The ecotoxicological data gathered from studies conducted in temperate climate soils are extrapolated to tropical and subtropical climate zones. Nevertheless, this decision sometimes proves to be inappropriate since the differences in climate conditions originate soils with contrasting characteristics. In this sense, the results presented here tend to overcome this constraint and, in an original way, clarify the Cu dose-effect relationship in natural subtropical Brazilian soils.
The assays carried out on natural soils with different chemical properties, as well as on artificial soil, indicated that the organisms were able to respond to the contamination in different ways, with the adverse effects being associated with greater bioavailability of Cu. According to Kabata-Pendias (2010), the bioavailability of trace elements is a result of the soil's adsorptive capacity, which is directly related to CEC, pH and clay content. In fact, Cambisol had lower clay content, CEC, and pH levels when compared to Nitisol. This finding is in line with the results of Duan et al. (2016), who reported that Cu toxicity in E. fetida is inversely associated with CEC and pH level. These attributes also tend to result in greater damage to other faunal species, given the dynamics of the contaminant (Natal-da-Luz; Römbke; Sousa, 2008). Furthermore, despite Cambisol having a lower CEC and clay content, the organisms were more susceptible to Cu contamination in TAS than in Cambisol. This observation suggests that other factors, such as the presence of Al (which is a toxic element to soil organisms) may influence the organisms vulnerability. These findings confirm the first hypothesis upon which this evaluation was built.
The results of the ecotoxicological assessments showed that Cu was able to reduce the reproduction of all organisms at distinct levels. The species most sensitive to the contaminant was the earthworm P. excavatus, followed by the enchytraeid E. crypticus (for the evaluations in TAS and Nitisol) and the earthworm E. andrei (in Cambisol), all oligochaetes with similar trophic habits and forms of exposure to the contaminant. On the other hand, the springtails were less susceptible to Cu exposure. These differences illustrate a scenario that emphasizes the importance of carrying out assessments with organisms from different taxonomic groups, especially when determining critical concentration limits for one or more contaminants in the soil.
In accordance with our findings, Mirmonsef et al. (2017), when assessing the effects of Cu on the reproduction of eight earthworm species collected in a contaminated area, found that the abundance of cocoons decreased as the concentration of the metal increased, thereby indicating a harmful effect on the reproduction of the organisms. Similarly, Maboeta and Fouché (2014), in a study aiming to determine the Cu toxicity in the species E. andrei, found a significant reduction in oviposition and cocoon hatching rates. Kwak and An (2021), in a 7-day assessment, were able to identify physiological abnormalities, such as mucous secretion, bleeding and swelling, for doses above 400 and 600 mg of Cu kg− 1 of dry soil in adults of P. excavatus and E. andrei respectively. Regarding the variation in LOEC which we have verified here between the two earthworm species evaluated, the results suggest that the P. excavatus species is more sensitive. This observation corroborates the findings of Spurgeon and Hopkin (1996), who, in an evaluation of ecologically relevant species, concluded that the Eisenia genus is the one with the greatest resistance to soil contamination by trace elements. Given the different responses, the importance of using native species in ecotoxicological assessments is therefore emphasized. This is because the use of tropical/subtropical species, such as P. excavatus (Silva & van Gestel, 2009), can better represent ecological risk analysis in Brazilian soils.
In terms of the damaging effects of Cu on enchytraeids, and in accordance with the results of this evaluation, Maraldo et al. (2006), when assessing the survival of E. crypticus in Danish soils contaminated with Cu, found a reduction in the survival of this organism at concentrations of 600 mg kg− 1. However, there have been few publications to this date on the effect of Cu on the reproduction of enchytraeids in tropical and subtropical soils. Nevertheless, Konečný et al. (2014), in a study assessing the influence of trace elements on E. crypticus, observed that the reproduction of the animals shows a negative correlation with the concentrations of Co and Cu, the latter being the most environmentally relevant contaminant, with its EC50 determined for the species at 351 mg kg− 1. These findings support the need to determine VP for Cu in different soils, given its effects on the reproductive capacity of organisms and the subsequent impact on the ecological functions of important biological groups.
The Cu exposure also had a negative effect on the springtails, although these were the organisms whose results indicate greater resistance to the effects of the metal. According to Renaud et al. (2020), the toxicity of trace elements in F. candida tends to be directly related to the solubility of the cations in the soil. In this sense, it should be noted that the changes followed the same pattern as for earthworms and enchytraeids, i.e. the effects in Cambisol were more significant than in the TAS and Nitisol. In contrast to the possibilities here suggested to justify the greater sensibility of the organisms in Cambisol, Sandifer and Hopkin (1996) concluded that there was no clear correlation between the increase in toxicity as a function of the reduction of pH when evaluations were carried out on F. candida. However, it should be considered that the assays conducted by the authors were carried out on OECD artificial soil (OECD, 1984), where the organic matter content (in the order of 10%) is too high, given that the great majority of tropical soils rarely exceed 5% (Amorim et al., 2005). According to Fountain and Hopkin (2001), although the species feeds on organic matter and is an important fragmenting agent, whose relevance to the provision of ecosystem services is of the utmost importance, it is highly tolerant to food contamination. Therefore, considering that the Cambisol had the highest levels of organic matter and that the most significant effects were recorded in this soil, it can be assumed that the factor with the greatest impact on Cu toxicity for these species was likely to have been CEC and pH.
Another outstanding aspect of this investigation, and in line with what was observed by Buch et al. (2016) when conducting assessments on the toxicity of Hg in tropical soils, was the greater sensitivity recorded for F. candida when compared to P. minuta. In this sense, it should be noted that this difference in responses to environmental stress justifies the need to include more than one species into de same group of organisms to ecotoxicological assessments; this is because, as pointed out by Salmon et al. (2014), springtails tend to show different interspecific responses due to the different habitats and trophic niches they occupy. Moreover, the greater tolerance of certain species may be related to their morphological characteristics, which reflect their ability to absorb, eliminate and immobilize metals (Janssens; Roelofs; van Straalen, 2009). Finally, it should be added that the summary choice of bioindicators should be based on their sensitivity to the stressor, considering the mechanisms of toxic action and exposure to the contaminant. Thus, to endorse the use of P. minuta in the current evaluation, it is worth noting that the species shows reasonable sensitivity to Cu (Nursita; Singh; Lees, 2005), despite its lower sensitivity when compared to F. candida.
The process of drawing up the SSD made it possible to assess the organisms' sensitivity to Cu by determining risk concentrations (i.e. HC5). In short, the graphical observation of adverse effects provides a better understanding of the results as it also represents a versatile approach to the development of practices and policies for the prevention, monitoring and mitigation of environmental degradation (Posthuma et al., 2019). Several studies have aimed to develop retrospective and predictive analysis of the effect of contaminants in Ecological Risk Assessment (ERA) and have employed the modeling of such curves, arguing positively for the use of the method (Princz et al., 2017; Posthuma et al., 2019; Bandeira et al., 2021). As stated before, the organisms were more susceptible to contamination in Cambisol, TAS and Nitisol, respectively, implying that the PV differed depending on the soil type. The decision to obtain PV for the TAS was taken in order to clarify which chemical characteristics of the soils, other than their clay content, have the greatest influence on copper toxicity, since Cambisol and TAS have similar textural compositions. The Al content and the greater amount of nutrients seemed to attenuate the effects of contamination in TAS.
The results presented here are pioneering in terms of the construction of the SSD and the concomitant derivation of PVs for subtropical soils. In this sense, it should be noted that in 2021, the State of Santa Catarina published its first quality reference values (QRV) in compliance with the Brazilian legislation. However, the QRV for Cu in Santa Catarina went beyond the PV present in the federal regulatory framework (Brasil, 2009; IMA, 2021); therefore, the results presented here tend to improve the ERA and make a unique contribution to the conservation of ecosystems in the Atlantic Forest Biome. The decision to base the PV estimation on the EC50 was driven by the need to safeguard ecosystems without being strictly restrictive, since the PV were estimated according to the HC5. However, if the amount of protection has to be greater, further assessments should be conducted so that the SSDs are generated with data from higher protective effect concentrations (ECx), such as EC20.
That said, it is worth noting that the results showed that the PV based on the EC50 data for Cu were higher than the stipulated in the CONAMA Resolution (Brasil, 2009), thus indicating the effectiveness of the legislation in protecting soil fauna organisms and refuting the second hypothesis here suggested. However, further assessments are required before assuming the efficacy of the national legislation since soils tend to have different capacities for retaining and making Cu bioavailable. The recently published work by Messias, Alves and Cardoso (2023) reinforce the discussion of the need to update the VPs for Cu, since the authors found adverse effects at concentrations below the limit established in the federal regulations. In this way, although it can be inferred that the VP set out in the CONAMA Resolution is protective for the soils evaluated, it cannot be concluded beyond the results presented here, given the influence of the chemical characteristics of the soils on the environmental dynamics of Cu.