Rare Earth Elements at different ratio (single or double), three in binary and seven in ternary mixtures, were assessed herein. Toxicity was categorized in relation to mixture ratio, either potentiating (synergism) or mitigating (antagonism) joint toxicity. So far, no study of this magnitude has been carried out, where La, Nd, and Sm mixture effects at different combinations and phytotoxicity parameters were broadly evaluated.
The results indicate that La, Nd and Sm significantly and negatively affect lettuce germination parameters and wet biomass. Lanthanum was the most toxic element concerning wet biomass in the AS and germination in the Ultisol (Tables 1 and 2), while Sm was the least toxic concerning biomass in AS (Table 1), and Nd, the least toxic regarding biomass in Ultisol (Table 2).
This contrasts with findings reported for lettuce in studies employing other REE (Ce, La, Gd and Yb) in the form of nanoparticles (Andersen et al. 2016; Cui et al. 2014; Ma et al. 2010, 2018; Zhang et al. 2015; Zhao et al. 2021), where germination inhibition was not observed. A difference between nano- and non-nano compounds was observed, which may be associated to greater nanoparticle toxicity due to their small size, greater surface area, greater reactivity, and dissolution with subsequent release of free ions (Gaiser et al. 2011; Ma et al. 2014; Rogers et al. 2010; Tiede et al. 2009).
Germination toxicity increased with increasing test concentrations in SA substrate (Tables S2 and S4), with a significant p-value obtained in the linear regression analysis (Table S3). In the Ultisol substrate, at lower concentrations, an increase in germination was observed, where were detected the hormesis phenomenon (Tables S5 and S6). At higher concentrations, there was a decrease in germination, with a significant p-value obtained in the linear regression analysis (Table S3).
The EC50 values were higher in AS than in Ultisol (Tables 1 and 2) for both single exposures and the binary mixtures Sm + La, Nd + La, and in ternary mixture containing one of the elements at double concentrations. Higher EC50 values in AS were also observed by Moreira and collaborators (2019), who tested the effect of Ce in different types of natural soil and one artificial soil. In that study, EC50 values varied in Oxisol (303.74 to 691.83 mg Ce.kg− 1), followed by Inceptsol (466.25 to 1325.18 mg Ce.kg− 1), and higher in artificial soil (997. 26 to 1524.81 mg Ce.kg− 1). Due to the granulometry of the investigated artificial soils, composed of inert materials, fine and coarse sand (ISO 2005), which favor less interaction with test substances, and consequently greater seedling availability through root absorption (OECD 2006), greater toxicity in AS is expected.
The IC50 results (Table 2) in Ultisol were higher than in the AS (Table 1) for Sm (612.58 mg.kg− 1) and Nd (857.06 mg.kg− 1) and in the three binary mixtures. In the ternary mixtures, all obtained Ultisol results were 10 -fold higher than in the SA (Table 1). The results reported herein also corroborate those presented by Moreira and collaborators (2019), as the IC50 values in artificial soil were lower than the EC50 values in natural soil (Table 1). The dry biomass parameter was the most sensitive for the observed phytotoxicity. The deleterious effects observed in this study occurred due to Ce accumulation in cells, causing structural damage and nutritional balance alterations, as well as decreased photosynthesis and growth or cell death (Moreira et al. 2019).
In addition to the internal plant factors, REE bioavailability and absorption are also influenced by physicochemical soil attributes, such as pH, cation exchange capacity (CEC), organic carbon content (organic matter), REE concentrations in the test solution and soil capacity to provide REE (Moreira et al. 2019; Ramos et al. 2016). Low pH and CEC values favor REE desorption from the soil mineral matrix into the soil solution, increasing REE bioavailability and phytotoxicity (Cheng et al. 2015; Moreira et al. 2019; Thomas et al. 2014). When soil pH values increase, REEs tend to precipitate, decreasing their release from the soil mineral matrix into the soil solution (Moreira et al. 2019; Ramos et al. 2016). Organic matter is an importer REE absorption factor, as it provides negative soil charges (Moreira et al. 2019; Ramos et al. 2016). The stability of this bond varies according to the molar ratio between REE and dissolved organic carbon, with REE concentrations inversely correlated with soil pH and directly correlated with dissolved organic carbon concentrations (Moreira et al. 2019; Ramos et al. 2016). In the natural soil used in the present study, soil physicochemical characteristics (Table S1), low CEC of 1.5 meq 100 g− 1 (< 4.5 – low; 4.5 to 10 – medium and > 10 high, Prezotti and Garçoni 2013), high organic matter content of 10.8 g.kg− 1 (< 1.5 – low; 1.5 to 3.0 – medium and > 3.0 high, Prezotti and Garçoni 2013), Ca and Mg concentrations from 2.5 to 1.4 cmolc.kg− 1 medium (< 1.5 – low; 1.5 to 4.0 – medium and > 4.0 high, Prezotti and Garçoni 2013) and high (< 0.5 – low ; 0.5 to 1.0 – medium and > 1.0 high, Prezotti and Garçoni 2013), respectively, and the acidic pH of 5.1, may also explain the lower toxicity concerning wet biomass observed in natural soil in comparison to the employed AS.
Among other REE (Ce, La, Gd and Yb) studies conducting exposure tests on lettuce seeds (Andersen et al. 2016; Barbieri et al. 2013; Cui et al. 2014; Ma et al., 2010, 2018; Zhang et al. 2015; Zhao et al. 2021), only Ma and collaborators (2018) employed natural soil, while the others used moist filter paper or agar medium, with a viscosity similar to soil. Avoiding a complex natural soil system containing organic and inorganic minerals, water, gases and living organisms (Cui et al. 2014), allows for root growth and cotyledon assessments the identification of initial development (Andersen et al. 2016). The small number of studies conducted with natural soils highlights the need to further assess natural plant REE exposure conditions.
Low-dose stimulation effect (hormesis) as observed herein, with increased germinations noted at lower test concentrations, in AS for the Sm + La 1:1 and 2:1 ratio at 100 mg.kg− 1, in the ternary mixtures 1:1:1 at 100 mg.kg− 1 and 1:2:2 at 100 and 200 mg.kg− 1, and in Ultisol for the Sm + Nd 1:1 ratio at 100 mg.kg− 1, and 2:1 ratio at 100 and 400 mg.kg− 1, Nd + La 1:2 ratio at 400 mg.kg− 1 and 2:1 ratio at 100 mg.kg− 1 and in the 1:1:1, 1:2: 1, 2:1:2 and 1:2:2 ternary mixtures at 100 and 200 mg.kg− 1, and germination inhibition observed above 400 mg.kg− 1 compared to the controls (Tables S4 and S6), were also observed following La exposure in rice (Oryza sativa) (Liu et al. 2013) and soybean (Glycine max) (Oliveira et al. 2015). In those studies, greater root growth was observed at lower concentrations of 0.05 mmol.L− 1 (6.946 mg.L− 1), 0.1 mmol.L− 1 (13.891 mg.L− 1) and 5 mmol.L− 1 (694.550 mg.L− 1), respectively, and inhibition at high concentrations of 1.0 (138.910 mg.L− 1) and 1.5 mmol.L− 1 (208.365 mg.L− 1) and 20 mmol L − 1 2778.200 mg.L− 1), respectively. In another assessment, exposure to Ce in cowpea plants (Vigna unguiculata) promoted increased leaf chlorophyll, dry mass and nitrate reductase activity at low concentrations (0.713–17.841 µmol.L− 1) and decreased chlorophyll content and increased proline and polyphenol oxidase activity at high concentrations (89,206 − 446.030 µmol.L− 1) (Shyam and Aery 2012).
Two biphasic curve models for the analysis of the hormesis phenomenon have been proposed, by Brain and Cousins (1989) and by Cedergreen and collaborators (2005) (Belz and Duke 2022). These have been recommended as the best way to demonstrate this effect, as they include biological parameters that allow for the deduction of the quantitative characteristics of the stimulus phase (sub-NOAEL). However, our results cannot be applied to these models because they present little data (points) in the stimulus phase, decreasing the probability that the curve reflects the true form of the dose-response relationship and must be used with care, as recommended by Belz and Duke (2022).
Germination inhibition increased at higher test concentrations, with a greater number of non-germinated seeds (Tables S2, S4, S5 and S6). In this study, toxicity categories were used as an indication of the test concentrations resulting in initial toxicity signs (moderately toxic), which may be used as a screening test as an alert to anticipate more severe events based on phytotoxicity tests or on ecological risk assessments.
The effects of REEs in single exposures were also observed for the investigated mixtures, such as germination inhibition at higher test concentrations (significant p-value) and wet biomass variations related to the tested substrate (only in Ultisol, with a significant p-value observed for the three binary mixture Nd + La ratio and all ternary mixtures). Concerning germination in the binary mixture Sm + La, double La (1:2) seems to increase the toxicity of this ratio in relation to the other two ratio in both substrates.
Concerning the AS, double Nd concentrations (1:2) in the binary Sm + Nd mixture, seem to attenuate the toxicity of this ratio concerning germination, while double Sm (2:1) in the same mixture seems to increase the toxicity of this ratio concerning wet biomass. In the AS, double La concentrations in the Nd + La 1:2 binary mixture seems to attenuate the toxicity of this ratio compared to the other two ratio with regard to wet biomass. Also in the AS, ternary mixtures with two elements present at double concentrations were more toxic than the other ratio concerning germination. In the AS, all mixtures and ratio were additive, while in Ultisol, 56% were additive (9) and 44%, synergistic (7). In the ternary mixtures, the ratio with synergistic interactions were those at single concentrations, and with two in double ratio, and with less toxic EC50 values than in the AS.
Artificial soil interactions were all additive (Table 1), indicating, according to the additive concentration model (AC), that the investigated REE can be replaced in the mixture without altering its toxicity, since, as long as the sum of the UTms is equal to 1, they do not interact at the physicochemical level or with regard to their toxicokinetics and toxicodynamics (Backhaus and Faust 2012). In the natural soil, on the other hand, interactions were additive, except the Nd + La binary mixture at all ratio and the ternary mixtures with all single elements or two in double, namely 1:1:1, 1:2:2, 2:1:2 and 2 :2:1 (Table 2), which were synergistic. In this interaction, one of the REEs in the mixture increases the effect of the other element at the physicochemical, toxicokinetic and toxicodynamic levels (Gong et al. 2020). Synergism only in the natural soil may be associates to soil physicochemical factors, such as organic carbon content, alkalinity and pH, all of which may influence REE speciation and bioavailability to plants, decreasing or increasing the amount of absorbed REE, as well as cellular uptake and internalization processes, influencing toxicokinetic and toxicodynamic processes (Gong et al. 2020).
No studies are available concerning the three REE investigates herein, in either single exposures or in mixtures, under the conditions investigated herein. IC50 values in the literature were found in a study with Nd and Sm in unitary exposure in artificial soil (10% moss, 20% kaolin and 70% silica sand) (Carpenter et al. 2015), and La with the same artificial soil, using the same plant species (Thomas et al. 2014). No comparison to other studies, however, can be performed with our findings, especially those obtained for the two mixtures. The understanding of the type of biological response induced by REE, in both single exposures and in mixtures, is still understudied (Gong et al. 2019; Warne 2010). The observed toxicity effects may result from different factors due to interactions at plasma membrane binding site(s), mainly with Ca2+ and Mg2+, inhibition of enzymes and proteins, physiological processes (absorption, translocation, distribution and detoxification) and chemical interactions with medium constituents, affecting their chemical speciation (free ions and/or complexes with ligands) and bioavailability (Gong et al. 2019; Gonzalez et al. 2014; Romero-Freire et al. 2018; Spurgeon et al. 2010; Thomas et al. 2014).