Agroindustrial by-products and residues from treatment of sewage sludge have been recently recycled as soil amendments. This study was aimed at assessing toxic potential of biosolid, obtained from a sewage treatment plant (STP), vinasse, a by-product of the sugar cane industry, and a combination of both residues using Allium cepa assay. Bioprocessing of these samples by a terrestrial invertebrate (diplopod Rhinocricus padbergi) was also examined. Bioassay assembly followed standards of the Brazilian legislation for disposal of these residues. After adding residues, 20 diplopods were placed in each terrarium, where they remained for 30 days. Chemical analysis and the A. cepa assay were conducted before and after bioprocessing by diplopods. At the end of the bioassay, there was a decrease in arsenic and mercury. For the remaining metals, accumulation and/or bioavailability varied in all samples but suggested bioprocessing by animals. The A. cepa test revealed genotoxic effects characterized by different chromosome aberrations. Micronuclei and chromosome breaks on meristematic cells and F₁ cells with micronuclei were examined to assess mutagenicity of samples. After 30 days, the genotoxic effects were significantly reduced in the soil + biosolid and soil + biosolid + vinasse groups as well as the mutagenic effects in the soil + biosolid + vinasse group. Similar to vermicomposting, bioprocessing of residues by diplopods can be a feasible alternative and used prior to application in crops to improve degraded soils and/or city dumps. Based on our findings, further studies are needed to adequately dispose of these residues in the environment.

The use of sewage mud in agriculture has generated several discussions due to the risks of soil contamination mainly by metals. Due to the intimate contact of the diplopods with soil, they have been successfully used in ecotoxicological analyses. The impact of the external environment in the physiological changes in organs and tissues of different organisms can be reflected by the ultrastructural changes of their cells. Using the transmission electron microscopy, this study aimed to analyze the ultrastructural alterations in the midgut of the diplopod Rhinocricus padbergi exposed to substrate containing sewage mud from a Sewage Treatment Station of São Paulo State, Brazil. Therefore, it was sought to identify cellular alterations resulting from the subchronic exposure (90 days of exposure) of the animals to complex and potentially toxic mixtures such as sewage mud at different concentrations (1%, 10%, and 50%). The control group presented integrity in the cells and tissues that constitute the midgut. The animals exposed to sewage mud at 10% and 50% died before the 90 days of exposure. The analysis of the midgut of the animals exposed to 1% of sewage mud showed alterations in the epithelial cells, cells of the fat body layer, and hepatic cells. The main alterations observed were cytoplasmatic vacuolization, loss of the plasmatic membrane, and nuclear envelope integrity. We conclude that the mud sample presents a mixture of complex substances of cytotoxic and genotoxic action to the diplopod, causing cell death, since both the nucleus and other cellular compartments were damaged.

Diplopods are components of the edaphic fauna, which makes them suitable bioindicators of soil quality. Some characteristics of the fat body of diplopods make it suitable for ecotoxicological studies. This organ, composed of diffuse tissue, fills the body cavity; it presents intense metabolic activity associated with lipid, glycogen, protein, and uric acid storage, being also responsible for storage, neutralization, and excretion of substances that are not useful to the organism. The present study aimed to investigate whether the perivisceral fat body of Rhinocricus padbergi can be used as a target organ in ecotoxicological studies and to identify possible histological and histochemical biomarkers in this organ. Upon examining the perivisceral fat body of individuals of R. padbergi exposed to different concentrations of industrial soil contaminated with polycyclic aromatic hydrocarbons (∑ 2,749.0 mg/kg) and metals (∑ 40,355.8 mg/kg), we have found evidence that this organ is sensitive to environmental pollutants present in the soil. The loss of integrity of the cell boundary associated with cytoplasmatic disorganization and depletion in total proteins, neutral polysaccharides, calcium, and lipids can be considered stress biomarkers for R. padbergi.

The destination of sewage sludge is a problem faced by sewage treatment plants (STPs). Many alternatives have been sought, such as the application of sewage sludge in degraded soils and in agriculture as fertilizer. However, due to the risk of contamination with pathogens and/or metals, the use of sludge should be done cautiously. By the habits that diplopods present, they have been considered good environmental indicators for soil analysis. In this study, animals from the Rhinocricus padbergi species were exposed to two sewage sludge samples from two STPs in the São Paulo State, for different periods. The midgut of the animals were removed and histologically processed and subjected to histochemical tests. It was detected the following tissular responses: clusters of haemocytes through the cells of the fat body layer, increase in the quantity of intracellular granules in the cells of the fat body layer, increase in the release of secretion vesicles of the intestinal epithelium, and intense vacuolization of the cytoplasm of epithelial cells. The results suggest the presence of toxic substances to the studied species in both sludge samples used.

Diplopods feed organic matter in decomposition; however, some environmental factors can promote changes in tissues of these animals. Sewage sludge has been applied for recuperation of physical structure of degraded soil. This work analyzed the influence of the sludge from a city of São Paulo in the midgut of the diplopod Rhinocricus padbergi. After the exposition to sludge, the midgut was prepared for histological and ultra-structural analyses. After 1 week of exposition, there were various glycoprotein globules in the fat body, which appeared, ultrastructurally, little electron dense. In the animals exposed for 2 weeks, there was an intensive renovation of the epithelium with the invasion of regenerative cells, which was observed in the histological and ultra-structural analyses. These data showed that the sludge present various substances that were very hazardous for these animals; more studies were necessary before the application of this in agriculture.

Large amounts of residues generated by agricultural, urban and industrial activities are dumped daily on the soil. This practice deserves special attention because it causes serious environmental problems. This study evaluated the toxic potential of the sugarcane vinasse, a by-product of the sugar-alcohol industry, and the biosolid, a residue produced by wastewater treatment plants, both widely used as fertilizers. The evaluation was performed through bioassays using a typical soil bioindicator, the diplopod Rhinocricus padbergi. The specimens were exposed to soils containing these residues in concentrations that are compatible with the Brazilian regulation for agricultural use. Semi-quantitative immunolabelling analyses of the stress protein HSP70 were performed on the midgut of the studied diplopods. There was a significant increase in the immunolabelling of HSP70 proteins as a response to xenobiotics from both residues, particularly in regions where the function of the cells is the detoxification of the organ (e.g. the hepatic cell layer and specific regions of the epithelium). Higher immunolabelling was observed in the specimens exposed to vinasse in comparison with the biosolid exposure. This demonstrates that the substances in the tested residues had proteotoxic action in the exposed animals and induced a cytoprotective response, which led to higher stress protein immunolabelling. Therefore, caution is needed for the use of such residues in agriculture.

Millipedes are organisms of the edaphic fauna and have been used as bioindicators for the evaluation of pollutants in the environment, as they are in constant contact with the soil. This study used the millipede Rhinocricus padbergi as surrogate to evaluate the toxicity of two metallic-insecticides that has been developed for leaf-cutting ants management. Millipedes were exposed in terrariums containing different concentrations of the metallic-insecticides and, after periods of 21 and 90 days, three individuals from each terrarium were dissected in order to remove the midgut, the organ where absorption of nutrients and, consequently, toxic substances occurs. The toxic action of the metallic-insecticides was analyzed through qualitative and semi-quantitative analysis of morphophysiological alterations and by quantitative analysis of the HSP70 stress protein. The results showed that the metallic-insecticides may increase HSP70 labeling, although not at all concentrations and periods of exposure. Histopathological alterations were not significant at any concentration, indicating that the cytoprotective action of HSP70 is able to prevent severe damage to the midgut. It is therefore suggested that the metallic-insecticides are not toxic to the species studied here as no toxicity was observed under the conditions tested. In addition, stress protein localization in midgut helps understand how morphophysiological processes can potentially be affected by pesticide exposure.

Urban forests are exposed to metals, such as manganese, copper, and zinc in the atmosphere that originate from anthropogenic activities, that include vehicle-related traffic, industries, construction sites, fossil fuel burning for heating and cooking purposes, and resuspension processes related to urban surfaces. Not only is the rich biodiversity of plant and animal species in forests under threat, but so are the biodiversity of soil, sustaining ecosystem functions, as well as human health. The objective of this study was therefore to determine the concentrations of manganese, copper, and zinc arising from urban, industrial, and traffic-related pollution in the remote and/or untouched urban indigenous forests using soil, leaf litter, and key forest organisms (mosses, lichens, and millipedes) in three forests (Platbos, Orange Kloof, and Newlands) in the Western Cape, South Africa. Elevated concentrations of these metals were found in the forests closest to the city, as well as at sites in close proximity of vehicle traffic.