Introduction

The expansion of industrial and urban regions has stimulated residues generation, which require an appropriated treatment. In this respect, the disposal of sewage sludge has become an extremely important subject for the scientific community, due to its high potential for being applied in the tropical agriculture and recuperation of degraded areas (Suthar and Singh 2008). Its use in such activities is mainly justified by its high levels of organic matter and nutrients, which are able to modify physical (permeability, porosity and aggregability) and chemical (fertility) properties of the soils (Selivanovskaya and Latypova 2003). Therefore, besides being able to decrease erosion rates in degraded areas, the application of sewage sludge on soils may also enable the cultivation of vegetables not adapted to the original environmental conditions (Borges and Coutinho 2004; Liu et al. 2005).

On the other hand, the presence of heavy metals, organic contaminants and pathogenic agents are the most important restrictions of applying sewage sludge in the agriculture (Schnaak et al. 1997; Obbard 2001; Bright and Healey 2003; Cai et al. 2007). In general, heavy metal contents in the sewage sludge are extremely higher than the concentrations naturally observed in tropical soils (Borges and Coutinho 2004). Consequently, metals uptake by soil fauna and plants may favor the insertion of those pollutants in the terrestrial trophic chain (Suthar and Singh 2008), causing damages on the health of those ecosystems. In this context, the ingestion of contaminated vegetables and the presence of pathogenic microorganisms may modify the pedomicrobial activity and cause damages on human health and biota. In addition, soils leaching, associated with runoff, can mobilize contaminants to the surrounding fluvial systems, thus causing serious adverse effects on aquatic biota (Parkpian et al. 2002; Selivanovskaya and Latypova 2003; Natal-da-Luz et al. 2009).

Over the last decades, the growing-interest on systemized methodologies for determining the risk of soil pollution and their effects on biota has been widely observed in the scientific literature. Most studies of environmental risk assessments have been based on total chemical analysis, chemical speciation and sequential extraction of geochemical fractions of high potential bioavailability (Crouau et al. 2002; Smith et al. 2007). However, those analytical approaches neither allow determining the mixture toxicity due to the simultaneous actuation of distinct pollutants in the environment nor take in consideration their ecological effects on the pedosphere. In this respect, bioassays can complement the results of chemical analysis by evaluating the environmental toxicity levels based on the monitoring of lethal and sub-lethal effects. Besides that, the quantification of contaminants’ concentrations in organisms can support, in a much better way, the estimation of bioavailable fractions in the environment (Straalen et al. 2005), as well as their potential risks on biota.

Bioassays are directly related to contaminants’ concentrations in the environment and its adverse effects on biota. Plants, edaphic and aquatic organisms (especially microorganisms, vertebrates and invertebrates) are commonly used for evaluating potential toxicity of soils, aquatic sediments and superficial waters (Crouau et al. 2002; Selivanovskaya and Latypova 2003; Burton et al. 2006; Fjällborg et al. 2006). In the case of soils, earthworms have been widely used as bioindicators (biomonitoring and/or bioassays), since they are well-studied organisms, are easily cultivated in the laboratory, ingest high amounts of soils, are extremely sensible to the presence of contaminants and play a crucial role in the terrestrial trophic chain (Liu et al. 2005; Nahmani et al. 2007; Hinton and Veiga 2008).

This paper proposes the investigation of the potential bioavailability of contaminants in tropical soils treated with different doses of sewage sludge, using distinct bioassays with earthworms (Eisenia andrei).

Materials and methods

Sampling and preparation of the soils

A sewage sludge sample was collected at the Ilha do Governador Effluents Treatment Station, located closer to the Guanabara Bay, at the Rio de Janeiro Municipality (RJ, Brazil). To investigate the influence of distinct soil classes and their properties in the contaminants bioavailability, samples of ferralsol and chernosol (B Horizon) were studied. Those soils were also collected at the Rio de Janeiro State, (chernosol: 22°51′22.5″S, 43°30′0.7″W; ferralsol: 22°41′34.2″S, 43°17′14.5″W).

Samples were collected using a soil auger. At the laboratory, samples were fractioned using nylon sieves of 1.7 mm, in order to remove stones, roots and other larger particles. The proportions of sewage sludge and soil (soil:sewage sludge mixtures) followed the recommendations of Tsutya 2000 apud (Alamino et al. 2007), who propose the dose of 112 ton sewage sludge/ha for recuperation of degraded areas. Thus, the ferrasol was initially treated with 6.66%, and the chernosol with 6.58%. In order to simulate a risk scenario of successive applications and accumulation of sewage sludge in the soil, other doses were also studies. Such doses followed an arithmetic progression of the initial values: for the ferraso—13.32, 19.98, 26.64 and 33.3%; for the chernosol—13.16, 19.74, 26.32 and 32.9%).

Soils properties

Cesar et al. (2008) and Alamino et al. (2007) indicated some aspects of the physical, chemical and mineralogical composition of these same soil samples used in this investigation (Table 1). Organic matter concentrations are similar and low in both soils, since they were collected in the B horizon. Chernosols presented abundance of 2:1 clay minerals in the clay fraction (especially vermiculite), very low permeability, high cation exchange capacity, high contents of Mg2+, Ca2+, K+ (high natural fertility) and more basic pH values. The chernosols are extensively distributed in southern Brazil and in temperate areas and widely used in the agriculture due to its high fertility.

Table 1 Some physical and chemical properties of ferralsols and chernosols used in bioassays
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Ferralsols are more acidic than the chernosols, contain high concentrations of total iron and are more permeable. Besides that, those soils contain high contents of kaolinite in the clay fraction and very low contents of nutrients (Table 1). Such differences between the soils are justified by the actuation of intense weathering processes on the ferralsols. The ferralsols have a great geographical distribution in tropical and sub-tropical regions and are intensively used in the Brazilian agriculture.

Determination of heavy metals

Total copper, lead, nickel, zinc, chromium and cadmium concentrations (CuT, PbT, NiT, ZnT, CrT and CdT) were determined in the sewage sludge sample, sewage sludge-amended soils and survival earthworms. The analytical procedure was based on the solubilization of 1 g of sample using an acid mixture composed by HF:HCl:HClO4 (2:1:1). Total contents in the analytical solution were quantified by ICP-OES (Horiba Jobin–Yvon, Ultima 2). Total mercury (HgT) determination in soils and biological samples was performed with the LUMEX equipment (RA—915+, Zeeman mercury spectrometer), an atomic absorption coupled to the thermodesorption technique.

Before being submitted to such procedures, the worms were previously allowed to void gut contents for a period of 24 h. After this step, the organisms were frozen and lyophilized. To guarantee the quality of analytical results, the values were compared with certified materials (NIST 2709 San Joaquin Soil and IAEA 407—fish homogenate, for non-biotic and biotic samples, respectively).

Metal contents in the sewage sludge-amended soils were compared with the values proposed by Brazilian legislation for soil environmental quality (CETESB 2005; CONAMA 2009) (Table 2). Metal contents in the sewage sludge sample were compared with the values established by CONAMA (2006), which regulates the use of sewage sludge as an input in the Brazilian agriculture (Table 2).

Table 2 Heavy metal concentrations recommended by CETESB (2005) and CONAMA (2006) for soil quality and application of sewage sludge on Brazilian soils, respectively
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Cultivation of test-organisms

The earthworms (Eisenia andrei) were cultivated at the Ecotoxicology Laboratory of the Centre for Mineral Technology (CETEM), in Brazil. Those animals have been cultivated for 4 years at the CETEM, and maintained under controlled temperature (20 ± 2°C) and humidity (60%). At the laboratory, the organisms are cultivated into plastic boxes containing fresh cow manure. The animals are separated according to their ages (cocoons, juveniles and adults). The cow manure is periodically exchanged, when the humus produced by the earthworms is removed. Animal’s health is monthly monitored by performing sensibility tests using chloroacetamide as test-substance.

Acute toxicity tests

Ten adult organisms of similar weight (to guarantee a more homogenous population) were inoculated to each replicate (3) containing 200 g of soil (ASTM 2004). Soil moisture was adjusted to the field capacity by adding distilled water. Before being used in the tests, the organisms were previously maintained on a humid absorbent paper, during 24 h, for elimination of their intestinal contents. The test-containers were covered with a transparent lid containing some holes, in order to prevent the worms from escaping. After 14 days of exposure, the amount of survival organisms in each replicate was counted. Total metal contents were quantified in survival organisms.

Artificial soils (70% of sand, 20% of kaolin and 10% of coconut shells dust) were prepared for the control samples (Garcia 2004). The lethal concentration of sewage sludge on 50% of the organisms (LC50) was estimated using the software Trimmed–Spearman–Karber Method. Dependence relationships between sewage sludge rate-increase, metal contents in earthworms and mortality levels were evaluated through linear regression analysis, using the software Statistica for Windows.

Potential bioavailability of heavy metals was evaluated by calculating the bioconcentration factors (BCF), a ration between total concentration in the organism and total content in the bulk soil. The BCF is an index which associates contaminants’ concentrations in the biotic and non-biotic compartments. Thus, the values of BCF are directly proportional to the increase of metal concentrations in worms’ tissues and to the decrease of metal contents in soils. The values of BCF ≤1 indicate that contaminant was only absorbed but not accumulated, while BCF values >1 suggest that the earthworms bioaccumulated the contaminant (Liu et al. 2005). The values of BCF are traditionally used as good indicators of bioavailability (Neuhauser et al. 1995; Liu et al. 2005), and widely applied by many authors.

Avoidance tests

Plastic boxes (20 cm length, 12 cm width and 5 cm height) were divided into two equal sections, using a plastic card (ISO 2008). About 500 g of test-soil was placed into one of the two sections. The other section was filled with the same amount of sewage sludge-non-amended soil. After removing the plastic card, ten adult earthworms were washed with distilled water, dried with absorbent paper and placed on the interface of the soils. The tests-containers were incubated at 20 ± 2°C with a photoperiod 12:12 h light:dark. After 48 h of exposure, the two different sections were again divided with a plastic card, and the number of organisms in each section was counted. When less than 20% of the organisms were not found in the test-soil, it was considered that such material had “limited habit function” (ISO 2008).

Acute toxicity tests with contact paper

The assay consists in the exposure of adult earthworms (of similar weight) on an absorbent paper (5 × 10 cm2) containing 2 mL of a test-solution, in order to evaluate its bioavailability through dermal contact. A piece of that paper was placed against the walls of a glass recipient (50 cm in volume, 8 cm in length and 3 cm in diameter) (OECD 1984). The test was performed in the dark and under a temperature of 22 ± 2°C. After 72 h of exposure, the number of survival animals was verified.

The tests were performed with soil leachates (elutriates). The mixtures (1: sewage sludge-amended soils—2.5 distilled water) were shaken (200 rpm) for 2 h, centrifuged and filtered. This procedure is an adaptation of the recommendations described by Baun et al. (2002), who propose the use of elutriates for evaluating their toxicity levels for aquatic biota.

Results

Determination of heavy metals in the sewage sludge-amended soils

Total concentration of toxic elements in the sewage sludge sample and in sewage sludge-amended soils are shown in the Table 3. The application of sewage sludge on the soils substantially increased heavy metal contents in those materials. In both soils, the concentrations of CuT, ZnT, PbT and HgT were higher than the reference limits for almost all studied doses (CETESB 2005). Cadmium concentrations were not detected at environmentally significant levels. Copper and mercury were the only elements whose concentrations were higher than the prevention limits (CETESB 2005; CONAMA 2009). On the other hand, total heavy metal contents in the sewage sludge sample were in agreement with the values proposed by Brazilian legislation for its agricultural use (CONAMA 2006) (Table 2).

Table 3 Concentrations of heavy metals (mg/kg) in soils treated with different doses of sewage sludge, survival earthworms (after 14 days of exposure in the acute toxicity test) and respective values of BCF, including the comparisons with the Brazilian legislation for soil quality
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Acute toxicity tests: lethal effects

Higher levels of mortality were observed in the treatments with ferralsols. Any of the sewage sludge doses applied on chernosols caused significant lethal effects on the organisms (Fig. 1). For this reason, the LC50 could not be estimated for such soil class. On the other hand, in the ferralsol all the organisms exposed on the doses of 19.98, 26.64 and 33.30% did not survive (Fig. 2) (LC50 = 14.37%, confidence interval of 95% = 13.04–16.06%). The ideal dose for agricultural application (6.66% for the ferralsol, and 6.58% for the chernosol) did not cause significant lethal effects on the exposed animals. In the case of the ferralsol, the results indicate the occurrence of lethal effects in hypothetic situations of successive applications of sewage sludge in the soil, as well as a positive significant relationship between the increase of sewage sludge rates and mortality levels (p = 0.001; r 2 = 0.843).

Fig. 1
figure 1

Lethality levels of the organisms exposed to sewage sludge-amended chernosols after 14 days of exposure

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Fig. 2
figure 2

Lethality levels of the organisms exposed to sewage sludge-amended ferralsols after 14 days of exposure

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Acute toxicity test: uptake of heavy metals

In the chernosol, metal concentrations in the worms’ tissues revealed preferential accumulations: Zn > Pb > Cu > Cr > Ni > Hg (Table 4). Such order was not so different for the ferralsol: Zn > Pb > Cu > Ni > Cr > Hg (Table 3). These sequences were independent to the rates of sewage sludge application. In general, metal contents in worms exposed to the chernosol were higher than that exposed to the ferralsol (Table 3), with independence of the sewage sludge rates. In addition, the values of BCF for metals also tended to be higher in the chernosol (Table 4).

Table 4 Mean BCF and metals average concentrations in earthworms exposed to the ferralsol (n = 81) and chernosol (n = 176) treated with different sewage sludge rates
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Mercury

The values of BCF of mercury were lower than one unit, for both soil classes (Table 3). Mercury contents in worm’s tissues were significantly lower than the concentrations obtained for the other metals. In the chernosol, a positive significant relationship was detected between the increase of mercury content in the soil and mercury concentration in worms’ tissues (p = 0.009; r 2 = 0.853). On the other hand, the values of BCF were inversely proportional to the sewage sludge rate-increase.

Zinc and copper

In general, the values of BCF for zinc were higher than one unit (especially at lower sewage sludge rates) (Table 3). In the chernosol, positive and significant relationships were found between: (i) zinc in worm’s tissues and zinc content in the soil (p = 0.004; r 2 = 0.904) and; (ii) copper in tissues and copper concentration in the soil (p = 0.007; r 2 = 0.868, Table 2). For both soil classes, most of copper BCF values were lower than one unit.

In the chernosol, zinc and copper concentrations in worms non-exposed to the sewage sludge-amended soil (dose of 0.00%) were almost twice and four times lower, respectively, than the values quantified in organisms exposed to the highest rate of sewage sludge application (32.9%), suggesting great accumulations of such metals in the tissues. Besides that, zinc concentrations in tissues were the highest ones in comparison with the other studied metals (Tables 3, 4).

Lead and chromium

The BCF values for lead were higher than one unit. In fact, lead presented the highest BCF values (between 0.5 and 3.6 units) among the studied metals. In chernosol, chromium BCF values were also higher than one unit at the two highest sewage sludge treatments, suggesting that the worms bioaccumulated it. On the hand, in the ferralsol the values of BCF were lower than one unit, indicating lower levels of bioavailability (Table 3).

In both soils, positive significant relationships were detected between the increase of lead concentrations in the soil and lead contents in worm’s tissues (ferralsol: p = 0.04; r 2 = 0.995, chernosol: p = 0.01; r 2 = 0.831). Moreover, in the chernosol positive relationships were verified between chromium concentrations in tissues and the increase of chromium contents in the soil (p = 0.003; r 2 = 0.917).

Nickel

In the chernosol, the values of BCF for nickel were higher than one unit (Table 3). A significant and positive relationship was also detected between the increase of nickel concentration in the chernosol and nickel contents in worms’ tissues (p = 0.005; r 2 = 0.889). Differently to the other studied metals, it was not possible to detect any inverse significant relation between the increase of sewage sludge rate and BCF in the chernosol. In the ferralsol, no significant differences were detected among nickel concentrations in earthworms exposed to distinct rates of sewage sludge (values between 1.5 and 1.8 mg/kg) (Table 3).

Avoidance tests

In the chernosol, more than 80% of the animals avoided the highest sewage sludge dose (32.9%) (Fig. 3). It is important to note that such dose did not cause significant lethal effects under an acute exposure, but it was able to cause significant avoidance effects. The other studied doses were not avoided by the organisms. In the ferralsol, the animals avoided the doses of 13.32, 19.98 and 33.3% (Fig. 4). Similar to the acute test, those results suggest the existence of a relationship between dose increase and avoidance responses, in hypothetic situation of successive additions of sewage sludge on soils.

Fig. 3
figure 3

Behavioral responses on the earthworms exposed to sewage sludge-amended chernosols in the avoidance tests after 48 h of exposure

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Fig. 4
figure 4

Behavioral responses on the earthworms exposed to sewage sludge-amended ferralsols in the avoidance tests after 48 h of exposure

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Acute toxicity tests with contact paper

Higher mortality levels were detected in ferralsols (Figs. 5, 6). Positive and significant relationships were observed between sewage sludge rate-increase and lethality levels in both soil classes (chernosol: p = 0.007, r 2 = 0.868; ferralsol: p = 0.02, r 2 = 0.789). No morphological damages were detected by the end of the assay.

Fig. 5
figure 5

Mortality levels of the organisms exposed to sewage sludge-amended chernosols after 72 h of exposure during the acute toxicity test with contact paper

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Fig. 6
figure 6

Mortality levels of the organisms exposed to sewage sludge-amended ferralsols after 72 h of exposure during the acute toxicity test with contact paper

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Discussion

Determination of heavy metals in the sewage sludge-amended soil

The reference limits proposed by CETESB (2005) describe the pedogeochemical background for São Paulo State (Brazil), while the prevention limits refer to the concentrations able to provoke damages on terrestrial biota. Thus, the concentrations of CuT, ZnT, PbT and HgT are anomalously higher in comparison with contents verified in the São Paulo State (reference limit). In addition, copper and mercury concentrations in the soils can offer risk on terrestrial fauna and flora (CETESB 2005; CONAMA 2009). The contents of heavy metals established by Brazilian legislation (CONAMA 2006) for agricultural use were not based on geo-ecological criteria. Thus, the execution of bioassays with sewage sludge-amended Brazilian soils becomes extremely important for evaluating toxic and ecological effects on terrestrial biota.

At this moment the Guanabara Bay Basin is submitted to the most important dreading intervention in Latin America, since its sediments are highly contaminated by domestic and industrial wastes. Its aquatic systems receive great amounts of contaminated materials from the Ilha do Governador district, where the sewage sludge sample was collected. The concentrations of heavy metals in the sewage sludge sample are similar to the contents determined in dredged sediments collected in estuarine systems from the Guanabara Bay Basin. Such sediments should be disposed on continental areas (soils) closer to Guanabara Bay (CH Serviços Ambientais 2008). In the near future, it is possible that our data can be used for evaluating potential ecological risks associated with terrestrial disposal of dredged sediments from the Guanabara Bay Basin. The importance of this action is recognized by the Brazilian government and it is essential for maintaining environmental quality and economy of the Rio de Janeiro State.

Acute toxicity tests: lethal effects

Soil properties played a very important role in the lethality levels. In this context, clay minerals, fertility and pH may be the most important attributes. Once both soils present similar organic matter contents, this parameter possibly did not influence mortality levels. Although the chernosols are sandier, the presence of 2:1 clay minerals makes them even more plastic and less permeable than the ferralsols. Therefore, texture probably did not play a crucial role in the bioavailability processes.

The abundance of 2:1 clay minerals in the chernosol may have stimulated the reduction of metal contents in the soil solution, due to its high potential of cationic adsorption. Matzke et al. (2008), e.g., described a similar behavior when studying the toxicity of organic substances for two species of vegetables (Triticum aestivum and Lepidium sativum). Such authors found lower levels of toxicity associated with the presence of expansive clay minerals, thus suggesting that those minerals are able to decrease not only the mobility of contaminants in the environment but also the fitotoxicity levels. In fact, Vijver et al. (2003) and (Nahmani and Navelle 2002 apud Natal-da-Luz et al. 2004) suggest that contaminant concentrations in the soil solution (soluble and exchangeable geochemical fractions) play a vital role in the bioavailability of contaminants for earthworms, since the dermal contact is one of the most important pathways of exposure for those organisms.

The chernosols present high levels of fertility, while the ferralsols contain very low nutrients contents and high concentrations of iron, since those last ones were submitted to more intense weathering processes. Thus, it is probable that the worms exposed to the chernosol were naturally healthier, due to the high availability of food (nutrients) in the soil. Thus, healthier animals (with more resistant immunological systems) could support higher levels toxic agents on the environment.

It is well known that pH can influence the oxidation and mobility of metals in the soil, affecting their bioavailability and toxicity. As the ferralsols are more acidic than the chernosols, heavy metal concentrations tend to be higher in the soil solution (geochemical soluble fraction). Consequently, metals also tend to be more available for biotic assimilation, especially through dermal contact. This is another aspect that could have favored the occurrence of higher levels of mortality in the ferralsol.

Acute toxicity test: uptake of heavy metals

Those results present some similarities (especially in respect to Zn, Cu and Hg) with the data obtained by Carbonell et al. (2009) in bioassays performed with agricultural soils treated with different sewage sludge rates: Zn > Cu > Cr > Ni > Pb > Hg. Differences of metal concentrations in tissues may be related to the food selectivity and differentiated metabolisms into earthworms (essential and non-essential elements) (Morgan and Morgan 1992 apud Suthar and Singh 2008). In this respect, zinc and copper play a crucial role in the metabolism and physiology of the earthworms. Therefore, those animals naturally have an affinity for bioconcentrate such metals. Another important biological aspect concerning metals uptake is the existence of competition sites of xenobiotics, since the organisms were simultaneously exposed to all pollutants contained in the sewage sludge (not only metals but also other contaminants).

Variations of potentially bioavailable contents in the soil (especially soluble and exchangeable fractions) may also play an important role in such processes. In this respect, Natal-da-Luz et al. (2004) also found positive relationships between the increase of heavy metal contents (especially extractable fractions) and avoidance responses in earthworms and collembolans (soil arthropods) exposed to soils from an abandoned mining area.

In terms of soil properties, the presence of expansive clay minerals in chernosols may strongly influence those mechanisms, due to the high cation exchange capacity associated with these materials. Organic matter is also able to form stable complexes with metals, but the concentrations are very similar in both studied soils. In respect to fertility, high concentrations of nutrients in chernosols indicate a high availability of food for the earthworms. Thus, it is expected that healthier animals would be able to bioconcentrate and tolerate higher levels of toxic metals in their tissues, explaining the observation of higher values of BCF in chernosols (Tables 3, 4). In fact, lower concentrations of heavy metals in the earthworm’s tissues were already able to provoke significant mortality levels in the ferralsol in comparison with chernosol (Figs. 1, 2). In this context, Suthar and Singh (2008) emphasize the scarcity of studies concerning the effects of soil properties (such as fertility, organic matter, pH and clay minerals) on metals accumulation by earthworms and the importance of further detailed investigations, especially in tropical soils.

Mercury

Liu et al. (2005) suggest that the values of BCF lower than one unit indicate that the organisms only absorbed the contaminant, but not bioaccumulated it. The values of BCF higher than one unit indicate that metals uptake involved bioaccumulation. The results suggest that mercury was only absorbed, but not bioaccumulated by the organisms. In fact, the low mercury contents determined in animal tissues suggest a low bioavailability in such materials. Cocking et al. 1991 apud (Burton et al. 2006) indicate that the values of BCF of mercury are usually lower than one unit, and just a few study cases report higher values.

It is also important to note that mercury is an extremely toxic element for the earthworms, besides being neurotoxic and teratogenic (WHO 1990). Thus, it is expected that mercury metabolism in earthworms present saturation mechanisms, in order to depurate the excess of mercury from its organism. The observation of an inverse proportionality between the values of BCF and sewage sludge-dose increase may suggest the existence of such mechanisms.

Zinc and copper

The values of BCF for zinc indicate that the animals were able to absorb and bioaccumulate this metal (Table 3). Such high zinc uptake rates (BCF >1) indicate that this metal was highly bioavailable in the soils. The values of BCF for copper values suggest that the organisms only absorbed the metal from the soil (Table 3).

In this context, it is important to emphasize that zinc and copper (especially zinc) are essential elements for the earthworms and then it could justify their high concentrations determined in animal tissue. Zinc plays a vital role in the cell metabolism, development, growing and regeneration of some tissues, while copper participates in the transport of substances among cells and tissues (Lukkari et al. 2005). Furthermore, those high concentrations of zinc and copper in the worm’s tissues may also be explained by the existence of an internal storage mechanism for future physiological demands. In addition, some investigations suggest that such storage mechanism is mainly regulated by zinc and copper binding to metallothioneins (Suthar and Singh 2008), which are proteins that have affinity with metals.

Although zinc and copper are essential micro-nutrients for the earthworms, those metals can provoke damages when they are present in very high concentrations. In this respect, the reduction of BCF for higher sewage sludge doses may be explained by the existence of a saturation mechanism, trying to eliminate the excess of zinc and copper from the metabolism. Neuhauser et al. (1995) and Spurgeon and Hopkin (2000) also describe an increase of zinc and copper uptake rates in soils contaminated by those metals, followed by an equilibrium between uptake and depuration rates, even in highly contaminated environments.

Lead and chromium

The values of BCF for lead indicate that the organisms absorbed and bioaccumulated this contaminant (BCF >1) (Table 3). In fact, such high values of BCF suggest a very high bioavailability of lead in the sewage sludge-amended soils. In this respect, it is important to note that lead is a non-essential and highly toxic metal, besides being neutoroxic and teratogenic (Paoliello et al. 2002). Other studies also report that the earthworms are able to bioaccumulate high amounts of lead, even in sewage sludge-amended soils (Suthar and Singh 2008; Carbonell et al. 2009; Nahmani et al. 2009). The principle of that metabolic process is not well understood yet, since lead does not play any role in the physiology of earthworms.

Some values of BCF (>1) for chromium indicate the bioaccumulation of that contaminant (BCF >1), suggesting a great incorporation of this metal by the earthworms. However, even at low uptake rates (FBC <1) chromium is able to provoke damages on exposed biota, especially when it is present in its hexavalent form (carcinogenic). Its trivalent species is essential for mammals and plays a vital role in the glucose metabolism (Katz and Salem 1993).

Those great lead and chromium bioaccumulations (BCF >1) suggest that these elements possibly played a crucial role in the toxicity (in comparison with the other investigated metals), since they are very toxic metals. High rates of lethality observed in ferralsols may be possibly related to the elevated bioavailability of theses metals in the soil.

Nickel

In the chernosol, the values of BCF suggest that the earthworms were able to bioaccumulate great amounts of nickel (Table 3). Since this metal does play any role in the worm’s metabolism, such results indicate possible physiological effects on those animals. However, the inverse proportionality between nickel in tissues and sewage sludge-dose increase suggests the absence of saturation processes (in contrast of other non-essential metals, such as mercury, lead and chromium) or at least the existence differentiated metabolic mechanisms. Thus, these animals may not be able to eliminate the excess of nickel from their organisms.

In the ferralsol, nickel seems to be less bioavailable than in chernosols, with independence of the increase of sewage sludge rates. Such process can be associated with preferential adsorption of metals on expansive clay minerals or iron and aluminum oxi-hydroxides, which are abundant in chernosols and ferralsols, respectively. Carbonell et al. (2009) found a similar behavior when evaluating nickel uptake by earthworms (values between 1.76 and 2.08 mg/kg) exposed to agricultural soils treated with different rates of sewage sludge. On the other hand, such authors found significant differences when determining nickel contents in tissues of plants (Triticum aestivum, Brassica rapa and Vicia sativa). Therefore, it is important to note that bioavailability mechanisms are equally related to soil properties and how the metal is metabolically processed by different ecological receptors.

Avoidance tests

The highest sewage sludge rate applied on chernosols seems to limit the habit function of the soil, as well as it was able to cause significant behavioral effects on the exposed worms. It is important to emphasize that such dose (32.9%) did not cause significant lethal effects under an acute exposure. The observation of higher levels of toxicity in ferralsols is probably again related to the influence of soil properties (especially clay minerals, fertility, pH and plasticity) in the bioavailability mechanisms and behavior of the earthworms into the soil. However, Natal-da-Luz et al. (2004) emphasize the importance of further detailed investigations concerning the study of the influence of soils properties (texture, field capacity, pH, organic matter, fertility, clay minerals, and electric conductivity) on the avoidance responses. Such specific studies do not exist for tropical soils yet.

The results also suggest that avoidance responses may be used as a very useful screening tool for monitoring the toxicity and bioavailability of contaminants in sewage sludge-amended soils. In fact, avoidances responses were more sensible indicators in comparison with mortality levels. The avoidance tests are simple assays and can be performed in a short period of time. Furthermore, the behavioral effects (avoidance responses) seem to be very sensible to the presence of toxic agents in soils treated with sewage sludge. Natal-da-Luz et al. (2004) and Hund-Rinke and Wiechering (2001) also demonstrated the feasibility of avoidance assays with earthworms as screening tools for evaluating heavy metals contamination in soils from abandoned gold mining sites.

Acute toxicity tests with contact paper

The results are in agreement with the other tests conducted with natural soils, suggesting that soil properties played a vital role in the toxicity. In the case of those soils, clay minerals and pH seem to be important parameters affecting the transference of metals to the aqueous phase (elutriates). More acid values of pH in ferralsols can favor the fractionation of metals to the dissolved fraction, increasing the bioavailability through dermal contact. Expansive clay minerals in the chernosol can play a crucial role in the reduction of the contaminants’ concentrations in the soil solution, thus decreasing their bioavailability and lethality levels.

Toxicity tests with contact paper using earthworms are traditionally performed with solutions of chemical substances. Roberts and Dorough 1984 apud (Miyasaki et al. 2002), e. g., studied the ecotoxicity of 90 chemical substances using this simple acute toxicity test. However, Miyasaki et al. (2002) report that those tests do not take in consideration soil properties when they are performed with solutions of chemical substances. On the other hand, this test can offer a good estimation of the content of toxic substances/elements in the soil solution (geochemical soluble fraction) able to cause adverse effects through dermal contact. In fact, dermal contact is one of the important pathways of metal exposure for the earthworms (Vijver et al. 2003).

In general, elutriates are used for evaluating toxicity of soils on aquatic organisms (Baun et al. 2002), in hypothetic situations where soil leaching and erosion processes could mobilize contaminants to surrounding fluvial systems. Elutriates are also commonly used for evaluating the ecotoxicity of water and aquatic sediment samples (Hall et al. 1996). The results indicated that the use of elutriates in tests with contact paper were able to demonstrate the influence of pedogeochemical aspects in the bioavailability processes. Thus, elutriates could be widely used in preliminary assessments of toxicity levels of sewage sludge-amended tropical soils. Those procedures are simple, require low amounts of soil and can be executed in a simple way and in a shorter period of time.

Conclusions

Soil properties played a vital role in the toxicity and bioavailability of metals for earthworms. Sewage sludge application caused more toxic effects in ferralsols than in chernosols. In this case, clay minerals, pH, fertility and plasticity greatly strongly influenced such mechanisms, in terms of liberation of metals to soil solution, behavior and immunological resistance of the earthworms. Therefore, the generation of toxic reference values of sewage sludge should be strongly based on physiological aspects and their relationships with soil properties and type of contaminant.

Earthworms were able to incorporate very high amounts of lead and chromium. Thus, those metals possibly played a very important role in the toxicity and should be carefully monitored. Avoidance tests with earthworms can be used as screening tools for evaluating the bioavailability of contaminants in sewage sludge-amended soils, since the responses seem to be very sensible to heavy metals contamination. In future investigations, such effects will be more detailed by quantifying biomarkers of metal exposure (metallothioneins).

Environmental standard values for sewage sludge application in tropical regions generally do not take in consideration their toxicological aspects and the use of bioassays with organisms of soil fauna as a monitoring tool of environmental quality. Such data can support the future establishment of sustainable rates of sewage sludge for the tropical agriculture and recuperation of degraded soils, as well as ecological risk assessments in areas treated with sewage sludge.