Prevention Values for Copper (Low Tier Approach) in Subtropical Acidic Soils

Prevention Values for Copper (Low Tier Approach) in Subtropical Acidic Soils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prevention Values for Copper (Low Tier Approach) in Subtropical Acidic Soils Daniela Aparecida de Oliveira, Thiago Ramos Freitas, Vanessa Mignon Dalla Rosa, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4485276/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Oct, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract Copper is a trace element in plants and animals whose importance can be understood due to its role in different essential metabolic processes. Anthropogenic activities such as agriculture and mining are potential sources of pollution due to the emission of copper into the environment. Brazilian legislation ties soil quality to guideline values, among which the Prevention Value indicates the critical environmental limit for trace elements. The aim of this study was to obtain PVs for copper for two subtropical soils (Cambisol and Nitisol), given that the pedological richness was not considered when deriving the PVs contained in the federal normative. Reproduction assays followed ISO guidelines with the earthworm species Eisenia andrei and Perionyx excavatus , the enchytraeids Enchytraeus crypticus and E. bigeminus and the springtails Folsomia candida and Proisotoma minuta . Results showed that the sensitivity of the organisms was greater in Cambisol. The most sensitive species were the earthworms, especially P. excavatus (EC 50 = 67.83 in Cambisol; EC 50 = 264.96 in Nitisol). The springtails, on the other hand, were the least sensitive to contamination. These findings reinforce the need to include organisms from different ecological groups in ecotoxicological assessments. It was also observed that the PV adopted in federal legislation (= 60 mg kg -1 ) is in fact protective for the species and soils we evaluated, since the PVs we obtained based on the EC 50 were 346.74 mg kg -1 in Nitisol and 134.05 mg kg -1 in Cambisol. It is important to note that our results do not exclude the need for evaluations with other subtropical soils, given the influence of their properties on the toxicity and bioavailability of copper to soil organisms. Ecotoxicology Soil Screening Values Trace Metals Soil Contamination Earthworms Enchytraeid Springtail. Figures Figure 1 1 Introduction Among the main contaminants of environmental interest, metals require some attention due to their metabolic involvement, because, although essential, in excess they can result in damage to organisms (Witkowska; Stowik; Chilicka, 2021). Copper (Cu) is a micronutrient for plants and animals that is naturally present in the soil due to the weathering processes taking place on the parent rock (Kabata-Pendias, 2010). However, the development of human activities, such as agricultural practices, has contributed to an increase in Cu concentrations in the environment, which has the potential to undermine the supply of environmental services because of contamination on key species' ecological dynamics. The group of invertebrates performs ecosystem functions that are extremely important for maintaining life in the soil, with their activities linked to the processes of organic matter and nutrient cycling, soil structuring and bioregulation (Decaëns et al., 2006 ; Briones, 2018 ; Heděnec et al., 2022 ). Therefore, it is essential to pay particular attention to the effects of Cu contamination on these organisms to preserve environmental quality. According to Sereni, Guenet, and Lamy ( 2021 ), soil fauna in Cu-contaminated sites exhibit morphological and behavioral alterations that can lead to trophic imbalance. Understanding the effects of this contamination has become essential for protecting biodiversity and ecosystem services connected with soil. Although the topic has been extensively researched in northern hemisphere soils (Spurgeon & Hopkin, 1996 ; Lukkari et al., 2005 ; Kupperman et al., 2006; Maraldo et al., 2006 ; Amorim & Scott-Fordsmand, 2012 ; Renaud et al., 2020 ), there are few reports on the effects of Cu in tropical and subtropical soils. The Brazilian federal legislation, in the form of Resolution 420/2009 of the National Environment Council (CONAMA), ties soil and groundwater quality to guideline values. Among these, the prevention value (PV) refers to the concentrations above which it can be inferred that the soil is polluted by a certain element or substance (Brasil, 2009 ). Due to the great pedological diversity, it is necessary to determine specific PVs for each of the states. The PVs currently adopted at the national level are those obtained only from soil analysis in the state of São Paulo, making it difficult to identify and manage contaminated areas in other states. The obtaining procedure of PV is closely linked to analyzing the effects of the exposure on key organisms such as arthropods and oligochaetes. These assessments are fundamental to understanding the effects of contamination and, as a result, to establishing effective prevention and management guidelines. The European Chemicals Agency (ECHA) recommends that such evaluations must be carried out with species representative of the different trophic levels (ECHA, 2008 ). The main methods commonly used for this purpose are described by the International Organization for Standardization (ISO) guidelines and aim to measure the dose-response relationship in parameters such as organism survival (lethality) and reproduction capacities when exposed to contaminated soils (van Gestel, 2012 ). Considering this scenario, the aim of the present research was to derive PVs for Cu for two subtropical representative soils of South Brazil through reproduction assessments with the earthworm species Eisenia andrei and Perionyx excavates , the enchytraeids Enchytraeus crypticus and E. bigeminus , and the springtails Folsomia candida and Proisotoma minuta . To attain these objectives, we hypothesized that (i) the effects of contamination measured on the reproduction of the organisms evaluated vary from soil type, and (ii) the PVs adopted at the national level are not fitting for the subtropical soils tested. The methodology employed was to produce species sensitivity distribution curves (SSDs) and estimate risk concentrations (HC 5 and HC50) based on the effect concentrations (EC 50 ) obtained in the exposure tests. 2 Materials and methods 2.1 Soils sampling and characterization Two natural soils representative of the subtropical region of Brazil and a tropical artificial soil (TAS) were used in the ecotoxicological assessments: an Cambisol (Cambissolo Húmico (Santos, 2018 )) was collected in Lages, SC [27º48'57''S; 50º21'45''W] and a Nitisol (Nitossolo Vermelho (Santos, 2018 )) was collected in Concórdia, SC [27º48'71''S; 51º59'34''W]. The soil samples were collected in the surface layer (0–20 cm) in areas under natural, unmanaged vegetation, free from fertilization and the use of agrochemicals. The TAS was made following the proportions proposed by Garcia (2004), i.e. 75% fine sand, 20% kaolinitic clay and 5% dried and sieved coconut fiber. The TAS' pH was corrected to 6.0 ± 0.5 by adding calcium carbonate (CaCO 3 ) (ISO, 2012). The characteristics of the soils were determined according to the procedures suggested by EMBRAPA (Teixeira, 2017 ) and are shown in Table 1 . Table 1 Chemical and physical properties of Nitisol, Cambisol, and TAS. Soil properties Nitisol Cambisol TAS Organic matter (g kg -1 ) 42 52 22 CEC 1 12.5 6.5 1.2 pH 2 5.8 4.7 6.2 Ca (cmol c dm -3 ) 11.7 2.6 2.0 Mg (cmolc dm -3 ) 2.5 1.4 0.9 P (mg dm -3 ) 47.1 6.9 11.1 K (mg dm -3 ) 162 52 262 Al (cmol c dm -3 ) 0.2 3.0 0.0 Cu (mg dm -3 ) 10.6 2.8 0.2 Zn (mg dm -3 ) 29.0 3.8 0.7 B (mg dm -3 ) 0.65 0.67 1.38 Mn (mg dm -3 ) 159 21.1 2.2 Clay (g kg -1 ) 543.7 133.7 94.9 Silt (g kg -1 ) 272.7 115.7 108.7 Sand (g kg -1 ) 183.5 750.5 796.4 Texture Clay Sandy Loam Sandy Loam 1 Cation Exchange Capacity at pH 7.0. 2 pH in water 2.2 Copper spiking procedure The soils were spiked with a Cu(NO 3 ) 2 solution (Table 2 ), following a three weeks incubation (Natal-da-Luz et al., 2011 ). Prior to the testing procedure, the moisture content was corrected to 50% of the soil's water holding capacity (WHC) in accordance with the ISO 11268-2 protocol (ISO, 2012). After the spiking procedure, soil samples were used in ecotoxicity assays. Soil Cu contents were evaluated through X-ray fluorescence spectrometry in accordance with the USEPA 3051A method (USEPA, 1999 ), as required by CONAMA Resolution n. 420/2009 (Brasil, 2009 ). The Standard Reference Material was a 2709a San Joaquin Soil. Table 2 Tested nominal concentrations of copper for each ecotoxicological assay depending on the soil type. Soil Organisms and copper nominal concentrations (mg kg − 1 ) Earthworms ( E. andrei and P. excavatus ) Nitisol and TAS 0, 100, 200, 400, 800, 1200, 1600 and 2000 Cambisol 0, 50, 100, 200, 300, 500, 700 and 1000 Enchytraeids ( E. crypticus and E. bigeminus ) Nitisol 0, 75, 150, 300, 450, 600, 750 and 1000 Cambisol and TAS 0, 25, 50, 75, 150, 250, 500 and 750 Springtails ( F. candida and P. minuta ) Nitisol, Cambisol and TAS 0, 75, 150, 300, 450, 600, 750 and 1000 2.3 Test organisms The earthworms were cultivated in a substrate consisting of equine manure (chemical-free), coconut husk fiber and fine sand (in the proportion of 7:2:1). The pH of the mixture was corrected to 5.7 ± 0.3 range with CaCO 3 . The organisms were fed weekly with oat flakes. The species Eisenia andrei was kept at 20 ± 2,0 ºC room and the Perionyx excavatus species at a 25 ± 2,0 ºC. The photoperiod for both was kept in 16 hours light. The enchytraeids were grown in TAS (Garcia, 2004) according to the ISO 16387 protocol (ISO, 2014a). The springtails were cultivated in a substrate composed of plaster, distilled water and activated charcoal (in a ratio of 11:7:1) maintained at a temperature of 20 ± 2.0 ºC and at a photoperiod of 16 hours light. 2.4 Experimental procedures The assays with the earthworm species E. andrei and P. excavatus followed the guidelines established in the ISO 11268-2 protocol (ISO, 2012). The experimental units (n = 5) consisted of containers filled with around 500 g of dry soil and in the presence of ten clitellate individuals weighing between 250 and 600 mg. The organisms were fed weekly with approximately 15 g of horse manure (free from pollutants) and the moisture was kept at 50% of WHC. After 28 days, the adults were removed, and the cocoons and juveniles were incubated for a further 28 days. At the end of the 56 days, the organisms were extracted using a temperature gradient in a laboratory water bath (60 ºC). The evaluations with enchytraeids were carried out according to the guidelines established in ISO 16387 (ISO, 2014a) with adjustments proposed by (Kuperman et al., 2006 ), and the assay was conducted for four weeks (28 days) for E. crypticus and three weeks (21 days) for E. bigeminus (Bandow et al., 2013 ). The criterion for selecting adult individuals for E. crypticus was the presence of a clitellum; for E. bigeminus , organisms were chosen according to their body size (8 to 12 mm), since this species is a fragmented reproducer (Bandow et al., 2013 ). Ten organisms were placed in containers with 30 g of soil (n = 4) and fed weekly with around 2 mg of rolled oats and the moisture content was kept at 50% of WHC. At the end of the test, 5 mL of pure alcohol, 1 mL of rose bengal dye solution (1% w/v) and 80 mL of water were added to each experimental unit for subsequent counting of the organisms. The springtails' assessments lasted 28 days and were carried out according to the procedures listed under ISO standard 11267 (ISO, 2014b). Ten organisms with 12 days old were kept in containers with 30 g of soil (n = 5). Food was added once a week and consisted of around 2 mg of dry biological yeast and the moisture content was kept at 50% of the WHC. At the end of the evaluation period, for the F. candida juveniles, each experimental unit was filled with about 80 mL of water and a few drops of black stamp ink (to increase the contrast between the organisms and the surface of the water). After gentle stirring, the number of adult and juvenile individuals was determined using ImageJ 1.54d software (NHI, 2023). To determine the reproduction rate of the P. minuta species, the method used was dry extraction in Berlese-Tüllgren funnels for a period of 24 hours with subsequent addition of water and red dye. This adaptation was necessary due to the smaller size and color of the species' exoskeleton (Hopkin, 1997 ; Mendonça; Queiroz; Silveira, 2015 ). Afterwards, the samples containing the organisms were carefully shaken and the number of individuals determined using ImageJ 1.54d software (NHI, 2023). 2.5 Data analysis The ecotoxicological data were subjected to the Shapiro-Wilk normality test (p > 0.05) and Bartlett's homogeneity test (p > 0.05) and the means were compared using the Dunnett test (p ≤ 0.05). The data was then submitted to a non-linear regression analysis to determine the dose of contaminant corresponding to a 50% effect on the population of organisms (EC 50 ). The EC 50 values were determined using the model that best fitted the data (Environment Canada, 2005 ). All analyses were carried out using STATISTICA 10.0 software (StatSoft, 2011). Using ETX 2.0 software (van Vlaardingen et al., 2004), sensitivity curves were developed for the species tested (SSDs). The SSDs were based on the EC 50 values for each organism in each soil evaluated (Aldenberg & Jaworska, 2000 ). The SSDs were employed to estimate the concentration of the contaminant capable of posing a risk to 5 and 50% of the species (HC 5 and HC 50 ), with a 95% confidence interval. Since the model assumes a log-normal distribution of the data, log-normality was tested using the Anderson-Darling, Kolmogorov-Smirnov and Cramer von Mises tests (p < 0.01). The PVs were obtained after the HC 5 had been calculated considering the Quality Reference Values (QRV, = 93.84 mg kg − 1 ) for soils in the state of Santa Catarina (IMA, 2021 ), according to the equation PV = QRV + HC5. 3 Results 3.1 Test validation The validation criteria were all met for the species E. andrei , P. excavatus , E. crypticus , F. candida and P. minuta (ISO, 2012; ISO, 2014a; ISO, 2014b). For the assays with the species E. bigeminus , the criterion adopted was the occurrence of 25 juveniles in the control groups with a coefficient of variation lower than 50% (Bandow et al., 2013 ). The choice of a specific criterion is justified due to the impossibility of identifying clitellate adult individuals as it is a fragmenting species. 3.2 Ecotoxicological assays The reproduction of the earthworm species E. andrei and P. excavatus was affected (p ≤ 0.05) in all the soils that were evaluated (Figure S1). Reductions in the number of E. andrei juveniles were observed starting at doses of 50 mg kg − 1 in Cambisol, 100 mg kg − 1 in TAS and 200 mg kg − 1 in Nitisol. For the species P. excavatus , the effects were observed at the lowest concentrations evaluated (50 mg kg − 1 in Cambisol and 100 mg kg − 1 in TAS and Nitisol). The most significant effect for both species was found in the Cambisol, since the EC 50 values for this soil (77.52 mg kg − 1 for E. andrei and 67.83 mg kg − 1 for P. excavatus) were lower than those found in Nitisol (428.01 mg kg − 1 for E. andrei and 264.96 mg kg − 1 for P. excavatus ) and TAS (173.81 mg kg − 1 for E. andrei and 110.46 mg kg − 1 for P. excavatus ). All the enchytraeid species evaluated were also affected (p ≤ 0.05) by soil contamination with Cu (Figure S2). The reduction in the number of juveniles of E. crypticus occurred from the concentration of 25 mg kg − 1 in TAS, 50 mg kg − 1 in Cambisol and 150 mg kg − 1 in Nitisol. For the species E. bigeminus , the effects were verified starting at a concentration of 250 mg kg − 1 in the TAS, 50 mg kg − 1 in the Cambisol and 75 mg kg − 1 in the Nitisol. As observed for the earthworms, the most significant effects for the enchytraeids were also observed in the Cambisol, since the EC 50 values obtained were lower than those found in the Nitisol and TAS. The springtails F. candida and P. minuta also had their reproduction affected (p ≤ 0.05) in all the soils evaluated (Figure S3). The reduction in the number of juveniles of the species F. candida was observed from the concentration of 75 mg kg − 1 in Nitisol and 300 mg kg − 1 in Cambisol and TAS. For the P. minuta species evaluations, the significant effects on reproduction occurred from the concentrations of 75 mg kg − 1 in Cambisol, 150 mg kg − 1 in Nitisol and 450 mg kg − 1 in TAS. The no effect concentration (NOEC), lowest effect concentration (LOEC) and EC 50 for each assessment are presented in Table 3 . In general, it can also be said that the earthworm species P. excavatus is the most sensitive of all the species evaluated, whereas the springtail P. minuta was the least affected by the contamination, independent of soil type. Table 3 Ecotoxicological endpoints (NOEC, LOEC and EC 50 ) for each evaluated species in Nitisol, Cambisol e TAS. NOEC (mg kg − 1 ) LOEC (mg kg − 1 ) EC 50 (mg kg − 1 ) Nitisol E. andrei 100 200 428.01 (300.58-555.44) P. excavatus < 100 100 264.96 (184.08-345.15) E. crypticus 75 150 366.61 (328.56-404.67) E. bigeminus < 75 75 444.71 (303.99-585.44) F. candida < 75 75 456.22 (305.94-606.51) P. minuta 75 150 636.00 (597.15-674.86) Cambisol E. andrei < 50 50 77.52 (66.16–88.87) P. excavatus < 50 50 67.83 (54.95–80.72) E. crypticus 25 50 142.25 (109.50–175.00) E. bigeminus 25 50 126.77 (56.30-197.24) F. candida 150 300 255.03 (223.52-286.53) P. minuta < 75 75 513.17 (453.78-572.56) TAS E. andrei < 100 100 173.81 (148.99-198.63) P. excavatus < 100 100 110.46 (73.82–137.10) E. crypticus < 25 25 171.71 (124.79-218.63) E. bigeminus 150 250 213.70 (181.36-246.03) F. candida 150 300 333.38 (297.35-369.42) P. minuta 300 450 578.74 (512.03-645.45) In parenthesis the confidence limits (lower and upper) are presented. 3.3 Chemical evaluations The data from the analysis of the Cu levels in the natural soils (Table 4 ) indicated an average percentage recovery (in relation to the nominal dose) of 83.8% in Nitisol and 86% in Cambisol, while in TAS the recovery was around 92%. For this reason, it was decided to employ the nominal doses in the construction of the SSD and the subsequent derivation of the PV. Table 4 Nominal concentration (mg kg − 1 ) and chemical measured concentration (mg kg − 1 ) of copper in subtropical natural soils and TAS. Nominal Real Nitisol Cambisol TAS 25 nd 16.7 20.9 50 nd 33.8 41.9 75 48.9 58.3 63.4 100 65.05 77.8 84.5 150 122.8 129.0 136.4 200 170.5 172.0 188.2 250 nd nd 290.1 300 260.9 261.0 nd 400 326.5 nd 384.0 450 375.2 429.1 nd 500 nd 477.5 422.6 600 539.4 551.0 nd 700 nd 668.5 nd 750 647.3 716.3 743.4 800 719.2 nd 792.8 1000 889.0 955.0 nd 1200 1078.8 nd 1185.6 1600 1438.5 nd 1586.1 2000 1798.1 nd 1982.4 nd: not determined once the nominal concentration was not evaluated in the ecotoxicological assay. 3.4 Species Sensibility Distribution and prevention values Once it was possible to assess the EC 50 for each of the tested organisms, the SSD were generated and the HC 5 and HC 50 were obtained for all the tested soils (Fig. 1 ). The highest HC 5 values were recorded in Nitisol (252.9 mg kg − 1 ), followed by TAS (76.12 mg kg − 1 ) and Cambisol (40.21 mg kg − 1 ). It can therefore be inferred that Cu contamination is more problematic in Cambisol, since the concentration capable of endangering the organisms is approximately six times lower than in Nitisol. Finally, based on the Cu QRV (93.84 mg kg − 1 ) published for Santa Catarina state soils (IMA, 2021 ), it was possible to estimate the PV via the HC 5 data (Table 5 ). We chose not to utilize HC 50 for this purpose because we intended to propose PVs that are ecologically safer. Table 5 Hazardous concentration for 5% of the species and prevention values for Cu based on EC 50 data for each evaluated soil. Soil LL HC 5 HC 5 UL HC 5 PV Nitisol 144.1 252.9 325.40 346.74 Cambisol 9.08 40.21 78.24 134.05 TAS 26.17 81.82 136.24 175.66 LL HC 5 : lowest limit for the calculated HC 5 (p ≤ 0,05); HC 5 : hazardous concentration for 5% of the species (p ≤ 0,05) based on the EC 50 values; UL HC 5 : upper limit for the calculated HC 5 (p ≤ 0,05); PV: prevention values (PV = QRV + HC 5 ). 4 Discussions 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 EC 50 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. HC 5 ). 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 EC 50 was driven by the need to safeguard ecosystems without being strictly restrictive, since the PV were estimated according to the HC 5 . 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 EC 20 . That said, it is worth noting that the results showed that the PV based on the EC 50 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. 5 Conclusions Based on the comparison between the results and the current Brazilian legislation, it can be assumed that the guideline values (i.e. PV) for Cu are protective for the different bioindicators in the soils evaluated. It is important nothing that the PV we derive were based on the concentration capable of inhibit in 50% the reproduction of the organisms. It was observed that the effects vary among soil types, likely as a result of their chemical properties, including pH and clay content. Hence, evaluations with other representative soil types from subtropical climate regions are required before it can be inferred that the PV included in the federal legislation is in fact protective to subtropical terrestrial ecosystems. The data here presented tends to support other similar investigations to build an ecotoxicological database that will allow a better understanding of the effects of trace element pollution on the biotic constituents of different Brazilian ecosystems. This represents a major step forward in the construction of public policies focused on environmental conservation, given that the organisms evaluated significantly play an important role in providing ecosystem services. Statements and Declarations Funding: This study was funded by thefinancial support from Santa Catarina State Research and Innovation Support Foundation (FAPESC, Grant Number: 2023TR000733 ). V.M.D.R. and T.R.F are grateful for the scholarship granted by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). Competing Interest: The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors Contribution: Daniela Aparecida de Oliveira: Investigation, Formal analysis, Writing – Original Draft, Validation, Visualization. Thiago Ramos Freitas: Formal analysis, Writing – Original Draft, Validation, Visualization. Vanessa Mignon Dalla Rosa: Formal analysis, Writing – Original Draft, Validation, Visualization. Luís Carlos Iuñes de Oliveira Filho : Supervision, Visualization, Validation, Writing – Review & Editing. Mari Lucia Campos: Conceptualization, Supervision, Writing – Review & Editing, Validation. Milton da Veiga: Conceptualization, Writing – Review & Editing. David José Miquelluti: Conceptualization, Supervision, Writing – Review & Editing,Validation, Project administration,Funding acquisition. Osmar Klauberg-Filho: Conceptualization, Supervision, Writing – Review & Editing, Validation, Project administration, Funding acquisition. Data Availability Statement: Datasets related to this article can be made available upon request to the corresponding author Osmar Klauberg-Filho ( [email protected] ) Ethical Approval: Not applicable. Consent to Participate: Not applicable. Consent to Publish: Not applicable. References Aldenberg T, & Jaworska J (2000). Uncertainty of the hazardous concentration and fraction affected for normal species sensitivity distributions. Ecotoxicology and Environmental Safety . https://doi.org/10.1006/eesa.1999.1869 Amorim MJB, Römbke J, Schallnaß HJ, Soares AMVM (2005) Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus , and Folsomia candida . Environmental Toxicology and Chemistry . https://doi.org/10.1897/04-505r.1 Amorim MJB, Scott-Fordsmand JJ. (2012). Toxicity of copper nanoparticles and CuCl 2 salt to Enchytraeus albidus worms: survival, reproduction and avoidance responses. Environmental Pollution . https://doi.org/10.1016/j.envpol.2012.01.015 Bandeira FO, Alves PRL, Hennig TB, Brancalione J, Nogueira DJ, Matias WG (2021). Chronic effects of clothianidin to non-target soil invertebrates: Ecological risk assessment using the species sensitivity distribution (SSD) approach. Journal of Hazardous Materials . https://doi.org/10.1016/j.jhazmat.2021.126491 Bandow C, Coors A, Römbke J (2013). 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In: 25th Annual Meeting of Society of Environmental Toxicology and Chemistry (SETAC) http://abstracts.co.allenpress.com/pweb/setac2004/document/?id=41943 Heděnec P, Jiménez JJ, Moradi J, Domene X, Hackenberger DK, Barot S, Frossard A, Oktaba L, Filser J, Kindlmann P, Frouz J (2022). Global distribution of soil fauna functional groups and their estimated litter consumption across biomes. Scientific Reports . https://doi.org/10.1038/s41598-022-21563-z Hopkin SP (1997). Biology of the Springtails. Oxford University Press EBook s , Oxford. https://doi.org/10.1093/oso/9780198540847.001.0001 IMA (2021). Instrução Normativa nº 45 : valores orientadores de qualidade dos solos e águas subterrâneas de Santa Catarina . ISO. International Organization for Standardization. (2012). Guideline 11268-2 . ISO. International Organization for Standardization. (2014a) . Guideline 16387 . ISO. International Organization for Standardization. (2014b) Guideline 11267 . Van Straalen NM, Janssens TKS, Roelofs D (2011). Micro-evolution of toxicant tolerance: from single genes to the genome’s tangled bank. Ecotoxicology . https://doi.org/10.1007/s10646-011-0631-3 Konečný L, Ettler V, Kristiansen SM, Amorim MJB, Kříbek B, Mihaljevič M, Šebek O, Nyambe I, Scott-Fordsmand JJ (2014). Response of Enchytraeus crypticus worms to high metal levels in tropical soils polluted by copper smelting. Journal of Geochemical Exploration . https://doi.org/10.1016/j.gexplo.2013.10.004 Kuperman RG, Amorim MJB, Römbke J, Lanno R, Checkai RT, Dodard SG, Sunahara GI, Scheffczyk A (2006). Adaptation of the enchytraeid toxicity test for use with natural soil types. European Journal of Soil Biology . https://doi.org/10.1016/j.ejsobi.2006.07.028 Kwak JI, An YJ (2021). Microplastic digestion generates fragmented nanoplastics in soils and damages earthworm spermatogenesis and coelomocyte viability. Journal of Hazardous Materials . https://doi.org/10.1016/j.jhazmat.2020.124034. Lukkari T, Aatsinki M, Väisänen A, Haimi J (2005). Toxicity of copper and zinc assessed with three different earthworm tests. Applied Soil Ecology . https://doi.org/10.1016/j.apsoil.2005.02.001 Maboeta M, Fouché T (2014). Utilizing an earthworm bioassay ( Eisenia andrei ) to assess a south african soil screening value with regards to effects from a copper manufacturing industry. Bulletin of Environmental Contamination and Toxicology . https://doi.org/10.1007/s00128-014-1302-x Maraldo K, Christensen B, Strandberg B, Holmstrup M (2006). Effects of copper on enchytraeids in the field under differing soil moisture regimes. Environmental Toxicology and Chemistry . https://doi.org/10.1897/05-076r.1 Mendonça MC, Queiroz GC, Silveira TC (2015). Two new species of Proisotoma Börner, 1901 from Southeastern Brazil (Collembola: Isotomidae). Soil Organisms . https://soil-organisms.org/index.php/SO/article/view/407 Messias TG, Alves PRL, Cardoso EJBN (2023). Are the Brazilian prevention values for copper and zinc in soils suitable for protecting earthworms against metal toxicity? Environmental Science and Pollution Research International . https://doi.org/10.1007/s11356-022-25106-x Mirmonsef H, Hornum HD, Jensen J, Holmstrup M (2017). Effects of an aged copper contamination on distribution of earthworms, reproduction and cocoon hatchability. Ecotoxicology and Environmental Safety . https://doi.org/10.1016/j.ecoenv.2016.10.012 Natal-da-Luz T, Ojeda G, Pratas J, van Gestel CAM, Sousa JP (2011). Toxicity to Eisenia andrei and Folsomia candida of a metal mixture applied to soil directly or via an organic matrix. Ecotoxicology and Environmental Safety . https://doi.org/10.1016/j.ecoenv.2011.05.017 Natal-da-Luz T, Römbke J, Sousa JP (2008). Avoidance tests in site-specific risk assessment—Influence of soil properties on the avoidance response of collembola and earthworms. Environmental Toxicology and Chemistry . https://doi.org/10.1897/07-386.1. Nursita AI, Singh B, Lees E (2005). The effects of cadmium, copper, lead, and zinc on the growth and reproduction of Proisotoma minuta Tullberg (Collembola). Ecotoxicology and Environmental Safety . https://doi.org/10.1016/j.ecoenv.2004.05.001 OECD (1984). Guidelines for testing of chemicals, 207 . Posthuma L, van Gils J, Zijp MC, van de Meent D, Zwart D (2019). Species sensitivity distributions for use in environmental protection, assessment, and management of aquatic ecosystems for 12386 chemicals. Environmental Toxicology and Chemistry . https://doi.org/10.1002/etc.4373 Princz J, Becker L, Scheffczyk A, Stephenson GL, Scroggins RP, Moser T, Römbke J (2017). Ecotoxicity of boric acid in standard laboratory tests with plants and soil organisms. Ecotoxicology . https://doi.org/10.1007/s10646-017-1789-0 Renaud M, Cousins M, Awuah KF, Jegede O, Sousa JP, Siciliano SD (2020). The effects of complex metal oxide mixtures on three soil invertebrates with contrasting biological traits. Science of the Total Environment . https://doi.org/10.1016/j.scitotenv.2020.139921 Salmon S, Ponge JF, Gachet S, Deharveng L, Lefebvre N, Delabrosse F (2014). Linking species, traits and habitat characteristics of Collembola at European scale. Soil Biology and Biochemistry . https://doi.org/10.1016/j.soilbio.2014.04.002 Sandifer RD, Hopkin SP (1996). Effects of pH on the toxicity of cadmium, copper, lead and zinc to Folsomia candida Willem, 1902 (Collembola) in a standard laboratory test system. Chemosphere . https://doi.org/10.1016/s0045-6535(96)00348-7 Santos, HG. (2018) Sistema Brasileiro de Classificação de Solos (5. ed.). Embrapa, Brasilia. Sereni L, Guenet B, Lamy I (2021). Does copper contamination affect soil CO 2 emissions? A literature review. Frontiers in Environmental Science . https://doi.org/10.3389/fenvs.2021.585677 Silva PMCS, van Gestel CAM (2009). Comparative sensitivity of Eisenia andrei and Perionyx excavatus in earthworm avoidance tests using two soil types in the tropics. Chemosphere . https://doi.org/10.1016/j.chemosphere.2009.09.034 Spurgeon DJ, Hopkin SP (1996). The effects of metal contamination on earthworm populations around a smelting works: quantifying species effects. Applied Soil Ecology . https://doi.org/10.1016/0929-1393(96)00109-6 STATSOFT, Inc. (2011). STATISTICA (data analysis software system), version 10.0. Teixeira, PC (2017). Manual de Métodos de Análise de Solo (3. ed.). Brasília, Embrapa. USEPA (1999). USEPA Method 3051A . Van Gestel CAM (2012). Soil ecotoxicology: state of the art and future directions. ZooKeys . https://doi.org/10.3897/zookeys.176.2275 Van Vlaardingen PLA, Traas TP, Wintersen AM, Aldenberg T (2005). ETX 2.0. A Program to calculate hazardous concentrations and fraction affected, based on normally distributed toxicity data. http://hdl.handle.net/10029/9005 Witkowska D, Słowik J, Chilicka K (2021). Heavy metals and human health: Possible exposure pathways and the competition for protein binding sites. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4485276","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321429707,"identity":"c70418d9-3743-4764-95d1-87c510d3475a","order_by":0,"name":"Daniela Aparecida de Oliveira","email":"","orcid":"","institution":"Santa Catarina State University: Universidade do Estado de Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"Aparecida","lastName":"de Oliveira","suffix":""},{"id":321429708,"identity":"f1b240f7-36ad-4dfa-92d0-c15c177b10f7","order_by":1,"name":"Thiago Ramos 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Catarina","correspondingAuthor":true,"prefix":"","firstName":"Osmar","middleName":"","lastName":"Klauberg-Filho","suffix":""}],"badges":[],"createdAt":"2024-05-27 13:13:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4485276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4485276/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-35271-w","type":"published","date":"2024-10-12T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60984188,"identity":"ce0d1bce-2115-4d84-953b-8501987739ee","added_by":"auto","created_at":"2024-07-24 09:43:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64001,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies sensitivity distribution based on the EC\u003csub\u003e50\u003c/sub\u003e data for the evaluated soils.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4485276/v1/ffc0620d5fcac384a073ce8c.png"},{"id":66597183,"identity":"959f119e-3bf6-444b-a1de-1dd3b9759f2e","added_by":"auto","created_at":"2024-10-14 16:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":908324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485276/v1/5b0f78aa-b8b9-42ca-9f89-5775166262cd.pdf"},{"id":60983612,"identity":"e8cb6f57-7ff1-4538-999b-9ef848227b8c","added_by":"auto","created_at":"2024-07-24 09:35:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":188270,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485276/v1/0f30a6743d754ecee89d396c.docx"},{"id":60984189,"identity":"9ada78e3-e06b-4c0b-b075-af27df471851","added_by":"auto","created_at":"2024-07-24 09:43:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":220441,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485276/v1/8bb94485fc9e67732173bb24.docx"},{"id":60983614,"identity":"4476d7bc-90a8-4e5c-bddf-cf2c04390665","added_by":"auto","created_at":"2024-07-24 09:35:06","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":229000,"visible":true,"origin":"","legend":"","description":"","filename":"FigS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485276/v1/17ebe4af16fa141505c1b97f.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePrevention Values for Copper (Low Tier Approach) in Subtropical Acidic Soils\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAmong the main contaminants of environmental interest, metals require some attention due to their metabolic involvement, because, although essential, in excess they can result in damage to organisms (Witkowska; Stowik; Chilicka, 2021). Copper (Cu) is a micronutrient for plants and animals that is naturally present in the soil due to the weathering processes taking place on the parent rock (Kabata-Pendias, 2010). However, the development of human activities, such as agricultural practices, has contributed to an increase in Cu concentrations in the environment, which has the potential to undermine the supply of environmental services because of contamination on key species' ecological dynamics.\u003c/p\u003e \u003cp\u003eThe group of invertebrates performs ecosystem functions that are extremely important for maintaining life in the soil, with their activities linked to the processes of organic matter and nutrient cycling, soil structuring and bioregulation (Deca\u0026euml;ns et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Briones, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Heděnec et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, it is essential to pay particular attention to the effects of Cu contamination on these organisms to preserve environmental quality. According to Sereni, Guenet, and Lamy (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), soil fauna in Cu-contaminated sites exhibit morphological and behavioral alterations that can lead to trophic imbalance. Understanding the effects of this contamination has become essential for protecting biodiversity and ecosystem services connected with soil. Although the topic has been extensively researched in northern hemisphere soils (Spurgeon \u0026amp; Hopkin, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lukkari et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kupperman et al., 2006; Maraldo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Amorim \u0026amp; Scott-Fordsmand, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Renaud et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), there are few reports on the effects of Cu in tropical and subtropical soils.\u003c/p\u003e \u003cp\u003eThe Brazilian federal legislation, in the form of Resolution 420/2009 of the National Environment Council (CONAMA), ties soil and groundwater quality to guideline values. Among these, the prevention value (PV) refers to the concentrations above which it can be inferred that the soil is polluted by a certain element or substance (Brasil, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Due to the great pedological diversity, it is necessary to determine specific PVs for each of the states. The PVs currently adopted at the national level are those obtained only from soil analysis in the state of S\u0026atilde;o Paulo, making it difficult to identify and manage contaminated areas in other states.\u003c/p\u003e \u003cp\u003eThe obtaining procedure of PV is closely linked to analyzing the effects of the exposure on key organisms such as arthropods and oligochaetes. These assessments are fundamental to understanding the effects of contamination and, as a result, to establishing effective prevention and management guidelines. The European Chemicals Agency (ECHA) recommends that such evaluations must be carried out with species representative of the different trophic levels (ECHA, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The main methods commonly used for this purpose are described by the International Organization for Standardization (ISO) guidelines and aim to measure the dose-response relationship in parameters such as organism survival (lethality) and reproduction capacities when exposed to contaminated soils (van Gestel, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering this scenario, the aim of the present research was to derive PVs for Cu for two subtropical representative soils of South Brazil through reproduction assessments with the earthworm species \u003cem\u003eEisenia andrei\u003c/em\u003e and \u003cem\u003ePerionyx excavates\u003c/em\u003e, the enchytraeids \u003cem\u003eEnchytraeus crypticus\u003c/em\u003e and \u003cem\u003eE. bigeminus\u003c/em\u003e, and the springtails \u003cem\u003eFolsomia candida\u003c/em\u003e and \u003cem\u003eProisotoma minuta\u003c/em\u003e. To attain these objectives, we hypothesized that (i) the effects of contamination measured on the reproduction of the organisms evaluated vary from soil type, and (ii) the PVs adopted at the national level are not fitting for the subtropical soils tested. The methodology employed was to produce species sensitivity distribution curves (SSDs) and estimate risk concentrations (HC\u003csub\u003e5\u003c/sub\u003e and HC50) based on the effect concentrations (EC\u003csub\u003e50\u003c/sub\u003e) obtained in the exposure tests.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Soils sampling and characterization\u003c/h2\u003e \u003cp\u003eTwo natural soils representative of the subtropical region of Brazil and a tropical artificial soil (TAS) were used in the ecotoxicological assessments: an Cambisol (Cambissolo H\u0026uacute;mico (Santos, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)) was collected in Lages, SC [27\u0026ordm;48'57''S; 50\u0026ordm;21'45''W] and a Nitisol (Nitossolo Vermelho (Santos, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)) was collected in Conc\u0026oacute;rdia, SC [27\u0026ordm;48'71''S; 51\u0026ordm;59'34''W]. The soil samples were collected in the surface layer (0\u0026ndash;20 cm) in areas under natural, unmanaged vegetation, free from fertilization and the use of agrochemicals. The TAS was made following the proportions proposed by Garcia (2004), i.e. 75% fine sand, 20% kaolinitic clay and 5% dried and sieved coconut fiber. The TAS' pH was corrected to 6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 by adding calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) (ISO, 2012). The characteristics of the soils were determined according to the procedures suggested by EMBRAPA (Teixeira, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical and physical properties of Nitisol, Cambisol, and TAS.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil properties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNitisol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCambisol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eTAS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic matter (g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCEC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa (cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg (cmolc dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e47.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e11.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e262\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAl (cmol\u003csub\u003ec\u003c/sub\u003e dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCu (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZn (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e29.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMn (mg dm\u003csup\u003e-3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e21.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClay (g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e543.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e133.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e94.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilt (g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e272.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e115.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e108.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSand (g kg\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e183.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e750.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e796.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTexture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSandy Loam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSandy Loam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003eCation Exchange Capacity at pH 7.0. \u003csup\u003e2\u003c/sup\u003epH in water\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Copper spiking procedure\u003c/h2\u003e \u003cp\u003eThe soils were spiked with a Cu(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e solution (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), following a three weeks incubation (Natal-da-Luz et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Prior to the testing procedure, the moisture content was corrected to 50% of the soil's water holding capacity (WHC) in accordance with the ISO 11268-2 protocol (ISO, 2012). After the spiking procedure, soil samples were used in ecotoxicity assays. Soil Cu contents were evaluated through X-ray fluorescence spectrometry in accordance with the USEPA 3051A method (USEPA, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), as required by CONAMA Resolution n. 420/2009 (Brasil, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The Standard Reference Material was a 2709a San Joaquin Soil.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTested nominal concentrations of copper for each ecotoxicological assay depending on the soil type.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrganisms and copper nominal concentrations (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEarthworms (\u003cem\u003eE. andrei\u003c/em\u003e and \u003cem\u003eP. excavatus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitisol and TAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 100, 200, 400, 800, 1200, 1600 and 2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCambisol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 50, 100, 200, 300, 500, 700 and 1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnchytraeids (\u003cem\u003eE. crypticus\u003c/em\u003e and \u003cem\u003eE. bigeminus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitisol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 75, 150, 300, 450, 600, 750 and 1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCambisol and TAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 25, 50, 75, 150, 250, 500 and 750\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpringtails (\u003cem\u003eF. candida\u003c/em\u003e and \u003cem\u003eP. minuta\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitisol, Cambisol and TAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0, 75, 150, 300, 450, 600, 750 and 1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Test organisms\u003c/h2\u003e \u003cp\u003eThe earthworms were cultivated in a substrate consisting of equine manure (chemical-free), coconut husk fiber and fine sand (in the proportion of 7:2:1). The pH of the mixture was corrected to 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 range with CaCO\u003csub\u003e3\u003c/sub\u003e. The organisms were fed weekly with oat flakes. The species \u003cem\u003eEisenia andrei\u003c/em\u003e was kept at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2,0 \u0026ordm;C room and the \u003cem\u003ePerionyx excavatus\u003c/em\u003e species at a 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2,0 \u0026ordm;C. The photoperiod for both was kept in 16 hours light. The enchytraeids were grown in TAS (Garcia, 2004) according to the ISO 16387 protocol (ISO, 2014a). The springtails were cultivated in a substrate composed of plaster, distilled water and activated charcoal (in a ratio of 11:7:1) maintained at a temperature of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 \u0026ordm;C and at a photoperiod of 16 hours light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental procedures\u003c/h2\u003e \u003cp\u003eThe assays with the earthworm species \u003cem\u003eE. andrei\u003c/em\u003e and \u003cem\u003eP. excavatus\u003c/em\u003e followed the guidelines established in the ISO 11268-2 protocol (ISO, 2012). The experimental units (n\u0026thinsp;=\u0026thinsp;5) consisted of containers filled with around 500 g of dry soil and in the presence of ten clitellate individuals weighing between 250 and 600 mg. The organisms were fed weekly with approximately 15 g of horse manure (free from pollutants) and the moisture was kept at 50% of WHC. After 28 days, the adults were removed, and the cocoons and juveniles were incubated for a further 28 days. At the end of the 56 days, the organisms were extracted using a temperature gradient in a laboratory water bath (60 \u0026ordm;C). The evaluations with enchytraeids were carried out according to the guidelines established in ISO 16387 (ISO, 2014a) with adjustments proposed by (Kuperman et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and the assay was conducted for four weeks (28 days) for E. crypticus and three weeks (21 days) for \u003cem\u003eE. bigeminus\u003c/em\u003e (Bandow et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The criterion for selecting adult individuals for E. crypticus was the presence of a clitellum; for \u003cem\u003eE. bigeminus\u003c/em\u003e, organisms were chosen according to their body size (8 to 12 mm), since this species is a fragmented reproducer (Bandow et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Ten organisms were placed in containers with 30 g of soil (n\u0026thinsp;=\u0026thinsp;4) and fed weekly with around 2 mg of rolled oats and the moisture content was kept at 50% of WHC. At the end of the test, 5 mL of pure alcohol, 1 mL of rose bengal dye solution (1% w/v) and 80 mL of water were added to each experimental unit for subsequent counting of the organisms. The springtails' assessments lasted 28 days and were carried out according to the procedures listed under ISO standard 11267 (ISO, 2014b). Ten organisms with 12 days old were kept in containers with 30 g of soil (n\u0026thinsp;=\u0026thinsp;5). Food was added once a week and consisted of around 2 mg of dry biological yeast and the moisture content was kept at 50% of the WHC. At the end of the evaluation period, for the \u003cem\u003eF. candida\u003c/em\u003e juveniles, each experimental unit was filled with about 80 mL of water and a few drops of black stamp ink (to increase the contrast between the organisms and the surface of the water). After gentle stirring, the number of adult and juvenile individuals was determined using ImageJ 1.54d software (NHI, 2023). To determine the reproduction rate of the \u003cem\u003eP. minuta\u003c/em\u003e species, the method used was dry extraction in Berlese-T\u0026uuml;llgren funnels for a period of 24 hours with subsequent addition of water and red dye. This adaptation was necessary due to the smaller size and color of the species' exoskeleton (Hopkin, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Mendon\u0026ccedil;a; Queiroz; Silveira, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Afterwards, the samples containing the organisms were carefully shaken and the number of individuals determined using ImageJ 1.54d software (NHI, 2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data analysis\u003c/h2\u003e \u003cp\u003eThe ecotoxicological data were subjected to the Shapiro-Wilk normality test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and Bartlett's homogeneity test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and the means were compared using the Dunnett test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). The data was then submitted to a non-linear regression analysis to determine the dose of contaminant corresponding to a 50% effect on the population of organisms (EC\u003csub\u003e50\u003c/sub\u003e). The EC\u003csub\u003e50\u003c/sub\u003e values were determined using the model that best fitted the data (Environment Canada, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). All analyses were carried out using STATISTICA 10.0 software (StatSoft, 2011).\u003c/p\u003e \u003cp\u003eUsing ETX 2.0 software (van Vlaardingen et al., 2004), sensitivity curves were developed for the species tested (SSDs). The SSDs were based on the EC\u003csub\u003e50\u003c/sub\u003e values for each organism in each soil evaluated (Aldenberg \u0026amp; Jaworska, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The SSDs were employed to estimate the concentration of the contaminant capable of posing a risk to 5 and 50% of the species (HC\u003csub\u003e5\u003c/sub\u003e and HC\u003csub\u003e50\u003c/sub\u003e), with a 95% confidence interval. Since the model assumes a log-normal distribution of the data, log-normality was tested using the Anderson-Darling, Kolmogorov-Smirnov and Cramer von Mises tests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The PVs were obtained after the HC\u003csub\u003e5\u003c/sub\u003e had been calculated considering the Quality Reference Values (QRV, = 93.84 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for soils in the state of Santa Catarina (IMA, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), according to the equation PV\u0026thinsp;=\u0026thinsp;QRV\u0026thinsp;+\u0026thinsp;HC5.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Test validation\u003c/h2\u003e\n \u003cp\u003eThe validation criteria were all met for the species \u003cem\u003eE. andrei\u003c/em\u003e, \u003cem\u003eP. excavatus\u003c/em\u003e, \u003cem\u003eE. crypticus\u003c/em\u003e, \u003cem\u003eF. candida\u003c/em\u003e and \u003cem\u003eP. minuta\u003c/em\u003e (ISO, 2012; ISO, 2014a; ISO, 2014b). For the assays with the species \u003cem\u003eE. bigeminus\u003c/em\u003e, the criterion adopted was the occurrence of 25 juveniles in the control groups with a coefficient of variation lower than 50% (Bandow et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). The choice of a specific criterion is justified due to the impossibility of identifying clitellate adult individuals as it is a fragmenting species.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Ecotoxicological assays\u003c/h2\u003e\n \u003cp\u003eThe reproduction of the earthworm species \u003cem\u003eE. andrei\u003c/em\u003e and \u003cem\u003eP. excavatus\u003c/em\u003e was affected (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in all the soils that were evaluated (Figure S1). Reductions in the number of \u003cem\u003eE. andrei\u003c/em\u003e juveniles were observed starting at doses of 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cambisol, 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in TAS and 200 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Nitisol. For the species \u003cem\u003eP. excavatus\u003c/em\u003e, the effects were observed at the lowest concentrations evaluated (50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cambisol and 100 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in TAS and Nitisol). The most significant effect for both species was found in the Cambisol, since the EC\u003csub\u003e50\u003c/sub\u003e values for this soil (77.52 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE. andrei\u003c/em\u003e and 67.83 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for P. excavatus) were lower than those found in Nitisol (428.01 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE. andrei\u003c/em\u003e and 264.96 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eP. excavatus\u003c/em\u003e) and TAS (173.81 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eE. andrei\u003c/em\u003e and 110.46 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for \u003cem\u003eP. excavatus\u003c/em\u003e). All the enchytraeid species evaluated were also affected (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) by soil contamination with Cu (Figure S2). The reduction in the number of juveniles of \u003cem\u003eE. crypticus\u003c/em\u003e occurred from the concentration of 25 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in TAS, 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cambisol and 150 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Nitisol. For the species \u003cem\u003eE. bigeminus\u003c/em\u003e, the effects were verified starting at a concentration of 250 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the TAS, 50 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the Cambisol and 75 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the Nitisol. As observed for the earthworms, the most significant effects for the enchytraeids were also observed in the Cambisol, since the EC\u003csub\u003e50\u003c/sub\u003e values obtained were lower than those found in the Nitisol and TAS. The springtails \u003cem\u003eF. candida\u003c/em\u003e and \u003cem\u003eP. minuta\u003c/em\u003e also had their reproduction affected (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in all the soils evaluated (Figure S3). The reduction in the number of juveniles of the species \u003cem\u003eF. candida\u003c/em\u003e was observed from the concentration of 75 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Nitisol and 300 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cambisol and TAS. For the \u003cem\u003eP. minuta\u003c/em\u003e species evaluations, the significant effects on reproduction occurred from the concentrations of 75 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cambisol, 150 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Nitisol and 450 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in TAS. The no effect concentration (NOEC), lowest effect concentration (LOEC) and EC\u003csub\u003e50\u003c/sub\u003e for each assessment are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eIn general, it can also be said that the earthworm species \u003cem\u003eP. excavatus\u003c/em\u003e is the most sensitive of all the species evaluated, whereas the springtail\u0026nbsp;\u003cem\u003eP. minuta\u003c/em\u003e was the least affected by the contamination, independent of soil type.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEcotoxicological endpoints (NOEC, LOEC and EC\u003csub\u003e50\u003c/sub\u003e) for each evaluated species in Nitisol, Cambisol e TAS.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNOEC (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLOEC (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNitisol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. andrei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e428.01 (300.58-555.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. excavatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e264.96 (184.08-345.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. crypticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e366.61 (328.56-404.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. bigeminus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e444.71 (303.99-585.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF. candida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e456.22 (305.94-606.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. minuta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e636.00 (597.15-674.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCambisol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. andrei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.52 (66.16\u0026ndash;88.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. excavatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.83 (54.95\u0026ndash;80.72)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. crypticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142.25 (109.50\u0026ndash;175.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. bigeminus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126.77 (56.30-197.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF. candida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e255.03 (223.52-286.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. minuta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e513.17 (453.78-572.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTAS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. andrei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173.81 (148.99-198.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. excavatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.46 (73.82\u0026ndash;137.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. crypticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e171.71 (124.79-218.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. bigeminus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e213.70 (181.36-246.03)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF. candida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e333.38 (297.35-369.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. minuta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e578.74 (512.03-645.45)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn parenthesis the confidence limits (lower and upper) are presented.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Chemical evaluations\u003c/h2\u003e\n \u003cp\u003eThe data from the analysis of the Cu levels in the natural soils (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) indicated an average percentage recovery (in relation to the nominal dose) of 83.8% in Nitisol and 86% in Cambisol, while in TAS the recovery was around 92%. For this reason, it was decided to employ the nominal doses in the construction of the SSD and the subsequent derivation of the PV.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNominal concentration (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and chemical measured concentration (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of copper in subtropical natural soils and TAS.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNominal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eReal\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNitisol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCambisol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTAS\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e170.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e172.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e188.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e290.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e260.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e261.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e326.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e384.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e375.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e429.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e477.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e422.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e539.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e551.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e668.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e647.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e716.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e743.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e719.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e792.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e889.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e955.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1078.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1185.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1438.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1586.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1798.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1982.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003end: not determined once the nominal concentration was not evaluated in the ecotoxicological assay.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Species Sensibility Distribution and prevention values\u003c/h2\u003e\n \u003cp\u003eOnce it was possible to assess the EC\u003csub\u003e50\u003c/sub\u003e for each of the tested organisms, the SSD were generated and the HC\u003csub\u003e5\u003c/sub\u003e and HC\u003csub\u003e50\u003c/sub\u003e were obtained for all the tested soils (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The highest HC\u003csub\u003e5\u003c/sub\u003e values were recorded in Nitisol (252.9 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), followed by TAS (76.12 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and Cambisol (40.21 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). It can therefore be inferred that Cu contamination is more problematic in Cambisol, since the concentration capable of endangering the organisms is approximately six times lower than in Nitisol.\u003c/p\u003e\n \u003cp\u003eFinally, based on the Cu QRV (93.84 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) published for Santa Catarina state soils (IMA, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), it was possible to estimate the PV via the HC\u003csub\u003e5\u003c/sub\u003e data (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). We chose not to utilize HC\u003csub\u003e50\u003c/sub\u003e for this purpose because we intended to propose PVs that are ecologically safer.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHazardous concentration for 5% of the species and prevention values for Cu based on EC\u003csub\u003e50\u003c/sub\u003e data for each evaluated soil.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLL HC\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHC\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUL HC\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePV\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNitisol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e144.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e252.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e325.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e346.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCambisol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eLL HC\u003csub\u003e5\u003c/sub\u003e: lowest limit for the calculated HC\u003csub\u003e5\u003c/sub\u003e (p\u0026thinsp;\u0026le;\u0026thinsp;0,05); HC\u003csub\u003e5\u003c/sub\u003e: hazardous concentration for 5% of the species (p\u0026thinsp;\u0026le;\u0026thinsp;0,05) based on the EC\u003csub\u003e50\u003c/sub\u003e values; UL HC\u003csub\u003e5\u003c/sub\u003e: upper limit for the calculated HC\u003csub\u003e5\u003c/sub\u003e (p\u0026thinsp;\u0026le;\u0026thinsp;0,05); PV: prevention values (PV\u0026thinsp;=\u0026thinsp;QRV\u0026thinsp;+\u0026thinsp;HC\u003csub\u003e5\u003c/sub\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussions","content":"\u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eThe 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. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who reported that Cu toxicity in \u003cem\u003eE. fetida\u003c/em\u003e 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; R\u0026ouml;mbke; Sousa, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eP. excavatus\u003c/em\u003e, followed by the enchytraeid \u003cem\u003eE. crypticus\u003c/em\u003e (for the evaluations in TAS and Nitisol) and the earthworm \u003cem\u003eE. andrei\u003c/em\u003e (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.\u003c/p\u003e \u003cp\u003eIn accordance with our findings, Mirmonsef et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), 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 Fouch\u0026eacute; (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), in a study aiming to determine the Cu toxicity in the species \u003cem\u003eE. andrei\u003c/em\u003e, found a significant reduction in oviposition and cocoon hatching rates. Kwak and An (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), 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\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of dry soil in adults of \u003cem\u003eP. excavatus\u003c/em\u003e and \u003cem\u003eE. andrei\u003c/em\u003e respectively. Regarding the variation in LOEC which we have verified here between the two earthworm species evaluated, the results suggest that the \u003cem\u003eP. excavatus\u003c/em\u003e species is more sensitive. This observation corroborates the findings of Spurgeon and Hopkin (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), who, in an evaluation of ecologically relevant species, concluded that the \u003cem\u003eEisenia\u003c/em\u003e 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 \u003cem\u003eP. excavatus\u003c/em\u003e (Silva \u0026amp; van Gestel, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), can better represent ecological risk analysis in Brazilian soils.\u003c/p\u003e \u003cp\u003eIn terms of the damaging effects of Cu on enchytraeids, and in accordance with the results of this evaluation, Maraldo et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), when assessing the survival of \u003cem\u003eE. crypticus\u003c/em\u003e in Danish soils contaminated with Cu, found a reduction in the survival of this organism at concentrations of 600 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 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, Konečn\u0026yacute; et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), in a study assessing the influence of trace elements on \u003cem\u003eE. crypticus\u003c/em\u003e, 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 EC\u003csub\u003e50\u003c/sub\u003e determined for the species at 351 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eThe 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. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the toxicity of trace elements in \u003cem\u003eF. candida\u003c/em\u003e 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 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) 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 \u003cem\u003eF. candida\u003c/em\u003e. However, it should be considered that the assays conducted by the authors were carried out on OECD artificial soil (OECD, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), 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., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). According to Fountain and Hopkin (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003eAnother outstanding aspect of this investigation, and in line with what was observed by Buch et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) when conducting assessments on the toxicity of Hg in tropical soils, was the greater sensitivity recorded for \u003cem\u003eF. candida\u003c/em\u003e when compared to \u003cem\u003eP. minuta\u003c/em\u003e. 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. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), 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 \u003cem\u003eP. minuta\u003c/em\u003e in the current evaluation, it is worth noting that the species shows reasonable sensitivity to Cu (Nursita; Singh; Lees, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), despite its lower sensitivity when compared to \u003cem\u003eF. candida\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe process of drawing up the SSD made it possible to assess the organisms' sensitivity to Cu by determining risk concentrations (i.e. HC\u003csub\u003e5\u003c/sub\u003e). 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., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Posthuma et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bandeira et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eThe 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, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; IMA, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); 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 EC\u003csub\u003e50\u003c/sub\u003e was driven by the need to safeguard ecosystems without being strictly restrictive, since the PV were estimated according to the HC\u003csub\u003e5\u003c/sub\u003e. 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 EC\u003csub\u003e20\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThat said, it is worth noting that the results showed that the PV based on the EC\u003csub\u003e50\u003c/sub\u003e data for Cu were higher than the stipulated in the CONAMA Resolution (Brasil, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), 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 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) 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.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eBased on the comparison between the results and the current Brazilian legislation, it can be assumed that the guideline values (i.e. PV) for Cu are protective for the different bioindicators in the soils evaluated. It is important nothing that the PV we derive were based on the concentration capable of inhibit in 50% the reproduction of the organisms. It was observed that the effects vary among soil types, likely as a result of their chemical properties, including pH and clay content. Hence, evaluations with other representative soil types from subtropical climate regions are required before it can be inferred that the PV included in the federal legislation is in fact protective to subtropical terrestrial ecosystems.\u003c/p\u003e \u003cp\u003eThe data here presented tends to support other similar investigations to build an ecotoxicological database that will allow a better understanding of the effects of trace element pollution on the biotic constituents of different Brazilian ecosystems. This represents a major step forward in the construction of public policies focused on environmental conservation, given that the organisms evaluated significantly play an important role in providing ecosystem services.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by thefinancial support from Santa Catarina State Research and Innovation Support Foundation (FAPESC, Grant Number: \u003cem\u003e2023TR000733\u003c/em\u003e). V.M.D.R. and T.R.F are grateful for the scholarship granted by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution: Daniela Aparecida de Oliveira:\u0026nbsp;\u003c/strong\u003eInvestigation, Formal analysis, Writing \u0026ndash; Original Draft, Validation, Visualization.\u003cstrong\u003e\u0026nbsp;Thiago Ramos Freitas:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing \u0026ndash; Original Draft, Validation, Visualization.\u003cstrong\u003e\u0026nbsp;Vanessa Mignon Dalla Rosa:\u0026nbsp;\u003c/strong\u003eFormal analysis, Writing \u0026ndash; Original Draft, Validation, Visualization.\u003cstrong\u003e\u0026nbsp;Lu\u0026iacute;s Carlos Iu\u0026ntilde;es de Oliveira Filho\u003c/strong\u003e: Supervision, Visualization, Validation, Writing \u0026ndash; Review \u0026amp; Editing.\u003cstrong\u003e\u0026nbsp;Mari Lucia Campos:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, Writing \u0026ndash; Review \u0026amp; Editing, Validation.\u003cstrong\u003e\u0026nbsp;Milton da Veiga:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing \u0026ndash; Review \u0026amp; Editing.\u003cstrong\u003e\u0026nbsp;David Jos\u0026eacute; Miquelluti:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, Writing \u0026ndash; Review \u0026amp; Editing,Validation, Project administration,Funding acquisition.\u003cstrong\u003e\u0026nbsp;Osmar Klauberg-Filho:\u0026nbsp;\u003c/strong\u003eConceptualization, Supervision, Writing \u0026ndash; Review \u0026amp; Editing, Validation, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eDatasets related to this article can be made available upon request to the corresponding author Osmar Klauberg-Filho ([email protected])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAldenberg T, \u0026amp; Jaworska J (2000). 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Bras\u0026iacute;lia, Embrapa. \u003c/li\u003e\n\u003cli\u003eUSEPA (1999). \u003cem\u003eUSEPA Method 3051A\u003c/em\u003e. \u003c/li\u003e\n\u003cli\u003eVan Gestel CAM (2012). Soil ecotoxicology: state of the art and future directions. \u003cem\u003eZooKeys\u003c/em\u003e. https://doi.org/10.3897/zookeys.176.2275 \u003c/li\u003e\n\u003cli\u003eVan Vlaardingen PLA, Traas TP, Wintersen AM, Aldenberg T (2005). ETX 2.0. A Program to calculate hazardous concentrations and fraction affected, based on normally distributed toxicity data. http://hdl.handle.net/10029/9005\u003c/li\u003e\n\u003cli\u003eWitkowska D, Słowik J, Chilicka K (2021). Heavy metals and human health: Possible exposure pathways and the competition for protein binding sites. \u003cem\u003eMolecules\u003c/em\u003e. https://doi.org/10.3390/molecules26196060 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ecotoxicology, Soil Screening Values, Trace Metals, Soil Contamination, Earthworms, Enchytraeid, Springtail.","lastPublishedDoi":"10.21203/rs.3.rs-4485276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4485276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCopper is a trace element in plants and animals whose importance can be understood due to its role in different essential metabolic processes. Anthropogenic activities such as agriculture and mining are potential sources of pollution due to the emission of copper into the environment. Brazilian legislation ties soil quality to guideline values, among which the Prevention Value indicates the critical environmental limit for trace elements. The aim of this study was to obtain PVs for copper for two subtropical soils (Cambisol and Nitisol), given that the pedological richness was not considered when deriving the PVs contained in the federal normative. Reproduction assays followed ISO guidelines with the earthworm species \u003cem\u003eEisenia andrei\u003c/em\u003e and \u003cem\u003ePerionyx excavatus\u003c/em\u003e, the enchytraeids \u003cem\u003eEnchytraeus crypticus\u003c/em\u003e and \u003cem\u003eE. bigeminus\u003c/em\u003e\u0026nbsp;and the springtails \u003cem\u003eFolsomia candida\u003c/em\u003e and \u003cem\u003eProisotoma minuta\u003c/em\u003e. Results showed that the sensitivity of the organisms was greater in Cambisol. The most sensitive species were the earthworms, especially \u003cem\u003eP. excavatus\u003c/em\u003e (EC\u003csub\u003e50\u003c/sub\u003e = 67.83 in Cambisol; EC\u003csub\u003e50\u003c/sub\u003e = 264.96 in Nitisol). The springtails, on the other hand, were the least sensitive to contamination. These findings reinforce the need to include organisms from different ecological groups in ecotoxicological assessments. It was also observed that the PV adopted in federal legislation (= 60 mg kg\u003csup\u003e-1\u003c/sup\u003e) is in fact protective for the species and soils we evaluated, since the PVs we obtained based on the EC\u003csub\u003e50\u003c/sub\u003e were 346.74 mg kg\u003csup\u003e-1\u003c/sup\u003e in Nitisol and 134.05 mg kg\u003csup\u003e-1\u003c/sup\u003e in Cambisol. It is important to note that our results do not exclude the need for evaluations with other subtropical soils, given the influence of their properties on the toxicity and bioavailability of copper to soil organisms.\u003c/p\u003e","manuscriptTitle":"Prevention Values for Copper (Low Tier Approach) in Subtropical Acidic Soils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 09:35:00","doi":"10.21203/rs.3.rs-4485276/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2024-09-18T00:31:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-15T16:47:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-01T23:44:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-06-13T17:18:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-05T04:52:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-06-03T09:01:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1bcc9e9-7436-44e2-adc0-7cb499647d9b","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-4485276","link":"https://doi.org/10.1007/s11356-024-35271-w","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-10-12 15:57:44","publishedOnDateReadable":"October 12th, 2024"},"versionCreatedAt":"2024-07-24 09:35:00","video":"","vorDoi":"10.1007/s11356-024-35271-w","vorDoiUrl":"https://doi.org/10.1007/s11356-024-35271-w","workflowStages":[]},"version":"v1","identity":"rs-4485276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4485276","identity":"rs-4485276","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}