Wastewater treatment plants (WWTPs) contribute to the release of significant quantities of microplastics into the aquatic environment. The facile identification of microplastics and an understanding of their occurrence and transport through WWTPs are essential for improving microplastic retention. Potential microplastic treatment technologies for both polymeric and ceramic membrane filtrations were systematically investigated to inform decisions on the optimal choice of membrane for effective microplastic retention. A blocking filtration model, based on a simple linear regression fitting, was used in experiments on the filtration of microplastic suspensions to determine the relative importance of individual fouling mechanisms. Unlike the commonly applied spectroscopic techniques, the facile identification approaches, that are closely related to the amounts of particles within wastewater samples, attempted to identify tiny microplastics (<1.0 µm) by comparing them against silica particles for reference. A larger decline in the normalized permeate flux was observed for 0.1 μm polystyrene microplastics, while standard pore blocking appeared to be the dominant fouling mechanism for all membranes. More microplastics based on turbidity and total solids were removed using the ceramic membrane than the other polymeric membranes. However, fewer microplastics, based on the particle size distribution analysis, were removed using the ceramic membrane as the pore size measurements gave a relatively large pore size for the ceramic membrane, compared with other polymeric membranes; even though a nominal pore size of 0.1 μm for all membranes were provided by the suppliers. The contribution of microplastic-containing synthetic wastewaters to overall flux decline was significantly greater than those of identical microplastic suspensions because of the aggregation of larger microplastics with dissolved organic matter in synthetic wastewater, leading to the formation of a cake layer on the membrane surface. Despite the challenges associated with the facile identification approaches, our findings provided deeper insights and understanding of how microplastics behave in membrane filtration, which could enable the application of potential microplastic treatment technologies. Microplastics are generally defined as small plastic pieces, less than 5 mm in length, that can be harmful to aquatic life ,2. Prima−1) and samples of 10–20 µm in size can rarely be analyzed [Microplastic analysis can be classified into physical and chemical characterizations . Chemicaanalyzed ,34. Howeanalyzed . Unlike analyzed . The tecanalyzed ,38,39.The present study addresses the potential of several engineered facile and indirect identification approaches by examining the removal efficiency and behavior of microplastics during wastewater treatment using membrane processes. Two tiny, differently sized PS and PE (0.1 and 1.0 µm) microplastics that cannot be measured by other spectroscopic techniques were chosen as the target microplastics. based on their high levels of prevalence in WWTPs, and their transport mechanisms were compared with standard silica particles. By applying several facile microplastic identification methods, the study aimed to fulfil the following specific objectives: (i) to investigate the effects of different types of particles (silica particle and microplastic), (ii) the effects of different sizes of microplastics, (iii) the effects of different types of microplastics (PS and PE), and (iv) the effects of synthetic wastewater samples on filtration and treatment behaviors. The results are expected to offer valuable insights into how we operate a membrane treatment system to improve the retention of microplastics, by better understanding the transport of these relatively tiny microplastics.w/w) aqueous suspensions. Monodispersed PS microplastics of two different sizes (0.1 and 1.0 μm) at 2% solids (w/w) aqueous suspensions  were used, since they are a particle size standard and are ideal for characterizing the particle size distribution of the samples. PE microplastics with 1.0 μm  in dry powder form were suspended in an aqueous solution with a surfactant  of 0.5 mg/L prior to being suspended in deionized (DI) water , since PE microplastics are hydrophobic. The concentrations of silica particles and microplastics were suspended in DI water at 50 mg/L for all experiments. For the wastewater samples, the same concentrations of each PS and PE microplastics were added to the prepared synthetic wastewater samples that were adapted from previous studies to maintain the consistency of wastewater composition [Non-functionalized silica microsphere particle with natural hydroxyl or silanol groups was purchased from EPRUI Biotech Co., Ltd.  as a reference particle. The silica microspheres of 0.1 μm nominal diameters were supplied in 10% solids  and the Anopore inorganic membrane , with a 13.1 cm2 effective surface area, were employed in a bench-scale membrane filtration system [Two polyvinylidene fluoride (PVDF) membranes  and wM is the MWCO (Da). The pore sizes given by the membrane suppliers were similar to each other. However, the actual membrane pore sizes were determined by measuring the retention of polyethylene oxides  with several different molecular weights, ranging from 100 kDa to 5000 kDa. The molecular weight cut-off (MWCO) of the membranes refers to the minimal molecular weight of organic solutes , where 90% of the solute can be retained . The memAtomic force microscopy  was used to measure the surface roughness of the membranes in non-contact mode with a 10 μm × 10 μm scale. The porosity was measured using an AutoPore IV 9500 mercury poremeter , based on the intrusion of mercury into a porous membrane structure under stringently controlled pressures. For these measurements the membranes were cut into small pieces, approximately 10 mm in length. The membrane filtration experiments were performed with identical silica particles and microplastic-containing aqueous solutions. A peristaltic pump provided an accurate constant feed flow during all the experiments at a constant transmembrane pressure (TMP) of 1.0 bar, which was maintained by a pressure gauge in front of the membrane. The permeate samples were collected in a glass beaker that was constantly being weighed, using an automated electronic scale , with the weight of the changing volume indicating flux changes. Throughout the experiment, 2 L of feed was stirred at 100 rpm, using a magnetic stirrer . All the experiments were carried out in duplicate. Firstly, the samples were filtered with DI water to remove contaminants from the system, then the feed water was replaced with ordinary water for the test. The subsequent filtration experiment lasted approximately 2.5 h and all experiments were performed at room temperature (20.8 ± 0.6 °C).Samples for analysis were collected regularly from the feed and permeate in the bench-scale membrane filtration unit. Several physicochemical parameters related to particle concentration were measured in the aqueous solutions. The feed and permeate concentrations were used to calculate the retention, which represents the amounts of particles retained by the membranes. Turbidity was measured using a portable turbidimeter . The TS were measured by weighing the amounts of solids present in a known volume of sample in accordance with the Standard Methods 2540 D/E . The method involved weighing a beaker, filling it with a known volume, evaporating the water in an oven and completely drying the residue, and then weighing the beaker with the residue. The TS concentration was equal to the difference between the weight of the beaker with the residue and the weight of the beaker without it. The size distribution of the particles was measured using a Mastersizer 3000 .V), and filtration time (t), for the individual fouling mechanisms are shown below [Q0 is the initial flow rate and K is the constant with the subscript indicating the blocking mechanism. Blocking filtration models describe the four mechanisms of membrane fouling by colloidal particles  as (a) cwn below .(2)KbV An alternative approach to identifying colloidal fouling mechanisms involves applying the blocking filtration models in their integrated forms, which involves a straightforward linear least-square fit and allows for facile identification of individual fouling mechanisms. Clear identification and differentiation between the pore blocking mechanisms has important practical implications, as knowing the fouling mechanism can determine the optimal choice of membrane. In this study, we performed dead-end filtration experiments, using polymeric and ceramic membranes for retaining tiny microplastics, before fitting Equations (2)–(5) to the experimental flux data using linear least-square fitting to identify the relative importance of individual fouling mechanisms.®) and a ceramic membrane (Anodisc) with the same nominal membrane pore size of 0.1 μm. The normalized permeate flux decline of PS microplastics was slightly greater than that of the silica particles for three different membrane filtrations, which could be explained by the steric (size) mechanism that predominates in microfiltration (MF) membranes. The distribution of particle sizes and membrane pores were thus investigated to obtain a better understanding of filtration and treatment behavior. The particle size distribution of the silica particles and the 0.1 μm-sized PS microplastics, provided by the manufacturer, is shown in ®), and PEO solutions of 3652.7 kg/mol (Anodisc). The computed nominal pore sizes for the membranes were empirically estimated to be 27.3, 45.6, and 73.7 nm according to Equation (1).We investigated the effects of two different types of particles (silica particles and PS microplastics) with the same average size (0.1 μm) on filtration and treatment performance to further understand microplastic transport compared with spherical silica particles, used for reference. 2 values indicated that a variety of fouling behaviors, such as complete, standard, intermediate pore blocking, and cake filtration were observed, because the D10 values of the silica particles and PS microplastics are similar to the Synder and Anodisc membrane pores, respectively –(5) with individual Rectively . For theEffluent guidelines for these particles are mandatory for wastewater discharged from domestic wastewater treatment facilities based on the performance of treatment technologies. We tried to determine whether some of the effluent guidelines could be used as an indirect measurement by evaluating any correlations with microplastics, based on the relatively facile and rapid measurements of water quality parameters in engineered systems, such as turbidity, TS, and particle size distribution. For example, linear correlations were observed between increasing feed concentration and higher values of turbidity and TS for silica particles, and PS microplastics. The turbidity values of feed water were 29.1 ± 0.1 NTU for silica particles and 105.3 ± 0.5 NTU for PS microplastic, despite being prepared with the same concentrations in an aqueous solution at neutral pH; while TS concentrations had similar values. The changes in turbidity and TS concentrations during the three different membrane filtrations were evaluated as retention performance, resulting in removal of over 90% for all membranes . The turParticle size distribution analysis, which can determine and report information about the size and range of particles representative of a given material, could be one of the facile and indirect measurement indicators with which to gain insight into the transport mechanisms in membrane filtrations. This approach would not be able to directly evaluate particle removal but could evaluate specific distributions of particles both before and after membrane filtration, suggesting that the retention mechanism of particles could be determined by understanding the interactions between particle size and membrane pore. The results of the particle size distribution analysis in feed and permeate water samples that contained silica and PS microplastic with the same concentrations are shown in ®, and Anodisc membranes with the same average pore size of 0.1 μm, as provided by the respective manufacturers. A relatively rapid flux decline was observed for small PS microplastics with 0.1 μm, with the normalized permeate flux suddenly decreasing after a few minutes and then consistently maintained for all membranes. These observations were attributed to standard or complete pore blocking, because the 0.1 μm PS microplastic is close to the average membrane pore size of 0.1 μm. Through blocking filtration model analysis, small PS microplastics satisfy the standard pore blocking because they can penetrate and attach to the inner wall within the membrane pores, leading to severe membrane fouling  of PS microplastics on filtration and treatment performance to understand their filtration and fouling mechanisms with polymeric and ceramic membranes.  fouling . For theThe changes in turbidity of the 0.1 and 1.0 μm PS microplastics, with the same concentration in aqueous solutions, during the three different membrane filtrations, are shown in ®, and Anodisc membranes. A relatively rapid decline in normalized permeate flux was observed for the PS microplastics at the relatively larger membrane pores (SteriLUX® and Anodisc membranes), while there was only a slight decrease in normalized permeate flux at the Synder membrane. For the PE microplastics, a rapid normalized permeate flux decline was observed at Synder membrane and gradual decreases at the other two membranes. In particular, the ceramic membrane (Anodisc) did not show a severe level of membrane fouling for the PE microplastics. These observations could be described through the particle size distribution of the PS and PE microplastics, with an average size of 1.0 μm. PS microplastics exhibited the narrowest particle size distribution, at around 1.0 μm, while the particle size distribution in PE microplastics was somewhat broader, ranging from 0.08–10.0 μm  on filtration and treatment performance were investigated to understand their filtration and fouling behaviors when using polymeric and ceramic membranes. –10.0 μm . As descplastics .The initial turbidity values were 683.6 ± 1.0 NTU for the 1.0 μm PS microplastics and 120.1 ± 3.2 NTU for the 1.0 μm PE microplastics, while TS concentrations were 4.6 ± 1.0 mg/L and 5.7 ± 0.5 mg/L, respectively. Altogether, 99% of the turbidity of 1.0 μm PS and PE microplastics was removed. However, TS retention increased slightly with increasing membrane pore size . AlthougA variety of physicochemical and biological compositions in wastewater samples can impact microplastic filtration behavior and retention performance in membrane filtrations. For example, dissolved organic matter in synthetic wastewater can exacerbate flux decline via membrane fouling, which increases filtration resistance because they contain relatively low concentrations of suspended particles. Our filtration and treatment experiments indicated that normalized permeate flux significantly decreased in the presence of microplastics in synthetic wastewater . SimilarIn this study, we used several facile water quality parameters that are widely applied in WWTPs to evaluate the filtration and treatment behaviors of microplastics. Our findings indicate that polymeric and ceramic membranes can remove a significant fraction of microplastics contained within the system by adopting the evaluation of facile water quality parameters. The reason why some microplastics remain after membrane filtration is still unknown, although it appears that a significant proportion of the microplastics remaining in the permeate can be analyzed with particle size distribution analysis. The particle size distribution analysis detailed in this study is a promising technique for quantifying the transport of tiny microplastics through wastewater treatment systems. Additionally, it can improve understanding of microplastic treatment mechanisms and give more precise estimations of the relative contribution to microplastic removal by membrane-based processes, which can subsequently assist engineers in choosing appropriate membranes for their particular objectives. Our results demonstrated that the diameter values obtained from the particle size distribution analysis could significantly promote microplastic retention between the polymeric and ceramic membranes through multiple fouling mechanisms. For example, microplastics in synthetic wastewater, which contains fewer suspended particles and more dissolved organic matter than identical microplastic-containing solutions, have a rapid flux decline curve because organic matter can more effectively limit fouling sources if they contain relatively large microplastics. The ceramic membrane was particularly effective at removing PE microplastics but was less effective in retaining PS microplastics. These facile and indirect measurement methods can be used to understand the transport of microplastics in membrane-based treatment processes as our results suggest that particle size distribution analysis could handle different sizes and types of microplastics. Currently, however, there are only limited options for directly quantifying microplastics associated with wastewaters, due to high concentrations of other suspended particles, nutrients, and organic contaminants. Additional studies involving direct quantification for a variety of microplastics are necessary to validate our findings.


The influence of climate change on civil conflict and societal instability in the premodern world is a subject of much debate, in part because of the limited temporal or disciplinary scope of case studies. We present a transdisciplinary case study that combines archeological, historical, and paleoclimate datasets to explore the dynamic, shifting relationships among climate change, civil conflict, and political collapse at Mayapan, the largest Postclassic Maya capital of the Yucatán Peninsula in the thirteenth and fourteenth centuries CE. Multiple data sources indicate that civil conflict increased significantly and generalized linear modeling correlates strife in the city with drought conditions between 1400 and 1450 cal. CE. We argue that prolonged drought escalated rival factional tensions, but subsequent adaptations reveal regional-scale resiliency, ensuring that Maya political and economic structures endured until European contact in the early sixteenth century CE. The influence of climate on premodern civil conflict and societal instability is debated. Here, the authors combine archeological, historical, and paleoclimatic datasets to show that drought between 1400-1450 cal. CE escalated civil conflict at Mayapan, the largest Postclassic Maya capital of the Yucatán Peninsula. The influence of climate change on civil conflict in the last century has also been the focus of compelling statistical studies12, and an important nexus for debate, revealing the importance of human agency and unexpected, non-linear relationships between climate and human behavior19. Longer-term climatic, archeological, and historical records can contribute to these contemporary debates, but demand a rigorous transdisciplinary framework that bridges natural and social systems18. We report a singular case study of the complexities of the natural and social systems at the Postclassic Maya capital of Mayapan  throughout its history and its ultimate demise. This occurred in the context of drought, civil conflict, and the collapse of the regional state. A period of political balkanization after 1450 cal. CE preserved complex organizational institutions and fostered a resilient, peninsular-wide market economy observed at European contact in the early 16th century CE. We examine climate stresses to Mayapan’s local and regional subsistence and economic systems, and the behaviors of human actors that included political violence within these dynamic social and political transformations.Archeological and historical studies have proposed linkages among global climate change, societal instability, violent conflict, and sociopolitical collapse20. Long-term climate reconstructions reveal that some climate variations in the past were of significantly greater magnitude than those experienced in the last 100 years. Furthermore, current and future anthropogenic influences are projected to amplify the severity of extreme events in the water cycle and lead to more intense and prolonged droughts than those that impacted agricultural productivity in the recent past22. Archeological and historical records are well-suited for examining past societal effects of climate crises over long-term cycles. Such effects in Pre-Columbian Mesoamerica may have included migration, demographic decline, geographic shifts in centers of political power, and warfare. We focus on the latter, specifically the initiation and consequences of societal conflicts that coincided with climate impacts on the agrarian food base and regional political economy. These subsistence and related economic foundations are well-known from the archeological record and from memories of descendant peoples in early Colonial period European accounts12. The magnitude and duration of the climate impacts in the northern Yucatán Peninsula (Mayapan) may have transcended established mechanisms to overcome modest inter-annual rainfall fluctuations23, given the limitations of cumbersome transport and the long-term storage of maize, the primary staple grain24.Climate assessment reports of the intergovernmental panel on climate change (IPCC) evaluate risk and model vulnerability during the last century at regional and global scales28. Large cities first appeared along the western edges of the Maya region by 1000–800 cal. BCE29 and numerous monumental centers and networks of villages developed in the heart of the Maya Lowlands by 500 cal. BCE30. One of the largest state capitals in all of Maya prehistory , El Mirador, was established between 100 cal. BCE and 250 cal. CE27. The Lowlands region was dotted with kingdoms large and small, amidst a densely populated countryside of towns and villages throughout the Classic period, with the Late Classic Period  witnessing the emergence of the largest number of competing states in the southern lowlands. Shifting centers of influence and the most dramatic sequential disintegration of these cities occurred during the Terminal Classic period, between 800 and 1000 cal. CE, with climate change argued to be a contributing factor to increasing conflict34. During this interval, the towns and cities of the northern and coastal peninsula  were magnets for Maya settlement, in turn experiencing political and demographic decline that coincide with multiple lines of evidence for drought during the eleventh century cal. CE.36. But northern and coastal Maya populations subsequently recovered quickly and the political capital of Mayapan arose and governed from at least 1100 to 1450 cal. CE37. Mayapan provides an appropriate test case for exploring the causes and impacts of civil conflict because of its temporal proximity to European Contact, and retrospective, Colonial-Period documentary accounts provide additional evidence that can be evaluated with chronological (radiocarbon [14C] dates) and human osteological data.The Maya region offers the breadth and depth of archeological, historical, and climate records essential for studying correlations between social change and fluctuating climate conditions. Here, we compare the archeological and ethnohistorical evidence for civil conflict with local and regional records of climate change at the last large Pre-Columbian Maya capital city of Mayapan  who governed the polity as members of Mayapan’s ruling council41. These lords established the city’s monumental center, replete with its principal pyramid, the Temple of K’uk’ulkan, and a nucleated set of other temples, colonnaded halls, and shrines covered in murals and sculptures that reflect the city’s mythical foundations42. Densely settled residential zones extend from the center in all directions within the city’s 9.1 km-long circumferential wall, which encloses an area of 4.2 km2, and housing sprawls at least a half kilometer beyond this boundary  to pan Fig. .Fig. 2Cu40. These intervals include: 1) a period of “terror and war” [1302–1323 cal. CE], 2) an interval of “inquisition against members of the Xiu nobility” [1361–1381 cal. CE]; 3) decentralization and population decline [1382–1401 cal. CE], 4) massacre of the Cocom noble family [1440–1461 cal. CE] and 5) political demise of the regional state and abandonment of the city [post–1450 cal. CE] , an easterly trade wind system. Variability of the CLLJ depends on the position and strength of the Bermuda High50, meridional sea level pressure (SLP), and sea surface temperature (SST). Stronger trade winds, caused by a high meridional SLP gradient, result in lower SSTs and drier conditions on the Yucatán Peninsula. The sea-level pressure gradient between the Caribbean and the eastern Pacific links the region in a complex fashion with the inter-tropical convergence zone (ITCZ) and the El Niño-Southern Oscillation (ENSO) system in the Pacific52, influencing Yucatán moisture dynamics indirectly. Such linkages tend to occur in the wet season, prior to El Niño events51, which reduce tropical depressions and hurricanes in the study region. Outside the rainy season, winter nortes occasionally bring rainfall and cold, windy weather from the Gulf of Mexico46.Rainfall over the Yucatán Peninsula is derived mainly from the Caribbean, Atlantic, and Gulf of Mexico sources, and only occasionally from the Pacific18O record2 shows a series of wetter intervals between 1100 and 1340 CE, an interval when populations at Mayapan were growing  identified here in a stalagmite (M1) collected from a cave located directly below the central Plaza at Mayapan, and analyzed for the present study  of directly radiocarbon-dated human skeletal material Fig.  from Mayals Fig. 40.58. In contrast, other large deposits of human remains in the monumental center  likely represent sacrificed (or otherwise killed) war captives from polities targeted by the Mayapan state elsewhere on the peninsula (beyond the northwest stronghold of the confederacy). One such deposit was found in an alleyway next to the site’s iconic Round Temple, which contained the crania and long bones of 25 individuals who were directly AMS-14C-dated to 1302–1362 cal. CE . Four of the individuals in MB1 suffered violent deaths, as indicated by stone knives embedded in their scapula, rib cage, or pelvis60. The bones of ~20 individuals in MB2 were chopped into pieces and burned. Both observations are consistent with historical records for the late 14th century CE, which indicate that one faction of the government persecuted, tortured, and executed rival factions. Following this turmoil, sources suggest a restoration of order and several decades of relative stability just before and after 1400 CE40. Direct AMS 14C dates on 10 individuals from MB1-MB2 are statistically indistinguishable from one another, with a modeled age of 1360 to 1400 cal. CE. This coincides with a major central Mexican drought in the Aztec-controlled highlands 61, which was the most extreme dry episode since 771 cal. CE. The historical records of massacres at Mayapan also correspond with the population decline that is evident in the decreasing number of directly AMS 14C-dated individuals ; both are adjacent to ceremonial edifices. Both features date to the late 14th century , were found mixed with debris from building collapse . This and other historical references are increasingly corroborated by paleoclimatic, paleoenvironmental, and archeological datasets, dated using AMS 14C and U-Th series methods. Like Mayapan’s ultimate demise, drought and famine are also described in the Book of Chilam Balam of Mani  flanks the northern staircase of the Temple of K’uk’ulkan, and is the best candidate for the Xiu-led massacre of members of the Cocom family that resulted in the city’s collapse and ultimate abandonment. The skulls and isolated limbs of nine individuals, many of them children  p < 0.001; Middle to Late  p = 0.002  were used to compare counts of deaths from internal conflict during Mayapan’s occupation Fig. . Results002 Fig. . Populat1 Rabbit  that caused severe famine in Aztec Central Mexico  to examine the relationships among climate change, fluctuations in population size/nucleation (SPD), and osteological data indicative of internal conflict during Mayapan’s entire occupational history 75, but found no significant relationship (p = 0.65).Despite the lack of association between climate and population throughout the occupational sequence, we do find a significant relationship between drought and population decline when we examine the period between 1350 and 1430 cal. CE, i.e., the time when the most substantial population decline occurred , ironically and meaningfully laid to rest at the base of the Temple of K’uk’ulkan, the iconic principal temple and ritual center of Mayapan. Our results suggest that rivalry among governing elites at Mayapan materialized into action in the context of more frequent and/or severe droughts. Comparatively, such climate challenges present a range of opportunities for human actors, from the development of innovative adaptations to the stoking of revolution76. These climate hardships and ensuing food shortages would have undermined the city’s economic base and enabled the Xiu-led usurpation. The unifying and resilient institutions that held the Mayapan state together until ~1450 CE were ultimately eroded, the confederation dissolved, and the city largely abandoned.Our findings support Mayapan’s storied institutional collapse between 1441 and 1461 cal. CE, a consequence of civil conflict driven by political rivalry and ambition, which was embedded in the social memory of Yucatecan peoples whose testimonies entered the written record of the early Colonial Period8. Mobility was an important component of social change as people dispersed from Mayapan to other parts of the Yucatán Peninsula77. This was a common indigenous response, observed historically during drought periods when Maya people abandoned Spanish towns and dispersed to settlements in the unsubdued frontier to the south79. The social and political landscape of the Yucatán Peninsula was also reorganized into a network of smaller states that were politically independent, but economically interwoven. Prosperous coastal towns, first observed by Spaniards,63 took advantage of a variety of freshwater features70 and marine resources that provided opportunities for subsistence and economic diversification. Maritime commerce thrived, with large canoes carrying salt, chocolate and other commodities around the peninsula80. At the time of European contact, the Yucatán Peninsula was divided into 15 states63, some with developed hierarchies and kings  and others more loosely organized; bureaucratic systems ranking nobles according to an array of religious and political titles81. The conflict between states occurred and each state had military leaders who could quickly mobilize armies, but there is little historical evidence for civil conflict within each state. Ironically, climatic impacts caused by drought and related pestilences continued in northern Yucatán with little respite in the century following Mayapan’s fall48. Yet economic, social, and religious traditions persevered until the onset of Spanish rule, despite the reduced scale of political units, attesting to a resilient system of human-environmental adaptations63.As a counterpart to strong archeological, historical, and climatic evidence for civil conflict and institutional collapse at Mayapan, we explored different pathways for resilience and societal transformation to regional climate change63. These complexities are important as we attempt to evaluate the potential success or failure of modern state institutions designed to maintain internal order and peace in the face of future climate change. Recent regional climate records and modeling results suggest drier conditions in Central America are associated with global warming83. Food insecurity, social unrest, and drought-driven migration are already of considerable concern in parts of Mexico and Central America. Our transdisciplinary work highlights the importance of understanding the complex relationships between natural and social systems, especially when evaluating the role of climate change in exacerbating internal political tensions and factionalism in areas where drought leads to food insecurity.Overall, we argue that human responses to drought on the Yucatán Peninsula during the 15th century CE were complex. On the one hand, drought stimulated civil conflict and institutional failure at Mayapan. However, despite decentralization, intervals of mobility, temporary impacts to trade, and political reorganization in the region, a resilient network of small Maya states persisted and was encountered by Europeans in the early 16th centuryThe primary bioarchaeological data for this paper derives from lnstituto Nacional de Antropología e Historia (INAH)-Yucatán’s projects in the excavation and restoration of Mayapan’s monumental zone (directed by Carlos Peraza Lope). Additional data originate from salvage archeological work conducted by Peraza Lope for INAH along the Merida-Chetumal highway that crosses through the Mayapan site, as well as National Science Foundation-supported collaborative research in the settlement zone. Permits were issued by the INAH-Consejo de Arqueología for excavation and the export of samples for analysis in the US . All work was conducted with local Yucatecan archeologists, field crews, support staff, and the community of Telchaquillo, located 1 km north of the archeological site of Mayapan. All excavation, including the excavation of human remains, was done with community permission and participation. All human remains that fall under the auspices of the lnstituto Nacional de Antropología e Historia of Mexico (Yucatan) are stored at a permanent repository located at the laboratory of the Proyecto INAH Mayapán, Telchaquillo, Yucatán, Mexico. Samples associated with the Carnegie Institution of Washington excavations are curated at the laboratory of the Centro INAH Yucatán, Mérida, Yucatán, Mexico.14C dates from 205 sets of human remains excavated at Mayapan  and a Thermo DeltaPlus Advantage isotope ratio mass spectrometer, respectively. Sample quality was evaluated by % crude gelatin yield, C%, N%, and C:N ratio. Ultrafiltered gelatin (∼2.1 mg) was combusted for 3 h at 900 °C in vacuum-sealed quartz tubes with CuO and Ag wire. Graphitization and AMS 14C measurements were done at the Keck Carbon Cycle Accelerator Mass Spectrometer facility and the Penn State University Accelerator Mass Spectrometer Laboratory. The 14C ages were corrected for mass-dependent fractionation using measured δ13C values and compared with backgrounds and known-age secondary standards. All dates were calibrated in OxCal version 4.486 using the IntCal20 curve87.We report 211 AMS 88 in the R environment to generate summed probability distributions (SPDs) on calibrated radiocarbon dates from our sample of 205 dated human skeletal remains. We then compared our SPDs to a Monte-Carlo simulation of a null exponential growth model , we derived summed probability values and paired them with δ18O values for every year both data were available. To pair SPD values with internal conflict data, we averaged values for the 2σ 14C probability distribution for each individual. For example, if an individual had a 2σ 14C range of AD 1100–1175, all SPD values from that temporal range were averaged. The resulting GLM was generated in the R programming environment.We fit generalized linear models (GLMs) with a binomial distribution and log link appropriate to proportional data and quasi-likelihood estimation to account for overdispersion. To generate the GLM comparing the Mayapan SPD (response variable) to the YOK-I δ18O values for the 2σ 14C probability distribution for each individual. The resulting dataset contains a sub-sample of 183 individuals, each with a death from civil conflict and YOK-I and Tzabnah δ18O designation, which was uploaded into the R programming environment to generate the GLMs.A GLM with a binomial distribution and log link appropriate to proportional data and quasi-likelihood estimation to account for overdispersion was estimated to compare precipitation with individual deaths from civil conflict. To do this we averaged the YOK-I and Tzabnah δFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Code 1Supplementary Code 2Reporting Summary


Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants. Ethically responsible research and development aim for the replacement of animal experiments. Hence, already in 1959, the 3R principles were introduced in the scientific community to refine, reduce, and ultimately replace animal experiments . Two-dimWith the advent of tissue engineering which is using cells, cellular products, materials, and engineering approaches to create functional tissue substitutes  a wide vEspecially in the field of skin tissue engineering, highly advanced skin equivalents have been developed that closely mimic native skin, including pigmentation  and vascDespite the improvement of sophisticated tissue equivalents for the investigation of pathophysiological processes, the investigation of the respective 3D equivalents constitutes the current limitation to replace animal models and exploit the full potential of tissue equivalents. Especially for complex 3D tissue equivalents, it is crucial to develop minimally invasive non-destructive analytical methods to facilitate continuous or repetitive analysis and monitoring of tissue viability, development, functionality, and cellular responses reliably, reproducibly, and preferably in real time ,21.There are commercially available systems to study the in vitro penetration of drugs and active substances to conclude on dosage forms. The Franz diffusion chamber is one of the most important methods for determining transdermal drug administration  in skin A change in traditional culture parameters, such as oxygen, pH, glucose, or lactate can have detrimental effects on cell behavior and thus on the relevance of the generated data . These pThe aim of the study was to develop a novel ex vivo incubator in combination with minimally-invasive open flow microperfusion (OFM) sampling technology , and an For penetration experiments, the OECD guideline  stipulatIt is also important that moisturizing prevents the skin from drying out. If the environment is too humid, the release rate will be too high, or if the environment is too dry, it will be reduced. Therefore, similar to testing medicines in the clinic, the humidity must be kept constant between 40 and 60% .Since no suitable device is commercially available, a mobile “ex vivo incubator” with three sliding doors for penetration tests with human skin explants or FTSE was developed for this study . The tecTM to prevent direct heat radiation and thus prevent the skin/ FTSE from drying out.The evaluation of the ex vivo incubator showed a run-in phase of approximately 50 min after which the temperature was regulated with an evaluated accuracy of 32.2 ± 0.67 °C and humidity of 53.2 ± 2.42%. The system comprises a modified climate control unit , two heaters , two humidifiers , and a ventilator . Both heaters were covered with StyrofoamOFM is a sampling technology for clinical and preclinical drug development studies and biomarker research. OFM was designed for continuous sampling of analytes from the interstitial fluid (ISF) of various tissues . It provides direct access to the ISF by insertion of a small, minimally invasive, membrane-free OFM probe with macroscopic openings . OFM is Online monitoring of oxygen, pH, glucose, and lactate was performed by connecting two individual flow-through cells consecutively after the outlet of the OFM probes . Flow-th®-PRO). Oxygen sensors showed a dynamic range from 0 to 100% air saturation, whereas pH sensors showed a range of 6 to 9. Data acquisition was performed using “PyroWorkbench” .The lead flow-through cell from PyroScience held optical sensor spots for oxygen and pH measurements and featured a minimized internal cell volume of less than 10 µL. In parallel, the same optical sensor spots were used to measure oxygen and pH in the Petri dish that contained culture medium to cultivate the FTSE . All senFor online monitoring of glucose and lactate, a second flow-through cell from Jobst  with tubing of 0.5 mm inner diameter was connected to the outlet of the PyroScience flow-through cell. The Jobst flow-through cell had to be re-evaluated for a flow rate of 1 µL/min because the manufacturer’s specifications only cover flow rates down to 5 µL/min. Successful re-evaluation showed suitable stability, accuracy, precision, and an acceptable response time (data not shown). The glucose sensor showed a range of <0.9 to 450 mg/dL, whereas the lactate sensor showed <0.18 to 135 mg/dL. Data acquisition was performed using the biosensor measuring instrumentation (Six) and software .The oxygen sensor was referenced using air-saturated water (100%) or oxygen-free water (0%) by using Oxcal . The pH sensor was referenced using calibration capsules pH 2 and pH 10 . Glucose and lactate sensors were tested individually, before and after the investigations, using 3 individual referenced stock solutions per analyte within the operation range, to determine drift, sensitivity loss, or sensor damage. For retrospective analysis, false values caused by air bubbles were excluded and linearly interpolated. Manual samples of the culture medium were taken frequently and analyzed offline for pH, glucose, and lactate. The removed volume was replaced, and unintentional evaporation was compensated with distilled water.Online readings of glucose and lactate in OFM samples from FTSE tissue and skin explants were referenced using a pre-evaluated analyzer . Offline pH values were estimated using test strips .® Full-Thickness LARGE Skin Models  with a diameter of 3.1 cm each were used as FTSE. This FTSE is a multilayer equivalent to human skin and consists of human keratinocytes and fibroblasts populated on a collagen matrix. For this study, the FTSE was cultivated in sterile conditions at Henkel Laboratories for an additional week, in order to increase the thickness of the stratum corneum and thus increase the penetration barrier .Phenion2) and prevent cross-contamination of the culture medium . OFM probes were implanted according to the manufacturer’s instructions and implanted FTSEs were placed into Petri dishes  inside the ex vivo incubator with integrated sensor spots for oxygen and pH, on top of spacers and filter paper. Petri dishes were filled with culture medium and placed in sampling units designed and manufactured by Joanneum Research. The CAD models for the sampling units were generated with SolidWorks® , 3D printed in clear resin using a Form 2  SLA 3D desktop printer. Then, implanted OFM probes were connected to OFM pumps, tubing, an online monitoring system, and sampling vials. FTSE online monitoring was performed in triplicates. For the penetration assay, triplicates were used for acyclovir, lidocaine, and diclofenac each.FTSEs were delivered on transport agar in well plates. An O-shaped patch  with an outer diameter of 3.1 and an inner diameter of 1.8 cm was bonded onto the FTSE surface to form round application sites  acyclovir, lidocaine, and diclofenac, which differ in their ability to penetrate dermal tissue . Creams Acyclovir was analyzed as described before  and 5% (v/v) formic acid in water and applied to a preconditioned solid-phase microelution plate . Loaded samples were washed with water and 50% (v/v) methanol in water prior to elution with 1% (v/v) formic acid in acetonitrile. Eluates were dried with compressed air and reconstituted in 30% (v/v) acetonitrile in water prior to quantitative analyses with a UHPLC/QqQ system [UHPLC 1290 Infinity II/QqQ 6495B ]. Isocratic separation of diclofenac (50% (v/v) acetonitrile in water) was achieved with an ACQUITY BEH C8 reversed-phase pre and analytical column  at a flow rate of 400 µL/min and 35 °C. Negative electrospray ionization was used prior to MS detection . The analytical range was from 0.9 to 186.0 ng/mL. For lidocaine, we diluted OFM samples, calibrators, and quality controls in the perfusate. Samples were then mixed with equal volumes of internal standard solution [lidocaine-d10  in 1.5% (v/v) formic acid in water] and applied to a preconditioned Strata™-X SPE plate (see above). Loaded samples were washed with water prior to elution with acetonitrile. Eluates were dried with compressed air and reconstituted in 5% (v/v) acetonitrile in water prior to quantitative analysis using HPLC coupled to a high-resolution mass spectrometer [HPLC Ultimate 3000/HRMS LTQ Orbitrap XL ]. Chromatographic separation of lidocaine was achieved using an Atlantis T3 reversed-phase column  and gradient elution with two mobile phases  at a flow rate of 300 µL/min and a column temperature of 25 °C within 7 min run time. The multistep gradient started with 5% mobile phase B, went up to 50% mobile phase B at 1.5 min and up to 90% at 2.0 min, and kept at 90% until 4 min. Positive-heated electrospray ionization was used prior to full scan mass detection from m/z 215 to 255. The analytical range was from 1 to 1000 ng/mL.All OFM samples were stored at −80 °C until further processing. d before . For dicp ≤ 0.01.Statistical analysis was performed using Microsoft Excel . Data are represented as the mean ± standard deviation (SD). API concentrations in FTSE and skin explants were compared by calculating the area under the curve AUC. Two group comparisons were performed with a non-parametric Mann–Whitney U test. Significance was accepted at We used OFM to monitor culture parameters in an in vitro FTSE and in ex vivo skin explants both situated in an ex vivo incubator under controlled conditions .OFM probes were perfused with the perfusate (pump head 1) which equilibrates with the ISF of the surrounding tissue and is withdrawn by the pump head 2 (OFM sample). To measure critical tissue parameters directly in tissue rather than in the culture medium, OFM samples were collected at the outlet of the OFM probe which was inserted directly in either FTSE or skin explants A. ConseqOxygen levels in the culture medium remained relatively constant at 190.7 ± 2.7 hPa, corresponding to the oxygen concentration of the ambient air  ( = 7.60) B.glucose level in the culture medium remained relatively constant at 328.8 ± 33.3 mg/dl. In contrast, glucose levels in the FTSE samples were around 171.5 ± 26.9 mg/dL, slightly decreasing over time . In general, API concentrations accumulated in the culture medium, whereas levels in FTSE tissue showed concentration maxima at different time points and then declined due to further penetration into the culture medium. For acyclovir, the maximum concentration in FTSE tissue was observed 6–12 h after application (The experimental setup  was usedlication D, wherealication E. The malication F.When comparing FTSE tissue to explanted skin, all OFM samples from FTSE tissue had higher API concentrations of acyclovir, diclofenac, and lidocaine relative to OFM samples from explanted skin A–C at anIn this study, we used OFM for continuous monitoring of culture parameters in the dermis of an FTSE. We also compared FTSE data to samples collected from the culture medium of the FTSE and to data from skin explants. Furthermore, we performed a penetration assay with acyclovir, diclofenac, and lidocaine and used OFM to monitor API concentrations in FTSE dermis and human skin explants relative to the culture medium.Standard cell culture parameters such as oxygen, pH glucose, and lactate are often neglected when using in vitro skin equivalents or skin explants for penetration assays. However, it is clear that shifts in the concentration of these parameters can have detrimental effects on cellular behavior. For example, a hypoxic culture environment has been shown to increase the lipid barrier formation of customized FTSE .2 transfer from the culture medium into ambient air, causing alkalization. In contrast, the pH in the FTSE samples remained relatively stable, which could be attributed to the production of lactate of the living cells in the FTSE to some extent. Especially oxygen and glucose supplies were significantly lower than indicated by measuring the culture medium samples. As expected, oxygen and glucose levels in the skin explants were completely depleted, indicating cell death and tissue degradation. In the skin explant samples, no glucose was detected which can be explained by the fact that the skin explant was not supplied with fresh nutrients and the glucose was already consumed at the time when the measurements were started. Living cells must be within 200 µm of blood supply to be properly supplied with oxygen and nutrients [We have demonstrated before that the actual values of these parameters in 3D-cultured in vitro equivalents differ significantly from the values measured in the culture medium . Knowingutrients . The extA recent review on alternatives to biological skin in permeation studies came to the conclusion that a huge problem is that skin equivalents or surrogates, such as skin-like membranes, fail to estimate the amount of drugs penetrated into different skin layers . By usinPenetration tests also showed higher API concentrations in FTSE compared to skin explant samples, which indicates that the barrier function of the FTSE is not yet comparable to that of skin explants. This observation has also been frequently made with non-vascularized in vitro skin models ,46. For In conclusion, the presented advanced approach for online monitoring of culture parameters allows an accurate characterization of in vitro skin equivalents and skin explants. This enables better control of in vitro cultures and the development of more physiological skin equivalents. Furthermore, API monitoring in the dermis of the FTSE and skin explants during penetration assays can determine the actual concentration of substances that cells in the tissue are exposed to. This might enable a more accurate characterization of pharmacological penetration assays in skin equivalents to reduce or avoid animal testing.


Boards made with the addition of biomass from grasses with the C3 photosynthetic pathway—tall wheatgrass (Elymus elongatus), tall fescue (Festuca arundinacea), and perennial ryegrass (Lolium perenne)—gave lower values of mechanical properties. The opposite results were obtained in the case of the formaldehyde content: the lowest value was measured for particleboards made with the addition of tall fescue biomass (0.1% less than the control), and the highest for switchgrass (0.9% greater than the control) and cordgrass (3.2% greater than the control). Future research should address the optimization of the manufacturing process of particleboards from perennial grasses, taking into account the needs and technical possibilities of the wood industry sector.Looking for new alternative raw materials is one of the key issues in line with a bioeconomy approach, particularly for particleboard manufacturing. In this framework, this paper presents a comparison of some physico-mechanical properties and the formaldehyde contents of particleboards made with 30% substitution of grass biomass from six perennial grass species. Our studies indicate relatively high values of mechanical properties for particleboards made with the addition of biomass from grasses with the C Particleboards are panels produced by combining wooden particles with a proper glue and they are among the most important value-added products in the wood sector ,2.2 [On the one hand, global consumption of wood is expected to increase in the near future ,4,5,6, b2 ,8,9.To avoid conflict between environmental requirements and the production of wood for energy, paper, furniture and construction materials, new biomass sources have to be found, evaluated, and converted to value-added products ,12,13,14Therefore, particleboard producers need to look for new, cheap, and available sources of raw material in order to ensure the sustainability of the manufacturing process without compromising the quality of the obtained boards ,17,18,19Apart from wood there are many possible feedstocks for producing particleboards, including post-consumer wood, forest, and agricultural residues ,20.Triticum spp.) and rapeseed (Brassica napus L.) straw was confirmed more recently, with internal bond strength values in the low range of 61–99 mN mm−2, notwithstanding the application of enzymes to increase particle surface energy [The first attempts to produce particleboards from cereal straw revealed the low suitability of such feedstock, due to its large capillaries, which cause the collection of an excessive amount of resin, and to poor adhesion, an effect of the high content of mineral substances . The lowe energy .Saccharum officinarum L.) bagasse agglomerated with castor (Ricinus communis L.) oil-based polyurethane resin [Elaeis guineensis L.) trunk attained acceptable physical and mechanical properties, even if this required the addition of ammonium dihydrogen phosphate [Other lignocellulose raw materials utilized for particleboard manufacture include bagasse and flax shives ,23,24. Ihosphate .Among the different sources of biomass, perennial grasses are used increasingly worldwide, and many of them can be grown with minimal maintenance on marginal soils , which iMiscanthus x giganteus Greef & Deuter), switchgrass (Panicum virgatum L.) and prairie cordgrass (Spartina pectinata Bosc ex Link), these being warm-season grasses with the C4 photosynthetic pathway (hereafter referred to as C4 grasses); and tall wheatgrass (Elymus elongatus (Host.) Runemark), tall fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium perenne L.), these being cool-season grasses with the C3 photosynthetic pathway (hereafter referred to as C3 grasses).In this study, among many grass species, focus was placed on certain species with relatively high biomass potential in different soil quality conditions: miscanthus  , fiber-rOne such species which has been briefly evaluated in that regard is miscanthus ,33,34,35Switchgrass may be a fiber source requiring fewer inputs for growth and may be economically more viable than traditional fiber crops such as jute and flax. In addition, about 25–30% of switchgrass can be obtained as long fibers for high-value applications, and another 20–25% consisting of short fibers and hemicellulose can still be used for ethanol production . A singlTall wheatgrass biomass can be used to produce certain kinds of paper, cardboard products, MDF, and particleboards . It has Perennial ryegrass clippings have been incorporated into particleboards together with eucalyptus . Boards No sufficient information is available on the evaluation of cordgrass and tall fescue biomass for particleboard production.Taking the above into account, there is a need to improve existing knowledge on the possibility of using perennial grass biomass for particleboard production. An additional argument supporting the intensification of research on the use of grass biomass in the particleboards production is the relatively low  impact of these crops on the natural environment. It results from their perennially, low expenditure on fertilization and chemicals, and most importantly, the possibility of growing on marginal lands unsuitable for food production.The objective of this study was to investigate some physical and mechanical properties, as well as formaldehyde contents of particleboards made using biomass from six perennial grass species with different photosynthetic pathways.Spartina pectinata, Festuca arundinacea, and Lolium perenne have not so far been widely tested for their suitability for particleboard manufacture.The novelty of our work lies in the comparison of two groups of grass species defined on the basis of their photosynthetic pathways, which may be one of the main factors determining a plant’s development, and therefore its final applications. Moreover, grass species as Seed accessions of tall wheatgrass cv BAMAR, perennial ryegrass cv BARONKA, and tall fescue cv BAROLEX were kindly provided by breeders . Seed ofKCl—5.17; Corg—0.35%; clay—12.7%, silt—40.0%, sand—47.3%; macronutrients (in mg·kg−1 of soil): NH4—8.0, P—63.0, K—80.0, Ca—330.0, Mg—58.5, Cl—14.5. Establishment details are given in 2 plot was established.To produce new planting material of miscanthus and cordgrass, three-year-old plants were split whilst dormant (late April 2014) and the rhizome pieces were collected for re-planting. At the end of February 2014, seeds of switchgrass cv MARDAN were sown on a germination tank, and after a few days, emerging seedlings were transplanted to plastic pots filled with a sand–soil–peat mixture. After 3 months of vegetation in an unheated glasshouse, plants were transferred to a field. Seed, seedlings, and rhizomes of all grass species selected for our experiment were placed in a field in Radzików, Central Poland  in sandy clay soil  with the following parameters: pH−1): 40 N, 60 P, and 60 K. No additional chemicals (herbicides or pesticides) were used. Each year, biomass was mechanically cut and removed: in autumn at seed maturity for the C3 species and at the end of winter for the C4 species. For particleboard testing, the biomass of C3 species was collected in 2016, and that of C4 species in 2017. Each plot was divided into three sub-plots. From each sub-plot, ca. 10 m2 was selected for biomass cutting for use in the experiment. The biomass collected from the sub-plots was further mixed together to produce a representative sample for each species. For each species, ca. 20 kg of air-dried biomass was used for particleboard testing. Biomass was harvested using an ALKO 5001 R-II reel lawn mower  and then dried to a humidity of 10% and chopped into pieces of 1.5 to 2.0 cm using an MTD 475 petrol-powered shredder  dedicated for the disintegration of tree branches.The plants were grown for the next three years to determine their yield potential. Weeds were hand-removed, and mineral fertilizers were applied in spring each year in the following amounts  adhesive was used in the production of the particleboards; its properties are given in Wood particles from a manufacturer of particleboards and six alternative raw materials were used in the research. With each alternative raw material, a slab was produced in which the grass particles accounted for 30% by weight of the lignocellulosic raw material. For purposes of comparison, a reference board was produced, which was made only of wood particles. The compositions of the boards produced are given in Modulus of rupture (MOR) and modulus of elasticity (MOE) according to EN 310:1994,Internal bond strength (IB) according to EN 319:1999,Thickness swelling (TS) and water absorption (WA) after 24 h based on EN 317:1999,Formaldehyde content (FC) according to EN ISO 12460-5:2016-02, andDensity according to EN 323:1999.Standard chemical, physical, and mechanical properties of the manufactured boards were evaluated, including:−1 until failure. Square samples with a side length of 50 mm were used to determine the internal bond strength (IB). The samples were sanded and glued between stainless steel blocks. Blocks were positioned in holders and preloaded at a tension of 5 N, and then a loading rate of 1 mm·min−1 was applied until failure.Three-point bending tests (EN 310) and internal bond strength (IB) tests were performed on a Zwick testing machine using testXpert software . Samples with dimensions (L × W × T) 370 mm × 50 mm × 16 mm were used to determine modulus of rupture (MOR) and modulus of elasticity (MOE), being subjected to a loading rate of 7 mm·minThe following standards were used: EN 312 Particleboards and fiberboards—determination of swelling in thickness after immersion in water, 1993; EN 310 Wood-based panels—determination of modulus of elasticity in bending and bending strength; EN 319 Particleboards and fiberboards—determination of tensile strength perpendicular to the plane of the board, 1993; and EN 312 Particleboards—Specifications.Thickness swelling (TS) and water absorption (WA) were determined based on EN 317. Square samples with a side length of 50 mm and thickness 16 mm were fully immersed in water at 20 °C. After an immersion time of 24 h, the samples were taken out of the water and excess water was removed. TS was measured using a thickness gauge, positioned in the center of the samples (with a precision of 0.01 mm). WA was determined by weighing the samples using laboratory scales before and after immersion (with a precision of 0.001 g).−1.Three square samples with side length 50 mm and thickness 16 mm were used to determine density profiles. An X-ray scanning device  was used to determine the density profile. Density measurements were made every 0.02 mm at a measurement speed of 0.4 mm·sFormaldehyde content was determined by means of extraction in a perforator, according to EN 12460-5. Approximately 110 g of 25 mm × 25 mm specimens were placed for 2 h in boiling toluene (600 mL). The refluxing toluene was bubbled through distilled water to extract any dissolved formaldehyde. The formaldehyde content of aqueous solution was determined photometrically and was expressed as milligrams of free formaldehyde per 100 g of dry board.® for Windows . Differences were regarded as significant at a level of 95% probability. Least significant differences (LSD) were calculated according to the Tukey test.All calculations were performed using STATISTICA 12 Biomass yields of C4 grass species increased from the second year after establishment and reached their peak after 4 years . The est4 and C3 grass biomass. In the case of the aforementioned traits as well as TS and WA, significant differences were also identified between grass species within the C3 and C4 groups.The statistical analysis showed that in the case of MOR, MOE, and IB there were significant differences between particleboards made with the addition of C4 grass species (13.7 MPa) differed significantly from the value obtained for C3 grass species (10.9 MPa). The results indicate that the addition of selected alternative lignocellulosic raw materials to particleboards may reduce the MOR and MOE values, except in the case of the board with the addition of miscanthus particles. The SP, LP, and FA boards do not meet the minimum bending strength requirements for P2 boards  according to EN 312.Based on the EN 312 standard, the minimum MOR and MOE values of particleboards for interior fitments including furniture are 11 MPa and 1600 MPa, respectively. For the SP, LP, and FA boards, these parameters are lower than for the other boards but the obtained values are above the minimum requirements.The lower MOR and MOE values may be due to the smaller specific surface area and less slender particles of the alternative raw materials added to the particleboards. Consequently, the SP, LP, and FA boards exhibited lower strength after the addition of 30% particles from alternative raw materials. The particle geometry used in particleboard production has a much greater influence on the board properties than the mechanical properties of the particles. The strength and stiffness of particleboards are affected by the quality of the bonds between the particles, which in turn are significantly influenced by their dimensions. In addition to differences in the size and shape of particles, the parameters of the board are also affected by their moisture content . The siz3 and C4 groups. Significant differences were also observed between the mean IB values of the C3 and C4 boards. The highest IB value for particleboards with added alternative particles was observed for MG board (0.56 MPa) and the lowest for FA board (0.28 MPa). The addition of 30% alternative particles reduced the IB value compared with the control board. The SP and FA boards did not reach the minimum internal bond strength value for boards for interior fitments including furniture, which should be at least 0.35 MPa according to EN 312.The obtained internal bond strength (IB) values are shown in The lower IB values for boards with the addition of alternative raw materials may be due to the larger particle surface area per unit mass compared with wood particles, which means that less adhesive is deposited on the surfaces of the particles from alternative raw materials . It is pIn the current literature, different studies presented trials to increase the mechanical properties of particleboards from alternative biomasses , for insComparing the obtained data with current literature, it is possible to notice that the presented value of IB for miscanthus-based particleboards were almost 45% higher than the values found by Klímek et al. (2018) . However3 and C4 indicate that the boards differ significantly in terms of the tested properties. Based on statistical analysis of the mean values of TS and WA for boards from the C3 and C4 groups, it was found that there were also significant differences between the groups. For particleboards with added alternative particles, the lowest TS value was observed for MG board (21.5%) and the lowest WA for PV board (72.1%). The highest values of TS (29.6%) and WA (92.1%) were recorded for FA board. The EN 312 standard does not define a maximum value of swelling in thickness after soaking in water for boards for interior fitments including furniture.The higher thickness swelling and water absorption of boards with the addition of alternative lignocellulosic raw materials may be caused by the presence of parenchyma cells in the pith of these plants. The high hygroscopicity of the parenchyma and its spongy structure compared with other cells means that it has high capacity for water absorption and storage in the tissue cell .3 vs. C4)  . This suThe density profiles of the tested boards  were fou−1 oven-dry board, respectively), being comparable to the value for the control board (5.1 mg·100g−1 over-dry board). The highest concentrations of formaldehyde were obtained for cordgrass and switchgrass . The mean value for C4 grass species (6.7 mg·100g−1 over-dry board) was significantly higher than the value for C3 grass species (5.3 mg·100g−1 over-dry board). It is worth highlighting that only the values for cordgrass were higher than the threshold for the E1 class according to EN 312.Mean values of formaldehyde content were significantly different between the tested grass species and between the groups of species . The lowThe formaldehyde issue should be further investigated in future research analyzing more samples, considering that there is no sufficient literature data of formaldehyde content of particleboards produced starting from biomass of perennial grass species available.There are multiple factors that can affect the formaldehyde content of particleboards. Among these, it is possible to list pressing parameters, type and amount of the hardener, feedstock, type of resin, and molar ratio , as well as the content of free formaldehyde in the resin . HoweverThe presented results confirmed the hypothesis that perennial grasses biomass can be a relevant feedstock for particleboards production. However, some drawbacks are still evident. Poorer wettability and reduced mechanical properties in comparison to control panel were highlighted, confirming what is reported in the current literature . One of Therefore, the future directions related to application of alternative lignocellulosic raw materials including perennial grasses to the production of particleboards should be focused on optimization of manufacturing parameters taking into account technical possibilities of the wood-based panels companies.Furthermore, agronomic studies are needed to further test and implement the possibility of cultivating such species in marginal conditions, thus avoiding the competition with food and feed crops.In the framework of looking for alternative feedstocks for particleboards’ production, the present study aimed to substitute 30% of wood particles with six perennial grass biomasses in particleboard production.4 photosynthetic pathway; and tall wheatgrass, tall fescue, and perennial ryegrass, being cool-season grasses with the C3 photosynthetic pathway, were tested.The following species: miscanthus, switch prairie, and cordgrass, being warm-season grasses with the CThis research represents the first assessment of comparison between the quality of the particleboards of perennial grass species of different photosynthetic pathways.4 grasses particleboards achieved sufficient mechanical properties, even if the formaldehyde content was higher than that of the control panel. On the other hand, particleboards made with C3 grasses exhibited not as high mechanical properties, but demonstrated lower formaldehyde content. The obtained results demonstrated that it is possible to obtain particleboards partially based on perennial grasses biomass that meet the standard requirements for boards’ quality.As demonstrated, it is possible to state that CFuture studies should be addressed to improve the overall quality of particleboards produced with perennial grasses biomass, including the application of alternative adhesives or pre-treatments, as well as to optimize the manufacturing parameters for the production of particleboards from perennial grasses biomass. Moreover, further agronomic studies regarding the cultivation of these grass species are needed to enable successful growing in marginal land conditions, in order to avoid competition with food crops, which is currently the main obstacle to the cultivation of many industrial crops.


In conclusion, NKA increased in the subjects with recurrent respiratory tract infections with PMBL treatment.Polyvalent mechanical bacterial lysate (PMBL) is used for the treatment and prevention of recurrent respiratory tract infections. Although PMBL is an immunostimulant, it remains unknown whether treatment with PMBL influences natural killer cell activity (NKA). Hence, this case-control study compared the changes in IFN-γ levels (surrogate index for NKA) following PMBL treatment or time passing between the PMBL-treated group and controls. The treatment group included adults who had a PMBL prescription for three months against recurrent respiratory tract infection from an outpatient clinic, while the control group had healthy adults visiting the health promotion center for periodic health check-ups. The control group (N = 506) showed no change in IFN-γ levels, while the treatment group (N = 301) showed a significant increase in mean from 462.8 to 749.3 pg/mL after PMBL treatment. In the subgroup with IFN-γ <500 pg/mL, IFN-γ levels significantly increased in both groups. However, the change in the treatment group (287 ± 822 pg/mL) was greater than that in the control group (58 ± 809 pg/mL), and the interaction between the visit and case/control was significant ( Natural killer (NK) cells play crucial roles in innate immunity; they recognize and eliminate virus-infected and neoplastic cells . MoreovePolyvalent mechanical bacterial lysate (PMBL) is prepared by the mechanical lysis of various types of bacteria . The lysIt remains unknown whether the efficacy of PMBL is associated with a change in NK cell function. Studies have shown activation in NKA in both animals ,15 and hThis case-control study was conducted based on the data between 2016 and 2020 from the Chaum Life Center . The treatment group included individuals treated in outpatient clinics. The inclusion criteria were as follows: (1) patients with recurrent respiratory tract infection under PMBL treatment for three months following the recommended protocol of 10 days of sublingual administration and 20 days of drug-free interval, and (2) those who have undergone NKA assays before and after PMBL treatment. A total of 311 patients were included in the treatment group according to these criteria. The control subjects were from the health promotion center belonging to the Chaum Life Center. The inclusion criteria for the control group were as follows: (1) people who have undergone NKA assays in the health promotion center periodically at 1-year intervals, and (2) those who were not treated with PMBL between the visits. The control group consisted of 549 people according to these criteria. The endpoint was the change in IFN-γ levels, a surrogate index for NKA, from baseline to follow-up.Those who had a history of malignancies or suspicious findings for any cancer were excluded (N = 16). We also excluded those with autoimmune disorders (N = 4), active allergic disease under treatment (N = 5), acute or chronic infectious disease (N = 7), and recent medication with antibiotics, immunosuppressants, or herbal medicines (N = 21). Finally, 301 patients and 506 controls were included in the analyses for the study .The study was conducted following the guidelines of Helsinki’s Declaration and was approved by the Institutional Review Board of CHA Bundang Medical Center (2021-04-034).®  was prescribed to the treatment group as a PMBL. Ismigen® was prepared from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus viridans, Klebsiella ozaenae, Haemophilus influenzae serotype B, Moraxella catarrhalis, and Streptococcus pneumoniae by mechanical lysis. The protocol involved the sublingual administration of one tablet of Ismigen® per day for 10 days, followed by a drug-free interval of 20 days for three consecutive months.IsmigenThe medical histories of the subjects were obtained. Height and weight were measured in a standing position without shoes and were recorded to the first decimal point in centimeters and kilograms, respectively. The body mass index (BMI) was defined as body weight in kilograms divided by the height squared in meters.The blood samples were obtained in the morning after the patient had fasted overnight for at least 8 h and were derived from the antecubital area. The serum samples were stored at 4 °C and were analyzed within a day of sampling. The neutrophil and lymphocyte counts were estimated as a proportion of the number of white blood cells (WBC). The neutrophil-lymphocyte ratio (NLR) was defined as the neutrophil count divided by the lymphocyte count.® Kit, NKMAX, Seongnam, Korea). Using a direct vacutainer system, a 1 mL sample of whole blood was directly transferred into a specific tube containing a patented stimulatory cytokine  for the tests. The collection tube was gently and repeatedly mixed, and within 30 min of collection, the tube was incubated for 20–24 h in a 37 °C chamber, following the manufacturer’s instructions. During the incubation period, the stimulatory cytokine caused the secretion of IFN-γ into the plasma; this secretion of IFN-γ predominantly occured through NK cells rather than other innate or adaptive immune cells [g for 3 min. The supernatant was then loaded onto enzyme-linked immunosorbent assay (ELISA) plates. The IFN-γ levels were measured using a designed ELISA, reported by the units of pg/mL. This study used the reference ranges given by the test-kit manufacturer; IFN-γ ≥ 500 pg/mL as normal and IFN-γ < 500 pg/mL as low [IFN-γ was measured with a recently developed blood test .The general features were expressed as mean ± SD or number (proportion). The differences between the treatment and control groups were compared using an independent sample To confirm the factors influencing NKA levels, linear regression models with a stepwise selection method were used on all the subjects at baseline.t-tests in the treatment and control groups. To compare the changes in NKA between the treatment and control groups, generalized estimating equation (GEE) models were formulated. In the GEE models, the difference between the treatment and control groups, between the baseline and follow-up visit, and their interactions were considered independent variables. Other factors, such as age, sex, medical history, and baseline laboratory data, were regarded as covariates, and significant variables were selected and included in the models. These statistical methods were repeatedly applied in the subgroups.The changes in NKA values from baseline to the follow-up were compared using paired p ≤ 0.05 were considered statistically significant.All of the statistical analyses were conducted using the SPSS statistical package, version 26 . The results with The baseline features of the subjects are indicated in Various factors such as age, sex, history, BMI, glucose, and complete blood count (CBC) parameters were considered covariates in the linear regression models . The steIn the treatment group, the mean IFN-γ levels significantly increased from 462.8 to 749.3 pg/mL . In the p = 0.003), and the interaction between visit and case/control was also significant (p < 0.001). This is in agreement with the results of the linear regression model, where age and NLR were also shown to be significant factors.To compare the changes in IFN-γ levels between the treatment and control groups, we created a GEE model that included variables of visit, case/control, their interaction, and covariates . In the p = 0.030). In this model, the other covariates were not significant.In the subgroup of low NKA, both the treatment and control groups showed a significant increase in IFN-γ levels from baseline to follow-up . The meaThe case-control study showed that the IFN-γ levels increased in the treatment group but not in the control group. In the subgroup with low NKA (<500 pg/mL IFN-γ), the mean increment was significantly higher in the treatment group than in the control group. Compared to the control group, the PMBL treatment group with recurrent respiratory tract infection showed a greater increase in IFN-γ levels, a surrogate index for NKA.Since PMBL is prepared from various pathogens, PMBL is involved in acquired immunity ,12. AddiInfections may induce NKA augmentation. Exposure to influenza virus-infected cells has been shown to increase NKA . CytokinOur previous report has shown that NLR is strongly associated with IFN-γ levels . In thisOur study has several limitations. First, since the study employed a case-control design, the findings are not confirmative. Moreover, the mechanism of the effect of PMBL remained unclear. Further studies including RCTs are required to find the effect of PMBL on NKA, and a basic investigation to unfold the molecular mechanism should be undertaken. Second, we do not have enough data on the long-term prevention of respiratory infection and adverse events. Further clinical information should be warranted in the next studies. Third, the control group did not include subjects with recurrent respiratory tract infections. The control group included individuals who had routine health check-ups and NKA examinations repeated in a year, which is different from the treatment group with three-month-interval assays. However, in the subjects with low NKA, the baseline IFN-γ levels were not significantly different between the groups. The analysis for the subgroup attenuated the heterogeneity between the groups. Fourth, NKA was measured using only IFN-γ assays. NKA is routinely measured by perforin and granzyme B production, degranulation, or in a direct cytotoxicity assay against K562 cells . AlthougPMBL treatment may increase IFN-γ levels, a surrogate index for NKA, in subjects with recurrent respiratory tract infections. It may suggest the influence of PMBL on innate immunity in the community setting. Further RCTs are required to find the effect of PMBL on the immunologic changes in the homogenous population.