Stéphane Berciaud

Laurent Cognet

Philippe Poulin

R Bruce

Brahim Lounis

Absorption spectroscopy of individual single-walled carbon nanotubes

Current methods for producing single-walled carbon nanotubes (SWNTs) lead to heterogeneous samples containing mixtures of metallic and semiconducting species with a variety of lengths and defects. Optical detection at the single nanotube level should thus offer the possibility to examine these heterogeneities provided that both SWNT species are equally well detected. Here, we used photothermal heterodyne detection to record absorption images and spectra of individual SWNTs. Because this photothermal method relies only on light absorption, it readily detects metallic nanotubes as well as the emissive semiconducting species. The first and second optical transitions in individual semicontucting nanotubes have been probed. Comparison between the emission and absorption spectra of the lowest-lying optical transition reveal mainly small Stokes shifts. Side bands in the near-infrared absorption spectra are observed and assigned to exciton-phonon bound states. No such sidebands are detected around the lowest transition of metallic nanotubes.

Single-walled carbon nanotubes (SWNTs) have attracted much attention for their remarkable physical properties, making them promising for applications in nanotechnologies. The diameter and chiral angle of SWNTs are defined by the two structural indices (n,m), which determine their electronic structure including their metallic or semiconducting nature [START_REF] Saito | Physical Properties of Carbon Nanotubes[END_REF][START_REF] Reich | Carbon Nanotubes: Basic Concepts and Physical Properties[END_REF] . The optical properties of SWNTs have received growing interest since the recent observation of near-infrared luminescence from well separated surfactant-suspended semiconducting SWNTs [START_REF] O'connell | [END_REF] . Subsequent photoluminescence excitation measurements resulted in the precise mapping of the transition energies for a large variety of specific semiconducting structural species 4,5 .

All current methods for producing SWNTs lead to heterogeneous samples containing mixtures of metallic and semiconducting species with a variety of lengths and defects. This diversity of SWNT structures complicates precise spectroscopic characterization. Single particle methods thus appear valuable for eliminating the heterogeneity and inhomogeneity present in bulk SWNT optical spectra 6,7 . For instance, photoluminescence studies performed on individual SWNTs revealed the single-nanotube linewidth of emission spectra and the presence of spectral variations within a given (n,m) type 6,8,9 . Luminescence measurements are however limited to semiconducting SWNTs. By contrast, Raman scattering has been used to study individual semiconducting and metallic SWNTs 6,10,11 , but such experiments remain constrained by the weakness of the signal and the need to use near-resonant laser sources.

Rayleigh scattering has also been studied to record optical spectra from individual structureassigned SWNTs 7,12 . It allowed probing of the third and fourth optical transitions of semiconducting SWNTs as well as the first and second transitions of metallic ones. So far, this technique has only been applied to studying long, large diameter (~2 nm) SWNTs that are individually suspended over open apertures.

Because the cross-sections for optical absorption decrease less rapidly than those for Rayleigh scattering as the nanotube diameter is reduced 13 , a detection method relying on the remarkable absorptive properties of carbon nanotubes would be valuable. Small diameter SWNTs, which are abundantly produced by the HiPco method [START_REF] O'connell | [END_REF] , are particularly appealing for optical studies because their first optical transitions lie in the visible region for metallic SWNTs and in the near-infrared for semiconducting species.

Here, we report the highly sensitive imaging and absorption spectroscopy of individual small diameter SWNTs, using Photothermal Heterodyne Imaging (PHI) 14,15 Semiconducting and metallic SWNTs can be equivalently regarded as ideal candidates for investigations with absorption based methods such as PHI 14 . First, carbon nanotubes are highly absorptive nano-objects with absorption cross-sections of typically 10 -18 cm 2 /carbon atom 16 and rapid inter-and intra-band carrier relaxation dynamics 17 . Metallic nanotubes have electron-electron and electron-phonon relaxation times in the subpicosecond range 17,18 , whereas semiconducting species have picosecond-scale nonradiative decay times associated with weak ensemble luminescence quantum yields (ca. 10 -3 ) [19][20][21] .

Our photothermal imaging and absorption spectroscopy apparatus has been described elsewhere 22 . In brief, it consists of a probe beam (HeNe laser, 633nm, output power of ~ 8 mW) overlaid with a modulated tunable cw absorption beam. For the latter we used either a dye laser (tunable from 2.07 to 2.34 eV) for spectroscopy of metallic SWNTs, or a cw Ti:sapphire laser (tunable from 1.20 to 1.31 eV or from 1.24 to 1.63 eV) for spectroscopy of semiconducting SWNTs. The absorption and probe beams were both focused onto the sample using a high NA objective. The absorption beam was linearly polarized and had an intensity Figure 1 shows typical luminescence (Fig. 1a and1b) and corresponding photothermal (Fig. 1c and1d) images acquired with two orthogonal polarizations of the absorption beam at a fixed wavelength (565 nm). Luminescent SWNTs represent only a small proportion of all observed SWNTs, and all of them co-localize with spots in the photothermal images (black circles on Fig 1). Interestingly, many more spots are detected in the photothermal images with high signal-to-noise ratios (from 10 to >1000), providing a more complete picture of the SWNTs present in the sample. The numerous non-luminescent peaks may arise from metallic SWNTs or may correspond to semiconducting species emitting either with very low quantum yields or at photon energies lower than our detection range. One can also not exclude the possibility that a few spots might arise from SWNTs aggregated into small absorbing bundles.

Considering that only ~20% of all semiconducting SWNTs have peak emission wavelengths within the range of our Si-APD, and that the metallic SWNT abundance is ca. 1/3 of the total, we estimate that photothermal images should exhibit ~10 times more spots than luminescence images, in good agreement with the data presented in Fig. 1. Almost all SWNTs appear as diffraction-limited spots, unresolved by the imaging method (Fig. 1a-d). This is consistent with the length distribution of nanotubes prepared using our ultrasonic dispersion process 19,20 .

However, as shown in Fig. 1e-f micrometer long nanotubes were occasionally observed.

The polarization-dependent absorption of SWNTs is evident in both luminescence and photothermal images (see circled SWNTs in Fig. 1a-d, and Fig. 1e-f); the highest absorption being achieved when the SWNT longitudinal axis is parallel to the incident laser field 6,7 . The corresponding to the radiative recombination of the lowest excitonic state 23,24 . From the distribution of the E S 11 values (data not shown), different sub-populations could be identified with mean values lying at 1.28, 1.25 and 1.20 eV. Based on the spectral assignments of Bachilo et al. 4 and the dominant species observed in bulk spectrofluorimetry of our sample, we identify these as the (8,3), (6,5) and (7,5) species, respectively. In a second step we recorded photothermal absorption spectra of such nanotubes, by scanning the absorption laser frequency with steps of ~2 meV (100 ms integration time per point). For normalization, the absorption beam power was measured at the sample during the acquisitions.

Figure 2a-c displays examples of luminescence and absorption spectra for three SWNTs with different chiralities, belonging respectively to the (8,3), (6,5), and (7,5) species, respectively.

An absorption peak A S 11 near the emission peak E S 11 is clearly visible for the (8,3) and (6,5) tubes (see insets of Fig. 2a and2b). The absorption peak of the (7,5) nanotubes can be measured with a different setting of the Ti-Sapphire laser, as exemplified in Fig. 2d. The influence of the cw probe beam was investigated by recording two successive spectra of the same nanotube, using a full and a four-fold attenuated probe intensity (Fig. 2e). No noticeable difference between the two normalized spectra was found, indicating that the probe had a negligible influence on the line shape of the absorption spectra.

The distribution of A S 11 values obtained from 62 absorption spectra is presented in Fig. 2f, where the positions of (7,5), (6,5) and (8,3) species are indicated.

We now consider the Stokes shift, We expect that such traps might be present initially or might be induced by laser irradiation during measurements 26 .

Finally, for all semiconducting species, we observe an absorption side-band lying ~200 meV above the 11 S transition (Fig. 2a-c). This energy difference matches within experimental error the energy of the Raman-active G band vibration. According to theoretical predictions 27 and room temperature ensemble spectra 28 , this sideband is assigned to exciton-phonon bound states. In principle, such a sideband could also be attributed to the second-lowest one-photonallowed excitonic transition, but its chirality-independent position rules out this possible assignment. This suggests that for linear processes most of the oscillator strength of the S 11 transition is contained in the lowest optically allowed excitonic state. One should also stress that, although exciton-phonon sidebands are detected on single tubes, exciton-phonon interactions remain in the weak coupling limit 29 , as confirmed by the small Stokes shifts measured here. Direct observation of such bound states in the absorption spectra of individual SWNTs supports the excitonic origin of optical resonances in SWNTs 23 .

The (6,5), and (8,3) species are difficult to fully discriminate by their first optical transition.

However, their second optical transitions, 22 S , are well separated 4 . We thus measured the absorption spectra of such nanotubes near the expected 22 S position of the (6,5) species (Fig. 3a-b). The observation of an absorption peak in Fig 3a and the absence of any feature in Fig. 3b allow us to unambiguously assign the 3a species as (6,5) and the 3b species as (8,3).

Interestingly, and in agreement with bulk measurements 30 , we find that 22 S transitions are systematically broader than 11 S transitions. This observation is consistent with time resolved photoemission measurements that show faster electronic relaxation with increased excitation photon energy 31 .

As mentioned above and illustrated in figure 1, PHI is a remarkable detection method for individual metallic SWNTs. Figure 4a-b presents absorption spectra of two non-luminescent SWNTs with peaks at 2.08 eV and 2.23 eV. Figure 4c shows the distribution of the peak energies found in the energy range of the laser for 23 non-luminescent SWNTs, revealing two subpopulations centred at 2.09 eV and 2.23 eV.

Although it might seem that these peaks could arise from S 22 transitions of semiconducting SWNTs (e.g. the (8,4) or (9,2) species) having peak emissions E S 11 falling outside the spectral range of our detector, the transitions are significantly narrower than all S 22 absorptions measured here (see Fig. 3a). Moreover, in contrast to the spectra shown in Fig. 2, we observed no exciton-phonon sidebands for any absorption peaks near 2.09 eV. This observation constitutes a strong indication that the spectra shown in Fig. 4 stem from the lowest optical transitions (M 11 ) of individual metallic nanotubes, for which excitonic effects are expected to be much weaker than in semiconducting tubes 32 . Using results from ensemble resonant Raman spectra 33,34 and numerical simulations 35 , we assign the lower and higher energy groups to species with 2n + m = 27 and 2n + m = 24, respectively.

The use of an appropriate source and detector to monitor absorption and emission in the nearinfrared, as well as complementary investigations of the trigonal warping effect 12,35 (a hallmark of metallic SWNTs), will allow unambiguous discrimination of metallic from semiconducting absorbers.

In conclusion, we have demonstrated the efficient detection of individual luminescent and nonluminescent SWNTs using a highly sensitive photothermal detection method. Absorption spectra of the lowest-lying optical transitions of individual SWNTs have been recorded. Our absorption technique has the unique ability to probe any optical absorption transition of individual SWNTs in common environments. We believe that it holds great promise for applications in nanotube characterization and sorting.

  . Because PHI probes light absorption, it enables identification of both semiconducting and metallic SWNTs. The chiral indices of individual semiconducting nanotubes are assigned by analyzing their absorption spectra around their first S 11 and second S 22 optical resonances. As expected for semiconducting nanotubes, exciton-phonon sidebands are observed near the first optical resonance. No such sidebands are detected around the lowest M 11 transition of metallic nanotubes.

1 -

 1 laser was also used at fixed wavelength (565 nm, i.e. photon energy of ~2.2 eV) and with an intensity of 2 20 ~cm kW to record confocal fluorescence images and emission spectra of individual luminescent SWNTs. In those experiments, the collected luminescence light was split between a silicon avalanche photodiode (APD) and a spectrometer equipped with a cooled Si-CCD camera. Due to the limited spectral sensitivity of Si-based detectors, only semiconducting nanotubes emitting in the range 1.19-1.45 eV were detected. We used SWNTs grown by the HiPco process and prepared following the procedure introduced by O'Connell et al. 3 . Dilute suspensions of SWNTs in aqueous sodium dodecylsulfate (SDS) were spin-coated onto clean microscope cover slips. We chose dilution factors and spinning rates to give a final SWNT density of less than 2 µm . A drop of viscous silicone oil was added on top of the samples to ensure homogeneous heat diffusion.

  variations in photothermal signal strengths can be explained by three factors: the rather broad distribution of nanotube lengths; their random orientations with respect to the linearly polarized absorption beam; and the varying mismatches between absorption beam wavelength and the transition peak among the numerous SWNT (n,m) species. The measured luminescence signal strengths can vary additionally because of the wavelength-dependent response of our Si-APD in the near-infrared and possible differences in numbers of defects and trap states among the nanotubes10,19 .Prior to measuring absorption spectra of individual semiconducting SWNTs, we recorded 117 individual nanotube luminescence spectra in the 1.19-1.33eV spectral window (10 s integration time per spectrum; examples are shown in Fig2a-c). Most luminescence spectra display a Lorentzian shape from which we extract the peak emission energy E S 11

  49(6,5) and(8,3) nanotubes.We find that this shift lies below our experimental accuracy (~5 meV) for most of the nanotubes (Fig 2g, see also for example insets in Fig 2a-b), in agreement with ensemble measurements 25 . As shown in Fig. 2g, no correlation between the position of the emission peak and the Stokes shift values is observed. Interestingly, ~25% of the SWNTs studied exhibit a larger Stokes shift (from ~10 to ~ 40 meV). Furthermore, absorption spectra of the S 11 transition are broader than emission lines (by a factor ~2). Those effects can be attributed either to spectral jumps as the absorption and luminescence spectra are not acquired simultaneously, or to the presence of trap states due to structural or chemical defects, or local environmental variations along the nanotube.

Figure 1 :

 1 Figure 1: (a-b) Luminescence and (c-d) photothermal absorption images of the same region

Figure 2 :

 2 Figure 2: Absorption and luminescence spectroscopy of the S 11 optical transition on

Figure 3 :

 3 Figure 3: Absorption spectroscopy of the S 22 optical transition on individual semiconducting

Figure 4 : 11 M

 411 Figure 4: Absorption spectroscopy of the M 11 optical transition on individual metallic

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 4

 4 Figure 4

Acknowledgement

This research was funded by CNRS (ACI Nanoscience and DRAB), Région Aquitaine, Agence Nationale de la Recherche (PNANO program) and the French Ministry for Education and Research (MENRT). RBW acknowledges support from the NSF (CHE-0314270) and the Welch Foundation (C-0807).


Estibaliz Lazaro

Gaelle Coureau

Jérémie Guedj

Patrick Blanco

Isabelle Pellegrin

Daniel Commenges

François Dabis

Jean-François Moreau

Jean-Luc Pellegrin

Rodolphe Thiébaut
email: rodolphe.thiebaut@isped.u-bordeaux2.fr

Change in T-lymphocyte count after initiation of highly active antiretroviral therapy in HIV-infected patients with history

Keywords: HIV infection, Mycobacterium avium complex infection, Immune restoration, CD4 Lymphocyte Count, Highly Active Antiretroviral Therapy

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

experience of antiretroviral treatment, AIDS clinical stage at the time of HAART initiation (baseline), age (+/-10 years) and gender. MAC+ and MAC-patients presented comparable median levels of total lymphocytes (488 vs. 688/mm 3 , p=0.09), CD4+ (11 vs. 16/mm 3 , p=0.15), CD8+ count (359 vs. 386/mm 3 , p=0.39) and plasma HIV RNA (5.3 vs. 5.1 log 10 copies/mL, p=0.22) at baseline. After 6 months on HAART, the median increase of CD4+ count was 28 cells/mm 3 (IQR 1; 63) in MAC+ and 72 cells/ mm 3 (IQR 34; 120) in MACpatients (p<0.0001) while the percentage of CD4+ cells was not significantly different between the two groups (p=0.13). Comparables differences were observed for total lymphocytes and CD8+ cells (p<0.001). The six months decline of plasma HIV RNA was not significantly different according to MAC exposure (-1.6 in MAC+ vs. -1.8 log 10 copies/mL in MAC-patients, p=0.65). Results were confirmed after adjustment for other characteristics than the matching variables and after taking into account potential informative bias due to unbalanced number of deaths between the two groups.

Conclusion: MAC infection at the time of HAART initiation is an important deleterious factor for immune reconstitution. A better understanding of the underlying mechanism and an evaluation of additional treatment strategies are necessary to help immune restoration in such circumstances.

Introduction

Highly Active Antiretroviral Therapy (HAART) allows an immune reconstitution even in patients with advanced HIV disease [START_REF] Autran | Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease[END_REF]. Although the increase in CD4+ T-lymphocyte count does not represent the whole immune reconstitution [START_REF] Lederman | Incomplete immune reconstitution after initiation of highly active antiretroviral therapy in human immunodeficiency virusinfected patients with severe CD4(+) cell depletion[END_REF], it is the main marker used in clinical practice and epidemiological studies to estimate immune reconstitution. The modifications of the CD4+ counts following HAART initiation are associated with T cell activation [START_REF] Hunt | T cell activation is associated with lower CD4+ T cell gains in human immunodeficiency virus-infected patients with sustained viral suppression during antiretroviral therapy[END_REF], plasma HIV RNA and proviral DNA [START_REF] Rouzioux | Early levels of HIV-1 DNA in peripheral blood mononuclear cells are predictive of disease progression independently of HIV-1 RNA levels and CD4+ T cell counts[END_REF] loads at the time of treatment initiation. A low nadir of CD4+ count or a sharp decline in CD4+ count before HAART initiation are important determinants of immune reconstitution [START_REF] D'amico | Lower CD4+ T lymphocyte nadirs may indicate limited immune reconstitution in HIV-1 infected individuals on potent antiretroviral therapy: Analysis of immunophenotypic marker results of AACTG 5067[END_REF][START_REF] Renaud | Determinants of paradoxical CD4 cell reconstitution after protease inhibitor-containing antiretroviral regimen[END_REF]. The other factors usually reported are an older age [START_REF] Viard | Influence of age on CD4 cell recovery in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy: Evidence from the EuroSIDA study[END_REF], AIDS staging [START_REF] Thiébaut | Determinants of response to first HAART regimen in naive patients with an estimated time since HIV seroconversion[END_REF], intravenous drug use [START_REF] Lucas | Detrimental effects of continued illicit drug use on the treatment of HIV-1 infection[END_REF], lack of adherence to HAART [START_REF] Carrieri | Impact of early versus late adherence to highly active antiretroviral therapy on immuno-virological response: a 3-year follow-up study[END_REF] and previous antiretroviral exposure [START_REF] Yamashita | Immunologic and virologic response to highly active antiretroviral therapy in the Multicenter AIDS Cohort Study[END_REF].

Profound immune deficiency, in particular of the CD4+ T helper cell compartment, limits the control of specific diseases such as Cytomegalovirus infection (CMV) [START_REF] Sacre | Repertoire, diversity, and differentiation of specific CD8 T cells are associated with immune protection against human cytomegalovirus disease[END_REF] or Mycobacterium Avium Complex (MAC) infection [START_REF] Havlir | Effect of potent antiretroviral therapy on immune responses to Mycobacterium avium in human immunodeficiency virus-infected subjects[END_REF]. Conversely, restoration of immune function has led to the recommendation of discontinuation of opportunistic infections prophylaxis, including MAC infection [START_REF] Zeller | Discontinuation of secondary prophylaxis against disseminated Mycobacterium avium complex infection and toxoplasmic encephalitis[END_REF][START_REF] Shafran | Successful discontinuation of therapy for disseminated Mycobacterium avium complex infection after effective antiretroviral therapy[END_REF]. Although profound immune deficiency is an important detrimental factor for immune reconstitution with HAART, little is known on the role of specific opportunistic infections often present in this context [START_REF] Cassol | Therapeutic response of HIV-1 subtype C in African patients coinfected with either Mycobacterium tuberculosis or human herpesvirus-8[END_REF]. For instance, the impact of HCV co-infection has been discussed [START_REF] Greub | Clinical progression, survival, and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection: the Swiss HIV Cohort Study[END_REF][START_REF] Antonucci | Role of hepatitis C virus (HCV) viremia and HCV genotype in the immune recovery from highly active antiretroviral therapy in a cohort of antiretroviral-naive HIV-infected individuals[END_REF][START_REF] Braitstein | Immunologic Response to Antiretroviral Therapy in Hepatitis C Virus-Coinfected Adults in a Population-Based HIV/AIDS Treatment Program[END_REF]. A potential mechanism of the detrimental effect on immune restoration may be the activation of apoptosis through the infection of macrophage and expression of Fas-ligand [START_REF] Graham | Why should hepatitis C affect immune reconstitution in HIV-1infected patients ?[END_REF][START_REF] Laskus | Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages[END_REF]. In the case of CMV infection, there is a known deficiency of hematopoiesis [START_REF] Maciejewski | Human cytomegalovirus infection of human hematopoietic progenitor cells[END_REF] due to (i) a direct cytotoxic effect of CMV which replicates inside hematopoietic progenitor cells and (ii) an inhibition of growth factors production [START_REF] Randolph-Habecker | Cytomegalovirus mediated myelosuppression[END_REF].

The direct effect on CD4+ T cells regeneration may also be mediated through apoptosis [START_REF] Goldmacher | Cell death suppression by cytomegaloviruses[END_REF].

Infection with MAC is also known to impair haematopoiesis [START_REF] Inderlied | The Mycobacterium avium complex[END_REF]. However, to our knowledge, few information are available on the response to HAART of patients with history of MAC infection [START_REF] Aberg | A study of discontinuing maintenance therapy in human immunodeficiency virus-infected subjects with disseminated Mycobacterium avium complex: AIDS Clinical Trial Group 393 Study Team[END_REF][START_REF] Macgregor | Clinical, microbiological, and immunological characteristics in HIV-infected subjects at risk for disseminated Mycobacterium avium complex disease: an AACTG study[END_REF]. In the ACTG protocol 393 study evaluating whether antimycobacterial therapy for MAC could be withdrawn in subjects with history of MAC, the CD4+ count rose slowly (6 cells/mm3 every 2 months) [START_REF] Aberg | A study of discontinuing maintenance therapy in human immunodeficiency virus-infected subjects with disseminated Mycobacterium avium complex: AIDS Clinical Trial Group 393 Study Team[END_REF]. We conducted a study within the ANRS Co3 Aquitaine Cohort of HIV-infected patients [START_REF] Thiébaut | Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment[END_REF], to compare change in CD4+, CD8+ and total lymphocytes counts between HAART treated patients with and without a recent history of MAC infection.

Methods

Design

The Aquitaine Cohort is a prospective hospital-based cohort of HIV1-infected patients, initiated in 1987 in the Bordeaux University Hospital and four other public hospitals in Aquitaine, South-western France [START_REF] Thiébaut | Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment[END_REF]. Clinical, biological and therapeutical information are collected prospectively at each visit under routine clinical management proceeding.

In the present study, we constituted two groups of patients who started for the first time a combination including at least three antiretroviral drugs (defined as HAART). The exposed group (MAC+) included patients with a diagnosis of MAC infection between 12 months before and 6 months after HAART initiation. We hypothesized that diagnoses of MAC infection made in the first six months on HAART corresponded to infections that were already present at the time of treatment start but clinically silent. Robustness analyses excluding those patients gave similar results (data not shown). The unexposed group (MAC-) included patients of the Aquitaine cohort without any history of MAC or CMV infection during their entire follow-up, who were individually matched on CD4+ count (+/-30 cells/mm 3 ), previous experience of antiretroviral treatment (naïve or not), AIDS clinical stage at the time of HAART initiation, age (+/-10 years) and gender. The matching ratio of exposed:unexposed was 1:3, with the exception of two exposed patient for whom this ratio was 1:2 and 1:1 for three exposed patients.

Diagnosis of MAC infection

Diagnosis of disseminated MAC infection was based on the isolation of MAC from cultures of blood, bone marrow, or other normally sterile tissue or body fluids. Diagnoses based only on compatible clinical signs and symptoms were not considered in the present study.

HIV RNA load and immunophenotyping 7/19 CD4+, CD8+ and total lymphocytes counts were performed by flow cytometry in a single laboratory. Plasma HIV RNA was quantified by different laboratory kits according to the period and the hospitals. The branched DNA assays (Chiron Quantiplex RNA HIV-1, Emeryville, CA) with lower limits of detection of 2.7 log 10 copies/mL (500 copies/mL) and 1.7 log 10 copies/mL (50 copies/mL) were most often used. The schedule of measurements was based on clinical practice, i.e. every 3 to 6 months.

Statistical methods

The primary analyses compared the different markers values at each time point by Wilcoxon test. Time points considered were baseline (date of HAART initiation), 6 months latter (M6), M12 and M24. For each time point and each patient, the measurement that was the closer to the time point in a 3 months window was analyzed. Also, we performed multiple linear regressions of CD4+ difference from baseline to six months follow up on treatment adjusted for all potential factors influencing markers levels at a given time point including the MAC exposure as the primary factor of interest. We observed an attrition of the number of patients followed due to early deaths and lost-to-follow-up. These monotone missing data may bias estimates because patients exposed to MAC infection were more likely to die. Therefore, we conducted robustness analyses by jointly modelling repeated measurements of CD4+ count and survival time. This type of model allows taking into account these biases due to informative dropout process [START_REF] Thiébaut | Joint modelling of bivariate longitudinal data with informative dropout and left-censoring, with application to the evolution of CD4+cell count and HIV RNA viral load in response to treatment of HIV infection[END_REF]. Briefly, the longitudinal model was a piecewise linear model explaining change in squared root of CD4+ count by a slope before HAART initiation, a slope between HAART initiation and the first 6 months and a slope after 6 months. The survival model was a lognormal survival model for time from HAART initiation to death or 24 months visit. The link between the two models was defined by the random slopes.

Results

Study population

Among 356 HAART treated patients followed in the Aquitaine Cohort and who presented a MAC infection during their follow-up, 51 patients were included in the present study because they started a combination of at least three antiretroviral drugs and they presented a MAC infection between 12 months before HAART initiation and 6 months after. Among these 51 patients, 13 MAC infections were diagnosed between 12 months and 6 months before HAART initiation, 13 MAC infections within 6 months before HAART initiation, nine at the time of HAART initiation and 16 within 6 months after HAART initiation. These MAC Characteristics of included patients are depicted in table 1. At the time of HAART initiation, there were no differences between the two groups according to matching factors as well as other characteristics.

Markers evolution

At the time of treatment initiation, MAC+ and MAC-patients presented comparable median levels of total lymphocytes (488 vs. 688/mm 3 , p=0.09), CD4+ count (11 vs. 16/mm 3 , p=0.15) and CD8+ count (359 vs. 386/mm 3 , p=0.39) (Table 1). During follow-up, the two groups presented a significant increase in total lymphocytes, CD4+ and CD8+ counts.

However, these increases were significantly more pronounced in MAC-than in MAC+ groups (Figure 1, panels a-c). After six months on HAART, the median increase of CD4+ count was +28 cells/mm 3 (IQR +1; +63) in MAC+ and +72 cells/mm 3 (IQR +34; +120) in MAC-groups (p<0.0001), while the percentage of CD4+ cells was not significantly different between the two groups (p=0.13) (Figure 1,panel d). Interestingly, plasma HIV RNA decreased significantly in the two groups and the decline was not significantly different according to MAC exposure (-1.6 in MAC+ group vs. -1.8 log 10 copies/mL in MAC-group at 6 months, p=0.65) (Figure 1, panel e). The proportions of patients reaching 500 copies/mL or less at 6 months were similar (48.8% and 49.5% in the MAC+ and MAC-, respectively, p=0.93). The effect of MAC exposure on the change of CD4+ count at 6 months was also confirmed after adjustment on other determinants (Table 2). Indeed, the factors significantly associated with a poorer CD4+ count increase were: MAC exposure, CD4 count nadir < 50 cells/mm 3 , higher baseline plasma HIV RNA and previous experience of antiretroviral treatment. Treatments including zidovudine and clarithromycin were associated with poorer CD4+ count increase at 6 months (p=0.03) but this effect did not remain after adjustment for other characteristics (p=0.51). The independent effect of MAC exposure was also confirmed at 12 and 24 months (data not shown). The effect of MAC exposure was also confirmed when modelling all repeated measures of CD4+ count taking into account potential informative drop-out due to death and adjusting for potential confounding factors. Thus, estimated CD4+ counts levels were significantly higher in the MAC-group compared to MAC+ patients at 6 11/19 months (p=<0.0001), 12 months (p=0.0002) and 24 months (p=0.0047) and not at M0 (p=0.17). The estimated slopes within the first 6 months were +19.4 cells/mm 3 /month ([15.9;22.8] ) in the MAC-group and +9.5 cells/mm 3 /month ([3.8 ; 15.1]) in the MAC+ group (p=0.003). After 6 months, the estimated slopes were +2.9 cells/mm 3 /month ([2.2;3.5]) and +2.7 cells/mm 3 /month ([1.5;3.8]), respectively (p=0.75).

There was no significant effect of MAC exposure on haemoglobin and platelets levels during follow-up although haemoglobin levels tended to be lower in patients exposed to MAC infection (Figure 1, panels f-g).

Discussion

In this study, we showed that patients who experienced a MAC infection during their follow up and started a HAART regimen, presented a weaker increase in their total lymphocytes, CD4+ and CD8+ counts despite similar virological response. MAC infection is known to compromise hematopoiesis and in particular erythropoiesis and granulopoiesis. This mechanism is thought to be multi-factorial. First, during the course of MAC disease, infection massively widespreads in bone marrow which must impair hematopoiesis [START_REF] Hafner | Correlation of quantitative bone marrow and blood cultures in AIDS patients with disseminated Mycobacterium avium complex infection[END_REF]. Second, the presence of mycobacteria induces the production of immunosuppressive cytokines by the host adaptative immune response, the secretion of soluble factors that inhibit erythroblastic progenitors [START_REF] Gascon | Impaired erythropoiesis in the acquired immunodeficiency syndrome with disseminated Mycobacterium avium complex[END_REF], the deleterious effect of TNF- on haematopoiesis [START_REF] Kremer | Ineffective cellular immune response associated with T-cell apoptosis in susceptible Mycobacterium bovis BCG-infected mice[END_REF] and Fas Ligand expression [START_REF] Isgro | Decreased apoptosis of bone marrow progenitor cells in HIV-1-infected patients during highly active antiretroviral therapy[END_REF] by infected macrophages known to induce the apoptosis of T lymphocytes.

Apoptosis is also one of the mechanisms explaining the deficiency of haematopoiesis in the course of CMV [START_REF] Goldmacher | Cell death suppression by cytomegaloviruses[END_REF] or HCV infections [START_REF] Graham | Why should hepatitis C affect immune reconstitution in HIV-1infected patients ?[END_REF][START_REF] Laskus | Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages[END_REF] as well as it impaired immune restoration in HIV-infected patients [START_REF] Benveniste | Mechanisms involved in the low-level regeneration of CD4(+) cells in HIV-1-infected patients receiving highly active antiretroviral therapy who have prolonged undetectable plasma viral loads[END_REF]. Treatment of MAC infection such as clarithromycin might also contribute to these disorders. In fact, the association of clarithromycin and zidovudine in HIVinfected patients is known to suppress lymphopoiesis and hematopoiesis [START_REF] Freund | Prophylactic clarithromycin to treat mycobacterium avium in HIV patients receiving zidovudine may significantly increase mortality by suppressing lymphopoiesis and hematopoiesis[END_REF]. Nevertheless, the impact of MAC infection and its management was not observed on haemoglobinemia and platelets, favouring the hypothesis of a specific alteration of the lymphocyte lineage.

This study presents three main limitations. First, we were not able to compare the effect of different opportunistic infections at the time of treatment initiation on the entire Aquitaine cohort because of the limited number of individual opportunistic infections in about 3000 HAART-treated patients. This calls for the need of analyses of this kind in multi-cohort collaborations [START_REF] Egger | Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies[END_REF]. Second, we do not considered the possible effects of adherence, ethnicity or social class which may vary in the two groups because it was not recorded in our database.

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Third, it would be useful to perform more specific measurements (apoptosis, naive and memory cells, activation…) to better characterize the mechanisms in this context [START_REF] Lederman | Incomplete immune reconstitution after initiation of highly active antiretroviral therapy in human immunodeficiency virusinfected patients with severe CD4(+) cell depletion[END_REF].

With respect to clinical care, hematotoxic drugs should be avoided in patients presenting MAC infection. Furthermore, immunotherapy may be indicated in such a context. The G-CSF and GM-CSF factors activate destruction of intracellular bacteria and stimulate the granulocyte colony. These factors have been successfully proposed to treat patients with MAC infection [START_REF] Bermudez | Treatment with recombinant granulocyte colony-stimulating factor (Filgrastin) stimulates neutrophils and tissue macrophages and induces an effective non-specific response against Mycobacterium avium in mice[END_REF]. Moreover, such treatment might help in immune restoration [START_REF] Nielsen | Highly active antiretroviral therapy normalizes the function of progenitor cells in human immunodeficiency virus-infected patients[END_REF]. Il-2 may also be a good candidate because of its stimulating effect on CD4+ proliferation [START_REF] Levy | Effects of interleukin-2 therapy combined with highly active antiretroviral therapy on immune restoration in HIV-1 infection: a randomized controlled trial[END_REF] and its inhibitory effect on cell apoptosis [START_REF] Estaquier | Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists[END_REF], thus increasing the survival of CD4+ T lymphocytes [START_REF] Kovacs | Induction of prolonged survival of CD4(+) T lymphocytes by intermittent IL-2 therapy in HIV-infected patients[END_REF].

In conclusion, MAC infection at the time of HAART initiation seems to be an important deleterious factor for immune reconstitution. A better understanding of the underlying mechanism and an evaluation of additional treatment strategies are necessary to help immune restoration in such circumstances.

  patients were mostly treated by a triple antibiotic combination including clarithromycin, ethambutol and rifabutin (N=17) or azithromycin, ethambutol and rifabutin (N=15). The others were treated by dual antibiotic combinations (ethambutol and rifabutin in 6 cases) or single therapy (N=6). The HAART regimen was mainly based on the combination of two nucleoside reverse transcriptase inhibitors and one protease inhibitor (88%). The most commonly used nucleosides were zidovudine (N=98) and lamivudine (N=138) and the most common PI was indinavir (N=92). Zidovudine was associated with clarithromycin in 11 cases. Two MAC patients died in the first 6 months on treatment, 10 between 6 and 12 months and six between 12 and 24 months. The 51 MAC+ exposed patients were individually matched to 145 MAC-patients. These unexposed patients, who had very low CD4+ count at HAART initiation due to the matching, had history of Kaposi sarcoma (N=21), pulmonary pneumocystosis (N=27), candidasis (but oral only) (N=22), toxoplasmosis (N=21), cryptococcosis (N=11), HIV encephalopathy (N=6), tuberculosis (N=11), lymphoma (N=5), cryptosporidiosis (N=3), recurrent bacterial pneumonia (N=1), progressive multifocal leucoencephalopathy (N=1) and salmonella septicaemia (N=1) but neither MAC nor CMV infection by design.

Table 1 .

 1 Baseline demographic and clinical characteristics at the time of initiation of HAART of patients exposed to Mycobacterium avium complex infection (MAC+) and matched patients unexposed to MAC infection (MAC-). ANRS Co3 Aquitaine Cohort.

	MAC+ (N=51)	MAC-(N=145)
	N or median % or IQR N or median % or IQR p value

Table 2 .

 2 Univariable and multivariable analyses of factors associated with change in CD4+level between baseline and 6 months after HAART initiation, adjusted for baseline CD4+.

	ANRS Co3 Aquitaine Cohort.
		Univariable	Multivariable*
	Characteristic	ß	P	ß	P
	MAC exposure (vs. no exposure)	-25.5 <0.0001 -24.7 <0.0001
	Age (for one year older)	0.3	0.53	_	_
	Women (vs. men)	-5.8	0.64	_	_
	Intravenous Drug Users (vs. other transmission groups) -4.5	0.69	_	_
	Naive of antiretroviral treatment (vs. experienced)	27.7	0.01	30.5	0.03
	AIDS clinical stage (vs. other clinical stages)	13.6	0.32	_	_
	Nadir of CD4+ count <50 cells/mm 3 (vs. ≥50)	-16.6	0.25	-55.8	0.007
	Baseline calendar period > 2000 (vs ≤ 2000)	22.3	0.11	_	_
	Baseline HIV RNA (for 1 log copies/mL higher)	21.2	0.0003	17.8	0.002
	ß : coefficient of the liner regression
	MAC : Mycobacterium avium complex

* All listed characteristics included

The ANRS Co3 Aquitaine Cohort and Jérémie Guedj are supported by the Agence Nationale de Recherches sur le SIDA (Action Coordonnée no.7, Cohortes and research grant for PhD).


Pierre Hennebert
email: pierre.hennebert@ineris.fr

Ismahen Samaali

Pauline Molina

A proposal for a test method for assessment of hazard property HP 12 ("Release of an acute toxic gas") in hazardous waste classification -experience from 49 waste

Keywords: European waste legislation, toxic gas release, gas measurement, gas identification, solid waste, liquid waste, EUH029, EUH031, EUH032, H2S, industrial air pollution control residue

Toxic gases are emitted by four waste: metallic dust from the aluminum industry (CO), two air pollution control residue of industrial waste incinerator (H2S) and a halogenated solvent (organic volatile(s) compound(s)). HF has not been measured in these trials started before the present definition of HP 12. According to the definition of HP 12, only the H2S emission of substances with hazard statement EUH031 is accounted for. In view of the calcium content of the two air pollution control residue, the presence of calcium sulfide (EUH031) can be assumed. These two waste are therefore classified potentially hazardous for HP 12, from a total of 49 waste. They are also classified as hazardous for other properties (HP 7, 10 and 14 for one of them, and HP 10 and 14 for the other one respectively) . Given these results, it can be assumed that few common household and industrial waste will be classified hazardous only by HP 12. Sample Plateau in 5 minutes ? Emission (Lg/kg) in 5 minutes SD (Lg/kg ) RSD Sludges from landfill leachate, after evapo-concentration no 27.1 7.7 0.28 APC residue from municipal waste after solid fuel, metals and organic matter separation no 13.3 7.5 0.56 Sludges from treatment of urban waste water no 10.6 7.5 0.71 Compost from organic fraction separately collected no 8.1 0.9 0.12 Bottom ash and slag from municipal waste after solid fuel, metals and organic matter separation -maturated and pretreated no 7.3 0.5 0.06 Pasty waste #1 no 6.6 0.9 0.13 Packages and materials C #5 no 6.6 0.7 0.11 Pasty waste #3 Mix of storage tank no 6.3 3.4 0.53 Surface treatement -sludges and filter cakes no 4.6 0.1 0.02 APC residue industrial waste #2 no 4.6 1.4 0.30 MSWI APC residue, lime process no 4.1 1.3 0.32 MSWI fly ash no 4.1 1.7 0.41 Compost from mixed municipal waste, fraction < 30 mm after crushing no 3.3 1.0 0.29 Wastes from transport tank cleaning, mixed sludge of food and chemical transport no 3.2 1.9 0.58 Metallic dust from aluminum industry no 3.2 0.2 0.05 Pasty waste #2 no 2.8 2.4 0.83 APC residue industrial waste #1 no 2.8 1.4 0.51 Packages and materials #3 no 2.4 0.3 0.14 Mixed municipal waste, fraction > 30 mm after crushing no 2.2 0.5 0.23 Bauxite residue no 2.2 0.3 0.15 Industrial waste bottom ash no 2.1 0.7 0.31 Municipal waste -Organic fraction separately collected no Table 3: Emission of gas by waste in 6 hours, sorted for the presence of a plateau and decreasing emission Sample Plateau between 1 h and 6 h ? Emission (Lg/kg) in 6 h Sludges from landfill leachate, after evapo-concentration no 95.8 Sludges from treatment of urban waste water no 79.3 Sediment Marseille (harbor, Mediterranean sea) no 56.6 Compost from organic fraction separately collected no 46.4 Packages and materials C #5 no 36.6 MSWI Air pollution control (APC) residue, bicarbonate process no 33.6 Pasty waste #1 no 31.2 Pasty waste #3 Mix of storage tank no 28.6 Wastes from transport tank cleaning, mixed sludge of food and chemical transport no 27.6 Mixed municipal waste, fraction > 30 mm after crushing no 22.6 APC residue industrial waste #2 no 20.7 MSWI fly ash no 16.0 Surface treatement -sludges and filter cakes no 14.0 Industrial waste bottom ash no 9.6 Scrapped tires 4 mm no 8.9 Municipal waste -Organic fraction separately collected no 8.2 Bauxite residue no 6.6 Packages and materials B #5 no 6.1 Active landfill leachate containing hazardous substances or landfill leachate other than those mentioned in 19 07 02 no 4.2 Halogenated solvent #1 no 3.9 Waste water #2, mixture of 13 wastes no 3.8 Waste water #1 no

ABSTRACT

A stepwise method for assessment of the HP 12 is proposed and tested with 49 waste samples. The hazard property HP 12 is defined as "Release of an acute toxic gas": waste which releases acute toxic gases (Acute Tox. 1, 2 or 3) in contact with water or an acid. When a waste contains a substance assigned to one of the following supplemental hazards EUH029, EUH031 and EUH032, it shall be classified as hazardous by HP 12 according to test methods or guidelines (EC 2014a, b). When the substances with the cited hazard statement codes react with water or an acid, they can release HCl, Cl2, HF, HCN, PH3, H2S, SO2 (and two other gases very unlikely to be emitted, hydrazoic acid HN3 and selenium oxide SeO2a solid with low vapor pressure). Hence, a method is proposed:

1 Measurement of the volume of gases emitted into contact with water or an acid (solid/liquid ratio of 10 L water or acid / kg raw waste, acid 2.5 M HNO3); 2 If gas is emitted at a rate higher than 0.1 L of gas/kg waste in 5 minutes (limit of quantification of the automated calcimètre used here), checking if one of the cited gases is emitted with electrochemical detection probes or simple qualitative colorimetric methods; 3 If one of these gases is detected, speciation of the emitting substance(s) (calculation by the "worst case" method from elements concentrations, or by specific analytic methods) and checking whether they have a hazard statement code EUH029, EUH031 or EUH032 (a list is provided). For a set of 49 waste, water addition did not produce gas. Nearly all the solid waste produced a gas in contact with hydrochloric acid in 5 minutes in an automated calcimeter with a volume > 0.1 liter of gas per kg of waste. Since a plateau of pressure is reached only for half of the samples in 5 minutes, 6 hours trial with calorimetric bombs or glass flasks were done and confirmed the results. Identification of the gases by portable probes showed that most of the tested samples emit mainly

INTRODUCTION

The Waste Framework Directive [START_REF] Ec | [END_REF] defines 'hazardous waste' as 'waste which displays one or more of the (15) hazardous properties listed in Annex III'. The property H 12 'Waste which releases toxic or very toxic gases in contact with water, air or an acid' was not further defined since that time. A Commission Regulation of 2014 redefined HP 12 (former H 12) as ("Release of an acute toxic gas": waste which releases acute toxic gases (Acute Tox. 1, 2 or 3) in contact with water or an acid. When a waste contains a substance assigned to one of the following supplemental hazards EUH029, EUH031 and EUH032, it shall be classified as hazardous by HP 12 according to test methods or guidelines (EC 2014a, b). There is still no standardized test for measure of gases emission from waste. The UN N.5 method characterizes substances which in contact with water produce flammable gases (UN 2003). It is mentioned in the Dangerous Goods Transport recommendations of the UN and in the CLP regulation. The volume of gas is measured manually by displacement of water in graduated flasks. It is poorly repeatable (according to INERIS experience with powders and chemicals) since the degree or type of wetting, a major parameter, is not defined [START_REF] Janes | Contribution à l'amélioration de la méthode d'essai ONU N.5, relative à la caractérisation des solides qui, au contact de l'eau, dégagent des gaz inflammables[END_REF][START_REF] Janes | Comparaison de dispositifs de mesure de dégagement gazeux associés à la méthode d'essai ONU N.5, relative à la caractérisation des solides qui, au contact de l'eau, dégagent des gaz inflammables RAPPORT[END_REF]. The Austrian experimental standard Önorm S 2120 also addresses only the flammable gas (contact with water and contact with a flame). The DG ENV has recently sent a mandate to Technical Committee TC 292 (Characterization of waste) of CEN to develop and validate a method for HP12.

Definition of HP 12

EU Member States have revised the definition of the hazard properties (EC 2014a, b). The HP 12 property is: "HP 12"Release of an acute toxic gas:"waste which releases acute toxic gases (Acute Tox. 1, 2 or 3) in contact with water or an acid. When a waste contains a substance assigned to one of the following supplemental hazards EUH029, EUH031 and EUH032, it shall be classified as hazardous by HP 12 according to test methods or guidelines.'. The waste must contain a substance or substances with the hazard statements (HSC) code EUH029, EUH031 and EUH032. There is no standardized test to date for measuring the gases emitted by the waste. These hazard statement codes are the following (CLP 2008): "EUH029 -'Contact with water liberates toxic gas' For substances and mixtures which in contact with water or damp air, evolve gases classified for acute toxicity in category 1, 2 or 3 in potentially dangerous amounts, such as aluminium phosphide, phosphorus pentasulphide. EUH031 -'Contact with acids liberates toxic gas' For substances and mixtures which react with acids to evolve gases classified for acute toxicity in category 3 in dangerous amounts, such as sodium hypochlorite, barium polysulphide. EUH032 -'Contact with acids liberates very toxic gas' For substances and mixtures which react with acids to evolve gases classified for acute toxicity in category 1 or 2 in dangerous amounts, such as salts of hydrogen cyanide, sodium azide."

The substances with these HSC have been extracted from the CLP tables (CLP 2008) (Table 1). The gas they can emit and their molecular formula have been added. Many substances are very reactive (specially EUH029 substances that react with water) and not likely to be found in waste because the chemical industry would destroy or neutralize them before release as waste. An exception is probably thionyl chloride present in the lithium-ion batteries (EUH029). These EUH029 substances emit in contact with water HCl and SO2, and for some H2S and PH3. Substances with hazard statement EUH031 include more common products (sulphides, polysulphides, hypochlorite, and carbamate derivatives of isocyanuric acid). They emit in contact with an acid mainly H2S and Cl2. Substances with hazard statement EUH032 mainly include cyanides (other than complex cyanides such as ferrocyanides, ferricyanides and mercuric oxycyanide), thiocyanate, and sodium fluoride. They emit in contact with an acid HCN and HF, and for some products PH3 and HN3. In practice, the substances that can be present in waste in the 'normal' waste management circuits are thionyl chloride, and some hypochlorites, fluorides, cyanides, sulfides and sulfites. As mentioned, the Li-ion batteries contain thionyl chloride. Sediment and organic media can accumulate sulphides in the absence of oxygen. Some contaminants can be insolubilized as sulfides when treating effluents or chemical waste. The cyanide and fluoride are present in chemicals. Hypochlorite is present in oxidants and disinfectants. But only some salts of these anions are targeted by the CLP. There are no routine methods of analysis in waste laboratories to identify and measure substances with HSC EUH029, EUH031 and EUH032. For example, EN 15875 determines the sulphide content in waste from the extractive industries by difference between the total sulphur and sulphate, and assumes that the sulphide is pyrite (FeS2). It is more easy and cost-effective to start by gas detection: if a specific gas is not emitted, substances that can emit that gas are not present in the waste. The objective is to detect qualitatively if any EUH029, EUH031 or EUH032 substances are present in the waste, by their gas emission.

The waste materials potentially of concern (containing critical substances in waste for HP12) are chemical waste reacting with an acid and containing hypochlorite (disinfectant, swimming pool chemicals), sulphide (mining residues with pyrite, precipitated elements from cleaning of aqueous liquid waste by treatment with Na2S), cyanide (salts from metal surface treatment), sodium fluoride. It is expected that the waste that react with water and produce a toxic gas is pretreated in situ in the chemical industry and neutralized before joining the waste flux. The objective of the paper is a starting point which is tested for some materials, and needs for improvements and further studies to become a full "ready" test. MATERIAL AND METHODS

Protocol

From Table 1, it can be seen that two gases a priori very unlikely to be met (hydrazoic acid HN3 and selenium oxide SeO2a solid with low vapor pressure). The gases to search (sorted by main element) are HCl, Cl2, HF, HCN, PH3, H2S and SO2. The principle of the method proposed in this article is the following:

1 Measurement of the volume of gases emitted into contact with water or an acid (solid/liquid ratio of 10 L water or acid / kg raw waste, acid 2.5 M HNO3); 2 If gas is emitted at a rate higher than the limit of quantification (in our case 0.1 L of gas at normal temperature and pressure/kg waste in 5 minutes), checking if HCl, Cl2, HF, HCN, PH3, H2S and SO2 is emitted with electrochemical detection probes or simple qualitative colorimetric methods; 3 If one of these gases is detected, speciation of the emitting substance(s) (either by direct method if they are in high concentration or by calculation by the "worst case" method if they are in low concentrations, or by specific analytic methods) and checking whether they have a hazard statement code EUH029, EUH031 or EUH032, or alternatively checking if the waste is likely to contain substances with these HSC (with information of the acid/base and redox status of the waste, and the origin or the process that has produced the waste).

Nitric acid is proposed because it is not emitted by the substances with hazard statement codes EUH029, EUH031 or EUH032. The proposed amount of acid is in excess even if the waste is composed of 100% of carbonate. Note that the assessment of the property HP 3 "Flammable" requires testing on emission of flammable gas on contact with water (UN Test N.5). If this test has already been done and showed no release of gas upon contact with water, this information can be used for the assessment of HP 12.

In this study, started before the 2014 decision, and using a preliminary and different definition of HP 12, the gases HCl, Cl2 and HF have not been looked for. The acid used in the present study was HCl, because that acid was compatible with the (preliminary) definition of HP 12 at that time. The results are still valid for the present definition of HP 12 for the following reason. The substances with hazard statement code EUH029 may emit HCl in contact with water (Table 1), and the substances with HSC EUH031 and EUH032 don't emit HCl in contact with an acid. Since no gas emission has been observed with contact with water (see Results), HCl has not been emitted. The substances that can emit Cl2 in contact with water or an acid are disinfectants and biocides acting by oxidation, and their presence should result in a very high redox potential (not observed, [START_REF] Hennebert | Classification of waste for hazard properties according to the recent propositions of the EC using different methods[END_REF]). HF has not been monitored in the present study. In the protocol, we recommend the use of HNO3 (despite its oxidative properties), rather than H2SO4 (also oxidant, but possibly emitting SO2).

Table 1

Contact with water or with an acid, measurement of gas volume and gas identification

A method of gas volume measurement that could be relevant was estimated to be the volumetric measure of CO2 produced by carbonates in soil in contact with hydrochloric acid (ISO 10693). Some biological methods measure also gas volume but during days (i. e. ISO 11734). In this study, gas volume was first measured by an automated agricultural calcimeter (Dream électronique, France) derived from ISO 10693. Weighted samples are put into flasks of 70 ml. The apparatus delivers a volume of water or acid, closes the flask and records the pressure for 5 minutes.

Different parameters (mass, granularity, liquid/solid ratio, water and acid addition, acid normality) were preliminary tested and fixed. A good wetting and a more homogeneous temperature in case of reactive waste is obtained with L/S = 10 L (water or acid plus water) / kg (waste). Lower L/S ratios are not always providing a complete wetting of the waste, since there is no agitation. Emission of gas from waste in contact with water was always very low (< 1 mbar pressure change) (results not shown). The subsequent tests were done only with an acid.

The apparatus can inject a volume up to 7.8 ml. The test sample was fixed to 1.6 g (the largest possible with that apparatus), and 7.8 ml of water were manually added. The apparatus added 7.8 ml of 5.1 N hydrochloric acid, giving a final L/S ratio of 10 L HCl 2.5 N/kg raw waste. Three replicates were done. Trials were done in an air-conditioned lab, and temperature was kept within 20°C ± 2°C.

For measurement of gas volume for a longer time, samples were put into 0.25 l glass flask. The stopper of the flask is connected to a U-shape tube filled with water. The water or acid is poured into a small test tube and the cap closed. The test tube is inverted. The difference of water level is recorded at periodic time, and converted to a volume of released gas at atmospheric pressure, taking into account the volume of the flask, the tube and the added water or acid. Stainless steel calorimetric bombs were also used but the security membrane (that burst in case of pressure > 160 bar) was corroded in some weeks by the acid vapors.

The results are expressed per kg of dry mass for solids, and per kg of raw mass for liquids. Gas identification by electrochemical methods was done with 3 Dräger portable apparatus (A-am 5000, X-am 5600 and X-am 7000). The three detectors were connected successively to a flask where a sample was in contact with an acid. The last detector is equipped with a pump and air is allowed to flow in the flask and through the system. The concentrations are not absolute concentration values but rather indication of the nature of the gas that is/are emitted.

Waste samples

A series of 49 waste (industrial, municipal, mine residue, sediments) has been tested. These waste have been assessed for other hazard properties in a previous paper [START_REF] Hennebert | Classification of waste for hazard properties according to the recent propositions of the EC using different methods[END_REF]. Ten waste were so-called 'mirror entries' in the European list of waste (can be hazardous or nonhazardous depending on the contaminant content). No information about gas emission were known, and HP 12 was not assessed in France before the present study. The frequency of occurrence of hazard by HP 12 was not known. Solid waste with volatile substances were used without pretreatment: a portion was taken in the flask. Samples without significant volatile content were pretreated according to EN 15002 (drying, crushing at 4 mm). Liquid waste were thoroughly mixed before sampling.

RESULTS

For blanks with water, the temperature was monitored and an increase of 2°C has been observed, due to heat of dilution of the acid. In the condition of the apparatus, it produces a mean pressure increase corresponding to an apparent gas emission of 0.06 liter of gas/kg sample (Lg/kg). The detection limit of the test was then set to 0.1 Lg/kg. For a test sample of 1.6 g of liquid or dry mass (MS) of solid, and an internal volume of 287 ml, an emission of 1 L of gas per kg of waste corresponds to a variation of pressure of 6 mbar above atmospheric pressure. It has been estimated that a plateau of pressure is reached if the relative difference in absolute pressure at 2 and 5 minutes time is lower than 5%.

Emission of gas by decreasing value and classified with or without a plateau in 5 minutes time in contact with hydrochloric acid is presented at Table 2. The group that does not reach a plateau in 5 minutes (24 samples) is composed only of solid waste and have all (but one) an emission rate > 1 Lg/kg. With the small test sample of 1.6 g, the relative standard deviation can be high for low values (Table 2), and the test should be repeated several times if the results is close to or lower than 2 Lg/kg. The relative standard deviations are quite high and further developments of the method could be done with larger aliquots. A longer duration test should be used for gas emission assessment for those waste. The group that does reach a plateau in 5 minutes (25 samples) is composed of all the liquid waste plus the sediments, one sample of air pollution control residue (APC) of municipal solid waste incinerator and the sample of scrapped tires. The sediment and the APC residue contain carbonates (information from other studies). Nine of the 25 have a gas emission rate > 1 Lg/kg.

Because a longer duration test needs to be implemented in order to better assess the gas emission and also the gas identification for some waste, emission of gas for 6 hours time of contact has been measured for some waste. Calorimetric bombs and glass flasks have been used, with a mass of 1.6 g, and L/S = 10 L (water or acid plus water) / kg (waste). The emission by decreasing value and classified with or without a plateau in contact with hydrochloric acid is presented at Table 3. The emitted volumes are higher than in 5 minutes, and for most of the waste (the relative pressure difference between 6 h and 1 h is greater than 5%), a plateau is still not reached. Since the definition of HP 12 is qualitative, the fact that the equilibrium is not reached (probably due to absence of pretreatment) is not invalidating the result.

Table 2 Table 3 Table 4 The gas identification results are presented at Table 4. Most waste emits CO2 and one waste (a municipal waste fraction) emits methane. Waste that emit gas toxic (for humans) are:

-Metallic dust from the aluminum industry (CO). This waste emit also H2 in contact with an acid; -Two air pollution control of industrial incinerators residue (H2S); -Halogenated solvents (volatile alcane). Only the emission of H2S is relevant for HP 12. The substances that can emit H2S in contact with an acid, and having the HSC EUH031 (Table 1), are sulfide (of Ba, Ca, K), polysulfide (Ba, Ca, K, Na, NH4), dithionite (Na), metabisulfite (Na), others (tosylchloramide sodium, di(benzothiazol-2-yl) disulfide). For the two industrial APC residues, the Ca content is between 15 and 30%, the Na content is between 1 and 2.5%, and the content of K and Ba is lower. It can be supposed that sulfide is in the form of CaS, as Ca is dominant. Those two waste are probably hazardous by HP 12. The APC residue of industrial waste #1 is classified for HP 7, HP 10 and HP 14, and the APC #2 is classified for HP 10 and HP 14 [START_REF] Hennebert | Classification of waste for hazard properties according to the recent propositions of the EC using different methods[END_REF].

CONCLUSION

The proposed stepwise method seems quite satisfactory. Further development could be done with larger aliquots. The test is suitabel for solid and liquid waste. Most of the tested samples emit gas in contact with concentrated hydrochloric acid, but it is mainly CO2. Human toxic gases are potentially emitted by three waste: metallic dust from the aluminum industry (CO), two air pollution control residue from industrial waste (H2S) and a halogenated solvent (gas with a response to alcane detector). The two waste that emit H2S are potentially classified for HP 12 if the solid sulfide they content is classified EUH031, like by example calcium sulfide. The two waste are nevertheless already classified hazardous for other hazard properties. Given these results obtained from a large panel of inert, municipal and industrial waste (Table 2), it can be assumed that few common household and industrial waste will be classified hazardous only by HP 12.

ACKNOWLEDGEMENTS

The professional unions of waste industries SYVED and SYPRED (F) are acknowledged for supplying samples of most of the waste used in this study. This research was financially supported by Ministère de l'Ecologie et du Développement Durable, France.

FIGURES

A proposal for a test method for assessment of hazard property HP 12 ("Release of an acute toxic gas") in hazardous waste classification -experience from 49 waste Pierre Hennebert a* , Ismahen Samaali a , Pauline Molina a

Figure 1 :Figure 1 :

 11 Figure 1: Automatic calcimeter, and example of emission of gas in contact with an acid

Table 1 :

 1 Substances with hazard statement codes EUH029, EUH031 and EUH032 with molecular formula and gas emission, sorted by gas (from Table3.2 of Annex VI of CLP).

Table 2 :

 2 Emission of gas by waste in 5 minutes, sorted for the presence of a plateau and decreasing emission

Table 3 :

 3 Emission of gas by waste in 6 hours, sorted for the presence of a plateau and decreasing emission

Table 4 :

 4 Identification of gases emitted by waste (neither NH3 nor HCN were detected)

Table 1 :

 1 Substances with hazard statement codes EUH029, EUH031 and EUH032 with molecular formula and gas emission, sorted by gas (from Table3.2 of Annex VI of CLP).

						Hazard	Molecula	Gas
	International Chemical					Statement	r	emissio
	Identification	Index N° EC No	CAS No Classification	Code	formula	n
	EUH029 Contact with water
						EUH014		HCl
					Acute Tox. 4	H332		SO2
	thionyl dichloride;	016-015-	231-	7719-09-	Acute Tox. 4	H302
	thionyl chloride	00-0	748-8	7	Skin Corr. 1A	H314	SOCl2
						EUH014		HCl
					Acute Tox. 2	H330
					Acute Tox. 2	H300
		015-007-	231-	7719-12-	STOT RE 2	H373
	phosphorus trichloride	00-4	749-3	2	Skin Corr. 1A	H314	PCl3
						EUH014		HCl
					Acute Tox. 2	H330
					Acute Tox. 4	H302
		015-008-	233-	10026-	STOT RE 2	H373
	phosphorus pentachloride	00-X	060-3	13-8	Skin Corr. 1B	H314	PCl5
						EUH014		HCl
					Acute Tox. 2	H330
					STOT RE 1	H372
		015-009-	233-	10025-	Acute Tox. 4	H302
	phosphoryl trichloride	00-5	046-7	87-3	Skin Corr. 1A	H314	POCl3
						EUH014		HCl
					Acute Tox. 3	H301		SO2
					Acute Tox. 4	H332
	disulphur dichloride;	016-012-	233-	10025-	Skin Corr. 1A	H314
	sulfur monochloride	00-4	036-2	67-9	Aquatic Acute 1	H400	S2Cl2
						EUH014		HCl
					Flam. Liq. 1	H224
					Pyr. Liq. 1	H250
					Acute Tox. 4	H332
		014-001-	233-	10025-	Acute Tox. 4	H302
	trichlorosilane HCl3Si	00-9	042-5	78-2	Skin Corr. 1A	H314	HCl3Si
						EUH014		HCl
					Acute Tox. 3	H331
					Acute Tox. 3	H311
					Acute Tox. 3	H301
					STOT RE 1	H372	Cl-CH2-
		607-080-	201-		Skin Corr. 1A	H314	COCl ;
	chloroacetyl chloride	00-1	171-6	79-04-9	Aquatic Acute 1	H400	C2H2Cl2O
						EUH014		HCl
					Skin Corr. 1A	H314		(hyp.)
	3-(4-fluorophenyl)-2-	602-099-	426-		Acute Tox. 4	H302	C10H9ClF
	methylpropionylchloride	00-1	370-7	-	Aquatic Chronic 3	H412	O
					Acute Tox. 3	H331		HF
					Skin Corr. 1B	H314		(hyp.)
					Acute Tox. 4	H302
	3,5-dichloro-2,4-difluorobenzoyl	607-181-	401-	101513-	Skin Sens. 1	H317	C7HCl2F3
	fluoride	00-0	800-6	70-6	Aquatic Chronic 3	H412	O
					Water-react. 1	H260		PH3
	calcium phosphide;	015-003-	215-	1305-99-	Acute Tox. 2	H300
	tricalcium diphosphide	00-2	142-0	3	Aquatic Acute 1	H400	Ca3P2
					Water-react. 1	H260		PH3
	magnesium phosphide;	015-005-	235-	12057-	Acute Tox. 2	H300
	trimagnesium diphosphide	00-3	023-7	74-8	Aquatic Acute 1	H400	Mg3P2
						EUH032		PH3
					Water-react. 1	H260
		015-004-	244-	20859-	Acute Tox. 2	H300
	aluminium phosphide	00-8	088-0	73-8	Aquatic Acute 1	H400	AlP

Table 2 :

 2 Emission of gas by waste in 5 minutes, sorted for the presence of a plateau and decreasing emission