Using treated municipal wastewater (TMW) for agricultural irrigation can help reduce stress on freshwater resources. However, using TMW could expose agricultural environments to antibiotic resistance determinants (ARDs, including antibiotics and antibiotic resistance genes [ARGs]), ultimately impacting human health. In this study, we investigated two filtration systems, conventional sand, and sand mixed with 2 % (w/w) dairy manure biochar, to remove the antibiotic trimethoprim (TMP) commonly present in TMW. While the performance of the sand filtration system ultimately declined to 20-40 % TMP removal efficiency, the sand/biochar filtration system maintained >94 % TMP removal efficiency throughout the study. This high performance was achieved using biochar derived from a common, low-cost agricultural waste (dairy manure) under realistic treated wastewater conditions, emphasizing economic and reuse feasibility. Despite the highly efficient TMP removal, both systems released bacteria and ARGs into the effluent after several weeks, raising concerns about the microbiological quality of the water. Subsequent microcosm studies indicated that sand/biochar filtration reduced the presence of TMP in soil following irrigation. Moreover, filtration effectively reduced the development of several ARGs that need a short period to develop (e.g., ttgA, ttgB), but did not significantly impact ARGs that need more time to develop. These findings offer practical and economical strategies for mitigating antibiotic dissemination and antibiotic resistance development following TMW irrigation while emphasizing the importance of appropriate design and operational management to limit ARGs dissemination.

Treated municipal wastewater (TMW) is an underutilized water source for crop irrigation that can reduce the pressure on fresh water. However, the source and quality of the TMW will determine its suitability for the safety of the produced crop. We determined the yield, mineral nutrients, antioxidants, and heavy metals in spinach and radish irrigated with TMW in soil amended or not with cow manure. Both tap water (TAPW) and TMW provided equivalent concentrations of macro and micronutrients. The treatments were TAPW control, TMW, TAPW + 2% manure, and TMW + 2% manure. Although TAPW and TMW alone did not affect leaf nutrient accumulation or yield for either species, manure significantly increased leaf accumulation of K and P, mainly in spinach. When combined with TAPW or TMW, manure significantly decreased leaf Na in spinach due to the increased accumulation of K leading to a significant increase in spinach yield. Results were similar for radish, although not significant. Neither treatment affected the antioxidant capacity of spinach or radish but radish leaves had over 3-fold the antioxidant capacity (ORAC) of spinach. The concentrations of heavy metals in TMW, soil, or manure were not of concern to consumers being below the levels of concern established by the EPA. Our results support the use of TMW as an alternative to fresh water, and cow manure as an organic soil amendment to boost the yield of irrigated vegetable crops cultivated in sandy loam soils. This information will be valuable for addressing concerns about the safety of TMW as an alternative to freshwater, and of manure as a safe source of organic matter used to mitigate salinity and to boost fresh vegetable yield in arid and semiarid soils poor in organic matter and in climates where freshwater is scarce.

Treated wastewater application in the environment to alleviate stress on traditional water sources introduces antibiotic resistance genes (ARGs) in extracellular DNA (eDNA) into the environment, enhancing antibiotic resistance risk. This study aimed to mitigate this risk by using biochar to immobilize eDNA from treated wastewater. The interactive role of biochar production conditions were probed across a range of pyrolysis temperatures (300-800 degrees C) and feedstock classes (e.g., grass clippings (GC), manure (MN), walnut shells (WS) and pine pellet (PP)) on eDNA immobilization. Characteristics varied widely across biochars, with pyrolysis temperature and feedstock controlling elemental composition, proximate analysis, surface area, functional group composition, surface area and pore geometries, translating to a range of eDNA removal efficiencies (30.4-84.2%) and Freundlich coefficients (0.16-2.06). Biochar aromaticity, polarity and volatile matter were parameters most related with eDNA adsorption. Pyrolysis temperature exerted greater influence on eDNA removal than feedstock, however, there were significant interactive effects between feedstock and pyrolysis temperatures. GC and MN biochars demonstrated temperature sensitivity for eDNA removal, with maximum adsorption observed at a minimum threshold of 500 degrees C, whereas WS and PP biochars yielded statistically equivalent removals across pyrolysis temperatures. Our results provide biochar production guidance for targeting eDNA removal based on available feedstocks and pyrolysis capabilities. These results will streamline biochar integration into wastewater treatment systems across scales to mitigate environmental risk of ARGs.

This study investigated the sources of fecal pollution in surface and groundwaters collected from three urban watersheds in Texas during dry and wet weather and identified the impact of precipitation on water quality. Water samples (n = 316 and 107 for dry and wet events, respectively) were collected biweekly from eight sampling sites (6 sites from creeks and ponds, and 2 well sites) during two-year monitoring and analyzed for six physico-chemical parameters and ten qPCR-based assays targeting general ( E . coli , Enterococcus , and universal Bacteroidales ), human (BacHum and HF183), animal (Rum2Bac, BacCow, BacCan), and avian (Chicken/Duck-Bac and GFD) fecal markers. Elevated concentrations of NO 3 -N and NO 2 -N were observed in ponds and creeks sites during wet weather. Fecal markers analysis indicated higher concentrations of Rum2Bac, BacCow, and BacCan markers in most of pond and creek sites under wet weather, suggesting stormwater runoff contributed to non-point sources of fecal contamination by animal sources. Furthermore, sporadically higher concentrations of these markers were detected at groundwater sampling sites, demonstrating the significant human health risk. Multivariate statistical analysis such as cluster analysis (CA) and principal coordinate analysis (PCoA) was performed to identify relationship between sampling sites; while CA majorly classified ponds, creeks, and well sites separately, PCoA identified similarities in water quality characteristics between waters of wells with ponds and creeks. Overall, results indicate ruminant and dog fecal contamination is a major concern during storm events, consequently impacting surface and groundwater quality of the study.

The spread of antimicrobial resistance (AMR) in agricultural systems via irrigation water is a serious public health issue as it can be transmitted to humans through the food chain. Therefore, understanding the dissemination routes of antibiotic resistance genes (ARGs) in agricultural systems is crucial for the assessment of health risks associated with eating fresh vegetables such as spinach and radish irrigated with treated municipal wastewater (TMW). In this study, we investigated the bacterial community structure and resistome in the soil -plant -earthworm continuum after irrigation of spinach and radish with TMW containing the antibiotics trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) using 16S rRNA gene sequencing and high throughput quantitative PCR (HT-qPCR). The study was conducted in two phases: Phase I involved eight weeks of spinach and radish production using TMW for irrigation, whereas Phase II entailed three weeks of earthworm exposure to contaminated plant material obtained in Phase I. The 16S data indicated that the rhizosphere bacterial community composition and structure were more resilient to antibiotic residuals in the irrigated water, with radish showing less susceptibility than spinach than those of bulk soils. The HT-qPCR analysis revealed that a total of 271 ARGs (out of 285) and 9 mobile genetic elements (MGEs) (out of 10) were detected in all samples. Higher diversity and abundance of ARGs were observed for samples irrigated with higher concentrations of antibiotics in both spinach and radish treatments. However, compared to spinach, radish ARG dynamics in the soil biome were more stable due to the change of antibiotic introduction to the soil. At the class level, multi -drug resistance (MDR) class was altered significantly by the presence of antibiotics in irrigation water. Compared to earthworm fecal samples, their corresponding soil environments showed a higher number of detected ARGs, suggesting that earthworms could play a role in reducing ARG dissemination in the soil environments. These findings will not only provide insight into the dissemination of ARGs in agricultural environments due to antibiotic residuals in irrigated water but could help understand the potential human health risks associated with ARGs.

Microcystis aeruginosa (M. aeruginosa) is the dominant cyanobacterial species causing harmful algal blooms in water bodies worldwide. The blooms release potent toxins and pose severe public health hazards to water bodies, animals, and humans who are in contact with or consume this water. The interaction between M. aeruginosa and heterotrophic bacteria is thought to contribute to the development of the blooms. This study strives to provide a specific answer to whether quorum sensing is also a potential mechanism mediating the interaction of different strains/species and the expression by gene luxS or gene mcyB in M. aeruginosa growth. The luxS gene in M. aeruginosa PCC7806 is associated with quorum sensing and was tested by q-PCR throughout a 30-day growth period. The same was performed for the mcyB gene. Heterotrophic bacteria were collected from local water bodies: Cibolo Creek and Leon Creek in San Antonio, Texas. Results revealed that in algal bloom scenarios, there is a similar concentration of gene luxS that is expressed by the cyanobacteria. Gene mcyB, however, is not directly associated with algal blooms, but it is related to cyanotoxin production. Toxicity levels increased in experiments with multiple algal strains, and the HSL treatment was not effective at reducing microcystin levels.

This study contributes to the collective evaluation of SARS-CoV-2 RNA persistence and removal along the wastewater treatment stages of a treatment plant in San Antonio, Texas.

This study used pilot-scale high-rate algae ponds to assess algal–bacteria biomass productivity and wastewater nutrient removal as well as the impact of mechanical and hydrothermal pretreatments on biomass disintegration, methane production kinetics, and anaerobic digestion (AD) energy balance. Mechanical pretreatment had a minor effect on biomass disintegration and methane production. By contrast, hydrothermal pretreatment significantly reduced particle size and increased the solubilized organic matter content by 3.5 times. The methane yield and production rate increased by 20–55% and 20–85%, respectively, with the highest values achieved after pretreatment at 121 °C for 60 min. While the 1st-order and pseudo-1st-order reaction equation models fitted methane production from untreated biomass best (R2 > 0.993), the modified Gompertz sigmoidal-type model provided a superior fit for hydrothermally pretreated algae (R2 ≥ 0.99). The AD energy balance revealed that hydrothermal pretreatment improved the total energy output by 25–40%, with the highest values for volume-specific and mass-specific total energy outputs reaching 0.23 kW per digester m3 and 2.3 MW per ton of biomass volatile solids. Additionally, net energy recovery (energy output per biomass HHV) increased from 20% for untreated algae to 32–34% for hydrothermally pretreated algae, resulting in net energy ratio and net energy efficiency of 2.14 and 68%, respectively.

Over the past 15 years, the field of oncology research has witnessed significant progress in the development of new cell culture models, such as tumor-on-chip (ToC) systems. In this comprehensive overview, we present a multidisciplinary perspective by bringing together physicists, biologists, clinicians, and experts from pharmaceutical companies to highlight the current state of ToC research, its unique features, and the challenges it faces. To offer readers a clear and quantitative understanding of the ToC field, we conducted an extensive systematic analysis of more than 300 publications related to ToC from 2005 to 2022. ToC offer key advantages over other in vitro models by enabling precise control over various parameters. These parameters include the properties of the extracellular matrix, mechanical forces exerted on cells, the physico-chemical environment, cell composition, and the architecture of the tumor microenvironment. Such fine control allows ToC to closely replicate the complex microenvironment and interactions within tumors, facilitating the study of cancer progression and therapeutic responses in a highly representative manner. Importantly, by incorporating patient-derived cells or tumor xenografts, ToC models have demonstrated promising results in terms of clinical validation. We also examined the potential of ToC for pharmaceutical industries in which ToC adoption is expected to occur gradually. Looking ahead, given the high failure rate of clinical trials and the increasing emphasis on the 3Rs principles (replacement, reduction, refinement of animal experimentation), ToC models hold immense potential for cancer research. In the next decade, data generated from ToC models could potentially be employed for discovering new therapeutic targets, contributing to regulatory purposes, refining preclinical drug testing and reducing reliance on animal models.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms

Treated municipal wastewater (TMW) can provide a reliable source of irrigation water for crops, which is especially important in arid areas where water resources are limited or prone to drought. Nonetheless, TMW may contain residual antibiotics, potentially exposing the crops to these substances. The goal of this study was to investigate the dissemination of antibiotics resistance genes (ARGs) in the soil-plant-earthworm continuum after irrigation of spinach and radish plants with TMW containing trimethoprim, sulfamethoxazole, and sulfapyridine in a greenhouse experiment, followed by feeding of earthworms with harvested plant materials. Our results showed that antibiotic resistance genes (ARGs) were enriched in the soil-plant-earthworm microbiomes irrigated with TMW and TMW spiked with higher concentrations of antibiotics. The number of ARGs and antibiotic-resistant bacteria (ARB) enrichment varied with plant type, with spinach harboring a significantly higher amount of ARGs and ARB compared to radish. Our data showed that bulk and rhizosphere soils of spinach and radish plants irrigated with MilliQ water, TMW, TMW10, or TMW100 had significant differences in bacterial community (p < 0.001), ARG (p < 0.001), and virulence factor gene (VFG) (p < 0.001) diversities. The abundance of ARGs significantly decreased from bulk soil to rhizosphere to phyllosphere and endosphere. Using metagenome assembled genomes (MAGs), we recovered many bacterial MAGs and a near complete genome (>90 %) of bacterial MAG of genus Leclercia adecarboxylata B from the fecal microbiome of earthworm that was fed harvested radish tubers and spinach leaves grown on TMW10 irrigated waters, and this bacterium has been shown to be an emerging pathogen causing infection in immunocompromised patients that may lead to health complications and death. Therefore, crops irrigated with TMW containing residual antibiotics and ARGs may lead to increased incidences of enrichment of ARB in the soil-plant-earthworm continuum.

Harmful algal blooms (HABs) have incurred numerous health problems through drinking water or exposure. A better understanding of the growth kinetics and toxin production under different environmental conditions will help develop tools for early‐warning indicators for predicting HABs. Lab‐scale experiments were performed with Microcystis aeruginosa PCC 7806 under varying N:P ratios and temperatures. We observed different growth behaviors for cultures growing at the three temperatures (20, 25, and 30°C). M. aeruginosa exhibited higher growth rates at higher N:P ratios during the incubation at 25 and 30°C. Microcystis 16S rRNA gene abundance correlated well with the cell growth parameters. Based on RT‐qPCR data, the transcripts of mcyA , mcyD , and mcyE were occasionally detected with N:P of 76.86 showing highest detections of all gene transcripts. Intracellular microcystin (MC) production corresponded well with the presence of gene transcripts, while no extracellular MC was detected. These findings would provide valuable insights and beneficial guidance to further explore and predict the impacts of cyanobacterial blooms in freshwater systems.

Fecal pollution of surface waters in the karst-dominated Edwards aquifer is a serious concern as contaminated waters can rapidly transmit to groundwaters, which are used for domestic purposes. Although microbial source tracking (MST) detects sources of fecal pollution, integrating data related to environmental processes (precipitation) and land management practices (septic tanks) with MST can provide better understanding of fecal contamination fluxes to implement effective mitigation strategies. Here, we investigated fecal sources and their spatial origins at recharge and contributing zones of the Edwards aquifer and identified their relationship with nutrients in different environmental/land-use conditions. During March 2019 to March 2020, water samples (n = 295) were collected biweekly from 11 sampling sites across four creeks and analyzed for six physico-chemical parameters and ten fecal indicator bacteria (FIB) and MST-based qPCR assays targeting general (E. coli, Enterococcus, and universal Bacteroidales), human (BacHum and HF183), ruminant (Rum2Bac), cattle (BacCow), canine (BacCan), and avian (Chicken/Duck-Bac and GFD) fecal markers. Among physico-chemical parameters, nitrate-N (NO3-N) concentrations at several sites were higher than estimated national background concentrations for streams. General fecal markers were detected in the majority of water samples, and among host-associated MST markers, GFD, BacCow, and Rum2Bac were more frequently detected than BacCan, BacHum, and HF183, indicating avian and ruminant fecal contamination is a major concern. Cluster analysis results indicated that sampling sites clustered based on precipitation and septic tank density showed significant correlation (p < 0.05) between nutrients and FIB/MST markers, indicating these factors are influencing the spatial and temporal variations of fecal sources. Overall, results emphasize that integration of environmental/land-use data with MST is crucial for a better understanding of nutrient loading and fecal contamination.

The COVID-19 pandemic has been challenging for various institutions such as school systems due to widespread closures. As schools re-open their campuses to in-person education, there is a need for frequent screening and monitoring of the virus to ensure the safety of students and staff and to limit risk to the surrounding community. Wastewater surveillance (WWS) of SARS-CoV-2 is a rapid and economical approach to determine the extent of COVID-19 in the community. The focus of this review is on the emergence of WWS as a tool for safe return to school campuses, taking into account methodological considerations such as site selection, sample collection and processing, SARS-CoV-2 quantification, and data interpretation. Recently published studies on the implementation of COVID-19 WWS on school and college campuses were reviewed. While there are several logistical and technical challenges, WWS can be used to inform decision-making at the school campus and/or building level.

The purpose of this study was to conduct a correlative assessment of SARS-CoV-2 RNA concentrations in wastewater with COVID-19 cases and a systematic evaluation of the effect of using different virus concentration methods and recovery and normalization approaches. We measured SARS-CoV-2 RNA concentrations at two different wastewater treatment plants (WWTPs) in the Bexar County of Texas from October 2020 to May 2021 (32 weeks) using reverse transcription droplet digital PCR (RT-ddPCR). We evaluated three different adsorption–extraction (AE) based virus concentration methods (acidification, addition of MgCl2, or without any pretreatment) using bovine coronavirus (BCoV) as surrogate virus and observed that the direct AE method showed the highest mean recovery. COVID-19 cases were correlated significantly with SARS-CoV-2 N1 concentrations in Salitrillo (ρ = 0.75, p < 0.001) and Martinez II (ρ = 0.68, p < 0.001) WWTPs, but normalizing to a spiked recovery control (BCoV) or a fecal marker (HF183) reduced correlations for both treatment plants. The results generated in this 32-week monitoring study will enable researchers to prioritize the virus recovery method and subsequent correlation studies for wastewater surveillance.

Hurricanes and extreme stormwater events can transport fecal contaminants and a wide range of bacterial pathogens to receiving rivers and streams, threatening public health. This study investigated the impact of flooding on bacterial diversity and the occurrence of fecal and potential bacterial pathogens in Texas Rivers over a short (3 weeks and 3 months) and long time (12 months) after Hurricane Harvey. Water samples were collected from 8 sampling sites of Guadalupe and San Antonio Rivers during three sampling events and bacterial community structure was evaluated using next-generation sequencing (NGS). Results showed that Proteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria were the predominant phyla in the water samples. Hierarchal cluster analysis and principal coordinate analysis indicated that bacterial community structure was significantly different in the water samples collected from flooded and non-flooded sites. At genus level, eight fecal-associated and twelve potentially pathogenic bacterial genera were detected in water samples, mainly from flooded sites collected during short-term sampling events. Overall, results suggest that NGS-based microbial water quality monitoring of environmental samples after flooding events could provide critical information about the wide range of pathogens, which can be further assessed by specific methods to identify the risk of exposure.

ABSTRACTThe purpose of this study was to conduct a preliminary assessment of the levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater at the Salitrillo Wastewater Treatment Plant in Texas during the initial peak of coronavirus disease 2019 (COVID-19) outbreak. Raw wastewater influent (24 h composite, time-based 1 L samples, n = 13) was collected weekly during June–August 2020. We measured SARS-CoV-2 RNA in wastewater by reverse transcription droplet digital PCR using the same N1 and N2 primer sets as employed in COVID-19 clinical testing. Virus RNA copies for positive samples (77%) ranged from 1.4 × 102 to 4.1 × 104 copies per liter of wastewater, and exhibited both increasing and decreasing trends, which corresponded well with the COVID-19 weekly infection rate (N1: ρ = 0.558, P = 0.048; N2: ρ = 0.487, P = 0.092). A sharp increase in virus RNA concentrations was observed during July sampling dates, consistent with the highest number of COVID-19 cases reported. This could be attributed to an increase in the spread of COVID-19 infection due to the Fourth of July holiday week gatherings (outdoor gatherings were limited to 100 people during that time). Our data show that wastewater surveillance is an effective tool to determine trends in infectious disease prevalence, and provide complementary information to clinical testing.

The vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body. In this paper, a novel modular microfluidic system with a vertical two-layered configuration is developed to generate large-scale perfused microvascular networks in vitro. The two-layer polydimethylsiloxane (PDMS) configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly. This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities. The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction, serving as the interface between the medium channels and tissue chambers. Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels. Various large-scale microvascular networks were generated and quantified in terms of vessel length and density. Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers. This platform enables the culturing of interconnected, large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications, including basic biological studies and drug screening.

Synthetic DNA tracers provide a promising approach for tracking water because they are unique, environmentally safe, and versatile and have low detection limits. This study aimed to develop and quantify synthetic double-stranded DNA tracers using a droplet digital PCR (ddPCR) platform and compared their performance with that of uranine dye, a widely used conventional tracer for hydrological investigations. The DNA tracers were stable in distilled water at 4 degrees C; however, their concentrations decreased at 25 and 40 degrees C. The DNA tracers degraded considerably in river water following a one-phase decay pattern at different temperatures (k = 0.125-0.071 h(-1)). In column experiments with sand and limestone media, the DNA tracers traveled at a greater speed (RV = 0.19) and showed less dispersion (RS = 0.05) and lower mass recovery (RB = 0.26) than uranine. To demonstrate concurrent tracing, two unique DNA tracers were injected simultaneously in the column, could be detected in the same sample, and exhibited similar peak concentration times. Finally, in a field experiment, DNA tracers were deployed, could be detected in a surface stream, and exhibited earlier breakthrough in comparison to uranine. The results of this study demonstrate the feasibility of using synthetic DNA tracers for tracking water and the effectiveness of ddPCR in quantifying the tracers.

The interaction of anaerobic microorganisms with carbon nanotube/polymer nanocomposites (CNT/PNC) will play a major role in determining their persistence and environmental fate at the end of consumer use when these nano-enabled materials enter landfills and encounter wastewater. Motivated by the need to understand how different parameters (i.e., polymer type, microbial phenotype, CNT characteristics) influence CNT/PNC biodegradation rates, we have used volumetric biogas measurements and kinetic modeling to study biodegradation as a function of polymer type and CNT properties. In one set of experiments, oxidized multiwall carbon nanotubes (O-MWCNTs) with a range of CNT loadings 0–5% w/w were incorporated into poly-ε-caprolactone (PCL) and polyhydroxyalkanoates (PHA) matrices and subjected to biodegradation by an anaerobic microbial community. For each CNT/PNC, complete polymer biodegradation was ultimately observed, although the rate of biodegradation was inhibited above certain critical CNT loadings dependent upon the polymer type. Higher loadings of pristine MWCNTs were needed to decrease the rate of polymer biodegradation compared to O-MWCNTs, an effect ascribed principally to differences in CNT dispersion within the polymer matrices. Above certain CNT loadings, a CNT mat of similar shape to the initial PNC was formed after polymer biodegradation, while below this threshold, CNT aggregates fragmented in the media. In situations where biodegradation was rapid, methanogen growth was disproportionately inhibited compared to the overall microbial community. Analysis of the results obtained from this study indicates that the inhibitory effect of CNTs on polymer biodegradation rate is greatest under conditions (i.e., polymer type, microbial phenotype, CNT dispersion) where biodegradation of the neat polymer is slowest. This new insight provides a means to predict the environmental fate, persistence, and transformations of CNT-enabled polymer materials.