Based on 158 existing and 351 proposed dams, we present a multi-objective optimization framework showing that low-carbon expansion of Amazon hydropower relies on strategic planning, which is generally linked to placing dams in higher elevations and smaller streams. Ultimately, basin-scale dam planning that considers GHG emissions along with social and ecological externalities will be decisive for sustainable energy development where new hydropower is contemplated.Hundreds of dams have been proposed throughout the Amazon basin, one of the world’s largest untapped hydropower frontiers. While hydropower is a potentially clean source of renewable energy, some projects produce high greenhouse gas (GHG) emissions per unit electricity generated (carbon intensity). Here we show how carbon intensities of proposed Amazon upland dams (median = 39 kg CO 2eq per year, and that if all proposed Amazon dams are built, annual emissions would increase 5-fold.Some dams produce large amounts of GHGs and it is important to see whether future dams will satisfy sustainable energy goals. Here the authors estimate the range of GHG emission intensities expected for 351 proposed and 158 existing Amazon dams and find that existing Amazon hydropower reservoirs collectively emit 14 Tg CO Globally, hydropower dam construction is expected to reach unprecedented rates in the coming decades, especially in countries with emerging economies2. One hotspot for future hydropower expansion is the Amazon5, the world’s largest river basin. Although dams have already been built in several regions of the basin, the Amazon hydropower potential remains largely untapped, and electricity generation is the primary motivation for new dam construction2. Existing evidence suggests that most global hydropower projects have total greenhouse gas (GHG) emissions per unit electricity generated  and methane (CH4) emissions to the atmosphere, and should thus be considered anthropogenic GHG sources14. CH4 is the most important GHG produced in reservoirs and originates from bacterial decomposition of organic matter in anoxic water and sediment environments created by impoundment13. GHG emissions  per unit of reservoir flooded area, or power density  can still have high carbon intensities if they produce low amounts of electricity per unit flooded area .GHG emissions from reservoirs stem primarily from the decomposition of organic matter that is either flooded, transferred to the reservoir via runoff and river input, or produced within the reservoir as aquatic plant and algal biomass22. The problem is compounded by the piecemeal nature of these studies where each project is evaluated independently without considering the integrated effect of all existing and planned dams on basin-wide emissions. Here, we use a database of GHG fluxes for existing tropical and subtropical reservoirs12 to calculate the range of carbon intensities expected for 351 proposed and 158 existing Amazon hydropower dams. To incorporate the time-related radiative forcing effect of CH4, a potent GHG with an approximate atmospheric residence time of only about a decade, we conducted analyses of carbon intensities considering 20-year and 100-year time horizons. We found that carbon intensities vary by over two orders of magnitude from the lowest to the highest emitting dam, with projects in lower elevations and larger rivers being associated with higher emissions per unit electricity generated. Using a basin-wide optimization approach, we show that strategic dam planning could minimize aggregate carbon intensity as hydropower generation expands. Our approach can be adapted to different scales and could help Amazonian countries achieve their energy goals more sustainably.Environmental impact studies for new dams rarely consider GHG emissions, especially in developing countries where hydropower is currently expanding2eq per year over a 100-year time horizon : 10–19), or ≈2% of the current total annual GHG emission from reservoirs globally12; if all 351 proposed dams are built, annual emissions from Amazon reservoirs would increase approximately fivefold , which would reduce the collective carbon intensity of the global electricity sector from the current ≈500 kg CO2eq MWh−1 to ≈80 kg CO2eq MWh−1 in 2040. Our analysis indicates that most proposed upland dams (92% for a 100-year time horizon and 60% for a 20-year time horizon) would likely result in carbon intensities below 80 kg CO2eq MWh−1 . In fact, over a 20-year time horizon about 25% of the proposed lowland dams would likely be more carbon-intensive than coal-fired power plants  World Energy Outlook 2017h−1 Fig. . By contnts Fig. .1, hydropower dams that can regulate inundation of downstream wetlands are typically those in lowland reaches, which generally implies lower power density and hence high carbon intensities for such projects. In addition, it is critical to understand whether lowland dams are more likely to create reservoirs enriched in nutrients such as phosphorus and nitrogen, which would increase aquatic primary production and consequently GHG emissions24, thereby increasing their carbon intensities.Lowland dams have significantly higher carbon intensities due to their typically larger reservoir areas and innately lower power densities, whereas the steeper topography of high-elevation areas favors hydropower projects with higher power densities. This explains why the largest number of Amazon dams with high carbon intensities occurs in Brazil, a predominantly lowland country, whereas dams with lower carbon intensities are concentrated in mountainous parts of Bolivia, Ecuador, and Peru Fig. . Notably25, which defines the set of solutions  that minimizes total basin-wide GHG emissions while satisfying varying hydroelectricity generation goals  Pareto frontier for 2351 (≈10105) possible combinations of proposed Amazon dams in very fast computational time (< 10 min) .Our findings suggest that Amazon hydropower must be developed strategically on a basin-wide scale to achieve low-carbon energy goals. We therefore performed a multi-objective optimization to determine the Pareto-optimal frontier2eq MWh−1 over a 100-year time horizon  of the total proposed electricity generation capacity while creating a portfolio of new dams with an aggregate carbon intensity below 80 kg COzon Fig. . Converszon Fig. . For inszon Fig. . Optimalnt7 Fig. . Thus, tons Fig. .Fig. 2Tr2eq MWh−1 (20-year time horizon) and ≈90 kg CO2eq MWh−1 (100-year time horizon)  Fig. . Optimalon) Fig. . After ≈on) Fig. . This woon) Fig. .Fig. 3Ch34. For instance, a study in a large tributary basin to the Mekong River, the largest river in Southeast Asia, has demonstrated that strategic planning would have allowed the exploitation of 70% of the basin’s hydropower potential while trapping only 20% of the river’s sand-sized (64 μm to 2 mm) sediment load, which is critical for downstream geomorphology, floodplain development and aquatic biota. However, project-by-project hydropower development implemented in the region has led to trapping of more than 90% of the sand load while exploiting only 50% of the hydropower potential33. Similar concepts of strategic planning have been applied to optimize dam removal strategies. For instance, a study in the Willamette River basin in the western US has shown that removing 12 existing dams would reconnect over 50% of the river network while sacrificing <2% of current hydropower and water-storage capacity28.The need for strategic planning to balance energy and water management benefits provided by dams with associated social and environmental externalities is becoming increasingly apparent21, power density is the criterion employed by the Clean Development Mechanism of the UN Framework Convention on Climate Change to finance and grant carbon credits to hydropower projects35. Projects with power densities above 4 MW km−2 are eligible for credits and GHG emissions from candidate projects with power densities above 10 MW km−2 are assumed to be negligible over 100-year horizons. While power density may provide a convenient sustainable energy metric, natural variability in GHG emissions observed in reservoirs can lead to differences in carbon intensities for dams with comparable power densities. We plotted power densities against our predicted carbon intensities to examine what densities may satisfy sustainable energy goals . For a 100-year time horizon, power densities above 6.7 MW km−2 (95% CI: 4.5–9.5) were associated with projects emitting <80 kg CO2eq MWh−1  suggests that the Clean Development Mechanism lending criterion of 4 MW km−2 avoids most carbon-intensive projects. The more conservative upper bound of the 95% CI indicates that projects are very likely to emit <80 kg CO2eq MWh−1 only when power densities exceed 9.5 MW km−2; about half of the proposed Amazon dams have power densities below 9.5 MW km−2 , information on flooded areas was unavailable. For existing reservoirs without reported flooded areas, we quantified flooded areas from satellite imagery (Google Earth Pro 7.3.2.5776). For proposed dams with missing information, we used available flooded areas as a training dataset to develop a multiple regression model including country, watershed area, installed capacity, and elevation as covariates to estimate flooded areas . We identified 351 proposed dams in various stages of inventory, planning and licensing . The proposed dams have a combined electricity generation capacity of 91,887 MW, on average 262 MW per dam (range: 1–6133 MW). Watershed areas above each dam were estimated from a digital elevation model of the region. Existing and proposed dams were categorized as upland or lowland using a cutoff of 500 m a.s.l.2. The most widely used time horizon for GWP of atmospheric gases is 100 years, but shorter time frames are particularly appropriate for interpreting the climate effects of certain activities when short-lived gases are to be prioritized. This is the case of CH4, which remains in the atmosphere for approximately a decade but has a large radiative forcing effect. Therefore, all of our analyses also consider a 20-year time horizon in addition to the commonly used 100-year time horizon. In terms of radiative forcing, CH4 is the predominant GHG emitted from hydropower reservoirs, and the general temporal pattern of GHG emissions from dams indicates that emissions peak in the first decade after damming and then fall to lower levels that remain somewhat constant over time13. Therefore, the high potential of dams to cause warming over short timescales gets underrepresented when the GHG footprint of dams is assessed only over long time horizons. We converted CH4 emissions to CO2-equivalents using a GWP of 34 over 100 years and 86 over 20 years50.To compare the radiative forcing effects of GHGs with different warming potentials and atmospheric residence times, an index termed Global Warming Potential (GWP) is typically used. The GWP measures the relative amount of energy the emission of a gas will absorb over a given period of time in relation to the same amount of CO2eq MWh−1). We combined project-specific data on flooded areas and installed capacity from our Amazon dams database with 48 CO2 and 38 CH4 published flux estimates for tropical and subtropical reservoirs12 to calculate carbon intensity ranges for all existing and proposed Amazon dams. To calculate the carbon intensity of a given dam, we first calculated total GHG flux as follows:dam is the total GHG flux (kg CO2eq d−1), with positive values denoting emission (water-to-atmosphere flux) and negative values denoting uptake (atmosphere-to-water flux); Adam is the reservoir flooded area (km2); FCO2,dam is the CO2 flux (kg CO2 km−2 d−1); FCH4,dam is the CH4 flux (kg CH4 km−2 d−1); GWPCH4 is a conversion factor for the global warming potential of CH4 over the corresponding time horizon (20 or 100 years) to transform kg CH4 km−2 d−1 to kg CO2eq km−2 d−1; Rdownstream is a constant representing the ratio of downstream emissions to reservoir-surface emissions, estimated to be 17%51. We multiplied CO2 fluxes by a discount factor of 0.25 (netCO2) and CH4 fluxes by 0.90 (netCH4) to account only for the net (anthropogenic) change in GHG emissions associated with reservoir creation (see details below). We then calculated total electricity generation as follows:dam is the total electricity generation of a given dam over a day (MWh d−1); Capdam is the installed capacity (MW), which was multiplied by 24 to obtain the energy output in 24 h and to have numerator and denominator units of Eq. (PCap is a constant representing the capacity factor (0.5727), which denotes the effective electricity generation as a proportion of installed capacity, and was derived from an empirical relationship between data in our database on existing Amazon dams. Carbon intensity  is then calculated as:construction is a constant representing the carbon intensity associated with construction and infrastructure of hydropower dams (19 kg CO2eq MWh−1 for a 100-year time horizon)7.The carbon intensity  of power sources measures the net GHG emission per unit electricity generated  and diffusion. Our bootstrapped ranges of carbon intensities therefore reflect project-to-project variability in GHG flux rates as observed for existing tropical and subtropical dams. The CO2 and CH4 fluxes measured for single dams12 were found to be uncorrelated , which allowed us to combine independently resampled CO2 and CH4 fluxes. Variation in calculated carbon intensity among dams is essentially driven by two parameters: installed capacity and flooded areas. Supplementary Fig. Uncertainty in estimated carbon intensities for proposed Amazon dams is largely influenced by variability in the GHG flux input data . Thus, 12. To assess the net change in GHG fluxes resulting from the creation of a reservoir, emissions that would have existed under pre-impoundment conditions have to be discounted from the gross fluxes. Although conceptually simple, disentangling natural and anthropogenic reservoir emissions is a complex task with limited empirical support13. A recent review suggested that it is reasonable to assume that practically all CH4 emissions from global reservoirs are new and therefore anthropogenic, whereas the majority of CO2 emissions (perhaps ≈ 75%) over a 100-year time horizon would take place even without the reservoir creation13. In our analysis, we conservatively assumed that 75% of reservoir CO2 emissions and 10% of CH4 emissions reflect natural pre-impoundment emissions, and thus we incorporated these corrections in Eq. (CO2 and netCH4). For a particular reservoir, the percentage of CH4 emissions that can be attributed to reservoir creation depends in part on the preexisting environments that become inundated; floodplains and other wetlands would have higher CH4 emissions rates than non-wetland environments53. We use the 10% estimate in our analysis because preexisting land cover information for all of the existing and proposed reservoirs in the Amazon is not available. We ran sensitivity analyses to verify how much these assumptions affect our results  can also occur in reservoirs; however, this gas was not considered in our analysis because N2O emissions generally represent < 5% of the total gross CO2-equivalent emissions from impoundments12, and because Amazon soils have naturally high rates of N2O emission, such that net increases in N2O emissions associated with dams are expected to be relatively low54.Our calculations incorporate the net change in GHG fluxes resulting from the transformation of a riverine landscape into a reservoir by dam construction. The most comprehensive review on GHG emissions from reservoirs, which we used to support our analysis, reported gross fluxes18 and therefore latitude17, with low-latitude dams generally emitting more GHG per unit area. Thus, we used flux information only from tropical and subtropical dams in the global reservoir emissions database to represent the latitudinal range of dam projects proposed in the Amazon12. Sensitivity analyses indicated that carbon intensities would not change substantially if fluxes from tropical dams only or dams from all climates (with most dams being located in northern temperate zones) were utilized instead of the subset that we adopted  for a given dam for which we predict a carbon intensity, based upon the emissions profile from an existing Amazon reservoir20.The increased rate of GHG emissions varies over the lifetime of a hydropower dam, with peak fluxes occurring in the first years after damming due to the decomposition of flooded biomass, followed by a protracted period of lower fluxes due to decomposition of soil organic matter, continuing river inputs, and new aquatic primary production2 and CH4 fluxes for operational Amazon dams in ref. 12 (n = 6). Our predictions were in reasonable agreement with observed carbon intensities .To assess the validity of our approach to generating predicted carbon intensities for Amazon dams, we compared our estimated carbon intensities against intensities calculated using reported measurements of CO23. To place proposed hydropower dams in the Amazon in a global energy production context, we used benchmarks from the IEA 2040 Sustainable Development Scenario, which portrays a decarbonized global electricity sector to meet the United Nations 2030 Agenda for Sustainable Development goals55. The IEA report suggests that a decarbonized global electricity sector should emit about 80 kg CO2eq MWh−1 in 2040, which is representative of a power mix sustained by renewables such as solar and wind power, as well as low-carbon hydropower plants. We also directly compared our calculated carbon intensities for Amazon hydropower dams against those reported for alternative energy technologies by the Intergovernmental Panel on Climate Change (IPCC), including coal-fired, combined-cycle natural gas-fired, and solar power plants7. The carbon intensities reported by the IPCC are for a 100-year time horizon. Owing to CH4 emissions, carbon intensities of natural gas and coal are at least 37 and 4% higher over a 20-year time horizon, respectively, compared with a 100-year time horizon56. We applied 37 and 4% correction factors to obtain carbon intensities for natural gas and coal over 20 years.The International Energy Agency (IEA) releases an annual report on the status and trends of global energy (World Energy Outlook), which includes carbon intensities anticipated under a range of global energy development scenariosTo analyze the tradeoffs between electricity generation capacity and GHG emissions, we computed the Pareto frontier with respect to the two criteria. The Pareto frontier is a function that identifies for a given installed capacity target the portfolio (or combination) of dams with the lowest amount of GHG emissions, or conversely, for a given GHG emission target, the portfolio of dams with the highest installed capacity. In our case, considering the 351 proposed dams in the Amazon basin, the possible portfolios of dams are: the empty portfolio that builds none of the proposed dams, 351 singleton portfolios with only one dam, 61,425 portfolios with two dams each 2eq MWh−1, whereas portfolio B has an installed capacity of 20,000 MW and carbon intensity of 100 kg CO2eq MWh−1; we say that portfolio A dominates portfolio B since portfolio A has a lower carbon intensity for the same electricity generation capacity. In Scenario 2, portfolio A has an installed capacity of 20,000 MW and carbon intensity of 90 kg CO2eq MWh−1, whereas portfolio B has an installed capacity of 18,000 MW and carbon intensity of 100 kg CO2eq MWh−1; in that case we say that portfolio A dominates portfolio B since portfolio A has lower carbon intensity and higher electricity generation capacity. In Scenario 3, portfolio A has an installed capacity of 20,000 MW and carbon intensity of 90 kg CO2eq MWh−1, whereas portfolio B has an installed capacity of 18,000 MW and carbon intensity of 85 kg CO2eq MWh−1; in this scenario neither portfolio dominates the other. The Pareto frontier is then defined as the set of all portfolios of dams that are not dominated by any other portfolio.The application of the Pareto frontier is illustrated in the following scenarios. In Scenario 1, portfolio A has an installed capacity of 20,000 MW and carbon intensity of 90 kg CO27. Our framework for computing the exact  and approximate  Pareto frontier exploits the tree structure of river networks27, extending previously proposed algorithms for single-objective optimization stochastic network design in bidirected trees58 to multi-objective optimization and computation of the Pareto frontier. In this approach, the river network is converted into a more abstract tree structure, whereby a node corresponds to a continuous section of the river uninterrupted by existing or proposed hydropower dams and an edge represents a proposed or an existing dam. This abstract tree structure is used by our dynamic-programming algorithm for the sequence of the merging and pruning of Pareto-optimal solutions.Computing the exact Pareto frontier is a challenging computational problem, referred to as non-deterministic polynomial-time hard (NP-hard) problem, which means that in the worst case the computational time increases exponentially as a function of the number of dams27. The key insight is that at a given node u, we only need to keep the Pareto non-dominated partial solutions and we can therefore eliminate suboptimal (dominated) solutions. To increase incremental pruning, we convert the original tree into an equivalent binary tree. Given a binary tree, we first compute non-dominated Pareto solutions for the two children of the given parent node u, enumerate the partial solutions from the children and consider the four possible different combinations of whether to include each of the dams associated with each edge from the children. We then compute the objective values for the different extended partial solutions and add them to the set of overall partial solutions. Finally, we remove all dominated partial solutions from this set, so that the remaining partial solutions are Pareto-optimal for the parent node. This procedure allows us to systematically explore the entire search space of possible Pareto-optimal solutions. To prevent memory overflow in response to the large number of partial Pareto solutions considered, the algorithm batches partial solutions at each node and is parallelized to speed up the approach. We do not assume spatial dependencies among reservoirs when optimizing hydropower for GHGs, but consideration of spatial dependence may be critical for other environmental criteria , and our algorithm has the ability to solve problems where spatial dependence is important to consider.The dynamic-programming approach recursively computes the Pareto-optimal partial solutions from leaf nodes up to the rootε and runs in time that is polynomial in the size of the instance and 1/ε27. The exact algorithm guarantees to find all optimal portfolios on the Pareto frontier. The approximate algorithm finds fewer portfolios but guarantees that every portfolio on the exact Pareto frontier is ε-approximately dominated by one of the portfolios on the approximate Pareto frontier. The algorithm used in our framework adapts and parallelizes a dynamic-programming based algorithm for the exact and approximate Pareto frontier. More computational details concerning our approach can be found in ref. 27, and the code is publicly available (see Code Availability section).In addition to computing the exact Pareto frontier, our dynamic-programming approach can provide a fully polynomial-time approximation scheme (FPTAS) by applying a rounding technique to the exact algorithm. The FPTAS finds a polynomially succinct solution set, which approximates the Pareto frontier within an arbitrary small factor ε-approximate Pareto frontier with 99% accuracy  for the 351 proposed dams takes 1.5 min wall-clock time  and produces 66,312 non-dominated portfolios. Except for Supplementary Fig. 105), we show only a subset of the suboptimal portfolios.Compared with previous approaches used to compute the Pareto frontiers for dam placement, our algorithm provides coverage optimality guarantees and runs faster. Importantly, we also show that the approximate version of our algorithm is guaranteed to run in polynomial time (Supplementary Fig. Supplementary InformationPeer Review FileSource Data


Once the highest PTV coverage was achieved dose escalation to a BED10Ten unselected LAPC patients were retrospectively included in the study. Two fractionation regimens were compared (5 and 15 fractions). The aim was to cover 95% of the Planning Target Volume (PTV) with a BED10In 5 fractions, 95% PTV coverage by both base and escalated doses could be achieved for one patient with PTV more than 1 cm away from OAR. 95% and 90% PTV coverage by the base dose was achieved in one and two patients respectively. In all other patients, coverage even by the base dose had to be compromised to comply with OAR constraints. In 15 fractions, 95% PTV coverage by the base dose was feasible for all patients except one. Dose escalation allowed improvement in target coverage by the base dose in both fractionation regimen whilst covering a sub-volume of the PTV with a BED1010 = 54 Gy to 90–95% of the PTV as commonly prescribed to this population. Combined with an adaptive approach, this may maximize PTV coverage by a high BED on days with favourable anatomy.LAPC patients with OAR close to the PTV are generally not eligible for hypofractionation with dose escalation. However, this planning study shows that it is possible to cover PTV sub-volumes with a BED Characterized by high dose per fraction and highly conformal dosimetry, these offer the convenience of shorter overall treatment time, reduced time off full dose systemic therapy and potentially, improved local control. Yet, the close proximity of radiosensitive organs at risk (OAR) has led to so-called ‘low dose’ SBRT becoming a favoured approach Although there have been no randomized control trials comparing CRT to SBRT, a systematic review of 19 trials suggests a median survival of 17 months with acceptable toxicity when using an SBRT approach 10 of 70 Gy increases survival from 15 months to 17.8 months (p = 0.03), whilst biophysical models suggest that this benefit can be further extended through dose escalation up to a BED10 of 100 Gy In an attempt to optimise the use of radiotherapy in LAPC, recent interest has focussed on the role of dose escalated treatments. A retrospective review by Krishnan et al.10The aim of this study is to investigate the potential for dose escalation using hypofractionated radiotherapy for a group of LAPC patients with a varying degree of OAR proximity and evaluate if there is a dosimetric benefit of moderate hypofractionation in 15 fractions compared to 5 fractions in achieving a BED22.1Following approval from the local institutional review board, CT planning scan images for ten consecutive LAPC patients treated with radical CRT (54 Gy in 30 fractions) between May 2016 and November 2017 at our institution were re-contoured. All patients had prospectively given consent for their anonymised data sets to be used for research purposes. The Planning Target Volume (PTV) was defined as the Gross Tumour Volume (GTV) plus a 5 mm isotropic margin, assuming treatment delivery in breath-hold using Active Breathing Coordinator (ABC) 2.210 = 54 Gy, based on an SBRT prescription of 33 Gy in 5 fractions Two fractionation regimens (5 and 15) were compared in this study. The base prescription dose was chosen to be equivalent to a BED1010 = 100 Gy) and 67.5 Gy in 15 fractions (BED10 = 98 Gy). The aim was to maximize the volume receiving the escalated dose whilst maintaining or increasing PTV coverage by the base dose (BED10 = 54 Gy). The maximum allowable dose to the PTV was 130% of the escalated dose.Once this was achieved, dose escalation to a BED2.3Volumetric Modulated Arc Treatment (VMAT) plans were designed according to our institutional standard for treatment on an Elekta linac with an Agility multi-leaf collimator (MLC)  with a single 6 MV Flattening-Filter Free (FFF) arc from 179° to 181° gantry rotation in Raystation 6.99 . The collimator rotation was set at 5° and the maximum MLC leaf speed was constrained at 0.6 cm/deg to limit plan modulation and increase treatment efficiency. The delivery time was restricted to approximately 100 s, such that treatment would be deliverable in 5–10 breath holds depending on patient compliance.2.4The achievable target coverage by the prescription dose was reported for all plans as well as the coverage by the base dose for the dose escalated plans. The Paddick conformity index, The overlapping volume between the PTV and duodenum, stomach, small bowel or large bowel relative to the PTV volume was calculated as:However, in some cases with little overlap, the OAR are abutting the target, therefore also limiting PTV coverage. In order to quantify this effect, the OAR proximity volume was calculated as the volume of OAR in the 1 cm periphery of the PTV divided by the total PTV volume:The Pearson’s correlation coefficient was calculated between PTV volume, 3Patients’ PTV volumes, limiting OAR, OAR = 0%) with OAR = 0.84%) with 33Gy = 83 ± 8%). PTV V33Gy was greater than 90% for patients 7 and 10  and the mean ± SD PTV coverage by the escalated dose was PTV V50Gy = 40 ± 19%.In 5 fractions, 95% PTV coverage by the base dose was achieved for patient 1    bottom.The PTV volume receiving the escalated dose was greater for 5 fractions than 15 fractions for four patients, greater for 15 fractions than 5 fractions for four patients and equivalent for both fractionation for patient 1 and 5 . Fig. 4 PTV volume, 410The benefit of a high BED for local control in LAPC has been previously demonstrated in tumours smaller than 5 cm and/or more than 0.5 cm away from GI OAR OAR and OARprox volumes leading to varying PTV coverage  although no significant correlation was found between target coverage and PTV volume. A 5-fraction regimen offers the advantage of a reduced overall treatment time and is less labour intensive in case of daily adaption. However, due to the risk of inter- and intrafractional anatomical changes, advanced adaptive technique should be used to ensure that OARs do not enter a high dose region. A 15-fraction regimen is more forgiving to interfractional errors, allows for concomitant chemotherapy and may be dosimetrically advantageous for large tumours. The longer overall treatment time may allow for response-based adaption. However, there is currently no prospective data for dose escalated 15 fraction regimens, and therefore it remains to be seen whether toxicity rates including rates of lymphopenia and immunosuppression differ between the two regimens.Despite the similar potential for dose escalation, target coverage by the base dose was more easily achieved in 15 fractions than in 5 fractions , Fig. 4.10The need to compromise the dose escalation to the PTV to comply with OAR constraints in dose escalated plans indicates that a heterogeneous dose distribution is most likely the best option to achieve a BED10 = 71.2 Gy) in 28 fractions to regions of vessels involvement with the aim to convert unresectable and borderline resectable cases to resectable 10 from 79.2 to 112.5 Gy) whilst the dose to the PTV was either 30 or 36 Gy (BED10 = 60 or 79.2 Gy). Dose limiting toxicity was not reached and eight of thirteen patients had R0 resection after SBRT.Previous studies on heterogeneous dose prescription in pancreas SBRT aiming at boosting sub-volumes of the PTV were limited to patients without duodenal involvement Gkika et al. used a de-escalation approach using simultaneous integrated protection (SIP). This involved reduced dose to PTV sub-volumes overlapping with OAR protection volumes In the present study, heterogeneous dose distributions were not prescribed but were accepted as a result of mandatory OAR constraints. Henke et al. used a similar strategy in 5 fractions 10 = 54 Gy was chosen from previous multi-institutional SBRT series 10 = 64 Gy), SBRT has been linked to better outcomes than conventional treatments It should be noted that the base dose of BEDA limitation of this study is the equivalence of OAR dose constraints in the two fractionation regimens. The constraints are not BED equivalent (α/β = 3 for GI OAR) but were based on currently recruiting trial protocols Other limitations of this study include the choice of 5 mm isotropic PTV margin. This was deemed sufficient in previous studies of residual liver motion under ABC In addition to target motion, OAR motion is an important consideration. Planning at Risk volumes (PRV) are sometimes used to ensure OAR protection Finally, the achievable target coverage by the base dose in 5 fractions and the coverage by the escalated dose in 5 and 15 fractions were all significantly correlated with the percentage of PTV volume overlapping with OAR and the proximity of OAR to the PTV. Interestingly, PTV volume did not show a correlation with target coverage in either fractionation schedule. These correlations are important as overlap and proximity may alter during treatment due to interfraction OAR motion. These changes can be capitalised upon using daily adaptive replanning to increase coverage by the prescription dose on days with favourable anatomy 510 = 54 Gy and maximize coverage by a BED10This study showed that dose escalation to sub-volumes of the PTV is dosimetrically feasible in 5 or 15 fractions for an unselected LAPC population. The proposed strategy to cover the PTV by a base dose with BED


Resistance to thyroid hormone (RTH) is a clinical disorder without specific and effective therapeutic strategy, partly due to the lack of structural mechanisms for the defective ligand binding by mutated thyroid hormone receptors (THRs). We herein uncovered the prescription drug roxadustat as a novel THRβ-selective ligand with therapeutic potentials in treating RTH, thereby providing a small molecule tool enabling the first probe into the structural mechanisms of RTH. Despite a wide distribution of the receptor mutation sites, different THRβ mutants induce allosteric conformational modulation on the same His435 residue, which disrupts a critical hydrogen bond required for the binding of thyroid hormones. Interestingly, roxadustat retains hydrophobic interactions with THRβ via its unique phenyl extension, enabling the rescue of the activity of the THRβ mutants. Our study thus reveals a critical receptor allosterism mechanism for RTH by mutant THRβ, providing a new and viable therapeutic strategy for the treatment of RTH.  •We identified a novel THR ligand that effectively binds to THRβ mutants•Structures revealed mechanisms for the RTH controlled by a key residue switch•Roxadustat retains unique hydrophobic interactions with THRβ mutants•We provide a promising approach to design THR ligands in treating RTH Biological Sciences; Molecular Biology; Structural Biology The thyroid hormone receptors (THRs) are nuclear hormone receptors regulated by endogenous thyroid hormones, including the inactive prohormone thyroxine (T4) and the bioactive hormone 3,3′,5-triiodothyronine (T3), which are critical for development regulation and metabolic homeostasis. Encoded by two different genes, THRα and THRβ are two main receptor subtypes with overlapping and differential characteristics in tissue distribution, ligand binding, and biological functions . NotablyThe negative feedback loop of the hormone active form T3 through binding to THRs plays important roles in the thyroid hormone homeostasis . As ligaIn search of novel ligands for THRs, we used THRβ LBD as bait to screen chemical libraries based on AlphaScreen biochemical assay, which determines the efficacy of small molecules in recruiting coregulator peptides to the THRβ LBD. Surprisingly, roxadustat (FG-4592), a first-in-class oral hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor  and rece3GWS)   showed t3GWS) D. Notabl3GWS)  (Nascime3GWS) . Surpris3GWS) C, sugges2H77)   revealed2H77) . Despite2H77) A. Simila2H77) B. In sumGiven the unique nature and scaffold of roxadustat as a THRβ-selective ligand, we next investigated its ability in activating THRβ associated with thyroid hormone resistance. As shown in Although all four mutant residues are located in the THRβ LBD, H435 is the only one that directly contacts ligands T3 or roxadustat, whereas the rest of the mutant residues are located outside the ligand-binding pocket, not in the range to form intimate contacts with the ligands , which mIn contrast to the reduced binding of T3 by THRβ mutants due to the loss of a critical hydrogen bond donor/acceptor contributed by H435, roxadustat retains the effective binding activity  owing toDespite the physiological and pharmaceutical importance of RTHβ, there is still specific therapeutic strategy available, partly owing to the limited understanding of the structural mechanisms for the defective ligand binding by THRβ mutants. Indeed, the lack of ligands that efficiently bind THR mutants makes the structural study of RTH difficult, given the critical roles of the ligand binding in the stabilization of THRs for biochemical and structural studies, which is evidenced by the unavailability of any apo-THR crystal structures. Here, we report the identification of an anemia drug roxadustat as a novel modulator for THRs with a therapeutic potential in the treatment of RTH, thereby providing a small molecule tool enabling the first probe into the structural mechanisms of RTH. In addition to a safe template, our structural and functional study reveals key structural features that define specific recognition of ligands by mutant THRβ and provides structural mechanisms for designing selective and potent ligands of THRβ for the treatment of RTH.The physiological function of roxadustat has been linked to HIF-PH signaling pathways. Since roxadustat also interacts with THRs, the structural mechanism may provide a basis for designing roxadustat-based compounds that can be used more specifically either for THR- or HIF-PH-regulated diseases, or for a combinatorial therapy. The beneficial and side effects arising from the cross-interaction with each target can be optimized by designing new roxadustat-based compounds with more selectivity. Further elucidation of these two disparate signaling pathways of THR and HIF should reveal specific molecular basis for pharmacological potentials of roxadustat.Notably, the allosteric regulation has been crucial in regulating receptor function and signal transduction, from inter-molecular domain interactions to intramolecular interactions among DNA , althougin vivo animal models will be the subject of future studies.Here we identified a small molecule tool enabling the first probe into the structural mechanisms of RTH and further revealed a molecular mechanism for the defective ligand binding of RTH, thus providing a structural template for designing small molecules in treating RTH. Since the study is based on biochemical and crystallographic assays, the pharmacological potentials of the ligands on All methods can be found in the accompanying


A major challenge to routine non-invasive, nanoscale magnetic imaging is the development of Hall sensors that are stable under ambient conditions and retain low minimum detectable fields down to nanoscale dimensions. To address these issues we have fabricated and characterised chemical vapour deposition (CVD) graphene Hall sensors with wire widths between 50 nm and 1500 nm, in order to exploit the high carrier mobility and tuneability of this material. The measured Hall voltage noise is in good agreement with theoretical models and we demonstrate that minimum detectable fields at fixed drive current are lowest in the vicinity of the charge neutrality point. Our best performing deep sub-micron sensors, based on a wire width of 85 nm, display the excellent room temperature resolution of 59 µT/√Hz at a dc drive current of 12 µA and measurement frequency of 531 Hz. We observe a weak increase in minimum detectable field as the active sensor area is reduced while the Hall offset field is largely independent of size. These figures-of-merit significantly surpass prior results on larger probes in competing materials systems, with considerable scope for further optimisation. Our results clearly demonstrate the feasibility of using CVD graphene to realise very high spatial resolution nanosensors for quantitative room temperature magnetic imaging. Although there are many different approaches to magnetic sensing, Hall-effect sensors have frequently been employed due to their high magnetic field sensitivities, quantitative linear response, non-invasive performance and fabrication versatility. This allows them to be used in a range of applications where other semi-quantitative and potentially invasive sensor types, such as magnetic force microscopy (MFM) cantilevers, may not meet requirements6. They are also much more compact and simple to use than the recently developed diamond Nitrogen vacancy (NV) centre magnetic microscope, which requires precisely fabricating a single crystal diamond with an NV center at the apex of an AFM tip as well as additional lasers and microwave excitation7. The fabrication of Hall probes based on nanoscale wire widths allows high spatial resolution mapping applications to be realised. This requirement is increasingly in demand due to the rapid miniaturisation of modern technologies, for example ultra-high density magnetic storage media or magnetic domain-wall racetrack memory. However, such probes have a much broader range of potential imaging applications including combined high-resolution topographic and magnetic imaging of vortices in superconductors and domains/domain walls in ferromagnetic films based on SHPM with scanning tunnelling microscopy (STM) or atomic force microscopy (AFM) surface tracking. Through the addition of field excitation coils they can also be used to perform highly local magnetic susceptometry.Magnetic field sensors are used for a very wide variety of purposes, including but not limited to; biosensing, instrumentation and process calibration as well as high precision magnetic field mapping such as Scanning Hall Probe Microscopy (SHPM) and magnetic susceptometryTo attain higher spatial resolution for these smaller scale applications, the active area of the Hall probe must be reduced while retaining sufficiently low minimum detectable fields. In Table 8. In addition, edge depletion effects make it very challenging to achieve suitably high spatial resolution devices on the order of hundreds of nanometres or below, with no Hall crosses yet demonstrated below 100 nm8. High quality epitaxial growth of InSb-based probes is challenging and active layers are typically buried ≥50 nm below the epilayer surface1. The device reported in ref.1 was also fabricated in a 320 nm thick InSb film leaving little scope for further reduction in size9. This is detrimental for applications such as SHPM, where the active layer must be as close to the sample surface as possible to achieve the highest spatial resolution. Bismuth probes have demonstrated reasonable 300 K resolutions, yet these suffer from poor chemical and mechanical stability and the reproducible growth of Bi films is challenging11, making them unsuitable for extended operation under ambient conditions.Probes based on GaAs heterostructures are the material of choice for low temperature imaging but, as reflected in Table 12. Being atomically thin it also allows the active probe to get extremely close to samples under study, enabling very high spatial resolution mapping. Looking towards the more routine fabrication of graphene-based Hall devices, chemical vapour deposition (CVD) holds promise as a facile growth technique for the scalable fabrication of large arrays of sensors14. In addition, once transferred to an appropriate insulating substrate, graphene is a much easier material to pattern at the nanoscale than alternative Hall probe materials; a lithography mask can readily be used to transfer a Hall cross pattern by etching in a simple O2 plasma. Monolayer CVD graphene is now readily available and is the obvious choice for the scaleable production of graphene-based Hall effect sensors15. Combined with a back gate dielectric such as SiO2, the carrier density and type can also be tuned, allowing an additional ‘tool’ for optimising minimum detectable fields that has not previously been available.In contrast, graphene’s low carrier density, tolerance to nanoscale pattering, mechanical and chemical stability and its unique band structure, whereby massless Dirac Fermions exhibit extremely high room temperature mobilities, make it an ideal contender for high-resolution nanoscale Hall probes19. Studies of the mobility of graphene devices with nanoscale dimensions have shown a decrease from ~3000 cm2/Vs for probes greater than 100 nm to <200 cm2/Vs for probes smaller than 20 nm. A rapid decrease in mobility due to edge scattering tends to set in below 60 nm20.Micrometer-sized graphene Hall probes have been extensively studied in recent years, showing impressive figures-of-merit, with some of the best reported probes shown in Table w, on the minimum detectable field, Bmin, and systematically investigated the impact of increasing drive currents and varying carrier density and carrier type. These critical variables have previously either been overlooked or studies have produced inconclusive results19. We establish the practical limitations for nanoscale CVD Hall sensors and ascertain the optimal measurement conditions for real-world high spatial resolution imaging applications.However, we demonstrate that the figures-of-merit of CVD graphene nanosensors significantly surpass those based on competing materials with much larger dimensions. We have studied the influence of wire width, 23, and it is widely accepted that it arises from ‘exchange noise’ due, for example, to carrier capture and release at traps in the SiO2 gate dielectric leading to fluctuations in the carrier density, n24. A second contribution arises from ‘configuration noise’ due to rearrangement of adjacent trapped charges which modify the disorder potential landscape, leading to fluctuations in the carrier mobility, µ25. However, as the measurement frequency is increased the 1/f noise power drops below the frequency-independent Johnson noise power at a characteristic noise corner as plotted schematically in Fig. The Hall voltage from our sensors contains several intrinsic sources of noise with different characteristic frequency dependencies. At low frequencies this is dominated by “1/f noise” whose power density is inversely proportional to the measurement frequency. The origin of 1/f noise in graphene Hall devices has been the subject of several experimental studieset al.26IH is the drive current, ρxx the longitudinal resistivity, α a dimensionless constant (expected to be on the order of 10−3), A is the contact-free surface area, F is a geometric factor (~1 for a cross with the aspect ratio of 5:1 used here) and f is the measurement frequency.The low frequency (below the 1/f noise corner) transverse voltage noise power limit for our 2D Hall sensors can be described using the conductivity fluctuation model proposed by L. K. J. Vandamme Rvv is the resistance between the Hall voltage contacts and T is the temperature. Comparing Eqs  varies with the sample current, moving to higher frequencies as the current is increased. As a consequence the minimum detectable field at a fixed measurement frequency will depend strongly on the current density and can be defined byRH is the Hall coefficient given byWell above the 1/f noise corner the Johnson noise power per unit bandwidth is given byVH is the measured Hall voltage and B is the applied magnetic field. Using the standard expression for the resistivity, μ, in the low frequency limit described by Eq. ) of the graphene Hall crosses were measured in 2-lead configuration across pairs of Hall voltage leads. A typical curve is shown in Fig. h ~ 1.7 × 1016–5.0 × 1016 m−2), with the charge neutrality point (CNP) up to and sometimes beyond VGS = +100 V before post-fabrication annealing.Transfer characteristics . For large sensors, that are initially closer to the thermal noise limit, Bmin starts to increase again if the current is raised beyond this optimum point6. Figure Bmin at 531 Hz as a function of JH for two different sized probes after scaling by a fractional power of n in order to collapse the data onto a single universal curve. For the 100 nm probe the same n0.1 density noise scaling is found as for the 400 nm probe in Fig. 0.4 scaling in this case. Both data sets show a decrease in Bmin as JH increases from zero, with a noticeably weaker dependence in the smallest sensor. We have quantified this by making fits to the data of the form 1/JHβ (dashed lines), and find β = 0.11 for the 100 nm probe and β = 0.41 for the 800 nm probe. In practice the Hall voltage offset saturated our low noise preamplifier at higher current densities before the regime of increasing noise levels could be accessed in the larger probe.In previous Hall probe architectures, it has been shown that Bmin in all our CVD graphene devices appears to be well-described by 1/JHβ, with values of β ranging from 0.87 for the largest Hall crosses to 0.08 for the smallest sensors studied. This is broadly consistent with our expectations that it will depend as 1/IH in very large sensors in the thermal noise limit  by more than an order of magnitudeRoff. This is the Hall voltage output when no magnetic field is present, arising due to misalignment of the Hall voltage contacts and inhomogeneous current flow in the active sensor area34. In practice it is highly desirable to minimise this to avoid saturating low noise preamplifier stages. In addition, by mixing the longitudinal resistance into the Hall voltage, the offset resistance contributes substantial new sources of noise that ultimately limit the best achievable minimum detectable fields.An aspect that is frequently overlooked in investigations of Hall sensors is the offset resistance, Figure Roff as a function of wire width in Fig. 2 substrate.The potential invasiveness of our Hall probes can be characterised by estimating the self-field using Ampere’s law. For a 85 nm probe carrying a 12 µA current, the self-field is estimated to be ~0.5 G at a distance of 50 nm, which is comparable to the earth’s magnetic field and hence relatively non-invasive. If the dominant cause of the offset resistance in our devices was linked to grain boundaries, one would expect a substantial reduction in sensors that are very much smaller than a characteristic grain size in CVD graphene (~10 µm). However, this is not supported by the data for 15 m−2, just either side of the charge neutrality point. Hall probe performance can be still further improved by increasing the drive current, leading to figures-of-merit that significantly surpass those of competing Hall sensor structures, e.g., a minimum detectable field of 59 µT/√Hz for an 85 nm wire width sensor at 531 Hz at a 12 µA drive current. Advances in highly quality CVD graphene growth and transfer methodologies, combined with the rather simple fabrication processes for deep sub-micron probes, makes them extremely attractive for high-resolution magnetic imaging applications such as SHPM and magnetic susceptometry. A dramatic improvement in the performance of exfoliated graphene devices after encapsulation is well documented and we expect to be able to achieve much better figures-of-merit in sensors encapsulated with hBN19, although achieving this with CVD graphene remains a major challenge. The introduction of these improvements should make nanoscale graphene Hall sensors the quantitative tools of choice for high resolution magnetic imaging under ambient conditions.We have demonstrated that CVD graphene can be used to fabricate nanoscale Hall sensors for state-of-the-art high spatial resolution magnetic imaging. Their minimum detectable fields can be optimised by tuning the carrier concentration with a back gate to values of about ±2.5 × 102 surface gate oxide. An optical micrograph of a CVD graphene Hall cross array and the Cr/Au inner contact leads is shown in Figure. Hall probe arrays were fabricated from CVD graphene purchased from Graphene Supermarket, which had been grown on a copper foil and wet-transferred onto a highly-doped Si substrate with a 285 nm thick SiOContact pads were defined by direct-write laser lithography in Shipley S1813 optical photoresist. The exposed CVD graphene in the developed window was then ICP-etched in an Oxygen plasma, followed by the deposition of 20 nm of Cr and 250 nm of Au by electron beam evaporation and lift-off. Inner contact leads were then defined by laser lithography, followed by the deposition of 5 nm of Cr, 70 nm of Au and lift-off.−2. After development in Tetramethylammonium hydroxide (TMAH) the unprotected PMMA and graphene were ICP-etched away in an O2 plasma. The HSQ mask was subsequently lifted-off by dissolving the PMMA layer in acetone under light sonication.To achieve the best lateral resolution, the fabrication of nanoscale Hall probes made use of a bilayer Poly(methyl methacrylate) (PMMA)/Hydrogen Silsesquioxane (HSQ) electron beam resist as an etch mask. PMMA was first spin coated at a thickness of 100 nm, and baked at 150 °C for 15 minutes. A 2% solution of negative tone HSQ from Dow corning was then spin coated to form a 30 nm thick layer and baked for 5 minutes at 150 °C. A 30 kV field emission Hitachi S-4300 scanning electron microscope (SEM) integrated with the Raith ELPHY Plus electron beam lithography system was used to pattern the Hall cross arrays in HSQ with an exposure dose of 500 µCcm35, but we find that such high temperatures lead to hardening of the gold contact pads making them very difficult to wire bond. This post-fabrication annealing step successfully reduces the extrinsic (hole) doping level, shifting the graphene CNP by up to −60V.We utilise a post-fabrication anneal in inert Argon gas at 200 °C for 6 hours to reduce extrinsic doping due to any low molecular weight contaminants adsorbed on the graphene during the fabrication process. Annealing temperatures up to 400 °C have been widely report in the literature−6 mbar to remove adsorbed water molecules and other volatile surface contaminants and all measurements were made at ambient temperature.A portion of the oxide layer on the bottom of each chip was scratched away to allow a contact to be made for the application of a back gate voltage. Each chip was glued into a 20-pin leadless ceramic package using conducting silver paint. Contact pads were bonded to the package pins with 25 µm diameter gold wires using an ultrasonic wedge bonder. The sample package was then inserted in to a spring-loaded holder at the end of a sample rod which fitted inside a commercial Oxford Instruments cryostat. A Cu-wound solenoid mounted on the tail of the cryostat was driven by a programmable bipolar Kepco power supply, generating a maximum magnetic field of 37.5 mT perpendicular to the plane of the Hall cross. A turbo pump was used to evacuate the sample space to ~10RDS) - back gate voltage (VGS) measurements were performed using a Stanford Research Systems SR830 digital lock-in amplifier to provide a 10 µA, 32 Hz AC current and detect the resulting AC voltage, and a Source Measure Unit (SMU) provided the back gate voltage. A Keithley 2450 (SMU) in 4-wire configuration was employed to characterise the Hall coefficient and Hall offset of devices using a 10 µA DC current. The Hall voltage noise was characterised using a battery-driven DC current source and an ultra-low-noise preamplifier with 104 gain, whose output was recorded at set Hall currents using a HP3561A dynamic signal analyser (DSA). Noise spectra were measured in the range 1 Hz – 1 kHz with a bandwidth of 1 Hz and averaged 100 times to reduce scatter.Two-lead Resistance (


The La Niña and El Niño phases of the El Niño-Southern Oscillation (ENSO) have major impacts on regional rainfall patterns around the globe, with substantial environmental, societal and economic implications. Long-term perspectives on ENSO behaviour, under changing background conditions, are essential to anticipating how ENSO phases may respond under future climate scenarios. Here, we derive a 7700-year, quantitative precipitation record using carbon isotope ratios from a single species of leaf preserved in lake sediments from subtropical eastern Australia. We find a generally wet (more La Niña-like) mid-Holocene that shifted towards drier and more variable climates after 3200 cal. yr BP, primarily driven by increasing frequency and strength of the El Niño phase. Climate model simulations implicate a progressive orbitally-driven weakening of the Pacific Walker Circulation as contributing to this change. At centennial scales, high rainfall characterised the Little Ice Age (~1450–1850 CE) in subtropical eastern Australia, contrasting with oceanic proxies that suggest El Niño-like conditions prevail during this period. Our data provide a new western Pacific perspective on Holocene ENSO variability and highlight the need to address ENSO reconstruction with a geographically diverse network of sites to characterise how both ENSO, and its impacts, vary in a changing climate. The impacts of changes in regional temperature and precipitation patterns associated with El Niño and La Niña phases of ENSO have wide-ranging environmental, societal and economic consequences. The El Niño phase manifests as a warming of central and/or eastern Pacific sea surface temperature (SST) with resulting increased precipitation in northern South America and western North America  describes variation in tropical Pacific Ocean temperatures and the resulting changes in atmospheric pressure gradients. The atmospheric changes widely propagate the effects of ENSO variability, making ENSO a major component of regional climate across much of the world7. The Holocene provides fruitful opportunities for this, with millennial-scale changes in orbital radiation forcing and centennial-scale global temperature changes, such as the Little Ice Age (~1450–1850)8. However, the evolution of ENSO through the Holocene remains unclear, with discrepancies between central Pacific SST proxies9 and eastern Pacific proxies of both precipitation10 and SST11, particularly during the mid-Holocene. Additionally, there are very few proxy ENSO records that can resolve centennial-scale trends in changing ENSO mean state. This is important as changes in the dominant phase of ENSO have been linked to solar irradiance12, orbital forcing13, average global temperatures14 and fresh water fluxes in the North Atlantic7.Given these wide-ranging effects, it is essential to understand how both phases of ENSO will respond to future climate change. Reducing predictive model uncertainties requires proxy data of ENSO behaviour under different background states, as well as in response to local and extra-regional influences from all ENSO-sensitive areas2  of leaves from the evergreen tree Melaleuca quinquenervia ((Cav.) S.T. Blake) preserved in the Holocene sediments of Swallow Lagoon on Minjerribah (North Stradbroke Island), the world’s second largest sand island. Swallow Lagoon (27°29′55″S: 153°27′17″E) is a small (0.27 ha), perched, freshwater lake that is isolated from the regional water table15. With no inflow or outflow streams, the balance of precipitation over evaporation determines lake level . Sediments from a 370 cm core were sieved at contiguous one-centimetre resolution for M. quinquenervia leaf fragments, yielding 284 samples. Each datum represents the δ13C of all leaf fragments at that depth and is an average for the period encapsulated by that centimetre of sediment, which ranges from two to 77 years . As such, these data do not represent El Niño or La Niña events, but represent mean conditions of individual time-slices. Age control is provided by 18 accelerator mass spectrometry 14C dates on short-lived terrestrial macrofossils, including M. quinquenervia leaves . In a novel approach, we utilise a relationship established specifically for M. quinquenervia using a 12-year collection of monthly litterfall samples from a nearby south-east Queensland wetland, which demonstrated a linear relationship  between the carbon isotope discrimination of M. quinquenervia leaves, relative to atmosphere, and mean annual rainfall17. We apply this calibration to sub-fossil M. quinquenervia leaf fragments from Swallow Lagoon to derive a quantitative estimate of mean annual rainfall. The linear nature of the model may skew precipitation estimates to the lower end and affect apparent variability; however, our calibration has advantages over other potential datasets as it uses location-specific climate data and is species-specific, as opposed to using modelled rainfall estimates16 or averaged data from all C3 plants at a location18. Comparing our results against various reconstructions from global datasets demonstrate that they consistently reconstruct higher precipitation estimates, however the patterns of change and variance, although accentuated, do not differ and our findings based on the species-specific calibration remain robust  and the standard deviation (σ) of the rainfall record. We find a statistically significant trend in σ. To test if this trend was influenced by sampling resolution, the estimated model was tested against a null model using 1000 simulated time series that follow the nonlinear trend estimated by our GAM-LS model but importantly with constant variance. This process demonstrates the range of trends in σ we might expect if there were no systematic change in variance. Simulation results demonstrate the estimated trend in σ is not an artefact arising from varying sampling resolution in time . Each simulation consists of a 1200-year model simulation  and differs only via changes in the Earth’s orbital parameters. The model reproduces the long-term trends in ENSO variability over the last 8000 years seen in proxy records, with lower variability during the mid-Holocene (8–5 kyr) and gradually increasing late Holocene variability , driven by high boreal summer insolation and a stronger monsoon system, as the primary driver of reduced ENSO variability evident in proxy records during the mid-Holoceneity Fig. . Modelleity Fig. . Though 25, is driven by increasing strength of the El Niño phase alone, rather than simply a more variable system. While this has previously been implied from a marked shift in vegetation across eastern Australia towards more drought tolerant species around 3 cal kyr BP29, the Swallow Lagoon record confirms the one-sided nature of late-Holocene ENSO intensification.As our record tracks both wet and dry anomalies, we can characterise the shift at 3.2 cal kyr BP in terms of changes in the distribution as well as the amplitude of extremes. Prior to 3.2 cal kyr BP, no events exceeded ±2σ of the record mean; after 3.2 cal kyr BP, there are 12 dry excursions greater than 2σ, but only one wet excursion of this magnitude. While an increase in resolution towards the top of the record will naturally lead to the preservation of more short-lived events, we note that dry anomalies dominate and the transition towards an overall drier mean state, as illustrated by the GAM-LS Fig. , remains11 from ~6–4 cal kyr and when Galápagos lake sediments suggest both phases of ENSO were less frequent21. Though some temporal smoothing is expected in the Galápagos record, as well as at Swallow Lagoon, the timing is also in general agreement with a “quiescent period” evident in high-resolution carbonate δ18O records from discrete periods between 5–3 cal kyr BP20. The GAM-LS model illustrates a period of very high rainfall at Swallow Lagoon around 3.5–3.0 cal kyr BP, which corresponds with a marked cool and dry period reflected in Galápagos lake sediments, also at 3.5–3.0 cal kyr BP21. Taken together, these findings suggest a centennial-scale period of enhanced zonal SST gradient, a persistently strong PWC and a more La Niña-like mean state.The mid-Holocene  at Swallow Lagoon is dominated by precipitation estimates above the mean  though some dry periods are evident. The most extensive of these are apparent around 6.9, 6.8 and 5.8 cal kyr BP, suggesting El Niño was still active at this time. During the period 5.5–3.5 cal kyr BP, rainfall at Swallow Lagoon was generally stable, around a wet La Niña-like mean state. This period closely corresponds with a time of low variance in eastern Pacific SSTs11 and the onset of more frequent El Niño events evident in sediment records from the Galápagos19 and Ecuador25 . Enhanced El Niño conditions in the west Pacific warm pool are evident in discreet coral records from Papua New Guinea around this time31, with notable prolonged and extreme events at ~2.5 and 2.04 ka corresponding with dry periods that exceed 2σ of the Swallow Lagoon record around 2.47 and 2.04 cal kyr BP. These events at Swallow Lagoon occur in a cluster of dry events during the ~2.6–2.0 cal kyr BP period, suggesting prolonged or extreme El Niño events, such as those evident in the coral records, may have occurred more regularly during this time. An absence of long coral records from the west Pacific precludes precise correlation with subsequent late-Holocene dry extremes in the Swallow Lagoon record, though a general agreement between rainfall trends (as illustrated by the GAM-LS) and west Pacific warm pool SSTs32 is evident through this period  suggest the pattern of reduced SST gradient described above is reminiscent of El Niño Modoki conditions; these can drive large-scale decreases in precipitation over northern Australia42, although they are unable to explain a wet southeastern40 or subtropical Australia . Given the critical impacts ENSO has on water resources in teleconnected regions, understanding this apparent disparity between SST and hydroclimate proxies highlights the need for further research into the response of ENSO to changes in global climate.A notable exception to the drier and more variable climate in the late Holocene at Swallow Lagoon is the stable high rainfall phase during the Little Ice Age (LIA: ~1450–1850)ca. 3 kyr cal BP, mirrors the variance evident in ENSO records from across the Pacific. The ~7.7–3.5 kyr cal BP period of low variability is characterised by predominantly wet climates at Swallow Lagoon, which implies a dominance of the La Niña phase during this time. After ~3 kyr cal BP, increasing variability is driven by the occurrence of extreme dry events, highlighting a strengthened El Niño phase as the primary driver of this change. Our climate model simulations implicate a progressive orbitally-driven weakening of Pacific Walker Circulation, particularly after 3 ka, as a contributing factor. At centennial scales, the record presents the first insights into subtropical Australian hydroclimates during the LIA, and we find that persistently high rainfall marks this period as anomalous in the context of the late Holocene. This contributes to a complex picture in which there is an apparent decoupling of SST and terrestrial hydroclimates during this interval. This requires further investigation as understanding ENSO response to radiative forcing is key to understanding the sensitivity of the system to anthropogenic climate changes35.Understanding ENSO variability is critically important because of its effects on precipitation regimes in teleconnected regions. The Swallow Lagoon precipitation record provides a new, quantitative, southwestern Pacific perspective of the influence of both ENSO phases over the mid- to late Holocene. The record has enabled, for the first time, an assessment of centennial- to millennial-scale variability in Australian subtropical rainfall. The pattern of low variability during the mid-Holocene, increasing after k parameter of 0.5 cm−1. The agreement index of the model Amodel of 76% was good as it is higher than the accepted level of 60%43. Radiocarbon calibration data used for calendar age conversion are the post-bomb 14C data for Southern Hemisphere zone 1–244 extended back in time by the SHCal13 calibration curve45. All calibrated ages are reported in cal. yr BP, with 0 yr BP being 1950 CE.An age model was developed based on 18 accelerator mass spectrometry (AMS) radiocarbon dates on short-lived terrestrial macrofossils Table , Fig. S213C values were calculated relative to the Vienna Pee-Dee Belemnite (VPDB) scale using within-run laboratory standards calibrated against NBS-18, NBS-19 and NBS-22. Replicate analysis of well-mixed samples indicated a precision of < 0.1‰ (1 SD).All leaf samples were freeze dried for 24 hours and ground to a homogenous fine powder. Carbon isotope analyses were performed at the Natural Environment Research Council’s Isotope Geosciences Laboratory at the British Geological Survey in Nottingham, United Kingdom, by combustion in a Costech Elemental Analyser on-line to a VG TripleTrap and Optima dual-inlet mass spectrometer. δr2 = 0.64) between annual mean M. quinquenervia Δleafsensu Farquhar et al. (ref.46) and mean annual rainfall17 which is improved slightly by taking into account the effect of atmospheric CO2 changes on Δleaf. Δleaf in the Swallow Lagoon record was calculated using the atmospheric δ13Catm from Elsig et al. (ref.47) between 7520 cal yr BP and 550CE and δ13Catm for the remaining period calculated using Ferrio et al. (ref.48). We inferred rainfall using the relationship between rainfall and a discrimination anomaly17, that is, the difference between Δleaf predicted using Farquhar et al. (ref.46) and that predicted from CO2 using Schubert and Jahran (ref.49). We utilised CO2 data from Monin et al. (ref.50) for the period to 245 cal yr BP, from Law Dome51 from 245 to −20 cal yr BP (1970 CE) and from Cape Grim (www.csiro.au) from 1971 CE to present.There is a significant relationship . Our GAM-LS model includes smooth functions of time to simultaneously estimate trends in mean and variance of the observed time series. Because the models use smooth functions to estimate trends they do not require the response variable to be regularly spaced in time55. Furthermore, they do not suffer from edge effects in the same way as moving window methods, thus allowing continuous estimates of changes in variance over the entire time series. Edge effects do lead to increased uncertainty in trend estimates from GAM-LS models, but this additional uncertainty is accounted for in the standard errors of estimates from the model which are used to produce credible intervals for the estimated trends.To simultaneously estimate trends in the mean and variance of the reconstructed rainfall time series requires a modelling approach that allows for linear predictors for each parameter of the conditional distribution of the response. Therefore, we chose to analyse the time series using a location scale generalized additive model (GAM-LS). Models of this type are also contained in the generalised additive model of location, scale, shape (GAMLSS) classith δ13C observation (yi) is distributed Gaussian with mean µi and variance µi as a smooth function (f1) of time  plus a constant term α (the model intercept). The linear predictor for σi is also modelled as a constant term, γ, plus a smooth function of time (f2), plus a linear parametric effect of the amount of time represented by each sample (tii). σi is modelled on the log scale with a small lower bound b (0.01) to ensure parameter estimates remain positive and avoid issues with singularities in the likelihood of the model56. Any deviation from the assumed Gaussian distribution was assessed using standard model diagnostic plots for GAMs.Rainfall values per year are typically observations of continuous random variables, bounded at 0. However, given the large rainfall values observed here, the Gaussian distribution is a close approximation for their conditional distribution. The Gaussian distribution is defined by two parameters; the mean (μ) and the standard deviation (σ). The GAM-LS approach allows both parameters to be modelled via separate linear predictors to capture variation in both the mean and the variance of the time series. The specific GAM-LS fitted here has the following form:f1 and f2 with 200 and 75 basis functions respectively, to allow for potentially complex fitted trends in mean and variance. A penalty on the second derivative of the fitted smooths was used to control the amount of wiggliness in the estimated functions. Smoothness parameters, used to balance the fit and complexity of the model, were estimated via penalized maximum likelihood57. This has the effect of slightly biasing downwards the estimated variance trend. Dropping f2 from the model allows a test to be performed for a trend in variance over and above that which we might expect to observe due to varying sedimentation rates and time averaging present in each sample. Additionally, AIC was used to select between these two models.Thin plate spline bases were used for both smooth functions σi were identified using the first derivatives of f2, calculated using the method of finite differences. Periods of significant change exist where the 95% confidence interval on the first derivative of the smooth does not include a value of zero slope.Periods of significant change in the estimated trend in σi was the result of variation in sedimentation rates, time averaging, and the uneven spacing of samples in time , we compared our estimated variance trend with those estimated from 1000 null models fitted to simulated time series. Simulated time series f2 . Each annual value was assigned to a cm sediment slice following the age-depth relationship of the observed sediment record, and averaged to provide a mean value of the response for each slice. This process approximates the time averaging of individual years in the observed record. We then fitted the same GAM-LS model to each simulated series, including the smooth f2; any trend in σi estimated by f2 would be spurious, the result of either stochastic variation or the time averaging process, because the data were initially simulated with constant variance. We then compared the observed trend in σi with those estimated from the simulated time series .Snapshot simulations of the state of the global climate at 8, 7, 6, 5, 4, 3, 2 and 1 kyr BP, conducted using the CSIRO Mk3L climate system model v1.1For this study, two metrics were derived: (i) the amplitude of variability in El Niño-Southern Oscillation (ENSO), and (ii) the strength of the Pacific Walker Circulation. The amplitude of ENSO variability was diagnosed by calculating the monthly-mean sea surface temperature (SST) for the Niño 3.4 region . A 2–7 year bandpass filter was then applied to extract ENSO variability. The ENSO amplitude was derived by calculating the standard deviation of the bandpass-filtered SST. The strength of the Pacific Walker circulation was diagnosed by calculating the monthly-mean strength of the zonal wind at 850 hPa for the Niño 4 region . For both metrics, a block bootstrap was used to derive the 95% confidence interval.Supplementary Information


Submerged forward osmosis (FO) is of high interest for bioreactors, such as osmotic membrane bioreactor, microalgae photobioreactor, food or bioproduct concentration where pumping through pressurized modules is a limitation due to viscosity or breakage of fragile components. However, so far, most FO efforts have been put towards cross flow configurations. This study provides, for the first time, insights on mass transfer limitations in the operation of submerged osmotic systems and offer recommendations for optimized design and operation. It is demonstrated that operation of the submerged plate and frame FO module requires draw circulation in the vacuum mode (vacuum assisted osmosis) that is in favor of the permeation flux. However, high pressure drops and dead zones occurring in classical U-shape FO draw channel strongly disadvantage this design; straight channel design proves to be more effective. External concentration polarization (ECP) is also a crucial element in the submerged FO process since mixing of the feed solution is not as optimized as in the cross flow module unless applying intense stirring. Among the mitigation techniques tested, air scouring proves to be more efficient than feed solution circulation. However, ECP mitigation methodology has to be adapted to application specificities with regards to combined/synergetic effects with fouling mitigation. Osmotic processes, such as forward osmosis (FO) and pressure retarded osmosis (PRO), have gained interests for the last two decades, following the commercialization of the first dedicated FO membrane by HTI  ,2. IntenAs for submerged microfiltration/ultrafiltration bioreactors, submerged FO operation could be of high interest, owing to its limited shear stress for applications, such as cultivation of bioproducts, and concentrations to avoid breakage of fragile biological compounds . As partMBRs, as a widely used mature technology, is usually adopted in submerged membrane systems; therefore, a large amount of studies have been published regarding MBR operation and optimization . The maiThe aim of this work was to bring better understanding on mass transfer limitations in the operation of submerged osmotic systems and to offer recommendations for optimized design and operation. At first, evidence of non-optimized mass transfer limitation of our formerly developed submerged module  versus tU-shape large-spacer module: U-shape draw channel design  commercially available FO membranes obtained from Toray Industries  membranes was used . Home-ma2 (only one side of each plate was covered by membranes); plates were realized with a membrane active layer facing the feed solution.A draw solution was circulated under negative pressure pumping conditions as required for the submerged plate . The wat−1 synthetic seawater (SW) as draw. Seasalts (>99.4% NaCl) was provided by Vicens i Batllori S.L. . Then, the effect of pumping was assessed; i.e., positive (pulsion) and negative (suction or vacuum) pumping, were performed by placing pump(s) before or after the CFC, respectively. These tests were realized for a range of flow rate varying from 0.12 to 0.36 L·min−1  connected to a Bluetooth-RS232 Adaptor. Bluetooth based pressure sensors (0–4 bar)  were used to record pressure in feed and draw channels. All instruments  data were recorded using the Bluetooth based system provided by Instrument Works .Membrane coupons were first evaluated in a CFC to evaluate membrane performances in optimized hydrodynamic conditions. Similar setups and operating conditions as in our former studies ,50 were  L·min−1  for both−1 SW was circulated in the draw channel under negative pressure pumping (suction). The plate was maintained at the bottom of the tank with the membrane facing the top. The water in the tank (feed) was stirred vigorously using a vertical stirrer. The same system as for the CFC setup was used for water flux and data collection. Comparative evaluations were performed with and without stirring. In addition, as for the CFC, the permeation flux was assessed using DI water as feed and 10, 20, 35 and 70 g·L−1 SW as draw; then pressure drop was assessed for a range of flow rate varying from 0.12 to 0.48 L·min−1 . The plate was submerged in 20 L of DI water (feed) and 35 g·L L·min−1 .2 in area) was operated as before; the other two plates were used to mimic stack operation. 35 g·L−1 SW was circulated in the draw channel at a pump flow rate of 0.3 L·min−1 under negative pressure pumping (−1 (0.67 to 3.33 m3·m−2·h−1) was tested. Feed recirculation was provided by a peristaltic pump by pumping out water from the bottom of the reactor and pumping in water at the top. A typical feed recirculation rate of 0.3 L.min−1, which was the same as applied in OMBR operation in our former study, was initially tested [−4 m·s−1), additional tests were performed by limiting the cross section of the reactor and increasing the pump speed to operate with a recirculation rate up to the observed rate in microalgae cultivation raceway ponds, i.e., 0.1 m·s−1.Finally, in order to assess the impact of aeration and of feed recirculation in the submerged system, FO plates were placed in a stack fitting 3 plates that was submerged in a 25-L vertical reactor . Only one FO plate  and defined as Equation (2):st is the thickness (m), τ is the tortuosity and ε is the porosity of the support layer and D is the self-diffusion coefficient of the solute. These parameters are characteristics of the membrane, and the S factor in which they are combined is commonly referred to as the structural parameter [−2·h−1·bar−1, 2 × 10−6 m·s−1 and 2 × 10−4 m for A, B and S respectively. Typical values found in the literature are slightly lower for A (6–9 L·m−2·h−1·bar−1) and higher for B (2.5–3.3 × 10−6 m·s−1) and S (2.15–2.7 × 10−4 m) [The theoretical water flux has been calculated based on existing solution diffusion models developed for FO by using Equation (1) as described in  to accouarameter . Membran 10−4 m) ,52,53,54−1. The resulting water permeation fluxes were compared with those from membrane coupon cut from this plate but operated in the CFC setup (A U-shape FO submerged plate was first tested using DI water as feed for a range of draw solution concentrations varying from 10 to 70 g·LFC setup .positive pressure pumping (feed and draw) for CFC versus negative pressure pumping for submerged plates (draw)straight draw channel for CFC versus U-shape design for platesdifferent CFVoptimized feed cross flow channel for CFC versus an unstirred submerged system for platesThe submerged plate module featured a permeation flux about 3 times lower than the one in the CFC for the whole range of draw concentration tested. Such severe loss of performance emphasizes the need of better understanding of mass transfer limitation in submerged FO systems. If both systems were operated with similar membranes, similar feed and draw solutions and similar draw solution flow rates, they also featured many distinct critical operating parameters that can influence system efficiency from both the feed and the draw sides, such as:The impact of each parameters mentioned above was systematically assessed in the following sections.Submerged MBR plate modules are typically operated using depression by negative pressure pumping (suction) in the permeate line, as it is the driving force for water permeation. Applying positive pressure on the permeate line is sometimes used as backwashing typically in hollow fiber configurations, but cannot be applied to plate modules due to practical and mechanical limitations (the membrane inflates and may break). In submerged osmotic processes (using plate and frame), pressure is not the driving force, but the draw has to be circulated between the plate and the membrane. Thus, as for submerged MBRs, circulating the draw solution in positive pressure (pulsion) is not desired due to similar membrane mechanical limitations (the membrane inflates and may break). Pumping operation in suction is unusual in osmotic processes normally operated in the cross flow mode with the positive pressure pumping mode (pulsion) . Therefo−1 CFV). TMP was dependent from the CFV with both feed and draw solutions . With operation in pulsion in the feed channel and suction in the draw, the TMP acts towards increase of the permeation flux (Equation (1)) and can be assimilated to pressure assisted osmosis [It was first observed that, depending on how feed and draw channels are operated in osmotic processes, significant differences of transmembrane pressure (TMP) can be expected a. Typicaolutions b, and co osmosis ,55 or va−1 SW as draw were compared  limiting ECP on the feed side.The interest of VAO was then assessed for different CFVs, and experimental values with model data obtained using Equation 1, with DI water as draw  flux regardless of the flow rate and the draw salinity used. The difference between U-shape and straight channel modules confirmed the limitation of U-shape design already pointed out in other FO modules with dead zones, leading to areas with intense concentration polarization and very low local fluxes [For the first time, fluxes above 15 L·msolution c, showinsolution d has beesolution c and drasolution d, indical fluxes . In addiThe impact of ECP was then evaluated by submerging modules in feed solutions with different salinities and by operating the system under no stirring or with intense mixing .−2·h−1, but flux was maintained constant under mixing conditions while a sharp decline down to 8 L·m−2·h−1 occurred in unstirred system, confirming a very intense and quick ECP buildup. Such ECP happened even when DI water was used as feed and thus indicates that observed ECP was the consequence of reverse salt diffusion (RSD). Feed salinity proved to have a crucial impact also on the permeation flux  submerged FO applications (1) due to the high shearing it produces and (2) the difficulty to implement it in a membrane stack. Other alternatives to promote turbulences, such as air scouring and recirculation, were then evaluated.At first, similar operation parameters (recirculation and aeration) as applied in our recent MBR/OMBR studies were tested and evaluated  for diff3·m−2·min−1). Above that value, minimal increase of permeation flux was observed, indicating that the typical aeration rate as commonly applied in MBR (0.6–1.5 Nm3·m−2·min−1) [The aeration rate was then evaluated for 2 types of feed water b. In bot2·min−1)  is suffi2·min−1) ,60.−2·h−1) and observed for saline feed water and the highest CFV tested (0.09 m·s−1). As such, ECP mitigation through CFV proved to be efficient only in the case of the high recirculation rate; such a configuration is not of application in OMBR but can be found, for example, in microalgae raceway ponds. Aeration appeared to be the preferred option for ECP mitigation, but high CFV can be applied if dedicated submerged OMBR designs are developed. Other options like mechanical shaking are still to be tested as other alternatives [CFV also proved to have an impact on permeation flux c but to rnatives .This study demonstrated that the knowledge learnt from MBR and existing cross flow FO modules cannot be directly applied to submerged FO configurations, where the draw recirculation rate, module design and ECP mitigation are three key issues largely affecting water permeation fluxes. High pressure drops and dead zones occurring in classical U-shape FO draw channel strongly disadvantage this design; straight channel design proved to be more effective and further improvement can be realized. Development and evaluation of submerged hollow fiber modules still needs to be explored. ECP is also a crucial element in the submerged FO process since mixing of the feed solution is not as optimized as in the cross flow module unless applying intense stirring. Among the mitigation techniques tested, air scouring proved to be most efficient. Further work is required to develop ECP mitigating strategies adapted to application specificities and with regards to combined/synergetic effects with fouling mitigation.


Macroporosity was higher in ZT (19.7%) compared to CT (14.3%). However the average number of pores was almost twice in CT than ZT. The largest contribution in both treatments was from very complex shaped pores, followed by triaxial and acircular shaped. Pore connectivity analysis indicated that the soil under ZT was more connected that the soil under CT. Soil under CT had larger values of tortuosity than ZT in line with the connectivity results. The results from this study indicate that long-term adoption of ZT leads to higher macroporosity and connectivity of pores which is likely to have positive implications for nutrient cycling, root growth, soil gas fluxes and water dynamics.Zero-tillage (ZT) is being increasingly adopted globally as a conservationist management system due to the environmental and agronomic benefits it provides. However, there remains little information on the tillage effect on soil pore characteristics such as shape, size and distribution, which in turn affect soil physical, chemical and biological processes. X-ray micro Computed Tomography (μCT) facilitates a non-destructive method to assess soil structural properties in three-dimensions. We used X-ray μCT at a resolution of 70 μm to assess and calculate the shape, size and connectivity of the pore network in undisturbed soil samples collected from a long-term experiment (~30 years) under zero tillage (ZT) and conventional tillage (CT) systems in Botucatu, Southeastern Brazil. In both systems, a single, large pore (>1000 mm •Soil macro-porosity was higher under zero-tillage than conventional tillage.•Conventional tillage increases the number of pores but decreases the connectivity between them.•Zero-tillage presented lower values of pore tortuosity, indicating better connectivity.•Long term zero tillage improves soil pore connectivity.•3D analyses of pore shape indicated difference between tillage systems. StudiesAdditionally, there remains a lack of knowledge regarding the effect of undisturbed soil structure on functional processes. A greater understanding of the role of structure on soil function can be achieved by a detailed characterisation of the spatial configuration of its components. In the context of global environmental change and agricultural expansion, there is an urgent need to assess how ZT, and in particular how this changes over the time-scale of years or decades, impacts on soil pore shape, size and connectivity. ZT systems have been associated with significant changes in soil porosity, especially in the upper few centimetres in comparison to CT soils . Sasal eMany previous assessments of the impact of land management on soil porosity have previously been either qualitative or limited to the analysis of bulk samples with field structure disturbed or removed. X-ray μCT provides an alternative tool for measurement of structural properties in three-dimensions (3D) and in a non-destructive manner. Previously this technique has been applied to characterising soil hydraulic properties e.g. , quantif22.1°49′ S, 48°25′ W altitude: 786 m). The climate is classified as mesothermal with dry winters, and the dry season is well defined from May to September, with yearly average rainfall of 1450 mm, distributed mostly between October and April. The soil type is a Typic Rhodudalf (Brachiaria grass and Safflower (Soil samples were collected from a long-term experiment (~30 years) managed under both zero-till and conventional tillage systems. The experiment was carried out in Botucatu, São Paulo, Brazil  using a randomized design. Since the five cores were collected in a single replicate of each treatment of the experiment, they comprise pseudo-replicates. The soil cores were sealed with paraffin wax at the top and bottom to prevent movement during transit and stored at 2 °C.2.3Samples were packaged very carefully and transported to the Hounsfield Facility at the University of Nottingham for μCT scanning. Prior to this a pilot study involving soil samples being μCT scanned in Brazil and then μCT scanned again in the UK was undertaken to confirm that the addition of paraffin wax prevented any sample disturbance . Samples were shipped to the UK as the μCT scanner available offered higher quality imaging. The soil cores were scanned using a G.E. V|Tomex|M Computed Tomography X-ray scanner. Each soil core was scanned for 45 min at 180 kV with an isotropic voxel size of 70 μm, with 2664 images per scan. The final image volume had 600 × 1400 × 600 voxels, corresponding to a volume of 42 × 98 × 42 mm.http://rsbweb.nih.gov/ij/) after cropping to exclude the area outside the soil column. The segmentation process was based on the non-parametric Otsu method of automatic thresholding  and the number of pores (NP) were calculated taking into consideration the image resolution. The 3D pore size distribution (sorted by pore volume) corresponded to the total number of disconnected volumes of pore space inside the total sample volume. For Ma and NP size distribution analysis, pores were classified in different volume intervals: 0.003–0.01; 0.01–0.1; 0.1–1; 1–10; 10–100; 100–1000 and >1000 mmThe classification proposed by G and LE represent the geodesic length between two connected points within the pore space and the Euclidian length between these two points. In this study, the average tortuosity was calculated considering three directions .The network tortuosity (τ) of the pores and connectivity were calculated using the computer program, Osteoimage . TortuosThe Euler-Poincaré characteristic (EPC) was utilized to estimate the degree of connectivity, which represents one of the Minkowski functions and a topological measure used for describing the connectivity of spatial structures . This pa2.4The statistical analysis was performed using the R software  to Ma in CT, which is associated with the connectivity of the pore system (3) accounting for 97% of Ma  as observed in 3 contributed to around 65% of NP  and 573 mm3 (0.1–1 mm3) in CT (3) and 249 mm3 (0.1–1 mm3)  in CT e. ZT pre1–1 mm3) f. The hies in ZT g, h.3) followed by the triaxial (161 mm3) and acircular (149 mm3) shapes (3) followed by the triaxial (87 mm3) and acircular (78 mm3) shaped pores (The contribution of the shape of pores to P and NP is presented in ) shapes a. Similaed pores b. The coed pores c, d, theWhen NC pores were excluded from the analysis in the CT treatment e, g, triSoils under CT had the largest pore volumes when different pore shapes were considered a, and a x = 1.48, τy = 1.46, τz = 1.48) compared to tortuosity in ZT . The average tortuosity considering the three directions was 1.47 and 1.34 for CT and ZT, respectively.The pore connectivity result indicated that soils under ZT (EPC = 297) were more connected than soils under CT (EPC = 931) . A small4These findings show that the porous system in both tillage systems for this soil type is characterised by highly connected pores. The effect of the conventional management contributed to an increase in the number of separate pore entities, with CT presenting almost twice the number of pores as ZT. This is mainly associated with the changes in the soil structure caused by the ploughing operations, which contribute to the disaggregation of the structure at the top soil . These oAn increase in soil porosity, especially in macroporosity, due to tillage has been frequently reported e.g. . De MoraThe comparison of the average Ma and NP between tillage systems demonstrates that the ploughing operation in the case of CT presents a high variability among samples in terms of Ma (CVs of around 25% - CT and 10% - ZT). On the other hand, this management system has the lowest variability in the amount of pores (CVs of around 8% - CT and 19% - ZT). This is mainly related to the homogeneity in soil structure that results from the preparation after ploughing operations as is required for seedbed preparation . PloughiThe soil under CT had larger pore volumes when the different pores were classified in terms of shape were considered, and a larger NP than ZT, which can be associated to aggregate breakdown induced by ploughing. Higher pore connectivity in ZT could enhance root penetration through a reduction in foraging and enhanced water and nutrient availability which might explain why high yields have been reported in some long-term ZT systems . This efThe pore connectivity analysis indicated that the soil under ZT (EPC = 297) is more connected that the soil under CT (EPC = 931). Soil under CT presented larger values of tortuosity  than ZT . These results indicate that long-term adoption of ZT leads to higher pore connectivity and greater macroporosity, which has implications in nutrient cycling, root growth, soil gas fluxes and water dynamics. We suggest that considering the wide range of morphological properties used to characterise soil structure, which is increasingly accessible as the use of image analysis software becomes more prevalent, that the soil pore connectivity is one, if not the most important measurements in this regard. Previous studies have suggested porosity is significantly reduced in the short term following conservation tillage practices e.g. , so it i5X-ray Computed Tomography was useful in assessing qualitative and quantitative differences between long-term conventional tillage and zero-tillage systems in soil pore volume, structure, connectivity, tortuosity, size and shape distribution.Soils under long-term zero-tillage present better pore connectivity and higher total porosity than conventional tillage. Long-term conventional tillage tends to increase the total number of pores compared to the zero-tillage system, which is likely related to changes in the soil structure caused by the ploughing operations.