Background: Patient-controlled epidural analgesia (PCEA) is well documented; however, it is unclear whether a high dosage of PCEA with a low dosage of background infusion during labor can be a safe and effective application. Methods: Group LH was administered a continuous infusion (CI) of 0.084 mL/kg/h with PCEA of 5 mL every 40 min. Group HL was given a CI of 0.028 mL/kg/h and PCEA of 10 mL every 40 min; Group HH was given a CI of 0.084 mL/kg/h and PCEA of 10 mL every 40 min. The primary outcomes were VAS pain score, the number of supplemental boluses, incidence of pain outbreaks, drug dose for pain outbreaks, PCA times, effective PCA times, anesthetic consumption, duration of analgesia, duration of labor and delivery outcome. Secondary outcomes included adverse reactions such as itching, nausea and vomiting during analgesia and neonatal Apgar scores 1 min and 5 min after birth. Results: A total of 180 patients, 60 in each group were randomly assigned to one of three groups included group LH, group HL or group HH. The VAS scores were obviously decreased in HL group and HH group in comparison with LL group at 2 h after analgesia and the time point of full cervical dilation and delivery of baby. The time for third stage of labor in HH group was increased compared with LH group and HL group. Incidence of pain outbreaks in LH group was obviously increased compared with HL and HH group. The effective PCA times in HL group and HH group were remarkably reduced compared with those in LH group. Conclusions: High dose of PCEA with a low background infusion can reduce effective PCA times, incidence of outbreak pain and the total amount of anesthetics without diminishing analgesia effects. However, high dose of PCEA with a high background infusion can enhance analgesia effect but increase the third stage of labor, instrumental delivery ratio and the total amount of anesthetics. While epidural labor analgesia is currently recognized as the safest and most effective type of labor analgesia, patient-controlled epidural analgesia (PCEA) combined with continuous infusion of local anesthetics and opioids (PCEA + CEI) is the most common type of drug administration. Manual bolus administration by PIEB  or CEI  is recognized to provide satisfactory analgesic effect and reduce the accumulation of the toxicity of local anesthetics; however, manual injection is time-consuming and laborious, and it is not realistic to popularize it in the clinic. The safety of PIEB in labor analgesia has not been confirmed for a long time. The greatest impediment to the implementation of PIEB analgesia is the lack of readily available epidural pumps designed to deliver timed boluses or time boluses with PCEA. The two-pump system we used is not clinically practical. Further studies are warranted to determine whether this technique has benefit in other clinical applications of epidural pain management. Outbreaks of pain are very common during labor analgesia. Programmed intermittent epidural bolus (PIEB) has demonstrated many advantages compared with background epidural infusion concerning breakthrough pain, by adding PIEB to background epidural infusion + PCEA improved labor analgesia by obviously decreasing the needs of rescue analgesia and extending the effect of sufficient analgesia. This combination led to a higher consumption of local anesthetic with no significant clinical complications .Previous studies have showed that PIEB can increase maternal satisfaction score and decrease the consumption of epidural drugs compared with a continuous epidural infusion ,3,4,5, wThe current ASA Practice Guidelines  do not pwww.chictr.org.cn . Written consent was obtained from all participating parturients. The 193 parturients were randomly assigned by a computer-generated list to one of three groups, group LH, group HL or group HH, which was included in a total of 180 patients. Sixty were in each group; thirteen women did not receive allocated intervention.This study was approved by the Ethics Committee of the International Peace Maternal and Child Health Hospital Shanghai Jiaotong University School of Medicine . VAS was still more than 3 at 30 min after the first dose; (2). Dural perforation, analgesia termination due to epidural catheter prolapsing into the bloodstream.All groups received an initial epidural drug before connecting to the PCEA pump. Group (LH) was administered a 5 mL PCEA  bolus with a continuous background infusion of 0.084 mL/kg/h via an analgesic pump. Group (HL) was given a 10 mL PCEA  bolus with a continuous infusion of 0.028 mL/kg/h. Group (HH) was given a 10 mL PCEA  bolus with a continuous infusion of 0.084 mL/kg/h based on the previous study [−1·h−1 lactated Ringer’s solution. The anesthetic solution mixture used in this study was prepared beforehand according to the following formula: dissolve 0.75% ropivacaine (AstraZeneca batch number: LBDX) 100 mg and fentanyl injection  0.2 mg in saline to form a 100 mL mixture containing 0.1% ropivacaine and 2 μg/mL fentanyl. The maternal heart rate, blood pressure, fetal heart rate and contraction intensity were monitored. After routine disinfection and local anesthetic, an 18G epidural catheter was inserted into the epidural space at the L2~3 or L3~4 interspace with the parturient lying down in the left lateral position. The epidural catheter was placed 4–5 cm towards the cephalic side, ensuring no blood or cerebrospinal fluid reflux; a test dose of 2% lidocaine 3 mL with 1:200,000 adrenaline was administered. After a 5 min observation showing that no general spinal anesthesia or extensive epidural block occurred, 10 mL of the mixture was injected via the epidural catheter, with 3–5 mL each time. If the VAS score of the parturient exceeded 3 after 10 min, an additional 5 mL bolus would be administered. If the parturient continued to have inadequate analgesia (VAS > 3), she would be excluded from this study and the anesthetic solution would be replaced with 0.15% ropivacaine plus 2 μg/mL fentanyl for better analgesic effects. If in the process of pushing the injection, the maternal patient had dizziness, nausea and other discomforts, the case should be eliminated. Loss of temperature sense was tested by rubbing alcohol on the skin; when the maternal temperature sense loss reached T10, T6 with VAS < 3, the Ogilvy & Mather Analgesic Pump (AM-3200) with the anesthetic solution intact would be connected. The parturients were told to press the demand button for analgesics when the VAS score exceeded 3. Drug administration would be terminated after the third stage of labor. The demand for drugs was considered invalid if the parturient pressed the button within 40 min of the previous dose. If the demand button was pressed twice within 20 min without pain relief, it was recorded as an incidence of pain outbreak. To ease the pain during the subsequent labor process, 0.1% ropivacaine plus can be manually injected. If the VAS was still greater than 3 scores after 10 min, the anesthesiologist would administer an additional 5–10 mL of 0.2% ropivacaine plus 2 μg/mL fentanyl. If during the analgesia process, the fetus heart > 180 bpm, or <100 bpm, or if late fetal heart deceleration, mutation deceleration or (and) baseline lack of variation occurs, suspend epidural injection and observe for more than 30 min, and then determine the cause of fetal heart mutation.After the parturients entered the delivery room for labor, the upper extremity intravenous channel was established, infusing a 10 mL·kgRandom numbers were placed in airtight envelopes, which were sealed; this was done by an anesthesiologist who was not involved with data analysis. Before inducing anesthesia, the same anesthesiologist who performed the randomization opened the envelopes in sequence. The clinical investigators, data collectors and patients involved in the study were blinded to the experimental group assignments and the drug-randomization sequence.VAS scores and the modified Bromage scale  of the parturients were assessed as the primary outcomes before analgesia, 10 min-, 30 min-, 1 h-, 2 h-post analgesia, during full cervical dilation and delivery. The number of supplemental boluses, incidence of pain outbreaks, drug dose for pain outbreaks, PCA times, effective PCA times, anesthetic consumption, duration of analgesia , duration of labor and delivery outcome were recorded as the primary outcomes. Adverse reactions such as itching, nausea and vomiting during analgesia and neonatal Apgar scores 1 min and 5 min after birth were noted as the secondary outcomes.The study was approved by the Ethics Committee of the International Peace Maternal and Child Health Hospital Shanghai Jiao Tong University School of Medicine, and all participants provided written informed consent for themselves and their infants.2 test or the Fisher exact probability method. p < 0.05 was considered statistically significant.Data were processed using SPSS 22.0  statistical software. The significance level was set at α = 0.05, and the power was 1–β = 0.8. Using PASS version 22.0 software for the analysis with 60 participants in each group, a possible 10% missing rate was considered. Thus, a total of 200 patients were included in this study. Numerical data that conformed to the normal distribution were expressed as mean ± standard deviation. Analysis of variance (ANOVA) with post hoc test was used for comparison among groups, and the count data were analyzed using the χA total of 200 women met the inclusion criteria for this study. Of these, the participants were randomized and 180 women received statistical analysis .n = 2); history of neuropsychiatric disorders(n = 2); high-risk pregnancy(n = 3).Seven women did not receive allocated intervention because of those patients suffering from some diseases including contraindictions for intraspinal anesthetic . The VAS values were obviously decreased in HL group in comparison with LH group at 2 h after analgesia (p < 0.01). Meanwhile, compared with LH group, the VAS values in HL and HH group were significantly reduced at the time point of full cervical dilation and delivery of baby (p < 0.001). The VAS value in HH group was lowest at the time of delivery of baby among three groups . The times for PCA boluses and effective times for PCA boluses in LH group were significantly higher than those in HL and HH groups . Cumulative amount of epidural infusion doses in HL group was lowest among the three groups (p < 0.001). Incidence of pain outbreaks in HL and HH group was obviously decreased compared with LH group. The PCA times and the effective PCA times in HL and HH group were remarkably reduced compared with those in LH group.The time for analgesic duration, first stage of labor and second stage of labor in three groups were of no significance as the primary outcomes . HoweverThere were no significances in three groups for Cesarean ration; meanwhile, instrumental and vaginal delivery ratio as the secondary outcomes were increased in HH group compared with HL group .The timing of labor analgesia has been debated by researchers around the world. Studies have shown that as long as the parturient was proven eligible for vaginal delivery by the obstetrics, when showing signs of regular contraction and normal cervix activities, labor epidural analgesia could be administered if required. However, prolonged epidural labor analgesia could cause anesthetics to reside in the epidural space, which may lead to local anesthetic and opioid accumulation. An excessive amount of local anesthetics can cause neural motor blockade, which in turn could reduce maternal exercise capacity, weakening the pelvic muscles and the force of “pushing down” during the second stage of labor, which may lead to dystocia or aided childbirth . Our stuThe mechanism of low-dose background infusion combined with high-dose PCEA was similar to that of epidural intermittent injection. The high pressure from the PCEA injection allowed the drug to be rapidly injected into the epidural space through the anterior and lateral orifice of the epidural catheter. The anesthetic would then be evenly distributed and more suited for the individual, thus improving the analgesia effects, reducing the consumption of anesthetics, shortening the second stage of labor compared with LH group, and avoiding anxiety from contractions that could have a negative impact on the newborn. This technique would not influence the analgesic effect during labor, nor would it affect the progress of labor. The high pressure from the infusion widens the diffusion range, inducing a higher level of anesthesia to alleviate the pain, resulting in a lower VAS score from the parturient. In recent years, there have been several research works regarding drug administration for labor analgesia, but the results were inconsistent. Some scholars indicated that no conclusion can be drawn regarding the risks or benefits of adding a continuous background infusion to PCEA compared with PCEA-only epidural labor analgesia .There are still controversies in terms of the optimal anesthetic concentration, delivery volume, administration interval and administration mode (on-demand or regular). Although the results in such studies are not completely consistent, one element is basically the same; the effect of epidural intermittent analgesic administration is generally better than high-dose continuous infusions . In a stAs for whether a background infusion dose was needed, studies have shown that PCEA+CI provides better analgesia effects with significant reduction in the incidence of pain outbreaks during labor compared to only PCEA. In a previous study, compared with background PCEA , PCEA required only  resulted in reduced local anesthetic consumption but increased incidence of breakthrough pain, higher pain score, shorter duration of effective analgesia and lower satisfaction level for parturients [In recent years, researchers have become focused on a new type of labor analgesia technique called programmable intermittent epidural bolus (PIEB). McKenzie et al.  believedTherefore, the safety of the application of PIEB during the whole process of labor, and its corresponding drug concentration and analgesic pump settings, remains to be further studied. We have acknowledged the limitations in this study. For instance, the sample size of this study is relatively small, and we failed to observe the whole process of labor analgesia. The settings of the analgesic pump in this study  were set according to our own experience. Although the results show that anesthetic consumption had significantly decreased without weakening the analgesic effects, it still may not be the optimal setting. For Chinese parturients, there is a risk that the level of anesthesia from 10 mL of PCEA may be too high for some individuals even if no obvious cardiovascular events or complications occurred such as hypotension and so on, Studies with larger sample size are needed to further investigate the optimal settings for a full-course labor analgesia.A low background infusion (0.028 mL/kg/h) combined with a high-dose PCEA  is a safe and effective clinical application for labor analgesia, which reveals an obvious advantage over high background infusion (0.084 mL/kg/h) combined with a high-dose PCEA  by decreasing instrumental delivery ratio, PCA times, effective PCA times, incidence of outbreak pain and the total amount of anesthetics without reducing analgesia effects. However, regardless of the analgesic method, rigorous monitoring, active and effective interventions by clinical doctors are still an indispensable part of a safer and ideal labor analgesia. Clinical strategies will be patient-oriented and constantly improve the clinical safety of maternal and infant patients and the satisfaction of parturients and their families.


While TFand EOF were not influenced by sex, women had higher UEOF comparedto men, opposite to target PFAS. This is the first FMB in human serumto include TOP, and it suggests that precursors with >4 perfluorinatedcarbon atoms make a minor contribution to EOF (0–4%). Additionaltools are therefore needed to identify substances contributing tothe UEOF in human serum.Of the thousandsof per- and polyfluoroalkyl substances(PFAS)known to exist, only a small fraction (≤1%) are commonly monitoredin humans. This discrepancy has led to concerns that human exposuremay be underestimated. Here, we address this problem by applying acomprehensive fluorine mass balance (FMB) approach, including totalfluorine (TF), extractable organic fluorine (EOF), total oxidizableprecursors (TOP), and selected target PFAS, to human serum samplescollected over a period of 28 years  in Tromsø,Norway. While concentrations of TF did not change between samplingyears, EOF was significantly higher in 1986 compared to 2007 and 2015.The ∑ Thecombined application of targeted and group-wise analyseson pooled serum samples enables the evaluation of the contributionof known and so far unidentified fluorinated compounds in human serumthrough time. Indeed, a growing number of fluorine massbalance (FMB) studies in human blood have quantified large fractionsof extractable organic fluorine (EOF) that cannot be explained bytargeted PFAS analyses.18 One possible explanation for this gap are perfluoroalkyl acids (PFAA)precursors, such as perfluorooctane sulfonamides, fluorotelomer alcohols,and polyfluoroalkyl phosphate esters. Many of these substances havebeen detected in human blood using targeted methodologies,19 but as-of-yet unidentified precursors may alsobe important. The total oxidizable precursor (TOP) assay, in whichPFAA concentrations are measured before and after controlled oxidation,20 offers a promising means for quantifying thetotal contribution from both known and unknown precursors. While theTOP assay has been used successfully to determine PFAA precursorsin environmental samples26 and consumer products,31 there are few examples of its application to human serum,33 and in particular no examples when used in conjunction with an FMB.Of the ∼4600 PFAS registered onthe global market in 2018,34 In the present study, serum samples from the Tromsø Study collectedin 1986, 2007, and 2015 were pooled and for the first time analyzedfor total fluorine (TF), EOF, TOP, and selected target PFAS. Throughthe combined application of a set of targeted and group-wise analyses,we aimed to evaluate exposure to total fluorine and known and unknownorganic fluorinated compounds over time with respect to sex and age.Here, we build upon previous analyses of PFAS time-trends in serumfrom the Tromsø Study, which showed that PFAS concentrationsin the Tromsø population changed according to the history ofproduction and use of these chemicals and that time-trends differeddepending on birth cohort, age group, and study design.2SI.Information onchemicals and consumables is provided in the 2.135 The studyobtained informed consent from all participants and was approved bythe Regional Committee for Medical Research Ethics .TheTromsø Study is a cohort study of the population of Tromsø,the largest city in Northern Norway. Details on the Tromsø Studyare provided by Jacobsen et al.n = 201),2007 (n = 198), and 2015 (n = 130). The samples were selectedbased on a case/control study design on type-2 diabetes: the cases were diagnosed between 2001 and 2007,while the controls  had no diagnosisrecorded in the local diabetes registry. The selected samples included104 women and 97 men in 1986, 113 women and 86 men in 2007, and 72women and 58 men in 2015. The age of the individuals ranged from 17to 61 years old in 1986 (mean: 46), from 38 to 81 in 2007 (mean: 67),and from 46 to 89 in 2015 (mean: 72).The present work utilized 529 individual TromsøStudy serum samples collected in 1986  were pooled basedon sampling year, sex, age, and type-2diabetes diagnosis (Table S1). Sampling year, sex,and age were chosen as variables for pooling because these are knownto influence PFAS concentrations in human blood. Type-2 diabetes diagnosiswas used as a variable for pooling because some studies have reportedassociations between this end point and PFAS concentrations, but itis important to note that evidence for these associations is contradictory.36 Pools 1 to 7 at each sampling year includedthe same individuals in 1986, 2007, and 2015. To have the largestpossible number of pools including the same individuals, these poolswere obtained mixing variable volumes  ofindividual serum samples but keeping the volume per individual constantthroughout the sampling years. For the remaining pools, it was notpossible to follow the same individuals through time and 15 participantswere included in each pool mixing 50 μL of serum per individual.From this selection, 472individual samples are reported in the SI.For TF measurements,100 μL of serum was transferred to a sampling boat for analysisusing a Thermo-Mitsubishi combustion ion chromatograph (CIC) withthe method described by Miaz et al.,2.2.215 Details about quality control measures  are reported in the SI.ForEOF determination, 500 μL of serum was transferred to Eppendorftubes and extracted once with 1 mL of ACN. Samples were vortexed andsonicated (10 min) 3 times, and after centrifugation at 10,000 rpmfor 10 min, supernatants were transferred to 2 mL glass vials. EOFanalyses were performed on 450 μL of the extracts with the sameCIC used for TF analyses and the method described by Miaz et al.2.2.332 Briefly, sampleswere spiked with 13C-PFAA and extracted with ACN. Aftervortexing, sonication, and centrifugation, the supernatant was collectedand split into two portions: one for target analyses before oxidationand one was oxidized for TOP determination. Prior to oxidation, ACNwas removed by evaporation, and the dry extracts were reconstitutedwith 0.8 M Na2S2O8 and 10 M NaOH.Post oxidation, the samples were acidified and extracted with MTBE.Aliquots of the organic phase were transferred to vials with insertand spiked with recovery standard and 2% ammonia in methanol. TheMTBE was evaporated prior analyses. Details about quality controlmeasures are reported in the SI.For the TOP assay, 150 μL of serum was processedusing a previouslypublished protocol.2.2.4Table S5) and were performed using a Dionex UltiMate 3000 Ultrahigh performanceliquid chromatograph coupled to a Q Exactive HF hybrid Quadrupole-Orbitrapmass spectrometer  asdescribed elsewhere.15 For these analyses,50 μL of EOF extracts was mixed with 10 μL of internalstandard and 50 μL of 4 mM NH4OAc in Milli-Q water.Since the internal standard was added after extraction, these concentrationswere not recovery corrected and were only used for FMB calculations.LODs and accuracy of these analysis are reported in the SI.Target analyses onthe EOF extracts included 54 PFAS  as described elsewhere.2 and C3-PFAA using a Raptor PolarX column. Details aboutthese analyses and the quality control measures (including blank concentrationsand LODs) are provided in the SI.After oxidation, the extracts were alsoanalyzed for C2.32.3.112PFAS concentrations obtained from the EOF extracts (Table S6) were subtracted from the EOF concentrations.For this comparison, PFAS concentrations were converted to fluorineequivalents using eq S1. PFAS concentrationsbelow the LOD were set to LOD/√2. The ∑12PFAS concentrations and detection frequencies from the EOF extractsare lower and less accurate than those from the TOP assay extracts(S6) because of the lack of recovery correctionfor procedural losses. However, the use of ∑12PFASconcentrations not corrected for recovery for FMB calculations providesa more representative and accurate result in terms of mass balance.This is because the EOF concentrations cannot be recovery correctedsince the addition of an internal standard before extraction wouldincrease the LOD and it is not possible to correct for the recoveryof unknown fluorinated chemicals present.EOF values were subtracted fromTF concentrations to estimate theamount of inorganic and nonextractable organic fluorine. For thiscomparison, samples with TF values below the LOD were excluded. Toestimate the unidentified portion of EOF (UEOF), the ∑extracts S6 were s33 To establish whetherthere was an increase in PFAA concentrations after oxidation, theratio between the concentration after oxidation and the concentrationbefore oxidation (PFAAafter-TOPA/PFAAbefore-TOPA) was calculated. To avoid the possibility that apparent changeswere influenced by analytical uncertainties, a cutoff of 20% changein PFAA concentrations was applied. Specifically, if the ratio was≥1.2, the difference (ΔPFAA) was calculated as the PFAAconcentration after oxidation minus the PFAA concentration beforeoxidation. If the ratio was <1.2, ΔPFAA was set to zero.The total amount ofoxidizable precursors (ΔPFAA) was estimated as described byCoêlho et al.eq S1). This comparison has someuncertainty because the TOP assay data are corrected for procedurallosses but the EOF data are not.To estimate the contribution of TOP to EOF, ΔPFAA concentrationswere converted to F equivalents with the same equation used for targetPFAS  groups were assessed by multiple linear regression using eq S2. When sex was a significant predictor,differences in concentrations between men and women at each samplingyear were assessed adding an interaction term (eq S3). The inclusion of the type-2 diabetes diagnosis (case/controlstatus) to the multiple linear regression model was tested by usingAkaike information criterion (AIC) model selection. Since the modelwith the lowest AIC score never included the diabetes diagnosis variable,this was not included. TF, EOF, and ∑12PFAS concentrationswere log-transformed before performing regression analyses. Statisticalsignificance was set at p < 0.05. Posthoc powercalculations were performed using the pwr package.Statisticalanalyses were performed using R version 4.1.2 (R Core Team). Priorto statistics calculations, concentrations below the LOD were substitutedwith LOD/√2. Differences in TF, EOF, ∑33.1Table S7). The percentage of pools with TF below the LOD (25ng F/mL) was 33% in 1986, 24% in 2007, and 7% in 2015. Based on multiplelinear regression, there were no significant differences in TF concentrationsbetween 1986, 2007, and 2015 and no significant effect of sex andage (Table S8). For TF, the time differencesobserved in the pools with the same individuals were not consistent,and this could be explained by TF being a sum parameter, includingboth inorganic and organic compounds containing fluorine for whichthe contribution might vary between individuals. In two of these pools,the concentration temporal changes clearly differed from the restof the pools with the same individuals because these were below orclose to the LOD at all sampling years .TF in pooled serumfrom the Tromsø Study ranged from <25 to 1330 ng F/mL, witha narrower range observed in 2015 compared to 1986 and 2007 (15 observed declining TF concentrationsin pooledserum samples from Swedish women collected between 1996 and 2017 (3.2%decline per year), although in that study the cohort was consumingPFAS-contaminated drinking water up until mid-2012.In contrast with the results of our study, Miazet al.Table S7). However, the mean concentrationsof TF in 1986, 2007, and 2015 were comparable to those reported forblood samples from China in 2008 and lower than those reported forserum from Japan in 2003–2004 and plasma from the USA in 2001(Table S7).The rangeof observed TF concentrations in 1986 and 2007 was widerthan those reported in the literature, but in 2015, it was comparable(3.2Table S7). Unlike TF, EOF wasdetected in all pools (LOD = 7 ng F/mL). EOF concentrations in 1986were significantly higher than those in 2007 and 2015, while no significantdifferences were found between 2007 and 2015 .EOFin serum from the Tromsø Study ranged from 12.6 to 45.3 ng F/mLacross all time-points , and asfor TF, this could be explained by EOF being a sum parameter, includingpotentially different PFAS and organofluorine chemicals.ForEOF, the time differences observed in the pools from the same individualswere not consistent  and pooledserum from Austria (2021) but lower than the EOF in plasma from theUSA (2001) and in whole blood from people living in Ronneby, wheredrinking water has been contaminated from firefighting foams (Table S7). However, apparent differences in EOFmeasurements between studies must be interpreted cautiously sincedifferent extraction methods may perform differently for individualfluorinated substances.38 In addition,different EOF values can be measured from different blood fractionssince some PFAS, for example, perfluorohexanoic acid (PFHxA) and perfluorooctanesulfonamide (FOSA), bind minimally to serum proteins and are usuallydetected in whole blood rather than serum or plasma.39TheEOF concentrations observed in our study were in the same rangeas those observed in plasma from Germany collected between 1982 and2009 and in pooled serum samples from Swedish women collected between1996 and 2017. However, no significant time trends were observed forEOF in the German (1982–2009) and Swedish (1996–2017)samples.13 On the contrary, EOF concentrations in samplesfrom Sweden in 2021 were higher in women compared to men.16Based on multiple linear regression, sex and agewere not associatedwith EOF. This observation agrees with EOF measurements in samplesfrom China that also showed no sex- and age-related differences.3.3Table S8).The pooledsamples from the Tromsø Study were also analyzed with the TOPassay to evaluate the contribution of oxidizable precursors. Evenif the increases in PFAA concentrations (ΔPFAA) were low (0.02–1.85ng/mL), all pools (except one from 2007) contained detectable oxidizableprecursors 1. No sigFigure S2).For TOP, the time differencesobserved in pools fromthe same individualswere not consistent, and this could be due to a higher variabilityin precursors exposure or to the low concentrations of precursorspresent. Additionally, for this method a higher variability comparedto target PFAS measurements is expected since the TOP concentrationsare estimated by comparing PFAA concentrations before and after oxidation. Thepattern of oxidation products differed from those observed for modelprecursors spiked into human serum32 andcould not be used to tentatively identify precursors in serum fromthe Tromsø Study. However, even if the structure of the precursor(s)is lost upon oxidation, the profile of the oxidation products offersclues about the chain length of the precursor and the presence ofsulfonic groups. For example, ΔPFDoDA points to the presenceof precursors with 11 or more perfluorinated carbons, while ΔPFBSand ΔPFHpS suggest the presence of precursors containing sulfonicgroups attached to four or seven perfluorinated carbons.32Increases in concentrationsafter oxidation were observed for eightPFCA and three PFSA 1. Perflu33 The patterns of PFAA that increased after oxidation were differentfrom those observed in this study. In contrast to our study, no increasesin the levels of PFDoDA and PFBS were observed. Also, in the TromsøStudy pools, the concentrations of branched isomers of PFOA and PFOSdid not increase after the TOP assay and the detection of ΔPFHpA,ΔPFNA, ΔPFDA, and ΔPFUnDA was limited, while inthe plasma collected from Norwegian women, seven PFAA increased afteroxidation  with the greatest concentration differences observed for PFHpA,branched PFOA, and PFDA. There are several possible explanations forthese differences. First, there could be differences in the exposureamong the studied groups. The samples in this study were collectedfrom both men and women living in Tromsø, while in the Coêlhoet al. study, samples were collected only from women but from allover Norway. Additionally, the sampling years were different in thetwo studies. Second, the use of serum in the present study and plasmain the other could lead to the detection of different precursors.Another possible explanation could be the different extraction methodsused in the two studies, resulting in different extraction effectivenessof the precursors present.The TOP assay has previously been appliedto plasma samples collectedbetween 2003 and 2006 from Norwegian women.3.442 However, someof these precursors have been widely detected in consumer products,such as cosmetics and food packaging.44 This discrepancybetween wide detection in consumer products and low detection in humanblood might be due to a low uptake potential, rapid metabolism, orelimination of precursors in humans, but the contribution of precursormetabolism to indirect PFAA exposure remains unknown.46Inthe pooled samples,12 out of 54 target PFAS were detected: six PFCA , three PFSA , andthree sulfonamidoacetic acids . Branchedisomers were above the LOD only for PFOS. It is interesting to notethat, in agreement with the TOP assay results showing low concentrationsof precursors, no precursors included in the target analyses otherthan the sulfonamidoacetic acids were detected in pooled serum. Other biomonitoring studies investigatingthe presence of these precursors in human blood have also reportedno detection or detection at trace levels (pg/mL).12PFAS concentrationsin 2007 were significantly higher than those in 1986 and 2015 (Table S8). Focusing on individualPFAS changes over time, concentrations of all PFAA in 2007 were higherthan in 1986, except for PFHpA. Between 2007 and 2015, PFSA  and PFOA concentrations decreased, as opposed tothe longer chained PFCA , for whichconcentrations increased. Concentrations of the sulfonamidoaceticacids increased from 1986 to 2007, but none was detected in 2015.PFHpA concentrations were comparable in 1986, 2007, and 2015 .Based on the analysis ofthe Tromsø Study pools, the ∑and 2015 S8. Focus12PFAS and individualPFAA concentrationsbetween 1986 and 2007 points to increased exposure between these years.However, we know from previous PFAS analyses in serum from the TromsøStudy, including individual samples from 1994 and 2001, that targetPFAS concentrations peaked in 2001 with an increase between 1979 and2001 followed by a decrease between 2001 and 2007.34 Divergenttrends between PFOA and longer chained PFCA were also reported inthe aforementioned studies: while PFOA concentrations peaked in 2001,long chained PFCA were increasing between 2001 and 2007. These trendsin the Tromsø Study samples have been shown to follow trendsof PFAS production and use.26The increase in ∑12PFAS concentrations in the pools withthe same individuals followed the temporal changes described by multiplelinear regression, except in one pool, that showed comparable ∑12PFAS concentrations in 1986 and 2007. This deviation couldbe due to this pool containing a lower number of individuals (10)compared to the other ones (11–14). With a lower number ofindividuals in a pool, even just one outlier could have a larger impacton the measured target PFAS concentrations .The∑12PFAS concentrations between 1986 and 2015 (Table S8). The highest ∑12PFASconcentrations were found in the pools with the highest mean age . This has been explained by higherexposure in the older birth cohorts compared to the younger ones dueto the history of changing PFAS production.8Mean age of the individuals in the pools was a predictorof the∑12PFAS concentrationsthan women (Table S8). When looking atthe difference in ∑12PFAS concentrations at eachtime-point, men had significantly higher concentrations only in 2007(Table S9). However, the difference mightnot be observed at all time-points due to limited statistical power.To obtain a power of 80% with a large effect size (0.35), at least39 samples are necessary, and the number of pools at each time-pointis lower than this value. Higher concentrations in men compared towomen were observed for most of the individual PFAS , but comparable concentrationswere observed for PFHpA and the three sulfonamido acetic acids  . HigherPFAS concentrations in men compared to women were already reportedin the Tromsø Study by Berg et al.,34 which also noted higher PFAS concentrations in women that had notgiven birth compared to multiparous women. Placental transfer,52 breast feeding,56 and menstruation60 are known PFAS elimination pathways in women and could all contributeto explain sex differences in PFAS concentrations.Men had significantly higher ∑3.5Table S10, The comparisonof EOF and target PFAS concentrations revealed the presence of unidentifiedorganofluorine at all time-points. This unidentified EOF (UEOF) rangedfrom 0.00 to 34.8 ng F/mL, accounting for 0 to 77% of the EOF .UEOF concentrations were highest in 1986 when thetarget PFAS concentrationswere lowest. In 2007, the UEOF portion was significantly lower thanin 1986, while between 2007 and 2015, a significant increase in UEOFwas observed  is in agreement with these findings and could be explainedby increasing exposure to novel PFAS that have not yet been identified.For comparison, the UEOF fractions from other studiesavailablein the literature are summarized in 62 Meanwhile, forpharmaceuticals, the percentage of globally used active substancescontaining fluorine increased from around 2% in 1970 to 25% in 2021.63 This percentage is expected to increase further since 25–30%of the newly approved drugs contain one or more fluorine atoms. Inaddition, among the most prescribed drugs, the proportion of fluorinatedpharmaceuticals is even higher.62 Whilewe are not aware of studies investigating the contribution of pharmaceuticalsand pesticides toward the EOF mass balance in human blood, a recentstudy determined that ∼22% of the EOF in wastewater treatmentplant sludge  was attributableto these substances, many of which do not contain fluoroalkyl functionalities.64However, fluorinated chemicals other than PFAS could also contributeto explain the elevated UEOF. Fluorine substitution is often usedin the agrochemical and pharmaceutical industry. Among the halogenatedagrochemicals available in the market between 1940 and 2003, around28% contained fluorine.Table S8, Table S9). The sex difference observed for UEOF is the oppositeof what we observed and what is reported in the literature for PFAA,for which concentrations are higher in men than in women.67 Higher UEOF in women compared to men has also been reported in wholeblood collected in Sweden, where the highest UEOF was reported inwomen aged 18–44.16 Two hypotheseswere proposed by Aro et al.16 to explainthe different UEOF concentrations between men and women. The firsthypothesis is that a more frequent use of cosmetics and personal careproducts containing precursors (like PAPs) and other unknown PFAS68 could lead to higher blood concentrations of unknown PFAS or precursormetabolism intermediates that are not investigated in the target PFASanalyses. This hypothesis is also supported by studies reporting associationsbetween PFAS concentrations in the blood and the use of cosmeticsand personal care products.70 In our study, the TOPassay showed only a minor contribution of precursors to the EOF inhuman serum with no differences between men and women, and therefore,this first hypothesis regarding precursor exposure can be discarded.However, the more frequent use of cosmetics might still be a possibleexplanation for the higher UEOF in women compared to men since cosmeticscould also lead to exposure to yet unknown PFAS that are not oxidizableand therefore nondetectable in the TOP assay. A second explanationcould lie in a difference in use of fluorinated pharmaceuticals betweenmen and women since sex differences in prescription are reported forseveral pharmaceuticals groups.76 Additionally, differences in elimination kinetics between men andwomen for these unidentified fluorinated chemicals could also playa role.Mean age was not a significant predictorof UEOF, but women hadhigher UEOF values than men , 2007 , and 2015 , thecontribution to the UEOF changed between time-points, ranging from1 to 7% in 1986 (median: 2%), from 0 to 100% in 2007 (median: 18%),and from 0 to 37% in 2015 (median: 3%).3 groups since these should be oxidizable toTFA, which was not detected after oxidation. However, Hammel et al.77 found that among the 360 organofluorine pharmaceuticalsapproved and used globally between 1954 and 2021, 50% of these chemicalscontained a single fluorine, 35% contained a single aromatic fluorine,and 10% contained more than three fluorine atoms. As most of thesefluorinated pharmaceuticals contain only one fluorine, this largenumber of substances would go undetected in the TOP assay, and fluorinatedpharmaceuticals could still contribute to the observed UEOF.The TOP assay resultssuggest the absence of pharmaceuticals containing−CFTable S8). The UTF can include both inorganicfluoride and organic fluorinated compounds not extracted or partiallyextracted with acetonitrile. Fasting plasma fluoride concentrationsreported in the literature range from 9.3 to 24 ng F/mL in areas withnonfluorinated water (water fluoride concentrations <0.3 mg/L).78 Water in Norway is not fluorinated and a study from 2017 found thatonly 4 of 201 registered waterworks had fluoride exceeding the regulatorylimit of 1.5 mg/L.79 In humans, the fluoridemetabolism is not homeostatically regulated and plasma concentrationsvary depending on levels of intake, deposition in hard tissues, andexcretion.80 After ingestion, plasma concentrationstake 3 to 6 h to return to baseline values.78 This could contribute to explain the variability observed in theUTF since the serum collected in the Tromsø Study is from nonfastingindividuals. Overall, these observations indicate the need for measuringfluoride when conducting FMB studies using TF.The EOF accounted for 20 to 99% of the TF and the unidentifiedTF (UTF) ranged from 5 to 1194 ng F/mL. This fraction did not changebetween time-points and was not influenced by sex and mean age  could not be assessedusing pools. In addition, the individuals in each pool covered a widerange of ages, and this limited the investigation of the influenceof age and birth cohorts on the different fluorine fractions measured.


Oocyte-secreted growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are critical paracrine regulators of female fertility. Recent studies demonstrated that serum concentrations are associated with the number of oocytes retrieved during IVF, and therefore potential clinical use as biomarkers. However, it is unknown if the presence of endometriosis affects serum GDF9 or BMP15. An exploratory case–control study was prospectively performed on 60 women who underwent laparoscopy between April 2017 and August 2018 at two hospitals. GDF9 and BMP15 were measured by validated immunoassays in pre-operative serum samples. Data were analysed relative to laparoscopic assessment of endometriosis and staging. There were 35 women with confirmed laparoscopic diagnosis of endometriosis and 25 controls with no evidence of endometriosis at laparoscopy. GDF9 was detectable in 40% of controls and 48% of cases. There was no difference in median GDF9 concentrations between controls  and cases . BMP15 was detectable in 48% of controls and 58% of cases, with no difference in median concentrations between controls  and cases . Furthermore, there were no significant differences in the proportion of detectable samples or concentrations of GDF9 or BMP15 with differing severities of endometriosis. In conclusion, serum concentrations of oocyte-secreted factors, GDF9 and BMP15 did not differ between control patients and patients with endometriosis. For clinical application in reproductive medicine, GDF9 and BMP15 serum biomarker quantitation is unlikely to be aberrant in the presence of endometriosis. Endometriosis is a chronic oestrogen-dependant gynaecological condition, presenting with persistent pelvic pain symptoms and infertility . One in Symptoms, clinical examination and imaging aid the diagnosis of endometriosis; however, currently the internationally accepted standard of diagnosis is direct visualisation of endometriosis with or without histology at laparoscopic surgery . Whilst As endometriosis has been reported to be associated with infertility , 12, oocThis was a prospective cohort study of 60 women, approved by the institutional ethical committee , with all participants providing written informed consent. Patients aged 18 to 45 years undergoing planned gynaecological laparoscopy between April 2017 and August 2018 at two university affiliated hospitals in Sydney, Australia, were involved in the study. Exclusion criteria were a previous history of cancer/chemotherapy, previous or current disease receiving gonadotoxic therapies, or inclusion in concurrent studies.Blood samples were drawn immediately pre-operative by venepuncture on sites contralateral to the upper extremity receiving IV fluid therapy. All blood samples were collected in serum-separating tubes , allowed to clot at room temperature for 30 min to 2 h following collection, then centrifuged and sera frozen. Frozen sera were defrosted, aliquoted for single use and stored at − 80 °C prior to analysis.An ELISA for detecting BMP15 in human serum was previously developed and validated . The AnsSerum anti-Müllerian hormone (AMH) was assayed using the Elecsys AMH Plus assay on the Cobas® e411 automated platform, with an electro chemiluminescent (ECL) method principle analyser . The assays were undertaken at the South Eastern Area Laboratory Services, Prince of Wales Hospital .Demographic information, past medical, surgical, gynaecological and family histories for all patients were recorded. All study data were entered onto and managed using Research Electronic Data Capture (REDCap) software .All patients underwent planned gynaecological laparoscopy, which included but was not limited to diagnostic surgery for the investigation of pain and/or fertility, excision of endometriosis, ovarian cystectomy and hysterectomy. The revised American Society of Reproductive Medicine (rASRM) 1996 classification of endometriosis score was employed. Endometriosis was excised and diagnosis was confirmed by histology. Patients with no evidence of endometriosis at laparoscopy were assigned to the control group.t-tests, whilst those that were non-normal were compared using a Mann–Whitney U test.Statistical analyses of patient demographics and gynaecological history were performed using the Statistical Package STATA 12.0 . Categorical variables were compared using a chi-square test. For continuous variables, the normality assumption was checked using the D’Agostino and Pearson test for normality. Variables that were approximately normally distributed were evaluated using independent The statistical package R version 4.0.4  was usedOf the 60 patients enrolled, 35 had confirmed intra-operative diagnosis of endometriosis and the 25 without endometriosis at laparoscopy were used as the control group. Demographics of the two groups are reported in Table There were no significant differences in the mean serum concentrations of GDF9 Fig.  between n = 14). The presence of other gynaecological pathogenesis, i.e. adenomyosis (n = 3), endosalpingiosis (n = 1) and leiomyomas (n = 9), did not affect the concentration of GDF9 and BMP15 (data not shown).There were no significant differences in biomarker concentrations when controls were compared to patients with an endometrioma (Follicle and oocyte quality may be impaired in patients with endometriosis, with inhibitory effects observed on follicle growth, steroidogenesis and granulosa cell function (reviewed in A limitation of this study was the large proportion of patients with undetectable concentrations of GDF9 and BMP15 in serum. This may be expected as these are oocyte paracrine factors that are exclusively secreted by oocytes and therefore appear in the circulation in very low concentrations and thus cannot always be readily detected using existing ELISAs. These findings are consistent with recent studies  and may This exploratory study demonstrated no significant difference in serum GDF9 and BMP15 concentrations between women with and without laparoscopically diagnosed endometriosis, suggesting that serum measurements of these oocyte-secreted proteins are unlikely to be affected by the presence of endometriosis or to be useful as a diagnostic test for endometriosis. Therefore, for clinical application of GDF9 and BMP15 as potential serum biomarkers in reproductive medicine, quantitation in serum is unlikely to be aberrant due to the presence of endometriosis. A limited number of other histologically diagnosed pathologies, i.e. adenomyosis, endosalpingiosis and leiomyomas, were present in this cohort and their presence did not affect the biomarker concentrations. Hence, it is unlikely that these conditions have affected our results. Furthermore, the novel assays utilised in this study were recently developed and validated [


The band observed near 3299 cm−1 widened and intensified with time due to the oxidation degree of samples, with this effect being higher in the control chips. The observed changes in fatty acid and hexanal content with storage time underlined the higher extent of oxidation in the control samples. This fact could suggest an antioxidant protectant action of OE in avocado chips during thermal treatment, which was attributed to the presence of phenolic compounds. The obtained chips incorporating OE represent a viable option for the development of a natural, healthy, and clean-label avocado snack at competitive cost and with low environmental impacts.Analysis of the oxidative stability of novel avocado chips with added natural extracts was carried out with the aim of reducing the chemical additive content in their formulation. Two different natural extracts were initially evaluated and characterized: one obtained from olive pomace (OE) and other from pomegranate seed waste. OE was selected due to its better antioxidant potential according to FRAP, ABTS, and DPPH assays as well as its higher total phenolic content. The formulations used were 0, 1.5 wt.%, and 3 wt.% of OE. A gradual disappearance of the band found around 3009 cm In recent times,, there has been an increasing interest in the consuming healthier natural products with functional properties and with a positive impact on human health ,2. One oIn recent years, there has been a growing concern about the need to reduce agro-industrial waste in the environment. In this context, the bioeconomy, which involves mobilizing biomass resources, is the key strategic innovation pillar in the European Union ,10,11. I−1 of semisolid mass in the olive oil industry, with a production estimation higher than 2.8 million tons/year worldwide. This residue is mainly composed of fatty acids, proteins, and polysaccharides, as well as polyphenols and pigments with antioxidant activity [Olives are a widely consumed fruit with high nutritional value. They are also used to produce extra virgin olive oil, a highly recognized product. The worldwide olive production in 2021 showed an increase from 21 million tons (Mt) in 2017 to around 23 Mt in 2021. Spain was the main producer with 35.8% of the total production followed by Italy (9.8%), Turkey (7.5%), Morocco (6.9%), and Portugal (6.0%)  The olivactivity ,16. Amonactivity . This byactivity .Punica granatum L.) fruit has been underlined in the literature with promising activity against inflammatory and chronic diseases [−1, and hydrolysable tannins, from 32 to 263 g kg−1 [Pomegranate  by-products. It was obtained after water extraction standardized with up to 40% wt.% of hydroxytyrosol. The other was an extract obtained after pressing the arils and seeds of the pomegranate fruit (Punica granatum L.) (PS) collected as residues from the industrial juice production. Avocado samples used to develop chips belonged to the Hass variety and were purchased in a local supermarket. Fruits were stored at 4 ± 1 °C under air atmosphere.Two naturals extracts kindly provided by Probeltebio  were used in the present study. One was an olive extract (OE) from Spanish olive fruit  reagent and ABTS ) diammonium salt, TPTZ , potassium persulfate, Folin−Ciocalteu reagent (2 N), sodium acetate trihydrate, Trolox, aluminum chloride and sodium nitrite). On the other hand, Panreac  supplied the sodium carbonate, sodium hydroxide, calcium chloride, ethanol, and iron trichloride.−1 was used as initial concentration of extracts diluted in distilled water and Trolox was used as reference standard. All methods were carried out in triplicate.DPPH radical scavenging assay, ABTS free-radical scavenging assay and ferric reducing antioxidant power (FRAP) were used to study the antioxidant activity of the extracts, as described elsewhere . In this−1 was mixed with 250 μL of Folin–Ciocalteu reagent and 3500 mL of distilled water and incubated, protected from light, for 3 min. Then, 1 mL of Na2CO3 solution at concentration of 20 wt.% was added. After 40 min at 40 °C in the dark, the absorbance at 750 nm was measured. The blank used was deionized water. Trolox was used as reference standard (50 to 500 mg kg−1).The Folin−Ciocalteu colorimetric method was used to determine the total phenolic content of extracts. A Biomate-3 UV/VIS spectrophotometer  was used in this work . The folA vegetable-based non-extruded snack was proposed in the present work based on avocado as matrix. A dehydration treatment was selected in order to make the avocado fruit more shelf-stable, without requiring any other preparation steps  . Avocado−1, using 64 scans and 4 cm−1 resolution. All spectra were corrected against the background spectrum of air.A Bruker Analitik IFS 66 FTIR spectrometer  was used to collect the FTIR spectra of samples. First, the grounded and homogenized chips were directly put on the instrument and the spectra were recorded from 4000 to 400 cmThe oil extraction of chips was previously necessary for the quantification of the major fatty acids. In this work, the use of a microwave-assisted extraction system following the procedure described in In this work a greener solvent to extract the oil from avocado by using MAE was proposed due to its relatively less toxic nature than the more commonly used solvents such as hexane, petroleum ether, or methanol ,40. Prev−1. Helium was used as carrier gas (1 mL min−1) and 1 µL was injected in the split mode (1:75). The tridecanoic acid methyl ester (700 mg kg−1) was used as internal standard whereas analytical standards were used for external calibration.Methylation of fatty acids present in oil samples was carried out as described elsewhere . Analysi−1) was added into the SPME vial with a micro-stirring bar. In accordance with the literature, a DVB/CAR/PDMS SPME fiber for the adsorption of polar and nonpolar volatile compounds was used  [−1 and (b) from 70 °C to 200 °C (hold 10 min) at 35 °C min−1. The carrier gas was helium at 1 mL min−1 and the FID temperature was 300 °C. Working solutions obtained from an hexanal stock prepared at 30 mg kg−1 in distilled water were used for the external calibration.A headspace-solid-phase microextraction (HS-SPME) followed by GC–FID analysis  was proposed in this work for the quantification of the hexanal content of chips . First, PA, USA) . The samp < 0.05.Statistical analysis of experimental data was performed with SPSS commercial software . ANOVA and Tukey test were assessed for In order to select the active extract with the highest antioxidant properties to be used as the active additive in the preparation of avocado chips, the antioxidant activity and TPC of the two studied extracts, OE and PS, were evaluated. The obtained results are shown in −1 extract, and TPC values ranging 36–53 mg Trolox g−1 extract [Both studied extracts showed an adequate antioxidant content according to the literature, between 56–71 mg Trolox g extract ,45. OE w extract . The edi extract . Phytoch extract ,48,49,5050 values obtained for both extracts by DPPH assay, since a lower IC50 value was obtained for OE (0.59 ± 0.05 µg Trolox g−1 extract) compared to PS (0.66 ± 0.03 µg Trolox g−1 extract). OE is a natural extract obtained from the fruit of the olive tree (Olea europaea L.) cultivated in the Mediterranean region of Spain through sustainable agriculture and extracted with a patented process which maintains the natural profile of the fruit. The olive pomace is rich in hydroxytyrosol, but also has a significant content in phenolic acids , secoiridoids , and flavonoids  [All studied antioxidant methods, except ABTS, showed statistically significant differences in results between OE and PS. OE showed higher antioxidant activity and total phenolic content. This behavior was also supported by IC others) . Current others) , enhance others) , and imp others) . Regardi others) . Among o others) . As a re others) . An init others) .p < 0.05) were found for some bands in the studied formulations with time, considering wavenumber and absorbance values (−1), lipids , and hydroperoxides, amylose, amylopectin, and amide A  showed significant differences at 0, 6, and 14 days of oxidative treatment. The presence of the band near 1559 cm−1 was due to the bending vibration of N-H bonds, which are typical of all proteins. The wavelength of this band slightly decreased with time in all samples. As was expected, noticeable differences were observed between samples with time for the bands linked to the fat fraction. Avocado fruit is rich in oleic (56 wt.%), palmitic (21 wt.%), linoleic (14 wt.%), and palmitoleic (9 wt.%) acids [−1 were attributed to tensile vibrations of CH2 for asymmetric and symmetric vibrations, respectively. High-frequency values in this absorption range indicate a sample rich in unsaturated and polyunsaturated acids [−1. The isomerization, cis to trans, of double bonds of unsaturated fatty acids has been reported in oxidative treatment progresses [−1 also showed statistically significant differences between samples at days 0 and 6. The protein fraction and carbohydrate constituents such as fiber, hemicellulose, and starch are significant compounds present in the seed but not in the pulp of avocado [−1, which could be related with the increased oxidation of all studied samples with time due to their degradation.Statistically significant differences  acids ,62. The ed acids . The strogresses . Therefo avocado ,64. Thus avocado . At the v/v) was chosen as potential candidate for greener extraction of the avocado-chip oil. The highest extraction yield was obtained for ethyl acetate followed by the mixture and, finally, the ethanol solvent with extraction yield values of 50 ± 4, 38 ± 2 and 19 ± 1%, respectively. The reason for this difference was mainly that the type of solvent plays an important role in MAE. Generally, the choice of solvent in MAE should be based on its solubility of the target compound [The effect of different solvents on yield extraction of oil by MAE were studied. Ethanol, ethyl acetate, and the mixture of both  by-products, and processed following a dehydration process in the oven at moderate temperatures. The addition of OE, as a strategy to improve the oxidative stability of the developed products, had different effects on the response variables evaluated in this study. In general, the formulation with 3 wt.% of added OE showed the best results, with no pronounced degradation process in these samples. Control chips without the addition of OE were less stable to the oxidative treatment. The elucidation of the degradation mechanism of fatty acids was linked to the production of hexanal volatile compounds and FTIR results, helping researchers to understand how the different technological practices available during avocado thermal processing  could affect the overall oxidative degradation of ‘Hass’ avocado chips. Further work will be needed to evaluate the sensorial behavior of the developed chips to improve the functional properties of the obtained formulations and their possible commercialization in the real market.The present work allowed us to increase the scientific knowledge about chemical changes occurring during the storage of avocado chips with the added natural antioxidant extract, OE, obtained from Spanish olive fruit (


A total of eighteen experiments have been conducted by varying three main machining parameters, i.e., cutting speed, feed rate, and depth of cut, at three levels per parameter. It has been found that tool flank wear increased with all three parameters and surface roughness decreased. At the longest dept of cut, surface roughness increased. An abrasion wear mechanism was found on the tool flank face at a high machining speed and adhesion at low speed. Chips with a helical shape and low serrations have been investigated. Turning SS 316 at optimum machining parameters of 170 m/min cutting speed, 0.2 mm/rev feed rate, and 1 mm depth of cut, as obtained by the multiperformance optimization technique grey relational analysis, resulted in the best values of all machinability indicators: 242.21 µm tool flank wear, 3.81 µm mean roughness depth, and 34,000 mm3/min material removal rate, at a single parameter setting. In terms of research achievements, the percentage reduction in surface roughness is approximately 30% and represents an almost ten-fold improvement in the material removal rate. The combination of machining parameters of 70 m/min cutting speed, 0.1 mm/rev feed rate, and 0.5 mm depth of cut is optimum for the lowest value of tool flank wear when considered for single parameter optimization. This paper presents a research investigation conducted on the turning of stainless steel 316 material under a dry environment using microwave-treated cutting tool inserts. Plain tungsten carbide WC tool inserts were exposed to microwave treatment for enhancement of their performance characteristics. It was found that a 20-min microwave treatment resulted in the best tool hardness and metallurgical characteristics. These tool inserts have been used to machine SS 316 material following the Taguchi L In recent years, researchers and engineers have focused on improving the physical and mechanical properties of cutting tool inserts instead of exploring new tool materials. Heat treatment is an important process in altering the material properties by heating and cooling metals under controlled environments . In the Nowadays, microwave heat treatment is becoming a popular technology to improve the capabilities of cutting tool inserts by improving mechanical properties such as wear resistance, hardness, and fracture toughness. Microwave heat treatment differs significantly from traditional heat-treatment methods in the sense that the heating is volumetric, selective, and internal . MicrowaStainless steel 316 is one of the most important engineering materials, being exposed to a wide variety of commercial applications. Stainless steel is considered difficult to machine due to its high hardness and low thermal conductivity ,8. Befora—3.436 μm, MRR—105,187 mm3/min average, flank wear—234.63 μm. Another important study reported by Sharma and Gupta is based on the machining of SS304 using coated carbide tools [Jhodkar et al. carried out a detailed investigation on the effectiveness of microwave treatment on tool performance while machining EN8 steel ,2. They From a detailed review of the important past attempts, it can be summarized that tool inserts with treatment, texture, and coatings can efficiently machine difficult-to-cut materials. Moreover, microwave-treated tools possess the capability to outperform plain tools and enhance machinability to a great extent. Previous attempts on the microwave-treated tool based machining of engineering materials, including stainless steel, are scarce and necessitate future investigations. The present study aims to fill such gaps by investigating the microwave-treated cutting tool based sustainable dry machining of stainless steel 316. It includes a detailed investigation on microwave tool treatment, the effect of cutting parameters on machinability indicators such as flank wear, the surface roughness of the SS 316 workpiece, and the material removal rate, and process optimization. The chemical composition of the stainless steel 316 (SS 316) work material is C: 0.08%, Cr: 18%, Ni: 14%, Si: 1%, P: 0.045%, the rest is iron. Its tensile strength and hardness were 480–620 MPa, 215 HV. The SS 316 bar with an initial diameter of 60 mm and a length of 400 mm was used, and the manual lathe Colchester Mascot 1600 UK was used as a machine tool for the turning operation. The microwave-heat-treated tungsten carbide cutting tool inserts equipped with hardness in the range of 2110–2127 HV were used for turning in dry environment. The percentage elemental composition for bare untreated tool insert is: carbide, 7.8%; Co, 9.6%; WC, 82.6% [In microwave heat treatment, the motions of ions and dipoles induces internal heating due to which WC grains are bound by liquified cobalt binders . The heaThe changes in the microstructural and mechanical properties of the microwave-heat-treated tool inserts were evaluated with the help of SEM and Vickers’s hardness test. SC’, feed ‘f’, and depth of cut ‘DoC’ at three levels per parameter, Taguchi’s L9 (33) orthogonal array design approach for the design of experiments (DoE) was employed to conduct the experiments. A total of nine turning combinations were designed and experiments were conducted with two replicates for each run. Thus, a total of 18 experimental runs were conducted by performing a turning operation on a SS 316 round bar using a microwave-treated (MWT) WC tool insert. The selection of turning input parameters and their levels, as presented in Based upon the selection of three input turning parameters, namely cutting speed ‘ZR’ is one of the most important roughness parameters and has been considered as a response/machinability indicator to indicate the quality of the machined surface after turning. The mean roughness depth is superior to the average roughness and maximum roughness in the sense that it gives a real indication of the surface irregularity, since it is an average of the maximum roughness  values of each of the five equal segments of the evaluation length. A TMTecK TMR200 tester was used to measure the ZR of the turned surfaces after each experimental run.Tool wear, surface roughness, and the material removal rate are the considered machinability indicators used to evaluate the success of microwave-treated-tool based machining of SS 316. The tool wear investigation was conducted on the flank face of the inserts. ISO recommendations were considered, in which the tool life ends at a flank wear value >600 microns. Flank wear and chip morphology were investigated using a Tescan VEGA3 scanning electron microscope (SEM). The mean roughness depth ‘VMRR’ indicates the removal of materials in mm3/min from the workpiece after machining. It is defined as the volume of material removed during the machining process to the total time taken for machining, and it is expressed by the following Equation (1):The volumetric material removal rate ‘beforeW, and afterW indicate the weight of the SS 316 round bar before and after turning respectively, ρ stands for the density of the SS 316 round bar, and t stands for the total machining time (fixed at 330 s) for turning the SS 316 bar for each experimental run.Here, 9 (33) orthogonal array was employed to identify the influence of the cutting speed ‘SC’ (70–170 m/min), feed rate ‘f’ (0.1–0.2 mm/rev), and depth of cut ‘DoC’ (0.5–1.5 mm) on flank wear ‘WF’ in µm; the mean roughness depth ‘ZR’ in µm; and the volumetric material removal rate ‘VMRR’ in mm3/min. The turning experiments on the SS 316 round bar of 60-mm diameter were conducted using microwave-heat-treated tungsten carbide inserts with hardness values in the range of 2110–2128 HV.This section presents the results of the microwave heat treatment of the WC tool inserts. The cutting tool insert treated for 20 min was found to be superior from the microhardness value and metallurgical point of view. The SEM image of this tool as shown in SC’, feed rate ‘f’, and depth of cut ‘DoC’ on flank wear ‘WF’; the mean roughness depth ‘ZR’; and the volumetric material removal rate ‘VMRR’, are discussed with the help of graphical representation as shown in DoC. Increasing the cutting speed and feed rate leads to a vanishing of the buildup edges, which consequently ensures smoother surface generation with less rubbing during chip-sliding action. Beyond a DoC value of 1.0, the roughness increases due to excessive friction between the tool work interface. It is obvious that high values of all machining parameters cause enhancement in the material removal rate. Higher values of cutting speed increase the rotational speed of the workpiece and higher values of feed increase the movement of the tool insert along the axis of the workpiece, thus increasing the amount of material removal from the workpiece during machining. In contrast, higher values of the depth of cut increase the thickness of the chip and thus remove more materials from the workpiece during machining.In this section, the effects of turning input parameters, namely the cutting speed ‘The study of the effects of machining parameters on machinability indicators itself is not enough to measure the success of any machining operation. Optimization of the machining parameters employing a suitable technique is essential to obtain the best machinability indicators . In thisFor minimizing the mean roughness depth and flank wear (lower-the-better)max(iY) and min(iY) are the highest and lowest values of the ith response among nine experimental runs as given in ith response of the jth experiment, grey coefficient ‘ij’GC values were obtained using Equation (4).’ refers to the distinguishing coefficient which has a 0 to 1 range of values. It is usually assigned 0.5 for giving equal weightage to all considered responses. ‘Δij’ indicates the inclination of the ith response of the jth experimental run from its anticipated value. It is equivalent to the absolute variation between the ideal normalized value ‘ii’Z and the normalized value ‘ij’Z of the ith response, i.e., min and Δmax are the minimum and maximum values of Δij. The grey relational grade (GRG) of the jth experiment ‘jG’ is computed by considering the average values of the grey coefficient ‘ij’GC of all of the responses corresponding to the jth experimental run in Equation (5).n’ is the total number of responses.For maximizing the material removal rate (higher-the-better)jG. For multiperformance GRA-based optimization, the optimum parameter combination is obtained from the third experiment with the following machining parameter combination: cutting speed 170 m/min, feed rate 0.2 mm/rev, and depth of cut 1.0 mm. 3/min) corresponding to Experiment 1 in For GRA-based multiobjective/performance optimization, Machining chips are divided into three types based on their morphology: aperiodic saw-tooth chips, continuous chips, and periodic saw-tooth chips ,14. The The microwave treatment caused the formation of a WC skeleton matrix that provided a favorable microstructure and extra hardness to the cutting tool inserts.The tool inserts which underwent microwave treatment for 20 min resulted in the best metallurgical characteristics, with an improved hardness ranging between 2110 and 2128 HV, suitable for turning SS 316.High flank wear has been observed with increasing cutting speed, feed rate, and depth of cut.The mean roughness depth decreased with increases in speed and feed rate, but increased at a high depth of cut.3/min for the material removal rate, while machining SS 316 at the optimum machining parameters of 170 m/min cutting speed, 0.2 mm/rev feed rate, and 1 mm depth of cut.A GRA-based multiperformance optimization results in values of 242.21 µm for tool flank wear, 3.81 µm for the mean roughness depth, and 34,000 mmHelical tubular type thick chip formation with fewer serrations takes place when machining at cutting speeds higher than 70 m/min. Flank wear occurred at higher speeds due to abrasion, whereas adhesion is observed for lower speed machining.In this research, plain tungsten carbide cutting tool inserts have been successfully treated by microwave to improve their cutting performance and utilized to turn the SS 316 material under a dry environment. A detailed investigation on turning SS 316 with microwave-treated tool inserts under a dry environment was performed, and machining parameter optimization was conducted to secure the best values of the considered machinability indicators of cutting speed, feed rate, and depth of cut. The following conclusions can be drawn from this investigation:Consequently, it is recommended that machining stainless steel (SS 316) material in a dry environment can result in better machinability if machined at a higher speed and feed rate and with a moderate value of depth-of-cut, using microwave-treated tools.


Higher ion levels were observed during the cold period, especially for Cl−, Na+, Mg2+ and K+. Dust intrusion caused significant increases in levels of Ca2+, Mg2+ and K+, while open-fire events increased the levels of K+. Deposition acidity showed seasonal differences as well as the influence of dust intrusion. Low ionic balance ratios indicated acidic deposition properties and the presence of organic anions. The highest neutralization ability was found for Ca2+, Na+ and NH4+. Several natural  and anthropogenic sources were determined, as well as the formation of secondary aerosols. Wet deposition was characterized by higher contribution of sea salt fraction compared to bulk deposition and lower contribution of crustal fraction.Atmospheric bulk and wet deposition samples were collected simultaneously at the background coastal site in the Eastern Middle Adriatic region in order to assess the impact of major ions (Cl Excessive anthropogenic activities such as combustion processes, agriculture, industry or traffic, as well as natural processes such as volcanic eruptions, events of open fire or dust storms are nowadays considered to be the main sources of different pollutants in air . Once in2+, Mg2+ or crustal-biological as K+  is the equivalent concentration of the anion, βi corresponding correlation coefficient of cation and βo the intercept. Alternatively, the relationship between [A−] and cations can be expressed using standardized regression coefficients β*i by equation [A−] = β*Na[Na+] + β*NH4[NH4+] + β*K[K+] + β*Mg[Mg2+] + β*Ca[Ca2+]. Standardized regression coefficients enable us to see which independent variable has the greatest effect on the dependent variable. However, the magnitude of the coefficient does not provide information on the importance of the predictor variable in explaining the variation in the dependent variable. Due to the relatively small number of samples, we included all the measured cations in the equation, except [H+]. The results are presented in Multiple linear regression (MLR) is a statistical tool that can be used to analyze the relationship between one dependent variable and several independent variables in order to see which independent variable is the best predictor of the dependent variable. Unlike simple (univariate) regression, MLR is sensitive to combinations of variables, i.e., whether a predictor is important or not depends on the other predictors in the set, so the obtained significances may differ from simple regression results. Following the procedure explained in Anatolaki and Tsitouridou , we appl42− none of the variables were statistically significant. For NO3−, the major neutralizing cation was NH4+, in both bulk and wet deposition. K+ was in significant negative correlation only in bulk deposition, which was probably more related to the influence of different sources (such as biomass burning) than neutralization. For Cl− in bulk deposition, the only significant neutralization cation was Na+, which is related with the marine character of the location. In wet deposition, Cl− correlated negatively with Ca2+, while the only significant neutralization cation was Mg2+ (marine source). In addition to the NF-approach, which identified cations Ca2+, NH4+ and Na+ as the most important neutralizers, MLR allowed us to see to which extent is the neutralization capacity of an individual cation related to the specific ion. Previous studies that used MLR for neutralization capacity assessment seawater = 0.121; [Cl−/Na+]seawater = 1.161; [Ca2+/Na+]seawater = 0.044; [Mg2+/Na+]seawater = 0.227; [K+/Na+]seawater = 0.022.Values of the theoretical equivalent ratio of each ion and Nae et al.  and Chene et al. : crustal = 0.0188; [Ca2+/Mg2+]crustal = 1.78; [K+/Ca2+]crustal = 0.504; [Cl−/Ca2+]crustal = 0.0031; [Na+/Ca2+]crustal = 0.569.The terrigenous  contribution of Ki et al. : [SO42−/−1) of the measured ions in bulk and wet deposition during overall sampling period are presented in Calculated marine and crustal EF factors , crustal source fraction (CF) as well as anthropogenic source fraction (AF) were calculated according to Equations (4)–(6) given by Chen et al. , KeresztHowever, in this context it should be kept in mind that AF actually represents all other sources, which are not necessarily all anthropogenic. For example, this approach cannot separate anthropogenic biomass burning from natural biomass burning sources, and it also includes products of gas-to-particle conversion  as well as combustion derived material.Results show that for the overall sampling period at Martinska station, the AF, SSF and CF sources contributed 33.1%, 38.9% and 28% to the bulk deposition, respectively. In wet deposition, contributions were 36.6%, 46.9% and 16.5% for AF, SSF and CF, respectively . SimilarThe AF contribution in wet deposition was lower than the overall average during the cold period but higher during the warm period and open-fire events, while during dust intrusion events the AF contribution did not differ significantly. During open-fire events and the warm period, the AF contribution was estimated to be 44.6% and 41.2%, respectively. In wet deposition, the contribution of CF was lower than in bulk, while the opposite was true for SSF. The contribution of the SSF fraction to wet deposition was higher than overall average only during the cold period, when it was estimated to be 54.1%. CF fraction contribution was slightly higher than the overall average during the warm period, while during dust intrusion events the contribution of the CF fraction was estimated to be 25%, an increase of about 9% compared to the overall period. The SSF enrichment in the cold period in both bulk and wet deposition samples at Martinska could be explained by meteorology. Frequent high-speed winds in the cold period at Martinska  can inte−, NO3−, SO42−, Na+, K+, NH4+, Mg2+, Ca2+) in bulk and wet deposition were measured simultaneously over a 6-month period at a coastal background location. The data were analyzed with respect to seasonal variations and special events such as dust intrusion and local open-fires, typical of Mediterranean coastal regions. To our best knowledge, this is the first study with parallel measurements of ionic content in both types of atmospheric deposition samples for the eastern middle Adriatic region.Major ions , with the highest values observed during the dust intrusion. Low ionic balance ratio indicated overall acidic deposition properties (more pronounced for wet deposition) and suggested that some other anions, which were not measured herein, were present in the atmospheric deposition. However, the ratio between neutralization and acidification potential indicated the dominance of alkaline ionic species preventing the acidity of deposition. The highest influence on the overall acidity of both bulk and wet deposition came from NORegression analysis and enrichment factor calculations were applied to estimate possible sources. The results indicated several natural  and anthropogenic sources of major ions in AD. The contributions of the sea salt source fraction and crustal source fraction differed in bulk and wet deposition. Wet deposition was characterized by a higher contribution of SSF (46.9%) compared to bulk deposition (38.9%) and lower contribution of CF. The anthropogenic source fraction was similar for both types of deposition samples (about 39%). The SSF contribution was higher than overall during the cold period and dust intrusion event, while the AF contribution was higher during warm period and open-fire events. The CF contribution was slightly higher in both bulk and wet deposition during the warm period, while during dust intrusion events its contribution increased by about 5% in bulk and 9% in wet deposition. For a better identification of sources, future studies should also include measurements of specific organic compounds.Given the limited project budget and duration, and relatively small number of collected samples, we could not carry out a more sophisticated source apportionment analysis. However, the results of this research, in addition to providing the first data on the ionic composition of wet and bulk deposition in the eastern middle Adriatic region, also clearly point out some specific sources that significantly affect ion levels and the acid-base balance in the environment. Furthermore, more detailed studies of the events such as desert dust intrusion and forest fires are needed in the future, as the research through this project has shown that they significantly affect air quality and the composition of deposited matter. These findings are in accordance with the latest WHO guidelines , which a