Such significant correlationsindicate SSA to be an important source of atmospheric PFAAs to coastalareas. The correlations in the samples from Andøya were observedfor more PFAA species and were generally stronger than in the samplesfrom Birkenes, which is located further away from the coast and closerto urban areas than Andøya. Factors such as the origin of theSSA, the distance of the sampling site to open water, and the presenceof other PFAA sources  can haveinfluence on the contribution of SSA to PFAA in air at the samplingsites and therefore affect the observed correlations between PFAAsand Na+.The effective enrichmentof perfluoroalkyl acids (PFAAs) in seaspray aerosols (SSA) demonstrated in previous laboratory studies suggeststhat SSA is a potential source of PFAAs to the atmosphere. In orderto investigate the influence of SSA on atmospheric PFAAs in the field,48 h aerosol samples were collected regularly between 2018 and 2020at two Norwegian coastal locations, Andøya and Birkenes. Significantcorrelations ( The field evidencepresented in the present study showssea spray aerosols (SSA) can be an important source of atmosphericperfluoroalkyl acids (PFAAs) in coastal areas. There are threemajor sources of PFAAs to the atmosphere: (1) direct emission frommanufacturing sources such as fluoropolymer plants,12 (2) formation in the atmosphere via degradation from volatile precursorslike fluorotelomer alcohols (FTOHs),14 and (3) water-to-airtransfer via sea spray aerosol (SSA) emission.16Perfluoroalkyl acids (PFAAs), including perfluoroalkyl carboxylicacids (PFCAs) and perfluoroalkanesulfonic acids (PFSAs), are a groupof persistent organic contaminants that have been found worldwidein abiotic environments, biota, and humans.17 When air is entrained into seawater by breakingwaves, surface active substances such as PFAAs can be scavenged bythe air–water interface of the bubbles and transferred to theatmosphere via SSA emission.17 LaboratorySSA simulation experiments have demonstrated that PFAA concentrationsin submicron SSA can be 4–5 orders of magnitude higher thanin the bulk water.18 On the basis of laboratory-derivedenrichment factors (EFs) and reported median concentrations in seawater,the estimated fluxes of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonicacid (PFOS) from SSA to the atmosphere were comparable with the othertwo sources of atmospheric PFAAs ,15 suggesting the potential of SSA as an important sourceof PFAAs to the atmosphere.SSA is emitted at the sea surface by bubble bursting.r80 (radius at 80%relative humidity) = 1, 2, and 5 μm, the estimated residencetimes with respect to dry deposition are ∼1.5 weeks, 2.3 days,and 10 h, and the transport distances are ∼104,2000, and 330 km, respectively.19 The removalof SSA by wet deposition depends on the frequency of precipitationevents, which is usually several days to a week in a marine environment.19 The modeling results by Johansson et al. showedthat the deposition of PFOA and PFOS to terrestrial environments viaSSA transport may impact large areas of inland Europe and not onlycoastal areas.15PFAAs can travel a great distancein the atmosphere via SSA. ForSSA particles with 20 evidence fromthe field remains elusive. Long-term air monitoring between 2006 and2014 showed significantly higher PFOA concentrations at two NorwegianArctic stations, Zeppelin and Andøya, compared to the CanadianHigh Arctic station of Alert.21 It wasspeculated that the two Norwegian stations might receive additionalPFOA transported via SSA since they are closer to open water (Andøya∼1.3 km and Zeppelin ∼ 2km) than Alert (∼4 kmfrom the coast and the surrounding water was covered by sea ice formost of the year).21 However, the PFOSconcentrations were not significantly different between the threeArctic stations.21 It is hypothesized that,if SSA was an important source of PFAAs to the atmosphere, significantcorrelations between the concentrations of SSA tracer ions  and PFAAs should be observed in aerosol samples obtainedin the marine atmosphere or at coastal locations where the atmosphericburden of SSA is highest. Casas et al. collected seven aerosol samplesat a site located at South Bay, Livingston Island, Antarctica, butobserved no such correlation in the samples,9 which might be due to the small sample size collected within a relativelyshort time period (one month).Despite increasingevidence that SSA may be an important sourceto the atmosphere from these laboratory studies,n = 10) around thecoastal area of Longyearbyen, Svalbard Archipelago, Norway, in May2006 and found that the concentrations of the sea-salt component SO42– were significantly correlated with perfluorohexanesulfonicacid (PFHxS) and PFOS concentrations  but not with PFCA concentrations.22 A series of field studies have investigatedsnow pit samples or ice core samples from ice caps that representedatmospheric deposition from multiple years and found no correlationsbetween the concentrations of PFAAs and SSA tracer ions.25 The lack of correlation may be because SSA only contributed to asmall portion of the PFAA deposition on the high altitude ice caps.Ice core samples represent atmospheric deposition from multiple yearsor decades. Compared with annual PFAA and PFAA-precursor emissions,27 the annual SSA deposition is expected to be less variable over decades.So the changes in yearly PFAA deposition may be mainly driven by thechanges in historical PFAA emissions rather than SSA deposition. Inaddition, other factors such as melting events during a temporarywarm period can lead to uncertainty25 whenusing deposition samples to examine the links between PFAAs and SSAtracer ions. Therefore, direct field evidence is still needed to understandthe influence of SSA on PFAAs in the atmosphere.Atmospheric deposition samplessuch as surface snow have also beenused to examine the relationship between PFAAs and SSA. Kwok et al.collected surface snow samples . The PFCAs were perfluoropentanoicacid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid(PFHpA), PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid(PFDA), perfluoroundecanoic acid (PFUnDA), and perfluorododecanoicacid (PFDoDA) and the PFSAs were perfluorobutanesulfonic acid (PFBS),PFHxS, and PFOS. Details of the target compounds, analytical standards,and reagents used can be found in Tables S1 and S2 in the Supporting Information. Sodium (Na+) and magnesium (Mg2+) ions wereanalyzed as tracers of SSA.A total of 11 PFAAswere investigated in this study, including C5–C12 PFCAs andC4, C6, and C8 PFSAs  was carried out between April2018 and July 2020 with 2–3 samples taken per month. The samplesfrom Birkenes (n = 58) were collected at a higherfrequency of about 5–8 samples per month between April andOctober in 2018 and between September and December in 2019. The sampleswere collected on prebaked (800 °C for 8 h) quartz fiber filters using a Digitel (DH77) high-volumeactive air sampler (HV-AAS) operated at a flow rate of ∼500L min–1. For each sample, ∼1500 m3 air was collected over 48 h, with a few exceptions. A complete samplelist including the start date, duration, and sample volume is providedin Table S3.Ambient aerosolsamples were collected at two Norwegian coastal sites, Andøya and Birkenes , as shown in 2.3+) and magnesium(Mg2+) analysis. The rest of the filter was used for PFAAanalysis. PFAAs were analyzed on an Acquity ultraperformance liquidchromatography system coupled to a Xevo TQ-S tandem mass spectrometer based on a previously published method.28 Na+ and Mg2+ ions wereanalyzed using an inductively coupled plasma-atomic emission spectrometer located atthe Department of Chemistry, Uppsala University, Sweden. Details regardingthe extraction of the samples and the analysis of PFAAs can be foundin the Supporting Information.Priorto extraction, a small hole (φ = 13 mm) was punched in eachQFF. The punch was stored for sodium (Na2.4n = 9) and Birkenes (n = 7) by loading the QFF on the HV-AAS and exposing themto ambient air for 1 min without turning the pump on. The field blankswere treated the same as the samples. Two blank QFFs (prebaked at800 °C for 8 h) were extracted as laboratory blanks and analyzedwith every 20 samples.Field blanks were producedroutinely at Andøya (n = 12) and analyzed for sodium and magnesium. Therelative standard deviations of triplicates were generally < 5%.Therefore, it can be assumed that SSA were evenly distributed on thefilters and the small punches were representative of the whole filters.Details regarding the blank levels, MDLs, MQLs, IS recoveries, andspike-recovery test can be found in Section S2 and Table S4.To check the distribution of SSA on thefilters, triplicate punches were randomly taken on 10% of the samples archive(https://www.ready.noaa.gov/archives.php). The HYSPLIT model was run using the ensemble method 10 days (240h) back in time. For each sample, the trajectory footprint was calculatedand used to determine the transport probability function plot , which represents the probability ofa backward trajectory passing through a certain grid cell . This was used to visualize the dominant movement pathof the air masses for any given period. The footprint analysis ofeach sample was further used to estimate the source attribution functionplot , which represents the average observed concentrationof PFAAs and Na+ if the trajectory spent time in a specificgrid cell . These source attributionfunction plots were used to identify potential source areas of PFAAs.Details regarding the trajectory analysis can be found in the Supporting Information.TheHYSPLIT4 model2.610[PFAA] and log10[Na]) before statistical analysiswas performed and values below the MDLs were not included, unlessotherwise stated. Values between the MDLs and MQLs were included withoutadjustment. Log–log linear correlations were evaluated usingthe Pearson’s correlation coefficient (r).To prevent a few extreme values biasing the correlations, only sampleswith PFAA and Na+ concentrations > MDLs and PFAA/Na+ ratios between the 5th and 95th percentiles at each locationwere included . Correlation analysis wasalso performed including the data with <5th percentile and >95thpercentile PFAA/Na+ values and substituting values <MDL by 1/2MDL. These different data treatments did not affect thesignificance of the correlations for PFSAs in the samples from bothsites and C7–C10 PFCAs in the Andøya samples but did affectthe PFCAs in the Birkenes samples. For example, when all samples wereconsidered, none of the PFCAs (p > 0.05) werecorrelatedwith Na+ at Birkenes. Details about the results of thecorrelation analysis and orthogonal regression with different datatreatments are included in Table S5.Statisticalanalysis was conducted using RStudio (Version 1.2.5033) with R version3.6.3. Concentration data were logarithm-transformed  and Birkenes (n = 58), with detectionfrequencies > 50%. PFHxA was found in 43% of the Andøya samplesbut in only 10% of the Birkenes samples. PFPeA was not reported dueto a matrix effect which resulted in a high chromatographic baseline.The median concentrations of ∑PFCAs were (ranges given in parentheses) 0.46 pg m–3 (0.045–3.4pg m–3) at Andøya and 0.22 pg m–3 (0.012–2.0 pg m–3) at Birkenes. The concentrationsof PFNA, PFDA, and ∑PFCAs were significantly higher in theAndøya samples than in the Birkenes samples .PFAAs were detectedin all samples, and the results are shown in –3 (0.004–0.16 pg m–3) and0.045 pg m–3 (0.007–0.46 pg m–3) at Andøya and Birkenes, respectively. No significant differences(p > 0.05) were observed for PFSAs between thetwosampling sites.For PFSAs, PFOS was detected in almost all samples and PFHxSwasdetected in >70% of the samples from both locations. PFBS was excludedfrom the following analysis due to its low detection frequency (<15%at both locations). The concentrations of ∑PFSAs  were about 1 order of magnitude lower than for ∑PFCAs,with median concentrations (ranges in parentheses) of 0.033 pg m–3 and range: < 0.003–1.3 pg m–3) and PFOS(median: 0.030 pg m–3 and range: < 0.003–0.14pg m–3) at Andøya were comparable to previouslyreported data for samples collected between 2010–2014 at thissite (median: 0.24 pg m–3 and range: < 0.12–5.5pg m–3 for PFOA and median: 0.072 pg m–3 and range: < 0.043–0.43 pg m–3 for PFOS).21 The detection frequencies of PFCAs with morethan 8 carbons (C > 8) at both locations in this study were muchhigherthan reported in previous studies (<25%).31The concentrations of PFOA (median: 0.11 pgmp < 0.05) were observed betweenall possible pairs of PFCAs atboth Andøya (r = 0.68–0.94) and Birkenes(r = 0.47–0.94) as shown in Figure S2. Additionally, PFHxS and PFOS were also found tobe positively and significantly correlated with PFCAs  with only a fewexceptions. The degradation of FTOHs in the atmosphere would forma series of PFCA homologues, while both PFCAs and PFSAs can be transportedvia SSA since they are all frequently detected in seawater.32 Although perfluoroalkyl sulfonyl fluoride (PASF)-basedprecursor compounds such as x-perfluorooctanesulfonamides/sulfonamidoethanols (xFOSAs/Es) can form both PFSAs and PFCAs in the atmosphere,14xFOSAs/Es were not frequentlydetected in the long-term air monitoring program at the two samplingsites (DF < 20%) and their concentrations were orders of magnitudelower than FTOHs.31 Therefore, such a correlationpattern may suggest the combined effect of both transport via SSAand degradation from FTOHs.Significant log–log linearcorrelations .The Na+ ion concentrations in the samples from Andøya(median: 0.21 μg m–3 and range: 0.022–3.2μg m–3) were significantly higher (p < 0.01) than in the samples from Birkenes . Significantcorrelations between Na+ and Mg2+ were observedat both locations . The Mg2+/Na+ ratiosdetermined by the slopes of the orthogonal regressions  were 0.135 and 0.141 at Andøyaand Birkenes, respectively, which are close to the observed ratioof 0.119 in seawater.33 The good correlationbetween the two elements and the close Mg2+/Na+ ratio to the Mg2+/Na+ ratio of seawater suggestthat sea spray aerosol was a major source of particulate matter atthe two sampling sites.The Na3.3+ ion concentrationswere observed in air samples from both locations . PFOA showed the strongest correlations with Na+  among the PFCAs.The correlations became weaker both as the chain length decreasedand increased relative to PFOA. PFHxS  showed a better correlation with Na+ than PFOS .At Birkenes, PFOA  and PFNA  were the only PFCAs that correlated with Na+ andthe correlations were weaker than at Andøya. In the case of thetwo PFSAs, the correlations with Na+ at Birkenes  were similar to those observed at Andøya.Positive log–loglinear correlations between PFAA and Naocations 2a, sugge18 Compared to PFOA and PFNA, laboratorystudies have shown that C < 8 PFCAs have ∼3–20 foldlower enrichment factors18 while field measurementshave shown that C > 9 PFCAs are usually present in seawater atconcentrationsorders of magnitude lower.37 Such laboratory and field observations may explain why PFOA andPFNA exhibit the strongest correlation with Na+ among thePFCAs at Andøya  and why theywere the only two PFCAs that showed significant correlations withNa+ at Birkenes .The amount of PFAAs transportedfrom the ocean to the atmosphereis determined by both their concentrations in seawater and the enrichmentfactor in SSA.+. PFAA seawater concentrationsvary over several orders of magnitude from populated coastal areasto the open ocean.39 SSA produced from areas with different PFAA concentrations wouldalso vary in the amount of PFAA transferred to air per pg of SSA (Na+), which could weaken the correlations.Different origins of SSA could be anotherfactor influencing thecorrelation between PFAA and Na21) can also weaken the correlationsobserved between PFAAs and Na+. For example, if the fluctuationin air concentrations of precursor compounds and/or the yield of thetransformation is large in relation to the contribution from SSA,the correlation between PFAAs and Na+ might be weak oreven difficult to observe. Birkenes has a lower SSA burden than Andøya(lower Na+ concentration) and is closer to an urban areawhere consumer products containing PFCA precursor compounds were likelyused widely.40 This may explain the weakercorrelation with Na+ for PFOA and PFNA in the Birkenessamples (r < 0.5) and the lack of correlationwith Na+ for the other PFCAs. Additionally, different SSAorigin and influence of other sources can lead to variability in the[PFAA]air/[Na+]air ratio and resultin the slope of the log–log linear relationship deviating from1. For example, for PFOA, the slope estimated for the Birkenes samplesis 0.419 while the slope for the Andøya samples is 1.06  and Birkenes  at the two samplingsites.31 Thus, the observed correlations for PFSAsand low PFSA precursor concentrations in air suggest that the contributionfrom SSA may be more important for PFSAs compared to PFCAs.In contrastto the PFCAs, the t test, p < 0.001, +, PFDA/Na+, and PFOS/Na+ were found to be significantly higher inthe Birkenes samples (p < 0.05). SSA originatingfrom more polluted areas  would havehigher PFAA/Na+ ratios since laboratory results show thatthe amount of PFAAs transferred via SSA emission is positively correlatedwith their seawater concentrations.18 Additionally,greater contributions from other sources such as transformation fromprecursors may also lead to a higher PFAA/Na+ ratio.Interestingly, while the overall PFNA and PFDA loadings were significantlyhigher in the Andøya samples  light intensity, and hydroxyl (OH) radical concentrations.14 These parameters all have a seasonal pattern, so the contributionof these two sources to atmospheric PFAAs may differ between seasons.To investigate whether the correlations between PFAAs and Na+ revealed seasonal differences, the samples from the two samplingsites were grouped as summer samples (collected between April firstand September 30th) and winter samples (collected between Octoberfirst and March 31st). Since the PFAA concentrations in air can besimplified as the combination of the contribution from SSA ([PFAA]SSA) and the contribution from other sources ([PFAA]other), in addition to log–log linear Pearson correlation analysisbetween PFAA and Na+, the data of the two seasons werefitted to a linear function of the form:kSSA (pgμg–1 Na+) represents the amountof PFAA transferred to the atmosphere per μg of Na+ and [PFAA]other (pg m–3) representsthe average of other sources. Results of the correlation analysisand the parameters of the fitted lines are included in Table S7.SSA productioncan be affected by a number of environmental variables including windspeed and the sea surface temperature.+ (p < 0.05) in both summer and winter samplescollected at Andøya except PFDoDA, for which the log–loglinear correlation was only observed in summer samples. For Andøyawinter samples, the fitted lines for each PFCA have greater kSSA (0.064–0.345) and lower [PFAA]other (0–0.012) than those of the summer samples (kSSA = 0.030–0.198 and [PFAA]other = 0.011–0.031). In other words, the PFCA concentrations inair at Andøya during winter appear more sensitive to changesin Na+ concentrations, but the contribution from othersources was lower compared to summer. PFOS showed a similar patternto PFCAs, but the difference between winter  and summer  was not as largeas observed for PFCAs.Almost all PFAAs were correlated with Na43 Concentrations of PFAA precursors in air were also found to be positively correlated withair temperature,21 and the lower air concentrationof precursor compounds in winter would further limit the degradationprocess to form PFAAs.The lower contribution from other sourcesin winter may be theresult of reduced yield of the indirect photolysis of PFAA precursors.In winter, the fewer daylight hours would lead to lower concentrationsof OH radicals in the atmosphere, which would limit the reaction rate.+ wereobserved for C8–C10 PFCAs (p < 0.05) inthe winter samples and for C7–C9 PFCAs (p <0.05) in the summer samples. The two PFSAs were correlated with Na+ (p < 0.01) in both sample groups. Similarto the Andøya samples, [PFAA]other obtained from thefitted lines for PFOA, PFNA, and PFOS at Birkenes were lower in winterthan in summer, suggesting a lower contribution from other sourcesin winter than in summer. However, contrary to the Andøya samples, kSSA of the Birkenes samples were similar betweenthe seasons, implying little variation of the contribution from SSA.The longer distance to the coast (∼20 km) than Andøya(∼1.3 km) may have limited the size of SSA that can be transportedto Birkenes, which may be one potential cause for the observed differences.However, since the PFAA enrichment in SSA under real-world conditionsis still not very well-understood, it is difficult to conclude thecauses of the observed seasonal differences. Further laboratory studieson the enrichment mechanisms of PFAAs in SSA  and field studies with size-resolved aerosolsampling techniques will help improve the understanding of the contributionof SSA to the atmospheric transport of PFAAs.For the Birkenes samples, correlationswith Na3.5i,j], the first row in The results of the backwardair mass trajectory analysis using theHYSPLIT4 model are presented in C̅ij,) of PFOA and PFOSare shown in 34 emissions from these sources may also have affectedthe observed PFAA concentrations at the two sites. In order to estimatethe accumulated source contribution to observed concentrations, thetransport probability function and source attribution function canbe combined in the Arctic region and North Atlantic than other areas, especiallyfor PFOS , suggestingPFAAs originated from these areas may be partly due to SSA emission.Plots for all PFAAs can be found in Figures S4–S6.Plots of correlations per grid cell  representsthe estimated amount of PFAA transferred to the atmosphere per μgof Na+. On the basis of laboratory-derived EFs and reportedPFAA seawater concentrations from the literature and assuming a constantNa+ concentration of 13.8 g L–1 in water, kSSA determined by the fitted lines for all samples from Andøya(Table S7) can be compared to kSSA_est estimated using 32 were used for the estimation. The average EFof individual PFAAs in particles with a dry aerodynamic diameter <10 μm from the previous laboratory study were used in the estimation.18 Details regarding the PFAA concentrations andEFs can be found in Table S8 and Table S9.In a previous laboratory study,kSSA and kSSA_est for Andøya are presented in kSSA_est followed a similar pattern to kSSA,but the differences between kSSA and kSSA_est can be 1–3 orders of magnitude.These estimates were greatly influenced by the PFAA seawater concentrations. The relative standard deviation ofthe laboratory-derived EFs are between 8–30% (Table S9), but the median PFAA seawater concentrations indifferent regions vary over orders of magnitude. For example, themedian seawater concentrations of PFOA between 2010 and 2014 are 43,103, 520, and 1090 pg m–3 for samples from the ArcticSea, North Atlantic, Baltic Sea, and North Sea, respectively.32 Such large differences highlight the importanceof accurate information on the geospatial variation of PFAA seawaterconcentrations when evaluating the contribution of SSA. It shouldbe noted that for PFCAs, kSSA_est is atleast 2 times lower than kSSA in mostcases, especially when the median PFAA seawater concentrations forthe time period of 2015–2019 were used. The causes of the underestimationare unclear since the enrichment process in the field is much morecomplicated than in controlled laboratory experiments. For example,other ions in seawater such as Mg2+ also contribute toSSA emission while only NaCl was used in the laboratory study; thepresence of organic matter in seawater may influence PFAAs emissionby SSA; wind speed at the sea surface may influence the amount andsize of SSA particles emitted; and the SSA size distribution at thesampling site can be very different from freshly emitted SSA, etc.As such, further research on the PFAAs enrichment mechanism in SSAis required to reduce the uncertainty.The comparison between kSSA determined from the fitted lines for all samples fromAndøya (Table S7), the global annualfluxes of PFOA and PFOS from the ocean to the atmosphere via SSA emissioncan be estimated askSSA of the Andøyasamples was used since the sampling site at Andøya is ∼1.3km to open water so the SSA size distribution may be more similarto nascent SSA above the sea surface than at Birkenes. For SSA annualproduction (fluxNa+), Textor et al. calculated 12 estimatesof SSA annual production using 12 chemical transport and general circulationmodels.44 The first and third quartileand the median values of these 12 estimates were used as the low,high, and median scenarios. These estimates of SSA annual productionhave recently been updated,45 but the valuesreported by Textor et al. were still used in the present study inorder to compare with the modeled PFOA and PFOS emission via SSA byJohansson et al.15 The estimated globalannual fluxes of PFOA and PFOS and comparison with Johansson et al.15 are shown in Table S10. Estimates of PFOA and PFOS fluxes using the updated SSA annualproduction are also included in Table S10 for reference.On the basis of modeled annual SSA production and –1 for the low,median, and high scenario, respectively)15 are all below the estimates based on the field measurements . Similarly, in the previous comparisonwith the laboratory result, kSSA_est ofPFCAs were also lower than kSSA in mostcases (–1) is close to the modelingresult of Johansson et al. (42 tonnes yr–1), whilethe median (96 tonnes yr–1) and high scenarios (149tonnes yr–1) are lower than the previous modelingresults .15 Asmentioned before, PFAA seawater concentrations have a large influenceon estimates of the amount of PFAA transferred to air via SSA emission(kSSA_est) based on laboratory-derivedEFs. So the PFAA seawater concentrations that are used as input inthe modeling may be one cause of the differences between the modelingresults and the estimated annual fluxes. In addition, artificial seawaterwithout organic matter was used to derive the EFs in the lab. Butthe composition of real seawater is very complex and may have influenceon the enrichment process,19 so the EFs measuredin the lab may differ from the field. It should also be noted thatthe model used the EF of three modes as input , while the estimate in the current study was based on thefield measurement of bulk aerosol samples. The EFs generally increasewith decreasing particle size.18 For example, the laboratory-derivedaverage EF of PFOS was 9.6 ± 1.4 × 103 for particles< 10 μm but increased to 2.9 ± 0.5 × 104 when only particles < 1.5 μm were considered (Table S8).For PFCAs, the modeling results from the threescenarios by Johanssonet al. , our results suggest that PFAAs transport via SSA may impact largeareas of inland Europe and other continents in addition to coastalareas. However, it is still challenging to evaluate the contributionfrom SSA on the global scale. Further laboratory experiments focusingon the enrichment mechanisms and more field evidence from variouscoastal locations are required before this process is well-parametrizedfor inclusion in global models.The observed significant correlationbetween PFAAs and Na


As a result, 75% of the hCG (n = 6/8) and 33% of GnRH (n = 1/3) inductions resulted in ovulation. Ovulation occurred when fecal estrogen concentration increased before and pregnane concentration after induction. Thirty-six percent of all treatments (n = 4/11) failed to induce ovulation. When ovulation induction by hCG/GnRH injection failed, estrogen and pregnane concentrations were significantly lower compared to ovulatory estrous (P < 0.001). Our results suggest that hCG and GnRH analog facilitate an easily applicable treatment to induce ovulation in females with behavioral but at times an-ovulatory estrous. Frequent use of hCG as an ovulation inducer might help to achieve pregnancies in genetically important but an-ovulatory GOH rhinoceroses.Despite a profound knowledge on reproduction biology in greater one-horned (GOH) rhinoceros, many individuals cope with sub or infertility or an-ovulatory estrous. At the same time, early and regular captive breeding is of high importance in female GOH rhinoceros due to their high prevalence to develop genital tract tumors with consequent cessation of reproduction. Thus, mature, an-ovulatory GOH rhinoceros represent a challenge for captive breeding programs and warrant for means of reliable ovulation induction. Here, we used hCG and GnRH analog histrelin to induce ovulation in an-ovulatory GOH rhinoceros. Upon ultrasound diagnosis of a preovulatory follicle hCG or GnRH were injected to induce ovulation ( Rhinoceros unicornis) is mono-ovulatory. Follicles grow in waves, from which the dominant, preovulatory follicle develops. The preovulatory, Graafian follicle measures an exceptional 10–12 cm in diameter, which is much larger than in other rhinoceros species. In fact, with a volume of 300–500 mL, it is the largest ovulatory follicle described for any mammal species (The mean estrous cycle length of the greater one-horned (GOH) rhinoceros is 43 ± 6 d , 2. LikeDespite available profound knowledge on reproduction biology in GOH rhinoceros, many individuals are affected by sub- or infertility, silent estrous, anestrous, early embryonic death, or still birth –5. EarlyEstrous synchronization in anestrous females to achieve ovulation and conception after natural mating or AI is an assisted reproduction tool so far described in black and white rhinoceros –8. For tIn GOH rhinoceros, irregular, and an-ovulatory estrous cycles with subsequent formation of hemorrhagic follicles represent a challenging problem in captive breeding programs. However, estrous synchronization has yet not been described for GOH rhinoceros. Only ovulation induction has been briefly mentioned being used during AIs , 5. HereWhile hormone activity and ovarian dynamics can be monitored closely , 2, therOvulation induction was performed in one anestrous, 12-year-old, captive female GOH rhinoceros (studbook no: 367) over a study period of 4 years. The study animal is a wild born, orphan transferred from Nepal to the Vienna Zoo at the age of 3 years. The female is housed with a fertile male in separate enclosures. Continuous hormone analysis had been conducted since this female was 4 years of age. From the age of 9 years, regular peaks of estrogen-precursors indicated follicular development, but no clear pregnane peaks were measured, suggesting that ovulation never occurred.Fecal samples were collected two times a week and analyzed using enzyme immunoassays for 20α-OH-pregnanes and 17-oxo-androstanes . Transrectal ultrasound monitored ovarian dynamics and development of preovulatory follicles. Weekly examinations were gradually increased toward daily ultrasounds when a dominant follicle emerged and grew >8 cm. Yet, without treatment pre-ovulatory follicles became atretic or hemorrhagic as previously described by Stoops et al. . A preovulatory follicle with a mean diameter of 11.9 ± 0.2 cm was imaged during eight silent estrous  were preceded by a significant, preovulatory increase of fecal estrogene concentration to >75 ng/ g feces. The presence of a 11.3 ± 0.9 cm corpus luteum .Successful ovulation inductions (s luteum  combinedn = 6/8). Injectable GnRH analog triggered ovulation only once (n = 1/3). Despite the rise in estrogen, the formation of a corpus luteum, and the increase in luteal activity as indicators for the induced ovulation, behavioral estrous was not observed before or after any of the hCG/GnRH treatments.HCG administered in the presence of a preovulatory follicle induced six ovulations resulting in an ovulation rate of 75%  indicate that GnRH analogs are similarly suitable to induce ovulation in an-ovulatory GOH rhinoceros than hCG.So far, GnRH has been used incidentally for ovulation induction in two GOH rhinoceros , 5. GnRHResults on the use of hCG for ovulation induction in GOH rhinoceros are consistent with results from white rhinoceros where 67% of estrous synchronizations using hCG resulted in ovulation . In whitOn one hand, hormone-induced ovulation is technically challenging for many facilities and does not appear as a comprehensive method for reproduction management of captive GOH rhinoceros. It is likely limited to facilities with an advanced animal training program and where critical hardware such as an ultrasound machine and a restraint chute is present. On the other hand, in females at advanced age, with “erratic” an-ovulatory estrous cycles and conception failure but yet important genetics for the captive population, the use of hCG/GnRH during behavioral estrous might present a new, practical reproductive management tool.In conclusion, our data shows that in presence of a preovulatory follicle hCG-induced ovulation in an-ovulatory GOH rhinoceros in 75% of treatments. Thus, injectable hCG represents an easy-to-apply and practical treatment to induce ovulation in GOH rhinoceros with behavioral but at times an-ovulatory estrous. As an-ovulatory GOH rhinoceros represents a challenge for captive breeding programs, frequent use of ovulation inducers, such as hCG or GnRH, might help to achieve late pregnancies in genetically important but an-ovulatory GOH rhinoceros.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Ethics Committee of the Leibniz Institute for Zoo and Wildlife Reserach. Written informed consent was obtained from the owners for the participation of their animals in this study.RH: study design, data anaylsis, and manuscript writing and edit. FB: study design, data aquisitition, data analysis, and mansuscirpt edit. SH: Data auqiuisition. ED: data acquisition. TH: supervision and manuscript edit. FS: study design, data aquisition, data analysis, and manusciprt writing and edit. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The essentiality of selenium (Se) and iodine (I) to human beings and the widespread areas of selenium and iodine deficiency determine the high significance of functional food production with high levels of these elements. In this respect, joint biofortification of agricultural crops with Se and I is especially attractive. Nevertheless, in practice this topic has raised many problems connected with the possible utilization of many Se and I chemical forms, different doses and biofortification methods, and the existence of wide species and varietal differences. The limited reports relevant to this subject and the multiplicity of unsolved questions urge the need for an adequate evaluation of the results obtained up-to-date, useful for developing further future investigations. The present review discusses the outcome of joint plant Se–I biofortification, as well as factors affecting Se and I accumulation in plants, paying special attention to unsolved issues. A particular focus has been given to the prospects of herb sprouts production enriched with Se and I, as well as the interactions between the latter microelements and arbuscular-mycorrhizal fungi (AMF). An adequate consumption of essential elements, such as Fe, Zn, Se, and I is one of the most important factors for maintaining good human health conditions. Among the above-mentioned elements, Se and I draw special attention due to the close relationship between their metabolism in mammals, where the fundamental function of Se is its presence in the active center of triiodothyronine deiodinases, involved in the biosynthesis of thyroid hormones ,2,3, in Based on the literature reports indicating a high variability of the results and the complexity of this topic, the present review is aimed both to provide the current status and to trigger future investigations relevant to the joint Se–I biofortification of agricultural crops, so as to develop and implement the technology of functional food production enabling to decrease, at least partially, the ecological risks connected with the deficiency of these two microelements. The main sources of Se and I for plants are soil and precipitation, with the latter providing a significant transfer of the two elements from the surface of sea and oceans . PhytoplIn this respect, the agrochemical biofortification of plants with Se and I is especially attractive, since it provides a chance to produce functional food products with a significant content of well-assimilated forms of trace elements and other natural antioxidants. Furthermore, most of agricultural crops belong to non-accumulators of these elements and therefore are highly sensitive to toxic Se–I levels, thus providing the so-called ‘buffer effect’ preventing human toxicities in case of overdosing during biofortification . In gene−1 d.w. without growth inhibition, depending on the Se form and dose [In general, Se content in most agricultural crops, known to be non-accumulators of Se, may be increased up to 0.2–3000 mg kgand dose , as it aand dose . ConsideSe and I are not essential for plants but, thanks to their antioxidant properties, they are able to protect plants from different forms of oxidant stress and at certain concentrations may serve as growth stimulators ,13. On tIn plants, the aforementioned threshold referred to Se and I is species- and variety-dependent, which determines significant differences in the enrichment efficiency with these microelements ,25,29,30As a sulfur analog, Se has several forms with different valences: selenates (+6), selenites (+4), organic derivatives  and elemental selenium . The known chemical forms of I are iodides (–1), iodates (+5) and derivatives of aromatic amino acids (–1) . +4 and later transformed to selenocystine (SeCys)2, Se-methyl-selenocysteine (MeSeCys), and selenomethionine (SeMet) [2, CH3I, KI, KIO3), and apparently via amino acid transporters for organic I forms [The assimilation of both Se and I is achieved with the participation of appropriate protein-transporters : sulfate (SeMet) . As far  I forms . Another I forms .All these forms of Se and I are used for producing vegetables fortified either with Se or I ,31.Under joint application of the two elements, the range of their chemical forms used is limited to selenates (+6), selenites (+4), and also iodates (+5) and iodides (–1) .The up-to-date results relevant to the joint plant biofortification with Se and I are presented in Attempts to quickly solve the deficiency problem of a whole set of trace elements, such as Se, I, Fe, Zn, in the conditions of different countries worldwide, were achieved in cereals (10 wheat cultivars and 7 rice cultivars) in 2019 and 2020, using foliar application of elements cocktail. These works revealed the efficiency of such biofortifications, but lack of significant effect on wheat yield and just a slight increase of rice yield ,30. In tThe success of Se enrichment of various agricultural crop seedlings ,51 and t3 and KIO3 + Na2SeO4 supply, indicating the development of oxidant stress , while both joint and separate application of potassium iodide and sodium selenate resulted in growth inhibition . It is st stress .In the aforementioned conditions, the Se–I interaction was also highly variable under the joint application of the two elements. Indeed, in pea and chervil seedlings there was no relationship between the two elements . Joint aHydroponics or soilless is another technological approach providing strict control of crop growing conditions and minimizing the effect of environmental factors. Four studies of joint Se–I biofortification in soilless conditions ,39,40,41Arabidopsis thaliana revealed that micromolar concentrations of I are beneficial for biomass accumulation and lead to early flowering, regulating the expression of several genes mostly involved in the plant defense response, and may be incorporated into proteins both of shoot, participating in the photosynthesis, and of roots associated with some peroxidase activities [Experiments with tivities . Unfortunately, no direct data on phytohormones participation in plant I accumulation are available, except the Smolen experiments with salicylic acid ,39 Tabl. Joint b2SeO4 supply compared to KIO3 and Na2SeO3 use. Despite a significant increase in SeMet and SeCys content in lettuce leaves due to joint Se–I application, the authors demonstrated a decrease in Se and I concentration in joint experiments compared to the separate application of the two elements [Great success in the utilization of Se-containing fertilizers in Finland  for optielements . Further−1 concentration was optimal for mature plants [−1 did not affect the sprout biomass.Among different methods of joint Se–I biofortification, foliar application of the two elements is maybe the most interesting, although the technology of such enrichment raises some risks due to Se toxicity. As shown in e plants ,62 and t+4) accumulation and decrease of selenate (Se+6), kohlrabi demonstrated antagonism between these elements contrary to pea with typical Se–I synergism. Se–I synergism in Indian mustard was demonstrated only in case of separate Se and I application but not in conditions of joint Se–I application [Notably, foliar application of the two elements resulted in significant Se–I interaction compared to the hydroponic conditions which did not cause significant effects of the joint Se–I supply, and only 50% of the investigations relevant to sprout Se–I biofortification revealed the existence of Se–I relationships. Foliar biofortification technology demonstrated that Se–I interaction is highly species-dependent. Indeed, while Se–I joint foliar biofortification of chicory resulted in the increase of selenite  [The assessment of joint Se–I effect on N, P, S, Si and V relationship should be the priority , conside n = 14) . A highlAs mushrooms belong to the plant kingdom, the relationship revealed in Furthermore, the protection effect of Se against plant biotic and abiotic stress including heavy metals uptake should be highlighted. This approach might become especially interesting in contexts of Se–I interaction and joint biofortification. A further focus should be given to the effect of joint Se–I biofortification on plant antioxidant status, including accumulation of sugars which are known also to participate in plant antioxidant protection .The results presented in this work indicate that a restricted number of species was used for joint Se–I biofortification, which entails the need for further determinations of the biofortification efficiency in widespread agricultural plants, such as tomato, onion, garlic, parsley, pepper, etc. Moreover, new discoveries relevant to organic Se forms and Se nanoparticles (NPs) efficiency in joint Se–I biofortification are expected. Finally, a special interest regards the interaction between Arbuscular Mycorrhizal Fungi (AMF) inoculation and Se–I biofortification in other agricultural crops. From a practical point of view the present knowledge of Se–I interaction in plants indicates the actual possibility of industrial production both of sprouts and microgreens and of several agricultural crops fortified with these elements, and emphasizes the importance of wide AMF utilization capable not only to increase plant yield and quality, but also to enhance the accumulation of Se and I.Despite the attractiveness of the joint Se–I biofortification of agricultural crops, unfortunately this issue has been poorly studied and further investigations are needed for identifying the mechanism of Se–I interaction and evaluating the main factors affecting its intensity. From a general point of view, this topic may be considered as a new field of novel discoveries both in human nutrition and plant physiology. However, it is important to highlight that the current research state-of-art already shows the actual possibility to achieve the joint Se–I biofortification of several plant species at industrial scale, targeting the human health improvement. Consumers’ willingness to purchase Se–I biofortified products compared to utilization of appropriate food supplements, and Se and I levels in fortified fruits and vegetables sufficient to significantly increase Se–I consumption levels may be considered as a basis for active development of functional food production with enhanced Se and I levels.


Foliar and soil Se application of either selenate or selenite significantly increased the Se content in different parts of wheat, while selenate had higher bioavailability than selenite in the soil. Regardless of Se application methods, the Se content of the first node was always higher than the first internode. Selenomethionine  and selenocystine  were the main Se species identified in grains of wheat. The percentage of SeMet increased by 6% in soil with applied selenite and selenate treatments at 0.5 mg kg–1 and decreased by 12% compared with soil applied selenite and selenate at 2 mg kg–1, respectively. In addition, flour processing resulted in losses of Se; the losses were 12–68% in white all-purpose flour compared with whole wheat flour. The Se bioaccessibility in whole wheat and white all-purpose flours for all Se treatments ranged from 6 to 38%. In summary, foliar application of 5 mg L–1 Se(IV) produced wheat grains that when grounds into whole wheat flour, was the most efficient strategy in producing Se-biofortified wheat. This study provides an important reference for the future development of high-quality and efficient Se-enriched wheat and wheat flour processing.A comprehensive study in selenium (Se) biofortification of staple food is vital for the prevention of Se-deficiency-related diseases in human beings. Thus, the roles of exogenous Se species, application methods and rates, and wheat growth stages were investigated on Se accumulation in different parts of wheat plant, and on Se speciation and bioaccessibility in whole wheat and white all-purpose flours. Soil Se application at 2 mg kg It was –1 . Conside–1 . Up to n–1 , mushroo–1 , maize  uaporins .–1, which provides insufficient daily Se for sustaining human health  and methylselenocysteine (MeSeCys) accounted for 2.6 and 0.3% of the total Se, respectively (Selenomethionine (SeMet) is the primary Se species in wheat grains . Lu et aectively . In geneectively , howeverThe production of Se-enriched wheat can be an important step in eliminating the negative impact of Se deficiencies in low Se areas. In this regard, bioaccessibility of Se from edible wheat tissues is important to understand. The bioaccessibility of Se using in vitro simulated gastrointestinal digestion test (PBET) refers to the portion of a nutrient, e.g., Se in a food product, that can be found dissolved in gastric (G) and intestinal (I) phases, and be potentially absorbed and utilized by organisms . The ordCurrently, the main methods used to explore the uptake, translocation, and transformation of Se in crops can be divided as: hydroponic experiment, pot experiment, and field experiment . The env–1; organic carbon, 8.53 g kg–1; cation exchange capacity, 23.34 cmol(+) kg–1; amorphous aluminum, 0.40 g kg–1; amorphous iron, 1.20 g kg–1; clay, 39.6%; and total Se, 0.139 mg kg–1.The pot experiment was carried out in a greenhouse at Northwest Agriculture and Forestry University in Yangling, Shaanxi from year 2018 to 2019. Tested soil was collected from the non-polluted farmland around Northwest A&F University, which has never received applied exogenous Se. After air-drying, homogenizing, and grinding, the soil was passed through 2 and 0.149 mm sieve for physical and chemical analysis determined according to Triticum aestivum L, Xiaoyan-22) were provided by a commercial seed company of Northwest Agriculture and Forestry University. Wheat seeds with full grains were selected for consistent size and no pest infestation damage, then disinfected with 5% (V/V) H2O2 for 30 min and washed thrice with deionized water.Winter wheat seeds , and Se(VI) was sodium selenate , both were analytical pure reagents. The organic Se  were all purchased from Sigma-Aldrich company and used for the determination of Se speciation in grain and leaves of wheat. Pepsin, sodium malate, sodium citrate, lactic acid, acetic acid, bile salt and trypsin, which were used for the determination of Se bioaccessibility, were all purchased from Yuanye Biological Technology Co., Ltd.Se(IV) was sodium selenite (Na–1) were selected in soil Se application. For foliar Se treatments, two species of exogenous Se (Se(IV) and Se(VI)), three application rates of Se  were applied at two growth stages of pre-flowering stage (F1) and pre-filling stage (F2). A total of 19 treatments were used in this experiment and each treatment was replicated three times.A complete block design was used in this study, two species of exogenous Se (Se(IV) and Se(VI)), three application rates of Se  and Se(IV) solutions were prepared according to the designed Se application rates and then evenly sprayed into the soil and mixed. The soil moisture was adjusted to 70% of the water holding capacity. After full mixing, the sprayed soil was allowed to equilibrate at 25°C for 30 days , and dei–1 nitrogen fertilizer was applied at regreening stage of wheat. 20 seeds were sowed into each pot. Two weeks after the emergence of seedlings, the seedlings were thinned to 10 plants per pot. The pots were weighed and watered every 4–14 days during the wheat growing season. For the foliar Se application, the Se solution was sprayed evenly on the plants during the pre-flowering stage (April 2019) and the pre-filling stage (May 2019) of growth. Specifically, 100 mL Se (IV) or Se (VI) solution  were mixed into water with 0.1% surfactant. Foliar Se was applied three times  in intervals of 5 days to ensure that Se was fully absorbed by wheat leaves. Each pot was sprayed with a total of 1.5, 3, and 6 mg Se(IV) and Se(VI), respectively. Moreover, during the foliar application process, the soil surface was covered with plastic film to avoid Se from dripping onto the soil.During the sowing period, 0.15 g N  and 0.033 g P  were applied to each kilogram of soil, and 0.15 g kgIn vitro simulated gastrointestinal digestion test”).The height and length of rachis and the effective ear number of wheat were measured after wheat harvest (June 2019). The harvested wheat was first washed with tap water thrice to remove dust and other impurities, rinsed with deionized water thrice, and then dried with absorbent paper. Meanwhile, each wheat plant was divided into nine parts: root, stem, leaf, glume, grain, sheath, first internode, first node, and rachis . After wThe total Se content was determined via hydride atomic fluorescence spectrometry  after wet-acid digestion. The specific procedure has been described by –1 for 10 min at 4°C. The supernatant was collected after pouring through a 0.22 μm filter membrane, and then analyzed using the HPLC-ICP-MS system. The instrument conditions are as follows: for the HPLC; Hamilton PRP-X100 anion exchange column  was used, the column temperature was room temperature, the mobile phase was 40 mmol L–1 diammonium hydrogen phosphate (pH = 6.0 adjusted with 10% formic acid) at a flow rate of 1.0 mL min–1, and the injection volume was 100 μL. For the ICP-MS; RF power was 1,550 W, RF matching voltage was 1.8 V, sampling depth was 8 mm, atomization chamber temperature was 2°C, plasma gas flow rate was 15.0 L min–1, the flow rate of carrier gas was 0.65 L min–1, the mode was high He collision mode, the flow rate of collision gas was 4.5 mL min–1, peristaltic pump speed was 0.3 r s–1. The detection mass number m/z = 78(Se), and the integration time was 0.5 s. At the same time, Se-enriched yeast of SELM-1 was used as the quality control sample, the measured content of SeMet in the quality control sample was 3,236 ± 21 mg kg–1, the standard value was 3,389 ± 173 mg kg–1, the recovery rate was 95.5%.Selenium speciation was determined by HPLC-ICP-MS. First, 0.2000 g freeze-dried grains or leaves was taken into a centrifuge tube, 20 mg protease XIV and 5 mL water were added, vortexed for 30 s, ultrasonic extraction for 3 h in a 37°C water bath and shook several times during the period. Second, the sample was centrifuged at 9,000 r minAccording to the method of (1)G: 1.000 g sample was accurately weighed into 100 mL polyethylene centrifuge tube, and 50 ml fresh gastric juice (pH 2.5) were added into a constant temperature (37°C) water bath for digestion at 150 rpm for 1 h. The obtained digestive juice was centrifuged at 4,000 rpm for 10 min and 10% of the supernatant was removed and stored at 4°C for Se content determination.(2)I: the pH of the remaining digestive juice was adjusted to 7.0 by 10% (m/v) NaOH. Then 5 mL intestinal fluid were added and digested in a constant temperature water bath  for 4 h. The obtained digestive fluid was centrifugated at 4,000 rpm for 10 min, and the supernatant was stored at 4°C for further Se content determination.Moreover, the total Se content in G and I sample (2 mL of digestive fluid) were determined by the method already described in section “Selenium content in various parts of wheat.” The composition of gastric juice and intestinal juice was the same as Pearson correlation analysis and variance analysis were performed by SPSS 20.0 . The data in the chart are the averages of three replicates, and the data were calculated using the following Eqs.a–b represents the Se translocation factor from part “b” to part “a” of wheat . “a” or part “b” refer to different parts of “root,” “first node,” “rachis,” “grain,” and “leaves.”where TFof wheat . Ca and –1. Se in sample indicates the Se content in the corresponding sample, mg kg–1.where BA% represents the Se bioaccessibility of whole wheat and white all-purpose flours. Selenium in G/I was the Se content in gastric or intestinal phase of the sample, mg kg–1) is the difference between the Se content of whole wheat and white all-purpose flours with different Se treatments.where LS% represents the Se lost proportion with different flour yield. The lost Se content (μg g–1 Se(VI) was soil applied, indicating that the 2 mg kg–1 Se treatment has little inhibition on wheat growth. However, the growth of wheat appeared to slow down due to the biological dilution effect at the later growth stage of wheat.p < 0.05) effects on the biomass and grain yield of wheat. Soil application with 2 mg kg–1 Se(IV) resulted in the highest grain yield of wheat, which was about 6% higher than control treatment. Compared with control, all soil Se application treatments reduced the yield of wheat (by 4–5% by Se(IV) treatments, except at 2 mg kg–1 Se(IV) (yield increased by 10% compared with 0.5 mg kg–1 treatments), and 4–62% in Se(VI) treatments). However, no significant effects (p > 0.05) were observed in the grain yield of wheat at different foliar Se application treatments, irrespective of the Se species, application rates, and application stages.p < 0.05) increased the biomass of wheat (7–11%), compared with control, while the application of Se(VI) increased Se application rates both significantly (p < 0.05) reduced wheat biomass (2–59%). Compared with 2 mg kg–1 Se(VI) treatment, the biomass of wheat treated with 0.5 mg kg–1 Se(VI) increased by 58%. No significant (p > 0.05) effects on the biomass of wheat were found among different foliar Se application treatments, irrespective of exogenous Se species, application rates, and application stages.We note that soil Se(IV) application treatments significantly (p < 0.05) increased the Se content in each part of wheat in comparison to control. The Se content increased with higher rate of Se application. A significant (p < 0.05) increase of 72 and 84% of the Se content in grains of wheat was observed at soil application 2 mg kg–1 Se(IV) and Se(VI) treatments, compared with 0.5 mg kg–1 treatment of either form. Meanwhile, the Se content in grains with foliar application rate of 20 mg L–1 Se(IV) and Se(VI) increased remarkably (p < 0.05) by 68–69% and 60–68% at pre-flowering and at pre-filling stages, respectively, compared with the corresponding 5 mg L–1 Se application treatments.The harvested wheat plants were divided into nine parts: root, stem, leave, sheath, first internode, first node, rachis, grain, and glume . We obsep < 0.05) increased the Se content in each part of the wheat (90–99.5%), compared with Se(IV) treatments (except foliar application of 20 mg L–1 Se(VI) applied at pre-filling stage). Compared with foliar Se(IV) treatment, foliar application of Se(VI) at pre-flowering stage and pre-filling stage significantly (p < 0.05) increased the Se content of wheat grains by 6–44% and 3–28%, respectively. In addition, the Se content of wheat grains from foliar Se(IV) and Se(VI) application at pre-filling stage significantly (p < 0.05) increased by 22–30% and 6–25% than that applied at pre-flowering stage, respectively.Regardless of application method of Se(IV) or Se(VI), the Se content of the first node of wheat was always higher than that of the first internode. Meanwhile, irrespective of the Se application rate and method, Se(VI) treatments significantly (p < 0.05) increased the Se content of wheat grains compared with corresponding soil application treatments, irrespective of the application stages. Specifically, the Se content of wheat grains sprayed with Se(IV) significantly (p < 0.05) increased by 45–54% and 61–68% at pre-flowering and pre-filling stages, respectively, compared with soil Se(IV) application. In contrast to foliar Se(IV) treatment, soil application of Se(VI) significantly (p < 0.05) increased the Se content of wheat grains compared with its foliar application. The Se content of wheat grains in the soil application treatments was significantly (p < 0.05) increased by 79–91% and 75–90% at the pre-flowering and pre-filling stages, compared with foliar application treatments, respectively.Foliar Se(IV) application significantly  foliar Se application: TFroot/first nodes, TFrachis/first nodes, TFgrains/rachis, and TFgrains/leaves.Translocation factor (TF) can be used to reflect the translocation capacity of plant from source to sink . Figure grain/rachis of wheat (1.2–2.1), while reduced the TFrachis/first nodes (0.6–1.1) and TFfirst nodes/root ). Moreover, when Se(IV) was soil applied, the TFgrains/rachis increased with the higher application rate of exogenous Se, and the TFgrains/rachis decreased when Se(VI) was soil applied. Although soil Se application reduced the TFfirst nodes/root, TFfirst nodes/root increased with a higher rate of exogenous soil Se applied, irrespective of the Se species. Regardless of the application stages, rates, and species of Se, foliar spraying of exogenous Se had no significant (p > 0.05) effects on TFgrains/leaves. Compared with control, foliar Se application of both forms of Se increased the TFgrains/rachis (0.1–0.8) in wheat. Compared with pre-filling stage, the TFgrains/rachis in wheat increased at pre-flowering stage ((Se(IV): 0.2–0.5; Se(VI): 0.1–0.9)). In addition, spraying Se(IV) at pre-filling stage significantly (p < 0.05) increased the TFrachis/first nodes (0.8–1.1) and the TFroot/first nodes (0.9–8.4), compared with control.Compared with control, soil Se application treatments significantly increased the TF2 (4–13%) ( (4–13%) , while S–1 increased by 6% and decreased by 12%, respectively, compared with the 2 mg kg–1 treatment, respectively. However, there was no significant (p > 0.05) differences among the foliar Se application rates.No significant differences were observed for the percentages of SeMet in wheat grains among soil (83–95%) and foliar (87–96%) Se application treatments, while the percentages of SeMet varied with Se application rate . The per–1, the percentage of SeMet in wheat grains increased by 7% in Se(VI) treatment compared with Se(IV) treatment, but decreased by 11% at 2 mg kg–1 Se(VI) soil treatment. However, the percentage of SeCys2 in wheat grains increased by 9% with 2 mg kg–1 soil Se(VI) treatment compared with 0.5 mg kg–1 Se(VI) soil treatment. Moreover, foliar Se(IV) application increased the percentage of SeMet in wheat grains, compared with Se(VI) application. We found that compared with Se(VI) treatments, the percentage of SeMet in wheat grains treated with Se(IV) increased by 3–7%. The percentage of SeMet measured in wheat grains produced from foliar Se treatment with high rate of Se(VI) at pre-flowering and pre-filling stages, increased by 9 and 5%, respectively, compared with soil Se treatments.The species of exogenous Se also affected the percentages of SeMet in grains. With soil application rate at 0.5 mg kg–1 Se(VI) treatment at pre-flowering stage and pre-filling stage, respectively, compared with foliar Se(IV) application.Regardless of the application methods, inorganic Se (50–100%) was the major Se species in wheat leaves in all the treatments, with Se(IV) and Se(VI) accounting for 1–71% and 18–99% of total Se, respectively, while SeMet (1–42%) was the main organic Se species . Moreove–1 Se(IV) rate was the highest, which was 35–49% higher than other treatments. Except for soil Se(IV) application at 2 mg kg–1, the percentage of organic Se in wheat leaves sprayed with 20 mg L–1 Se(VI) increased by 8% and 11% at pre-flowering and pre-filling stages, respectively. The percentage of SeMet in wheat leaves with soil Se(IV) application at 2 mg kg–1 increased by 40% compared with the corresponding foliar application treatments at pre-flowering stage. The percentage of Se(VI) in wheat leaves with soil application at 20 mg L–1 Se(VI) increased by 8% and 9% applied at pre-flowering stage and pre-filling stages, respectively, compared with foliar application treatments.Irrespective of the application methods of Se, the percentage of organic Se in wheat leaves with soil application at 2 mg kg–1 Se(IV) and Se(VI) treatments, the percentages of Se lost in wheat flour were 4% and 8%, respectively, compared with the 0.5 mg kg–1 Se(IV) and Se(VI) treatments. The percentages of Se lost in wheat flour produced from plants sprayed with 5 mg L–1 Se(VI) increased by 40% (pre-flowering stage) and 23% (pre-filling stage), compared with 20 mg L–1 Se(VI) treatment.The whole wheat (100% wheat) and white all-purpose flour (70% wheat flour and 30% bran) were obtained by controlling different proportions of flour and bran of wheat. Selenium lost was calculated as the difference between the Se content of whole wheat and white all-purpose flour. Irrespective of the Se application methods, the percentage of Se lost in wheat flour process among the different Se treatments ranged from 12 to 68%. For soil Se application at 2 mg kgIn general, flour produced from foliar Se application had a higher percentage of Se lost in flour produced from soil Se application. The percentage of Se lost in wheat flour treated with foliar Se(IV) application was 2–12% (pre-flowering stage) and 43–51% (pre-filling stage) higher than that of the soil Se treatments (except for spraying Se(VI) at pre-filling stage). When Se(VI) was sprayed, the percentage of Se lost in wheat flour was 28% (at pre-filling stage) and 42% (at pre-filling stage) higher than the soil Se(VI) treatment.The bioaccessibility of Se in whole wheat and white all-purpose flours are shown in –1 increased by 6–13% compared with 0.5 mg kg–1 treatments, except for the soil application of 2 mg kg–1 Se(VI) treatments. Compared with the 0.5 mg kg–1 Se(VI) treatment, the Se bioaccessibility in whole wheat and white all-purpose flours decreased by 13% (G) and 16% (I), and 15% (G) and 17% (I) in soil at 2 mg kg–1 Se(VI) treatment, respectively. In addition, the Se bioaccessibility in Se(VI) treatments in both foliar and soil Se application was higher than that in Se (IV) treatment, except soil application at 2 mg kg–1 Se(VI). In soil Se application, the bioaccessibility of Se in wheat flour of Se(VI) treatment increased by 4% in both G and I (in whole wheat flour), compared with Se(IV) treatment. Compared with foliar Se application at pre-flowering stage, foliar Se application at pre-filling stage increased the bioaccessibility of Se in whole wheat (3–4%) and white all-purpose flours (2–8%) in G and I .Irrespective of the Se application methods and Se species, the Se bioaccessibility in wheat flour  produced from soil at 2 mg kg–1 Se(VI) significantly reduced plant height, effective ear number, and rachis length of wheat compared with 0.5 mg kg–1 Se (VI) treatment (–1 Se(VI) has a certain toxic effect on wheat growth. Based on this observation, it appears that 1 mg kg–1 can be used as the tolerance limit of Se(VI) in a wheat Se biofortification strategy. The grain yield of wheat in 0.5 mg kg–1 Se(VI) treatments increased by 61% compared with 2 mg kg–1 Se(VI) treatments. Similarly, this study also found that the highest grain yield was significantly (p < 0.05) obtained in soil applied with 2 mg kg–1 of Se(IV), which was about 6% higher than the control treatment  difference between foliar Se application and control treatment. p > 0.05). However, in soil Se application, compared with the selenite application (1 mg kg–1), the grain and the biomass yield of ZM-9023 significantly (p < 0.05) increased by about 15% for selenate application  increased the Se content in each part of wheat (90–99.5%), and spraying Se(VI) increased the Se content of wheat grains (3–44%) compared with Se(IV) treatment. This increase in Se accumulation with selenate may be attributed to the different transport mechanism of Se(VI) and Se(IV) in plants. The uptake and translocation of these two inorganic forms of Se by plants is an energy-consuming process   process . However process .Rachis is the organ connecting the stem and grain of wheat . SeleniuThis study found that irrespective of the Se application methods, the Se content in the first node was higher than that of the first internode. Except for wheat grains and leaves, the Se content in first internodes and nodes was relatively high, which was consistent with findings reported by rachis/first nodes increased when exogenous Se was applied at pre-flowering stage compared with pre-filling stage  were the main Se species in wheat grains  and MeSeCys (0.3%). More than 50% of Se was stored in edible parts such as grains, beans, and leafy vegetables as organic Se, when different species of Se(VI) or Se(IV) were applied . This st93–100%; , which iSe(VI) is difficult to be converted into organic Se compared with SeIV; . TheoretIn general, most of the exogenous Se was accumulated in wheat leaves after foliar Se application . The perPleurotus ostreatus and Pleurotus florida had high Se bioaccessibility, which reached 70–92% , the Se bioaccessibility of Se(IV) treatment was slight higher than Se(VI) treatment (63 vs 56%), although this difference was not significant  had higher Se bioaccessibility than Se(IV) treatments. Regardless of the Se application methods, the Se bioaccessibility in intestinal phase of whole wheat and white all-purpose flour was higher than that in gastric phase. The results are consistent with the study of –1 Se(IV), since Se(VI) has a higher Se bioavailability than Se(IV), there was an increased translocation of Se in wheat from the rachis to the grain. Both foliar and soil Se application can effectively increase the Se contents of wheat. The Se species applied to soil or to plant, application rates and growth stages applied, all influenced the Se content of wheat. Irrespective of Se application methods, the Se content of the first node was always higher than the first internode, indicating that the first node plays an important role in Se translocation in wheat. SeMet and SeCys2 were the main Se species in grains of wheat, indicating that wheat can efficiently convert applied inorganic Se into organic Se within the plant. In addition, flour milling process will cause losses of Se in wheat. The percentages of lost Se in white all-purpose flour were 12–68% higher compared with whole wheat. The Se bioaccessibility of whole wheat and white all-purpose flour with different Se treatments ranged from 6 to 38%, and white all-purpose flour had higher Se bioaccessibility than whole wheat. Future studies should also focus on the speciation changes, genotypes, and influence of the nodes on the mechanisms of Se translocation within wheat.This research is the first systematic study conducted to explore Se bioassessibility in wheat Se fortified with different Se application methods. The wheat was separated into nine parts . The grain yield was the highest in plants treated with soil application at 2 mg kgThe original contributions presented in this study are included in the article/MW and DL created the hypothesis, objectives, outline the draft, and wrote the manuscript. FZ, NC, and PC performed the statistical analysis. YM, HZ, MQ, NL, YL, and LM performed the experiment. GB and DL revised the manuscript. All authors contributed to the article and approved the submitted version.


Shigella vaccines is critically dependent on development of an international standard serum and harmonized ELISA, demonstration of field efficacy in young children in low- and middle-income countries, and early engagement with regulators and policy makers.Advancing new O-antigen-based  Shigella are in clinical development. Historical efficacy studies identify serum O-antigen immunoglobulin G as a correlate of protection, leading to the suggestion that accelerated licensure could be achieved using the Shigella-controlled human infection model (CHIM). We discuss the role of the Shigella CHIM alongside critical imperatives needed for accelerated licensure and uptake of Shigella vaccines: (1) development of an international standard serum and harmonized enzyme-linked immunosorbent assay; (2) demonstration of field efficacy in young children in low- and middle-income countries; and (3) early engagement with regulators and policy makers.New O-antigen-based vaccines against Shigella is a major cause of diarrhea and dysentery disproportionately affecting children in low- and middle-income countries (LMICs) and responsible for 164 300 deaths annually [Shigella vaccine preferred product characteristics concluded the following: “the primary goal is to develop a safe, effective and affordable vaccine to reduce mortality and morbidity due to dysentery and diarrhea caused by Shigella in children under 5 years of age in LMICs” [Shigella species express different O-antigens. A vaccine will need to protect against Shigella sonnei and Shigella flexneri 2a, 3a, and 6 to cover the most prevalent globally circulating strains [n LMICs” . A vacciS sonnei O-antigen conjugated to recombinant exotoxin of Pseudomonas aeruginosa (rEPA) conferred 74% protection against S sonnei shigellosis in Israeli military [Over 20 years ago, a National Institutes of Health (NIH) glycoconjugate vaccine of military . Loss ofmilitary .Shigella sonnei-rEPA used random coupling for conjugation resulting in a heterogenous lattice structure. Variable amounts of free polysaccharide may have interfered with the immunologic response, as seen in mice where admixing free Streptococcus pneumoniae type 4 polysaccharide with S pneumoniae type 4 polysaccharide-protein conjugate decreased polysaccharide antibody responses [esponses . Subsequesponses . Short sShigella conjugates could increase immunogenicity, overcoming lack of protection in young children. A S flexneri 2a bioconjugate developed by Limmatech (GSK), with O-antigen covalently coupled in sun-type format to rEPA, was immunogenic in US adults [Shigella-controlled human infection model (CHIM) study [Enhanced immunogenicity with reduced saccharide length and site-specific conjugation is seen with dextran conjugates . PneumocS adults , and it M) study , providiShigella outer membrane vesicle (OMV) vaccine, the vesicles serving as a delivery vehicle for O-antigen. An S sonnei OMV candidate was well tolerated and immunogenic in French, United Kingdom (UK) [S flexneri 2a synthetic O-antigen conjugated to tetanus toxoid induced significantly higher O-antigen IgG levels in Israeli adults than an NIH S flexneri 2a O-antigen-rEPA vaccine [The GSK Vaccines Institute for Global Health (GVGH) developed a dom (UK) , and Kendom (UK) , but it dom (UK) . A vacci vaccine . It is uShigella. Possible protection against shigellosis through other mechanisms including mucosal IgA and Shigella protein antibodies presents a confounder to this approach. Efforts are underway to confirm serum IgG as a correlate of protection and establish protective thresholds [By accepting serum O-antigen IgG as a correlate of protection against shigellosis , an analresholds , but theShigella vaccines [Shigella ELISA collaborative study by the National Institute of Biological Standards and Controls (NIBSC), UK, was a first step towards ELISA harmonization. In 2020, a decision was made to harmonize a Shigella ELISA protocol at the Walter Reed Army Institute for Research (WRAIR) and develop a first international standard serum at NIBSC with the goal of submitting it to the WHO Expert Committee on Biological Standardization for approval and adoption.An international standard reference serum and harmonized ELISA are required for comparability between studies. The standard serum should cover each serotype within multivalent vaccine formulations and be generated from vaccinees. Such a serum and ELISA will underpin clinical development of all O-antigen-based vaccines . In 2019Shigella CHIMs are conducted in adults in high-income countries (HICs). Therefore, such studies could potentially be used to license new-generation Shigella vaccines for travelers [ravelers . There iravelers  for travravelers . A CHIM ravelers , 23.Shigella vaccine in this population. It is not known whether serum O-antigen IgG is a correlate of protection in LMIC children, nor whether a threshold of protection determined in HICs will be applicable in these children. Therefore, efficacy needs to be demonstrated in this population and can only be achieved with a phase 3 field study. Such a study could confirm correlate of protection status and protective titers for serum O-antigen IgG in this population, enabling subsequent immunobridging and accelerating licensure for other Shigella vaccines.However, a typhoid conjugate vaccine was efficacious in young LMIC children 20 years ago . EfficacShigella vaccine. In addition, CHIM studies may identify serum IgG thresholds of protection, although the artificial nature of infection and low numbers of participants present limitations. Finally, if CHIM for S flexneri 3a and 6 become available, they may confirm that Shigella vaccines can protect against these serotypes. The low incidence of shigellosis due to these serotypes means it is difficult in a field setting to demonstrate efficacy against them. Moreover, after a successful phase 3 efficacy study in LMIC infants and children, an established correlate and threshold of protection could leverage a large phase 2 study as basis of licensure for subsequent Shigella vaccines. If this is acceptable to the regulators, the strategy could accelerate licensure, which reduces the cost of late-stage clinical development.Nevertheless, CHIM studies could assist licensure for children through licensure for travelers, by facilitating earlier use, awareness, and generation of safety data for a Shigella vaccines, allowing comparability between studies, confirming serum O-antigen IgG as a correlate of protection, and determining thresholds of protection. Currently, almost all data informing correlates and thresholds of protection are from HIC adults and older children. A field-efficacy study among LMIC infants and young children will be critical to advance a first vaccine to licensure, WHO policy recommendation, and prequalification, which are prerequisites for Gavi-supported introduction.In conclusion, an international standard serum and harmonized ELISA are needed to advance new-generation global health Shigella vaccine candidates advance to phase 3 clinical evaluation.Proactive scenario planning of regulatory strategies by vaccine developers and planning of data to be collected in late-stage product development to support future policy recommendation will help mitigate delays to postlicensure implementation. Early and parallel engagement with LMIC national regulatory authorities and policy makers, as well as global policy and financing bodies, such as WHO and GAVI, is critical before designing pivotal phase 3 efficacy studies. This is the proposed approach of the authors as