Dependence of Positive Bias Stress Instability on Threshold Voltage and Its Origin in Solution-Processed Aluminum-Doped Indium Oxide Thin-Film Transistors.

The initial electrical characteristics and bias stabilities of thin-film transistors (TFTs) are vital factors regarding the practical use of electronic devices. In this study, the dependence of positive bias stress (PBS) instability on an initial threshold voltage (VTH) and its origin were analyzed by understanding the roles of slow and fast traps in solution-processed oxide TFTs. To control the initial VTH of oxide TFTs, the indium oxide (InOx) semiconductor was doped with aluminum (Al), which functioned as a carrier suppressor. The concentration of oxygen vacancies decreased as the Al doping concentration increased, causing a positive VTH shift in the InOx TFTs. The VTH shift (∆VTH) caused by PBS increased exponentially when VTH was increased, and a distinct tendency was observed as the gate bias stress increased due to a high vertical electric field in the oxide dielectric. In addition, the recovery behavior was analyzed to reveal the influence of fast and slow traps on ∆VTH by PBS. Results revealed that the effect of the slow trap increased as the VTH moved in the positive direction; this occured because the main electron trap location moved away from the interface as the Fermi level approached the conduction band minimum. Understanding the correlation between VTH and PBS instability can contribute to optimizing the fabrication of oxide TFT-based circuits for electronic applications.

Evidence of soil particle-induced ecotoxicity in old abandoned mining area.

Ecotoxicity of heavy metals in soil is primarily associated with their bioaccessibility and bioavailability in the soil media. However, in some exceptional cases, soil ecotoxicity has been observed despite high total metal concentrations and low extractable metal concentrations in contaminated field sites; therefore, other exposure pathways must be considered. Therefore, the aim of this study was to evaluate the soil-particle induced ecotoxicity in an old mining area. We hypothesized that heavy metals, strongly adsorbed onto soil particles of consumable size for soil organisms, exhibit ecotoxicity, especially on soil particles ∼1 µm to 300 µm in size. A plant seedling assay, in vivo cytotoxicity assay using earthworm immune cells, and a metal bioconcentration assessment were performed. The results of soil particle toxicity revealed that the soil from the study area (A1-A4) had a low contribution to the soil ecotoxicity of extractable metals. For instance, the concentration of extractable arsenic was only 1.9 mg/kg soil, despite the total arsenic concentration reaching 36,982 mg/kg soil at the A1 site. The qualitative and quantitative analyses using SEM-EDX and ICP-OES, as well as principal component analyses, supported the hypothesis of the present study. Overall, the study results emphasize the importance of soil particle-induced ecotoxicity in long-term contaminated field soils. Our study results can inform on effective site-specific soil ecological risk assessment as they suggest the inclusion of soil particle-induced ecotoxicity as an important criterion in old, contaminated field sites, even when the extractable metal fraction in the field soil is low. ENVIRONMENTAL IMPLICATION: Bioaccessibility and bioavailability are primary factors contributing to the soil ecotoxicity of heavy metals. However, in some cases, such as long-term contaminated field sites, soil ecotoxicity has been confirmed even when low extractable metal concentrations were detected alongside high total metal concentrations. The findings of this study reveal that soil particles of edible size could be sources of soil ecotoxicity in the case of long-term contaminated fields with low extractable metal concentrations. The results of this study would contribute to the area of site-specific soil ecological risk assessment.

Microplastic ingestion in aquatic and soil biota: A comprehensive review of laboratory studies on edible size and intake pattern.

Microplastic contamination is ubiquitous and can be transferred through the food chain to humans. However, studies on microplastic size have mainly focused on large animals with a body length >20 mm. To address this gap, we conducted a comprehensive review of 169 laboratory studies to determine the edible size of microplastics for macrofauna and flora in aquatic and soil biota. Our findings indicate that microplastics with a size of <300 µm and 1 µm, respectively, are edible for these organisms, which are positioned at the base of the food chain. We also analyzed intake and depuration patterns and identified factors affecting microplastic ingestion. Our study fills an important knowledge gap by identifying the range of microplastic sizes that can enter the food chain and be transferred to humans. The study findings have strong implications for the ecological risk assessment of microplastics and suggest a starting point for mitigating this threat.

Evidence of parental transfer of nanoplastics in pea (Pisum sativum) plants.

The increasing abundance of nanoplastics in the environment is a cause of serious concern and its acute and chronic effects on ecosystems need to be thoroughly investigated. Toward this end, this study investigated the parental transfer of nanoplastics by chronically exposing Pisum sativum (pea) plants to nanoplastics through soil medium. We observed the presence of nanoplastics in harvested fruits and a subsequent generation of plants replanted in uncontaminated soil using confocal laser scanning microscopy. The fluorescence was located in the cell wall of the vascular bundles, but not in the epidermis, indicating the parental transfer of nanoplastics. In addition, we determined the effects of nanoplastics on the health of subsequent plant generations by estimating the reproductive factors and measuring the content of individual nutrients in peas. Decreases in crop yield and fruit biomass, in addition to changes in nutrient content and composition, were noted. The transgenerational effects of nanoplastics on plants can profoundly impact terrestrial ecosystems, including both plant species and their predators, raising critical safety concerns. Our findings highlight the evidence of parental transfer of nanoplastics in the soil through plants and shows that the chronic effects of nanoplastics on plants may pose a threat to the food supply.

Analyzing species sensitivity distribution of evidently edible microplastics for freshwater biota.

Assessing the ecological risks of microplastics is difficult because of the limited availability of reliable ecotoxicity data. Although freshwater is a valuable sink for microplastics, the current framework for ecological risk assessment using traditional toxicity data is not applicable to freshwater ecosystems. Herein, species sensitivity distribution (SSD) curves were compared for edible and all microplastics exposed to aquatic organisms based on traditional endpoint-based and all-endpoint-based databases. Freshwater toxicity data for microplastics were screened after verifying microplastic presence in test species (56 toxicity datapoints for one microalga, three water fleas, one fish, and one crab; 0.02-100 µm-sized microplastics). SSD and curve parameters were compared with or without non-traditional toxicity endpoints. The HC50 in all endpoint databases was more sensitive than that in the traditional endpoint database and showed a good fit. SSD curves derived from the database for all microplastics were compared and analyzed with edible microplastics. HCx increased for edible microplastics (0.02-100 µm-sized) than for all microplastics (0.02-200 µm-sized), and the size of edible microplastics was lower than of all microplastics. Thus, using non-traditional toxicity data, the SSD approach compensates for the limited ecotoxicity data on microplastics while considering the internalization of microplastics in biota.

Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor.

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. RESULTS: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. CONCLUSIONS: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by 'silent' BGCs.

Atomically Thin Amorphous Indium-Oxide Semiconductor Film Developed Using a Solution Process for High-Performance Oxide Transistors.

High-performance oxide transistors have recently attracted significant attention for use in various electronic applications, such as displays, sensors, and back-end-of-line transistors. In this study, we demonstrate atomically thin indium-oxide (InOx) semiconductors using a solution process for high-performance thin-film transistors (TFTs). To achieve superior field-effect mobility and switching characteristics in TFTs, the bandgap and thickness of the InOx were tuned by controlling the InOx solution molarity. As a result, a high field-effect mobility and on/off-current ratio of 13.95 cm2 V-1 s-1 and 1.42 × 1010, respectively, were achieved using 3.12-nanometer-thick InOx. Our results showed that the charge transport of optimized InOx with a thickness of 3.12 nm is dominated by percolation conduction due to its low surface roughness and appropriate carrier concentration. Furthermore, the atomically thin InOx TFTs showed superior positive and negative gate bias stress stabilities, which are important in electronic applications. The proposed oxide TFTs could provide an effective means of the fabrication of scalable, high-throughput, and high-performance transistors for next-generation electronic applications.

Low-Temperature Enhancement-Mode Amorphous Oxide Thin-Film Transistors in Solution Process Using a Low-Pressure Annealing.

The interest in low processing temperature for printable transistors is rapidly increasing with the introduction of a new form factor in electronics and the growing importance of high throughput. This paper reports the fabrication of low-temperature-processable enhancement-mode amorphous oxide thin-film transistors (TFTs) using the solution process. A facile low-pressure annealing (LPA) method is proposed for the activation of indium oxide (InOx) semiconductors at a significantly low processing temperature of 200 °C. Thermal annealing at a pressure of about ~10 Torr induces effective condensation in InOx even at a low temperature. As a result, the fabricated LPA InOx TFTs not only functioned in enhancement mode but also exhibited outstanding switching characteristics with a high on/off current ratio of 4.91 × 109. Furthermore, the LPA InOx TFTs exhibit stable operation under bias stress compared to the control device due to the low concentration of hydroxyl defects.

One-Stop Strategy for Obtaining Controllable Sensitivity and Feasible Self-Patterning in Silver Nanowires/Elastomer Nanocomposite-Based Stretchable Ultrathin Strain Sensors.

In this study, selective photo-oxidation (SPO) is proposed as a simple, fast, and scalable one-stop strategy that enables simultaneous self-patterning and sensitivity adjustment of ultrathin stretchable strain sensors. The SPO of an elastic substrate through irradiation time-controlled ultraviolet treatment in a confined region enables precise tuning of both the surface energy and the elastic modulus. SPO induces the hydrophilization of the substrate, thereby allowing the self-patterning of silver nanowires (AgNWs). In addition, it promotes the formation of nonpermanent microcracks of AgNWs/elastomer nanocomposites under the action of strain by increasing the elastic modulus. This effect improves sensor sensitivity by suppressing the charge transport pathway. Consequently, AgNWs are directly patterned with a width of 100 µm or less on the elastic substrate, and AgNWs/elastomer-based ultrathin and stretchable strain sensors with controlled sensitivity work reliably in various operating frequencies and cyclic stretching. Sensitivity-controlled strain sensors successfully detect both small and large movements of the human hand.

Improvement in Switching Characteristics and Bias Stability of Solution-Processed Zinc-Tin Oxide Thin Film Transistors via Simple Low-Pressure Thermal Annealing Treatment.

In this study, we used a low-pressure thermal annealing (LPTA) treatment to improve the switching characteristics and bias stability of zinc-tin oxide (ZTO) thin film transistors (TFTs). For this, we first fabricated the TFT and then applied the LPTA treatment at temperatures of 80 °C and 140 °C. The LPTA treatment reduced the number of defects in the bulk and interface of the ZTO TFTs. In addition, the changes in the water contact angle on the ZTO TFT surface indicated that the LPTA treatment reduced the surface defects. Hydrophobicity suppressed the off-current and instability under negative bias stress because of the limited absorption of moisture on the oxide surface. Moreover, the ratio of metal-oxygen bonds increased, while the ratio of oxygen-hydrogen bonds decreased. The reduced action of hydrogen as a shallow donor induced improvements in the on/off ratio (from 5.5 × 103 to 1.1 × 107) and subthreshold swing (8.63 to V·dec-1 and 0.73 V·dec-1), producing ZTO TFTs with excellent switching characteristics. In addition, device-to-device uniformity was significantly improved because of the reduced defects in the LPTA-treated ZTO TFTs.

Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin.

Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.

Microplastics enhance the toxicity and phototoxicity of UV filter avobenzone on Daphnia magna.

Microplastics (MPs) and ultraviolet (UV) filters cause pollution in aquatic ecosystems. Moreover, regulations on the use and discharge of UV filters in personal care products are lacking. Therefore, the combined toxicity of MPs (virgin polystyrene (PS) spheres; size: 200 nm) and avobenzone (AVO; a UV filter) on Daphnia magna were assessed. The exposure groups were AVO, AVO + UV irradiation for 6 h [AVO (UV)], AVO with MPs (Mix), and AVO with MPs + UV irradiation for 6 h [Mix (UV)]. The daphnids were exposed to these treatments for 48 h and observed for an additional 6 h. Energy reserves of all treated groups increased compared to that of the control group. Growth in the Mix group was inhibited despite a high food uptake, and food uptake and growth inhibition were validated in the Mix (UV) group. Additionally, the food uptake of the AVO (UV) and Mix (UV) groups decreased during the recovery period, possibly owing to a decrease in the normal feeding ability resulting from an increase in abnormality. These results indicate that the combined toxicity of MPs+AVO can be exacerbated under natural conditions; the complex toxicity should be considered when assessing aquatic environment pollution.

Species sensitivity distributions of micro- and nanoplastics in soil based on particle characteristics.

Micro- and nanoplastics are released into the soil through various anthropogenic activities; however, research on ecological risk assessment (ERA) of soil microplastics is limited. In this study, the species sensitivity distributions (SSDs) of representative groups of soil biota were analyzed to determine their sensitivity to microplastic properties. A total of 411 datasets from apical endpoint data within 74 studies were classified and utilized in SSD estimation. The hazardous concentrations for 5% of species for microplastics was 88.18 (40.71-191.00) mg/kg soil. It has been established that small-sized microplastics are more toxic to soil organisms than larger microplastics. Most microplastics were spherical and polystyrene, exhibiting the most adverse effects among all the microplastic types assessed herein. The results suggest that physical characteristics of microplastics are important toxicity determinants in soil ecosystems. Given the potential for adverse environmental effects, further effective management strategies should urgently be employed in these areas. This study provided an integrated perspective of microplastic ecotoxicity in soil. In addition, SSDs were estimated using larger datasets and for more species than in previous studies. This is the first study to consider microplastic properties for estimating SSD.

Importance of Structural Relaxation on the Electrical Characteristics and Bias Stability of Solution-Processed ZnSnO Thin-Film Transistors.

Effect of structural relaxation (SR) on the electrical characteristics and bias stability of solution-processed zinc-tin oxide (ZTO) thin-film transistors (TFTs) were systematically investigated by controlling the annealing time of the ZTO semiconductor films. Note that SR was found to increase with increased annealing time. Due to the increased SR, the ratio of oxygen vacancies (VO) increased from 21.5% to 38.2%. According to increased VO, the mobility in the saturation region was exhibited by a sixfold increase from 0.38 to 2.41 cm2 V-1 s-1. In addition, we found that the threshold voltage negatively shifted from 3.08 to -0.95 V. Regarding the issue of bias stability, according to increased SR, positive-bias stress of the ZTO TFTs was enhanced, compared with reverse features of negative-bias stress. Our understanding is expected to provide a basic way to improve the electrical characteristics and bias stability of rare-metal-free oxide semiconductor TFTs, which have not been sufficiently studied.

Proximity of maxillary molar apexes to the cortical bone surface and the maxillary sinus.

Objectives: This study aimed to analyze the proximity of maxillary molar roots to their overlying cortical bone surfaces and the maxillary sinus. Materials and Methods: Cone-beam computed tomographic images of 151 patients with completely erupted upper molars that had 3 separate roots were studied. The following distances were measured: from the root apex to the cortical plate and maxillary sinus floor, and from the apical 3-mm level of the root to the cortical plate. Differences between groups were analyzed with 1-way analysis of variance and the Scheffé post hoc test, the significance of differences between cone-beam computed tomography views with the paired t-test, and the significance of differences among age groups with linear regression analysis. The significance level was set at p < 0.05. Results: The mesiobuccal and distobuccal root apexes of maxillary second molars were more distant from the buccal cortical plate than the maxillary first molars (p < 0.05). The apical 3-mm level of the mesiobuccal root of the first molar was closer to the buccal cortical bone than the second molar (p < 0.05). In the maxillary first molars, the thickness of the buccal cortical bone decreased in all roots with age (p < 0.05). In all root apexes of both molars, the difference in the vertical level between the maxillary sinus floor and the root apex increased with age (p < 0.05). Conclusions: Awareness of the anatomical profile of maxillary molar apices in relation to the cortical bones and maxillary sinus will be beneficial for apical surgery.

Translocation and chronic effects of microplastics on pea plants (Pisum sativum) in copper-contaminated soil.

Microplastics (MPs) released into soil environments, along with the existing pollutants in soil, may have adverse effects on plants. However, the chronic effects of MPs in soils contaminated with heavy metals on crop plants remain unidentified. This study aimed to investigate the chronic effects of MPs (polystyrene, 20 nm) on the reproductive and nutritional status of pea crop plant (Pisum sativum) grown in Cu- (40 mg/kg) and MP-contaminated soils (40, 20 mg/kg). The crop yield reduced in all groups, with an evident decrease in the complex exposure group (comprising MPs and Cu). Moreover, significant changes in plants were identified regarding the weight, color, amino acids, and protein content of peas. Nutrient content in beans increased by MP exposure in single and complex exposure groups. Cu accumulation did not differ in the presence and absence of MPs. Additionally, MPs that infiltrated into incomplete casparian strips during root formation translocated into aerial parts via the apoplast pathway along the cell walls of the vascular bundle. Therefore, long-term exposure to MPs in soil can significantly affect plants while collective application of Cu and MPs imposed severe damage. The changes in the crop quality and nutrient contents may in turn affect human health through the food chain.

Perilla frutescens Leaf Extract Attenuates Vascular Dementia-Associated Memory Deficits, Neuronal Damages, and Microglial Activation.

Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation.

Comparative genomic analysis of Streptomyces rapamycinicus NRRL 5491 and its mutant overproducing rapamycin.

Streptomyces rapamycinicus NRRL 5491 is a well-known producer of rapamycin, a secondary metabolite with useful bioactivities, including antifungal, antitumor, and immunosuppressive functions. For the enhanced rapamycin production, a rapamycin-overproducing strain SRMK07 was previously obtained as a result of random mutagenesis. To identify genomic changes that allowed the SRMK07 strain's enhanced rapamycin production, genomes of the NRRL 5491 and SRMK07 strains were newly sequenced in this study. The resulting genome sequences of the wild-type and SRMK07 strains showed the size of 12.47 Mbp and 9.56 Mbp, respectively. Large deletions were observed at both end regions of the SRMK07 strain's genome, which cover 17 biosynthetic gene clusters (BGCs) encoding secondary metabolites. Also, genes in a genomic region containing the rapamycin BGC were shown to be duplicated. Finally, comparative metabolic network analysis using these two strains' genome-scale metabolic models revealed biochemical reactions with different metabolic fluxes, which were all associated with NADPH generation. Taken together, the genomic and computational approaches undertaken in this study suggest biological clues for the enhanced rapamycin production of the SRMK07 strain. These clues can also serve as a basis for systematic engineering of a production host for further enhanced rapamycin production.

Soil algae as a potential carrier for nanoplastics: Adsorption and internalization of nanoplastics in algal cells.

Although plastics are widely distributed in soil environments, few studies have assessed their effects on different test species. In this study, the responses (adsorption or internalization) of two green algal species (Chlorococcum infusionum and Chlamydomonas reinhardtii) to nanoplastics in soil were evaluated via microscopic analysis for nine weeks; additionally, the effects of polystyrene (PS) nanoplastics on their photosynthetic activity and growth were assessed. The results showed that PS nanoplastics and C. infusionum or C. reinhardtii cells were aggregated with the formation of palmelloid colonies; additionally, internalization of PS nanoplastics to C. reinhardtii cells was verified. However, no significant effects of PS nanoplastics on photosynthetic activity and growth of soil algae were observed. Algae were more likely to be threatened by plastic aggregation in the soil environment than in the aquatic environment due to the formation of a mucilaginous sheath on their cell. Our results highlighted that soil algae are a potential carrier of nanoplastics to other organisms in the food chain due to plastic aggregation through adsorption or internalization. This study will enhance our understanding of nanoplastic distribution in soil algae.

Validation of the paper-disc soil method using soil alga Chlorococcum infusionum to quantitatively determine the toxicity of heavy metals.

The paper-disc soil method is a soil algal bioassay used to assess soil water transfer in polluted soils. Although this ecotoxicological method was first established in 2016 to assess soil pollution, quantitative toxicity assessments of Cu- or Ni-treated soil and in situ or ex situ soils polluted with other heavy metals (HMs) have not been widely conducted. In this study, the paper-disc soil method was validated under different test conditions. The validated paper-disc soil method was used to quantitatively investigate the toxicity of four HMs (As, Cd, Cr, and Zn). Based on the results, the test species, initial inoculation concentration in the algal-dispersed solution, water content, light intensity, and exposure duration were proposed as Chlorococcum infusionum, 3 × 106 cells/mL in Bold's basal medium, 80% water-holding capacity, 4000 ± 500 lx, and 6 days, respectively. The toxicity of HMs was quantified using the validated paper-disc soil method, and the half-maximal effective concentration values for biomass were calculated as 22.49 (21.26-23.78) mg Cr/kg dry weight (dw) soil, 42.72 (38.64-47.24) mg Cd/kg dw soil, 57.79 (55.46-60.21) mg As/kg dw soil, and 183.06 (175.38-191.06) mg Zn/kg dw soil. The paper-disc soil method using the soil alga C. infusionum was validated by quantitatively evaluating the test conditions and toxicity of HMs. Our results provide important quantitative toxicity data for soil algae exposed to HMs and a basis for standardizing the paper-disc soil method using soil algae.