Interactions between earthworms, trees, soil nutrition and metal mobility in amended Pb/Zn mine tailings from Guangdong, China

This paper reports research that attempts to rehabilitate toxic Pb/Zn mine tailings, in Guangdong, China, to achieve a healthy functional soil that supports sustainable vegetation. We studied the effects of the earthworm Pheretima guillelmi on the growth of a woody legume Leucaena leucocephala on Pb/Zn mine tailings diluted with varying amounts of mineral soil in pot experiments. L. leucocephala grew successfully on tailings with a 25% (w/w) soil amendment, but P. guillelmi only survived and actively burrowed with a 50% soil amendment. The presence of earthworms improved the yield of plants by 10-30%. Whilst earthworms marginally increased available N and P in soil, they increased uptake of phosphorus (by about 10%) to above-ground plant tissues. Six-month-old plants were more sensitive than 10-month plants to metal stress. P. guillelmi increased bioavailable metal concentrations in the amended spoils, accompanied by a direct increase of metal uptake by the plants. Increased metal uptake by plants was largely due to the higher dry matter production stimulated by earthworm activity, but this increased the rate of metal uptake into plants from spoil by at least 16% and as much as 53%. These results demonstrate that we should broaden the ecological context of phytoremediation by considering the plant-soil-animal interactions that influence metal mobility.     

Impacts of earthworms and arbuscular mycorrhizal fungi (Glomus intraradices) on plant performance are not interrelated

Earthworms and arbuscular mycorrhizal fungi (AMF) might interactively impact plant productivity; however, previous studies reported inconsistent results. We set up a three-factorial greenhouse experiment to study the effects of earthworms (Aporrectodea caliginosa Savigny and Lumbricus terrestris L.) and AMF (Glomus intraradices N.C. Schenck & G.S. Sm.) on the performance (productivity and shoot nutrient content) of plant species (Lolium perenne L., Trifolium pratense L. and Plantago lanceolata L.) belonging to the three functional groups grasses, legumes and herbs, respectively. Further, we investigated earthworm performance and plant root mycorrhization as affected by the treatments. Our results accentuate the importance of root derived resources for earthworm performance since earthworm weight (A. caliginosa and L. terrestris) and survival (L. terrestris) were significantly lower in microcosms containing P. lanceolata than in those containing T. pratense. However, earthworm performance was not affected by AMF, and plant root mycorrhization was not modified by earthworms. Although AMF effectively competed with T. pratense for soil N (as indicated by δ¹⁵N analysis), AMF enhanced the productivity of T. pratense considerably by improving P availability. Remarkably, we found no evidence for interactive effects of earthworms and AMF on the performance of the plant species studied. This suggests that interactions between earthworms and AMF likely are of minor importance.     

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

The effects of soil disturbance and residue retention on the functionality of the symbiosis between medic (Medicago truncatula L.) and arbuscular mycorrhizal fungi (AMF) were assessed in a two-stage experiment simulating a crop rotation of wheat (Triticum aestivum L.) followed by medic. Plants were inoculated or not with the AMF, Glomus intraradices and Gigaspora margarita, separately or together. The contribution of the arbuscular mycorrhizal (AM) pathway for P uptake was determined using ³²P-labeled soil in a small hyphal compartment accessible only to hyphae of AMF. In general AM colonization was not affected by soil disturbance or residue application and disturbance did not affect hyphal length densities (HLDs) in soil. At 4 weeks disturbance had a negative effect on growth and phosphorus (P) uptake of plants inoculated with G. margarita, but not G. intraradices. By 7 weeks disturbance reduced growth of plants inoculated with G. margarita or AMF mix and total P uptake in all inoculated plants. With the exception of plants inoculated with G. margarita in disturbed soil at 4 weeks, the AM pathway made a significant contribution to P uptake in all AM plants at both harvests. Inoculation with both AMF together eliminated the negative effects of disturbance on AM P uptake and growth, showing that a fungus insensitive to disturbance can compensate for loss of contribution of a sensitive one. Application of residue increased growth and total P uptake of plants but decreased ³²P in plants inoculated with the AMF mix in disturbed soil, compared with plants receiving no residue. The AMF responded differently to disturbance and G. intraradices, which was insensitive to disturbance, compensated for lack of contribution by the sensitive G. margarita when they were inoculated together. Colonization of roots and HLDs in soil were not good predictors of the outcomes of AM symbioses on plant growth, P uptake or P delivery via the AM pathway.     

Earthworms can modify effects of hydrochar on growth of Plantago lanceolata and performance of arbuscular mycorrhizal fungi

Hydrothermal carbonization (HTC) is a method to produce carbonized material at relatively low temperatures (180–250 °C) under pressure and aqueous conditions. The product is called hydrochar and can be used as a soil amendment. However, applied in high dosages it may have detrimental effects on plants or soil biota. The potential impact of hydrochar amendment on beneficial soil organisms such as arbuscular mycorrhizal fungi (AMF) and earthworms and their interactions are not well understood. The goal of the present study was to determine effects of hydrochar on plant growth and soil biota and to evaluate interactions of earthworms and hydrochar on plant and AMF performance and to identify underlying mechanisms. In a greenhouse experiment, we investigated the effect of hydrochar at different addition rates (control, 1% and 10%, v/v) with or without the earthworm Aporrectodea caliginosa on the growth of Plantago lanceolata L. and the performance of its AMF. We observed a positive interaction between earthworms and 10% hydrochar on shoot and root biomass: added as a single treatment hydrochar had a negative effect on plant growth at this dosage, but plant biomass increased significantly when hydrochar was added together with earthworms. Root colonization by AMF increased significantly with increasing concentration of hydrochar, but was not affected by earthworms. Contrastingly, extraradical hyphal length of AMF was reduced by earthworms, but not affected by hydrochar. Thus, hydrochar and earthworms affected the performance of AMF, albeit of different AMF structures and in different directions. Our results indicate that earthworms may play an important role in alleviating the negative impacts of high dosages of hydrochar on plant growth; such interactions should move into focus of future research on potential effects of HTC materials.     

Toxicity of Pb/Zn mine tailings to the earthworm <Emphasis Type="Italic">Pheretima</Emphasis> and the effects of burrowing on metal availability

Lead and zinc mine tailings from Guangdong, China were found to be toxic to two common species of the earthworm Pheretima, unless the spoil was diluted by at least 20% with an artificial uncontaminated soil. With different soil dilutions, there were significant differences in mortality, growth and Zn uptake between the two species. Zinc and arsenic were principally responsible for the toxicity of the tailings. LC₅₀ values for Zn, Pb, Cu and As for Pheretima were similar to those reported in the literature for Eisenia fetida. Following inoculation with the earthworms, spoil pH, EC and available metals increased; available-Pb and -Zn concentrations increased by up to 48.2% and 24.8% respectively. The findings suggest that earthworms may play an important role in reclamation of mining spoils to achieve a healthy soil supporting sustainable vegetation. Potentially increased toxicity of metals to plants and animals, through increased bioavailability in the presence of earthworms, is discussed in the context of their role in modifying the efficiency of phytoremediation treatments.     

Plant type differently promote the arbuscular mycorrhizal fungi biodiversity in the rhizosphere after revegetation of a degraded, semiarid land

It is suggested that the diversity of arbuscular mycorrhizal fungi (AMF) and their association with distinct plants species are crucial in the early stages of revegetation procedures since the AMF roots colonisation plays an important role improving plant establishment and growth. We carried out a study where we analyse the AMF community composition in the roots of Ephedra fragilis, Rhamnus lycioides, Pistacia lentiscus and Retama sphaerocarpa fourteen months after revegetation in a Mediterranean semiarid degraded area of southeast Spain in order to verify whether different plant species can variably promote the diversity of AM fungi in their rhizospheres after planted. We analysed a portion of approximately 795 bases pairs of the small-subunit ribosomal DNA by means of nested PCR, cloning, sequencing and phylogenetic analyses. Eight fungal sequence types belonging to Glomus group A and B and to the genus Paraglomus were identified. The different plant species had different AM fungal community composition. Thus, R. lycioides harboured the highest number of four fungal sequence types while from E. fragilis only two types could be characterized that were specific for this plant species. P. lentiscus and R. sphaerocarpa harboured each one three sequence types and two of them were shared. All AMF sequence types were found in the natural soil. These results show that one effective way of restoring degraded lands is to increase the number of plant species used, which would increase the AMF diversity in the soil and thus the below-ground, positive interactions.     

Competition between invasive earthworms (Amynthas corticis, Megascolecidae) and native North American millipedes (Pseudopolydesmus erasus, Polydesmidae): Effects on carbon cycling and soil structure

Invasive earthworms can have significant impacts on C dynamics through their feeding, burrowing, and casting activities, including the protection of C in microaggregates and alteration of soil respiration. European earthworm invasion is known to affect soil micro- and mesofauna, but little is known about impacts of invasive earthworms on other soil macrofauna. Asian earthworms (Amynthas spp.) are increasingly being reported in the southern Appalachian Mountains in southeastern North America. This region is home to a diverse assemblage of native millipedes, many of which share niches with earthworm species. This situation indicates potential for earthworm-millipede competition in areas subject to Amynthas invasion.In a laboratory microcosm experiment, we used two ¹³C enriched food sources (red oak, Quercus rubra, and eastern hemlock, Tsuga canadensis) to assess food preferences of millipedes (Pseudopolydesmus erasus), to determine the effects of millipedes and earthworms (Amynthas corticis) on soil structure, and to ascertain the nature and extent of the interactions between earthworms and millipedes. Millipedes consumed both litter species and preferred red oak litter over eastern hemlock litter. Mortality and growth of millipedes were not affected by earthworm presence during the course of the experiment, but millipedes assimilated much less litter-derived C when earthworms were present.Fauna and litter treatments had significant effects on soil respiration. Millipedes alone reduced CO₂ efflux from microcosms relative to no fauna controls, whereas earthworms alone and together with millipedes increased respiration, relative to the no fauna treatment. CO₂ derived from fresh litter was repressed by the presence of macrofauna. The presence of red oak litter increased CO₂ efflux considerably, compared to hemlock litter treatments.Millipedes, earthworms, and both together reduced particulate organic matter. Additionally, earthworms created significant shifts in soil aggregates from the 2000-250 and 250-53 μm fractions to the >2000 μm size class. Earthworm-induced soil aggregation was lessened in the 0-2 cm layer in the presence of millipedes. Earthworms translocated litter-derived C to soil throughout the microcosm.Our results suggest that invasion of ecosystems by A. corticis in the southern Appalachian Mountains is unlikely to be limited by litter species and these earthworms are likely to compete directly for food resources with native millipedes. Widespread invasion could cause a net loss of C due to increased respiration rates, but this may be offset by C protected in water-stable soil aggregates.     

Using native plants to evaluate the effect of arbuscular mycorrhizal fungi on revegetation of iron tailings in grasslands

A pot experiment was conducted to evaluate native plant species associated with exogenous AMF for their suitability in the revegetation of iron mine tailings of Inner Mongolia grassland. Agropyron cristatum (L.) Gaertn. and Elymus dahuricus Turcz. associated with AMF, Glomus mosseae, or Glomus versiforme, were grown on iron mine tailings to assess the mycorrhizal effects on plant growth, mineral nutrition uptake, C:N:P stoichiometry, and heavy metals uptake. The symbiotic associations were successfully established between exogenous AMF and two native plants, and root colonization rates of G. versiforme were significantly (P?<?0.05) higher than those of G. mosseae. G. versiforme was more effective than G. mosseae in promoting plant growth by significantly (P?<?0.05) increasing the concentrations of N, P, and K and decreasing the ratios of C:N:P. The shoot and root dry weights of A. cristatum and E. dahuricus were increased by 51–103 %. The N, P, and K concentrations of shoots and roots of two plants were increased by 18–236 %. Inoculation with AMF also significantly (P?<?0.05) decreased concentrations of heavy metals in the shoots and increased those in the roots, indicating that AMF could confer some degree of heavy metal tolerance to plants. The results indicated that plant inoculation with G. versiforme was more suitable than inoculation with G. mosseae for the revegetation of iron mine tailings. The experiment provided evidence for the potential use of local plant species in combination with exogenous AMF for ecological restoration of metalliferous tailings in arid and semi-arid grassland.     

The effect of feeding behavior on Hg accumulation in the ecophysiologically different earthworms Lumbricus terrestris and Octolaseon cyaneum: A microcosm experiment

 A soil microcosm experiment was performed to assess the uptake of Hg from various Hg-spiked food sources (soil, leaf litter and root litter of Trifolium alexandrinum) by two earthworm species, Lumbricus terrestris (anecic) and Octolaseon cyaneum (endogeic). Treatments were applied in which one of the three food sources was Hg spiked and the other two were not. Additional treatments in which all or none of the food sources were Hg spiked were used as controls. Uptake of Hg from soil into tissues of both earthworm species was significantly higher than uptake of Hg from leaf litter or root litter, indicating that soil may be the most important pool for the uptake of Hg into earthworms. In addition, the anecic L. terrestris significantly accumulated Hg from all Hg-spiked food sources (leaf litter, root litter and soil), whereas the endogeic O. cyaneum took up Hg mainly from soil particles. Interestingly, there was no further increase in Hg in L. terrestris when all food sources were Hg spiked compared to the single Hg-spiked sources. This may be attributed to the relatively high Hg content in the soil, which may have influenced the feeding behavior of the earthworms, although their biomass did not significantly decline. We suggest that, in addition to the physiological differences, feeding behavior may also play a role in the contrasting uptake of Hg by the two earthworm species.     

Effects of arbuscular mycorrhizal fungi on maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) grown in rare earth elements of mine tailings

Rare earth elements (REE) of mine tailings have caused various ecological and environmental problems. Revegetation is one of the most cost-effective ways to overcome these problems, but it is difficult for plants to survive in polluted tailings. Arbuscular mycorrhizal fungi (AMF) can provide biotic and abiotic stress tolerance to its host plant and has widely adopted for the revegetation of degraded ecosystems. However, little is known about whether AMF plays role in facilitating the revegetation of REE of mine tailings. The objective was to investigate the uptake of nutrients and REE when plants are inoculated with AMF. A greenhouse pot experiment was conducted on the effects of Glomus mosseae and Glomus versiforme for the growth, nutritional status, and uptake of REE and heavy metals by maize (Zea mays L.) or sorghum (Sorghum bicolor L. Moench) grown in REE of mine tailings. The results indicated that symbiotic associations were successfully established between AMF and the two plant species. G. versiforme was more effective than G. mosseae at promoting plant growth by significantly increasing the uptake of nitrogen (N), phosphorus (P), and potassium (K) and decreasing carbon:nitrogen:phosphorus (C:N:P) stoichiometry. The shoot and root dry weights of the two plant species were increased by 211–387% with G. versiforme inoculation. Maize and sorghum exhibited significant differences in the REE concentrations in response to the colonization by AMF. The shoot and root lanthanum (La), cerium (Ce), praseodymium (Pr), and neodymium (Nd) concentrations of the maize inoculated with G. versiforme were decreased by approximately 70%, whereas those in the roots of sorghum were increased by approximately 70%. G. mosseae only significantly decreased the La, Ce, Pr, and Nd concentrations in the maize shoots. Inoculation with AMF also significantly decreased the concentration of certain heavy metals in the shoots and roots of maize and sorghum. These findings indicate that AMF can alleviate the effects of REE and heavy metal toxicity on plants and enhance the ability of plants to adapt to the composite adversity of REE in mine tailings.     

The effect of two endogeic earthworm species on zinc distribution and availability in artificial soil columns

The objective of this study was to determine the impact of earthworm bioturbation on the distribution and availability of zinc in the soil profile.Experiments were carried out with Allolobophora chlorotica and Aporrectodea caliginosa in 24 perspex columns (∅ 10 cm), filled with 20-23 cm non-polluted soil (OM 2%, clay 2.9%, pH 0.01 M CaCl₂ 6.4), that was covered by a 3-5 cm layer of aged zinc spiked soil (500 mg Zn/kg dry soil) and another 2 cm non-polluted soil on top. After 80 and 175 days, columns were sacrificed and each cm from the top down to a depth of 15 cm was sampled. Earthworm casts, placed on top of the soil, were collected. Each sample was analyzed for total and CaCl₂-exchangeable zinc concentrations.Effects of earthworm bioturbation were most pronounced after 175 days. For A. chlorotica, total and CaCl₂-exchangeable zinc concentrations in the polluted layers were lower with than without earthworms. Total zinc concentrations in the non-polluted layers were higher in columns with earthworms. Casts of A. chlorotica collected on the soil surface showed slightly higher total zinc concentrations than non-polluted soil. Casts were found throughout the whole column. For A. caliginosa there were no differences in total zinc concentration between columns with and without earthworms. CaCl₂-exchangeable zinc concentrations in the polluted layers were lower for columns with earthworms. Casts were mainly placed on top of the soil and contained total zinc concentrations intermediate between those in non-polluted and polluted soil layers.This study shows that different endogeic earthworm species have different effects on zinc distribution and availability in soils. A. chlorotica transfers soil throughout the whole column, effectively mixing it, while A. caliginosa decreases metal availability and transfers polluted soil to the soil surface.     

A Laboratory Study on Revegetation and Metal Uptake in Native Plant Species from Neutral Mine Tailings

Lygeum spartum, Zygophyllum fabago and Piptatherum miliaceum are typical plant species that grow in mine tailings in semiarid Mediterranean areas. The aim of this work was to investigate metal uptake of these species growing on neutral mine tailings under controlled conditions and their response to fertilizer additions. A neutral mine tailing (pH of soil solution of 7.1–7.2) with high total metal concentrations (9,100 and 5,200 mg kg^?1 Zn and Pb, respectively) from Southern Spain was used. Soluble Zn and Pb were low (0.5 and <0.1 mg l^?1, respectively) but the major cations and anions reached relatively high levels (e.g. 2,600 and 1,400 mg l^?1 Cl and Na). Fertilization caused a significant increase of the plant weight for the three species and decreased metal accumulation with the exception of Cd. Roots accumulated much higher metal concentrations for the three plants than shoots, except Cd in L. spartum. Shoot concentrations for the three plants were 3–14 mg kg^?1 Cd, 150–300 mg kg^?1 Zn, 4–11 mg kg^?1 Cu, and 1–10 mg kg^?1 As, and 6–110 mg kg^?1 Pb. The results indicate that neutral pH mine tailings present a suitable substrate for establishment of these native plants species and fertilizer favors this establishment. Metal accumulation in plants is relatively low despite high total soil concentrations.     

Inoculating maize fields with earthworms (Aporrectodea trapezoides) and an arbuscular mycorrhizal fungus (Rhizophagus intraradices) improves mycorrhizal community structure and increases plant nutrient uptake

Earthworms and arbuscular mycorrhizal fungi (AMF) are important macrofauna and microorganisms of the rhizosphere. The effect of the inoculation of soil with earthworms (Aporrectodea trapezoides) and mycorrhiza (Rhizophagus intraradices) on the community structure of mycorrhizal fungi and plant nutrient uptake was determined with split plots in a maize field. Maize plants were inoculated or not inoculated with AMF, each treated with or without earthworms. Wheat straw was added as a feed source for earthworms. Inoculating AMF significantly increased maize yield (p?<?0.05), and the yield was further enhanced by the addition of earthworms. Alkaline phosphomonoesterase activities, soil microbial biomass carbon (SMBC) and nitrogen (SMBN) increased with the addition of both earthworms and AMF. Soil inorganic N and available K were positively affected by earthworms, while available P showed a negative relationship with AMF. Treatment with both AMF and earthworms increased shoot and root biomass as well as their N and P uptake by affecting soil phosphomonoesterase and urease activities, SMBC, SMBN, and the content of available nutrients in soil. The applied fungal inoculants were successfully traced by polymerase chain reaction with novel primers (AML1 and AML2) which target the small subunit rRNA gene. The amplicons were classified by restriction fragment length polymorphism and sequencing. Moreover, field inoculation with inocula of non-native isolates of R. intraradices appeared to have stimulated root colonization and yield of maize. Adding earthworms might influence native AMF community, and the corresponding abundance increased after earthworms were inoculated, which has positive effects on maize growth.     

Earthworm-mediated maternal effects on seed germination and seedling growth in three annual plants

Many ecological studies have pointed out maternal effects in plants and shown that plant maternal environment influences germination of their seed and subsequent seedling growth. However, few have tested for maternal effects induced by soil macroorganisms. We tested whether two earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) trigger such maternal effects on seed germination and seedling growth of three plant species (Veronica persica, Poa annua and Cerastium glomeratum). Our results show that, through maternal effects, A. caliginosa enhanced seed germination (V. persica and P. annua) and seedling growth (C. glomeratum and P. annua) while L. terrestris reduced seed germination only in V. persica. In some cases, the increase in germination rates of seeds produced in the presence of earthworms was associated with a reduction of nitrogen content in seeds. These results show that earthworms induce maternal effects in plants and that the size and direction of these effects depend on the combination of plant and earthworm species.     

Soil properties and metal accumulation by earthworms in the Siena urban area (Italy)

This paper reports the results of a study focused on the metal (Cd, Co, Cr, Cu, Ni, Pb, Sb, U and Zn) distribution in soils and uptake and accumulation by earthworms Nicodrilus caliginosus (Savigny) from urban, peri-urban, green-urban and non-urban zones of Siena municipality (central Italy). The main goal was to define the influence of soil properties and metal soil contents on the uptake of these contaminants by earthworms. Data indicated that Cd, Cu, Pb, Sb and Zn soil contents increased in the following order: non-urban < green-urban < peri-urban < urban soils, suggesting that vehicular traffic affects the distribution of these metals. Pb and Sb were the main soil contaminants and their highest enrichments were found in urban sites where stop-and-go traffic occurs. Concentrations of these traffic-related metals in earthworms showed a distribution pattern similar to that in soil, suggesting that soil contamination influenced the uptake of Cd, Cu, Pb, Sb and Zn by N. caliginosus. There were significant positive correlations between Cd, Pb and Sb earthworm concentrations and their soil contents. The lack of correlation for Cu and Zn could be due to the physiological regulation of these elements by earthworms. Statistical analysis pointed out that the uptake and accumulation of Cd, Cu, Pb, Sb and Zn by earthworms were affected by some soil physicochemical properties such as the organic carbon and carbonate contents that are able to rule the bioavailability of metals in soils.     

Field inoculation effectiveness of native and exotic arbuscular mycorrhizal fungi in a Mediterranean agricultural soil

In sustainable agriculture, arbuscular mycorrhizal (AM) fungal inoculation in agronomical management might be very important, especially when the efficiency of native inocula is poor. Here, we assessed the effect of native and exotic selected AM fungal inocula on plant growth and nutrient uptake in a low input Trifolium alexandrinum-Zea mays crop rotation. We evaluated the effects of four exotic AM fungal isolates on T. alexandrinum physiological traits in greenhouse. Then, the field performances of T. alexandrinum inoculated with the exotic AMF, both single and mixed, were compared to those obtained with a native inoculum, using a multivariate analysis approach. Finally, we tested the residual effect of AM fungal field inoculation on maize as following crop. Multivariate analysis showed that the field AM fungal inoculation increased T. alexandrinum and Z. mays productivity and quality and that the native inoculum was as effective as, or more effective than, exotic AM fungal isolates. Moreover, the beneficial effects of AMF were persistent until the second year after inoculation. The use of native AMF, produced on farm with mycotrophic plants species, may represent a convenient alternative to commercial AM fungal inocula, and may offer economically and ecologically important advantages in sustainable or organic cropping systems.     

铅锌矿废弃地尾矿砂对银合欢幼苗定居的影响

研究了广东省仁化县韶关凡口铅锌矿废弃地尾矿砂对银合欢(Leucaena glauca)幼苗定居的影响。结果表明，尾矿砂中Pb、Zn、Cu、Cd等重金属含量极高，Pb、Zn比对照土壤高100倍左右，Cu、Cd高4～5倍，植物对重金属的吸收、积累量较大，且多集中于根部。尾矿砂中硝态氮(NO^-₃-N)和铵态氮(NH⁺₄-N)含量很低，仅为对照土壤的13%～23%。银合欢幼苗在尾矿砂中能生长，但其叶片光合色素含量较少，株高、复叶数、植株干物质量等生物量显著降低，这主要是由于重金属含量过高导致根系活力降低，限制了植物对水分和营养物质的吸收，其次是土壤贫瘠的影响。     

Earthworm avoidance of biochar can be mitigated by wetting

Biochar has a great potential for enhancing soil fertility and carbon sequestration while enabling beneficial waste disposition. Because of the potential for widespread application, it is essential to proactively assess and mitigate any unintended consequences associated with soil biochar amendment. We conducted soil avoidance tests, growth and reproduction tests, and oxidative stress assays with the earthworm Eisenia foetida to assess the potential toxicity of soil amended with biochar produced from apple wood chips. Earthworms avoided soils containing 100 and 200 g/kg dry biochar at statistically significant levels (p < 0.05), and after 28-day incubation, these earthworms lost more weight than those in control (unamended) soil. However, biochar did not affect the reproduction of earthworms. We investigated whether the observed avoidance was due to nutrition deficiency, desiccation, or the presence of toxic polynuclear aromatic hydrocarbons (PAHs) formed during biochar production by pyrolysis. Nutrition deficiency was excluded by the lack of earthworm avoidance to soil amended with nutrient-deficient sand instead of biochar. Although traces of PAH were detected in the tested biochar (e.g., 25.9 μg/kg fluorene, 3290 μg/kg naphthalene, and 102 μg/kg phenanthrene), the lack of lipid peroxidation and no increase in superoxide dismutase activity in biochar-exposed earthworms suggests that presence of toxic compounds was not a likely reason for avoidance. Furthermore, wetting the biochar to its field capacity resulted in statistically undetectable avoidance relative to control soil, indicating that insufficient moisture could be a key factor affecting earthworm behavior in soil amended with dry biochar. To avoid desiccation of invertebrates and enable their beneficial ecosystem services, we recommend wetting biochar either before or immediately after soil application.     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.