A review on photocatalytic attribution and process of pyrolytic biochar in environment.

Biochar has attracted significant attention due to its excellent environmental benefits and extensive applications. Recently, a consensus has been accepted that biochar can act as a photocatalyst and trigger effective photocatalytic reactions in the environment, which is important to energy conversion and the cycle of elements. However, its photocatalytic processes and the corresponding environmental impacts need to receive more and due attention. In this review, we provide a comprehensive summary of the underlying correlations among the pyrolytic evolution of biomass, the structure characteristic of biochar, and the resultant photocatalytic performance. Moreover, the photocatalytic processes and the influence of environmental factors were elaborately investigated on biochar. Finally, future tendencies and challenges in the photocatalysis of biochar have been prospected in the environmental field. This review has offered innovative insights into the photocatalytic essential of biochar and highly enhanced the understanding of its environmental impact.

Different photoreduction processes of Cr(VI) on cellulose-rich and lignin-rich biochar.

In this study, a series of biochar were prepared via pyrolyzing cellulose-rich pakchoi (PBC) and lignin-rich corncob (CBC) to explore the photoreduction process of Cr(VI). X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy confirmed higher oxygenated functional groups in PBC (48.9%-57.1%), whereas CBC exhibited more aromatization properties due to the stable aromatic network in lignin. For PBC, the valence bands decreased from 1.42 eV to 1.20 eV with the increase of pyrolysis temperature from 300 °C to 500 °C; however, an opposite trend was observed for CBC. The photoreduction of Cr(VI) clearly showed that both PBC and CBC had the best performance at the carbonization temperature of 300 °C (named PBC300 and CBC300). It is noted that PBC300 exhibited the most effective photoreduction of Cr(VI), which was about 1.3 times higher than that of CBC300. The maximum reduction capacities of Cr(VI) were 68.2 mg g-1 on PBC300 and 66.1 mg g-1 on CBC300 at pH∼2.0. Compared with the insoluble char substances, dissolved black carbons made more contributions for Cr(VI) photoreduction, ∼70% in PBC and almost 100% in CBC, which suggested that in the case of PBC, the insoluble char and the corresponding dissolved black carbons play an important role in the photoreduction of Cr(VI). However, only dissolved black carbons contributed to Cr(VI) photoreduction on CBC. As the key reaction pathway, the interfacial electron transport dominated Cr(VI) reduction on PBC and CBC. Moreover, the radical of •O2- had some contribution to the reduction of Cr(VI) only in the PBC system. Interestingly, •OH could promote the photoreduction of Cr(VI) in both PBC and CBC systems, which might be due to the fact that •OH facilitated the formation of small molecule fragments. These findings provide an essential basis for evaluating the environmental impact of photocatalytic behaviors of biochar.

Heterogeneous photo-Fenton catalyst α-Fe2O3@g-C3N4@NH2-MIL-101(Fe) with dual Z-Scheme heterojunction for degradation of tetracycline.

A novel photo-Fenton catalyst α-Fe2O3@g-C3N4@NH2-MIL-101(Fe) (FGN) with dual Z-scheme heterojunction was successfully prepared by hydrothermal method to degrade tetracycline (TC). The preparation conditions were optimized by orthogonal test, and the successful synthesis was confirmed by characterization analyses. The prepared FGN showed better light absorption performance, higher photoelectrons-holes separation efficiency, lower photoelectrons transfer resistance, and higher specific surface area and pore capacity compared with α-Fe2O3@g-C3N4 and α-Fe2O3. The effects of experimental conditions on the catalytic degradation of TC were investigated. The degradation rate of 10 mg/L TC could reach 98.33% within 2 h when the dosage of FGN was 200 mg/L, and the degradation rate could remain 92.27% after 5 times of reuse. Furthermore, the XRD spectra and XPS spectra of FGN before and after reuse were compared to explore the structural stability and catalytic active sites of FGN, respectively. According to the identification of oxidation intermediates, three degradation pathways of TC were proposed. Through H2O2 consumption experiment, radical-scavenging experiments, EPR results, the mechanism of the dual Z-scheme heterojunction was proved. The improved performance of FGN was attributed to the dual Z-Scheme heterojunction effectively promoting the separation of photogenerated electrons from the holes and accelerating the electrons transfer, and the increase of the specific surface area.

Interactions between micaceous minerals weathering and cesium adsorption.

The environmental behavior of radioactive cesium (RCs) in contaminated areas is generally governed by soil and sediment components and natural weathering conditions. In this study, desorption tests and spectroscopic approaches were used to explore the interaction between the weathering of micaceous minerals (i.e., biotite and phlogopite) and the adsorption of Cs+ and the critical role of weathering in the environmental behavior of RCs. Results showed that the reaction sequence between weathering and Cs+ adsorption significantly affected the surface species of Cs+ and the structure of biotite and phlogopite. Regardless of whether it occurred before, after, or during Cs+ adsorption, weathering generated more high-affinity adsorption sites, namely, interlayer sites (ITs) and frayed edge sites (FESs), to different extents, and then facilitated the uptake of Cs+ at FESs and ITs on micaceous minerals in a poorly exchangeable state. Cs+ stabilized the micaceous mineral structure once it was absorbed within collapsed interlayers by hindering cation exchange and preventing further destruction during weathering. As important weathering factors, high temperature and Ca2+ content promoted the binding of Cs+ in the interlayers of biotite and phlogopite by enhancing interlayer cation exchange. These findings are beneficial for a better understanding of the environmental behaviors of RCs in the hydrosphere and pedosphere.

Remediating petroleum hydrocarbons in highly saline-alkali soils using three native plant species.

Phytoremediation of total petroleum hydrocarbons (TPHs) contamination is a process that uses the synergistic action of plants and rhizosphere microorganisms to degrade, absorb and stabilize pollutants in the soil, and has received increasing attention in recent years. However, this technology still has some challenges under certain conditions (e.g., highly alkaline and saline environments). The present study was selected three native plant species (alfalfa, tall fescue, and ryegrass) to remediate petroleum pollutants in greenhouse pot experiments. The results indicate that TPH contamination not only inhibited plant growth, soil chemical properties and soil fertility (i.e. lower plant biomass, chlorophyll, pH, and electrical conductivity), but also increased the malondialdehyde, glutathione, and antioxidant enzyme activities (catalase and polyphenol oxidase). Further, correlation analysis results illustrated that TPH removal was strongly positively correlated with chlorophyll, soil fertility, and total organic carbon, but was negatively correlated with dehydrogenase, polyphenol oxidase, pH, and electrical conductivity. The highest TPHs removal rate (74.13%) was exhibited by alfalfa, followed by tall fescue (61.79%) and ryegrass (57.28%). The degradation rates of short-chain alkanes and low rings polycyclic aromatic hydrocarbons (PAHs) were substantially higher than those of long-chain alkanes and high rings PAHs. The findings of this study provide valuable insights into petroleum decontamination strategies in the highly saline - alkali environments.

A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence.

Legumes establish symbioses with rhizobia by forming nitrogen-fixing nodules. Nitrate is a major environmental factor that affects symbiotic functioning. However, the molecular mechanism of nitrate-induced nodule senescence is poorly understood. Comparative transcriptomic analysis reveals an NAC-type transcription factor in Lotus japonicus, LjNAC094, that acts as a positive regulator in nitrate-induced nodule senescence. Stable overexpression and mutant lines of NAC094 were constructed and used for phenotypic characterization. DNA-affinity purification sequencing was performed to identify NAC094 targeting genes and results were confirmed by electrophoretic mobility shift and transactivation assays. Overexpression of NAC094 induces premature nodule senescence. Knocking out NAC094 partially relieves nitrate-induced degradation of leghemoglobins and abolishes nodule expression of senescence-associated genes (SAGs) that contain a conserved binding motif for NAC094. Nitrate-triggered metabolic changes in wild-type nodules are largely affected in nac094 mutant nodules. Induction of NAC094 and its targeting SAGs was almost blocked in the nitrate-insensitive nlp1, nlp4, and nlp1 nlp4 mutants. We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate-induced expression of SAGs. Our study fills in a key gap between nitrate and the execution of nodule senescence, and provides a potential strategy to improve nitrogen fixation and stress tolerance of legumes.

New Strategy for the Persistent Photocatalytic Reduction of U(VI): Utilization and Storage of Solar Energy in K+ and Cyano Co-Decorated Poly(Heptazine Imide).

The photocatalytic conversion of soluble U(VI) into insoluble U(IV) is a robust strategy to harvest aqueous uranium, but remains challenging owing to the intermittent availability of solar influx and reoxidation of U(IV) without illumination. Herein, a dual platform based on K+ and cyano group co-decorated poly(heptazine imide) (K-CN-PHI) is reported that can drive persistent U(VI) extraction upon/beyond light. K-CN-PHI achieves the photocatalytic reduction of U(VI) with a reaction rate of 0.89 min-1 , being 47 times greater than that over pristine carbon nitride (PCN). This system can further be triggered by light to form long-living radicals, driving the reduction of U(VI) in the dark for over 3 d. The flexible structural K+ as counterions stabilize the electrons trapped by cyanamide groups, enabling the long lifetime of the generated radicals. The results collectively prove K-CN-PHI to be a novel and efficient photocatalyst enabling persistent U(VI) extraction around the clock, and broadening the practical applications of the photocatalytic extraction of U(VI).

Influence mechanism of organic matter and low-molecular-weight organic acids on the interaction between minerals and PAHs.

Investigate the effect of soil organic matter (SOM) and low molecular weight organic acids (LMWOAs) on minerals adsorption of PAHs. Batch adsorption experiments have been carried out to study the adsorption of PAHs (Naphthalene (NaP), Phenanthrene (Phe) and Pyrene (Pyr)) by minerals (Montmorillonite (Mnt), kaolinite (Kln) and calcite (Cal)). This research found that compared with Kln and Cal, Mnt showed the maximum adsorption capability for PAHs. And the order of PAHs adsorption by Mnt was: Pyr > Phe > Nap, which corresponds to the octanol-water partition coefficient (Kow) of different PAHs. The adsorption kinetic and isotherm were well fitted by Pseudo-second-order kinetic model, Freundlich and Linear isotherm model. Furthermore, inorganic ions (Ca2+) impacted PAHs adsorption by competitive adsorption and cation-π interactive. Cal has the maximum desorption of PAHs among three minerals, and there was desorption hysteresis phenomenon. Field emission-scanning electron microscope (Fe-SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) analysis indicated that SOM enhanced the sorption of PAHs by van der Waals, hydrogen bonding, π-π interactions, and chemical bonding. LMWOAs significantly inhibited PAHs adsorption and promote PAHs desorption from the minerals. As a result, LMWOAs increased of PAHs bioavailability, which provide a new strategy to improve PAHs cleanup efficiency.

Photoreduction behavior of Cr(VI) on oxidized carbon nanoparticles: From photocatalytic efficiency to oxygenated groups.

Clarifying the reaction process and specific mechanism between variable-valence elements and oxidized carbon nanoparticles is essential to evaluate the environmental impact of carbon nanomaterials. In this study, the photocatalytic reduction of Cr(VI) on oxidized carbon nanotubes (OCNTs), oxidized graphene ribbons (OGRs), and graphene oxide sheets (GOs) was explored by batch experiments and spectroscopic analyses. The reaction efficiencies strongly depended on the number of oxygenated groups in the oxidized carbon nanoparticles. The abundant oxygenated groups enabled the GOs to exhibit the highest photocatalytic activity, followed by the OGRs and OCNTs. As a result, the photoreduction efficiency of Cr(VI) reached 96% for GOs, whereas those of OGRs and OCNTs were only 40% and 13%, respectively. In addition, different types of oxygenated groups exhibited various activities based on molecular model tests, following the sequence carboxylic > hydroxyl > carbonyl > ether > aldehyde > edge. Based on the underlying relationship between the oxygenated groups, topological structures, and mechanical strain in the carbon nanoparticles, we speculate that mechanical strain plays a critical role in the formation of oxygenated groups, thereby regulating their photocatalytic activities. The findings in this work provide novel insights into the roles of oxygenated groups and the mechanical strain of carbon nanoparticles in their environmental behavior.

New Insights into the Cellular Toxicity of Carbon Quantum Dots to Escherichia coli.

In this study, the cytotoxicity and toxic mechanism of carbon quantum dots (CQDs) to E. coli were evaluated in vitro. The synthetic CQDs were extremely small in size (~2.08 nm) and displayed strong fluorescence. The results demonstrated that CQDs showed good biocompatibility with E. coli within a short culture time. However, when the exposure time exceeded 24 h, the toxicity of CQDs became apparent, and the contents of reactive oxygen species, lactate dehydrogenase, and the crystal violet absorption rate increased significantly. To further explore the cytotoxic mechanism, approaches including confocal laser scanning microscopy, scanning electron microscopy, and biological transmission electron microscopy combined with zeta potential tests, osmotic pressure measurement, and comet assays were performed. On the one hand, the CQDs altered the surface charges of cells and induced lipid peroxidation by adhesion on the surface of E. coli, leading to an increase in the permeability of the cell wall. On the other hand, when the concentration of CQDs reached 200 µg/mL, the osmotic pressure of the extracellular environment was significantly reduced. These are the main factors that lead to cell edema and death. Finally, the comet assays confirmed that CQDs could induce DNA damage, which could inhibit the proliferation of E. coli.

Spatial distribution, composition, and source analysis of petroleum pollutants in soil from the Changqing Oilfield, Northwest China.

Petroleum contamination surrounding oilfields has attracted more concerns. However, the levels, distribution and source of petroleum of Changqing Oilfield soil still remain lots of knowns, which is important for local environmental protection. Given soil contamination issues in Changqiong Oilfield were investigated. The maximum concentrations of total petroleum hydrocarbons (TPHs), N-alkanes (TNAs) and polycyclic aromatic hydrocarbons (PAHs) were determined to be 1960.29, 96.13 and 0.82 mg/kg, respectively. TPHs were higher in the north than the south of the study area. TPHs decreased in the horizontal and vertical distribution as soil depth and distance from oil wells increased. Source analysis showed that TNAs mainly originated from petroleum, PAHs were controlled by petroleum spills, combustion and traffic. Correlation analysis implied that TPHs residues had an effect on soil environmental quality. This study have important implications for understanding the environmental behavior of petroleum and can provide support for petroleum remediation and risk control.

Oxygen-containing groups in cellulose and lignin biochar: their roles in U(VI) adsorption.

The adsorption behaviors of cellulose and lignin biochar depend on the evolution of their oxygen-containing groups to some extent. In this study, cellulose-rich pakchoi and lignin-rich corncob were selected to prepare the pyrolytic biochar at variable temperatures, named PBC and CBC, respectively. Their structure-function relationships were in-depth studied via the combination of the adsorption experiments of U(VI) and comprehensive spectral analyses. The maximal adsorption capacity of PBC 300, obtained at 300 °C, was measured as 46.62 mg g-1 for U(VI), which was ⁓1.3 times higher than 35.60 mg g-1 of CBC 300. U(VI) adsorption on PBC and CBC were predominantly ascribed to the coordination interaction between oxygen-containing groups and U(VI). Interestingly, the main complexation groups were distinct in both biochars due to the different inherent evolutions of cellulose and lignin. Volatile d-glucose chains in cellulose were apt to degrade rapidly, and the formed carboxyls acted as the most important sites in PBC. However, the stable aromatic network in lignin led to a slow degradation, and more hydroxyls thus remained in CBC, which controlled U(VI) adsorption. In this study, we obtained greatly cost-effective adsorbents of U(VI) and provided some essential insights into understanding the structural evolution-function relationship of cellulose and lignin biochar.

Light Promotes the Immobilization of U(VI) by Ferrihydrite.

The environmental behaviors of uranium closely depend on its interaction with natural minerals. Ferrihydrite widely distributed in nature is considered as one main natural media that is able to change the geochemical behaviors of various elements. However, the semiconductor properties of ferrihydrite and its impacts on the environmental fate of elements are sometimes ignored. The present study systematically clarified the photocatalysis of U(VI) on ferrihydrite under anaerobic and aerobic conditions, respectively. Ferrihydrite showed excellent photoelectric response. Under anaerobic conditions, U(VI) was converted to U(IV) by light-irradiated ferrihydrite, in the form of UO2+x (x < 0.25), where •O2− was the dominant reactive reductive species. At pH 5.0, ~50% of U(VI) was removed after light irradiation for 2 h, while 100% U(VI) was eliminated at pH 6.0. The presence of methanol accelerated the reduction of U(VI). Under aerobic conditions, the light illumination on ferrihydrite also led to an obvious but slower removal of U(VI). The removal of U(VI) increased from ~25% to 70% as the pH increased from 5.0 to 6.0. The generation of H2O2 under aerobic conditions led to the formation of UO4•xH2O precipitates on ferrihydrite. Therefore, it is proved that light irradiation on ferrihydrite significantly changed the species of U(VI) and promoted the removal of uranium both under anaerobic and aerobic conditions.

Interaction behaviors of Cr(VI) at biotite-water interface in the presence of HA: Batch, XRD and XPS investigations.

The interaction behaviors of heavy metals and micaceous minerals are extremely important to understand the environmental behaviors of heavy metals. In this work, the interaction behaviors of Cr(VI) and biotite in the presence and absence of HA were studied combining batch and spectroscopic approaches. Batch experiments showed that biotite had the ability to remove Cr(VI) from the water and the removal markedly increased with decreasing pH. However, sorption of total Cr onto biotite increased with increasing pH (2.0-4.0), whilst quickly decreased above pH âˆ¼ 4.0. It was worth noting that redox process of Cr(VI) to Cr(III), caused by structural Fe(II) on biotite, was another important factor for the high removal of Cr(VI) in a pH range of 2.0-4.0. Ionic strength also influenced Cr(VI) removal that Cr(VI) removal became higher with increasing ion strength. The presence of HA did not show obvious macroscopic effect on Cr(VI) removal, however, HA could cover biotite surface, and promote the sorption of total Cr onto biotite and attenuate the reduction effect caused by Fe(II) on biotite. Spectroscopic approaches, like FT-IR, XRD and XPS further confirmed the existence of Cr(III) on biotite interacting with Cr(VI) and the reduction of Cr(VI) to Cr(III) was drove by the Fe(II) dissolving from biotite to Fe(III). Further, sorption effect and reduction effect competitively contributed to the Cr(VI) removal by biotite, and reduction effect played a more important role at lower pH.

New insights into the sorption of U(VI) on kaolinite and illite in the presence of Aspergillus niger.

The regulation effect of Aspergillus niger to the sorption behavior of U(VI) on kaolinite and illite was studied through investigating the enrichment of U(VI) on kaolinite-Aspergillus niger and illite-Aspergillus niger composites. Kaolinite- or illite-A. niger composites were prepared through co-culturation method. Results showed that U(VI) sorption on kaolinite and illite in different pH ranges could be attributed to ion exchange, outer-sphere complexes (OSCs), and inner-sphere complexes (ISCs), while only the ISCs on the bio-composites. Moreover, micro-spectroscopy tests revealed that U(VI) coordinate with phosphate, amide, and carboxyl groups on illite- and kaolinite- A. niger composites. X-ray photoelectron spectroscopy (XPS) further found that U(VI) was partly reduced to non-crystalline U(IV) by A. niger in the bio-composites, occurring as phosphate coordination polymers or biomass-associated monomers. The findings herein provide further insight into the immobilization and migration of uranium in environments.

Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation.

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjßCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjßCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjßCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjßCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations.

Ultrasound-assisted electrodeposition synthesis of nZVI-Pd/AC toward reductive degradation of methylene blue.

A novel composite (nZVI/Pd-AC) was prepared by loading nanoscale zero-valent iron (nZVI) and Pd on activated carbon (AC) electrode under electrodeposition with ultrasound, which was used to reductive degradation of methylene blue (MB). The loading contents of Fe and Pd in composite materials were 15.84% and 2.06%, respectively. XPS results further confirmed that the as-prepared material contained Fe0 and Pd0. Without external conditions, MB could be degraded in the presence of nZVI/Pd-AC and reached equilibrium within 180 min. To investigate the reusability, the re-electrodeposition strategy was effective to refresh the active sites of nZVI/Pd-AC, and the removal efficiency only reduced by 4.51% in five circles indicating the good reusability of nZVI/Pd-AC composites. GC-MS was used to identify possible degradation pathways of MB; the results showed that the degradation products were mainly N, N-dimethylaniline and 2-amino-5-dimethylamino-benzenesulfonic acid. And the S-C, C-N bonds are the sites easier to be attacked.

Cytotoxic Effect of Graphene Oxide Nanoribbons on Escherichia coli.

The biological and environmental toxicity of graphene and graphene derivatives have attracted great research interest due to their increasing applications. However, the cytotoxic mechanism is poorly understood. Here, we investigated the cytotoxic effect of graphene oxide nanoribbons (GORs) on Escherichia coli (E. coli) in an in vitro method. The fabricated GORs formed long ribbons, 200 nm wide. Based on the results of the MTT assay and plate-culture experiments, GORs significantly inhibited the growth and reproduction of E. coli in a concentration-dependent manner. We found that GORs stimulated E. coli to secrete reactive oxygen species, which then oxidized and damaged the bacterial cell membrane. Moreover, interaction between GORs and E. coli cytomembrane resulted in polysaccharide adsorption by GORs and the release of lactic dehydrogenase. Furthermore, GORs effectively depleted the metal ions as nutrients in the culture medium by adsorption. Notably, mechanical cutting by GORs was not obvious, which is quite different from the case of graphene oxide sheets to E. coli.

Circular RNA circ_0128846 promotes the progression of osteoarthritis by regulating miR-127-5p/NAMPT axis.

BACKGROUND: Mounting evidence indicates that circular RNAs (circRNAs) participate in the occurrence and development of various diseases, including osteoarthritis (OA). However, the effects and molecular mechanism of circ_0128846 in OA have not been reported. METHODS: The expression levels of circ_0128846, microRNA-127-5p (miR-127-5p), and nicotinamide phosphoribosyltransferase (NAMPT) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was examined by flow cytometry and western blot assay. Inflammatory response and cartilage extracellular matrix (ECM) degradation were evaluated by western blot assay. The relationship between miR-127-5p and circ_0128846 or NAMPT was predicted by bioinformatics tools and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. RESULTS: Circ_0128846 and NAMPT were upregulated and miR-127-5p was downregulated in OA cartilage tissues. Knockdown of circ_0128846 increased cell viability and inhibited apoptosis, inflammation and ECM degradation in OA chondrocytes, while these effects were reversed by downregulating miR-127-5p. Moreover, circ_0128846 positively regulated NAMPT expression by sponging miR-127-5p. Furthermore, miR-127-5p promoted cell viability and suppressed apoptosis, inflammation, and ECM degradation in OA chondrocytes by directly targeting NAMPT. CONCLUSION: Circ_0128846 knockdown might inhibit the progression of OA by upregulating miR-127-5p and downregulating NAMPT, offering a new insight into the potential application of circ_0128846 in OA treatment.