This paper investigates the correlation between climate, environment and human land use in the Westernmost Mediterranean on both sides of the Strait of Gibraltar during the Late Glacial. Using a multi-proxy approach on a sample of 300 sites from the Solutrean and Magdalenian of the Iberian Peninsula and from the Iberomaurusian in Morocco, we find evidence for significant changes in settlement patterns and site density after the Last Glacial Maximum. In Southern Iberia, during Heinrich Stadial 1, hyperarid zones expanded drastically from the south-eastern coast to the West through the Interior. This aridification process heavily affected Magdalenian settlement in the South and caused a strong decline of hunter-gatherer population. Southern Iberia during Heinrich Stadial 1 turned out to be a high-risk environment when compared to Northern Iberia. At the same time, the Late Iberomaurusian of Morocco, although considered to be situated in a high-risk environment as well, experiences an increase of sites and expansion of settlement area. Climatological research focusing on the coring of ice sheets, marine and lake sediments undertaken during the last decade has given evidence of rapid climate changes during Late Pleistocene Europe . These rIn the Westernmost Mediterranean, there is a long research history of Palaeolithic sites  that is Evidence for climate change is documented in a multitude of marine and terrestrial proxy records published during the last decades. As summarized by a series of excellent reviews –20, closDuring the last years, our research group compiled a rich dataset from fieldwork at archaeological sites and at geo-archives and combined this empirical data in a meticulously controlled database of secondary archaeological data . In a prConcerning Morocco, a strong decrease of site numbers after the Middle Palaeolithic resulting in a settlement gap is also evident. A very slow increase of site numbers begins only in the LGM . TherefoThe site location and present-day environmental data  suggest These regional and diachronic disparities in site density could be the result of changes in habitat suitability or in patterns of land use and adaptive strategies, and therefore are the starting point of our analysis. In order to quantify diachronic and regional changes in occupation density, different methodological approaches have been applied ,4,28,29.The study is based on a database of archaeological sites from Iberia and Morocco dated to the interval between Heinrich Stadial 2 (HS 2) and the end of the Pleistocene  Tables. geographical coordinates (recorded in WGS84)site type archaeological composition (sites with settlement during one or multiple techno-complexes)stratigraphic information (single-layered or multi-layered occupation in the same techno-complex)radiocarbon datessensu lato in the following analyses.We only selected sites with reliable stratigraphies in which layers possess a clear cultural attribution given by the excavators. Additionally, we collected all reliable radiocarbon dates corresponding to these layers. Some authors assume the presence of a Badegoulian as a random diffusion from France onto the IP. The number of sites is very low and the inventories are small as well ,31. Due After filtering, our analyses were carried out using 143 Solutrean, 176 Magdalenian and 38 Iberomaurusian sites. The sites are grouped into three geographical areas: the North and the South of Iberia divided by 40°N and North-Western Africa between 30°N to the South and 0° to the East. Although the IP displays a climate gradient from the Mediterranean Southeast to the Atlantic Northwest and is morphologically divided into dry basins and humid mountains, which occur to the South as well as to the North of 40° N, we prefer a macro-scale subdivision of the Westernmost Mediterranean in three units: Northern Iberia, Southern Iberia and Morocco Further subdivisions are not suggested due to low sample size and lack of regionalized palaeoenvironmental proxy records. Although we expect that this division will not sufficiently reflect small-scale regional patterns, we deem our large-scale approach appropriate for the supra-regional analytical scale discussed in this paper.Our starting point is the site distribution plot on the modern ombrotype map , based oThe distribution of known archaeological sites may be biased by factors such as the easy recognition of diagnostic tools, research history and preferences, site preservation potential or certain geomorphological processes. Large dune fields and cover sands may have buried archaeological sites. In Iberia, large inland dune fields are known, including the Tierra de Pinares sand of the Duero Basin  the MancDespite these potential biases, the compilation of sites in our database represents an exceptionally high-quality record available for analysing human occupation during the Solutrean, Magdalenian and Iberomaurusian in the Western Mediterranean. Due to this, we regard the results obtained from the following analyses as relatively robust and the data adequate to infer certain properties of prehistoric settlement and land use dynamics.Within this paper, we use 14C data and their Summed Calibrated Date Probability Distribution (SCDPDs) to recognize patterns in population dynamics . The appA first presupposition to use dates-as-data is the validation of the radiocarbon dates. This concerns both the archaeological validity, i.e. the stratigraphic integrity, as well as the methodological validity, concerning the quality of the 14C measurement itself. Hence, our data underwent a strict quality protocol. We excluded all dates with unclear methodology, bad preservation of bone collagen, sample contamination resulting in a rejuvenated date, and dates from rock art, as these samples lack stratigraphical context. In addition, we removed all dates with a standard deviation greater than 350 years to ensure a high precision of our analysis. As previously established, single dates from Iberomaurusian sites with unclear stratigraphic sequences, but with a clear association with the cultural material were kept in the database. The exclusive use of confirmed dates should guarantee a minimum level of reliability of the emerging patterns.Another aspect concerns the number of dates. Several authors ,52 argueCalPal version 2016.8  was usedAnalysis of diachronic and regional change in site numbers and site density have resulted in useful characterizations of population dynamics and land use in Late Pleistocene Iberia ,14,55 asIn a previous study , we tookSimilarly, we analyse repeated settlement of a site in broader temporal entities. If a Solutrean site is reoccupied in the Magdalenian, it may represent enduring continuity in the role that site played in the overall land use pattern. If sites are not reoccupied in the later techno-complex, it could reflect changes in resource availability , changes in mobility or the complete abandonment of a (micro-)region.The sites in our database are therefore classified by their overall stratigraphic composition (i.e. techno-complex presence or absence) and the number of stratigraphic units per techno-complex (techno-complex sub-phases). We separate multi- or single-layered sites as well as sites with material from single or multiple techno-complexes; the schematic is presented in Site density analyses can be applied in a similar way to the reconstruction of Palaeolithic settlement patterns. Morgan , SchmidtThe NNA analyses site density by measuring distances between sites and their closest neighbouring site . Mean diThe resulting overall density not only gives a first impression of site distribution, but likewise yields average distances between sites, which can be used to characterize and compare mobility. However, the NNA only analyses the pattern on one scale; in order to analyse site density on multiple scales, we also apply Ripley’s K analysis to our dataset ,65. We uAfter the statistical site density analyses, we turn to the location of the sites in the landscape. Kernel Density Estimation (KDE) is a tool often used in archaeological studies to visualize and interpret site clusters –69. HereGrove follows Isaac  in acknoA final step in the analysis arises from the site density analyses. As the archaeological sites represent the remnants of human subsistence activity, we test to see if the distribution of these sites would have the potential to form stable social networks which guarantee the long-term survival of the Pleistocene populations. Thus, a modified version of Wobst’s Mating Network Model  was applWobst divides hunter-gatherer populations into minimum bands forming a network with other minimum bands. The purpose of these networks is communication, trade, and ensuring mating partners. Each minimum band comprises of about 25 individuals  should increase the stability of a mating network at least to the point when population growth leads to resource shortage. To assess the social stability of our four archaeological techno-complexes, we simulated networks with steadily increasing artificial population densities . Archaeological evidences are indeed limited to few sites, which are mainly concentrated in the mountainous North-East. Only during HS 1 does Morocco display a first slight increase of Early Iberomaurusian occupation. After HS 1, during the Late Iberomaurusian, the strong increase of data corresponds to a general increase in site numbers and to a development, in many locations, of rich and thick shell midden deposits, so called “escargotières” . The numThe Early Iberomaurusian overlaps with the Solutrean and the first part of the Magdalenian. During this phase, the Moroccan radiocarbon record is at its weakest, while the contemporaneous Solutrean in Iberia shows intensive human occupation. This changes after the LGM, when Southern Iberia now evidences weak human occupation until the end of HS 1, while the data increases slightly in Morocco. The record in northern Iberia is only very slightly affected by the breakdown of radiocarbon data seen in the South. After HS 1, all regions experience a peak phase of radiocarbon data, which breaks down again during the Younger Dryas.The chronology of the techno-complexes under study is not synchronous from Iberia to Morocco. This makes the comparison between the four techno-complexes difficult, as they overlap slightly. However, a crosscheck between Southern Iberia and Morocco is of special interest because we expect similar dry environmental conditions on both sides of the Strait of Gibraltar. For this reason, the following results from both sides of the Western Mediterranean will be discussed together, but all direct comparisons must keep this temporal overlap in mind.The regional and diachronic distribution of the 300 archaeological sites in our database shows two prominent trends: an increase of site numbers from early to late periods in Northern Iberia and Morocco, and a decrease of sites in Southern Iberia . While tTo get further insight into settlement dynamics, we compare the distribution of single- and multi-layered sites in the different regions of the IP . As descDuring the Solutrean, settlement dynamics in Iberia seem quite balanced in both the North and the South; single-layered sites are the most frequent category in both regions, and the relationship between single-layered and multi-layered sites is similar. This indicates comparable conditions on the whole IP. During the Magdalenian, the situation is noticeably different. The boom of Magdalenian sites in the North is accompanied by a boom of multi-layered sites. In Southern Iberia, the general decrease of site numbers does not change the relationship between single- and multi-layered sites. Both changes in site numbers and archaeological composition point to the emergence of a new settlement system in Iberia during the Magdalenian.In a second step, the re-use of sites and settlement areas in succeeding techno-complexes are analysed see . 39 of tIn addition to this, few sites with occupations during multiple techno-complexes are multi-layered in both time frames . In the North, only one of these sites (Cardina I) is located in the interior of the Peninsula, while the other 9 are along the Cantabrian Coast, a region densely settled during both time frames. In the South, 4 of these sites are also located in large clusters on the Atlantic and Mediterranean coasts, with only Nerja being more isolated. In Morocco, 3 of the 5 multi-layered sites from multiple techno-complexes are located in the Eastern Rif, another densely inhabited region, while the other two are located close to neighbouring sites (Kehf el Hammar and Dar es-Soltan 2). Such sites, often amidst clusters of other sites, therefore seem to play a special role in the settlement system.In conclusion, multiple changes in settlement pattern during the Western Mediterranean Late Pleistocene are apparent. Before and during the LGM, site numbers are high in Northern and Southern Iberia, while they are extremely low in Morocco. After the LGM, in the Magdalenian, site numbers increase significantly in Northern Iberia as well as in Morocco, the latter albeit on a much smaller scale, while they decrease markedly in Southern Iberia. This increase of site numbers in Northern Iberia goes hand-in-hand with an increase of multi-layered sites, while the relationship between single-layered and multi-layered sites in the South remains unchanged. In addition to this, more Solutrean sites in the North are reoccupied during the Magdalenian. Sites that yield multi-layered occupations during both techno-complexes are regularly located in the middle of site clusters and along the coasts, and probably represent core areas of human occupation.For the following density analyses, the Early and Late Iberomaurusian time frames were combined. As outlined above, only a few sites in our database could be attributed to the Early Iberomaurusian, so that statistical analyses are not expected to yield reliable results. Most of these sites were reoccupied in the Late Iberomaurusian, and there is a large number of sites where the attribution to either phase is unclear. For this reason, all Iberomaurusian sites were analysed together for the Nearest Neighbour and Ripley’s K analyses.The results of the Nearest Neighbour Analyses are presented in All site groups are significantly clustered. In the northern Iberian Peninsula, the Solutrean is markedly more clustered than the Magdalenian, evidencing a clear difference in settlement pattern. The increase of Magdalenian sites goes hand-in-hand an expansion of settlement areas. In the southern Iberian Peninsula, the values are almost identical, demonstrating that the decrease of Magdalenian sites did not change the overall settlement structure. In Iberia, the observed mean distances are in higher in the South and especially in the Magdalenian, pointing to an increased mobility compared to Northern Iberia. The Iberomaurusian, with the fewest number of sites, also shows significant clustering, but displays the largest observed mean distance of all techno-complexes. This finding suggests highest mobility in the Iberomaurusian.To follow this, we did Multi-Scale Ripley’s K Cluster Analysis . This giTo translate these results onto a map and analyse the geographical distribution of the site clusters, we calculated the KDE. The KDE bandwidth was derived from the mean of all mean nearest neighbour distances (17.942 km); the resulting figures can be interpreted as models of the intensity of human influence on the landscape based on inter-site mobility.Following the previous discussion of multi- and single-layered sites, we think it adequate to incorporate a weighting factor into the model to reflect more or less intense use in accordance to the archaeological data. Multi-layered sites are weighted with 2 instead of the default weighting 1, to reflect repeated occupation of the site by people during one techno-complex. For the Late Iberomaurusian, the situation is somewhat more complicated. Due to the more rapid sediment accumulation of the escargotière, a multi-layered Iberomaurusian site is not comparable to a multi-layered site from the Iberian Peninsula. Because of this, we derived more intense use from the thickness of the escargotière in published site stratigraphies. Settlement layers thicker than our (arbitrary) limit of 50cm were weighted like multi-layered sites with a value of 2; all other sites had the same default weighting as single-layered sites. Sites without reliable information on stratigraphy were weighted as a single-layered site. The unsure dating of the Iberomaurusian is also problematic. As we aim to examine the increase in site numbers from the Early to Late Iberomaurusian, we cannot combine the time frames as we did in the previous site density analyses. To incorporate this problem in our model, instead of grouping all Iberomaurusian sites into one, we recognize the uncertainty and display all “unknown” sites with half transparency next to the securely dated sites without transparency. The calculations were however undertaken together.Using Qgis 2.8, we calculated a bivariate KDE for each time frame separately and combined them into one diagram. The result is shown in Figs In the Solutrean , we can In Morocco in the Early Iberomaurusian clustering is low  and we cThe site density analyses show clear differences in land use pattern over time and space in Iberia. In the North, settlement expands and becomes more homogeneous through time, while it is more constricted in the South. In North-West Africa, settlement structure is very dispersed with low site density and gets more stable only in the Late Iberomaurusian; this difference to Southern Iberia will be subject to further discussion.To study possible effects of these changes in settlement pattern on the population, we applied the Mating Network Model Figs  and 12. 2 shows large stable networks during the Solutrean in all settlement areas except for the Interior  and steppe vs. forest vegetation in pollen records of Padul [Ericaceae and Pinus point to relatively humid conditions not necessarily dryer than today. The d18O-speleothem record of Eagle cave, located in Central Iberia, suggests that the LGM was drier than the end of MIS3, when Iberian glaciers reached their maximum extent [Most proxy records suggest that the LGM was colder and drier than today. The reduction in moisture is, for instance, clearly documented by increased percentages of semi-desert taxa . Dry clof Padul . Pollen of Padul . Howeverm extent . The samm extent ,22. HoweNeoglobigerina pachyderma (s) and in ice rafted debris (IRD), concomitant minima in reconstructed sea surface temperatures (SST), e.g. [Quercus type pollen and a reduction in Artemisia, a regional climatic pattern, which is closely mirrored in the MD95-2043 core. After GI-1, the climate got dryer and colder during the Younger Dryas, clearly reflected by multiple records covering the Late Glacial/Holocene transition in northern and southern Spain. The general picture of cold and arid climatic conditions during HS 2, HS 1, and the Younger Dryas (YD), interrupted by comparatively moist phases of the LGM and GI-1, is also reflected in proxy data for Northern Iberia [HS 1  was probT), e.g. , and incT), e.g.  and in tT), e.g.  show a tn Iberia ,23,94,95Artemisia percentages. The Younger Dryas is apparently not represented in the pollen diagrams of Northern Africa. The speleothem of La Mine, Tunisia, shows an abrupt onset of the YD with a decrease in delta13C, suggesting a colder climate than afterwards. Its continuous growth does not point to drier climatic conditions than before or after the YD event [The climatic conditions in North-West Africa are less well known, but proxy records from marine cores retrieved from the North-Western African margin suggest that HS 1 was the driest period between HS 2 and the Younger Dryas in North-West Africa –101. LesYD event . So far,Although a long tradition deals with cost-benefit ratios of hunter-gatherer subsistence, it remains difficult to define thresholds at which environmental constraints become too risky for human occupation and hunter-gatherer resilience collapses ,107. SucConsidering the LGM in Iberia, it apparently did not cause a gap in human occupation. On the contrary; although human presence in Central Iberia, the North-East and the North-West was weak during the Solutrean, the rest of the Peninsula experienced an increase in human presence in the landscape and in stable settlement systems. Considering site numbers and distribution, this changes dramatically during the Magdalenian in the South.Proxy data and palaeoclimatic modelling shows that dry spells during HS 1 considerably increased the extension of dry areas from the South-East to the rest of Southern Iberia, only leaving small refugia of more favourable conditions . During In Morocco, after a gap in human occupation following the end of Middle Palaeolithic, radiocarbon data increases steadily from the LGM into HS 1. From a very low presence of humans in the LGM, stable clusters seem to have developed in the course of the Early Iberomaurusian. After HS 1, however, this region, like the Iberian regions, evidences a simultaneous peak phase in the radiocarbon record which is matched by site numbers and an expansion of settlement areas. Modern ombrotype distributions show more semiarid and dry conditions than Southern Iberia, but the palaeoclimate data record is very sparse and, so far, does not support the notion of a more critical environmental situation in Morocco. An unmodified transference of HS 1 climate simulation from Southern Iberia to Northern Morocco would probably result in even stronger aridity-indexes, but further research is required to clarify this topic.Compared to the Early Iberomaurusian and the Magdalenian, a marked difference in human adaptation during the Late Iberomaurusian can be identified. After HS1, groups in Morocco heavily exploited land snails as a food resource. Although this has so far only been studied in detail for the Neolithic , exploitOur data from the Westernmost Mediterranean indicates significant changes in settlement patterns and site density after the LGM on both sides of the Strait of Gibraltar. During the Solutrean, major parts of Iberia, with the exception of the Interior and some coastal areas, are consistently populated by hunter-gatherer groups. This pattern abruptly changes with HS 1, as hyperarid zones drastically expand throughout Southern Iberia, from the south-eastern coast via the Interior to the West. High resolution climate modelling indicates that this aridification process is especially severe due to a precipitation minimum at the beginning of the growing season. Magdalenian settlement in the South is probably heavily affected by this climatic impact. The mainly coastally orientated site distribution makes settlement systems and mating networks highly vulnerable to dry spells. During HS 1, networks were probably interrupted by arid zones for several hundreds of years or even shorter periods, causing a strong decline in hunter-gatherer population. Southern Iberia turns out to be a high-risk environment during HS 1 when compared to Northern Iberia, where climate change is less severe and even a contemporaneous expansion of settlement areas is evident. Interestingly, after HS 1 a recovery of the Magdalenian population, marked by site numbers and distribution, is not discernible.Southern Iberia and Northern Morocco show similar current climate regimes and are thus assumed to be high-risk environments, at least during HS 1. However, the development of human demography is different in both areas during the LGM and in the Late Glacial. In the LGM, with a stable habitation of Southern Iberia, Morocco is weakly occupied. During HS 1, both regions are affected. However, the decrease of human presence as seen in Southern Iberia after the Solutrean is not visible in Morocco, where evidences of the Early Iberomaurusian still slightly expand. After HS 1, the radiocarbon record displays an increase of data, but the overall number of sites in Southern Iberia is still significantly lower when compared to the North and single-layered sites are more abundant. At the same time, we see a constant increase of sites in Morocco during the Late Iberomaurusian. If our site data and the conclusions derived are correct, these differences could be explained by three alternative scenarios:Climate differences between regions are stronger or more complex than our data suggestDifferences in the history of human demography of the two areas are responsibleHuman adaptation to cope with climate stress was different in the two areasAt the moment, it is still difficult to decide which scenario is most likely; further research is needed. However, this study illustrates the high potential of the Westernmost Mediterranean for modelling Late Pleistocene hunter-gatherer behaviour in relation to climate change. The data presented shows changes in settlement patterns in the three macro areas of the Westernmost Mediterranean through time, following clearly different trends. The impact of HS 1 was probably a major driving factor behind changes in the settlement system. While Northern Iberia remains stable and evidences even an expansion of human population, Southern Iberia and Morocco can both be classified as temporary high-risk environments that required special adaptive capacity and adjustment.S1 TableThe region, site type, geographical coordinates and presence or absence (–) of archaeological complexes are noted for each site, as is the presence of single (s), multi (m) and unknown (?) stratigraphical subunits for each complex. The ID number corresponds to the labels of (XLSX)Click here for additional data file.S2 TablePublished radiocarbon dates  for Late Glacial archaeological sites from Iberia and Morocco (n = 542). IBM = Iberomaurusian; SOL = Solutrean; MAG = Magdalenian.(XLSX)Click here for additional data file.


Here, using a method designed for the long-term coupling of climate and cryosphere models and pCO2 scenarios from different studies, we highlight the pivotal role of pCO2 on the GrIS expansion across the Plio-Pleistocene Transition , in particular in the range between 280 and 320 ppm. Good qualitative agreement is obtained between various IRD reconstructions and some of the possible evolutions of the GrIS simulated by our model. Our results underline the dynamism of the GrIS waxing and waning under pCO2 levels similar to or lower than today, which supports recent evidence of a dynamic GrIS during the Plio-Pleistocene.It is generally considered that the perennial glaciation of Greenland lasting several orbital cycles began around 2.7 Ma along with the intensification of Northern Hemisphere glaciation (NHG). Both data and model studies have demonstrated that a decline in atmospheric pCO 2 control on the development of the Plio-Pleistocene Transition  Greenland Ice Sheet (GrIS). Here based on transient ice sheet experiments, the authors demonstrate the pivotal role of modern-like CO2 on the onset and dynamism of the PPT GrIS.Previous work has argued for a CO A set of reconstructed SST records4 spread across the globe describes a gradual cooling trend for the middle to late Pliocene and early Pleistocene (3.6–2.2 Ma), associated with a progressive increase in high δ18O peaks of benthic foraminifera5, reflecting both lower deep-water temperatures and increased ice volume. Terrestrial data from Lake El’gygytgyn are also consistent with long-term ocean cooling6. During this period, the first marked peak of IRD was found around 3.4–3.3 Ma (MIS MG2 and MIS M2) East of Greenland (ODP site 9077) and is interpreted as evidence of an important glacial event occurring before the establishment of the cyclic NHG around 2.7 Ma8. Significant amounts of IRD were recovered in different ocean drilling sites in the North Atlantic during the PPT9. These deposited IRD likely originated from Greenland, Iceland, North America and Scandinavia, suggesting large land ice expansions to the coast during this interval9. In addition, records based on volcanic and sedimentary facies from Iceland also indicate the onset of a large glaciation after 2.6 Ma11. The onset of a perennial GrIS and of the cyclic NHG at the end of the PPT stands out as a tipping point in the climate evolution of the Earth. Indeed, it marks the beginning of a low pCO2 world with perennial ice sheets in both hemispheres, an infrequent occurrence in the Earth’s history12, thereby creating specific geologic and climatic conditions allowing the development of glacial/interglacial cycles.The long-term trend that led to the initiation of the cyclic Northern Hemisphere glaciations (NHG) can be dated from 3.6 Ma15. In particular, the last large Northern Hemisphere glaciation prior to the major intensification at 2.7 Ma occurred during MIS-M2 (3.312–3.264 Ma)18. This 50 kyrs glaciation was followed by the well-established Mid-Pliocene Warm Period (MPWP) from 3.3 Ma to 3.0 Ma for which numerous observational and modelling studies performed in the framework of the PlioMIP project19 have demonstrated that the warmer conditions led to a reduced GrIS22 . Most climate/cryosphere studies on the GrIS onset during the PPT using GCM have generally produced snapshot climatic simulations subsequently used to force an ice sheet model (e.g. ref. 23). For instance, Lunt et al.23 demonstrated, based on a series of fully coupled GCM snapshot experiments with different forcing factors, that pCO2 decline was the major driver of the GrIS glaciation. However, this result was obtained from equilibrium simulations at 2.7 Ma and the underlying GCM simulations included a pre-existent Pliocene GrIS21. A more recent study, also using snapshot simulations at 2.7 Ma, demonstrated that pCO2 values had to remain low to counterbalance the increasing summer insolation in order to maintain the GrIS after the initial onset24. In contrast, low resolution, conceptual and intermediate complexity models have been used to perform transient long-term experiments27, but these models remain simplified with respect to many processes and do not have the spatial resolution to focus specifically on Greenland. Willeit et al.28 have recently worked on increasing the resolution but their simulations of the GrIS across the PPT still used pre-defined pCO2 forcings. Therefore, although important insights into the dynamics of the cryosphere across the PPT have emerged from these studies, simulation of the GrIS evolution across PPT with a forward physically-based model driven by realistic forcings remains a major challenge. Here, we use a recent numerical interpolation method29 (see Methods), which couples climate simulations obtained with the fully coupled IPSL-CM5A model30 and the 15-km resolution version of the ice sheet model GRISLI31, in order to investigate the transient evolution of the GrIS across the PPT. The major advantage of our approach is the ability to directly test the response of the climate-ice sheet system to different pCO2 evolution scenarios (see Methods) in order to define plausible pCO2 scenarios that led to the GrIS inception and variability. We thus apply various reconstructions of pCO2 evolution across the PPT from both proxy records and inverse modelling studies (e.g. ref. 35), as well as constant pCO2 evolutions.Greenland, however, may have experienced waxing and waning of ice before the intensification of NHG, as suggested by Eocene, late Miocene and early Pliocene IRD records2 values ranging from 220 to 405 ppmv using realistic orbital variations36 . Our results show that for extreme pCO2 values (220 or 405 ppm) the simulated ice sheet evolution does not depend upon the initial configuration of the ice sheet at 3.0 Ma , orbital and pCO2 forcings have comparable influence on the simulated GrIS volume but these simulations demonstrate that the initial configuration of the ice sheet also controls the GrIS response. Indeed, bedrock vertical motion driven by the presence or absence of ice can alter topography for tens or hundreds of meters, which, at pCO2 levels close to the threshold, may lead to differences in ice volume and extent. However, the general evolution of the GrIS volume remains similar in the scenario starting with an ice-free Greenland and in that starting from a full ice sheet. More importantly, during major deglaciation episodes driven by high summer insolation, the modelled GrIS in both cases recedes to identical configurations. As there is evidence for a much reduced, perhaps even non-existent, GrIS during interglacials of the MPWP , we initialize the ice sheet model with an ice-free Greenland. Indeed, the starting date of our simulation (3 Ma) is preceded by a very high summer insolation maximum . During the 2.9–2.7 Ma interval, the low variability in insolation prevents the GrIS from growing in spite of pCO2 levels dropping to 300 ppm. The simulated GrIS evolution then shows a significant increase at 2.7 Ma and pronounced orbital scale variability between 2.7 and 2.5 Ma, but importantly, there is no full retreat of the GrIS during the insolation maximum after 2.6 Ma because this maximum is associated with pCO2 levels below 300 ppm  are similar. However, the simulation forced with the mean pCO2 estimates of Martinez-Boti et al.33 shows an early attempt of GrIS expansion (~2.98–2.94 Ma) before a progressive onset from 2.8 Ma onwards, with a significant increase in GrIS volume around 2.72 Ma and 2.6 Ma  that are relatively well correlated in time with decreases in North Atlantic SSTs and large modelled GrIS variations  therefore seem to correlate relatively well with IRD records because the GrIS expands preferentially from the south and east regions and intervals of large GrIS variations are synchronous with IRD peaks.There is variable agreement between our reconstructed GrIS volume scenarios and inferred GrIS extension as implied by IRD records. For instance, the GrIS evolution forced by the pCO Ma Fig. . Around 307 Fig. . The shaons Fig. , in part41). Although by model design ice growth in other regions of the Northern Hemisphere is not simulated, simple inferences of the impact of North American and Scandinavian ice sheets after 2.7 Ma would tend to suggest that the large GrIS retreat around 2.6 Ma might have been more limited. Indeed, the cooling effects of such ice masses on regional and global climate would presumably have partly counterbalanced the combined increase in pCO2 and summer insolation. Still, the evidence for other NH ice sheets can tentatively be used to suggest that the GrIS evolution driven by the Martinez-Boti et al.33 mean pCO2 estimates is the most plausible pCO2 scenario among those that are the most consistent with IRD records , because it leads to a much larger GrIS between 2.8 and 2.6 Ma  with a resolution of 3.75°×1.875° and 39 vertical layers. More details about the physical parameterization can be found in46. The ocean model is NEMOv3.247, which integrates the dynamical model OPA, the LIM2 sea ice model and the PISCES biogeochemical model. NEMO runs on a tri-polar grid (one pole in the Southern Hemisphere under Antarctica and two poles in the Northern Hemisphere under North America and Asia) in order to improve the representation of ocean dynamics in the northern high-latitudes. There are 31 unequally spaced vertical levels and a nominal resolution of 2° that is refined up to 0.5° in the equatorial area. The atmosphere and ocean models are linked through the coupler OASIS48, ensuring energy and water conservation. Additional details about the IPSL-CM5A model can be found in Dufresne et al.30.The climate model used in this study is the IPSL-CM5A GCM31 and ref. 49. In this study, the GRISLI model is nested to the Greenland region on a cartesian grid of 15 km × 15 km. Over the grounded part of the ice sheet, the ice flow resulting from internal deformation is governed by the shallow-ice approximation50. The model also deals with ice flow through ice shelves using the shallow-shelf approximation51 and predict the large-scale characteristics of ice streams using criteria based on the effective pressure and hydraulic load. At each time step, the velocity and vertical profiles of temperature in the ice are computed, as well as the new geometry of the ice sheet. The isostatic adjustment of the bedrock in response to the ice load is governed by the flow of the asthenosphere, with a characteristic time constant of 3000 years, and by the rigidity of the lithosphere. The temperature field is computed both in the ice and in the bedrock by solving a time-dependent heat equation. Here, as the ice sheet model GRISLI is not synchronously coupled with the IPSL-CM5A model, temperature and precipitation fields are asynchronously passed from IPSL-CM5A to GRISLI. The surface mass balance is defined as the sum between accumulation and ablation computed by the positive degree-day (PDD) method52.The ice sheet model used in this study is the GRenoble Ice-Shelf and Land-Ice model (GRISLI). GRISLI is a three-dimensional thermo-mechanical model that simulates the evolution of ice sheet geometry (extension and thickness) and the coupled temperature–velocity fields in response to climate forcing. A comprehensive description of the model can be found in ref. 53. In this work, we use an improved version of this method, which has first been applied to the EO glaciation29 and that we have adapted to Greenland. Specifically here, we build a three-dimensional matrix to account for the three main drivers of an ice sheet evolution that are: (1) realistic insolation variations, (2) the atmospheric pCO2 evolution and (3) the ice sheet feedbacks on itself. The matrix hence comprises temperature and precipitation fields that are obtained from reference IPSL-CM5A runs initialized with different combinations of orbital parameters, pCO2 and ice sheet size. In a second step, the temperature and precipitation fields that force GRISLI can be computed by interpolating between the reference IPSL-CM5A climatic states, based on the current value taken by the 65°N summer insolation and by the atmospheric pCO2 and on the instantaneous size of the ice sheet in GRISLI. This ensures that the climatic fields passed to GRISLI are appropriately updated at each time step to follow the evolution of both external (insolation and pCO2) and internal (ice sheet geometry) forcings.In the absence of synchronous coupling between GRISLI and IPSL-CM5A, we utilise an interpolation that asynchronously couples both models and offers the possibility of carrying long-term numerical integration of the ice sheet model while accounting for the time evolution of the main climate forcings. This method consists of building a matrix of possible climate states that are generated by IPSL-CM5A under various combinations of forcings, which are chosen from the range of possible values taken by forcings. The ice sheet model GRISLI can then be continuously forced by temperature and precipitation fields obtained by interpolation between the different IPSL-CM5A climatic states, interpolation based on the time evolution of the forcings. The principle of this method has been described in details in Pollard (2010) and an early version of it has been applied to the Eocene-Oligocene Transition (EOT) in Antarctica36. At each time step of the ice sheet model simulations for the 3–2.5 Ma period, the impact of the summer insolation can be included by appropriately interpolating between reference IPSL-CM5A runs with warm or cold orbits.In this study, we have chosen two orbital configurations that produce respectively the maximal (warm orbit) and the minimal (cold orbit) mean summer insolation at 65°N between 3.0 and 2.5 Ma, according to the astronomical solution calculated by the model of ref. 2 concentrations: 220 ppmv, 280 ppmv, 360 ppmv and 405 ppmv. Late Pliocene pCO2 records indeed document a range of variation of the atmospheric pCO2 comprised between ~200 ppmv and ~400 ppmv . We initialise IPSL-CM5A with standard PlioMIP phase 1 conditions54, which are then modified to start from an ice-free Greenland and extremely favourable conditions for glacial inception represented by the cold orbit described above and a pCO2 concentration of 220 ppmv. GRISLI is then force with constant temperature and precipitation fields from the IPSL-CM5A simulation until the ice sheet reaches equilibrium. The ice sheet geometry is then fed back to IPSL-CM5A while pCO2 and orbital parameters are kept identical. New climatic fields are thus obtained and GRISLI further simulates the ice sheet gain until a new equilibrium is reached. The geometry of the ice sheet can then be passed again to IPSL-CM5A. This process is repeated until a new iteration does not markedly increase the ice volume. Here, seven iterations allow us to obtain seven Greenland ice sheet sizes ranging from very small to nearly full size ice sheet  × 7 (for Greenland ice sheet sizes) simulations are run in parallel to provide reference T and P fields that cover the range of possible variations of the three main drivers that are insolation, pCO2 and ice sheet size. A continuous T and P forcing can then be calculated based on the prescribed (for insolation and pCO2) and emerging (for ice sheet) evolutions of these drivers. Although complex and fastidious to implement, one particular advantage of this method is that it allows virtually any pCO2 scenario to be tested without additional GCM runs.The matrix of reference IPSL-CM5A climatic states is then built using the forcings described above. A total of 2 (for the insolation) × 4  in which the pCO2 evolution is very well constrained.We assess our model performances and our modelling strategy by presenting first a comparison between the simulated surface mass balance (SMB) obtained from the CMIP5 historical IPSL-CM5A experiment57), such as South Greenland here; 2) to biases in simulated temperatures and precipitations in the Greenland region in IPSL-CM5A30 and/or MAR. Yet overall, the simulated SMB using IPSL-CM5A is reasonably close to that of MAR.Supplementary Fig. 2 scenarios (185 ppmv and 220 ppmv) and our two extreme orbital configurations. We also restrict the ice sheet simulation to Greenland. Because the climate states added in the matrix have extensive ice sheet and sea ice covers over the Northern Hemisphere, we allow the ice sheet model to use variable basal melting factors following the multi-proxy index of Quiquet et al.58. Basal melting factors thus vary linearly between 0 and 5 m/year in regions where depth are lower than 1300 m and are fixed to 10 m/year where depth greater than 1300 m. In addition, we use the sea level curve of Waelbroeck et al.59 as a forcing to determine which ice model grid points are floating or grounded. These two processes are added to take into account the climatic influence of the LIG deglaciation outside Greenland because we do not explicitly model this ice sheet evolution. Finally, the ice sheet model is initialized with the LGM GrIS.In order to simulate the Eemian deglaciation, we have performed four additional climate simulations with a LGM ice sheet configuration, two low pCO2 leads to the waning of the GrIS between 133 and 123 Ka. The timing of the deglaciation is consistent with results from other studies . Stone et al.60 “best score” simulation places the start of the GrIS deglaciation at 135 Ka  and the end around 123 Ka. Goelzer et al.62 restrict their figures to the interval 130–115 Ka so that the start of the deglaciation in their simulations is unclear but the lowest GrIS volume is also found around 123 Ka. Finally, Bradley et al.61 also place the beginning and end of the GrIS deglaciation at 133 and 123 Ka, respectively  simulated by GRISLI under preindustrial IPSL-CM5A forcings30. Compared to other studies, our simulated LIG GrIS does not reach a volume low enough. However, considering that we just adapted a method that is designed for the PPT and not specifically for the Eemian, we argue that the model does a reasonable job in reproducing the Eemian deglaciation. In summary, the reasonable agreement between our modelled deglaciation and the results from other transient LIG simulations provides confidence in our modelling strategy.The results from this sensitivity experiment are shown on Supplementary Fig. The codes of the IPSLCM5 and GRISLI models are available on request to the authors.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Bellamya aeruginosa was caged in situ at two locations representing different pollution levels for different exposure times . At each of these time points, biochemical parameters, i.e., phase I biotransformation enzymes ethoxyresorufin-O-deethylase (EROD), the antioxidant enzymes superoxide dismutase and catalase, reactive oxygen species, protein carbonyl content and lipid peroxidation, were evaluated in the hepatopancreas of snails. In addition, surface sediments were collected for analysis of contaminants of concern, including inorganic pollutants, organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers.Freshwater sediments have been recognized as a long-term sink and potential source for environmental pollutants released into the aquatic ecosystems. In this study, the sediment quality of Taihu Lake, which is susceptible to anthropogenic contamination, was assessed by a combination of chemical analytical and biological end points. Specifically, the snail B. aeruginosa were subjected to significant environmental stress, which diminished during later sampling time points.Chemical analyses revealed that sediments from Taihu Lake were contaminated with trace elements and organic pollutants. Concentrations of trace elements  and organochlorinated pesticides  exceeded their corresponding threshold effect level according to the sediment quality assessment values for freshwater ecosystems in Canada, indicating that adverse biological effects may occur. All biomarkers, except EROD activity, were induced in snails during all exposure times. The integrated biomarker response index (IBR) indicated that during the initial exposure phase (7 days), Results showed that IBR correlated well with the levels of environmental contaminants, demonstrating the applicability of this biomonitoring approach to complex environmental exposure scenarios.The online version of this article (10.1186/s12302-018-0164-y) contains supplementary material, which is available to authorized users. In recent years, water quality of many freshwater lakes has been shown to progressively deteriorate with the rapid economic growth in many developing countries. Main contributors to the contamination of lake ecosystems are runoff from agricultural activities and industrial and municipal effluents , 2. SediField toxicity tests using caged organisms present numerous advantages over laboratory toxicity testing and indigenous community surveys . In situBellamya aeruginosa  is commonly found in various freshwater habitats of lakes (including Taihu Lake), reservoirs, rivers, streams, ditches and ponds throughout China. This species is a dominant community member of freshwater aquatic systems [B. aeruginosa ingests sediment particles, organic detritus, algae and bacteria on the surface of sediments or other substrates [B. aeruginosa is a primary food item of the black carp (Mylopharyngodon piceus), which is also consumed by humans. Therefore, it represents a key organism involved with the transfer of contaminants through the food web in Chinese surface waters and, thus, it can be recommended as a key organism in the assessment of contaminant risks to ecosystems in China.Benthic gastropods are essential members of aquatic systems and relatively sensitive to contaminants . Bellamy systems  and it ibstrates  and, thebstrates , 24. B. O-deethylase) represents the activity of the phase I biotransformation enzyme, cytochrome P4501A (CYP1A), which is essential for detoxification and excretion of chemicals. Measurements of ROS (reactive oxygen species) production and antioxidant defense in invertebrates are also applied widely for monitoring of pollution of aquatic environments [The impacts of sediment contamination on biota can be determined by the measurement of biomarkers . Biomarkronments , 30. Manronments . ROS canronments . To interonments , is wideronments , 34. Theronments .Taihu Lake, located in the delta region of the Yangtze River, is the third largest freshwater lake in China and an important drinking water source for surrounding cities , 37. WitB. aeruginosa as a tool to assess exposure to contaminated sediments under field conditions; and (2) to evaluate the sediment quality of Taihu Lake using a combination of chemical analysis and in vivo toxicity assessments. Specifically, the battery of biomarkers utilized by this study consisted of activities of EROD, antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT), reactive oxygen species (ROS), the protein carbonyl content (PCC) and lipid peroxidation (LPO). Finally, levels of trace metals, OCPs, PCBs and PBDEs were investigated in sediments of the lake.In this study, caged snails were employed to assess sediment quality in the eastern part of Taihu Lake. The objectives were (1) to determine the suitability of a battery of biochemical biomarkers analyzed in caged cis-heptachlor epoxide, trans-heptachlor epoxide, methoxychlor and mirex were purchased as a mixture from AccuStandard .Reference standards of OCPs, including 2,4′-dichlorodiphenyldichloroethylene , 2,4′-dichlorodiphenyldichloroethane , 4,4´-dichlorodiphenyltrichloroethane , 4,4′-dichlorodiphenyldichloroethylene , 4,4′-dichlorodiphenyldichloroethane , alfa-hexachlorocyclohexane (α-H-CH), beta-hexachlorocyclohexane (β-HCH), gamma-hexachlorocyclohexane (γ-H-CH), delta-hexachlorocyclohexane (δ-HCH), aldrin, hexachlorobenzene (HCB), pentachloroaniline (PCA), dieldrin, α-endosulfan, β-endosulfan, endrin, heptachlor, The PCB congeners including CB-18, 28, 31, 44, 52, 101, 118, 138, 149, 153, 170, 180, 194 and 209 were also purchased from AccuStandard . PBDE congeners: BDE-28, 47, 66, 99, 100, 153, 154 and 183 were bought as a mixture from Wellington Laboratories Inc. . A mixture with octa- to deca-BDEs  was also bought from Wellington Laboratories Inc. . Methoxylated polybrominated diphenyl ethers (MeO-PBDEs) congeners, including 2-MeO-BDE68 and 2-MeO-BDE47, were purchased from AccuStandard . All solvents used for sample processing and analyses were of pesticide grade.B. aeruginosa were obtained from clean sediments of artificial ponds in Wuhan Botanic Garden  and selection of the correct species was confirmed taxonomically [B. aeruginosa were fed with commercial aquarium fish food  [n = 120) was sampled for the verification of baseline levels of biomarkers for this species, and as no significant difference was detected among baseline levels determined for this group. This group was named basal group (Control). 720 snails were transported to the selected experimental sites in plastic bags containing water and oxygen for the caging exposure tests. Snails with an average shell length of 20.00 ± 1.68 mm and average body weight of 2.05 ± 0.20 g were used in the experiments. The experiments were conducted in accordance with the Animal Ethics Committee at Tongji University.Adults of omically . Before omically . The con, China) . After aIn situ exposure tests were performed at two experimental sites in Taihu Lake, site A (31°13′51′′N–120°22′7′′E) and site B (31°6′2′′N–120°13′55′′E)  composed of nylon mesh (3 mm) and a polypropylene baffle were used for the exposures. At each site, three cages, containing 120 snails each, were immersed into the water and fixed in direct contact with the sediments. One cage from each site was sampled each after 7, 14 and 21 days, respectively, and surviving organisms were counted. A subset of snails was immediately frozen in liquid nitrogen for biochemical analysis. However, one cage located at site A disappeared unexpectedly for unknown reasons after 21 day of exposure, and thus it was impossible to recover the snails from this cage. The caged snail samples exposed for 7 and 14 days, and 7, 14 and 21 days at sites A and B were defined as Surface sediment samples (0 to 5 cm depth) were collected from each caging site at the beginning of the encaged experiment using a Van Veen grab and were brought to the laboratory immediately. Sediments were freeze-dried, homogenized, ground and sieved with a 200-mesh nylon screen and kept below − 20 °C until chemical analysis.Total organic carbon (TOC) contents of the sieved and freeze-dried sediments were determined using a total organic carbon analyzer .3, HCl and HF according to the EPA Method 3052 [4 was added to remove HF and then adjusted to a volume of 10.0 mL with 2% (v/v) HNO3 before instrumental analysis [The sieved and freeze-dried samples (0.2 g) were microwave digested in Teflon vessels using a mixture of concentrated HNOhod 3052 . HClO4 wanalysis . Target The procedures for the extraction and purification of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) have been published in detail elsewhere –52. So tg for 10 min at 4 °C and the section of supernatants was collected for determination of protein carbonyl content. The remaining content was continuously centrifuged at 9000×g for 20 min at 4 °C and the resulting supernatant was separated for the analysis of other biochemical parameters. Total protein concentrations were determined with the total protein kit  based on the Bradford method [Thirty snails from each cage were dissected and their hepatopancreas was removed for biomarker determination. Hepatopancreas tissue was weighed and homogenized (1:4 w/v) in ice-cold Tris–HCl buffer  and 1.0 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor using a glass homogenizer. The homogenates were centrifuged at 1000×d method . All bioEROD activity of the supernatant (S9 fraction) was measured following the hydroxylation of 7-ethoxyresorufin according to the method of Burke and Mayer  as adaptROS  were detected with the Fluorometric Intracellular ROS Assay Kit  following the manufacturer’s protocol using fluorometric sensors localized to the cytoplasm, resulting in a fluorometric product proportional to the amount of ROS present. The fluorescence intensity was determined at λex = 540 nm and λem = 570 nm. Fold increases in ROS levels were determined by comparison with the control group.SOD activity was determined by the SOD assay kit . SOD activity was quantified by measuring the decrease in the color development at 440 nm with a colorimetric method, which is proportional to the amount of superoxide anion. Results were expressed as the inhibition rate (%) of the reduction with molecular oxygen.Changes in CAT activity were evaluated by the catalase assay kit . CAT activity was quantified by measuring the hydrogen peroxide substrate remaining after reaction for 15 min by an absorbance spectrophotometer at 520 nm. Results were expressed as mmole/min/mL.LPO was determined by the lipid peroxidation assay kit  by measuring the colorimetric product (532 nm) formed by the reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA) and which is proportional to the concentration of MDA present. Results were expressed as nmole/μL.PCC assay kit  was applied to detect the production of stable carbonyl groups induced by oxidative stress. Carbonyl content was determined by the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of stable dinitrophenyl (DNP) hydrazone adducts, which can be detected spectrophotometrically at 375 nm, proportional to the carbonyls present. Results were expressed as nmole/mg protein.X0) were used as a reference value.The biomarker results were applied into the “Integrated Biomarker Response Index” (IBRv2) described by Beliaeff and Burgeot  and modiXi) obtained at each caging site and the respective mean baseline (X0) was log-transformed (Yi)  Eq. :1\documeμ) and standard deviation (s) of Yi were computed. Then, Yi values were standardized by the formula  and mean of reference biomarker data (Z0) were used to define the biomarker deviation index (A) (Eq. A parameters for each biomarker.The differences between the mean of standardized biomarker response ( (A) Eq. :3\documep < 0.05.Statistical analysis of data was carried out by SPSS 19.0. Biomarker results were tested for normality and homogeneity of variance with the Shapiro–Wilks and Levene test, respectively. Analysis of variance (ANOVA) followed by a Dunnett’s test was used to compare the biomarkers obtained in snails from the two caging sites after different exposure times with those obtained in snails acclimated in the laboratory. The significance level was set at There were no differences in water quality between the two sampling sites, with water temperatures of 26.8–33.1 °C, pH of 8.2–8.7 and dissolved oxygen (DO) concentrations of 6.57–9.16 mg/L.Chemical characterization of the sediment samples from the caging sites identified 7 trace elements and 55 organic compounds  , while no significant differences were observed between snails confined at the site A compared with the control group .The levels of ROS in the hepatopancreas of caged snails at both sites A and B showed a time-dependent and significant increase compared to snails from the control group Fig. a. Specifp < 0.05; Fig. p < 0.01; Fig. p < 0.01; Fig. Significant and time-dependent increases in activities of antioxidant enzymes were observed in snails exposed at both sampling sites Fig. b, c. SODp < 0.01 and p < 0.05, respectively). Then the levels of LPO and PCC decreased on day 21 at site B. While LPO was still significantly higher in snails from site B than the control group (p < 0.01), no such difference was observed for the levels of PCC in the hepatopancreas of caged snails after 21 days at this site (p < 0.05).During the initial 14 days of exposure, levels of LPO and PCC in the hepatopancreas of caged snails at both sites increased with the increasing exposure time Fig. . SignifiA) of CAT activity was greater than that of all other biomarkers, which indicated that CAT was the most responsive biomarker in this in situ exposure experiment. The IBR values for both sites A and B were 10.2 and 10.8 after caging for 7 days, respectively, as minor variations in the analyzed biomarkers were observed. After exposure for 14 days, both caging sites A and B showed the highest IBR value . The IBR value for site B decreased to 14.1 after 21 days of exposure.All biochemical biomarkers showed a significant induction in the hepatopancreas of snails as a function of exposure time, with the exception of EROD activity at site A Fig. . The bioB. aeruginosa caged in situ, integrating multiple biochemical responses and chemical analysis.Biomarkers are important tools for understanding linkages between external contaminant exposures, internal doses and associated potential impacts on health status . In thisB. aeruginosa collected at 20 locations in Taihu Lake. Thus, there is the potential for chemical compounds in sediments from Taihu Lake to have adverse effects on aquatic organisms. In addition, all concentrations of chemicals analyzed in the sediment collected from site A, except for PBDEs, were greater than those at site B. This is in accordance with the result that the survival rate of B. aeruginosa caged at site A was lower than that of snails caged at site B after different exposure times (7 and 14 days), indicating that B. aeruginosa was a relatively sensitive species for biomonitoring in Taihu Lake.Analyses of selected contaminants in sediments of Taihu Lake showed that metals , OCPs, PCBs and PBDEs were detectable at both caging sites. Sediment quality assessment values for freshwater ecosystems in Canada  were conp-dioxins and dibenzofurans (PCDD/Fs) [Xenobiotics, especially lipophilic pollutants, can induce adaptive changes in exposed organisms such as induction of phase I enzymes that can oxidize xenobiotics to make them more polar, and thus allow them to be conjugated and excreted. In this study, the mixture of contaminants in sediments of Taihu Lake significantly induced EROD activity in hepatopancreas tissues of snails from both sites after exposure for 7 days. Dioxin-like contaminants, including some PCBs , certainPCDD/Fs)  have beePCDD/Fs)  also demB. aeruginosa were significantly increased after exposure to 51 mg/kg of Cu for 28 days. These concentrations were similar to those measured during our study (Cu concentrations ranged between 17.7 and 39.5 mg/kg). Thus, together with the other elements measured in sediments from Taihu Lake, we hypothesize that metals were likely to have caused the oxidative stress observed in caged snails during the exposure.Pollutants such as PCBs, lindane and certain heavy metals are important triggers of oxidative stress and the formation of ROS in biological systems , 61. In Lipid peroxidation is the process of oxidative degradation of lipids, which is regarded as a measure of oxidative stress induced by environmental pollutants including heavy metals and wide ranges of organic pollutants , 64. In B. aeruginosa could get enough food during the caging experiment [B. aeruginosa increased in Suzhou Creek for 0, 15 and 30 days and did not show significant difference (p < 0.05). Abiotic factors such as temperature, salinity and dissolved oxygen content also influence biomarker responses in an organism [Variations in biochemical biomarkers depend on food availability and spawning period besides exposure to pollutants . Prelimiperiment . Xiong eperiment  have alsorganism . The anaorganism . So the B. aeruginosa [Theba pisana) [B. aeruginosa initially increased and then weakened, which might be an adaptation mechanism for B. aeruginosa to toxic effects of environmental contaminants. The IBR values were higher in animals from site A on the 14th day of exposure compared to site B, indicating that site A had a greater impact on B. aeruginosa. As higher contamination levels of metals, OCPs and PCBs were found in the sediments collected from site A compared to site B, the IBR index proved to be in agreement with the levels of environmental contamination in different sediments from Taihu Lake. Thus, it could be inferred that the integration of multiple biomarker responses found in caged snails could reflect the contamination levels measured at different sites and used as an efficient biomonitoring approach [The Integrated Biomarker Response Index (IBRv2) has been found capable of discriminating scores to describe toxically induced stress based on the specific responses of a number of biomarkers across different sampling sites . In thisruginosa  and land pisana) . In geneapproach , 34. HowB. aeruginosa were consistent with the chemical contamination in the sediments of the lake, indicating that in situ exposures with caged snails can serve as an efficient biomonitoring approach to evaluate sediment quality.The biochemical responses in caged snails deployed at two sites in Taihu Lake as well as the chemical analysis of selected organic and inorganic pollutants indicated significant anthropogenic pollution and potential risks to resident organisms. Multiple biomarkers including EROD, ROS, SOD, CAT, LPO and PCC were induced during caging exposure experiment. The IBRv2 results showed that the CAT activity was the most responsive biomarker. The results of the biochemical biomarkers in Additional file 1: Table S1. Chemical analysis of surrogate sediments used in the laboratory culture of Bellamya aeruginosa.Text S1. Organic pollutants.


Cuban sugarcane wax acids (SCWA) and policosanol (PCO) are mixtures of higher aliphatic acids and alcohols, respectively, purified from sugarcane wax with different chief components. Although it has been known that they have antioxidant and anti-inflammatory activities, physiological properties on molecular mechanism of SCWA have been less studied than PCO. In this study, we compared antiatherogenic activities of SCWA and PCO via encapsulation with reconstituted high-density lipoproteins (rHDL). After reconstitution, SCWA-rHDL showed smaller particle size than PCO-rHDL with increase of content. PCO-rHDL or SCWA-rHDL showed distinct inhibition of glycation with similar extent in the presence of fructose. PCO-rHDL or SCWA-rHDL showed strong antioxidant activity against cupric ion-mediated oxidation of low-density lipoproteins (LDL), and inhibition of oxLDL uptake into macrophages. Although PCO-rHDL showed 1.2-fold stronger inhibition against cholesteryl ester transfer protein (CETP) activity than SCWA-rHDL, SCWA-rHDL enhanced 15% more brain cell (BV-2) growth and 23% more regeneration of tail fin in zebrafish. PCO and SCWA both enhance the beneficial functions of HDL to maximize its antioxidant, antiglycation, and antiatherosclerotic activities and the inhibition of CETP. These enhancements of HDL functionality by PCO and SCWA could exert antiaging and rejuvenation activity. Dyslipidemia is global health risk causing cardiovascular disease (CVD), the first leading cause of death in the world. A higher level of serum HDL-cholesterol is inversely correlated with the incidence of the CVD and hypertension , 2. InhiIn our previous studies, policosanol (PCO) has been found to have potent cardioprotective properties based on molecular basis, such as CETP inhibitory activity, antiglycation, and anti-inflammatory activities . In animSaccharum officinarum L.), namely, octacosanol, triacontanol, and dotriacontanol, hexacosanol, and tetratriacontanol as major components -cholesteryl oleate . The CE-transfer reaction was conducted in 300-μL reaction mixtures each containing human HDL3  as a cholesteryl ester transfer protein (CETP) source, the apoA-I-rHDL  as a CE-donor, and the human LDL  as a CE-acceptor. PCO-rHDL or SCWA-rHDL was provided to the reaction mixture as an inhibitor. The extent of CETP inhibition was calculated as follows:An apoA-I-rHDL containing cholesteryl ester was synthesized as previously described  with [3H2 and 95% air) as our previous report [Human monocyte THP-1 cells were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS). To induce differentiation into macrophages, monocyte cells that had undergone no more than 20 passages were incubated in medium containing phorbol 12-myristate 13-acetate  in 24-well plates for 48 h at 37°C in a humidified incubator , and either SCWA-rHDL or PCO-rHDL for 48 h at 37°C in a humidified incubator. After incubation, the cells were stained with oil red O solution (0.67%) to visualize the amounts of lipid species after uptaken. In order to determine amount of oxidized species, the cell media (0.25 ml) were then analyzed by TBARS assay using malondialdehyde (MDA) standard.The differentiated and adherent macrophages were then rinsed with warm PBS and incubated with 400 2 and 95% air). After incubation, the cell number was counted using Cellometer K2 image cytometer Microglial cells in human brain (BV-2) were cultured in a Dulbecco's modified Eagle medium (DMEM) and maintained at 70% confluency as our previous report . Cells wWildtype zebrafish (AB strain) and its embryos were maintained according to standard protocols as in our previous report , 10. TheμL of rHDL alone (7 μg of apoA-I), PCO-rHDL, or SCWA-rHDL was injected into the tail muscle of each zebrafish near the urostyle (n=9 for each group). After the injection, the fish were maintained in a 28°C system incubator. Images of regenerating fins of live zebrafish were taken at 24-h intervals for up to 168 h under a stereomicroscope  using a Motic cam 2300 CCD camera with Image-Pro Plus software version 4.5.1.22 .In order to compare ability for tissue regeneration by PCO or SCWA, fins were tested using an STZ-induced adult zebrafish as in our previous reports , 10, 28. p<0.05.All data were expressed as the mean±SD of at least three independent experiments with duplicate samples. Data were evaluated via one-way analysis of variance (ANOVA) using SPSS , and the differences between the means were assessed using Duncan's multiple-range test. Statistical significance was defined asSCWA-rHDL and PCO-rHDL were synthesized  with a sFluorospectroscopy measurement revealed that lipid-free and lipid-bound apoA-I showed WMF of 341 and 337 nm, respectively, suggesting that Trp in apoA-I moved to a more hydrophobic phase via interaction with phospholipids as shown in Conjugated diene detection assay revealed that SCWA-rHDL and PCO-rHDL showed similar antioxidant activity against cupric ion-mediated LDL oxidation after 2 h incubation .2+, PCO-rHDL retained more antioxidant activity than SCWA-rHDL-treated LDL and showed a more distinct band with slower mobility.After 6 h incubation with cupric ion, oxidized LDL showed the fastest electromobility on 0.5% agarose gel lane O,  compared3 showed a 3.4-fold increase in glycation compared to HDL alone based on yellow fluorescence  also resulted in approximately 15% and 26% inhibition, respectively , a well-known CETP inhibitor, resulted in 41% and 62% inhibitionectively . They shμM SCWA) showed 26 and 45% inhibition, respectively, while PCO-rHDL (6 and 30 μM PCO) showed 36% and 55% inhibition, respectively. Interestingly, PCO-rHDL showed 1.2-fold higher CETP inhibition activity than SCWA-rHDL.However, in rHDL state, SCWA-rHDL and PCO-rHDL showed remarkably higher inhibitory activity against CETP than SCWA and PCO in ethanol . SCWA-rH5 cells from H&E staining, a 33% increase in cell numbers compared to PBS-treated cells (13.8 x 105 cell), indicating that native-rHDL facilitated cell replication with cytoprotective properties. Interestingly, SCWA-rHDL and PCO-rHDL treatment increased more cell numbers. Treatment with SCWA-rHDL (1:1) and SCWA-rHDL (1:5) resulted in the highest cell number with 22.1 x 105 cells and 26.7 x 105 cells, respectively, corresponding to 9 and 45 μM SCWA. Treatment with PCO-rHDL (1:1) and PCO-rHDL (1:5) resulted in 19.1 x 105 cells and 23.1 x 105 cells, respectively, corresponding to 9 and 45 μM PCO.SCWA-rHDL and PCO-rHDL treatment increased growth of brain glial cells in a dose-dependent manner as shown in As shown in Quantification of oxidized species in cell media revealed that cells treated with oxLDL alone contained 2-fold more MDA than PBS-treated cells. As a control, rHDL-treated cells had 20% less MDA than those treated with oxLDL alone; however, SCWA-rHDL- or PCO-rHDL-treated cells contained 45% or 42% less MDA than cells treated with oxLDL alone.With normal diet (ND) consumption, rHDL-injected group showed 1.2-fold higher tissue regeneration activity than the PBS-injected group over a period of 168 h . The SCWAlthough PCO is known as a lipid-lowering agent that increases HDL-cholesterol levels  via CETP in vitro study on suppression of LDL oxidation due to the antioxidant activity of PCO and SCWA  also resulted in 15% and 26 % inhibition, respectively (Interestingly, as shown in ectively . They exα-helical region and helps in the transfer process [α-helix and block CE/TG transfer from HDL to LDL by interfering with CETP and LDL binding. In our current study, we showed that CETP inhibitory activity was enhanced by incorporation of PCO and SCWA into rHDL. Collectively, PCO and SCWA may interfere with HDL and LDL binding to CETP to form a hydrophobic channel.Regarding the detailed mechanism of CE transfer, a recent study reported that CETP binds to HDL via hydrophobic interactions because the HDL surface lipid curvature generates a hydrophobic environment . Many re process . A compoEnhanced LDL catabolism and reduced TGs metabolism can be accelerated by inhibition of CETP. It has been asserted that CETP inhibition is a potent antiatherogenic strategy. Because of higher CETP activity, low HDL-C levels and prevalent dysfunctional HDL become risk factors for autoimmune disease with inflammation and other risk factors , 41. TheFew studies on SCWA, which is a mixture of long chain aliphatic primary acids also known as D-003, have discussed its ability to lower lipid levels and its anti-inflammatory activities , 21, 42.In conclusion, PCO and SCWA both enhance the beneficial functions of HDL to maximize its antioxidant, antiglycation, and antiatherosclerotic activities and the inhibition of CETP. These enhancements of HDL functionality by PCO and SCWA could exert antiaging and rejuvenation activity.


In the case of H2O ice, H2O2 is formed at the temperatures below 60 К. In the case of H2O:O2 ice, H2O2 is formed in the 20–140 К range. For H2O:O2 = 9:1 ice, we derived H2O2 photochemical quantum yield as a function of sample irradiation temperature. The obtained data can be used for evaluation of H2O2 photoproduction at the surface of astrophysical water ice bodies and inside the particles of Noctilucent Clouds in the Earth’s atmosphere.We report the results of laboratory measurements of H The characteristics of these processes greatly depend on the characteristics of irradiation, ice type, its thickness, temperature, additives, and others. The end products such as H2O2, O2, O3, etc., both in solid and gas phase are of considerable interest for astrophysics as they are all oxidizing agents and may provide a source of chemical energy as a fuel for extraterrestrial life5.It is well known that surfaces of most icy bodies in the outer Solar System and interstellar space consist mainly of water and are regularly bombarded with energetic particles and photons. This irradiation triggers a spectrum of physicochemical processes inside solid phase2O2 on Europa’s surface6 and the discussed existence or absence of H2O2 on Enceladus, Ganymede, and Callisto7 stimulated extensive laboratory studies of the mechanisms of formation and measurement of the parameters of producing concentrations of this component in high-purity H2O ice and H2O ice with different additives irradiated by energetic particles14. In particular, it was shown that, as compared to H2O ice, the presence of О2 greatly increases the production of H2O2 and other products , especially at relatively high irradiation temperatures of 80–120 К. At the same time, except for a few works, significantly less attention was paid to investigation of H2O2 production by VUV photons. In particular, Gerakines et al.15 and Schriver et al.16 reported about H2O2 formation in H2O ice at 10 K irradiated by microwave discharge hydrogen flow lamps. Yabushita et al.17 found out H2O2 presence in H2O ice after its irradiation by 157 nm photons at 90 K. Shi et al.18 measured H2O2 in porous H2O + O2 ice irradiated by 193 nm photons at 40–78 K. Thus, many details of H2O2 production by VUV photons, the temperature dependence in the first place, are still understood poorly.The established detection of H2O2 production inside thin H2O and H2O:O2 ice samples irradiated by 121.6 nm photons in the temperature range of 20–140 K. We discuss possible implications of the obtained results at the surface of astrophysical water ice bodies and inside the particles of Noctilucent Clouds in the Earth’s atmosphere.This work reports the first results of laboratory measurements of H21. Since the clouds discovery22, there were many discussions about their nature (see reviews by Gadsden & Schröder19 and by Thomas20). Only recently, the infrared spectra of clouds showed23 that NLCs consisted mainly of water ice. Thus, it is not doubt now that clouds form by condensation of water vapour and can influence on gas-phase chemistry of this region due to water vapour is its key parameter.There exists at least one analog of such water icy bodies regularly irradiated by VUV photons in the Earth’s atmosphere. Each summer at polar and middle latitudes, one can observe the highest atmospheric clouds called Noctilucent Clouds (NLCs). They appear in mesopause region  at the temperatures of 120–150 Kα line) and the reaction H2O + hv → H + OH provides the main chemical source of the family of odd hydrogen 24. In turn, the reactions with participation of HOx components  remove the components of the odd oxygen family (Ox: O(1D), O(3P), and O3). Therefore, the appearance of NLCs is expected to reduce the concentrations of water vapour and HOx and to increase Ox. However, rocket measurements of the concentration of atomic oxygen made during several campaigns demonstrated unpredicted O depletion around NLCs25. A possible explanation of the revealed effect was proposed by Murray and Plane26 who noticed that there also occurs photolysis of H2O molecules inside NLCs particles. The photoproducts (H and OH) may release into gas phase and additional source of HOx leads to increase of Ox removal. In the work by Kulikov et al.27 this hypothesis was verified by laboratory measurements of the photodesorption rate from thin water ice samples irradiated by 121.6 nm photons in the temperature range of 120–150 K. It was found that most photoproducts did not leave the solid phase and tended to recombine in water molecules back. Basing on the results of ice irradiation by energetic particles at relatively high temperatures of 80–120 К14, we can assume that H2O2 is the principal photoproduct that may accumulate in NLCs.In the conditions of daytime mesopause, water vapour is subjected to intensive solar VUV radiation  taken in different seasons. The model estimates of the concentration of those ions formed as a result of the H2O2 + O2+ → H2O2+ + O2 reaction agreed well with the results of measurements made in different seasons, except summer. The authors conjectured that the discrepancy was caused by the increased H2O2 concentration in the summer time. Later measurements29 revealed positively charged clusters containing H2O2 in the presence of NLCs particles. The researchers arrived at the conclusion that for such clusters to be formed, the concentration of H2O2 must be much higher than the expected level.Note that the experimental evidence of H−1 appeared in FTIR spectra of H2O and H2O:O2 ice samples as the result of their irradiation by calibrated source of 121.6 nm photons at high vacuum conditions. For more details please see Methods.Hydrogen peroxide was found by detecting the IR absorption band of 2850–2860 cmα photons at the photon flux intensity Iα = 5·1014 photons/(cm2·s) are presented in Fig. 2O ice  ≤ 60 К, and the temperature rise from 20 to 60 К results in a monotonic decrease of the integrated area of the 2850–2860 cm−1 band  cm−1. Forth, H2O2 peak position lays at 2850 cm−1 in the range of 20–100 K and shifts to 2852–2854 cm−1 at the temperatures 120–140 K. Note also, in both cases, there are no new features at 1039, 1142, and 1259 cm−1 . Note firstly, the present results are in a qualitative agreement with H2O2 temporal evolution obtained in earlier laboratory studies of the formation of this component in H2O ice irradiated by energetic particles14 and photons15. The temporal evolution of 2O ice, the growth stage continues for ~20–30 min and can be described by a quadratic function of irradiation time (VUV fluence). This corresponds to the results of irradiation of H2O ice by Lyman-α photons at 10 K obtained by Gerakines et al.15. In the case of H2O:O2 ice, the growth stage continues for ~10 min and can be described by a linear function of irradiation time (VUV fluence). Such behavior of 2O:O2 ice was found at other photon flux intensities. It means that, in the case of H2O:O2 ice, the H2O2 production during the growth stage can be fit successfully to a (pseudo) first-order reaction. We can conclude that the rate of H2O2 production is proportional to Iα and can determine the H2O2 photochemical quantum yield (2O2 generated per a Lyman-α photon absorbed by ice) as a function of Tir following, for example, Cooper et al.12 and Hand and Carlson14. For this, we carried out special experiments with H2O:O2 ice at lowIα = 3·1013 photons/(cm2·s) at which the growth stage continued more than 100 min. The Fig. Tir with corresponding linear fits whose values of slope are presented on Fig. Figure 2O2 production after irradiation of different ices36 used the values of −1 band absorption) from two sources10. Moore & Hudson8 measured −17 cm·molecule−1 and this value used for all temperatures. Loeffler et al.10 found out that −17 cm·molecule−1 at 20 K, 5.2·10−17 cm molecule−1 at 80 K, and 4.9·10−17 cm·molecule−1 at 110 K. Thus, for estimation of H2O2 column density (relative concentration) and H2O2 photochemical quantum yield from the results presented in Figs et al.8 data were interpolated into the temperature regions of 20–80 K and 80–110 K, and extrapolated to the temperature region of 110–140 K.All known studies of H2O ice, the maximum of relative concentration H2O2/H2O at 20 K is ~2% at 8 and ~1% at et al.10. In the case of H2O:O2 ice, the maximum of H2O2/H2O at 100 K is ~9% at 8 and ~4.5% at et al.10. In that time, the maximum of 8 and ~(0.0053–0.0175) molecules/photon at et al.10.One can see from Fig. see Fig. . Note al2O2 formation during VUV irradiation of H2O ice was discussed by Gerakines et al.15 and pointed by Loeffler et al.10 in comparison with H2O2 formation by ions. This component is formed due to reaction OH + OH → H2O2. The quadratic character of 2O2 molecule. These photons should produce two OH close to each other due to extremely low mobility of OH in water ice below 80 K38. The saturation of 2O2 + hv → 2OH and H2O2 + OH → H2O + HO2.The possible mechanism of Hsee Fig.  indicate2O:O2 ice, the linear character of 2O2 formation differs from the previous case. Note, Loeffler et al.10 and Hand and Carlson14 found out the same behavior of 2O ice irradiated by high-energy ions and electrons correspondingly. It was proposed for explanation of this, in particular, that two OH could be produced in an ion track caused by an ion10. Evidently, this mechanism cannot be transferred on our situation. But, following Loeffler et al.10 and Hand and Carlson14, we can speculate that the rate of H2O2 formation in H2O:O2 ice irradiated by VUV photons is proportional to VUV intensity and the concentrations of H2O and O2. In other words, one photon, one H2O molecule and one O2 molecule are needed for appearance of one H2O2 molecule. On the other hand, O2 is impurity of H2O ice and typically two O2 molecules are separated from each other by a “paling” of H2O molecules. So, it is hard to imagine that these O2 molecules can participate in formation of one H2O2 molecule due to extremely low mobility of intermediates (such as OH and HO2) in water ice below 80 K as above mentioned. Thus, we can conclude as a first approximation that H2O2 photochemical quantum yield inside VUV irradiated H2O:O2 ice is proportional to the relative concentration of O2 (O2/H2O), when O2/H2O In the case of Hsee Fig.  shows us2O:O2 ice, temperature dependences of H2O2 column density  H2O2 + hv → 2OH and (2) H2O2 + OH → H2O + HO2. So, R1,2 are the corresponding reaction rate coefficients. Note, Loeffler et al.39 measured the loss of H2O2 in H2O:H2O2 at temperatures between 21 and 145 K initiated by UV photons (193 nm). They obtained that the temperature dependences of H2O2 photodestruction cross section  varied in the range of 0.2–0.4 molecules/100 eV at 50–100 K, that is close to G-value of Lyman-α photons at 50–100 K obtained in our paper. Thus, contribution of Lyman-α photons to H2O2 production is defined by the ratio between energy fluxes of photons (EFph) and energetic particles (EFep). In the case of EFph~EFep, photons produce approximately the same amount of H2O2 inside ice as particles. At that, as it was noted by Gerakines et al.40, the penetration depth for Lyman-α photons (~45 nm41) in water ice is essentially less, than for protons which depends on its energy . So, one would expect the high relative H2O2 concentration in the top few tens of nm caused by Lyman-α photons. It means that H2O2 production by VUV photons can be important at EFph/EFep ≥ 10−2.In astrophysics, VUV irradiation competes with energetic particles bombardmentEFph EFep. But, at this moment, there is no information about containing of O2 inside water ice of Noctilucent Clouds. It is well-known that NLCs are formed as a result of gas-kinetic collision of H2O molecules with the surface of mesospheric aerosols, including adsorption and desorption properties. It is, generally, a relatively slow process, with the characteristic time (2–20 hours) depending on temperature43. In the real conditions of a summer mesopause, H2O concentration in gas phase is more than 4 orders of magnitude less than the concentration of O2 in ground (triplet) state and less than daytime concentration of O2 in singlet state ((2–4)·109 cm−3 at 80–85 km44). The molecules of O2 in ground and excited states, like those of H2O, continually bombard the surface of the forming particles of clouds, adsorb on their surface and can be trapped inside the ice matrix as the NCL particles are being covered by new ice layers. This suggests that a small part of O2 molecules from gas phase may be uptaken by the forming matrix of the cloud. It is certainly hard to believe that the relative concentration of O2 inside NCL particles  measurements with the ALOMAR RMR-lidar in Northern Norway , at the altitudes of 81–86 km SNLC varies within the (3–6)·10−8 cm2/cm3 range45. Taking into consideration that at these heights in the conditions of average solar activity Iα~3 · 1011 photons/(cm2·s)24, we obtain 3·3·s). In the real conditions of the mesopause, the NLCs particles are also irradiated by UV solar photons, which leads to H2O2 photodissociation in solid phase with the efficiency close to this process in gas phase39. For the considered range of altitudes of 81–86 km, the photodissociation constant of H2O2 in gas phase −4 s−1. Thus, the equilibrium concentration of H2O2 that may be accumulated in cloud particles irradiated by Lyman-α photons is 7·−3. Gumbel et al.25 reported the maximum of SNLC~10−7 cm2/cm3, which gives the estimate for the maximum values of 2O2 to be 7.5 · 103·3·s) and 5 · 107·−3, correspondingly. Under the conditions of NLCs existence, typical value of gas-phase concentration of H2O2 is ~2 · 105 cm−3 at 81 km and decreases with increasing height down to the values less than 104 cm−3 at 86 km26. Thus, the minimum of 2O2 concentration is equal to 104 cm−3, corresponds to 2 · 10−4 molecules/photon. Following our above conclusions, H2O2 photochemical quantum yield inside H2O:O2 ice is proportional to the relative concentration of O2. Taking into consideration the measured values of 2O:O2 = 9:1 ice at the temperatures of 120–140 K, we estimate that the minimum of −4 molecules/photon corresponds to 2 in NLCs ~(1.4–4.9)·105 cm−3) at 8 and ~(0.11–0.38)% (the absolute concentration ~(2.5–8.3)·105 cm−3) at et al.10. Note, that the estimated values of the O2 absolute concentration in NLCs are several orders of magnitude less than typical gas-phase concentrations of this component in ground and singlet states at the altitudes of 81–86 km.Let us assume that 2, the physicochemical processes occurring in them may remarkably affect the chemical composition of the mesopause region. On the one hand, it may be a possible explanation of the results of early rocket mass-spectrometer measurements29 indicating increased H2O2 concentration in the clouds. On the other hand, H2O2, product of its UV photodissociation (OH), H2O, O2 and other impurities  can participate in subsequent reactions producing more complex chemical compounds inside NLCs as it takes place, for example, in the bulk of supercooled water particles46. We hope that this research stimulates further experimental and theoretical investigations of the chemical composition of cloud particles. Note also that the obtained results are interesting for astrophysical applications, for example, for assessing the contribution of VUV irradiation to H2O2 production in the outer Solar System and interstellar space depending on temperature.To conclude, we have demonstrated for the first time that, if NLCs particles contain ≥0.1% Oet al.27, the apparatus consisted of a Fourier Transform Infrared Spectrometer (Bruker IFS 66 v)), a closed-cycle He refrigerator (Leybold ROK 10–300), a gas preparation and inlet system (further briefly GPIS), and a high-vacuum chamber with a volume of about 1000 cm3 pumped continuously by a turbomolecular pump system (Leybold-Heraeus) securing a high vacuum in the chamber down to the 10−8 mbar range. Inside the chamber, at the cold end of the cryostat there was a vertically mounted aluminium mirror (2.5 × 4 cm in size) as a substrate whose temperature was precisely regulated by a temperature controller . The mirror temperature could be selected in the 10–300 K range. The GPIS was equipped with baratrons and needle valves. The upper part of the high vacuum chamber had two ports, one of which was equipped with a MgF2 (5 mm thick) input window for a VUV lamp. The second port had a KBr window for the IR beam of the FTIR spectrometer. The input for the VUV lamp made an angle of incidence of ~450 to the mirror surface and, according to the estimates of the manufacturer, MgF2 transmitted about 60% of the quantum flux at the wavelength of 121.6 nm. As a VUV source (Lyman–α) we used a resonance hydrogen lamp (Opthos Instruments) containing a mixture of 10% H2 and 90% Ar excited by a microwave generator  with a frequency of 2450 MHz. The lamp intensity was determined by the power supplied by the microwave generator . The FTIR spectrometer was placed on rails allowing precise positioning of the instrument with respect to the cryostat with the sample. This was important for achieving a good overlap of the areas of the light spots from both, infrared (from spectrometer light source) and vacuum ultraviolet irradiation (from VUV lamp) of the ice film sample on the substrate. The operation of the FTIR spectrometer was PC controlled by means of software (OPUS) that permitted scanning spectra over a wide range (from 6000 to 500 cm−1) and analyzing the obtained spectra. The spectra were recorded with a spectral resolution of 0.2–2 cm−1 in the RAIRS mode (reflection absorption infrared spectroscopy) where the IR beam passes through the sample twice.The experimental set-up was the same as we used under laboratory measurements of the photodesorption rate from water ice. As described by Kulikov et al.27, each experiment with a particular sample of ice was conducted in two stages. At the first stage, two background spectra with different resolution were recorded at a mirror temperature of 20 K and at a temperature of subsequent irradiation (Tir). At that time, H2O or H2O + O2 gas was got ready in GPIS. We used the oxygen (Air Liquide 5.5) with purity better than 99.9995 Vol% and triply distilled water with resistivity better than 107 ohm cm, additionally degassed by freeze/thaw cycles in vacuum conditions. As described in our previous study27, then, an ice film was prepared by depositing H2O or H2O + O2 vapour onto the mirror at 20 K. The deposition speed and sample thickness were controlled by a needle valve and internal baratron of GPIS and by tracking the evolution of absorption bands in the FTIR spectra. More specifically, a sample was prepared by successive slow depositions of small vapour portion which were accompanied by the integrated area measurements of the water ice 3275 cm−1 band  of 10.2 eV photons in water ice and close to typical radii of NLC particles48. After the sample preparation, the mirror temperature was set at Tir (in the 20–140 K range) and several IR spectra of unirradiated ice were recorded. At Tir = 120 K and above, the IR spectra showed crystalline features of all ice samples that was in accordance with composition of NLCs49. Note also, Bartels-Rausch et al.50 discussed recently the simulations of disorder on pure ice at different temperatures below melting point (Tm) and showed that, at the temperatures 10 K below Tm, disorder affected the first molecular layer of ice only. In current study, the samples consisted of more than 100 layers of water and the highest temperature in our experiments (140 K) was about 20 K below than characteristic Tm of water ice in our vacuum chamber. Thus, we can conclude that interface processes could not influence essentially on the studied processes inside ice samples.The experimental procedures were almost the same as we used under laboratory measurements of the photodesorption rate from water ice. As described by Kulikov 27, at the second stage, the vacuum ultraviolet lamp was switched on and the ice films were exposed to VUV radiation with intensity set by microwave generator. After each photolysis period, IR spectra of the irradiated ice films were recorded. For improving the signal-to-noise ratio we used the spectral resolution of 2 cm−1 and a large amount of scans (2000). Hydrogen peroxide was found by detecting the IR absorption band of 2850–2860 cm−1 in difference spectra (before and after irradiation). The band was exuded by subtracting the baseline from the spectrum in manner described, for example, in Hand & Carlson14. Values of H2O2 column densities were determined as −1 band, As described in our previous studyet al.27. Before an experiment with specific H2O or H2O + O2 ice sample, we performed series of measurements of the absolute magnitude of the flux of Lyman-α photons that reach the ice sample at different adjustments of the microwave generator power. The widely used “ozone method”51 was applied for the procedure. The intensity of the lamp was determined by measuring the O2 → O3 conversion rate in a VUV photolyzed sample of solid O2 at 16 K. The ozone formation as a function of photolysis time was monitored with the FTIR spectrometer via the O3 absorption band at about 1040 cm−1. More specifically, for finding the VUV intensity at the mirror for a specific generator output power (GOP) we made successive measurements of the integrated area of the 1040 cm−1 absorption band . After that, the lamp intensity at this GOP was determined as Y was the quantum yield for the formation of O3 from O2, and et al.52 and was equal to 8.4 · 10−18 cm·photon−1. We obtained that, depending on GOP varied within the range of 4–120 W, the photon flux intensity varied within the range 5 · 1012–1015 photons/(cm2•s). The stability of lamp intensity at the fixed GOP was checked by means of photodiode SXUV300 .The calibration was carried out in the same manner as it was described by Kulikov