Several samples proved to have metrologically different microplasticcontamination. This work represents a contribution to the objectivityof the assessment of environmental contamination with microplastics.The quantification and comparison of microplastic contaminationof sediments are affected by sample heterogeneity and the systematicand random effects affecting sample analysis. The quantification andcombination of these components in the measurement uncertainty allowsthe objective interpretation of analysis results. This work presentsthe first detailed evaluation of the uncertainty of microplastic contaminationquantification in sediments. The random and systematic effects affectingmicroplastic counts are modeled by the Poisson-lognormal distributionwith inputs estimated from duplicate sediment analysis and the analysisof sediments spiked with microparticles. The uncertainty from particlecounting was combined with the uncertainty from the determinationof the dry mass of the analytical portion by the Monte Carlo method.The developed methodology was implemented in a user-friendly spreadsheetmade available as the Supporting Information. The contamination ofsediment samples collected in various inland Portuguese waters wasdetermined, ranging from  to  kg This work describes and applies a novelapproach for thebottom-up evaluation of the uncertainty of microplastic contaminationquantification in sediments where random and systematic componentsare quantified and combined. Riverinewaters and sediments are a major pathway for plastic litter towardoceans.The presence of largeamounts of plastics in the aquatic environmentis well recognized and widely debated by the scientific community,governmental bodies, and the population.3 Microplastic is mistaken for food by smaller organisms being a vehiclefor accumulating several contaminants in the food chain.4 According to their shape, microplastics can becharacterized as fragments, pellets, fibers, or filaments.4 The color of particles can also be used to guesstheir origin since transparent particles are frequently produced fromdisposable bottles and cups. The micro- and nano-particles can bemanufactured with that size or result from the fragmentation of largerparticles by environment agents being designated primary or secondarymicroplastics, respectively.Plastic contamination affects the ecosystems differently,dependingon their size and shape. Macro-, nano-, and micro-plastics are plasticparticles larger than 5 mm, smaller than 1 μm, and with a sizebetween these two limits, respectively.5 Typically,the studies on the presence of microplastics on a specific matrixinvolve the characterization of these particles regarding their chemical and physical properties. However, the uncertainty associated with contaminationquantification has not been reported, making the comparison of similarcontamination levels impossible.7 The quality of a measuredquantity can be expressed by the measurement uncertainty (MU), whichallows assessing if the measurement is fit for purpose and an objectiveand sound interpretation of the measurement result.Research on the levels of microplasticcontamination in differentenvironmental compartments worldwide  has been reported.The evaluationof the measurement uncertainty, in particular thequantification of uncertainty components, can follow different approachesthat can be divided into “top-down” or “bottom-up”,which provide different knowledge on the measurement quality.8 In both “top-down”alternatives, the uncertainty sources are grouped in associated withrandom and systematic effects quantified from precision and truenesstests, respectively. In precision tests, the same sample/s is/areanalyzed by different laboratories (SLA) or on different days by thesame laboratory (SAA), and in trueness tests, it is assessed the agreementbetween the reference and the mean measured value from the analysisof a reference or various reference materials.14 Top-down evaluations are not particularly useful for measurementoptimization and involve an overvaluation of the measurement uncertaintyby double-counting measurement precision in the evaluation of systematiceffects.The top-down approaches can be further divided based on interlaboratorydata, also known as “supralaboratory” (SLA), and basedon in-house validation data, known as the “supra-analytical”(SAA) approach.15 the uncertainty sources should be individuallyidentified and, subsequently, quantified and combined. The quantificationof the uncertainty components by this approach can be hampered bymodel complexity and component correlation. However, bottom-up assessmentsare particularly useful if measurement uncertainty and cost must bereduced by managing major and minor uncertainty components, respectively.Therefore, the evaluated uncertainty is a function of the measurementperformance, the collected performance data, and how uncertainty ismodeled from available information.8On the other hand, in a “bottom-up” or“subanalytical”uncertainty evaluation, whose principles are described in the Guideto the Expression of Uncertainty in Measurement (GUM),Counting the number of microplastics of a sample originates discretequantitative data instead of continuous quantitative data such asthe mass of a species. However, after dividing the number of countedparticles by the analytical portion, typically a mass or volume, acontinuous variable is obtained. Nevertheless, understanding and handlingthe discrete nature of particle counting is essential for the detailedmodeling of the uncertainty of microplastic contamination quantificationof environmental samples.16 One of the lognormal distribution features that makes it particularlyadequate to model λ is that it produces rational values largerthan zero. A Poisson distribution with a mean value modeled or generalizedby a lognormal distribution is called Poisson-lognormal.Poisson distribution is adequate tomodel discrete count variableswith a known mean value, λ, where this mean is a rational valuelarger than zero. However, since the distribution mean is known withsome uncertainty, it can be modeled assuming a lognormal distributionwith a median equivalent to the best estimate of λ and a standarddeviation expressing λ uncertainty.19 Poisson regressionwas used to determine microplastics  and environmental characteristics thatcontributed to microplastic ingestion by African catfish.20 The only relevant limitation of this distributionis the mathematical complexity of its application. This limitationcan be overcome by the development of user-friendly and validatedsoftware.Poissonand Poisson-lognormal distributions have been used to modelmicrobiological colonies counting in the microbiological analysisof foodstuffs and environmental samples.16 Thus, the generalized Poisson-lognormaldistribution can be used to model systematic and random componentsaffecting microplastics counting. The uncertainty from the estimatedmass of the analytical portion of the sediment, divided into microplasticscounting, can be evaluated by combining relevant systematic and randomuncertainty components.12 The distributionof the ratio between the number of microplastics and the dry massof sediment can be simulated by the Monte Carlo method (MCM) basedon simulated distributions of the inputs. The MCM, described in asupplement to the Guide to the Expression of Uncertainty in Measurement(GUM),21 is used to simulate the distributionof an output quantity by determining N output valuesfrom N simulations of the input quantities. The distributionof the output quantity can be characterized by adequate statisticalparameters of the simulated values, such as the mode and relevantpercentiles.The generalized Poisson-lognormal distribution complieswith allfive suitability criteria proposed by I. Jongenburger et al. (2012)for microbiological analysis of foodstuffs. It is a non-negative anddiscrete distribution that allows zeros values and converges exactlyto the Poisson or the Lognormal distribution, assuming appropriateparameters.Supporting Information. The comparison of microplasticcontamination of a pair of sediment samples involved the MCM simulationof the difference in results.This work aims at the bottom-up evaluation of theuncertainty ofmeasurements of microplastics in sediment samples where uncertaintycomponents are combined by the MCM. This research also includes theapplication of the uncertainty models to estimate and compare microplasticcontaminations of sediments collected from four Portuguese inlandwaters. The developed tool was applied to the quantification of (i)total (T) microplastics less dense than an aqueous solution of saturatedNaCl, (ii) polypropylene (PP) microparticles with fragment shape,and (iii) poly (PET) microparticles independentlyof the shape, in sediment samples. A user-friendly and validated spreadsheetfor quantifying the measurement uncertainty is made available as the As far as the authors know, thisis the first bottom-up evaluationof the uncertainty of microplastic quantification in environmentalsamples. This work contributes to the objective interpretation oflevels and trends of microplastic contamination of environmental samplesconsidered a priority by the scientific community dedicated to thisresearch area.22.1Sixty-three sediment samples were collected from previously definedlocations of Portuguese inland waters, namely, Ria de Aveiro (RA),Ria Formosa (RF), Mondego River (RMo), and Mira River (RMi). RA andRMo are located in the center/north, and RF and RMi at the south ofmainland Portugal 1. Three 2 and 5 kg sediment areaand mass were collectedfrom a boat using a Petite Ponar Grab or a Ponar Grab sampler. Thesamplers penetrated the bottom by approximately 5 cm, depending onthe sediment type.22 Samplers were slowlyraised to minimize any particle losses from the sample edges.About 0.05 mTo minimize the overestimation of microplastic content in sedimentsby the deposition of additional particles during sample manipulation, it was cleaned boat deck before collection,reduced the number of people during sample manipulation, homogenizedsediment in an aluminum tray using a wooden spoon and covered about2 kg of sediment in aluminum containers. The transport of sampleswas carried out using cool boxes to the laboratory. In the laboratory,subsamples of approximately 300 g were transferred to a glass containerand frozen below −18 °C until preparation. No blanks testor spikes were performed during sampling.2O2, 30or 35%, Merck)—the digestion efficiency of 30% or 35% H2O2 is not significantly different; (iv) separatingthe lighter microparticles from the denser matter of the samples bymixing with 200 mL of sodium chloride saturated solution  in a measuring cylinder and filtrating the solutionabove the settled matter to a filter  or polycarbonate membranes ; (v) Repeating the densityseparation and supernatant filtration of the matter settled in thebottom of the cylinder; (vi) Storing filters in closed Petri dishesbefore (micro-)ATR-FTIR (Fourier Transform Infrared) identification Sediment wet sieving using ultrapure water with a mesh size of5.6, 5, 2 mm, and 50 μm to isolate sediment matter with thesize range of microplastics; (ii) weighing 8.0–100.0 g of sievedsediment in a Mettler PE 1600 analytical balance; (iii) digestingthe aliquot using peroxide hydrogen  set at 10× to25× amplification to examine and isolate the microparticles suspectedto be microplastics. The 150 membranes obtained were entirely examinedto minimize the under- or over-estimation of the number of microplasticsin the samples, contrary to the suggested by some authors.25 The spectra obtainedby the micro-FTIR were acquired in reflectance mode from 4000 to 600cm–1 using eight scans or in micro-ATR mode from4000 to 500 cm–1 using four scans to reduce analysistime.27 For 94% of the particles, a 100 × 100 μm2 aperture wasused for spectrum acquisition, but the remaining smaller particleswere identified using reduced apertures. The four spectra from microparticleswith sizes larger than 1000 μm, obtained by the Spectrum TwoFTIR, were acquired in universal ATR mode from 4000 to 600 cm–1 using eight or four scans.27 A strong Norton–Beer apodization with 1 and 4 cm–1 wavenumber and resolution intervals, respectively, were used inall operational modes. The instrumental signal was acquired in transmittanceunits (%T). The scan of the background was acquiredbefore the analysis.The isolated suspicious microparticles were mainly analyzedby a PerkinElmer micro-FTIR spectrometer Spotlight 200i MicroscopeSystem equipped with a mercury–cadmium–telluride (MCT)detector or by a PerkinElmer Spectrum Two FTIR. The instrumental conditionsof the FTIR analysis are described following guidelines proposed byAndrade et al..28The infrared spectra were collected bythe PerkinElmer Spectrumsoftware version 10.6.2.1159, also used to measure the size of themicroplastics by taking the distance between the two farthest pointsof the microparticle. No further handling  was considered for spectra identification. Spectraprocessing and comparison for particle identification were performedusing a user-friendly and validated Microsoft Excel spreadsheet developedby Morgado et al.29 ensuring true and false identification rates are not lower or higherthan 95 or 5%, respectively. After determining the match between micro-FTIRspectra of manually identified microparticles and a reference spectrumof the same polymer, the bootstrap numerical sampling method is usedto estimate the match value lower than 95% of the determined match, setting the true positive rate of identificationsto 95%. The minimum match is used in future identifications of unknownparticles. Afterward, it is assessed if the minimum match is not lowerthan more than 5% of the match between the reference spectrum andspectra of particles from a different polymer to ensure the false-positiverate is lower than 5%. Different unweighted and weighted match algorithmsand spectrum requirements were tested. The false identification ofparticles in samples used to validate microplastics quantificationin sediments impacts on estimated measurement precision and truenessand on the analysis of samples by the validated method. The microplasticidentification can be reported with uncertainty as a likelihood ratiodetermined as the ratio between true- and false-positive rates.30The validationof the method of microplastics identification, whichincludes instrumental parameters and spectra comparison algorithmsand criteria, was performed as described by Morgado et al.,2.3Wd, of the sedimentsamples, required to report the number of microplastics present ina dry sample mass, was determined. The Wd was estimated by drying a sediment portion in an oven at (105 ±2) °C following EN 12880:2000.31Wd.mP is themass of the empty Petri dish, mI is theinitial gross mass of the sample, and mF is the dry and final gross mass of the sediment sample.The dry weight fraction, 2.4For determining the measurement precision, 12 sediment samplesfrom different inland waters were prepared and analyzed in duplicate:one sample from RA1 and RMi, two samples from RF and RMo2, and sixsamples from RMi2. To 12 analytical portions of sediment, it was addedfive films smaller than 3 mm of translucent polyethylene (PE), andfive fragments each smaller than 3 mm of green poly(PET), translucent polypropylene (PP), and pink polystyrene (PS) beforesample preparation to check their recovery during digestion, densityseparation, and counting, designated “positive controls”.Airborne particles contamination of areas where samples were manipulated was monitored called “negativecontrols”.Supporting Information. The guidelines proposed by Hermsen et al.32 for classifying the extent of quality control measures were followed,after adequate adjustments to sediment analysis, being scored 15 outof 20 points for procedure adequacy. The nonmaximum score resultsfrom precision being estimated from duplicate instead of triplicatepositive controls, and tests were performed on the few types of studiedsediments. The deviation from the defined ideal quality control procedureis not considered relevant or is justified by the defined analyticalmethod scope.The quality control measures implemented aredescribed in detail in the 33.136 Measurement results are expressed in kg–1 units, although some authors prefer to express the same value notfollowing the rules of the International System of Units as “numberof particles per kg”.37The evaluationof the measurement uncertainty starts with the definition of the quantityto be measured, i.e., the measurand, and the algebraic relationshipbetween the measured value and the parameters on which it depends. The measurand is the number of specificor various types of microplastics in dry mass of the sediment sample.The type of microplastic(s) is defined by the polymer type, particlesize, and shape. The way measurand is defined excludes the samplingprocess from the measurement, being only characterized the sampleand not the sampled population.Threetypes of particles were quantified in this work: (i) microplasticsless dense than saturated NaCl solution—T; (ii) microfragmentsof polypropylene—PP, and (iii) microparticles of polyethyleneterephthalate with different shapes—PET.3.2The second stage of measurement uncertainty evaluation is the identificationof the sources of uncertainty that contribute with random and systematiceffects to the measured quantity. These effects are responsible forpossible deviations between the measured and conventional “truevalue” of the measurand. The random errors vary with replicatemeasurements typically following known distribution models. On theother hand, the systematic effects produce error components that remainconstant or vary in a predictable manner in the measurement interval,with the time, type of analyzed item, or another factor.3.33.3.1C is the number ofparticles }), P(C) is the mass probability of observing C number of particles in a sample, e isthe Euler’s number, and λr is the mean ofobserved particle counting .The random errors,ε, were modeled from the measured values of 12 duplicate samplescollected from the distinct inland water compartments: RMo1, RA1,RMi1, RF, and RMi2. The difference in the number of T, PP, and PETbetween duplicate samples was determined depending on the studiedmeasurand. Since the number of microplastics in a sample is a discretevariable, the probability mass function (PMF) of Poisson distribution,presented by r would be the mean of particles counted in equivalent portions ofthe sediment. One of the particularities of this distribution is thatit does not presume any variability of λr, and assumesthat λr is equal to the expected value, E(C), and variance, Var(C), of the counts.Since the estimated λr has its own uncertainty, theprevious model was fed with an uncertain λr valuewith expected E(λr) value and variance Var(λr) producing a generalized Poisson distribution. In this work,the considered generalization assumes λr has a lognormaldistribution with impossible zero value and a wide distribution tailabove E(λr). The resulting distribution is called“Poisson-lognormal”.38If the studied sediment is analyzed several times, λr) is equal tothe number of particles observed in the analyzed sample  = C), and Var(λr) is estimatedfrom duplicate counting observed in duplicate analyses of sedimentsamples. The variance, Var(d(C)),of the difference of C, d(C), observed in samples is used to estimate Var(λr)(Var(λr) = Var(d(C))/2) since it is known that the variance of the differenceof two variables with Poisson distribution is 2 times larger thanthe variance of individual counts.39 Therandom effects considered in this calculation comprise all analyticalsteps, including those associated with the measurement of the massof the analytical portion, although this contribution should be negligible.For modeling the random effects affecting the determination ofparticles counts, it was assumed E) , and the variance Var(ln(λr)) of the natural logarithm of λr, i.e., ln (λr). r)) and Var(ln(λr)),40 respectively.To simulate the Lognormal distribution of λr)) and variance Var(ln(λr)), Normal (E(ln(λr)); Var(ln(λr))), are converted into simulatedλr with lognormal distribution, Lognormal (E(λr); Var(λr)), by determining the natural exponentof each simulated ln(λr). The simulated C values are microplastic counts not corrected for losses in samplepreparation but expressing random effects. The λr values were simulated 49 999 times, its distribution beingcharacterized by the 0.5th and 99.5th percentiles .The simulated values of the normaldistribution with mean E representing the opposite of the observed mean counts error (N – n̅) normalized by themean number of recovered particles, n̅  type(s)). The corrected counts, CC, are estimated by adding CC = C + C·F). The variance of recovered particle counts, Var(n), was calculated to express their dispersion.The systematic component of countserror, cles, n̅ 5.5where s), varies withthe C  = C·F) (where the “s” in λs stands for “systematic”). The variance Var(λs) is estimated by C·F, knowing that N is a constant without uncertaintysince the added number of particles is known without any doubt. Inthis equation, C is also a constant used to convert F into n̅) is thevariance of mean n, n̅ (Var(n̅) = (Var(n))/M) being M the total number of n estimates. The s)) and variance Var(ln(λs)) of the natural logarithm of λs ) calculated using r is changed for λs. The ln(λs) was simulated 49999 times, being its distribution characterizedby P0.5 and P99.5 percentiles.Analogousto the study of random effects, the systematics effectswere modeled by the Poisson-lognormal distribution that considersthe uncertainty of systematic effects determination. In this case,the expected value of the systematic error, 3.3.3m,41 determined in weighing by difference of sediment analyticalportion was simulated by the MCM. um is thestandard uncertainty of m, and ub and sr are the standarduncertainty associated with systematic effects and the standard deviationof mass measurements repeatability, respectively. These terms arecounted twice, assuming the same value for both the tare and grossmasses. The ub value is quantified bythe maximum admissible error, EM, consideredin balance calibration assuming a rectangular distribution.12 To simulate the measurement repeatability components,it was considered a scaled and shifted t distributionfor the degrees of freedom of sr.The uncertainty associatedwith the conventional mass, mF – mP) and (mI – mP) consideredin the determination of Wd of the sedimentsample is simulated equivalently to m. In the evaluationof Wd uncertainty, it is assumed the dryingprocess in the oven is responsible for a negligible uncertainty component.The uncertainty of the differences  are simulatedcombined as described in mWd are simulated as described in The uncertainty components were combined by the MCM applicable toboth continuous and discrete variables. Supporting Information, was developed to combine the uncertainty components and estimatemicroplastic contamination in sediments with uncertainty. This toolsimulates and combines random and systematic errors in microplasticcounts and the dry mass of the analytical portion. The distributionof ω was estimated from 49 999 simulations. The firstsheet of the spreadsheet presents use instructions.A user-friendly MS Excel spreadsheet, madeavailable as the P0.5and P99.5 were used to define the confidence intervalfor 99% confidence level, which has a 99% probability of includingthe true value of the measurand.The modeof simulated ω represents the most probable or thebest estimate of the value of the measurand. C + ε; (b) systematic error in particles count, mWd; and (d) T microplastics per dry sedimentmass, ω. The distribution of mWd (n of mWd 3c has an of mWd 3d is con4d(C)),equals to 12.0, 2.50, and 0.967, estimating a random error variance,Var(λr), of 5.98, 1.25, and 0.483, respectively )/2). From the analysisof 12 fortified samples with five PE, PET, PP, and PS particles, itwas observed mean numbers of recovered particles, n̅, of 3.97, 3.58, and 1.67 with a variance, Var(n), of 0.0372, 0.143, and 0.505, for T (PE + PP + PS), PP, and PET,respectively. The recovery of T was estimated as the combined recoveryof PE, PP, and PS since the studied polymers are less dense than saturatedNaCl solution.The 12 sedimentsanalyzed in duplicate (24 tests) presented quantitative microplastic contaminations. For the determinationof the studied three parameters , the varianceof counts differences, Var in the sixty-three sediments samplescollected from the studied environmental compartments . P0.5 and P99.5,indicating that the sample contaminations are metrologically equivalentor different for 99% confidence level, respectively. Sample S01 ismore contaminated than sample S02 since P0.5 is largerthan zero. When P99.5 is smaller than zero, the secondsample of the difference is less contaminated. The simulated differencesfrom To compare the microplastic contamination levelof a pair of samples,it was simulated the difference in estimated microplastic contaminationby the MCM since it takes the complex distribution of estimated valuesinto account. However, since the estimated microplastic contaminationof sedimentsdoes not consider the sampling uncertainty, this comparison is notrepresentative of a larger fraction of river sediments.44Althoughthe evaluation of the uncertainty of microplastic contaminationestimated for each sample is important, since it objectively informson the expected true value of the contamination, it is in the comparisonof the contamination of two samples that this concept is particularlyrelevant. If a maximum threshold for microplastic contamination isdefined, the uncertainty from the sample analysis could be used toassess the conformity with that limit.Supporting Information. In thiswork, it was also simulated the mean of the estimated microplasticcontamination of all samples collected from the same environmentalcompartment and sampling occasion to allow mean comparisons by calculatingthe difference of the means. The meaning of the mean contaminationis even more difficult to describe since it can be not representativeof the heterogeneity of compartment contamination.The microplasticcontaminations of sediment samples collected ineach compartment and sampling campaign were compared. The contaminationsof samples collected in the same GPS coordinates in different campaignswere also compared. The results from these comparisons are made availableas the Regarding the contamination level of PP, the meanvalue of RF samplesis greater than the mean of RMi2, RA1, and RA2. Nevertheless, themean value of RA2 is lower than for the RMi1. Microplastic contaminationon RA result from industrial and agricultural sources, RMo tide influence,RMi agricultural activities, and RF urban sites proximity.Tables S1–S33). Only the samples of RA2 campaign present equivalent T values.Nevertheless, in the other campaigns, some samples have significantlydifferent contamination levels. For instance, S01 and S10 of RA1 areequivalently contaminated but more contaminated than the remainingsamples of the same campaign (see Table S1). Regarding the samples collected from the same coordinates, mostsamples have equivalent T levels, except for S04 of RMi1, which ismore contaminated than S03/S03II of RMi2 (see Table S9). In this case, sediment contamination was reducedover time. To infer the representativeness of samples and of thisconclusion about sediments contamination, the microplastic contaminationheterogeneity and sampling uncertainty should be considered.Thetables with the comparison of the microplastic contaminationof samples collected from the same sampling campaign or collectedfrom the same coordinates in different campaigns are made availableas the Supporting Information (Table S15). The S09 of RMo1 has a contaminationlevel greater than S01/S01II, S02, and S06, and S07 is more contaminatedthan S01 of the same campaign (see Table S18). The samples collected at the same coordinates on different occasionshave equivalent contamination levels of PP.Concerning PP contamination, most of the samples from the samesampling campaign present equivalent contamination levels, exceptfor some samples of RMo1 and RF. The S07 of RF has a contaminationlevel equivalent to the samples S02 and S05, and a contamination levellower than the sample S11 of the same campaign. Nevertheless, S11is only equivalent to sample S02. Besides, S07 and S11 of RF havea contamination level greater than the remaining samples . After some adaptation, this tool can also be used tocharacterize analytical portions determined by volume, such as waterof different types and origins. Results with uncertainty from microplasticcontamination of water and sediments from the same location can beused to better understand how different microplastics distribute inthe environment.


Although children and adolescents with hearing impairment are at risks of falls from balance problems, reliable information on effects of interventions are scare. Therefore, the purpose of this review is to systematically summarize studies on the evidence of interventions to improve balance ability in children and adolescents with hearing impairment. A systematic literature search was conducted on five major electronic databases. Studies were included if: 1) interventions or trials focusing on improving balance in children and adolescents with hearing impairment; 2) research targeting children with hearing impairment (samples with a mean age below 18 years); 3) studies were published in English peer-reviewed journals due to language barriers and resource limitations; and 4) study designs were randomized controlled trial or quasi-experiment. A nine-item tool adapted from the Consolidated Standards of Reporting Trials Statement was used to assess the quality of the studies. Through the search strategy, 373 articles were identified, and 15 studies published between 1981 and 2021 met the inclusion criteria. Most of the studies reviewed were categorized as medium or low quality, and only three were identified as high quality. Exercise interventions were adopted in 80% of the included studies, whereas studies that employed music + vibration, motor, and game as the intervention modalities accounted for the remaining 20.0%. The results of this review showed that the included trials with exercise interventions had a positive influence on the balance among children and adolescents with hearing impairment (the post-intervention scores were significantly higher than the pre-intervention or the control group scores). In addition, the interventions with duration of 8–16 weeks were more effective than those with less than 8 weeks. However, due to most of the reviewed studies were of low methodological quality, the trials results analyzed by this systematic review should be interpreted with caution. Further investigations of high-quality studies are therefore needed to prove the effectiveness of interventions on improving balance performance in children and adolescents with hearing impairment.Systematic Review Registration: [https://www.crd.york.ac.uk/PROSPERO/], PROSPERO [308803]. Balance is an essential prerequisite for most daily life activities in children . It is tvia physical activity-related parameters, such as mobility function and balance  Statement .The researchers systematically searched studies with the following databases from inception to December 2021: PubMed, PsycINFO, Scopus, Education Resources Information Centre (ERIC), and Web of Science (WoS) Core Collection. The search strategy included four groups of keywords: 1) hearing impairment* OR hearing disability* OR deaf OR hard of hearing; 2) child* OR adolescent* OR teenager* OR youth* OR youngster* OR student* OR pupil*; 3) motor ability* OR motor skill* OR balance OR posture control OR postural stability; 4) intervention OR trial OR experiment. In addition, the snowballing technique was used to identify potential studies by scanning the references of all the included articles.Studies meeting the following criteria were included in this review: 1) interventions or trials focusing on improving balance in children and adolescents with hearing impairment; 2) research targeting children with hearing impairment (samples with a mean age below 18 years); 3) studies were published in English peer-reviewed journals due to language barriers and resource limitations; and 4) study designs were randomized controlled trial (RCT) or quasi-experiment (QE). Studies were excluded if they 1) were pertaining to other topics; 2) participants’ mean age was above 18; 3) were unpublished articles, comments, conference proceedings, reviews, and dissertations; and 4) failed to report the detailed outcomes of trials. Two authors independently screened the returned articles according to the inclusion and exclusion criteria. Any disagreements between authors were resolved through discussion.To determine the methodological quality of the included studies, the researchers used a nine-item tool adapted from the Consolidated Standards of Reporting Trials Statement , which hThe first author completed the data extraction and then the second author verified the data. Discrepancies were resolved through a consensus discussion. The researchers extracted the following information: the first author, publication year, geographic location, and intervention detail.Given the heterogeneity of the included studies, meta-analysis was not conducted. Instead, the intervention characteristics of each study were first summarized and analyzed and then recorded on a standardized form created by the authors. The effective rate of the interventions was calculated using the following equation: effective trials (the post-intervention scores were significantly higher than the pre-intervention or the control group scores)/. Similarly, data analysis was performed by the first author and then verified by the second author.n = 15; WoS Core Collection, n = 179; PubMed, n = 106; PsycINFO, n = 55; Scopus, n = 373). Of these articles, 186 duplicates were removed. An additional article was identified from a citation search of relevant published literature reviews. After screening the titles and abstracts, 126 studies were excluded. The remaining full-text articles were read, and 45 others were excluded. Therefore, a total of 15 studies were included in this review. The PRISMA flowchart is shown in The initial search identified 373 studies , whereas studies that employed music + vibration, motor, and game as the intervention modalities accounted for the remaining 20.0%. Exercise intervention mainly used a series of structured course or training, such as vestibular-specific neuromuscular training , exercisThe total duration of interventions ranged from 10 days to 16 weeks, 80% of which opted for a duration of 8–16 weeks. In terms of intervention frequency, 66.7% of the included studies performed their experiments three times a week, whereas 20.0 and 6.7% conducted their experiments twice and once a week, respectively. Sessions of 30–45 min per time were selected by 46.7% of the trials, whereas sessions of more than 45 min and less than 15 min per time accounted for 26.7 and 13.3%, respectively. However, 13.3% of the trials failed to report the duration of each session.A total of 10 studies evaluated the static balance, among which the most common assessment was one-leg standing test, such as One-leg Standing Balance Test, Stork Balance Test, Flamingo Balance Test, and Single-limb Standing Test. Six studies tested dynamic balance, two of which used Y Balance Test. Five other studies tested subjects’ postural stability, and another one measured overall balance using the Pediatric Balance Scale.vs. 75.0%). And trials in children with hearing impairment were more effective than adolescents with hearing impairment (91.7 vs. 75.0%). Besides, among 10 studies that measured static balance, the effective rate was 90%, whereas the efficiency of six studies that assessed dynamic balance was 100%. Five studies that evaluated postural stability had an effective rate of 80%, whereas the effective rate of one study that measured overall balance was 100%.Overall, 13 out of 15 trials (86.7%) showed a positive effect on improving balance among children and adolescents with hearing impairment (the post-intervention scores were significantly higher than the pre-intervention or the control group scores). Interventions with a total duration of 8–16 weeks were more effective than those with less than 8 weeks  conducted independent t-test or paired t-test, and 20.0% of which performed U-test. Repeated-measures ANOVA and its combination with t-test accounted for 13.3% of the total, whereas t-test combined with Wilcoxon signed rank tests accounted for 6.7% of the studies.For this review, we identified 15 studies that used the intervention program to improve the balance of children and adolescents with hearing impairment. The results of methodological quality assessment showed that most of the studies reviewed were categorized as medium or low quality, and only three were identified as high quality. Most research used QE designs, being one of the reasons that reduce the methodology quality. Even in the RCT designs reviewed, the random assignment, allocation concealment, and assessor blinding procedures were not fully described. RCT designs pose methodical, practical, and ethical challenges to researchers , especiaOne area that must be pointed in the reviewed trials is the relatively small sample sizes. Most studies (86.7%) had sample sizes less than 40, and none of the authors specified if they used calculations to establish such sample sizes. Theoretically, sample size depends on three aspects: the main measurement variable, the variance in the primary variable, and the acceptable error. Another shortcoming of some trials analyzed in this review is not controlling the types of hearing impairment. According to the American Speech–Language–Hearing Association, hearing loss has three basic types: conductive hearing loss, sensorineural hearing loss (SNHL), and mixed hearing loss . HoweverIn terms of the instruments for the evaluation of the outcomes, future studies should fully consider the applicability of measurement tools to children and adolescents with hearing impairment. The use of unvalidated instruments would reduce the reliability of the evidence . RombergThe results from this review showed that the included trials with exercise interventions had a positive influence on the balance among children and adolescents with hearing impairment (the post-intervention scores were significantly higher than the pre-intervention or the control group scores). Given that most studies employed structured exercise training with an emphasis on balance and coordination that present wide displacements of the gravity center, the experimental group therefore may obtain further practice on the balance ability . MoreoveAnother important finding of this review showed that the balance interventions were more effective in participants with an average age of 6–12 years than those with an average age of 13–18 years. This finding expanded, as well as affirmed, previous studies, that the most critical period in motor development occur in the first decade of life with balance control usually established at 7–10 years old . HoweverRegarding the duration of training, interventions with duration of 8–16 weeks were more effective than those with less than 8 weeks. Short-duration programs may not suffice to support the physical and cognitive integration of new skills to achieve the long-term modification of balance ability . PlanninStudies that employed music + vibration, motor, and game as the intervention modalities had an effective rate of 100, 50, and 0%, respectively. Considering that each of these three modalities was adopted by only one study, drawing a conclusion on the effectiveness of these interventions on enhancing balance among children and adolescents with hearing impairment is difficult. Additional studies are needed to confirm the results of interventions adopting these modalities.Based on our findings, we conclude that the exercise interventions were effective on improving balance in children and adolescents with hearing impairment. In addition, the interventions with duration of 8–16 weeks were more effective than those with less than 8 weeks. However, most of the reviewed studies were of low methodological quality; thus, the trial results analyzed in this systematic review should be interpreted with caution. Further investigations of high-quality studies are therefore needed to prove the effectiveness of interventions on improving balance performance in children and adolescents with hearing impairment.Two limitations inherent within the current review should be noted. First, although we conducted an extensive literature search on five major databases to identify potential studies, a few published studies were possibly missed because our search was limited to English journal articles. Certain studies, which could have added relevant information to the field, might have been discarded. Second, due to the heterogeneity of reviewed studies, such as participant characteristics and outcome measures, a meta-analysis could not be conducted.


Fully bio-based poly(lactic acid) (PLA) and poly(3-hydroxybutyrate) (PHB) blends plasticized with tributyrin (TB), and their nanocomposite based on chitin nanoparticles (ChNPs) was developed using melt mixing followed by a compression molding process. The combination of PHB and ChNPs had an impact on the crystallinity of the plasticized PLA matrix, thus improving its oxygen and carbon dioxide barrier properties as well as displaying a UV light-blocking effect. The addition of 2 wt% of ChNP induced an improvement on the initial thermal degradation temperature and the overall migration behavior of blends, which had been compromised by the presence of TB. All processed materials were fully disintegrated under composting conditions, suggesting their potential application as fully biodegradable packaging materials. Several biodegradable bio-based polymers have attracted special interest in the packaging industry since they have comparable properties to those of conventional polymers and allow for a reduction environmental impact . A numbeThe addition of nanoparticles into the polymeric packaging material makes it possible to modulate the physicochemical properties and leads to improvements in specific features such as barrier capability, mechanical resistance, and thermal stability . In partNano-fillers can be classified according to their origin  . In thisThe use of polysaccharide nanoparticles has attracted considerable attention in a wide range of areas and especially in packaging applications. Polysaccharides are the most abundant macromolecules in the biosphere and have remarkable features such as biocompatibility, biodegradability, renewable origin, and facile modification . These cIn the particular case of the production of films based on PLA-PHB, where the use of plasticizers is required to modulate the compatibility and flexibility of the blend, the barrier performance is usually affected by the presence of the plasticizer. Therefore, strategies such as the development of nanocomposites can be used to obtain materials that have a good balance between structural and functional properties.Scientific articles of PLA-PHB polymeric matrices reinforced with cellulose nanocrystals (CNC) have been published ,14,15,16Chitin is the second most abundant polysaccharide in nature after cellulose, with global reserves of 100 billion tons . It is wIt is a fairly recent trend that has embraced the importance of chitin as a promising source of new materials, especially as a nanostructured material in the form of chitin nanocrystals (ChNC) and/or nanofibers (ChNF) . One of Different techniques have been reported to prepare chitin nanoparticles (ChNP) with different morphologies , althougThe purpose of the present study was to evaluate the role of chitin nanoparticles on the structural and functional properties of thermoplastic nanocomposite PLA-PHB-based films prepared by melt mixing and a compression molding approach. A number of previous works describing the PLA-PHB system have already been published ,34,35. Tw = 236,000 g mol−1, 96 wt% L-isomer) was supplied by NatureWorks®  and poly(3-hydroxybutyrate)  was provided by PHB Industrial S.A.  under the name Biocycle® L-61. Chitin from shrimp shells  and tributyrin  were purchased from Sigma–Aldrich® . Hydrochloric acid  was acquired from Anedra .Poly(lactic acid)  were synthesized from purified chitin powder by a hydrochloric acid hydrolysis following a process based on the method reported by Gopalan Nair et al. . Hydrolycle size ,37. AftePrior to any processing, PLA, PHB, and ChNP were dried to avoid any moisture trace and undesirable hydrolysis reactions during the melt blending. Materials were dried overnight at 60 °C in a Cole-Parmer StableTemp vacuum oven (USA).PLA/PHB blends were prepared by mixing PLA and PHB pellets in a double screw Haake mixer at 185 °C, a screw speed of 60 rpm, and a mixing time of 3 min. The weight ratio of PLA:PHB used was 60:40, and the proportion of plasticizer (TB) incorporated was 15 wt% of the final weight mixture, in accordance with previous works ,34.Plasticized PLA/PHB/TB blend was reinforced with 2 wt% of ChNP in order to manufacture the chitin nanocomposite, using a temperature of 185 °C and a screw speed of 60 rpm for a total time of 5 min. In this case, a suspension of ChNP and TB was used as feeding during the processing. The suspension was ultrasound-treated at room temperature for 1 h, stopping every 20 min to homogenize the mixture, in order to facilitate the dispersion of the nanoparticles in the plasticizer. Firstly, PLA pellets were put in the mixer until completely melted. Secondly, PHB pellets were incorporated. Finally, the ChNP-TB suspension was fed into the mixer after 3 min of blending PLA/PHB. The nanocomposite was mixed for two additional minutes. The process was performed in an inert atmosphere using nitrogen gas to avoid possible oxidation of the ChNP.Blends were compression molded in an EMS AMS 160/335DE hydraulic press (Argentina) to obtain the films. The processing temperature was 190 °C and the pressure was kept 1 min at 0.1 MPa and 2 min at 5 MPa. Lastly, films were quenched at room temperature.Neat and plasticized PLA and PHB films were also produced for comparison. The proportion of each component of the obtained binary, ternary, and quaternary formulations is summarized in The resulting ChNP morphology was characterized by field emission scanning electron microscopy . A drop of highly diluted nanoparticles suspension was placed on a silicon plate and was left to dry. The sample was sputtered with a thin gold layer. The dimensions of the nanoparticles were obtained using SEM images and the ImageJ software, evaluating 250 representative items of the chitin nano-whiskers.The microstructure of blends and bio-nanocomposite films’ cross-sections were observed by scanning electron microscopy . The samples were previously frozen in liquid nitrogen, cryofractured, and then sputtered with a gold layer.−1 by performing 32 overlapping scans at a resolution of 4 cm−1 at room temperature.Fourier Transform Infrared spectroscopy analysis in attenuated total reflectance (FTIR-ATR) was performed using a Thermo Scientific  Instrument in the range of 4000–400 cmWide angle X-ray scattering (WAXS) measurements were carried out using a X’PertPro diffractometer  equipped with a fast solid-state X’Celerator detector, operating at 40 kV and 40 mA, with CuKα radiation (λ = 1.54 Å). Data were acquired in the 3–60° 2θ interval .The surface hydrophobicity of films was studied by measuring the static water contact angle (WCA) by means of a KSV CAM101 goniometer  at ambient conditions by recording the side profiles of deionized water drops for image analysis. Ten drops were observed on different areas for each film, and contact angles were reported as the average value ± standard deviation. Each drop (4 uL) was deposited on the films by placing it in contact with the polymeric surface using a syringe needle (100 uL/min).0) were calculated at 5% mass loss, while thermal degradation temperatures at the maximum rate (Tmax) were determined from the first derivatives of the thermogravimetric curves (DTG).Thermogravimetric analysis (TGA) was carried out using Perkin Elmer TGA7  apparatus under nitrogen atmosphere (gas flow: 40 mL/min) by heating from 30 to 700 °C at 10 °C/min. The specimen mass was in the range of 6–10 mg. The initial degradation temperatures (Tg) was taken as the midpoint of the endothermic step associated to the glass-to-rubber transition. The melting temperature (Tm) and the crystallization temperature (Tc) were determined as the peak value of the endothermal and the exothermal phenomena in the DSC curve, respectively. The heat of fusion  was carried out on a Perkin Elmer DSC7 instrument  under a nitrogen atmosphere (gas flow: 20 mL/min). The external block temperature control was set at −90 °C. The samples, of about 6 mg each, were encapsulated in aluminum pans and exposed to the following thermal treatment: (i) heating scan from −70 °C to 200 °C at 20 °C/min (first scan); (ii) isothermal scan at 200 °C for 1 min; (iii) cooling scan to −70 °C at 100 °C/min; (iv) isothermal scan at −70 °C for 14 min; (v) heating scan from −70 °C to 200 °C at 20 °C/min (second scan). The glass transition temperature (Tctively) ,39, and The absorption spectra of film samples were obtained in the 190–1000 nm region using a UV–vis spectrophotometer . To convert absorbance values (A) to percent transmittance (Op), and the average of three measurements at random positions over the film surfaces were reported. Total color difference  with an 8 mm diameter measuring area, calibrated against a white standard tile. Color coordinates, max), and elongation at break (εb) were calculated. Reported values were the average of at least five valid tests.Uniaxial tensile tests were performed under ambient conditions using a universal testing machine . Dumbbell shape specimens were investigated according to ASTM D1708-93, with a crosshead speed of 1 mm/min and a load cell of 500 N. Young’s modulus (E), tensile strength (σ2) as a desiccant agent. The chamber allows controlling temperature and relative humidity with continuous air circulation to maintain uniform conditions at all test locations. Weight measurements were made using an analytical balance at regular time intervals until a steady state was reached. The WVP of the films was calculated according to Equation (4).Water vapor permeability (WVP) of the films was evaluated following the methodology described by ASTM E96-00. Teflon capsules of 5 cm of diameter containing the film samples were placed in a chamber at 65% relative humidity and at an average temperature of 18 °C, using anhydrous calcium chloride (CaClWVTR is the water vapor transmission rate through the film (g/m2.s), d is the average film thickness (m), and ΔP is the difference in partial vapor pressure (Pa) between both sides of the film. Reported values were the average of three tests.2) and carbon dioxide (CO2) transmission rate was assessed by a manometric method using a Permeance Testing Device, type GDP-C , according to ASTM 1434-82 (Standard test Method for Determining Gas Permeability Characteristics of Plastic Film and Sheeting), DIN 53 536 in compliance with ISO/DIS 15 105-1 and according to Gas Permeability Testing Manual . Method A was employed in the analysis, as reported in the Brugger manual, with the evacuation of top/bottom chambers. The film was placed between two chambers. The amount of gas flowing through the membrane was determined from the pressure variation due to the gas accumulation in the closed downstream chamber. All the measurements have been carried out at 23 °C. The operative conditions were a gas stream of 100 cm3·min−1; 0% RH of gas test, food grade; sample area of 0.785 cm2. The gas transmission rate (GTR) was determined considering the increase in pressure in relation to the time and the volume of the device. Gas transmission measurements were performed at least in triplicate and data was normalized for the thickness of the film samples.Oxygen (O2 (mass of migrant per dm2 of film) that should not be exceeded.Overall migration (OM) tests were performed according to the Commission Regulation EU No. 10/2011 on plastic materials and articles intended to come into contact with food . A totalv/v) (simulant A) and isooctane . Specifically, food simulant A is designated for food products that have a hydrophilic character; instead, simulant D1 is designed to simulate the behavior of food products that have a lipid character. Rectangular strips with a 25 cm2 total area of each film formulation were immersed in a glass tube with 25 mL of food simulant. Samples in 10% ethanol (v/v) were kept in a controlled chamber at 40 °C for 10 days, while samples in isooctane were kept at 20 °C for 2 days, according to EN-1186 standard. After the incubation period, the film samples were removed from the tubes, and the simulants were totally evaporated. Blank tests for each simulant under the same incubation conditions but without samples were also run. The non-volatile residue was determined by using an analytical balance. The overall migration (OM) values were expressed in mg dm−2 of film using Equation (5).Two liquid food simulants were selected for analysis: 10% ethanol . Vessels were incubated in an SW22 Julabo water bath at 58 °C and 90 %RH.The study of the disintegration of the materials under aerobic composting conditions was carried out by a laboratory-scale test. Each film sample with dimensions 15 × 15 mmD) was determined gravimetrically according to Equation (6).Prior to degradation experiments, the samples’ dry weight was measured to obtain the sample initial mass. At different time intervals , duplicate sacrificial specimens of each sample were recovered, washed, and dried until the constant was weighed. Photographs of samples were taken for visual comparison. The disintegration degree , diameter (d), and its respective average aspect ratio (L/d) are summarized in −1 are attributed to the −OH and −NH2 groups’ stretching vibration and intermolecular hydrogen bonding; the bands at 1656 and 1620 cm−1 correspond to the amide I region (stretching of the C=O group of the peptide bonds); the peak at 1554 cm−1 corresponds to the amide II band (N-H bending); and the peak at 1309 cm−1 is attributed to the amide III vibration (C-N stretching) [The FTIR-ATR spectrum of dry ChNP powder is shown in etching) . All theThe crystalline structure of ChNP was studied by WAXS B. The diTGA assays were carried out to investigate the thermal stability and degradation profile of chitin nanocrystals. −1 region. The characteristic peaks corresponding to the asymmetric stretching of the carbonyl group (C=O) in the polyesters were observed at 1747 and 1718 cm−1 for pure PLA and PHB, respectively [The FTIR spectra of PLA- and PHB-based materials are shown in ectively . As expeIn order to investigate the crystalline structure of the developed materials, X-ray diffraction tests were performed. The diffractograms are shown in In general, the diffraction patterns of all the PLA/PHB blends E–G were The presence of ChNP in a PLA/PHB/TB/ChNP nanocomposite G was eviSince materials intended for food packaging are required to protect foodstuff from humidity, the wettability of films was evaluated. The hydrophilic/hydrophobic behavior of the surface film samples was investigated by static water contact angle (θ) measurements. Results are shown in The contact angles for PLA/TB, PHB/TB and PLA/PHB/TB films were around 6% higher than their un-plasticized counterparts, suggesting that the addition of 15% of TB produced a reduction in materials’ surfaces wettability. Similar results were observed in PLA-PHB matrices plasticized with acetyl tributyl citrate (ATBC) or D-limonene ,50, and Color and optical properties are of importance for materials intended for food packaging purposes. The transmission of UV and visible light through polymers is one of the main factors affecting food quality. For instance, light has been found to affect the flavor and the nutritional content of some products . In thisNeat PLA sample A showed Despite of the different transmittance percentages obtained within the visible light range (400–700 nm), all developed films showed good transparency since the logo situated under them in the pictures of Op is indicative of its high transparency . Additionally, the PHB sample presented a %Op two-fold higher than pure PLA due to its crystalline nature. The PLA/PHB sample turned out to be the least transparent among the materials studied with an 18%Op. Opacity in immiscible polymer blends can be originated from different refractive indexes between the two domains and/or due to the scattering of light due to interfacial voids [Op, indicating an improvement in transparency compared with the PLA/PHB blend. The subsequent incorporation of ChNP caused a slight increase in the E for the PLA/PHB/TB/ChNP sample—a possible result of the typical yellowish coloration of chitin.Neat PLA showed the highest sparency . Similaruman eye . Lightneal voids . Its L* These results indicate that the PLA/PHB/TB and PLA/PHB/TB/ChNP nanocomposites have better UV barrier properties than PLA or PLA/TB, demonstrating their potential applicability as fully biomass packaging and/or coating systems characterized by transparency and exceptional UV-protection capability, which are mainly significant for light-sensitive products .Differential scanning calorimetry was carried out to investigate the film samples’ thermal characteristics and the role of ChNP on bio-nanocomposite thermal behavior. g value for the neat PLA sample was found at 62 °C, while weak peaks corresponding to the exothermic crystallization and endothermic melting of PLA were observed at around 124 and 152 °C, respectively. The addition of plasticizer induced a significant depression in Tg and Tc values and a slight reduction in Tm for the PLA/TB film sample due to the higher molecular mobility of the polymer chains, confirming the efficiency of TB as a plasticizer. Moreover, the presence of TB increased the ability of PLA to crystallize, revealed not only by the shift of Tc to lower temperatures, but also by an exothermic crystallization enthalpy value ten times higher than the one of the neat PLA , presenting a double melting peak behavior [The Tneat PLA . The PLA samples C,D confi samples A,B that behavior ,61, as im value in the PHB/TB sample [g and Tc of PHB at around 1 and 50 °C, respectively, could be detected, as could the subsequent depression of these temperatures in the PHB/TB sample due to plasticization. Therefore, it can be inferred that TB is completely miscible and effective as plasticizer for both PLA and PHB, as has already been shown in our previous works [c values of PHB/TB in respect to neat PHB , and multi-step melting, with the first peak being due to PLA and the second one corresponding to PHB. Moreover, the DSC curves displayed no considerable variations of the thermal characteristic properties with respect to individual neat polymers  of both polymers in the PLA/TB and PHB/TB samples, respectively, in good agreement with other plasticized PLA [Thermogravimetric analysis of processed materials was also conducted. The TG and DTG curves and the main thermal parameters obtained from them are shown in ized PLA  and plasized PLA .0 to upper temperatures. It should be noted that no degradation takes place within the temperature range from ambient temperature to 200 °C, where the biodegradable nanocomposite and blends are processed and intended to be used.PLA/PHB, PLA/PHB/TB, and PLA/PHB/TB/ChNP materials degraded in two-step processes. Once again, the initial thermal stability of the blend was compromised by plasticization but the addition of highly stable ChNP slightly shifted the TMechanical properties of PLA/PHB-based materials plasticized with TB have been extensively studied in our previous published works ,34. In og value than the plasticized PLA counterpart  and carbon dioxide (CO2) transmission rates through the films, were evaluated, and the results are shown in The barrier properties are considered important parameters in food packaging manufacturing due to the role of water vapor and various gas transmission in deteriorative reactions and microbial and mold growth . Hence, c makes the polymer less permeable [Neat PHB-processed film presented greater resistance to the transmission of dry gases and water vapor due to its crystalline nature. The gas and vapor permeability of semi-crystalline polymers is mainly affected by the percentage of a crystalline phase which is impermeable to gases, therefore a higher χermeable . The incPlasticization of PLA, PHB, and PLA/PHB samples increased the gas and vapor transmission through the PLA/TB, PHB/TB, and PLA/PHB/TB films as a result of the increase in free volume and chain mobility ,62,73,742 was the gas that propagated fastest through the films, despite its larger dimension. Similar results were found by some of us when studying the highly hydrophobic polymer matrix [2 and CO2 transmission rate values for the PLA/PHB/TB blend and the PLA/PHB/TB/ChNP nanocomposite are clearly lower than those of commercial low-density polyethylene (LDPE), namely 19.5 and 78 cm3 cm m−2 d−1 atm−1, respectively [For all samples under consideration, COr matrix . It shouectively , therefov/v) as food simulants. Overall migration (OM) tests were carried out on PLA/PHB bio-nanocomposite films using isooctane and 10% ethanol (−2). Yet, it was observed that the migration levels increased when samples were plasticized with TB. On the other hand, migration tests performed in the polar simulant showed that PLA/TB, PHB/TB, and PLA/PHB/TB formulations exceeded the OML. This behavior could be related to the plasticizing effect of TB, resulting in an increase in free volume and the mobility of polymer chains within the matrix, as was previously reported in our preceding work [In the case of isooctane (non-polar), all materials showed overall migration values lower than the current limit (10 mg dming work  and as iv/v), respectively, compared to the unfilled counterpart blend (PLA/PHB/TB film). Then, the incorporation of 2 wt% of ChNP into the polymer matrix shifted the OM values in the 10% ethanol (v/v) simulant to a value below the OML. The interaction between the ChNPs and the polymeric matrix could have caused a restriction in the polymer chains’ mobility, lowering the migration [The PLA/PHB/TB/ChNP nanocomposite was detected to have reductions of 18 and 34% in the migration values in isooctane and 10% ethanol  into the PLA matrix improved its degradation kinetics, being the observed trend PLA < PLA/PHB < PLA/PHB/TB < PLA/PHB/TB/ChNP. Chitin nanoparticles (ChNPs) sped up the disintegration rate of the PLA/PHB/TB blend mainly during the first 3 weeks of incubation. As aforementioned, the disintegration in the compost of the PLA matrix starts by a hydrolysis process ; thus, tIn brief, all formulations were fully disintegrated under composting conditions within one and a half months, suggesting their respective advantages in industrial applications when short biodegradation times are required.Plasticized nanocomposite films based on PLA/PHB/TB reinforced with synthesized ChNP were developed and deeply characterized. Chitin nanoparticles were successfully obtained by hydrochloric acid hydrolysis.The resulting nanocomposite film was optically transparent to visible light and opaque in the UV region, which is particularly advantageous for packaging light-sensitive products. The presence of ChNP on the surface of the specimens was detected by a reduction in the WCA measurements. The addition of ChNP to the PLA/PHB/TB blend contributed to a reduction in the melting temperature of PHB as well as to an increment in the initial thermal degradation temperature of the film, thus leading to an improvement in the typically narrow processing window of the neat PHB. The synergic effect of PHB and ChNP enhanced the crystallization of the PLA matrix and improved both the gas barrier properties and the overall migration behavior of plasticized PLA/PHB blends. The composting test confirmed the biodegradable character of all film formulations. Moreover, the tributyrin and chitin nanoparticles were able to speed up the disintegration process of the produced materials. The tensile properties of the films showed a reinforcing effect of chitin nanoparticles by increasing their rigidity and strength, while their elongation was greatly reduced. The absence of a ductile behavior was explained by the poor interfacial adhesion between the components that would be insufficient to withstand interfacial stresses generated during tensile deformation. An adequate amount of filler and/or a modification in nanoparticles’ surface polarity would enhance the interfacial adhesion and thus the mechanical properties.These results suggest that chitin nanoparticles are promising filler for the preparation of multifunctional materials based on plasticized PLA/PHB blends and that may offer good perspectives for food packaging applications. Further investigation is currently in progress to modify chitin whiskers’ surfaces, leading to higher hydrophobicity in order to increase their dispersion in the non-polar polymer matrix to promote interfacial adhesion and to enhance the final properties of nanocomposite systems while taking into account the proposed field of application.


The objectives of this study were to test differences in lying and stepping behaviors in lactating cows with altered gait or other signs of pain in the day preceding either corrective claw trimming (CCT) or therapeutic claw trimming , including healthy controls (HC) as a reference group, and to analyze the associations between categories of lying and stepping behaviors at d −1 and subsequent submission to CCT or TCT (d 0). In addition, we characterized variations in lying and stepping behaviors within 7 d relative to CCT or treatment for a specific foot condition (TCT). A total of 310 Holstein cows in a USDA-certified organic dairy herd in northern Colorado were enrolled. Cows were affixed with an accelerometer  below the fetlock of one hind leg at 12 ± 8 d in milk and monitored for 6 mo for daily activity . Cows with altered gait or other signs of pain were submitted to claw trimming  and differentiated as receiving only corrective interventions (CCT) or as being treated for a lameness disorder (TCT). Concurrent activity from cows not submitted to CT was considered a reference from HC. Daily averages for each activity within ±7 d relative to CT were compared among the 3 groups using linear mixed models . Logistic regression analyses with a logit link function (PROC GLIMMIX of SAS) were used to test the associations between categories  of lying and stepping behaviors  preceding CT (d −1) and subsequent submission to CCT or TCT (d 0). On d −1, average (± standard error) LY was greater in TCT (631 ± 34 min/d) than in CCT (568 ± 22 min/d) and HC (581 ± 13 min/d). Overall, LB were not statistically different among groups . Daily ST were lower in TCT  than in CCT  and HC . The odds  of TCT were greater in cows in the high LY [3.27 (2.47–4.33)] and high LB [2.31 (1.77–3.02)] categories at d −1. The low ST category was associated with increased odds of CCT [3.32 (1.96–5.65)] and TCT [8.48 (6.49–11.1)]. We concluded that, under the current experimental conditions, cows requiring TCT evidenced greater overall LY and lower daily ST the day before CT. However, these differences were not consistent throughout days in milk categories within the lactation. Type of CT was partially associated with category of activity the day preceding CT, and the associations were more evident for TCT. The magnitude of the behavioral changes before and after CT and the recovery times posttreatment to approach HC behavioral levels varied depending on specific conditions. CT) and monitoring and treatment of locomotion dysfunctions  compared with cows with identifiable foot disorders receiving therapeutic CT (TCT). Therefore, the objectives were to test the differences in daily lying time (LY) and number of lying bouts (LB) and steps (ST) preceding either CCT or TCT (d −1) in cows identified with altered gait or other signs of pain and to analyze the associations between categories of lying and stepping behaviors preceding CT (d −1) and subsequent submission to CCT or TCT (d 0). In addition, we characterized variations in LY, LB, and ST within 7 d relative to CCT or treatment for a specific foot condition (TCT). Concurrent activity data from a static group of cows that did not experience any claw disorder during the study period and were not submitted to CT were used as a reference from healthy controls (HC).Our hypothesis was that cows with abnormal gait or other signs of pain would have altered lying and stepping behaviors the day preceding CT and that the magnitude of the changes would be different in cows requiring corrective CT . The sample size was limited to the number of activity sensors available. A total of 310 Holstein cows (69 primiparous and 241 multiparous) calving between November 2017 and January 2018 were enrolled within 20 DIM and monitored for foot disorders and lameness until June 2018. No criteria other than the calving date were considered in the selection of the study cows. Cows were housed in freestall barns with sand-bedded stalls and had free access to a contiguous dry lot. Average number of cows per pen was 250. Cows were fed a TMR twice a day to meet or exceed the nutritional requirement for a lactating Holstein cow producing 30 kg of milk/d with 3.5% fat and 3.1% true protein . During DD) and interdigital dermatitis or foot rot (FR), as identified by an experienced claw trimmer working full time on the farm. Cows were also reported with foot wounds and unspecific lameness conditions (LAM). Briefly, DD was defined as a painful papillomatous digital lesion, mostly in plantar or palmar skin bordering the interdigital space (Cows received CT at least once every 6 mo (approximately at dry-off and again around 180 DIM), and the study herd had a history of lameness associated with digital dermatitis , cows were affixed with an accelerometer  below the fetlock of one hind leg using a hook-and-loop band. The accelerometer validated by The study pens were monitored daily by an experienced claw trimmer to detect cows with altered gait or other signs of pain, such as an arched-back posture or reluctance to bear weight on one or more limbs or feet, which would be equivalent to a lameness score >2 from Animals diagnosed with foot disorders were treated according to the farm protocol using therapies approved for use in USDA-certified organic dairies . The maiTHI) values were calculated , and cow information was retrieved from PCDART herd management software (Dairy Records Management Systems). Ambient temperature and humidity were continuously measured using Hobo UX100-011 temp/RH 2.5% loggers (Onset Computer Corp.) installed in 6 different points through the farm, and daily temperature-humidity index , early lactation (22–150 DIM), and mid lactation (151–212 DIM). Access to pasture, THI category , and concurrent estrus were also tested in the models to account for the potential effects of theses variables on activity. The differences in lying and stepping behaviors on d −1 by CT category were tested by ANOVA (PROC MIXED). The final model included CT category, parity category , concurrent estrus, access to pasture, and THI . LogistiRepeated-measures ANOVA (PROC MIXED) were used to assess daily activity behaviors before and after CT (±7 d), and daily comparisons were completed. The model included CT category (considering specific disorders within TCT), time relative to CT, parity category, concurrent estrus, access to pasture, THI, DIM category, and the interaction between CT category and time relative to CT as fixed effects and cow as random effect . The TukP ≤ 0.10. For model building, individual explanatory variables were initially tested in univariable models. Variables associated at P < 0.25 in the univariable analyses with each outcome variable were included in the multivariable models. The manual backward elimination method was used to remove any variables with P > 0.1. Significance and tendency levels were declared at P < 0.05 and P ≤ 0.1, respectively. As the sample size was limited by the availability of activity sensors and the moderate number of study cows, results should be interpreted with caution.Pearson correlation coefficients were calculated for all explanatory variables that were considered for inclusion in the multivariable models to detect issues with collinearity, and signs of strong collinearity were not found (−0.32 < r < 0.48). Two-way interactions between all the predictor variables were tested and kept in the final models if P < 0.001 and P < 0.001, respectively). Overall, LB were not statistically different among groups . However, TCT cows had the fewest LB during early and mid lactation. Daily ST were lower in TCT cows  than in CCT  and HC  cows  and were excluded from the analyses. No cows experienced 2 CT events during the study period. On d −1, LY (min/d) was greater for TCT cows  than for CCT  and HC  cows , with power = 80% and confidence = 95%, the difference required to determine significance was 2.3 bouts/d. Although sample size and variation were also limitations for the analyses of LY and ST, the size of the differences in LY and ST between TCT and the other groups was sufficient to establish statistical significance.In our study, ST was the most consistent behavior across all stages of lactation and was smallest for TCT at all the DIM categories. Notably, at d −1, TCT cows had on average 993 and 732 fewer steps/d than CCT and HC cows. In agreement, Results from this study indicated that activity behaviors on the day before CT differed for cows having CCT or TCT, which could be an indication of the predictive potential of these behavioral changes. Relative to HC, LY increased by 8.6% in TCT cows and ST decreased by 28%, leading to the possibility of detection of cows needing therapeutic assistance. Notably, this was not the case for CCT cows, which did not show statistical differences from normal cows. These results could be valuable in prioritizing treatment application on the farm and identifying animals that urgently required treatment.To avoid the effect of management and displacements at the day of CT, we analyzed the associations between categories of activity behaviors during the day preceding CCT or TCT. Our results indicated significance for the association between changes in the 3 behaviors and TCT . On the contrary, only the reduction in ST was associated with subsequent CCT (odds ratio: 3.32; These findings support the potential of automated lameness detection systems that are already in use on commercial farms. However, sensitivity values to predict CT using the suggested categorization of behaviors on d −1 were low, whereas specificity values were moderate. This suggest that the selected cut-off value for the classification of positive and negative cows should be re-examined and that approaches combining multiple variables should be considered.Notably, our comparison of activity behaviors within ±7 d of CT evidenced some differences in the timing and magnitude of the behavioral changes for distinct claw conditions. Moreover, times to recover and approach levels similar to those of HC were also distinct for some disorders, such as FR . Miguel-In our study, behavioral changes were most evident for FR, where LY, LB, and ST clearly deviated from CCT and normal cows in a large magnitude and for extended time . SpecifiWe concluded that, under the current experimental conditions, cows requiring TCT evidenced greater overall LY and lower daily ST the day before CT. However, these differences were not consistent throughout lactation periods. Type of CT was partially associated with category of activity the day preceding CT, and the associations were more evident for TCT. The magnitude of the behavioral changes before and after CT and the recovery times required posttreatment to approach HC behavioral levels varied depending on specific conditions.Monitoring behavioral variables has the potential to detect cows needing interventions for locomotion disorders. However, the detection of specific lameness disorders using activity variables in dairy cows requires further consideration. Future research may be warranted to validate these variables and explore the use of combinations of multiple behaviors.


TM microcellular injection molding process and were tested for their impact resistance, dynamic properties, and vibration response. Material constants were compared to the properties of unfoamed door panels. The changes in mechanical behavior were explained through the underlying differences in their respective microstructures. The specific storage modulus and specific elastic modulus of foamed components were within 10% of their unfoamed counterparts, while specific absorbed energy was 33% higher for the foamed panel by maintaining the panel’s mass/weight.To maximize the driving range and minimize the associated energy needs and, thus, the number of batteries of electric vehicles, OEMs have adopted lightweight materials, such as long fiber-reinforced thermoplastics, and new processes, such as microcellular injection molding. These components must withstand specific loading conditions that occur during normal operation. Their mechanical response depends on the fiber and foam microstructures, which in turn are defined by the fabrication process. In this work, long fiber thermoplastic door panels were manufactured using the Ku-Fizz The global demand for battery electric vehicles (BEV) has grown over the past decade to meet climate action targets and governmental energy policies . With a Ku-Fizz’s concept and gas pressure operating window (<3 MPa) significantly differs from the leading foaming technology MuCell (<20 MPa). Upgrading traditional IM machines to offer MuCell foaming capabilities involves installing a specially designed screw to generate a single-phase solution and a super critical fluid (SCF) metering, delivery, and dosing system b, while Both foaming and LFTs have demonstrated their potential in the automotive industry ,13,14,15For this purpose, the mechanical response of a SCANIA AB inner truck door panel was evaluated and the difference in response of the foam and compact  part was assessed. This door panel is used in all SCANIA trucks and is assembled to the cab door. Several components, such as an electric control unit, a door lock, a door window winder mechanism, and motors, are assembled to the door panel. The principal design attributes for these assembled door bodies are static stiffness, strength, fatigue durability, crashworthiness, and vibration frequency response . The typDoor panels also need to withstand a side impact collision to protect the occupant’s chest and pelvis from door contact . During Door panels respond dynamically to loads, which is also measured in terms of their vibration . VehicleThis study determines the mechanical performance of lightweight foamed SCANIA AV truck door panels and shows its microstructural and mechanical differences to compact panels. First, the fiber and foam MS were measured in key locations to aid in the explanation of the observed mechanical behavior. Second, DMA testing was performed for coupons at various fiber orientations (FO). Third, a side pole impact event was simulated by conducting drop-weight impact tests to determine the energy absorption and evaluate failure behavior characteristics. Fourth, free–free vibration hammer tap tests were performed to obtain the vibration frequency response. The door panel was tested in isolation without any interaction with other components. Testing frequencies were based on loading scenario door panel encounters during normal operation .TM). Pultruded pellets have a nominal fiber length (FL) of 15 mm. The fiber diameter was measured to be 19 ± 1 μm using an optical microscope. The material was dried at 80 °C for 4 h to remove any moisture.The material used in this study is a commercially available 30 wt% long glass fiber-reinforced polypropylene (SABIC STAMAX1400-5200 IM machine equipped with a Ku-Fizz hopper unit. The processing settings are summarized in SCANIA AB R-Series truck door panels (813 mm × 457 mm × 2.2/2.0 mm) were molded by Idé-Pro on a 1000-ton Demag Ergotech 1000/2 (L × T) were cut out, as shown in The foam morphology and its representative values, CD and CS, were determined by the procedure outlined in . The tec2 × 2.0/2.2 mm were scanned with an industrial μCT system . The resolution of the μCT system is 4 µm. Throughout the reported studies, the voltage was set to 80 kV, the current to 100 µA, the integration time to 1000 ms, the gain to 8, the number of projections to 2200, and the voxel size was set to 5 μm.FO and fiber concentration (FC) were obtained with the X-ray microcomputed tomography (µCT) approach described in . Discs oThe FL measurement procedure presented in  was emplViscoelastic and damping properties were analyzed with the TA Instruments Rheometric Series RSA 3 DMA equipped with a 35 N load cell. Tests were performed using a 3-point bending configuration with a 40 mm span. Specimens (44 mm × 12.7 mm × 2.06 or 2.15 mm) were extracted at various angles with respect to the flow direction, as shown in Rectangular specimens (152.4 mm × 101.6 mm × 2 mm) cut from the door panel  were tesTo estimate the modal properties of the door panels, a free–free vibration hammer tap test was performed. The panels were hung on an adjustable steel frame with elastic strings as shown in For each hammer input location, the hammer signal and the three-accelerometer signals were recorded with a data acquisition system (Bruel and Kjaer Lan-Xi 3160 A-042 Generator Module). The time domain signals were processed to estimate the frequency response functions (FRF) for each of the three accelerometer locations versus the hammer input location. This was repeated for five hammer taps at each input location, and the resulting FRFs were averaged. Each hammer tap that contributed to the average was verified to ensure the hammer made a single contact with the door panel. The algorithm of mode isolation (AMI) was used to determine the natural frequencies and damping ratios of modes in the response . First, MAC) analysis was conducted to examine the similarity between compact and foamed mode shapes [MAC was calculated according to Equation (1), where mode A modal assurance criterion  as the flow distance increases [YY) to the weld line [The presence of gas changes the matrix rheology, effectively modifying the velocity profile and its effect on FO. Additionally, bubble growth displaces and re-aligns fibers ,50,53. Tncreases . Locatioeld line . Foamed panels presented slightly less alignment in the flow direction when compared to their compact counterparts. These findings agree with published studies. Kim et al., 2019, found that cell growth induced the randomization of the FO in MuCell foamed glass fiber-reinforced PP plates . Yang etCell growth in the core region tends to displace fibers towards the mold surface, effectively changing the FC profile . This phThe behavior experienced during the dynamic strain sweep can be seen in To study the difference in E’ between compact and foamed panels, coupons were cut in various orientations with respect to the flow direction to randomize the effect of FO. Measurements were taken until 46 Hz, as samples would lose contact with the bending fixture. Results from the isothermal frequency sweeps provide valuable insight into the behavior of door specimens at different time scales . E’ incrAverage orced PP . Both compact and foamed specimens absorb impact energy by a complete failure. Crack propagation was evaluated using micro-CT scans. Hammer-tap tests were conducted to determine the effect of MIM on the vibration response. Natural frequencies from the modal analysis of acceleration FRFs were estimated by fitting prominent peaks in the composite FRF with AMI. The first two peaks in the range of zero to five Hz were not fitted, as these modes were identified as rigid body modes of the panels on the suspension system and were omitted. Following all the individual fits, AMI refines a multi-mode model that includes all the individually fit peaks. The composite FRF for the compact and the foamed door panels is shown in Regarding the damping of composite plates, it can be noted that compact door panels exhibited better damping properties for modes 1, 3, 4, and 7–9 than their microcellular counterparts. This phenomenon could be explained by the presence of localized large voids introduced by the dynamic nature of MIM . In addiPlate mode shapes for compact and foamed panels are shown in It is important to note that the composition and structure of the object, material anisotropy, part density, L/T ratios, and the manufacturing process all influence the vibration response at various degrees, and thus, changes in modes and their shapes cannot be reduced to one physical characteristic. Vibration response characteristics and thus mode shapes are affected by cell generation, which in turn affects the fiber microstructure. This work has shown that the presence of gas results in an increase of FL, modifies the FC profile, and increases normal to flow fiber alignment due to reduced matrix viscosity. Longer fibers and higher fiber volume fractions increase the stiffness of foamed panels and, therefore, increase natural frequencies . During Beam cutout samples were also used to reveal differences between foamed and compact door panels. The frequency span was limited to the first three natural frequencies, and the corresponding composite FRFs are shown in The estimated elastic modulus is shown in W), increased randomization of FO, and fiber displacement towards the mold surface were observed. Cell MS analysis revealed high CDs and small CSs close to the cavity wall and decreased CD and larger cells in the core region of the part. CD values up to 1.14 × 105 were recorded. The impact of gas on the MS varies in magnitude and is dependent on the gas pressure operating window, which is one of the main differences between traditional MIM Mu-Cell and the more recent technology Ku-Fizz. The MS resulting from the Ku-Fizz process caused relevant changes in the mechanical response of door panels. The introduction of gas affects structural variables in two ways; first, it generates a cell distribution; second, it modifies the matrix rheology; and thus, it impacts the main fiber characteristics . For foamed panels, a 14% increase in FL (LFor DMA testing, a 3-point bend configuration and 0.05% strain were used. For these conditions, compact samples showed a 20% higher E’ over a 1–50 Hz frequency range. Ku-Fizz is a cost-effective competitor to the leading MIM technology, MuCell. Upgrading an IM machine to offer Ku-Fizz foaming capabilities requires an upfront capital investment of USD 40–60,000 depending on the IM machine size. In comparison, the upgrade costs for MuCell are approximately USD 180–300,000 or 35% of the IM machine cost . The addAs vehicles become lighter, even percentual weight reductions can represent important savings in battery life and fuel efficiency. In this study, there was a 10% weight reduction for foamed door panels. However, weight reduction comes at the cost of decreased mechanical performance. Although it is difficult to draw direct correlations between individual MS variables and the mechanical response due to the complex geometry and mold filling pattern, it is useful to look at generalized properties to assess the impact of MIM.


The global consumption of herbal medicine is increasing steadily, posing an extinction risk to medicinal plants. Uganda is among the top ten countries with a high threat of herbal medicine extinction, and Traditional Medicinal Knowledge (TMK) erosion. This might be attributed to the inadequate documentation, plus many more unclear hindrances. In this study, plant species used to treat human diseases in Butaleja district in Eastern Uganda and their associated TMK were documented. The conservation methods for medicinal plants were also evaluated. The rationale was to support the preservation of ethnopharmacological knowledge.Data were collected from 80 herbalists using semi-structured questionnaires, from July 2020 to March 2021. Additionally, guided field walks and observations were conducted. Quantitative indices such as, use categories and informant consensus factor (ICF) were evaluated to elucidate the importance of the medicinal plants. Data were analyzed using STATA version-15.0 software.In total, 133 species, belonging to 34 families and 125 genera were identified. Fabaceae (65%), and Solanaceae (29%) were the dominant families. Leaves (80%), and roots (15%), were the commonest parts used in medicinal preparations; mostly administered orally as decoctions (34.6%) and infusions (16%). The commonest illnesses treated were cough (7.74%), gastric ulcers (7.42%), and malaria (4.52%). The informant consensus factor was high for all disease categories (≥ 0.8), indicating homogeneity of knowledge about remedies used. Only 73% of the respondents made efforts to conserve medicinal plants. The commonest conservation strategy was preservation of forests with spiritually valued species (100%), while compliance with government regulations was the rarest (4.5%). Overall, efforts to stop the extinction of medicinal plants and TMK were inadequate.There was enormous dependency on a rich diversity of medicinal plant species and TMK for healthcare and income generation. The potential for medicinal plant biodiversity loss was evident due to habitat destruction. Inclusion of traditional cultural norms in conservation strategies, and laboratory-based efficacy tests for the species identified are necessary, to promote the conservative and utilization of validated herbal medicines and TMK in rural settings.The online version contains supplementary material available at 10.1186/s41182-022-00428-1. Plants have been used for their therapeutic properties since antiquity , 2. ConsIn Uganda, the prevalence of herbal medicine use was estimated to be 60% , 11. TheIn the recent past, Uganda has been listed among the countries that are facing the greatest risk of future extinction of medicinal plants , 16. UnfThe study was conducted in three villages namely; Budoba, Bubuhe and Mulanga, located in Busaba sub-county, in Butaleja district. Communities in the selected villages have limited access to medical facilities and thus heavily dependent on medicinal plants for primary health care. The district is located in the remote part of Eastern Uganda, 38 km from Mbale city and 247.8 km from Kampala, the country’s capital and major commercial city. Butaleja district lies between of 33° 45″ E to 33° 53″ E and 0° 51″ N to 0° 55″ N  and Luganda (Lug). With the help of key informants, voucher specimens of all mentioned medicinal plant species were collected and prepared following standard procedures . SpeciesNur is the total number of plant-use reports (citations), for a disease category, and Nt is the total number of plant species used for that ailment category.The ailments treated were categorized basing on the eight systems of the human body, cause of disease, and the medication characteristics of the people of Butaleja. The informant consensus factor (ICF) was then calculated to assess the homogeneity in the ethnomedicinal information documented from the traditional health practitioners based on diseases treated and plant species used  using th-15.0. The results were summarized in form of bar graphs and tables. The graphs were generated with GraphPad Prism 8.0 software .Simple descriptive statistics were adopted for data analysis using STATA versionAll participants were Ugandans with the majority, 59 (74%) being male. Most participants, 36 (45%) were in the age category of 31–40 years while the minority were of ages ≥ 71 and ≤ 30 years. Seventy-nine (99%) of the respondents were married while 1 (1%) was single. Most of the respondents, 49 (62%), had attained the primary level of education while the minority, 1 (1%) were had tertiary education (Table Mangifera indica L. (TFM = 98), Momordica foetida Schumach (TFM = 96), Aloe wollastonii Rendle (TFM = 81), Tamarindus indica L (TFM = 80), Cannabis sativa L. (TFM = 73) Aristolochia elegans Mast. (TFM = 72), Vernonia amygdalina Delile (TFM = 62), Bidens pilosa L. (TFM = 61), Aloe vera (L.) Burm.f. (TFM = 59) and Carica papaya L. (TFM = 55). The major plant part used in herbal medicine preparations was the leaf; 81 (80%) followed by the root; 15 (15%). Out of the 133 medicinal plant species recorded, herbaceous species were dominant. There was wide variation in the number of diseases treated by each species. For instance, Persea americana Miller, Rumex usambarensis (Dammer), Allium sativum L., Mangifera Indica L., Aristolochia elegans, Aloe vera (L.) were reported to treat more than four diseases each.A total of 133 medicinal plant species belonging to 34 families and 125 genera were identified , Blood pressure (FM = 54) cough (FM = 49) and malaria (FM = 42), respectively. There were similarities and variations in the use of plant species in the different villages in the study area. Diseases whose frequency of citation was between 1 and 4 were categorized among “others” of which 44, 25, 9 and 5 had citation of 1, 2, 3 and 4, respectively. Most herbalists believed in spiritualism and witchcraft, and thus some plants, such as Dyschoriste radicans (Hochst. ex A. Rich.) Nees, and Chenopodium ambrosoides L., were being used in the management of complications related these two beliefs. Such complications included evil spirit possession, madness, swollen legs and bad luck among others ; followed by drinking of infusions, 16%); and the use of concoctions, (8.3%) . Others included; distant forests, (10%); markets, (9%); and spiritually protected forests, (Engolo). Wetlands were the rarest source of medicinal plants (6%). All the THP mentioned obtaining TMK as an inheritance from parents and grandparents. Experimentation , was the least mentioned source of TMK (6%) Fig. .Fig. 2SoFifteen disease categories were identified Table . The higTo ensure sustainability of herbal medicine use, most of the TMP 73%) mentioned that they made efforts to conserve medicinal plants compared to 21 (27%) did not besides abiding to the communal restrictions. Preservation of spiritually protected forests, locally known as “Engolo” was the most frequently mentioned conservation strategy, 80 (100%); while compliance with government laws and policies was the least mentioned approach, 4 (5%) . The high ICF values mean that there is a high level of agreement regarding the plant species used to treat various ailment categories in the study area and that such species are effective . The facOn the other hand, the low ICF values observed in the current study may possibly be attributed to, (1) the fact that herbalists are often secretive with their traditional medicinal knowledge leading to differences in the species they cite for a particular disease category; (2) availability of a rich diversity of medicinal plants species; and (3) differences in the sources of medicinal traditional knowledge in herbalists’ communities.Though the conservation of medicinal plants was highly threatened by multiple factors such as habitat loss, population pressure and the increasing dependence on herbal medicine as a source of income, their conservation was promoted through cultural beliefs and norms. These included; gender-restricted harvesting, mouth harvesting, night and naked harvesting, and the gazetting of spiritually protected forests (Engolo) for each clan, from which harvesting is only authorized by the clan head. Further, there was a common belief among many herbalists that the onus to conserve medicinal plants and the associated knowledge was a divine duty entrusted on them by ancestral spirits. Spiritual beliefs have been reported to play a major role in conservation of resources , 60. In Though peer learning was also cited, it is likely to become minimal with increased commercialization of the herbal medicine industry, due to fear of competition among the herbalists. Notably missing was the adoption of technical training of the young generation as a means of conserving TMK. This points to complete lack of integration of herbal medicinal use into the health care system. Therefore, there is no reliable method of indigenous medicinal knowledge conservation in the study community. This calls for the need to devise reliable conservation measures to arrest the possible loss of TMK in the rural communities as previously reported elsewhere . CompliaThis study showed that Butaleja district has a high diversity of plant species used as remedies for several ailments. The high dependency on herbal medicine for primary healthcare and income generation in Butaleja district calls for preservation of this resource and the associated TMK. A majority of the medicinal plant species used belong to the Fabaceae and Solanaceae, and they were most frequently used in managing some of the world’s most killer diseases, such as; respiratory tract ailments and malaria. There was a general consensus on the plant species used to manage diseases in different categories which is a clear indicator that such plants are effective and worth further investigation for bioactive ingredients and their associated pharmacological activities. This will be instrumental in establishing the dosages, toxicity and efficacy levels of different herbal preparations and could act as a springboard to discovery on new plant-based drugs.The conservation of medicinal plants, and the associated indigenous traditional knowledge, was strongly attributed to traditional cultural beliefs and norms. Therefore, traditional cultural values should be incorporated into the national biodiversity conservation agenda, owing to the extraordinary role of culture in inspiring the conservation of medicinal plants and the TMK observed in this study. There is need for laboratory-based experiments on the plants identified in this study to confirm their safety and efficacy. This would support the adoption of these plants in the global fight against the deadly diseases they were claimed to treat.Additional file 1. Table S1. Medicinal plants used for treatment of common ailments in Butalejja District, Eastern Uganda.