The distinguishing of the IgG4-related disease (IgG4-RD) from among other rheumatic diseases has brought attention to the IgG4 subclass of immunoglobulins. It is the least numerous subclass among immunoglobulins G. In general, IgG4 is considered to be non-inflammatory and tolerance inducing, due to its unique structure. However, in IgG4-RD this antibody plays a pathogenic role in activation of the fibrinogenesis and of the inflammatory process; there are also suggestions that it may be a marker of an abnormal inflammatory response. The importance of IgG4 for the pathogenesis of allergic diseases, with a vital role of its ratio to immunoglobulin E (IgE/IgG4 ratio), has been known for years. The role of IgG4 in the course and pathogenesis of rheumatic diseases is still being researched and is not yet fully understood. Increased IgG4 levels have been revealed in rheumatoid arthritis, although no clear link between this phenomenon and disease activity has been demonstrated. There are articles on the potential importance of IgG4 concentration (of both elevated and decreased serum levels) in Sjogren’s syndrome. Additionally, anti-nuclear IgG4 antibody significant titers have been detected in SLE patients, and it has been suggested that the effect of these antibodies on complement consumption and the production of proinflammatory cytokines may play a role in inhibiting the progression of SLE. IgG4 plays a role in autoimmune diseases other than rheumatic diseases, such as pemphigus, bullous pemphigoid, idiopathic membranous glomerulonephritis, or myasthenia gravis, but also in helmints infections. Research shows the importance of IgG4 in malignancy of neoplasms. Melanoma cells are known to stimulate IgG4 production through a modified Th2-based inflammatory response. The role of this immunoglobulin in cholangiocarcinoma is also considered as possible. The aim of this review article is to discuss the current knowledge of IgG4 not only from the perspective of the IgG4-RD but also from a point of view of other autoimmune diseases with particular emphasis on rheumatic diseases. IgG4 is a subclass of immunoglobulin G. Its role in the inflammation is still being defined, as the significance of its anti-inflammatory activity and tolerance-inducing properties is counteracted by its pathogenic features present in recently identified IgG4-dependent diseases. IgG4 is the least numerous of four IgG subclasses and accounts for only about 5% of IgG immunoglobulins in the human body. Its unique properties, dissimilar to other IgGs, are the lack of influence on the classical complement component pathway and, through a property called “fab-arm exchange” (FAE), inhibition of the formation of large immune complexes .These specific qualities of IgG4 are related to its varied affinity to specific Fc gamma receptors (FcγRs); in fact, IgG4 has no affinity to the receptors FcγRIIIa and FcγRIIIb , 2.via variable domains (IgG4 may mimic rheumatoid factor (RF) activity by interacting with other immunoglobulins G, although IgG4 binds to their constant domains, while a classical RF acts  domains , 4. Zack domains  also fouIgG4 blocking of the binding of IgG1 to C1q and thus inhibiting the activity of IgG1 underlie anti-inflammatory properties of IgG4 , 6.Over the years, these properties of IgG4 have been analyzed, revealing its vital role in a number of autoimmune diseases, including pemphigus, bullous pemphigoid, idiopathic membranous glomerulonephritis, or myasthenia gravis, as well as its association with parasitic infections . The impImmunoglobulins G are important in secondary immune response, particularly in response during infections and allergy. Four subclasses of immunoglobulin G have different abilities and affinity to type of FcR receptor, as well as the possibilities for activating the complement pathways.in vitro, while in vivo it can be caused by certain drugs  integrin) and it can run spontaneously  and two light chains (consisting of two domains: one constant—CL and one variable—VL). The antigen-binding region of IgG4 consists of both constant (CL) and variable (VL) domains of the light chain and the heavy chain, a variable domain of the heavy chain (VL) and one of its constant domains (CH1). The IgG molecule is bonded by disulfide bridges inside the heavy and light chains and between heavy chains in the hinge region. IgG4 can dissociate into two halves, each consisting of one heavy and one light chain. Such semi-molecules are able to join corresponding semi-molecules deriving from other IgG4—this unique property being called “Fab-arm exchange” (FAE). A bispecific antibody with two antigen-binding sites is thus generated. This mechanism is possible due to the replacement of proline for a serine at position 331  and a proline for a serine in the hinge region at position 228 (P228S) compared to IgG , 10. Theaneously . FigureThis phenomenon causes high variability of IgG4 that generally prevents it from forming immune complexes under chronic antigen stimulation, which possibly plays a role in inhibiting inflammatory reactions. However, the formation of immune complexes with IgG4 seems likely under certain conditions—this phenomenon being described in RA, IgG4-RD, and membranous nephropathy , 12. ItsApart from FAE mechanism, IgG4 does not have the ability of bivalent antigen binding, suggesting that it should not play an important role in the context of inducing autoimmunity. However, IgG4 is associated with various autoimmune diseases in which it is thought to have diverse range of functions—as it is discussed further below.Immunoglobulins G bind FcγRs by its crystallizable fragment Fc; FcγRs and these receptors are on most effector cells of the immune system such as monocytes, macrophages, NK cells, mast cells, eosinophils, neutrophils, basophils, dendritic cells, and platelets , 14. TheIgG4 presents affinity to FcγRI, FcγRIIa, and FcγRIIIav, low affinity to FcγRIIb/c, and no affinity to FcγRIIIaF and FcγRIIIb receptors. All immunoglobulins G, including IgG4, present high affinity to the FcγRn receptor. This receptor is responsible for the transport of IgG across the intestinal mucosa, placenta, and mammary gland, and due to this activity, the balance of IgG and albumin in the body remains stable. These functions of FcγRn depend on the intracellular signal transduction and activation caused by the combination of its extracellular domain and the IgG Fc domain , 16. As via release of cytokines  in the course of chronic exposure to the antigen  cause a cellular cytotoxicity, opsonization, phagocytosis, cell damage, and inflammatory process. As it has been described above, the unique structure of IgG4 causes that this antibody is an exception from this pathophysiological picture. Still, this does not rule out this antibody from being a causative agent of some autoimmune diseases. There are autoimmune diseases, in which autoantibodies consisted mainly of the IgG4 subclass of immunoglobulins G (IgG4-AID). These include diseases such as pemphigus vulgaris and foliaceus , thromboThe diagnosis of IgG4-AID is based on the presence of antigen-specific autoantibodies with combination of specific clinical symptoms. In contrast to this, the diagnosis of IgG4-related diseases (IgG4-RD) is based on the presence of elevated serum concentrations of IgG4, pseudotumors, infiltrations of IgG4-positive plasma cells, fibrosis, and obliterative phlebitis .in vitro , and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), it was concluded that IgG4 is involved in the processes responsible for meeting at least two of Witebski’s condition .Less certain, although not devoid of evidence, is the effect of this antibody on the development and severity of diseases such as membranous nephropathy (proteinuria and nephrotic syndrome with immune deposits predominantly with IgG4) and AIDP (acute idiopathic demyelinating polyneuropathy) as well as anti-LGI1 autoimmune encephalitis and Morvan’s syndrome . In this group, IgG4 takes part in mechanisms, which result in meeting first of Witebsky’s conditions , 29.The weakest data link IgG4 with, e.g., such rare diseases as Goodpasture syndrome (antibodies against type IV collagen) or Iglon 5 parasomnia (Iglon-5 Ag). In this group, there is a proof of a presence of IgG4 autoantibodies, but there is no proof of their involvement in the pathological process .What matters in the context of the topic under discussion in autoimmune diseases of the central and peripheral nervous system with importance of CASPR2, CASPR1, LGI1, or neurofascin 155 antibodies is that the shared feature is the predominance of antigen-specific antibodies of the IgG4 subclass , 31.What can be seen are the main areas of the IgG4 ARD which are the skin, kidneys, and the central nervous and peripheral systems.The IgG4-related diseases form a still growing group of fibroinflammatory diseases with special clinical features such as pseudotumors, storiform fibrosis, obliterative phlebitis, and organ damage. Their main histopathological feature is a presence of infiltrations by IgG4-producing plasma cells and eosinophils.Apart from the above described diagnostic criteria, there has been suggestion by Hubers et al.  that theThe inflammatory infiltrates which include IgG4-positive plasma cells probably activate myofibroblasts and lead to a storiform fibrosis, characteristic for IgG4-RD. It can be assumed that the inflammatory process with the activation of B cells and activation of fibrosis (myofibroblasts) may proceed simultaneously. Changes in the histopathological image can also be expected over time—from the predominance of cellular infiltration to a weakly cellular image with dominant fibrosis . Della-TIn IgG4-RD, obliteration of venous vessels with their inflammation, i.e., lymphoplasmacytic infiltration of their walls, is found. The occlusion of these vessels without inflammation in the vascular wall is not a clue for the diagnosis of IgG4-RD . ArterieAccording to the classification criteria for IgG4-RD, the ratio of IgG4-positive plasma cells to total IgG plasma cells should be over 40% . The groRecent publications suggest candidates for potential new biomarkers in Ig-G4 RD, such as serum and tissue IgG2 or soluble IL-2 receptor (sIL2R)—for inflammation intensity and cc–chemokine ligand 18 (CCL18) for fibrosis . The IgGIt was found that in AIP associated with the IgG4-RD, the serum concentration of laminin 511-E8 was significantly higher in comparison with healthy controls . AnotherThe introduction of a concept of IgG-4 RD means that some diseases were treated until now as one uniform entity, such as autoimmune pancreatitis (AIP); a subtype concerning IgG-4 involvement was distinguished. In case of AIP, AIP-1 is a subtype regarded as IgG-4 RD. AIP 1 is characterized by lymphoplasmacytic sclerosing pancreatitis (LPSP) in histopathological assessment, with storiform fibrosis, IgG4-positive plasma cell infiltrations, and presence of other Ig-G4 RD features, including extraintestitial symptoms . In caseThe history of the discovery of IgG4 RD begun since the first description of the association of the elevated serum concentration of IgG4 with clinical features of autoimmune pancreatitis (AIP), by Hamano et al. in 2001. These observations and conclusions were confirmed and extended by other researchers primarily from Japan , 51. HowThe scheme of the pathogenesis of IgG4-RD assumes the increased activity of T regulatory cells (Treg) with overexpression of IL-10, transforming growth factor β (TGF-β) production, and upregulation of Th2 response, in which predominant roles play interleukins 4, 5, and 13  Figure 2Presently, the knowledge of IgG4-RD is still expanding and the list of these diseases is constantly being supplemented. Several questions concerning IgG4-RD still remain to be answered, such as the following:- is the presence of an increased amount of IgG4 in the patients’ sera a result of the activation of their production as an anti-inflammatory antibody in response to other factors, e.g., environmental such as infections, trauma, and other inflammatory processes ? , 57- does the possibility of IgG4 complement activation other than the classic pathways (the mannose binding lectin pathway) better explain the role of this antibody in IgG4-RD?- does IgG4 play a pathogenic role in IgG4-RD or is its increased concentration only an epiphenomenon related to other immunological processes taking place in IgG4-RD?- does the effectiveness of therapies directed against B cells (anti-CD-20 and anti-CD-19) in IgG4-RD derive from the results they have on the IgG4 overproduction or from other effects of the depletion of B cell populations?In recent years, the importance of the IgG4 subclass in RA has been raised. There was an increase in the concentration of this immunoglobulin in RA patients compared to the control group. Among antibodies to cyclic citrullinated peptides (ACPA), IgG4 levels exceeded IgG2 and IgG3, and among immunoglobulins G belonging to the class of rheumatoid factor (RF), IgG4 levels were second only to IgG1. It is assumed that the persistent autoimmune stimulation in the course of active RA gives a boost to the production of IgG4 , 61. ThiPrimary Sjögren’s syndrome (pSS) is a chronic autoimmune disease in which under certain conditions, the tolerance is broken and B cells are stimulated depending on and independently of the activity of T lymphocytes, which causes their hyperreactivity and the production of antibodies mainly for ribonucleoproteins: anti-SSA (Ro52 + Ro60) and SSB/La. A further consequence of these events is the accumulation of mononuclear cells in infiltrates in the exocrine glands, which leads to their damage and clinical symptoms, such as dryness  . This diThere are contradictory reports on IgG4 in pSS; an increased concentration of this immunoglobulin in serum was reported in some of pSS patients. Mavgrani et al.  noted thThere are reports about increased IgG4 levels in some vasculitides, especially in eosinophilic granulomatosis with polyangiitis (EGPA)—formerly known as Churg-Strauss syndrome—and in granulomatosis with polyangiitis (GPA), associated with anti-neutrophil cytoplasmic antibodies (c-ANCA).In EGPA with eosinophilia and other allergic features such as asthma, allergic rhinitis, and sinusitis, the increase in IgG4 levels can be explained as being analogous to allergic diseases. However, misdiagnoses of IgG4 RD are also possible. EGPA represents necrotizing vasculitis affecting medium-sized small vessels, while in IgG4-RD an involvement of larger vessels, such as the aorta, is expected. In case of doubts in the diagnosis, these diseases will be differentiated mainly by histopathology. The presence of ANCA antibodies may also point against IgG4-RD . Differein vitro constructed study using IgG4 healthy controls and IgG4 antibodies from SLE patients that the latter can inhibit complement consumption by the activity of autoantibody–autoantigen immune complexes (ICs). The authors concluded that SLE IgG4 inhibited the activation of the alternative pathway by other subclasses of autoreactive IgGs (The function of IgG4 antibodies in systemic lupus erythematosus (SLE) is still being discussed. Significant anti-nuclear IgG4 antibody titers (ANA-IgG4) have been detected in SLE patients, and it has been suggested that the effect of these antibodies on complement consumption and the production of proinflammatory cytokines may play a role in inhibiting the progression of SLE. The presence of anti-dsDNA IgG4 antibodies was also reported and was associated with skin lesions and kidney involvement in patients with SLE . Pan et ive IgGs .via IgG4 to IgE. In this case, the inhibition of that switch at the IgG4 level disturbs the ratio of IgG4 to IgE in favor of the former. The second proposed mechanism assumes a competition between IgG4 (with low affinity for Fcγ receptors) and IgE for epitopes on the helmints surface or in a close environment. The similar situation is described in reactions to allergens. “Blocking antibody” activity explains the finding that IgE-mediated hypersensitivity reactions are rare in patients with chronic parasitic infections . As more often the ratio of IgE to IgG4 (IgE/IgG4) is used, its lower value indicates higher tolerance to the allergen , 78.The ability of IgG4 to compete with IgE for the same epitopes in this case prevents the IgE-dependent acute reaction with degranulation of mast cells and release of histamine and bradykinin. In IgG4-RD, the presence of allergy, atopy, eosinophilia, and increased serum levels of IgE and IgE-positive mast cells in lymphoid, biliary, and pancreatic tissues is quite often described . Higher In allergic diseases, specific immunotherapy (SIT) relies on the administration of allergen extracts to produce the emergence of tolerance to those allergens. This method implies an impact on Treg cells and switching of allergen-specific B-cells toward IgG4 production . This caThe IgG4 serum level may be elevated in IgG4-RD but may also be within a normal range—the latter does not exclude the diagnosis of IgG4-RD. On the other hand, the elevation of IgG4 may be observed in case of malignancy .It is not uncommon that a pseudotumor lesion in an organ is treated as neoplastic, when in fact it is IgG4-RD. Therefore, in the differential diagnosis, IgG4-RD as a cancer mimicker should be taken under consideration. In 2.6% of patients with suspected pancreatic cancer, in whom the surgery was performed, AIP was a final diagnosis . In bothIn some malignancies, tumor cells escape host control, by inducing the switch of the production of autoantibodies toward IgG4, thus taking the advantage of the protective properties of IgG4. Such a phenomenon has been demonstrated for a number of neoplasms, including melanoma, extrahepatic cholangiocarcinoma, pancreatic cancer, or glioblastoma . The knoIn a Chinese study presented in 2020, researchers found increased incidence of malignancy in patients with IgG4-RD. The authors pointed to risk factors, such as autoimmune pancreatitis, but also highlighted potential protective role of other phenomena, such as eosinophilia . Also, oAnother interesting conclusion presented by Wallace et al.  is that An interesting research and conclusions were presented by Zhou et al.  in a stuTo summarize, increased serum levels of IgG4 may in some instances may be useful in the differentiation of clinical conditions. Maślińska et al. reported  the loweThere is also an increase in the concentration of IgG4 in chronic inflammation, which takes place, for example, in active RA . It can An interesting work was recently published by Olejarz et al.  showing From the clinical point of view, other diseases located in the eyeball and the periocular area such as orbital pseudotumor, orbital neoplasms , infections, orbital myositis, sarcoidosis, and inflammatory orbitopathy in the course of vasculitides especially granulomatosis with polyangiitis (GPA) should be considered in the differential diagnosis. The above considerations indicate the scale of difficulties in the proper diagnosis of IgG4-RD in everyday clinical practice.IgG4 antibodies found a place in immunotherapy, as their specific anti-inflammatory and protective abilities make them an advantageous subclass in the design of therapeutic antibodies, especially when there is a need to block other antibodies. The interesting therapeutical option is a pembrolizumab-humanized anti-receptor monoclonal antibody programmed cell death-1 (PD-1) which consists of IgG4/kappa isotype with stabilizing modification of the Fc region sequence. This monoclonal antibody found use in therapy of several neoplasms, among others melanoma, cervical cancer, esophageal cancer, colorectal cancer, and triple-negative breast cancer) . In JuneAnother interesting drug is nivolumab—a fully human immunoglobulin G4 and a PD-1 immune checkpoint inhibitor antibody. It has demonstrated efficacy in the treatment of patients with advanced non-small-cell lung cancer (NSCLC) as well as melanoma, urothelial cancer, renal cell carcinoma (RCC), or malignant pleural mesothelioma (MPM) . Nivolumper se in IgG-RD is unclear) and in neoplasms, in which the pathogenic role of IgG4 is overwhelming.The disbalance between IgG4 and other classes or subclasses of immunoglobulins, depending on how it is shaped and in which disease it occurs, may lead to various outcomes tilting the clinical picture toward inhibition of inflammation or its activation, e.g., in allergic diseases where the IgG4 role is strongly inhibitory and protective, in IgG4 RD where IgG4 is an indicator of inflammation and uncontrolled fibrosis  in the human body and disbalance between this and other immunoglobulins may be, depending on circumstances, interpreted in different ways, e.g., in malignances and allergic diseases. The elevation of IgG4 levels by itself is not always an indication of an ongoing pathologic process, as the role of IgG4 is very diverse. The unique properties of this immunoglobulin, such as FAE and its influence on the other immunoglobulins, most likely determine such a variability of its impact. The mere increase in the concentration of IgG4 in the patient’s serum without taking into account all the accompanying phenomena and tests, including histopathological ones, cannot form the basis for the diagnosis of a specific disease. The recent establishment of IgG4-RD has brought IgG4 into the spotlight and raised interest in its involvement in rheumatic and autoimmune diseases. As we learn more about IgG4, the need for taking its presence into consideration during diagnostic and therapeutic process will grow.MM—first author, author for correspondence, concept of the work, writing, literature review, final acceptance. JD-C—writing, literature review, final acceptance. MJ—writing, literature review, final acceptance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Antibiotic pollution has become an increasingly serious issue due to the extensive application of antibiotics, their resistance to removal, and the harmful effects on aquatic environments and humans. Breeding wastewater is one of the most important sources of antibiotics in the aquatic environment because of the undeveloped treatment systems in breeding farms. It is imperative to establish an effective antibiotic removal process for breeding wastewater. This paper reviews the treatment methods used to remove antibiotics from breeding wastewater. The mechanisms and removal efficiency of constructed wetlands, biological treatments, advanced oxidation processes (AOPs), membrane technology, and combined treatments are explained in detail, and the advantages and disadvantages of the various treatment methods are compared and analyzed. Constructed wetlands have high removal rates for sulfonamide (SM), tetracycline (TC), and quinolone (QN). The antibiotic removal efficiency of biological treatment methods is affected by various processes and environmental factors, whereas AOPs and combined treatment methods have better antibiotic removal effects. Although it has broad application prospects, the application of membrane technology for the treatment of antibiotics in breeding wastewater needs further research. Antibiotics are synthesized with the aim to kill or inhibit other microorganisms and their derivatives. Because antibiotics can inhibit bacterial infection and promote animal growth, they are widely used in the livestock and aquaculture industries ,2. The gThe main antibiotics used in the breeding industry in China are chloramphenicol (broad-spectrum antibiotic), tetracycline (inhibits Gram-negative and -positive bacteria), macrolides or MACs (inhibit Gram-positive bacteria), sulfonamide or SM (treatment of furunculosis and vibriosis), and nitrofurans (inhibit and kill bacteria and protozoa). Because some farmers have used antibiotics in large quantities, serious environmental pollution has occurred alongside their economic benefits. Most antibiotics cannot be fully metabolized by animals, and large amounts are excreted into the environment in their original form. Various processes were developed to remove antibiotics in urban water/wastewater treatment plants ,6. The tWith the development of the breeding industry and the widespread application of antibiotics, a large amount of wastewater containing antibiotics is produced. If this is discharged into the environment without effective treatment, it will cause serious pollution, altering the ecological balance and endangering human health. Therefore, the removal of antibiotics from breeding wastewater is an important issue. This paper reviews the existing treatment methods for removing antibiotics from breeding wastewater. The mechanism, removal efficiency, and advantages and disadvantages of each removal process are evaluated, providing a basis for further studies of antibiotic pollution remediation in breeding water.A constructed wetland wastewater treatment system is an artificial sewage treatment ecosystem, which uses the combined action of soil, plants, and microorganisms to treat the wastewater that enters the wetland. The wastewater is purified by filtration, adsorption, co-precipitation, ion exchange, plant adsorption, and microbial decomposition . ConstruConstructed wetlands can be classified as free water surface flow constructed wetlands (FWS CWs), horizontal subsurface flow constructed wetlands (HSF CWs), and vertical subsurface flow constructed wetlands (VSF CWs) according to the direction of water flow. In vertical subsurface flow wetlands, aerobic and anaerobic activities coexist, and they have a long hydraulic retention time (HRT), which can enhance the degradation of antibiotics by microorganisms . TherefoAzolla is 56%–86%  [Eichhornia crassipes is higher than 70%  [The performance of constructed wetlands is mainly affected by plant species, fill material type, water depth, hydraulic loading rate (HLR), HRT ,24,25 an50 mg/L) . The rem50 mg/L) . The TC .0 mg/L) .The fill material functions in interception and adsorption, which can enhance the degradation of antibiotics by microorganisms in the fill material . The porThe HLR refers to the amount of wastewater per unit volume or unit area that a constructed wetland can treat per day. The greater the HLR, the shorter the HRT . The dai4+-N [In addition, pH affects antibiotic activity, temperature affects the reproduction of microorganisms, and light affects plant growth; thus, these factors all indirectly affect antibiotic removal efficiency . Liao et4+-N . The tre4+-N .The main antibiotic removal processes during biological treatment include sludge adsorption and biodegradation . In geneThe BAF system is a new type of sewage treatment process that combines biological contact oxidation and filtration . It consBecause the packing has a strong biofilm adhesion ability and large specific surface area, it promotes the adsorption of sludge; therefore, the antibiotic removal efficiency is rather high. For example, with an HRT of 40–48 h and HLR of 2.8 cm/h, the total antibiotic removal rate with the BAF process can be as high as 89%–91% .3·d, an operating temperature of 37 ± 1 °C, and an HRT of 16 d, the TC and QN removal rates from swine wastewater by AD have been reported to be 65% and 85%, respectively [AD includes four stages: hydrolysis, acidification, hydrogen production, and acetic acid and methane production . The AD ectively .The SBR system consists of one or more aeration reaction tanks, and the sewage enters the tank in batches. The SBR reactor operates in five sequences: influent feeding, anoxic phase, aerobic phase, sludge settling, and effluent discharge . The opeAn MBR is a type of wastewater biological treatment technology that combines modern membrane separation technology and biological technology. The MBR has the advantages of long sludge retention time, flexible operation, low sludge output, and high nitrification performance, but the disadvantages are its high energy consumption and operating costs . A previImprovements in existing technology in terms of combining different processes have also enhanced the removal of antibiotics from breeding wastewater. For example, some researchers have used biofilm MBRs (BF-MBRs) and compared the effectiveness of this technology in removing antibiotics from piggery wastewater with that of a conventional MBR. When the HRT was 5–4, 3–2, and 1 d, the antibiotic removal rates associated with the BF-MBR were 87%, 80%, and 45%, respectively, whereas those associated with the MBR were only 84%, 57%, and 26%, respectively .The main factors affecting the efficiency of biodegradation are HRT, sludge concentration, membrane type, water quality , and the property of the antibiotics ,52,53. TThese studies have shown that the biological method is selective in the removal of antibiotics, and the removal efficiency is greatly affected by process parameters and environmental parameters. It therefore has certain limitations for removing antibiotics from breeding wastewater. Therefore, improvement of the removal efficiency when biological methods are applied to remove antibiotics needs to be further studied.2 and H2O [AOPs are oxidation technologies that use strong oxidizing hydroxyl radicals (·OH) produced in reactions as the main oxidant to decompose and mineralize organic pollutants in water . The ·OH and H2O . The mos2·, and O2− to degrade pollutants through electrode reactions [2SO4 as the electrolyte and Ti/IrO2 as the anode [4+-N and COD, were completely removed by the flow-through electro-oxidation process [Electrochemical oxidation is a technology that produces strong oxidants such as ·OH, HOeactions . Miyata he anode . Antibio process . The rem process .Wang et al. also found that coexisting substances in wastewater affect antibiotic removal efficiency . When thThere are two mechanisms for ozone-based antibiotic degradation: the direct oxidation of ozone and indirect oxidation through the generation of free radicals (Equation (1)) . As an e2O2), or catalysts can generate large amounts of ·OH (Equations (2)–(6)), and then degrade organic pollutants [2O2 coupling system compared with ozone oxidation alone [In addition, the combined processes of ozone oxidation with ultraviolet light (UV), hydrogen peroxide ), which then oxidize and decompose antibiotics [2O2 concentration of 40 mg/L, Fe2+ concentration of 12 mg/L, initial pH of 4, microwave radiation time of 2 min, and microwave radiation power of 445 W.In the Fenton process, the reagents, i.e., Hibiotics . Microwaibiotics . The OTC2O2 and Fe2+ affect the efficiency. Apart from that, the pH also affects the reaction rate. Lin et al. found that the H2O2 input, Fe2+ concentration, and initial pH of the water all affected antibiotic removal efficiency in a study of microwave-assisted Fenton oxidation of antibiotics in biogas slurry [2O2 input, the removal efficiency of OTC, TC, CTC, and OLA increased at first and then levelled off. Studies have shown that an appropriate increase in the H2O2 concentration contributes to the production of ·OH, thereby improving antibiotic removal efficiency [2+ concentration, the ·OH production increased, and therefore, the removal efficiency of the four antibiotics increased. By contrast, an increase in the initial pH of the water sample caused the removal efficiency of the four antibiotics to decrease. This is because the oxidation-reduction potential of Fenton’s reagent is affected by the initial pH.It is easy to understand that the dosages of Hs slurry . With anficiency . With an2O2 method, H2O2 is decomposed to produce ·OH with UV irradiation (Equation (8)) [2O2 process is relatively stable in operation and can degrade most organic pollutants. It produces less solid waste during the treatment process than the alternative methods and is widely used in organic wastewater treatment [2O2 treatment process under the conditions of pH 5.0, UV wavelength of 254 nm, and H2O2 concentration of 7.0 mM [In the UV/Hion (8)) . The UV/reatment . The remf 7.0 mM .(8)H2O22O2 concentration, and reaction time affected antibiotic removal efficiency [2O2 is easily decomposed into water and oxygen (Equation (9)). Therefore, the ·OH production decreases, which in turn affects antibiotic removal efficiency. Under alkaline conditions, part of the H2O2 reacts with OH− to form HO2− (Equation (10)), which can react with ·OH (Equation (11)). Therefore, the amounts of ·OH and degradation efficiency reduced. The antibiotic removal rate was highest when the H2O2 concentration was 7.0 mM. An excessive input of H2O2 reduced the efficiency of antibiotic removal. Because H2O2 plays a dual role in the oxidative degradation of antibiotics, an appropriate amount of H2O2 can produce ·OH, whereas excessive H2O2 will consume ·OH (Equation (12)). The antibiotic removal rate will also be higher with longer reaction times. For example, when the reaction time was extended from 20 to 60 min, the SM removal rate increased from 80% to 95%.Li et al. found that pH, Hficiency . They alThese studies have shown that AOPs can effectively remove antibiotics from breeding wastewater; especially, that high removal efficiency can be achieved in a short reaction time. The AOPs therefore have good potential in antibiotic treatment in breeding wastewater.With membrane technology, pollutants are intercepted as wastewater passes through small membrane pores. The methods mainly depend on microfiltration, ultrafiltration, nanofiltration, and reverse osmosis . MembranIn addition to applying a single method to treat antibiotics in breeding wastewater, some combined treatments to improve removal efficiency were developed. For example, Ben et al. studied the degradation of antibiotics in swine wastewater using a combined biological–Fenton process. In this method, SBR was used to perform the biological treatment, and then the Fenton process was used for further treatment . After pApart from the above technologies, other promising remediation technologies, e.g., the microalgae technique, were also explored . Sun et The fate and mechanisms of treatment processes for removing antibiotics from breeding wastewater, including constructed wetlands, biological treatments, AOPs, and combined treatments, are reviewed in this paper.(1) Constructed wetlands can be operated economically and are characterized by a strong decontamination ability, high efficiency, easy maintenance and management, and some level of selectivity in the removal of antibiotics.(2) The degradation of antibiotics by biological treatments is affected by factors such as process parameters, water quality conditions, and environmental factors.(3) AOPs and combined treatments show a high removal efficiency of antibiotics. They have broad development and application prospects in the treatment of breeding wastewater.(4) Membrane technology can be used to effectively remove antibiotics, but it is currently seldom used in the removal of antibiotics from breeding wastewater. The application of membrane technology to remove antibiotics from breeding wastewater remains a viable option.The development of more efficient, low-cost, and easy-to-operate treatment processes for removing antibiotics from breeding wastewater will be a pressing research topic in the future. Faced with the increasingly serious problem of antibiotic pollution, it is necessary to not only continuously improve wastewater treatment processes but also curb the abuse and excessive discharge of antibiotics at their sources. In China, there are clear regulations on the amount of antibiotics to be used in animal husbandry feed, but there are no clear standards for the levels of antibiotics allowed to be discharged in breeding wastewater. Therefore, it is imperative to issue such standards for breeding wastewater as soon as possible.


Self compacting concrete (SCC) is special type of concrete which is highly flowable and non-segregated and by its own mass, spreads into the formwork without any external vibrators, even in the presence of thick reinforcement. But SSC is also brittle nature like conventional concrete, which results in abrupt failure without giving any deformation (warning), which is undesirable for any structural member. Thus, self-compacting concrete (SCC) needs some of tensile reinforcement to enhance tensile strength and prevent the unsuitable abrupt failure. But fiber increased tensile strength of concrete more effectively than compressive strength. Hence, it is essential to add pozzolanic materials into fiber reinforced concrete to achieve high strength, durable and ductile concrete. This study is conducted to assess the performance of SCC with substitutions of marble waste (MW) and coconut fiber (CFs) into SCC. MW utilized as cementitious (pozzolanic) materials in percentage of 5.0 to 30% in increment of 5.0% by weight of binder and concrete is reinforced with CFs in proportion of 0.5 to 3.0% in increment of 0.5% by weight of binder. Rheological characteristics were measured through its filling and passing ability by using Slump flow, Slump T50, L-Box, and V-funnel tests while mechanical characteristics were measured through compressive strength, split tensile strength, flexure strength and bond strength (pull out) tests. Experimental investigation show that MW and CFs decrease the passing ability and filling ability of SCC. Additionally, Experimental investigation show that MW up to 20% and CFs addition 2.0% by weight of binder tend to increase the mechanical performance of SCC. Furthermore, statistical analysis (RSM) was used to optimize the combined dose of MW and CFs into SCC to obtain high strength self-compacting concrete. SCC is particular types of concrete which is flowable, non-segregated and by its own mass spread into place and fill the formwork even in the presence of congested reinforcement2. SCC can be defined in terms of four characteristics such as flowability, viscosity, passing ability and segregation resistance. If the concrete possess all the above four characteristics are term as SCC3. Historically, the concept of SSC was first proposed In 19864. But 1988 Japan was first country which was successfully able to develop the prototype of (SCC).In civil engineering, increased in interest in Self compacting concrete (SCC) has been observed. SCC is still developing a wide range of application and properties5. In the cement production industry, there are several opportunities to utilized waste raw material in concrete6. Waste can be utilized as fine or coarse aggregate or cement in concrete.The concept of environmental development believes that environmental reserves must be considered as restricted goods and the wastes must be reasonably controlled. Growing quantities of accumulated garbage, up to 2500 million tons per year over the world which promote the scholar to a built modern technique of dumping7 show that, aggregate plays major role on the performance of concrete. Unluckily, the majority of the natural sand used in concrete is either rolled sand, sand of river, dune sand, and sea sand is chosen for the economy and the accessibility8. Properties of sand disturb the strength performance of concrete, as sand is an important ingredient of concrete.For a good quality concrete mix, fine aggregates must be clean, hard, strong, and free of immersed chemicals and more fine materials that may possibly produce the weakening of concrete. Rajan and Singh9. The aim of this article is to utilized waste marble as a partially substitution of sand which takes from the marble factory. Real estimate about the amount of waste generated in Pakistan from the marble industry are unavailable since it is not analyzed or supervised by the government or any other political party. More references assess that 20 to 25% of the marble delivered in the form of slurry through the cut off procedure10. These marble wastes are present in the environment and cause to pollution.Various productions are the cause of waste which are created as a byproduct during the production procedure. It is recommended that Marble be able to be utilized in the building production to make Cement Concrete11. Management of the marble powder in various factories regions particularly the paper, agriculture, glass, and building factories would assist to care for the environment12. Through the quarrying procedure and in the cleaning of marble dust, marble powder is seen as a rubbish material13.Subsequently ancient occasions marble has been commonly utilized as a cementitious material in cement concrete. The factories disposing of the marble residue material, which contain of a extremely fine ash, currently comprises one of the ecological problem throughout the world14. Let consider the example of Japan in visible of us, a nation that effectively improved the reprocessing rate of concrete waste up to 98% utilizing waste material as aggregate15. It has been identified that the Marble powder delivered through treating corresponds to almost 40 percent of the finishing product from the marble factory16. They also described that workability of concrete reduce with the supplement of marble powder.Many developed countries have put in motion legal regulations concerning the recovery of structural waste aiming to reduce the amount of waste and to ensure waste recycling17. They detected that compressive strength is enhanced up to 50 percent substitute of fine aggregate with marble dust which is almost 12 percent greater than from reference concrete18. Compressive strength and flexural strength of concrete are improved almost 28 percent and 13 percent correspondingly at 50 percent substitution and then steadily decline with the supplement of marble dust19. Resistance to the acid of concrete made with waste marble was slightly low in comparison to that of control concrete20. It has been Indicated that Marble dust can be easily utilized in the cement concrete production9. A study shows that durability aspects of concrete such corrosion and drying shrinkage improved with addition of marble waste21. It has been also reported that marble waste increased mechanical performance concrete due to micro filler22. Although marble increase mechanical performance of concrete considerably, but still concrete has less tensile strength which result brittle failure. Therefore, concrete still some of tensile reinforcement to improve tensile capacity. Fibers is one of the most prevalent method to enhance tensile capacity of concrete.Katuwal et al. also suggest that marble as a fine aggregate reduce workability of concrete26. Improving the production technique of SCC is increasing day by day in concrete production27. ACI 544.5R-10 stated that thick fibers are lower efficient in reducing the plastic shrinkage cracks width as compare to thin fibers28. Most thin diameter microfibers are particularly effective in reducing plastic shrinkage cracking of concrete because larger surface area of fibers29. Moreover, the use of fibers helps in reducing the porosity and bleeding of concrete33.Through extensive research, it has been determined that the performance of concrete were significantly increased because of the addition of fibers to concrete34. The choice of the kind of fibers is depends on different aspects such area, length, tensile strength modulus of elasticity and the material from which it is made. Furthermore the amount to which these fibers effect the performance of concrete35. Mostly fibers are classified into two types, one is metallic and the other one is nonmetallic. Metallic fibers are mainly steel fibers whereas nonmetallic are glass fiber, propylene fiber, carbon fibers36. It has been reported that mechanical performance of foam concrete considerable improved with addition of propylene fibers24. Research shows that, rheological properties (filling and passing ability) of self-compacting decreased with concrete of fibers. Furthermore, mechanical performance of concrete improved considerably up to 2.0% addition of PPFs25. A studies show that, fibers improve mechanical performance in terms of compressive strength, split tensile strength as well as flexure strength of concrete34. Steel Fibers improved concrete performance against acid attacks4.Various kinds of fibers are utilized to reinforce concrete such organic and inorganic fibers37 used steel fiber in concrete and reported steel fiber increased tensile strength more effectively than compressive strength. Further research was recommended37 that pozzolanic materials must be added to fiber reinforced concrete to achieved high strength, durable and ductile concrete. Also reported that steel fibers are too costly as well as easily corroded. Therefore, the primary objective of this research is to utilize marble waste in SCC. Secondary objective of this research is utilizing coconut fibers instead of steel fibers which are costly as well as easily corroded. Third objective of this is research was to optimized substitution ratio of marble waste and coconut fibers with help of statistical analysis (RSM) to achieved high strength self-compacting concrete. Finally experimental results and predicted results from statistical analysis (RSM) will be compared.In existing literature shows that the fibers increased mechanical performance of concrete particular tensile strength more effectively than compressive strength. Ahmad et38, were used as binding material throughout in this study. Furthermore, physical properties and chemical composition of cement used in this study were given Table Normal setting time cement as per according to ASTM C150Coconut fibers having 80 to 100 mm long length with a diameter of 0.5 to 1.0 mm were utilize in this study. Its properties are given in Table 39 respectively. Furthermore, different parameter which influence the performance were evaluated in laboratory and presented in the following Table Nearby accessible rive sand in saturated surface dry (SSD) condition were utilized as a fine aggregate throughout in this study. Normal weight limestone crush aggregate in saturated surface dry (SSD) condition were utilized as coarse aggregate in this study. Both (fine and coarse aggregate) were collected from Margallah Taxila Pakistan. Figures Marble waste (Mw) were procured from the national marble factory industrial zone Risalpur Pakistan and grinded at Pakistan council of scientific and research lab Peshawar Khyber Pakhtunkhwa, Pakistan. Table 40 and ASTM C-494 Type F41 as well as other known practices. Table Chemrite-530 has been utilized as a super plasticizer since it is a high-range water-reducing mixture that is also considered non-toxic and non-hazardous in compliance with appropriate health and safety regulations. The superplasticizer satisfies the requirements of EN 934–2 T 3.1/32 Six mixes were created by various proportions of marble waste (MW) and coconut fibers (CFs) in the second stage to find out the the impact of marble waste (MW) and coconut fibers (CFs) on self-compacting concrete (SCC), which were developed on the basis of the first stage. Table To attain the project's objective, an experimental study program was created, and it was segregated into two phases. In the first phase, trial mixes were created to meet the technical requirements for SCC42. moreover, as per ASTM C39/C39M43, standard-sized cylinders (150 mm in diameter and 300 mm in length) have been used to test the compressive strength at seven, fourteen, and twenty-eight-day intervals. The tensile strength of similar cylinders of standard size (150 × 300 mm) has also determined by casting and testing them in the laboratory. A beam with dimensions of (150 × 150 × 500 mm) would be casted and tested to determine its flexure strength in accordance with ASTM44.Considering ASTM45, a cube of standard dimensions (150mm3) has been used to determine bond strength. For each test, three specimens have been tested at seven, fourteen, and twenty-eight days, and the mean value of the specimens have opted as the strength. Eight different mixtures were created to investigate the impact of marble wastage and coconut fibers on the performance of solidified and fresh concrete. Table Slump flow, Slump T50, L-Box, and V-funnel tests were used to assess rheological characteristicsIn the first phase, eight different mixes have created to obtain the best feasible blend that would meet the requirements of Technical Specification for the product (SCC). As a result of the research, Table Mix 6 (SCC) was chosen for beyond investigational work according to the results obtain in first stage. Hence, the next stage of the investigational work was performed on Mix 6 (SCC) with differing proportions of Mw and CFs with constant water binder ratio (0.60). Twelve mix were planned at the next phase after attained the requirement of technical guideline for self-compacting concrete (Mix 6). Different proportions of Mw and CFs were incorporated to fresh SCC. The proportions of Mw were from 5.0% to 30% by weight of cement increment in 5.0% and CFs were incorporated from 0.5% to 3.0% by weight of cement in increment of 0.5%. Table 46. However, all of the SCC mixtures are within the limits defined by the technical standard for SCC and have excellent flowability2.Figures As for CFs, the Slump flow value decreases as the proportion of CFs in the mixture increases. As indicated in Fig. 2 having good passing ability. Figure 47 concluded that blocking ratio up to 0.60 possess good passing ability. However, technical specification for SCC states that the SCC has a good passing ability when the blocking ratio value (H2/H1) in the L box test is more than 0.848.The ability of self-consolidating concrete to pass the L-Box test was determined using this method. A similar trend to slump flow was observed in the passing ability of concrete, with the proportion of marble waste increasing from the level of 0 to 30% with respect to weight of cement as illustrated in Fig. 2 and have good flowability. As for coconut fibers, maximum flowability was found at 0% substitution of CFs, whereas lowest flowability was found at 3.0% substitution in coconut fiber. Flow time for mix (0 to up to 2.0 percent coconut fibers by weight of cement) is between 6 and 12 s, which is within the permissible range specified by the technical standard for SCC  was checked through V-Funnel test. Flow time of V funnel test indicate the flowability of SCC. Increased flowability associates to shorter flow time. Self-compacting concrete flow decreases as percentages of marble waste and coconut fibers increased as illustrated in Fig. 43.Compressive stress is the measure of the concrete's ability to resist the highest compressive load divide by cross sectional area of sample. A cylindrical specimens of standard size 150 mm diameter and 300 mm length were casted and tested for compressive strength under the standard procedure of ASTM C39/C39M43. At a dose of 20% of the marble waste shows maximum compressive strength as compared to the blank or reference concrete. However, beyond 20% of marble waste compressive strength decreased gradually. The positive impact of marble waste on compressive strength is due to pozzolanic reaction in which Sio2 (silica) in marble waste react with CH  of cement yields more C-S–H  gel which is additional cementitious compounds49. At higher dose of marble waste beyond (20% by weight of cement), strength is reduced owing to dilution effect, resulting in alkali-silica reaction due to the presence of more unreactive silica. Also a higher dosage, compaction becomes more difficult due lack of workability, which results in increased porosity of concrete, leading to porous concrete which ultimate results lower compressive strength50. Based on result, it is suggested that marble waste can be utilized as binding material up to a maximum of 20% by weight replacement.Figure 53. Hence X-ray diffraction (XRD) results confirm the performance and strength development of marble waste is due to the pozzolanic reaction which produce secondary C-S–H gel. C-S–H gel possess binding properties which increased the strength of concrete.X-ray diffraction (XRD) analyses were conducted at the age of 28 days to investigate the mineralogical composition and to assess the measure calcium silicate hydrate (C-S–H) in control and marble substituted batches. Peaks of C-S–H 27° were selected for analysis. These peaks and their pattern with 10% 20% and 30% substation of marble waste can be observed in Fig. 54. In comparison with blank or reference concrete, a 2.0 percent dose of Coconut fiber yielded maximum compressive strength at 28 days of curing. The compressive strength, however decreased by further addition of CFs (beyond 2.0%). The confinement of the fiber reinforcement on the specimen results positive influence on compressive strength, Compression causes lateral expansion, which is restricted by the CFs, leading to more compressive strength. The fibers are able to withstand tension and shear because of their strength37. Compaction process become difficult at higher dose (beyond 2.0%), due lack of workability which results lower strength. it has been reported that 1.5% of the fibers increased compressive strength almost 15%, as compare to reference concrete55. Fibers at 1.0% by volume produce a significant increase in the initial as well as the later ages mechanical performance of concrete. The highest improvement in 28-days strength was seen to be 29.15%56. Therefore, there is an optimal limit for Coconut fiber. According to the results of the tests, the optimal dose of Coconut fiber for strength is 2.0% by weight of cement.As for coconut fiber concern, Compressive strength increased up to 2.0% addition of CFs and then decreased gradually as shown in Fig. Figure 57. In this study dependent variable are dose of Mw and CFs while independent variable is strength.Response surface technique is a statistical tool whose primary goal is to maximize a response or output that may be affected by a variety of factors or input variables, such as the number of input variables. When there are several responses, it is critical to determine the combined optimal dose of both compounds that does not maximize just one reactionTo investigate the combined effects of Mw and CFs on the compressive strength, a 3D response surface and contour plot were developed in the Minitab software for the Mw and CFs verse compressive strength at the age of 28 days curing. To assess best possible dose MW and CFs in SCC, 3D response surface was transformed into contour plot in which 18% Mw and 1.8% CFs were chosen from contour plot showing compressive strength 23 Mpa as given in Figs. 58, at the ages of 7, 14 and 28 days of curing.Split tensile strength can be defined as the tensile stresses produced as a result of applying tensile force to the concrete sample in such a way that cylindrical sample split at vertical diameter. It is an indirect technique for examining the tensile strength of the concrete. split tests were executed on cylindrical samples of 150 mm diameter and 300 mm height in accordance with ASTM C496-71 20. The pozzolanic reaction of marble waste, which results in the formation of extra cementitious compounds, is responsible for the beneficial impact on split tensile strength53 According to certain reports, marble waste exhibits pozzolanic characteristics if the particle size that is smaller than that of cement particles, which is 75 microns or less11. It is possible for marble waste concrete to continue to acquire strength over time because of the extra binder generated by the interaction of marble waste with accessible lime. Aside from that, marble waste is the filler material because of its finesse, as it may be used as a micro-filler in cement aggregate matrix to produce dense concrete59. Higher dosages (30 percent) make the compaction process more difficult since the material lacks flowability, resulting in porous concrete with reduced strength.Figure 60. The strength, however, was decreased when the 2.0 percent dose was exceeded. In concrete, fibers are used to improve the flexibility of the concrete by delaying the appearance of tension fractures or avoiding the formation of cracks in such a way that the tensile strength of (SFRC) steel fiber reinforced concrete is greater to that of ordinary concrete. Fibers have a crack-stopping effect rather than a crack-prevention effect. It is important to note that CFs have a greater impact on tensile strength than they do on compressive strength when compared to other materials. Several studies have shown that fibers increase the tensile capacity of post-cracking behavior61. Fibers have been found to have more substantial impacts on flexural tensile strength at volume fractions ranging from 0.5 to 2.0 percent, which were involved in this study62.As for coconut fibers (CFs), similar to the compressive strength split, tensile strength was also enhanced as the proportion of CFs rose up to 2.0 percent and subsequently decreased, as shown in Fig. 2 higher than 90 percent, as illustrated in Fig. As previously stated, split tensile strength exhibits the same pattern as compressive strength in a split configuration. As a result, there was a significant correlation between compressive and split strength. A regression model with an RAs illustrated in Fig. The optimal dosage of marble waste (MW) and coconut fibers (CFs) for split tensile strength in SCC is determined by selecting 18 percent marble waste (MW) and 1.8 percent coconut fibers (CFs) from the contour plot that gives split tensile strength of 10 Mpa, as displayed in Figs. 63. Flexure tests were performed on beam specimens with dimensions of 150 × 150 × 500 mm at 7, 14 and 28 days curing.Flexural strength, also known as bend strength or modulus of rupture, is a material property that may be specified as the stress in a material immediately before it fails in a flexure test11. It is due to pozzolanic reaction of marble waste. The pozzolanic reaction of Sio2 in marble waste with CH of cement results in the formation of extra cementitious compounds (C-S–H), which has a beneficial impact on flexure strength49.Increased dosages of marble waste (more than 20 percent by weight of cement) result in a reduction in strength owing to dilution effect, which cause to an alkali-silica reaction due to a greater amount of unreactive silica accessible due to the increased quantity of marble waste used in the mix. A greater dose also makes the curing process more problematic owing to a lack of workability, which results in porous concrete with a lower strength50.Figure 54. When flexure strength was measured after 28 days of curing, the greatest value was achieved with a 2.0 percent dose of Coconut fiber when compared to blank or reference concrete. The strength, however, was decreased when the CFs exceed 2.0 percent dose. The confinement of the fiber reinforcement on the specimen has a beneficial impact on flexure strength. Compression causes lateral expansion, which results in tension and shear as a result of the expansion. The fibers are able to withstand tension and shear due to their strength. This confinement has the potential to decrease the specimen's transverse deformation while simultaneously increasing its flexural strength. When the percentage of coconut fiber is increased, particularly at a greater dose, the process of compaction becomes more difficult, resulting in porous concrete and a reduction in flexural strength, as previously stated. It has been observed that increasing the flexure strength by up to 1.5 percent of the total volume of fibers increases it from 20 to 25 percent55. Steel fibers, at a concentration of 1.0 percent by volume, produce a significant increase in both the early and long-term strength of concrete. The highest increase in 28-day strength was found to be 29.15 percent with the maximum increase being 29.15 percent56.Considering Coconut fiber (CFs), flexure strength rises as the proportion of Coconut fiber grew up to 2.0 percent and subsequently dropped, as shown in Fig. 2 of more than 90 percent.Figure Additionally, a comparison  is carried out, in which the 28-day flexure strength of control mix is taken into consideration as the reference mix, from which various mixes with varied percentages of marble waste and Coconut fiber are compare, as shown in Fig. Figures 45. For this test, the #4 bar is kept 100 mm from the top as shown in Fig. Pull out the test was carried out on a cube of size 150 mm to determine bond strength between concrete with reinforcing bar and could be performed according to ASTM C-23464 which gives dense mass, and the bond between reinforcement bar and surrounding concrete improved due to the confinement effect. Also, due to its finesse, marble waste acts as a micro-filler in cement aggregate matrix which dense concrete leading to more force is required to pull out reinforcement of concrete59. It has been also reported that bond strength mainly depends on the strength of surrounding concrete i-e before pulling reinforcement concrete laterally expands65. By using marble waste, more force is required to expand concrete laterally which results in more bond strength. However, at a higher dosage of marble waste (beyond 20% by weight of cement) strength reduces due to dilution effect which leads to alkali-silica reaction due to higher quantity of unreactive silica available due to high quantity of marble waste. Also, at a higher dosage, the compaction process become more difficult due to lack of workability which results in porous concrete, leading to lower strength50.Bond strength with varying dosages of marble waste was presented in Fig. 65. By using fibers, more force is required to expand concrete laterally as fiber prevents propagation of cracks which results in more bond strength. However, at a higher dosage, the compaction process becomes more difficult due to lack of workability which results in porous concrete, leading to lower concrete strength leading to less force is required to pull out reinforcement from concrete.As for Coconut fiber concerns, bond strength improved as the percentage of Coconut fiber (CFs) enhance up to 2.0 percent and then decreased as displayed in Fig. 2 greater than 90%.The correlation between compressive strength and bond strength with the incorporation of waste marble is shown in Fig. A relative analysis is also performed in which the 28-day bond strength of the control mix is taken into account as the reference mix as shown in Fig. To evaluate optimum dosage marble waste and Coconut fiber for bond strength in SCC, 18% marble waste and 1.8% Coconut fiber are selected from the contour plot giving flexure strength 5.0 Mpa (at 28 days) as shown in Figs. When the proportion of marble waste (Mw) and coconut fibers (CFs) is raised, the flowability and passing ability of the concrete decreased. However, all the mixes, except for the 2.5 percent and 3.0 percent CFs, demonstrate excellent filling and passing capabilities within the limits specified by the practical requirement for SCC.Mechanical performance improved as the proportion of Mw was increase up to 20 percent addition and then reduce as compared to the reference concrete. It is because of micro-filling voids in concrete ingredients as well as pozzolanic reaction of Mw.Coconut fibers (CFs) improved mechanical performance up to 2.0 percent addition by weight of cement and then declines gradually as compared to the reference concrete. The positive influence on mechanical performance is because of confinement coconut fibers (CFs) around the sample. However, at higher dosage (beyond 2.0% substitution), the compaction of concrete become more difficult due lack flowability, resulting pore in harden concrete which decreased mechanical performance od SCC.Based on work results, the predicted value from statistical analysis and the experimental value were comparable.The maximum mechanical performance was achieved at the substitution ratio of 18 percent Mw and 1.8 percent CFs respectively, having a maximum compressive strength of 23 Mpa which was almost 37 percent greater than from control mix. Hence 18 percent marble waste (Mw) and 1.8 percent coconut fibers (CFs) were optimal dosage for combined substitution.In this study, a step towards utilization of marble waste (from 0 to 30% by weight of cement) and coconut fibers (from 0 to 3.0% by weight of cement) has been made in the manufacturing of sustainable self-fiber compacting concrete. The following conclusion has been drawn based on experimental work:Finally, the current research indicates that marble wastes and coconut fibers (CFs) are excellent, abundant, local eco-materials that are low-cost and may be utilized for SCC production from the point of view economic and environmental limitations. SCC has several advantages in terms of production, placement and compaction as compared to conventional concrete namely, removal of external or internal vibration for compaction, better flowability, passing ability and pumpability, as well as increased bonding with congested reinforcement. Moreover, the placement of SCC is faster and requires less labor. The surface finish, mechanical performance and durability aspects of SCC can be considerably better than traditional concrete.


There is significant comorbidity of opioid use disorder (OUD) and post-traumatic stress disorder (PTSD) in clinical populations. However, the neurobiological mechanisms underlying the relationship between chronic opioid use and withdrawal and development of PTSD are poorly understood. Our previous work identified that chronic escalating heroin administration and withdrawal can produce enhanced fear learning, an animal model of hyperarousal, and is associated with an increase in dorsal hippocampal (DH) interleukin-1β (IL-1β). However, other cytokines, such as TNF-α, work synergistically with IL-1β and may have a role in the development of enhanced fear learning. Based on both translational rodent and clinical studies, TNF-α has been implicated in hyperarousal states of PTSD, and has an established role in hippocampal-dependent learning and memory. The first set of experiments tested the hypothesis that chronic heroin administration followed by withdrawal is capable of inducing alterations in DH TNF-α expression. The second set of experiments examined whether DH TNF-α expression is functionally relevant to the development of enhanced fear learning. We identified an increase of TNF-α immunoreactivity and positive cells at 0, 24, and 48 h into withdrawal in the dentate gyrus DH subregion. Interestingly, intra-DH infusions of etanercept (TNF-α inhibitor) 0, 24, and 48 h into heroin withdrawal prevented the development of enhanced fear learning and mitigated withdrawal-induced weight loss. Overall, these findings provide insight into the role of TNF-α in opioid withdrawal and the development of anxiety disorders such as PTSD. Post-traumatic stress disorder (PTSD), a devastating mental illness, is highly comorbid with substance use disorder (SUD), as nearly 40% of individuals diagnosed with PTSD are diagnosed with SUD . SpecifiHeroin use and withdrawal may give rise to long-term neurobiological changes believed to underlie major symptoms of PTSD, such as the fear learning, hyperarousal, and/or re-experiencing events, and increased vulnerability future stressors. Opioid-dependent patients display increased distress on the perceived stress scale, and interestingly, abnormally high cortisol levels have been correlated with an individual’s increased discontinuation risk for recovery . ConsistOur laboratory has previously shown that dorsal hippocampal (DH) interleukin-1 (IL-1) signaling is responsible for the development of stress-enhanced fear learning (SEFL), a reliable and reproducible animal model of fear-related features of PTSD . AlthougWe have recently developed an animal model for chronic escalating heroin administration and withdrawal that we use in combination with the enhanced fear learning paradigm . This moAlthough our research has primarily focused on IL-1β, central cytokines function in concert to facilitate learning processes , and theThe current studies test the hypothesis that chronic heroin and withdrawal are capable of inducing alterations in TNF-α expression in the DH, and that this DH TNF-α expression is functionally relevant to the development of future enhanced fear learning and withdrawal symptoms, as indicated by weight loss. To this end, experiment 1 determined the consequence of chronic heroin administration and withdrawal on TNF-α expression in the DH. Our analysis focused on the DH as this region has been shown to be critical to context-dependent fear learning and conditioning –37. SpecAdult male Sprague Dawley rats  were individually housed under a reversed 12-h light-dark cycle. Rats were given ad libitum access to food and water, and were regularly handled throughout experimentation. All procedures were conducted with approval from the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.Heroin  Drug Supply Program, Bethesda, MD, USA) was dissolved in sterile 0.9% saline to produce 1.0, 2.5, 5.0, 7.5, or 10.0-mg/mL solutions and stored at 4°C until time of injection.For stereotaxic surgery, animals were anesthetized with a 1.0-mL/kg intraperitoneal injection of 9:1 (vol/vol) ketamine hydrochloride (100 mg/mL) mixed with xylazine (100 mg/mL). Guide cannulae  were directed bilaterally at the DH 325 . Animals were given 2 weeks for postoperative recovery prior to the start of experimental procedures. Animals were randomly assigned to receive heroin or saline in the chronic escalating heroin administration and then either a saline or etanercept infusion treatment. Etanercept , a TNF-α inhibitor, was dissolved in sterile saline (2.5 μg/μL). Animals underwent the chronic heroin escalating administration and were given three infusions of etanercept or saline at 0, 24, and 48 h into withdrawal . Forty-eight hours prior to the first infusion, animals were given a sham microinjection to allow for habituation to the injection experience. Animals were microinfused with 1.25 μg of etanercept or saline vehicle per hemisphere at a rate of 0.25 μL/min, and the injectors were left in place for 1 min to allow for drug diffusion away from the injection site.In experiment 1, animals were sacrificed by transcardial perfusion 0, 24, 48, and 72 h into withdrawal. Animals that were perfused 1h following their last heroin injection were classified to be in the 0-h withdrawal group, as they were considered to have heroin in their system and, therefore, had not undergone withdrawal. Animals perfused at 24 h following the last injection are considered to be in the 24-h withdrawal group, while the same applies for the animals perfused at 48h and 72h groups respectively. Animals were terminally anesthetized with 9:1 (vol/vol) ketamine hydrochloride (100 mg/mL) mixed with xylazine (100 mg/mL), and transcardially perfused with ice-cold phosphate buffer  followed by 4% paraformaldehyde in 0.1 M PB. Brains were extracted and post-fixed in 4% paraformaldehyde for 6 h, and used 30% sucrose for cryoprotection with 0.1% sodium azide at 4 °C. Once the brains were saturated with sucrose, brains were cut into 40-μm coronal sections on a cryostat . For experiment 2, animals were sacrificed by rapid cervical dislocation and DH cannula placement was verified.Experiment 1 used fluorescent immunohistochemistry (IHC) to examine alterations in DH TNF-α in the DG. The IHC protocol used here has been described previously , 21. Brin = 1 0-h saline and n = 1 24-h saline) that yielded high nonspecific background which interfered with thresholding was dropped from the analysis. In these perfusions, gross inspection of the brain did not reveal a fixed appearance—void of blood in circulatory system , but instead was found to be reddish, indicative of blood present. Moreover, automatic thresholding failed on this tissue and resulted in exclusion from analysis. The decision to exclude these two samples was made blind to the treatment group. Publication images were compiled with the Adobe Photoshop CS software . Color levels and background were reduced for optimal representation with level tools. Images from all experimental groups were treated equally.Fluorescent microscopy  was used to capture color images. Positive fluorescence in images was quantified using automatic Image J (NIH) triangle thresholding feature. The implementation of the ImageJ automatic triangle algorithm has been previously described . BrieflyAnimals were randomly assigned to drug  treatment and heroin withdrawal timepoint  and underwent chronic escalating heroin administration as described previously . BrieflyThis procedure has been previously described at length . Brieflyt tests determine specifically whether drug treatment altered TNF-α immunoreactivity and cell counts between the 0-h, 24-h, 48-h, and 72-h timepoints. In experiment 2, a one-way  ANOVA was used to analyze baseline freezing data. A 2  × 2  × 4 (test day) repeated-measures ANOVA was used to analyze freezing behavior. A 2  × 2  × 3 (timepoint) repeated-measures ANOVA was used to analyze weight change across withdrawal. Significant interactions for both freezing behavior and weight change were examined using Tukey’s post hoc comparisons.Experiment 1 was run using separate cohorts for each of the withdrawal timepoints. Therefore, planned comparisons using unpaired, two-tailed Student’s t(18) = −2.727, p = .029)  = −3.416, p = .003)  = −3.190, p = .008)  = −.277, p = −0.603)  = 12.107, p < .001)  = −2.542, p = .024)  = 5.651, p = .002)  = 1.583, p = −0.174)  Fig. , 24-h he03) Fig. , and 48-08) Fig.  relative03) Fig. . Additio01) Fig. , 24-h he24) Fig. , and 48-02) Fig.  relative74) Fig. . These rF = 2.216, p = .111), indicating that there is no generalized fear to the novel context. A 2 × 2 × 4 repeated-measures ANOVA revealed a significant main effect of heroin treatment  = 15.366, p < .001) and a significant main effect of etanercept treatment  = 8.772, p = .007). These main effects of heroin treatment and etanercept treatment were on contextual freezing. There was also a significant effect of test day  = 42.100, p < .001), indicating that conditioned freezing behavior diminished over time, thus suggesting extinction of contextual conditioning. Importantly, there was a significant heroin treatment by etanercept infusion interaction  = 13.195, p < .001). On test days 1, 2, and 7, Tukey’s post hoc comparisons revealed heroin-withdrawn, vehicle-treated animals exhibited significantly higher freezing behavior compared to animals that were saline controls and vehicle-treated on test days 1 (p = .001), 2 (p = .005), and 7 (p = .042) replicating enhanced fear learning. Heroin-withdrawn, etanercept-treated animals exhibited significantly less freezing than withdrawn controls that received intra-DH vehicle on test days 1 (p < .001), 2 (p = .016), and 7 (p = .036). Furthermore, heroin-withdrawn, etanercept-treated animals exhibited a comparable amount of freezing behavior (no significant difference) to both saline control groups (p > .05)  Fig. . These rF = 25.410, p < .001). Weight loss in rodents is a hallmark symptom of opioid withdrawal [F = 5.333, p = .029) indicating that intra-DH etanercept infusions may have prevented the heroin withdrawal decrease in weight. There was also an overall effect of withdrawal time  = 41.192, p < .001), indicating over the course of withdrawal animals started to gain weight again . Importantly, heroin-withdrawn, vehicle-treated animals had a significantly higher weight change compared to both the saline control groups (p < .001) and heroin-withdrawn, etanercept-treated animals also had a significantly higher weight change compared to both the saline control groups (p < .001). Strikingly, heroin-withdrawal, vehicle-treated animals had a significantly higher weight change in comparison to heroin-withdrawn, etanercept-treated animals (p < .05), indicating that etanercept treatment mitigated the withdrawal-induced weight loss at 24-h withdrawal . Importantly, heroin-withdrawn, vehicle-treated animals had a significantly higher weight change compared to both the saline control groups (p < .001) and heroin-withdrawn, etanercept-treated animals also had a significantly higher weight change compared to both the saline control groups (p < .001). Strikingly, heroin-withdrawn, vehicle-treated animals had a significantly higher weight change in comparison to heroin-withdrawn, etanercept-treated animals (p < .05), indicating that etanercept treatment mitigated the withdrawal-induced weight throughout the 48-h withdrawal timepoint. During the 72-h withdrawal timepoint, both the saline control groups did not differ in weight change (p > .05), and heroin-withdrawn, vehicle-treated animals had a significantly higher weight change compared to both the saline control groups (p < .001)  Fig. . OverallThe current study demonstrates for the first time that DH TNF-α signaling mediates some long-lasting maladaptive behavioral responses induced by chronic heroin and withdrawal. We have shown that exposure to chronic heroin administration and withdrawal induces TNF-α immunoreactivity and TNF-α positive cell counts within the DG, and disrupting DH TNF-α signaling during withdrawal blocks the development of enhanced fear learning. Critically, this manipulation also mitigates heroin withdrawal-induced weight loss, a hallmark sign of withdrawal in rodent models. These findings provide new evidence that heroin withdrawal-induced TNF-α is necessary for the development of future enhanced fear learning and is a potential mechanism by which opioids and opioid withdrawal can elicit fear-related and arousal-related features of PTSD symptomatology.The present findings suggest that neurobiological changes, specifically increases in TNF-α immunoreactivity, leave the animals hypersensitive to future stressors. Exposure to chronic heroin administration and withdrawal increases TNF-α immunoreactivity within the DG up to 48 h into withdrawal. This finding complements our recently published IL-1 findings ; howeverA receptors [The present study demonstrates that heroin and withdrawal-induced enhanced fear learning is driven by hippocampal TNF-α signaling, as inhibiting DH TNF-α signaling disrupts future exaggerated fear conditioning. A significant body of literature implicates TNF-α signaling as an integral component of the learning and memory processes. Specifically, a local increase of TNF-α in the hippocampal dentate gyrus activates TNF receptor type 1, which triggers an astrocyte-neuron signaling cascade resulting in persistent functional modification of hippocampal excitatory synapses . Moreoveeceptors , 50. Thieceptors , 51, andeceptors . These sThe focus of the current work examined the role of hippocampal TNF-α in heroin withdrawal for enhanced fear learning and weight loss, and directly compliments our previous work indicating that IL-1 signaling is involved in heroin withdrawal and enhanced fear learning . EvidencEvidence suggests that glial activation leads to the release of proinflammatory molecules that modulate neuronal activity crucial to the complex syndrome of opioid dependence and withdrawal. In particular, glial cells appear to be primarily responsible for TNF-α release , which mThe present work focused on the DH, a brain region critical to learning and memory processes. As disruption of TNF-α signaling mitigated withdrawal-induced weight loss, future studies should investigate TNF-α expression in other areas involved in opioid abuse, such as the ventral tegmental area, locus coeruleus, or periaqueductal gray (PAG). Specifically, the PAG can be targeted to better understand the effects of TNF-α in heroin withdrawal and withdrawal symptomology. Functional studies have implicated the PAG in the expression of symptoms of opioid withdrawal, but the molecular mechanisms involved are not fully understood. Further studies should investigate the role of TNF-α expression in other brain regions involved in opioid abuse, such as the PAG, that may also contribute to enhance fear learning and alter heroin withdrawal-induced weight loss or other withdrawal-related behaviors.Strikingly, intra-DH etanercept infusions mitigated heroin withdrawal-induced weight loss. This intriguing finding demonstrates a role for DH TNF-α signaling in both learning processes and withdrawal behaviors, such as weight loss. Although this study evaluated weight loss and freezing behavior separately, it would be interesting to study the correlation between these two behaviors. As regards to weight loss, specifically, we believe TNF-α may be exerting an anorexic effect in opposition to ghrelin. Studies have shown that lower levels of total TNF-α promoter methylation had higher success with a weight loss program , and theIn summary, our exciting findings demonstrate that heroin use and withdrawal increase expression of hippocampal TNF-α, and inhibition of TNF-α signaling disrupts future enhanced fear learning, as well as mitigates heroin withdrawal-induced weight loss. Collectively, these data provide important new evidence that chronic heroin administration and withdrawal alter hippocampal neuroimmune signaling and downstream behavioral responses, such as enhanced susceptibility to future stressors and withdrawal-induced weight loss. The current study identifies neuroimmune targets that can be used to alleviate long-term maladaptive responses stemming from a history of chronic heroin use and protracted withdrawal.


Objectives: Understanding factors influencing inappropriate antibiotic use can guide the design of interventions to improve antibiotic practices and reduce antimicrobial resistance (AMR). Methods: A nationally representative cross-sectional survey (N = 2004) was conducted between November 2020 and January 2021. Knowledge of antibiotic use and AMR using the World Health Organization’s Multi-Country AMR Survey questionnaire, and antibiotic practices were examined. Multivariable logistic regression was performed to identify factors associated with inappropriate antibiotic use and examine effect measure modifications. Results: After adjusting for potential confounding, poor knowledge of antibiotic use was associated with a 3x increased odds of inappropriate antibiotic use in adults aged ≥50 years , 5× increased odds in those aged 35–49 years , and 7× increased odds in those aged 21–34 years . While there was no statistically significant association in adults aged ≥50 years, poor knowledge of AMR increased the odds of inappropriate antibiotic use by 4 times in adults aged 35–49 years  and 5 times in those aged 21–34 years . Conclusions: Targeted educational interventions for specific age groups are needed in conjunction with empowering the public with knowledge of antibiotic use and AMR. Unlike influenza and coronavirus disease 2019 (COVID-19), which often result in explosive epidemics and pandemics, antimicrobial resistance (AMR) poses a silent but major threat to global health, with a projected annual mortality rate of 10 million deaths per year by 2050 ,4,5.A multi-country survey conducted by the World Health Organization (WHO) to assess public awareness of AMR amongst 12 countries in 2015 reported a lack of knowledge in both aspects . More thPopulation-based studies thus far have not assessed the knowledge gaps concerning antibiotics and AMR, stratified by socio-demographic factors. Purposive sampling from different sub-populations of the same local context is efficient but could lead to contradictory and non-generalisable findings due to selection bias ,19. HencWe conducted a nationally representative cross-sectional, community-based survey on a random sample of Singapore residents (citizens and permanent residents) aged 21 years and over between November 2020 and January 2021, using the sampling frame as described below:Addresses with at least 1 Singapore resident were first stratified according to the ethnic group of the residents at the address. The number of addresses selected from each predominant ethnic group was proportionate to the distribution of the ethnic group in Singapore, with an additional oversampling of Malays and Indians to ensure representation from these minority groups in the final dataset. In addition, within each predominant ethnic group, the addresses were further stratified by their broad dwelling type. Within each detailed house type, addresses were randomly selected.To avoid cluster bias, only one member of each household was invited to participate in the study. The person with the most recent birthday within each household was selected to avoid selection bias and to achieve a truly random sample of the population.The survey was self-administered and disseminated using a drop-off/pick-up method. Each survey package containing instructions on how to complete the hardcopy or online questionnaire was delivered in person via door-to-door visits. Any household uncontactable at the first visit was approached up to twice more at different times and on different days of the week before the household was replaced with a neighbouring household of the same housing type.We developed the survey instrument through adaptation of questions and validated scales from various cross-sectional surveys reported in the literature ,22,23,24The survey questionnaire focused on these main points: knowledge of antibiotics, antibiotic use and AMR, attitude towards antibiotics, antibiotic use and AMR, and self-reported antibiotic practices and experiences. Only the question items on knowledge and practice were used in this study. Knowledge questions on antibiotics, antibiotic use, and AMR were designed in the form of Yes/No/Don’t know or True/False/Don’t know, whilst practice questions were presented in a 5-point Likert scale: . Respondents were determined to have good knowledge of antibiotic use and AMR after they had correctly answered all of the respective question items–7 items on antibiotic use and 8 on AMR . OtherwiSix proxy statements on the inappropriate use of antibiotics were selected based on the US Centers for Disease Control and Prevention’s advisory on appropriate antibiotic use : (1) “I Proportions were tabulated for categorical variables, whereas means  were calculated for continuous variables and responses on a 5-point Likert scale. Chi-squared test and t-test were used to compare differences between proportions and means, respectively.p-value < 0.05. Statistical analyses were conducted in Stata version 14.0 .Multivariable logistic regression was performed to determine the independent factors associated with inappropriate use of antibiotics. Covariates were selected through stepwise regression and included in the final regression model to adjust for potential confounding. Interactions between covariates were individually explored, and product terms were also included in the final model. Effect measure modification due to socio-demographic factors was further assessed. Statistical significance was defined as a Out of 4791 households approached, 2004 (41.8%) responses were collected with demographics presented under The distribution of residency status , gender , ethnic group ; age ; housing type ; marital status ; and religious affiliation  of our study population was representative of the Singapore population in 2020 . Nearly Of those who had a religious affiliation, one-third (33%) reported an influence of their religious beliefs on their health-seeking behaviour. Pertaining to health-related questions, 32% self-reported the presence of chronic illnesses, and 97% reported having taken antibiotics in their lifetime. Of those who had taken antibiotics, most had obtained them from a general practice clinic in Singapore (66%), 92% recalled receiving advice from a doctor, nurse, or pharmacist on how to take the antibiotics, and 81% reported completing them as prescribed.The top 3 statements of knowledge answered correctly by respondents were “overuse of antibiotics can cause antibiotics to lose effectiveness in the long term” (77%), “antibiotics can treat bacterial infections” (72%), and “bacteria can be resistant to antibiotics” (68%) . By contA total of 1188 Singapore residents (59%) answered correctly to all 3 knowledge statements on antibiotic use .The majority were aware that it is inappropriate to “use antibiotics that were given to a friend or family member, as long as they were used to treat the same illness” (89%), or “to buy the same antibiotics or request for them from a doctor, if they had helped you get better previously when you had the same symptoms” (68%), and 1703 (85%) respondents were aware that antibiotics should only be stopped when one has taken all of them as directed.p = 0.876) (data not shown). Interestingly, 57% did not know that antibiotics could not be used to treat COVID-19, which is also a viral infectious disease.More than half responded correctly that bladder infection or urinary tract infection (64%) and skin or wound infection (57%) could be treated with antibiotics . HoweverThe top 3 terms familiar to the respondents were “antibiotic resistance” (76%), “drug resistance” (69%), and “antibiotic-resistant bacteria” (64%) . While oMore than half of the respondents answered correctly to these statements: “if bacteria are resistant to antibiotics, it can be very difficult or impossible to treat the infections they cause” (67%), “antibiotic resistance is an issue that could affect my family or me” (61%), “many infections are becoming increasingly resistant to treatment by antibiotics” (59%), “antibiotic resistance is an issue in other countries but not here” (53%), and “antibiotic-resistant infections could make medical procedures like surgery, organ transplant, and cancer treatment much more dangerous” (51%) . HoweverNinety percent of respondents reportedly professed to taking antibiotics according to instructions on the label, while 85% reported to have checked the expiry date of antibiotics before taking them . More thp = 0.023) expected antibiotics from doctors for a cold or the flu  during the COVID-19 pandemic than before.To some extent, antibiotic practices regarding the common cold and flu were influenced by the COVID-19 pandemic . RespondKnowledge of antibiotic use, knowledge of AMR, gender, ethnic group, age, and education level were independent factors associated with the inappropriate use of antibiotics .p < 0.001) and on AMR  were three to four times as likely as those with good knowledge to be inappropriate users of antibiotics. There was an inverse dose-response relationship between inappropriate antibiotic use and age, with respondents aged 35–49 years  being 42% more likely and those aged 21–34 years  being twice as likely to be inappropriate users of antibiotics, compared to respondents aged 50 years and above.Respondents with poor knowledge of antibiotic use  and poor knowledge of AMR , and the interaction terms were included in Model 2. Males , non-Chinese , and lower educated respondents  were more likely to be inappropriate users of antibiotics.Age was found to interact positively with poor knowledge of antibiotic use , by 5 times in respondents aged 35–49 years , and by 7 times in respondents aged 21–34 years , respectively .Whilst there was no significant association between poor knowledge of AMR regarding inappropriate antibiotic use in respondents aged ≥ 50 years, poor knowledge of AMR increased the odds of being an inappropriate user of antibiotics by 4 times in respondents aged 35–49 years , and 5 times in respondents aged 21–34 years , respectively .The top 3 promotional methods perceived to be effective by respondents regarding public education on AMR were posters or pamphlets in clinics or hospitals (66%), television and radio advertisements (63%), and annual AMR campaigns (61%) .p = 0.001), newspaper articles (p < 0.001), and magazine articles (p < 0.001) to be effective for public education on AMR, while younger adults aged 21–34 years were more likely to perceive contemporary methods such as YouTube (p < 0.001), movie advertisements (p = 0.001) and mobile applications (p = 0.001) as effective educational tools. Interestingly, respondents aged 35–49 years were more likely than their younger (21–34 years) and older (≥50 years) counterparts to perceive annual AMR campaigns to be effective (p = 0.013).Older adults aged ≥ 50 years were more likely to perceive traditional means such as posters or pamphlets in clinics or hospitals , and did not know that antibiotic resistance does not occur when the body (instead of the bacteria) becomes resistant to antibiotics (91%). Compared to the aggregated findings from the WHO Antibiotic Resistance: Multi-country Public Awareness Survey [5%, and dAwareness regarding medical conditions warranting antibiotics was also lacking, and therefore major knowledge gaps on antibiotic use and AMR need to be addressed. In particular, two consecutive years of consistent messaging through national AMR campaigns to correct the misuse of antibiotics for flu were carried out, but awareness that antibiotics cannot treat the common cold and flu remained low (39%), and the intervention showed no significant improvement in knowledge, supporting Price et al.’s argument that a “one-size-fits-all” public education was ineffective .Another broader finding concerns the differences in antibiotic practices before and during the COVID-19 pandemic. With the majority of Singapore residents (61%) being inappropriate antibiotic users, there was also a slight increase of 3% in respondents reportedly using and expecting antibiotics to be prescribed inappropriately for cold or flu symptoms during the pandemic. This could be due to uncertainties in the clinical management of COVID-19, for which patients tended to present with acute respiratory symptoms. Notably, nearly half of the respondents did not know if antibiotics were required for COVID-19 treatment or not. More public education on COVID-19 is required to address the knowledge gaps.Age played a pivotal role between knowledge and antibiotic practices. Poor knowledge of antibiotic use was associated with inappropriate antibiotic use in all age groups, with those aged 21–34 years having the strongest association. However, poor knowledge of AMR only had significant effects on those aged 35–49 years and 21–34 years, with increased odds of 4 and 5 times, respectively. As knowledge of AMR had no effect on the antibiotic use practices of individuals aged ≥50 years, addressing the knowledge deficit in AMR can be focused and tailored to adults below the age of 50. Whereas for older adults, as with their younger counterparts, education is needed to improve knowledge of the proper use of antibiotics. This finding is novel and not reported by previous studies.Annual campaigns remained favourable for people across all ages (61%), but the roll-out of interventions could take on new forms leveraging technological advances and social networks. Traditional mass media, such as newspaper and magazine articles, were preferred by adults aged 50 years and above. Newer platforms such as YouTube were preferred by younger adults aged 21–34 years, as are advertisements during movies, mobile applications such as games, and online information portals. In particular, serious games or rules of “gamification” could be opportunistically used as they were shown to be successful in increasing knowledge of antibiotic use and AMR in both young children and adults ,29,30. OHealthcare workers, in particular, doctors or nurses, could be tapped to provide public and patient education on appropriate antibiotic use and AMR. They have been reported as the top sources of information for terms related to AMR. Since the most preferred intervention was posters or pamphlets in clinics or hospitals (66%), they could be handed out during consultations as decision aids . PreviouPrimary healthcare providers are pivotal to providing education during a consultation, and shared decision-making concerning antibiotic uptake between patients and attending doctors should be encouraged, as recommended by the VALUE model . In partThis study has several strengths. Instead of convenience sampling, we adopted a robust methodology to avoid selection bias and cluster bias by proportionally stratifying the population by ethnic group (with oversampling of minority groups) and housing type to ensure representation, and randomly selecting one individual within each selected household. Despite a response rate of 41.8%, our study population was representative of the Singapore Census 2020, and therefore selection bias (if any) was likely to be minimal. As the population is heterogeneous in terms of education levels and language proficiencies, the survey booklets were provided in 3 other main languages  to avoid recruiting only English-literate residents and provide options to illiterate participants for survey administration by bilingual surveyors. Surveyors were all trained by the study team prior to data collection to avoid interviewer bias.Furthermore, this is the first-ever nationally representative survey conducted in Singapore to assess residents’ knowledge and practices related to antibiotics and AMR. The adaptation of validated questions from the WHO Antibiotic Resistance: Multi-country Public Awareness Survey enabled international comparisons, and will allow future studies to assess the effectiveness of subsequent educational interventions. However, we cannot exclude the possibility of respondents underreporting inappropriate antibiotic practices due to social desirability bias, although it is likely to be minimal as the survey was anonymous and self-administered. Also, there could be unknown confounders which were not adjusted for in the final multivariable logistic regression model.Knowledge deficits on antibiotic use and AMR are associated with the inappropriate use of antibiotics and how their effects are modified by age. Whilst education on antibiotic use should be considered for all adults, education on AMR could be tailored and targeted for adults aged below 50 years. Opportunistic education on antibiotic use at primary care clinics could be effective in improving antibiotic practices and reducing AMR.