48, 72 and 96 h after application of the pesticide (Boccardo and Fernandes, 2000). Boccardo and Fernandes (2001) also found low toxicity of the pyretroid deltamethrin to diplopods of the same species. These responses were attributed to the protection of a calcified exoskeleton, which might have interfered with pesticides penetration. The effects of the fungicide dithane M-45 (mancozeb) on several arthropods were studied by Adamski et al. (2007), who reported high mortality rates for diplopods. The short period of exposure to the fungicide (24 h) revealed a low value for LC (LC50 ¼0.351 and LC95 ¼6.13). According to the authors, these results are likely to represent an underestimation of the effects of the fungicide in the field, where entire diplopod populations could be wiped out.
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Weber and Nentwig (2006) did not find higher mortality rates for individuals of Allajulus latestriatus fed with N4640Bt corn, which expresses the Bt Cry1Ab protein, used as an herbicide. The authors however found that the diplopods excreted considerable amounts of the active protein, making it available for other soil organisms. Regarding food intake and biomass, the authors did not find a significant difference between specimens of A. latestriatus fed with transgenic N4640Bt corn, and the control group. Merlini et al. (2012) evaluated different concentrations of trifluralin herbicide in the midgut of Rhinocricus padbergi and reported the disruption or total loss of the epithelium and others histological alterations describe in Section 2.5. 2.4. Aromatic hydrocarbons Induction of the cytochrome P450 has been accepted as a wellestablished sublethal stress biomarker that results from exposure to xenobiotics. Studies on the organization and distribution of the cytochrome P450 of invertebrates are restricted to only a few species (Livingstone, 1990). Zanger et al. (1997) reported the induction of cytochrome P450 compounds in J. scandinavius (Diplopoda) fed with β-naphthoflavone contaminated litter. However, a larger sample is required to substantiate the results of authors, because their enzymatic analyses were performed on a statistically small pool of specimens (10 diplopods). Moreover, the authors noted that individual analyses would be necessary, as well as the identification of the enzymes produced, in order to reveal similarities and differences with the P450 system of vertebrates. 2.5. Complex mixtures Fontanetti et al. (2012) analyzed diplopods behavior and the survival rate by log-rank test (p ¼0.05). The analysis showed that the animals exposed to pure sewage sludge presented higher mortality index than the specimens exposed to different concentrations of the sludge mixed with soil. Ultrastructural alterations in midgut of R. padbergi showed that nucleus and other cellular compartiments were damaged after exposure to sewage sludge sample (Nogarol and Fontanetti, 2011). Tissue alterations in the midgut of the diplopod R. padbergi were studied after exposure to soil contaminated with different proportions of residues of a complex nature, such as sewage sludge (Godoy and Fontanetti, 2010; Nogarol and Fontanetti, 2010, 2011; Perez and Fontanetti, 2010; Christofoletti et al., 2012; Bozzato and Fontanetti, 2012; Souza and Fontanetti, 2012) and soil from a deactivated bioremediation system from a petroleum refinery, contaminated with metals and polycyclic aromatic hydrocarbons (Souza and Fontanetti, 2011). Collectively, the results obtained by the above research indicated the presence of toxic substances in the samples and imply that the exposed animals display defense mechanisms that attempt to neutralize or eliminate the effects of toxic residues. In summary, the main tissue biomarkers observed in those papers were: (a) Death of the cell: Vacuolization of the cytoplasm of epithelial and hepatic cells, nuclear chromatin condensation and dilatation of the intracellular space. According to Nogarol and Fontanetti (2010), cell death could represent a physiological response that facilitates the discharge of the altered tissues, being important in the maintenance of the organ structure. However, cell death is a direct result of the environmental contaminants on the tissue and extensive cell death can lead to total or partial organ failure (Souza and Fontanetti, 2011). (b) Increase in the renewal rate of the epithelium: The process of epithelium renewal involves the removal of damaged cells to the lumen of the intestine, where they are digested. An
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increase in the renewal rate indicates that at least a portion of the toxic compounds are being eliminated through epithelium renewal. (c) Increase in the number of secretory vesicles: Apocrine secretory vesicles (Fantazzini et al., 2002), which discharge their contents together with a portion of their cytoplasms, were observed all over the epithelium of R. padbergi. The glycoproteic nature of these vesicles was interpreted as evidence that they correspond to a defense against the entrance of potentially toxic substances. However, in some treatments, the secretion vesicles display vacuolization, indicating that the toxic compounds present in the soil reach the cytoplasm of the main epithelial gut. Also, an increase in the amount of secretion vesicles released toward the lumen could represent a response of the organism to the effects of the pollutants, since the excretion of toxic compounds stored in the epithelium of the gut of diplopods would be accelerated. (d) Increase in the number of hemocytes between the hepatic cells layer: Hemocytes are cells of the arthropod immune system that are able to detect, encapsulate, or perform phagocytosis of pathogens and alien substances (Xylander, 2009). The total number of circulating hemocytes can be affected by changes in environmental conditions. Different stressors can activate the proliferation and migration of hemocytes to the affected tissues. A large number of isolated and lumped hemocytes have been observed between the hepatic cells of R. padbergi, indicating tissue lesion and possible inflammation. Therefore, they represent a defense mechanism aimed to destroy toxins and also participate in tissue renewal, through absorption of the damaged tissue. (e) Increased number of cytoplasmatic granules in the layer of hepatic cells: In diplopods, as in other invertebrates, the accumulation of potentially toxic materials as insoluble and inactive substances is an important detoxification mechanism that ensures homeostasis (Hopkin and Read, 1992). The biological origin of these cytoplasmic granules is not yet completely understood. It has been suggested that they come directly from the Golgi complex or associated cisterns; or that they are produced in the rER. Granules that appear in vacuoles or vesicles of undetermined origin are a third possibility. A fourth possible place of origin of these granules is the internal portion of the mitochondria (Köhler, 2002). In some species, the cytoplasmic granules may remain in the cell for long periods of time before being excreted. After precipitating, they can be transported to the intestinal lumen and subsequently freed (Vandenbulcke et al., 1998). Molting is also an opportunity for detoxification, once the intestinal epithelium disrupts and the contents of the main cells are eliminated together with the feces (Hubert, 1979b). An increase in the amount of intracellular granules in the tissues of aquatic and terrestrial invertebrates as a function of high concentrations of metal in the substrate has been used as a biomarker for environmental contamination (Köhler et al., 1996; Barka, 2007). This seems to be true for the diplopod R. padbergi. The mobilization of detoxification processes and survival in stressful situations require utilization of energy reserves (Köhler et al., 1996). It is possible to measure the usage of these reserves to detect substances such as glycogen, proteins and lipids, and to use these results to complement morphological analyses. Lately, the depletion of these substances has been used in ecotoxicologic evaluations when diplopods are used as bioindicators (Souza and Fontanetti, 2011). The perivisceral fat body of diplopods presents intense metabolic activity and functions in lipid, glycogen, protein and uric acid storage, as well as in the neutralization and storage of substances
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that are neither needed nor useful (Hubert, 1979a, 1979b; Hopkin and Read, 1992; Fontanetti et al., 2006). Histological and histochemical analyses have been conducted in the perivisceral fat body of R. padbergi exposed to landfarming derived from refinery oil by Souza et al. (2011). The loss of integrity of the cell boundary, the cytoplasmic disorganization, and depletion of neutral polysaccharides, in these diplopods, can be used as environmental contamination markers. Souza and Fontanetti (2012) analyzed the parietal and perivisceral fat body of R. padbergi exposed to substrate containing sewage sludge. The parietal fat body showed no alterations after variations of time exposure and concentrations. Addition to the changes reported by Souza et al. (2011) the perivisceral fat body showed increase in the quantity of spherocrystals and nuclei with altered morphology.
3. Sensivity of diplopods: comparison with others soil bioindicators Isopods (Köhler et al., 1996; Lemos et al., 2010), nematodes (Sochová et al., 2006), earthworms (Natal Da Luz et al., 2004; Sissino et al., 2006; Pérès et al., 2011) and collembolans (Crouau et al., 2002; Natal Da Luz et al., 2004; Eom et al., 2007) are established models of soil ecotoxicology. Despite their ecological importance, however, diplopods have seldom been used as bioindicators, when compared with other saprophagous terrestrial invertebrates. Due to a growing number of contamination sources, however, the potential utility of this group has gained increased attention in recent years. Metal bioaccumulation in various arthropod groups was investigated by Heikens et al. (2001). Diplopoda and Isopoda bioaccumulated copper and zinc significantly. Lead concentration was extremely high in Isopoda and Collembola, intermediate in Diplopoda, low in Chilopoda and Coleoptera. As in diplopods, heavy metals have been promoted ultrastructural and histological alterations in earthworms (Eudrillus eugeniae) (Sharma and Satyanarayan, 2011), mites (Nothrus silvestris) (Triebskorn et al., 1991), centipedes (Lithobius forficatus) (Vandenbulcke et al., 1998) and isopods (Porcellio scaber) (Köhler et al., 1996). Also, Khöler and Triebskorn (1998) found similar patterns of cellular responses to cadmium and lead in the tissues of isopods (P. scaber) and diplopods (J. scandinavius). The diplopod was more sensitive to cadmium than slugs (D. reticulatum). From these comparisons, it would appear that sensitivity of diplopods to heavy metals is comparable with other soil bioindicators when bioaccumulation and morphological alterations are employed as biomarkers. Already, Zanger et al. (1997) provides evidence of the induction of the cytochome P450 system in diplopods (J. scandinavius) and isopods (O. asellus) fed with β-naphthoflavone contaminated litter.
4. Conclusions and future prospects The response of diplopods to environmental contaminants can be evaluated in several ways such as at the community level (e.g. abundance, biomass species and functional structures) as well as through the individual response of selected species (e.g. measuring the biochemical biomarkers and histopathology). In diplopods the effects of heavy metals have been largely studied and sensitivity to these compounds has been reported. Even though the number of diplopod species may be affected by high amounts of metal contamination, some species will develop strategies that minimize the effects of these substances on their survival such as decrease in food intake, decrease in nutrient assimilation, or both. This review also clearly indicates that diplopods have efficient potential for bioaccumulation heavy metals in their tissues which
can be used as an ecological indicator of soil pollution. The assessment of structural alterations on the cell and tissue is the early stage of a reaction to cell injury, before an integrated cellular response would manifest at level of whole animal physiological processes, and long before there were any changes at the population level. Diplopods also good bioindicators of soils contaminated with radioactive elements and complex mixtures. Pesticides are potentially toxic for non-target species however there has been little progress in understanding of the effects those organic pollutants in diplopods. Most of the studies related in this review, conducted in laboratory, showed that the pesticides caused low toxicity in diplopods. But, considering that these compounds are widely used in agricultural soils, future research requires attention to understanding the biological effects of pesticides. Also, the effects of other persistent organic pollutant need to be clarified.
Acknowledgments We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Process nos. 06/52383-7 and 09/50578-3, the Fundação para o Desenvolvimento da UNESP (FUNDUNESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support.
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