M John Chapman¹

Stefan Blankenberg

Ulf Landmesser

M John Chapman
email: john.chapman@upmc.fr

The Year in Cardiology 2015: Prevention

Improved prevention of cardiovascular disease (CVD) is of critical importance, as coronary heart disease still represents the most common cause of death worldwide, engendering inestimable socioeconomic cost. The year 2015 has witnessed dramatic progress in CVD prevention on several fronts. Notably, this includes (i) event reduction in high risk patients in general practice following introduction of a comprehensive strategy to attenuate modifiable risk factors, including lifestyle and dietary habits; (ii) the use of hybrid imaging to detect subclinical atherosclerosis, with potential improvement in risk prediction/management; (iii) the demonstration that culprit plaque rupture and thin-cap fibro-atheroma predominate (70-77%) in ST-elevation myocardial infarction; (iv) the emergence of "omics" technologies to identify new causal biofactors; (v) the validation in clinical trials of the efficacy of monoclonal antibodies targeted to proprotein convertase subtilisin / kexin type 9 (PCSK9) in markedly reducing levels of low-density lipoprotein-cholesterol (LDL-C) across a spectrum of patients at high risk of premature CVD, with preliminary findings strongly suggestive of reduction in cardiovascular events; (vi) significant reduction of cardiovascular and all-cause mortality in diabetic patients in the EMPA-REG OUTCOME trial with the anti-hyperglycaemic agent, empagliflozin, a selective sodiumglucose co-transporter (SGLAT2) inhibitor; (vii) new pharmacotherapeutic strategies for superior control of hypertension emanating from the PATHWAY-2 and PATHWAY-3 clinical trials involving spironoloactone add-on therapy in resistant hypertension, and amiloride plus hydrochlorothiazide in hypertensive patients requiring a diuretic, respectively; and finally (viii) a reduced mortality associated with a lower blood pressure target of 120 mm Hg in patients at high cardiovascular risk in the SPRINT trial. Considered together, such progress augurs well for the future control of dyslipidaemia, hyperglycaemia and hypertension, and with it, progressive Norfolk prospective population study, in which even small improvement in modifiable risk factors led to substantial reduction in cardiovascular events (5). These important findings indicate not only that an integrated approach to modifiable risk factor control is feasible, but equally that it is achievable in general practice. Finally, imaging technologies for detection of subclinical atherosclerosis may be invaluable in adding incremental value to strategies for diagnosis, risk stratification and early initiation of prevention (see below).

reduction in atherosclerotic vascular disease and associated cardiovascular events in high risk patients.

Preamble

The prevention of cardiovascular diseases (CVD) represents an enormous challenge to health professionals on a global scale. Indeed, on the basis of the 2015 World Health Organization database for the European region, and calculating age-standardized mortality rates with the new European Standard population, CVD remains the most common cause of death among Europeans, accounting for 40% in males and 49% in females, and equating to more than 4 million deaths per year [START_REF] Townsend | Cardiovascular disease in Europeepidemiological update 2015[END_REF]. While mortality from coronary heart disease (CHD) and stroke have decreased overall across Europe over the past decade, CHD continues to represent the single most common cause of death [START_REF] Townsend | Cardiovascular disease in Europeepidemiological update 2015[END_REF]. Importantly, morbidity data reveal that population-based rates of hospitalization for both CVD and stroke have increased; considered together with ever increasing rates of cardiovascular interventions, greater use of medications, and expanding needs for rehabilitation for disabilities, these overwhelming socioeconomic costs present a major burden to healthcare systems across Europe [START_REF] Townsend | Cardiovascular disease in Europeepidemiological update 2015[END_REF].

How can we address this insurmountable challenge? Clearly lifestyle and diet represent our first line of action as currently recommended in recent guidelines [START_REF] Perk | European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts)[END_REF][START_REF] Reiner | ESC/EAS Guidelines for the management of dyslipidaemias: the Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS)[END_REF], and early identification and management of modifiable risk factors is paramount. Indeed, Avanzini and colleagues [START_REF] Avanzini | Improving cardiovascular prevention in general practice: Results of a comprehensive preventive strategy in subjects at high risk[END_REF] have recently demonstrated that application of a comprehensive personalized preventive strategy in more than 12,000 high risk subjects in general practice, but with suboptimal baseline risk factor control, led to gradual and significant improvement in global cardiovascular risk profile over a five year period. Thus, improvement in risk factor profile in the first year, (including physical inactivity, hypertension, hypercholesterolaemia, diabetes, and an unhealthy diet), was independently and significantly associated with lower rates of cardiovascular events in subsequent years. These findings are entirely consistent with new observations from the EPIC-

Introduction

The year 2015 isand continues to be -a vintage one for seminal progress in our knowledge of the pathophysiology underlying acute coronary syndromes (ACS), and of the epidemiology, diagnosis, and prognosis of CVD, thereby reflecting concerted efforts in our quest to prevent the global scourge of atherosclerotic vascular disease and its thrombotic complications. Such advances have been paralleled by the successful and rapid development of highly efficacious, innovative therapeutics to markedly lower circulating levels of low-density lipoprotein cholesterol (LDL-C). Indeed, in the landmark INTERHEART study of risk factors for the first myocardial infarction across 52 countries worldwide, atherogenic cholesterol transported as LDL predominated, accounting for the majority of population-attributable risk [START_REF] Yusuf | Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): casecontrol study[END_REF]. In this context, it is especially relevant that recent genetic findings, involving Mendelian randomization strategies which integrate lifelong and therefore cumulative risk exposure, have consolidated the evidence base for a causal role of LDL in the pathophysiology of atherosclerosis and CVD (7-9) (Table 1).

Moreover, the IMPROVE-IT trial [START_REF] Cannon | Ezetimibe added to statin therapy after acute coronary syndromes[END_REF] has now demonstrated that a mechanism of LDL lowering distinct from that of statins translates into clinical benefit. Ezetimibe-mediated inhibition of intestinal cholesterol absorption yielded incremental lowering of LDL-C on a background of statin treatment in this trial, (involving 18,144 patients hospitalized for an ACS over 7 years), and translated into moderate improvement in cardiovascular outcomes, i.e. a 7.2% lower rate of major vascular events. Baseline levels of LDL-C were low (1.8 mmol/L or 70 mg/dL), with a 24% further reduction when ezetimibe was added to simvastatin; that cardiovascular benefit is proportional to the degree of LDL-C reduction is of critical relevance in this context [START_REF] Treatment Trialists | Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials[END_REF].

Cardiovascular mortality was not modified, a finding which may result from several factors, and particularly the need for post-trial, long term follow-up data on clinical benefit. Indeed, it is increasingly evident that such follow-up reveals legacy benefits of LDL lowering beyond the active intervention period in randomized, placebo-controlled statin trials, typically featuring decrease in cardiovascular death rates [START_REF] Packard | Long term follow-up of lipid-lowering trials[END_REF]. Clearly then, a new paradigm is appearing in which LDL lowering therapies may alter the pathophysiological course of atherosclerotic vascular disease and its thrombotic complications, potentially by inducing lesion stabilization, or lesion regression, or both.

In this condensed distillate of advances in prevention of CVD over the past year, three key areas stand out. First, the evolution from emphasis on the ruptured, vulnerable coronary plaque to coronary plaque erosion in the context of ACS, with immediate relevance to approaches searching for "vulnerable" plaques [START_REF] Libby | Requiem for the 'vulnerable plaque[END_REF]. Second, the appearance of advanced molecular methodologies for identification of biomarkers with potential for high predictive value [START_REF] Hoefer | Novel methodologies for biomarker discovery in atherosclerosis[END_REF].

Third, the advanced development, based on the molecular genetics of familial traits for cholesterol dysmetabolism associated with premature atherosclerosis, of monoclonal antibodies targeted to proprotein convertase subtilisin/kexin type 9 (PCSK9) for marked reduction in LDL-C levels [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF]. Importantly, progress in all three areas holds great promise to positively impact the care pathway for patients at high risk of CVD.

Plaque imaging and cardiovascular risk prediction

A recent hybrid imaging study to evaluate the systemic extent of atherosclerotic disease in the carotid, abdominal aortic, iliofemoral and coronary arteries in a middle-aged population (the PESA Study, Progression of Early Subclinical Atherosclerosis) revealed subclinical atherosclerosis in 63% of participants (71% men, 48% women), who ranged from low to high risk [START_REF] Fernández-Friera | Prevalence, vascular distribution, and multiterritorial extent of subclinical atherosclerosis in a middleaged cohort: the PESA (Progression of Early Subclinical Atherosclerosis) Study[END_REF]. With a similar approach, the BioImage Study (A Clinical Study of Burden of Atherosclerotic Disease in an At-Risk Population) evaluated the predictive value of carotid plaque burden (as examined by 3-D ultrasound) and coronary artery calcification for cardiovascular risk assessment in a population of approximately 6,000 asymptomatic adults who underwent multimodality vascular imaging of both coronary and carotid arteries. Both imaging methods suggested that higher detected plaque burden was associated with adverse cardiovascular events; furthermore, both imaging methods improved cardiovascular risk prediction to a similar degree [START_REF] Baber | Prevalence, impact, and predictive value of detecting subclinical coronary and carotid atherosclerosis in asymptomatic adults: the BioImage study[END_REF].

Novel insights into coronary plaque pathobiology and mechanisms leading to progression towards acute coronary syndromes

Over recent years, coronary atherosclerotic plaque rupture and subsequent thrombus formation has been widely considered as the mechanism causing ACS. Subsequently, imaging studies have aimed to reveal the "vulnerable plaque". High-resolution intracoronary imaging studies using optical coherence tomography (OCT) have now revealed that a significant proportion of ACS events are caused by coronary plaque erosion (on an intact fibrous cap) and subsequent intracoronary thrombus formation, in addition to those "classically" resulting from coronary plaque rupture of vulnerable thin-cap fibroatheroma rich in lipid [START_REF] Hoefer | Novel methodologies for biomarker discovery in atherosclerosis[END_REF]. Indeed, Libby and Pasterkamp [START_REF] Libby | Requiem for the 'vulnerable plaque[END_REF] have highlighted this consideration in an editorial entitled "The requiem of the vulnerable plaque", in which they discuss different plaque pathobiologies leading to ACS. Moreover, Niccoli et al. [START_REF] Niccoli | Plaque rupture and intact fibrous cap assessed by optical coherence tomography portend different outcomes in patients with acute coronary syndrome[END_REF] reported that ACS caused by coronary plaque erosion may have a better prognosis as compared to those due to coronary plaque rupture, as such events appear to result from late thrombi suggestive of less intense thrombotic stimuli, thereby allowing time for thrombus dissolution caused by spontaneous fibrinolysis Finally, a recent meta-analysis of OCT studies suggested that the mean prevalence of culprit plaque rupture and thin-cap fibroatheroma was almost 50% across different clinical subsets of patients; importantly, such events were most prominent in ST-elevation myocardial infarction (70-77%) [START_REF] Iannaccone | Prevalence and predictors of culprit plaque rupture at OCT in patients with coronary artery disease: a meta-analysis[END_REF].

Innovative methodologies for novel biomarker identification to assess cardiovascular risk

Although current risk models allow for increasingly precise risk equations in the general population, predicting life-threatening cardiovascular events at the level of the individual remains a challenge. More precise risk stratification, ideally based on causal factors, and personalization both of risk factor assessment and management is increasingly needed. A number of strategies have been employed to search for novel biomarkers of CVD. Unbiased technologies, including genomics, proteomics and metabolomics, all utilize a "big data" approach for novel biomarker discovery, but to date these technologies have failed to deliver on their initial promise, yielding no new clinically useful biomarkers in cardiac care. A genetic risk score has been analyzed recently in clinical cohorts and data from randomized clinical statin trials and may identify individuals at increased risk for both incident and recurrent CHD events.

People with the highest burden of this genetic risk derived the largest relative and absolute clinical benefit from statin therapy [START_REF] Mega | Genetic risk, coronary heart disease events, and the clinical benefit of statin therapy: an analysis of primary and secondary prevention trials[END_REF].

An alternative strategy is to focus on known proteins reflecting mediating pathways to ensure a higher probability of association with CVD, an approach that can now be implemented on a massive scale using new multiplex immunoassay techniques that allow conservation of sample volume. This approach yielded promising results as recently tested in individuals with dysglycaemia [START_REF] Gerstein | Identifying novel biomarkers for cardiovascular events or death in people with dysglycemia[END_REF]. Further, non-coding RNAs including microRNAs are considered a potential biomarker which might support diagnosis and prognosis in different cardiovascular conditions [START_REF] Zeller | High population prevalence of cardiac troponin I measured by a high-sensitivity assay and cardiovascular risk estimation: the MORGAM Biomarker Project Scottish Cohort[END_REF]. Irrespective of big data approaches, single plasma biomarker assessment might be attractive to improve risk prediction models. Sensitive techniques to assess low concentrations of troponin I might open avenues to improve risk prediction in the general population by use of a cardiac specific biomarker [START_REF] Zeller | High population prevalence of cardiac troponin I measured by a high-sensitivity assay and cardiovascular risk estimation: the MORGAM Biomarker Project Scottish Cohort[END_REF][START_REF] Everett | High-sensitivity cardiac troponin I and B-type natriuretic Peptide as predictors of vascular events in primary prevention: impact of statin therapy[END_REF]. Indeed, in the Bypass Angioplasty Revascularisation Investigation in Type 2 Diabetes trial, cardiac troponin T concentration measured with a high sensitivity assay was an independent predictor of death from CV causes, myocardial infarction or stroke in patients who had both type 2 diabetes and stable ischemic heart disease [START_REF] Everett | Troponin and cardiac events in stable ischemic heart disease and diabetes[END_REF]. Nevertheless, development of new strategies to identify causal biofactors is warranted in biological fluids, circulating cells and tissues, and it is in this framework that emerging "omics" technologiesmetabolomics, lipidomics, proteomics, transcriptomics and miRNAomics -augur well [START_REF] Hoefer | Novel methodologies for biomarker discovery in atherosclerosis[END_REF].

Prevention of atherosclerotic vascular disease and cardiovascular events in dyslipidaemia

Statin intolerance

As recommended in current European guidelines, statins constitute first line therapy in standard care for dyslipidaemic patients at high and very high cardiovascular risk in primary and secondary prevention [START_REF] Perk | European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts)[END_REF][START_REF] Reiner | ESC/EAS Guidelines for the management of dyslipidaemias: the Task Force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS)[END_REF]. While the Cholesterol Treatment Trialists' meta-analyses of randomized controlled trials involving statins strongly substantiate their clinical efficacy [START_REF] Treatment Trialists | Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials[END_REF], nonetheless, the profile of statin-associated adverse effects has been progressively clarified to reveal not only that statin associated muscle symptoms (SAMS) predominate in observational studies, registries and clinical practice (range of prevalence 7-29%), but also that they are the primary cause of statin discontinuation [START_REF] Stroes | Statin-associated muscle symptoms: impact on statin therapy.-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management[END_REF]. To this end, the European Atherosclerosis Society (EAS) Consensus Panel recently issued a statement providing clinical guidance in the form of a flow-chart for management of patients with SAMS, and recognized the central role of attenuated mitochondrial energy production in skeletal muscle in its pathophysiology; it is noteworthy that inefficient first pass statin uptake into the liver may critically underlie SAMS (Figure 1) [START_REF] Stroes | Statin-associated muscle symptoms: impact on statin therapy.-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management[END_REF]. It is equally relevant that SAMS are a central feature of "statin intolerance", which also includes adverse events at the level of the liver, kidney, peripheral tissues and potentially the central nervous system, but whose frequency is markedly less than that of SAMS [START_REF] Stroes | Statin-associated muscle symptoms: impact on statin therapy.-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management[END_REF].

Interindividual variability in response to statin therapy

Interindividual variability in response to statin treatment has received little attention until late, when a pharmacogenetic meta-analysis of genome wide association studies from randomized controlled trials and observational studies was reported, identifying the implication of two new genetic loci, SORT1/CELSR2/PSRC1 and SLCO1B1, in addition to those of APOE and LPA, in variation in LDL-C response [START_REF] Postmus | Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins[END_REF]. These findings take on added significance when it is considered that a substantial proportion of patients with incident CHD are hyporesponders to statin therapy, show minimal LDL-C reductions, and most importantly, greater atheroma progression as compared to responders [START_REF] Kataoka | Atheroma progression in hyporesponders to statin therapy[END_REF]. Under such circumstances, follow-up monitoring of LDL-C levels after initiation of statin becomes primordial in order to ensure goal attainment.

Familial hypercholesterolaemia

Alarmingly, the proportion of patients with familial hypercholesterolaemia (FH) at LDL-C goal on statin treatment has been reported to be as low as 20% in the seminal Dutch experience; such patients are characterized by accelerated and premature atherosclerotic vascular disease and CHD [START_REF] Nordestgaard | European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Cuchel | Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society[END_REF]. Several reasons may underlie this situation, some of which arise from the markedly elevated LDL-C levels frequently encountered at baseline in such patients. A maximally-tolerated dose of an intensive statin is therefore the order of the day in FH, potentially in combination with ezetimibe, a synergistic association [START_REF] Catapano | IMPROVE-IT and genetics reaffirm the causal role of LDL in cardiovascular disease[END_REF][START_REF] Nordestgaard | European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Cuchel | Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society[END_REF][START_REF] Fazio | The role of PCSK9 in intestinal lipoprotein metabolism: synergism of statin and ezetimibe[END_REF]. Despite currently available therapies, however, FH in both its homozygous and heterozygous forms is widely underdiagnosed and undertreated, as emphasized by the EAS FH Consensus Panel [START_REF] Nordestgaard | European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Cuchel | Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society[END_REF]. Indeed, the recent revelation from population genetic studies that FH is the most commonly inherited metabolic condition, with a population frequency approaching 1:200 persons, has warranted a call to action, with widespread creation of patient registries and FH patient advocacy groups [START_REF] Nordestgaard | European Atherosclerosis Society Consensus Panel[END_REF][START_REF] Wiegman | European Atherosclerosis Society Consensus Panel[END_REF]. The underdiagnosis of FH is especially critical in children and adolescents, as emphasized recently by Wiegman et al [START_REF] Wiegman | European Atherosclerosis Society Consensus Panel[END_REF]. The evidence base in FH children treated with statins indicates not only that intervention with lipid lowering therapy may be safely initiated as early as 8 years of age, but also that when treated early in childhood, children born to FH families can anticipate normal life expectancy [START_REF] Wiegman | European Atherosclerosis Society Consensus Panel[END_REF].

The need for therapeutic innovation: PCSK9 inhibition

From the above, it is evident that innovative lipid lowering therapies have been -and remainurgently needed, always on a background of statin treatment whenever possible, in order to fully translate the exceptional evidence base for reduction in cardiovascular events concomitant with LDL-C lowering into reality for many dyslipidaemic patients at high risk. Such patients include those with FH, those in secondary prevention and those who are statin-intolerant; additional patient populations may include individuals with diabetes, chronic kidney disease, and non-FH hypercholesterolaemia [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF]. It is in this context that the recent approval in the USA and Europe of two humanized monoclonal antibodies to PCSK9, alirocumab and evolocumab, is especially pertinent; the development of a third, bococizumab, which is partially humanized, is ongoing [START_REF] Ballantyne | Results of bococizumab, a monoclonal antibody against proprotein convertase subtilisin/kexin type 9, from a randomized, placebo-controlled, dose-ranging study in statin-treated subjects with hypercholesterolemia[END_REF]; all are well tolerated with a satisfactory safety profile [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF][START_REF] Dadu | Lipid lowering with PCSK9 inhibitors[END_REF][START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF][START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF]. As exemplified by alirocumab, these antibodies act in vivo primarily by accelerating the fractional catabolic rate of LDL [START_REF] Reyes-Soffer | Effects of a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor, alirocumab, on lipid and lipoprotein metabolism in healthy subjects[END_REF]. An alternative approach to reduction of plasma PCSK9 concentrations involves direct inhibition of its hepatic production. A novel RNA interference drug, ALN-PCSsc (given as a subcutaneous formulation), has demonstrated the feasibility of this modality in phase 1 studies, resulting in a dose-dependent reduction in circulating PCSK9 levels of up to ≈80%, and a mean reduction in LDL-C of 40% for periods of one month or more, with favourable safety and tolerability [START_REF] Fitzgerald | A phase 1, randomized, placebo-controlled, single ascending and multiple dose study of subcutaneously administered ALN-PCSSC in subjects with elevated low density lipoprotein cholesterol[END_REF].

Monoclonal antibodies to PCSK9

The decade required for the development of monoclonal antibodies to inhibit PCSK9 has been driven by novel genetic and mechanistic insights into the role of this protein in the regulation of the availability of surface LDL receptors primarily in the liver, its relation to the regulation of circulating LDL-C levels, and ultimately to cardiovascular morbi-mortality [START_REF] Shimada | PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future[END_REF]. Quasi-complete removal of plasma PCSK9 by antibody binding results in highly efficacious lowering of LDL-C in the range of 40-70% as a function of dose across dyslipidaemic patient phenotypes in monotherapy or on a statin background, with uptake of LDL-antibody complexes by cells of the reticuloendothelial system; the duration of antibody action is dose-dependent for both alirocumab and evolocumab, whose (single dose) pharmacokinetics and pharmacodynamics resemble each other [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF][START_REF] Dadu | Lipid lowering with PCSK9 inhibitors[END_REF][START_REF] Shimada | PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future[END_REF]. Moreover, anti-PCSK9-mediated LDL lowering is additive to that of statins and ezetimibe [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF][START_REF] Dadu | Lipid lowering with PCSK9 inhibitors[END_REF][START_REF] Shimada | PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future[END_REF]. Importantly, the efficacy of these antibodies is independent of the specific class of the mutation of the LDL receptor (receptor negative, defective, unclassified, or no mutation detected) in heterozygous FH [START_REF] Raal | RUTHERFORD-2 Investigators. PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): a randomised, double-blind, placebo-controlled trial[END_REF]; this effect attests to the fact that PCSK9 action in vivo typically leads to the premature degradation of a major proportion of LDL receptors, a pathway largely neutralized by PCSK9 antibody treatment [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF].

In the "Year in Cardiology 2014", De Backer and colleagues comprehensively reviewed extensive data from the Phase III randomized controlled trials with alirocumab and evolocumab [START_REF] Backer | The year in cardiology 2014: prevention[END_REF]; clinical trial updates for 2015 are currently available in recent reviews [START_REF] Chapman | PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era[END_REF][START_REF] Shimada | PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future[END_REF]. Of late, the ODYSSEY FH I and FH II (heterozygous FH) trials included the option to increase the antibody dose to 150 mg every 2 weeks when LDL-C goal was not attained on the starting dose (75 mg every 2 weeks). In this way, some 59-68% of patients achieved an LDL-C goal of <1.8 mmol/L (70 mg/dL) [START_REF] Kastelein | ODYSSEY FH I and FH II: 78 week results with alirocumab treatment in 735 patients with heterozygous familial hypercholesterolaemia[END_REF]. Discontinuation due to treatment-emergent adverse events occurred in 3.4% of antibody-treated patients vs. 6.1% on placebo, while injection site reactions were reported for 12.4% in FH I and 11.4% in FH II (versus 11.0% and 7.4%, respectively for placebo), thereby attesting to satisfactory tolerability. Importantly and overall, these findings are consistent with those reported in FH heterozygotes upon treatment with evolocumab in the RUTHERFORD-2 trial, albeit involving a distinct dosing regimen from that above for alirocumab [START_REF] Raal | RUTHERFORD-2 Investigators. PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): a randomised, double-blind, placebo-controlled trial[END_REF]; furthermore, additional novel trial data have recently been reported in FH homozygotes in the TAUSSIG and TESLA trials (comprehensively reviewed in reference 15).

Safety of PCSK9 inhibition: vitamin E, gonadal hormones, cognitive function, very low LDL-C and anti-drug binding or neutralizing antibodies

As lipophilic vitamin transport and steroidogenesis are intimately linked to LDL-C metabolism, it was critical to provide safety data for the potential impact of these innovative therapeutics on vitamin E and steroid hormone levels [START_REF] Blom | Effects of evolocumab on vitamin E and steroid hormone levels: results from the 52-week, phase 3, doubleblind, randomized, placebo-controlled DESCARTES study[END_REF]. Thus, in the 52 week, double-blind randomized placebo-controlled DESCARTES study, evolocumab, on a background of statin, did not affect gonadal hormone levels up to 52 weeks of treatment, while changes in vitamin E paralleled those in lipoproteins; erythrocyte vitamin E levels were unchanged [START_REF] Blom | Effects of evolocumab on vitamin E and steroid hormone levels: results from the 52-week, phase 3, doubleblind, randomized, placebo-controlled DESCARTES study[END_REF]. Equally, adrenocorticotrophic hormone (ACTH) levels and the cortisol/ACTH ratio did not change, even when LDL-C levels were very low (<0.88 mol/L or 15 mg/dL).

Given that long term statin therapy is associated with new onset diabetes, particularly in individuals presenting with features of prediabetes and the metabolic syndrome [START_REF] Cederberg | Increased risk of diabetes with statin treatment is associated with impaired insulin sensitivity and insulin secretion: a 6 year follow-up study of the METSIM cohort[END_REF], it is imperative to exclude potential effects of PCSK9 inhibition on glucose homeostasis. Recent findings in the OSLER trial over a period of 52 weeks, involving subjects with impaired fasting glucose, metabolic syndrome and type 2 diabetes, demonstrate convincingly that PCSK9 inhibition (as evolocumab) was without effect on fasting plasma glucose and glycated haemoglobin (HbA1c) levels [START_REF] Sabatine | Open-Label Study of Long-Term Evaluation against LDL Cholesterol (OSLER) Investigators. Efficacy and safety of evolocumab in reducing lipids and cardiovascular events[END_REF]. Recent data with alirocumab equally indicate the lack of any adverse signal on glycaemic control [START_REF] Colhoun | Alirocumab effect on glycemic measures in patients without diabetes at baseline[END_REF][START_REF] Ginsberg | Efficacy and safety of alirocumab: Pooled analyses of 1048 individuals with diabetes mellitus from five placebo-controlled Phase 3 studies of at least 52 weeks duration[END_REF]. Practitioners frequently express two lingering concerns with respect to marked lowering of circulating LDL-C concentrations, firstly that low LDL-C levels may raise a range of safety issues, and secondly, prompted by concerns of the US Food and Drug Administration, that low LDL-C on statin treatment may lead to deterioration of cognitive function. Importantly, patients who achieved very low LDL-C levels on statins displayed lower risk for major cardiovascular events [START_REF] Boekholdt | Very low levels of atherogenic lipoproteins and the risk for cardiovascular events: a metaanalysis of statin trials[END_REF]. Furthermore, recent data from the OSLER trial have documented the absence of any safety signal as a function of on-treatment LDL-C levels down to 0.65 mmol/L (25 mg/dL) [START_REF] Shimada | PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future[END_REF].

Similarly, ODYSSEY LONG TERM showed no increase in the incidence of AEs in patients attaining very low LDL-C levels (<0.65 mmol/L or 25 mg/dL) [START_REF] Robinson | Efficacy and safety of alirocumab in reducing lipids and cardiovascular events[END_REF]. Moreover, no significant signal concerning cognitive function has been detected to date in either the ODYSSEY or PROFICIO clinical trials programme [START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF][START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF]. In addition, new findings from a Mendelian randomization study do not support a causal link between low LDL-C (<1.5 mmol/L) and dementia, Parkinson's disease, or epilepsy [START_REF] Benn | Low PCSK9 and LDL cholesterol and risk of dementia, Parkinson's disease, and epilepsy -A Mendelian Randomization Study[END_REF]. Notwithstanding these findings, the EBBINGHAUS trial, a substudy of the FOURIER outcomes trial, will examine the effect of evolocumab-induced low LDL-C levels on cognitive function using objective assessments [START_REF]EBBINGHAUS: Evaluating PCSK9 Binding antiBody Influence oN coGnitive HeAlth in High cardiovascUlar Risk Subjects[END_REF].

Finally, composite findings to date in the ODYSSEY and PROFICIO clinical trials programmes have revealed a low incidence of anti-drug binding or neutralizing antibodies, with incidence varying from 0.1 -7.3% (placebo corrected) of patients; the presence of such antibodies is typically transient [START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF][START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF][START_REF] Kastelein | ODYSSEY FH I and FH II: 78 week results with alirocumab treatment in 735 patients with heterozygous familial hypercholesterolaemia[END_REF]. Long term follow-up data will be essential to evaluate this key question fully, as it may be relevant to instances when a contingency for patients to switch antibodies may arise.

A word of caution is in order when considering the nature of "very low LDL-C levels".

Typically, such levels are calculated on the basis of the Friedewald equation, and therefore include the cholesterol content of lipoprotein(a) [Lp(a)], thereby overestimating true LDL-C. In subjects with elevated Lp(a) levels and "very low LDL", however, LDL may be effectively absent from plasma, and thus the readout potentially corresponds to Lp(a) cholesterol; the clinical implications of this concept are indeterminate [START_REF] Yeang | LDL-C' = LDL-C + Lp(a)-C: implications of achieved ultra-low LDL-C levels in the proprotein convertase subtilisin/kexin type 9 era of potent LDL-C lowering[END_REF]. Under these conditions, ultracentrifugal isolation of LDL provides an accurate readout.

Cardiovascular outcomes trials

It is encouraging that exploratory analyses of ODYSSEY LONG TERM (alirocumab, n=2,341) and OSLER (evolocumab, n=4,465) indicate diminution in cardiovascular outcomes of 50-55% over treatment periods of up to 78 weeks [START_REF] Sabatine | Open-Label Study of Long-Term Evaluation against LDL Cholesterol (OSLER) Investigators. Efficacy and safety of evolocumab in reducing lipids and cardiovascular events[END_REF][START_REF] Robinson | Efficacy and safety of alirocumab in reducing lipids and cardiovascular events[END_REF][START_REF] Koren | Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients with hypercholesterolemia: 52-week results from the Open-Label Study of Long-Term Evaluation Against LDL-C (OSLER) randomized trial[END_REF]. Moreover, a recent meta-analysis of 24 trials of PCSK9 antibody therapy, involving more than 10,000 patients, highlighted a 55% reduction in all-cause mortality (p<0.015), with similar decrements in cardiovascular mortality and myocardial infarction [START_REF] Navarese | Effects of Proprotein Convertase Subtilisin/Kexin Type 9 antibodies in adults with hypercholesterolemia: a systematic review and meta-analysis[END_REF]. Together with the SPIRE clinical trial programme for bococizumab [START_REF];RN316)[END_REF][START_REF]RN316)[END_REF], the FOURIER (patients with a history of CVD and at high risk of recurrent events) [START_REF]FOURIER: Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk[END_REF] and ODYSSEY OUTCOMES (patients recently hospitalized for ACS) [START_REF]ODYSSEY Outcomes: Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab SAR236553[END_REF] trials involve more than 70,000 high risk, dyslipidaemic patients. While the findings are fully anticipated to confirm the preliminary observations discussed above, they will be essential elements in the evaluation of the long term efficacy, tolerability and cost-effectiveness of PCSK9 inhibition. We should not forget, however, that the trajectory of CVD over time is not limited to a single cardiovascular event, and that lowering LDL-C exerts cumulative, long term arterial benefit, modifying the pathophysiological trajectory of atherosclerotic vascular disease [START_REF] Packard | Long term follow-up of lipid-lowering trials[END_REF]. Therefore, critical appraisal of these agents should integrate their cumulative, long term health benefits both for the individual and potentially for healthcare systems. In this light, we summarize future perspectives for PCSK9 inhibition in Table 2.

Beyond the LDL-C target: Triglyceride-rich lipoproteins and lipoprotein(a)

In addition to LDL-C, PCSK9 inhibition, by virtue of its marked enhancement of LDL receptor number, may impact components of the atherogenic lipid profile beyond LDL-C, including triglyceride-rich lipoproteins and remnants (TGRL); such action may equally modulate levels of both high-density lipoprotein (HDL) and apolipoprotein (apo)AI via intravascular remodelling mechanisms. As exemplified by early results from OSLER, atherogenic TGRL levels are significantly reduced when PCSK9 is inhibited, while those of HDL/apoAI may increase (44); similar findings have been made across the ODYSSEY phase III studies [START_REF]The Endocrinologic and Metabolic Drugs Advisory Committee Meeting[END_REF]. Further information on these actions as a function of baseline lipid profile will be of special interest, as we cannot exclude the possibility that they may enhance clinical benefit gained from LDL-C reduction alone.

The lack of therapeutic effect of statins on a potent atherothrombogenic lipid risk factor, Lp(a) has been perplexing, especially as abundant evidence now supports the contention that it is a causal, genetically-determined and independent risk factor for premature CVD [START_REF] Nordestgaard | Lipoprotein(a) as a cardiovascular risk factor: current status[END_REF][START_REF] Tsimikas | Lipoprotein(a) as a potential causal genetic risk factor of cardiovascular disease: a rationale for increased efforts to understand its pathophysiology and develop targeted therapies[END_REF]). Moreover, Mendelian randomization studies have documented a key role for Lp(a) in calcific aortic valve disease, an observation supported by new mechanistic insights intimately linked to its content of oxidized phospholipids [START_REF] Capoulade | Oxidized phospholipids, lipoprotein(a), and progression of calcific aortic valve stenosis[END_REF][START_REF] Bouchareb | Autotaxin derived from lipoprotein(a) and valve interstitial cells promotes inflammation and mineralization of the aortic valve[END_REF]. The finding then that PCSK9 inhibition reduces circulating Lp(a) levels by up to 35% [START_REF] Raal | Reduction in lipoprotein(a) with PCSK9 monoclonal antibody evolocumab (AMG 145): a pooled analysis of more than 1,300 patients in 4 phase II trials[END_REF][START_REF] Gaudet | Effect of alirocumab, a monoclonal proprotein convertase subtilisin/kexin 9 antibody, on lipoprotein(a) concentrations (a pooled analysis of 150 mg every two weeks dosing from phase 2 trials)[END_REF], and that this effect may reside at least partially in the supraphysiological availability of LDL receptors for its catabolism, represents a major mechanistic advance [START_REF] Romagnuolo | Lipoprotein(a) catabolism is regulated by Proprotein Convertase Subtilisin/Kexin Type 9 through the low density lipoprotein receptor[END_REF]. The ongoing cardiovascular outcomes studies for PCSK9 inhibitors may reveal whether Lp(a) reduction contributes to overall reduction in events. Ultimately, however, the answer to this question may require an outcomes trial involving antisense inhibition of hepatic apo(a) production in patients at high cardiovascular risk displaying elevated Lp(a) levels; such a scenario has entered the realm of possibility with the ongoing development of ISIS-APO(a) Rx, which can reduce Lp(a) concentrations by up to 80% dose-dependently [START_REF] Tsimikas | Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study[END_REF].

Unmet clinical needs in dyslipidaemia: The therapeutic horizon

Clinical needs in moderate hypertriglyceridaemia are largely unmet to date, and are a central target on our therapeutic radar screen, especially the highly atherogenic mixed dyslipidaemia involving elevated levels of TGRL and subnormal HDL-C, a profile typical of insulin resistance [START_REF] Hegele | The polygenic nature of hypertriglyceridaemia: implications for definition, diagnosis, and management[END_REF][START_REF] Chapman | Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management[END_REF]. Molecular genetics has clearly identified the majority of such dyslipidaemic states as polygenic, upon which environmental influences are superimposed [START_REF] Hegele | The polygenic nature of hypertriglyceridaemia: implications for definition, diagnosis, and management[END_REF][START_REF] Rosenson | Genetics and causality of triglyceride-rich lipoproteins in atherosclerotic cardiovascular disease[END_REF]. Nonetheless, in the light of new genetic insights indicating that a loss-of-function mutation in apo CIII leads to concomitant fall in levels of TGRL and in cardiovascular risk, novel targeting of the apo CIII gene by antisense inhibition brings considerable optimism to this arena [START_REF] Gaudet | Antisense inhibition of apolipoprotein C-III in patients with hypertriglyceridemia[END_REF]. Indeed, dose-dependent reductions attaining ≈ 80% in hypertriglyceridaemic patients (baseline triglycerides approximately 4.0-22.6 mmol/L or 350-2000 mg/dL) were found using a weekly injection protocol in phase II studies [START_REF] Gaudet | Antisense inhibition of apolipoprotein C-III in patients with hypertriglyceridemia[END_REF]. No safety concerns were identified.

Patients with chronic kidney disease (CKD) are at high CV risk (3); preliminary findings suggest that PCSK9 inhibition is as efficacious in LDL-C lowering in those with moderate CKD as in those with mild or without CKD, with no evidence of safety issues [START_REF] Toth | Alirocumab LDL-C-lowering efficacy in patients with moderate CKD[END_REF].

Cardiovascular prevention in diabetes

After numerous cardiovascular outcome studies over the past years in patients with diabetes, suggesting no short-and medium-term risk reduction with anti-hyperglycaemic agents, the EMPA-REG OUTCOME trial reported a significant reduction of cardiovascular and all-cause mortality using a selective sodium-glucose co-transporter-2 (SGLAT-2) inhibitor, empaglifozin in patients with type 2 diabetes at high cardiovascular risk [START_REF] Zinman | Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes[END_REF]. These observations will have a significant impact on the future management of cardiovascular prevention in patients with type 2 diabetes.

Novel insights into better control of hypertension

The PATHWAY-2 study has suggested that spironolactone is a particularly effective add-on drug for the treatment of resistant hypertension [START_REF] Williams | Spironolactone versus placebo, bisoprolol, and doxazosin to determine the optimal treatment for drug-resistant hypertension (PATHWAY-2): a randomised, double-blind, crossover trial[END_REF]. The results of the PATHWAY-3 study support the first-line use of amiloride plus hydrochlorothiazide in hypertensive patients who need treatment with a diuretic [START_REF] Brown | British Hypertension Society's Prevention; Treatment of Hypertension with Algorithm-based Therapy (PATHWAY) Studies Group. Effect of amiloride, or amiloride plus hydrochlorothiazide, versus hydrochlorothiazide on glucose tolerance and blood pressure (PATHWAY-3): a parallel-group, double-blind randomised phase 4 trial[END_REF]. The DENERHTN study examined 106 patients with well defined resistant hypertension and suggested that renal denervation plus an standardized stepped-care antihypertensive treatment (SSAHT) decreased ambulatory blood pressure more than the same SSAHT alone at 6 months [START_REF] Azizi | Renal Denervation for Hypertension (DENERHTN) investigators. Optimum and stepped care standardised antihypertensive treatment with or without renal denervation for resistant hypertension (DENERHTN): a multicentre, open-label, randomised controlled trial[END_REF], raising hope that renal denervation may lower blood pressure in well-selected patients.

Importantly, the SPRINT study [START_REF] Group | A randomized trial of intensive versus standard bloodpressure control[END_REF] demonstrated that among patients at high risk for cardiovascular events but without diabetes, targeting a systolic blood pressure of less than 120 mm Hg, as compared with less than 140 mm Hg, resulted in lower rates of fatal and nonfatal major cardiovascular events and death from any cause, although significantly higher rates of some adverse events were observed in the intensive-treatment group. This trial was larger than the previous ACCORD study, where a trend for a lower rate of cardiovascular events was observed with more intensive blood pressure lowering.

Summary and Conclusion

The year 2015 has seen dramatic progress in the control of dyslipidaemia, hyperglycaemia and hypertension. These risk factors exert their nocivity throughout the course of the atherogenic process. Dyslipidaemia may, however, be unique as a target to attenuate progression of advanced plaques, and it is in this context that the marked efficacy of PCSK9 inhibition in lowering LDL-C to levels below the critical value of 1.8-2.1 mmol/L (70-80 mg/dL) required to stop progression in the majority of patients may present major therapeutic interest [START_REF] Nicholls | Effect of two intensive statin regimens on progression of coronary disease[END_REF][START_REF] Nissen | Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: the ASTEROID trial[END_REF]. Indeed, could rapid reduction of LDL-C to very low levels post-cardiovascular event result in rapid lipid depletion and enhanced fibrous matrix content across diffuse plaques in the arterial tree, and with it, irreversible -or long term-plaque stabilization with subsequent reduction in cardiovascular events? Could rapid attenuation of dyslipidaemia by PCSK9 inhibitors attenuate endothelial erosion on complex plaques, indirectly diminishing thrombotic complications? Such questions challenge cardiology, obliging us to determine the most efficacious pharmacotherapeutic strategies for CVD prevention. Finally, the first large cardiovascular outcome data of SGLAT-2 inhibition will have a major impact on the future treatment of diabetes, and in hypertension, the PATHWAY and SPRINT studies have provided valuable insights into optimization of treatment. encoded by the SLCO1B1 gene. Thus, variant forms of the protein may exhibit low binding affinity for the statin. Under these conditions, first pass hepatic uptake of the statin is incomplete, leading to elevated levels of statin in the circulation with prolonged residence time. At high statin doses, accumulation of statins in plasma correlates with a poor low-density lipoprotein cholesterol lowering response and a distinct trend to increased frequency of SAMS and myopathy [START_REF] Stroes | Statin-associated muscle symptoms: impact on statin therapy.-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management[END_REF]. As a consequence, peripheral tissues such as skeletal muscle are exposed to high statin concentrations with the potential for enhanced uptake; several mechanisms appear to contribute to statin-induced reduction in ATP production and mitochondrial function in muscle cells [START_REF] Stroes | Statin-associated muscle symptoms: impact on statin therapy.-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management[END_REF]. High demand for energy production in muscle, as occurs in intense exercise, may potentiate SAMS. Figure 2. A schematic summary of the ongoing cardiovascular outcome trials for the three monoclonal antibodies to PCSK9 (proprotein convertase subtilisin/kexin type 9), on a background of human LDL particles visualized by negative stain electron microscopy (copyright Dr M.J.Chapman). Overall, some 70,000 dyslipidaemic patients at high risk will be included in these multicentre, international trials. The primary endpoints in these trials, which are expected to report over the period of 2016 to 2017 are as follows: FOURIER: cardiovascular death, myocardial infarction, hospitalization for unstable angina,

  .C has received research funding from CSL, Kowa, MSD, Pfizer and Randox Laboratories, and honoraria for participation in Speakers Bureaux and Advisory Boards of Amgen, AstraZeneca, Kowa, Merck, Pfizer, Sanofi-Regeneron and Unilever. U.L. has received lecture/advisory board honoraria or research funding from Roche, Sanofi, Amgen, MSD, Pfizer, Servier, Menarini, Astra, St Jude, Orbus&Neich and Terumo. S.B. reports no disclosures.

Figure 1 .

 1 Figure 1. Statin-associated muscle symptoms (SAMS) predominate as adverse effects among

Table 1 .

 1 Abbreviations: HDL high-density lipoprotein; LDL low-density lipoprotein; LDL-C LDL

Acknowledgements

None

Funding

stroke, or coronary revascularization, whichever occurs first [START_REF]FOURIER: Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk[END_REF] ODYSSEY OUTCOMES: coronary heart disease death, any non-fatal myocardial infarction fatal and non-fatal ischaemic stroke, unstable angina requiring hospitalization (57) SPIRE 1, SPIRE-2: major cardiovascular event, a composite endpoint that includes cardiovascular death, non-fatal myocardial infarction, non-fatal stroke, and hospitalization for unstable angina needing urgent revascularization [START_REF];RN316)[END_REF][START_REF]RN316)[END_REF] Abbreviations: ACS acute coronary syndrome; CV cardiovascular; CVD cardiovascular disease; HDL-C high-density lipoprotein cholesterol; LDL-C low-density lipoprotein cholesterol


Boram Kim

Mathieu Gautier
email: mathieu.gautier@insa-lyon.fr

Pascal Molle

Philippe Michel

Rémy Gourdon

Influence of the water saturation level on phosphorus retention and treatment performances of vertical flow constructed wetland combined with trickling filter and FeCl 3 injection

Keywords: vertical flow constructed wetland, trickling filter, ferric chloride coagulation, redox potential, phosphorus

The aim of the study was to determine the effects on treatment efficiency of oxidationreduction (redox) conditions that are caused by different water saturation levels within vertical-flow constructed wetlands (VFCW), with specific attention to phosphorus (P) retention. The study was conducted by monitoring over 18 weeks a pilot-scale system consisting of a biological aerobic trickling filter as a biological pre-treatment step, followed by ferric chloride (FeCl 3 ) addition for phosphate removal and a stage of VFCW. By adjusting the water saturation level, the VFCW was operated successively under fully unsaturated, partly saturated, completely saturated (flooded), and then again unsaturated conditions.

Redox potentials (Eh) were measured at three different levels within the VFCW. Results revealed that Eh was logically affected by the water-saturation level and the feeding-resting periods. Treatment efficiency was very good under fully unsaturated and partly saturated conditions. Under flooded conditions, the performance of the filter declined when the flood was maintained for around one week. However, VFCW regained its previous efficiency after the effluent was drained out and aerobic conditions were restored, indicating that the system was resistant and robust enough that periodical flooding did nott affect its performance afterwards.

1.

Introduction

Raw domestic wastewater treatment by vertical flow constructed wetlands (VFCW) has become very well developed in small communities in France [START_REF] Molle | Effect of reeds and feeding operations on hydraulic behaviour of vertical flow constructed wetlands under hydraulic overloads[END_REF][START_REF] Torrens | Impact of design and operation variables on the performance of vertical-flow constructed wetlands and intermittent sand filters treating pond effluent[END_REF]. Numerous studies have proven the efficiency of the system in carbon removal and nitrification [START_REF] Abou-Elela | Municipal wastewater treatment in horizontal and vertical flows constructed wetlands[END_REF][START_REF] Brix | The use of vertical flow constructed wetlands for on-site treatment of domestic wastewater: New Danish guidelines[END_REF][START_REF] Kadlec | Constructed Wetlands for Pollution Control[END_REF]. Other authors introduced various alternative compact systems [START_REF] Heistad | A high-performance compact filter system treating domestic wastewater[END_REF][START_REF] Prigent | Biological characterization and treatment performances of a compact vertical flow constructed wetland with the use of expanded schist[END_REF] or highlighted some limitations of VFCWs such as low denitrification rate and poor phosphorus removal [START_REF] Prochaska | Performance of pilot-scale vertical-flow constructed wetlands, as affected by season, substrate, hydraulic load and frequency of application of simulated urban sewage[END_REF][START_REF] Verhoeven | Wetlands for wastewater treatment: Opportunities and limitations[END_REF]. Indeed, the release of nitrates and phosphorus into sensitive aquatic ecosystems may promote eutrophication [START_REF] Lowe | Managing phosphorus-based, cultural eutrophication in wetlands: a conceptual approach[END_REF][START_REF] Schindler | Evolution of Phosphorus Limitation in Lakes[END_REF][START_REF] Tiessen | Phosphorus in the global environment[END_REF], and therefore phosphorus (P) and total nitrogen (TN) concentrations in the treated effluents must satisfy increasingly low regulatory limits.

In order to improve TN and P removal, various combined processes and tertiary treatments were developed. Hybrid systems combining vertical and horizontal flow (HF) constructed wetlands were investigated for TN removal by successive nitrification and denitrification [START_REF] Molle | Potential for total nitrogen removal by combining vertical flow and horizontal flow constructed wetlands: A full-scale experiment study[END_REF][START_REF] Vymazal | Horizontal sub-surface flow and hybrid constructed wetlands systems for wastewater treatment[END_REF]. Good efficiencies were obtained but with increased environmental footprint. Regarding phosphorus removal, most studies investigated systems using various filter media with high phosphorus sorption capacity [START_REF] Martín | Phosphorus and nitrogen removal from tertiary treated urban wastewaters by a vertical flow constructed wetland[END_REF][START_REF] Molle | Phosphorus removal by the use of appatite in constructed wetlands: Design recommendations[END_REF][START_REF] Vohla | Filter materials for phosphorus removal from wastewater in treatment wetlands-A review[END_REF]. Although very efficient, these approaches may not provide cheap and durable solutions on a long term.

An alternative approach, the AZOE-NP ® process, was developed by the French company SCIRPE (EP1857419A1; WO2012150296). The treatment line combines (i) a screening at 3 mm mesh, (ii) a biological aerobic trickling filter, (iii) ferric chloride (FeCl 3 ) addition for dissolved phosphate removal and (iv) two stages of partly saturated VFCWs. The use of a trickling filter as a first biological treatment step allows to reduce the required surface of VFCW down to a maximum of 1.5 m 2 per Population-Equivalent (PE) as compared to 2 m 2 in the general operational recommendations in France for classical two-stage VFCWs [START_REF] Molle | How to treat raw sewage with constructed wetlands: an overview of the French systems[END_REF].

One of the particularities of the AZOE-NP ® system is the succession of different levels of redox conditions along the process line. Firstly, the trickling filter is passively aerated by its bottom part and therefore stimulated aerobic microbial processes such as nitrification. Then, within each VFCW stage, the upper zone is not saturated and therefore aerobic, whereas the lower zone is saturated with anoxic conditions allowing denitrification process to occur. The depth of the saturated zone can be adjusted to optimize TN removal efficiency. Another particularity of the AZOE-NP ® process concerns phosphorus removal. Dissolved reactive P is firstly precipitated by FeCl 3 injection at the outflow of the trickling filter. The particulate forms of P are subsequently retained by filtration though the first stage of VFCW [START_REF] Kim | Physical-chemical characterization of sludge and granular materials from a vertical flow constructed wetland for municipal wastewater treatment[END_REF]. The deposits, which progressively accumulate at the surface of VFCW, are usually removed every 10-15 years.

Redox conditions are known to influence microbial processes within constructed wetlands [START_REF] Laanbroek | Bacterial cycling of minerals that affect plant growth in waterlogged soils: a review[END_REF][START_REF] Reddy | Biogeochemical indicators to evaluate pollutant removal efficiency in constructed wetlands[END_REF]. In the case of the AZOE-NP ® process, redox conditions may affect the fate of the ferric species within the VFCW, and consequently the behaviour of P species associated to them. However, no study actually measured Eh levels within the granular medium in VFCW [START_REF] Faulwetter | Microbial processes influencing performance of treatment wetlands: A review[END_REF]. In this study, a pilot-scale VFCW was implemented to monitor Eh at three different depths within the porous medium with the objectives to determine (i) the influence of VFCW saturation level on the performances of the system and (ii) the effects of Eh within the VFCW on the retention of phosphorus.

Materials and methods

Description of pilot's design

Pilot-scale unit of VFCW was installed within a greenhouse in Irstea's experimental hall located at La Feyssine in the metropolitan area of Lyon (France). A schematic diagram of pilot-scale installation is shown in Fig. 1. This experimental setup is meant to simulate the operational units of a real-scale AZOE-NP ® plant but focused only on the first stage of filters as previous studies had shown that the efficiency of the process mainly occurred within the 1 st stage [START_REF] Kim | Field study of wastewater treatment with partially flooded vertical-flow constructed wetlands using AZOE® process[END_REF]. The pilot-scale system was designed as a combination of four sub-units as shown in Fig. 1:

1) A pumping station used for the successive following treatment steps: collection of screened wastewaters, feeding of trickling filter in a closed loop mode, mixing of the solution after ferric chloride injection, and injection of the solution at the surface of the VFCW.

2) A pilot-scale trickling filter of 0.29 m 3 fully packed with cross flow plastic media (ordered) developing a specific surface area of 200 m 2 .m -3 .

3) A dosing pump (DDE, Grundfos) for ferric chloride injection.

4) A pilot-scale VFCW with a surface of 2 m 2 and a depth of 0.7 m. It was filled with 4 successive layers of granular materials which were taken from a real scale AZOE-NP ® system in operation since 8 years: a 20 cm upper filtration layer was made of 2-4 mm gravels; two intermediate layers (20 cm with 4-8 mm gravels and 7 cm with 8-16 mm gravels) and a 6 cm lower drainage layer made of 20-60 mm pebbles, corresponding to two thirds of the real-scale plant depth. The pilot was also equipped with a siphon to adjust the water saturation level. In addition, a layer of 10 cm of surface deposit sludge (sampled from the real scale plant) was disposed at the top surface of the VFCW. The characteristics of the sludge deposit were described in a previous study [START_REF] Kim | Physical-chemical characterization of sludge and granular materials from a vertical flow constructed wetland for municipal wastewater treatment[END_REF]. Finally, 8 young common reeds (Phragmites australis) per square meter were planted in the surface deposit layer.

Description of pilot's operation

The pilot system was fed with real domestic wastewater obtained directly from the nearby wastewater treatment plant of La Feyssine. To reduce the risks of clogging of the distribution pipes however, wastewater was passed through a 1 mm sieve before being used. The pilot system was then operated automatically as described below.

Sieved wastewater was introduced into the pumping station's tank. The feeding pump was started to feed the trickling filter downward in a closed-loop mode. Large openings in the top and bottom of the bioreactor allowed a good circulation of air though the packing medium.

When a pre-determined volume (50 L) of wastewater was reached in the pumping station, the trickling filter's feeding was stopped and the pre-treated water was drained into the pumping station's tank. The desired volume (7.0 mL) of a 574 g.L -1 ferric chloride aqueous solution was then precisely injected into the tank with a dosing pump, corresponding to a total added iron concentration of 0.45 mmol.L -1 within the tank. The mixture was mixed by closed-loop circulation within the tank using the feeding pump. Finally, the content of the tank was spread at the surface of the VFCW using the feeding pump through one feeding pipe positioned in the centre part of the VFCW.

Experimental protocol

The trickling filter pilot was put in operation 2 months before the beginning of the experiments, to reach stable biological conditions, with a continuous feeding of 0.9 m 3 .day -1 .

The VFCW pilot was then operated by alternating feeding periods of 1/3 of week and resting periods of 2/3 of week in order to simulate the operational conditions of real scale systems.

In order to study the influence of redox conditions on performance of the system, particularly The study started under unsaturated condition (first phase) which was monitored over 6

weeks. The second phase (partly saturated conditions) was then implemented for 6 weeks as well, followed by the last phase (totally saturated also called flooded conditions) which was only conducted over 4 weeks because of the extreme condition. Finally, the effluent was drained out of the VFCW pilot to monitor over 2 more weeks the re-establishment of aerobic conditions and thereby investigate the resilience of the system.

Sampling and analyses

Influent and effluent samples were taken twice a week at the beginning and the end of each Dissolved reactive phosphorus (DRP) was analyzed by the colorimetric molybdenum blue method [START_REF] Murphy | A modified single solution method for the determination of phosphate in natural waters[END_REF]. Total soluble elemental concentrations (Fe, Ca, Al, Mn and Si) were determined by ICP-AES (Ultima 2; Horiba JobinYvon SAS). Sulphate and chloride concentrations were determined by ion chromatography using a Dionex Ion Pac ® AS17C column preceded by an AG17 guard-column.

Redox conditions within the VFCW pilot were monitored by a method described by [START_REF] Veneman | Salt bridge for field redox potential measurements[END_REF] using redox probes and the salt bridge. The salt bridge was made by filling 1.25 cm diameter Plexiglas ® pipes with saturated KCl agar-agar gel (3%).

The gel was spiked with phenol to prevent microbial growth within the salt bridge. The top part of the salt-bridge pipe received a single junction Ag/AgCl (KCl 3 M) reference electrode and the bottom of the tube was sealed. On the salt bridge tube, three 6-mm diameter holes were drilled face to the placements of each platinum electrode and covered with filter paper to maintain the gel inside. Three platinum electrodes were placed in the VFCW pilot respectively at 10 cm, 34 cm and 58 cm from the bottom. The salt bridge was installed close to the Pt electrodes. Redox potentials were taken every five minutes using a potentiometer Component Analysis (PCA) were performed using the R software package v 3.0.2.

Results and discussion

The average concentrations of measured parameters at different steps of the treatment line are gathered in During unsaturated VFCW configuration, good removals of SS and dCOD were observed. As the whole treatment line was under aerobic condition, high nitrification rate with concomitant nitrate production were obtained. Excellent dissolved phosphate retention was also observed.

Under partly saturated VFCW configuration, excellent SS removal rate was observed.

Ammonium and dCOD removal rates were in the same order of magnitude as in the unsaturated phase. Dissolved TN removal rate increased, indicating the installation of denitrification process within water saturated zone.

Under flooded condition, only SS removal was observable. Releases of dissolved COD and phosphate were obtained, suggesting anaerobic biodegradation of organic matters.

Performance of trickling filter pilot and ferric chloride injection

The results obtained for measured parameters at first three sampling points (Inlet-TF, Outlet-TF and Inlet-VFCW; Ferric chloride injection showed a good performance in P removal with an average of 98% (SD: 2). The residual total dissolved iron amounts were only 1.2% of total iron initially added. Almost all dissolved iron was therefore found to react with the reactive species during the treatment, indicating that the dosage of FeCl 3 was not in large excess.

Performance of VFCW pilot

Influence of the water saturation level within the VFCW pilot

The evolution of redox potential (Eh) at three different depths within the VFCW pilot during the study is shown in Fig. 3. Four different redox conditions were considered based on the approach followed in soil sciences [START_REF] Diáková | The Distribution of Iron Oxidation States in a Constructed Wetland as an Indicator of Its Redox Properties[END_REF][START_REF] Laanbroek | Bacterial cycling of minerals that affect plant growth in waterlogged soils: a review[END_REF][START_REF] Reddy | Soil precesses regulating water quality in wetlands[END_REF][START_REF] Szogi | Characterization of oxidationreduction processes in constructed wetlands for swine wastewater treatment[END_REF], namely: aerobic (oxygen reduction zone; > 300 mV), anoxic (nitrate and Mn 4+ reduction zone; 100 to 300 mV), reduced (Fe 3+ reduction zone; -100 to 100 mV) and anaerobic (sulphate reduction zone; -200 to -100 mV). The influence of the level of water saturation can be clearly observed. Under unsaturated conditions (first phase of the study), Eh ranging between 400 and 670 mV were measured, indicating that aerobic conditions prevailed within the filter. During the second phase of the experiment (partly saturated conditions), the Eh measured at the bottom of the filter (within the saturated zone) decreased progressively down to -250 mV after 2 weeks, revealing anaerobic conditions. In the unsaturated upper part of the filter however, Eh remained between 400 and 670 mV as previously, thereby confirming aerobic conditions. Finally in the last phase (flooding), Eh values at the top and the middle of the filter also decreased rapidly down to around -250 mV within one week, indicating that the whole filter was then under anaerobic conditions.

The influence of feeding and resting periods on the Eh within the VFCW was also clearly observed in Fig. 4. It can be seen that during feeding periods, the Eh values measured in the unsaturated zone decreased by approximately 200 mV, and a reverse phenomenon occurred in saturated zone as a consequence of the impact of fresh wastewater flowing in the different zones. This phenomenon was not observed during the flooded phase, suggesting the installation of anaerobic condition within the entire depth of the VFCW pilot which was unaffected by feeding and resting periods. Regarding suspended solids (SS), despite high variations of inlet concentration, quite stable and low outlet concentrations were maintained at the successive phases of the treatment line.

Evolution of relevant parameters at each phase of the experiment

Outlet SS concentrations varied between 30-98 mg.L -1 , 4-13 mg.L -1 , and 26-69 mg.L -1 during unsaturated, partly saturated and flooded phases, respectively. The best SS removal rate (97.8%) was found during partly saturated phase. It could be explained by a settling effect within the water saturated zone. For flooded phase, despite of an important height of water settling zone within the filter, SS removal rate decreased compare to the previous condition.

This could be explained by a physical degradation of organic matters within the sludge layer due to anaerobic conditions.

Regarding dissolved COD and ammonium, their outlet concentrations varied within similar ranges under unsaturated and partly saturated conditions (11-58 mg.L -1 and 1.7-27.7 mg.L -1 respectively). On the other hand, during the flooded phase, the outlet concentration of both dissolved COD and ammonium were found to increase, suggesting degradation of some organic matters under anaerobic condition. This observation could indicate a negative performance of the filter due to the extreme conditions simulating a severe malfunctioning of the system. However, when aerobic conditions were re-established by draining the filter at the end of the 4-week flooded phase, the concentrations of both parameters rapidly decreased, thereby revealing the good resilience of the system. Two hypotheses may be proposed to explain this good resilience of the VFCW pilot:

-a rapid recovery of the activity of facultative aerobic bacteria after inhibition by flooding.

-a continuous provision of aerobic bacteria from the trickling filter.

For nitrates concentrations, strong increases were observed between inlet and outlet during the unsaturated phase. In addition, very high concentrations of nitrates at the beginning of feeding periods were noted. This phenomenon could be explained by a continuous nitrification during resting period causing discharges of organic nitrogen and adsorbed ammonium within the sludge layer of VFCW pilot [START_REF] Morvannou | Modeling nitrogen removal in a vertical flow constructed wetland treating directly domestic wastewater[END_REF]. Starting from the very beginning of the partly saturated phase, a decrease of outlet nitrate concentrations was observed while outlet ammonium concentrations stayed stable: the nitrates formed by nitrification were denitrified in the saturated zone. On the other hand, during the flooded phase, no more nitrates were detected at the outlet because of lack of nitrification. Once the unsaturated zone re-established, an observable production of nitrates after one week can be seen. Finally, Fig. 4 shows that dissolved phosphate outlet concentration varied between 0.30-0.93 mg.L -1 , 0.34-2.31 mg.L -1 , and 3.25-7.44 mg.L -1 under unsaturated, partly saturated and flooded conditions respectively. The fact that P concentration increased during the partly saturated phase indicated firstly the presence of P species at the bottom of the VFCW pilot, suggesting the vertical transfer of P species through the porous medium from the surface sludge layer to the bottom of the VFCW. Secondly this observation also showed that reduced conditions within this zone induced the solubilisation of present P species. Dissolved iron concentration followed a similar pattern (as also Mn concentration) as P during the first two phases (unsaturated and partly saturated VFCW pilot), suggesting the dissolution of reduced iron species, with concomitant P releases. Numerous studies reported the release of P by the reductive dissolution of P-bearing ferric species [START_REF] Hantush | Nutrient Dynamics in Flooded Wetlands. I: Model Development[END_REF][START_REF] Moustafa | Influence of hydropattern and vegetation on phosphorus reduction in a constructed wetland under high and low mass loading rates[END_REF][START_REF] Patrick | Phosphate Release and Sorption by Soils and Sediments: Effect of Aerobic and Anaerobic Conditions[END_REF][START_REF] Shenker | Redox reactions and phosphorus release in re-flooded soils of an altered wetland[END_REF].

On the other hand, the strong release of P during the flooded phase was not accompanied by an equivalent increase of iron release at the outlet of VFCW pilot. Furthermore, decrease of dissolved iron concentration was even observed after two weeks of flooded phase. This observation could suggest that the conditions within the filter induced the precipitation of solubilised iron species. For example, the presence of sulphides probably caused FeS formation as already described by other authors (Chen et al., 2013;[START_REF] Erler | Observations of nitrogen and phosphorus biogeochemistry in a surface flow constructed wetland[END_REF][START_REF] Jakobsen | Redox zoning, rates of sulfate reduction and interactions with Fe-reduction and methanogenesis in a shallow sandy aquifer, Rømø, Denmark[END_REF]. Indeed, due to the anaerobic conditions (Eh below -250 mV), sulphides were likely produced by sulphate reducing anaerobic bacteria. Fig. 4 shows that dissolved sulphate outlet concentrations were relatively stable during unsaturated and partly saturated phases, but decreased down to very low concentrations during the flooded phase, thereby confirming the hypothesis of sulphate reduction.

When aerobic conditions were re-established at the end of the 4-week flooded phase, the outlet concentrations of dissolved phosphate and iron rapidly decreased, confirming the good resilience of the system.

3.4. Principal components analysis (PCA) of the data gathered for the VFCW pilot

In order to investigate the influence of the redox conditions measured at three different depths of the pilot on the numerous parameters followed in the study, a principal components analysis (PCA) was performed. Preliminary study (results not shown) allowed to select 9 relevant parameters for the final analysis, namely: Eh values measured at the bottom, middle and surface of the filter (Eh.B, Eh.M, Eh.S respectively), VFCW pilot outlet concentrations for SS, dCOD, ammonium, nitrate, phosphate and iron (SS, dCOD, NH4-N, NO3-N, PO4-P and Fe, resp.). According to the results of PCA, only first two principal components had statistically significant Eigen values (> 1) and they accounted for 80.7% of the inertia.

Therefore, the analysis shown below refers only to these two components which were considered to be sufficiently informative. First of all, it can be seen that the redox potentials at the surface and in the middle of the VFCW pilot varied together. Among the parameters of water analyses, only outlet concentration of SS seemed to be independent of the redox conditions within the VFCW pilot. Nitrates outlet concentration was positively correlated with the Eh at the bottom of the filter.

Releases of dissolved iron and phosphate were found to be negatively correlated with the combination of redox conditions within the entire depth of VFCW pilot, thereby confirming the reduction of P-bearing ferric species as a significant mechanism of P release in the studied system. The fact that the dissolved iron release was not as much anti-correlated to the surface Eh as P release could be explained by FeS formation under anaerobic condition as described before (cf. 3.3.2). However, this observation may also suggest other mechanisms of P release in the VFCW pilot. Release of organic P may have occurred from the autolysis of strictly aerobic bacterial cells due to the brutal shift to anaerobic conditions [START_REF] Turner | Potential contribution of lysed bacterial cells to phosphorus solubilisation in two rewetted Australian pasture soils[END_REF], or from the anaerobic breakdown of organic nitrogen and phosphorus compounds [START_REF] Reddy | Soil precesses regulating water quality in wetlands[END_REF]. Strong negative correlations between the surface Eh and releases of dissolved COD, ammonium and phosphate could support this hypothesis.

The plot of individual data points (Fig. 5,b) revealed the existence of 3 distinct groups of data, defined with respect to water saturation level within the VFCW: 0 cm, 30 cm and 70 cm. In addition, the point representing the return to unsaturated condition [R0cm] joined the initial unsaturated [0cm] data group. This could also indicate indirectly a good resilience of the VFCW pilot.

Conclusion

This study was conducted at the pilot scale to determine the influence of water-saturation level on redox conditions within the VFCW and on the treatment efficiency with specific attention to P retention. Redox potentials monitored at three different depths clearly showed time-course changes of Eh according to water saturation levels and feeding-resting cycles.

Statistical analysis of experimental data using PCA showed that Eh was the key parameter controlling phosphorus release from the sludge.

When the VFCW was under aerobic condition (unsaturated phase), significant nitrification and a low release of dissolved P were observed. During the following experimental phase, when the VFCW was partly saturated, a decrease of the Eh was observed at the bottom of the VFCW (saturated zone) about 2 weeks after the experimental condition was changed from unsaturated to partly saturated. During this phase, very good performance in SS removal and denitrification were observed. On the other hand, iron reduction with concomitant P release (1.08 ± 0.57 mg P.L -1 ) was observed. Since only the bottom part of the VFCW was saturated in this experimental phase, the migration of P species from the surface sludge layer to the bottom of the VFCW but also suggested that the reduction of P-bearing ferric species was a significant mechanism of P release.

In the last experimental phase, when the VFCW pilot was completely flooded (including the surface sludge layer), one week was needed until anaerobic conditions were established in the whole depth. During this phase, the performance of the VFCW declined for all parameters.

While dissolved P release increased, dissolved iron release did not exhibit an equivalent increase. This could suggest either the re-precipitation of reduced iron species (due for example to FeS formation) and/or the occurrence of other mechanisms of P release such as the autolysis of strictly aerobic bacterial cell material due to the shift to anaerobic conditions.

Flooded conditions should therefore be avoided in practice to prevent phosphate release.

However, in this study, a sharp increase in the dissolution of Fe-bound phosphates was observed only after one whole week of flooding. This delay maybe attributed to the role of the trickling filter which provided relatively stable sludge organic matters but also non negligible quantities of nitrates to the VFCW. A fresh biodegradable sludge would probably have caused a higher consumption of oxygen resulting in a more rapid Eh drop and phosphates dissolution. These phenomena would have been accelerated without the nitrates provided by the trickling filter. Therefore, in the present study, the use of a trickling filter as pretreatment stage contributed to the good resistance of the system to unfavourable conditions as flooding.

Finally, when the whole water within the VFCW was drained out at the end of the experiment, aerobic conditions and good treatment efficiency were rapidly re-established, underlining that VFCW was resilient enough that the changes in the saturation level affected its performance only temporarily.

Fig. 1 .

 1 Fig. 1. Simplified diagram of pilot-scale installation.

  phosphorus retention, investigations were done with three different water-saturation levels within the VFCW: (1) unsaturated phase [0 cm] corresponding to a free drainage with no effluent resting at the bottom of the filter; (2) partly saturated phase [30 cm] corresponding to the classical mode of operation of AZOE-NP ® process, and (3) saturated phase[70 cm] where the water level was set to completely flood the filter. During the first phase of the study, aeration within VFCW was possible from the top and the bottom. During the second phase, aeration would be only provided form the top of the filter, resulting in the establishment of aerobic conditions in the 23 cm upper part and a 30 cm anoxic to anaerobic lower zone. The last phase of the study, where even the surface deposit sludge layer was flooded, would correspond to extreme conditions resulting from a malfunctioning of the wastewater collect system during long and heavy raining period.

  feeding period of the VFCW pilot. Four sampling points were monitored, namely: (i) inlet of trickling filter (sieved wastewater) [I-TF], (ii) outlet of trickling filter (before ferric chloride injection) [O-TF], (iii) inlet of VFCW (after ferric chloride injection) and (iv) outlet of VFCW stage. Each sample was immediately divided into two aliquots. The first aliquot was analysed for total suspended solids (TSS) according to French standard methods (AFNOR, 2005). The second aliquot of each sample was immediately filtered through 0.45 µm syringe filter (Sartorius Minisart). The filtrate was stored at 4°C for a maximum of 6 h before being analyzed as follows. Dissolved chemical oxygen demand (dCOD) was photometrically determined after sample oxidation with a hot sulphuric solution of potassium dichromate (analogous to standard methods ISO 15705; C4/25 and C3/25 cell tests; WTW). Ammonium and nitrate were analyzed by colorimetric techniques (14739 and 114542 cell tests; WTW).

(

  Consort; C861) which was connected to the Pt and reference electrodes. Recorded redox potential values Eh m were then expressed with respect to the standard hydrogen electrode (SHE) reference using the following equation:Eh = Eh m + 222 mVwhere Eh is the corrected value of redox potentials in mV with respect to SHE, and Eh m is the experimental reading in mV with respect to the Ag/AgCl reference. Though the SHE correction factor is temperature dependent, corrections were not made as the error resulting from the variation of temperature in the study was considered negligible as compared to other experimental uncertainties. No supplementary pH correction was made because pH measurements at inlet/outlet of the VFCW pilot were close to neutrality (7.20 ± 0.34).2.5. Statistical data analysesThe 95% confidence interval was determined by means values ± 1.96 times standard error of the mean (SEM), which was calculated as the standard deviation (SD) divided by the square root of the sample size. For data analyses and visualisation, ANalysis Of VAriance (ANOVA, least significant differences [LSD] at p < 0.05 level), Pearson correlation coefficients and Principal

Table 1 .

 1 Results are shown below in terms of inlet and outlet concentrations measured at each individual step of the treatment line, in order to evaluate their respective contribution to the performance of the whole treatment line. The major focus is made on the monitoring of the VFCW stage and the effect of its mode of operation on P retention. 3.1. Effect of water saturation level in VFCW on the overall performance of the treatment line Treatment performances with respect to each parameter were calculated in the form of "(inletoutlet) / inlet" concentrations of the considered parameter expressed in percentage. Results are gathered in

Fig. 1 )

 1 along the treatment line are shown in Fig. 2. The total number of monitoring was 38 and its results are presented by box plots which indicate the median (band inside the box), quartiles (top and bottom of the box), minimum and maximum values. In the first place, significant variations were observed at the inlet (untreated wastewater) for almost all parameters.Regarding the trickling filter pilot, mean input-output removal rates, for dissolved COD, 53% (SD: 12), 41% (SD: 19), and 40% (SD: 15), respectively. For dissolved COD, the trickling filter allowed to smooth the relatively high variation of inlet concentrations. The removal of ammonium (also observed at this step of the treatment line, with about 40% (. This observation may partly be attributed to microbial consumptions within the bioreactor, and also to physicochemical sorption onto organic matters and/or residual iron species in the bottom of the pumping station.

Fig. 2 .

 2 Fig. 2. Concentrations of different parameters at three different points along the process line:

Fig. 3 .

 3 Fig. 3. Eh evolutions at three different depths of the VFCW during the successive phases for

  figure clearly highlighted that the water saturation level had influence on the observed results.

Fig. 4 .

 4 Fig. 4. Concentrations of different parameters at inlet/outlet of the VFCW pilot. White, light

Fig. 5

 5 Fig. 5 shows selected parameters on the correlations circle (a) and a plot of individual data

Fig. 5 .

 5 Fig. 5. PCA plots in the subspace defined by the first two principal components: (a) variables

Table 2 .

 2 The sum of dissolved ammonium and nitrate concentrations was used to calculate

	total dissolved nitrogen ([NO 3	-+ NH 4	+ ]-N) removal.

Table 2 .

 2 Overall removal rate (%) of the whole pilot-scale installation with different water saturation level within VFCW pilot expressed as Mean ± SEM × 1.96 (sample size).

	Parameter	Unsaturated VFCW	Partially saturated VFCW	Totally saturated VFCW
						0 cm	30 cm	70 cm
	SS				71.4 ± 7.5 (15)	94.9 ± 1.8 (12)	70.9 ± 10.1 (11)
	d COD				69.4 ± 5.5 (15)	69.6 ± 6.0 (11)	-*
	d N-NH 4	+	78.4 ± 8.2 (15)	60.9 ± 11.4 (11)	-**
	d N-[NO 3	-+ NH 4	+ ]	-*	44.8 ± 11.0 (11)	-**
	d P-PO 4	3-		90.5 ± 3.3 (15)	77.3 ± 6.6 (11)	-*
	d dissolved concentrations

* Negative removals indicating a production or release of concerned parameter.

** No significant differences (ANOVA, p value > 0.05) between concentrations before and after concerned treatment step.

Acknowledgements

Financial support for this work was provided by SCIRPE and CIFRE (Conventions Industrielles de Formation par la REcherche). The authors wish to thank O. Collache, R.

Poncet and H. Perier-Camby for the installation of the pilot system and redox potential measurements equipments. They are also grateful to N. Dumont and D. Lebouil of LGCIE for chemical analyses (ICP-AES and ion chromatography) and to reviewers for their contribution to improvement of this manuscript.


Julien Alexandre

Dit Sandretto

Alexandre Chapoutot

Dynibex

DynIBEX: a Differential Constraint Library for Studying Dynamical Systems

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