Valérie Forest
email: vforest@emse.fr

Agathe Figarol

Delphine Boudard

Michèle Cottier

Philippe Grosseau

Jérémie Pourchez

Adsorption of lactate dehydrogenase enzyme on carbon nanotubes: how to get accurate results about the cytotoxicity of these nanomaterials

Keywords: Carbon nanotubes, cytotoxicity, LDH assay, nanomaterial/assay interferences

Carbon nanotubes (CNT) cytotoxicity is frequently investigated using in vitro classical toxicology assays. However these cellular tests, usually based on the use of colorimetric or fluorimetric dyes, were designed for chemicals and may not be suitable to nano-sized materials. Indeed, due to their unique physico-chemical properties CNT can interfere with the assays and bias the results. To get accurate data and draw reliable conclusions, these artifacts should be carefully taken into account. The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between CNT and the commonly used lactate dehydrogenase (LDH) assay. Experiments in cell free conditions were performed and it was clearly demonstrated that artifacts occurred. They were due to the intrinsic absorbance of CNT on one hand and to the adsorption of LDH at the CNT surface on the other hand. The adsorption of LDH on CNT was modeled and was found to fit the Langmuir model. The Kads and neq constants were defined, allowing the correction of results obtained from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

Introduction:

The use of nanomaterials in general and of carbon nanotubes (CNT) in particular has incredibly increased during the last decades. Due to their exceptional physico-chemical properties CNT exhibit promising applications, in fields as varied as material sciences, microelectronics and even medicine 1,2 … This extensive use results in an increased risk of exposure for workers and consumers therefore leading to a growing need for accurate cytotoxicity assays. For that purpose in vitro cellular assays are usually used. Although insufficient to draw firm conclusions they are useful for a rapid screening of the cellular response in terms of cytotoxicity, pro-inflammatory effect, oxidative stress, genotoxicity… but only if they are conducted properly. Indeed, in nanotoxicological studies, standard toxicological assays are used but these latter were developed for chemicals and they may not be suitable for these particular nanomaterials [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Han | Validation of an LDH Assay for Assessing Nanoparticle Toxicity[END_REF][START_REF] Kroll | Current in Vitro Methods in Nanoparticle Risk Assessment: Limitations and Challenges[END_REF] . These assays use colorimetric or fluorimetric dyes as markers to determine cell viability assessing membrane integrity or cell metabolism.

Due to their high surface area, nanomaterials can interfere with the assay components or products and bias the results, potentially leading to misinterpretations. Besides, this has been proposed as a partial explanation for the conflicting results reported in the literature about the cytotoxicity of nanomaterials [START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF] and especially CNT.

For example, Wörle-Knirsch et al. [START_REF] Wörle-Knirsch | Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays[END_REF] have demonstrated that CNT could trigger false-positive results with one of the most popular cytotoxicity assay: the MTT test. This assay is based on the conversion of a yellow tetrazolium salt: the MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] into an insoluble purple formazan precipitate. This reduction is carried out by the mitochondria of viable cells and therefore the amount of formazan formed (quantified by measuring the absorbance at 570 nm) is directly proportional to cell viability. CNT have been shown to interact with the tetrazolium salts used in this test (but not with that used in variants of this assays such as the WST-1, INT or XTT assays) [START_REF] Wörle-Knirsch | Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays[END_REF] making impossible the MTT conversion into formazan. This interference was confirmed by Belyanskaya et al. [START_REF] Belyanskaya | The Reliability and Limits of the MTT Reduction Assay for Carbon Nanotubes-cell Interaction[END_REF] and Casey et al. [START_REF] Casey | Spectroscopic Analysis Confirms the Interactions between Single Walled Carbon Nanotubes and Various Dyes Commonly Used to Assess Cytotoxicity[END_REF] further demonstrated that CNT interacted, with a variable degree, with all the cell viability dyes they used in their study (i.e.

Commassie Blue, Alamar Blue, Neutral Red, MTT and WST-1), arguing for the inadequacy of these assays to report CNT cytotoxicity. Similarly, Monteiro-Riviere et al. [START_REF] Monteiro-Riviere | Limitations and Relative Utility of Screening Assays to Assess Engineered Nanoparticle Toxicity in a Human Cell Line[END_REF] tested a panel of six classical dye-based assays and found that most of them produced erroneous results.

Similarly, it was observed that nanomaterials could interfere with classical genotoxicity assays (micronucleus or comet assays), oxidative stress assay (measurement of reactive oxygen species), or with inflammatory response assays (evaluation of the production of various cytokines or mediators) [START_REF] Hirsch | A Novel Comprehensive Evaluation Platform to Assess Nanoparticle Toxicity in Vitro[END_REF][START_REF] Tournebize | Pitfalls of Assays Devoted to Evaluation of Oxidative Stress Induced by Inorganic Nanoparticles[END_REF][START_REF] Oostingh | Problems and Challenges in the Development and Validation of Human Cell-Based Assays to Determine Nanoparticle-Induced Immunomodulatory Effects[END_REF][START_REF] Herseth | The Challenge of Obtaining Correct Data for Cellular Release of Inflammatory Mediators after in Vitro Exposure to Particulate Matter[END_REF][START_REF] Stone | Development of in Vitro Systems for Nanotoxicology: Methodological Considerations[END_REF] . As an example, a previous study from our group focused on the artifacts occurring during the assessment of TNF production after a contact between macrophages and boehmite nanoparticles [START_REF] Pailleux | New Insight into Artifactual Phenomena during in Vitro Toxicity Assessment of Engineered Nanoparticles: Study of TNF-Α Adsorption on Alumina Oxide Nanoparticle[END_REF] . It was observed that 6 wt.% of TNF adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. This suggests that data from this assay can be underestimated.

The interference in biological assays are far from being systematically taken into consideration, as reported by Ong et al. [START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF] . In this paper a review of the existing nanotoxicology literature was carried out to assess whether appropriate assay controls were performed to ensure that nanomaterials did not interfere with the assays and that the results of these tests were accurate. It turned out that in 2010, 84% of papers in the nanotoxicology field used at least one type of colorimetric or fluorescence assay and of these, 95% were published without reporting controls for nanomaterial interferences. In 2012, notwithstanding the increasing number of published reports on nanomaterial-assay interferences, this rate had only marginally improved: 90% of papers were published without some type of assay control. This may explain the lack of consistency of some results reported in the literature. These interferences may be so important that they can make some commonly used tests totally irrelevant to report nanomaterials toxicity [START_REF] Wörle-Knirsch | Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays[END_REF][START_REF] Casey | Spectroscopic Analysis Confirms the Interactions between Single Walled Carbon Nanotubes and Various Dyes Commonly Used to Assess Cytotoxicity[END_REF][START_REF] Belyanskaya | The Reliability and Limits of the MTT Reduction Assay for Carbon Nanotubes-cell Interaction[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] . But as the nature and intensity of artifacts also depend on the nanomaterial type, many authors recommend that interferences should be carefully assessed for each nanomaterial type in an acellular system. In other words, the suitability of the assay for assessing nanomaterial toxicity should be examined case by case. Only this approach will allow selecting the optimal assay for different types of nanomaterials [START_REF] Han | Validation of an LDH Assay for Assessing Nanoparticle Toxicity[END_REF][START_REF] Monteiro-Riviere | Limitations and Relative Utility of Screening Assays to Assess Engineered Nanoparticle Toxicity in a Human Cell Line[END_REF][START_REF] Breznan | Non-Specific Interaction of Carbon Nanotubes with the Resazurin Assay Reagent: Impact on in Vitro Assessment of Nanoparticle Cytotoxicity[END_REF] .

In this context, we focused our attention on the cytotoxicity of CNT, in particular the loss of membrane integrity was evaluated by the commonly used released lactate dehydrogenase (LDH) quantification. The principle of this toxicological assay is illustrated in Figure 1. Briefly, the LDH released from cells in the culture supernatant catalyzes the hydrolysis of lactate in pyruvate. This reaction is accompanied by the stoichiometrical reduction of Nicotinamide Adenine Dinucleotide (NAD + ) in NADH. This latter is used as a limiting reagent in a second reaction catalyzed by the diaphorase. A tetrazolium salt: INT (2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tertrazolium) is transformed in red formazan which formation can be spectrophotometrically quantified at 490 nm and which amount is proportional to that of LDH present in the medium. But in this assay, artifacts can be due to three parameters: 1) the CNT intrinsic absorbance, 2) the interactions between CNT and reagents from the assay or 3) the interactions between CNT and LDH.

The aim of this study was to evaluate qualitatively and quantitatively the interferences occurring between the CNT and the LDH assay. To that purpose, experiments in cell free conditions were performed, and from the data obtained the adsorption isotherm of LDH on CNT was studied. This allowed taking into account the interferences occurring in the assay and consequently correcting results from cellular experiments to get more accurate data and lead to proper conclusions on the cytotoxicity of CNT.

Experimental:

 CNT: Multi-walled carbon nanotubes (MWCNT, NC7000) were provided by Nanocyl (Sambreville, Belgium). They measured 9.5 nm in diameter and 1.5 µm in length (according to the manufacturer). CNT were dispersed in culture medium: Dubelcco's modified Eagle's medium (DMEM, Invitrogen) complemented with 10%fetal calf serum (FCS, Invitrogen), 1% penicillin-streptomycin (penicillin 10,000 U.mL -1 , streptomycin 10 mg.mL -1 , Sigma-Aldrich).

Concentrations from 15 to 120 µg.mL -1 were used. A sonication was carried out until the complete dispersion of the suspension (5 min, 30 %, 3 mm probe, Branson Sonifier S-450D).

The dispersion and suspension stability were established by measuring zeta potential and particle size distribution by means of the light scattering technique (Zetasier Nano ZS). After dilutions, the suspensions were kept at 4°C and used in the following 48 h.

 LDH assay in cell free conditions:

The LDH assay was performed using the CytoTox-96  non-radioactive cytotoxicity assay kit (Promega). Within this kit a LDH solution was provided as a positive control solution. This LDH was extracted from bovine heart and its initial concentration was 0.8 U.µL -1 . U is the unit of enzymatic activity, defining the speed of consumption of a substrate by unit of time.

For example, in the LDH case, 1U corresponds to the oxidation of 1 mM.min -1 of NADH. A protocol to assess the interferences in the determination of the LDH release was established in cell free conditions. The absorbance of solutions containing LDH alone (concentrations from 0.00005 to 0.004 U.µL -1 ), CNT alone (concentrations from 15 to 120 µg.mL -1 ) or a mixture of LDH and CNT was assessed using a microplate reader (Multiskan GO, Thermo Scientific) set at 450 nm. Negative controls were also included (solutions with no LDH or no CNT).

 LDH assay in cellular conditions:

The RAW264.7 cell line, commonly used in nanotoxicology studies was provided by ATCC Cell Biology Collection (Promochem LGC) and derived from mice peritoneal macrophages transformed by AMLV (Abelson Murine Leukemia Virus). Cells were cultured in complemented DMEM and incubated at 37°C under a 5% carbon dioxide humidified atmosphere. Cells were seeded in a 96-well plate (100,000 cells/well) and were incubated for 24h with CNT (concentration ranging from 15 to 120 µg.mL -1 ). Cell viability was then evaluated by quantifying the LDH released in culture supernatant from cells with damaged membranes, using the CytoTox-96  non-radioactive cytotoxicity assay according to the manufacturer's instructions. Detection was performed using a microplate reader (Multiskan GO, Thermo Scientific) at 450 nm. The activity of the released LDH was reported as a percentage of the total cellular LDH (measured after the complete lysis of control cells corresponding at the maximal amount that can be released by cells, therefore 100%).

 Expression of the results and statistical analysis:

Results are expressed as means of 3 independent experiments, with each measure performed in triplicate. Statistical analysis was carried out using a Student test. Difference was declared significant when p<0.05.

Results:

 Cell free condition assays:

The absorbance at 450 nm of solutions containing various concentrations of LDH (from 0 to 0.004 U.µL -1 ) was measured in presence of various concentrations of CNT (from 0 to 120 µg.mL -1 ). Results are reported in Figure 2. It clearly appeared that CNT alone induced a dose-dependent increase in absorbance at 450 nm. The addition of low doses of LDH (0.00005 or 0.0005 U.µL -1 ) did not influence this increase. But with higher concentrations of LDH (0.001 or 0.004 U.µL -1 ) a peculiar behavior was observed: solutions containing 30 or 60 µg.mL -1 CNT exhibited an absorbance significantly lower than that containing 15 or 120 µg.mL -1 and even than that without CNT. No CNT

[CNT] = 15 µg.mL⁻¹

[CNT] = 30 µg.mL⁻¹

[CNT] = 60 µg.mL⁻¹

[CNT] = 120 µg.mL⁻¹

1. An optical artifact occurred, induced by the partial extinction of light due to the diffusing nature of CNT.

2. An artifact due to a variation of the LDH concentration consecutive to its adsorption at the CNT surface might be implicated.

3. A combination of the two above-mentioned artifacts occurred.

To test the first hypothesis, the absorbance of CNT alone was further investigated. CNT were diluted either in PBS (Phosphate Buffered Saline) or in culture medium in presence of the reagents from the assay, and the absorbance was measured as indicated in Figure 3. where A represents the absorbance, ε the molar extinction coefficient (L.mol -1 .cm -1 ), l the length of the optical path travelled by the light beam (cm) and C the concentration (mol.L -1 ).

Similarly, the absorbance of LDH alone follows a linear relationship (y = 942.1x + 0.3239; R²=0.9997, see Supplementary Figure 1).

In theory, the absorbance of a mixture of two solutions is equivalent to the sum of the absorbance of each solution. In other words, we should observe that ACNT+LDH = ALDH + ACNT.

However, this was not the case: the absorbance of the solution containing both CNT and a known concentration of LDH was lower than that of the control solution containing only LDH, without CNT. An example (for the 0.001 U.µL -1 LDH concentration) is detailed in Figure 4 but the same conclusion was reached for each LDH concentration as reported in Supplementary Figure 2. [LDH] = 0.004 U.µL -1

[LDH] = 0.001 U.µL -1

[LDH] = 0.0005 U.µL -1

[LDH] = 0.00005 U.µL -1

Equation 2

where  free LDH represents the amount of LDH free in solution (mol),  theoretic LDH is the theoretic amount of LDH in solution (i.e. the introduced amount of LDH, in mol),  theoretic LDH is the awaited absorbance of the LDH solution (calculated by the Beer-Lambert law, Equation 1),

A LDH+CNT the measured absorbance of the solution containing both LDH and CNT and A CNT the absorbance of the solution with CNT only.

The amount of LDH adsorbed on the CNT can then be deduced simply by subtracting the amount of LDH free in solution (and that has formed the colored INT formazan) from the initially introduced LDH, as reported in Figure 5B. [LDH] = 0.004 U.µL -1

[LDH] = 0.001 U.µL -1

[LDH] = 0.0005 U.µL -1

[LDH] = 0.00005 U.µL -1

B)

Figure 5: A) Free LDH depending on the CNT concentration calculated for different LDH concentrations (concentration from 0.00005 to 0.004 U.µL -1 ). B) LDH adsorbed on CNT calculated for different LDH concentrations (concentration from 0.00005 to 0.004 U.µL -1 ).

The thermodynamic equilibrium was reached leading to the determination of the adsorption isotherm. Figure 6 represents this adsorption isotherm, i.e. the amount of adsorbed LDH by mass of CNT depending on the amount of free LDH reported by mass of CNT. The same conclusion was reached when the data were expressed depending on CNT surface instead of CNT mass (Supplementary Figure 3). The results confirmed the existence of a bias due to the adsorption of LDH on CNT, providing an explanation for the difference observed between the theoretical absorbance of a mixture of CNT and LDH and the absorbance experimentally measured (Figure 4). where n ads corresponds to the amount of proteins adsorbed by mass of solid, n eq the maximal amount of proteins adsorbed by mass of solid, K ads the adsorption constant, n free the amount of free protein.

Here, the equation is adapted to normalize the data by the mass of CNT: Rigorously, it should be specified that although the Langmuir model proposed allows a satisfactory mathematical fit of the adsorption data, the basic assumptions of the model were not fully verified. Indeed, we did not check if all conditions implied in the Langmuir model were met (e.g. the reversibility of adsorption, the monolayer coverage, the site-binding model were not demonstrated). But the understanding of the adsorption mechanism was beyond the scope of this study and it was observed that our experimental data mathematically fitted best with the Langmuir model. Moreover, other models (Hasley, Temkin-Pyzhev, Freundlich) were tested but the results were not satisfactory as illustrated in Supplementary Figure 5.

Finally, the actual concentration of LDH in solution can be calculated by combining the following equations: Langmuir model with and keeping in mind that was already calculated: Equation 7It is therefore possible to determine the actual LDH concentration in solution after the calculation of the adsorption constants as determined by a cell free assay.

 Correction of data from cellular assays:

This approach was adopted to correct results from previous experiments, in which macrophages from the RAW264.7 cell line were incubated with CNT for 24 hours and the LDH release was assessed using the same assay to detect the membrane integrity loss [START_REF] Figarol | Biological Response to Purification and Acid Functionalization of Carbon Nanotubes[END_REF] .

Figure 8 compares the values before and after correction. The differences ranged from 9 to 12% depending on CNT concentration. Only the highest concentration of CNT triggered a LDH release significantly different from that of the control but the same conclusion was reached either with or without correction. Even though the correction did not change the conclusion of the assay in this particular case, it clearly appeared that data were underestimated. Results are more accurate and trustable after correction.

Discussion:

Current in vitro toxicity tests used for nanomaterial risk assessment rely on standard viability assays that were developed for chemicals or macro-scale substances and that are frequently based on absorbance or fluorescence measurements. However, nanomaterials display unique physicochemical properties that can interfere with or pose challenges to the use of classical

LDH release (%)

Before correction of the bias After correction of the bias toxicity assays [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] . They can even lead to false-positive or false-negative results resulting subsequently in misinterpretations. Nanomaterial interferences with toxicity assays should not be neglected as they have been reported for most spectroscopic analyses [START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF] .

Possible interactions from particles are: 1) particle optical properties that interfere with light absorption or fluorescence used for detection, 2) chemical reactions between the particles and assay compounds, and 3) adsorption of assay molecules to the particle surface [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] .

In this study focused on the cytotoxicity of CNT as determined by the LDH release assay, we clearly demonstrated an optical artifact (due to the intrinsic absorbance of the CNT) resulting in an over-estimation of the LDH release as well as a chemical bias (due to the adsorbance of LDH on the CNT) leading to an under-estimation of the LDH release. Therefore in this particular case the two kinds of artifacts seem to compensate and the final result has a poor impact on the conclusion of the toxicological assay as illustrated by the comparison of the results before and after correction of the bias (Figure 8). But it may not be always the case and the interferences should not be neglected but on the contrary should be carefully considered.

Our results are in agreement with those of Wang et al. [START_REF] Wang | Understanding and Correcting for Carbon Nanotube Interferences with a Commercial LDH Cytotoxicity Assay[END_REF] who observed a decrease in the absorbance at 490 nm of LDH solutions with increasing CNT concentrations. Our observations are also concordant with those of Breznan at al. [START_REF] Breznan | Non-Specific Interaction of Carbon Nanotubes with the Resazurin Assay Reagent: Impact on in Vitro Assessment of Nanoparticle Cytotoxicity[END_REF] who, although using another detection system (the fluorescent resorufin), reported that CNT could decrease the fluorescence signal through optical interference. This effect was not surprising as CNT are highly optically dense materials that prevent transmitted/emitted light from reaching the detector as already reported by Oostingh et al. [START_REF] Oostingh | Problems and Challenges in the Development and Validation of Human Cell-Based Assays to Determine Nanoparticle-Induced Immunomodulatory Effects[END_REF] . This quenching of fluorescence by CNT was also reported by others [START_REF] Casey | Spectroscopic Analysis Confirms the Interactions between Single Walled Carbon Nanotubes and Various Dyes Commonly Used to Assess Cytotoxicity[END_REF][START_REF] Monteiro-Riviere | Limitations and Relative Utility of Screening Assays to Assess Engineered Nanoparticle Toxicity in a Human Cell Line[END_REF] whereas other nanomaterials such as quantum dots or C60 fullerene did not interact with the resorufin fluorimetric assays [START_REF] Breznan | Non-Specific Interaction of Carbon Nanotubes with the Resazurin Assay Reagent: Impact on in Vitro Assessment of Nanoparticle Cytotoxicity[END_REF] . Alternatively (and sometimes additionally) the bias could originate from a physical interaction between the fluorescent/colorimetric dye and the CNT. Indeed, it is now well admitted that nanomaterials, due to their large surface area, can physically adsorb the assay components (e.g. substrates, dyes…) or the molecule to dose [START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF][START_REF] Jones | In Vitro Assessments of Nanomaterial Toxicity[END_REF] . In this case, nanomaterial characteristics (such as hydrophobicity, surface charge…) are expected to play an important role in the adsorption process [START_REF] Kroll | Current in Vitro Methods in Nanoparticle Risk Assessment: Limitations and Challenges[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] .

The lack of sensitivity and reliability of existing methods clearly argue for the urgent need for the development of alternative and validated assays, best adapted to nanomaterial specificities as supported by many authors [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Kroll | Current in Vitro Methods in Nanoparticle Risk Assessment: Limitations and Challenges[END_REF][START_REF] Casey | Spectroscopic Analysis Confirms the Interactions between Single Walled Carbon Nanotubes and Various Dyes Commonly Used to Assess Cytotoxicity[END_REF][START_REF] Stone | Development of in Vitro Systems for Nanotoxicology: Methodological Considerations[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF][START_REF] Wang | Understanding and Correcting for Carbon Nanotube Interferences with a Commercial LDH Cytotoxicity Assay[END_REF][START_REF] Jones | In Vitro Assessments of Nanomaterial Toxicity[END_REF] or as demonstrated by international efforts to discuss and determine which protocols might be standardized [START_REF] Stone | Development of in Vitro Systems for Nanotoxicology: Methodological Considerations[END_REF] . Recently, some new methods and modified versions of pre-existing methods have been developed for assessing the toxicity of nanomaterials [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF] . One strategy may be to limit the interferences. To that purpose, Ali-Boucetta et al. 22 have chosen to assess the intracellular LDH instead of the extracellular LDH to avoid the interactions between CNT and the components used in the test. After incubation with cells, CNT are rinsed off from the supernatant and the impact on the absorbance measure should therefore be eliminated. Cells are then lysed, centrifuged, and the amount of LDH present in the supernatant is determined reflecting the relative amount of viable cells instead of the amount of damaged cells as determined with the classical LDH assay. However, this approach has some limitations for instance it is difficult to rinse off all CNT, some remaining attached to cell membranes and some potentially uptaken in the cytoplasm [START_REF] Monteiro-Riviere | Limitations and Relative Utility of Screening Assays to Assess Engineered Nanoparticle Toxicity in a Human Cell Line[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] . It may therefore be technically difficult to assess the bias and potentially not completely reliable. Moreover, particle induced artifacts are still controversial, especially they are thought to depend on the type of CNT considered. This is why the systematic consideration of the interferences in the CNT cytotoxicity assays remains rare [START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF] .

Another way to mitigate the interactions between nanomaterials and CNT consists in limiting nanomaterials concentrations below levels that interfere or by adding compounds such as FBS which can decrease the adsorption of cytokines to particles [START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] .

As an example of alternative method, Wang et al. [START_REF] Wang | Understanding and Correcting for Carbon Nanotube Interferences with a Commercial LDH Cytotoxicity Assay[END_REF] propose a variant in the LDH assay, they recommend to measure absorbance at 580 nm instead of 490 nm, as they observed that while the broad 490 nm absorbance peak was decreased in intensity with increasing CNT concentrations, the smaller absorbance peak at 580 nm also varied, but considerably less.

Although reducing the bias, this solution remains imperfect as it does not eliminate the interferences.

Until methods that avoid nanoparticle interferences have been fully developed, it appears necessary to be very cautious when using usual cytotoxicity assays not specifically designed for nanomaterials. One should be aware of their limitations and should try to understand how nanomaterials might interfere in the toxicological assay and if it is possible should quantify these biases. Indeed, if the interference is well characterized for the exposure conditions, a correction factor may be applied to the results [START_REF] Stone | Development of in Vitro Systems for Nanotoxicology: Methodological Considerations[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF] . To that purpose the evaluation of adsorption isotherms may be useful. But, it should be kept in mind that the adsorption of LDH on CNT is very likely to depend on the CNT physico-chemical features, especially the structural defect rate and the surface chemistry. Thus, adsorption isotherms should be calculated for each type of CNT.

To summarize, most researchers are now aware of the nanomaterial interference issue and many of them have emitted recommendations:

 the compatibility of an assay must be verified for each type of nanomaterial, in other words, it must be checked that the nanoparticles under investigation do not interfere with a specific assay at the expected concentrations. And to that purpose relevant controls must be used [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF][START_REF] Wörle-Knirsch | Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays[END_REF][START_REF] Belyanskaya | The Reliability and Limits of the MTT Reduction Assay for Carbon Nanotubes-cell Interaction[END_REF][START_REF] Stone | Development of in Vitro Systems for Nanotoxicology: Methodological Considerations[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF][START_REF] Wang | Understanding and Correcting for Carbon Nanotube Interferences with a Commercial LDH Cytotoxicity Assay[END_REF][START_REF] Jones | In Vitro Assessments of Nanomaterial Toxicity[END_REF] .

 if interferences are found to occur with an assay and if alternative systems are not available the interferences should be limited (for example by using low concentrations of nanomaterials as the highest the concentration, the greater probability of interferences).

Alternatively, interferences should be carefully characterized to be taken into account.

Artifacts should be quantitatively evaluated and a corrective factor may be applied to the data [START_REF] Ong | Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing[END_REF][START_REF] Holder | Particle-Induced Artifacts in the MTT and LDH Viability Assays[END_REF][START_REF] Breznan | Non-Specific Interaction of Carbon Nanotubes with the Resazurin Assay Reagent: Impact on in Vitro Assessment of Nanoparticle Cytotoxicity[END_REF][START_REF] Wang | Understanding and Correcting for Carbon Nanotube Interferences with a Commercial LDH Cytotoxicity Assay[END_REF][START_REF] Jones | In Vitro Assessments of Nanomaterial Toxicity[END_REF] .

 two or more independent test systems should be performed to validate the results before concluding on the nanomaterial toxicity [START_REF] Dhawan | Toxicity Assessment of Nanomaterials: Methods and Challenges[END_REF][START_REF] Kroll | Current in Vitro Methods in Nanoparticle Risk Assessment: Limitations and Challenges[END_REF][START_REF] Wörle-Knirsch | Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays[END_REF][START_REF] Jones | In Vitro Assessments of Nanomaterial Toxicity[END_REF] .

 new or adapted methods must be developed to get new standards for nanoparticle risk assessment [START_REF] Kroll | Current in Vitro Methods in Nanoparticle Risk Assessment: Limitations and Challenges[END_REF] .

Conclusion:

Interferences occur in the LDH assay due to the intrinsic absorbance of CNT on one hand and to the adsorption of LDH at the CNT surface on the other hand. Even if the final artifacts are low, a correction is necessary to be rigorous. Further investigations with other kinds of nanomaterials are required to better understand this phenomenon and to adapt the correction to each case. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1 :

 1 Figure 1: Schematic representation of the principle of the LDH release assay. The LDH assay

Figure 2 :

 2 Figure 2: Absorbance at 450 nm of solutions containing CNT and LDH (acellular condition). *

From

  these observations three assumptions could be made:

Figure 3 :

 3 Figure 3: Absorbance at 450 nm of CNT diluted in culture medium in presence of the kit

Figure 4 :

 4 Figure 4: Difference between the theoretical and experimental absorbance of a LDH solution

Figure 6 :

 6 Figure 6: Adsorption isotherm of LDH on CNT for different LDH concentrations. Results

Figure 7 :

 7 Figure 7: Adsorption isotherm of LDH on CNT fitting with the Langmuir model.

Figure 8 :

 8 Figure 8: LDH release expressed as a percent of the positive control (lysed cells) determined

( 22 )

 22 Ali-Boucetta, H.; Al-Jamal, K. T.; Kostarelos, K. Cytotoxic Assessment of Carbon Nanotube Interaction with Cell Cultures. Methods Mol. Biol. Clifton NJ 2011, 726, 299-312.Supporting information available.Five supplementary figures illustrate respectively: 1) the standard curve of absorbance at 450 nm of LDH solutions depending on the LDH concentration, 2) the absorbance of solutions containing CNT and LDH depending on the CNT concentration, 3) the adsorption isotherm of LDH on CNT for different concentrations of LDH reported by surface of CNT, 4) the linear regression for the determination of the parameters of the Langmuir model and 5) the adsorption isotherm of LDH on CNT with the Hasley, Temkin-Pyzhev and Freundlich models.

Equation 4

The equation is rearranged assuming that . Consequently, .

By replacing this constant in the previous equation, the following expression of the Langmuir equation is obtained: Equation 5and n eq are determined using a linear regression: Equation 6From the plot, reported in Supplementary Figure 4, it was determined that and therefore =0.07 and =30.14.

This fitted with the adsorption isotherm reported in Figure 6, as illustrated in Figure 7. [LDH] = 0.001 U.µL -1

[LDH] = 0.0005 U.µL -1

[LDH] = 0.00005 U.µL -1

Supplementary Figure 4: Linear regression for the determination of the parameters of the Langmuir model.


Fabien Jobard
email: fabjob@cesdip.fr

Police Autonomy

The police usually enjoys a great amount of autonomy. One must distinguish between two levels of autonomy. The first level (micro-level) is called police discretion, which is used by the rank-and-file officers. Daily police matters call for quick if not split-second decisions by the individual officers on the beat, like bringing someone to the police station, interrupting an apparent source of disorder, following or abandoning the task given by his or her superior in order to help someone on the field. The second level (macro-level) is the autonomy exercised by the police as an institution. This autonomy is conceptualized in relation toward the authorities (usually political or judicial authorities), which give the police their mandate. With respect to these two levels of autonomy, crowd control operations are specific in that the first level of autonomy has been vanishing more and more in the course of the militarization process that characterizes the maintenance of order in most contemporary democracies. Individual officers are expected not to act on their own initiative: today, almost no discretion is tolerated in the context of highly professionalized forms of protest policing, even in the cases of self-defence.

Two different conceptions of police autonomy are at stake. In common law countries, the key notion is the police chief's "independence" in all law enforcement operations, specifically in maintaining order. This was once expressed by the Royal Commission on the Police in Britain in 1962, and re-affirmed in what [START_REF] Stenning | The Idea of the Political 'Independence' of the Police: International Interpretations and Experiences[END_REF] calls the « most oftquoted statement of the doctrine in Britain, Canada, Australia, New Zealand »: the « Blakburn doctrine », which states that « [t]he responsability for law enforcement lies on him [the chief constable]. He is answerable to the law and to the law alone ». An opposite conception can be found in "continental" countries, especially as far as protest policing is concerned. There, the police is seen as the long arm of the government. For instance, in France, the central government handles the police operations during protests, riots or collective violence through its local representatives, the préfet. The government's préfet give the orders and police commissioners receive and implement these orders on the field.

It is true that in both protest policing regimes, the government's intervention in protest policing operations can lead to large-scale political scandals. Nevertheless, the public response is different in each system. The Canadian Prime Minister's interference with the police operations during the APEC meetings held in Vancouver in 1997 lead to the formation of a commission which deplored that the police had "succumbed to government influence and intrusion". In France, events like the massacre of more than one hundred peaceful Algerian protestors in 1961, the massacre of communist protestors the year after, or, more recently, the debates over police provocation through over-intervention or nonintervention never generated any public discussion on the police subordination to the government's stakeholder (the préfet or the ministry of Interior), but only with regards to the intents or the final results of the political intervention. Policing protest is always in France government's matter: No parliamentary, independent or judicial commission ever investigated such issues, which simply remained part of the political debate between the government, its opposition and the voters.

These two opposite notions of police autonomy actually rely more on different patterns of policing than on a strict division between continental law and common law systems. Countries like New Zealand or Northern Ireland have, like France, a centralized police force headed by the government, and the notion of "police independence" appears almost fictitious there.

One should, however, not overestimate the size of the differences between the two police systems. First, operational autonomy is not the only one at stake. Resources, nominations, promotions, working conditions, transactions with police unions, as well as general police policies are always defined by the government or the regional or local political authority, which therefore has indirect control capacities. Facing a political protest, police chiefs keep that in their minds. From this point of view, recent changes in police policies tend to increase central controls, via fiscal constraints, audits, new public management doctrine in the organisation of the police. Moreover, the focus on police agencies by "law-and-order" governments favours the intermingling of government interests and police matters. The uniformization of management guidelines and the growing importance of police issues in contemporary democracies, contribute to erasing the differences between the two police systems outlined in the beginning.

The second reason why the differences between the two systems are less clear-cut hinges on the fact that national police organisations display their own (sub-)cultures and collective identities. A universal aspect of police subculture seems to be that it favours more repressive orientations towards groups identified as specifically weak or threatening, like racial minorities, foreigners, students, urban youths, prostitutes, etc. This kind of police autonomy is the source of a police-centred approach introduced by many scholars of social movements, which examine institutional factors in how the police is handling protest. From this perspective, the problem is no longer the dependence of police chiefs on government or politicians, but, on the contrary, the lack of adaptiveness of police organisations vis-à-vis political change. This can be illustrated with the difficult transition from escalation to negotiated management strategies. During some periods (at the end of the 1980s and again since the end of the 1990s), the local political government in Berlin (Germany) has tried to discipline the inclination of the local police force towards repressive tactics against left-wing and anarchist 1 st of May demonstrations, and finally succeeded in it. In contrast, police autonomy was so strong in France during the 1950s-60s that the government's only hope were damage control strategies in order to limit the abuse of force by order maintenance units in Paris. The police's power even went so far that many local politicians in Paris and its surroundings, and also many civil servants in charge of immigration and colonial issues were former police chiefs back from Algeriawhich reverses the question of police independence and political influence.

SEE ALSO:


Fabien Jobard

Chapter 11 POLICE DEVIANCE IN FRANCE

France holds a somewhat unique place in the European political space when seen through the prism of the police institution. France shares with Turkey the dubious distinction of having been twice censured for "torture" by the European Court of Human Rights (in view of the European Convention on Human Rights). Moreover, these two condemnations did not occur during as disturbed a period as, for example, the Algerian war of independence , but consecutively in 1999 and 2002 (in the cases of Selmouni vs France and Mouisel vs France).

. Our intention here is to try and assess the extent of police deviance, apart from its articulation as a "public problem". Our aim is to try and "measure" acts whose primary characteristic is their very opacity and considerable difficulty in enumerating them; the same applies to other deviant phenomena (whether criminally reprehensible or not), but often for different reasons, such as white-collar crimes, sexual delinquency, consumption of alcohol or cannabis, etc.

In order to do this, we will demonstrate the considerable difficulty created by the problem of measurement through the medium of a conflict which for some years put the French government at odds with one of the institutions of the Council of Europe. This will help us, in passing, to get a better idea of the magnitude of the problem of police brutality.

Then we will examine the problem of discriminatory practices on the part of the police in France because the problem of police violence is difficult to dissociate from police interactions with ethnic minorities.

I -Counting violence committed by the police: raw facts and narratives

"A non-negligible risk of being mistreated": this is the conclusion reached by the European Committee for the prevention of torture and inhuman or degrading punishment or treatment (CPT) following its visit to France (from October 27 to November 8, 1991) to determine the probability of occurrence of improper use of force by the police. The CPT is an independent organization created by the signatory States of the Council of Europe to ensure the effective enforcement of paragraph 3 of the European convention of human rights ("No one may be subjected to torture or to inhuman or degrading punishment or treatment"). The CPT has the rare privilege of being allowed to make surprise visits to police stations and on the basis of its first-hand testimony, to challenge the government. Whereas associations such as Amnesty International must rely on the publicly available press as their source of information, the members of the CPT have complete freedom to uncover actual practices, behind the clauses in official documents on the legal use of force.

"A non-negligible risk of being mistreated". This sounds like both an evaluation and a warning: like a value judgment (a moral judgment) and a factual judgment (a scientific judgment). Is the approximation "non-negligible" a sufficient expression of the gap between recorded facts and those acts that are unknown but believed to exist ? The CPT assessment raises the issuer of its particular way of evaluating the probability that unlawful violence is exerted by some law enforcement officers. Let us look at how this judgment was generated: what are its foundations ? Next, we will follow the conflicts of interpretation between the French government, opposed to the verdict, and the CPT, which maintained it. What are the rules governing the calculations of probability used on each side ? Finally, we will attempt to determine whether the two views may be reconciled, and at what cost. '(...) The delegation heard a considerable number of allegations of more or less serious mistreatment inflicted on individuals in police custody. The allegations voiced mainly accused the police. They included: punching and slaps; hitting on the head with a phone book; psychological pressure; insults; deprival of food and medication (...). The existence of this type of mistreatment was corroborated by several reliable sources. To illustrate this, the CPT mentions the case of a woman drug abuser seen during its visit to the Marseille-Baumettes prison who had allegedly been beaten during questioning by the police early in 1991. Her medical record showed that when she arrived at the prison she was heavily bruised and presented hematomas consonant with her allegations. The woman claimed to have filed a complaint. (...) The CPT was led to the conclusion that a person taken into custody by the police runs a non-negligible risk of being mistreated' (CPT 19 jan. 1993, 15).

-Probabilistic vs. judicial approaches in counting police violence cases

Here, then, are the grounds on which the CPT founded its evaluation of the risk of unlawful violent acts: "a considerable number of allegations" (...) "corroborated by reliable sources" and by one testimony cited "to illustrate this".

The French government made its response known promptly. Citing the conditional terms used by the Committee (the lady "had allegedly been beaten" and "claimed to have filed a complaint") it recalled that a duly founded judgment requires proof, which distinguishes it from a simple slanderous charge. It was therefore opposed to this formulation. In its opinion, the guarantees surrounding the use of force by police officers in France, and especially the code of ethics of the National Police department (1986) and the Declaration of human and citizen"s rights (1789) considerably reduce the risk of mistreatment. Mistreatment is subject to preventive action defined by official texts, thanks to which it is quite infrequent, and the French government stressed the small number of allegations of violent acts reported to the board of control and disciplinary action of the national police force (at this time, about 250 to 300 annually) in France. Consequently, the government did not feel obliged to take action against this supposed risk:

'The Committee does point out that the expression 'non-negligible risk' was intentionally used instead of the expression 'serious risk' employed for other countries, so as to relativize its conclusion; nonetheless, the minute number of cases of this type brought to the knowledge of the judicial authorities in comparison with the total number of officers of the police and gendarmerie and the number of individuals held in police custody necessarily further accentuates this relativization. Moreover, for lack of details on the elements on which the Committee bases its assertion, the French government is unable to provide any valid response, and this leads it to express its reservations with respect to this assessment, which should be more explicit and given detailed discussion, at the very least' (CPT 19 jan. 1993, 4).

The conflict between the European Committee and the government went on for months and years in the same terms. The government waited for the Committee to furnish proof that the woman had definitely been the victim of materially identifiable assaults and that similar assaults had been observed on a sufficient number of victims for the expression "nonnegligible risk" to be attested by material facts. In response, the Committee could only brandish its conviction of the probability of all sorts of other unlawful forms of assaults on the body or breaches of the dignity of individuals held in police custody. In its last follow-up report on the 1991 inspection, the government draws the following conclusion: "The French government notes that the Committee has still not given it the means to provide concrete evidence so that it may defend itself against allegations which continue to be formulated in very elliptic terms" (CPT 17 feb. 1994).

A charge that is not supported by any element of proof is an allegation, and an unlawful one if it insults the honor or the dignity of the person concerned. The French government, accused by the CPT, asserts the need for proof as a guarantee that individuals are accountable and responsible for their acts. It refuses, and rightly so, to conceive of a charge unless an act has been committed. And the charge must then involve a duly recorded offense, and not a probability adducing a risk. This defines the gap between two judgments on a particular state of affairs: the illegitimate use of force by police officers in France. On the one hand, the government refuses anything other than recorded facts. On the other hand, the European Committee wishes to establish probabilities.

-Inference from data sampling

This disagreement led the CPT to refine its evaluation tools. During its visit in 2000, in particular, it went to the Paris medical/judicial emergency unit. "Of the 2,980 individuals brought there by the police between December 1999 and January 2000, 137 showed injuries of traumatic origin and at least 39 showed injuries (hematomas, bruises, scratches, fractures) compatible with their allegations of mistreatment by the police". The report actually added "that a significant number of imprisoned individuals seemed frightened and refused to explain the causes of their injuries". By concentrating its investigations on this unit in charge of providing first aid for individuals taken in by the police in Paris, the Committee proceeded by pre-selection, thus obtaining a double zoom effect. Firstly, it focused on a period coming immediately after interaction with the police, thus leaving little time for the different parties to advance any post hoc reconstructions. The Committee thus was able to correct the temporal distance separating it from the time when the individual was taken in. Secondly, it investigated individuals defined by two specific features: they had been victims of violence, and had been taken in by the police. This gave the Committee the possibility of learning that a non-negligible number of individuals taken in by the police impute that violence, precisely, to their interaction with the police.

A non-negligible number? Let us return to the precise figure for complaints filed for "unlawful violence": between 250 and 300 annually in France (289 in 1995France (289 in , 269 in 1996)).

There is undeniably a difference here. If we extrapolate from the CPT findings (39 physical problems compatible with the allegations of unlawful violence over a two-month period in Paris and the three surrounding suburban districts, which all gather a bit less than 10 percent of French population), and if we set aside the number -unspecified, of course, but "significant" -of individuals too "frightened" to make such allegations, we may estimate that over the year, for these four districts, approximately 240 individuals showed bodily damage compatible with their allegations of unlawful violent acts. We are far from the 250-300 complaints filed in all of France at the time of the visit of the Committee. But we are even farther from the number of substantiated cases (by criminal courts or by the internal affairs) at that time, for around 80 percent of the alleged cases were dismissed by the judiciary. The risk of having a complaint filed for unlawful violence result in dismissal of one sort or another is therefore considerable in the case in point.

It is these various aspects specific to allegations of unlawful violence that must be pieced together. On the one hand, according to the CPT, "a non-negligible risk of being mistreated". According to the government, a small number of filed complaints (250-300) and an almost negligible number of substantiated acts (20-25). On the other hand, a number found for individuals who are victims of violence and taken in by the police (for Paris only) and whose allegations are made plausible by the immediate visibility of their bodily injuries and the fact that the action had taken place recently, which is five to six times as high as the number of proved cases established by the justice system for the entire country.

It is precisely by following the repeated exchanges between the French government with its insistence on convincing proof and the CPT with its desire to go beyond appearances that we may identify the line that separates light from shadow, the visible from the invisible.

The decisive gap, of course, is the difference between the twenty-odd proved cases and the 500 plausible ones, which only represent a bottom estimation, since they do not consider frightened individuals who are afraid to testify, and are restricted to four districts (France"s administrative system encompasses 100 districts). If we examine the distinguishing features between those cases of unlawful violence that remain in the shadows and those that come into the limelight, those that manage to achieve recognition by the justice system or by public opinion via the press, we find that the latter are subjected to serious constraints on the way.

In so doing, we can also provide some idea of scale. Firstly, the expression "police violence" or "unlawful violence" refers to the overwhelming majority of cases of the type of violence noted by CPT inspectors during the nights spent at the Paris high security ward where injured or sick persons under police custody are kept. Cases of death are rare; by this we mean that for the past 25 years there have been some ten cases of death per year [START_REF] Jobard | Bavures policières ? La force publique et ses usages[END_REF]. For the rest, mistreatment mainly consists of blows, probably inflicted when the individual was being arrested rather than when he was in police custody, although unfortunately we do not have sufficient proof of this. This assessment is made partly on the basis of personal observations in the spring of 2004 during some 250 hours of participation in police activities in so-called "sensitive urban areas" (Jobard, 2006a(Jobard, , 2006b)). It is also based on the fact that since the law of June 15, 2000 (prepared by the law of January 24, 1993), the lawyer of the person held in police custody has right of access in the very first hour of detention of the accused at the police station and that all remarks he considers important are forwarded to the prosecutor responsible for both overseeing the conditions of police custody and the investigation into the actions of the person taken for questioning. Typically, we would thus have the report of the physician who has to pay a visit to the individuals in police custody, and note the non-negligible neuropathic compression caused by the application of handcuffs. Indeed, as the authors say, "the frequency of these complications is unknown [but] twelve of 190 (6.3 percent) consecutive subjects kept in police custody presented distal neurological symptoms related to handcuff application" [START_REF] Chariot | Focal neurological complications of handcuff application[END_REF].

Generalizing by extrapolating data collected during surveys and applying it to the whole of French territory is difficult. The four "Parisian" districts represent 10 percent of the population, but are at the same time those where police strength is the highest (about a quarter of the total number of police officers in France). Are we to imagine that there are about 4,000 incidents of police excess occurring every year which necessitate moving persons to a medical facility ? If we are to continue this extrapolation, this (speculative) figure should be compared to the number of individuals in police custody reported in France, approximately 470,000 in 2004, to conclude that one in one thousand detainees in France needs medical intervention connected with the circumstances of questioning or, much rarer no doubt, of police custody. Let us however remember that these figures are anyway extrapolations and, as such, they should be treated with caution. They simply help to specify the scale of things.

II -Police and discrimination

We now have to examine a second point. One of the reasons for the fear that forces people interrogated in the Paris hospital by the CPT to remain silent, is the presence in their midst of strangers or people of foreign origin. The report adopted by one of the monitoring bodies of the French police, the Commission Nationale de Déontologie de la Sécurité (CNDS, 2005, p. 496see further) states that a "large nucleus" of complainants of police mistreatment (total number = 36) 1 are "youth of 18 to 35 years of Maghrebian migrant origin, called in for questioning in the disadvantaged Paris suburbs" (the four districts mentioned earlier on). It is this issue of discrimination or discriminatory practices that we have to now examine.

The question of relations between ethnic minorities and the police has only become a "public issue" in the last twenty years, even though police action in urban residential zones in which migrant workers are to be mostly found, especially Maghrebian workers, was much more violent during the entire period of the Algerian war and subsequent years. But during this time, two social phenomena made their appearance. Firstly, the birth on French soil of the children of migrants, who underwent the greater part of their education and their socialization in France and then, at the age of 18, acquired French nationality. Secondly, the growing economic disparity between territories where the migrants lived, on the periphery of towns, a disparity aggravated by the industrial crisis of the mid-seventies [START_REF] Zauberman | Police, Minorities and the French Republican Ideal[END_REF]Bonelli, 2007). Public opinion suddenly turned to the specific problem of these deprived urban areas (known in French as "banlieues") when, the same year the Left came to power (in 1981), large scale violence2 broke out in the housing estates of Lyon, bringing into the limelight a population of young children of impoverished workers, most of whom were of foreign origin and who remained outside the ambit of the dominant political debate (Beaud & Pialloux, 2001;Duprez & Hebberecht, 2002;Jobard, 2009 ;[START_REF] Lagrange | The French Riots and Urban Segregation[END_REF]Mucchielli, 2009).

To simplify an extremely complex debate, we can roughly say that there are two distinct modalities of analysis. The first is qualitative and attempts to illustrate either through observations or through interviews, the racist conduct or attitude of the police. Scientific research has explored this avenue briefly, and has highlighted the significance of a racist culture among the French police (Lhullier 1987;[START_REF] Wieviorka | La rance raciste[END_REF][START_REF] Hodgson | French Criminal Justice: A Comparative Account of the Investigation and Prosecution of Crime in France[END_REF], which has been very well summarized by Zauberman and Lévy, "People do not enter the incapacity lasting less then a month, and one-fifth were complainants who suffered a lasting infirmity or incapacity of more than a year, or else plaintiffs of deceased persons (CNDS, 2005:498).

However, these events are not comparable, in extent and intensity, to the full-scale urban riots that raged in the United States or even in Great Britain. They remain confined to small territories and mostly consist of attacks on private and public property. Cars are burned at random, stones are thrown at the police, and petrol bombs are thrown at police stations or other official buildings. It should be emphasised that there is typically no use of lethal weapons on either side during these riots, and thus, they never result in deaths. police because they are racist; rather, they acquire racial prejudice through a process of professional socialization. In other words, the habit of judging individuals on the basis of their supposed ethnic characteristics is acquired on the job. " (2003: 1076). This rough assessment amongst police does not however exhaust, on the one hand, the diversity of available opinions highlighted chiefly by recent researches [START_REF] Monjardet | La culture professionnelle des policiers. Une analyse longitudinale[END_REF][START_REF] Pruvost | Etrangers. Quels policiers prônent la vigilance ?[END_REF] and, on the other, the gap that still exists between the expression of an opinion and on the job conduct (Manning 1976, Mastrosky, Reisig, McCloskey, 2002;Jobard, 2008). It is however not this first modality of analysis that we are going to discuss here, but the second, which is quantitative in nature.

On the quantitative study front, there is very little statistical data available in comparison with the English-speaking countries. The main reason for this is legal: in France the law forbids the inclusion of any ethnic and religious categorization of the population in public statistics and, more generally, does not allow the processing of "personal data revealing racial origin" based on public records [START_REF] Weil | The History of French Nationality. A lesson for Europe[END_REF][START_REF] Simon | The measurement of racial discrimination: the policy use of statistics[END_REF]. And since most children of foreigners born and brought up in France take French nationality on reaching their majority, they "disappear" from national statistics that only retain a dichotomous indicator: French/foreign. This clause in French law, relative to the protection of individual data has the indisputable effect of rendering racial problems invisible and is strongly discussed in academic circles.

At present, quantitative research that demonstrates discrimination against individuals of foreign origin by the French police is rare. Police statistics show an arrest rate for foreigners that is much higher than for French nationals, and this even when immigration offences are taken apart. Zauberman & Lévy thus mention that the arrest rate among foreigners was about 2.3 times higher in 2001 than the proportion of foreigners in the total French population [START_REF] Zauberman | Police, Minorities and the French Republican Ideal[END_REF]Lévy 2004: 1310). In addition, [START_REF] Lévy | Du suspect au coupable: le travail de police judiciaire[END_REF] had already established that the risk of being taken to court (by the prosecution) and condemned (by the sitting judge) is higher when the person being questioned is of Maghrebian origin, even when controlling for type of offence and social profile of the offender. Lastly, in a recent study, Pager (2008) has demonstrated a close correlation between tribunals where a majority of the harshest decisions were taken (in this case police custody, legal restrictions pending trial and criminal sanctions) and the districts where young men of Maghrebian origin were in the majority: "it is the Maghrebian population that reveals the strongest association with local crime control. While ethnicity cannot be directly measured using the available data, these results are suggestive of a strong ethnic component in the local fashioning of punishment".

-Investigation into the judicial handling of 'offences against the police'

In this section, I shall summarize results from a personal study on offences against police officers (OAPO) which highlights some important aspects of the relationships of police and minorities (Jobard, Nevanen, 2008). Data presented here stems from statistics taken from the district court of a town in the outlying suburbs of Paris, where I had also conducted qualitative surveys that showed the considerable and growing tension in the town since 1997, when a young man was shot dead by the police (Jobard & Linhardt, 2008). In the course of these inquiries, it appeared that the offences against police officers were considered by the young Maghrebians there as a particularly discriminatory instrument of repression used by the police against migrant youth.

OAPO are definitely revealing tensions between police and those which they label as "youths known to the police", among whom we find mainly young men of foreign origin living in deprived urban areas. On the one hand, these offences are used by the police forces as indicators measuring urban violence. But on the other hand, the people who record them are also those who claim to be the victims, they are viewed by the accused as embodying the discretionary element of police power. These offences therefore reveal a crucial part of what goes on between police and the population.

Three offences are involved here: contempt, obstruction and assault on an officer. Our data cover 864 adults judged at the criminal court and charged with OAPO. I have collected a sample of over 1,500 cases judged between 1965 and 2005 at a tribunal de grande instance (a district court) in an outlying Paris area district. The data cover 864 adults judged at the criminal court and charged with OAPO, plus 268 defendants judged by a juvenile court. These documents do not give any information as to where the offences were committed, nor as to whether the suspect had any previous conviction, nor on social, occupational and marital status, although studies have shown these to be decisive factors in determining the sentences handed out.

a) Broad trends for the offences judged

The evolution of offences judged by the TGI between 1965 and 2005 shows a definite rise in the number of defendants: The OAPO rates were multiplied by two between 1965 and 1975, then leveled off, and doubled again between 1990 and 1999. A major change definitely occurred in the 1990s. No such rise can be found in drugs, property offences, violent offences: the rate of cases judged between 1990 and 1999 was only multiplied by 1.4.

The rise in the number of OAPO judged affects all categories, but differently depending on the type of OAPO (contempt, obstruction, contempt and obstruction, assault).

The proportion of assaults on officers dropped consistently, then rose a bit in the 2000s (one fourth of OAPO were assaults from 1965 to 1984 and only one sixth today). Now, between 1960 and 1980 few cases were taken to court, and the fact that assault represented a large portion of these shows that at the time the police only transmitted those cases they felt were particularly serious. Today we have quite the opposite: the police send all cases to the courts. This leads us to assume that in the past cases other than "assaults" were handled outside the courts, on the spot, by anything from verbal admonition to a couple of smacks. This trend is most probably the other side of the gradual reduction of police brutality.

b) Defendants

In order to get around the lack of information on the origin of the accused, I have coded names and birth places, to set up what we will call, for want of a better name, "race group".

For the entire period, sixty two percent of the adult defendants are "Europeans", twenty percent are "Maghrebians", fifteen percent "Blacks" (two percent others). This situation was of course fed by major demographic trends within the considered district. If we look at groups for juvenile court defendants, we find that thirty nive percent were "Europeans", forty five percent "Maghrebians" and twelve percent "Blacks". Thus, over the last decade the proportion of the latter two groups was extremely high among those accused of OAPO, especially for juveniles.

A more detailed view of age groups shows a very large proportion of youthful defendants in the "North-African" group. These defendants are strikingly young: for the period as a whole, fifty percent are under twenty two and twenty five percent are under eighteen. If we focus on 2002 and 2003 (data for the juvenile court judge"s chambers were missing for 1999, 2000 and 2001), the median age drops to twenty one. The majority of individuals prosecuted for OAPO are young adults and juveniles, just as were the people tried for unrest during the 2005 riots in a comparable Parisian suburb court. There is no main difference between the youngsters implied in daily conflicts with the police and the ones who took part in the November 2005 disorders.

c) Criminal sentencing and discrimination

The forthcoming analysis concentrates on adults and OAPO only, that is, with no other misdemeanours involved in the same trial (n=864).

Sentences are largely different with regard to the different "race groups", since 13.6 percent of the "Europeans" face an unsuspended imprisonment, but 23.6 percent of the "Maghrebians" and 25.4 percent of the "Blacks". Differences also appear with respect to average length of imprisonment: fourteen percent of imprisoned "Europeans" get less than one month jail, but only seven percent of their "Maghrebians" and three percent of their "Blacks" counterparts; similarly around thirty three percent of "Maghrebians" and "Europeans" defendants were sentenced to more than two months of jail, but the proportion of "Blacks" to forty four percent3 . What accounts for these differences? The most important factor is the type of offence get. Indeed, ten percent of "contempt" cases unsuspended imprisonment as opposed to around twenty percent of contempt and obstruction or cases and around forty percent of assault cases (***). Moreover the types of offences prosecuted are definitely not the same for the different groups alone: 61.5 percent of "European" defendant are tried for contempt, only 15.5 percent for assault. Proportions are completely different among the two other groups: around forty two percent are judged for contempt versus, around nineteen percent for assault (***).

Another reason is the type of trial: in French law, immediate hearing trial (IHT) is prescribed for misdemeanours incurring sentences of at least two years in prison, or one year in case of recidivism [START_REF] Hodgson | French Criminal Justice: A Comparative Account of the Investigation and Prosecution of Crime in France[END_REF][START_REF] De Cavarlay | The prosecution service function within the French criminal justice system, in Collectif, Coping with Overloaded Criminal Justice Systems[END_REF]. Given the fact that the prescribed sentence is doubled in case of recidivism, it may safely be said that people who are given IHT for contempt, obstruction or contempt-obstruction (prescribed sentences = six months) are judged as "recidivists" by definition. Now, defendants facing a IHT proceeding are usually exposed to a greater risk of imprisonment, and our data show no exception to this general statement: forty six percent of IHT-defendants are sentenced to jail against fourteen percent of others (***); half of the defendants charged with contempt only are sentenced to jail if facing a IHT trial. Again, the differences among the groups are significant in this respect as well: only nine percent of the "Europeans" face an IHT trial, against twenty one percent of the "Maghrebians" and twenty eight percent of the Africans (***).

As we can see, this study documents potential discrimination engendered by the judicial system rather than by the police; however, the penalties meted out are directed at misdemeanours whose particularity is that it is the police who say they are the victims. The rapid growth of this type of case in the suburban court examined points to growing of conflicts with the police in these areas, which are being taken up, by the courts.

Beside it, our data do indicate a series of significant differences (more unsuspended prison sentences, longer prison sentences, more frequent requests of civil damages by police officers)4 . There is thus a series of significant differences, but the variable "race group" does not appear to be significant variable when included in a multivariate analysis with all the others. "Maghrebians" and "Blacks" populations are tried for particular kinds of offences within the OAPO category and more often tried as recidivists... Court decisions pitilessly echo and multiply the singularities of a population which differs in its origins, social status and relations with the criminal justice system.

We now turn on a second aspect of possible discrimination towards minorities in France, which is constituted by stop and searches operations (called "identity checks") conducted by police officers in Paris.

II Identity checks in Paris

Law and jurisprudence in France on id checks by police officers are rather fuzzy (art. 78.1 to 78.6 of the Code of Criminal Procedure). officers cannot base their checks on the person"s physical appearance, and must allege a presumption that the person is a foreigner, based either on the person"s own admission or on some other « objective » fact (which case law has great difficulty defining). The legislation leaves some room for institutional discrimination here, since the public prosecutor may very well demand that the police target the offense of undocumented migration within an area defined by him.

Profiling may be encouraged by explicitly discriminatory policing policies, but it may also be the fruit of decisions by individual police officers, produced by shared stereotypes as to what should be viewed as suspicious, or as the propensity of any particular minority group to break the law. It may also originate in policies targeting specific kinds of criminal offenses and/or some geographic areas, with no regard for the disproportionate effects of those policies on particular minorities.

Due to the lack of any kind of official statistics on id checks made by police officers in France, René Lévy, John Lamberth and I conducted a research funded by the Open Justice foundation in order to know about who is checked in Paris places and if there is a discrimination between the people checked by the police and the people available on the places where the checks are conducted (Lévy & Jobard, 2010).

In order to do so, we first benchmarked the available population in the places we examinated. Around 37.000 people " characteristics were recorded ; these characteristics are : (approximate) age, gender, outfit, (alledged) race. In fact, our aim was to know if the people stopped and searched by the police share the same characteristics like the ones available on the different places. Race, age, gender and outfit are characteristics based on perceptions by the observers we had on the places.

The result is a massive case for discrimination, but race is far from being the only concerned variable. Age and gender are the most important variabe at stake: young male are overstopped. Maghrebians and Blacks are also overstopped. For instance, Blacks are overstopped in places where they are only a few among the available population (like on international train peers at North Railway Station in Paris, where they are 7,5% of the available population, but 31% of the checked), and they are also overstopped in places where they are quite numerous (in Châtelet place in the very centre of Paris, which is a place where young people gather and chat, Blacks are 29% of the people available, and 62% of the those checked). In overall, odds ratios of Blacks compared to Whites are comprised between 4 and 11,55 ; odds ratio for Maghrebians compared to Whites between 2 and 15 -which are very high numbers.

But the influence of the "outfit" variable appears to be also massive on the respective risks of being stopped. In fact, being dressed in typical youth culture outfit (like hip-hop clothes) seems to influence the stops made by the police in a comparable proportion in comparison to the ones dressed "casual" or "business". That is to say that police officers seem to target the young male minorities and/or the young male hip-hop youths… which are, in fact, quite indistinguishable: two third of the available population who are dressed in "youth culture" outfit have been sorted in minorities groups.

As a matter of fact, the police in Paris concentrate on a population which has always been a central target: the young male hanging out in public places, where the proportion of minorities is very high. The only belonging to a minority seems to influence the decision to stop made by police officers in Paris, especially as far as Blacks are concerned; but it is the combined belonging to a minority group and to a youth" group which is over-predictive in the decision to stop. The case for blank racism from the police (or the political authority which drives the police) is then hard to make on quantitative basis, even if the numbers strongly suggest such a problem in the ranks of French police.

Conclusion

French research, especially in comparison to Anglo-American research, does not give precise quantitative information either in relation to frequency of police abuse nor to frequency of discriminatory practices. This confession of weakness should also be interpreted as an imperative of modesty: much of what we know is taken from interviews and very little from observations of concrete situations and even less from quantitative data. From the available quantitative data, we can nevertheless draw a certain number of conclusions.

1)

Police work in deprived urban areas is characterized by growing dissent over a period of twenty Five years, spectacularly highlighted in November 2005. This situation of conflict is greater when the police interacts with the young, in fact the very young (around twenty years and even less) from a migrant milieu.

2)

The conflicts are increasingly absorbed by the judicial system. This signifies that cases of settlement of conflicts by the use of physical violence are probably becoming rarer, but on the other hand, the population involved in conflicts with the police is coming increasingly within the ambit of the judicial system, thus reinforcing its character as "property" (Lee 1981) of penal institutions.

3) When violence is used ill-advisedly by the police, it remains at an infra-lethal level.

The cases of deaths during questioning and above all in police custody have now decreased considerably. This is probably due to greater judicial control exercised on police custody since 1993, including the right to access to a physician and to a lawyer.

4)

The reports addressed by the Council of Europe to France suggest that the conditions of custody, police opacity and lack of diligence in the handling of judicial matters, implicating policemen, are more prejudicial in France than they are in Great Britain or in the North European countries, including Germany.

5)

Police discrimination is an issue which cannot be properly assumed at present. It appears that the issue of discrimination is as much territorial as strictly racial. In France there is a vast difference between areas accessible to the middle and upper classes and those inhabited by the working classes, which today comprise in a very large part of people of foreign origin, the ones who are targeted by the police.

Consequently, it is not certain that a detailed investigation into the nature of discriminations will enable us to clearly separate territorial, social and racial discriminations.

6)

Despite this, there exists among the French police a quite unanimously shared stock of prejudices against people of migrant origin. We cannot, however, scientifically establish the link between, on the one hand, an expressed attitude, and, on the other hand, a behaviour on the job and, on this point, probably cannot even distinguish the French police from its European counterparts.

From these facts, various political responses have emerged these last few years. A limited recruitment of officers of Maghrebian origin in the police force has been attempted [START_REF] Duprez | La tradition, un frein à l"intégration[END_REF][START_REF] Zauberman | Police, Minorities and the French Republican Ideal[END_REF]. We also note the creation in 2000 of a very interesting institution, the Commission Nationale de Déontologie de la Sécurité, whose task is to investigate any alleged violation of deontology committed by security personnel, whether public or private (CNDS 2005). Unfortunately, the Commission is not in a position (just like any institution or NGO working on data gathered from testimonies) to establish with precision indirect discrimination on the part of the police and moreover government"s aim is now to abrogate this institution.

Lastly, it should be noted that the issue of police abuse and discrimination acquires a new place in French society. Carried by a wave of "law and order" measures, the questions of security and of the police have acquired since a decade and in particular since 2002, considerable importance. This has two, somewhat paradoxical consequences. The first is increased repression of petty street crimes, targeting people exposed to risks of discrimination. But the second, unexpected consequence, is the greater attention paid by the press and public opinion to everything pertaining to the police, with raises the stakes of dissimulation (within the agencies and vis-à-vis society as a whole) of possible deviance. It is on this very narrow path that the issue of police deviance in France will evolve in the coming years.

Let us right away say, in relation to the earlier remarks about the intensity of violence, that about 40 percent of the 36 complainants did not suffer any medical repercussions, approximately one third experienced a short-lived

Here too, when talking of urban violence, what Anglo-Saxon researchers call "riots", the nature and the intensity of the violence are not comparable to what this term stands for in the United States or in India. As[START_REF] Zauberman | Police, Minorities and the French Republican Ideal[END_REF] Lévy (2003: 1073) stressed even before the 2005 unrest (see alsoJobard, 2009):

Differences are statistically significant concerning the differences in sentences (chi2, <0.01), but non significant concerning the length of imprisonment. In what follows, (***) stands for <0.01 significance; (**) for <0.05 significance, (*) for <0.1 significance, (n.s.) for non-significance.

Fifty one percent of the "Maghrebian" defendants and 46 percent of "Blacks" face police officers asking civil litigation, but only thrity seven percent of the "Europeans" (***). In regards with a logistic regression analysis, this difference is the only one where the group as such seems to have an influence on the observed differences(Jobard, Nevanen, 2008).

Odds ratios compare the respective probabilities of being checked for different groups in relation to the composition of the available population. Blacks have a risk of being checked compared to the the risk faced by Whites to be checked between 4 and 11,5 times more, with respect to the overall composition of the population.