Here, we show that previously inferred poorly ventilated conditions in the North Atlantic were linked to enhanced pre-aged organic carbon (OC) input during Heinrich Stadial 1 (HS1). The 14C age of sedimentary OC was approximately 13,345 ± 692 years older than the coeval foraminifera in the central North Atlantic during HS1, which is coupled to a ventilation age of 5,169 ± 660 years. Old OC was mainly of terrigenous origin and exported to the North Atlantic by ice-rafting. Remineralization of old terrigenous OC in the ocean may have contributed to, at least in part, the anomalously old ventilation ages reported for the high-latitude North Atlantic during HS1.Changes in ocean ventilation have been pivotal in regulating carbon sequestration and release on centennial to millennial timescales. However, paleoceanographic reconstructions documenting changes in deep-ocean ventilation using  Ocean ventilation plays on global climate evolution. Here, the authors suggest that previously inferred poorly ventilated conditions in the North Atlantic were linked to enhanced pre-aged organic carbon input. Old organic carbon was mainly of terrigenous origin and exported by ice-rafting Many paleoceanographic reconstructions are consistent with a more poorly ventilated high-latitude deep North Atlantic Ocean during the last ice age and early deglaciation, with ventilation ages typically ranging between ~1000 and ~5000 yr (the benthic-atmosphere radiocarbon age offset)3, significantly exceeding modern values (~400 yr)4. Reduced ventilation-defined here as the degree of air-sea equilibration - in the North Atlantic has commonly been attributed to dynamic changes in the Atlantic Meridional Overturning Circulation (AMOC) and the northward advection of poorly ventilated water masses from the Southern Ocean6. When pronounced local ventilation anomalies are taken into consideration, such as ventilation ages exceeding 5000 years in the intermediate-depth eastern equatorial Pacific, the dominant paradigm involving the advection of poorly ventilated water masses becomes, however, potentially inconsistent8. Indeed, 14C-depleted  carbon sources, such as inputs of 14C-dead hydrothermal carbon to the abyssal ocean9, may account for some of the old ventilation ages inferred based on marine authigenic carbonate phases7. As changes in ocean circulation dynamics have been pivotal in modulating abrupt climate variability since the Last Glacial Maximum (LGM)12, it is essential to understand the mechanisms that may have caused changes in ocean ventilation in the past and in particular across the last glacial termination.Rapid changes in ocean-circulation dynamics during the last deglaciation may provide fundamental constraints for understanding the past and predicting future variations in atmospheric 13, abrupt fluvial runoff14, and ice-rafting events16. The most prominent of these perturbations relate to Heinrich Events (HEs), during which large amounts of freshwater and terrigenous material16 surged to the high-latitude North Atlantic and were discharged through icebergs17, which transiently altered the intensity of the AMOC18 and led to abrupt cooling in the northern hemisphere16. Modern oceanographic studies indicate that melting glaciers and ice sheets export large volumes of dissolved organic carbon  and particulate organic carbon  to the ocean19, with both fractions being prone to bacterial degradation21. Given the widespread influence of HEs in the high-latitude North Atlantic22, the impact of the pre-aged terrigenous OC supply and subsequent remineralization may significantly have affected the regional (radio)carbon inventory. Enhanced terrigenous OC supply associated with HEs could thus provide a potential source of 14C-depleted carbon to the ocean and account for, at least in part, the anomalously old ventilation ages reported for the high-latitude North Atlantic during the last deglaciation.Paleoceanographic evidence gleaned through the testimony of Northwestern Atlantic sediment cores indicates that catastrophic events episodically occurred during the last deglaciation, including outburst floods14C ages of sedimentary OC and paired planktic and benthic foraminifera in three North Atlantic sediment cores  foraminifera in the central North Atlantic. Thus, our findings provide a perspective for understanding the multi-facetted mechanisms underlying reduced ocean ventilation in the high-latitude North Atlantic and their implications for the oceanic carbon cycle.In this study, we measured the 14C ages of sedimentary OC vary between 18,450 ± 150 and 30,200 ± 310 yr during HS1. These 14C ages are significantly older than those of co-occurring foraminifera, which typically range between 13,000 ± 50 and 18,950 ± 500 yr   during HS1 in northern North Atlantic  and northern (U1314 Site) North Atlantic, correspond to reduced ocean ventilation reconstructed based on B-P  yr Fig.  and 8. A Site and14C ages amongst the three sites highlights regional sedimentological characteristics . This may be related to the location of Site U1308, which lies within the IRD belt 36 . Relatively well-ventilated intermediate-depth (<2000 yr) waters prevailed from low- to mid-latitudes37 in the North Atlantic basin across the last deglaciation . Emerging evidence indicates that the ventilation of the South Atlantic, and more generally, the Southern Ocean, began to recover at the onset of HS142. Would SSW have substantially intruded the deep North Atlantic during HS1, it would have resulted in ventilation ages (B-P 14C ages) ranging between ~2000 and 4000 yr, which is arguably incompatible with a sluggishly ventilated high-latitude North Atlantic at that time.To further document the potentially obfuscating influence of old terrigenous OC remineralization on ventilation-age reconstructions, we compile the ventilation ages available for the entire North Atlantic basin Fig. . Taking ion Fig. . In contion Fig.  and inte 38 Fig.  depths d43 during the last ice age; however, whether it can also influence the deep North Atlantic remains questionable. To some extent, sedimentary 231Pa/230Th and εNd evidence is consistent with a weaker AMOC state and reduced North Atlantic Deep Water (NADW) formation during HS146  may have contributed to reduce ventilation in the high-latitude North Atlantic146 Fig. , which m146 Fig.  and Arct146 Fig.  coincide146 Fig.  and low-146 Fig.  and Sout146 Fig. , albeit −1 during HS1 – 50 times the OC-input flux by the modern Greenland Ice Sheet (~1 Tg·yr−1)20 – sustained for ~1500 yr47, we estimate that ~75 Pg (1 Pg = 1015 g) of terrigenous OC may have been exported to the North Atlantic during HS1. We estimate that at least ~18 Pg terrigenous OC would have been remineralized in the North Atlantic during HS1. Thus, the remineralized OC may have significantly impacted the 14C ages of benthic foraminifera (14CBenthic), which are commonly used for ventilation-age reconstructions. Based on our semi-quantitative framework  events and its subsequent remineralization and release may have contributed to the rapid rises in atmospheric CO2 that characterised previous glacial terminations52. Further investigations are needed to better quantify the impact of catastrophic outflow events on ocean ventilation and circulation during deglaciations and their implication for the global carbon cycle.In addition, the terrigenous OC supplied by ice-rafting to the high-latitude North Atlantic may have provided a hitherto unaccounted for source of carbon to the atmosphere during the last deglaciation. Were the respired carbon derived from the remineralization terrigenous OC released to the atmosphere, then it may have contributed, at least in part, to the rapid rise in atmospheric CO53. Site U1308  is located in the central North Atlantic and Ruddiman’s IRD belt, making it a key site for investigating ocean–atmosphere–ice sheet interactions, which have a profound impact on global climate change. Site U1302  is located on the crest of a small ridge, east of a fault scarp marking the eastern side of Orphan Knoll. Site U1302 is bathed by Labrador Sea Water which may transport terrestrial materials from the North American continent55.Integrated Ocean Drilling Program (IODP) Site U1314 is located on the southwest flank of the Reykjanes Ridge (Iceland) on the southern Gardar Drift  , in order to investigate to which degree old terrigenous OC input may have affected 14C dating of planktonic foraminifera in the three IODP cores. The age model used for U1314 was based on seven planktic foraminifera (G. bulloides) 14C dates and Bacon modelling56. The reservoir ages and standard errors at Site 1314 were obtained from the literature57. The stratigraphy for site U1308 used here was also published previously47, with 24 planktic foraminifera (G. bulloides) used for 14C dating at sites U1308A47 and 60958. We recalculated calendar ages based on the latest Marine20 curve and Bacon modelling59. We used magnetic susceptibility to graphically align U1308A, U1308B, and U1308C and propose a revised metre composite depth (rmcd) model. The age model for U1302 was based on four planktonic foraminifera (N. pachyderma) 14C dates. Owing to the lack of constraints on the reservoir age during the last deglaciation in the vicinity of site U1302, modern ΔR values were used to determine calendar ages using the Bacon model. See the The age models developed in this study were primarily based on 13Corg data are reported relative to the Vienna Pee Dee Belemnite (V-PDB) standard with an external analytical precision of ±0.1 ‰, and the δ15N data are reported relative to air with an external analytical precision of ±0.5 ‰. The external analytical precisions of the TOC and TN data were ±5 ‰ and ±10 ‰, respectively.The samples were freeze-dried, weighed (0.5 g), homogenised, and acidified using 1 M HCl to remove carbonate until no bubbles were present. The samples were then rinsed repeatedly with Milli-Q water until the pH reached neutrality, after which the samples were freeze-dried again. All glassware used was pre-combusted at 450 °C for 5 h prior to use. Carbon and nitrogen content and isotope analyses were performed with an EA-IsoLink elemental analyser coupled to a MAT253 plus isotope ratio mass spectrometer via a ConFlo IV universal interface  at the Key Lab of Submarine Geosciences and Prospecting Techniques . The δ14C contents of TOC and foraminifera at the National Ocean Sciences Accelerator Mass Spectrometry Facility at Woods Hole Oceanographic Institution (WHOI). Acid pre-treatment was conducted at the WHOI. Planktonic foraminifera (Globigerina bulloides and Neogloboquadrina pachyderma (sin.)) were subjected to five interations of ultrasonic treatment in Milli-Q water to remove excess clay minerals60, as shown in Supplementary Dataset 14C analysis included Cibicidoides wellerstorfi, Uvigerina spp. and Melonis spp.In this study, we measured the 13C was used to identify the OC source  for δ15N and C/N values were used to constrain terrigenous OC input. According to previous studies, C/N ratios >1064 and δ15N values of <1‰65 are diagnostic for a terrigenous provenance for the OC.Furthermore, the bulk sedimentary δ40, namely B-P foraminifera age differences and the 14C offset between benthic foraminifera and the contemporary atmosphere (B-atm). To investigate the potential contrasts in ventilation age reconstructions among the three sites in this study, we used the B-P approach to avoid the uncertainties related to constraining calendar ages. When comparing ventilation ages across the entire Atlantic, the B-atm approach was used to unify the available datasets and overcome the drawback pertaining to the lack of planktonic data, which was reported as a limitation in previous studies43. In this study, when considering the western North Atlantic39, we used atmospheric 14C ages derived from IntCal2059 (see the data in Supplementary Dataset 14C yr) and did not affect our argumentation.In this study, two methods were used to quantify past changes in ocean ventilation14C were applied to quantify the 14C contribution from OC to the benthic foraminifera14CBenthic, Δ14COC, and Δ14CBW represent Δ14C values derived from benthic foraminifera, OC, and bottom water, respectively; fOC and fBW represent the relative Δ14C proportions of OC and the bottom water to benthic foraminifera. Based on the 14C age offsets between the OC and benthic/planktic foraminifers shown in Fig. 14CBenthic (−905.8%) and Δ14COC (−976.9%) to analyse the relationship between fOC and Δ14CBW. These values for Δ14CBenthic and Δ14COC were defined at Site U1308 where the ventilation ages were the oldest .First, two end-member models (OC and bottom water) for ΔOC and Δ14CBW, using Eq.   4) to obsing Eq. .5\docume14C age) was considered as representing the offset in 14C age between bottom and surface waters, and AgeSW was considered a fixed value for the 14C age of planktonic foraminifera during HS1.BW and AgeSW represented the 14C age of the bottom water and surface water, repsectively. AgeBW would be expressed as follows:The theoretical ventilation age , an, an7), aOC and model and 14C-dead contribution of OC to benthic foraminifera; Fig. Finally, we resolved the relationship between f14COC, Δ14Cdead carbon and Δ14CAgeHS1 represented fixed Δ14C dates for OC, 14C dead carbon, and the 14C age model, respectively; fdead carbon represented the percentage of dead 14C in OC.To quantify the amount of pre-aged OC exported into the North Atlantic that caused the largest ventilation age during HS1 at U1308, we adopted Eqs.  and 11)11):10\doWe derived that OC was composed of 82.9% dead carbon during HS1.Furthermore, we estimated the pre-aged OC flux based on Eq. :12\documSupplementary InformationPeer Review File NEWDescription of Additional Supplementary FilesSupplementary Dataset S1Supplementary Dataset S2Supplementary Dataset S3Supplementary Dataset S4Supplementary Dataset S5


Antarctica’s continental margins pose an unknown submarine landslide-generated tsunami risk to Southern Hemisphere populations and infrastructure. Understanding the factors driving slope failure is essential to assessing future geohazards. Here, we present a multidisciplinary study of a major submarine landslide complex along the eastern Ross Sea continental slope (Antarctica) that identifies preconditioning factors and failure mechanisms. Weak layers, identified beneath three submarine landslides, consist of distinct packages of interbedded Miocene- to Pliocene-age diatom oozes and glaciomarine diamicts. The observed lithological differences, which arise from glacial to interglacial variations in biological productivity, ice proximity, and ocean circulation, caused changes in sediment deposition that inherently preconditioned slope failure. These recurrent Antarctic submarine landslides were likely triggered by seismicity associated with glacioisostatic readjustment, leading to failure within the preconditioned weak layers. Ongoing climate warming and ice retreat may increase regional glacioisostatic seismicity, triggering Antarctic submarine landslides. Changes in climate preconditioned large-scale, recurrent Miocene to Pleistocene Antarctic submarine landslides through variations in biological productivity, ice proximity and ocean circulation, posing tsunami risk to Southern Hemisphere populations. These landslides can generate tsunamis, which may have significant socio-economic consequences through destroying human life, seafloor equipment and infrastructure3. For example, the 1929 Grand Banks submarine landslide-generated tsunami off Canada generated tsunami waves with a 13 m runup that killed residents along the Newfoundland coast, impacted the coast of Portugal, and caused significant economic damage by severing trans-Atlantic telecommunications cables5. In 1998, a submarine landslide near Papua New Guinea generated tsunami waves that killed 2200 people6. In the mid-Holocene, the Storegga submarine landslide off Norway produced tsunami waves with 20 m runup that likely impacted populations along the southern North Sea coast, Iceland and Greenland, >900 km away7. In the southern hemisphere, tsunami waves arriving from South America, New Zealand, and Southeast Asia have been observed around Antarctica and models indicate that centimetre-to-metre scale tsunami waves may impact Antarctica’s margins;9 thus, tsunami waves originating from Antarctica could reverse this path. Because few submarine landslides are documented around Antarctica10, little is known about the probability and potential of southern hemisphere socio-economic impacts of tsunami waves originating from Antarctica’s continental margins. This potential geohazard risk, coupled with increased international interest in subsea internet cable connections to Antarctica, highlights a critical need to improve understanding of downslope processes specific to glaciated continental margins11.Submarine landslides are global geohazards that can displace huge volumes of sediment, exceeding the size of their terrestrial counterparts by several orders of magnitude12. It is not clear if the low numbers of Quaternary submarine landslides on the Antarctic margin reflect the region’s limited geophysical data coverage or if Antarctica’s slopes are relatively stable due to more homogenous margin sediments, lower regional sedimentation rates, or increased sediment compaction via ice sheets10. In contrast, the extensively surveyed northern hemisphere high-latitude margins host some of Earth’s largest Holocene submarine slope failures, which may result from changes in marine sedimentation rates and lithologic properties, gas hydrate dissociation, ice-proximal glacial dynamics, seismicity due to glacioisostatic rebound, and relative sea-level changes19. Because glacial ice transports large volumes of poorly sorted sediment towards the shelf edge, changes in glacial dynamics can influence ice-proximal submarine landslide occurence16. Rapid sedimentation decreases pore fluid dissipation, leading to overpressure, under-compaction and a reduction in the sediment effective stress, causing slope failure18. As many high-latitude submarine landslide triggers  are sensitive to climate perturbations, it is hypothesised that ongoing and future climate warming may increase the likelihood (or frequency) of high-latitude submarine landslides and associated tsunamis19. Thus, understanding the factors that precondition slopes to fail and mechanisms triggering high-latitude submarine landslides is essential for predicting the timing and location of future slope failures with ongoing climate warming20.Glacially influenced continental shelves make up a fifth of Earth’s continental margin area and are particularly sensitive to climate changes23. During interglacials in the circum-Antarctic, diatom productivity and/or sedimentation has been observed to increase within the seasonal sea ice zone while sedimentation rates are typically lower (e.g. few cm kyr−124) and under the influence of contouritic, plumitic, hemipelagic/pelagic processes23. Recent studies highlight the importance of climate-influenced diatom production/sedimentation (e.g. diatom oozes) in weak layer formation and submarine landslide occurrence, where climatic changes such as variations in sea-ice cover and ocean temperatures have been shown to affect diatom abundance26. Failure along weak layer planes results from loading and overpressurisation by younger, rapidly deposited glaciogenic or glaciomarine sediments26. Differences in the strength and sediment composition of overlying layers is recognised as a key factor preconditioning slope failure23. However, because of the challenges associated with characterising and constraining weak layers , failure planes are often difficult to identify23. Thus, establishing the environmental conditions that predispose slopes to fail on glacial continental margins remains a challenge.On glacial-interglacial timescales, observations indicate that changes in sedimentation on high-latitude margins can result in weak layer formation27. The eastern Ross Sea is part of the West Antarctic Rift System, where extension in West Antarctica likely initiated during the late Cretaceous (~100 Ma) and ceased by the early Miocene28. This part of the margin is now considered tectonically passive. The Ross Sea continental shelf is cut by glacially carved troughs eroded by repeated glacial advances since the early Miocene , adjacent to the southwest-northeast trending Hillary Canyon Fig. , consisttaceous ~00 Ma and−1 38 moves onto the continental shelf via bathymetric lows at the shelf edge39 and mixes with surface and shelf waters to form modified CDW 40. Cold dense Ross Sea Bottom Water 40 forms via brine rejection during sea-ice formation and cools and mixes during sub-ice shelf circulation. Hillary Canyon, adjacent to Iselin Bank, forms a major conduit for RSBW, which flows down the canyon at velocities up to 1 m s−1 and mixes with CDW to form Antarctic Bottom Water 40.Presently, the Antarctic Slope Current (ASC) flows westward along the continental shelf break and across the Iselin Bank, with average bottom-current velocities of ~0.1–0.3 msHere, we provide a high-resolution , multidisciplinary analysis of Neogene to Quaternary (<23 Ma) submarine landslide preconditioning and triggering on the Antarctic continental margin. We integrate downhole-log data with lithologic, chronologic, and seismic data recovered from the Ross Sea continental margin during International Ocean Discovery Programme (IODP) Expedition 374 to identify multiple weak layers beneath a large submarine landslide complex and provide minimum ages for these submarine landslides obtained by dating stratigraphic horizons immediately overlying submarine landslide scarps. Chronologic data indicate that long-term climatic shifts in the Neogene and Quaternary may have played a critical role in the formation of distinct lithological contrasts that form weak layers prone to failure. Our insights will inform future investigations of geohazards associated with continued climate warming and Antarctic ice retreat.2 , and ~13 km3  cross the headwalls of two exposed scarps (S1 and S2) and one buried scarp (S1b) in the southern submarine landslide region36 Figs. . Landwar36 Figs. . Limited2, has a maximum thickness of ~0.3 s TWT (~240 m) and volume of ~230 km3. The buried MTD (~4.1 s TWT), is rectangular in shape, has an area of ~50 km2 and is 0.17 s TWT thick (~136 m), indicating a minimum volume of ~7 km3.Seismic line IT17RS301 shows a thick chaotic sedimentary unit at the base of the slope, partially buried by stratified parallel subhorizontal reflections and interlayered with opaque tabular lenses and mounds, representing small mass-transport deposits , bulk density (~1.6 g cm-3), and shear strength (1.65 kg cm−2). The core scanning XRF-derived element log-ratio of silica to aluminium counts (ln(Si/Al)) shows high values (~3.35) with low Zr/Rb (~1.3) and Ti/Al (~2.1) ratios indicating high sediment opal content. Sediment grain size, XRF, natural gamma radiation (NGR) and MS data indicate bigradational grading with bioturbation and pyrite infilling observed , MS (128 × 10−5 SI) and shear strength (3.5 kg cm−2), and a decrease in porosity (~52%) relative to WL1. Grain size analysis shows that the clay fraction decreases in the overlying package from ~19% (WL1) to ~10% and that the ln(Si/Al) record declines (~3.1), while the Ti/Al (~2.45) and Zr/Rb (~2.1) records increase.The sediments immediately overlying WL1 are interbedded muds and clast-poor sandy diamicts , NGR ( ~ 27 counts s−1), bulk density (~1.5 g cm-3) and shear strength (0.6 kg cm−2) and high porosity (63%). The XRF data reveal high log-ratios of ln(Si/Al). The muddy sand bed overlying WL1b is coarser-grained  and there is a stepwise increase in MS (89 × 10−5 SI), NGR ( ~ 40 counts s−1), bulk density (~1.9 g cm−3), shear strength (1.5 kg cm−2), and decreased porosity (61%).WL1b lies beneath buried S1b and occurs within core U1523B-5F  and MS (~2070 counts) as well as moderate-high porosity (~42%). Formation MicroScanner (FMS) resistivity images show a low resistivity interval, with submeter-scale variations in low to medium resistivity, indicating interbedded diatom-bearing to -rich muds. High-resistivity spots indicate occasional clasts. Vp/Vs for WL2 is ~3.96, indicating shaly intervals with elevated porosity (see Methods). The package immediately above WL2  and MS (~2260 counts) and decreased porosity (~41%). FMS images indicate this interval has generally high resistivity, with common high resistivity spots indicative of clasts in diamict and/or gravel. The Vp/Vs ratio decreases slightly, indicating decreased porosity.WL2 is located at ~267 mbsf and its physical properties are only available from downhole-log data due to extremely low core recovery below ~155 mbsf Fig. . WL2 is Denticulopsis lauta (~13.0 Ma) and first appearance datum (FAD) of diatom Nitzschia denticuloides (13.5 Ma) in the deepest diatom bearing sample at 221.55 mbsf35. If the sedimentation rate remained constant and the age model linearly extrapolated to the WL2 depth of 267 mbsf, this surface would correlate to ~14.8 Ma. However, the validity of this assumption remains equivocal due to limited recovery 36 occur at the Iselin Bank slope base  reveal thick packages of muddy diatom ooze and diatom-rich mud Fig. , with do−1; Fig. -146) in comparable fine-grained hemipelagic muds is also low, with conditions unlikely to result in slope failure without an external trigger47. Numerical modelling shows that sediment compressibility is a critical factor in the stability of low gradient slopes46, where compressibility increases with increasing biogenic opal content48. Diatom oozes and diatom-rich muds, such as those observed in the Iselin Bank weak layers, are more compressible than siliciclastic muds; therefore the process of compaction could release excess fluid that might accumulate if bounded by a lower-permeability unit, leading to weak layer formation47. Excess pore pressure can also be generated by crushing of in-situ microfossils present in the hemipelagic sediments during compaction49, and the expulsion of intraparticle waters stored in their tests50. Water released during diagenetic alteration of biogenic silica may also result in slope failure, although this process typically occurs between 300 and 800 mbsf25, below the depth of observed Iselin Bank weak layers (between 60 and 270 mbsf) and the opal CT transition zone at IODP continental shelf Site U1521 (~285 mbsf)35.Sedimentation rates associated with WL1 and WL1b deposition are relatively low , the mid Pliocene Warm Period (mPWP: 3.264–3.025 Ma) and the Miocene Climate Optimum (MCO: 17–14.5 Ma) where there were reduced Antarctic ice volumes, higher sea levels, warm Southern Ocean surface waters and regional open marine conditions56.Based on our revised age model for Site U1523, we suggest that abundant, well-preserved diatoms and fine-grained siliciclastic sediments that characterise diatom oozes and diatom-rich muds of regional weak layers Fig. , were de58. Within the WL1b interval (~2.93–2.82 Ma), subtropical and temperate planktic foraminifera dating to ~2.9 Ma indicate distinct warm-water incursions into the Ross Sea57. WL1 (~3.07 Ma) corresponds to the mPWP (3.264–3.025 Ma), an interval when mean temperature was 2–4 °C warmer than today59 and numerical modelling shows higher sea levels (~25 m higher than today) and reduced Antarctic ice-sheet extent61. These weak layer intervals correspond with negative foraminiferal and biogenic silica oxygen isotope data62, and peaks in the XRF data (ln(Si/Al)), a proxy for sedimentary biogenic opal content63 are coincident with deep-sea stable isotope data and models which suggest that climate was warmer and ice volume was reduced during WL1 and WL1b diatom ooze deposition65. Ice-proximal evidence from ANDRILL AND-1B in the southwestern Ross Sea indicates reduced summer sea-ice extent during peak interglacials prior to 2.58 Ma, and a significant interval of meltwater-rich ice retreat near the G17 interglacial (~2.95 Ma)60. Continental shelf deglaciation and warming may have resulted in enhanced productivity and greater accumulation of diatom ooze relative to periods of increased summer sea-ice duration before 2.6 Ma53.Analysis of the planktic foraminiferal assemblages and planktic foraminiferal and diatom geochemistry of Site U1523 provides further evidence of reduced ice volume and warmer-than-present ocean temperatures at the site when WL1b and WL1 were deposited (between ~3.07 and 2.82 Ma)35. The interpretation of gravel-rich strata above WL1b is supported by the inference of hard layers at ~46 mbsf indicated by drilling parameters35. This increase in grain size likely reflects a shift towards enhanced sediment delivery from expanded ice sheets and iceberg rafting, combined with enhanced winnowing by invigorated along-slope bottom currents as global and regional climates cooled36. Evidence from AND-1B sediments show glacial-interglacial grounding-line oscillations between 3.3 Ma and 2.0 Ma that are associated with stepwise cooling from 2.8 Ma to 2.6 Ma, a cooling trend also observed in deep oxygen records53. A significant cooling event occurred at 2.6 Ma, with increased polynya activity, sea-ice presence, and grounding-line advance leading to reduced diatom ooze deposition53.WL1b and WL1 are overlain by packages of sandy muds, gravels, and diamict Fig. 35. The i66. This interval of enhanced diatom deposition occurred at the end of the MCO when Southern Ocean temperatures were the warmest of the Neogene and ice volume was reduced69. MCO-age records from IODP Site U1521 and Deep Sea Drilling Project Site 273 recovered metres-thick sections of diatom oozes and diatom-rich muds, providing additional support for our interpretation of the Site U1523 downhole-log data67.WL2 (~14.8 Ma) is characterised by downhole-log data suggesting the presence of diatom oozes and diatom-rich muds were deposited under seasonally open marine conditions with minimal terrigenous sediment input35, this change in sedimentation reflects increased bottom-current activity leaving behind coarser-grained lag deposits consisting of IRD, glacimarine outwash, and mass-wasting deposits35. The sedimentary package corresponds to RSS-5, above RSU-4 (~14.5 Ma), based on regional seismic correlation68. Contourite mound growth and slope progradation were also enhanced from ~14.5 Ma to 8 Ma, suggesting that increased sediment discharge, down-slope flows, and bottom-current activity occurred as Antarctic ice sheets expanded and climate cooled36. This observation is consistent with western Ross Sea evidence for a major discontinuity at ~14.6 Ma coincident with a 30-m decrease in eustatic sea level and cooling of Southern Ocean bottom water temperatures70.The package overlying WL2 is characterised by a diamict-dominated sequence with ice rafted debris (IRD), suggesting an ice-proximal to glacimarine depositional environment. As seismic, lithologic and geomorphological data from the Iselin Bank suggest that grounded ice did not override Site U1523 during full glacial conditions36  are fast enough to deposit bedload and transport fine-grained suspended load71. A decrease in average current intensity (to ~0.03 m s−1) occurs over the outer shelf to continental rise, leading to sediment deposition and construction of contourite mounds36  finer grained material, consistent with observed grain size increases above the weak layers. As ice did not ground at the Iselin Bank shelf edge, increased terrigenous material in sediments overlying the weak layers likely reflects a combination of changes in the proximity of the ice margin, increased availability and transport of IRD and sediment-laden meltwater35. These environmental conditions likely reduced diatom productivity at the shelf break73.In sediment packages overlying the weak layers, changes in sediment type, shown in the elemental composition (XRF), physical properties and downhole-log data, may reflect a northward shift in the pathway and strength of regional bottom currents and the sea-ice edge due to a more northerly position of the core of easterly winds during glacials36. This may have caused over-steepening that facilitated slope failure and undercutting at the base of the slope by current scouring, as indicated by the channel observed at the continental slope base, where the present-day ASC has maximum velocity 79. Columnar blankings observed on Ross Sea continental rise seismic profiles may indicate gas-associated fluid escape36. However, no columnar blanking is observed on the shelf or slope surrounding the Iselin Bank scarps. Further, there is no evidence of methane gas or gas hydrates in regional seismic profiles, suggesting that gas hydrate dissociation was unlikely to have triggered slope failure80. Even if gas hydrates were present in this area in the past, they were likely stable at the Iselin Bank submarine landslides depths81.On the Iselin Bank, slope gradients are enhanced by locally mounded contourite geometries resulting from bottom current-driven sediment deposition Fig. 36. This ity Fig. . Contour47. Thus an external trigger was likely required to destabilise Iselin Bank sediments. Submarine landslide ages on the Iselin Bank may be correlated with broad-scale climate events: S1: < 400 kyr, S1b: ~1.72 Ma, and S2: ~ 12.14 Ma (see Methods for uncertainties). This is based on unique chronologic ties at 21.7 mbsf (C1r.3r/2n: 1.78 Ma) and 48.52 mbsf (C2r/C2An: 2.58 Ma) and FAD and LAD events (see Methods). Direct correlations between the submarine landslides and MTDs on the continental rise are limited by seismic correlation down-slope of U1523. However, regional core-seismic correlations41 show that S1/S1b may correlate with the Iselin MTD (<2.5 Ma), and S2 with the buried MTD (~11–14.5 Ma)36.Modelling studies of similar sediments and sedimentation rates show that excess pore pressure alone is unlikely to result in slope failure82. S1b and S2 follow the Plio-Pleistocene transition (3.2–2.6 Ma)53 and middle Miocene Climate Transition (~14 Ma)66. Direct evidence of major ice sheet advances across the Ross Sea continental shelf at these times are unequivocally provided by drill sites on the continental shelf69, followed by periods of extensive ice sheet retreat as climate rebounded in the late Miocene and early Pleistocene83. The S1 event postdates the Mid-Pleistocene Transition , low density diatom-rich weak layers are deposited. During periods of climatic cooling , more proximal grounded ice increases erosion and terrigenous sediment supply to the outer shelf. On Iselin Bank, diamicts deposited above diatom-rich weak layers preconditioned its slope to fail. Glacial retreat, glacio-isostatic readjustment and unloading may also have led to rapid local uplift82, increasing earthquake frequency19 that could trigger slope failure via liquefaction of weak layers when bounded by lower permeability layers84.We propose that submarine landslide triggers on the Iselin Bank may be associated with changes in sedimentation following periods of climatic deterioration and/or rapid local uplift following glacial retreat, glacio-isostatic readjustment, and unloadingnce Fig. . This fo65, preventing the build-up, and subsequent failure, of weak sedimentary packages over multiple glacial cycles, resulting in over-compacted, lithologically uniform and overall stable depositional sequences. The conditions that led to recurrent failure on the Iselin Bank are likely to be present in other high-latitude margins, suggesting that these margins are likely unstable and may pose a significant hazard risk43. Our results emphasise the need for future research aimed at understanding the extent and presence of weak layers around Antarctic margins and highlight increased risk of slope instability with ongoing and future rapid warming and ice retreat.Our study has global implications for large-scale mass-wasting events, as biogenic opal deposition is prevalent in the Southern Ocean and circum-Pacific upwelling zones. However, Iselin Bank’s unique environmental setting may explain the region’s abundance of submarine landslides. Here, continuous along-slope sediment deposition of relatively uncompacted diatomaceous sediments occurs alternating with mud, sand, and gravel-rich sediments. Bathymetric, seismic and core data show that scarps occur at similar depths along-slope and are likely coeval, with weak layers extending >15 km along-slope beneath the scarps  on the North Scotia Ridge generated tsunami waves ranging from 1-40 m amplitude85. Similarly, the Grand Banks submarine landslide (185 km3) caused 3–8 m wave amplitudes5 and the Storfjorden submarine landslide, Norway (40 km3) caused 1–2.5 m wave amplitudes86. Recent studies demonstrate teleconnections between Antarctica and Southern Hemisphere landmasses, with centimetre-to-meter scale tsunami waves reaching Antarctica within hours of generation9. These teleconnections show that tsunami waves originating from Antarctica have the potential to reverse these pathways.Our study also has implications for predicting submarine landslide frequency and occurrence, the potential for submarine cable breaks, and global tsunami risks. Tsunami wave characteristics are largely controlled by submarine landslide volume, slope gradient, and submergence depthRecurrent slope failure occurred along >100 km of the Iselin Bank slope during the Neogene. Core-log-seismic data show that three submarine landslides occurred above recognisable weak layers characterised by distinct packages of water-rich diatom oozes overlain by dense, water-poor coarse-grained diamicts. Sedimentological and chronologic analyses indicate that past warm climate events, including the MCO and the mPWP, led to enhanced diatom deposition. The weak layers are overlain by low permeability glacigenic sediments likely deposited by down-slope transport of glacimarine outwash, debris-rich icebergs, and winnowing by along-slope currents. We propose that the combination of undisturbed packages of weak, water-rich sediments overlain by dense coarser grained glacigenic sediments increased pore pressure in the biogenic weak layers making the slope susceptible to failure. While the ages of the weak layers correspond to warm climate intervals, the ages of the submarine landslides are not correlated to specific climate events. The trigger required to destabilise the slope was most likely seismicity generated by glacial loading or unloading during periods of rapid marine-based ice-sheet retreat. This study has important implications for understanding the influence of global climate changes on factors influencing slope failure. Given increasing interest in deploying submarine cable networks to Antarctica, similar morphological settings should be prioritised for study elsewhere around Antarctica’s margins to better understand the causes and consequences of large submarine landslides. This is especially pertinent given predictions of future rapid global warming.Explora. Bathymetric data were acquired using a hull-mounted 12 kHz Reason SeaBat 7150 and 8111 MBES and were processed using PDS2000 and Caris Hips and Sips v8.0 to a 30-m cell size. Sound-velocity data were acquired from two sound velocity probe stations. ArcGIS v10.7 was used to create a slope-gradient map (◦) with a 30-m cell size from the bathymetric data. Single-channel seismic-reflection lines IT17RS301, IT17RS302, IT17RS303, IT17RS316 and IT17RS315 were acquired using a linear array of two 210 cu.in GI guns spaced 2 m apart, towed at 4 m water depth, and shot in harmonic mode. The vertical resolution is ~3 m based on a frequency of 130 Hz and sound velocity of 1600 ms−136. A shot point interval of 8 s was used, corresponding to a shot distance of 13–15 m. A 9.5 mini-streamer receiver array was towed 35 m from the source, which included 10 hydrophones spaced 0.625–1 m apart. Processing and interpretation were done using Schlumberger Vista and Petrel software and included bandpass filtering, deconvolution, horizontal stacking of the 10 traces to produce a single channel configuration, amplitude gain normalisation and time-migration.Multibeam echosounder (MBES) and high-resolution single-channel seismic data were acquired during the 2017 ANTSSS and ODYSSEA expeditions on RV OGS Explora in 1980 which used a 2400-m, 58-trace streamer and 23.4 l, 24-gun source array. Seismic reflection line BGR80-009A was reprocessed for this study using the open-source Seismic Unix® software and Echos/Geodepth (Paradigm®) software. Processing included static corrections, band-pass filtering, geometrical spreading correction, automatic gain control, horizontal stacking and time migration. The vertical resolution is ~17 m based on a frequency of 23 Hz and sound velocity of 1600 m s−1.Multi-channel seismic reflection line BGR80-009A was acquired by RV OGS Explora in 1993–94 using a 3000-m, 120 trace streamer and a 74.8 l, 2 × 20-gun source array. The seismic line was reprocessed for this study using the open-source Seismic Unix® software and Echos/Geodepth (Paradigm®) software, with processing including resampling in time and space, and band-pass filtering. Seismic line IT94A127A has a vertical resolution of ~13 m based on a frequency of 30 Hz and sound velocity of 1600 m s−1.Multi-channel seismic reflection line IT94A127A was acquired by RV OGS −1 was used to calculate the dimensions of seismic features in metres, including MTDs on the continental rise (not crossing site U1523)36.A sound velocity of 1600 m sSubmarine landslide volumes were calculated by creating a digital elevation model representing the smooth pre-submarine landslide surface for each submarine landslide scarp  drilled in close proximity35. Stratigraphic correlation and the composite depth scale incorporates Holes A, B and E and is based on high-resolution  core scanning X-ray fluorescence (XRF) analysis87. High-resolution  shipboard physical property measurements were made on the recovered core material35. Measurements included: gamma-ray attenuation bulk density (2.5 cm resolution), MS (2.5 cm resolution), compressional (P-wave) velocity (2.5 cm resolution), spectral gamma ray (2.5 cm resolution) and colour reflectance and colorimetry (2.5 cm resolution). Undrained shear strength measurements were made using a handheld Torvane. Moisture and density analyses measured wet and dry bulk density, porosity, and grain density on discrete ~10 cm3 sediment samples every ~75 cm down-core. Post-expedition core scanning XRF measurements were made using the Avaatech XRF fluorescence core scanner at the Gulf Coast Repository (1–2 cm sampling resolution). Elemental ratios Zr/Rb, Ti/Al and ln(Si/Al) were used as proxies to show variations in grain-size and sediment opal content88.Site U1523 was drilled in 828 m water depth on the outermost Ross Sea continental shelf during IODP Expedition 3743 subsample was dry sieved using a 1-mm mesh sieve, a 10% H2O2 solution was added to remove organic material and the samples were left overnight in a water bath at 60 °C. A 10% solution of sodium hexametaphosphate was added and ultrasound applied before measurement. To analyse the coarse fraction (>1 mm), subsamples of 1 cm3 were manually dry-sieved in a nested stack at half-phi intervals from 1.4–16 mm. Grain-size data were analysed using GRADISTAT software according to the following grain-size intervals: very fine-coarse sand (1 mm–62.5 μm), silt (62.5–4 μm) and clay (<4 μm). The volume mean grain-size diameter is presented . These data alongside post expedition core descriptions have been used to refine the high-resolution stratigraphic columns presented here, using the same classification scheme as ref. 35.Grain size of the <1 mm fraction was measured every 15 cm down-core using a Malvern Mastersizer 2000 laser particle size-analyser. Each 5 mm35. Down-hole acoustic porosity was derived indirectly from the sonic log . Vp/Vs was derived from the ratio of compressional to shear wave velocity. The log curves were depth matched using the total gamma-ray log from the triple combo tool string and depth-shifted to the seafloor35. The data were processed at Lamond Doherty Earth Observatory following standardised procedures, including removing depth offsets between logging runs, data conversion and image creation35. Data were visualised using Schlumberger Petrel and Techlog software. Discrepancies in the extent of individual tool measurements downhole are due to different tool positions along the ~50 m-long tool string35.Two downhole-logging runs were carried out in Hole U1523D using a modified triple combo and FMS-sonic tool strings35.Downhole-log data were combined with core analysis from Holes U1523A, B and E and seismic-reflection data. Lithostratigraphic and core measurement depths were converted from depth in metres to two-way-travel time to create a depth-travel time relationship using the Petrel software package, using P-wave calliper point-source velocity data from physical property measurements and in-situ and continuous sonic velocity data from the Dipole Sonic Imager collected during downhole-logging operations35, and two magnetic polarity reversals35 were also identified shipboard  and Last Appearance Datum (LAD) events are plotted on Supplementary Fig. 35.A series of four holes at Site U1523 resulted in the development of a composite section−1 (~72% core recovery). The lower interval between 21.70 and 48.52 m CSF-A is interpreted to represent the time interval from 1.78 Ma to 2.58 Ma, with an average sediment accumulation rate of 3.352 cm kyr−1 (~52% core recovery).Two segments of a linear age-depth line of correlation are presented in Supplementary Fig. Most of the samples examined for diatom biostratigraphy contain poorly preserved diatoms with an abundance of inferred and obvious reworking. Of note are two diatom-rich intervals with excellent preservation at 61 and 62.1 m CSF-A. In selecting the depth of FAD and LAD events, preference was given to the biostratigraphic information derived from the discrete, rare, and discontinuous samples that yielded rich and diverse diatom assemblages. These were interpreted to result from primary sedimentation from surface waters to the seafloor, with only minimal modification by glacial and transport (reworking) processes . These rich diatom-rich samples are inferred to reflect seasonally open marine conditions above Site U1523.35.The coincident occurrence of numerous biostratigraphic LAD and FAD events at the boundary between Lithological Units II and III indicates the presence of a disconformity spanning the time interval from 8.2 to 4.1 Ma. The position and slope of the segment of the age-depth correlation in Unit III is transferred from that of the shipboard age model35. The minimum submarine landslide ages are based on the ages of the post-failure reflectors  traced into the undisturbed sedimentary sequence to Site U1523. The U1523 age model, alongside the published record from ANDRILL60, were used to constrain the weak layer and submarine landslide ages.The minimum weak layer ages are based on core-seismic correlation by tracing the horizons directly beneath the submarine landslide scarps into the undisturbed sedimentary sequence to Site U1523. Where an age range is presented, this represents the minimum-maximum weak layer age range, based on dating constraints Fig. 35. The m35. Uncertainty is also introduced during core-seismic integration due to seismic resolution  and low sediment deposition rates (e.g. 1.219–3.352 cm kyr−1; Fig. 20. We use the U1523 age model tie points closest to each of the submarine landside scarps to calculate the rate of sediment deposition of the overlying deposits (Fig. Chronologic uncertainty intervals for the weak layers and minimum submarine landslide ages are shown on the U1523 age model as vertical lines highlighting uncertainty Fig. . UncertaSupplementary Material


It has been widely recognized that site-specific attributes such as infrastructure, personnel, and participant recruitment-related factors are critical to effective and successful conduct of clinical trials –4. IndusAdoption of site readiness practices aims to improve sites’ success in initiating and sustaining clinical trials and streamlining operations. Nevertheless, there are several potential challenges sites could face in implementing the site readiness practices. With robust centralized infrastructure and sophisticated personnel expertise, Clinical and Translational Science Awards (CTSA) hubs are well-positioned to lead the adoption, documentation, maintenance, and further evaluation of site readiness practices. Furthermore, the framework defines three cross-cutting principles essential for site readiness: a culture of quality, clinical research literacy, and person-centeredness. The work of the CTSA hubs inherently encompasses these qualities, further priming them for leading these efforts in their institutions. In this paper, we highlight several anticipated challenges, opportunities, and recommendations to assist CTSA hubs in planning for and implementing site readiness practices for clinical trials.The process of standardizing operations as required to implement the site readiness practices will present challenges and is likely to face adoption delays and covert or frank opposition. Often, at large academic medical centers — where CTSA hubs are typically based — there is variability in standards and processes between various clinical units, departments, and locations. This variability is even greater in healthcare systems that often have multiple facilities and practices, sometimes with distinct governance. However, the CTSA hubs’ focus on innovation, efficiency, and quality, as well as experience in enabling enterprise-wide collaborations, allows them to understand existing differences in processes and to identify optimal local standards. Additionally, CTSA personnel are likely to be viewed as more neutral than personnel affiliated with a specific department or practice, and therefore potentially more successful in identifying suitable standards and subsequently implementing standardized processes that have historically been characterized by wider variability.Implementing the site readiness practices may cause some disruption for site personnel as operations are standardized and processes change. CTSA hubs can minimize this disruption through their leadership across institutions, education, celebration of local successes, engagement of early adopters, and through the expansion and dissemination of existing resources or via training programs developed and vetted as part of the CTSA infrastructure.The process of working across complex organizations to identify and define promising approaches to adoption will require significant and persistent effort. Simply documenting information from various units or locations will require coordinated effort, while working to establish  new standards will require considerably more. CTSA hubs can leverage the advantage of existing personnel resources and technical infrastructure to refine approaches to adoption and document the site readiness practices in a centralized and accessible location, according to a set of defined organizational standards and principles of team science, including stakeholder engagement and shared decision-making.de novo . Once identified or established, the site readiness practices will need to be documented and continually reviewed for appropriateness, accuracy and optimal use, especially given the rate of change across the national clinical research enterprise and local conditions. In order to realize optimal efficiency and ease in developing and maintaining site readiness practices, we make the following recommendations for CTSA hubs:Develop institutional standard operating procedures (SOPs) based on federal and local regulations according to the domains defined in the site readiness practices;Create a standardized template for work instructions that individual units or locations may implement to supplement institutional SOPs;Develop a robust SOP implementation plan that includes access to resources and trainings beneficial to study teams as they transition to new standard procedures;Establish a rigorous review schedule and plan to appropriately update and maintain the SOPs; andHarmonize documentation of and access to the site readiness practices across the institution.Most of the recommended site readiness practices are policies and standardized procedures based on regulatory requirements. Some of the site readiness practices likely already exist , but may need to be harmonized across locations or units. Other site readiness practices may need to be established Close engagement with leadership of the University, healthcare system, and/or School of MedicineAlignment with institutional compliance offices Collaboration with the healthcare system, especially spanning distinct sitesGiven that CTSA hubs are established and supported in various manners across institutions, some may be better-positioned to lead this charge. There are several factors that we believe will bolster the ability of CTSAs wishing to implement the site readiness practices across large and complex institutions:Implementation of the site readiness practices to demonstrate a site’s ability to conduct a clinical trial may be challenging for diverse and complex institutions. However, the anticipated benefits of increased engagement with clinical trial sponsors, greater trial opportunities for patients and communities, reduced burden for study teams related to initiating and executing clinical trials, and improved efficiency and quality are substantial.Given the collaborative nature of the CTSA consortium and its rich expertise in all aspects of clinical trial design and conduct, implementation of the site readiness practices by CTSA hubs offers the unique opportunity of additional value and efficiency through sharing methods and feedback over time. With engagement from CTSA hubs, the site readiness practices may be further refined, and novel methods and processes for demonstrating and maintaining the readiness factors may be developed and shared across institutions.


Escherichia coli, Staphylococcus aureus, and Sphingobacterium multivorum. Varying dose, core composition, surface coating, and bacterial species revealed that nanoparticle surfaces accounted for most of the variation in antibacterial activity. In all experiments, dose produced a linear inhibitory effect. Surprisingly, bacterial species reacted similarly regardless of evolutionary relatedness. There is a high degree of consistency, effectiveness, and efficacy among PVP silver and copper nanoparticle. These findings have implications for the intentional use of nanotechnology in environmental systems.Nanoparticles have been proposed as tunable delivery vehicles for targeted treatments and, in some cases, the active therapeutic agents themselves. Despite the promise of such customizable impacts, little evidence exists to support these claims in the realm of antibiotics. Exploration of the silver and copper nanoparticle antibacterial impacts have been reported with inconsistent results. Here, we investigate the physical, chemical, and bacterial properties of silver and copper core particles stabilized with commonly used surface coatings, namely, polyvinylpyrrolidone , cetrimonium bromide , citrate , and ascorbic acid (AA, negatively charged surface for copper nanoparticles. The impacts of these potential antibacterial nanoparticles are measured against three bacterial species spanning deep divisions in the bacterial tree of life and include  Targeted vehicles are needed to chaperone therapeutics through biological systems as diverse as humans and plants for the treatment of disease. Nanoparticles can serve as a customizable platform for the delivery of payloads and in some cases, the particles themselves can also function as the active therapeutic agent . For exaAn understanding of the interactions between nanoparticles and bacteria is required to characterize the antibiotic effects. Because of the particle’s size, the interaction often occurs with the cell wall of bacteria. The interaction is dynamic involving the surface of the nanoparticle with distinct properties and the fluidic nature of the cell’s surface made of lipids, receptors, and/or cellulose . The intvia electrostatics , Staphylococcus aureus (Firmicutes), and Sphingobacterium multivorum (Bacteroidota). The findings show that nanoparticles can be customized for diverse antibacterial strategies.The study summarized in this paper investigated the antibacterial properties of silver and copper particles stabilized with citrate (Cit) and ascorbic acid (AA), respectively, to confer a negatively charged surface; polyvinylpyrrolidone (PVP) to confer a neutrally charged surface; and cetyltrimethylammonium bromide (CTAB) to confer a positively charged surface. Three bacterial species representing deep divisions in the bacterial tree of life were used for endpoint analysis: Reagents. Trisodium citrate  and polyvinylpyrrolidone  were purchased from Alfa Aesar  and silver nitrate  was purchased from Ricca Chemical Company . Sodium hydroxide , copper (II) nitrate trihydrate (Cu(NO3)2·3H2O, CAS# 10031-43-3), ethanol , and cetyl trimethylammonium bromide  were purchased from Sigma-Aldrich . L-ascorbic acid , copper (II) chloride  and hydrazine hydrate  were purchased from Acros Organics . Mueller Hinton agar was purchased from Oxoid Ltd. , while Mueller Hinton broth, Nutrient agar, Nutrient broth and 0.5 McFarland standard were purchased from Remel (Thermo Fisher Scientific). Bacterial culture Escherichia coli (E. coli) 25992 and Staphylococcus aureus (S. aureus) 6538 were purchased from Microbiologics . Sphigobacterium. multivorum (S. multivorum) 5,011 was collected from Mexican fruit fly colonies.Nanoparticle synthesis. Citrate coated silver nanoparticles were synthesized via trisodium citrate (10 mL of 1%) added dropwise to silver nitrate (500 mL of 1 mM) under vigorous magnetic stirring for 1 h at 150°C until a grey-colored particle suspension, termed ‘Cit-AgNPs’, was observed (via silver nitrate (2 mL of 5%) added dropwise to PVP (100 mL of 2%) under vigorous magnetic stirring at 100°C (observed . Similarat 100°C . The reavia silver nitrate (50 mL of 0.01 M) added dropwise into CTAB (50 mL of 0.01 M) under vigorous magnetic stirring until a milky white opalescent suspension slowly formed. In a separate reaction vessel, sodium hydroxide (50 mL of 0.01 M) was added to glucose (25 mL of 5.0 mM) under vigorous magnetic stirring (Cetyltrimethylammonium bromide coated silver nanoparticles were synthesized stirring . Solutiovia ascorbic acid (250 mL of 1.0 M) added dropwise to copper chloride (250 mL of 10 mM) while being stirred vigorously at 80°C for 17 h until the formation of brown-colored particle suspension, termed ‘AA-CuNPs’ was observed (via 1:1 ratio by volume of freshly prepared PVP (0.8 M) and copper (II) nitrate trihydrate (0.01 M) and stirred at 45°C for 3 h until the formation of pink-colored particle suspension, termed ‘PVP-CuNPs’ (Ascorbic acid coated copper nanoparticles were synthesized observed . SimilarP-CuNPs’ .via 1:1 ratio by volume of copper (II) chloride  and CTAB (0.01 M) mixed with hydrazine hydrate (0.08) (Cetyltrimethylammonium bromide coated copper nanoparticles were synthesized e (0.08) . SolutioNanoparticle spectroscopic characteristics. Optical absorption spectra of the synthesized nanoparticles were examined using ultraviolet/visible spectroscopy  to confirm the presence of nanoparticle colloids. The surface coating was confirmed via Fourier-transform infrared spectrophotometry . Nanoparticle surface charge was assessed using zeta potential measurements collected via dynamic light scattering .In addition to spectroscopy, morphological characteristics were observed using microscopy. Transmission electron microscopy (TEM) was performed on JEOL JEM-1010 TEM  at an accelerating voltage of between 10 and 100 kV. Briefly, a drop of the prepared particle suspension was added to the surface of carbon-coated copper grids  and allowed to dry in a hot air oven set at 160°C. Atomic force microscopy (AFM) was performed using Bruker Dimension Icon Atomic Force Microscope  in tapping mode.Antibacterial assay. The antibacterial efficiency of the nanoparticles synthesized were tested against E. coli, S. aureus, and S. multivorum using the agar disc diffusion method  to approximate a normal distribution. Values were transformed for meaningful presentation.After incubation, the zone of bacteria growth inhibition around each disc was measured to the nearest millimeter. Zone of inhibition is traditionally measured as the diameter of the zone defined by no bacterial growth on a bacterial lawn. That is, the measurement is taken from the edge of bacterial growth on one side through the filter paper disk to the edge of bacterial growth on the other side. Zero inhibition is indicated by bacterial growth directly at the edge of a filter paper disc soaked in antibacterial agent being tested. Because the zone of inhibition includes the diameter of the filter paper disc, this classification system results in a distribution of inhibitory diameters that is not continuous. That is, the smallest value is zero and the second smallest value is larger than the disc size with no possible values between those two points. To better fit a continuous distribution, we subtracted the diameter of the filter paper disc from the diameter of the zone of inhibition measurements. Furthermore, because the data were bounded by zero and positively skewed, we used a square root transformation  to fit a model that includes the following independent factors: nanoparticle core, surface coating, dose, and species. Comparison of means between levels of each factor was performed using Tukey HSD test (α = 0.05). To estimate the amount of effect of each independent factor , we calculated eta-squared, η2, using the formula©, Version 13 .which indicates large effects at 0.14, medium at 0.06, and small effect sizes at 0.01 . All sta). Atomic force microscopy (AFM), a type of scanning probe microscopy, has a demonstrated resolution on the order of fractions of a nanometer and was used to measure the height (z-axis) of each nanoparticle suspension  confirmed particle dimensions in the x-y plane and indicates the nanoparticle’s propensity to agglomerate . Atomicspension . Dynamicdnp of 64 nm (dcore of 48 nm) and 57 nm (dcore of 45), respectively. The thickness of the citrate coating is 8 nm while the thickness of the ascorbic acid coating is 6 nm. PVP-AgNP and PVP-CuNPs have a dnp of 120 nm (dcore of 81 nm) and 127 nm (dcore of 89n m), respectively. The thickness of the PVP coating ranges 19–20 nm. CTAB-AgNP and CTAB-CuNPs have a dnp of 59 nm (dcore of 28 nm) and 49 nm (dcore of 19 nm), respectively. The thickness of the CTAB coating ranges 15–16 nm. All nanoparticles appear spheroidal in shape. Zeta potential data show that Cit-AgNPs and AA-CuNPs have negatively charged surfaces (−23.9 mV vs. −26.0 mV), while PVP-AgNPs and PVP-CuNPs have values close to 0 (−5.84 mV vs. −0.57 mV). Zeta potential data show that CTAB-AgNPs and CTAB-CuNPs have positively charged surfaces (+18.2 mV vs. +18.6 mV).When inferring particle size from both TEM (to measure the core) and DLS (to measure the entire nanoparticle system), the size data shows trends. Cit-AgNP and AA-CuNPs have a Ultraviolet-visible (UV-Vis) spectroscopy was used to confirm the presence of silver and copper nanoparticles in the suspensions (data not shown). All six nanoparticle suspensions produced a single peak indicating one distinct size population per sample. The UV-Vis absorbance peaks of the nanoparticles were observed between 300 and 450 nm . The absFourier-transform infrared spectroscopy (FTIR) was used to verify the surface coating by identifying chemical composition . This inEscherichia coli (Proteobacteria), Staphylococcus aureus (Firmicutes), and Sphingobacterium multivorum (Bacteroidota). E. coli is Gram-negative bacterium and is a common human bacterial pathogen. S. aureus is a Gram-positive bacterium and is the leading cause of soft tissue infection. S. multivorum is a Gram-negative bacterium and grows in antiseptics and disinfectants. Each of these bacteria species were used in the Kirby-Bauer disk diffusion susceptibility test.Three bacterial species representing deep divisions in the bacterial tree of life were used for endpoint analysis: The disk diffusion susceptibility method tests the effectiveness of antibiotics on a specific microorganism . The zonE. coli, S. aureus, or S. multivorum at any concentration tested  were assessed on the size of the bacterial zone of inhibition. There was significant variation between groups; the statistical model explained a large proportion of variation in bacterial inhibition  = 97.31, p < 0.0001, R2adj. = 0.91) . However, the proportion of variation explained by each factor, that is, the size of the effect , differed greatly. Overall, surface coating had the largest effect on bacterial inhibition  = 397.28, p < 0.0001, η2 = 0.11).Antibacterial sensitivity tests showed that Cit-AgNPs did not inhibit growth of n tested ; Table 1 = 0.91) . Each fa = 223.64, p < 0.0001, η2 = 0.09). Significantly greater inhibition occurred at the 8 nM dose (least squares mean ± SD: 8 nM = 2.87 ± 0.0005 mm) and significantly reduced inhibition occurred at the 1 nM dose (least squares mean ± SD: 1 nM = 0.89 ± 0.0005 mm). However, the 2 and 4 nM doses did not differ from each other . While statistically significant, core and species alone had only a slight effect on inhibition  = 25.71, p < 0.0001, η2 = 0.003 and F = 3.76, p < 0.02, η2 = 0.001, respectively).Tukey’s HSD (honestly significant difference) test identified significant differences between the three different nanoparticle surface coatings with bacterial growth inhibition increasing with zeta potential values . Dose explained a moderate proportion of variation in bacterial inhibition with  = 201.01, p < 0.0001, η2 = 0.05). Cit-AgNPs induced almost no inhibition in bacterial growth while PVP-AgNPs and CTAB-AgNPs induced significantly greater levels of inhibition . All CuNPs, on the other hand, induced bacterial inhibition, but PVP-CuNPs induced significantly less inhibition than AA-CuNPs and CTAB-CuNPs .Interactions among the main factors were statistically significant . Core inE. coli, PVP-AgNPs showed a relatively constant zone of inhibition over the dosing concentrations , while the zone of inhibition within CTAB-AgNP treatment exhibited the greatest inhibition at the lowest concentration tested. The zones of inhibition within all CuNP treatments where greatest at the highest concentration. Most notably, the mean zone of inhibition within AA-CuNP treatment is 4.53 mm at 8 nM and within PVP-CuNP treatment is 3.39 mm at 8 nM, which are some of the largest inhibition zone mean values observed in this study. Dose influenced the relationships between surface coating and core with inhibition. In S. aureus. PVP-AgNPs zone of inhibition increased gradually from 2.88 to 7.61 mm as the concentration increased. CTAB-AgNPs zone of inhibition slightly increased from 3.88 to 4.88 mm when the concentration reached 8 nM. Cit-CuNPs showed no growth inhibition against S. aureus at the lowest concentration tested, with little growth inhibition at 2 and 4 nM concentrations (1.42–1.49 mm), but this drastically increased to 10.23 mm at 8 nM concentration.All particles tested except for Cit-AgNPs showed a dose-dependent inhibition against the growth of S. multivorum, were also relatively constant, ranging 1.0–2.53 mm and 2.74–4.0 mm over the dosing concentrations , increasing only slightly as concentration increased. Bacterial inhibition by CTAB-CuNPs and AA-CuNP were dose-dependent with the highest zones of inhibition  produced at the highest concentration. No growth inhibition was observed by PVP-CuNPs at the lowest concentration; however, a mean zone of inhibition of 3.4 mm was observed at 2 nM, with decreasing growth inhibition as concentration increased. Most notably, the mean zone of inhibition after AA-CuNP treatment was 8.15 mm at 8 nM and after PVP-AgNP treatment is 2.53 mm at 8 nM.The antibacterial action of PVP-AgNPs and CTAB-AgNPs against the growth of Nanoparticle-induced inhibition of bacterial growth can be thought of from three different perspectives: the influence of the surface coating, the core, and the species. When reviewing the effects of surface coating on inhibition, overall CTAB performed the best. CTAB-AgNPs and CTAB-CuNPs induced inhibition (at 1 nM) and showed a consistent dose-response (up to 8 nM) in all bacteria species tested. PVP also performed well in that a high degree of consistency, effectiveness, and efficacy was observed between PVP-AgNPs and PVP-CuNPs. These nanoparticle systems were mostly inhibitory (at 1 nM) and showed a gradual dose-response relationship (up to 8 nM).In terms of core, the suite of copper nanoparticles produced greater zones of inhibition as compared to the suite of silver nanoparticles. AA-CuNPs produced the highest zones of inhibition among all nanoparticle systems and all bacteria species used in the study. S. aureus and S. multivorum) than between the two Gram-negative species (E. coli and S. multivorum).Each nanoparticle system induced remarkably similar patterns of responses in all three bacterial species. While species identity did influence the degree of inhibition, it did so mainly through interactions with other variables and the size of this effect was substantially smaller than the effect of surface coating or dose. Moreover, the peptidoglycan layer of Gram-positive bacteria has been hypothesized to influence the impact of nanoparticles on bacterial growth . Howeveri.e., animals, plants, fungi) as well as on other nanoparticle organic (or inorganic) surface coatings that may induce inhibition. The variable that explained most variation in bacterial growth inhibition was nanoparticle surface coating. This result provides guidance on the design of nanoparticles with specific antibiotic results in mind. Citrate coated silver nanoparticles might be chosen as a drug delivery vehicle when preservation of bacterial communities is needed. Alternatively, when bacterial inhibition is the goal, CTAB coated copper nanoparticles would be preferred. This critical finding offers a path forward for the long-promised goal of designer nanoparticles.Ultimately, the similarity in species’ responses suggests that the interactions between nanoparticles and bacteria impact cellular structures, or alter cellular processes, that are highly conserved across the bacterial tree of life. Further exploration should focus on similarities of response among more diverged branches of the tree of life (Previous studies have demonstrated antibacterial properties of silver and copper in a variety of different formulations . ResultsThe patterns that emerged among the six nanoparticle systems and the three bacteria species are unique between the metal core and the surface coating but consistent among bacterial species. Each species responded similarly to each surface coating independent of the metal core. Given the distant evolutionary relationships between these species, we hypothesize that these molecules impact anciently evolved cellular pathways and, thus, predict that these effects may be universal among bacteria. Such ancient traits may be subject to strong purifying selection and thus resistant to selection imposed by antimicrobials The emergence of patterns such as these demonstrate the utility of systematically investigating the antibacterial properties of nanoparticles.The findings have implications for the intentional use of engineered nanoparticles in environmental systems. Importantly, we have demonstrated that the tunability of nanoparticle-based antibacterial agents are primarily dependent on the surface features of the particle system. This may be because it is the surface of the particle that interacts with the bacteria’s cell membrane; therefore, surface interactions ought to be the focus of any design of new antibacterial materials. Of note, however, is that the data presented here reflect immediate and short-term interactions. Yet, nanoparticles are likely to degrade through time. Work is needed to explore the impacts of degradation and the timeline over which the metal core may become more important. This information will be critical to developing time-released formulations and understanding the ecological and environmental impacts of nanoparticle-based therapeutics.


A comparison between the hoof print temperature values was performed between the horses used for leisure and those used for cross-country. The studied horses were selected based on the following criteria: no alterations in posture and no muscle group asymmetry during visual examination, no lateral or medial deviation of the carpus or hock, no reaction to the flexion tests, negative reactions to the hoof tester, no lameness during walking, trotting or lunging, no anti-inflammatory medication in the last three weeks and rectal temperature between 37 °C and 38 °C. The thermal patterns of the hoof print show no difference among the four limbs and the mean temperature of the selected areas presents no significant statistical differences. Also, there was no statistical differences between the mean temperature of the selected areas from the forelimbs and hindlimbs from the horses used for leisure and those used for cross-country. Thermography can detect the hoof print on a flat surface and the mean temperature for each studied area can be proposed as a reference temperature value. There were no differences in the mean hoofprint temperature between leisure and cross-country Warmblood Horses. Further investigations are required to clarify whether there are any differences in the thermal pattern of hoof prints from other breeds or from horses with musculoskeletal conditions.The role of thermography as a detection method of the hoof print in non-lame Warmblood horses as well as its use in temperature determination in six areas from its surface was investigated in this pilot study. The study included sixty non-lame horses, and all four limbs of each horse were taken into consideration (n = 240). The selection criteria for the horses were: no alterations in posture and no muscle group asymmetry during visual examination, no lateral or medial deviation of the carpus or hock, no reaction to the flexion tests, negative reactions to the hoof tester, no lameness during walking, trotting or lunging, no anti-inflammatory medication in the last three weeks prior to examination and rectal temperature between 37 °C and 38 °C. The hoof print of each hoof was measured with the horse in the standing position, all four limbs on the ground, using a FLIR E50 thermal camera. Six areas of temperature from the hoof print were taken into consideration, and for each of them, the mean value was identified using FLIR Tools software for photo interpretation. The One-Way ANOVA test was used to test the differences between the mean temperatures obtained for each selected area from all limbs and to compare the hoof print temperature values between the leisure horses and cross-country horses. Data were statistically processed using SAS Studio. Results: Thermography can detect the temperature emitted by the hoof but the thermal patterns of the hoof print show no difference for all four studied limbs. No significant statistical differences were noticed between the mean temperatures identified for each studied area. Also, there were no statistical differences between the mean temperature of the selected areas from the forelimbs and hindlimbs from the horses used for leisure and those used for cross-country. Based on this aspect, the mean temperature of one selected area can be determined in any of the four limbs, without visible variations. Conclusions: Thermography can detect the hoof print on a flat surface and the mean temperature for each studied area can be proposed as a reference temperature value. There were no differences in the mean temperature of the hoofprint between leisure and cross-country Warmblood Horses. Further investigations are required to clarify whether there are any differences in the thermal pattern of hoof prints from other breeds or from horses with musculoskeletal conditions.Background: The field of veterinary medicine lacks information on equine thermal hoof printing, and few data on the same subject are available in dogs. In human medicine, thermography is used to detect heat emitted by the foot when it comes in contact with a flat surface to detect the abnormalities of the foot balance. The hypothesis states that the thermal pattern of the hoof print in Warmblood horses is detectable and it does not vary among the four limbs in leisure and cross-country Warmblood horses in terms of mean temperature of the hoof print surface. A pilot study was conducted to investigate the accuracy of thermography in temperature detection of the hoof print and to investigate the occurrence of possible differences in the mean value of six selected areas and whether there are any differences in the mean temperature of the hoof print between leisure and cross-country Warmblood horses. Methods: The study included sixty non-lame Warmblood horses with all limbs taken into consideration ( The use of horses has changed over the last hundred years, moving from a central role in agriculture, industry and transportation to the new social assignment which is primarily represented by their use for sporting, leisure and recreational purposes . WarmbloChanges in temperature and blood flow can be visualized by thermal imaging. However, there is no information in veterinary medicine regarding the thermal hoof print in horses , but theVarious methods of how to evaluate whether the horses are sound or show lameness are currently available; thus, the veterinarian can use clinical and lameness examination and can complete them with imaging scans  ,10,11. AIn contrast to radiography or computed tomography, the thermographic examination does not use any penetrating radiation and expensive devices. Also, the anatomical part that is examined does not receive X-rays, like in radiography; it is not placed in an electromagnetic field as in magnetic resonance imaging and does not use any radioactive substances, as in scintigraphy ,16. Thusto investigate the hoof print of non-lame horses;to calculate and propose a temperature reference value for six areas from the hoofprint surface;to check for any differences in temperature between the horses used for leisure and those trained for cross-country;to compare the results obtained in different locations.Based on the information obtained from human medicine  and veteThe hypothesis was that the thermal pattern and the mean temperature obtained from the hoof print surface will not present differences among the four limbs in leisure and cross-country Warmblood horses.The study was conducted in accordance with the guidelines of the Declaration of Helsinki, and approved by the Bioethics Commission of Banat University of Agricultural Sciences and Veterinary Medicine “King Michael I of Romania” from Timisoara with No. 51/07.06.2021.The study was performed in two private horse-riding centers (Location A and Location B) and in the Surgery Clinic of the Faculty of Veterinary Medicine from Timisoara (Location C) during the summer season of 2021. All horses were Warmblood horses: thirty-five of the horses were used for cross-country sports and fifty-two were used for leisure. The horses ranged in age from 4 to 16 years (mean 7.63 years and median 7.0 years), in weight from 510 to 615 kg (mean 545 kg) and height from 152 to 168 cm (mean 160 cm). There was no history of lameness throughout the last month prior to examination and no anti-inflammatory therapy within the last three weeks for all examined horses. The rectal temperature was between 37 and 38 °C for all horses and was measured at the beginning of the examination.Before examination, the shoe was removed and the hooves were trimmed and balanced by a skilled qualified farrier with more than 15 years of experience of normal and therapeutic shoeing, and all the horses included in the study were trimmed by the same farrier. The hooves were trimmed according to barefoot trimming principles, which involved leveling the hoof to the live sole, lowering the heels and the frog, sole with bars remaining intact . The norThe inclusion criteria were as follows: no alterations in posture and no muscle group asymmetry at visual examination, no lateral or medial deviation of the carpus or hock, no reaction to the flexion tests, negative reactions to the hoof tester, and no lameness during walking, trotting and lunging. Measured rectal temperatures were between 37 °C and 38 °C. The exclusion criteria consisted in muscle group asymmetry, lameness during walking, trotting and lunging, positive reaction to the flexion tests, positive reaction to the hoof tester.The horses were clinically examined following the agreement of two veterinarians with 11 and 15 years of experience in musculoskeletal injuries, with a lameness exam performed in accordance with the American Association of Equine Practitioners AAEP lameness system, with a 0 score for horses with no lameness under any circumstances and a 5 score for horses with non-weight-bearing lameness . MedicalThe horses were examined visually, during and through manipulative maneuvers and after the movement evaluation ,28,29,30From the eighty-seven examined horses, sixty displayed soundness and twenty-seven presented different scores of lameness; In those sixty horses selected for thermographic investigation, no vertical movements of the head or rotation of the pelvis were observed during lunging, and the horses moved symmetrically with equal movement of the head, trunk and limbs during walking, trotting or lunging on both the left and right sides; The intensity of infrared spectrum radiation in the hoof print  was evalThermographic measurements were performed in the consultation room, at the Faculty of Veterinary Medicine and in the sheds of the riding horse centers.All measurements for each studied horse were conducted observing the same environmental conditions for all examination spaces, i.e., dry, 12 mm-thick rubber floor at 18 °C, air temperature of 21 °C, humidity between 60% and 70%, and no air currents . The floSix sections of the hoofprint were considered: El1—toe, El2 and El3—hoof wall, El4 and El5—sole, El6—frog apex, El7—frog, El8 and El9—heels. The mean temperatures for the toe (El1), frog apex (El6), and frog (El7) were taken into account, and for the remainder parts, the average between the mean areas was used, so for the hoof wall it was (El2 + El3/2), for the sole it was (El4 + El5/2) and for the heels it was (El8 + El9/2) . Each coFor each temperature area from the thermal hoofprint of the hoof taken into study, the mean temperature was determined using the FLIR Tools software for photo interpretation.p > 0.05.The One-Way ANOVA test was used to test the differences between the mean temperature obtained for both the left and right hindlimbs, respectively, between the left and right hindlimbs, between left hindlimb and right hindlimb, right hindlimb and left hindlimb, as well as between all four limbs in all horses. Comparisons were made between the mean hoofprint temperatures of leisure and cross-country horses as well as between the locations where thermography was conducted. SAS Studio was used to carry out statistical analysis on the data, with statistical significance set at The hoof print was well defined on both the left and right limbs. There was an area of increased temperature in the region of the frog, the apex of the frog and the corresponding region of the hoof wall. The thermal shape of the frog was identifiable on the hoof print. The temperature of the sole’s thermal hoof print was consistent across its entire surface. The temperature in the heel area was less representative than in the other areas; The hoof print was well defined on both hindlimbs. Areas of increased temperature were detected along the frog, frog apex and hoof wall. The temperature of the frog and its apex were identifiable, having the characteristic shape of the frog. The hoof wall temperature was higher than that of the other structures and could be identified on the edges of the hoof. The temperature of the sole hoof print was even across the entire surface. The temperature in the heel area was less detectable than in other areas; n = 240) was calculated, and since there were no significant differences between the mean temperatures of the forelimbs and hindlimbs, we propose the mean temperature as a reference value for each studied area. For the toe area, the mean temperature obtained from the four limbs proposed as a reference value was 20.30 °C, with a 95% probability that this value lies in the range from 19.96 °C to 20.63 °C. For the sole area, the mean temperature obtained from the four limbs proposed as a reference value was 20.17 °C, with a 95% probability that this value lies in the range from 19.83 °C to 20.52 °C. For the frog area, the mean temperature obtained from the four limbs proposed as a reference value was 20.44 °C, with a 95% probability that this value lies in the range from 20.10 °C to 20.78 °C. For the frog apex area, the mean temperature obtained from the four limbs proposed as a reference value was 20.26 °C, with a 95% probability that this value lies in the range from 19.92 °C to 20.60 °C. For the hoof wall area, the mean temperature obtained from the four limbs proposed as a reference value was 20.12 °C, with a 95% probability that this value lies in the range from 19.77 °C to 20.46 °C. For the heel area, the mean temperature obtained from the four limbs proposed as a reference value was 20.01 °C, with a 95% probability that this value lies in the range from 19.66 °C to 20.35 °C.The mean temperature of each area of the four limbs  in mean toe temperature derived from the forelimbs and hindlimbs (n = 240). The boxplot in There were no statistically significant differences (p = 0.89) between the means obtained (n = 240) from forelimbs and hindlimbs. The boxplot in In the case of mean sole temperature, there were no statistically significant differences (p = 0.73) between the means obtained (n = 240) from fore limbs and hindlimbs. The boxplot in In the case of mean frog temperature, there were no statistically significant differences (p = 0.81) between the means obtained (n = 240) from forelimbs and hindlimbs. The boxplot in In the case of the mean frog apex temperature, there were no statistically significant differences (p = 0.81) between the means obtained (n = 240) from forelimbs and hindlimbs. The boxplot in In the case of mean hoof wall temperature, there were no statistically significant differences (p = 0.86) between the means obtained (n = 240) from forelimbs and hindlimbs. The boxplot in In the case of mean heels temperature, there were no statistically significant differences  can be taken into account as well. For further studies, we recommend comparisons of hoof prints after hoof contact with several different support surfaces. The fact that we did not perform radiological assays nor did we evaluate the changes in serum biochemical parameters to establish the possibility of a metabolic disorder or chronic inflammatory disease as a cause of lameness is also a limitation. In further studies, we recommend blood tests to obtain the full blood count and biochemistry. Hoof conformation, namely thickness of the sole and frog in horses, represent another limitation. Thus, we recommend performing latero-medial radiographs in future studies to correctly measure the anatomical structures. The final limitation that we can take into consideration is represented by the contact time of the hoof with the floor surface, so future research must include a comparison between several periods of contact time of the hoof with the floor surface. Finally, comparisons among various contact periods between the hoof and the floor surface must be included in order to obtain a comprehensive view .Thermography can detect the hoof print on a flat surface, and this study is the first investigation of the thermal hoof print in Warmblood horses. The mean temperature for each studied area of the hoof print did not vary between the forelimbs and hind limbs in leisure and cross-country Warmblood horses; thus, based on the results obtained in this pilot study, we propose the mean temperature of each selected area as a reference temperature value. Further investigations to clarify whether there are any differences in the thermal pattern of hoof prints from other breeds or from horses with musculoskeletal conditions are required.