Halichoerus grypus) population in the Baltic Sea has created conflicts with local fisheries, comparable to similar emerging problems worldwide. Adequate information on the foraging habits is a requirement for responsible management of the seal population. We investigated the applicability of available dietary assessment methods by comparing morphological analysis and DNA metabarcoding of gut contents , and tissue chemical markers i.e. fatty acid (FA) profiles of blubber and stable isotopes (SIs) of liver and muscle . The methods provided complementary information. Short-term methods indicated prey species and revealed dietary differences between age groups and areas but for limited time period. In the central Baltic, herring was the main prey, while in the Gulf of Finland percid and cyprinid species together comprised the largest part of the diet. Perch was also an important prey in the western Baltic Proper. The DNA analysis provided firm identification of many prey species, which were neglected or identified only at species group level by morphological analysis. Liver SIs distinguished spatial foraging patterns and identified potentially migrated individuals, whereas blubber FAs distinguished individuals frequently utilizing certain types of prey. Tissue chemical markers of adult males suggested specialized feeding to certain areas and prey, which suggest that these individuals are especially prone to cause economic losses for fisheries. We recommend combined analyses of gut contents and tissue chemical markers as dietary monitoring methodology of aquatic top predators to support an optimal ecosystem-based management.The growing grey seal ( Halichoerus grypus) population has recovered from the low numbers in the 1980s, caused by extensive hunting and environmental toxins, to about 30 000 counted animals [Increasing seal populations worldwide have created resource competition and conflicts between the seals and local commercial fisheries, leading to culling programs with uncertain benefits ,2. Thus, animals . Consequ animals  but also animals . Selecti animals ,7.Clupea harengus), is the most important prey, followed by cod (Gadus morhua) and sprat (Sprattus sprattus) in the Baltic Proper, and common whitefish (Coregonus lavaretus) and vendace  in the Gulf of Bothnia. However, it is not known how representative this information is. The traditional diet estimation method, based on morphological identification of the prey remains in the gut, only represents the most recent diet, and might be biased towards prey with long-retained hard parts (HP). Currently, the HP analysis could be complemented with DNA analysis of the gut contents which may reduce bias caused by digestive erosion [Targeted hunt of problem seals is feasible if individual preferences to certain feeding areas and prey species exist. Baltic grey seals, as a population, have been considered opportunistic predators, an interpretation based on analysis of gut contents –10. Acco erosion  and reve erosion . ICES ge erosion . Since p erosion , the sea erosion , althoug erosion .e.g. δ13C, δ15N or δ34S) and light element isotopes in prey leads to predictable changes in the stable isotope (SI) values in predator tissues and different tissues can provide SI-based dietary information in different time scales, e.g. weeks for liver samples and months for muscle samples [Recent studies have suggested that individual grey seals, instead of being opportunistic, have specialized feeding areas and behaviours ,15,16. A samples ,24. To bBy using data from a variety of methods it is possible to get estimates on short-, mid- and long-term diets of individual seals. The first aim of the study was to compare the short-term diet estimates obtained from HP and DNA analysis of grey seal gut contents, and to investigate the complementarity of these two methods. Second, we compared the power of tissue FA and SI profiles in assessing mid- and long-term feeding habits and examined whether these methods are able to reveal individual, or age- and sex-group related specialization. We hypothesized that the results from HP and DNA analyses would differ from each other. Further, we hypothesized that significantly different chemical marker profiles refer to individuals specialized in a certain foraging area and/or diet, whereas similar marker profiles would mean no preferential use of habitat or prey. Growing seal populations may adopt new foraging areas and resources, and adequate information on the spatial and temporal dietary variability clarifies the ecological role of marine mammals and may offer means for mitigating conflicts between seals and fisheries. In addition, dietary shifts and tissue chemistry of top predators are integrated proxies of food web changes, thus indicating the dynamics and health of the ecosystem ,26.http://www.swedishepa.se) and the Agency for Marine and Water Management (http://www.havochvatten.se) and carried out by the University of Agricultural Sciences (http://www.slu.se) and Museum of Natural History (http://www.nrm.se); in Finland conducted by the Natural Resources Institute (http://www.luke.fi). The samples were collected during 2011 and 2012 and covered the ICES SDs 27, 29, 30 and 32 of the Baltic Sea , sea trout  and common white fish, whereas the bottom fykes were placed at the bottom, had various traditional structures, and were meant to catch perch (Perca fluviatilis), pikeperch (Sander lucioperca), European eel (Anguilla Anguilla) and cyprinids. The gear type information was used in this study to define the specific habitat of the area where the seal was collected. Since 5 years is the most likely age of the first birth of the grey seal [Blubber, muscle, liver (n = 108 for each) and gut samples  from grey seals  were collected during 2011 and 2012. The SD29 and 30 include pelagic and coastal areas, and the west and east coast ecosystems could provide the seals with different diet having distinct chemical markers. However, all the individuals from SD29 were collected in the archipelago between Åland Islands and Turku, and thus formed an ecologically uniform sample. The seals collected in SD 30 were mainly from the west coast  but specimens of the east coast  were included as well, and thus the seals were subgrouped into western SD30 and eastern SD30. Seal sex and age  were rerey seal , the 0–4Esox lucius and perch) and demersal  fish was demonstrated. European eel, being a migrating species was not categorized into any aforementioned habitat.Whole fish were stored in freezer (-25°C) before homogenized and sampled in the laboratory for the 4 types of analyses. The fish tissue library created consisted of 26 species but the profound species-level analyses of the whole data with regional comparisons remain out of the scope of this study and will be published separately. This full fish material of 433 individuals were at first used to address the chemical marker variability of the Baltic fishes, and subsequently the 11 most probable prey species (for FA n = 233 and for SI n = 216) were chosen for the comparative analyses of this study (Tables A-D in Saduria entomon specimens) ingested per individual seal were estimated by considering all prey HPs, known relationships between otolith size and fish size, and compensating for digestive erosion of otoliths [The morphological HP analysis followed the methodology described by Lundström et al. ,29. Brie5’-CGTGCRAAGGTAGCG-3’) and reverse primer 16sPreyR (5’-CCTYGGGCGCCCCAAC-3’) were designed by aligning and identifying variable sections of 16s sequences from various marine vertebrates present in the Baltic Sea, including seals and aquatic birds. The 3’ nucleotide of the forward primer mismatches the 16s sequence of seals, which inhibits the amplification of seal DNA, maximizing the prey DNA amplification.DNA was extracted individually for every stomach, intestine and colon content sample using a QIAmp DNA stool minikit  following the manufacturer’s “protocol for Human DNA”. An approximately 270 base-pair long fragment of the 16s rDNA gene (hereby 16s) was amplified by polymerase chain reaction (PCR) to be used as a “DNA barcoding” marker for prey species identification. PCR primers, forward primer 16sPreyF (Lampetra fluviatilis and Petromyzon marinus), all samples produced equally strong PCR products as visualized in agarose gels (data not shown). Eight forward and eight reverse primers were synthesized containing unique combinations of six nucleotides at the 5’ end. Such primers were used to produce 64 unique “barcode” identifier combinations to facilitate multiplexing of individuals in parallel sequencing and subsequent de-multiplexing of the output data, as described by [The primer pair was tested initially using reference DNA template from 47 different fish species and eight bird species from the Baltic Sea region. With the exception of Agnatha species (ribed by .Taq master mix (Qiagen), 1 μL of each PCR primer (10 μM concentration), and 2 μL or DNA extract. Cycling conditions included an initial 5 minute (min) denaturing step at 95°C; 40 cycles of denaturing at 94°C for 30 seconds (s), 54°C for 30 s and 68°C for 60 s; and ending with a final extension step of 72°C for 10 min.PCR reactions were carried out in volumes of 25 μL containing 12.5 μL HotStart PCR products were pooled in groups of 64 barcoded individuals. Pooled reactions were then used to construct DNA libraries for sequencing following the “Rapid library preparation method manual” for GS junior Titanium series  with the following modifications: the nebulization step was omitted, the RLdNTP, RL T4 polymerase and RL Taq polymerase were not included in the fragment end-repair reaction, and the small fragment removal was carried out by agarose-gel size selection and excision. Each of the pooled 64 individual reaction libraries was prepared using a different molecular identifier adapter (MID). DNA libraries were sequenced in two different runs in a GS-Junior instrument (Roche), following the emPCR amplification manual- Lib-L” and the “Sequencing method manual GS junior Titanium Series” protocols (Roche).https://github.com/nylander/454_tag_sorting). Comparisons were performed using the BLASTn algorithm and species identification from the output sequences was carried out using the BLAST+ program [The DNA sequence data output in FastA format and its respective quality scores were combined into a FastQ file using Galaxy . Sequenc program , with th program ,37.Blubber samples were consistently collected from above sternum. In addition, the accurate sampling location has been reported to have negligible influence on the FA composition of pinniped blubber . The blue.g. 20:5n-3). When studying the fish homogenates , 9 FAs  explained the most part of the interspecific variation and thus these were used as dietary markers for the seals. These FAs showed the largest relative standard deviations among the FAs present with levels not affected by methodological variation , and they also were responsible for the main part of the data variation in the Principal Component Analysis  using as loadings standardized mol% data of either the full 26 species or the selected 11 main prey species.The FA composition in the seal and fish tissue samples was analyzed by gas chromatography according to previously published procedures ,38 using13C, δ15N and δ34S analyses. A maximum of 0.6 mg of each sample was loaded into a 4x6 mm tin capsule and combusted in Elementar Vario Pyrocube elemental analyser  connected to Isoprime 100 CF-IRMS (Isoprime UK) mass spectrometer. Differences in the isotope values were measured relative to standards and expressed as per mil (‰) deviation from Vienna PeeDee belemnite (VPDB) for carbon, from atmospheric N2 (AIR) for nitrogen, and Vienna Canon Diablo Meteorite Troilite (V-CDT) for sulphur [13C, δ15N, δ34S (‰) = (R sample / R standard– 1) × 103, where R sample is the ratio between the heavy isotope and its lighter counterpart for the sample, and R standard is the ratio for the international standard [Seal muscle and liver samples, and the reference fish homogenates were frozen, freeze-dried and powdered for δ sulphur ,40. Morestandard .e.g. CH6, N2 and sphalerite NBS 123) provided by the International Atomic Energy Agency . FSS has known values of δ13Cstd = -26.39 ‰, δ15Nstd = 13.08 ‰ and δ34Sstd = 12.45 ‰ and was used as working standard to examine isotopic drift within and throughout the run. Elemental analysis standard reference material, sulfanilamide  was used to correct the % C, % N and % S data. As lipids are known to be 13C-depleted (having lower δ13C relative to other major tissue constituents as proteins) [13C) were ‘lipid normalized’ for both seal tissues and all fish samples using the C/N ratio according to [For standard reference materials pike muscle (FSS) standard was used as the internal laboratory standard, calibrated against isotopic standards (roteins) ,43, carbrding to .13C, δ15N and δ34S values from seal liver reflect the dietary elements 2–3 weeks prior to sampling, and the isotope values from muscle tissue should represent the elements assimilated up to 2–3 months prior to sampling.Information on the turnover rates of SIs and FAs in the studied seal tissues  ,45,46 suIn mammals, clearance of circulating chylomicrons and absorption of FAs begins in min scale and the lipids not immediately needed for energy metabolism are stored in the adipose tissue ,52. Thisarcsine (of the square root) transformed to improve data normality, and all FA and SI variables were standardized to prevent large components from dominating the analysis. Since systematic small differences in the relative concentrations of diet-reflecting small components of the FA profile may carry equally important dietary information as the differences in large components [P < 0.05 significant. SIMCA is a supervised classification method building multiple PCA-based class models, and as a “soft” method it can classify a sample into several overlapping classes. SIMCA uses F-test to evaluate the sample Euclidean distances from the models, and it is regarded as a robust method, which can be applied to data having non-normal distribution, although it performs ideally with data having normal distribution or transformed for better normality [FA and SI data were subjected to multivariate PCA  to assess compositional differences between the samples and highlight the marker FAs and SIs mainly responsible for the variation in the data. Prior to the analysis, FA data were mponents , the stamponents ,56 and rormality . HoweverGasterosteus aculeatus and cod to the diet compared to HP analysis . DNA metabarcoding also detected the presence of important dietary species not identified by HP analysis: bream Abramis brama (6.0%) and burbot Lota lota (3.4%), and other 5 species with a DNA sequence proportion < 1% . The isopod cructacean Saduria entomon was detected by HP analysis (2.9%) but not by DNA analysis.In the diet of subadult males, morphological HP analysis (n = 37) and DNA metabarcoding (n = 42) identified similar number of prey taxa  . Both meLeuciscus sp. and 0.2% for white bream. In addition, HP analysis reported a mass proportion of 9.2% for common whitefish while 13.2% of the DNA sequences belonged to Coregonus species, i.e. common whitefish or vendace. The contribution of Atlantic salmon, pikeperch, sprat, sea trout and ruffe Gymnocephalus cernua differed markedly between the methods with larger proportions indicated by the DNA analysis . In the adult males, turbot Scophthalmus maximus, four-horned sculpin Myoxocephalus quadricornis, common dab Limanda limanda, tench Tinca tinca, black goby and the benthic isopod Saduria entomon were only detected by the morphological analysis (listed in the order of descending proportion).In the diet of adult males, morphological analysis (n = 51) distinguished higher number of prey taxa than the DNA analysis (n = 44) (23 species + 6 taxa vs 18 species + 4 taxa) . Both mei.e. sculpins were also only detected in the DNA analysis but with DNA sequence proportions < 1%.The HP analysis of female subadults (n = 26) distinguished a slightly lower number of prey taxa (11 species + 4 taxa) than DNA analysis  . Both meCoregonus spp. (12.8 vs 7.6%) as other major items. The small contributions of roach, sprat, bream, pikeperch, flounder/plaice Platichthys flesus/Pleuronectes platessa and sculpins were only detected by the DNA analysis.Also in the adult females, there were variability in the taxa identified by the HP  and DNA analysis  but still the total number of taxa was the same . Both meThe short-term diets indicated by the HP and DNA analyses also differed between the Baltic ICES SDs  Tables  and 4. IIn subadult males , herringIn adult males , herring15N and δ13C.The FAs and SIs of 11 key prey species representing pelagic, coastal or demersal habitats were analysed by PCA and the mean compositions in each area were plotted for each species . The firResults from inter- and intraspecies comparisons by PCA and SIMCA using the FA and SI data  in each ICES area are presented as supporting information  showed no significances in any comparisons between ICES areas. However, if an age group is studied separately, statistically significant separations can be found. When the subadult males collected from bottom fykes and shot in open water area , the individuals grouped according to ICES area . The adult males are addressed below but the low sample numbers prevented similar comparisons with the females.At first, the data of all grey seal individuals (n = 108) studied for tissue chemical markers were subjected to PCA and possible differences were examined in their inner blubber FAs , liver Sy groups . If the Fig A in . Within To compare the separation power of the FAs and SIs, data from seals with detailed information about sex, age, area and type of fishing gear (defines the recent feeding habitat) were subjected to PCA. Such strictly defined groups could only be formed of adult males from the Finnish coast caught from trawls or different types of fykes . Thus, three groups were formed from these males that were from the same area and had been caught there with the same type of gear .34S) versus coastal fyke groups  when using muscle SIs as variables. When using Liver SIs, PCA followed by SIMCA showed no statistically significant differences. The individuals that were bycaught in the pelagic surface or coastal bottom fykes (S vs B) did not differ from each other in any of the comparisons by FAs and SIs, alone or combined. Further comparisons on the separation power of FAs and SIs using a larger set of adult male individuals are presented in Fig B in The segregation of these groups of adult males were examined by using inner blubber FAs  and liveFor consistency, all the multivariate comparisons were performed by using metric PCA and SIMCA. To ensure that the results of the comparisons using the limited data of 11 adult males were not biased, these PCA and SIMCA comparisons  were repIn the current study, the power of different methods to determine marine mammal diet or feeding ecology was compared. Studies using several complementary methods, allowing to confirm the results of one dietary proxy with another, have not been conducted on any seal species in the Baltic Sea previously, and there are no studies applying all the proxies included in this work from any other marine mammal population either. Hence, in the absence of reference studies, our study is unique. The first published surveys of Baltic grey seal diet used gut prey remains and were based on material from the 1960-70s ,59. LundMorphological identification of undigested prey remains relies on expertise and reference collections but enables estimates of ingested prey sizes and biomass. DNA metabarcoding of gut contents provides more exact prey identification but does not provide information about prey size, and experience in converting DNA sequence proportions to biomass does not yet exist. Thus, the estimated mass proportions from the HP analyses and the DNA sequence proportions are not fully comparable to each other. Despite these limitations, the DNA metabarcoding clearly demonstrated prey taxa that are underestimated by the HP analyses.Salmo species were found to be underrepresented by the HP analysis in male seals  [Regardless of accurate prey identification, no short-term method reveals the integrated average prey of a free-ranging marine mammal, which may migrate and thus at different times exploit different habitats and prey. Attempting to attain data of temporal representativeness from short-term gut samples would require frequently repeated hunt. Diet assessment using FAs and SIs offer long-term dietary estimates but with the drawback of failing to reach firm prey species identification. Baltic grey seals have so far been studied little for tissue FAs ,67, and othnica) .15N values and in this work also the δ13C values, analysed by using delipidated samples [Successful food web studies require a representative reference library of prey FAs and SIs and that the prey species have characteristically different chemical markers. Used together, FAs and SIs are complementary since they are proxies of different dietary components. While the FAs are derived from dietary lipids, the δ samples , represe samples ,18 but t samples . The hab samples ,72. The 15N and δ13C but high δ34S may come from freshwater input, sediments, precipitations and various anthropogenic sources and thus may offer information on foraging area [34S was enriched in all studied Salmo species. To our knowledge, the δ34S has not been used in the studies of Baltic food webs before but should become a frequently analyzed element since aquatic primary producers have been reported to have large variation of δ34S values, and their level of trophic‐step fractionation is low, which makes the element useful in revealing food web relations [Salmo species spent in river or their estuarine dietary sources, with sulphur supply different from the pelagic sea areas, may have influence on this value  by separating the predators salmon and trout  from the planktivorous herring and sprat  during a couple of months’ time [Provided that no prior subgrouping of the seal individuals was made to diminish biological variation, the only chemical tissue marker that grouped the seals according to the studied parameters ) was the liver SIs (with the time window of weeks and reflecting dietary protein component), which indicated that the individuals from SD27 and western SD30 had similar signatures differing from those in the SD29 and SD32 samples. The SD29 seals were all from the area between Åland Islands and Turku. This west-east pattern was broken by 7 seals (6.5%) caught on the west coast of SD27 and SD30 but with liver SI signatures similar to most of the individuals caught in the east, which suggests westward migration. These possibly migrated individuals were of different age and sex, and had blubber FA compositions similar to those of the other SD27 and SD30 western individuals. This leaves similar foraging area in the past, better indicated by SIs than FAs ,75, as ahs’ time .e.g. herring, are consumed by both young and adult seal individuals. The tissue chemical markers, however, revealed individual long-term specialization for a certain habitat and type of prey for the adult males, and the DNA analyses, especially, confirmed that Atlantic salmon was included in their diet. These findings imply that the adult males are the most likely individuals to cause local and sustained loss of catch of the most valuable fish, and gear damage.In the case of blubber FAs, the lack of distinct sample groups in the PCA of all 108 individuals made it difficult to recognize the dietary origin of the variation in blubber FA profiles. However, reducing biological variation among the individuals studied may help in relating tissue FA profile differences to feeding area and diet, and indeed among the subadult males the inner blubber FA and liver SI profiles grouped individuals according to the SD area they were collected in Fig A in . When st34S and blubber 18:4n-3 in the adult male seals bycaught in trawls. Thus, by this first use of the δ34S to study foraging ecology of the seals we could define δ34S as a marker of consuming SD30 pelagic fish. The fact that both mid-term and long-term markers separated the seal individuals in similar ways suggests individual long-term specialization for a certain type of prey and habitat.Available information on the rates of absorption and turnover of FAs and SIs in seal tissues ,21,75 suAnalysis of gut contents was required to identify prey species, and both morphological analysis of prey hard parts and DNA metabarcoding showed clear dietary differences between age groups and areas. Concerning the foraging ecology of the seals, these proxies of the very recent diet cannot reveal potential specialization of individuals for certain feeding area or prey type. For this purpose the mid- and long-term markers can be used. The bycaught adult males formed distinctive groups having similar FA and SI markers, which resembled the marker patterns of the fish caught in the area by the gear type in which the seals were found , S2 Fig.This study suggests a combination of multiple diet estimation methods as the optimal protocol to assess as detailed information as possible about feeding habits of aquatic top predators. Efficient use of the dietary methods, however, sets high requirements for recording detailed background information on the studied individuals, which is a prerequisite for discovering dietary subgroups in large diverse data sets.S1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The rice arable system is of importance to both society and the environment. The emergence of rice paddies was a crucial step in the transition from pre-domestic cultivation to systematic land use and management. However, many aspects of the formation of rice farming systems remain unclear. An important reason is the lack of reliable methods for identifying early rice paddies. One possible means of remedying this knowledge deficit is through analysis of phytolith assemblages, which are closely related to their parent plant communities. In this study, phytolith assemblages from 27 surface soil samples from wild rice fields, 91 surface soil samples from modern rice paddies, and 50 soil samples from non-rice fields were analysed to establish a discriminant function. This discriminant function enabled classification of 89.3% of the samples into appropriate groups. Further, the results suggested that phytolith assemblages can be used to identify rice fields and differentiate between wild rice fields and domesticated rice fields. The method was demonstrated to be an effective way of utilising the large amounts of unidentifiable phytoliths discovered at archaeological sites to provide a modern analogue that may be a valuable key to unlocking the past. Eastern Asia, Southern Asia, and South-Eastern Asia account for almost 90% of rice production and 88% of the rice cultivation area2. The history of Asian societies, human populations and the evolutionary history of rice as a crop are inseparable3; rice production was a prerequisite for the rise of civilization in monsoon Asia. The earliest confirmed rice paddy cultivation began in the coastal wetlands of Eastern China about 7,700 cal yr B.P.4; however, phytolith evidence showed that rice domestication might have begun as early as 9,400 cal yr B.P. in the lower Yangtze Basin6. Exploring the transformation from pre-domestic cultivation to current systematic, large-scale land use and management is the key to understanding the formation of rice farming systems; however, when, where, and how rice paddies emerged remains unclear, largely because of the lack of reliable methods to identify early rice paddies.Rice paddies are crucial for global food security because rice is the staple food of nearly half the world population8, such as micromorphological features9, organic matter12 , forms of iron28, mineral characteristics29, bacterial communities34, soil fertility35, pollen features36, and phytolith accumulation37. Although these methods are effective for analysing the characteristics of paddy fields, they can only be applied to known paddy fields.Currently, the methods for studying ancient and present-day rice paddies mainly focus on the physical and chemical properties of paddy soil38. Phytoliths can be used to identify particular genera or species based on shape, size, and other anatomical features40. Rice bulliform phytoliths42 and double-peak phytoliths44, which differ between wild and domesticated rice, have been employed to study the origin and domestication of rice45. Fujiwara et al.46 established the paddy identification standard as 5000 rice bulliform phytoliths in 1 g of dried soil sample and this standard has been applied to identify Neolithic paddies in the Shandong and Zhejiang Provinces48. However, careful consideration is required when applying this standard because if the crop is harvested by uprooting or basal cutting, only a few bulliform phytoliths will remain in the field50.Phytoliths are siliceous bodies present in certain plant tissues; plants absorb silica in a soluble state from groundwater and accumulate solid silica in both intracellular and extracellular locations54 and the environment57. As rice arable systems are associated with specific grasses, weed assemblages61 can be used to determine changes in arable systems. Recently, phytolith assemblages, based on the proportion of fixed and sensitive types have been used to distinguish between wet and dry cultivation systems64; however, similar studies using phytolith assemblages to differentiate wild and domesticated rice paddies are rare.Notably, a rice system does not contain paddy soil alone; it is a relatively independent ecosystem including other plant species that might be of use for identification. Rice arable systems are of importance to both the societyIn the present study, a detailed discriminant analysis was conducted on modern soil samples Fig.  to develA total of 170 soil samples were collected and 168 of these contained abundant phytoliths. Twenty-one phytolith morphotypes were identified Fig. , includiAll surface soil samples from the wild rice fields were characterised by high rates of square (22.17%), bilobate (18.19%) and elongate psilate (17.62%) phytoliths. Furthermore, the proportion of bulliform phytoliths with ≥9 fish-scale decorations was 0.55%, whereas that for bulliform phytoliths with <9 fish-scale decorations was 1.55%. The average phytolith concentration in these samples was ~1.18 million particles/g; the highest phytolith concentration was 6.34 million particles/g (WN-BT8), whereas the lowest concentration was 5,200 particles/g (WN-BT6).Among 93 soil samples from domesticated rice paddies, no phytoliths were observed in samples DTZJ9 and DTZJ11. Phytolith assemblages from the other 91 domesticated rice paddy samples were characterised by fairly high rates of bilobate (30.54%), square (15.96%), and elongate psilate (15.90%) phytoliths. The proportion of bulliform phytoliths with ≥9 fish-scale decorations was 1.43%, whereas that for bulliform phytoliths with <9 fish-scale decorations was 1.14%. The average phytolith concentration in these samples was ~3.03 million particles/g. The highest concentration was 6.61 million particles/g (HL-OS1), whereas the lowest concentration was 15800 particles/g (DTZJ3).Phytolith assemblages from the 50 non-rice field samples were different from those of rice field samples in that high rates of square (22.14%), elongate psilate (13.67%), bulliform (12.78%) and bilobate (11.47%) phytoliths were present. In particular, four samples  in this group contained a low rate of rice bulliform phytolith particles. Although the four soil samples did not come from wild rice or domesticated rice fields, their sampling locations were extremely close to wild or domesticated rice fields. Therefore, it is possible that these samples contained rice bulliform phytoliths because of soil tillage or other soil-disturbing activities.We used the data on 21 phytolith types  of the original grouped cases were classified correctly  was observed in the soil samples from the domesticated rice paddies, whereas a higher rate of elongate psilate (a sensitive type) phytoliths was obtained from the wild rice fields. This probably indicates that wild rice habitats are swampier than those of domesticated rice. The proportion of rice bulliform phytoliths is informative, although it is relatively low. Rice bulliform phytoliths are unique to rice plants and the decorations on the edges of rice bulliform phytoliths can be used to differentiate wild rice from domesticated rice42. However, because of the limited number of samples from Southern China, the results were not compared with samples from Northern China, South Asia, and Southeast Asia, which are also major rice-producing areas.This study demonstrated that rice paddies could be distinguished by their phytolith assemblages. Soil samples from both the wild rice fields and domesticated rice paddies were characterised by high proportions of bilobate, square and elongate psilate phytoliths, whereas the non-rice field samples exhibited diverse phytolith assemblages dominated by the rondel type. Furthermore, according to phytolith grouping for fixed and sensitive typesThis study analysed the phytoliths present in 168 soil samples collected from wild and domesticated rice fields and non-rice fields to establish a discriminant function, and demonstrated that these phytolith assemblages correctly classified 89.3% of the samples. The results statistically support the development of a robust method for identifying rice paddies and distinguishing between wild and domesticated rice fields. The method was demonstrated to be an effective way of utilising the large numbers of unidentifiable phytoliths from archaeological sites and this modern analogue should provide a valuable key to unlocking the past.65). The geographic distribution of rice fields is 18.23°N–30.91°N and 109.5°E–121.33°E, so we chose non-rice fields samples located at 18.3°N–32°N and 106.6°E–122.6°E to ensure all studied samples came from similar climatic regions. Surface soils with a depth of 0–5 cm  were collected and mixed evenly before analysis. Leaves, roots, and plant litter were removed prior to sampling. Details of all the samples are presented in Supplementary Table In this study, a total of 170 soil samples were collected for phytolith analysis  to separate the phytoliths, which were subsequently mounted on a slide with Canada Balsam. The phytoliths were counted and identified under a Leica microscope at 400X magnification. More than 400 phytolith particles from grass were counted in each sample. A few woody phytoliths were identified in this study; however, because these can be highly influenced by local environment and climatic factors, they were excluded to minimize possible bias73. Identification of phytoliths was performed using previous studies as references74.Phytoliths were extracted from the soil samples according to the procedure described by Zhang76. These discriminant functions ensure maximum separation among priori sample groups and can also be used to classify new samples with unknown group memberships into one of the priori groups77. In this study, 168 samples were initially divided into three priori groups ; subsequently percentage data of 21 phytolith types were used to establish the discriminant functions and the samples were classified into predicted groups.Discriminant analysis was performed using SPSS version 22.0. It is a method of statistical inference for deriving linear combinations of variables, called discriminant functions, which are mutually independentDataset 1


Employee innovation is the cornerstone of the organization, and the motivation for employee innovative behavior largely depends on the leadership style of the leader. With the economic development of society, the traditional authoritative style of leadership can no longer adapt to the psychological characteristics of employees, who use new-era work concepts, techniques, and social rules . Inclusive leadership is based on the concept of “fully inclusive and equitable” in traditional Chinese culture, and it can adapt to the independent needs of new generation employees. At present, the research on the relationship between the traditional leadership style and employee innovative behavior is relatively extensive, but there is little research on the relationship between inclusive leadership style and employee innovative behavior, and this needs further exploration. This paper takes new generation employees as the sample and uses psychological capital as an intermediary variable to explore the influence of inclusive leadership style on the innovative behaviors of new generation employees. We found that inclusive leadership is significantly and positively related to new generation employees’ innovative behavior. Theoretical and practical implications are discussed. Innovation drives enterprise development, and companies are relying more and more on their employees’ innovative contributions to maintain and improve their competitiveness . “New geThe effects of certain leadership styles may have different consequences for different generations in the workplace . To dateFurthermore, new generation working styles require new governance approaches, which depart from concrete, tangible, pre-defined rules to more diverse, flexible, and intangible motivators . For exaInclusion is written into the UN Millennium Development Goals and is a historical feature of Chinese civilization . InclusiThe inclusive leadership style was initially studied in the field of Western education. People of different races and abilities should be educated inclusively. Ryan believed that inclusive leadership in education requires an equal collective leadership process and defined inclusive leadership in education as the presence of a learning leader . For theChinese scholars started late in the study of inclusive leadership, but many explorations are still ongoing. Based on the literature about inclusive leadership, this paper integrated the concept of “inclusiveness” from Chinese traditional culture into that of inclusive leadership. The concept of inclusiveness in the West was mainly derived from the ideas of democracy and justice. In Chinese culture, “inclusiveness” is more about the “tolerance and greatness” of the mind and moral cultivation. The inclusive leadership that integrates Chinese traditional culture emphasizes equal opportunity and fair distribution, in line with higher psychological pursuits and the respected needs of the new generation employees, is a new type of democratic leadership. Inclusive leaders are able to treat employees with recognition, respect, and tolerance, listen to and recognize the opinions and contributions of subordinates , and proIn the design of their questionnaire scale, Based on the previous literature and empirical research, we introduced the concepts of recognition, encouragement, and inclusiveness into the leadership practices of leaders in the new era. (1) Leaders should listen to the opinions of employees, attach importance to encouragement of employees, and show their recognition when employees make achievements; (2) Leaders should respect and treat employees fairly. That is, the leaders can treat employees fairly, justly respect the employees’ suggestions, and let employees work more to receive more; (3) Leaders should rationally understand employees and tolerate their failures. That is, when employees make mistakes, leaders can rationally tolerate and understand them.The concept of innovative behavior began in the 1970s. Innovative behaviors consist of three levels: organizational, team, and individual innovative behavior. This paper studies the individual innovative behavior of enterprise employees. Scholars in China has also conducted research on employee innovative behavior. New generation employees are more active in their work and have stronger willingness and ability to learn at work than previous generations . New genCultivating innovative behavior is one of the most important leadership functions of today’s organizations . EmployeLike those positive leadership styles, inclusive leadership also has beneficial effects from the Chinese cultural perspective. Employees are more innovative when working at a higher level of engagement because they think their efforts have won the leaders’ accolades . EmployeFang  put forwHypothesis 1: Inclusive leadership has a positive impact on new generation employees’ innovative behavior in China.Hypothesis 1.1: Inclusive leadership style has a positive impact on new generation employees’ innovation outcomes.Hypothesis 1.1.1: Leaders’ encouragement and recognition of new generation employees have a positive impact on their innovation outcomes;Hypothesis 1.1.2: Leaders’ respect and fair treatment of new generation employees have a positive impact on their innovation outcomes;Hypothesis 1.1.3: Leaders’ rational understanding and tolerance of new generation employees’ failures have a positive impact on their innovation outcomes.Hypothesis 1.2: Inclusive leadership has a positive impact on new generation employees’ innovative thinking.Hypothesis 1.2.1: Leaders’ encouragement and recognition of new generation employees have a positive impact on their innovative thinking;Hypothesis 1.2.2: Leaders’ respect and fair treatment of new generation employees have a positive impact on their innovative thinking;Hypothesis 1.2.3: Leaders’ rational understanding and tolerance of new generation employees’ failures have a positive impact on their innovative thinking.Psychological capital reflects an optimistic attitude toward work and life . The conThe discussion of psychological capital can be divided into two categories: those based on economics and psychology or organizational behavior. The concept of psychological capital based on economics emphasizes the relatively stable psychological tendencies or characteristics that individuals develop in their early years of life , 1998. TInclusive leadership positively impacts employee self-efficacy . When leHypothesis 2: Inclusive leadership has a significant impact on employees’ psychological capital.As an important psychological resource of organizational collectives, psychological capital could play a mediating role that transforms organizational-wide force/interventions  and organizational consequences. Psychological capital plays mediating roles between organizational innovation atmosphere and employees’ innovative behavior  and betwHypothesis 3: Psychological capital plays a mediating role between inclusive leadership and employees’ innovative behavior.Hypothesis 3.1: Psychological capital plays a mediating role between inclusive leadership and employees’ innovation outcomes.Hypothesis 3.1.1: Psychological capital plays a mediating role between leaders’ encouragement and recognition of employees and employees’ innovation outcomes.Hypothesis 3.1.2: Psychological capital plays a mediating role between leaders’ respect and fair treatment of employees and employees’ innovation outcomes.Hypothesis 3.1.3: Psychological capital plays a mediating role between leaders’ rational understanding and tolerance of employees’ failures and employees’ innovation outcomes.Hypothesis 3.2: Psychological capital plays a mediating role between inclusive leadership and employees’ innovative thinking.Hypothesis 3.2.1: Psychological capital plays a mediating role between leaders’ encouragement and recognition of employees and employees’ innovative thinking.Hypothesis 3.2.2: Psychological capital plays a mediating role between leaders’ respect and fair treatment of employees and employees’ innovative thinking.Hypothesis 3.2.3: Psychological capital plays a mediating role between leaders’ rational understanding and tolerance of employees’ failures and employees’ innovative thinking.nearly 60% had earned an educational degree of bachelor’s or above, and over 20% of them used continuing education as a major tool to update work concepts and skills. In terms of job positions, over 20% of them were working as experts/professionals, and over 50% were managers. Both of those job types were characterized by high-level and fast-changing knowledge bases, and those sampled employees were therefore required to update themselves to fit new work concepts, models, and trends.We adopted a random questionnaire survey method, taking enterprise employees of Zhejiang as the research sample. We sent out a total of 372 questionnaires, and 351 valid ones were returned, resulting in a return rate of 94.35%. Among these people, 177 male and 174 female workers were questioned: 43.59% were aged less than 30 years; 19.37% were aged 30–39 years; 27.64% were aged 40–49 years; 7.69% were aged 50–59 years; and 1.71% were aged over 60 years. Most employees had a bachelor’s degree or above: 57.55% undergraduate, 10.54% master’s, and 3.70% doctorate. General staff accounted for 29.63% of the sample, followed by 19.09% middle layer managers, and 7.12% senior professional and technical personnel. Focusing on those who were aged above 40 years in the sample , This article contains three scales. The Inclusive Leadership Scale was designed based on the pilot study and in-depth interviews. The Employee Innovative Behavior Scale and the Psychological Capital Scale were adopted from the questionnaires by Based on previous literature and previous surveys, we conducted questionnaire surveys and interviewed employees and leaders. The Inclusive Leadership Scale based on the questionnaire by The KMO value of the Psychological Capital Scale was 0.904, as obtained by analysis of the exploratory factor, and the results of the Bartlett spherical test were also significant at the 0.01 level. The Psychological Capital Scale includes four factors that accounted for a total of 63.89% of the variation: hope accounted for 20.33%, optimism accounted for 15.41%, toughness accounted for 15.16%, and self-efficacy accounted for 12.99%. The factor loading of each item was higher than 0.551 and lower than 0.868. The alpha coefficient of this scale was 0.888, showing good reliability.The Innovative Behavior Scale uses Likert’s five-point scoring method. Factor analysis of this scale showed two factors that accounted for a total of 67.12% of the variation. The factors of employees’ innovation outcomes and their innovative thinking accounted for 34.03 and 33.09% of the variation, respectively. The factor loading of each item was higher than 0.690 and lower than 0.917. The alpha coefficient of this scale was 0.890, showing good reliability.The results of the descriptive analysis are shown in The results of the correlational analysis in To verify the influence of inclusive leadership on employees’ innovative behaviors, we used regression analysis. We also tested psychological capital as the mediating variables, and the results are shown in F = 23.700, p < 0.001). Leaders’ encouragement and recognition of employees , leaders’ respect and fair treatment of employees , and leaders’ rational understanding and tolerance of employees’ failures  were significantly associated with psychological capital. Therefore, Hypothesis 2 could be tested.The dependent variable in model 1 was employees’ psychological capital. The three dimensions of controlling variables and inclusive leadership collectively accounted for 34.2% of the variation in the dependent variable of employees’ psychological capital . Among these, leaders’ encouragement and recognition of employees  and leaders’ respect and fair treatment of employees  positively influenced employees‘ innovation outcomes, while leaders’ rational understanding and tolerance of employees’ failures  could not significantly account for employees’ innovation outcomes. Therefore, Hypothesis 1.1.1 and 1.1.2 could be verified. In model 3, the controlling variables of inclusive leadership and psychological capital accounted for 35.2% of the variation in employees’ innovation outcomes . Leaders’ encouragement and recognition of employees  significantly accounted for the employees’ innovation outcomes, while leaders’ respect and fair treatment of employees  and leaders’ rational understanding and tolerance of employees’ failures  could not significantly account for this. However, psychological capital could account for the employees’ innovation outcomes . F2 could significantly account for the psychological capital, but F1 had a weaker association. Therefore, leaders’ respect and fair treatment of employees influenced employees’ innovation outcomes through the mediating function of psychological capital, while leaders’ encouragement and recognition of employees partially mediated innovation outcomes through psychological capital. Therefore, Hypothesis 3.1.1 and 3.1.2 could be verified.The dependent variable of models 2 and 3 was employees’ innovation outcomes. In model 2, the three dimensions of controlling variables and inclusive leadership collectively accounted for 22.0% of the variation in the dependent variables representing employees’ innovation outcome . Among these, leaders’ encouragement and recognition of employees  had a positive influence on employees’ innovative thinking, while leaders’ respect and fair treatment of employees  and leaders’ rational understanding and tolerance of employees’ failures  could not significantly account for employees’ innovative thinking. Therefore, Hypothesis 1.2.1 could be verified. In model 5, the controlling variables of inclusive leadership and psychological capital accounted for 36.1% of the variation in employees’ innovative thinking . Leaders’ encouragement and recognition of employees  significantly influenced the employees’ innovative thinking, and psychological capital  significantly accounted for employees’ innovative thinking. F1’s explanatory ability for employees’ innovative thinking was weak. Therefore, leaders’ encouragement and recognition of employees partially mediated innovative thinking through psychological capital. Therefore, Hypothesis 3.2.1 could be verified.The dependent variable of models 4 and 5 was employees’ innovative thinking. In model 4, the three dimensions of controlling variables and inclusive leadership collectively accounted for 25.0% of the variation in the dependent variables representing employees’ innovative thinking  of innovative behaviors. Among the three dimensions of inclusive leadership style, leaders’ encouragement and recognition of employees has a significant influence on new generation employees’ innovation outcomes and innovative thinking. Leaders’ respect and fair treatment of new generation employees has a significant influence on their innovation outcomes. Further, we provide detailed information about practical implications. Leaders adopting an inclusive style know more precisely how to generate different innovative results for new generation employees. Third, psychological capital plays a mediating role between leaders’ respect and fair treatment of employees’ innovation achievements. It also played a partial mediating role between leaders’ encouragement and recognition of employees’ innovation outcomes and innovative thinking. Such results add to the existing literature by clarifying psychological capital’s differentiated effects on different inclusive leadership-innovative behavior relationships. Fourth, different from the concept of fairness and justice in Western countries, this article integrates the traditional Chinese cultural aspect of “tolerance as a virtue” into the connotation of inclusive leadership style and emphasizes the concepts of tolerance and leniency. Combining the characteristics that differ between traditional employees and new generation employees, we used empirical research to verify how inclusive leadership style affects employees’ innovative behavior. The results promote the reasonable use of the inclusive leadership style, adaptation to the characteristics of new generation employees, and giving a full audience to new generation employees. The results ultimately provide a basis for boosting the innovative thinking and behavior of the new generation employees.This paper provides theoretical bases and implications for future studies. It also supplements and expands the research on the relationship between inclusive leadership style and employees’ innovative behavior and provides new ideas for human resource management approaches among new generation employees.Our results have practical significance for enterprise human resource management and the development of entrepreneurial leadership style in the new era. The results give leaders clear guidance regarding leadership style that can accommodate new generation employees’ characteristics, give full play to their advantages, and stimulate their innovative behaviors to facilitate enterprise development. In addition to caring about leadership style, leaders should simultaneously focus on cultivating employees’ psychological capital to create more advantages and value for enterprises resulting from innovation . EncouraThis article has some deficiencies. The questionnaires about inclusive leadership, psychological capital, and innovative behavior were all filled in by the same person, which may cause homologous data errors. However, we have made some efforts to lower potential biases. Before we collected the data, we re-ordered the questionnaire items according to the independent and dependent variables, preventing the raters from guessing the causal relationships between variables; thus, we reduced the possible bias caused by having the same person provide answers for both independent and dependent variables . After dThe datasets generated for this study are available on request to the corresponding author.Y-CF conceived and designed the research and provided guidance throughout the entire research process. J-YC wrote and supplemented the English manuscript. M-JW and C-YC participated in the data processing and writing of the Chinese Papers.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.


Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit  protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus. Flavivirus genus, emerged in the Americas in 2015 and is a significant public health concern.3 The virus has been known to circulate with limited reported activity in the Old World since before the middle of the twentieth century.3 It first appeared in the continental New World in Brazil in March 2015, initiating an explosive outbreak that resulted in millions of infections throughout South, Central, and North America, including the U.S. territory of Puerto Rico, within 1 year.3 Most of the clinical cases reported in the U.S. states have been associated with travel to the affected areas.4 Since 2015, more than 5,000 such travel-associated cases have been documented, mostly in 2016. The number of locally acquired ZIKV cases in the United States is about 20 times lower than travel-associated cases, with limited autochthonous transmission confined to Florida and south Texas.4Zika virus (ZIKV), a member of Aedes spp. mosquitoes.3 Humans develop a level of viremia high enough to propagate the transmission cycle.3 Other routes of ZIKV transmission in humans have been documented, including sexual, congenital, breast milk, and blood transfusion.8 Transmission through organ transplantation is a possibility for ZIKV as it was reported for another flavivirus, West Nile virus (WNV).9Zika virus is mosquito-borne, transmitted to humans by a bite of 3 Clinical manifestations of ZIKV infection are typically mild with acute onset of fever with maculopapular rash, arthralgia, conjunctivitis, myalgia, and headache, lasting from several days to 2 weeks.3 In rare cases, ZIKV infection can result in serious complications, including Guillain– Barré syndrome.3 The most severe ZIKV infection complications occur during pregnancy, leading to severe birth defects, death, and poor clinical outcomes in the newborn, with the condition collectively known as congenital Zika syndrome.10 The U.S. Zika Pregnancy Registry reported ZIKV-associated birth defects, including congenital microcephaly, in up to 5% of neonates born to mothers with possible recent ZIKV infection during pregnancy.11About 80% of infections with ZIKV are asymptomatic.3 Therefore, nucleic acid tests on paired serum and urine samples are recommended for the laboratory diagnosis of acute ZIKV infection.2 Detection of viral RNA in sera or urine provides conclusive diagnosis of infection, but the timeframe for it is limited to 1–2 weeks post onset of symptoms because of fast decline of virus presence in serum and urine.3 Tests for ZIKV-specific IgM antibodies in serum are also used as a diagnostic evidence for ZIKV infection and might increase the window for diagnosis to several months, but ZIKV serology is complicated by considerable cross-reactivity with other related flaviviruses, particularly in dengue-endemic areas.2 Considering these diagnostic constraints, it would be ideal to optimize the ability to detect RNA for a longer window of time, particularly in high-risk individuals , or individuals with serious complications such as Guillain–Barré syndrome. We recently reported our laboratory’s experience in detecting ZIKV RNA in saliva and whole blood up to 14 and 81 days post onset of symptoms in one acute ZIKV case, respectively.12 Given these findings, the purpose of this study was to identify viral RNA extraction methods that maximized ZIKV RNA recovery from a given sample type to further strengthen diagnostic protocols for detection of ZIKV in various body fluids.Zika disease onset usually coincides with viremia and viruria.12 plasma was separated and discarded, and the remaining blood cells and a small portion of plasma were mixed. Analogous to the setup and description from a previous WNV study,13 this fraction is referred to as whole blood from this point forward, as it contains all major components of whole blood: erythrocytes, leukocytes, thrombocytes, and a portion of plasma. Urine specimens were spun at 1,000 × g and the supernatant was collected for testing. Saliva swabs and vaginal secretions swabs were obtained with BD BBL CultureSwab Sterile Single Swab (BD), which is free of media or preservatives. Semen specimens were collected in a sterile urine cup, allowed for liquefaction, and used without separation. Breast milk specimens were collected using the subject’s own breast pump, then transferred to a sterile specimen cup.Collection of clinical specimens was performed by standard procedures under a research protocol enrolling suspected or confirmed cases of ZIKV infection in U.S. residents. The studies were approved by Baylor College of Medicine Institutional Review Board, protocols H-30533 and H-39865. Serum specimens were obtained by collection and processing of venous blood in BD SST vacutainers . Whole blood specimens were obtained by collection and processing of venous blood in BD K2EDTA vacutainers (BD). As it is shown that ZIKV remains associated with red blood cell fraction of the whole blood,Zika virus–positive control, strain MEX I-7, was provided by University of Texas Medical Branch World Reference Center for Emerging Viruses and Arboviruses. The strain was isolated in 2016 from mosquito samples collected in Mexico and passaged a total of five times on Vero cells.Ten aliquots of each sample matrix were prepared, with 200 µL volume for liquid samples. AVE buffer, the elution buffer from the QIAamp MinElute Virus Spin Kit , was used as the spiking reference control buffer to generate expected cycle threshold (Ct) values. The aliquots were mixed with 200 µL of the lysis buffer from the kit, which is AL buffer containing 28 ng/µL of carrier RNA. The aliquots were spiked in triplicates with 10 µL of viral cell culture supernatant containing three levels of viral load, and a single aliquot was left unspiked to confirm ZIKV-negative status of the samples from the donors. To obtain high, medium, and low spiking loads, the original viral cell culture supernatant was diluted in culture media so that the reference buffer samples’ final Ct values were in the range of 19–21, 27–29, and 33–35, respectively. Spiked samples were stored at −80°C before proceeding to RNA extraction.To facilitate lysis of 200 µL of packed blood cells specimens, which approximate 400 µL of the original venous blood specimens, a total of 400 µL of AL along with 200 µL of phosphate buffered saline (PBS) were added to whole blood samples . AL for Saliva and vaginal secretions swabs were first incubated in 250 µL of AL/carrier RNA for 10 minutes at room temperature and were then compressed against the inside of the tube to expel the liquid before removing and discarding. Remaining volume of the lysis buffer was about 200 µL, and 200 µL of PBS was added to adjust the total sample volume according to the QIAGEN protocol.14 with elution in 30 µL of AVE buffer. All centrifugations were performed at maximum speed , except where indicated below. Each extraction batch included positive (ZIKV MEX I-7 diluted supernatant) and negative (AVE) extraction controls. Depending on the sample type, three sets of adjustments to the manufacturer’s protocol (Optimization A through C) were shown to be beneficial for ZIKV RNA recovery. The details of the three optimizations are described in the following text and The baseline method of RNA extraction from the study specimens was the QIAamp MinElute Virus Spin Kit (QIAGEN) according to the manufacturer’s protocolg , followed by 1 minute centrifugation at maximum speed. Elution incubation , followed by 1 minute centrifugation at maximum speed. The column was washed and dried per QIAamp MinElute Virus Spin Kit protocol, and the elution incubation step  was added, and the sample was incubated at room temperature for 5 minutes. After adding 1.4 mL of ethanol, the sample was loaded onto the RNeasy Mini Spin Column (QIAGEN) at 900 × Step 14,  was perfg , and pellet was resuspended in a mix of 200 µL AVE, 200 µL AL/carrier RNA, and 25 µL Protease. The sample was further processed starting with Step 4 of the manufacturer’s protocol  of spiking material was added directly to freshly collected urine. Seventy microliters of Urine Conditioning Buffer  was mixed in. Samples were centrifuged at 3,000 × protocol . Two intSupplemental Tables 1 and 2.Additional procedures and reagents/supplies were tested to improve viral RNA recovery from semen and urine samples, including buffers and columns of RNeasy Plus Mini Kit or QIAamp Viral RNA Mini Kit, QIAzol, QIAshredder columns, ATL buffer , Direct-zol RNA MiniPrep Kit (Zymo Research), RNasin Plus RNase Inhibitor , SUPERase In RNase Inhibitor , Protector RNase Inhibitor , ProtectRNA RNase Inhibitor , and Aptima Urine Specimen Transport Tubes . The summary of these experiments is presented in 15 The following controls were included in each RT-qPCR run: six points of ZIKV RNA standard curve (in vitro transcribed oligonucleotide), extraction negative control, extraction positive control, and no template control, all in duplicate.Five microliters of extracted RNA were used in a duplicate 20-µL reaction with TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) on ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The previously published TaqMan ZIKV 1107 assay was used.Ct values and copies/µL values from a duplicate of RT-qPCR for each sample were used to calculate mean Ct values and mean copies/µL values for each extracted sample. Each triplicate of mean copies/µL values per viral load was used to calculate average concentration in spiked matrix for each level of viral load.Supplemental Table 3).To determine expected Ct values and average concentration in spiked reference control, three sets of 10 spiked reference controls samples  were independently extracted and run on RT-qPCR (one set was run twice). Expected Ct value was calculated as average across all mean Ct values from a duplicate of RT-qPCR for each reference sample of a given viral load. Average concentration in spiked reference control was calculated as average across all mean copies/µL values from a duplicate of RT-qPCR for each reference sample of a given viral load  × 100%.12 was subjected to five freeze/thaw cycles: an aliquot was taken after each thaw cycle and kept at 4°C in AL buffer/carrier RNA until extraction later on the same day. Each freeze cycle was for 20 minutes at −80°C.A known ZIKV-positive serum specimen aliquotSupplemental Table 4). Because most of the tested matrices contained high amounts of cellular DNA/RNA, the extraction protocol was optimized by addition of 5 minutes incubation at 56°C at the elution step. Slow loading on the extraction column  was also added to the protocol to potentially improve recovery of sample nucleic acids. These modifications restored recovery of ZIKV RNA in whole blood, vaginal secretions, and breast milk samples and maintained full recovery in saliva samples .Initial testing of the spiked matrices with RNA extracted by the QIAamp MinElute Virus Spin Kit manufacturer’s protocol yielded poor recovery values for all sample types, except for serum and saliva . A series of alternative extraction protocols were tested for semen specimens and eventually one was selected that yielded 89% of the replicates within two Ct units of the expected Ct values and an average of 29% ZIKV RNA recovery .The extraction protocol optimized as before did not improve the recovery of ZIKV RNA in urine and semen specimens . The UCB protocol was applied to spiked urine specimens from two additional negative control donors. In these urine samples, the protocol did not yield expected Ct values, but the results were still the most favorable compared with other available urine preservation methods tested . However, an abbreviated single spiking load test of two previously available urine specimens, which were stored frozen, showed complete recovery of ZIKV RNA even with the original protocol . These data can be explained by variation in urine properties from donor to donor.Similarly, a number of additional extraction protocols were tested for urine specimens. The most successful protocol for RNA extraction from urine specimens included addition of UCB and was shown to work with 1-mL aliquots of fresh urine, or frozen “as is.” Spiked urine specimens from a negative control donor were tested and showed 89% of the replicates within two Ct units of the expected Ct values, and an average of 51% ZIKV RNA recovery .Spiked urine specimens from negative control donors #1 and #3 were tested to compare addition of UCB to a urine specimen stored frozen as is with the addition of UCB to a fresh UR specimen and then freezing and storing the resulting pellet of nucleic acids. There was no difference between these two urine processing methods . The adjustments also included slow load of the sample onto the column, but the main effect of the RNA recovery restoration was achieved because of the modification of the elution step. The specimen types that benefited from this Optimization A protocol have significant cellular content, and it was assumed that more stringent elution conditions would be required to release high amount of cellular nucleic acids  from the column filter. Despite excellent performance of the original protocol for saliva specimens, optimized extraction protocol would be recommended for this matrix because of potential variation in the amount of cellular DNA/RNA content.Whole blood, vaginal secretions swabs, and breast milk specimens required extended incubation at elevated temperature at the elution step for optimal recovery of viral RNA . Of the several tested methods, these two simple modifications proved to be the best technique of managing extremely high DNA content in semen specimens. Although we could not reach full recovery of ZIKV RNA from this matrix, our result (29%) was very similar to that of a recently published study from the Centers for Disease Control and Prevention (CDC) group in Puerto Rico.16 The investigators showed an average of 31% ZIKV RNA recovery from semen specimens using Trioplex Real-time RT-PCR Assay testing platform, with magnetic beads–based automated sample RNA extraction.Semen specimens required the use of high volume of RLT Plus buffer and RNeasy Mini Spin Columns . However, because the extractions with this protocol are performed from 1 mL of urine sample, compared with 0.2 mL in the original protocol, expected Ct values are decreased by 2.3 units. In that regard, although average recovery was 51%, Ct values of all replicates were actually lower than expected Ct values when extracted from 0.2 mL of urine sample, suggesting that this extraction protocol offers a more sensitive test. We also showed that addition of UCB could be performed before or after freezing the specimen for storage, with the same effect on RNA recovery. However, in our experiment, specimens were kept as is at −80°C only for 1 day. Therefore, storing of urine specimens preserved with UCB right after the sample collection should be preferable because of potential decrease in recovery of low levels of ZIKV RNA when stored as is for prolonged time, as it was shown in a recent study.18 On the other hand, we used the UCB method with archived ZIKV-positive urine specimens (stored for more than 9 months at −80°C without any preservatives), and were able to achieve better Ct values when compared with extractions performed right after specimen collection by the standard protocol ,20 in saliva up to 60 DPO,26 in vaginal secretions up to 14 DPO,28 and in breast milk up to 8 days post delivery.29 A recent Puerto Rican prospective cohort study of 150 symptomatic participants who tested positive by RT-PCR at presentation showed the following medians and 95th percentiles for the time until loss of ZIKV RNA detection: 14 and 54 days, respectively, in serum; 8 and 39 days in urine, and 34 and 81 days in semen.16 In animal models, ZIKV RNA was shown to persist for several weeks after infection in saliva, lymph nodes, and colorectal tissue in cynomolgus and rhesus macaques.25Our long-term goal is to apply these methods for optimal RNA recovery to determine shedding time in different body fluids among a cohort of ZIKV-infected patients to identify opportunities for a longer window of time to diagnose infection. In addition to specimen type and timeframe recommendations for viral RNA tests in laboratory diagnosis of acute infections, cerebrospinal fluid (CSF) and amniotic fluid specimens are also acceptable for testing, but must have a paired serum specimen as recovery of RNA from these alternative specimens can be highly variable. Zika virus RNA has been detected in other body fluids and, for some matrices, far past the acute stage of the disease. This RNA persistence was shown in clinical studies and in animal models.16 However, even the same study showed only about 30% recovery of ZIKV RNA from semen specimens with their assay. Semen, as well as urine and whole blood, are known for their complex biochemical and cellular composition that present challenges for complete and PCR inhibitor-free extractions of RNA or DNA.35 Several studies have compared the effectiveness of different nucleic acid extraction methods from these body fluids,36 rarely showing actual viral RNA recovery compared with buffer control.37Studies that generated aforementioned results on prolonged detection of ZIKV RNA in tissues and body fluids mostly used the generalized RNA extraction protocols provided by the extraction kit manufacturers, without confirming (or at least without reporting) the level of viral RNA recovery for a given specimen type and a given RNA extraction method. One exception is the Puerto Rican cohort study where ZIKV RNA detection was performed by Trioplex Real-time RT-PCR Assay, which is validated by CDC for ZIKV for use with serum; and with urine, CSF, and amniotic fluid only alongside the patient-matched serum specimen.14 Another concern of adding a step of plasma separation in the whole blood testing procedure is an increased risk of contamination of the sample. This risk increase can be alleviated by strict adherence to high standards of laboratory practices.A main limitation to our study was that we have not tested stability of ZIKV in various body fluids as applied to time from collection to time of addition of lysis buffer and following processing of the sample. Our study was focused on effect of a matrix in terms of its composition on RNA extraction and coupled RT-qPCR. Hence, the specimens were first lysed, and then spiked, to differentiate form any effect of processes that might happen in a clinical specimen in its natural state. We were also limited in testing variations of RNA extraction as applied to a single commercially available kit. Although this QIAamp MinElute Virus Spin Kit is widely used in viral research, there are many other RNA extraction kits on the market, including automation equipment/kits used in clinical diagnostic laboratories. Our results might provide guidance in optimizing RNA recovery when using these alternative methods. Finally, our study design was limited in testing whole blood specimens that lack most of the plasma component. Different amounts of plasma in separated versus complete whole blood specimens might change the performance of the RNA extraction kit. However, we would expect the effect to be minimal as plasma is one of the specimen types for which the QIAamp MinElute Virus Spin Kit was developed.Optimal methods for ZIKV detection in various body fluids will allow accurate monitoring of viral RNA presence during the disease progression in the future studies aimed to improving our understanding of natural history of ZIKV infection and duration of viral persistence in various body fluids. Optimized methods can potentially be successfully used with other arboviruses, as improved diagnostics are crucial to timely detection and response to emerging infections such as ZIKV.Supplemental figure


Scientific development has increased the awareness of water pollutant forms and has reawakened the need for its effective purification. Oxyanions are created by a variety of redox-sensitive metals and metalloids. These species are harmful to living matter due to their toxicity, nondegradibility, and mobility in aquatic environments. Among a variety of water treatment techniques, adsorption is one of the simplest, cheapest, and most effective. Since metal-oxide-based adsorbents poses a variety of functional groups onto their surface, they were widely applied in ions sorption. In this paper adsorption of harmful oxyanions by metal oxide-based materials according to literature survey was studied. Characteristic of oxyanions originating from As, V, B, W and Mo, their probable adsorption mechanisms and comparison of their sorption affinity for metal-oxide-based materials such as iron oxides, aluminum oxides, titanium dioxide, manganium dioxide, and various oxide minerals and their combinations are presented in this paper. The perironment .Adsorption technologies represent an innovative and economic approach to the arsenic removal problem. Metal oxide-based materials were successfully applied for arsenic adsorption from environment since its pollution problem was noticed. In the 2O3), also called activated alumina (AA), is produced by thermal dehydration of aluminum hydroxide, so that the surface can exchange contaminants for hydroxyl groups. It is characterized by a relatively high surface area (about 200 m2/g) and diverse pore distribution of macro and micropores. It can be regenerated with sodium hydroxide, followed by neutralization with sulphuric acid trimethylammonium chloride) in boron removal by means of adsorption. Pure magnetite was obtained by coprecipitation of Fe(II) and Fe(III) ions in aqueous solution with ammonia. For Fe3O4-TSPA composite, to 5.0 g of wet magnetite dispersed in water 2.5 mL of TSPA (Gelest) was added and stirred within 30 min. Composite material was removed from reaction environment using magnet and washed to inert pH. Second composite was prepared analogically, the flocculating agent was 0.5 g/L 1010f . For adsorption experiments 3.0 g of the wet particles were dispersed in 50 mL solution at the desired initial boron concentration, pH, and ionic strength. All of them were carried out using a SHA-C shaking water bath  with a shaking speed of 80 rpm at 22 °C. Experimental data revealed that boron adsorption is the most favorable in pH = 6 . From the boron speciation chart  with boric acid and borate, which can promote the adsorption. The authors determined that three types of interactions determine boron adsorption onto Fe3O4–TSPA: (i) electrostatic interaction; (ii) hydrogen bonding; and (iii) hydrophobic interaction and proposed specific mechanism of it.Adsorbents with magnetic properties are a promising technology for the future of water treatment. Such sorbents reveal the great potential of functionalization and the effective sorption of different substances . Magnetirability . Considerability  tested Fon chart  it can bÖztürk and Kavak  investig3. Nevertheless, this process is effective only for medium-high concentration of boron in the water and adsorbent can be used only in 3 cycles due to irreversibility of boron adsorption onto magnesium oxide.In their work de la Fuente García-Soto et al.  investig4−. Maximum adsorption capacity was obtained for Mg-Al oxide when the Mg/Al = 2 and was equal to 80 mg/g. Recycling of the adsorbent was proposed in two steps: (i) borate intercalated Mg-Al LDH treatment with carbonate ions in water in order to enable anionic exchange between carbonate and borate, to produce CO3·Mg-Al LDH, (ii) CO3·Mg-Al LDH calcination in 400–800 °C in order to recover Mg-Al LDH. After regeneration tests the adsorbent maintained the ability to remove boron from aqueous solution, but efficiency declined significantly—boron concentration at 480 min of adsorption for the first cycle was 32.3 mg/L, and for the regenerated adsorbent 62.1 mg/L. Such a low performance of Mg-Al oxide is connected to the decreased crystallinity and remains of adsorbed boron into the Mg-Al oxide structure. To consider the commercial use of Mg-Al oxide as boron adsorbent, the regeneration procedure needs to be refined and the contact time should be significantly reduced.Considering the need to design recyclable adsorbent for boron removal Kameda et al.  decided The adverse effects of boron pollution for living organisms could not be ignored. Research community developed some efficient borate sorbents based on metal oxides. Borates are preferably sorbed in alkaline pH. At pH below 8 the main boron compound is orthoboric acid and due to its lack of charge adsorbed quantity is insignificant. Thus, neutral pH could not be optimal one for boron species adsorption. The data gathered during the literature survey is shown in 4 ions. Tungstate is able to occur in various complexes depending on the pH of the environment and total W concentration. In the range from neutral to alkaline conditions, oxyanion WO42− with tetrahedral coordination is the predominant form. In acidic media, tungsten aims for the creation of polymeric compound such as paratungstate  with octahedral coordination [Bacillus subtilis and Pseudomonas fluorescen) in 3 months, and caused death of ryegrass plants and red worms. Such tungsten concentrations also inhibit the growth of bacterial colonies, what could possibly deteriorate process performance of biological wastewater treatment systems. Tungsten (W) is a heavy metal which creates compounds in oxidation states ranging from −4, to +6, while the most stable state is +6. In the environment, it occurs naturally in soils and sediments. In solution W is oxidized to soluble WOdination ,109,110.dination . Its avedination . The resdination . This phdination ,114. Itsdination . Some phdination ,114,116.dination  reported4 increases more in alkaline than in acidic conditions due to the occurrence of increased repulsive force between the negatively-charged mineral surface and the tungstate oxyanion [4 is higher for metal oxide minerals  than for montmorillonite. Generally it follows the order of Al hydroxide or Fe (oxyhydr)oxides  > Mn oxide (birnessite) > phyllosilicate (montmorillonite) in the whole pH range. The best adsorption capacities were obtained for acidic conditions (pH 4). Aside from adsorption capacities, researchers evaluated an influence of the presence of phosphate and molybdate oxyanions on tungstate sorption. Oxyanions of PO4 and MoO4 revealed higher affinity than tungstate in neutral-alkaline conditions. In acidic media, tungstate is more preferably adsorbed on clay minerals. Unfortunately, adsorption–desorption tests had not been conducted, so the reusability of adsorbents remains unknown. Hur and Reeder [Sorption processes onto the surface of minerals are crucial for regulating the distribution and mobility of trace metals in natural aquatic environments and soils, and it is hardly dependent on environmental pH. The mobility of tungstate oxyanion WOoxyanion . Gustafsoxyanion  investigoxyanion  had adsod Reeder  investig0.5Zn0.5Fe2O4 prepared according to the chemical co-precipitation method at room temperature for four different oxyanions’ removal, including W(IV). The adsorption capacity was in the order W(VI) > Cr(VI) > Mo(VI) > V(V) and for W(IV) was significantly higher than for other investigated oxyanions—72 mg/g. Desorption efficiency exceeded 98% while process was carried using 2 mol/L NaOH, as the most effective eluent and its equilibrium was reached in 15 min. Thus recovery of such adsorbents should be possible. Prepared nanomaterial can be easily dispersed in water, and due to its magnetic properties can be easily removed from the adsorption environment. The authors predict that Ni0.5Zn0.5Fe2O4 nanocomposites could be candidates for the removal of trace amounts of chromium, molybdenum, vanadium, and tungsten ions.Due to their large surface area and small diffusion resistance, magnetic adsorbents are of great interest. Afkhami et al.  investigDue to the late discovery of tungsten’s harmful properties, only few works consider its removal via means of adsorption onto metal oxide-based materials. Researchers are at the beginning of the road to find effective sorbents for W contamination’s removal. Specific data gathered during literature studies were presented in 43−, HMoO42−, H2MoO4−, HMoO4−, and MoO42− depending on the pH of the environment [42−. In more acidic conditions molybdate is protonated to the less charged anionic species (HMoO4−) and in strongly acidic media the neutral molybdic acid MoO3(H2O)3 is created. In high molybdenum concentrations at pH below 5–6 it is able to form isopolymetalates such as Mo7O246− or Mo8O264− [Molybdenum is a transitional metal occurring on oxidation states from −II to +VI , which aironment . In pHs Mo8O264− . The dis42−, as the most common oxyform of this element, occurs in many types of industrial wastewaters i.e., wastewater from a styrene monomer plant (1000 mg/L), scrubber effluent of a municipal solid waste incinerator (0.95 mg/L) and mining water (0.1–2.2 mg/L) [However, though its presence is essential for life on Earth, due to its ability to form active sites for many enzymes , that c.2 mg/L) . Thus ef42−, as they may acquire positive charge at low pH [Adsorption as a relatively cheap and effective method for many oxyanions removal had also been considered for molybdenum oxyanions elimination. Iron, aluminum, and, to some extent, titanium oxides may be important adsorbent minerals for MoOt low pH .2O3 < gibbsite < amorphous Al2O3. Adsorption of molydbdeum oxyanions onto aluminum oxide was a subject of research of many scientists [2O3 catalysts for hydrotreatment of petroleum fractions. Luthra and Cheng [7O246− and MoO42−) onto γ-alumina via Molybdenum-95 NMR Study. Both oxyanions were attracted to the positively charged alumina surface in pH lower than the isoelectric point of the alumina (pH 8.5). NMR studies showed that Mo7O246− after the contact with alumina decomposes to MoO42− in minutes. The decomposition was believed to be caused by the increase in the pH of the impregnation solution inside the pores of the alumina. Wu et al. [2O3 surface: molybdate, selenite, selenate, chromate, and sulfate. Specific surface area of obtained γ-Al2O3 was measured by BET method and was equal to 100 m2/g. Molybdate and selenite are both strongly binding adsorbates—molybdate depresses selenite sorption at acidic pH and selenite suppresses molybdate sorption at alkaline conditions. The affinity for aluminum oxide surface was the highest for molybdate oxyanions, followed by selenite, selenate, sulfate, and chromate, respectively.Goldberg et al.  were intientists ,128,129 nd Cheng  examined2), which maintained oxygen concentration on the 1 mg/L level. Goethite slurry was synthesized by the researchers. Results indicated that MoO42− adsorption on goethite obeys the Langmuir model. Adsorption capacity of MoO42− on goethite amounted 25.9 mg/g at pH 4. Competition tests revealed that an affinity for goethite follows the order phosphate > tungstate > molybdate > silicate > sulfate. Adsorption of Mo seems to be more affected by the presence of the phosphate than the tungstate anion, which is attributable to increased repulsion between Mo and more negatively charged surface sites after phosphate adsorption onto goethite surface. The goethite surface probably contains adsorption sites common for Mo, P and W anions, as well as specific for each element. Gustafsson [Xu et al. ,123 investafsson  examined3O4) were used in water purification due to its magnetic properties. The main drawback of magnetite usage is its dissolution in water. High iron concentrations are toxic for humans and other living organisms. Consider that Keskin [3O4-embedded 1,3,5-triacryloylhexahydro-1,3,5-triazine-acrylamide hybrid polymer for molybdenum oxyanions removal. Triazine compounds are able to create complexes with iron ions which connected with polymeric network prevent iron release. The triazine molecules were used as a crosslinker and the polymeric material was synthesized by free radical polymerization. Adsorption tests were performed for pure Fe3O4, Fe3O4 embedded hydrolyzed, and nonhydrolyzed polymers. The highest removal efficiency was obtained at pH 2.5 and pure magnetite was subtly more effective than both composite materials (97% for Fe3O4 and 96.87% and 96.36% for hydrolyzed and nonhydrolyzed polymers). The iron release between those three materials varied insignificantly—the highest value was noted for magnetite—1.16 mg/L, followed by magnetite embedded by nonhydrolyzed polymer—1.02 mg/L, and the lowest value of 0.22 mg/L characterized magnetite-embedded by hydrolyzed polymer. Kinetic studies and isotherms were investigated only for an Fe3O4-embedded hydrolyzed acrylamide -1,3,5-triacryloylhexahydro-1,3,5-triazine polymer. The experimental data was fitted well to the pseudo-second-order kinetic model and Langmuir isotherm. Maximum Langmuir adsorption capacity was equal to 0.213 mg/g. Author performed reusability tests of obtained material by HNO3, CaCl2, NaOH, EDTA, HCl, and HCl/HNO3 solutions. The best results were obtained by CaCl2 treatment—sorption efficiency for molybdenum removal after regeneration increased to 97%, while its mass decreased from 0.25 g to 0.21 g, which throws into question if the adsorbent surface was chemically modified by the regenerating medium.Iron oxide (Fet Keskin  decided 2MoO4·2H2O in ultrapure water. Sorption was the most effective in a strongly acidic environment (pH = 3), where maximal adsorption of MoO42− and minimal adsorbent dissolution . The adsorption capacity for molybdenum in optimal conditions (pH = 3 and 25 °C) is 17.9 mg per gram of adsorbent. In the absence of molybdenum the point zero charge of the obtained composite lies around the pH = 4, so below that value its surface is charged positively. In the presence of Mo the point zero charge is no longer observed and magnetite-zeolite surface is negatively charged into whole studied pH range (2–10). That facts indicated a chemical adsorption of MoO42−, which causes the shift of pHpzc to a lower value. Negatively charged Mo species (HMoO4− and MoO42) are pulled toward composite surface via electrostatic forces and bind chemically to the Fe(III) present on magnetite surface. Such a connection results in a negative charge of the composite, even below pH 4, and lowering of the zeta potential. In pH 4–6.5 where the composite surface is negatively charged, the attractive specific sorption forces still overcome the repulsive electrostatic forces, which indicate that molybdenum adsorption onto the magnetite-zeolite surface is a specific chemisorption process. The maximum adsorption capacity increases from 13.6 mg/g at 4 °C to 20.2 mg/g at 40 °C, which indicates the endothermic adsorption. Kinetic data fits well to pseudo second order equation. Experimental results indicate that Mo adsorption is better fitted by the Langmuir isotherm, which indicates chemical bonding between the adsorbent and adsorbate, assuming the formation of a Mo monolayer onto the magnetite-supported zeolite surface. In conclusion, the molybdenum is adsorbed onto the magnetite-supported zeolite composite via formation of an inner-sphere FeOMoO2(OH)·2H2O complex. Such a sorption mechanism was verified for different iron oxides, like goethite and feriihydrite, by Goldberg et al. [An innovative, attractive, and economical approach for oxyanion removal is the usage of sorbent materials consisting of a matrix and a proper adsorbent. Verbinnen et al.  decided g et al.  and Gustg et al. . In theig et al.  had testMolybdenum sorption onto metal oxide-based adsorbents has not been a subject of research of many scientists. Adsorption data gained by literature survey is gathered in In spite of the fact that metal and metalloid oxyanion pollution is a real phenomenon and their elimination is the subject of increased interest of scientific communities, due to the lack of data in published papers the results are incomparable to each other. Authors are unwilling to share exact results of their work, which cause replication of the same studies over and over again by different research teams. In addition most of the research is carried out only on model solutions, so proposed methods might not be applicable as such for the removal of oxyanions from industrial wastewaters, because they are more complex systems containing other oxyanions and compounds, that can compete and interact with each other. Some research in the field of competitive adsorption was held, but the exact mechanisms of oxyanion behavior in solution are still unknown. Thus single sorbate and single mineral adsorption studies in the laboratory may not be directly applicable .n+ ion removal process indicates the optimal conditions for its realization, which depend on many factors such as the type of cation, the type of sorption material and its physicochemical parameters, as well as the parameters of the adsorption process. The key element seems to be the pH of the adsorption environment—for metal cations, in most cases analyzed, the optimal pH at which the highest removal efficiency is observed is pH = 5–6. This is related to the Pourbaix diagrams, indicating pH, beyond which precipitation of appropriate forms of metal hydroxides will occur. In this aspect, one should also remember the influence of pH on the nature of the sorbent functional groups, which can be protonated  or deprotonated . This is important when defining the adsorption mechanism, which in the case of Mn+ is based on electrostatic interactions—attraction when the charge of functional groups is different from the Mn+ charge or repulsion when these charges are the same. In this respect, the oxyanion adsorption is definitely more complex. The probable mechanism of oxyanions adsorption on the surface of (hydr)oxides has been presented in The presented literature review unambiguously indicates the complexity of the process of removing metal oxyanions from aqueous systems via adsorption. In contrast to metal cations, which have been extensively studied and described in scientific papers, oxyanions studies are not entirely clear. Analysis of the MMoreover, a significant majority of papers pertain to arsenic removal, which is visible on the statistic presented in The environment pH influences as well metal chemistry in solution (occurrence of various oxyforms) as the metal (hydr)oxide surface’s protonation/deprotonation . Thus inThe spectrum of speciation forms of oxyanions of the relevant metal is very wide, which translates into their random behavior in aqueous solutions, especially those with varying pH. Unfortunately, the aspect of the effect of pH on the efficiency of oxyanion-binding by various sorbents, e.g., those presented in the literature review, is treated fairly generally, and in some cases even overlooked. This hinders the interpretation of the dependencies obtained and introduces confusion regarding the comparison of the behavior of various sorption materials towards the removal of these types of impurities from aqueous solutions. The presented comparisons unequivocally do not indicate optimal conditions for the removal of a particular oxyanion group using the available sorption materials. It seems that the mechanism of their binding should be at least similar for different sorbents, and as a result it is completely random. Analyzing at least the effect of the pH discussed earlier, one would expect such an environment pH, which would enable a strong attraction between the positively charged surface of the sorbent and the negative form of most metal oxyanions. On the other hand, paying attention to the type of sorption material, it would be important to use one which after synthesis or preparation would exhibit a significant positive charge that could ensure strong interaction with oxyanions. These issues should not be problematic when conducting research on model solutions, which are different to real wastewaters as their composition is diverse (high concentrations of components and their diversity) and can affect the selective adsorption of individual components. All oxyanions presented in this review, excluding borates, are preferably adsorbed in acidic media, due to the positive electrical charge present on adsorbents’ surfaces. Electrostatic attraction is one that the most important mechanism of oxyanions bonding.The role of sorption material, and in fact its physicochemical parameters designed at the synthesis stage—the presence of functional groups exhibiting a positive charge, facilitating the binding of negatively charged oxyanions;The influence of the pH of the adsorption environment on the nature of functional groups of the sorption material and the form of oxyanions in aqueous solutions, so important analyzing their potential interactions;Selectivity tests of sorption materials towards various metal oxyanions, which in the scientific papers published so far are effectively omitted, inversely as in case of sorption of metal cations,The effect of the presence of other components of wastewater on the sorption efficiency of a particular oxyanion group.The following issues seem to be worth analyzing here:In these aspects, the presented scientific papers leave considerable dissatisfaction, and on the other hand, they open a wide range of activities to optimize the process of removal of these particularly “uncomfortable” inorganic impurities present in water systems. This all is more justified when analyzing the number of works published on this subject.