3.1 Biomass and Ce content of earthworms
Figure 1 shows the biomass and Ce content of earthworms exposed to different nCeO2 concentrations. The initial Ce concentration of earthworm in clay soils was higher than in sandy loam soils. In sandy loam soils, the Ce content in earthworms was not significantly changed by the addition of low nCeO2 concentrations (10–100 mg/kg), except 500–1000 mg/kg nCeO2 treatments, in which the Ce content significantly increased compared with the control. In contrast, in clay soils, the Ce contents in earthworms ranged from 1.4 to 2.7 mg/kg, increasing along with nCeO2 concentration (0–50 mg/kg), after which the content was stable at 2.3 to 2.4 mg/kg, independent of the added nCeO2 concentration (100–1000 mg/kg). Moreover, nCeO2 had no adverse effects on earthworm survival within the range of the tested concentrations in clay soil, whereas in sandy loam soils, significant reductions in earthworm biomass occurred, compared with the control group, after exposure to nCeO2 treatments. In particular, the Ce contents of earthworms in clay soils treated with 50 mg/kg nCeO2 reached as high as 2.7 mg/kg, an increase of 91.7% compared with the control (1.4 mg/kg), but there was no significant effect on the earthworm biomass. Thus, the bioaccumulation and toxicity effects of nCeO2 on the earthworms in the two soils were inconsistent.
There are two main metal bioaccumulation pathways in earthworms: (1) the ionic form of the soluble metal passes into the tissue by dermal uptake though the process of diffusion and then accumulates in the circular and longitudinal muscles of earthworms; or (2) the metal particles entrapped in soil and pore water are consumed by earthworms and then undergo degradation and absorption under the actions of intestinal enzymes. The unused part finally being excreted in the form of earthworm feces (Li et al. 2019, Yuvaraj et al. 2021). Thus, the uptake of nCeO2 by earthworm is different from that of plants, in which nanoparticles enter through cell walls and root epidermal cell membranes (Tripathi et al. 2017). For plants, soil bioavailability metals are mainly in the ionic state, the complex dissolved organic matter state, and the acid-soluble fraction (Lewis et al. 2021). Yet, the Ce accumulated in earthworms includes not only easily available fraction, but also the residual fraction, owing to the ways earthworms ingested metals. It can be inferred from the BCR sequential extraction analysis (Fig. 5). Meanwhile, residual fraction is the main Ce form in clay soil, and its toxicity is much less than that of non-residual fraction (A et al. 2016). So, even if the Ce content in earthworms in clay soil was higher, the biomass of earthworm would not be significantly affected.
3.2 Ultrastructural characterization of earthworm intestines
The intestinal mucosal epithelial cells of earthworms were observed as a columnar epithelial monolayer using TEM (Fig. 2A&C). The cell surfaces were densely covered with short microvilli, and the cell structures include a small number of lysosomes and a high electronic density. The cell structures of earthworms treated with nCeO2 were fuzzy and appeared to contain cavities. The organelle structures were decomposed, autolyzed, and diffuse (Fig. 2B&D). Earthworms accumulate pollutants through passive epidermal absorption and active intestinal absorption, which could cause morphological damage to the coelom tissues and intestinal epithelia in the earthworms (Tang et al. 2020). The intestinal mucosal epithelial cells of earthworms were morphologically damaged, indicating that nCeO2 caused toxic reactions in the earthworm at the tissue or organ level (Kwak &An 2021).
3.3 Oxidative stress responses of earthworms
The ROS produced by mitochondria can transmit information and maintain organ functions, and the production and elimination of ROS in the body are in a balanced state under normal conditions (Roy et al. 2020). When stimulated by external pollutants, cells produce excessive oxygen anions and hydrogen peroxide, potentially causing mitochondrial damage, that could interrupt protein synthesis and transport, inhibit cell proliferation, and induce apoptosis (Li et al. 2003). The percentages of free-radical change in earthworms exposed to nCeO2 are shown in Fig. 3. On the whole, in sandy loam soils, the free-radical changes in earthworms were evidently greater than in clay soils. The ROS contents of the exposed groups in sandy loam soils, compared with the control, significantly increased by 17.6% − 46.5%. However, in clay soils, the free-radical levels in the nCeO2 treatment groups were not much different from that of the control, except for 100 mg/kg nCeO2 group, which increased by 27.3%.
To minimize the degree of oxidative damage, CAT is induced by cells to catalyze peroxides into harmless O2 and H2O (Zhu et al. 2020). Excessive ROS likely caused oxidative stress, which resulted in the continuous increase in the CAT activity of earthworms exposed to nCeO2 in sandy loam soils (Fig. 3b). However, in clay soils, the CAT activity was obviously reduced.
When generated free radicals cannot be completely neutralized by the antioxidative enzyme system, they eventually cause oxidative damage that decreases proteins solubility and causes lipid peroxidization in the organism (Zhang et al. 2021). Protein carbonylation (PCO) is the most frequent form of protein oxidation, and it is stable and irreversible (Márquez-Lázaro et al. 2021). Malondialdehyde (MDA) is the most abundant reactive carbonyl compound generated through the degradation of unstable lipid hydroperoxide (Maity et al. 2018). Thus, PCO and MDA indirectly reflect the influence of free radicals on proteins and lipids, respectively. As shown in Fig. 3c, in clay soils, the MDA contents of the exposed groups significantly decreased compared with the control. However, in sandy loam soils, the MDA content of exposed groups increased by a certain amount compared with the control. In particular, the MDA content after exposure at 10 mg/kg nCeO2 reached 12.8 nmol/ (g Pr). The fluctuations in the PCO contents were similar to those of the MDA contents (Fig. 3d). Compared with the control, the PCO contents of the exposed groups in clay soils significantly decreased to between 3.2 and 4.1 nmol/ (mg Pr), whereas, in sandy loam soils, it increased by a certain amount. These results demonstrated that, in sandy loam soils, excessive ROS caused oxidative stress, which induced CAT activity in earthworms exposed to nCeO2, and finally led to an increase in their PCO and MDA contents to varying degrees. However, the CAT activities, MDA contents and PCO contents of the exposed groups in the clay soils significantly decreased compared with the control. Thus, the toxicity effects of nCeO2 on earthworms were different in the two soils.
3.4 The MT contents and GSH/GSSG ratio of earthworms
The MT concentrations in earthworms from the two soils are shown in Fig. 4a. MT is a metal-binding peptide with a molecular weight of 6–7 kDa that can chelate various metals ions, such as Zn2+, Cu2+, Cd2+, and Cr3+, through mercaptide linkages and transport the bound metals to chloragogenous tissues to complete the detoxification process (Blindauer &Leszczyszyn 2010, Homa et al. 2016, Hussain et al. 2021). The Ce ions that accumulate in the circular and longitudinal muscles of earthworms cause stress, which can trigger MT synthesis to reduce damage to the epidermal and midgut cells (A et al. 2020).
The Ce bioaccumulation in earthworms in the two soils increased under nCeO2 treatments (Fig. 1b), but the MT contents in earthworms did not increase significantly. Even in earthworms in clay soils exposed to the 50 mg/kg nCeO2 treatment (Ce content, 2.7 mg/kg), the MT content did not significantly increase. This indicated that the concentration of Ce ions in the earthworms in the two soils did not increase and, in clay soils, the Ce bound form may exist in earthworm body with high Ce content. Nanoparticles can penetrate directly into the tissue when the external cuticle layer of organism is eroded or damaged and may also be attached to the inner wall of intestinal lumen (Unrine et al. 2010). Therefore, we speculated that the bioaccumulation and biological effects of nCeO2 on earthworms are related not only to the Ce ion accumulation in the body but also to the unused Ce bound form. Moreover, in addition to the high available acid-soluble, reducible, and oxidizable fraction, the residual fraction can be consumed by earthworms. Therefore, the traditional BCR analysis method, being used to determine the bioavailability of heavy metals for plants, which is not suitable for analyzing the bioaccumulation of nanoparticles in earthworms.
Glutathione (GSH) can further combine with free radicals or heavy metals through its sulfhydryl moiety to excrete harmful substances from the body, thereby maintaining a healthy immune system (Ighodaro &Akinloye 2017). The GSH/GSSG ratio can be used as an indicator of oxidative stress to signify the health and detoxification capability of cells (Colacevich et al. 2011). In our study, the GSH/GSSG ratios of earthworms in clay soils were significantly greater than in sandy loam soils. Additionally, there was a decreased GSH/GSSH ratio in earthworms exposed to nCeO2 in sandy loam soils (Fig. 4b), which may be related to the production of antioxidative enzymes under oxidative stress conditions. This was not enough to alleviate the damage caused by nCeO2 and the inhibition of enzyme activities, indicating that the detoxification capability of the antioxidative defense system was weakened (Yin et al. 2017).
3.5 Ce distribution in soils
As shown in Fig. 5, the major portions of Ce, 80.8% and 83.7%, in the original sandy loam and clay soils, respectively, were associated with the residual phase. With as the nCeO2 concentration added to the two soils increased, the acid-soluble, oxidizable, and reducible fraction continued to decrease, whereas the residual fraction increased to 98.1%-98.9%. This may be related to the increase in the soil pH caused by the nCeO2 concentration, which resulted in the nCeO2 transforming into lower-activity residue. Under neutral or alkaline conditions, the precipitation reactions of hydroxides, sulfides, phosphates, and carbonates that generate heavy metals owing to pH account for larger proportions (García-Gómez et al. 2018), which was also reflected in the sandy loam treatment groups (pH = 7.4).
In the nCeO2-treatment groups, only very small fractions of Ce (0.24–0.29 mg/kg) were present as acid soluble fractions in the sandy loam soils. However, in clay soils, the range of extractable Ce in the acid-soluble fraction was observed as 0.48 -3.0 mg/kg, which was slightly greater than in sandy loam soils. This may be because the low pH of the clay soils (pH = 4.6, Table 1) increased nCeO2 solubility (Cornells et al. 2011). In addition, a high clay content also increases the retention of available Ce in the soil (García-Gómez et al. 2018).
The oxidized Ce fractions in the sandy loam soils were higher than that those in the clay soils at low and very high Ce concentrations. Sandy loam soil has strong water and fertilizer retention capabilities, and its high organic matter (18.4 g/kg) content can convert organic matter in the carbonate-bound state to the organic bound state, thereby reducing the bioavailability of Ce. This type of soil has a large number of colloids and a large specific surface area and surface charge. It is prone to obligate adsorption to form the iron-manganese oxidation-combined state (Li et al. 2021). Natural organic matter makes nCeO2 highly stable in soil suspension owing to the positive surface charged (Van Koetsem et al. 2018). Additionally, surface charge influenced by pH can affect particle-particle interactions as well as particle-soil interactions (Saleh et al. 2008).
The bioaccumulations of heavy metals in earthworms may be significantly affected by soil characteristic (such as pH and organic matter) (Wang et al. 2013). The high clay contents (51.5%, Table 1) of clay soils may enhance the aggregation of nCeO2 and lead to the formation of clusters (Khushi et al. 2018, Van Koetsem et al. 2018), reducing ion release and its toxic effects on earthworms. This might explain why the biomass of earthworms in clay soils is not obvious negatively affected even when there is high Ce concentration in the organisms. Similarly, even after 28 days of exposure to 10,000 mg/kg concentration, nCeO2 has no effects on the survival and reproduction of earthworms (Lahive et al. 2014).
The differences in the toxicity levels of heavy metal ions are also largely the result of the different reducing capabilities of soils (Shahid et al. 2017). Although nCeO2 has a nanotoxicity level, the high clay contents of clay soils will directly or indirectly lead to spontaneous redox reactions involving Ce ions, producing properties similar to those of SOD and CAT that reduce the toxicity of free radicals (Ashraf et al. 2017, Lin et al. 2019). If this property is dominant during the whole process, then oxidative damage is alleviated, and the MDA and PCO contents in earthworms can be reduced (Tassi et al. 2017, You et al. 2021). In addition, under acidic conditions, nCeO2 exists as spherical particles instead of rod-shaped particles, which increases migratory capacity and reduces toxicity because of the reduced radii (Darlington et al. 2009). Thus, the Ce bioaccumulation in earthworms in clay soils was higher, whereas nCeO2 was more toxic to earthworms in sandy loam soils as assessed by their biomass, morphology, and antioxidative damage.