In Iran, river buffalo is of great importance. It plays an important role in the economy of the Country, because its adaptation to harsh climate conditions and long productive lifespan permitting its farming across the Country and to convert low-quality feed into valuable milk. The genetic variability in Iranian buffalo breeds have been recently studied using SNPs genotyping data, but a whole genome Copy Number Variants (CNVs) mapping was not available. The aim of this study was to perform a genome wide CNV scan in 361 buffaloes of the three Iranian river breeds  through the analysis of data obtained using the Axiom® Buffalo Genotyping Array 90 K.n = 302) mapped a total of 409 buffalo genes, some of which resulted associated with morphological, healthy, milk, meat and reproductive traits, according to Animal Genome Cattle database.CNVs detection resulted in a total of 9550 CNVs and 302 CNVRs identified in at least 5% of samples within breed, covering around 1.97% of the buffalo genome. and A total of 22 CNVRs were identified in all breeds and a different proportion of regions were in common among the three populations. Within the more represented CNVRs (This work provides a step forward in the interpretation of genomic variation within and among the buffalo populations, releasing a first map of CNVs and providing insights about their recent selection and adaptation to environment. The presence of the set of genes and QTL traits harbored in the CNVRs could be possibly linked with the buffalo’s natural adaptive history together to a recent selection for milk used as primary food source from this species.The online version contains supplementary material available at 10.1186/s12864-021-07604-3. Bubalus bubalis) and the African wild buffalo or Cape buffalo (Syncerus caffer) are the two main species of buffalo in the world [Bubalus bubalis bubalis 2n = 50) and the swamp  buffalo, occurred about 3000–6000 years before present in the Indo–Pakistani area and in the vicinity of borders of China, respectively [The Asian water buffalo (he world . The domhttp://www.fao.org/faostat/en/?#data/QA accessed 2020/10/08) the proportion of buffalo population in Iran respect to cattle species was 2.5%. A very similar data is reported by Beldman et al. [According to the FAOSTAT data  the ability of buffalo to convert low-quality feed to valuable milk, (ii) adaptation to harsh climate conditions and resistance to local parasites, (iii) long productive lifespan , and (ivCopy number variants (CNVs) are a source of structural variability that have been utilized to identify genetic variability among breeds in several species. Redon et al.  defined ST results based on the SNPs genotypes confirms that the three populations are clearly differentiated  on all 24 autosomes were detected in the 361 samples  for AZE, KHU and MAZ, respectively  are the regions defined by CNVRs mapped in at least 5% of samples . Non redundant regions include pop_CNVRs (i.e. non singleton regions within population) plus the common_CNVRs (i.e. common CNVRs across populations), counting these latter only once when found in more than one population. All the following statistics and graphical representation have been obtained using the pop_CNVRs, covering 28.40 Mb (1.14%), 29.91 Mb (1.21%), and 11,44 Mb (0.46%) of the buffalo autosomes total length, respectively.For each population, a graphical representation of CNVRs frequencies on autosome is shown, together with the mean CNVRs coverage length Fig. . The numWithin the 203 non redundant CNVRs a different proportion of common_CNVRs were observed among the three populations .A total of 234, 365, and 158 genes were annotated within AZE, KHU and MAZ CNVRs, respectively  .In Iran, breeding of river buffaloes plays an important role in the economy of the country, also impacting on social and cultural activities . In thisST statistics here calculated as in [ST values: the lowest between AZE vs KHU  obtained from the Affymetrix 90 K SNP genotypes  showed aed as in , shows a = 0.021 ); an intTo improve the knowledge on these populations, we investigated the genomic structure of the Iranian buffaloes through the analysis of the CNVs, in order to provide additional information that could be used for breeding and conservation programs of these populations, as concluded by Clop et al. .A genome wide CNVs detection has been here performed resulting in a total of 9550 CNVs and 2.995 CNVRs in the three breeds. The AZE and KHU show about 50% of their CNVR as singleton, while MAZ breed 73%. The large singleton_CNVRs proportion for MAZ is most likely due to the reduced sample size that does not permit to identify a larger proportion of pop_CNVRs in this breed. Excluding singleton_CNVRs the CNVRs resulted 1605 in the three breeds. Among the 1605 pop_CNVRs, 302 were identified across breeds in at least 5% of individuals within breed, covering about 1.97% of the buffalo genome . Out of these latter CNVRs, 22 (10.8%) are those shared by all populations, identifying a common genomic structural background of among these buffaloes. In fact, when considering the common_CNVRs counts (those shared among breeds), the highest number of common_CNVRs are found in AZE having the lowest proportion of pop_CNVRs (30.7%) respect to those identified for KHU 48.9%) and for MAZ (44.1%). MAZ breed shared a higher number of CNVRs with AZE respect to those resulted in common with KHU. This result suggest that the moderate level of admixture identified for MAZ and AZE by [8.9% and ABCC4). This gene appears to be involved in several basic metabolic pathways including resistance/susceptibility to intestinal nematodes [ABCC4 gene it has been also highlighted its possible contribution on reproduction traits, resulting among the top genes associated with number of services per conception [ABCC4 gene increases in pregnant cattle and pigs’ endometrium and it could be important to support pregnancy given its role in prostaglandin efflux from cells [The most represented common_CNVR, defined by the CNVs identified in 209 buffaloes of all the three breeds Table , mapped ematodes , feed efematodes , and marematodes . Recentlnception . Additioom cells , 26.ABCC8, USH1C, MYOD1, OTOG, KCNC1, SERGEF), which all resulted close (i.e. surrounding 200 kb) to the significant SNPs affecting Nellore age at first calving [MYOD1 gene is a component of myogenic regulatory factors involved in myoblast differentiation and in concordance with [Other two common_CNVRs were defined by CNVs called in more than 100 individuals. The first region mapped on chr8  and the second one on chr16 . Only this latter region, resulted complex in AZE and KHU and gain in MAZ, harbors genes . For these samples the CNVs defining CNVRs also resulted with a large proportion of complex state  and in mouse [FYN gene, considered by [PCSK6 and SNRPA1, together with PLXNA2, mapping within an AZE gain CNVR, resulted lying within a positive selection signature region identified using SNP as genetic markers in creole breeds [PLXNA2 gene has been also associated with cattle temperament [EPHB3, a gene resulted associated with muscling at weaning (MW) and muscling at yearling (MY) in Bos indicus populations [EPHB3 maps in a selection signature region identified by VST analysis based on CNVs performed through a comparison between Valdostana Red Pied vs Italian Brown Swiss, a double proposal and a dairy cattle breed, respectively [Within the CNVRs here found in gain in state , several annotated genes with a well-known associated phenotype in cattle or in other species were found in addition to those reported in Tables in human , while fn cattle , with san cattle , and witn cattle . For thein mouse , respectdered by  as therme breeds . The PLXperament . For theulations . Also, Eectively .n = 334 recognized IDs) mainly involved general biological processes  and may be occurred because these bovid in recent time have started a selection program for milk yield, that is a primary food source from this species.A total of 384 Iranian Buffalo raw genotyping data (i.e. cel.files) obtained using the Axiom® Buffalo Genotyping Array 90 K  were available for the three breeds , 9 and uwww.affymetrix.com) was performed in order to guarantee a high quality of obtained data. Individuals with a value of call rate less than 97% and Dish Quality Control less than 82% were removed. After quality control a total of 9 low quality samples have been identified and not used.A quality control of raw intensity files using the standard protocol in the Affymetrix Power Tools package (https://www.ncbi.nlm.nih.gov/assembly/GCF_003121395.1).The marker positions of the Genotyping Array 90 K array were recently updated and based on the newly released University of Adelaide water buffalo assembly  genome assembly.A total of 35,114 SNPs was filtered out, in part for their undefined chromosomal locations, and in part because a proportion of SNPs had more than one nearby probeset: for these latter the Axiom Analysis suite picked the best performing probeset per each SNP. A total of 70,230 SNPs was then retained in the analysis mapped on the To confirm the clustering of individuals to the three populations, the genetic diversity within and among breeds was explored using SNP genotypes by Principal Component Analysis (PCA) and by the pairwise Fixation Index obtained according to the pipelines of Golden Helix (SVS) 8.8.4 software .This study did not require approval from the Animal Care and Use Committee as we use already available data obtained in previous researches compliant , 6, 9 wiwww.affimetrix.com). A quality assurance of LRR raw data and filtering of outlier samples was performed before CNV calling with the SVS software through: i) the overall distribution of derivative log ratio spread (DLRS) values as described by [p-value cut off of 0.005.The CNV detection was performed on the 24 autosomes, using the Copy Number Analysis Module (CNAM) of SVS , through the univariate analysis based on LRR values obtained using the Axiom® CNV Summary Tool software  theribed by . A totalCNV regions (CNVRs) at population level were obtained by merging CNVs that overlapped by at least 1 bp in at least two animals using the -mergeBed command of Bedtools . CNVRs whttps://www.ncbi.nlm.nih.gov/genome/browse/#!/proteins/791/374666|Bubalus%20bubalis/). Genes with official “gene name ID” and LOC genes associated with a protein coding gene name (excluding uncharacterized ones) were annotated within the detected CNVRs using the Bedtools “-intersectBed” command. Gene Ontology terms (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the DAVID Bioinformatic Database (https://david.ncifcrf.gov).The complete list of buffalo protein coding genes was downloaded from NCBI online Database  by gene name, using the “Search by associated gene” option of QTLdb.As the Quantitative Trait Loci (QTL) database for the buffalo species is not available, the QTL associated to the genes here found in the CNVRs were identified in the cattle QTL database  using Past software . Sample-Additional file 1: Supplementary Table S1 List of CNVs identified for each Iranian river buffalo breed.Additional file 2: Supplementary Table S2 List of all CNVRs identified for each Iranian river buffalo breed.Additional file 3: Supplementary Table S3 List of 203 non redundant CNVRs. Samples count for each breed, genes and QTL are also reported.Additional file 4: Supplementary Table S4 Gene annotation according to DAVID Database.Additional file 5: Supplementary Figure S1 PCA and FST based on SNP genotypes.


In recent years, consumers have become increasingly aware of the nutritional benefits brought by the regular consumption of fresh fruits and vegetables, which reduces the risk of health problems and disease. High-quality raw materials are essential since minimally processed produce is highly perishable and susceptible to quality deterioration. The cutting, peeling, cleaning and packaging processes as well as the biochemical, sensorial and microbial changes that occur on plant tissue surfaces may accelerate produce deterioration. In this regard, biological contamination can be primary, which occurs when the infectious organisms directly contaminate raw materials, and/or by cross-contamination, which occurs during food preparation processes such as washing. Among the many technologies available to extend the shelf life of fresh-cut products, ozone technology has proven to be a highly effective sterilization technique. In this paper, we examine the main studies that have focused on the effects of gaseous ozone and ozonated water treatments on microbial growth and quality retention of fresh-cut fruit and vegetables. The purpose of this scientific literature review is to broaden our knowledge of eco-friendly technologies, such as ozone technology, which extends the shelf life and maintains the quality of fresh produce without emitting hazardous chemicals that negatively affect plant material and the environment. Fruits and vegetables are essential components of a healthy, balanced diet, as they are important sources of both macro and micronutrients. In fact, regular fruit and vegetable consumption is known to reduce the risk of several degenerative cardiovascular diseases ,2 as theAccording to European Community standards, fresh-cut fruits and vegetables, which are essential to human health and wellbeing, are defined as minimally processed, ready to eat and fresh-cut products, i.e., subject to reduced technology for direct consumption without further handling or with minimal handling .Some examples of ready-to-eat (RTE) fresh-cut produce are broccoli and cauliflower florets, riced cauliflower, cut celery stalks, cut melon, peeled baby carrots, salad mixes, sectioned grapefruit, shredded cabbage, shredded lettuce, sliced bell peppers, sliced mushrooms, sliced pineapple, sliced tomatoes and cut zucchini squash, while cubed butternut squash and cubed sweet potatoes are not ready-to-eat (NRTE) fresh-cut produce .Minimally processed fresh-cut fruits and vegetables are foods that have been washed, peeled and cut into 100% usable products, which are then packaged into retail-size PET, PVC and PP polymers containers with selective gas permeability, sometimes associated with modified atmosphere  11]..11].According to the Produce Marketing Association , the US market for fresh-cut vegetables and fruit is estimated at USD 27 billion per year with bagged salads accounting for 61% of the market, other fresh-cut vegetables 27% and fresh fruit 11%.The global marketing of such minimally processed fruits and vegetables in 2017 was USD 245.97 billion and is projected to reach USD 346.05 billion by 2022 . Fresh-cut produce accounts for 16% of total fruit and vegetables sales. It is difficult to estimate the post-marketing losses of fresh-cut produce; however, given the high perishability of fresh-cut produce compared to intact produce, the total retail loss caused by wastage of fresh-cut produce may exceed USD 9–10 billion.For at least two decades, the European fresh-cut market has shown a clear preference for fresh-cut, packaged lettuce, which represents approximately 50% of the total fresh produce market volume, while only 10% is represented by fresh-cut fruit and the remaining 40% by ready-to-cook and crudités products. More specifically, between 2009 and 2014, a significant increase of approximately 19% was observed in fresh-cut fruit consumption, although it is only a small percentage of the overall fruit and vegetable market (approximately 10%). The countries with the highest fresh-cut produce growth rates are the Western European nations , while fresh produce consumption growth in southeastern European countries is still negligible . In addiSeveral food commodities, including fresh-cut produce, have been associated with foodborne illness outbreaks, and many fresh-cut products are consumed raw. Other foods are cooked and safe to eat because heat destroys pathogenic microorganisms.Preventive controls to significantly minimize the risk or prevent the contamination of fresh-cut produce with pathogens are essential food safety practices . These cw) values above 0.80 and 0.98 and pH values between 3.0 and 7.5, which can be a suitable environment for the development of undesirable microorganisms. Fungi are the main microbial spoilage organisms in fruits and vegetables  and bacteria  [Cut fruit and vegetables are classified as potentially hazardous foods because they have water activity (aia spp.) ,16,17. Hia spp.) .As previously mentioned, the preliminary treatments the raw materials undergo  cause mechanical and physiological damage to the produce leading to a series of chemical and enzymatic stress reactions induced by oxidative processes. As a result of these reactions, undesirable phenomena occur in the exposed wound areas, such as loss of tissue firmness, enzymatic browning and primarily microbial infection facilitated by the initiation of oxidation ,20,21. I2). Chlorine is also applied as hypochlorite (NaOCl), but it has been observed that in the presence of organic matrices, it is likely to form halogenated by-products (trichloromethane) and other by-products that are potentially dangerous to the environment and human health [Cutting and washing operations are the most important steps in the RTE fresh produce preparation process. As already reported, when cutting fresh produce, it is important to limit the effects of wounding stress in order to reduce oxidation phenomena, enzymatic activities and microbial development . Using sn health . For thiOther “eco-friendly” sanitizers for disinfecting cut products and preserving their quality and safety include peroxyacetic acid, hydrogen peroxide, weak organic acid , calcium, sodium and potassium derived salts, plant essential oils , electrolyzed water and ozone ,28,29,302, CO2 and N2, and the concentration depends on various factors, mainly on the type of produce. The different MAP component gas concentrations used for packaging vary according to the physiological properties of the fresh-cut produce , the storage temperature adopted and the gas and water vapor permeability values of the films employed for packaging [While whole fruits and vegetables have a longer shelf life, fresh-cut produce can only be stored at temperatures below 8 °C for a short time (EUR-LEX Document 32007R1580). The commercial shelf life of sliced vegetables is a few days (on average 4–7 days), while for sliced fruit, it is a little longer (7–10 days). The conventional technologies used to preserve fresh-cut products and maintain the stability of the organoleptic properties and product quality are based on refrigeration in cold storage rooms ,35. Howeackaging ,36,38,39Today’s consumers expect healthy produce, ideally free of chemical residues, which has led to the development of innovative technologies, which are mainly used to reduce the microbiological load of minimally processed produce. For example, electron bean irradiation has obtained promising results despite regulatory restrictions , while more recently, ultraviolet C light used in the 200 to 280 nm spectrum and pulsed light (PL) generated by xenon lamps are techniques that have effective germicidal properties, are relatively simple to use and require low-cost equipment ,41,42,43Post-harvest shelf life and microbial contamination control strategies are often based on the use of chemical compounds that are harmful to human health and the environment. Today, it is of fundamental importance to find a viable alternative to traditional technologies in order to ensure safety and quality . Ozone i2) is divided and each of the two oxygen radicals (O) react with another diatomic oxygen to form the triatomic ozone molecule (O3). The rearrangement of atoms to form the ozone molecule requires a large amount of energy that is usually generated by ultraviolet irradiation, electrochemical processes or electrical discharges . The first two methods are seldom used due to very high costs and low ozone yields [The chemical reaction required for ozone formation was described by Rice et al. ; a diatoe yields .In 1997, a group of experts from the US Food and Drug Administration (FDA) assigned ozone generally recognized as safe (GRAS) status . In 2001Salmonella and Norovirus in Leafy greens eaten raw as salads and berries)” suggests using ozone, as well as other sanitizing molecules, for disinfecting leaf vegetables and berries [However, to date, the European Food Safety Authority (EFSA) are still validating the use of ozone in the fresh produce industry. The EFSA publication “Scientific Opinions on the risks posed by pathogens in food of non-animal origin  is very short, usually 30–40 min in water and 2–3 h in air, although these parameters may vary depending on temperature and pH values .2, without leaving residues on the produce. Although it reacts with some organic compounds present in food matrices, the possible by-products are aldehydes, ketones or carboxylic acids, which do not pose a threat to human health. Contrastingly, other chemicals used as sanitizing compounds for washing fruit and vegetables, such as chlorine (and its derivatives), have proven to produce harmful by-products , which trigger cell damage and are strongly associated with the onset of degenerative diseases in humans [The sanitizing and biocidal properties of ozone has attracted the interest of the food and fruit and vegetable sector because the molecule rapidly decomposes into On humans .Ozone treatments can be applied in both gaseous and aqueous phases. Once produced, ozone can be continuously or intermittently added to the environment in which the produce is stored, or it can be dissolved into water to produce aqueous ozone used for washing and sanitizing; however, it is important to note that it is relatively stable in the gaseous state and highly unstable in aqueous solution . The choThe equipment costs for the ozone treatment are higher than the costs for the conventional methods of sanitization or fumigation. However, they are largely compensated for long-term applications of the ozone treatments since electrical energy is the main component of cost basis. The power consumption of the ozone generators varies according to the capacity of the oxygen flow in the concentrator and of the gas flow output (it can vary from a few tens of W to a few tens of KW) and is lower in the case of aqueous ozone generators than in gaseous ones .Scientific studies have widely demonstrated the effectiveness of ozone technology, in particular its efficacy in preserving fruits and vegetables ,53,57,58Ozone’s sanitizing effectiveness can be influenced by various factors as described in the studies published by Aslam et al.  and TzorAlthough studies on the effectiveness of ozone-based treatments have obtained contrasting results, ozone has proven to be an efficient method for fruit and vegetable preservation due to its strong antimicrobial and antioxidant properties. ,61. TherThe papers included in the bibliographical review are the studies on aqueous ozone treatments used for washing minimally processed fruits and vegetables, while few studies deal with the use of gaseous ozone in food processing. In this case, ozone treatments are applied to fresh produce cut before or during the packaging process.Our aim is to discuss the current progress of the research studies on ozone treatments for assuring the quality and microbiological safety of minimally processed fruits and vegetables.For each scientific study, the tables below show the main qualitative and microbiological characteristics of all fresh-cut produce (firstly for vegetables and secondly for fruits) treated with either aqueous  and/or g−1 for 1, 5 and 10 min (cabbage) and 1 and 5 min (apple) stimulated the initial respiratory metabolism of cabbage and reduced ethylene production in fresh-cut apple and cabbage, thus enhancing the overall quality of the produce [3 treatment with 6.34 mg m−3 for 4 h and storing for 12 days at 5 °C in commercial packaging, low respiration (0.61 μLC O2 kg−1 h−1) and ethylene production rates (1.04 μL kg−1 h−1) were observed in minimally processed melon fruits [TM solution at 300 ppm for 4 min). No significant differences between the two treatments regarding the atmosphere created inside the polypropylene packages were found, and the barrier layer did not affect the respiration rate pattern [3 h−1) for 10 min followed by cold storage at 6 °C under controlled atmosphere .A major challenge for the fresh-cut industry is the cutting process that causes mechanical and physiological damage to plant tissues known as “wound injuries”. As reported in previous studies, these injuries occur by inducing a stress response in the vegetal cells involved in this stress and those nearest the wound, with a physiological stress response that causes an increase in ethylene production (stress ethylene) and the rapid deterioration of the produce. Wounding may cause chlorophyll degradation and yellowing of leafy green vegetables and stems. Moreover, wounding caused by the cutting, peeling and trimming processes increases the respiration rate of fruits and vegetables ,106. Wou produce ,94. Folln fruits . The dec pattern . Chauhan pattern  observed3 immediately following treatment , and no surface discoloration was observed during storage at 10 °C for 14 days [−1 ozonated water and stored at 5 °C for 9 days [−1), electrolyzed water and ozonized water (4 mg L−1). They were then stored in polypropylene bags at 5 °C for 12 days, and color was evaluated by comparing the hue angles of the various treatments during storage. The study revealed that long ozone contact times resulted in slightly lower hue angle values than short contact time, which is a positive effect [Color is a factor that significantly influences consumer purchase behavior towards RTE fruit and vegetables. In fact, it is usually visual color evaluation that affects consumer perception of freshness, firmness, taste and sweetness as well as the expected degree of ripeness . Moreove 14 days . Furtherr 9 days . Fresh-ce effect .3 treatment reduced the activities of the cell wall enzymes α-arabinopyranosidase, β-galactopyranosidase and polygalacturonase, which delayed fruit softening and maintained the quality of cantaloupe melon. The main issues encountered in fresh-cut processing are dehydration and tissue shrinkage, which can lead to quality defects such as texture alterations. Texture is a critical quality parameter, and loss of firmness may be due to tissue degradation [The main cause of detrimental changes in the texture of fresh-cut produce is physiological cell wall degradation that occurs during and after ripening, which in cut fruit is accentuated by the wounding stress caused by cutting and by metabolic phenomena triggered by the greater freshly exposed surface of plant tissue. Pectinolytic enzymes are mainly responsible for fruit softening as they act on the lamella mediana component, which rapidly leads to increases in porosity and intercellular spaces. Among these enzymes, pectin methylesterases (PMEs) and polygalactorunases (PGs) are those mainly responsible for the loss of firmness. PMEs and PGs act sequentially: PMEs demethylate pectins, while PGs act on these demethylated polymers to break glucosidic bonds and degrade the cell wall ,110. In radation .−1), organic acids (5 g L−1 citric acid and 5 mL L−1 lactic acid) and ozonated water (4 mg L−1) during storage for 12 days [−1 ozone at 18–20 °C, 15 g L−1 calcium lactate at 50 °C and a combination thereof—which were kept in cold storage for 10 days. It was observed that the ozone treatment exhibited a negative effect on textural properties, since the decrease in texture-related PME activity was correlated with a lower crispness coefficient. It is possible that the controlled activation of PME may improve texture, as it increases cross-linking between pectin chains and cations [−1, respectively, for 1, 3 and 5 min and then stored under refrigerated conditions at 5 °C in polypropylene packages, it was observed that ozone treatments had no negative effect on the firmness of the pepper fruits during storage. These findings are consistent with those obtained by Botondi et al. [No significant changes were observed in the texture and moisture content of fresh-cut “iceberg” lettuce samples washed in chlorinated water  within a few minutes .Maintaining the quality of fruit and vegetables depends on the ability of the plant tissues to preserve their nutritional and safety characteristics for a long time. As previously mentioned, minimally processed produce deteriorates rapidly due to the damage triggered by cutting processes, which cause metabolic stress  leading to reduced shelf life and quality loss. Therefore, any negative impact on nutrient content caused by ozone can be assumed to be limited to surface only .−1), the TSS contents proved to be similar amongst ozone-treated samples and control samples during storage. However, Amaral et al. [−1 of ozonated water were statistically equal with Brix values of 4.25°, while a different value (4.0°Brix) was obtained for the samples treated with ozonated water at 1.6 mg L−1.In general, the total soluble solid (TSS) content of fruit is an important quality parameter, in close parallel with the content of organic acids (titratable acidity). Increased TSS values and reduced titratable acidity during the shelf life of minimally processed products depend mainly on the water loss and hydrolysis of polysaccharides such as starch to form soluble sugars and increased respiration of organic acids . Regardll et al.  noticed When fresh-cut produce is cut and exposed to air oxidization occurs, depending on its structural and molecular composition, since there is a considerable increase in enzyme activity on the surface of fresh produce. As a result, dark pigments generated by the oxidation of polyphenols appear, which are the products of reactions catalyzed by the enzyme phenylalanine ammonium lyase (PAL). PAL activity is closely related to browning. Browning occurs when injured plant tissue (caused by cutting and slicing procedures) comes into contact with oxygen and a key enzyme named polyphenol oxidase (PPO) changes phenolic compounds into quinones through oxidation, which then polymerize to form dark pigments .Another enzyme that acts as a “scavenger” of the free radicals formed in this process is peroxidase (POD). The intensity of browning phenomena is influenced by the level of enzyme activity and by the polyphenol content of the tissue.2, O2− and O3− ions, which leads to increased degradation. Ozone also binds to the double bonds of molecules such as carotenoids, chlorophylls and anthocyanins and oxidizes them, which can result in changes in tissue color [As ozone is a strong oxidizing agent, it tends to react with the components in contact with the cut tissue, thus creating high reactive species referred to ROS such as OH∙, ∙HOue color . This de®-ReduSal, Mantrose-Haeuser Co., Inc., Westport, CT, USA) with or without an additional 2 ppm aqueous ozone dip treatment for 2 min. The results obtained during cold storage at 4 °C for 28 days showed that the ozone-treated samples of fresh-cut potatoes showed reduced enzymatic browning than the samples that had not undergone ozone dip treatment. However, ozone did not appear to have any effect on aerobic plate counts or PPO activity. In contrast to previous studies on potato slices and fresh-cut lettuce, it was observed that ozone has no anti-browning properties. After 14 days of storage at 4 °C under MAP conditions, fresh-cut potato strips dipped in ozonated water or ozone–Tsunami exhibited a significant increase in browning due to prolonged storage [Previous studies have revealed that ozone has an inhibitory effect on the enzymes responsible for browning in fresh-cut celery and lettuce, probably due to ozone’s high oxidation potential ,90,112.  storage . Similar storage .−3 for 15 min and stored under MAP conditions at 5 °C for 21 days) and PPO activity (inhibited by ozone and MAP treatment). These authors concluded that both ozone and MAP can enhance the effectiveness of the antioxidant defense system. The same trend was observed in fresh-cut carrots treated with ozone and stored in controlled atmosphere for 30 days. Reductions in ascorbic acid, carotenoids and oxidative enzymes such as PPO and POD were observed in the samples. In this case, the reduction in POD activity by ozonation implies detoxification, which may be linked to the surface decontamination process, and the reduction in PPO activity could be associated with lower respiration rates detected in the experiment [Antioxidants are important compounds commonly found in fruit and vegetables. These molecules play an important role in defending plant tissue against oxidative stress by binding to free radicals and preventing damage. Antioxidant defense systems generally include both enzymatic and non-enzymatic systems. The main non-enzymatic antioxidants include ascorbic acid, polyphenols, carotenoids and anthocyanins, while enzymatic antioxidants include superoxide dismutase (SOD), peroxidases  and catalase. All of these antioxidants enable plant cells to scavenge ROS and help to prevent cellular damage . In thisperiment .Ozone can increase plant stress tolerance by stimulating the ROS scavenging system in cells, which enhances the synthesis of the antioxidant enzymes. It is assumed that ozone induces loss of antioxidant compounds due to its strong oxidizing ability. However, ozone oxidative stress may induce defense mechanisms in plant tissue. The sensitivity of fresh produce to ozone varies according to the type and species of vegetable crop .−1). The suggested mechanism of action indicates that ascorbic acid can act either directly by oxidizing molecules or through the formation of free radical intermediates [Ascorbic acid and phenolic compounds are undoubtedly the most important antioxidants found in fresh-cut fruit and vegetables. Numerous studies have reported that the total phenolic and ascorbic acid content during storage of different minimally processed produce treated with different concentrations of gaseous ozone and ozonated water tend to decrease compared to their respective controls ,99 and 2 and 2−1.mediates .−1 of minimally processed bell pepper for 14 days significantly reduced the microbial load and showed better retention of ascorbic acid, firmness and color. Similar trends were observed for fresh-cut pineapple fruits treated with aqueous ozone at 0.6, 0.9 and 1.5 ppm and stored for 20 days at 2 °C [However, ozone can also have a positive effect on ascorbic acid levels, which can be explained by preserving its content caused by stimulating the natural plant defense mechanisms through the stress generated by the oxidizing molecule and by increased synthesis and accumulation of antioxidant compounds such as ascorbic acid in the cells. Ummat et al.  proved t at 2 °C  and for  at 2 °C  who note at 2 °C  and Hass at 2 °C  in fresh−1 for 10, 20 and 30 min to investigate the effect of ozone on antioxidants following a 20 min ozone treatment showed a 10.3% increase in total phenolic content and a 2.3% decrease in ascorbic acid content. Contrastingly, a 30 min ozone treatment caused a decrease in total phenol content [−1 and with NaOCl (100 mg L−1) treatment and then stored for 21 days at 5 °C, total polyphenol content proved to be 23–50% higher in Thompson Seedless and 18.5–28% higher in Black seedless grapes washed in ozonated water  compared to the NaOCl samples [−1 [A study on fresh-cut papaya treated with ozone at 9.2 μL L content . The inc content  who foun content  showed t content  obtained samples . However samples , lettuce samples , apple [ samples , spinach samples  and parsples [−1 .Consumer acceptability depends on several critical quality parameters described by four different sensorial attributes: attractive color (appearance), acceptable aroma and taste (flavor), appropriate texture . ExternaSeveral studies demonstrated that ozone treatment is an alternative method to extend the shelf life of fruit and vegetables, maintaining the sensory quality during storage. Olmez and Akbas  evaluate−1 min and 10 mg L−1 min activated by ultraviolet C) on fresh-cut lettuce showed that ozone samples maintained an excellent visual quality, including the appearance features of gloss, freshness and color uniformity and intensity for up to 13 days. At the end of the storage, the fresh-cut produce provided a moderate crispy texture and a typical aroma [Another study on the effects of different concentrations of ozone water treatment . Erwinia, Pseudomonas, Xanthomonas and Pectobacterium genera are the main spoilage microorganisms that cause qualitative alterations and off-odors, by altering the chemical composition and physical structure of plant products [E. coli, Salmonella spp., Yersinia enterocolytic, Shigella spp., L. monocytogenes, Aeromonas hydrophila and Pseudomonas aeruginosa [Norovirus and Hepatitis A Virus (HAV), Rotavirus and Astrovirus [Fresh-cut products generally have aproducts . The mairuginosa . Moreovetrovirus Microbiological limits are regulated by ISO standards and the normative reference in Europe is the Regulation (CE) 2073/2005 of the European Commission, issued on 15 November 2005 related to microbiological criteria applicable to food products.Fresh-cut processing of fruit and vegetables can induce physiological, biochemical and morphological alterations in plant tissues, especially during cutting operations, which damage the plant tissue and promote the entry of microorganisms, thus increasing the risk of microbial contamination. Moreover, cutting the fruits allows the leakage of plant cellular fluids that provide a nutrient medium for bacterial survival and growth .L. monocytogenes [Leuconostoc spp. against Listeria spp. [The shelf life of fresh-cut fruit and vegetables is quite short, approximately 4–10 days, which depends mainly on the type of produce. There are several techniques that can effectively control the development of microorganisms in minimally processed fruit and vegetable produce: refrigerated storage; acidic solutions that are mainly used to control the growth of ytogenes ; antimicytogenes ; bio preria spp. ,131.Ozone technology is an innovative sanitation technique used for the decontamination of fresh-cut produce due to its antimicrobial action. Ozone effectively controls microbial growth due to its high reactivity and strong oxidizing potential and is a more efficient biocide than other chemical substances used . SeveralMicro-nano-bubbles (MNBs) are an innovative method for prolonging the reactivity of aqueous-phase ozone and enable us to reduce the growth of bacteria, total yeasts and molds in food and food processing environments with the aim of maintaining freshness and quality and extending the shelf life of fresh-cut produce .The efficiency of aqueous ozone depends on the different ozone concentrations, temperature and mainly the contact time between the ozonated water and the produce during the washing process.Lactuca sativa) and bell peppers (Capsicum annuum) in pre-saturated ozonated water (0.5 mg L−1) resulted in a lower microbial reduction compared to immersion in continuously ozonated water (0.5 mg L−1), which leads to a reduction of 2 log after 15 min and 3.5 log after 30 min of exposure [Immersing fresh-cut lettuce (exposure .Brassica oleraceae L.) treated with aqueous ozone (2.5 mg h−1) for 5, 10 and 15 min. The most effective ozone concentrations used were 0.56, 1.00 and 1.50 ppm. The results showed reductions in total coliform bacteria, molds and yeasts of 1.20, 2.50 and 1.80 log10 CFUg1, respectively, compared to the control treated with pure water. Several studies have reported the influence of ozonated water (1.4 mg L−1) for different treatment times on fresh-cut apple slices (5 and 10 min) [Renumarn et al.  analyzed 10 min) , onion ( 10 min)  and cabb 10 min) . All ozoE. coli O157:H7 counts by 1.78 log10 CFUg1, compared to the control sample, and inhibits the growth of bacteria, yeast and mold after 12 days of storage.The results of the study conducted by Nie et al.  showed t−1 for 30 s, did not reduce the bacterial count of Escherichia coli after 5 days of storage at 5 °C. Bacterial colonies were counted after 0, 3 and 5 days, and the average logarithmic reduction in microbial load was approximately 0.8 log CFUg−1 [However, ozone combined with other antimicrobial treatments did not always obtain positive results; for example, fresh-cut lettuce treated with 1% lactic acid for 90 s, followed by aqueous ozone treatment of 1 mg Lg CFUg−1 .−1 ozonated water was tested at two different temperatures, 4 and 15 °C. The treatment carried out at 4 °C significantly reduced the microbial load of Salmonella enterica serovar Typhimurium and Escherichia coli inoculated onto the surface of the lettuce leaves without altering their visual and sensory properties [Several studies have demonstrated that treatment temperature is another important parameter to consider. Fresh-cut lettuce treated with 2 mg Loperties .−1) for 1, 3 and 5 min, which were then stored for 14 days and destined for the fresh-cut industry. The 3 min contact time proved to be the most effective for reducing total bacterial count values: the mesophilic, psychrotrophic and yeasts’ load was reduced by at least 1CFU g−1, compared to the control sample. In this case, mold growth remained the same showing values of approximately 2.1CFU g−1 for all samples. Long treatment times do not improve microbiological safety, probably due to the presence of organic compounds with high ozone demand on fruits and vegetables surface that may inactivate the ozone before it can reach microorganisms [−1) for 1, 2 and 3 min, Listeria innocua in fresh-cut peppers, total mesophilic bacteria in fresh-cut strawberries and total coliforms in fresh-cut watercress were tested, resulting in microbial load reduction of 2.8, 2.3 and 1.7 log CFUg−1, respectively, compared to the control. A 1.7 microbial log reduction was also observed in fresh-cut celery, treated with ozonated water at 0.18 mg L−1 by Zhang et al. [In another study, low temperatures (5 °C) were used to wash samples of tomato slices with ozonated water  than 10 and 30 min treatments [Gaseous ozone treatments require longer contact time than ozonated water treatments; in a study conducted on fresh-cut papaya, gaseous ozone treatment for 20 min proved to be more effective for reducing total coliform count , respectively.Selma et al.  reported−1 for 30 min every 3 h was applied to fresh-cut “Thomas” tomatoes, stored at 5 °C for 15 days. Gaseous ozone was more effective at reducing bacterial load (from 1.1 to 1.2 log10 units) than fungal count (0.5 log10 units). A greater level of ozone antimicrobial effectiveness was observed when produce was exposed to a higher ozone concentration (7 μL L−1), which extended produce shelf life, and the organoleptic and biochemical characteristics of the produce remained unchanged [In another experiment, a flow of ozone-enriched air at 4 ± 0.5 μL Lnchanged .Gaseous ozone treatments on food products can be combined with other sanitizing technologies to enhance disinfection potential. Ozone treatments combined with UV-C radiation were applied to fresh-cut rocket for 12 days at 5 °C in order to monitor qualitative, sensory and microbiological parameters. The synergistic antimicrobial activity of these technologies does not affect the sensory quality of the produce, which remained unchanged throughout the storage period; however, it induced a reduction in microbial load from storage day 8, compared to gaseous ozone and chlorine treatments .There is an increased demand for RTE products, particularly fresh-cut fruits and vegetables, especially in the most industrialized economies, which is mainly due to the availability of technologies that preserve the quality and safety of fresh produce and ensure maximum shelf life.Several studies have investigated the sanitation potential of ozone for processing fresh-cut produce with the aim of preserving the nutritional and sensory quality characteristics and obtaining microbiological control to stop or slow down food spoilage. Sanitizing treatments reassure consumers that the treated produce is hygienically clean and safe.Although in some studies the results were conflicting, in accordance with several papers included in this review, ozone treatments applied at appropriate concentrations and contact times (in aqueous and/or gaseous form and/or in combination with other compounds/technologies), adjusted according to the different RTE produce and combined with good agricultural practices, GMP and HACCP methods appear to provide promising qualitative and biological results.


Background: Occupational contact with dust particles is a well-known phenomenon, particularly in developing countries of the world. Crystalline silica present in marble dust is the main etiology of a rising prevalence of obstructive lung diseases in marble stone workers, who are in direct contact with marble dust in the surrounding environment during their regular work.  The purpose of this study was to compare the pulmonary function parameters of workers in marble workshops and age matched healthy individuals in the Lahore District of Pakistan.Methods: The study included 164 male individuals, 82 individuals working in marble workshops and 82 healthy individuals from the same community. Data were collected through in-person interviews using a structured questionnaire after obtaining written consent. A Spiro Lab spirometry for pulmonary function tests was used to identify any change in the lung function parameters. FVC% , FEV 1 (forced expiratory volume in first second) and FEV1 / FVC ratio were evaluated.Results: Mean age in the exposed group (marble workers) and non-exposed group  were 29.92 ± 6.19 and 30.58 ± 6.37 years, respectively. The mean years of work experience of the exposed group was 11.92 ± 5.67 years. A statistically insignificant difference was observed between marble exposed workers & healthy individuals from the demographic variables. Lung function parameters in marble workers exhibited a highly significant (P < 0.001) decrease in FVC%, FEV1 & FEV1 / FVC ratio when compared to healthy individuals. Seventy-one percent of marble workers had abnormal pulmonary parameters whereas 34% of workers had restrictive pulmonary impairment. Marble workers who had worked for more than 15 years had a highly significant risk of developing abnormal pulmonary function (P < 0.001).Conclusion: Continuous exposure to marble dust deteriorates the lung function of marble workers. Marble dust mainly contains free silica, which means “free of elements” as it is not combined with other elements or silicon dioxide (SiO2). Silica is a common ingredient of the Earth’s crust and can be found in alpha quartz, beta quartz, moganite, granite, slate, sandstone, and keatite, and it is toxic to the human respiratory system. Occupational exposure to dust containing crystalline silica occurs in the stone, granite, construction, mining, metal foundry, ceramic production and glass industries. Airborne dust (free silica) is produced in marble workshops during quarrying, grinding, mining, cutting, and polishing activities and is the main causative factor for many occupational lung diseases such as pneumoconiosis, chronic obstructive pulmonary disease (COPD), silicosis and asthma.Toxic elements, fumes, and respirable dust produced in marble workshops can cause a health-related risk to the workers in different units of marble workshops. Marble is a semi-translucent very fine to coarse grained crystalline rock. It mainly contains calcite, serpentine and dolomite and is mostly used in making monuments, headstones, and floors. Finely ground marble is used as a whitening agent in toothpastes, paint, and paper. Deposited crystalline silica particles cause respiratory mucosal irritation, mucosal hypersecretion in the large airways, mucosal gland hypertrophy of trachea and bronchi, an increase in the number of goblet cells in small airways, and excessive mucus formation, which results in the formation of a mucous plug in the lumen, and fibrosis of small airways. Obstruction in the air flow in airways results in decreased FVC , FEV1 (forced expiratory volume in first second) and FEV1/FVC ratio airways. The results of recent research showed a significant association between pulmonary problems and inhalation of silica dust in Bangladesh. Previously published data from Australia demonstrated that stone workers working in factories that perform cutting and grinding activities produce higher contents of respirable silica, which is associated with a higher rate of severe silicosis. A previous cross-sectional study among Indian stone-crush workers reported a noteworthy decrease in FEV1, FVC and FEV1/FVC parameters. Several previous studies conducted in Austria, Nigeria, and Lebanon demonstrated that longer exposure to occupational dust (silica) as well as smoking leads to a gradual decrease in Pulmonary Function Tests (PFTs).Continuous inhalation of respirable silica causes many diseases including silicosis characterized by inflammation and pulmonary fibrosis. Silica dust particles enter the alveoli, increase the production of inflammatory mediators in the peripheral airway, and cause emphysema. The particles are mainly deposited on airway surfaces where air flow changes direction. Silica particles having a size 0.2 to 2 micrometers get deposited on the walls of the airway and particles less than 0.2 micrometers enter the terminal respiratory epithelial surfaces and finally diffuse into alveolar gas. and over ten thousand silicosis associated deaths were reported in South African miners. Despite better dust control safety measures and advancements in developed countries, there is a constant need to control dust due to a recent pneumoconiosis outbreak in Australia and the USA.In low and middle economic countries, silica associated lung illness remains a major health threat. In China more than half a million silicosis cases were documented between 1991 and 1995 Sharmaet al. reported that silica is a key factor for other autoimmune diseases such assystemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, Caplan syndrome, and Erasmus syndrome, which derives from silica exposure and cytoplasmic antibody related vasculitis.Risk of developing lung diseases among silica exposed workers was higher with prolonged exposure to respirable silica dust. The incidence of silica associated silicosis was 12% among the workers who worked for 30 years or more.Marble workers are at a greater risk because of illiteracy, low socioeconomic status, poor knowledge of personal protective measures (PPM), lack of safety rules and their enforcement, and exploitation by employers. Thousands of individuals work in small to medium sized marble workshops in a poorly ventilated hazardous environment. The marble workers working in marble workshops are among the most neglected and work in poorly organized areas. The present study measured the impact of silica dust exposure on pulmonary function tests of these workers. The objective of the current study was to compare pulmonary function parameters among the workers of Lahore based marble workshops and age matched healthy individuals.In developing countries such as Pakistan, plenty of evolutionary projects such as roads, flyovers, underpasses, housing schemes, hospitals etc. are commissioned by the local government and federal agencies. These projects demand people working in the marble sector. Vast research has been done in the stone cutting sector globally but data on pulmonary function parameters for marble industry workers is scarce.The study was carried out in the Ichra and Township areas of the Lahore District, which are heavily populated commercial areas having the majority of small to medium sized marble workshops. The study unit consisted of marble workers working in wet cutting and dry cutting (grinding) units of the marble workshops. The study group (exposed) comprised male marble workers, aged 18 to 40 years inclusive exposed to marble dust for more than one year, and working 30 hours or more per week. Adult male workers with a diagnosed respiratory system disease , those with thoracic deformity, those who underwent chest surgery, those who had history of pneumothorax, hemoptysis, recent abdominal or eye surgery, known respiratory malignancy, unstable cardiovascular status , and those who were unfit for spirometry were excluded.Normal healthy male individuals from the same community residing near each study site having similar socio demographic status but free from respiratory problems were selected as the control (non-exposed) group.The sample size was calculated with the WHO sample size calculator using the formula:1-β = 90% power of study, population variance (σ2) = 0.5329, anticipated mean for study group I , anticipated mean for study group II , the calculated sample size (n) for one group was 82. The total required sample size for both groups was 164.Assuming 90% power of study, 5% significance level, ZA total of 164 subjects were selected, 82 marble workers (study exposed group) by simple random sampling (lottery method) and 82 healthy individuals (study control group) were selected by convenience sampling. and the pulmonary function tests of each subject were addressed as outlined in the Medical Research Council Questionnaire (https://mrc.ukri.org/documents/pdf/questionnaire-on-respiratory-symptoms-1986/). Some items of ATS proforma, like some symptoms and family history, were not included in the questionnaire as these did not relate to our study objectives. The first section of the functional proforma captured information on gender, area of residence, date of birth, marital & educational status. While the second section of the questionnaire documented information regarding occupational history, nature of work, working hours, and total duration of work in years. The height in centimeters and weight in kilograms were measured for all study participants. Spirometry was performed by using a Spirolab spirometer . Spirolab spirometry was used throughout the study period for both study groups for PFTs. Spirometry pre-requisites  were followed in accordance with the standard protocols and guidelines by ATS. The purpose of spirometry and the procedure were explained to each participant. The spirometry values obtained were reported with correction for body temperature at ambient pressure, saturated with water vapor (BTPS). Lung volumes (FVC and FEV1) were measured in a standing position from a sequence of a minimum of three readings, having adequate start. After probing the data from whole usable curves, the highest FVC & FEV1 were noted even though they didn’t belong to the same curve. The FEV1/FVC was also calculated from the same tracing.The study was approved by the Ethical Committee of the Institute of Public Health (IPH), Lahore, vide letter number 478, dated July 23, 2018. The data was collected from February to April 2019. The data collection techniques  were similar for both study groups . The study procedures were explained to every study subject and a written consent was obtained from each individual who agreed to participate in the study. All participating subjects were interviewed in-person using functional proforma developed by the American Thoracic Society (ATS):•FEV < 80% - predicted normal•1 < 80% - predicted normalFEV•1/FVC ratio < 0.7FEVSpirometry designates the presence of pulmonary impairment, if any of the following recordings were observedet al.’s following formula:The percentages of the predicted values were calculated by BellamyFVC  × 100% = %age of predicted value1 Reading/ × 100% = %age of predicted valueFEV1/FVC Reading/Reading %FEV:•1 more than 80%Mild airflow obstruction - FEV•1 between 50–80%Moderate airflow obstruction - FEV•1 between 30–49%Severe airflow obstruction - FEV•1 <30% predictedVery Severe airflow obstruction - FEVThe airflow obstruction can be defined as follows according to the NICE COPD guideline Narrowing of airways is the main reason of obstructive type impairment and is typically found in asthma and COPD (http://www.irishthoracicsociety.com). Restrictive Type Impairment: The lung capacity is lower than the predicted value for age, sex & size. In this type of impairment, the capacity of lung volume is lower because the lungs are firm & less compliant. There is a decrease in the level of lung parenchymal mobility or chest wall. Pneumoconiosis and pulmonary fibrosis are examples of restrictive types of impairment, which causes scarring (fibrosis) of the lungs. Combined Restrictive/Obstructive: In combined type of impairment, all the values of FVC, FEV1, & FEV1/FVC are decreased. It may be associated with two conditions i.e. asthma and another lung pathology or some pulmonary conditions (http://www.irishthoracicsociety.com).Obstructive Type Impairment: is characterized by a complete or partial narrowing of airways at any point, which results in an increased resistance and air flow.SPSS version 24 and organized in tabulated form according to the frequency distribution table. The continuous variables such as age, duration of dust exposure, pulmonary function values were summarized in mean, median, standard deviation (SD), and categorical variables like nature of work were depicted as frequencies and percentages. The SD was calculated to understand the variation in the study data. The student t-test was employed to compare mean pulmonary function indicators  between marble workshop workers and healthy community controls. The ANOVA was applied to compare pulmonary function indicators  by years of exposure; Tukey’s method was employed for post-hoc pairwise comparisons. ANCOVA was employed to control and test for confounding due to smoking on pulmonary function indicators. Chi-square test was used to test the difference in the proportion of smokers and pulmonary impairment between marble workshop workers and healthy community controls. The p-value of 0.05 or less was taken as statistically significant for all analyses.The data were analyzed inThe current study showed that the average age, height, and weight of the study exposed group (participants exposed to marble dust) were 29.92 ± 6.19 years, 172.84 ± 10.2 cm and 74.39 ± 13.77 kg respectively and for the control group (non-exposed group) were 30.58 ± 6.37 years, 170.45 ± 9.56 cm and 74.89 ± 12.54 kg respectively. There was no statistically significant difference in the sociodemographic characteristics among marble dust exposed workers and the control group (p > 0.05). About 89% of the exposed group had education levels up to matriculation or below, as compared to 13.4% of the non-exposed group . In the study exposed group, 48% of the subjects were married compared to 39% of participants among the non-exposed group (p = 0.353). Among the exposed group, 19.5% were smokers compared to 4.9% of non-exposed , and this was statistically significant (p = 0.007) .About 71% of workers from the exposed group had statistically significant (p < 0.001) abnormal spirometry results whereas none of the healthy individuals (non-exposed) had abnormal spirometry results. The vast majority of lung impairments were of the restrictive type 40.25%; including mild (34.15%) and moderate (6.1%) restriction respectively followed by obstructive type 25.62% of impairment; which includes mild obstruction (10.98%), moderate obstruction (7.32%), moderate-severe obstruction (6.1%), and very severe obstruction (1.22%) respectively, while combined type of impairment was 4.88%.1% and FEV1/FVC ratio of non-exposed  were 95.68 ± 10.66, 92.85 ± 9.78, and 80.73 ± 4.71 respectively and in the marble dust exposed group were 85.36 ± 14.80, 75.93 ± 15.65 and 74.34 ± 12.68, respectively   .Comparison of pulmonary function parameters (mean ± SD) in the study population showed the average duration of work in marble workshop workers was 11.2 ± 5.677 years, and 51.2% had been working for more than 10 years .1% and FEV1/FVC ratio) among marble workers by years of exposure showed a statistically significant reduction with increase in duration of work (p < 0.001). The maximum reduction in pulmonary function indices was observed among marble workers who had been working for 16–20 years . Compared to participants with 1–5 years’ work exposure, FEV1/FVC ratio was significantly less among workers with 16–20 years’ work exposure. No other significant differences between groups were observed.et al. concluded in his study that airborne crystalline silica significantly reduced the FVC and FEV1 values in workers who were engaged in marble factories when they were exposed to marble dust as an air pollutant. These particles cause respiratory irritation mechanically and release of histamine mediators, which leads to obstruction in the airway.PFTs are the main tool to help identify pulmonary abnormalities. PFTs also provide information about airways, pulmonary bed integrity, and parenchymal size. Sharma1% and FEV1/FVC ratio) of marble dust exposed workers as compared to the non-exposed  group. These results were also in agreement with data reported by Ophiret al. in his recent study, who found that ultrafine silica particles are strongly associated with and responsible for the deterioration of pulmonary function parameters among stone factory workers. In another study, Ullahet al. reported that exposure to high concentration silica particles by stone crushing workers significantly affects their lungs. Researchers also observed that carbon containing particles have a contrary effect on the dysfunction of pulmonary functions in addition to silica particles.The current study showed a significant reduction (p < 0.001) in mean values of PFT indices  were statistically significant (p < 0.01) and had similar differences between the control group and marble workers to our study. Furthermore, many previous studies in India, Thailand, Iran, Egypt, Nigeria, and Libya reported similar findings and mean values of pulmonary function parameters were found to be remarkably deteriorated resulting in pulmonary dysfunction.Ahmed The change in the elasticity and viscosity of mucous influences its clearance and results in a luminal mucous plug, which is responsible for obstruction to the air flow resulting in decrease in FVC, FEV1 and FEV1/FVC ratio.The inhaled dust particles get deposited in lungs, cause irritation, and cause an inflammatory reaction leading to fibrosis, faulty oxygen diffusion and abnormal lung functions.1 and FVC are indications of obstructive and restrictive types of lung changes respectively. The deviations of pulmonary function indicators  are the major characteristic features of restrictive lung disease. In our study, 71% of marble workers had abnormal PFTs while 29% had normal PFT values. Among abnormal PFTs, 40.25% had the highest proportion of pulmonary impairment labelled as restrictive lung pathology, which was followed by 25.62% obstructive type, while 4.88% of cases demonstrated a mixed type of impairment. Our current study findings are quite similar to past studies where researchers reported that 20.1% and 24.3% of lung impairments were of restricted type and 1.5% and 5.8% were of obstructive type. Similar results (restriction 23.3% and obstruction 6.2%) were also reported in a previous study among stone crushing industrial workers. In contrast to our study, notably less restrictive (2.5%) and obstructive (6.7%) types of pulmonary impairment were reported in a recent study on stone crushing workers. The major causes of restrictive type impairment are chest wall limitations, neuromuscular disorder, fibrosis of lung parenchyma, and pleural disorder, whereas obstructive type of pulmonary impairment is commonly seen in chronic bronchitis, asthma and emphysema.Reduced values of FEV Previously published data showed that the anthropometric parameters have a strong association with lung function parameters. The present study showed an equal distribution of age group among marble dust exposed workers and non-exposed healthy individuals, both groups were insignificant (p < 0.491). Similar studies on marble stone workers found no statistically significant (p > 0.05) differences in the ages of marble workers and control groups.Age of the study subject could be considered an important causal factor for variation in lung functions and it accounts for a 1.41 fold higher incidence rate using a fixed ratio of variation of lung function parameters. Shaiket al., and Dostbilet al. for case control studies among marble stone workers.In our study, the results of sociodemographic characteristics showed no statistical difference in age (p < 0.491), weight (p < 0.808) and height (p < 0.1274). Our findings for the mean age, height, and weight are similar to previously reported data by Vyas, although other authors, Rathodet al., Sheikhet al., in India, Ullahet al., in Pakistan, and Jaberet al., in Cairo reported a significant association (p < 0.05) of smoking on pulmonary function impairment, which contradicts our study. In our study the possible reason of these non-significant effects could be due to shorter smoking duration, the low intensity (only four packs per year) of smoking in the study population, a fewer number of study participants, and less inhalation during smoking.In the current study, only 20 (12.2%) of the total study participants were smokers, while the proportion of marble workers who smoked were 16 (19.50%) only, compared to four healthy individuals (4.90%). The smoking effect on all PFT parameters was statistically not significant (p > 0.05). Similar findings were reported in a previous study, which showed that smoking did not (p < 0.98) affect PFTs1% and FEV1/FVC ratio, showed significant reduction with increase in duration of work (p < 0.001).The mean years of work experience of our participants (marble workers) was 11.20 ± 5.67 years, which was statistically highly significant (p < 0.001). The majority of the marble workers  were working for a period of more than 16 years and their PFT values markedly decreased due to the high duration of exposure (p < 0.001). The maximum reduction in pulmonary function indices were noted among the marble workers who had been working for 16–20 years. All three pulmonary functions parameters, FVC%, FEV He claimed that the duration of exposure to silica is most important and documented predictor of decreased pulmonary functions in the workers exposed to silica dust.Our study results are analogues to a previous study in which the study author reported a statistically highly significant (p < 0.001) reduction in PFT values. The researchers also reported that the deterioration of PFTs is strongly associated with increased duration of exposure to silica.Our study results are parallel to an earlier study in which the researchers documented a statistically highly significant (p < 0.05) reduction in pulmonary function parameters with longer duration of work exposure in marble factories.1 (p < 0.05), and lower lung function values were reported. In concurrence to our study, researchers  also reported a significant reduction in FVC, FEV1 and FEV1/FVC ratio (p < 0.001) in quarry and granite workers having greater than 10 years of silica dust exposure.Similar types of results were also reported among quarry workers in Nigeria where the mean values of PFTs, the negative correlation between longer work exposure and FEVThere is a possibility of confounding due to age and smoking. Age matched participants from the community (control group) were selected, hence confounding due to age is unlikely. No differences in the pulmonary function test values between smokers and non-smokers were observed. Also, on ANCOVA analysis, smoking was not statistically significant, therefore, confounding due to smoking is also unlikely.Pulmonary function parameters were found to be significantly decreased among marble workers who have marble dust exposure compared to healthy individuals from the same community. The current study disclosed that the continuous occupation of the marble industry for more than 15 years resulted in further reduction of workers’ pulmonary function parameters, which ultimately would have harmful effects on their health status.Exposure to marble dust is not the only reason for pulmonary function deterioration. There could be other possible factors such as toxic materials in the work environment as well as undetectable concentrations of dust particles other than silica. Dust exposed particle sampling was not used to quantify its composition and concentration, and a comparison to silica dust was not made in both study groups. In addition to this, further respiratory procedures like chest radiographs should be carried out for comparison with previous literature in order to verify findings.https://figshare.com/s/7a24edab6fc0d64c526c.Figshare: Underlying data for ‘Pulmonary function parameters among marble industry workers in Lahore, Pakistan’,•This project contains the following underlying data:Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of theWritten informed consent was taken from each study participant, the informed consent form was in Urdu, the national language. Under the confidentiality section of the informed consent, it was clearly mentioned that confidentiality will be strictly maintained at all levels, and in reporting the results, no personal information will be shared, and data will be reported in aggregate form. Specific permission was obtained for the publication of participants' data. COMMENTS It is one of the best efforts made by the authors to conduct such a study on workers of the marble industry. As reported by the authors, the study setting where this study was carried out is thickly populated, located in a residential area, hence named cottage industry where marble crushing/cutting activities have been going on in houses since long ago. Being located in a thickly populated place, lots of people are directly and indirectly exposed to silica dust. It would have been a remarkable effort if particulate matter (PM), both PM 2.5 and PM 10 were also measured as mentioned in observation 3. Secondly, as silica particles (being heavy in weight) are suspended in the atmosphere for a short distance around these plants, it would have been a great effort if a pulmonary function test was performed in people living at least 50 meters from the marble industry as noted in observation 1. Respiratory symptoms in these people should also be recorded. In addition to that, one of the limiting factors of this study was its short duration. It would have been better if this study was carried out in a larger time period, 6 months or one year, as the weather conditions can affect the outcome of the result. It is usually seen that in hot weather, workers try to stay and work at the point where some fans are plying with the result that dust spread is more in such cases and chances of getting affected is more than in winter season. Technology being used in the marble industry is also very important. In those places where wet technology is used for cutting marbles, the spread of dust particles is far less than in those places where marble is cut in dry conditions.2, etc, are emitted from other sources, like vehicles, burning of waste, etc., which is very common practice, especially in an industrial area. These pollutants might have augmented bad effects on the lungs of workers of the marble industry and people residing nearby and so should have been measured. In addition to that, few workers were smokers as the result of this study reported, but also many people might have been addicted to other substances prepared locally like opium, niswar, battery, etc., which can also enhance the respiratory symptoms and decrease lung function test. These workers should have been included in the study. Regarding confounding factors like the presence of pollutants around the marble industry, it is pertinent to mention that lots of pollutants, like NOX, SOX, CO Looking at the prevailing situation in the industrial area in general and study setting in particular, it is the need of an hour that Environmental Monitoring, namely Environmental Impact Assessment (EIA) for larger marble cutting units and Environmental Examination (EE) for small industries, cottage units by the Environmental Protection Department (EPD) should be done. Although these measures have been in place but unfortunately not strictly followed.           COMMENTS ABOUT QUALITY OF PAPERAre sufficient details of methods and analysis provided to allow replication by others? Methods of data collection have been given in detail and were quite comprehensive. A list of inclusiveness and exclusiveness is also complete. One can say that this has increased the understanding of the matter even for a layman. Another good thing is the comprehensiveness of data analysis. So, I will say that sufficient details of methods and analysis have been provided and it has added the knowledge of the reader significantly. It is also a very good source of reference for other studies.The status you would give this article: I can say with confidence thatthe article is scientifically valid in its current form. The experimental design, including controls and methods, is adequate; results are presented accurately and the conclusions are justified and supported by the data. Revisions/modifications suggested are minor in nature, and are considered to be non-essential.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Public Health, Environmental and Occupational HealthI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The study "Pulmonary function parameters among marble industry workers in Lahore, Pakistan" is a methodologically sound case-control study. The researchers have taken all the necessary steps to ensure adherence to the application of basic protocols of research. The study finds the negative impact of the long duration of silica dust exposure on the lungs of the marble industry workers. Findings are in line with similar studies conducted around the world on occupational exposure to dust, particularly stone or marble dust. However, a brief justification about the selection of the research site, the absence of female participants in the study, and the incentive provided to the participants, if any, will strengthen the generalizability of the study further.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Respiratory diseases, mental health, and health inequitiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.