Importantly, during the production of the biochars with the highestnon-NAP PAH contents, the conditions in the post-pyrolysis area, wherepyrolysis vapors and biochar are separated, favored condensation anddeposition of PAHs on biochar. Under these conditions, NAP condensedto a lower degree because of its high vapor pressure. In biocharsnot contaminated through this process, the average non-NAP contentwas only 2 ± 3 mg kg–1 (median 0.5 mg kg–1). Uneven heat distribution and vapor trapping duringpyrolysis and cool zones in the post-pyrolysis area need to be avoided.This demonstrates that the most important factor yielding high contentsof toxic PAHs in biochar was neither a specific pyrolysis parameternor the feedstock but the pyrolysis unit design, which can be modifiedto produce clean and safe biochar.The content of polycyclicaromatic hydrocarbons (PAHs) in biocharhas been studied extensively; however, the links between biomass feedstock,production process parameters, and the speciation of PAHs in biocharare understudied. Such an understanding is crucial, as the healtheffects of individual PAHs vary greatly. Naphthalene (NAP) is theleast toxic of the 16 US EPA PAHs but comprises the highest proportionof PAHs in biochar. Therefore, we investigate which parameters favorhigh levels of non-NAP PAHs (∑16 US EPA PAHs without NAP) ina set of 73 biochars. On average, the content of non-NAP PAHs was9 ± 29 mg kg Productionof safe biochar, which is essential for environmentalapplications, can be ensured through adequate design of biochar productionunits. The aim wasto develop recommendations for the production of biochar with lowPAH-related risk for human health and the environment. We thereforeinvestigate whether high contents of non-NAP 16 US EPA PAHs in biocharscan be linked to particular reactor design and pyrolysis conditions.To do so, the 10 biochars with the highest non-NAP PAH contents werestudied thoroughly to pinpoint the reasons for the high PAH contents,based on a detailed and in-depth understanding of the production processes.(i)28The “Stage I” pyrolysisunit is a small-scale, batch pyrolysis reactor with a vertical quartztube (inner diameter 50 mm) that has a sample bed depth of around200 mm and is heated up by a 12 kW infrared gold image furnace . A condensation system was assembled tocollect different fractions of condensable gases in cold traps, andnoncondensable gases were collected in a gas bag for further analysis.A schematic of the unit with more details about the pyrolysis unitsetup was published elsewhere.(ii)29The “Stage II”pyrolysisunit is a continuous-screw pyrolysis unit (auger reactor) that usesan electrically heated split tube furnace with an inner diameter of100 mm. After an initial nitrogen purge, the feed hopper transportsthe feedstock to a rotary valve, where it drops onto the furnace screw.The discharge chamber, where pyrolysis vapors and solids (biochar)are separated, was actively heated up with heating tapes (heatingtape (HT) I and III), which were set to 500 and 400 °C, respectively.A third heating tape (HT II) heated the pipes that connect the dischargechamber with the afterburner, where the pyrolysis vapors are combustedusing propane. At various parts of the pyrolysis units, pressure andtemperature were measured. A schematic of the unit with more detailsabout the unit can be found in Buss et al.(iii)30The “StageIII” pyrolysisunit is a pilot-scale rotary kiln with a heat tube length of 2.8 m,an inner diameter of 244 mm, an angle of 0.5°, and a rotationalspeed of 1–7 rpm. A biomass hopper with a feed screw deliversthe feedstock to the rotary kiln, where it is heated to a maximumtemperature of 850 °C. The discharge chamber separates pyrolysisvapors from solids that drop on a cooling screw that transports thechar to a nitrogen-purged discharge drum. The vapors are channeledinto an afterburner, where they are combusted with propane and theexhaust gases are released. Temperature and pressure were monitoredat different entry points within the heat tube. A schematic of theunit and more details are available in Buss et al.Three different pyrolysis units atthe UK Biochar Research Center, University of Edinburgh, were usedfor biochar production.Supporting Information. In addition, four differentfeedstock pretreatments  and one biochar post-treatment (heating at 200 °C for20 h) were applied, which were part of previous studies. The biocharswere chosen to relate PAH content and composition, and in particular,PAH toxicity, to feedstock properties and biochar production conditions. All 73 biochars and their productionconditions are displayed in Table S1.Seventy-threebiochars were produced from14 different feedstocks in the three different pyrolysis units describedin the section above. The parameters controlled during the productionincluded the highest treatment temperature (HTT), residence time atHTT, and carrier gas flow rates. Besides changes in the productionconditions, changes to the pyrolysis unit setup were implemented.More details on biochar production can be found in the 31 Accurate and precise determination of PAHs inbiochar largely depends on the homogeneity of the material or ratheron the reduced heterogeneity. This is important for PAH analysis becausethe smaller the particle, the higher the PAH content.32 Furthermore, the relative standard deviation (RSD) of PAHlevels is concentration-dependent31 inthe way that the lower the concentration, the higher the RSD. Hence,the biochar needs to be thoroughly mixed prior to analysis (describedbelow) to obtain a representative subsample. Under these circumstances,an RSD between 10 and 20% is considered good for PAHs in biochar.31Every solid material witha different particle size is considered heterogeneous.Representative samples were obtained bymixing the biochar samples manually, taking a subsample of around1/10 of the total amount produced, grinding it with mortar and pestle,taking a homogenized subsample (2 g), and transferring it into a sampletub. Of this sample, ∼1 g was finally used for the extractionand PAH analysis.33 The analyseswere conducted by Northumbrian Water Scientific Services . As previously described,20 100 mLtoluene was added to 1 g of ground biochar that was subjected to Soxhletextraction for 36 h, and the resulting extract was reduced to 1 mLand analyzed by GC-MS (Agilent 6890 GC plus 5975c MS). A six pointcalibration curve  was runwith all 16 US EPA PAHs anthracene,chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, (B(a)P), indenopyrene, dibenzanthracene, benzo(ghi)perylene). Five deuterated PAHs  compensated for the losses of the respectivenative compounds and were spiked into the solvent after extractionat 20 mg L–1. The correlation coefficient of allPAHs was >0.997. The limit of detection (LOD) was 0.10 mg kg–1. Extraction blanks (<3× LOD) were analyzedwith each batchof samples and subtracted from all samples. The precision of extractionand GC-MS analysis was obtained by measurement of three replicatesof some biochar samples and was <20%.Due to the strong sorption of organic substancesby biochar, themethod recommended for PAH extraction from biochar is a 36-h Soxhletextraction using toluene.Table S2. Total PAH contents for 46 biochars20 and individual PAH contents for 6 biochars34 werealready reported previously. Throughout the manuscript, the∑16 US EPA PAH content without NAP is referred to as “non-NAPPAHs.”The individual PAHcontents for all 73 biochars are reported in –1 with an average of 27 ±36 mg kg–1 . Therefore, in –1) and in biochars in other studies.36 The contents of the other 3-ring PAHs and more toxic5- and 6-ring PAHs were typically slightly lower than the contentsof 4-ring PAHs  was even broader, which inparts can be explained by the way the percentage of NAP of the sumof PAHs was calculated with individual PAH contents smaller than thelimit of detection (LOD) (0.10 mg kg–1) not takeninto account. If the LOD is used instead of a value of zero, the averageproportion of NAP decreases to 72 ± 21%, which is well in therange reported for rotary kiln biochars.38 The reason for the high content of NAP in many biochars and whyits content fluctuates so widely is unclear. To investigate this further,mechanistic studies are needed.Taken all 73 biochartogether, NAP was by far the most dominatingPAH with 83 ± 22% of the total PAH contents 2, which n = 63,low non-NAP content), there is a linear relationship of NAP with thetotal 16 US EPA PAHs , there is a linear relationship of non-NAP levels with total16 US EPA PAHs . This suggeststhat the pathways of contamination for NAP and non-NAP PAHs are different.While in most of the biocharsin our study, NAP comprised a veryhigh proportion of the sum of 16 PAHs (>90%) 2, in somEPA PAHs 3A, whileEPA PAHs 3B. In biEPA PAHs 2. There To reveal the cause for this distribution of PAHs in these biocharsand to present options for avoiding high non-NAP PAH biochar production,the production process of some of the biochars with the highest non-NAPcontents are discussed in detail in the following section.Table S3), it becomes apparent that the high contents of PAHscannot be associated with a particular feedstock type, which is inline with other studies, e.g., Bucheli et al.38 A large variety of feedstocks, from plant residues , sewage sludge, and demolitionwood yielded biochars with elevated levels of non-NAP PAHs under certain,but not all, production conditions. The increased non-NAP PAHs contentcould also not be unanimously ascribed to a particular pyrolysis temperaturenor pyrolysis unit.When comparingthe production conditions and feedstocks of the 10biochars with the highest non-NAP PAH contents (30 The VC (vapor-contaminated) and LC (liquid contaminated) biochars,named due to their respective way of contamination during pyrolysis,were produced from softwood pellets at 550 °C with the StageIII pyrolysis unit .30 During the “VC biochar”-production,a connecting pipe to the combustion chamber was restricted and eventuallyblocked due to a buildup of tars, resulting in backflow of pyrolysisvapors that contaminated the biochar (vapor-contaminated biochar).During the production of “LC biochar,” the dischargechamber walls (separation of pyrolysis vapors and biochar) were muchcooler than the pyrolysis vapors due to insufficient insulation and,therefore, the vapors in the pyrolysis gas condensed on the walls,and the liquids contaminated the biochar (liquid contaminated biochar).This caused a considerable increase of PAH levels and in particular,elevated levels in non-NAP PAHs over a sample produced under the sameproduction conditions and from the same feedstock but after the dischargechamber and the previously blocked tube were cleared and insulated.9Two of the biochars were produced during StageIII pyrolysis unitruns , where irregularities led to the contactof biochar with pyrolysis vapors/liquids.20 showedthat the biochar produced in the Stage II unit at 750 °C hadsignificantly higher total PAH contents than the biochar producedat lower temperatures in the same unit. This marked increase in PAHcontent with HTT increase to 750 °C becomes even more pronouncedwhen only non-NAP PAHs are considered , ∼100-fold (DNX), ∼2.6-fold(MC) and ∼10-fold (WC) higher contents. This trend is not supportedfor biochar produced in the other continuous pyrolysis unit (StageIII unit), where the non-NAP PAH content in biochar produced at 750°C was lower (5.0 mg kg–1) than that in thebiochar produced at 350 °C (7.2 mg kg–1) . Analysis of total PAHs in these and other biocharsamples, as reported in Buss et al.nsidered 4. Comparmg kg–1) 4.20 we identified the temperature of the heating tapes in thedischarge chamber of the Stage II pyrolysis unit as the cause of thehigh PAH content in biochar. The discharge chamber is the area wherepyrolysis vapors and solids are separated. The temperatures of theheating tapes (between 400 and 500 °C) were too low to preventthe condensation of pyrolysis vapors at the discharge chamber wallswhen the pyrolysis zone was operating at 750 °C. The condensedvapors subsequently deposited on biochar. Although PAH condensationand deposition will happen at all HTTs, this effect is only apparentin biochars produced at 750 °C because the PAH formation duringpyrolysis is substantially higher at temperatures >700 °C43 and, therefore, pyrolysis vapors produced at 750 °C containmuch more PAHs than vapors produced at lower HTT. Since the temperaturein the discharge chamber of the Stage II unit is fixed at 400–500°C irrespective of the HTT in the pyrolysis zone, there is astrong temperature gradient between the pyrolysis zone and dischargechamber when the pyrolysis zone operates at 750 °C and, therefore,PAH condensation in the discharge chamber is high . In contrast, the discharge chamberof the Stage III unit is passively heated through insulation of thepost-pyrolysis zone .Therefore, the higher the pyrolysis temperature in the Stage III unit,the higher the discharge chamber temperature, which lowers the temperaturegradient and reduces vapor condensation.After detailed investigations, in Buss et al.,–1 with a proportionof NAP of 63% (Table S1). The levels werecomparable to the total PAH content in biochar produced at 550 °Cfrom the same feedstock  and the undigested sewagesludge biochar . However, SS I pyrolyzed at 700 °Chad a total PAH content of ∼232 mg kg–1 , of which non-NAP PAHs comprised∼207 mg kg–1. After investigations, we noticedthat during the production of this biochar sample, one of the heatingtapes in the discharge chamber of the Stage II pyrolysis unit wasfaulty (HT I) and consequently, the temperature measured by a thermocouplelocated between the inner and outer wall of the discharge chamberwas much lower (113 °C) than in comparable pyrolysis runs . The temperature doesnot reflect the internal temperature within the discharge chamber,but it does indicate the relative difference between the two pyrolysisruns. As a result of the considerably lower temperature in the dischargechamber, it is very likely that PAHs condensed and deposited on biochar.This example illustrates that PAH contamination through condensationand deposition might be a common phenomenon in biochar productionand can occur even due to small modifications or faults during thepyrolysis unit setup or its operating parameters.The biochar produced from anaerobically digested sewagesludge at 700 °C (“AD–700”) had a totalPAH content of ∼22 mg kg39 Under hightemperatures in the pyrolysis zone, most PAHs are either evaporatedor react with the biochar in the so-called secondary char formationprocess that is responsible for a significant part of the biocharproduction.45 However, when pyrolysis vaporscondense in the post-pyrolysis stage, in areas much cooler than thefurnace and PAHs deposit on biochar, they are not converted into butinstead physically sorb onto biochar and thus contaminate the material.This still does not explain why the proportion of NAP is smaller inthese biochars.As the above examplesillustrate, the biochars with high contents of non-NAP PAHs were mostlikely contaminated with pyrolysis vapors in the post-pyrolysis zone.The pyrolysis process itself is very effective in separating PAHsfrom pyrolysis solids; <1% of the PAHs synthesized during pyrolysisare typically found in biochar, and the remaining proportion residesin the pyrolysis liquids and gases.27 At lower temperatures (<∼300 °C), such as those thatcan occur in cool zones in the post-pyrolysis stage (discharge chamber),less formation and transformation reactions of organic compounds occurthan at high temperatures in the pyrolysis zone; instead, condensationand deposition of PAHs present in the pyrolysis gas and vapors onbiochar dominate. This depends on (i) the content of the PAH in thegas phase at the respective pyrolysis temperature; (ii) the (equilibrium)vapor pressure of the PAH (boiling point); and (iii) the temperaturedifference between the gases in the reaction chamber and the surfacefor potential condensation . NAPhas a much higher vapor pressure than higher molecular weight (HMW)PAHs, such as B(a)P  and hence is morevolatile and will remain in the gas phase to a higher degree . This means that, of theB(a)P and NAP present in the gas phase, a larger proportion of B(a)Pwill condense and deposit as a liquid when the temperature in thedischarge chamber falls. Therefore, in biochars contaminated via vaporcondensation, as observed, it is expected that the proportion of HMWPAHs will increase relative to the amount of NAP.The processes determining the content and compositionof PAHs in biochar within the furnace area (formation and evaporation)are complex and depend on a number of parameters.45 Overall, this work emphasizes the importance of monitoring and controllingthe pyrolysis process beyond just simple parameters such as HTT inthe pyrolysis reactor to achieve production of good quality biochar.Here, we demonstratethat the high levels of non-NAP PAHs weremainly the result of condensation and deposition of PAH in cool zonesof the post-pyrolysis area rather than the result of the pyrolysisprocess itself. Therefore, the pyrolysis unit needs to be carefullydesigned to avoid cool zones along the whole biochar production path.A suitable design of biochar discharge chamber arrangements is crucial.Where the pyrolysis gases and solids travel concurrently through thepyrolysis unit, the discharge chamber, which separates pyrolysis solidsand vapors, needs to be maintained at temperatures as close as possibleto the HTT. Another option is to separate pyrolysis vapors from solidsalready within the pyrolysis reactor, e.g., using a counter-currentarrangement, where pyrolysis gases are extracted close to the feedstockentry point, i.e., on the opposite end from the biochar discharge.Such a counter-current arrangement would not only have an impact onthe quality of the biochar—yielding biochar with lower PAHscontents—but also could even increase the yield of biochardue to increased secondary char formation.46 Despite significant efforts, these studies have notprovided a general relationship between biochar PAH contents and HTT.Based on our investigations, we suggest this is the case since importantaspects of biochar production have not been sufficiently addressedby these studies, to allow development of such general understanding.In this study, we demonstrated that weaknesses in the design or inthe operation of biochar production units can have a striking effect,resulting in high contents of non-NAP PAHs in biochar and which alsosignificantly increases the total PAH content. This condensation effectof pyrolysis vapors in cooler zones during pyrolysis operations isof great significance and indeed surpassed the effects of HTT, carriergas flow, or feedstock.In the literature, effects of various pyrolysis parameters on PAHcontents in biochar were reported; in particular, numerous differenteffects of HTT on PAHs in biochar have been observed.8 or in “traditionalkiln or soil mounds,”47 where heat distributionis uneven and vapor release can be inhibited and cause condensationof PAHs on biochar. In De la Rosa et al., the highest levels of total(and toxic) PAHs were found in biochars produced at lower temperatures(400 °C) in a muffle furnace setup that did not allow for pyrolysisliquid release (nor sufficient PAH incorporation into the biocharpolyaromatic matrix due to the low temperatures).23 The second-highest level of PAHs in De la Rosa et al. werefound in biochars from traditional kilns, while biochar from a high-tech,continuous pyrolysis reactor demonstrated low levels of PAHs.23 It is important to note that PAH contents inbiochars made from simple “Kon-Tiki” flame curtain pyrolysis—wherevolatiles can freely escape and hence are unlikely to condense—werealso low.48 This demonstrates that safebiochar can be produced in the field with the right technology.Our observation is supported by variousstudies that report thehighest levels of PAHs in biochars produced under “uncontrolledfield conditions”We recommend that future investigations of the relationships betweenbiochar PAH contents closely monitor the temperatures in the differentzones of the pyrolysis unit, in particular, the discharge chamber.This ensures that the biochars are compared on the same basis so thatcertain processes, such as deposition of PAHs, are not misinterpretedas effects of, for example, pyrolysis temperature or feedstock.49 However, since both environmental legislation andbiochar guidelines values are typically based on the sum of the 16US EPA PAHs, the content of NAP often decides about compliance/noncompliancewith PAH threshold values.Fora risk-based assessment of biochars, changes in the content of NAPare of little relevance due to NAP’s low carcinogenicity andtoxicity and rapid degradation in soil.–1, EBC-FEED or EBC-AgroBio) and the highest   values for biochar soil use are 6 mg kg–1 and 300 mg kg–1).51 When NAP isconsidered, 24 (33%) and 51 (70%) of the set of 73 biochars exceededthe EBC upper and lower guideline values, respectively. However, whenNAP is excluded, only six biochars (8%) exceeded the 30 mg kg–1 threshold and 20 biochars (27%) the 4 mg kg–1 threshold, respectively. This shows the disadvantage of using thesum of 16 US EPA PAHs for evaluating the potential risk of PAHs inbiochar, where all PAHs are weighted equally. Consequently, alternativeways of evaluating the risk of PAHs in biochar should be used.In 15 Its average content in all 73 biochars was 0.59± 1.82 mg kg–1, but most biochars showed contentsbelow the limit of detection of 0.10 mg kg–1 (Table S2). The B(a)P contents correlate wellwith the non-NAP PAH contents in the higher and lower end of non-NAPPAH levels in this study  and, consequently, could be used asan indicator for non-NAP PAHs and PAH-associated risk as already establishedfor food products in the EU.52 The EBChas a threshold value for B(a)P in place for animal feed additive(0.025 B(a)P mg kg–1), yet in addition, it has athreshold value for ∑16 US EPA PAHs that needs to be met.Benzo(a)pyrene is the most investigated PAH and is often used asa reference point to compare the toxicities of all 16 US EPA PAHs.49 used B(a)Pas a reference to set up toxicity equivalent factors (TEFs), normallyof 1, based on the carcinogenicity of B(a)P. For comparison, NAP hasa TEF of 0.001.49 Multiplying the TEFswith their respective PAH contents in biochar and summing up the valuesfor all PAHs results in the toxicity equivalent quantity (TEQ). Themean TEQ based on the 16 US EPA PAHs for all 73 biochars in this studywas 1.4 mg kg–1 B(a)P-TEQ (–1 B(a)P-TEQ. The TEQs are similar to the values in Wang et al.,53 who report an increase in TEQ with pyrolysistemperature. In our biochars produced in the temperature range 350–750°C, we cannot confirm a temperature effect, except for higherTEQs in the 750 °C biochars produced in the Stage II pyrolysisunit , where contamination ofbiochars with vapors occurred. In Wang et al.,53 the number of outliers with higher TEQ increasesin the biochars produced at higher pyrolysis temperature. It is likelythat these biochars were contaminated through condensation and depositionof non-NAP PAHs in the post-pyrolysis area as our 750 °C biocharsfrom the Stage II unit. When the outliers are included and the meanis calculated, this shows a misleading trend.As an alternative to threshold values based on the sum of the USEPA 16 PAHs, Nisbet and LaGoy(a)P-TEQ 2 and var51 a thresholdvalue based on the TEF approach was included,though only taking into account the eight most toxic PAHs (3 mg kg–1 B(a)P-TEQ). This value is only exceeded by the sixmost contaminated biochar of the total of 73 biochars analyzed inour study and shows that guide values based on the TEF method alreadyexist but are, however, not legally binding.In the IBI biocharguidelines (update 2015),54 The mean TEQ in group 1 (8.3 B(a)P-TEQkg–1) P-TEQ kg–1) was lower than the mean TEQ of PAHs in rural soils(0.44 B(a)P-TEQ kg–1). Importantly, the median ofthe entire set of biochars in our study was only 0.05 mg kg–1, highlighting that the vast majority of biochars are safe for applicationto soil.To bring our TEQvalues for all 16 US EPA in context, we comparedthem to urban and rural soil in the U.K. as published in Creaser etal.EQkg–1) 2 was hig55 Overall, it does not seem advisable to use threshold values basedon the sum of the content of 16 US EPA PAHs as they do not discriminatebetween the toxicity of the compounds, for instance, such as NAP andhighly carcinogenic compounds, such as B(a)P, for humans and the environment.Treating each PAH equally does not seem fit for purpose. For biochar,this is a particular issue as NAP is the dominant compound and itscontent fluctuates widely, and hence total PAH contents do not appropriatelyreflect the toxicity and, together with exposure, the risk associatedwith biochars. Instead, the content of B(a)P or the TEF approach shouldbe used. Alternatively, different threshold values could be establishedindividually for NAP and the sum of the remaining 15 US EPA PAHs asdone in the German Federal Soil Protection Ordinance for the soil-groundwaterinterface.56 In the German Federal SoilProtection Ordinance, the threshold value for NAP is 10 times higherthan the value for the sum of 16 US EPA PAHs without NAP. Using suchan approach, the lower EBC threshold value for PAHs in biochar, forexample, could remain at 4 mg kg–1 but re-definedas 16 US EPA PAHs without NAP, and the NAP only threshold value couldbe set at 40 mg kg–1. This methodology would betterreflect the hazards associated with PAHs in biochar.The 16 US EPA PAHs were set up for wastewater originally,whereother PAHs rather than NAP tend to dominate.57 All methods seem tobe suitable for biochar; however, since the TEF approach is widelyused in practice for assessing the risk of dioxins, we suggest thatit should also be the method of choice for setting up future guidelinesand legislation thresholds for PAHs in biochar.To assessthe bioavailability or bioaccessibility of PAHs in biocharrepresents yet another approach.The analysis of PAHs in 73 biochars, together with a detailed investigationof the production conditions, showed that post-pyrolysis contact ofpyrolysis vapors with biochar was the most important factor determiningthe content of non-NAP PAHs in biochar in this study. The NAP contentwas only marginally, if at all, influenced by this process, whichcan be explained by NAP’s high vapor pressure and therefore,low chance of condensation in cool zones of the pyrolysis unit. Post-pyrolysiscontamination by condensation and deposition of PAHs was much moreimportant than the effects of HTT, carrier gas flow rate, or feedstockchoice in determining the non-NAP PAH levels in biochar. These findingsare novel and of great significance for biochar research, applications,and relevant regulations.To ensure the production of biocharwith low non-NAP content and,thus, low PAH-related hazard, the pyrolysis unit design and operationmode must be modified to avoid conditions suitable for depositionof PAHs on biochar. We found the most common issue in our continuousproduction units (auger and rotary kiln) was insufficiently high temperaturein the area where pyrolysis vapors and biochar are separated. Thisdischarge chamber either needs to be actively heated and/or well insulatedso that the furnace passively heats the area. In both cases, the temperaturein the discharge chamber should be close to the temperature in theheated zone to minimize vapor condensation. The discharge chamberset up in a moving bed pyrolysis unit would be comparable to our augerand rotary kiln units, and therefore, although we did not test biocharsfrom such units, our conclusions still hold true for other continuouspyrolysis units. Condensation and trapping of pyrolysis vapors on/withinbiochar in batch units, for example, in traditional kilns or mufflefurnaces, caused by uneven heat distribution and because vapors cannotescape freely, also need to be avoided. Our study highlights thatbiochar with low total and non-NAP PAH contents can, in principle,be produced from different feedstock, on different scales, and usingdifferent technologies.Lastly, with biochar PAHs being dominatedby low-toxicity NAP,the use of threshold values in biochar standards and environmentalregulations based on the sum of 16 US EPA PAHs that was introducedwith wastewater in mind is not fit for purpose. New standards andregulations should set PAH limits based on their toxicity and perhapseven availability, e.g., using the TEF approach to appropriately reflectthe hazards of different PAHs.


Cynodon dactylon). However, the role of hormonal reprogramming and crosstalk in regulating root growth and salt tolerance in bermudagrass was not reported. Here, we examined the physiological and hormonal responses of two contrasting bermudagrass genotypes; ‘C43,’ salt-tolerant ‘C198’ salt-sensitive. Under salt stress, ‘C43’ had better membrane stability and higher photosynthetic activity than the ‘C198.’ Salt stress promoted root growth and improved root/shoot ratio and root activity in ‘C43,’ but the root growth of ‘C198’ was inhibited by salt stress, leading to diminished root activity. The two bermudagrass genotypes also showed critical differences in hormonal responses, especially in the roots. The root contents of indole-3-acetic acid (IAA), cytokinin derivatives, such as trans-zeatin riboside (tZR) and dihydrozeatin riboside (DHZR) were increased in ‘C43,’ but decreased in ‘C198’ when exposed to salt stress. The root growth rate was positively correlated with the root IAA, tZR and DHZR, indicating their crucial role in root growth under salt stress. The expressions of TAA/YUCCA and CYP735A involved in IAA and tZR biosynthesis were induced by salt stress in ‘C43,’ but inhibited in ‘C198,’ leading to reduced hormone accumulations. Salt stress decreased the iP, tZ, and DHZ content in the roots of both genotypes, and no significant difference was observed between the two genotypes. Salt stress reduced the content of GA3 in both genotypes by inhibiting GA20ox and GA2ox genes, which could be attributed to the reduced shoot growth in both genotypes. The increased ABA level by salt stress was significantly higher in ‘C198’ than ‘C43.’ Furthermore, there were positive and negative correlations between different hormones and root growth, suggesting that root growth could be regulated by complex hormonal reprogramming and crosstalk. This study provides a foundation for understanding the underlying mechanisms of hormonal-mediated root growth and salt tolerance in bermudagrass.Salt stress is one of the major abiotic factors limiting the productivity of bermudagrass ( Pers.) is a warm-season turfgrass species widely used to establish sports fields, lawns, and golf courses  to characterize the accumulations of different phytohormones in two contrasting bermudagrass genotypes  in response to salt stress. Furthermore, to understand how phytohormones are regulated at the molecular level, we investigated the mRNA expression levels of critical genes involved in different phytohormones’ metabolism. Overall, this study shed light on how hormonal reprogramming and crosstalk are involved in root growth and salt tolerance in bermudagrass.–2 s–1 at canopy height for 14 h for 4 weeks. During the establishment period of shoot and root, the shoots were cut weekly at 4-cm height, and roots were clipped back to the bottoms of the pots at the beginning of the salt treatment to allow the plants to reach full maturity and develop uniform and equal size roots and shoots.Plant materials were prepared according to our previous studies , 2015. BAfter 4 weeks of cultivation, plants were subjected to 0 mM (control), 180 mM and 300 mM NaCl  for 10 days and then canopy height and root length were determined. Roots elongated from nylon screen into nutrient solution were sampled after 4 h light. Plants were removed from the nutrient solution and roots were gently washed for 30 s with distilled water. The root tips, encompassing the meristem and the elongation zone, were excised with a scalpel from the remaining root system and immediately frozen in liquid nitrogen and stored at −80°C for further analysis. Four independent biological replicates, each containing a pool of twenty different plants, were sampled for hormone and qPCR analysis. The salt treatments and grass genotypes were arranged in a randomized complete block design with four replicates.Shoot and root growth rates were determined as the difference in growth before and after salt treatment . The roo2HPO4, pH 7.0 for 20 h. The colored root samples were blotted dry and rinsed in deionized water, then incubated in 10 ml of 95% ethanol at 60°C for 4 h under gentle agitation during the extraction. The absorbance of the extract solution was recorded at 530 nm and root activity was calculated according to the standard curve with the following equation:The root activity was detected by the TTC method as described previously . BrieflyD represents the deoxidizing amount of the TTC (mg); W is the fresh weight of roots (g); t stands for the time of coloration (h).Where Ci) was determined using a conductivity meter . Subsequently, the tubes were autoclaved at 121°C for 20 min, and the second conductivity (Cmax) was determined after the solution cooled to room temperature. Relative EL was calculated using the formula: EL (%) = (Ci/Cmax) × 100.Cell membrane stability was determined as electrolyte leakage (EL) according to our previous study . Briefly–1 CO2, 500 μmol s–1 flow rate) and a Licor 6400 LED external light source providing a photosynthetic photon flux density of 600 μmol m–2s–1. Net photosynthetic rate (Pn) and stomatal conductance (Gs) were measured for three subsamples in each pot, with each subsample consisting of five leaves  with the controlled atmosphere (400 μmol mole leaves .3), and ABA were extracted and purified according to the method of g for 15 min, and re-extracted for 1 h in an additional 2 ml of the same extraction solution at 4°C. After centrifugation, extracts were combined with the first supernatant. Pooled supernatants were passed through a Sep-Pak tC18 cartridge  on an automated solid-phase extraction system  to remove interfering lipids and plant pigments. The column was washed with 0.3 ml of extraction solvent and then combined with elutes. The combined elute evaporated in a vacuum concentrator at 40°C  and then reconstituted with 2 ml of 1 M formic acid. The hormone-containing fraction was passed through an Oasis MCX 96-Well Plate 150 mg (Waters) preconditioned with 2 ml of methanol followed by 2 ml of 1 M formic acid. After washing with 1 M formic acid, ABA and auxins were eluted with 2 ml of methanol, and cytokinins were eluted with 2 ml of 0.35 M ammonia in 60% (v/v) methanol as indicated in Hormones were analyzed as previously described . Cytokin–1. Mobile phase A consisted of water with 0.1%formic acid, and mobile phase B (methanol), was used for the chromatographic separation. The elution program maintained 70% A, 30% B for 3 min, then a linear gradient from 30 to 70% B in 7 min, followed by another linear gradient from 70 to 95% B in 10 min, and finally 95% B maintained for another 5 min. The column was equilibrated with the starting composition of the mobile phase for 30 min before each analytical run. The eluting ions were subjected to multiple reaction monitoring (MRM). The mass spectrometer was operated in the positive mode for cytokinins and IAA, and negative mode for ABA with a capillary spray voltage of 3500 V and a scan speed of 22,000 (m/z)/s from 50 to 600 m/z. Instrument control, data acquisition and processing were performed with Xcalibur 2.1 software . The level of plant hormones  in the samples were quantified using the calibration curves constructed for each analyzed component  and corrected with the labeled forms of each compounded as internal standards at 0.1 μg.ml–1. The recovery percentages ranged between 90 and 92%.Analyses were carried out on a high-performance liquid chromatography (HPLC)-mass spectrometry (MS) system  coupled to a triple quadrupole mass spectrometer  and equipped with a heated electrospray ionization source (HESI-I). For each sample, 10 μl was injected into a Zorbax SB-C18 HPLC column  maintained at 35°C and eluted at a flow rate of 200 μl min–ΔΔCt method  according to the user’s manual. After extraction, the RNA pellet was dissolved in 100 μl of RNase-free water. RNase-free DNase I was added to the total RNA to remove DNA contamination. The total RNA concentration was then determined by absorbance at 260 nm and RNA quality was evaluated on a 0.8% agarose gel. The first-strand cDNA fragments were synthesized from 2 μg of total RNA using oligo(dT)12-18 primer using a cDNA synthesis kit . The gene-specific primers  were dest method .P = 0.05 level of probability.All data were subjected to two-way ANOVA according to the general linear model procedure of SAS  to determine the effects of genetypes, salinity and their interactions. Treatment means were separated using the Duncan’s multiple range test at the Salt stress caused severe damage to shoot growth in both bermudagrass genotypes, but the effect was more pronounced in the ‘C43’ genotype, as evidenced by severe leaf damage and reduced shoot growth . Both geWe measured electrolyte leakage in the leaves and roots of the two bermudagrass genotypes to investigate the degree of salt stress-induced membrane damage . The twoPn and Gs in the leaves of both genotypes after salt stress. There were no significant differences in these parameters between the two bermudagrass under normal conditions. But, salt stress significantly reduced Pn , cytokinins  and GAarations . Under tarations . Salt starations .Both genotype’s leaf iP and iPA content showed decreasing trends with increasing NaCl concentrations . Relativ3 content was higher in ‘C198’ than in ‘C43.’ The root GA3 content significantly decreased with salt treatments in both genotypes  and 9-cis-epoxycarotenoid dioxygenase (NCED). Salt stress did not affect the expression of ZEP in both genotypes. However, the expression of NCED was markedly increased in the roots of the two bermudagrass genotypes, more profoundly in ‘C198’ than in ‘C43’ (IPT) and CYP735A genes, encoding the key enzymes involved in the biosynthesis of tZR. Salt stress did not affect the expression level of IPT in the roots of both genotypes. Interestingly, CYP735A exhibited contrasting expression between the two bermudagrass in response to salt stress, increased in ‘C43’ and decreased in ‘C198’ (CKX). Salt stress caused a remarkable increase in the expression level of CKX in ‘C198,’ particularly under severe salt stress (300 mM). In comparison, only a slight increase in the expression level of CKX was observed in ‘C43’ under severe salt stress (300 mM)  .TAA and YUCCA, were up-regulated in ‘C43’ and down-regulated in ‘C198’ following salt stress (GA20ox) and GA-degradation genes (GA2ox) were significantly down-regulated under salt stress in the roots of the two genotypes  is a key enzyme that catalyzes the de novo synthesis of CKs and the biosynthesis of the major derivatives of CKs, iP- and tZ is catalyzed by IPT. Overexpression of IPT has enhanced the biosynthesis of CKs , an enzyme that catalyzes the degradation of CK, was upregulated by salt stress in both genotypes, but ‘C198’ had significantly higher expression in both leaves and roots, resulting in lower CK contents in both tissues. The upregulation of IPT in the salt-tolerant bermudagrass was responsible for the accumulation of CK, especially tZ, and may be involved in the salt tolerance of bermudagrass  and YUCCA (YUC) enzymes-mediated (TAA)/YUCCA (YUC) pathway. In Arabidopsis, the inactivation of several YUC genes dramatically decreased endogenous IAA  and 9-cis-epoxycarotenoid dioxygenase (NCED) are critical enzymes in the biosynthesis of ABA. In this study, salt stress did not affect the expression of ZEP in the roots of both genotypes.It is well documented that salt-induced osmotic stress could increase the concentration of ABA in both roots and shoots of salt-treated plants, providing a mechanism of salt stress tolerance . Here, sNCED was induced by salt stress in bermudagrass. The expression was significantly higher in the roots and leaves of the C198 genotype, consistent with the elevated levels of ABA in both tissues of the salt-sensitive genotype. The accumulation of ABA is primarily associated with the upregulation of NCED genes  and GA2-oxidase (GA2ox) are key enzymes involved in the biosynthesis and degradation of GAs, respectively. Overexpression of OsGA2ox5 improved rice tolerance to salt stress (Gibberellin is crucial for plant growth and development, and its deficiency is associated with plant dwarfism . Stress t stress .GA20ox2 was involved in root elongation and root branching by regulating IAA synthesis and transport in Arabidopsis (3 in bermudagrass could be related to the downregulation of GA2ox and GA20ox by salt stress. Under normal conditions, the GA3 content was higher in the salt-sensitive genotype, but relatively ‘C43’ had significantly higher GA3 content under salt stress. At optimal concentration, GA3 is beneficial for the physiology and metabolism of plants under abiotic stresses (3 to maintain normal functioning, thus improving root growth and salt tolerance.bidopsis . Thus, tstresses . These rWhen exposed to a stressful condition, plants reprogram the biosynthesis and transport of hormones in a way to cope with the stress. Hormonal crosstalk is a crucial process for the understanding of the role of phytohormones, because hormones may not act independently to regulate plant growth and development . To provIPT expression and conversely increase the expression of CKX to inhibit CK biosynthesis (IPT was higher and CKX was lower in the ‘C43,’ suggesting that auxin may not antagonistic to CK in bermudagrass.Studies have shown that the crosstalk and regulation of hormones by each other occurs at the transcription level through regulating biosynthesis and degradation genes. For instance, auxin could inhibit ynthesis . Despiteynthesis . In our ynthesis , 10. AddThe negative effect of ABA in seed germination was improved by GAs through antagonistic crosstalk . In thisThe contents of IAA, GA3 and IBA were decreased in the leaves of the two bermudagrass in response to salt stress. Interestingly, IAA, GA3 and IBA contents were significantly higher in ‘C43.’ Moreover, we found a significant positive correlation between the contents of these hormones and shoot growth under control and salt stress conditions . Thus, tDue to the complex nature of plant hormone interactions, integrating physiological and molecular levels to the whole plant response is a key to uncovering the detailed mechanisms of hormonal crosstalk . It has 3 in the roots, which are crucial for plant growth and development. The expressions of hormones related genes involved in the biosynthesis of these hormones were induced by salt stress in the salt-tolerant genotype. The antagonistic interaction of two hormones might be affected by the third hormone to synergistically improve root growth and salt tolerance in bermudagrass. Moreover, the crosstalk of hormones and the outcomes depend on the stress condition, the plant species, and the hormones concentration. Thus, further study is necessary to elucidate how hormonal crosstalk regulates root growth and salt tolerance in bermudagrass at the molecular level.This study demonstrates the role of hormonal reprogramming and crosstalk in regulating root growth and salt tolerance in bermudagrass. In addition, to the morphological and physiological differences, the two contrasting genotypes showed an apparent hormonal regulation, including biosynthesis and degradation in response to salt stress. With improved root growth and root activity by salt stress, the salt-tolerant genotype ‘C43’ had a higher accumulation of IAA, CK, and GAThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YY, QX, and L-XH conceived and designed the experiments. YY and MW performed the experiments and wrote the manuscript. YY, MW, and N-FL analyzed the data. HD and Y-BZ helped with manuscript reviewing. QX and L-XH revised the manuscript. All authors read, revised and approved the final manuscript.


This study investigated the effect of 5% rapid weight loss on hydration status and performance in elite judo athletes. The study demonstrated that rapid weight loss performed in 48 h induced dehydration and increased heart rate responses during the exercise. However, it did not negatively affect performance during judo-specific tasks. Despite this finding, rapid weight loss may lead to hazardous effects on the health and performance of judo athletes in the long term. Thus, more precautions should be taken to stop judo athletes from resorting to rapid weight loss before competitions with better in-season weight management.p < 0.001). In addition, athletes’ SJFT total throw numbers differed among measurements . Athletes presented worse SJFT index after weight loss . HR changed significantly among measurements days and times . Conclusion: RWL induced dehydration and impaired heart rate recovery in highly trained judo athletes, and they could not rehydrate between competition simulated weigh-in and 15 h of recovery.Background: This study aimed to investigate the effect of 5% rapid weight loss on hydration status and judo performance in highly trained judo athletes. Methods: Eighteen male judo athletes participated in the study and were divided into two groups: control and rapid weight loss (RWL). RWL athletes were given 48 h to cut 5% of their body mass while the control group followed their routines. Athletes performed three measurements, including hydration, body mass and three consecutive special judo fitness tests (SJFTs). At the 1st and 6th minutes following each SJFT and 1st, 6th and 15th minutes following the last SJFT, blood lactate and heart rate (HR) was monitored. Results: The effect of RWL on variables was tested with split-plot ANOVA. RWL significantly affected urine specific gravity with a higher value following weight loss compared to baseline and recovery (F2-32 = 13.2,  Judo is a grappling Olympic combat sport where athletes compete according to weight categories . The weiThere are conflicting findings in the literature related to the effects of RWL in combat sports. Some studies suggested that RWL resulted in impaired repeated aerobic and anaerobic performance and a decrease in strength gain ,13,14, wRWL induces dehydration in combat sports athletes. Dehydration may cause physiological changes in the body, diminishing exercise performance and putting one’s health at risk . PreviouAlthough there are numerous studies related to the effect of RWL, a large number of studies did not include a control group/exercise protocol specific to judo match demands and format and were executed under the previous IJF Refereeing Rules, where athletes were weighed almost 2 h before the competition ,24. UndeEighteen highly trained male judo athletes competing at the national and international level voluntarily participated in this study. The sample size was justified using G*Power software  with a priori test with an effect size of 0.25 and 0.85 statistical power using repeated-measures ANOVA, within–between factors test . The stuThe current study aimed to simulate a real competition environment to test our hypothesis. Therefore, the athletes from both RWL and control groups participated in 3 measurements: anthropometry, hydration status and performance tests. The first measurement was made 48 h before reference weigh-in time at 19:00), the same time the official weigh-in is held during official competitions. The second measurement was carried out after 48 h at 19:00 , during which RWL group athletes had to meet the study’s criteria of losing 5% of their body mass. Finally, the next day, the third measurement was made after 15 h of recovery (RWG) at 10:00–11:00, where the athletes of the RWL group were instructed not to gain more than 5% of their body mass. This simulated the competition environment and the official weigh-in procedures under the current IJF Refereeing Rules [9:00, theThe summary of the study design can be found in The sequence of the tests performed by the athletes can be found in −1) [Urine specific gravity (USG) is an easy and reliable method to monitor the hydration status of combat sports athletes with urine concentration ,28. Each−1) .Body compositions of the athletes were determined using a bioelectrical impedance (BIA) device , which uses a dual-frequency method (50 kHz/6.25 kHz). Body composition measurements were performed in standing position, barefoot, with legs and thighs not touching. The skin and the electrodes were precleared and dried before the measurement. The general measurement guidelines  for BIA Resting blood lactate (LA) and heart rate (HR) of the athletes were measured following a 30 min rest in the supine position. HR and LA were also monitored at the 1st and 6th minutes following each SJFT application and 1st, 6th and 15th minutes following the last SJFT application . LA was ippon-seoi-nage technique as many times as possible. Performance was determined based on the total throws completed during each of the three periods (A + B + C). The heart rate (HR) was measured immediately after the test and then 1 min later to calculate the index using the following equation:SJFT was used to simulate judo match performance as it presents the same physiological responses following a judo match and also has positive relationships with performance parameters in competition  ,33. ThreReliability values (ICC) of the number of throws and index have been previously reported as 0.73 and 0.88, respectively .2) was used to determine effect size (ES), with 0.0099, 0.0588 and 0.1379 considered as small, medium and large effect sizes [p < 0.05.The data were presented as mean ± standard deviation with 95% confidence intervals (CI). The data normality was checked with the Shapiro–Wilk test and descriptive methods using skewness and kurtosis coefficients . Indepenct sizes . Analysi2 = 1.67, ES = large, p < 0.001) while there was no significant effect of group factor . When the difference among the measurement days was investigated, there was no significant difference in the control group (p > 0.05), whereas there was a significant difference in the RWL group with lower values at baseline and following recovery compared to RWL (p < 0.001). Although there was no significant main effect of group on USG changes, there was a significant interaction effect of time and group on USG . In other words, the differences in USG values of RWL group were found different from those obtained from the control group. Related to USG, a significant main effect of time was found  throughout the measurements, and the RWL group presented dehydration (USG > 1.020 g·mL−1) after the RWL process and 15 h of recovery, although they started the study in a hydrated state. 2 = 0.056, p = 0.37, ES = small). There was also no significant main effect of group on body mass changes .As illustrated in 2 = 0.12, p = 0.11, ES = medium). Moreover, RWL and measurement times did not affect total throw numbers during each SJFT . However, a significant main effect of time on SJFT total throw numbers was found . The data related to throw numbers in SJFT applied by athletes at baseline, after RWL and after 15 h of recovery are presented in p < 0.05). According to one-way repeated-measures ANOVA results, the RWL group showed worse performance following RWL with lower throw numbers after RWL compared to after 15 h of recovery . SJFT index differed between groups and at different measurement times , while it did not differ among three SJFTs implemented consecutively . According to one-way repeated-measures ANOVA results, there was significant effect of RWL group on changes in SJFT index . The differences related to SJFT index between measurements after RWL and after recovery in RWL group can be seen in SJFT index did not differ between groups among different measurement times at baseline, after RWL and after 15 h of recovery . Moreover, there was no significant interaction effect of time and test, time and group, and test and group factors . While LA did not differ between groups at baseline, after RWL and after 15 h of recovery , it differed during SJFT applications implemented consecutively .LA changes were not affected by time, test and group factors . Moreover, there was no significant interaction effect of time and group or test and group factors . However, HR differed following each SJFT performance at baseline, after RWL and after 15 h of recovery . In addition, HR showed difference at baseline, after RWL and after 15 h of recovery . Furthermore, there was significant main effect of test and group factors separately on HR changes . HR did not differ between groups following each SJFT at baseline, after RWL and after 15 h of recovery . RWL group also presented higher values 1 min after the third SJFT performance . HR changes can be found in Following RWL, a significant difference was found in HR values between RWL and control groups. RWL group presented higher HR values 1 min after the second SJFT compared to the control group  RWL resulted in a high level of dehydration in the RWL group, which did not change even after 15 h of recovery and 5% weight gain, while the hydration level of the control group stayed unchanged throughout the measurements; (2) the RWL group presented better performance following 15 h of recovery compared to second measurements following RWL; and (3) the RWL group demonstrated higher HR values during recovery following RWL compared to control group.Fluid loss and decreasing fluid intake are the most applied weight loss methods among combat sports athletes ,25. In tPrevious research demonstrated that insufficient recovery following dehydration led to decrements in aerobic and anaerobic performance and thus led to worse performance ,43,44. WSJFT has the same physiological responses following a judo match and also has positive relationships with performance parameters in competition  ,33 and cIn practice, RWL should be avoided as much as possible to avoid increased strain on the cardiovascular system. Therefore, coaches and athletes (C&A) should pay more attention to better pre- and in-season weight management. In addition, C&A should strive to reduce weight cycling and the difference between training and competition bodyweight as much as possible. C&A should also focus their attention on developing personalized strategies of RWG to achieve proper regeneration and hydration status before the first fight. They should keep in mind that RWG of 5% in 15 h before the competition does not equal a normal body hydration status. Future studies should also try to address the aforementioned issues. This study had some limitations. The food and fluid intakes of the athletes were not recorded and monitored throughout the study. It would provide an insight into the recovery process of judo athletes between weigh-in and the start of the competition if the food and fluid intakes of the athletes were recorded. In addition, athletes trained during the 48 h of the weight-cutting process, but training intensities and weight-cutting methods of the athletes were not monitored and were self-reported. Future studies could include such variables. Additionally, the fact that BIA provides accurate results depends on the guidelines proposed by the manufacturer; however, they could not be completely followed due to the nature of the study with a simulation of the competition environment. RWL procedures induced dehydration in judo elite athletes, and the athletes could not rehydrate between RWL and the last measurement following 15 h of recovery. This study showed that RWL did not affect judo-specific performance but partially increased physiological load during the exercise. However, it is important to note that athletes did not rehydrate enough even after 15 h of recovery and despite up to 5% weight gain. Therefore, future studies are warranted to focus on the effects of RWL and long-term dehydration by monitoring athletes’ performance changes as well as health-related variables.


With advancing maturity, phenolic acids and antioxidant activity were found to increase, whereas flavonoids and limonoids decreased in concentration. There was a transient increase in the concentration of some polyphenolics such as hesperidin, naringin, narirutin, naringenin, neoeriocitrin, rutin, nobiletin and tangeretin, and limonoid aglycones such as limonin and nomilin at mid-maturity stage (M3) which coincided with prevailing low temperature and frost events at growing locations. A higher concentration of limonin and polyphenolics was observed for fruit grown under STH climates in comparison to those grown under STA climates. The data indicate that fruit metabolism during advanced stages of maturation under distinct climatic conditions is fundamental to the flavor, nutrition and processing quality of Kinnow mandarin. This information can help in understanding the optimum maturity stage and preferable climate to source fruits with maximum functional compounds, less bitterness and high consumer acceptability.In this study, we investigated the impact of harvest maturity stages and contrasting growing climates on secondary metabolites in Kinnow mandarin. Fruit samples were harvested at six harvest maturity stages (M1–M6) from two distinct growing locations falling under subtropical–arid (STA) and subtropical–humid (STH) climates. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technique was employed to identify and quantify secondary metabolites in the fruit juice. A total of 31 polyphenolics and 4 limonoids, with significant differences ( Citrus fruits are among the most widely consumed natural products in the world. In recent years, there was an increase in worldwide citrus production and consumption due to an increasing understanding of their health benefits. Citrus fruit draws the attention of researchers due to the presence of metabolites with plentiful health benefits such as antioxidant ,2, anti-The accumulation of these metabolites in citrus is affected by various factors such as species, variety, climate and soil conditions of a location and rootstock and development and maturation . Among tMetabolomics has provided a rapid and realistic method for monitoring changes in metabolites in different plants and plant products. Analytical strategies for metabolomics research aimed to characterize the whole metabolite complement and then relate their concentrations to features and properties of the sample ,37. LiquCitrus nobilis) and Willow leaf mandarin (Citrus deliciosa). Kinnow has become a commercial fruit crop in the Punjab state of India and contributed 1.33 million tonnes in the year 2019-20 [Although there are numerous metabolomic studies in citrus, to the best of our knowledge, there is no information on the comprehensive profiling of secondary metabolites at different harvest maturity stages and growing climates in Kinnow mandarin, a hybrid of King tangor  unless mentioned otherwise. HPLC grade formic acid (FA) was obtained from Merck Limited . Milli-Q synergy system  was used for obtaining LC-MS grade water (resistivity 18.2 mΩ). Nomilin and obacunone were procured from ChromaDex . All the required reagents for the determination of antioxidant activity were obtained from Analytik Jena  Instruments India Pvt. Ltd., Delhi, India).C. jambhiri). The fruit was harvested from each location separately at a regular interval of 10 days, starting from mid-December (M1) to early February (M6). Overall, there were six harvest maturity stages from M1 to M6. At each location, fruit was collected from six selected healthy trees for each maturity stage. Uniform cultural practices were given to all the trees. Ten fruit were harvested from different parts of each tree covering the entire canopy and randomized with fruit samples of other trees. Sixty fruit were harvested from six selected trees at each maturity stage and transported in plastic crates to the laboratory at National Agri-Food Biotechnology Institute (NABI), Mohali, within 6–8 h. The fruit was thoroughly washed to remove adhering dirt and allowed to dry before being peeled off carefully for juice extraction by hand-squeezing. The juice sample was passed through a strainer to remove seeds and fibrous material and then stored in 50 mL-polypropylene tubes at −80 °C until further analyses.Two different locations under contrasting growing climates located in the Punjab state of India were identified for collecting fruit samples: STH—Chhauni Kalan and STA—Abohar . The comv/v) and the supernatant obtained after centrifugation was filtered through a 0.2 µm polytetrafluoroethylene (PTFE) membrane filter  and used for polyphenolics analysis. Zorbax Eclipse plus C–18 column  maintained at 40 °C was used to achieve separation of metabolites using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. An Agilent 1260 HPLC coupled to an ion trap mass spectrometer QTRAP®® 5500  was used for identification. Quantification was based on calibration curves of external standards and results were expressed as mg/L.Samples for polyphenolic analysis were extracted as previously reported by Vrhovsek et al.  with min®® R-215  and reconstituted in 2 mL of acetonitrile. The extracts were filtered using a 0.2 µm PTFE membrane filter and used for the analysis of limonoid aglycones. Aglycones were eluted using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B.Limonoid aglycones were extracted as described by Manners et al. . Chromatv/v) and solvent B and water: acetonitrile . Zorbax Eclipse Plus C-18 column maintained at 35 °C was used for the separation of both limonoid aglycones and limonoids glucosides. The identification of limonoids (aglycones and glucosides) was achieved by coupling an Agilent HPLC system with a QTRAP®® 5500. Calibration curves based on corresponding external standards were used for quantification, and results were expressed as mg/L.Limonoid glucosides extraction was carried out following the method described by Jayaprakasha et al. . ChromatThe MS/MS conditions used were as reported in previously published papers ,47. The The limits of detection (LOD) and quantification (LOQ) for each metabolite were determined from the corresponding calibration curves with LOD = 3 √S/a and LOQ = 10 √S/a  (IHT guideline). The LOD, LOQ, r and calibration range for the metabolites are shown in ®  as described previously [The antioxidant activity of the juice sample was determined by a photochemiluminescence (PCL) assay using the Photochemeviously  and exprp < 0.05) were noted. Tukey’s Honest Significant Difference (HSD) test was conducted for pair-wise comparison of the maturity stages, different locations and their interaction. Mean values along with Standard Error of Mean (SEM) and significantly different effects/means  are presented in The statistical and multivariate analysis procedure was the same as described in a previously published paper for primary metabolites . All thee transformed to reduce heteroscedasticity followed by principal component analysis (PCA) using MarkerView™ software . Heap map based on quantitative changes of secondary metabolites was made using Multi Experiment Viewer (MeV) software .For multivariate analysis, centered data were logm/z), MS/MS fragments, collision energy and declustering potential used for identification and quantification are given in the Citrus nobilis) × Willow Leaf (Citrus deliciosa), the former parent being a cross between mandarin and pummelo. The second most abundant flavonoids identified were flavonols which are known to be free radical scavengers and powerful antioxidants [The major flavonoids and phenolics identified and quantified by LC-MS/MS using MRM analysis are summarized in oxidants . Flavonooxidants ,56,57. TIn addition to flavonoids, phenolics in Kinnow constituted benzoic and cinnamic acid derivatives and dihydrochalcones. Sinapic acid was the most abundant benzoic and cinnamic acid derivatives (phenolic acid) in Kinnow, and average values increased with harvest maturity (M1 to M6) from 2.1 to 3.3 mg/L under STA and 1.8 to 2.2 mg/L under STH growing climates. The other compounds in this group included ferulic, ellagic, syringic, p–coumaric acid, t–cinnamic acid, chlorogenic acid, benzoic acid and caffeic acid. Generally, ferulic acid is abundant in mandarins, but sinapic acid is more abundant in Kinnow, which could be attributed to the parentage of one of its parents King mandarin, to Pummelo . The valCitrus limon [Citrus Unshiu Marc indicated a higher accumulation of flavonoids and expression of flavonoid biosynthetic genes in young fruits suggesting the synthesis of flavonoids in the initial developmental stage [The total flavonoids and phenolic concentration appeared to follow a predictable pattern over fruit maturity, occurring at the highest levels at the initial stages and declining with maturity. However, a sharp increase was observed in the concentration of some flavonoids  at mid-maturity stage (M3) in Kinnow which coincided with prevailing low temperatures and frost events at growing locations. The declining trend in flavonoids is in agreement with the trend reported in red grapefruit , sweet ous limon  and Ponkus limon . Citrus us limon . Howeverus limon . Flavonous limon . PAL is al stage . Citrus al stage . The indal stage . Zhang eal stage  reportedal stage , while lal stage .m/z), MS/MS fragments, collision energy and declustering potential used for the identification and quantification of limonoids are given in the Limonoids, the predominant triterpenoids responsible for delayed bitterness in citrus, are known to follow a dynamic pattern during fruit development and maturity . LimonoiStudies in Thai tangerines  and ValeThe total antioxidant activity (AOX) is expressed as the sum of antioxidant activity due to lipid-soluble (ACL) and water-soluble compounds (ACW) . AntioxiPCA score plots revealed the overall metabolome pattern of the combined effect of growing climates and six different maturity stages , and a cIn addition, individual PCA score plots based on harvest maturity stages under each climatic condition were plotted. Principal components 1 (PC1) and 2 (PC2) accounted for 72.9% and 15.1% of the data variance in Kinnow with harvest maturity under the STA growing climate  and 59.1To the best of our knowledge, this is the first report on the characterization of flavonoids, phenolics and limonoids in Kinnow as affected by harvest maturity and growing climates. With advancing maturity, flavonoids and limonoids were found to decrease. However, there was an increase in phenolics and antioxidant activity as harvest maturity progressed. There was a transient increase in the content of some polyphenolics , ascorbic acid, limonin and nomilin at the mid–maturity stage which coincided with prevailing low temperature and frost events. Kinnow from the STA growing climate with lower concentrations of limonin and optimum polyphenolics, and maturity stage M2 with low limonin and optimum polyphenolics seemed to be better suited for the fresh and processing industry. The information generated might help to identify maturity markers to determine the optimum maturity stage in Kinnow. Furthermore, the knowledge gained from this study helps to understand the optimum harvest maturity stage and preferable climatic conditions to source fruits with maximum functional compounds, less bitterness and high consumer acceptability.


Irisin, encoded by the FNDC5 (fibronectin type III domain containing 5) gene, is a novel myokine that has been implicated as an essential mediator of exercise benefits. Effects of irisin on heart physiology is still ambiguous. This study aimed to evaluate the impact of exogenous administration of irisin on heart physiology and the pharmacokinetic profile of pump-administered irisin. To do so, Sprague Dawley rats were implanted with an irisin-loaded osmotic pump (5 μg/kg/day) for 42 days, and other animals were administered with single bolus subcutaneous injections of irisin (5 µg/kg). Body weights and blood samples were collected weekly for 42 days for serum irisin quantification and histopathology. Clinical biochemistry analyses were performed. Heart mRNA expression was assessed in 26 selected genes. Chronic interventional exogenous irisin significantly reduced body weight without affecting the heart myocyte size and significantly reduced creatine kinase enzyme level. Blood CBC, serum biochemistry, and heart morphology were normal. Gene expression of FNCD5, Raf1, CPT1, IGF-1, and CALCIN, encoding for heart physiology, increased while PGC1, Nox4, and Mfn1 significantly decreased. Nevertheless, irisin increased the expression of cardioprotective genes and inhibited some genes that harm heart physiology. Administration of irisin promotes myocardial functions and could be translated into clinical settings after preclinical profiling. Physical exercise is correlated with hemodynamic changes and alters the loading conditions of the heart. The major components of cardiac output—stroke volume and heart rate—are the main hemodynamic features that change an athlete’s heart . These aIrisin is a novel hormone secreted by myocytes and has been suggested to mediate some of the beneficial effects of exercise . It is aIn the present study, we examined the potential effects of interventional exogenous irisin administration (chronic model) on gene expression of proteins associated with adaptive physiological cardiac changes and myocardial health. In addition, a superimposition simulation model of repetitive subcutaneous administrations of irisin was generated and compared to a pump-released experimental model. The potential cardiometabolic impact of chronic irisin administration is also discussed. Changes in percent bodyweight of study animals are shown in Blood analysis for CBC is shown in As shown in Serum irisin levels of study animals are shown in Levels of serum irisin were determined for intervals of the dosing periods. For animals receiving irisin via the osmotic pump, sampling was conducted at intervals from 7 days to 42 days. As for animals receiving irisin as subcutaneous bolus injections, sampling was conducted at intervals of up to 4 h. In comparison to levels of irisin in non-treated rats, alterations of increase in irisin were noted on multiple days throughout the dosing period, thus confirming the consistent release of irisin from the osmotic pump.The administration of irisin via an osmotic pump consistently released irisin throughout the 42 days at levels higher than the baseline. Baseline levels were estimated as 24 ng/mL in control groups and after 4 h from subcutaneous administration. Simulation data were built using a subcutaneous pharmacokinetics profile and compared with the pump-released irisin profile. Daily administration of irisin is needed to establish a profile similar to the pump.Our study investigated the potential change in gene expression of 28 selected heart genes shown in . ChronicSince 2012, the discovery of irisin has raised questions about its role in longevity and maintaining a healthy life through its capacity of mimicking exercise action and offering a novel therapeutic intervention for many ailments such as obesity, type 2 diabetes, cardiovascular disease, and neurodegeneration. Several previous animal studies reported the valuable role of irisin in metabolic homeostasis and maintaining body mass index (BMI) , and agaIn this study, chronic administration of interventional exogenous irisin for 42 days showed a valuable role in reducing body weight without affecting the heart myocyte size. At the day of termination, change in body weight was no longer observed, and that could be linked to the uptake of irisin in the pump, demonstrating a direct effect of irisin on reducing body weight. Nevertheless, such observation warrants further investigation in the impact of irisin on adipocyte and skeletal muscle mass. In the current study, irisin significantly reduced creatine kinase and thus reducing the chance of heart muscle damage. In addition, irisin increased the expression of some genes protecting cardiac myocytes and cardiac function such as FNDC5, CPT1, IGF-1, and Calcin while inhibiting the expression of some genes that harm heart physiology, such as PGC1, NOX4, and Mfn1.The presented pharmacokinetic and protein experiments confirmed a consistent release of irisin from the osmotic pump. It seems that the exogenous interventional irisin has no toxic effect in the animal model study, i.e., no clinical significance was detected on CBC parameters after irisin administration. The exception is platelet distribution width (PDW), where the change was considered statistically significant but of negligible clinical impact since the platelet count was not affected. Moreover, most serum biomarkers were considered clinically normal and untriggered upon administration of irisin, especially ALT, AST, and ALP. However, creatine kinases and LDH were decreased in rats administered with irisin, thus supporting the beneficial effects of irisin on health. Previous research did not report any toxic effects or inverse impact on animal health . The negThe present study reported a considerably clinically significant decrease in percent change of body weight in animals treated with irisin compared to animals of the non-treated group. A previous study in obese mice showed a significant decrease in BMI , while aOur present study investigated the level of heart gene expression of rats exposed to continuous exogenous irisin for 42 days (0.15 µL/h = 5 µg/day). The results showed a significant increase in essential genes that have a substantial role in cardiac energy and cardiac myocyte contractility and hypertrophy, such as IGF-1, Raf1, CPT1, and CALCIN. On the other hand, irisin inhibited the expression level of many genes that pose potential harm to heart physiology, such as NOX4 and Mfn1. Together, these results suggest an opportunity to develop irisin as a novel pharmaceutical compound for a healthy heart.Our result showed that chronic intervention of exogenous irisin significantly increased the expression level of the IGF-1 gene. According to a previous experimental study, IGF-1 inhibits the progression of cardiomyopathic disease in a transgenic mouse model of heart failure, suggesting that heart failure may benefit from early treatment with interventional IGF-1 . This adCalcineurin (CN) is a calcium- and calmodulin-dependent protein phosphatase that provides an essential contractual point for coordination between two fundamental modes of intracellular communication: protein phosphorylation and calcium signaling cascade . It is wFatty acids and acyl-CoAs cannot enter the mitochondrial membrane unless they conjugate with creatinine. In the heart, long-chain fatty acids form a high-energy ester bond with carnitine facilitated by carnitine palmitoyltransferase 1 (CPT-1), located in the inner aspect of the outer mitochondrial membrane . ActivatSeveral previous studies on Raf-1 knockout mice have suggested that Raf-1 plays an essential role in preventing apoptosis ,35; howeResults showed not only increased expression of the genes that positively impact heart function and development, but also inhibition of the expression of genes that might be harmful to cardiac physiology, such as Nox4 and Mfn1. This suggests that irisin might have a role in protecting the heart against I/R via inhibition of the gene expression of Nox4. We recommend further investigation on the interventional exogenous irisin for the treatment of I/R. Previously, it was suggested that Nox4 plays a very important role in mediating oxidative stress and myocardial injury after I/R . InteresIrisin inhibits Mfn1 gene expression in heart tissue, and according to previous studies, deficient Mfn1 hearts are protected against acute myocardial infarction due to impaired mitochondria/SR tethering . It was As concluded in a systematic review, two out of eleven studies report that chronic exercise induces the expression of both genes. However, nine studies reported no change in gene expression after chronic exercise . On the Despite the documented benefits of irisin on cardiac health and clinical management, a controversy around its impact on a coronary heart disease patient is still unsolved. Moreover, during heart failure, irisin was reported to influence cellular metabolism, mitochondrial energetics, and heart failure prognosis. The benefits of irisin on muscle and cardiac health are multiple, yet some controversies are still raised considering its safety in clinical settings. By contrast, high levels of irisin in myocardial infarction might be associated with more cardiovascular risks. Findings reported by Hsieh et al. showed that serum concentrations of irisin were increased in patients post-ST-elevation myocardial infarction . HoweverThe impact of irisin on cardiovascular disease has also been reported as effective for diabetic subjects, showing a reduction in the complications that may arise in such sensitive patients. Multiple studies on diabetic patients concluded that serum irisin levels are associated with enhanced prognosis in patients with cardiovascular comorbidities. A study by Khorasani et al. showed that irisin levels were relatively lower in diabetic patients with cardiovascular complications in comparison to uncomplicated diabetic patients . On the Twenty adult female Sprague Dawley rats with an average weight of 250 ± 40 g were supplied by the Animal Research Unit of the University of Petra Pharmaceutical Center (UPPC), University of Petra . Female animals were selected due to the complexity of the female endocrine system and for being preferable in toxicity assessment if any. Rats were maintained under climate-controlled temperatures (22–24 °C), humidity (55–65%), and a 12-h light/dark cycle.n = 10): a sham group that received a plain surgery and an irisin-treated group that received an irisin-loaded osmotic pump. The dorsal area of the rats was shaved using an electronic clipper and prepared for surgery. Animals were placed over a surgical platform  and anesthetized by isoflurane  (5% induction and 2.5% maintenance) carried by oxygen  using a low-flow anesthesia system . After reaching the surgical depth of anesthesia, povidone–iodine was applied for preoperative skin preparation, and an incision was created for the implantation of the pump . The pump was filled with 150 μL of 0.25 μg/μL recombinant human fibronectin type III domain-containing protein 5 (FNDC5)  reconstituted with PBS . The pump, as claimed by the manufacturer, releases 0.15 μL/h, thus delivering a dose of 5 μg/kg/day. Incisions were closed with surgical staple sutures and observed closely during the first day and daily throughout the study for mortality, general assessment, and any abnormal clinical signs.After acclimatization, rats were randomized into two groups . Afterwards, animals were subjected to blood sampling from the tail tip at specific time intervals  and serum was collected for quantification using ELISA analysis.g for 10 min. At the day of study termination, specifically day 42, animals were subjected to euthanasia by 5% isoflurane, and blood from the ocular sinus was collected into EDTA tubes and plain tubes. Whole blood in EDTA tubes was tested for CBC within 24 h and serum separated following centrifugation was stored at −20 °C for further biochemical analysis at MedCare Labs . The heart was collected for histopathology and RNA extraction.Body weights and blood samples were collected weekly for 42 days. Less than 100 µL blood was withdrawn from the ocular sinus using a heparinized capillary tube into plain tubes. Then, serum was collected for hormone quantification and biochemical analysis through centrifuge at 200× According to the manufacturer’s instructions, serum irisin levels were quantified using the irisin ELISA kit .Simulation of daily administration of exogenous irisin was based on data acquired through parenteral administration. Data were processed considering a dose of 1.25 µg to 250 g rat mass every 1440 min (24 h). Analysis was run using WinNonlin Noncompartmental Analysis Program (Version 5.2). Non-parametric superposition methodology was employed to predict the drug concentration in blood or plasma after multiple doses based on concentration data from a single dose. This was achieved by fitting the data based on the principle of superposition, which does not assume any pharmacokinetic (PK) model. To predict the drug concentration resulting from multiple doses, the concentration–time profile of a single dose was processed. Two assumptions about the data are required: independence of each dose–effect and linearity of the underlying pharmacokinetics. The former assumes that the effect of each dose can be separated from the effects of other doses. The latter, linear pharmacokinetics, assumes that changes in drug concentration will vary linearly with dose amount. The required input data are time, dosing regimen, sort variables, and drug concentration after a single dose. The method of computation was set to log without defining the terminal phase.® solution tubes . A Bead Ruptor Elite-Bead Mill Homogenizer was used to homogenize tissue samples . Homogenized samples were then processed with Direct-ZolTM RNA MiniPrep (Zymo Research Co.) and TRI Reagent® to isolate total RNA (Zymo Research Co.). Qubit 4 Fluorometer , BioTek PowerWave XS2 Spectrophotometer , and 1% agarose gel was utilized to quantify and qualify the RNA. Then, cDNA was synthesized for each sample using a high-capacity cDNA reverse transcription kit .On day 42 following the administration of irisin, cardiac samples were taken from all animals and snap-frozen on-site with liquid nitrogen to avoid RNA degradation. These samples were then kept at 20 °C in TRI ReagentIn a Rotor-Gene Q MDx 5 plex instrument , TB Green™ Premix Ex Taq™ II kit  was used. Briefly, the 20 µL reaction mix was prepared from 10 µL of the master mix, 1.2 µL forward primer (4 pmol), 1.2 µL reverse primer (4 pmol), 2 µL cDNA of the sample, and 5.6 µL of nuclease-free water. Cycling parameters were 50 °C for 2 min, 95 °C for 15 min, 40 cycles of 95 °C for 15 s followed by 40 s at 57 °C, and 72 °C for 20 s, with final melting at 95 °C for 20 s. Duplicates from each cDNA were analyzed, fluorescence emission was detected, and relative quantification was computed automatically. The fold changes in gene expression were normalized using GAPDH and β-actin as internal controls, with which the melting curve confirmed the single-target amplification specificity.https://www.ncbi.nlm.nih.gov/nucleotide/; accessed on: 1 October 2021). All primers were designed using IDT PrimeQuest™ Tool version (2.2.3) , Integrated DNA Technologies Inc. . The primer sequences are presented in The cDNA sequence for each gene utilized in the primer design was obtained from the NCBI’s Nucleotide Database . One-way ANOVA followed by post-hoc Tukey’s HSD test was performed to calculate the statistical significance of gene expression, whereas Mann–Whitney non-parametric analysis was used for CBC and Clinical Biochemistry analysis using IBM SPSS Statistics 25, IBM Corporation Values with A chronic intervention model of exogenous irisin in rats showed a valuable role for irisin in reducing body weight without affecting myocyte size and significantly reducing creatine kinase and, thus, heart muscle damage. Irisin also increased the expression of some genes protecting cardiac myocytes and cardiac function and inhibited some genes that harm heart physiology. Our results shed light on the importance of performing further clinical and physiological studies. Improved insight into the role of irisin on heart health and pathophysiology promises to lead to more innovative treatment strategies that will improve care of affected patients.


Celluloid artifacts are known by conservation professionals to be prone to degradation, threatening their own integrity and that of nearby heritage collections. Celluloid alteration can have a heterogeneous nature, and this research topic is still in its infancy for heritage science. This article investigates degradation gradients, both along depth and width, of artificially aged celluloid sheets, and compares them to three-dimensional (3D) historical objects with the aim of gaining a better insight into the nature and evolution of their decay. ATR-FTIR was used to systematically study different sampling points of the artificially and naturally aged specimens and allowed us to recognize better-preserved surfaces and more deteriorated cores. ATR-FTIR was found suitable for assessing the molecular changes induced by degradation, particularly denitration and formation of carbonyl-containing degradation products in severely aged specimens. Even though the severely artificially aged sheets displayed unusual alteration phenomena, they present a degradation gradient similar to the one observed for the naturally aged 3D objects under study. This research underlines that sampling at different depths and/or widths is relevant for characterizing the heterogeneity of degraded celluloid, and further investigation with chromatographic techniques would greatly benefit the understanding of the complex degradation of celluloid artifacts. For the total weight of a CN chain, the mass percentage attributable to nitrogen (N) would be equal to 14.14%, 11.1% or 10.5% if, respectively, all three, two or only one of the hydroxyl groups per monomeric unit would have been substituted with nitro groups [Cellulose nitrate (CN) is the cellulose derivative resulting from the substitution of side hydroxyl groups (OH) with nitro NO groups. o groups ,4) with Celluloid was employed for the industrial production of transparent films, sheets and three-dimensional (3D) artifacts. It could also be mixed with appropriate fillers or pigments to imitate expensive materials, such as ivory, mother-of-pearl and tortoiseshell in jewelry cases, brooches, dolls, etc.Celluloid artifacts are widely spread in archives, cinematheque and modern museum collections. These objects are well-known by conservation professionals for being highly sensitive to degradation ,6. YelloAs a consequence, efforts have been made to study celluloid degradation. As it would not be practical to wait for several years to observe their natural aging, artificial aging protocols, including CN and celluloid samples, have been adopted to simulate their natural degradation in shorter times. Past artificial aging experiments studied the thermal stability of highly nitrated cellulose by subjecting it to high-temperature conditions (100 °C–200 °C) ; or expoMost recent studies have preferred instead photodegradation, especially under UV-visible light filtering wavelengths below 300 nm, to represent outdoor weathering due to the faster rate of degradation obtained when compared to pure thermal aging at low temperatures . These sFew artificial aging studies have employed humidity coupled with high temperature. These include early works on the aging of cinematographic films with CN bases ,16 and tBesides the sublimation of camphor, the loss of nitro groups and the creation of carbonyl (C=O) functions can be typically linked to the two main pathways that rule the degradation of celluloid with moderate N mass percent content when the temperature remains below 100 °C and illumination is kept above 300 nm ,12,18.2 bonds (The first pathway begins with the homolytic scission (homolysis) of RO-NO2 bonds , a react•NO2) and alkoxy macro radicals (RO•) [Nitrogen dioxide radicals (ls (RO•) ,22 resulls (RO•) ,21. The ls (RO•) . Reactiols (RO•) .RONO2 →From this stage onwards, several radical reactions can take place at the different C positions ,12,13. ISumming up, photo-thermal degradation reactions lead to the complete denitration of the cellulose ring, to the creation of C=O functions and to the decreasing molecular weight of the CN chain.x). The reaction of highly reactive nitrogen dioxide (NO2) with moisture in the air forms nitric acid (HNO3) [The second degradation pathway of celluloid involves the hydrolysis of ester links ,22. Thisd (HNO3) ,9,19.2 and HNO3) are among the most dangerous products which can be formed from •NO2 radicals, either by •H or reaction with water [2 detachment [It is important to notice that both nitrous and nitric acids  has been used for the investigation of celluloid historical objects and artificial aging experiments in different acquisition modes ,21,27,32Racemic camphor 96%  and CN membranes  were used as reference materials to characterize the main components constituting celluloid.Transparent celluloid industrially produced by Rothko and Frost™  was used and saw-cut into square sheets (4 × 4 cm). A hole was drilled in one corner of each sheet so they could be hung on a glass rod to ensure good air circulation during aging.In total, 80 Celluloid sheets were aged at 70 °C and 75% RH in a fan-assisted dynamic climate chamber , ensuring good aeration of the samples by hanging them on glass rods. After 10 days, 40 sheets achieved a moderate condition of being slightly yellowed. After 13 days, 40 sheets showed severe discoloration and physical alteration . In part®) with permanent filtered  air ventilation.After aging, both aged and unaged sheets were kept in the dark at room temperature inside a safety storage cabinet  to its corner (D) . Micro-sTwo historical artifacts were selected. The first was a transparent eyeglass temple a–c, formAs both historical objects appeared to retain a homogeneous condition along their moderately damaged surfaces, sampling was performed only considering their section. Two micro-samples were taken from the eyeglass temple, one at the surface and another at the core. Since the organ key was thicker, three sampling depths were chosen along its section: at the surface, middle and core, following the gradient of visual degradation.−1 range with a 4 cm−1 spectral resolution. At least two spectra were acquired from each micro-sample. Enough material was collected from the historical objects to allow the acquisition of at least three spectra per micro-sample.The ATR-FTIR spectra were acquired with an Alpha FTIR Spectrometer (Bruker) equipped with a diamond ATR crystal controlled through the OPUS 8.1 software (Bruker). The spectra were collected by integrating 64 scans for each measurement in the 4000 to 400 cmThe ATR-FTIR spectra of the two components of celluloid, cellulose nitrate (CN) and camphor, are depicted in Three sheets of each aging time were randomly selected for conducting the analyses because they showed similar conditions among each other.−1, νaNO2 at 1634 cm−1, νsNO2 at 1273 cm−1 and νNO at 826 cm−1) against the absorbance of the reference band (νCOC at 1053 cm−1) [−1 for νaNO2 and νC=O bands; from 1503 to 1187 cm−1 for νsNO2 band; from 1187 to 930 cm−1 for νCOC band; and from 1189 to 771 cm−1 for νNO band. In the spectra of the eyeglass temple and organ key, the baselines were drawn differently: from 1187 to 881 cm−1 for the νCOC band and from 1187 to 785 cm−1 for νNO band because they showed significant variations that required tailored intervals for detecting the peak height. The standard deviation (SD) among original measurements was used to assess the statistical difference between the obtained averaged values.The absorbance of each probe band was measured using the peak height tool of the OMNIC 7.2 software (Thermo Electron Corporation) without any correction or further manipulation of the spectra. An average ratio was calculated between the absorbance of each probe band (νC=O at 1730 cm53 cm−1) . The abssNO2 at 1273 cm−1 and νNO at 826 cm−1 was more descriptive in registering molecular variations with aging than the νaNO2 signal at 1634 cm−1.The evolution of molecular changes and related gradients along both depth and width at different aging times was successfully characterized by ATR-FTIR . GeneralIn accordance with visually evident alteration, ATR-FTIR detected statistically significant modifications between surface and core after 13 days, identifying a higher degree of denitration at the core. A gradient along width was also observed from the border (point D), showing higher signals of the nitro groups, towards the center (point A) with lower nitro group absorption. The carbonyl group increased significantly after 13 days, highlighting the greatest molecular change at the core, even for the border areas (point D), not affected by severe visual degradation. Just as for the nitro groups, a gradient of carbonyl signal intensity along the sheet’s width was detected, being more intense at sampling point A than at sampling point D, in correspondence with the severity of degradation. In summary, the analytical evidence correlates well, i.e., higher denitration along a more intense signal of carbonyl groups, highlighting the severest extent of degradation at the core of the sheet center (point A) after 13 days.•NO2 is formed by initial thermolysis, and by H abstraction at the RH groups of CN, it gives rise to an R• radical, which leads to further oxidation and the creation of HNO2, which can trigger acid hydrolysis. This reaction (2) is enhanced by the relatively low solubility of NO2 in CN [The mechanisms responsible for denitration can be both thermolytic and hydrolytic, due to the simultaneous presence of high temperature and humidity, influencing one another and giving rise to fast, complex and intertwined degradation reactions. For example, O2 in CN .RH + •N−1 [2 detachment due to the presence of free OH groups [It is possible that hydrolysis of CN occurred at an early stage because a certain amount of water was already adsorbed by the celluloid sheets due to CN’s slight hygroscopicity ,19 and r−1 ,42) at r2 group at the C6 position [As mentioned in the Introduction, thermal degradation leads to the creation of cyclic ketones, gluconolactones and anhydrides as carbonyl-containing degradation products, but hydrolytic degradation has been suggested to also give rise to the formation of aldehyde (RCHO) functions (3), containing C=O groups, upon hydrolysis of the NOposition , or by rposition ,43 (4).−1 [The formation of degradation products containing carbonyl groups during artificial aging of CN has been previously reported by UV spectroscopy , synchro−1 ,15. Howe−1 : the los−1 ,11,12,35After 13 days, the artificially aged celluloid sheets displayed unexpected degradation effects. To pose hypotheses about the mechanisms that might have induced such degradation, it was necessary to correlate infrared data with visual appearance, considering the sampling points.The volume expansion at the center of the sheets could be related to the formation of volatiles, which would remain trapped and induce internal pressure. The accumulation of volatiles in the bulk of celluloid has already been reported to occur in naturally aged 3D objects . Such exx species [2O4 in particular due to the reddish-brown discoloration [The development of brown color in the core can be attributed to the accumulation of NO species  and to Nloration   of celluloid would greatly support the selection of T and RH for the aging protocol. Tg measurement requires specific method development and data interpretation via dynamic mechanical analysis (DMA) [The artificial aging induced bubbling and extreme volume expansion in the core of the severely aged samples, which were not identified in naturally aged 3D museum objects so far. Nonetheless, this study can serve to plan future aging studies. From a future perspective, the measurement of the glass-transition temperature (Tis (DMA) ,50, as tis (DMA) ), the sais (DMA) ), the cris (DMA) , plasticw), the nitrogen and camphor contents. In detail, size exclusion chromatography (SEC) can be used for determining the Mw of CN [The mechanisms responsible for the observed degradation gradients deserve future investigation. In particular, chromatographic techniques could be applied, following the same in-depth sampling strategy implemented in this work, to measure the molecular weight (MMw of CN ,51,52 anMw of CN  along saMw of CN . This muMw of CN . ATR-FTI