Toxicokinetics of selenate in earthworm sub-tissues and potential bio-accessibility assessment of earthworm-derived selenium.

Selenium (Se) pollution is mainly caused by anthropogenic activities, and the resulting biosecurity concerns have garnered significant attention in recent years. Using one-compartmental toxicokinetic (TK) modelling, this study explored the kinetic absorption, sub-tissue distribution, and elimination processes of the main Se species (selenate, Se(VI)) in the cultivated aerobic soil of the earthworm Eisenia fetida. The bio-accessibility of earthworm-derived Se was assessed using an in vitro simulated gastrointestinal digestion test to evaluate its potential trophic risk. The results demonstrated that Se accumulated in the pre-clitellum (PC) and total tissues (TT) of earthworms in a time- and dose-dependent manner. The highest Se levels in the PC, post-clitellum (PoC), and TT were 70.54, 57.93, and 64.26 mg/kg during the uptake phase, respectively. The kinetic Se contents in the earthworms PC and TT were consistent with the TK model but not with PoC. The earthworm TT exhibited a faster uptake (Kus = 0.83-1.02 mg/kg/day) and elimination rate of Se (Kee = 0.044-0.049 mg/kg/day), as well as a shorter half-life time (LT1/2 = 15.88-14.22 days) than PC at low soil Se levels (≤5 mg/kg). Conversely, the opposite trend was observed with higher Se concentrations (10 and 20 mg/kg). These results are likely attributable to the tissue specificity and concentration of the toxicant. Earthworms PC and TT exhibited a higher kinetic Se accumulation factor (BAFk) than steady-state BAF (BAFss), with values ranging from 8 to 24 and 3-13, respectively. Furthermore, the bio-accessibility of earthworm-derived Se to poultry ranged from 66.25 % to 84.35 %. As earthworms are at the bottom of the terrestrial food chain, the high bio-accessibility of earthworm-derived Se poses a potential risk to predators. This study offers data support and a theoretical foundation for understanding the biological footprint of soil Se and its toxicological impacts and ecological hazards.

Selenium toxicity, bioaccumulation, and distribution in earthworms (Eisenia fetida) exposed to different substrates.

Selenium (Se) is an essential microelement for human or animal health. At high concentrations, it can cause Se poisoning. Human activities (such as coal burning and mining) threaten soil biota by mobilizing high levels of Se. We used the earthworm Eisenia fetida as a bio-indicator of environmental pollutants to investigate Se acute toxicity, enrichment, and distribution through exposure tests using filter paper, artificial soil and cow manure. The 24 h- and 48 h-LC50 for the filter paper contact test were 2.7 and 1.52 µg/cm2. In artificial soil test, the 14 d-LC50 and 14 d-biomass inhibition concentration (IC20) were 63.86 and 59.81 mg/kg, respectively. The cow manure resulted in a 2.2- and 2.6-fold higher LC50 and IC20 than artificial soil results, respectively. Earthworms accumulated the largest Se load (89.47 mg/kg) in artificial soil containing 80 mg Se/kg and only accumulated 90.3 mg/kg in cow manure containing 160 mg Se/kg; 46.6-60.59% of the total Se was distributed in the tail of E. fetida. The Se enrichment rate (SERSe) and bioaccumulation factor (BAFSe) scored higher in artificial soil than in cow manure with the same Se concentration exposure, and the highest SERSe was 6.21 and 6.31 mg Se/kg earthworm/d, respectively. The highest BAFSe was 1.49 in artificial soil and 0.75 in cow manure. Our results demonstrate that selenite is more toxic to earthworms living in artificial soil than in cow manure. E. fetida possesses certain Se detoxification mechanisms by distributing Se in the tail.

Responses and resistance capacity of Solanum nigrum L. mediated by three ecological category earthworms in metal-[Cd-As-Cu-Pb]-contaminated soils of North China.

Earthworms play vital functions affecting plant growth and metal accumulation from downground to aboveground. Soil metal mobilization may be combined with use of earthworm and hyperaccumulator-Solanum nigrum to improve its remediation efficiency. Understanding the effects of specific-species earthworm belonging to different ecological categories on mechanisms underlying of S. nigrum is critical for metal-polluted remediation. However, seldom studies concerned earthworm-assisted phytoremediation of metal contaminated soil in Northern China. This study investigated the effects of earthworm (Eisenia fetida, Amynthas hupeiensis and Drawida gisti) on S. nigrum with exposure to uncontaminated and [Cd-As-Cu-Pb]-contaminated soil (referred to as S0 and S1) for 60 days, respectively. In S1 soil, A. hupeiensis (anecic) had stronger effects on growth and metal accumulation in the organs (root, stem, and leaf) of S. nigrum than D. gisti (endogeic) and E. fetida (epigeic), attributing to their ecological category. The BAF values of S. nigrum were generally ranking in Cd (0.66-5.13) > As (0.03-1.85) > Cu (0.03-0.06) > Pb (0.01-0.05); the BAFCd values were ranking in leaf (2.34-5.13) > root (1.96-4.14) > stem (0.66-1.33); BAFAs, BAFCu, and BAFPb were root (0.04-1.63) > stem (0.01-0.09) ≈ leaf (0.01-0.06). A. hupeiensis decreased the TF values of S. nigrum from the roots to the shoots. Co-effects of metal stress and earthworm activity on metal uptake by shoots suggested that A. hupeiensis increased the uptake of As, Cu, and Pb (by 56.3 %, 51.5 %, and 16.2 %, p < 0.05), but not Cd, which appeared to remain steady for prolonged durations. Alterations in the integrated biomarker response index version 2 (IBRv2) values demonstrated that A. hupeiensis (12.65) improved the resistance capacity (stimulated GSH, SnGS1, and SnCu-SOD) of S. nigrum under metal-containing conditions, compared with E. fetida and D. gisti (IBRv2 were 9.61 and 9.11). This study may provide insights into the patterns of 'soil-earthworm-plant system' on improving remediation efficiency of S. nigrum, from the perspective of earthworm ecological niche partitioning.

Eisenia fetida impact on cadmium availability and distribution in specific components of the earthworm drilosphere.

Although the potential of vermiremediation for restoring metal-contaminated soils is promising, the effects of earthworms on the availability of soil metals are still debatable. Most previous studies considered the soil as a "whole black box." Mobilization or immobilization of metals are affected by earthworm activities within drilosphere hotspots under different soil conditions, which has not been specifically studied. Therefore, an improved 2D terrarium was designed to study the impact of earthworm activities on cadmium (Cd) fate in the drilosphere hotspots (burrow wall soils, burrow casts, and surface casts) of different artificially spiked Cd treatments (CK: 0 mg kg-1; LM: 1 mg kg-1; and HM: 5 mg kg-1) with different organic amendments (2% and 10%). The results revealed that Cd increased earthworm activities with the highest cast production in HM and the highest burrow length in LM. Earthworms exhibited a stronger tendency to reduce total Cd concentration by 4.48-13.58% in casts of LM soils, while 3.37-5.22% in burrow walls under HM treatments. Overall, earthworms could increase the availability of Cd in casts under all conditions (55.46-121.01%). The organic amendments decreased the total Cd concentration and increased the availability of Cd in the disturbed soil. A higher amount of organic amendment significantly decreased total Cd concentration of the drilosphere by 1.16-5.83% in LM and HM treatments, while increasing DTPA-Cd concentrations in all components by 23.13-55.20 %, 14.63-35.11%, and 3.30-11.41% in CK, LM, and HM treatments, respectively, except for earthworm non-disturbed soil and no-earthworm soil in HM treatments. Redundancy analysis (RDA) revealed that the moisture, pH, and total carbon contents in soil are the main factors affecting Cd bioavailability. In this study, we decoded the "black box" of soil by making it relatively simple to better understand the effects and mechanisms of earthworm activities on soil metal availability and consequently provided comprehensive insights for using earthworms in soil vermiremediation.

Reproductive damage and compensation of wild earthworm Metaphire californica from contaminated fields with long-term heavy metal exposure.

Reproduction is a significant biological process for organisms responding to environmental stresses, however, little is known about the reproductive strategies of invertebrates under long-term exposure to contaminations. In this study, earthworm Metaphire californica (Kinberg, 1867) from contaminated fields with an increased metal gradient were collected to investigate their reproductive responses. The results showed heavy metals (Cd, Cu, Zn, and Pb) induced histological damage to earthworms' seminal vesicles, including tissue disorders and cavities, and decreases in mature spermatozoa. Sperm morphology analysis indicated deformity rates were up to13.2% (e.g. head swollen or missing) for worms from the most contaminated site, which coincided with DNA damages. Furthermore, the computer-assisted sperm analysis (CASA) system was employed for the evaluation of sperm kinetic traits. Results suggested earthworms exposed to higher contamination showed a lower sperm viability rate but faster sperm velocity after re-exposure with Cd solution (like the curvilinear velocity and straight-line velocity paraments) compared with those from relatively clean sites. The activities of lactate dehydrogenase and sorbitol dehydrogenase showed the highest 32.5% and 12.5% up-regulation respectively with the increased metal gradient. In conclusion, this study elucidated the earthworm reproductive toxicity, underlying reproductive compensation, metal stress-induced damages, and adaptive responses caused by heavy metal exposure, while also providing the possibility of sperm trait analysis (CASA) for related earthworm toxicological studies.

Impact of progressively cumulative exposure of AgNPs on earthworms (Eisenia fetida) and implication for eco-toxicological risk assessment.

Hazardous pollutants released into the real environment mostly own long-lasting cumulative characteristics and have progressively negative impacts on organisms, which are always neglected in laboratory toxicological tests. Here in this study, the different ecotoxicity of Ag nanoparticles (AgNPs) on earthworm Eisenia fetida was compared via various endpoints and transcriptional sequencing between the 28-day progressively repeated (from 60 to 80, final 100 mg/kg) and one-step (directly to 100 mg/kg) exposure. The results showed that earthworms under progressively repeated exposure showed significantly less biomass loss and reproductive inhibition, as well as lower Ag bioaccumulation (15.6 mg/kg) compared with one-step exposure (17.9 mg/kg). The increases in enzyme activities (superoxide enzyme and catalase) and gene expression (metallothionein) also implied higher antioxidant and genetic toxicity in one-step exposed earthworms compared with those from progressively repeated exposure. Furthermore, the transcriptomic analysis identified 582 and 854 differentially expressed genes in the treatments of one-step and repeated exposure respectively compared with the control group. The results of pathway annotation and classification suggested similar enrichments of damage induction but different in toxic stress responses, whereas earthworms from repeated exposure possessed more detoxification-related pathways like translation and multicellular organismal processes. This study innovatively took into account the impacts of processive exposure occurring in the real environment and elucidated distinctions of toxicity and adaptation caused by different exposure patterns, which provided the theoretical basis for real risk identification under the framework and guidance of traditional toxicology, also the implication for the improvement of eco-toxicological risk assessment.

Visual mapping of global nanoplastics research progresses and hotspots: a scientometric assessment analysis.

Environmental plastic wastes are continuously degraded into microplastics (MPs) and nanoplastics (NPs); the latter are more potentially harmful to organisms and human health as their smaller size and higher surface-to-volume ratio. Previous reviews on NPs mainly concentrate on specific aspects, such as sources, environmental behavior, and toxicological effects, but few focused on NPs-related scientific publications from a global point of view. Therefore, this bibliometric study aims to summarize the research themes and trends on NPs and also propose potential directions for future inquiry. Related papers were downloaded from the Web of Science Core Collection database on NPs published from 2008 to 2021, and then retrieved information was analyzed using CiteSpace 6.1 R2 and VOSviewer (version 1.6.). Research on NPs mainly involved environmental behaviors, toxicological effects, identification and extraction of NPs, whereas aquatic environments, especially marine systems, attracted more attentions from these scientists compare to terrestrial environments. Furthermore, the adsorption behavior of pollutants by NPs and the toxicological effects of organisms exposed to NPs are the present hotspots, while the regulation of humic acid (HA) on NPs behaviors and the environmental behavior of NPs in freshwater, like rivers and lakes, are the frontier areas of research. This study also explored the possible opportunities and challenges that may be faced in NPs research, which provide a valuable summary and outlook for ongoing NPs-related research, which may be of intrigue and noteworthiness for relevant researchers.

Genetic evidence behind the Cd resistance of wild Metaphire californica: The global RNA regulation rather than specific mutation of well-known gene.

Heavy metal contamination presents a profound threat to terrestrial biodiversity, yet the genetic adaptation and evolution of field organisms under persistent stress are poorly understood. In this study, the Cd-resistant earthworms Metaphire californica collected from the control (Meihua, MHC) and elevated-pollution (Lupu, LPC) pairwise sites were used to elucidate the underlying genetic mechanism. A 48-h acute test showed that LPC worms exhibited 2.34 times higher LC50 (50% lethal concentration values) compared to MHC ones. The Cd bioaccumulation, metallothionein (MT) protein contents, and MT gene expression of LPC M.californica were all significantly higher than those of MHC worms. The well-known MT gene of M.californica was successfully cloned and identified, however, the encoding nucleotide and amino acids displayed non-observable mutations and the phylogenetic tree also revealed that different populations clustered together. Additionally, the results of transcriptomics sequencing demonstrated 173 differentially expressed genes between LPC and MHC worms, primarily involved in stress-response and detoxification pathways, including signal transduction, material metabolism, and protein exports. The above results confirmed that the crucial MT gene did not undergo genetic mutations but rather exhibited global mRNA regulation responsible for the Cd resistance of M.californica. The current study partially disclosed the stress adaptation and evolution of organisms under long-term in situ contamination, which provides insights into maintaining biodiversity under adverse environment.

Uptake, distribution, and elimination of selenite in earthworm Eisenia fetida at sublethal concentrations based on toxicokinetic model.

Natural and anthropogenic causes have promoted the rapid increase in environmental selenium (Se) levels, and the complex Se metabolism and dynamic in organisms make it challenging to evaluate the toxicity and ecological risks. In this study, the kinetics of selenite in earthworm Eisenia fetida were investigated based on toxicokinetic (TK) model (uptake-elimination phases: 14-14 days). The results showed the highest sub-tissue Se concentrations in pre-clitellum (PC), post-clitellum (PoC) parts, and total earthworms were 95.71, 70.40, and 79.94 mg/kg, respectively, which indicates the distinctive Se uptake capacities of E. fetida. Se kinetic rates in PCs were faster than that of the total E. fetida for both uptake (Kus = 0.30-0.80 mg/kg/day) and elimination phases (Kee = 0.024-0.056 mg/kg/day). Longer half-life times (LT1/2) were observed in the total earthworms (17.85-47.15 d) than PCs (12.28-29.22 d), while non-significant difference was found for the kinetic Se bioaccumulation factor (BAFk) in PC and total earthworms (12-19), which demonstrates that Se can be efficiently bioaccumulated and eliminated in earthworm PC part. Besides, the significant increase Se concentration in PoC with rapid elimination in PC also illustrates that earthworms can alleviate the Se stress by the transformation strategy of Se from the head to tail tissues. In conclusion, the investigation of Se kinetic accumulation and elimination characteristics in this study is helpful for understanding the metabolism and detoxification processes of Se in earthworms, and also providing a theoretical basis for further Se risk assessment using TK model.

Evidence for the metal resistance of earthworm Eisenia fetida across generations (F1 and F2) under laboratory metal exposure.

Environmental stressors are persistent but most toxicological studies always evaluate the risk via short-term acute toxicity, while continuous toxicity and biological resistance across generations are relatively unknown. Here, earthworm Eisenia fetida was laboratory-reared and exposed to historically contaminated soils with an increasing metal gradient (CK, LM and HM), to investigate cross-generation toxicity and resistance of F1 and F2 worms. The results elucidated that biomass and juvenile hatching rate of F2 E. fetida showed maximum decreases of 20.8% and 38.5% than those of F1, which indicated severer toxicity of earthworm offspring. However, metal bioaccumulation in F2 E. fetida showed maximum increases of 150%, 49.2%, 19.7% and 25.5% than F1 for Cd, Cu, Zn and Pb, respectively. F2 E. fetida suffered less oxidative stress because the activities of superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH), and malondialdehyde (MDA) contents were basically lower than that of F1. Meanwhile, the detoxification genes of metallothionein and heat shock protein 70 in F2 E. fetida showed maximum of 296% and 78.9% up-regulations, respectively, which suggested greater metal resistance of F2 E. fetida. This study confirmed the cross-generation toxicity and resistance of earthworms, which provides novel insights to reveal specific contaminant risks from longer lifecycles. CAPSULE: Earthworms under cross-generation exposure can develop metal resistance despite suffering worse toxicity effects.

Microplastics aggravate the joint toxicity to earthworm Eisenia fetida with cadmium by altering its availability.

Microplastics (MPs) have become a global environmental issue, however, the threats of metal-associated MPs to soil ecosystems and their involved processes have not been fully disclosed. In this study, a microcosm experiment with co-exposure of polyethylene and cadmium was conducted to determine their joint effects on the earthworm Eisenia fetida and to explore their relationship with the soil Cd availability that affected by MPs. The results showed that 28-day co-exposure of MPs and Cd significantly induced higher avoidance responses, weight loss and reduced reproduction of earthworms with the increasing content of pollutants. MPs and Cd jointly inhibited the superoxide enzyme (SOD) and peroxidase (POD) activities while increasing the glutathione (GSH) and malondialdehyde (MDA) activities in E. fetida. Histopathological changes and DNA damage to earthworm sperm also occurred in an MPs-dose-dependent manner. In addition, the presence of MPs significantly increased the soil diethylenetriaminepentaacetic acid (DTPA)-Cd concentrations by 1.20-fold and 1.43-fold while increasing the Cd bioaccumulation in E. fetida by 2.65-fold and 1.42-fold in low- and high-Cd-contaminated soil, respectively, which potentially contributed to the aggravation of the joint toxicity to E. fetida. In conclusion, this study demonstrated that microplastics could enhance the cadmium availability in the co-exposure soil which resulted in the joint toxicity of metal-associated MPs to soil organisms. CAPSULE: MPs increased soil Cd availability and potentially aggravated the joint toxicity with Cd to Eisenia fetida.

Impact of soil metals on earthworm communities from the perspectives of earthworm ecotypes and metal bioaccumulation.

The current study elucidates the impact of soil metal contamination on earthworm communities at the ecotype level. A total of 292 earthworms belonging to 13 species were collected in metal-contaminated soils from Wanshou (WSC), Daxing (DXC) and Lupu (LPC) plots (1.40-6.60, 29.4-126, 251-336 and 91.9-109 mg/kg for soil Cd, Cu, Zn and Pb, respectively) in Hunan Province, southern China. The results showed that the total earthworm density and biomass significantly decreased along the increasing metal-contaminated gradient while epigeic earthworms became more dominant than anecic and endogeic earthworms. Redundancy analysis (RDA) showed that soil pH, total nitrogen and Cd concentration were the primary factors influencing earthworm communities, explaining 33.7%, 29.1% and 26.7% of the total variance, respectively. In addition, epigeic earthworm Metaphire californica bioaccumulated more Cd (0.27-0.60 mmol/kg), while endogeic earthworm Amynthas hupeiensis and anecic earthworm Amynthas asacceus bioaccumulated more Cu (0.55-1.62 mmol/kg) and Zn (2.86-6.46 mmol/kg) from soil, respectively, which were related to their habit soils and showed the species-specific bioaccumulation features. Our study discovered the diverse responses of earthworm ecotypes to metal contamination and their specific features of metal bioaccumulation, provide insight for soil risk assessments and for biodiversity conservation from a niche partitioning perspective. CAPSULE: Earthworms of different ecotypes showed different responses to soil metal contamination and species-specific features of metal bioaccumulation.

Reveal the metal handling and resistance of earthworm Metaphire californica with different exposure history through toxicokinetic modeling.

Toxicokinetic (TK) model provides a new approach to mechanistically elucidate the natural variation of metal handling strategy by adaptive and sensitive earthworm populations. Here, TK model was applied to explore the metal handling and resistance strategy of wild Metaphire californica with different historical exposure history through a 12-day re-exposure and another 12-day elimination incubation. M. californica populations showed different kinetic strategies for non-essential metals (Cd and Pb) and essential metals (Zn and Cu), which were closely related to their exposure history. M. californica from the most serious Cd-contaminated soil showed the fastest kinetic rates of both Cd uptake (K1 = 0.78 gsoil/gworm/day) and elimination (K2 = 0.23 day-1), and also had the lowest Cd half-life (t1/2 = 3.01 day), which demonstrated the potential Cd-resistance of wild M. californica from Cd-contaminated soils. Besides, the comparative experiment showed totally different metal kinetics of laboratory Eisenia fetida from field M. californica, suggesting the impacts of distinct exposure history and species-specifical sensitivities. These findings provide a novel approach to identify and quantify resistance using TK model and also imply the risk of overlooking existing exposure background and interspecies extrapolation in eco-toxicological studies and risk assessments.

Use of integrated biomarker response for studying the resistance strategy of the earthworm Metaphire californica in Cd-contaminated field soils in Hunan Province, South China.

Research was conducted to study the response and detoxification mechanisms of earthworms collected from Cd-contaminated areas in Hunan Province, South China. Metaphire californica, the dominant earthworm species in fields, referred as earthworm-A and -B that collected from low- (0.81 mg kg-1) and high-Cd soil (13.3 mg kg-1), respectively, for exchanging incubation in laboratory. The results showed that earthworm-A gradually accumulated higher Cd when exposed in the high-Cd soil, whereas Cd concentration of earthworm-B decreased after being transferred to low-Cd soil (albeit BAFCd >20). The integrated biomarker response index was calculated with the biomarkers of antioxidant systems (e.g., superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH), glutathione peroxidase (GPx), glutathione-S transferase (GST), and malondialdehyde (MDA)) and energy index (e.g., protein and glycogen) in M. californica. GSH, GPx, and GST contributed the most to the integrated biomarker response (IBR) in earthworm-A when exposed in high-Cd soil for 14 d. Earthworm-B responded with higher GST and GPx activities and decreased protein content in low-Cd soil. For 28 d, the response of earthworm-A was not evident in either low- or high-Cd soil, and the inductive effect of metal stress on earthworm-B tended to be stable, except for the higher MDA content (p < 0.05) when exposed in low-Cd soil. The IBR index of earthworm-B (2.93 and 3.40) in low- and high-Cd soil, respectively, was higher than that of earthworm-A (0.89 and 1.0). Overall, earthworm-A exhibited a detoxification process to resist high-Cd toxicity from low-to high-Cd soil. Earthworm-B exhibited a physiological resilience once its habitat had changed to a normal or low-Cd soil environment, possibly owing to the cost of its resistance adaptation to the historical highly contaminated soil in fields.

Role of biochar and Eisenia fetida on metal bioavailability and biochar effects on earthworm fitness.

Biochar has gained extensive attention due to its remediation role in soil pollution. However, its hazardous effects on the soil fauna in contaminated soil and its remediation efficiency affected by soil organisms are still obscure. The individual and combined effects of biochar and earthworms (Eisenia fetida) on soil properties, metal bioavailability, and earthworm fitness were investigated in historically heavy metal (HM)-contaminated soil. The results showed that biochar increased the soil pH by 0.31, decreased DTPA-extractable Cd, Cu, Zn and Pb contents by 11.9%, 14.3%, 5.27% and 23.8%, respectively, and immobilized the HMs from a bioavailable fraction to a residual fraction. The co-incubation of biochar and E. fetida decreased soil pH by 0.11 and increased DTPA-extractable Cu, Zn, and Pb contents by 3.75%, 20.9% and 4.43%, respectively. The results of the correlation analysis showed that soil pH was significantly negatively correlated with HM bioavailability, and it was a potential factor contributed to this opposite effect. Furthermore, biochar decreased the biomass growth of E. fetida and inhibited the activities of SOD, CAT and GSH in E. fetida by 31.1%, 51.3% and 29.6% after 28 days of incubation. Overall, biochar and E. fetida showed the opposite effects on the soil remediation, and biochar also led to a negative effect on earthworms. These findings provided insights on verifying the actual remediation effects of biochar and its ecological risk in situ soil remediation.

The effects of biochar and AM fungi (Funneliformis mosseae) on bioavailability Cd in a highly contaminated acid soil with different soil phosphorus supplies.

Due to the increase of cadmium (Cd)-contaminated land area worldwide, effective measures should be taken to minimize the Cd bioavailability in crops. A study was performed to explore the effectiveness of biochar pyrolyzed from rice straw at 400 °C alone or combined with AM fungi (Funneliformis mosseae) on the corn growth and Cd uptake in corn in Cd-contaminated soil with different levels of phosphorus supplies. The results showed that biochar significantly reduced 66% and 38% of Cd uptake in shoot and root respectively (P < 0.001) attributed to the increase of soil pH and dissolved organic matter. In contrast, AM fungi inoculation of corn plants had little effect on Cd bioavailability due to the AM was suppressed by the highly contaminated acid soil (31.76 mg/kg), and had neither synergistic effect with biochar on decreasing the Cd bioavailability with high or low phosphorus supplies. This study demonstrated that biochar application could be a promising method to immobilize Cd in the contaminated soil to ensure the safety of agro-product while high Cd-contaminated soil would suppress the growth of mycorrhizae, so this remains an open question to be further studied.

Long-term effects of intensive application of manure on heavy metal pollution risk in protected-field vegetable production.

Heavy metal contamination in protected-field vegetable production has aroused widespread concern and manure is considered to be one of the contamination sources. Little is known about its long-term effects on heavy metal pollution in uncontaminated soils. A 15-year protected-field vegetable production experiment was carried out with three manure treatments (chicken manure: cattle manure = 3:1) with high (HMAR), medium (MMAR) and low (LMAR) application rates to evaluate the long-term risks of heavy metal pollution. It was found that continuous and high manure application rates significantly increased the total concentrations of soil Cd, Zn, Cr, and Cu rather than Pb, Ni or As. The high application rate of manure also increased soil available heavy metals although the soil organic matter was increased as well. Though total soil Cd under the HMAR exceeded the threshold of national soil standard, Cd content in tomato and fennel still complied with the food safety requirements of vegetables. Generally, the accumulation rates of soil Zn, Cu, and Cr with 1 t⋅ha-1 of manure application in three treatments were ranked by HMAR < MMAR < LMAR. Based on the results of the ratio of heavy metal accumulation risk (RAR), Zn, Cu, and Cr under HMAR and Cd and Zn under MMAR would exceed their soil threshold values within 100 years and RAR could be a useful indicator for monitoring the long-term risk of soil heavy metal pollution. Recommended manure application rates to guarantee a 100-year period of clean production were 44, 74, and 63 t⋅ha-1⋅yr-1 for Zn, Cu, and Cr, respectively. Measurements should be taken to minimize the risk of heavy metals (Cd, Zn, Cr, and Cu) pollution sourced from manure to ensure food safety and 'cleaner' protected-field vegetable production.