Juan Sebastian Ulloa
email: juan.ulloa@mnhn.fr

Thierry Aubin

Diego Llusia

Élodie A Courtois

Antoine Fouquet

Philippe Gaucher

Sandrine Pavoine

Jérôme Sueur

Explosive breeding in tropical anurans: environmental triggers, community composition and acoustic structure

Keywords: acoustic diversity, anuran community, ecoacoustics, biodiversity monitoring

Background

Anurans largely rely on acoustic communication for sexual selection and reproduction. While multiple studies have focused on the calling activity patterns of prolonged breeding assemblages, species that concentrate their reproduction in short-time windows, explosive breeders, are still largely unknown, probably because of their ephemeral nature. In tropical regions, multiple species of explosive breeders may simultaneously aggregate leading to massive, mixed and dynamic choruses. To understand the environmental triggers, the phenology and composition of these choruses, we collected acoustic and environmental data at five ponds in French Guiana during a rainy season, assessing acoustic communities before and during explosive breeding events.

Results

We detected in each pond two explosive breeding events, lasting between 24 and 70 hours.

The rainfall during the previous 48 hours was the most important factor predicting the emergence of these events. During explosive breeding events, we identified a temporal factor that clearly distinguished pre-and mid-explosive communities. A common pool of explosive breeders co-occurred in most of the events, namely Chiasmocleis shudikarensis, Trachycephalus coriaceus and Ceratophrys cornuta. Nevertheless, the species composition was remarkably variable between ponds and for each pond between the first and the second events. The acoustic structure of explosive breeding communities had outlying levels of amplitude and unexpected low acoustic diversity, significantly lower than the communities preceding explosive breeding events.

Conclusions

Explosive breeding communities were tightly linked with specific rainfall patterns. With climate change increasing rainfall variability in tropical regions, such communities may experience significant shifts in their timing, distribution and composition. In structurally similar habitats, located in the same region without obvious barriers, our results highlight the variation in composition across explosive breeding events. The characteristic acoustic structure of explosive breeding events stands out from the circadian acoustic environment being easily detected at long distance, probably reflecting behavioural singularities and conveying heterospecific information announcing the availability of short-lived breeding sites. Our data provides a baseline against which future changes, possibly linked to climate change, can be measured, contributing to a better understanding on the causes, patterns and consequences of these unique assemblages.

Background

Amphibians are currently the most endangered group of vertebrates, with more than 32% of species classified as at risk of extinction [START_REF] Stuart | Status and Trends of Amphibian Declines and Extinctions Worldwide[END_REF][START_REF] Hoffmann | The Impact of Conservation on the Status of the World's Vertebrates[END_REF][START_REF] Alroy | Current extinction rates of reptiles and amphibians[END_REF]. Recent investigations on the causes of amphibian declines have identified the role of climate change on a global scale [START_REF] Carey | Climate change and amphibian declines: is there a link?[END_REF][START_REF] Pounds | Widespread amphibian extinctions from epidemic disease driven by global warming[END_REF][START_REF] Lips | Riding the Wave: Reconciling the Roles of Disease and Climate Change in Amphibian Declines[END_REF][START_REF] Bellard | Will climate change promote future invasions?[END_REF]. In addition to the climate-linked epidemic hypothesis, research has focused on the effect of climate change on behaviour, reproduction and distribution of amphibians [START_REF] Llusia | Calling behaviour under climate change: geographical and seasonal variation of calling temperatures in ectotherms[END_REF][START_REF] Araújo | Climate warming and the decline of amphibians and reptiles in Europe[END_REF]. As ectotherms, alterations on temperature and rainfall regimes might strongly affect key aspects of amphibian life cycles, even jeopardizing their survival [START_REF] Duarte | Can amphibians take the heat? Vulnerability to climate warming in subtropical and temperate larval amphibian communities[END_REF]. Both theoretical and experimental studies suggest that low latitude ectothermic species are more vulnerable to climate changes than their higher latitude counterparts [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF]. Tropical species indeed tend to have narrower thermal tolerance [START_REF] Bonetti | Evolution of climatic niche specialization: a phylogenetic analysis in amphibians[END_REF] and their actual habitat temperatures are closer to their upper thermal limit [START_REF] Duarte | Can amphibians take the heat? Vulnerability to climate warming in subtropical and temperate larval amphibian communities[END_REF][START_REF] Sunday | Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation[END_REF]. Even slight changes in environmental conditions might therefore have a strong effect on these tropical species [START_REF] Foden | Identifying the World's Most Climate Change Vulnerable Species: A Systematic Trait-Based Assessment of all Birds, Amphibians and Corals[END_REF].

Anurans largely rely on acoustic communication for sexual selection and reproduction [START_REF] Gerhardt | Acoustic communication in insects and anurans: common problems and diverse solutions[END_REF][START_REF] Narins | Hearing and Sound Communication in Amphibians[END_REF]. Studies have revealed that temporal patterns of calling and breeding activity of anurans are influenced by multiple environmental factors, such as temperature, humidity or light intensity [START_REF] Brooke | Environmental and social factors influence chorusing behaviour in a tropical frog: examining various temporal and spatial scales[END_REF][START_REF] Oseen | Environmental factors influencing calling in sympatric anurans[END_REF][START_REF] Llusia | Environmental and social determinants of anuran lekking behavior: intraspecific variation in populations at thermal extremes[END_REF]. Moreover, recent findings have also shown that photoperiod might be an important driver of the calling activity of numerous anuran species [START_REF] Both | An austral anuran assemblage in the Neotropics: seasonal occurrence correlated with photoperiod[END_REF][START_REF] Canavero | Clues supporting photoperiod as the main determinant of seasonal variation in amphibian activity[END_REF][START_REF] Iwai | Relationship between chorusing activity and number of oviposition events in the Otton frog[END_REF][START_REF] Schalk | Environmental drivers of anuran calling phenology in a seasonal Neotropical ecosystem: Anuran Temporal Partitioning[END_REF]. The response to abiotic environmental factors may vary between species and according to the reproduction strategy [START_REF] Oseen | Environmental factors influencing calling in sympatric anurans[END_REF][START_REF] Wells | The social behaviour of anuran amphibians[END_REF]. While some anurans show long periods of calling activity and mating, known as prolonged breeders, others concentrate their reproduction during short time windows, even a few hours per year, and are known as explosive breeders [START_REF] Wells | The social behaviour of anuran amphibians[END_REF]. In tropical regions, massive aggregations of explosive breeders generally involve multiple species simultaneously, leading to highly-diverse anuran communities [START_REF] Aichinger | Annual activity patterns of anurans in a seasonal neotropical environment[END_REF][START_REF] Duellman | Temporal Fluctuations in Abundances of Anuran Amphibians in a Seasonal Amazonian Rainforest[END_REF][START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF][START_REF] Prado | Breeding activity patterns, reproductive modes, and habitat use by anurans (Amphibia) in a seasonal environment in the Pantanal, Brazil[END_REF]. Such phenomena typically occur in ephemeral ponds, which are sparsely distributed in tropical forests and are likely triggered under particular weather conditions. Yet, the structure and dynamics of these unique acoustic communities are still largely unknown probably because of their ephemeral nature, density and complexity. To our best knowledge, few studies have documented broad and generic patterns in explosive neotropical anurans, observing correlations between peaks of activity and the occurrence of heavy rainfall at the beginning of the rainy season [START_REF] Aichinger | Annual activity patterns of anurans in a seasonal neotropical environment[END_REF][START_REF] Duellman | Temporal Fluctuations in Abundances of Anuran Amphibians in a Seasonal Amazonian Rainforest[END_REF][START_REF] Prado | Breeding activity patterns, reproductive modes, and habitat use by anurans (Amphibia) in a seasonal environment in the Pantanal, Brazil[END_REF], and only two studies have analysed their fine scale dynamics [START_REF] Schalk | Environmental drivers of anuran calling phenology in a seasonal Neotropical ecosystem: Anuran Temporal Partitioning[END_REF][START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF]. In the former study, the data collection was done by human calling surveys through a four-month fieldwork in French Guiana. [START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF] identified temporal partitioning within the anuran community according to their reproductive modes [START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF]. In particular, the group of species with aquatic oviposition presented sporadic acoustic activity following heavy rainfall, a phenomenon that occurred twice during the study.

But their study focused on two close-by ponds, less than 240 m apart, limiting the interpretation of the results. Replications at spatial and temporal dimensions are crucial to examine the constitution and diversity of these communities, to decipher their dynamics and to identify their link with environmental factors. In the later study, Schalk and Saenz in 2016 examined the calling phenology of anurans in the Gran Chaco ecoregion at seven ponds during nine months with passive acoustic sensors. For explosive breeding species they found that calling activity was positively and significantly correlated with at least two abiotic factors, rainfall and photoperiod [START_REF] Schalk | Environmental drivers of anuran calling phenology in a seasonal Neotropical ecosystem: Anuran Temporal Partitioning[END_REF]. Calling individuals gathering around breeding points form dense choruses characterized by a complex acoustic structure, broad masking interference and high sound pressure level [START_REF] Gerhardt | Acoust. Commun. Insects Anurans Common Probl. Diverse Solut[END_REF]. Choruses formed by tropical anurans in explosive breeding events are extreme on these features due to the extraordinary species diversity and density of calling males [START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF]. Such assemblages constitute unique examples of multi-species choruses presumably eliciting complex interspecific interactions.

The technical difficulty in monitoring simultaneously these ephemeral communities has been one of the reasons for the lack of a wider geographic coverage. Traditional fieldbased observations are not scalable, thus it is crucial to adapt and test cost-effective methods.

More than 20 years ago the idea of using automated data acquisition methods to monitor amphibians was already proposed [START_REF] Peterson | Automated data acquisition[END_REF], but it is only recently, thanks to the development of reliable passive acoustic sensors, that this method has gain popularity [START_REF] Browning | Passive acoustic monitoring in ecology and conservation[END_REF][START_REF] Saenz | Abiotic correlates of anuran calling phenology: the importance of rain, temperature, and season[END_REF][START_REF] Gibb | Emerging opportunities and challenges for passive acoustics in ecological assessment and monitoring[END_REF][START_REF] Acevedo | Using automated digital recording systems as effective tools for the monitoring of birds and amphibians[END_REF][START_REF] Obrist | Bioacoustics approaches in biodiversity inventories[END_REF][START_REF] Servick | Eavesdropping on Ecosystems[END_REF]). These acoustic sensors can be programmed to record for days or even months in a non-invasive and cost-efficient way, so that replication in time and space is now possible. Most anuran amphibians produce loud, stereotyped, and species-specific advertisement calls for mate attraction. These acoustic signals can be therefore remotely recorded to monitor populations as testified by several studies on temperate (e.g. [START_REF] Llusia | Calling behaviour under climate change: geographical and seasonal variation of calling temperatures in ectotherms[END_REF][START_REF] Oseen | Environmental factors influencing calling in sympatric anurans[END_REF][START_REF] Bridges | Temporal variation in anuran calling behavior: implications for surveys and monitoring programs[END_REF]) and tropical species (e.g. [START_REF] Brooke | Environmental and social factors influence chorusing behaviour in a tropical frog: examining various temporal and spatial scales[END_REF][START_REF] Hilje | Recovery of amphibian species richness and composition in a chronosequence of secondary forests, northeastern Costa Rica[END_REF][START_REF] Jansen | Asymmetric frequency shift in advertisement calls of sympatric frogs[END_REF][START_REF] Pereyra | Diurnal? Calling activity patterns reveal nocturnal habits in the aposematic toad Melanophryniscus rubriventris[END_REF]).

Using automated sensors, we collected for the first time acoustic and environmental data to monitor simultaneously and regularly explosive breeding events in tropical anuran communities, at five temporary ponds located along the Kaw Mountain in French Guiana. This systematic passive acoustic monitoring allowed us to tackle key ecological questions related to the patterns, causes and consequences of such a striking phenomenon. We specifically addressed four questions: [START_REF] Stuart | Status and Trends of Amphibian Declines and Extinctions Worldwide[END_REF] What are the main meteorological factors that trigger the emergence of explosive breeders? (2) Which species co-occur before and during explosive breeding events? (3) What is the variation in the acoustic community composition within and between sites? (4) What are the main acoustic patterns, spectral characteristics and diversity before and during explosive breeding events? Answering these questions may shed light on the potential selective pressures shaping these complex acoustic communities.

Methods

Study site

We monitored explosive breeding assemblages in the lowland tropical rainforest of French Guiana, along the Kaw Mountain (4°36′N; 52°16′W). As in most regions located close to the equator, seasonal climatic variations in the study site were primarily due to changes in rainfall and humidity. The climate regime is characterized by two periods of rainfall: the main rainy season takes place from mid-November to the end of February and a less marked rainy season occurs from April to July. For this study, we collected acoustic and environmental data from the end of the dry season (10 November 2015) to the end of the main rainy season (16 February 2016).

We focused the sampling on five seasonal ponds along a 30.4 km transect corresponding to the departmental road D6 (Fig. 1). These temporary shallow water bodies are flooded during the rainy seasons and then dry out predictably during periods of low rainfall, July to November. The ponds were surrounded by dense tropical forest, located between 236 and 313 meters above the sea level, and had distinct sizes, from 224.8 m 2 to 2240.2 m 2 (Table 1).

Sampling protocol

We monitored anuran calling activity and weather conditions simultaneously in each pond using automated sensors with a regular sampling schedule. To record the acoustic communities, we placed on the edge of each pond at breast height an automated sound recorder equipped with an omnidirectional microphone (SM2, Wildlife Acoustics, Inc., Concord, MA, USA). The device was set up to record data 1 minute every 29 minutes, at 44.1 kHz and 16 bit resolution, so that we obtained 5,616 recordings for each pond.

To register local abiotic environmental data, we placed next to the sound recorder a data logger (H21-002, Onset) equipped with sensors to measure three weather variables: rainfall (Onset, S-RGB-M002), temperature, and relative humidity (Onset, S-THB-M008). In addition, we retrieved two global environmental variables, atmospheric pressure (PTB220, Vaisala) and solar radiation (CMP6, Kipp and Zonen), from the nearest weather station at the Félix Eboué airport (4°50′N; 52°22′W), 19 km from the study site.

Time-series analysis

Because of the emergence of a great number of males from multiple amphibian species, explosive breeding events are known to produce a remarkably loud chorus. Therefore, we identified the occurrence of explosive breeding events in the audio recordings by searching for outlier amplitude peaks. The overall amplitude of each recording was measured by computing the root-mean-square of the signal amplitude envelope. Then, we applied a median filter with a 24-hour window and we searched for outliers in the resulting smoothed time series. The outliers were defined as values distributed one-and-a-half times the inter-quartile range (IQR) above the third quartile (Q3 + 1.5 × IQR). Every outlier event was inspected by listening to the recordings to confirm the presence of an explosive breeding event.

Preliminary analyses showed clear and steep increase on the sound pressure level during explosive breeding events resulting from the increase in calling activity from anuran communities. While the beginning of the explosive breeding events exhibited constant and exceptionally high call rate for around 24 hours, calling activity later presented multiple oscillations before ceasing or returning to common levels. In order to have comparable sections for each event and compare pre-and mid-explosive breeding communities, we focused our subsequent analysis on a 48 h window, starting 24 h before the onset and ending 24 h after the onset of explosive breeding events.

We used a machine-learning framework to test whether the occurrence of the explosive breeding events could be predicted by abiotic factors. Weather conditions were considered as predictor variables and the triggering dates of the explosive breeding events as a binary response variable. The abiotic variables comprised low-level and high-level features. Lowlevel features were the raw quantitative meteorological measurements from the on-site sensors and the weather station, namely temperature, temperature variation, relative humidity, rainfall, atmospheric pressure, atmospheric pressure variation, photoperiod and solar radiation. Since the emergence of the breeding events can also be due to previous environmental conditions, we also included high-level features in the statistical analyses calculated based on the raw climatic data. These high-level features were the laggedvariables, previous 24, 48, and 72 hours, and past-cumulative variables from the previous 48 and 72 hours. The final predictor matrix included 48 variables with 466 observations. We measured prediction accuracy and variable importance on classification using the Random Forest statistical classifier [START_REF] Breiman | Random Forests[END_REF]. We assessed the importance of the predictor variables by comparing the difference in misclassification error (mean decrease accuracy) between the original data and a permuted set of data. The modified data for each predictor variable consisted in randomly permuted observations that are passed down the Random Forest. The higher the decrease in accuracy between the original and the modified data, the higher the importance of the predictor variable [START_REF] Cutler | Random forests for classification in ecology[END_REF].

Community diversity analysis

We investigated temporal and spatial variation on the diversity and composition of the acoustic communities of explosive breeding events. We define a community as the set of species heard at a given time interval before or after a specified explosive event, on a given pond. For each event, we systematically discretized the temporal gradient of 48 hours into four temporal periods of 12 hours. A first period (t1) ranged from 24 to 12 hours before the explosive breeding event, a second period (t2) ranged from 12 hours before to the onset of the event, a third period (t3) enclosed the first 12 hours of the event, and a fourth period (t4) ranged from 12 to 24 hours after the onset of the event.

We then sub-sampled our database by choosing one recording every two hours, for a total of 240 recordings of 60 seconds. Three of us (EC, AF and PG), who are highly trained in aural identification of anuran species of French Guiana, scrutinized each recording and annotated the occurrence of calling species. A final presence-absence vector was derived for each recording by majority voting, thereby, potential observer bias was prevented while the accuracy of the annotations enhanced. This phase led to the identification of a total of 25 species.

We used the crossed-DPCoA [START_REF] Pavoine | A New Technique for Analysing Interacting Factors Affecting Biodiversity Patterns: Crossed-DPCoA[END_REF], an ordination method that provides an approach for analysing the effects of crossed factors on the diversity of communities, to identify the effects of external factors on community composition. Here we analysed the effect on the species composition of amphibian communities of the time period before or after the event (t1, t2, t3, t4), and the event (an event is one of the two breeding explosions observed at a given pond).

The time period and the event are two crossed factors. The aim of crossed-DPCoA is to visualize the pattern of diversity due to a factor A knowing the existence of a crossed factor B. DPCoA helps to visualize the main effect of factor A, here species composition, and the effect of the interaction between A and B, removing the main effect of factor B. The method first defines a space where species, communities and the levels of the two factors are visualized as points. Then, the communities are positioned at the centroid of their constitutive species, and the levels of the factors at the centroid of communities associated with them. The method then searches for principal axes of the levels of factor A, retaining potential effects of the interaction between A and B, but removing the main effect of factor B. In particular, we used the first version of DPCoA, which eliminates the effect of factor B by moving this factor to the centre of the space. We analysed first the effect of the events on the species composition of amphibian communities given the time period and then the effect of the time periods given the event.

Acoustic diversity analysis

To further compare the anuran acoustic assemblages of the pre-and mid-explosive breeding events, we followed the same previous procedure while adding information related to the acoustic dissimilarities between species. We used the same community data and repeated the ordination analysis. However, here we did not consider species as equidistant in the space of the crossed-DPCoA, we used the acoustic properties of the calls of the species to define acoustic dissimilarities between pairs of species. In this defined space, the distance between two species-specific points is a measure of the acoustic dissimilarity.

We estimated the acoustic dissimilarity between two species using focal recordings of each species-specific call available from personal field recordings (PG, EC, AF, JSU; n=17) and from commercial recordings ([44], n=8). We selected recordings that met two criteria: [START_REF] Stuart | Status and Trends of Amphibian Declines and Extinctions Worldwide[END_REF] the call had to be emitted by an isolated individual, and (2) the signal-to-noise-ratio (SNR) of the signal had to be higher than 30, where SNR = 20 log 10 (RMS signal /RMS noise ) and RMS is the root-mean-square amplitude of the signal. Then, the spectral composition of each call was quantified by computing a short-time Fourier transform (FFT length of 512, no overlap, Hanning window), averaging the columns of the subsequent matrix (the temporal dimension), and applying a log-transformation. The acoustic dissimilarity between the species call was assessed by computing the cumulative dissimilarity of the spectral distributions or index D cf [START_REF] Lellouch | Monitoring temporal change of bird communities with dissimilarity acoustic indices[END_REF].

In addition, we analyzed the spectral profiles of the recordings collected in the field to investigate the changes in the acoustic environment before and during explosive breeding events. We first calculated the mean spectrum of each file. Then, we compared the spectral profiles at different moments of the explosive breeding event using a Random Forest procedure. We quantified and evaluated the classification accuracy and the importance of each feature, here each spectral profile, for the classification using the Random Forest importance measure [START_REF] Cutler | Random forests for classification in ecology[END_REF].

Finally, we estimated the α diversity of each acoustic community by computing the species richness, the Gini-Simpson coefficient, and the quadratic entropy. The richness is the number of species in the community. The Gini-Simpson index takes into account the number of species and their proportions [START_REF] Gini | Contrib. Allo Studio Delle Distrib. E Delle Relazioni Stat[END_REF][START_REF] Simpson | Measurement of Diversity[END_REF]. The quadratic entropy, or Rao's diversity coefficient [START_REF] Rao | Diversity and dissimilarity coefficients: A unified approach[END_REF], is based on the number of species, their proportions and incorporates a between-species dissimilarity matrix (here the pair-wise acoustic dissimilarities). For each diversity index, we tested the differences among periods of the explosive breeding event (i.e. t1, t2, t3 and t4) and between events (i.e. the first and second event per site), as well as the interaction between both factors, with repeated-measures ANOVA. Shapiro-Wilk and Mauchly tests revealed no violation of the assumptions of normality and sphericity, respectively, when using ANOVA tests (in all cases: W > 0.76, df = 5, p > 0.05; X 2 < 0.02, df = 5, p > 0.05). Tukey test with

Bonferroni correction was finally performed as post-hoc procedure to examine pairwise comparisons between time periods. The type I error was set at a nominal level of 5%.

Acoustic and statistical analyses were computed using the R software [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]. In particular, spectral audio features and dissimilarity matrices were computed using the seewave Rpackage [START_REF] Sueur | Seewave: a free modular tool for sound analysis and synthesis[END_REF], community and diversity ordination analyses were calculated with the adiv Rpackage [START_REF] Pavoine | Analysis of Diversity[END_REF], and statistical classification was computed with the randomForest R-package [START_REF] Liaw | Classification and Regression by randomForest[END_REF].

Results

Time series analysis

Sound pressure level showed regular 24-hour cycles during the study (Fig. 2). Yet, this regularity was interrupted by abrupt and steep increases in the amplitude lasting between 24 and 70 hours that occurred at the end of December 2015 and the beginning of February 2016.

Rainfall was irregularly distributed during the study showing two major rainfall events, the first one between 19 December 2015 and 4 January 2016, and the second one from 23 January to 15 February 2016. During those periods, daily fluctuations in temperature were less pronounced, solar radiation was lower, and relative humidity remained close to 100% (Fig. 2).

Applying an amplitude filter, we detected in each pond two major explosive events, i.e. 10 in total, lasting between 24 and 70 hours, later confirmed by aural evaluation. Using the combined meteorological variables (instant, lagged and past-cumulative) and the Random Forest classifier, we were able to accurately predict the emergence of all (100%) explosive breeding events with a low false positive rate of 9.6 % for out-of-the-bag estimates, that is using observations that were not used to build the predictive model. Variable importance ranking showed that rainfall was the most influential weather determinant, in particular, the amount of rain during the previous 24 hours and most importantly the past-cumulative rainfall during the previous 48 to 72 hours (Fig. 3). The rest of the variables (temperature, relative humidity, atmospheric pressure, photoperiod and solar radiation) had minor predictive power.

Community diversity analysis

We first analysed the species composition of explosive breeding events using crossed-DPCoA, which allowed to focus on the explosive breeding events removing the effect of the crossed factor linked to the time period before or after the event. The first two principal axes expressed respectively 34.8% and 30% of the main effect variability of the factor site (Fig. 4A). Neither the first nor the second axis presented a particular pattern, the explosive breeding events having largely overlapping communities. Nevertheless, some sites (Patawa, Arlesienne and Petite) presented high between-event diversity, each explosive breeding event having a particular and unique combination of species (Fig. 4B). Inter-site and intra-site variability of the explosive breeding events for these sites had the same order of magnitude.

Then, to reveal the temporal variability in the communities, we eliminated the crossed effect of factor 'event' with the DPCoA. The calling activity of the anuran communities was structured along the temporal dimension (Fig. 5A). The first axis of the DPCoA, with 84.3 % of variance explained, clearly discriminated two assemblages: the pre-explosive community (t1 and t2 on the negative side) and a characteristic explosive breeding community (t4 on the positive side). A transitional community with species from both sides appeared near the origin (t3). While the pre-explosive communities (t1 and t2) were partly similar in their species composition, t3 and t4 had clear and unique species composition. The species that characterized the pre-explosive community (t1 and t2) were Phyllomedusa tomopterna, Leptodactylus mystaceus, and Dendropsophus counani (Fig. 5B). Because they had positive coordinates on the first axis, the species that characterized the explosive breeding community (t4) were Chiasmocleis shudikarensis, Trachycephalus coriaceus and Ceratophrys cornuta (Fig. 5B). The transitional community (t3) showed an intermediate place on the ordination;

these communities had a balanced mixed of pre-explosive and explosive breeding species.

Acoustic diversity analysis

As in the previous community analysis, we initially removed the effect of the crossed factor time. The first principal axis, with 87.6 % of variance explained, was strongly correlated with the peak frequency of the calls (r = 0.96, Pearson correlation; Fig 6). The crossed-DPCoA ordered the species with low frequency sounds on the left of the axis and species with high-pitched calls on the right. Distributed in this new space, the sites presented largely overlapping acoustic communities with a balance between high and low frequencies.

Yet, the ponds Patawa, and Arlesienne had a high between-event acoustic diversity (Fig. 6A).

At both ponds, the first explosive breeding event was characterized with lower frequencies than the second one.

Subsequently, we removed the effect of the cross factor event to show the temporal variability of the acoustic signals. Again, the first and second axes were strongly correlated with the peak frequency of the calls (r = 0.91 and r = 0.96, Pearson correlation). For both axes, low frequency calls lied on the negative side of the axis and high frequency calls on the positive one (Fig. 7). In this bi-dimensional space the acoustic community was structured along the temporal dimension (Fig. 7A). The first axis of the ordination analysis, with 60.1 % of explained variance, showed a progression from t1 (negative side) to t4 (positive side), a progression toward mid-frequencies dominance. The levels t1 and t2 presented elongated ellipses, showing a dispersed range of frequency calls, with low and high-pitched sounds (Fig. 7A). This elongated shape was much less pronounced for levels t3 and t4, which was mainly characterized by calls in the mid-frequency range. The sounds that characterized, by their higher proportions, the explosive breeding event acoustics were the calls of C. shudikarensis and T. coriaceus (Fig. 7B). The calls of these anurans were in the middle range of the acoustic community, 3.4 kHz and 1.8 kHz for C. shudikarensis and T. coriaceus respectively.

Further spectral analyses at the soundscape level supported the previous results obtained with isolated vocalisations. Using a statistical classifier we were able to classify explosive breeding recordings with high accuracy, using only their spectral profile (Random Forest, 89% out-of-the-bag accuracy). The feature importance analysis showed that mid frequencies, between 2 and 4.4 kHz, were clearly the most important predictor variables (Fig 8).

The temporal pattern observed using the species richness and the Gini-Simpson index was similar, with maximal values during the first hours of the explosive breeding event (period t3; Fig. 9). Differences in acoustic diversity among periods were statistically significant when measured as species richness (ANOVA, F 3,12 = 5.86, p = 0.010) and marginally significant when measured by Gini-Simpson index (ANOVA, F 3,12 = 3.21, p = 0.062). Post-hoc test revealed that the period t3 showed significantly higher species richness (2.8 ± 0.8) than the previous period t2 (Z = 3.51, p = 0.003), being others not statistically different. Rao's diversity coefficient, which includes the acoustic dissimilarity matrix, also varied according to the time periods (ANOVA, F 3,12 = 5.72 p = 0.011). This index was significantly higher at t1 than at t4 (0.15 ± 0.05; Z = 3.24, p = 0.007), indicating a progressive decrease in acoustic diversity as the explosive breeding community predominates (Fig. 9). No effect of the season nor its interaction with the periods of the event were identified in all cases (ANOVA, F 1,4 < 4.48, p > 0.101), and hence the two explosive breeding events recorded per site, during each of the two rainy seasons, were equivalent in terms of acoustic diversity.

Discussion

Time series analysis

We found that environmental variables could predict the emergence of explosive breeding events, with rain as the most important predictor variable. While rain is abundant during the whole season, it is relevant to note that explosive breeding species respond to two specific patterns of rain: consistency during the previous 48 to 72 hours and amount during previous 24 hours. Our results are in agreement with those of Gottsberger and Gruber [START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF] who found that rainfall for the previous 24 hours contributed the best, among other environmental variables, to explain the calling activity of the explosive breeding species. As we included more derived variables of the rain in our analyses, we complement previous results asserting that the consistency of the rain is also crucial. Having replicated this observation at several sites, we confirm that species participating in explosive breeding events are highly tuned to specific rainfall patterns. Recent studies have identified the photoperiod as an important predictor of anuran activity [START_REF] Both | An austral anuran assemblage in the Neotropics: seasonal occurrence correlated with photoperiod[END_REF][START_REF] Canavero | Clues supporting photoperiod as the main determinant of seasonal variation in amphibian activity[END_REF][START_REF] Iwai | Relationship between chorusing activity and number of oviposition events in the Otton frog[END_REF][START_REF] Schalk | Environmental drivers of anuran calling phenology in a seasonal Neotropical ecosystem: Anuran Temporal Partitioning[END_REF], but our statistical analyses showed no clear links between this factor and explosive breeding events. Our study site was very close to the equator (4º36'N), were the difference between maximum and minimum day length across the year is less than 32 minutes. Former studies on photoperiod were conducted at latitudes were the difference in day length are much more pronounced (at least 4.4 times stronger), which probably explains why this factor was so important.

This apparently high dependency of explosive breeders' reproduction not only to the amount of precipitation but also to the timing of rain events raises the question of the vulnerability of explosive breeders to climate changes. While other factors such as programmed annual migration might be involved in triggering explosive breeding events, our study suggests that the two specific patterns of rain (i.e. consistency during the previous 48 to 72 hours and amount during previous 24 hours) are key parameters for the initiation of reproduction. With climate change increasing rainfall variability in tropical regions [START_REF] Feng | Changes in rainfall seasonality in the tropics[END_REF],

reproductive events might be shifted or disrupted. Moreover, these species rely on very specific habitats (temporary reproductive ponds) for their reproduction that are very fragile and particularly vulnerable to climate changes [START_REF] Foden | Identifying the World's Most Climate Change Vulnerable Species: A Systematic Trait-Based Assessment of all Birds, Amphibians and Corals[END_REF]. Finally, the high number of individuals from several species at the time of reproduction might increase probability of intra and interspecies infection at the breeding ponds and therefore increase the sensitivity of these species to emerging infectious disease, in particular the fungus Batrachochytrium dendrobatidis [START_REF] O'hanlon | Recent Asian origin of chytrid fungi causing global amphibian declines[END_REF].

These combined factors, may lead to significant shifts in the timing, distribution and composition of explosive breeding communities, which may desynchronize phenology and other biological responses throughout several trophic levels in the ecosystem [START_REF] Schwartz | Phenology: an integrative environmental science[END_REF].

Community diversity analyses

In structurally similar habitats, located in the same region without obvious barriers, we expected to have homogeneous amphibian communities. Yet, our results highlight the variability of species composition in explosive breeding events. The ordination diagram showed differences in species composition both between ponds and for a given pond, between the two observed events. In other words, each explosive breeding event, while often sharing a common pool of species, had a unique combination of species. When controlling for the differences between explosive breeding communities, a clear temporal factor structured the acoustic community during explosive breeding events, showing pronounced differences between pre-explosive and explosive breeding communities. The main species characterising the explosive breeding event, C. shudikarensis, T. coriaceus and C. cornuta, were also found as predominant species in explosive breeding events in the Arataï river, more than 100 km away from our study site [START_REF] Gottsberger | Temporal partitioning of reproductive activity in a neotropical anuran community[END_REF]. While other species are also present during these aggregations, these species seem particularly representative of the acoustic community.

It remains to explain the species turnover between events in space (ponds) and time (for each pond between the first and the second event). This turnover could be due to stochastic factors or related to multiple combined determinants, such as ecological and behavioural traits. As in many other sampling techniques in ecology, rare and elusive species are difficult to detect. It is also possible that the dense chorus of the louder species masked the vocalisations of more silent species, inducing detection errors and causing community variations in space and time.

Acoustic diversity analyses

Regarding the acoustic environment of explosive breeding events, we found outlying levels of activity with a characteristic spectral signature. This signature stands out from the circadian acoustic environment and can be easily detected at long distance. Acoustic signatures convey information that could be exploited by conspecifics (or heterospecific) for general orientation within a landscape [START_REF] Slabbekoorn | Soundscape orientation: a new field in need of sound investigation[END_REF]. Fish and crustacean larvae [START_REF] Montgomery | Sound as an orientation cue for the pelagic larvae of reef fishes and decapod crustaceans[END_REF], birds [START_REF] Mönkkönen | Numerical and behavioural responses of migrant passerines to experimental manipulation of resident tits (Parus spp.): heterospecific attraction in northern breeding bird communites?[END_REF], and frogs [START_REF] Gerhardt | Phonotactic responses and selectivity of barking treefrogs (Hyla gratiosa) to chorus sounds[END_REF][START_REF] Bee | Selective phonotaxis by male wood frogs (Rana sylvatica) to the sound of a chorus[END_REF] are known to use sounds in the environment for spatial orientation. Indeed, acoustic cues might gain importance for anuran explosive breeding species since sounds may signal availability, in space and time, of short-lived breeding sites [START_REF] Bee | Selective phonotaxis by male wood frogs (Rana sylvatica) to the sound of a chorus[END_REF][START_REF] Swanson | Do female frogs exploit inadvertent social information to locate breeding aggregations?[END_REF].

Alpha diversity indices, measured with species richness and Gini-Simpson, showed temporal communities with similar values between pre-explosive (t1-t2) and the explosive breeding community (t4). The transitional community (t3) had higher values, probably because it had species from both communities, pre-and explosive breeding. More surprisingly, Rao's diversity coefficient showed a significant diminution of the spectral diversity during explosive breeding events (t4). Even when the number of calling species was similar, we observed more frequency overlap in signals for the explosive breeding community than for the pre-explosive community.

Species belonging to a community may compete to access acoustic resources, that is to a free acoustic channel. It has been therefore hypothesized that species calling in a chorus should exhibit frequency dispersion. Formulated under the acoustic niche hypothesis, organisms would have evolved to occupy specific spectro-temporal 'niches', decreasing the risk of heterospecific mating and information masking [START_REF] Krause | The niche hypothesis: a virtual symphony of animal sounds, the origins of musical expression and the health of habitats[END_REF]. Acoustic partitioning has been observed in multiple taxa, such as insects [START_REF] Sueur | Cicada acoustic communication: potential sound partitioning in a multispecies community from Mexico (Hemiptera: Cicadomorpha: Cicadidae)[END_REF][START_REF] Schmidt | Spectral niche segregation and community organization in a tropical cricket assemblage[END_REF], birds [START_REF] Planqué | Spectral Overlap in Songs and Temporal Avoidance in a Peruvian Bird Assemblage[END_REF] and amphibians [START_REF] Villanueva-Rivera | Eleutherodactylus frogs show frequency but no temporal partitioning: implications for the acoustic niche hypothesis[END_REF]. However, recent studies also presented limitations of such hypothesis, showing no significant spectral divergence in cricket assemblages [START_REF] Schmidt | No phenotypic signature of acoustic competition in songs of a tropical cricket assemblage[END_REF] and more similarity in signal design that expected by chance for tropical forest birds [START_REF] Tobias | Species interactions and the structure of complex communication networks[END_REF]. Our results are in line with these last studies; contrary to our prediction, the species did not show frequency dispersion but frequency overlap.

Multiple hypotheses might explain this observation. First, the study ponds had similar habitat characteristics and hence similar acoustic properties that might have an effect on the features of anuran sounds. Following the Acoustic Adaptation Hypothesis [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF][START_REF] Marten | Sound transmission and its significance for animal vocalization[END_REF][START_REF] Ey | The "acoustic adaptation hypothesis" -a review of the evidence from birds, anurans and mammals[END_REF], the habitat might impose limits (e.g. signal attenuation and degradation) for sound propagation at high and low frequencies, resulting in an adaptation of explosive breeding species to produce sounds at mid frequencies, where they can maximize propagation. Indeed, for sounds produced at ground level, a window suitable for acoustic long range communication have been found at mid-frequencies (1-4 kHz) during experiments in an Amazonian rainforest in southern Venezuela [START_REF] Ellinger | Habitat acoustics of a neotropical lowland rainforest[END_REF]. Second, compared to prolonged breeders that show long periods of calling activity, explosive breeders share the acoustic space for very brief moments. As discussed by Wells [START_REF] Wells | The social behaviour of anuran amphibians[END_REF], due to the short time window for exchanging vocal signals between individuals, males would rather compete physically and not acoustically. The selective pressures acting on the acoustic space of these species might be weaker, which could explain the high frequency overlap of the explosive breeding events. Finally, a convergence of signals, not only in time but also in frequency, could serve to better synchronise the sporadic emergence of multiple anuran species. A signal with common features across taxa would allow recruiting a larger number of individuals at precise location and time, aggregating organisms at densities that exceed the potential number of local predators. Indeed, studies on a variety of animals [START_REF] Sweeney | Population Synchrony in Mayflies: A Predator Satiation Hypothesis[END_REF][START_REF] Williams | Emergence of 13-Yr Periodical Cicadas (Cicadidae: Magicicada): Phenology, Mortality, and Predators Satiation[END_REF][START_REF] Santos | The antipredator role of within-nest emergence synchrony in sea turtle hatchlings[END_REF] and plants [START_REF] Donaldson | Mast-seeding in the cycad genus Encephalartos: a test of the predator satiation hypothesis[END_REF][START_REF] Xiao | Long-term seed survival and dispersal dynamics in a rodentdispersed tree: testing the predator satiation hypothesis and the predator dispersal hypothesis[END_REF] have shown that sporadic synchronous reproduction within a population significantly reduces levels of predation. However, to confirm a convergence on the signal, additional data should be included in the analysis, such as phylogenetic and functional traits. Moreover, sound propagation and playback experiments should be performed to shed light on the selective pressure driving widespread chorusing behaviour.

Conclusions

In this study, we coupled biotic and abiotic variables, revealing community changes at multiple spatiotemporal scales and their tight link with the environment. Such data provides a baseline against which future changes can be measured, contributing to a better understanding and hopefully to a better management of such unique communities. Acoustic signatures could be used as a suitable way to monitor wildlife, not only at the individual or population level, but also at the community level, one of the main task of ecoacoustics [START_REF] Farina | Ecoacoustics: The Ecological Role of Sounds[END_REF]. A more widespread use of standardized methods combining passive acoustic recorders with a monitoring of key environmental parameters would become a comprehensible and costefficient framework to improve our knowledge and manage rich animal communities of tropical forests. explosive breeding communities (t4), Rao' s diversity index, which includes spectral distances between species, showed a significant diminution during explosive breeding events. Asterisk indicates significant differences between time periods.

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • -0.2 -0.1 0.0 0.1 0.2 0.3 -0.2 -0.1 0.0 0.1 0.2 PC1 34.8 % PC2 30 % • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Ca1 Ca2 Bl1 Bl2 Pa1 Pa2 Ar1 Ar2 Pe1 Pe2 A • • • • • • • • • • • • • • • • • • • • • • • • • -0.

Fig. 1 13 Fig. 2 Fig. 3

 11323 Figures

Fig. 4 Fig. 5 Fig. 7 Fig. 8

 4578 Fig. 4 Diversity of the species composition in explosive breeding events across sites. The two principal axes (64.8 % of variance explained) of the crossed DPCoA analysis are plotted. (A)Diversity between and within communities. Each point is a community. The communities were color-coded with the levels of the factor event. Code names for events are: Ar =

Fig. 9

 9 Fig. 9 Diversity measures within each of the temporal community (t1, t2, t3 and t4) at each explosive breeding event (n=10). Three diversity indices are compared: species richness, Gini-Simpson diversity and Rao's diversity coefficient. While alpha diversity indices (species richness and Gini-Simpson index) showed similar values for pre-explosive (t1-t2) and

Table 1 .

 1 Altitude, location, and area of the five study ponds. Altitude is given in meters above sea level (m a.s.l.) and area in m 2 .

	Local name	Code name	Altitude	GPS coordinates	Area
	Caïman	Ca	313	4°34'10"N; 52°13'11"W	1192.3
	Blanc	Bl	236	4°40'14"N; 52°18'22"W	399.5
	Patawa	Pa	295	4°31'41"N; 52°07'14"W	2240.2
	Arlesienne	Ar	269	4°32'44"N; 52°14'11"W	672.0
	Petite	Pe	289	4°35'59"N; 52°15'59W	224.8

Acknowledgements

The authors would like to thank Dorothée Deslignes from the CNRS Cayenne for the fieldwork logistics operations. In addition, authors would like to warmly thank Jeannot and Odette Morvan from the Gîte Patawa for their help during field work in French Guiana. We acknowledge support from an 'Investissement d'Avenir' grant managed by Agence Nationale de la Recherche (CEBA, ref.ANR-10-LABX-25-01). Support from the Marais de Kaw Reserve.

Funding This research was supported by the Labex CEBA (Centre d'Étude de la Biodiversité Amazonienne), which provided financial and logistic support for the data collection. JSU was supported by COLCIENCIAS (Doctoral Scholarship of the Colombian government, 2014 call #646). DLL was supported by a Global Marie S. Curie fellowship (European Commission, program H2020, EAVESTROP-661408) and a postdoctoral grant Atracción de Talento Investigador (Comunidad de Madrid, CAM, Spain, 2016-T2/AMB-1722). None of the funders had any role in the design, analysis, interpretation of results or writing the manuscript.

Availability of data and material

The environmental audio recordings were deposited at the sound library of the Muséum national d'Histoire naturelle (https://sonotheque.mnhn.fr/).

Abbreviations

Declarations

Ethics approval and consent to participate

There was no requirement to seek ethical approval to carry out the work described above.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests Authors' contributions JSU, JS, EC and PG participated in the deployment and maintenance of acoustic sensors for data acquisition. EC, PG and AF manually scrutinised audio recordings and provided personal recordings for the analyses. JSU, JS and TA made substantial contributions on the conception and design of the acquisition of data, analysis of the data and drafting of the manuscript. DLL and SP assisted in the analysis and interpretation of the community diversity data. All authors participated in revising critically the article contents. All authors read and approved the final manuscript.


T Voisin

A Erriguible

C Aymonier

Influence of multiphasic systems on salt(s) solubility in supercritical water: the case of NaCl and NaCl-Na 2 SO 4

Keywords: SCWO, supercritical water, salt precipitation, multiphasic systems, solubility

Supercritical water oxidation processes (SCWO) have been developed as an alternative technology to treat toxic and/or complex chemical wastes with a very good efficiency.

However, one main limitation of the SCWO process comes from the precipitation of inorganic compounds. When dealing with supercritical water conditions (T ≥ 374°C, p ≥ 22.1 MPa), the polarity of water drops and inorganics, such as salts, are no longer soluble. This precipitation phenomenon results in a solid salt deposition in the reactor, which can lead to clogging and interruption of the continuous process. Considering a specific salt, beside its intrinsic solubility in supercritical water, the presence of other salts or other compounds in the media can modify its precipitation behaviour. With the use of a specific experimental set-up, we propose in the present work to study the solubility of NaCl (type I salt) in supercritical water, and the influence of its phase diagram on its precipitation behaviour as well as on the solubility of a second salt, Na2SO4 (type II salt). The complex and dynamic aspect of NaCl precipitation has been studied through the use of a continuous solubility measurement set-up, revealing unexpected results.

Introduction

Research regarding Supercritical Water Oxidation (SCWO) process has become an important field of development over the past 30 years [START_REF] Loppinet-Serani | Supercritical water for environmental technologies[END_REF][START_REF] Kruse | Water -A magic solvent for biomass conversion[END_REF][START_REF] Brunner | Chapter 12 -Corrosion in Hydrothermal and Supercritical Water[END_REF][START_REF] Vadillo | Problems in Supercritical Water Oxidation Process and Proposed Solutions[END_REF][START_REF] Bermejo | Supercritical water oxidation: A technical review[END_REF]. With high degradation rates and efficiencies for the treatment of industrial wastewater and chemical wastes, SCWO processes are part of the technologies bringing environmental waste treatment alternatives [START_REF] Loppinet-Serani | Supercritical water for environmental technologies[END_REF][START_REF] Brunner | Supercritical process technology related to energy and future directions -An introduction[END_REF]. However, supercritical water (T ≥ 374°C, p ≥ 22.1 MPa) exhibits particular properties among which a very low polarity, leading to the capacity to dissolve organics compounds, with the drawback of precipitation of inorganic compounds. This precipitation phenomenon is one of the main limitation regarding SCWO industrialization as a continuous process [START_REF] Hodes | Salt precipitation and scale control in supercritical water oxidation-Part A: fundamentals and research[END_REF][START_REF] Marrone | Salt precipitation and scale control in supercritical water oxidation-part B: commercial/full-scale applications[END_REF][START_REF] Xu | Salt deposition problems in supercritical water oxidation[END_REF]. As precipitation occurs, inorganic compounds are deposited on the reactor wall, leading to the formation of clogs and interruption of the process.

Although this limitation is well-known, a limited amount of data is available in the literature concerning the solubility limits for inorganic compounds in supercritical water, especially for salts present in most of the real wastewater to be treated [START_REF] Voisin | Solubility of inorganic salts in sub-and supercritical hydrothermal environment: Application to SCWO processes[END_REF]. Few research works studied the salt solubility in supercritical water, and fewer the solubility of salt mixtures [START_REF] Voisin | Solubility of inorganic salts in sub-and supercritical hydrothermal environment: Application to SCWO processes[END_REF][START_REF] Dipippo | Ternary phase equilibria for the sodium chloride-sodium sulfate-water system at 200 and 250 bar up to 400 C[END_REF][START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 1: Type 1 salts[END_REF][START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 2. Type 2 salts and mixtures of two salts[END_REF][START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 3: Interesting effects in processing type 2 salt mixtures[END_REF][START_REF] Reimer | Influence of anions and cations on the phase behavior of ternary salt solutions studied by high pressure differential scanning calorimetry[END_REF]. Indeed, SCWO processes rarely deal with simple water/salt wastes, but most of the time with complex mixtures of several compounds including organics, inorganics, water and oxidizing agent (air, O2, H2O2...). Mixture of different salts can influence the solubility of each salt and some research works have been conducted regarding the change in solubility with salt mixtures [START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 1: Type 1 salts[END_REF][START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 2. Type 2 salts and mixtures of two salts[END_REF][START_REF] Schubert | Continuous salt precipitation and separation from supercritical water. Part 3: Interesting effects in processing type 2 salt mixtures[END_REF][START_REF] Reimer | Influence of anions and cations on the phase behavior of ternary salt solutions studied by high pressure differential scanning calorimetry[END_REF]. We also have to mention the important work of classification performed by Valyashko et al. regarding the types of salt and their behaviour according to it [START_REF] Valyashko | Phase equilibria of water-salt sytems at high temperatures and pressures[END_REF]. Different interaction phenomena can take place with salt mixtures, and influence the solubility of each salt. One effect frequently mentioned in the literature is the common ion effect. If two salts share a common ion, then the less soluble salt sees a decrease of its solubility. In addition, it is mentioned that mixing a type I salt with a type II salt (less soluble in supercritical water than the type I salt) increases the solubility of the type II [START_REF] Valyashko | Phase equilibria of water-salt sytems at high temperatures and pressures[END_REF][START_REF] Valyashko | Fluid phase diagrams of ternary systems with one volatile component and immiscibility in two of the constituent binary mixtures Presented at the International Bunsen Discussion Meeting of the Deutsche Bunsen-Gesellschaft für Physikalische Chemie[END_REF]. Thus, considering a type I salt such as NaCl, and a type II salt like Na2SO4, with the sodium as a common ion, it is difficult to predict if the Na2SO4 solubility is increased or decreased. Very few research works achieved a quantitative study of the changes in solubility due to a mixture of different salts [START_REF] Voisin | Solubility of inorganic salts in sub-and supercritical hydrothermal environment: Application to SCWO processes[END_REF]. The present work intends to study on one hand, the solubility of a common type I salt, sodium chloride (NaCl), and its influence on the solubility of a type II salt, sodium sulfate (Na2SO4), when they are mixed together in sub-and supercritical water.

Regarding the study of the NaCl solubility in supercritical water, the literature data are restricted to two research works [START_REF] Leusbrock | Quantitative approaches for the description of solubilities of inorganic compounds in near-critical and supercritical water[END_REF][START_REF] Armellini | Solubility of sodium chloride and sulfate in sub and supercritical water vapor from 450-550 °C and 100-250 bar[END_REF] which used an experimental method consisting in dissolving NaCl salt crystals into supercritical water and then measuring the salt concentration. We decided to use a reverse method, consisting in injecting a salted water solution in a continuous reactor to precipitate the salt and measuring the remaining salt concentration in water. The benefits of such process are double. First, working in continuous enables a fast screening of NaCl solubility in large temperature domains. Second, continuous measurements can give access to new information on the dynamic of salt precipitation in supercritical water conditions.

Materials and methods

To perform the solubility measurements of NaCl and of the mixture NaCl-Na2SO4, a continuous conductivity analysis set-up was used, coupled with Inductively Coupled Plasma (ICP) analysis. The set-up used for the solubility measurements has been described in details elsewhere [START_REF] Voisin | Investigation of the precipitation of Na 2 SO 4 in supercritical water[END_REF].

It is composed of a 600 mm long Inconel 625 reactor, heated with two heating cartridges inserted in a brass support, with a large filter inside the second half part of the reactor. The filter is composed of amorphous carbon particles, to sustain supercritical water conditions and insure that salt particles are not pulled along the reactor with water, but remain in the hot part (c.f. Figure 1). The water flow is then cooled down with the remaining salt ions and is analysed in an in-flow conductivity cell. Samples are also recovered at the outlet to perform ICP measurement. When studying one pure salt in water, conductivity can be used to measure the salt solubility in a continuous way. However, for salt mixture, conductivity only indicates when precipitation occurs but cannot be used for a quantitative analysis. This is why ICP analysis was mainly performed for the NaCl-Na2SO4 mixture, through the quantification of the sulfur and sodium content. Sulfur content will then be used to determine Na2SO4 concentration, whereas the remaining sodium content will give the NaCl concentration. Salted solutions have been prepared with ultra-pure water, NaCl powder (ALFA AESAR, crystalline powder, PDV, 99+%) and Na2SO4 powder (SIGMA-ALDRICH, ACS reagent, ≥99.0%, anhydrous, granular). mol/L.

NaCl solubility measurement using conductivity is performed according to the same methodology described in our previous work [START_REF] Voisin | Investigation of the precipitation of Na 2 SO 4 in supercritical water[END_REF]. A power law is used to fit the molar conductivity of NaCl (c.f. Figure 2) [START_REF] Bernard | Conductance in electrolyte solutions using the mean spherical approximation[END_REF]. This empirical law is then combined with the expression of the conductivity σ to result in a polynomial equation. Solving this polynomial expression gives access to the NaCl concentration.

Results and discussion

This result section is composed of two different subsections. First is presented the solubility study of NaCl, in order to know the reference system and then proceed to the second section, dedicated to the study of the NaCl-Na2SO4 system.

Solubility of sodium chloride

Several previous research works studied the NaCl solubility in water at high temperatures, but few in supercritical water conditions at 25 MPa [START_REF] Voisin | Solubility of inorganic salts in sub-and supercritical hydrothermal environment: Application to SCWO processes[END_REF][START_REF] Leusbrock | Quantitative approaches for the description of solubilities of inorganic compounds in near-critical and supercritical water[END_REF][START_REF] Armellini | Solubility of sodium chloride and sulfate in sub and supercritical water vapor from 450-550 °C and 100-250 bar[END_REF]. However, the NaCl-H2O binary diagram has been determined in these conditions (c.f. Figure 3) [START_REF] Bischoff | The system NaCl-H2O: Relations of vapor-liquid nar the critical temperature of water and of vapor-liquid-halite from 300°C to 500°C[END_REF][START_REF] Bischoff | Liquid-vapor relations for the system NaCl-H2O : summary of the P-T-x surface from 300°C to 500°C[END_REF], and one should know that a liquid-vapor diphasic domain appears, beginning at about 390°C and ending at 450°C. This diphasic domain means that instead of a solid precipitation, two phases are first formed.

One "vapor" phase consists in a low-density water phase, with a low salt concentration, and one "liquid" phase which corresponds to a high-density brine with a higher salt concentration.

As temperature increases, the salt concentration in the vapor phase decreases whereas the liquid phase becomes more and more concentrated, but also less dense. One should notice that the only NaCl solubility data available at 25 MPa, presented in Figure 3, have been determined using a salt dissolution process, meaning salt crystals deposited in a reactor filled with supercritical water and progressively dissolved up to the saturation [START_REF] Armellini | Solubility of sodium chloride and sulfate in sub and supercritical water vapor from 450-550 °C and 100-250 bar[END_REF][START_REF] Leusbrock | Quantitative approaches for the description of solubilities of inorganic compounds in near-critical and supercritical water[END_REF]. Therefore, the dynamic of the NaCl precipitation cannot be evidenced. The first interesting result obtained from our continuous set-up for the NaCl concentration measurement, is due to the impact of the diphasic domain on the solubility of the system.

Indeed, as the liquid-vapor equilibrium is formed, instabilities in temperature and concentration (conductivity) are detected in the system (c.f. Figure 4), thanks to a K-type thermocouple placed inside the reactor (c.f. Figure 1) and the continuous conductivity measurement cell. One can then determine the beginning and ending temperature of these instabilities and report them on the H2O-NaCl binary diagram for different initial concentrations (c.f. Figure 5).

At first glance, one would think that instabilities begin with the appearance of the new phase, as described by the binary diagram. Although the detection of the first instabilities well matches with the lower limit of the liquid-vapor domain (around 390°C), the upper limit is lower than the one which could be expected from the diagram. As it can be seen from Figure 5, the detection of the first instabilities corresponds to the lower limit of the diphasic domain, at about 390°C.

A more unexpected result is related to the end of these instabilities. While the NaCl-H2O binary diagram predicts the end of the liquid-vapor domain at a temperature of 450°C, experimentally the instabilities disappear around 415-420°C, with an abrupt decrease in NaCl concentration, indicating the precipitation of the salt (c.f. Figure 4).

However, it appears that between the upper limit of instabilities (415°C) and the end of the diphasic domain (450°C), the measured NaCl concentrations vary, for a given temperature, depending on the initial concentration (c.f. Figure 6). This unexpected behaviour suggests that the system is still located in its diphasic domain, as predicted, and the measured concentrations are then influenced by the proportions between the liquid phase and the vapor phase. As this proportion may depend on the initial concentration, it would explain the different concentration values obtained for a given temperature. Consequently, the measured concentrations These results clearly show how the existence of this high temperature diphasic domain, between NaCl and water, makes more complex the determination of the NaCl solubility in this temperature range. The results suggest that a first diphasic limit exists between 390°C and about 415-420°C, then an event occurs at 420°C, which may be due to a significant change in the phase proportions, and also to the precipitation of the first NaCl crystals. A triphasic domain would then prevail between 420°C and 450°C (as illustrated in Fig. 7). Above 450°C, a standard diphasic equilibrium between water and solid salt is present. The solid precipitation occurring between 420 and 450°C has never been predicted before, especially by the reported binary H2O-NaCl diagram.

H2O-NaCl-Na2SO4 system

The Figure 8 presents the values of solubility of Na2SO4 in SCW [START_REF] Voisin | Investigation of the precipitation of Na 2 SO 4 in supercritical water[END_REF]. The experimental data were obtained using the same experimental set-up used in the present study.

Results of the concentration evolutions for NaCl and Na2SO4 obtained from the ICP analyses are presented in Figure 9. Different phenomena can be observed from these evolutions, particularly three temperature domains I, II and III.

The first domain concerns the temperature from 370°C to 380°C (area I in Figure 9). Regarding the Na2SO4 concentration evolution, it can be seen that the precipitation occurs at a lower temperature compared with the solubility of pure Na2SO4 (dashed curved in Figure 9, from [START_REF] Voisin | Investigation of the precipitation of Na 2 SO 4 in supercritical water[END_REF]). This difference can be explained by the common ion effect. Indeed, as the two salts have in common the sodium ion Na + , the precipitation of Na2SO4 is favoured due to the larger quantity of sodium ions. The second observation is related to the temperature domain between 380°C and 400°C (area II on Figure 9). It can clearly be seen that the Na2SO4 concentration is stabilized to a value ten times higher than the solubility of pure Na2SO4. This temperature domain also corresponds to the beginning of the NaCl diphasic domain with water, meaning that a dense brine phase appears in the system. This dense phase could probably dissolve a part of the Na2SO4 solid crystals, preventing the complete precipitation. This explain why the sodium sulfate concentration remains so high compared to its solubility at these temperatures for pure Na2SO4.

The last result is located in the third temperature domain above 400°C (area III on Figure 9).

At this point, both concentration of NaCl and Na2SO4 get back to their "normal" solubility values. Yet, NaCl concentration begins to decrease around 400°C, instead of 415-420 °C as previously exposed. This premature precipitation can be explained by the fact that, as sodium sulfate is dissolved by the NaCl brine, the saturation concentration in the brine is reached for lower temperatures as if it was pure NaCl. Instead of reaching saturation for temperatures around 415-420 °C, the sodium sulfate saturates the dense phase around 400°C. Thus, NaCl precipitation occurs at a lower temperature than for pure NaCl, and as soon as the brine phase disappears, Na2SO4 get back to its solubility for pure Na2SO4.

These results evidence the influence of the dense brine phase from NaCl, on the solubility of a second salt with a lower solubility, such as Na2SO4. However, the presence of sodium sulfate has also influenced the precipitation of NaCl, as the dissolution of sodium sulfate in the brine phase decreases the temperature at which NaCl precipitates. Despite the small impact of the common ion effect between two salts, the main mixture effect comes from the dense phase.

This phenomenon brings quantitative evidences of the impact of a type I salt (NaCl) on the solubility of a type II salt (Na2SO4), as it was predicted by Valyashko and his classification.

Conclusion

Understanding the precipitation behaviour of a salt in supercritical water is of main interest for SCWO processes and hydrothermal applications. But the salt solubility can be influenced by numerous parameters, especially when mixing several salts together. From the common ion effect, to the influence of a type I salt on a type II salt, it is not always easy to determine the global effect on the solubility. This work showed, through the extensive study of the NaCl precipitation, its complex diphasic behaviour and the study of the NaCl-Na2SO4 system, that one decisive parameter on the solubility of salts is the intervention of a dense phase. Indeed, as seen on the example of the NaCl-Na2SO4 system, the main effect on the solubility of sodium sulfate is the formation of the dense brine phase by NaCl. Due to the high concentration in NaCl in the water brine phase, a high polarity is maintained and enables to dissolve sodium sulfate among it. This leads to a visible increase in sodium sulfate solubility. Despite the small decrease in solubility which can be explained by the common ion effect, it is mainly the dissolution by the NaCl brine which impacts the sodium sulfate precipitation. This supports the statement according to which a type I salt increase the solubility of type II salt.

This work also exposes the interest in using continuous quantification measurements as it reveals dynamic aspects which can otherwise be missed. Regarding SCWO investigations, it is of great interest to perform studies in conditions as close as possible to the process reality.

Considering the high temperature and high pressure of the supercritical water, any existence of a dense phase in these conditions is often synonym of a high polarity, leading to the dissolution of salt and influencing the solubility. It is likely that other type I salt with the existence of a diphasic domain, like NaCl, would influence the solubility of other salts. For a better understanding of these diphasic behaviours, and to obtain accurate solubility measurements within this temperature domain, one should try to look closely into the chemical/physical explanation of this brine phase formation.

Figure 1 :

 1 Figure 1: Scheme of the principle of the experimental set-up and the continuous conductivity measurement used for the salt solubility analyses. Illustration with NaCl.

Figure 2 :

 2 Figure 2: Molar conductivity data for NaCl at 25 °C from the literature and empirical power law used to fit the data .

Figure 3 :

 3 Figure 3: NaCl-H2O binary diagram, with solubility data from the literature, at 25 MPa.

Figure 4 :

 4 Figure 4: Example of the detection of instabilities between 385 and 415°C, before precipitation of NaCl, for an initial concentration of 0.05 mol/L.

Figure 5 :

 5 Figure 5: Representation of the beginning and ending temperatures of instabilities on the phase diagram of NaCl-H2O, for different NaCl initial concentrations.

Figure 6 :

 6 Figure 6: NaCl concentrations as a function of temperature, at 25 MPa. The grey area represents the instabilities domain.

Figure 7 :

 7 Figure 7: Schematic illustration of the proposed H2O-NaCl diagram As a type I salt, NaCl behaviour in supercritical water conditions differs from Type II salt such

Figure 8 :

 8 Figure 8: Na2SO4 solubility measurement in SCW as a function of temperature at 25 MPa (reprinted with permission from Voisin et al. [17]. Copyright © (2017) Elsevier)

Figure 9 :

 9 Figure 9: Concentration results for NaCl (dots) and Na2SO4 (squares) from ICP analysis. Comparison with the solubility curve of Na2SO4

Acknowledgement

The authors acknowledge financial support from the INNOVEOX Company, the French Environment and Energy Management Agency (ADEME) and the French Region Nouvelle Aquitaine.


Kelsey Flanagan
email: kelsey.flanagan@enpc.fr

Philippe Branchu
email: philippe.branchu@cerema.fr

Lila Boudahmane

Emilie Caupos

Dominique Demare

Steven Deshayes

Philippe Dubois

Meriem Kajeiou

Laurent Meffray

Chandirane Partibane

Mohamed Saad

Maria Vitart De Abreu Lima

Marie-Christine Gromaire

Mass balance of micropollutants over the first year of operation in a stormwater biofilter: a field approach integrating water and soil

Keywords: Biofiltration, fate mass balance, micropollutants, performance

Mass balance of micropollutants over the first year of operation in a stormwater biofilter: a field approach integrating water and soil

Bilan de masse de micropolluants au cours de la première année de fonctionnement dans un ouvrage d'infiltration végétalisé : une approche in situ sur l'eau et le sol

INTRODUCTION

As techniques used for urban stormwater management evolve from approaches based on rapid drainage toward centralized management systems to source-management techniques, the technique of biofiltration, also referred to as bioretention, has gained in popularity, especially in contexts where pollution control is an important objective. A biofiltration device consists of a vegetated depression that receives and stores runoff water, which then filters through a filter media made of engineered soil, before being collected by an underdrain or exfiltrated to the surrounding soil (Davis et al., 2009). In this type of system, pollutants are expected to undergo various processes including sedimentation, physical filtration, sorption, biodegradation, volatilization and plant uptake. A biofilter's pollution management performance may be considered on two levels: its ability to intercept pollution and, in the case of pollutants subject to biodegradation, volatilization and photodegradation, such as organic micropollutants, its ability to dissipate the intercepted pollution. The first type of performance has been evaluated in a field context for a variety of pollutants, including total suspended solids (TSS), nutrients, trace metals and polycyclic aromatic hydrocarbons (PAH) (Liu et al., 2014), while the potential for dissipation has mainly been studies at the laboratory (LeFevre et al., 2012) or mesocosm scale (Leroy et al., 2015). The two aspects are rarely considered together and few studies have accounted for the performance relative to emerging micropollutants common in the urban context, such as alkylphenols and phthalates.

The establishment of an in situ mass balance for a stormwater biofilter, accounting for inlet and outlet loads, as well as pollutant accumulation in the filter media, would allow for an integrated evaluation of both types of efficiency over a given period. The objective of the present work is to evaluate an integrated mass balance for several micropollutants-Cu, Zn, pyrene (Pyr), phenanthrene (Phen), bisphenol-A (BPA), octylphenol (OP), nonylphenol (NP) and bis(2-ethylhexyl)phthalate (DEHP)-over the first 13 months of operation of a biofiltration swale in order to (i) consider the integrated pollutant load reduction over the period and (ii) compare the intercepted mass with that accumulated in the soil. This is achieved by combining results of a field campaign with a stochastic modeling approach to integrate the data and account for uncertainties.

METHODS

Study site

The study site includes a biofiltration swale (BFS), which drains water from the RD 212, a suburban highway with 11,000 vehicles/day/direction traffic, located in Compans, France in the Paris region. The system is both drained and lined and was constructed in March 2016. Water was collected from a drain beneath the filter media (a sandy loam) at a depth of 50 cm. Inlet quality was evaluated using a reference catchment on the same road and with identical traffic, located 68 m from the BFS catchment (Figure 1). Flow was measured in the BFS drain and RR manhole using tipping bucket flow meters; overflow from the BFS was measured using a V-notch weir. Turbidity was also measured continuously in the RR manhole.

Figure 1: The Compans study site

Sampling and analysis

Water was collected at each point in proportion to water flow in order to measure the event mean concentration (EMC). 15 rain events were sampled between May 2016 (two months after the construction of the BFS) and July 2017. While simultaneous sampling of all points was attempted, due to technical difficulties, not all data is paired. Samples were collected within 24 h of each rain event and sent to partner laboratories within 24 h of collection, where they were immediately filtered to distinguish between dissolved and particulate phases for a wide range of micropollutants. Campagnes and water quality results are discussed thoroughly elsewhere (Flanagan et al., 2018); the integrated mass balance was evaluated for a selection of these pollutants (Cu, Zn, Pyr, Phen, BPA, OP, NP and DEHP).

In order to evaluate the mass of pollutants accumulated in the filter media, an initial sample was taken before installation and a sampling campaign was undertaken 13 months after the beginning of operation. In this campaign, seven composite filter media cores were sampled in the BFS from zones with similar Cu, Pb and Zn surface contamination, as determined from a cartography produced by soil contamination at 90 points using X-ray fluorescence spectrometry.

Mass balance and uncertainty calculations

Five mass balance terms were evaluated using a stochastic approach: the mass entering the biofilter (Min), the mass exiting the biofilter through the drain (Mdrain), the mass overflowing from the biofilter (Mover), the initial mass in the soil (Msoil,i) and the final mass in the soil (Msoil,f). The evaluation of each mass balance term was done stochastically, varying uncertain values randomly within a supposed distribution, and repeating the calculations 10,000 times.

The masses associated with water flows were evaluated according to Eq. 1. Measured event volumes and event mean concentrations (EMC) were used when available, stochastically accounting for uncertainties in the measurements. When measured values were not available, volumes were reconstituted using simple hydrologic models, stochastically varying the parameters of the model, while inlet and drain concentrations were reconstructed stochastically from observed distributions and the overflow concentration was derived from the inlet concentration accounting for dilution.

𝑀 = ∑ 𝑉 𝑒𝑣 𝐶 𝑒𝑣 (Eq. 1)
Where M is the load over the full period, Vev is the flow volume for a given event and Cev is the event mean concentration for the event.

Soil masses was also evaluated stochastically, accounting for uncertainties associated with soil sample representativity, soil concentration analysis, density and the composition of the soil.

The evaluated masses were then used to calculate an integrated mass reduction over the 13-month period (Eint, Eq. 2), as well as to compare the intercepted mass (Mint=Min-Mdrain-Mout) with that accumulated in the soil (ΔMsoil=Msoil,f-Msoil,i).

𝐸 𝑖𝑛𝑡 = 1 - 𝑀 𝑑𝑟𝑎𝑖𝑛 +𝑀 𝑜𝑣𝑒𝑟 𝑀 𝑖𝑛 (Eq. 2)
Where Eint is the proportion of pollutant mass intercepted by the system, 𝑀 𝑑𝑟𝑎𝑖𝑛 is pollutant load in the outflow from the drain, 𝑀 𝑜𝑣𝑒𝑟 is the pollutant load in the system overflow and 𝑀 𝑖𝑛 is the pollutant load from road runoff.

RESULTS AND DISCUSSION

Integrated mass reduction

The integrated mass balance calculations showed mass reductions of between 27-72% for the pollutants studied, with the highest reductions observed for PAH molecules Pyr and Phen, followed by heavy metals Zn and Cu, then TSS, OP, BPA, NP and DEHP (Table 1). Mass reductions of all pollutants were higher than the reduction of water volume, which was only 20%, with 35% overflow and 45% exiting through the drain. Despite the fact that drained water accounted for a greater volume than overflow, overflow accounted for a greater mass for most pollutants than drained water, due to the much lower concentrations observed in the drain than in the untreated road runoff. For example, because PAH concentration reductions generally exceeded 90%, only 7% of Pyr and Phen mass exited through the drain. On the contrary, DEHP concentrations were not significantly reduced and 51% of pollutant mass exited through the drain. The high overflow from this system indicates that increasing surface storage could improve performance.

Comparison of intercepted mass and mass accumulated in the soil

When comparing intercepted mass (Mint) with the mass accumulated in the soil (ΔMsoil), no significant dissipation could be observed for any of the pollutants studied. Among the conservative trace metals studied, Mint was within (case of Cu, Figure 2) or slightly below (case of Zn) the 95% confidence interval for ΔMsoil. The higher mass of Zn found in the soil may be due to a galvanized barrier between the swale and the road, which did not have contact with water on the reference catchment. Among organic micropollutants, two different issues prevented the observation of dissipation. For PAH molecules, the accumulation was low compared to the initial mass in the soil, leading to very large relative uncertainties in ΔMsoil, such that the accumulation was not significantly different from zero and dissipation could not be demonstrated even if it occurred. In the case of BPA, OP, NP and DEHP, Mint was much smaller than ΔMsoil, indicating that an additional source of these pollutants were present. Indeed, leaching tests on synthetic materials (liner, drain, drain filter fabric, asphalt) used for BFS construction demonstrated that sources of each of these pollutants were present. These emissions may also partially explain the lower mass reductions observed for these pollutants.

Figure 2 :

 2 Figure 2: Comparison of intercepted mass to accumulation in the soil for Cu, Phen and DEHP.

Table 1 :

 1 Median and 95% confidence interval of mass reduction Eint, proportions of inlet mass accounted for in overflow (Mover/Min) and in drained water (M,drain/Min)

	Parameter	Eint (%)	Mover/Min (%)	Mdrain/Min (%)
	Water	20 (15, 24) 35 (31, 37) 45 (41, 52)
	TSS	67 (56, 74) 20 (13, 34) 13 (9, 16)
	Cu	63 (54, 69) 23 (17, 33) 14 (10, 20)
	Zn	65 (51, 73) 21 (15, 34) 13 (8, 24)
	Pyr	72 (60, 79) 21 (14, 34) 7 (4, 13)
	Phen	70 (58, 77) 23 (16, 37) 7 (4, 11)
	BPA	45 (38, 51) 29 (23, 36) 26 (21, 33)
	OP	53 (40, 62) 26 (20, 34) 20 (14, 33)
	NP	36 (15, 48) 28 (22, 36) 35 (24, 57)
	DEHP	27 (3, 44)	21 (13, 38) 51 (33, 77)

CONCLUSIONSThe present study demonstrates the utility of a stochastic approach, which may be coupled with experimental measurements in order to evaluate integrated mass balances at the annual scale. A significant mass reduction was observed for all of the considered pollutants (between 27-72%) in the studied biofiltration system, although large overflow volumes limited the performance. In addition, the study showed some of the methodological difficulties associated with evaluating dissipation in the field. Uncertainties in the accumulated soil mass were very high for all pollutants, underlining the importance of accounting for uncertainties when attempting to interpret soil sampling campaigns. The potential of synthetic materials commonly used in biofiltration devices to emit some organic micropollutants, polluting the soil, was also demonstrated. These emissions pose a problem for demonstrating dissipation of these pollutants in situ, but also contribute to contamination of the urban environment and may limit the ability of the system to intercept pollutants over the long term.

LIST OF REFERENCES

Davis, A.P., Hunt, W.F., Traver, R.G., Clar, M. (2009)


M Valette
email: mvalette@laas.fr

M Bouguelmouna

M Mariotte

R Courson

M C Blatché

A Girousse

C Sengenès

K Reybier

A M Gué

ADIPOSE STEM CELLS (ASCs) ISOLATION BY ON-CHIP PRE-TREATMENT OF BIOLOGICAL SAMPLES

Context

Step 1: Hydrodynamic Filtration ASCs Lymphocytes of diameter 5 or 10 or 15 or 20 μm

The size of target cells is an essential criterion for the choice of cell sorting techniques. Up to now, we studied samples of adipose tissue from mice.

The size of cells has been determined by using a FACS (Fluorescent Activated Cell Sorting) calibrated with beads of different diameters : 5, 10, 15 and 20 μm. A sample of Stromal Vascular Fraction (SVF = white blood cells, blood cells and ASCs) from a mouse has been analysed via FACS  10 < diameter(ASCs) < 40 μm.

ASCs are not distinguishable from leucocytes by their size

3D Microfabrication

Early diagnosis or therapeutic follow-up (circulating tumour cells -CTC), regenerative medicine (mesenchymal stem cells) or the control of bacteriological risks (pathogens detection) are major topics of cell isolation. The techniques currently dedicated to cell isolation are very complicated. Lab-on-chip, a miniaturized system integrating several laboratory functions, achieves quick analysis while consuming a small volume of sample. This cost-effective tool is easy of use and can be used in any area. It represents an attractive alternative to other common cell isolation techniques.

The goal of this study is to develop a miniaturized analysis system (lab-on-chip) able to isolate and identify different cell types present in very small quantity in biological samples. It potentially concerns a large variety of cell species : mesenchymal stem cells, bacteria, etc. Cells of interest are stem cells from the adipose tissue (Adipose stem cells -ASCs). These cells are relevant for regenerative medicine and may be a key in the prevention of type 2 diabetes.

To isolate them, we propose an original approach combining two complementary steps: Step 2 Step 1

Step 3

1)•Final

  Scheme of the principle of hydrodynamic filtrationScheme of the principle of hydrodynamic filtration