Mechanistic exploration of COVlD-19 antiviral drug ritonavir on anaerobic digestion through experimental validation coupled with metagenomics analysis.

Aggregation of antiviral drugs (ATVs) in waste activated sludge (WAS) poses considerable environmental risk, so it is crucial to understand the behavior of these agents during WAS treatment. This study investigated the effects of ritonavir (RIT), an ATV used to treat human immunodeficiency virus infection and coronavirus disease 2019, on anaerobic digestion (AD) of WAS to reveal the mechanisms by which it interferes with anaerobic flora. The dosage influence results showed that methane production in AD of WAS decreased by 46.56 % when RIT concentration was increased to 1000 µg/kg total suspended solids (TSS). The AD staging test revealed that RIT mainly stimulated microbial synthesis of the extracellular polymeric substance (EPS), limiting organic matter solubilization. At 500 µg/kg TSS, RIT decreased CHO and CHON levels in dissolved organic matter by 23.12 % and 56.68 %, respectively, significantly reducing substrate availability to microorganisms. Metagenomic analysis of microbial functional gene sets revealed that RIT had greater inhibitory effects on protein and amino acid metabolism than on carbohydrate metabolism. Under RIT stress, methanogens switched from hydrogenotrophic and acetotrophic methanogenesis to methylotrophic and acetotrophic methanogenesis.

Evaluation of the texture characteristics and taste of shrimp surimi with partial replacement of NaCl by non­sodium metal salts.

Ionic strength plays a significant role in the aggregation behavior of myofibrillar proteins. The study investigated the effects of KCl or CaCl2 as substitutes for NaCl on the gel properties and taste of shrimp surimi at a constant ionic strength (IS = 0.51). Increased KCl substitution ratio resulted in a reduction in α-helix content and an increase in ß-sheet content of myofibrillar proteins, thereby enhancing water holding capacity. Optimal KCl substitutions (1.5% NaCl +1.94% KCl) contributed to maintaining the desired taste and improving gel properties. CaCl2 facilitates the extraction and dissolution of myofibrillar proteins, resulting in an organized and dense gel network with significant water-holding capacity. However, excessive additions (>1.27%) resulted in a notable decrease in taste and gel strength due to excessive aggregation and precipitation of myofibrillar proteins. These findings provide a solid theoretical foundation for production of high-quality, low-salt shrimp surimi.

Novel strategy for efficient energy recovery and pollutant control from sewage sludge and food waste treatment.

Considering the high organic matter contents and pollutants in sewage sludge (SS) and food waste (FW), seeking green and effective technology for energy recovery and pollutant control is a big challenge. In this study, we proposed a integrated technology combing SS mass separation by hydrothermal pretreatment, methane production from co-digestion of hydrothermally treated sewage sludge (HSS) centrate and FW, and biochar production from co-pyrolysis of HSS cake and digestate with heavy metal immobilization for synergistic utilization of SS and FW. The results showed that the co-digestion of HSS centrate with FW reduced the NH4+-N concentration and promoted volatile fatty acids conversion, leading to a more robust anaerobic system for better methane generation. Among the co-pyrolysis of HSS cake and digestate, digestate addition improved biochar quality with heavy metals immobilization and toxicity reduction. Following the lab-scale investigation, the pilot-scale verification was successfully performed (except the co-digestion process). The mass and energy balance revealed that the produced methane could supply the whole energy consumption of the integrated system with 26.2 t biochar generation for treating 300 t SS and 120 t FW. This study presents a new strategy and technology validation for synergistic treatment of SS and FW with resource recovery and pollutants control.

Mechanistic insights into Fe3O4-modified biochar relieving inhibition from erythromycin on anaerobic digestion.

Anaerobic digestion (AD) of antibiotic manufacturing wastewater to degrade residual antibiotics and produce mixture of combustible gases has been investigated actively in the past decades. However, detrimental effect of residual antibiotic to microbial activities is commonly faced in AD process, leading to the reduction of treatment efficiency and energy recovery. Herein, the present study systematically evaluated the detoxification effect and mechanism of Fe3O4-modified biochar in AD of erythromycin manufacturing wastewater. Results showed that Fe3O4-modified biochar had stimulatory effect on AD at 0.5 g/L erythromycin existence. A maximum methane yield of 327.7 ± 8.0 mL/g COD was achieved at 3.0 g/L Fe3O4-modified biochar, leading to the increase of 55.7% compared to control group. Mechanistic investigation demonstrated that different levels of Fe3O4-modified biochar could improve methane yield via different metabolic pathways involved in specific bacteria and archaea. Low levels of Fe3O4-modified biochar (i.e., 0.5-1.0 g/L) led to the enrichment of Methanothermobacter sp., strengthening the hydrogenotrophic pathway. On the contrary, high levels of Fe3O4-modified biochar (2.0-3.0 g/L) favored the proliferation of acetogens (e.g., Lentimicrobium sp.) and methanogen (Methanosarcina sp.) and their syntrophic relations played vital role on the simulated AD performance at erythromycin stress. Additionally, the addition of Fe3O4-modified biochar significantly decreased the abundance of representative antibiotic resistant genes (ARGs), benefiting the reduction of environmental risk. The results of this study verified that the application of Fe3O4-modified biochar could be an efficient approach to detoxify erythromycin on AD system, which brings high impacts and positive implications for biological antibiotic wastewater treatment.

Antiviral drugs in wastewater are on the rise as emerging contaminants: A comprehensive review of spatiotemporal characteristics, removal technologies and environmental risks.

Antiviral drugs (ATVs) are widely used to treat illnesses caused by viruses. Particularly, ATVs were consumed in such large quantities during the pandemic that high concentrations were detected in wastewater and aquatic environment. Since ATVs are not fully absorbed by the human or animal body, this results in large amounts of them being discharged into the sewage through urine or feces. Most ATVs can be degraded by microbes at wastewater treatment plants (WWTPs), while some ATVs either require deep treatment to reduce concentration and toxicity. Parent and metabolites residing in effluent posed a varying degree of risk when entering the aquatic environment, while increasing the potential of natural reservoirs for environmentally acquired antiviral drug resistance potential. There is a rising research on the behavior of ATVs in the environment has surged since the pandemic. In the context of multiple viral diseases worldwide, especially during the current COVID-19 pandemic, a comprehensive assessment of the occurrence, removal, and risk of ATVs is urgently needed. This review aims to discuss the fate of ATVs in WWTPs from various regions in the world with wastewater as the main analyzing object. The ultimate goal is to focus on ATVs with high ecological impact and regulate their use or develop advanced treatment technologies to mitigate the risk to the environment.

Density and row spacing of short-season cotton suitable for machine picking in the cotton region of Yellow River Basin.

To determine the suitable planting density and row spacing of short-season cotton suitable for machine picking in the Yellow River Basin of China, we conducted a two-year field experiment in Dezhou during 2018-2019. The experiment followed a split-plot design, with planting density (82500 plants·hm-2 and 112500 plants·hm-2) as the main plots and row spacing (equal row spacing of 76 cm, wide-narrow row spacing of 66 cm+10 cm, equal row spacing of 60 cm) as the subplots. We examined the effects of planting density and row spacing on growth and development, canopy structure, seed cotton yield and fiber quality of short-season cotton. The results showed that plant height and LAI under high density treatment were significantly greater than those under low density treatment. The transmittance of the bottom layer was significantly lower than under low density treatment. Plant height under 76 cm equal row spacing was significantly higher than that under 60 cm equal row spacing, while that under wide-narrow row spacing (66 cm +10 cm) was significantly smaller than that under 60 cm equal row spacing in peak bolling stage. The effects of row spacing on LAI varied between the two years, densities, and growth stages. On the whole, the LAI under the wide-narrow row spacing (66 cm+10 cm) was higher, with the curve declining gently after the peak, and it was higher than that in the two cases of equal row spacing in the harvest time. The change in transmittance of the bottom layer presented the opposite trend. Density, row spacing, and their interaction had significant effects on seed cotton yield and its components. In both years, seed cotton yield was the highest (3832 kg·hm-2 in 2018, 3235 kg·hm-2 in 2019) under wide-narrow row spacing (66 cm+10 cm), and it was more stable at high densities. Fiber quality was less affected by density and row spacing. To sum up, the optimal density and row spacing of short-season cotton were as follows: density with 112500 plants·hm-2 and wide-narrow row spacing (66 cm+10 cm).

Compost-derived indole-3-acetic-acid-producing bacteria and their effects on enhancing the secondary fermentation of a swine manure-corn stalk composting.

Composting, as an effectively bio-oxidative process, has been widely used for converting organic waste to organic fertilizer. However, the low fertilizer efficiency of composting product limited its application in agriculture. To improve the growth-promoting effect of composting product, the present study investigated the bioaugmentation strategy of inoculating indole-3-acetic-acid (IAA)-producing bacteria. Firstly, two IAA-producing bacteria (Bacillus safensis 33C and Rhodococcus rhodochrous YZ) were isolated from composting products with high IAA yields of 39.18 and 16.32 µg mL-1, respectively. Secondly, the microbial inoculants were prepared with 33C, YZ and a previously isolated IAA-producing strain Corynebacterium stationis 29B. To increase the accumulation of microbial secondary metabolites, microbial inoculants were amended at the secondary fermentation stage of composting. Physicochemical characterization showed that the maturity of composting product was significantly promoted by inoculating microbial inoculants prepared with 33C and 29B (single and combined inoculants). Finally, bioaugmentation with 33C and 29B increased the IAA contents of composting products by 2.9-5.2 times, which benefited the germination and early vegetative growth of plants. In summary, inoculating proper IAA-producing bacteria during secondary fermentation of composting could improve the quality of composting product and expand its application.

pH and hydraulic retention time regulation for anaerobic fermentation: Focus on volatile fatty acids production/distribution, microbial community succession and interactive correlation.

Enriching suitable fermentative products by optimizing operation conditions could effectively improve the efficiency of anaerobic digestion. In the present study, pH (5.0-6.0) and hydraulic retention time (HRT) (2 h-12 h) were regulated for volatile fatty acids (VFAs) production during glucose fermentation in acidogenic continuous stirred tank reactor (CSTR). Results showed that acetate and butyrate dominated during pH regulation. HRT reduction favored butyrate production and formate retainment. Maximum total VFAs production with highest acetate content was achieved at pH of 6.0 and HRT of 6 h. Microbial analysis revealed that Clostridium_sensu_stricto_1 was predominant butyrate producer during pH regulation, and Bacteroides was main contributor when HRT shorter than 6 h. In addition to acetyl-CoA pathway, acetate could also be produced via homoacetogenesis by Parabacteroides, UCG-004 and norank_f__Acidaminococcaceae. These results would give guidance for enhancing targeted VFAs products by optimizing operational parameters or bio-augmentation with specific bacteria.

Carbon- and metal-based mediators modulate anaerobic methanogenesis and phenol removal: Focusing on stimulatory and inhibitory mechanism.

In this study, anaerobic batch experiments were conducted to investigate the effect of carbon-based (biochar) and metal-based (nanoscale zero-valent iron, NZVI and zero valent iron, ZVI) mediators on the AD process treating phenolic wastewater. Fresh apricot shell- and wood-derived biochar (BiocharA, BiocharB) could remove the phenol efficiently (77.1% and 86.2%), suggesting that biodegradation cooperated with adsorption had advantage in phenol removal. BiocharB, NZVI and ZVI enhanced the methane production by 17.6%, 23.7% and 23.2%, respectively. Apart from serving as carrier for microbial growth, BiocharB might promote the direct interspecies electron transfer (DIET) since the Anaerolineaceae/Clostridium sensu stricto, which have potential for DIET, were enriched. NZVI and ZVI added systems mainly enhanced the abundance of Clostridium sensu stricto (24.5%, 37.6%) and Methanosaeta. Interestingly, BiocharA inhibited the methanogenesis completely. An inhibitory mechanism was proposed: the exposure of absorbed microbes on the BiocharA to the highly concentrated phenol in biochar' pores resulted in the inhibition of methanogens, especially for Methanosarcina. In conclusion, this study showed that suitable biochar (BiocharB) could serve as an alternative redox mediator for realizing simultaneously the efficient phenol removal and methane production.

Biosynthesis of polyhydroxyalkanoate from food waste oil by Pseudomonas alcaligenes with simultaneous energy recovery from fermentation wastewater.

Bioconversion of food waste oil (FWO) into biodegradable plastic is a promising method for converting waste into high-value products. In this study, a strain (Pseudomonas sp. H3) was isolated for polyhydroxyalkanoate (PHA) synthesis from FWO. After 72 h of cultivation with 20 g/L of FWO, the high cell dry weight (CDW) of 3.6 g/L, PHA yield of 2.4 g/L, and PHA content of 65 wt% were obtained under the optimal temperature (25 °C) and inoculum amount (6% (v/v)). Fed-batch fermentation was conducted in a 5 L bioreactor with a maximum CDW of 16 g/L, PHA content of 54 wt%, and PHA productivity of 0.23 g/(L·h) after 36 h. The PHA had a molecular weight of 54 782 Da and a low polydispersity index of 1.41 with glass transition, melting, and degradation temperatures of -20 °C, 34 °C, and 210 °C, respectively. To further utilize the wastewater after PHA production, anaerobic digestion was employed for CH4 production, and the CH4 yield was 284 mL/g volatile solids. Microbial community analysis showed that the abundance of acetate-oxidizing bacteria and Methanobacterium significantly increased during anaerobic digestion. This study describes a new strain for the economical synthesis of biodegradable plastics and presents a novel framework for fully utilizing FWO with the production of PHA and CH4.

Deeper investigation on methane generation from synthetic wastewater containing oxytetracycline in a scale up acidic anaerobic baffled reactor.

Acidic anaerobic digestion attracted much attention and interest due to its significant advantage in wastewater treatment. In the present study, methanogenic fermentation was successfully operated under acidic condition during treating wastewater containing oxytetracycline (OTC) in a scale up anaerobic baffled reactor (ABR). After start-up process, the pH value in the first compartment was 4.60 with high activity of methanogenesis. After stabilization, different OTC loading of 1.0, 3.3 and 5.0 g/m3/d was added in the influent for OTC removal. The resulted showed that OTC addition had little impact on the methane generation with whole COD and OTC removal rate of 95% and 60%, respectively. The microbial analysis, OTC addition could significantly influence the bacteria and archaea communities. To be more specific, Methanosaeta showed the highest relative abundance and tolerance to OTC under acidic condition. The present work supplied deeper insights into methane generation from acidic condition during wastewater containing OTC treatment.

Syntrophic butyrate-oxidizing methanogenesis promoted by anthraquinone-2-sulfonate and cysteine: Distinct tendencies towards the enrichment of methanogens and syntrophic fatty-acid oxidizing bacteria.

Interspecies electron transfer (IET) between syntrophic fatty-acid oxidizing bacteria (SFOBs) and methanogens decided the performance of anaerobic digestion. Electron shuttles, as potential IET accelerators, were controversial concerning their influences on methanogenesis. In this study, concentration-dependent effects of anthraquinone-2-sulfonate (AQS) and cysteine on glucose digestion were firstly demonstrated: low dosage of AQS and cysteine (50 and 100 µM, respectively) had highest methane yield (133.5% and 148.6%, respectively). Using butyrate as substrate, distinct tendencies towards the enrichment of methanogenic community were further revealed. Cysteine just acted as a reductant which lowered ORP quickly and enriched most methanogens. It benefited methanogenesis right until methanogenic substrates accumulated. AQS, however, showed characteristic features of electron shuttles: it was firstly oxidized by SFOBs and then reduced by hydrogenotrophic methanogens, which accelerated methanogenic butyrate degradation. This study showed wide spectrum of SFOBs and methanogens benefited from the addition of electron shuttles, which laid foundation for future application.

Novel strategy for enhancing acetic and formic acids generation in acidogenesis of anaerobic digestion via targeted adjusting environmental niches.

Optimization of acetic acid and formic acid production efficient methanogenesis is always the research hot spot in anaerobic digestion. It is a promising approach to adjust the operation parameters to influence the functional microorganisms for better acetic acid and formic acid production in acidogenesis. Herein, the effects of pH, oxidation-reduction potential (ORP) and carbon-nitrogen (C/N) ratio were determined in batch experiments to probe acetic and formic acids production, and were further verified in continuous stirred tank reactor (CSTR). The results revealed that the content of volatile fatty acids (VFAs) reached to maximum at pH 6.0 or ORP -350 mV, while the production of acetic and formic acids was the highest at pH 7.0 or ORP -450 mV in 9 h fermentation. Also, fermentation products dominated by acetic and formic acids were adjusted in the CSTR under the operating conditions of pH 7.0 and ORP -450 mV. Microbiological analysis from batch test showed that fermentation at pH value of 7.0 enriched the diversity of microorganism, and provided a niche for microbes (Petrimonas, norank_f__Synergistaceae, vadinBC27_wastewater-sludge_group, and Trichococcus) to produce acetic and formic acids. Correspondingly, 78.70% of the carbon was converted to acetic and formic acids in pH 7.0. This study provides a promising strategy for the targeted regulation of acetic and formic acids production in acidogenesis of anaerobic digestion.

Deeper insights into effect of activated carbon and nano-zero-valent iron addition on acidogenesis and whole anaerobic digestion.

Conductive materials presented promising advantages for enhancing anaerobic digestion (AD) performance. This study evaluated the effects of activated carbon (AC) and nano-zero-valent iron (nZVI) on the acidogenesis and whole AD to explore their potential mechanisms. AC increased the content of lactic and propionic acids in acidogenesis. nZVI increased the production of formic acid, acetic acid and H2 in acidogenesis, thus significantly promoted the methane yield in the whole AD. Mechanism exploration proved that AC enriched Trichococcus, and norank_f__Bacteroidetes_vadinHA17, and then improved the activity of enzymes involved in the production of lactic and propionic acids. nZVI buffered the pH to increase the activity of pyruvate formate-lyase (PFL) in formic acid production. Furthermore, nZVI enriched the Methanobacterium which use H2 and formic acid as substrate. The research paves pathway for the efficient enhancement of conductive materials added novel AD process.

Novel strategy for relieving acid accumulation by enriching syntrophic associations of syntrophic fatty acid-oxidation bacteria and H2/formate-scavenging methanogens in anaerobic digestion.

Aiming at relieving acid accumulation in anaerobic digestion (AD), syntrophic associations of syntrophic fatty acid-oxidation bacteria and H2/formate-scavenging methanogens were enriched by feeding propionate, butyrate and formate in an up-flow anaerobic sludge blanket (UASB) reactor. Results showed that methane yield increased by 50% with increasing formate concentration (0-2000 mg COD/L). In addition, the abundance and quantity of SFOB (Syntrophobacter, Smithella and Syntrophomonas) and H2/formate-scavenging methanogens (Methanobacteriales and Methanomicrobiales) were increased after microbial acclimation. The enriched syntrophic associations showed higher propionate and butyrate removal efficiencies of 98.48 ± 1.14% and 99.71 ± 0.71%, respectively. Furthermore, encoding genes of formate dehydrogenase and hydrogenases presented higher abundances after microbial enrichment, which suggested that the enhancements of interspecies formate transfer and interspecies hydrogen transfer between syntrophic associations benefited volatile fatty acids (VFAs) conversion. This research provided an effective strategy to relieve acid accumulation.

Anaerobic digestion of sludge filtrate using anaerobic baffled reactor assisted by symbionts of short chain fatty acid-oxidation syntrophs and exoelectrogens: Pilot-scale verification.

The growing amount of sewage sludge from wastewater treatment plant is an emerging challenge in China. The efficient anaerobic digestion of sludge filtrate generated from hydrothermally pretreated sewage sludge can promote the disposal of sewage sludge. Herein, a pilot-scale anaerobic baffled reactor (ABR) assisted by symbionts of short chain fatty acid-oxidation syntrophs (SFAS) and exoelectrogens was developed to improve its stability and efficiency for filtrate treatment. The results demonstrated that the symbionts of exoelectrogens and SFAS, which were enriched by introduction of electrodes in the ABR system, promoted the degradation of butyric, propionic and acetic acids. Therefore, the COD removal efficiency increased from 74.1% to 86.6% and the methane content increased from 81.5% to 92.2% with methane production rising from 241 to 282 mL/g CODremoved. Furthermore, the economic evaluation indicated that the energy consumption of electrodes was 0.600 kWh/m3 of sludge filtrate, the net energy profited from increased methane was 2.344 kWh/m3 of sludge filtrate. These results confirmed that the ABR system assisted by symbionts of SFAS and exoelectrogens was feasible for treatment of sludge filtrate in terms of both technical and economic level through pilot-scale verification.

Simultaneous biogas and biogas slurry production from co-digestion of pig manure and corn straw: Performance optimization and microbial community shift.

Anaerobic co-digestion (AcoD) is proved as an effective approach to solving a bottleneck problem of the low biogas yield in agricultural biomass waste treatment with anaerobic digestion (AD) technology. The present study investigated the effect of C/N radio, organic loading rate (OLR) and total solids (TS) contents on reactor performance in AcoD of pig manure and corn straw for simultaneous biogas and biogas slurry production. It was found that the highest biogas production was obtained at C/N ratio of 25, while the best biogas slurry performance was achieved at C/N ratio of 35. And high OLR and TS resulted in good performances in both biogas production and biogas slurry. At last, the microbial community analysis suggested that Bacteroidetes played a significant role in AcoD process. Acetoclastic methanogenesis was the main pathway for methane production in the stable system. And changing operational parameters could transform and shift the microbial community.

Assessment of multiple exposure to chemical elements and health risks among residents near Huodehong lead-zinc mining area in Yunnan, Southwest China.

The contents of chemical elements (Cd, Cr, Cu, Pb and Zn) in 11 kinds of crop/vegetables and soils around the Huodehong lead-zinc mining area in Yunnan, Southwest China were determined by using inductive coupled plasma emission spectrometry (ICP-MS). Results showed that element contents in soils decreased in the order of Zn > Pb > Cr > Cu > Cd. The high geo-accumulation indexes (Igeo) showed that cultivated soils near mine were practically polluted by Cd, Pb and Zn. The contents of Cd, Cr and Pb in crop/vegetables samples were significantly higher than the maximum permissible standard set by China. The potential health risk assessments among local residents were evaluated by the hazard index (HI), the total carcinogenic risk (TCR), the target hazard quotient (THQ) and carcinogenic risk (CR), respectively. The results showed that diet was the dominant exposure pathway. The results of HI for adult and child were 6.21 and 6.08, respectively. TCR values of Cr and Cd were more than 10-4. The THQ decreased in the following order: Cd > Pb > Cu > Zn > Cr. Among all kinds of crop/vegetables, leafy-vegetables were the major source of Cd and Pb exposure, root-vegetable were the important factors for Cu and Zn exposure, but tuber-vegetable were the factors for Cr exposure. The contents of Cd and Pb in human scalp hairs near Huodehong mine were higher than that in S20km area. Females possessed a higher risk for Cd, Cr, Cu and Pb exposure than males in study area. Significant differences between ages were found for Cd, Cu and Pb (p < 0.01). This study provided a powerful basis for the coordination of local environmental protection and economic sustainable development and assessing chemical elements risk to human health.

Study on Adsorption and Desorption Characteristics of Cd2+ and Cu2+ on the Surface Sediments of Sanhuhekou of Yellow River by Using ICP-MS.

In order to study the interaction mechanism between Cd2+, Cu2+ and surface sediments in the upper reaches of the Yellow River, the surface sediment of Sanhuhekou (YRSSM) was chosen as research object with inductively coupled plasma mass spectrometry (ICP-MS) as analysis method. The adsorption reaction condition such as liquid-solid ratio, reaction time and pH were optimized, and the adsorption and desorption characteristics of Cd2+ and Cu2+ onto the surface sediments under the optimized experimental conditions were studied. The results showed that the adsorption capacity of Cu2+ was greater than that of Cd2+, the equilibrium absorption capacity were 0.88 and 0.13 mg·g-1 under each optimum experimental condition, respectively. The adsorptions of Cu2+ and Cd2+ were in accord with the pseudo-second-order kinetic, while adsorption rate of Cu2+ was also greater than that of Cd2+. The adsorption thermodynamics data were in accordance with the Freundlich model and the fitting. Results showed that the adsorption process of Cu2+ and Cd2+ belonged to the preferential adsorption, and were endothermic and spontaneous processes. The desorption process showed that the Elovich equation were suitable for Cd2+ and Cu2+ and belonged to the heterogeneous diffusion. Multi-ions competitive adsorption and desorption experiments indicated that Cu2+ was influenced more by co-existing ion. The study revealed not only the mechanism of adsorption and desorption between Cd2+, Cu2+ and surface sediment in Sanhuhekou, but also the influence of coexisting ions on the adsorption and desorption characteristics. The results demonstrated that the distribution mechanism of heavy metals between solid-liquid phases, and provided a theoretical basis for the migration ability of heavy metals. It also had a guiding significance for establishing heavy metals preventive and control measures of the study area.