Toxicity of Aged Paint Particles to Soil Ecosystems: Insights from Caenorhabditis elegans.

Despite the extensive global consumption of architectural paint, the toxicological effects of aged exterior paint particles on terrestrial biota remain largely uncharacterized. Herein, we assessed the toxic effect of aged paint particles on soil environments using the nematode Caenorhabditis elegans (C. elegans) as a test organism. Various types of paint particles were generated by fragmentation and sequential sieving (500-1000, 250-500, 100-250, 50-100, 20-50 µm) of paint coatings collected from two old residential areas. The paint particles exerted different levels of toxicity, as indicated by a reduction in the number of C. elegans offspring, depending on their size, color, and layer structure. These physical characteristics were found to be closely associated with the chemical heterogeneity of additives present in the paint particles. Since the paint particle sizes were larger than what C. elegans typically consume, we attributed the toxicity to leachable additives present in the paint particles. To assess the toxicity of these leachable additives, we performed sequential washings of the paint particles with distilled water and ethanol. Ethanol washing of the paint particles significantly reduced the soil toxicity of the hydrophobic additives, indicating their potential environmental risk. Liquid chromatography-mass spectrometry analysis of the ethanol leachate revealed the presence of alkyl amines, which exhibited a high correlation with the toxicity of the paint particles. Further toxicity testing using an alkyl amine standard demonstrated that a paint particle concentration of 1.2% in soil could significantly reduce the number of C. elegans offspring. Our findings provide insights into the potential hazards posed by aged paint particles and their leachable additives in the terrestrial environment.

Species sensitivity distributions for ethylparaben to derive protective concentrations for soil ecosystems.

Ethylparaben is used as an antifungal preservative. Although some countries have implemented regulations for human exposure to parabens, environmental regulations for ethylparaben have not been established. This study provides new toxicological data for ethylparaben, for which data regarding soil organisms were previously lacking. Although ethylparaben toxicity has been reported in other species, we present herein the first comprehensive study of its toxicity in soil organisms. We used 12 test species (Lycopersicon esculentum, Vigna radiata, Hordeum vulgare, Oryza sativa, Eisenia andrei, Folsomia candida, Lobella sokamensis, Caenorhabiditis elegans, Chlamydomonas reinhardtii, Chlorococcum infusionum, Chlorella sorokiniana, Chlorella vulgaris) from eight taxonomic groups for acute bioassays and nine test species (L. esculentum, V. radiata, H. vulgare, O. sativa, C. reinhardtii, C. infusionum, C. sorokiniana, and C. vulgaris) from five taxonomic groups for chronic bioassays. A suite of acute and chronic toxicity tests, using 21 soil species, was conducted to estimate EC50 values, which facilitated the construction of species sensitivity distributions (SSDs) and the calculation of protective concentrations (PCs). Acute and chronic PC95 values (protective concentration for 95% of species) for ethylparaben were estimated to be 14 and 5 mg/kg dry soil, respectively. To the best of our knowledge, this is the first study to evaluate the toxicity of ethylparaben to soil species and derive PCs for soil ecosystems based on SSDs. Therefore, the data presented in this study can be used as a basis for further investigations of paraben toxicity to the soil environment.

Effects of Different Microplastics on Nematodes in the Soil Environment: Tracking the Extractable Additives Using an Ecotoxicological Approach.

With increasing interest in the effects of microplastics on the soil environment, there is a need to thoroughly evaluate the potential adverse effects of these particles as a function of their characteristics (size, shape, and composition). In addition, extractable chemical additives from microplastics have been identified as an important toxicity pathway in the aquatic environment. However, currently, little is known about the effects of such additives on the soil environment. In this study on nematodes (Caenorhabditis elegans), we adopted an ecotoxicological approach to assess the potential effects of 13 different microplastics (0.001-1% of soil dry weight) with different characteristics and extractable additives. We found that poly(ethylene terephthalate) (PET) fragments and polyacrylicnitrile (PAN) fibers show the highest toxicity, while high-density polyethylene (HDPE), polypropylene (PP), and polystyrene (PS) fragments induced relatively less adverse effects on nematodes. In addition, low-density polyethylene (LDPE) induced no toxicity within our test concentration range for the acute period. Acute toxicity was mainly attributed to the extractable additives: when the additives were extracted, the toxic effects of each microplastic disappeared in the acute soil toxicity test. The harmful effects of the LDPE films and PAN fibers increased when the microplastics were maintained in the soil for a long-term period with frequent wet-dry cycles. We here provide clear evidence that microplastic toxicity in the soil is highly related to extractable additives. Our results suggest that future experiments consider extractable additives as key explanatory variables.

Application of a soil quality assessment system using ecotoxicological indicators to evaluate contaminated and remediated soils.

The deterioration of soil quality owing to human activities results in adverse effects on the soil ecosystem. This study developed a systematic method to quantitatively evaluate soil quality based on physical, chemical, biological, and ecotoxicological indicators and proposed the soil quality assessment and management system. This system consists of step-by-step processes, including indicator classification, indicator measurement, scoring and weighting, and soil quality index (SQI) calculation. The novel strategy included the usage of authentic ecotoxicological indicators for realistically interpreting soil quality assessment results. This study used five ecotoxicological indicators, including earthworm survival, enzyme activities, nematode reproduction, plant germination and growth, soil algal biomass, and soil algal photosynthetic capacity. Relatively higher SQI values than those corresponding to the actual soil quality status would be obtained without considering the ecotoxicological indicators. We conclude that the use of ecotoxicological indicator can help in soil quality assessment even under extreme soil quality conditions, such as highly contaminated or physically and chemically remediated soils.

Changes in soil properties after remediation influence the performance and survival of soil algae and earthworm.

Previous research on soil remediation focused on soil properties and not on its effects on soil ecosystems. The present study investigated the adverse effects of soil physicochemical changes due to remediation on the biological indicators Chlorococcum infusionum and Chlamydomonas reinhardtii (algae) and Eisenia andrei (earthworm). Soil physicochemical properties, concentrations of total, bioavailable, and water-soluble heavy metals in soil were measured before and after remediation. Changes in soil pH, electrical conductivity, total nitrogen, and total phosphorous immediately after soil remediation were the primary causes of the biomass and photosynthetic activity inhibition observed in C. infusionum and C. reinhardtii, and the survival, normality, and burrowing behavior decrease observed in E. andrei in remediated soils showing dramatic changes in those properties. These findings suggest that remediated soils need some time to recover before restoring their functions, although heavy metals are no longer contaminating the soil.

Rapid in situ assessment for predicting soil quality using an algae-soaked disc seeding assay.

The soil quality of remediated land is altered and this land consequently exerts unexpected biological effects on terrestrial organisms. Therefore, field evaluation of such land should be conducted using biological indicators. Algae are a promising new biological indicator since they are a food source for organisms in higher soil trophic levels and easily sampled from the soil. Field evaluation of soil characteristics is preferred to be testing in laboratory conditions because many biological effects cannot be duplicated during laboratory evaluations. Herein, we describe a convenient and rapid algae-soaked disc seeding assay for assessing soil quality in the field based on soil algae. The collection of algae is easy and rapid and the method predicts the short-term quality of contaminated, remediated, and amended farm and paddy soils. The algae-soaked disc seeding assay is yet to be extensively evaluated, and the method cannot be applied to loamy sand soil in in situ evaluations. The algae-soaked disc seeding assay is recommended for prediction of soil quality in in situ evaluations because it reflects all variations in the environment. The algae-soaked disc seeding assay will help to develop management strategies for in situ evaluation.

Simultaneous detection of bioavailable arsenic and cadmium in contaminated soils using dual-sensing bioreporters.

Whole-cell bioreporters (WCBs) have attracted increasing attention during the last few decades because they allow fast determination of bioavailable heavy metals in contaminated sites. Various WCBs to monitor specific heavy metals such as arsenic and cadmium in diverse environmental systems are available. However, currently, no study on simultaneous analysis of arsenic and cadmium has been reported, even though soils are contaminated by diverse heavy metals and metalloids. We demonstrated herein the development of dual-sensing WCBs to simultaneously quantify bioavailable arsenic and cadmium in contaminated sites by employing the promoter regions of the ars and znt operons as separate metal-sensing domains, and egfp and mcherry as reporter genes. The dual-sensing WCBs were generated by inserting two sets of genes into E. coli DH5α. The capability of WCBs was successfully proved to simultaneously quantify bioavailable arsenic and cadmium in amended Landwirtschaftliche Untersuchungs und Forschungsanstalt (LUFA) soils, and then, it was applied to contaminated field soils collected from a smelter area in Korea. As a result, it was noticed that the bioavailable portion of cadmium was higher than that of arsenic while the absolute amount of bioavailable arsenic and cadmium level was opposite. Since both cadmium and arsenic were assessed from the same E. coli cells, the data obtained by using dual-sensing WCBs would be more efficient and convenient than that from comparative WCB assay. In spite of advantageous aspects, to our knowledge, this is the first report on a dual-sensing WCB for rapid and concurrent quantification of bioavailable arsenic and cadmium in contaminated soils.

A new and sensitive method for measuring in vivo and in vitro cytotoxicity in earthworm coelomocytes by flow cytometry.

This study describes a new and sensitive method for measuring the in vivo and in vitro cytotoxicity of 2 earthworm species, Eisenia andrei and Perionyx excavatus, exposed to copper. Specifically, we measured the number of coelomocyte cells that were affected by copper following in vivo and in vitro exposure by flow cytometry, after calcein acetoxymethyl ester (calcein-AM) staining. We found that the coelomocyte viability of both earthworm species was noticeably reduced in the in vivo cytotoxicity test at concentrations of 100mg/kg copper in dry soil. However, pathological symptoms, such as mucous secretion and bleeding, swelling, thinning, and fragmentation, and burrowing symptoms were not evident following exposure to copper levels of <400mg/kg dry soil. In conclusion, the present study demonstrates that calcein-AM is a more sensitive test of earthworm coelomocyte cytotoxicity compared to the traditional individual level toxicity test. Therefore, this test could be used to detect low levels of metal contamination in soils.

Interaction of citrate-coated silver nanoparticles with earthworm coelomic fluid and related cytotoxicity in Eisenia andrei.

Understanding the interaction of nanoparticles with biological fluid is important for predicting the behavior and toxicity of nanoparticles in living systems. The earthworm Eisenia andrei was exposed to citrate-coated silver nanoparticles (cAgNPs), and the interaction of cAgNPs with earthworm coelomic fluid (ECF), the cytotoxicity of cAgNPs in earthworm coelomocytes was assessed. The neutral red retention assay showed a reduction in lysosomal stability after exposure. The toxicity of silver ions dissolved from cAgNPs in the soil medium was not significant. The aggregation and dissolution of cAgNPs increased in ECF, which contains various electrolytes that alter the properties of nanoparticles, and their subsequent toxicity. Microscopic and dissolution studies demonstrated that the aggregation of cAgNPs rapidly increased, and readily dissolved in ECF. The bioavailability of cAgNPs to earthworms induced lysosomal cytotoxicity. This is the first report to test the interaction and lysosomal cytotoxicity of nanoparticles in earthworm biofluids.

The soil plastisphere.

Understanding the effects of plastic pollution in terrestrial ecosystems is a priority in environmental research. A central aspect of this suite of pollutants is that it entails particles, in addition to chemical compounds, and this makes plastic quite different from the vast majority of chemical environmental pollutants. Particles can be habitats for microbial communities, and plastics can be a source of chemical compounds that are released into the surrounding environment. In the aquatic literature, the term 'plastisphere' has been coined to refer to the microbial community colonizing plastic debris; here, we use a definition that also includes the immediate soil environment of these particles to align the definition with other concepts in soil microbiology. First, we highlight major differences in the plastisphere between aquatic and soil ecosystems, then we review what is currently known about the soil plastisphere, including the members of the microbial community that are enriched, and the possible mechanisms underpinning this selection. Then, we focus on outlining future prospects for research on the soil plastisphere.

Multigenerational study of gold nanoparticles in Caenorhabditis elegans: transgenerational effect of maternal exposure.

In this study, the generational transfer and multigenerational effect of gold nanoparticles (AuNPs) on Caenorhabditis elegans were investigated by observing the parental generation (F0) to the fourth offspring generation (F4) using food-exposure approaches. There were no significant changes on survival rate under all generations by AuNP maternal exposure to the F0 generation. However, reproduction rate was clearly affected in the F2 generation but then gradually recovered in the F3 and F4 generations. The abnormalities of the reproductive system showed a close relationship with reproduction rates. These phenomenons may be due to the germ-line transfer. The germ line of F0 generation such as gonad and embryo germ cell may be affected during their development by maternal exposure of AuNPs, and this generation caused transgeneration effect on future generations. To the best of our knowledge, this is the first study to provide the evidence of transgenerational effects by maternal exposure of nanoparticles to the next generations.

No evidence of the genotoxic potential of gold, silver, zinc oxide and titanium dioxide nanoparticles in the SOS chromotest.

Gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs), zinc oxide nanoparticles (ZnO NPs) and titanium dioxide nanoparticles (TiO2 NPs) are widely used in cosmetic products such as preservatives, colorants and sunscreens. This study investigated the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO2 NPs using the SOS chromotest with Escherichia coli PQ37. The maximum exposure concentrations for each nanoparticle were 3.23 mg l(-1) for Au NPs, 32.3 mg l(-1) for Ag NPs and 100 mg l(-1) for ZnO NPs and TiO2 NPs. Additionally, in order to compare the genotoxicity of nanoparticles and corresponding dissolved ions, the ions were assessed in the same way as nanoparticles. The genotoxicity of the titanium ion was not assessed because of the extremely low solubility of TiO2 NPs. Au NPs, Ag NPs, ZnO NPs, TiO2 NPs and ions of Au, Ag and Zn, in a range of tested concentrations, exerted no effects in the SOS chromotest, evidenced by maximum IF (IFmax) values of below 1.5 for all chemicals. Owing to the results, nanosized Au NPs, Ag NPs, ZnO NPs, TiO2 NPs and ions of Au, Ag and Zn are classified as non-genotoxic on the basis of the SOS chromotest used in this study. To the best of our knowledge, this is the first study to evaluate the genotoxicity of Au NPs, Ag NPs, ZnO NPs and TiO2 NPs using the SOS chromotest.

Effect of ZnO and TiO2 nanoparticles preilluminated with UVA and UVB light on Escherichia coli and Bacillus subtilis.

We evaluated the effects of zinc oxide (ZnO) and titanium dioxide (TiO2) nanoparticles (NPs) preilluminated with ultraviolet light on Escherichia coli and Bacillus subtilis. The experiments were conducted using three different types of light: visible, Ultraviolet A (UVA, 315-400 nm), and Ultraviolet B (UVB, 280-315 nm). The bacteria were exposed to NPs, either as liquid suspensions for growth inhibition assays or on agar plates for colony forming unit (CFU) assays. We found that the ZnO NPs were more toxic when preilluminated with UVA or UVB light than with visible light in both growth inhibition and CFU assays. TiO2 NPs were not toxic to the bacteria under UVA or UVB preillumination conditions. The photo-dissolution of ZnO NPs increased with UV preillumination, which could explain the observed toxicity of ZnO NPs. We detected oxidative stress elicited by photoactive nanoparticles by measuring superoxide dismutase activity. The results of this study show that the toxicity of photoactive nanoparticles can be increased by UV preillumination by dissolution of toxic ions, which suggests the potential for preillumination-dependent toxicity of nanoparticles on soil environments in low light or darkness.

Interaction of silver nanoparticles with biological surfaces of Caenorhabditis elegans.

Silver nanoparticles (AgNPs) are being used in an increasing number of industrial and commercial applications; this has resulted in an increased release of AgNPs into the environment. Understanding the interaction of AgNPs with biological surfaces is important, as such understanding will facilitate predictions of the further effects of nanoparticles on biological systems. This study highlights the interaction of citrate-coated silver nanoparticles (cAgNPs) with the biological surfaces of the nematode C. elegans. General toxicity, as proxied by factors such as mortality and reproduction, was evaluated in nematode growth medium (NGM), which provides a more homogeneous distribution of cAgNPs than in K-medium. The survival and reproduction of C. elegans evidenced a clear reduction in up to 100 mg/L and 10 mg/L of cAgNPs, respectively. We also noted significant interactions of cAgNPs with the biological surfaces of C. elegans. Severe epidemic edema and burst were detected in the exposure group, which may be associated with secondary infections in soil ecosystems. We observed no evidence of cAgNPs intake by C. elegans. This is, to the best of our knowledge, the first report to investigate the nanotoxicity of cAgNPs as related to biological surfaces of C. elegans; further research is needed to study the fate of cAgNPs inside of C. elegans.

Soil Storage Conditions Alter the Effects of Tire Wear Particles on Microbial Activities in Laboratory Tests.

In this study, we focused on the fact that soil storage conditions in the laboratory have never been considered as a key factor potentially leading to high variation when measuring effects of microplastics on soil microbial activity. We stored field-collected soils under four different conditions [room-temperature storage, low-temperature storage (LS), air drying (AD), and heat drying] prior to the experiment. Each soil was treated with tire wear particles (TWPs), and soil microbial activities and water aggregate stability were investigated after soil incubation. As a result, microbial activities, including soil respiration and three enzyme activities (ß-glucosidase, N-acetyl-ß-glucosaminidase, and phosphatase), were shown to depend on soil storage conditions. Soil respiration rates increased with the addition of TWPs, and the differences from the control group (no TWPs added) were more pronounced in the AD TWP treatment than in soils stored under other conditions. In contrast, phosphatase activity followed an opposing trend after the addition of TWPs. The AD soil had higher phosphatase activity after the addition of TWPs, while the LS soil had a lower level than the control group. We suggest that microplastic effects in laboratory experiments can strongly depend on soil storage conditions.

Assay-dependent effect of silver nanoparticles to Escherichia coli and Bacillus subtilis.

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.

Microplastics disrupt accurate soil organic carbon measurement based on chemical oxidation method.

Microplastics are widespread contaminants in soils and terrestrial ecosystems in many areas worldwide. In this study, we measured soil organic carbon (SOC) and soil organic matter (SOM) in microplastic-treated soils to determine if the presence of microplastics could affect the accuracy of carbon-based soil quality indicator measurements. Six different sizes and types of microplastics were selected, and six soil samples were used to evaluate the impacts. Treating soil with polyethylene and low-density polyethylene significantly increased SOC (p < 0.05) when measured with the modified Walkley & Black method; microplastic addition (0.01%, v/v) increased SOC by >40% compared to control organic carbon-poor soil (<10.0 g kg-1). We conclude that the microplastics can disrupt the accurate measurement of SOC. Likely, the physicochemical treatment used in the SOC measurement process can cause the organic compounds and/or carbon complexes to be extracted from microplastics, and this can affect the results. Considering that SOC is a main indicator for assessing soil quality and the global carbon cycle, overestimations caused by microplastic contamination should be further discussed to identify appropriate ways to deal with microplastics as a new carbon source in the environment.

Selection of Ovalbumin-specific Binding Peptides through Instant Translation in Ribosome Display Using E. coli Extract.

In vitro selection has been widely used to generate molecular-recognition elements in analytical sciences. Although reconstituted types of in vitro transcription and translation (IVTT) system, such as PURE system, are nowadays widely used for ribosome display and mRNA/cDNA display, use of E. coli extract is often avoided, presumably because it contains unfavorable contaminants, such as ribonuclease. Nevertheless, the initial speed of protein translation in E. coli extract is markedly faster than that of PURE system. We thus hypothesized that E. coli extract is more appropriate for instant translation in ribosome display than PURE system. Here, we first revisit the potency of E. coli extract for ribosome display by shortening the translation time, and then applied the optimized condition for selecting peptide aptamers for ovalbumin (OVA). The OVA-binding peptides selected using E. coli extract exhibited specific binding to OVA, even in the presence of 50% serum. We conclude that instant translation in ribosome display using E. coli extract has the potential to generate easy-to-use and economical molecular-recognition elements in analytical sciences.