Environ Sci Pollut Res DOI 10.1007/s11356-015-5376-8
RESEARCH ARTICLE
Histopathological changes in the perivisceral fat body of Rhinocricus padbergi (Diplopoda, Spirobolida) triggered by biosolids Annelise Francisco 1 & Cintya Aparecida Christofoletti 2 & Nilton Righetto Neto 1 & Carmem Silvia Fontanetti 1
Received: 9 February 2015 / Accepted: 6 September 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Human activities generate a great amount of sewage daily, which is dumped into the sewer system. After sewage-treatment processes, sewage sludge is generated. Such byproduct can be treated by different methods; the result of treatment is a stabilized compost of reduced pathogenicity that has a similar inorganic chemical composition to the raw sewage sludge. After such pretreatment, sewage sludge is called a biosolids, and it can be used in agriculture. In this contest, the present study evaluated the effects of a sample of biosolids on the perivisceral fat body of a diplopod. These invertebrates are soil organisms that play an important role in the dynamics of terrestrial ecosystems, and as a consequence, they are in contact with xenobiotics present in this environmental compartment. Special emphasis is given on the interpretation of the effects of complex mixtures in target organs of diplopods. A semiquantitative analysis for the evaluation of histopathological changes in the perivisceral fat body was proposed. The sample-induced histopathological and ultrastructural changes in individuals exposed to it, and the severity of the effects was positively related to the exposure time, resulting in the deaths of exposed individuals after 90 days. Thus, the results indicate the need for caution in the use of biosolids as well as the need Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-5376-8) contains supplementary material, which is available to authorized users. * Carmem Silvia Fontanetti
[email protected] 1
UNESP, São Paulo State University, Av. 24-A, n°1515, CP 199, CEP 13506-900 Rio Claro, SP, Brazil
2
Hermínio Ometto Foundation (FHO/UNIARARAS), Dr. João Maximiliano Baruto Avenue, 500, CEP 13607-339 Araras, SP, Brazil
for improving waste management techniques, so they will produce environmentally innocuous final products. Keywords Electron microscopy . Histopathology . Millipede . Oenocyte . Sewage sludge . Spherocrystals
Introduction In large urban and industrial centers, a great amount of sewage is generated daily and dumped into the sewer system. This waste is often dumped indiscriminately into the environment, but due to growing social and political demands for maintaining and improving the environment, there are a growing number of municipalities and industries that treat their sewage (Camargo et al. 2008). Sewage treatment, in turn, generates a mud rich in organic matter, macronutrients, and micronutrients called sewage sludge. This may be subject to several processes that generally aim to increase the solid content, reduce the load of microorganisms and pathogenic organisms, and reduce its attractiveness to vectors; the result of this process is a residue called biosolids, which are considered more innocuous than raw sewage sludge (Artuso et al. 2011; Bertelli 2007; Baldwin et al. 2001; Vanzo et al. 2000). Biosolids have variable chemical composition because the composition depends on the origin of the wastewater from which it came; however, overall, biosolids are rich in organic matter and nutrients that are essential to plants and microorganisms (Lambais and Carmo 2008). Therefore, biosolids have been cited as an excellent organic fertilizer, one that promotes the growth of plants, improves the level of soil fertility, and increases the soil’s ability to supply nutrients to plants. This residue has been applied to a wide variety of crops around the world, such as in coffee, maize, soybeans, peas,
Environ Sci Pollut Res
and canola; it has also been used in forestry (USEPA 1999; Vanzo et al. 2000; Baldwin et al. 2001; Currie et al. 2003; Bañuelos et al. 2004; Granato et al. 2004). However, biosolids may contain persistent toxic organic compounds and trace metals because the treatment processes employed are not always capable of removing such compounds (Baldwin et al. 2001; Hébert 2010; Shinbrot 2012). According to Camargo (2006), even sludge from households contains higher trace metal concentrations than those normally found in the soil. Thus, there is a need for proper management of this waste to avoid environmental contamination. Biological assays can be used to verify the real effects and toxicity of compounds on organisms and ecosystems; these assays can be used in conjunction with analyses of cellular and histological changes as a metric for assessing the effects of stressors on different organisms (Fontanetti et al. 2010; Kammenga et al. 2000). Several ecotoxicological studies have affirmed that diplopods are good bioindicators to assess soil quality (Fontanetti et al. 2011, 2012; Souza et al. 2014). Diplopods selectively accumulate heavy metals and other harmful compounds mainly in midgut, hepatic cells, and fat body. In this sense, such organs are suitable as target organs in ecotoxicological evaluations (Dallinger et al. 1992; Köhler 2002). Given the above, this study aimed to evaluate the effects of a biosolids sample on the perivisceral fat body of a member of the soil fauna. The millipede Rhinocricus padbergi was chosen as a bioindicator once it is available in the region, and previous studies have shown that they are good bioindicators.
Materials and methods Soil and biosolids samples The soil sample, which was used both as a control and for mixing with the biosolids, came from the same site where the millipedes were collected at the São Paulo State University (UNESP), Rio Claro campus (22° 24′ 36″ S/47° 33′ 36″ W), São Paulo, Brazil. In order to assemble the bioassays, the soil was collected in 2011 at a depth of 0–20 cm, dried at room temperature, sieved with 4 mm mesh, and subjected to chemical characterization. The biosolid sample was collected in 2011 from a sewage treatment plant in the state of São Paulo, Brazil. The plant is managed by the Basic Sanitation Company of the State of São Paulo (SABESP). The biosolid sample was collected and stored in plastic boxes wrapped with dark plastic bags; it was maintained in a cold room (i.e., 4 °C) until use. To properly apply the biosolids, data related to agronomic potential and control of soil fertility were necessary. The sample was characterized for chemical and physicochemical characteristics by the Instituto Campineiro de Análise de Solo e Adubo (ICASA), Campinas, São Paulo, Brazil, and by
TASQA laboratory, Paulínia, São Paulo, Brazil, according to the methodology described by Christofoletti et al. (2013). Metals (As, Ba, Cd, Cu, Cr, Hg, Mo, Ni, Pb, Se, and Zn) and the 16 priority polycyclic aromatic hydrocarbon compounds (PAHs) defined by the US Environmental Protection Agency (EPA) (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenz(ah)anthracene, benzo(ghi)perylene, and indeno(1,2,3-cd)pyrene) were also measured by TASQA Laboratory for control soil and biosolids samples. The metal analyses were performed by inductively coupled plasma emission spectrometry (ICP), while PAHs analyses were performed by gas chromatography. The analyzed parameters followed the Standard Methods for the Examination of Water and Wastewater 21th Edition 2005 (SM21) and EPA 8270D, respectively.
Bioassays with R. padbergi Adult specimens of R. padbergi with a mean size of 6.0 cm were used to avoid intraspecific differences related to either diplopod size or age. The specimens were collected at the campus of the São Paulo State University (UNESP), Rio Claro. After collection, the specimens were kept in the laboratory for 3 weeks for acclimation; they were stored in a terrarium containing soil, tubercles, and decomposing branches from the capture area. The room temperature was controlled (i.e., 21±2 °C) as was the photoperiod (i.e., 12-h light/12-h dark). Two glass terraria, both measuring approximately 25 cm× 20 cm×45 cm (length×width×height), were used to carry out the bioassays. One was supplied with 5 kg of control soil (SC), and the other was filled with 5 kg of control soil plus a fixed amount of biosolids; the amount was calculated based on the Brazilian law for the application of biosolids in agriculture (SB). After chemical characterization of the sample and fertility analyses of the control soil sample (Table 1), calculations for the application of biosolids were made and resulted in the application of 234.4 g of biosolids. Each terrarium received 20 millipedes. The animals were exposed to the biosolids sample for 7, 30, and 90 days to assess acute responses (7 days), subchronic responses (90 days), and responses at an intermediate period (30 days). After each exposure period, three animals from each group were anesthetized with chloroform and dissected in a saline solution for insects to remove the fat body, in accordance to the procedures described by Souza et al. (2011). Fractions of the fat body were fixed using different solutions (i.e., Bouin, calcium formaldehyde, and paraformaldehyde) such that different techniques could be applied. After fixing, the material was placed in a sodium phosphate buffer (pH 7.4) for 24 h and stored in the refrigerator.
Environ Sci Pollut Res Table 1
Physicochemical analysis of control soil and biosolids samples
Parameter
Methodology
Control soil (mg/kg)
Biosolids (mg/kg)
Organic carbon (g/kg)
SSSA Cap34
12.6
590
Total phosphorus
SM21 3120B
182 4.40 0.06 31.8
18156
