Rébecca Bonnaire

Woo-Suck Han

Paul Calmels

Reynald Convert

Jérôme Molimard

Parametric study of lumbar belts in the case of low back pain: effect of patients' specific characteristics

Objective: A numerical 3D model of the human trunk was developed to study the biomechanical effects of lumbar belts used to treat low back pain. Methods: This model was taken from trunk radiographies of a person and simplified so as to make a parametric study by varying morphological parameters of the patient, characteristic parameters of the lumbar belt and mechanical parameters of body and finally to determine the parameters influencing the effects of low back pain when of wearing the lumbar belt. The loading of lumbar belt is modelled by Laplace's law. These results were compared with clinical study. Results: All the results of this parametric study showed that the choice of belt is very important depending on the patient's morphology. Surprisingly, the therapeutic treatment is not influenced by the mechanical characteristics of the body structures except the mechanical properties of intervertebral discs. Discussion: The numerical model can serve as a basis for more in-depth studies concerning the analysis of efficiency of lumbar belts in low back pain. In order to study the impact of the belt's architecture, the pressure applied to the trunk modelled by Laplace's law could be improved. This model could also be used as the basis for a study of the impact of the belt over a period of wearing time. Indeed, the clinical study shows that movement has an important impact on the distribution of pressure applied by the belt.

Introduction

Low back pain is a frequent disease which induces high cost to society in developed countries. So, from prevention to disability, a lot of treatments are proposed.

Low back pain is clinically described by physio-pathological characteristics and its duration, from acute to chronic form [START_REF] Stucki | Approche multidisciplinaire de la lombalgie commune subaiguë et chronique : expérience suisse romande[END_REF]. One of the treatments for low back pain is the use of a lumbar orthotic device. The objectives of this device are to reduce mobility, decrease pain [START_REF] Calmels | Effectiveness of a lumbar belt in subacute low back pain: an open, multicentric, and randomized clinical study[END_REF] and medication used which could induce some risks of iatrogenic complications.

The clinical efficacy of lumbar belt has been shown in the event of subacute and chronic low back pain [START_REF] Calmels | Effectiveness of a lumbar belt in subacute low back pain: an open, multicentric, and randomized clinical study[END_REF]. However, few studies exist that clearly elucidate all the mechanisms of action proposed for this orthotics. After these studies, we could conclude that lumbar orthoses:  limit the motion range of the trunk [START_REF] Axelsson | Effect of lumbar orthosis on intervertebral mobility. A roentgen stereophotogrammetric analysis[END_REF][4] [START_REF] Fidler | The effect of four types of support on the segmental mobility of the lumbosacral spine[END_REF];  reduce disc stresses by increasing the abdominal pressure [START_REF] Andersson | Lumbar disc pressure and myoelectric back muscle activity during sitting. 3. Studies on a wheelchair[END_REF][7] [START_REF] Nachemson | In vivo measurements of intradiscal pressure[END_REF];  correct the posture by modifying lumbar lordosis [START_REF] Cholewicki | The effects of a 3-week use of lumbosacral orthoses on proprioception in the lumbar spine[END_REF][10] [START_REF] Thoumie | Effects of a lumbar support on spine posture and motion assessed by electrogoniometer and continuous recording[END_REF];  have no negative impact on muscle strength [START_REF] Fayolle-Minon | Effect of wearing a lumbar orthosis on trunk muscles: study of the muscle strength after 21days of use on healthy subjects[END_REF][13][14] [START_REF] Warren | Effect of soft lumbar support belt on abdominal oblique muscle activity in nonimpaired adults during squat lifting[END_REF];  have muscle-relaxing effects [START_REF] Holmström | Effects of lumbar belts on trunk muscle strength and endurance: a follow-up study of construction workers[END_REF][17] [START_REF] Valle-Jones | Controlled trial of a back support ('lumbotrain') in patients with non-specific low back pain[END_REF] [START_REF] Willner | Effect of a rigid brace on back pain[END_REF];  facilitate trunk mobility for daily living activities [START_REF] Calmels | Effectiveness of a lumbar belt in subacute low back pain: an open, multicentric, and randomized clinical study[END_REF] [START_REF] Valle-Jones | Controlled trial of a back support ('lumbotrain') in patients with non-specific low back pain[END_REF].

No overall study of the mechanisms of action of lumbar belts and no study based on numerical modelling of their effects on the trunk to reduce low back pain were found through the review of the literature. However, numerous models exist for the human trunk and, more specifically, the spine. These models can be divided into three types: detailed models, simplified models, and hybrid models, i.e. containing both highly detailed zones and highly simplified zones. Numerical models have been used, for example, to compare the influence of parameters for surgical treatments or other techniques [START_REF] Carrier | Biomechanical modelling of growth modulation following rib shortening or lengthening in adolescent idiopathic scoliosis[END_REF] [START_REF] Nagasao | Dynamic effects of the nuss procedure on the spine in asymmetric pectus excavatum[END_REF] [START_REF] Pankoke | Determination of vibration-related spinal loads by numerical simulation[END_REF], to analyze the deformation of the spine ( [START_REF] Huynh | Simulation of progressive spinal deformities in Duchenne muscular dystrophy using a biomechanical model integrating muscles and vertebral growth modulation[END_REF][24] [START_REF] Villemure | Biomechanical simulations of the spine deformation process in adolescent idiopathic scoliosis from different pathogenesis hypotheses[END_REF]) or the effect of carrying a load ( [START_REF] Arjmand | Predictive equations for lumbar spine loads in load-dependent asymmetric one-and two-handed lifting activities[END_REF] [START_REF] Bazrgari | Soft tissue wobbling affects trunk dynamic response in sudden perturbations[END_REF]). One study on the mechanisms of action of rigid orthosis in the treatment of scoliosis appears to be similar to the present study and report that biomechanical analysis using computational modelling could be useful in the design of rigid orthosis [START_REF] Clin | Virtual prototyping of a brace design for the correction of scoliotic deformities[END_REF].

In this Finite Element Analysis (FEA), a lumbar orthosis, named lumbar belt, was studied. This orthosis is a common treatment of subacute or chronic low back pain. The main objective of this study is to evaluate the parameters that can influence the choice of lumbar belts types to treat low back pain, using a finite elements model. The studied parameters will be related to the patient's morphology, the belt and the mechanical characteristics of the human trunk.

Methodology

The aim is to perform a screening of the factors influencing the global mechanical effect of the belt. For this purpose, a simplified numerical model was built so as to change easily trunk geometry according to different typical patients. Unfortunately, this induces a compromise between computing time and precision on the re-sults. The model might not render properly local effects but must be reliable on global ones. Indeed, a special attention must be done on the spine modeling, because pain is supposed to be caused from intervertebral discs. The geometry is simplified on trunk and vertebrae. Finally, the mechanical behavior is assumed to be linear and a simple Laplace law simulates the pressure applied by the belt. All these assumptions make a parametrical study possible, even if they track important limitations.

Trunk modelling

Construction of geometric model

A simplified 3D model of the human trunk was developed for FEA. This model is built using frontal and sagittal radiographies of the trunk of one person. Using these radiographies, the following elements are measured:

 the lateral inclination of each intervertebral endplate in the sagittal plane,  the height at their center of the thoracic and lumbar vertebrae,  the diameter of the vertebral anterior segment at thoracic and lumbar level,  the height at their center of the intervertebral discs in the thoracic and lumbar regions,  the width of the trunk at the chest, below the chest, at the waist and hips in the frontal plane,  the thickness of the trunk at the chest, below the chest, at the waist and hips in the sagittal plane.

Each vertebra is modelled as a cylinder based on the radiographs. Lumbar lordosis and thoracic kyphosis are constructed using two circle arcs obtained thanks to the lateral inclinations of each vertebral endplate. It is assumed that the vertebra have parallel lower and upper endplates. The posterior segment of vertebra is not represented in this model since their action is assumed to be negligible for this study. The intervertebral discs are also modelled as a cylinder having two distinct parts: a central circular part corresponding to 30% of the volume representing the nucleus and a peripheral part representing the annulus [START_REF] Chagnon | Biomechanical influence of disk properties on the load transfer of healthy and degenerated disks using a poroelastic finite element model[END_REF].

The tissues composing the trunk, excluding the spinal column, are divided into three zones in order to represent the abdomen, the iliac crests and one other part including the thorax and the soft tissues in the back zone (see Fig. 1). This geometric model is simplified to easily change the trunk morphology in the simulation and realize the parametric studies. The surface of the trunk, corresponding to the skin, has different mechanical properties from the rest of the volume. This skin has a thickness of 4 mm; the value was determined by ultrasound on the trunk of two human subjects (see Fig. 2).

Finite Element modelling of the trunk

The geometry is meshed using ten-node tetrahedral second-order elements (C3D10H) using ABAQUS. Figure 3 shows a meshed model of the trunk. To determine the appropriate number of elements, a convergence study was done. This study consists in determining the mean abdominal and intradiscal pressure in the lumbar region as a function of the mean size of the elements. This study was carried out on the eight subjects chosen in this study, described in the next paragraph 2.2.

For the purpose of ensuring model simplicity, all the structures have elastic linear mechanical properties. As this study is a first approach, it would be interesting to modify this hypothesis in a future work. Mechanical properties can vary as a function of the age and musculature of the subject modelled. They are therefore considered as design parameters during this study.

After studies on different sets of boundary conditions, these were determined in order to obtain realistic movement of the trunk under the loading of the belt. When a patient wears a belt, the pelvis can rotate slightly relative to the transverse axis.

In addition, the patient tends to straighten up. It was therefore decided to block the upper plate of the trunk in translation in the transverse plane. The top of the trunk can translate in the frontal plane. The lower plate of the trunk is restricted in order to enable rotation of this plate only in the transverse axis. This restriction is obtained by embedding the two points at the intersection of the ellipse, representing the extremity of the lower plate of the trunk, and the frontal plane of the trunk. The boundary conditions are represented in Fig. 3.

Modelling of the lumbar belt

Lumbar belts are medical devices composed primarily of fabric in whom four steel whales are inserted in the dorsal part and a flexible whale in the front part. They may consist of one or several layers of fabric. In this finite element model, it was opted to represent the lumbar belt globally as application of pressure to the lumbar region of the trunk. The bottom of the belt is located at the lower endplate of lumbar vertebra L4. This pressure is obtained by the following Laplace's law [START_REF] Dubuis | Patient-specific numerical model of soft tissues in the compressed leg: application to six subjects[END_REF]:

(

) 1
where T is the linear tension of the belt on the trunk and R the radius of curvature of the trunk at the pressure application point. The linear pressure of the lumbar belt used in this study was obtained by tensile tests on the belts [START_REF] Bonnaire | Caractérisation mécanique des orthèses : Application aux ceintures de soutien lombaire dans le cadre de la lombalgie[END_REF]. This overall belt model does not enable to simulate specific actions of certain structures of the belt, such as the back stays, but provides an adequate simplified model for the purposes of this study.

Assessment of belt modelling

In parallel of this simulation, a clinical study was carried out on fifteen low back pain subjects [START_REF] Bonnaire | Caractérisation mécanique des orthèses : Application aux ceintures de soutien lombaire dans le cadre de la lombalgie[END_REF]. The interface pressure between the belt and the trunk was measured using piezoresistive pressure mapping system of 32×12 sensors [START_REF] Bonnaire | Characterization of a pressure measuring system for the evaluation of medical devices[END_REF].

The mean pressure measured during the clinical study could therefore be compared to the pressure by Laplace's law.

Parametric study 2.2.1. Input parameters

To show how lumbar belt wearing influences mechanical conditions, a numerical design of experiments was built. Its input parameters are:

 the morphological parameters of the patient: height, corpulence and lumbar lordosis angle,  the characteristic parameters of the lumbar belt: belt type and height,  the mechanical properties of the structures composing the model: skin, abdomen, annulus, nucleus, bone and others soft tissues.

To vary the patient's morphological parameters, a basic geometry was constructed as described in the previous paragraph 2.1 "Trunk modelling". This geometry was obtained from radiographies of one patient, without 3D deformation of the spine.

This geometry was proportionally modified in order to obtain two trunk's geometries: one corresponding to a person with the height of 1.60m and one corresponding to a person with the height of 1.80m. The basic geometry corresponded to a person with a small waist circumference. This basic geometry was therefore considered as "slim". The trunk thicknesses in the sagittal plane at the waist and hips levels were doubled in order to obtain the geometry of a "fat" person. The inclination angles of the lumbar vertebrae were modified to obtain subjects with a low lumbar lordosis of 33° or a high lumbar lordosis of 53°. Subjects with hypo-and hyper-lordosis could therefore be modelled. Finally, eight characteristic geometries were obtained by varying the height (tall/small), stature (slim/large) and lumbar lordosis (hypo/hyper-lordosis). With linear elastic assumptions, we only need Young's moduli and Poisson's ratios to describe mechanical behavior of materials. Poisson's ratios, used in the model and taken from the literature [START_REF] Clin | Virtual prototyping of a brace design for the correction of scoliotic deformities[END_REF][33][34] [START_REF] Sylvestre | Finite element modeling of the growth plate in a detailed spine model[END_REF], are shown in Table 1. Only Young's moduli will vary in this parametric study. They are defined by continuous parameters, in contrast with the other parameters, and may vary within a range on the basis of the data in the literature [START_REF] Clin | Virtual prototyping of a brace design for the correction of scoliotic deformities[END_REF][33] [34][35].

This parametric study therefore includes 11 input parameters (see Table 2).

Output parameters

The following three output parameters were observed:

 the variation in abdominal pressure, since it characterizes the direct impact of belts on the trunk,  the variation in lordosis, characterized by measurement of the mean displacement of the lumbar vertebrae in the sagittal plane to determine the impact on the spine posture,  the variation in intradiscal pressure in the lumbar spine.

Design of experiments

The design method used is a stratified 100-experiment Latin hypercube design. It was taken since it makes possible to cover the entire study space with a relatively small number of experiments and at the same time, enabling binary or continuous input parameter analysis.

Results

Convergence study

Fig. 5 represents the variation in mean abdominal pressure as a function of the size of the elements for the most slim and smallest patient with hypolordosis in the study. Convergence is obtained for an element size of less than 15 mm, corresponding to a little over 80,000 elements in this case. Similar results were found for the other seven geometries modelled. The convergence study done with the eight used subject geometries finally shows that convergence is achieved for all the cases when the elements have a mean size of 10 mm. This mean size corresponds to models including 242,230 to 481,850 elements. It was therefore selected to perform the numerical design of experiments.

Assessment of belt modelling

In the clinical study, the mean pressure applied is 1.12 kPa or 2.03 kPa, respectively, for a single or double fabric thickness lumbar belt. In our model, the mean pressure applied to model by the belt is 1.06 kPa or 2.16 kPa, respectively, for a single or double fabric thickness lumbar belt. The mean error is about 6% and resulting from simplified modelling of the belt by Laplace's law. The belt model is acceptable.

Example of detailed results for a modelled subject

Fig. 6 shows the pressure and displacement results for two subjects modelled in this study.

The displacements are very different between the subject's abdomen and back. However, the displacements are symmetrical in the sagittal plane. At the iliac crests, displacements are almost zero.

The pressure inside elements is the highest at the iliac crests. This pressure is also high in the abdomen. It is negative in the lumbar region.

Parametric study

The stratified Latin hypercube design made it possible to construct a linear model according to Equation 2:

(2)

where:

 -Y = the output variable, which may be either the variation of abdominal pressure or the variation of intradiscal pressure, or the mean displacement of the lumbar vertebrae in the sagittal plane,  -β 0 = the mean value of the linear model,  -β i = the linear coefficients of the model and have an index s for the patient's height, c for the patient's corpulence, l for the patient's lordosis, bt for the belt type, h for the belt height, pab for Young's modulus of the abdomen, pan for Young's modulus of the annulus, pn for Young's modulus of the nucleus, ps for Young's modulus of the skin, pb for Young's modulus of the bone and ot for Young's modulus of the other soft tissues,  -X = is the input variable with the same indices as for the β coefficients.

X is reduced in a [-1 +1] range. The mean displacement of the vertebrae is taken to be positive when the lordosis increases. The model chosen was a linear one without interaction since the interaction parameters are weak compared to the main parameters. Fig. 7 plots the linear coefficients of the model for the variation in abdominal and intradiscal pressures and the mean displacement of the lumbar vertebrae, related to the variation in lordosis. Because of the input parameters reduction to [-1 +1], this graphic representation of the linear model developed enables to determinate the parameters with the greatest influence. It shows that greater is the linear coefficient compared to the mean value of the output parameter, greater is the influence of the input parameter corresponding to this linear coefficient.

Hence the parameters with the greatest influence on displacement of the lumbar vertebrae are waist circumference, belt height and type, and lumbar lordosis before wearing the belt; the parameters with the least influence are the subject's height and Young's modulus of the annulus.

With respect to the variation in abdominal pressure, the parameters with the greatest influence are belt height and type as well as lumbar lordosis; the parameters with the least influence are the waist circumference and the mechanical properties of the skin and the annulus.

Finally, the parameters with the greatest influence to the variation in intradiscal pressure are the belt type, the subject's height and waist circumference, while the parameter with the least influence is Young's modulus of the skin.

A summary of the influence of input parameters on output parameters is presented in Table 3.

Discussion

Trunk model

The developed FE trunk model is geometrically very simple but includes numerous structures, such as the abdomen, iliac crests and spine. Its main advantage lies in the fact that it enables rapid modelling of different morphologies solely from two complete radiographies of the trunk. This model can also be adapted to only simulate part of the trunk, such as the lumbar region. Another advantage of this model is a reliable calculation time, which means that it is possible to test numerous configurations in a reasonable computing time: a few hours for one configuration on a 2-core computer.

During the construction of this model, several hypotheses were made.

Firstly, deformations were assumed to be small, making it possible to model the structures using elastic linear properties. This hypothesis is confirmed since strains less than 1% for each component make it possible to stay in the linear zone of the biomechanical structures behavior.

Secondly, in order to simplify the model and not to model the posterior parts of the vertebrae present in other models [START_REF] Wagnac | Finite element analysis of the influence of loading rate on a model of the full lumbar spine under dynamic loading conditions[END_REF], it was assumed that rotation of the vertebrae in the transverse axis is small on application of the pressure representing the belt wearing. The rotation of the vertebrae observed in the model is less than 5°. This rotation is small compared to the lumbar lordosis and thoracic kyphosis of the patients prior to application of pressure. This second hypothesis is also validated therefore.

Thirdly, the initial stresses in the trunk due to gravity and the presence of the head and legs are not taken into account in the model, in contrast with other models [28][37][38]. This hypothesis is correct since the study only concerns the pressure variation and displacement of vertebrae due to the belt wearing and not absolute pressure or deformation values.

Fourthly, it was opted to represent the action of the lumbar belt by a pressure range obtained by Laplace's law, as has already been proposed for compression socks in another study [START_REF] Dubuis | Patient-specific numerical model of soft tissues in the compressed leg: application to six subjects[END_REF]. The mean pressures applied by lumbar belts obtained in a clinical study in fifteen subjects with low back pain [START_REF] Bonnaire | Caractérisation mécanique des orthèses : Application aux ceintures de soutien lombaire dans le cadre de la lombalgie[END_REF] demonstrate that this simplistic model is acceptable. The difference in the interface pressure applied by the lumbar belt between simulation and clinical measurement is only 6%.

In the context of our study, the hypotheses adopted are therefore all justified. In addition, the clinical study demonstrates that the variation in trunk volume measured in humans during wear of a lumbar belt is almost identical to the variation in volume obtained in this model. This proves that, for an overall study of the mechanism of action of lumbar belts, our model is valid.

Mechanism of action of a lumbar belt

Observation of the pressure and displacement variations on a patient can explain some mechanisms of therapeutic action of lumbar belts. The belt applies pressure on the skin, and greatly increases abdominal pressure. This increase leads to a slight change in lordosis and to reduce constraints on vertebrae and intervertebral structures. The variation in intradiscal pressure is low, at around 1%. This lowpressure variation is close to pressure variation obtained in other studies on rigid orthotics [START_REF] Munoz | Évaluation biomécanique des orthèses lombaires -Application à l'orthèse Lordactiv®[END_REF].

Pressure is the highest in the iliac crests. It can be explained by the fact that the iliac crests are highly rigid structures compared to the soft tissues of the trunk.

High pressure is linked to the feeling of well-being. If the pressure applied by the belt is too high, the patient will find it more difficult to accept his/her belt [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee braces based on computational modelling[END_REF].

The parametric study made it possible to determine some parameters influencing the variation in abdominal and intradiscal pressure and displacement of the lumbar vertebrae, related to the modification in lumbar lordosis, which can play an important role in pain relief. These three output parameters are complementary to contribute to the therapeutic effects of lumbar belts in the treatment of low back pain.

Finally, the results of this parametric study raise the following points with respect to the clinical use of lumbar belts in the treatment of low back pain:

 the choice of belt is very important;  it is necessary to take into account the patient's morphology;  the belt treatment is independent of mechanical properties of soft tissue, excluding the mechanical properties of intervertebral discs.

As a matter of fact, all the effects of a lumbar belt cannot be described by the model. In particular, pressure induces proprioceptive effects on the body and an active change in the posture. This approach only shows a passive effect of the belt on the trunk.

Conclusion

Using a simplified model of the human trunk obtained on the basis of two radiographies, a parametric study made it possible to determine some parameters influencing the efficiency of lumbar belt in the therapeutic treatment of low back pain. These parameters are the patient's morphology and the type of lumbar belt.

The numerical model used is a simple one. It can serve as a basis for more indepth studies concerning the analysis of efficiency of lumbar belts in low back pain. In order to study the impact of the belt's architecture, modelling of the pressure applied to the trunk using Laplace's law could be improved. For example, the linear tension used in Laplace's law could vary depending on the application zone. This linear tension could be higher at the dorsal part of the trunk, to reflect the presence of the back whales. For a more precise solution, it would be possible to model the belt using a volume structure including the different existing parts, i.e. the fabrics, the back and front whales, etc. This numerical model could also be used as the basis for a study of the impact of the belt over a period of wearing time. Indeed, the clinical study shows that movement has an important impact on the distribution of pressure applied by the belt. This dynamic study would require modelling of the back muscles using volumetric structures with specific dynamic characteristics or beams.

Fig. 1 .

 1 Fig. 1. Representation of modelling of trunk geometry for this study: trunk geometry before modelling a. from the back b. in profile; modelled trunk geometry c. and e. from the back, d. and f. in profile.

Fig. 2 .

 2 Fig. 2. Ultrasound image used to measure the thickness of the skin at the trunk.

Fig. 3 .

 3 Fig. 3. Representation: a. of meshing of the finite trunk element model and b. of conditions at the limits.

Fig. 4 .

 4 Fig. 4. Examples of lumbar belts used in this study provided by Thasne®: a) Lombacross Activi-ty®, b) LombaSkin®.

Fig. 5 .

 5 Fig. 5. Result of the convergence study: variation in mean abdominal pressure in the model as a function of the number of elements.

Fig. 6 .

 6 Fig. 6. Displacement result for a fat, tall subject with hyperlordosis applied by Lombacross Ac-tivity®: a. in profile and b. from the front, and pressure result inside element for a slim and small subject with hypolordosis: c. in profile and d. from the front.

Fig. 7 .

 7 Fig. 7. Representation of the linear coefficients obtained in absolute values according to the analysis by experimental design for: a. displacement of the lumbar vertebrae, b. variation in abdominal and intradiscal pressures.

Table 1 .

 1 Poisson's ratio for the different components in the model.

	Model structure	Poisson's ratio
	Abdomen	0.45
	Annulus	0.45
	Nucleus	0.48
	Skin	0.2
	Bone	0.3
	Other soft tissues	0.45

Table 2 .

 2 Morphology of the subjects modelled, characteristics of the belts studied and variation range for the mechanical properties of the structures for the parametric study

	Parameters	-1	1
	Subject's height (cm)	160	180
	Subject's corpulence	slim	large
	Subject's lordosis (°)	33	53
	Belt type	LombaSkin®	LombaCross Activity®
	Belt height (cm)	21	26
	Young's modulus of the abdo-	0.01	1
	men (MPa)
	Young's modulus of the annu-	4	14.9
	lus (MPa)
	Young's modulus of the nucle-	3	8
	us (MPa)
	Young's modulus of the skin	1	5
	(MPa)
	Young's modulus of the bone	1,000	12,000
	(MPa)
	Young's modulus of the other	0.55	1

Table 3 .

 3 Influence of input parameters on output parameters.

		Mean abdominal	Mean intradiscal	Variation in lum-
		pressure	pressure	bar lordosis
	Belt height	++	+	+
	Belt type	++	++	+
	Subject's height	+	++	+
	Lumbar lordosis	++	+	+
	Subject's waist circumference	--	++	++
	Mechanical prop-erties of the skin	--	--	-
	Mechanical prop-
	erties of the ab-	+	-	-
	domen
	Mechanical prop-
	erties of the soft	-	+	-
	tissues
	Mechanical prop-erties of the bone	-	-	-
	Mechanical prop-
	erties of the annu-	--	+	--
	lus
	Mechanical prop-
	erties of the nu-	+	+	-
	cleus


Thomas Thiebault
email: thomas.thiebault@ephe.psl.eu

Mohammed Boussafir

Laëtitia Fougère

Emilie Destandau

Lucie Monnin

Claude Le Milbeau

Clay minerals for the removal of pharmaceuticals: Initial investigations of their adsorption properties in real wastewater effluents

Keywords: Pharmaceutical Products, Clay minerals, Adsorption, Wastewater Treatment, Organic Matter

The adsorption of pharmaceutical products (PPs) onto kaolinite and raw and sodium-exchanged montmorillonite was investigated in real wastewater effluents (WWE). The important role of the charge state of the PPs in controlling the adsorption extent was highlighted. Whereas cationic PPs were mostly adsorbed through cation exchange, the adsorption of neutral and anionic PPs appeared to be controlled by the nature of compensating inorganic cations and/or the simultaneous adsorption of organic moieties onto clay minerals. In raw WWE, the concentration of PPs is indeed far lower than the concentration of other organic molecules. Among the adsorbents tested, kaolinite displayed the lowest adsorption capacity of both PPs and other organic molecules, compared to raw montmorillonite which presented the highest adsorption capacity. The sodium-exchanged montmorillonite displayed intermediate adsorption properties, highlighting the key role of divalent inorganic cations in the adsorption of non-cationic PPs and other organic molecules. Hence, raw montmorillonite appears to be the most promising adsorbent for further investigations aiming to test the practicability of a clay-based adsorbent for the removal of organic contaminants, such as PPs, in WWE.

Introduction

The occurrence of pharmaceutical products (PPs) in numerous environmental compartments (da Silva et al., 2011;[START_REF] Patel | Pharmaceuticals of Emerging Concern in Aquatic Systems: Chemistry, Occurrence, Effects, and Removal Methods[END_REF] raises serious concerns about their potential hazard for living organisms [START_REF] Richmond | A diverse suite of pharmaceuticals contaminates stream and riparian food webs[END_REF][START_REF] Saaristo | Direct and indirect effects of chemical contaminants on the behaviour, ecology and evolution of wildlife[END_REF]. Contamination by PPs is mostly generated by excretion through urine and feces following both human and veterinary uses [START_REF] Baker | Illicit and pharmaceutical drug consumption estimated via wastewater analysis. Part A: Chemical analysis and drug use estimates[END_REF][START_REF] Choi | Wastewater-based epidemiology biomarkers: Past, present and future[END_REF]. Then, PP-contaminated wastewaters are transferred toward wastewater treatment plants, in which current treatment chains remain inappropriate for their complete removal [START_REF] Grandclément | From the conventional biological wastewater treatment to hybrid processes, the evaluation of organic micropollutant removal: A review[END_REF][START_REF] Petrie | A review on emerging contaminants in wastewaters and the environment: Current knowledge, understudied areas and recommendations for future monitoring[END_REF]. This contamination therefore challenges the scientific community and water treatment operators in order to find efficient and economically practicable removal solutions. Among the innovative treatments for the removal of organic contaminants, adsorption is considered as a promising way if the selected adsorbent displays a high adsorption capacity and moderate cost [START_REF] De Andrade | Adsorption of pharmaceuticals from water and wastewater using nonconventional low-cost materials: a review[END_REF]. Most of the literature focuses on activated carbons, which are currently used for the treatment of drinking water [START_REF] Cuthbertson | Does Granular Activated Carbon with Chlorination Produce Safer Drinking Water? From Disinfection Byproducts and Total Organic Halogen to Calculated Toxicity[END_REF][START_REF] Wong | Recent advances in applications of activated carbon from biowaste for wastewater treatment: A short review[END_REF], even though the potential of clay minerals for the removal of organic contaminants has already been demonstrated [START_REF] Thiebault | Raw and modified clays and clay minerals for the removal of pharmaceutical products from aqueous solutions: state of the art and future perspectives[END_REF][START_REF] Zhu | Adsorbents based on montmorillonite for contaminant removal from water: A review[END_REF]. Moreover, clay minerals can be considered as cheap and widely available materials. Yet, several studies pointed out their limitations for the adsorption of non-cationic PPs (Gao and Pedersen, 2005;[START_REF] Zhang | Sorption and desorption of carbamazepine from water by smectite clays[END_REF]. These limitations, however, were mostly found during batch experiments, in which the starting concentration of PPs wase much higher than their environmental occurrences, and in idealized solutions (i.e. pure water, no competing compounds). Several studies have recently demonstrated that the affinity of anionic and neutral PPs with clay minerals can be improved in the presence of other organic moieties [START_REF] De Oliveira | Adsorption of diclofenac onto organoclays: Effects of surfactant and environmental (pH and temperature) conditions[END_REF][START_REF] Wu | Adsorption of sulfamethoxazole and tetracycline on montmorillonite in single and binary systems[END_REF]. Therefore, it appears necessary to assess as precisely as possible the potential of a clay-based solution for the removal of PPs in real WWE in order to determine the affinity of such contaminants with adsorbents and to evaluate which type of clay minerals would be the most versatile for the design of a removal solution. The purpose of this work was therefore to investigate the PP removal potential of three clay minerals in raw WWE.

Material and methods

2.1.Chemical reagents and adsorbents

Standards for atenolol (ATE), bezafibrate (BZB), carbamazepine (CBZ), codeine (COD), diazepam (DIA), diclofenac (DCF), doxepin (DOX), gemfibrozil (GEM), ketoprofen (KET), naproxen (NAP), metoprolol (MET), norfloxacin (NOR), oxazepam (OXA), sulfamethoxazole (SUL), tramadol (TRA) and trimethoprim (TMP) were purchased from Sigma-Aldrich with a purity > 98%. Separation solvents, methanol (MeOH) and acetonitrile (AcN) were purchased from Fisher-Scientific, assuming an analytical grade (purity higher than 99.95 %). The selected adsorbents were Georgia kaolinite KGa-2 and Wyoming smectite SWy-2 both obtained from the Source Clay Minerals Repository, University of Missouri (Columbia, MO). The two adsorbents were fractionated < 2µm by gravity sedimentation prior to use in order to limit the impact of mineralogical impurities [START_REF] Chipera | Baseline studies of the clay minerals society source clays: Powder X-ray diffraction analyses[END_REF]. In order to investigate the impact of compensating inorganic cations on the removal capacity of SWy-2, this adsorbent was used raw (~20% of Ca 2+ and 80% of Na + ) and Na-exchanged through well-established procedures (Le [START_REF] Forestier | Textural and hydration properties of a synthetic montmorillonite compared with a natural Na-exchanged clay analogue[END_REF].

The resulting materials, labelled KGa2, SWy2 and Na-SWy2 display a cation exchange capacity of 3.7, 85 and 85 meq.100g -1 respectively, and a specific surface area of 18, 32 and 32 m².g -1 respectively with the N2 BET method [START_REF] Forestier | Textural and hydration properties of a synthetic montmorillonite compared with a natural Na-exchanged clay analogue[END_REF][START_REF] Lv | Uptake and retention of amitriptyline by kaolinite[END_REF].

2.2.Sample collection and batch experiments

10 L of WWE were sampled at the outlet of a vertical-flow constructed wetland in one intake (central part of France). The design of this sewage treatment plant consists of two stages of unsaturated vertical-flow constructed wetlands in series, with the first one planted with Phragmites Australis [START_REF] Paing | Vertical flow constructed wetlands for municipal wastewater and septage treatment in French rural area[END_REF]. After collection, WWE were filtered with glass-fiber filters (GF/F, Whatman) prior to storage at 4°C in amber glass bottles.

Batch experiments were systematically conducted in triplicate on the day following the sampling.

The WWE solutions (200 mL) were put in 250 mL bottles, mixed with 100 or 200 mg of adsorbents (i.e. KGa2, Na-SWy2 and SWy2) and then wrapped in aluminum foil in order to prevent lightinduced degradation. The solutions were stirred for 2 hours at 500 rpm with a magnetic stirrer. This time was considered as appropriate to reach equilibrium in such conditions (Thiebault et al., 2016a). A volume of 100 mL of supernatant was recovered by centrifugation at 8,000 rpm during 10 minutes prior to the extraction and analysis of the PPs. In parallel, blank experiments (i.e. Blank)

were conducted in the same conditions without adsorbent, in order to estimate the initial PP concentration and avoid any misinterpretation due to degradation.

2.3.Quantification and validation

The quantification procedure of PPs, and its validation, were already presented in detail (Thiebault et al., 2019). Briefly, a solid phase extraction was performed on the supernatant using Chromabond HR-X cartridges (6 mL x 500 mg, Macherey-Nagel) prior to analysis and quantification in high pressure liquid chromatography coupled with tandem mass spectrometry. The equipment, analytical procedures and quantification performances are detailed in the supplementary material (Table S1-2).

2.4.Processing of the results and analysis of the adsorbents

The removal of PPs after interaction was calculated using the following equation:

Removal = 100 -( 100 𝑥 𝐶 𝑒𝑞 𝐶 𝑠𝑡 )
with the Removal in %, Ceq the equilibrium concentration in µg.L -1 and Cst the starting concentration in µg.L -1

In order to evaluate the amount of organic matter adsorbed onto the clay minerals after interaction, carbon analyses were performed on powdered samples by using a Thermo Scientific Flash 2000 organic analyzer. These analyses were systematically triplicated. XRD and FTIR analysis conditions are given in the supplementary material.

Results and Discussion

3.1.Wastewater effluent basic parameters

The concentrations of PPs and the physico-chemical characterization of the raw WWE were determined in triplicate experiments. The most concentrated PPs were TRA and DCF with concentrations of 12.48 and 8.23 µg.L -1 , respectively. The concentrations of the other PPs mostly ranged between 0.03 and 1.32 µg.L -1 (Table 2).

The analysis of the WWE provided the following values: pH = 6.4, Total Organic Carbon content was 27.6 mg.L -1 , P-PO4 content was 12.0 mg.L -1 , N-NO2 content was 59.2 mg.L -1 , Suspended solids concentration was 5 mg.L -1 and the conductivity was 950 µS.cm -1 .

3.2.Removal of PPs from wastewater effluents

Due to the important role of the speciation on the affinity between PPs and clay minerals, the removal values after interaction with clay minerals are hereafter detailed according to the theoretical speciation of the PPs (Table 1).

Positively charged PPs

The removal values of cationic and zwitterionic PPs are presented in Figure 1. Except for NOR, which shows a high removal rate whatever the adsorbent, the removal rates of cationic PPs display the same pattern. The removal is lower after interaction with KGa2, whereas very similar removal values are found for SWy2 and Na-SWy2. Moreover, increasing the mass of adsorbent increases the removal values of PPs (Figure 1). In general, with 200 mg of montmorillonite, the removal rate ranges from 58 to 100 %. Yet, for several cationic PPs such as DOX and TRA, it was expected that this removal would be total within this range of concentration due to the high affinity between these PPs and clay minerals, and the adsorption through cation exchange [START_REF] Chen | Tramadol hydrochloride/montmorillonite composite: Preparation and controlled drug release[END_REF][START_REF] Thiebault | Adsorption mechanisms of emerging micropollutants with a clay mineral: Case of tramadol and doxepine pharmaceutical products[END_REF]. However, the complexity of WWE (e.g. inorganic and organic compounds) associated with the low starting concentration of PPs in comparison to total organic carbon (i.e. < 0.1%), appear to strongly hinder the adsorption of cationic PPs, suggesting that the cation exchange mechanism between compensating inorganic cations and cationic PPs is limited due to the competition with other compounds for cation exchange (Thiebault et al., 2016b;[START_REF] Wang | Role of interlayer hydration in lincomycin sorption by smectite clays[END_REF].

Neutral PPs

The removal rates of neutral PPs are presented in Figure 2. Various patterns are exhibited as a function of the PP and/or the mass of adsorbent. TMP is a particular case as its speciation in this pH range is partially neutral and partially protonated (Table 1). As a result, its behavior appears to be intermediate between neutral and cationic molecules. CBZ and OXA are poorly adsorbed onto clay minerals whatever the clay type and the mass of adsorbent; the only significant removal rates displayed are onto SWy2 (Figure 2). DIA is conversely significantly adsorbed onto the three adsorbents with higher removal values on increasing the mass of adsorbent, and higher removal values on SWy2 (i.e. between 5 and 20%) in comparison with the other two adsorbents (i.e.

between 0 and 11%). This variation between PPs with closely related properties highlights the important role of the starting concentrations of PPs in order to assess their affinity with clay minerals [START_REF] Thiebault | Adsorption mechanisms of psychoactive drugs onto montmorillonite[END_REF], and of the affinity of PPs with organic moieties for the adsorption onto clay minerals in effluent solutions [START_REF] Torrents | The sorption of nonionic pesticides onto clays and the influence of natural organic carbon[END_REF].

Anionic PPs

The removal rates of anionic PPs are displayed in Figure 3. Only SUL displays a total removal whatever the adsorbent, mostly due to its very low concentration in the selected effluent (Table 2).

The other PPs present a similar pattern with lower removal rates onto KGa2 and higher rates after interaction with SWy2. Weak removal variations are displayed between KGa2 and Na-SWy2

highlighting the limited affinity of anionic PPs for these two clay minerals. The higher removal rates observed after interaction with 200 mg SWy2 (i.e. between 38 and 78% except for SUL) emphasize the impact of the compensating inorganic cations. The only variation between SWy2

and Na-SWy2 is the homogenization of the compensating cations in the latter adsorbent. As a result, the significant amount of Ca 2+ in SWy2 (i.e. ~20%) appears to significantly enhance the adsorption extent of anionic PPs, or the adsorption of organic moieties which may adsorb such PPs in a second phase.

3.3.Adsorbent characterization

Prior to characterization, the clay minerals were separated from the supernatant after the interaction experiment by centrifugation. XRD patterns and FTIR spectra exhibited no significant layer expansion or band stretching, respectively (data not shown). The organic carbon content adsorbed onto clay after interaction was estimated from the elemental analyses. This bonded organic carbon can be either PPs or organic complexes present in the WWE.

All the clay minerals display an increase in the carbon content in comparison with the initial composition (Table 3). Moreover, due to the weak concentration of PPs in the raw effluents in comparison with the total organic carbon content (i.e. < 0.1%), it is highly possible that most of the adsorbed organic carbon was generated by other organic compounds, thus masking the PP contribution to the whole carbon content. The carbon content after interaction ranges from 0.34 % (or 0.08 % if we consider the carbon content of the raw adsorbent) for KGa2 to ~1 % (or 0.7% if we consider the carbon content of the raw adsorbent) for SWy2 (Table 3). On increasing the mass of adsorbent from 100 to 200 mg, the carbon content of the adsorbents did not significantly vary, highlighting that the adsorption of organic moieties onto these adsorbents may be controlled by the solid/water partition. Therefore, an increase in the mass of adsorbent proportionally increases the amount of organic carbon adsorbed. From a global point of view, the displayed pattern is equivalent for PP removal and carbon analyses. The adsorption is weak onto KGa2 whereas it is higher onto SWy2, emphasizing the important role of the properties of the adsorbent, and the impact of the inorganic compensating cations.

3.4.Comparative efficiency of clay minerals

The removal capacity of the three selected clay minerals appears to be enhanced when a higher mass of adsorbent is used. This is consistent with previous studies, which emphasized the contribution of the solid liquid partition coefficient in the adsorption of non-cationic PPs [START_REF] Stein | Analysis and sorption of psychoactive drugs onto sediment[END_REF]. Hence, the higher the solid/liquid ratio of adsorbent, the higher the removal rate.

However, significant variations are found in the efficiency of the tested adsorbents. KGa2 presents the lowest adsorption capacities of all the targeted PPs, whatever their charge state. This is probably due to its weak CEC and SSA, limiting the affinity with organic moieties in comparison with montmorillonites. Between SWy2 and Na-SWy2, the adsorption performance of the latter is slightly better for the removal of cationic PPs whereas the opposite pattern is displayed for neutral and anionic PPs. This can be attributed to the fact that the compensating inorganic cations of SWy2

are not only Na + , but also Ca 2+ in a significant proportion, allowing the formation of cationic bridges with negatively charged organic moieties such as anionic PPs, or organic complexes present in the effluents [START_REF] Aristilde | Enhanced interlayer trapping of a tetracycline antibiotic within montmorillonite layers in the presence of Ca and Mg[END_REF][START_REF] Zhang | Interfacial reaction between humic acid and Ca-Montmorillonite: Application in the preparation of a novel pellet binder[END_REF]. These results on PPs are confirmed by the elemental analyses, in which SWy2 displays the highest adsorbed amount of carbon. The latter adsorbent, therefore, presents the most suitable adsorption properties for the removal of PPs and potentially other organic contaminants, from WWE.

Conclusion

In this study, the potential of three clay minerals for the removal of PPs from raw WWE was studied for the first time in batch experiments, thereby deepening our understanding of the affinity between such adsorbents and PPs. The results clearly show that among the three adsorbents tested, KGa2

presents the lowest adsorption capacity for both PPs and other organic moieties present in the effluent. Secondly, the homogenization of the compensating cations of SWy2 (i.e. Na-SWy2) is not favorable for the adsorption of neutral and anionic PPs, which may interact with Ca 2+ cations through cationic bridges. This can be considered as a positive outcome, given the need to use as raw as possible material. Hence, SWy2 presents suitable adsorption properties in batch experiments for the removal of PPs from wastewater effluents. Finally, this work demonstrates that, in contrast to many studies on idealized solutions, the organic compounds present in raw WWE contribute to improving the adsorption capacity of neutral and anionic PPs by clay minerals in comparison to experiments conducted in pure water. These adsorbed organic moieties increase the hydrophobicity of the adsorbent and the number of adsorption sites. However, further studies remain necessary in order to transfer these results from batch experiments to dynamic ones, closer to the design of a field solution.

Figure 1 :

 1 Figure 1: Removal percentages of cationic (green bars) and zwitterionic (purple bars) PPs as a function of the mass of adsorbent (i.e. 100 and 200 mg), n=3

Figure 2 :

 2 Figure 2: Removal percentages of neutral PPs as a function of the mass of adsorbent (i.e. 100 and 200 mg), n=3

Figure 3 :

 3 Figure 3: Removal percentages of anionic PPs as a function of the mass of adsorbent (i.e. 100 and 200 mg), n=3

Table 1 :

 1 Physico-chemical properties of the targeted PPs, with Abb. the abbreviation, Mw the molecular weight in g.mol -1 , pKa the acid dissociation constant, log Kow the octanol/water partition coefficient, Sw the solubility in water at 25°C in mg.L -1 , and Charge the dominant form at the effluent pH (i.e. = 6.4)

	PP	Abb.	Formula	CAS-

Number Mw pKa a log Kow b Sw a Charge

	Norfloxacin	NOR	C16H18FN303	70458-96-7 319.3	5.7-8.7	-0.31	1,010 +/-
	Oxazepam	OXA	C15H11ClN2O2	604-75-1 287.0	1.7-11.6	3.34	88 0
	Sulfamethoxazole SUL	C10H11N3O3S	723-46-6 253.3	6.2	0.48	459 -
	Tramadol	TRA	C16H25NO2	27203-92-5 263.4	9.4	3.01	750 +
	Trimethoprim	TMP	C14H18N4O3	738-70-5 290.3	7.2	0.73	615 +/0
	With a drugbank.ca and b chemspider.com (predicted KOWWIN v.1.67)
	Atenolol	ATE	C14H22N2O3	29122-68-7 266.3	9.6	0.16	430 +
	Bezafibrate	BZB	C19H20ClNO4	41859-67-0 361.8	3.9	4.25	1.5 -
	Carbamazepine	CBZ	C15H12N2O	298-46-4 236.3	13.9	2.45	152 0
	Codeine	COD	C18H21NO3	76-57-3 299.4	8.2	1.28	577 +
	Diazepam	DIA	C16H13ClN2O	439-14-5 284.7	3.4	2.70	50 0
	Diclofenac	DCF	C14H11Cl2NO2	15307-79-6 296.1	4.2	4.02	4.5 -
	Doxepin	DOX	C19H21NO	1229-29-4 279.4	9.0	3.86	32 +
	Gemfibrozil	GEM	C15H22O3	25812-30-0 250.3	4.8	4.33	28 -
	Ketoprofen	KET	C16H14O3	22071-45-4 254.3	4.4	3.00	21 -
	Metoprolol	MET	C15H25NO3	56392-17-7 267.4	9.6	1.69	502 +
	Naproxen	NAP	C14H14O3	22204-53-1 230.3	4.2	3.10	16 -
							4

Table 2 :

 2 Concentrations of the targeted PPs (in µg.L -1 ) in the raw effluent (Blank) and after interaction with 100 and 200 mg of the selected clay minerals, with LOD the limit of detection

	PP	Blank	100mg	KGa2	200mg	Na-SWy2 100mg 200mg	100mg	SWy2	200mg
	ATE	0.55 ± 0.10	0.52 ± 0.16	0.46 ± 0.10	0.40 ± 0.13	0.25 ± 0.00	0.42 ± 0.15	0.23 ± 0.10
	BZF	0.19 ± 0.08	0.14 ± 0.03	0.13 ± 0.04	0.12 ± 0.06	0.14 ± 0.09	0.11 ± 0.05	0.05 ± 0.01
	CBZ	0.34 ± 0.03	0.34 ± 0.04	0.33 ± 0.04	0.33 ± 0.03	0.34 ± 0.02	0.31 ± 0.06	0.31 ± 0.07
	COD	0.03 ± 0.01	0.02 ± 0.01	0.01 ± 0.00	0.00 ± 0.01	< LOD	0.00 ± 0.00	0.00 ± 0.00
	DIA	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.01	0.02 ± 0.01	0.02 ± 0.01	0.02 ± 0.01	0.01 ± 0.01
	DCF	8.23 ± 1.69	7.46 ± 0.86	6.87 ± 1.59	8.05 ± 0.59	7.84 ± 1.99	6.91 ± 0.50	5.19 ± 0.42
	DOX	0.84 ± 0.09	0.80 ± 0.08	0.67 ± 0.36	0.74 ± 0.26	0.30 ± 0.07	0.50 ± 0.41	0.07 ± 0.02
	GEM	0.06 ± 0.02	0.06 ±0.01	0.06 ± 0.02	0.06 ±0.02	0.05 ± 0.01	0.05 ± 0.03	0.03 ± 0.01
	KET	0.62 ± 0.04	0.58 ± 0.09	0.53 ± 0.11	0.58 ± 0.04	0.53 ± 0.14	0.51 ± 0.06	0.20 ± 0.16
	MET	0.07 ± 0.03	0.06 ± 0.01	0.05 ± 0.02	0.04 ± 0.01	0.03 ± 0.02	0.05 ± 0.01	0.03 ± 0.01
	NAP	1.32 ± 0.10	1.43 ± 0.31	1.35 ± 0.31	1.38 ± 0.18	1.13 ± 0.87	1.16 ± 0.15	0.58 ± 0.26
	NOR	0.92 ± 0.78	0.03 ± 0.03	< LOD	0.12 ± 0.21	< LOD	0.28 ± 0.49	< LOD
	OXA	0.16 ± 0.01	0.15 ± 0.01	0.14 ± 0.03	0.15 ± 0.02	0.15 ± 0.03	0.14 ± 0.01	0.12 ± 0.03
	SUL	0.00 ± 0.01	< LOD		< LOD	< LOD	< LOD	< LOD		< LOD
	TRA	12.48 ± 1.04	11.55 ± 0.61	10.6 ± 2.11	2.83 ± 1.35	2.87 ± 0.46	8.37 ± 7.78	3.47 ± 4.30
	TMP	0.05 ± 0.02	0.05 ± 0.03	0.05 ± 0.04	0.03 ± 0.02	0.03 ± 0.01	0.04 ± 0.04	0.02 ± 0.01

Table 3 :

 3 Carbon percentages of raw adsorbents (Raw) and after interaction (i.e. 100 and 200 mg) with the

	effluent, n=3
		KGa2	Na-SWy2	SWy2
	Raw	0.256 ± 0.02	0.214 ± 0.03	0.312 ± 0.05
	100 mg	0.336 ± 0.03	0.648 ± 0.04	1.027 ± 0.05
	200 mg	0.341 ± 0.05	0.581 ± 0.07	0.959 ± 0.06

Acknowledgements

This study received financial support from the HArPE Project (2012-00073536) funded by the Région Centre Val de Loire. M. Hatton is gratefully thanked for carbon elemental analyses.


Blandine Courel
email: bcourel@britishmuseum.org

Philippe Schaeffer

Clément Féliu
email: clement.feliu@inrap.fr

Yohann Thomas
email: yohann.thomas@inrap.fr

Pierre Adam
email: padam@unistra.fr

Birch bark tar and jewellery: the case study of a necklace from the Iron Age (Eckwersheim, NE France)

Keywords: birch bark tar, conifer resin, Early Iron Age, necklace, jewellery, lupane-related triterpenoids

come

Introduction

Archaeological evidence of birch bark tar, an adhesive substance obtained by dry pyrolysis of birch bark (e.g. [START_REF] Regert | Identification of Neolithic hafting adhesives from two lake dwellings at Chalain (Jura, France)[END_REF][START_REF] Koller | High-tech in the Middle Palaeolithic: Neandertalmanufactured pitch identified[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF], is abundantly documented in the literature. Considered as one of the first man-made organic material, birch bark tar is frequently present among archaeological finds dated from the Neolithic period in Europe and its first use by Neanderthalians is dated back to the Palaeolithic [START_REF] Koller | High-tech in the Middle Palaeolithic: Neandertalmanufactured pitch identified[END_REF]. This sticky and hydrophobic material has been used for many purposes such as the hafting of lithic or bone tools (e.g. [START_REF] Regert | Identification of Neolithic hafting adhesives from two lake dwellings at Chalain (Jura, France)[END_REF][START_REF] Koller | High-tech in the Middle Palaeolithic: Neandertalmanufactured pitch identified[END_REF], the reparation and waterproofing of pottery (e.g. [START_REF] Binder | Identification de brai de bouleau (Betula) dans le Néolithique de Giribaldi (Nice, France) par la spectrométrie de masse[END_REF][START_REF] Version Avant ; Charters | Identification of an adhesive used to repair a Roman jar[END_REF][START_REF] Connan | Apport de la chimie moléculaire à la connaissance des enduits utilisés pour le traitement des bois et le calfatage des bateaux romains de la Saône[END_REF]Urem-Kotsou et al., 2002;[START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF], the decoration of ceramics (e.g. [START_REF] Vogt | The birch as a source of raw material during the Stone Age[END_REF][START_REF] Trąbska | Black painted pottery, Kildehuse II, Odense County, Denmark[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF] and, more surprisingly, as chewing-gum (e.g. [START_REF] Aveling | Chewing tar in the early Holocene: an archaeological and ethnographic evaluation[END_REF][START_REF] Van Gijn | Birch bark tar[END_REF][START_REF] Karg | Vegetal grave goods in a female burial on Bornholm (Denmark) from the Late Roman Iron Age period interpreted in a comparative European perspective[END_REF]. The identification of birch bark tar mainly relies on the detection of lupane-related triterpenoids as diagnostic molecular biomarkers. These compounds possess indeed ideal features since lupane-related triterpenoids are abundant in birch tar and their assemblage can be considered as highly specific [START_REF] Hayek | A bicentennial of betulin[END_REF][START_REF] Schnell | Contrasting diagenetic pathways of higher plant triterpenoids in buried wood as a function of tree species[END_REF]. In addition, these compounds are relatively resistant to various alteration processes such as biodegradation which probably accounts for their good preservation even in ancient archaeological samples dating back to the Palaeolithic and Mesolithic Periods [START_REF] Koller | High-tech in the Middle Palaeolithic: Neandertalmanufactured pitch identified[END_REF][START_REF] Aveling | Identification of birch bark tar at the Mesolithic site of Star Carr[END_REF].

We report here the investigation by gas chromatography-mass spectrometry (GC-MS) of the lipid content of a dark organic substance of unknown nature that has served to assemble two bronze half-spheres in order to form the pendant of a necklace (Fig. 1) dating to the Hallstatt D1 period (625-550 BC). Due to the predominance of lupane-related triterpenoids in the lipid extract, their origin (genuine biological compounds vs. triterpenes altered by natural or anthropic processes) is discussed in the light of the molecular investigation of reference samples of birch bark and birch bark tars.

Materials and method

Archaeological sample

Sample A (INRAP -Institut National de Recherches Archéologiques Préventives -reference number: PRL BG 6008-02) corresponds to a dark substance found between two small bronze

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half-spheres which were assembled to create the pendant of a necklace (Fig. 1). The jewellery was uncovered among the furniture and artefacts of the tomb 6008 unearthed at Eckwersheim (Alsace, NE France) by an INRAP team led by Dr. Clément Féliu and Dr. Yohann Thomas.

The tomb was dated to the Early Iron Age and the necklace between 625 BC and 550 BC (Hallstatt D1 period).

Reference samples

The reference samples comprise: (1) the lipid extract of pieces of birch bark (Betula pendula) from a trunk in an advanced state of alteration (sample B); (2) the lipid extract of pieces of the same birch bark sample pyrolysed with a heat gun during 10 minutes under N 2 atmosphere (sample C); (3) a birch bark tar from Betula pendula prepared by descending distillation (sample D, reference number 162/5 20 18 00, Dr. Andreas Kurzweil, Museumsdorf Düppel, Berlin, Germany).

Lipid analysis

Samples A -D were extracted by sonication using a mixture of dichloromethane/methanol (CH 2 Cl 2 /CH 3 OH, 1:1, v/v) followed by filtration of the supernatant through celite and removal of the solvent under reduced pressure. An aliquot of the extract in CH 2 Cl 2 was acetylated (Ac 2 O, N-methylimidazole, 30 min, ambient temperature) and, after removal of the solvents and excess reagents, treated with a solution of diazomethane in diethylether to methylate the carboxylic acids. The derivatised crude extract was fractionated on a silica gel column into an apolar fraction eluted with CH 2 Cl 2 /EtOAc (8:2, v/v) which was analysed by GC-MS and a more polar fraction eluted with CH 2 Cl 2 / CH 3 OH (1:1, v/v) which was not further investigated.

GC-MS

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GC-MS analyses were carried out using a Thermo Trace gas chromatograph (Thermo Scientific) equipped with an autosampler Tri Plus, a programmed temperature vaporizing (PTV) injector and a HP5-MS column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) using He as carrier gas (constant flow rate at 1.1 ml/min). Temperature program: 70 °C -200 °C (10 °C/min), 200 °C -300 °C (4 °C/min), isothermal at 300 °C (40 min). The mass spectrometer was operating in the electron ionization (EI) mode at 70 eV with a scan range of 50 to 700 m/z. The data were investigated using Xcalibur Software and mass spectra were compared with the NIST library and literature data.

Results and discussion

Triterpenoids of the lupane series: birch bark as main ingredient

GC-MS analysis of the organic extract of the substance from the pendant (sample A) led to the identification of specific triterpenoids from the lupane series, indicating a predominant contribution from an angiosperm source. These triterpenoids comprise, notably, lupeol 1 (numbers refer to structures presented in the Appendix), betulin 2, lupenone 3, betulone 4, lupanone 5 and lupan-3,28-diol 6 (Fig. 2) which are important triterpenes of birch bark [START_REF] Hayek | Identification of archaeological and recent wood tar pitches using gas chromatography/mass spectrometry and pattern recognition[END_REF][START_REF] Krasutsky | Analytical characterization of diterpenoid resins present in pictorial varnishes using pyrolysis-gas chromatography-mass spectrometry with on line trimethylsilylation[END_REF][START_REF] Schnell | Contrasting diagenetic pathways of higher plant triterpenoids in buried wood as a function of tree species[END_REF]. The predominance of the same compounds among the lipids of the reference birch bark samples B-D (Fig. 3) was thus not unexpected. Betulin 2, in particular, largely predominates the gas chromatogram of the lipids from the birch bark sample B, representing ca. 60 % of the apolar lipids. The identification of triterpenoids from the lupane series in the archaeological sample A points thus clearly to birch bark tar which may have been used as an adhesive material. It can be proposed that bark of either Betula pendula (silver bitch) or Betula pubescens (downy birch) served as raw material for the preparation of the adhesive since they are the most common Betula species in Europe [START_REF] Beck | Betula pendula, Betula pubescens and other birches in Europe: distribution, habitat, usage and threats[END_REF].

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Altered triterpenoids as markers for ageing and thermal treatment

The distribution of the triterpenoids in the archaeological sample (Fig. 2) is, however, significantly different from that observed with fresh birch bark (Fig. 3). This can likely be explained by the various alteration processes affecting triterpenoids upon the thermal treatment used for the preparation of the substance, in addition to other processes induced by oxidative ageing and/or by diagenetic alteration during burial in the soil.

Such processes may notably account for the lower proportion of betulin 2 in the archaeological sample as compared to the large predominance of 2 in fresh birch bark (Fig. 3; [START_REF] Hayek | A bicentennial of betulin[END_REF], as well as for the absence of betulonal 7 and betulinic acid 8 -two native triterpenoids from birch bark (cf. sample B; Fig. 3)-in the archaeological material (Fig. 2).

In parallel with the absence of some genuine lupane-related triterpenoids from birch bark, three families of triterpenes likely resulting from alteration processes were detected in sample A. They comprise A-neo-triterpenoids (e.g., 9, 10),  2 triterpenoids (e.g., 12, 13), and allobetulane derivatives (e.g., 14-16).  2 lupane-derivatives 12 and 13 detected in samples A, C and D most likely originate from the thermally-induced dehydration of lupeol 1 and betulin 2 and can be considered as typical pyrolytic compounds. Their formation was observed, notably, during a controlled pyrolysis experiment of birch bark by [START_REF] Regert | Molecular characterisation of birch bark tar by headspace solid-phase microextraction gas chromatography-mass spectrometry: a new way for identifying archaeological glues[END_REF] and they generally occur in archaeological birch bark tars (e.g. [START_REF] Binder | Identification de brai de bouleau (Betula) dans le Néolithique de Giribaldi (Nice, France) par la spectrométrie de masse[END_REF][START_REF] Version Avant ; Charters | Identification of an adhesive used to repair a Roman jar[END_REF]Avelling and Heron, 1998;[START_REF] Regert | Adhesive production and pottery function during the Iron Age at the site of Grand Aunay (Sarthe, France)[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF].

Allobetulane derivatives (11, 14-16; mass spectra in Fig. S1) were also present in samples A, C and D. These compounds are postulated to be formed by an acid-catalysed intramolecular rearrangement of ring E [START_REF] Green | Isolation of betulin and rearrangement to allobetulin. A biomimetic natural product synthesis[END_REF][START_REF] Salvador | Bismuth triflate-catalyzed Wagner-Meerwein rearrangement in terpenes. Application to the synthesis of the 18-oleanane core and A-neo-18-oleanene compounds from lupane[END_REF]. Since these compounds exclusively occur in tars (sample D) and not in birch bark (sample B), they are thus considered, like  2 derivatives, to be closely associated to the thermal alteration undergone by

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birch bark during tar preparation (cf. [START_REF] Heron | Chromatography in archaeology[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF]. The acid catalysis necessary for the intramolecular rearrangement of ring E might have been provided by phenols formed by the pyrolytic degradation of lignin [START_REF] Faix | Thermal degradation products of wood[END_REF].

The A-neo-triterpenoids found in sample A (9 and 10; MS data shown in Fig. S2 in Supplementary data) and in the reference thermally-treated samples C and D (9-11) derive, respectively, from lupeol 1, betulin 2 and allobetulin 16 by contraction of ring A. Their formation most likely involves the acid-catalysed loss of the C-3 alcohol moiety followed by a Wagner-Meerwein rearrangement [START_REF] Salvador | Bismuth triflate-catalyzed Wagner-Meerwein rearrangement in terpenes. Application to the synthesis of the 18-oleanane core and A-neo-18-oleanene compounds from lupane[END_REF]. To our knowledge, these compounds have not been reported from fresh birch bark and might have two distinct origins.

They can be formed during the preparation of the tar as shown by [START_REF] Rageot | Les substances naturelles en Méditerranée nord-occidentale (VI ème -I er Millénaire BCE): Chimie et archéologie des matériaux exploités pour leurs proprietés adhésives et hydrophobes[END_REF] in the case of birch bark tars prepared in the laboratory, the acid catalysis necessary for their formation being possibly induced, as mentioned above, by phenols formed upon lignin pyrolysis [START_REF] Faix | Thermal degradation products of wood[END_REF]. In addition, [START_REF] Rageot | Les substances naturelles en Méditerranée nord-occidentale (VI ème -I er Millénaire BCE): Chimie et archéologie des matériaux exploités pour leurs proprietés adhésives et hydrophobes[END_REF] has shown that their relative abundance is correlated with the experimental conditions, higher temperatures or longer heating periods likely favoring their formation. Nevertheless, the detection of A-neo-triterpenoids, even in small amounts, in the altered birch bark sample B indicates that natural alteration processes may also account to some extent for their formation [START_REF] Ten Haven | Early diagenetic transformation of higher-plant triterpenoids in deep-sea sediments from Baffin Bay[END_REF].

Input of conifer resin attested by diterpenoids

Besides the predominant lupane-related triterpenoids reported above, GC-MS analysis of the organic extract of the substance from the pendant (sample A) revealed an additional contribution of small amounts of early eluted compounds corresponding to diterpenoids.

They comprise di-dehydroabietic acid 17, dehydroabietic acid 18, 7-oxodehydroabietic acid 19 along with the related compounds 20 and 21 bearing a hydroxyl function at C-15. All of them derive from abietic acid 22, a diterpenic acid ubiquitously found in conifer resins (Otto

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and Wilde, 2001) and especially abundant in resins from Pinaceae [START_REF] Otto | Terpenoids as chemosystematic markers in selected fossil and extant species of pine (Pinus, Pinaceae)[END_REF]. The contribution of these diterpenoids revealed thus the presence of conifer material in the archaeological substance which could either correspond to a raw resin or a tar made by dry distillation of conifer wood (e.g. [START_REF] Evershed | Pine wood origin for pitch from the Mary Rose[END_REF][START_REF] Connan | Conifer tar on the keel and hull planking of the Ma'agan Mikhael Ship (Israel, 5 th century BC): identification and comparison with natural products and artefacts employed in boat construction[END_REF][START_REF] Bailly | Caractérisation moléculaire et isotopique de goudrons et résines archéologiques dérivés de conifères en contexte maritime[END_REF]. However, in the case of conifer tars, aromatic diterpenoids like retene 23 resulting from the thermal transformation of resinic acids upon pyrolysis are generally abundant. In the present case, such compounds could not be detected, suggesting that the use of a conifer tar can be ruled out. Thus, the ingredient corresponds most likely to a conifer resin. In addition, the identification of oxidized derivatives of 18, comprising compounds with a ketone at C-7 (19 and 21) or a hydroxyl group at C-15 (20 and 21), suggests that the resin was severely altered by oxidative processes during the ageing of the adhesive substance [START_REF] Colombini | Direct exposure electron ionization mass spectrometry and gas chromatography/mass spectrometry techniques to study organic coatings on archaeological amphorae[END_REF]Osete-Cortina and Doménech-Carbó, 2005;[START_REF] Bailly | Caractérisation moléculaire et isotopique de goudrons et résines archéologiques dérivés de conifères en contexte maritime[END_REF].

Mode of preparation of the adhesive -Origin of the conifer resin component

The presence of lupane-related biomarkers which have obviously undergone thermal alteration (formation of  2 -triterpenoids and allobetulane derivatives, notably; see above) in sample A clearly indicates that the organic substance corresponds to an adhesive material (birch bark tar) used to assemble the pendant. However, the contribution of small proportions of conifer resin raises the question whether the addition of the latter was intentional or was the result of a "contamination" during birch bark tar preparation. Indeed, a few examples of mixtures of archaeological birch bark tar containing additional substances have been reported by several authors. These substances comprise animal fat, beeswax, plant oil [START_REF] Dudd | Unusual triterpenoid fatty acyl ester components of archaeological birch bark tars[END_REF][START_REF] Regert | Adhesive production and pottery function during the Iron Age at the site of Grand Aunay (Sarthe, France)[END_REF][START_REF] Van Gijn | Birch bark tar[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF] and, in some rare cases, conifer resin/tar [START_REF] Stacey | Evidence for the use of birch-bark tar from Iron Age Britain[END_REF][START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF]. The input of additional substances can sometimes be intentional in order to improve the properties of the material.

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For instance, the addition of beeswax to birch bark tar leads to a substance which is less brittle than pure birch bark tar [START_REF] Regert | Adhesive production and pottery function during the Iron Age at the site of Grand Aunay (Sarthe, France)[END_REF][START_REF] Van Gijn | Birch bark tar[END_REF]. It is interesting also to note that [START_REF] Stacey | Evidence for the use of birch-bark tar from Iron Age Britain[END_REF] and [START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF] reported the identification of adhesives very similar in molecular composition to sample A, and which consisted of small amounts of conifer resin mixed with birch bark tar. They were used, respectively, for gluing coral studs on strap unions from harness fittings [START_REF] Stacey | Evidence for the use of birch-bark tar from Iron Age Britain[END_REF] and for the reparation of pottery [START_REF] Rageot | Exploitation of beehive products, plant exudates and tars in Corsica during the Early Iron Age[END_REF]. This supports the hypothesis that the admixture of small amounts of conifer resin to birch bark tar might possibly improve its performances as an adhesive and that such mixtures have been prepared on purpose for this specific application.

However, based on the relatively small contribution of the conifer resin as compared to the birch bark tar in the case of sample A, it cannot be excluded that the container used for the storage or the production of birch bark tar may have served previously to collect or store a conifer resin and might have thus contaminated the birch bark tar. It should however be mentioned that the relatively low proportion of diterpenes relative to triterpenes observed in sample A does not necessarily reflect the initial proportions of conifer resin and birch bark tar making up the adhesive. Indeed, resinic acids might be significantly more sensitive to oxidative alteration given the presence of reactive benzylic positions on dehydroabietic acid 18 and related structures as illustrated by the detection of several oxidized dehydroabietic acid derivatives (19-21) in sample A. The presence of several oxygenated functionalities on these structures might thus be responsible for an enhanced water solubility as compared to lupane-related triterpenoids, thus favouring their progressive removal by leaching, notably after burial.

Conclusion

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Identification of triterpenoids from the lupane series in the organic material found on an archaeological necklace pendant clearly indicates that this substance was made predominantly of birch bark tar, together with a small contribution of conifer resin. This material likely corresponds to an adhesive that could have served to fix a decorative item on the pendant. The question remains regarding the role of the conifer resin which could either be part of the ingredients added intentionally to improve the quality of the adhesive material, or correspond to a "contamination" introduced during birch bark tar preparation.

Figure captions

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Figure 1 .

 1 Figure 1. Necklace (a) with a pendant (b) found among the furniture and artefacts of a tomb

Figure 2 .

 2 Figure 2. Gas chromatogram of the lipid extract of the adhesive of the necklace pendant

Figure 3 .

 3 Figure 3. Gas chromatograms of the organic extract of (a) altered birch bark sample (sample

Figure S2 :

 S2 Figure 1

Acknowledgement

We thank A. Kurzweil for the birch bark tar reference sample produced according to traditional techniques (Museumsdorf Düppel, Berlin). The INRAP is thanked for providing the archaeological sample. B.C. thanks the French Ministère de l'Enseignement Supérieur et de la Recherche for a doctoral fellowship.

Appendix

Structures cited in the text

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"And now, speak:" Emotional Manipulation in The Taming of the Shrew, The Winter's Tale and Macbeth

Dominika Kecsöová, Charles University, Prague Emotional manipulation as a form of violence, despite beginning to be widely discussed in recent years, still belongs among secondary characteristics of domestic abuse; since it is, by definition, "an interpersonal emotional construct," 1 its lasting effects remain invisible, but often prove more damaging than physical injuries. The harm is inflicted through "symbolic acts, humiliation, and verbal threats." 2 Identifying emotional manipulation and reacting to it adequately presents a complex issue even these days, despite abundant examples in literature. 3 This essay shall analyse emotional manipulation within partnerships in three plays of William Shakespeare: The Taming of the Shrew, The Winter's Tale and Macbeth.

Each of these plays falls within a different genre, which not only proves the ubiquity of such manipulation, but also reveals the underlying power structures fuelling these behaviours.

Emotional manipulation is used by both male and female characters as a vehicle of power; the violence inflicted on the other partner relies entirely on language, and therefore emotional manipulation should be considered in relation to rhetoric. This becomes apparent especially within Brummett's understanding of rhetoric as violence, in his words "an act that influences and manages the meanings victims experience through the course of their relationships, whether done through physical means or not." 4 The experience of the victim is thus dependent on the contrast between reality and the way it is constructed by the manipulator. These three main themes -power, rhetoric and reality -appear in all instances of emotional manipulation in The Taming of the Shrew, The Winter's Tale and Macbeth and provide the basis for analysing the manipulation in these plays.

The Taming of the Shrew, one of Shakespeare's "problem plays" may not even seem like a comedy to 21 st century audiences -as Elizabeth Hutcheon states, if we are reading Katharina as "cowed into submission by Petruchio's tactics, it is hard to participate in the comedic happiness of the play's ending." 5 Before the play itself, the induction presents manipulation (or, to be more particular, gaslighting) 6 as a device to entertain the audience; however, this very device makes it possible to consider the play in a slightly different light.

As shown in the induction, the main contrast informing The Taming of the Shrew is reality versus illusion. This contrast then raises the question of Katharina's shrewishness (Is the perception of her as a shrew real?) and of the success of Petruchio's manipulation (Is she really "tamed" by the end of the play?). The text offers only partial answers, and of course, its interpretation depends on staging. Nevertheless, this demonstrates that the key to emotional manipulation is perception, and the rest of the characters in the play perceive Katharina as she seems in Hortensio's description in Act I: "Her only fault, and that is faults enough, / Is that she is intolerable curst / And shrewd and forward" (I.ii.86-9). 7 The rhetoric power wielded by the characters has the potential to influence reality; Petruchio begins his manipulation of Katharina by manipulating her image, as he openly states in his monologue in Act II: I will attend her here, And woo her with some spirit when she comes. Say that she rail; why then I'll tell her plain She sings as sweetly as a nightingale: Say that she frown, I'll say she looks as clear As morning roses newly wash'd with dew: Say she be mute and will not speak a word; Then I'll commend her volubility, And say she uttereth piercing eloquence: If she do bid me pack, I'll give her thanks, As though she bid me stay by her a week: If she deny to wed, I'll crave the day When I shall ask the banns and when be married. But here she comes; and now, Petruchio, speak. (II.i.167-180) 6 The Cambridge Dictionary defines gaslighting as "the action of tricking or controlling someone by making them believe things that are not true." This tactic appears most prominently in The Taming of the Shrew and constitutes a psychological abuse. S.v. "gaslighting", Cambridge Advanced Learner's Dictionary and Thesaurus (2008) The Cambridge Dictionary Online <https://dictionary.cambridge.org/dictionary/english/gaslighting> 27 July 2019. 7 This and all following quotations are from The Complete Works of William Shakespeare, The Alexander Text (London: HarperCollins Publishers, 2006).

Petruchio's manipulation is based on his rhetorical ability and his goal is to convince both the audience and Katharina: the audience should stop seeing Katharina as a shrew, while she herself is supposed to change her behaviour on the basis of this rhetorical re-description, despite the fact that Katharina's shrewishness is a rhetorical construct perpetuated by other characters to begin with. Katharina, however, proves a worthy opponent in this verbal sparring, so that while her "shrewishness" is connected to her being "an inappropriate speaker,"8 her speeches are not as unintelligible as Elizabeth Hutcheon would have them.

Hutcheon interprets Petruchio's behaviour as education in rhetoric, claiming that by the end of the play Katharina becomes a speaking subject;9 this education, however, is conducted through emotional manipulation. Rather than providing Katharina with rhetorical power, Petruchio intends to overturn her presentation of herself.10 Act IV consequently abounds with scenes of manipulation, as Petruchio begins his project of "taming" proper, which he envisages as a sort of guide for other husbands. To assist his manipulation, Petruchio uses physical means such as lack of food and sleep: "She eat no meat to-day, nor none shall eat; / Last night she slept not, nor to-night she shall not...This is a way to kill a wife with kindness; / And thus I'll curb her mad and headstrong humour" (IV.i. 181-2, 192-3). The label of the "shrew" may at this point be attached to him as well, since he mistreats not only Katharina (though he may call it "kindness"), but also his servants: PETRUCHIO Enter one with water Come, Kate, and wash, and welcome heartily. You whoreson villain! will you let it fall? Strikes him KATHARINA Patience, I pray you; 'twas a fault unwilling. (IV.i.138-140) Katharina, on the other hand, defends the servants against Petruchio in all instances; when not constructed as a shrew, her behaviour is far from what the audience would expect if the label were true. In the following scenes Petruchio "imposes activity, mobility, dress, thoughts" 11 in a way that, based on a study by Josie Queen, Margaret H. Brackley, and Gail B. Williams, would constitute abuse. Despite Petruchio's gaslighting, Katharina refuses "to enter the fictional space that Petruchio has constructed, she holds onto her experience of reality in the face of his presentation of it," 12 insisting on her right of speech: "I am no child, no babe: / Your betters have endured me say my mind, / And if you cannot, best you stop your ears" (IV.iii.74-6). Speech is the defining element of the "shrew" label in the play and also a way to resist manipulation; no wonder then that Katharina exclaims "I will be free / Even to the uttermost, as I please, in words" (IV.iii.79-80). She is soon stripped of this as her last freedom -but through her acceptance of Petruchio's perception of the world, the play highlights its own illusory nature. It is not important what Katharina thinks, as long as her behaviour and speech are the image of the "tamed" wife. After Petruchio gains his ends, he begins to present reality: KATHARINA Forward, I pray, since we have come so far, And be it moon, or sun, or what you please: An if you please to call it a rush-candle, Henceforth I vow it shall be so for me. PETRUCHIO I say it is the moon. KATHARINA I know it is the moon. PETRUCHIO Nay, then you lie: it is the blessed sun. KATHARINA Then, God be bless'd, it is the blessed sun: But sun it is not, when you say it is not; And the moon changes even as your mind. What you will have it named, even that it is; And so it shall be so for Katharine. (IV.v.12-22)

The manipulation in The Taming of the Shrew is based on opposites and is made possible by rhetorical construction. Katharina's final speech is the most divisive aspect of the whole play: the text presents us with what seems to be Petruchio's rhetorical victory, even though it is greatly helped by physical effects of lack of sleep and food. Yet Katharina's speech is curiously unconvincing in comparison with the rest of the play -the nature of her "taming" remains largely dependent on decisions of director and actors staging the play.

Unlike The Taming of the Shrew, the emotional manipulation in The Winter's Tale is closely linked to jealousy, as Leontes, king of Sicilia, attempts to sacrifice his wife in order to achieve rest, relief and social order; 13 the reason for his behaviour is that Leontes believes his wife, Hermione, has been unfaithful to him with his friend, Polixenes, king of Bohemia.

Leontes' manipulation places Hermione in the position of an adulteress, so that he (similarly to Petruchio) manipulates not only his wife, but also the audience and the court within the play. His jealous conviction is, however, dependent upon witnesses: LEONTES You, my lords, Look on her, mark her well; be but about To say 'she is a goodly lady,' and The justice of your hearts will thereto add ''Tis pity she's not honest, honourable:' (…) Ere you can say 'she's honest:' but be't known, From him that has most cause to grieve it should be, She's an adulteress. HERMIONE Should a villain say so, The most replenish'd villain in the world, He were as much more villain: you, my lord, Do but mistake. (II.i. 64-8, 76-80) Leontes in the rhetoric construction of his wife places, similarly to Renaissance painters, one hidden flaw in Hermione's perfection: he formulates a complex argument in which his wife is the ideal woman, but due to a loss of honesty he cannot respect her perfection anymore. This construction of Hermione serves not only to manipulate Leontes' audience, but also to convince himself; in fact, the amount of obsessively jealous soliloquies points to Leontes' need to literally speak his wife's unfaithfulness into reality. The manipulation directed towards Hermione is not as clear as Petruchio's was, partially because Hermione refuses to yield -her death and resurrection can be interpreted as a kind of emotional manipulation too, conducted vicariously through Paulina. The queen's words "How will this grieve you, / When Both Leontes and Hermione manipulate the emotions of the audience using variety of tactics, including "public performance, persuasion, the strategic use of amplification of coalitions and outrage, and emotional entrepreneurship" 14 all of which can be seen in Act II; however, it is Leontes who is less sure of his power. When Hermione says "The king's will be perform'd!" (II.i.115) Leontes replies in the same line by asking "Shall I be heard?" (II.i.115).

This once again reveals that at the centre of emotional manipulation is a power structure considered destabilised by one of the characters: emotional manipulation presents a means of gaining power in a relationship. Hermione's political and personal power should not be underestimated, though it depends upon the support she has as a queen: she affirms her position through her refusal to accept Leontes' fantastic accusations. The conflict between Leontes and Hermione is not only that of illusion and reality (in relation to Hermione's adultery), but also a battle for power conducted through rhetoric virtuosity. Public performance of such rhetoric in court (both judicial and royal) highlights the nature of manipulation as a power-based interpersonal structure. The illusory nature of this power is articulated by Hermione in her defence in Act III: HERMIONE Sir, You speak a language that I understand not: My life stands in the level of your dreams, Which I'll lay down. LEONTES Your actions are my dreams; You had a bastard by Polixenes, And I but dream'd it. As you were past all shame, -Those of your fact are so -so past all truth: Which to deny concerns more than avails; for as Thy brat hath been cast out, like to itself, No father owning it, -which is, indeed, More criminal in thee than it, -so thou Shalt feel our justice, in whose easiest passage 14 Rose McDermott., "Leadership and the Strategic Emotional Manipulation of Political Identity: An Evolutionary Perspective," The Leadership Quarterly (2018): 5, ScienceDirect <https://www.sciencedirect.com/science/article/pii/S1048984318301310?via%3Dihub> 20 July 2019.

Look for no less than death. (III.ii.76-89) After Hermione's presumed death, Paulina assumes her role as a voice of conscience; she accuses Leontes of tyranny and murder -reminded of his acts and the tragedy they lead to, Leontes repents through Paulina's manipulation. Emotional manipulation, therefore, is not necessarily only malevolent: while Leontes uses manipulation to make certain illusory perceptions seem true, Paulina's manipulation ensures the happy ending of the play.

In Macbeth, on the other hand, emotional manipulation leads to real death: while all the previous marriages could be described as psychosexual wars, this one is particularly striking, as lady Macbeth manipulates her husband without him being aware of it. When she argues for the murder of Duncan, she does not reference power (already attained or to be still attained) as a motivating factor to spur her husband into action; rather, she constantly mocks and devalues his manhood. Macbeth acts because he perceives a lack of power as a threat to his selfhood: LADY MACBETH Was the hope drunk Wherein you dress'd yourself? hath it slept since? And wakes it now, to look so green and pale At what it did so freely? From this time Such I account thy love. Art thou afeard To be the same in thine own act and valour As thou art in desire? Wouldst thou have that Which thou esteem'st the ornament of life, And live a coward in thine own esteem, Letting 'I dare not' wait upon 'I would,' Like the poor cat i' the adage? MACBETH Prithee, peace: I dare do all that may become a man; Who dares do more is none. (I.vii.35-47) Lady Macbeth deliberately describes violence as valour and aggression as sexual prowess: her manipulation is highly effective, since Macbeth echoes her ideas for the rest of the playwhen he is about to commit regicide, he does so with "Tarquin's ravishing strides" (II.i.55), describing his act as a rape. Lady Macbeth aligns violence with masculinity and then presents it as desirable: "When you durst do it, then you were a man; / And, to be more than what you were, you would / Be so much more the man" (I.vii.49-51). To achieve this, she manipulates Macbeth's anxiety and highlights threats to their shared identity. 15 She assumes a position of masculinity: in the context of Galenic one-sex model, her call to spirits to "unsex" her is perceived as a possibility -she can become a man not only through her behaviour, but also physically. If Macbeth does not dare to act, thus forfeiting his masculinity, she will assume it (and the power related to it) for herself. In this particular marriage, masculinity is the symbol of power and Macbeth and lady Macbeth seem unable to wield it both at the same time.

The banquet scene in Act III most clearly demonstrates the way lady Macbeth uses manipulation to gain power; when Macbeth reacts to Banquo's ghost, she mocks this reaction as unmanly, going as far as to outright ask Macbeth "Are you a man?" (III.iv.58) As in the previous plays, lady Macbeth attempts to control the way her husband sees reality, when she denies the existence of the ghost. Thus, when the ghost enters the second time, Macbeth feels compelled to repeat several times that his reaction is not due to lack of manliness and compares his behaviour to that of lady Macbeth:

You make me strange even to the disposition that I owe, When now I think you can behold such sights And keep the ruby of your cheeks When mine is blanched with fear. (III.iv.112-116) Lady Macbeth in this particular scene assumes not only his masculinity, but also Macbeth's position as a host and a husband, rendering him practically useless. It is no surprise then that Macbeth wants to keep his wife in the dark about the other murders, so that she can be "innocent of the knowledge" (III.ii.45), until she can "applaud the deed" (III.ii.46). Lady Macbeth manipulates her husband's fear of being perceived as less than a man so well that Macbeth continues conflating manhood with violence even after her death. He uses the same techniques towards other male characters in the play, repeatedly challenging Malcolm by calling him a "boy". The emotional manipulation used by lady Macbeth is based on "using cues that in other circumstances would indicate a particular emotion as an appropriate response" 16 so that she can "shape emotional responses, even if those cues are false, manufactured or misrepresented."17 As this and previous examples prove, emotional manipulation is not limited to men only, though the research suggests that "the consistent effect of masculine gender roles in the prediction of emotional manipulation for both sexes reveals that perceptions of agency and dominance are key to emotional manipulation." 18 As was demonstrated in all three plays, power is the key concept connected to emotional manipulation. The power structures informing intimate partnerships in The Taming of the Shrew, The Winter's Tale and Macbeth are connected with the social position of the main characters; the manipulation functions as a form of rhetoric 19 and is used to realize certain constructs through speech. As Christy-Dale L. Sims states:

The destruction of the victim's identity and sense of self may be accomplished rhetorically by the abuser through the use of displaced or symbolic violence, or any combination of some or all of the following behaviors: jokes and teasing, blaming, belittling, ridiculing, criticizing, insulting, name-calling, derogatory comments, bickering, quarreling, silence, ignoring, gestures, and threats. 20 The difference between literature and accounts of emotional abuse reported by the victims is relatively small: Shakespeare's characters use gaslighting well before the term denoting this type of manipulation even existed. The techniques remain the same, even though nowadays we may evaluate emotional manipulation differently. Nevertheless, emotional abuse is still hard to identify, as the victims' stories are excluded from newspapers, so that "their experiences are effectively marginalized and made invisible by the focus given to physical forms of violence, such as battery and sexual abuse." 21 In this regard the possibility to see these plays as stories of emotional manipulation allows for a recognition of such behaviours in oneself or in others; such a recognition often presents the first step in dealing with the trauma of emotional abuse. The point of this essay, however, is not to provide a solution (since the only possible solution is a deep change within society), but to point out the prevalence of emotional manipulation in relationships both real and staged, past and present,

  you shall come to clearer knowledge, that / You thus have publish'd me!" (II.i.96-8) turn prophetic in the latter part of the play.

1

  Rachel Grieve et al., "Masculinity might be more toxic than we think: The influence of gender roles on trait emotional manipulation," Personality and Individual Differences 138 (2019): 158, Science Direct <https://www.sciencedirect.com/science/article/pii/S0191886918305282?via%3Dihub> 18 July 2019. 2 Josie Queen et al., "Being Emotionally Abused: A Phenomenological Study of Adult Women's Experiences of Emotionally Abusive Intimate Partner Relationships," Issues in Mental Health Nursing 30.4 (2009): 238, Taylor & Francis Online <https://doi.org/10.1080/01612840802701257> 21 July 2019. 3 It must be noted that there is a significantly smaller number of examples in the media, though the issue is being talked about more and more.

4 Christy Dale L. Sims, "Invisible Wounds, Invisible Abuse: The Exclusion of Emotional Abuse in Newspaper Articles," Journal of Emotional Abuse, 8.4 (2008): 380, Taylor & Francis Online <https://www.tandfonline.com/doi/full/10.1080/10926790802480422> 23 July 2019.

Hutcheon, 323.

Hutcheon, 323.

Hutcheon, 326.

Queen et al., 240.

McDermott, 2.

McDermott, 7.

McDermott, 7.

Grieve et al., 160.

Sims, 379.

Sims, 381.

Sims, 377.