Moussa Bounahkla
email: moussabounakhla@yahoo.fr

Zahra Bounakhla

Mounia Tahri

Fatiha Zahry

Mustapha Zghaid

Anass Doukkali

Ouafae Benkirane

Naïma Saïdi

Essaïd Bilal

EVALUATION OF THE METALLIC CONTAMINATION IMPACT ON 'MYTILUS EDULIS' MUSSEL AT THE LEVEL OF THE MOUTH OF SEBOU'S ESTUARY, MAROCCO

Keywords: Mytilus edulis, Trace metals, Bioaccumulation, Bioindicator

The understanding of the bioaccumulation phenomenon of heavy metals in the living substance is of an extreme complexity. This is due to the interpenetration of several parameters which influence this bioaccumulation such as: the physico-chemical characteristics of the study area, the chemical properties of the contaminant and the biologic factors of the organism. This work constitutes, using Mytilus edulis as bioindicator, a contribution to study the metal contamination at different points of the mouth of Sebou's estuary. The samples were collected during the spring and the summer of the year 2005. The obtained results reveal a contamination of the study area by all the measured metals especially by lead, cadmium and chromium. This study allowed also discerning the existence of a variation in the accumulation of heavy metals according to Mytilus edulis size, as it was reported in the literature. Indeed, it showed that the small mussels accumulate heavy metals more than the big, mainly for manganese, chromium and cadmium. Seven trace metals (Mn, Cu, Cr, Pb, Co, Fe and Cd) were detected in the four studied organs (hepatopancreas, gills, gonads and muscle); the variations was least or more significant according to the organ. So, we conclude that the bioaccumulation does not make in a strictly selective way.

INTRODUCTION

Oued Sebou draining basin is situated in the North West of Morocco and collects waters from the southern slopes of the Rif range and from the north western slopes of the middle Atlas. It lies between the 33 rd and 35 th north parallels, and being in the north western part of Africa has Mediterranean type weather. Oued Sebou is a main water body in Morocco. It waters serves important towns (Sidi Slimane, Sidi Kacem, Bel Ksiri, Kenitra, etc...) and receives, without any treatment, waste waters from many industrial plants (sugar mills, paper mills, petrol refinery, canneries, etc.). The excessive and uncontrolled use of pesticides also contributes to the degradation of Sebou water quality.

Thus, the study of pollution of the estuary and its effects on the ecosystem has become the major concern of several researches on ecotoxicology with an increased attention accorded to the analysis of biological materials [START_REF] Bouachrine | L'étude de la contamination métallique d'un cours d'eau marocain, Cas du Bas Sebou[END_REF][START_REF] Bouachrine | Distribution of Selected Metals in Tissue Samples of Fish from an Industrially Contaminated Stream (the River Sebou, Morocco)[END_REF][START_REF] Benkirane | Niveau de l'Acétylcholinestérase chez quatre espèces de poissons de l'estuaire du Sebou[END_REF][START_REF] Bouhali | Etude Ecotoxicologique et Biochimique de Gambusia Halbrooki utilisé comme biomarqueur de l'état de l'environnement dans la région de Gharb[END_REF][START_REF] Benkirane | Etudes Ecotoxicologiques, Ichthyoparasitologiqus et Histopathologiques dans l'Estuaire du Sebou[END_REF]. Indeed, considering the difficulty of the collection of valid samples for trace metal analysis in water as well as the low spatial and temporal representativeness of these, the use of the quantitative bioindicators of the chemical contamination attracts more and more attention in ecotoxicology.

It is in this context that we have elaborated this work to estimate the degree of the metal contamination at the level of Sebou's mouth and this by dosage of seven trace metals (Cu, Mn, Fe, Co, Cr, Pb and Cd) in mussels (Mytilus edulis). In fact, Mytilus edulis have been found to be suitable indicator species for trace metals [START_REF] Goldberg | The Mussel Watch -A first Step in Global Marine Monitoring[END_REF][START_REF] Phillips | The Common mussel Mytilus edulis as an Indicator of Pollution by Zinc, Cadmium, Lead and Copper. II. Relationship of Metals in the Mussel to those Discharged by Industry[END_REF][START_REF] Phillips | The Use of Biological Indicator Organisms to Monitor Trace Metal Pollution in Marine and Estuarine Environments-a Review[END_REF][START_REF] Brown | Use of the Euryhaline Bivalve Potamorcorbula Amurensis as a Biosentinel Species to assess Trace Metal Contamination in San Francisco Bay[END_REF]Jumshamn & Grahl-Nielsen, 1996). It is capable to accumulate trace metals such as cadmium, mercury and lead to a larger extent than for example fish and algae (Jumshamn & Grahl-Nielsen, 1996;[START_REF] Riget | Influence of Length on Element Concentrations in Blue Mussels (Mytilus edulis)[END_REF][START_REF] Airas | Copper, zinc, arsenic, cadmium, mercury, and lead in blue mussels (Mytilus edulis) in the Bergen harbor area, Western Norway[END_REF]. It has wide geographical distribution and tolerance range for different salinities and temperatures. In addition, it has sufficient size, sessile life form and is robust in laboratory conditions.

METHODS AND MATERIALS

Sampling Sites

The sampling of mussels was elaborated at four stations belonging to the mouth of the Sebou estuary (Fig. 1):

• S1: Monegasque; near the discharges of the "Monegasque" factory for the canning of fishes; • S2: Camping; very important domestic discharges especially in summer;

• S3: the extremity of the mouth; important fishing activity;

• S4: Mehdia beach. These four stations were chosen for their socio-economic interest (main zones for harvesting mussels by fishermen). The mussel samples were collected at low tide. They were placed in plastic flasks filled with water of each station, then immediately transported to the laboratory.

Sample Preparation

The shells were carefully opened so as not to damage tissues. Four organs were taken from each mussel: gills, gonads, muscle and hepatopancreas for organotropisme study, a sample of measure corresponds to ten units of the same organ.

The samples were conserved in sterilized petri dishes, weighed and stored at a temperature of -18°.

S 1 S

The procedure adopted for the preparation of mussels was as bellow:

• Lyophilization during 48 hours under vacuum and a temperature of -50°C, and under a pressure of about 4000 bar; • Grinding of each sample in a porcelain mortar until the obtaining of a fine powder; • Recovery of 0.5 g of broyats in Teflon bombs;

• Addition of 3 ml of concentrated HNO3 (69°C) in each bomb to solubilise the metals and 1 ml of H2O2 to oxidize the organic matter; • Digestion in a microwave oven at high pressure and high temperature.

Analysis of final solutions for all trace elements detected by Atomic Absorption Spectroscopy in graphite furnace. For each series of analysis, a negative control reagent was prepared in the same way as the samples, except that no sample has been added in the flask of digestion. The results are expressed in μg/g of dry weight (ppm).

RESULTS AND DISCUSSION

Preferential Distribution of Heavy Metals in the Studied Tissues

In table 1, we present the concentrations of the heavy metals (Fe, Mn, Cu, Co, Cr, Cd and Pb) analyzed in the four studied organs.

Table 1: Average heavy metal concentrations (in μg/g) in the four studied organs.

According to table 1, all the measured metals are detected in the four studied organs with significant but variable concentrations according to the accumulator organ. So, from this study it appears that the gonads concentrate more Mn, Cu, Pb and Cr; the gills accumulate preferentially Cr, Mn and Cu; the hepatopancreas is the preferred location for almost all the studied metals; and finally the muscle concentrates more Cu, Mn, Cr and Pb.

Access to the internal compartments of the body and the processes of accumulation in tissues are extremely complex and depend on several factors including the properties of the receiving tissue (vascularization, membrane properties, chemical composition, active processes, etc.) [START_REF] Engrand | Recherches en écotoxicologie expérimentale sur la bioaccumulation et les transferts de mercure dans un système "sédiment-eau-macrophytes[END_REF]; the nature of the contaminant (molecule size, chemical speciation, etc.) [START_REF] Bowen | Trace elements in biochemistry[END_REF] and the intra and extracellular environment (temperature, pH, etc.).

Contrary to fishes, the organs of the mussel are all exposed to chemical pollutants suspended in the water; direct contamination could have the same significance as the trophic contamination which could explain the wide repartition of metals for all the studied organs.

According to the bibliography, bivalves accumulate Cd mainly in hepatopancreas and in kidney in the form of deposits in lysosomes (Serafin et al., 2002), and accumulate Cu mainly in hepatopancreas, gonads and gills [START_REF] Reference Adami | Levels of cadmium and zinc in hepatopancreas of reared Mytilus galloprovincialis from the Gulf of Trieste (Italy)[END_REF] what does not are in contradiction with our results. Indeed, in molluscs, the blood contains a respiratory pigment based on copper, the hemocyanine, which explains its distribution in these richly vascularized organs.

Figure 2 shows the variations of heavy metals contents in the various studied organs. Cr shows an accumulation significantly low in gonads compared to other organs. Indeed these greasy organs have more affinity to divalent elements [START_REF] Simkiss | Kipid solubility of heavy metals in saline solution[END_REF], which is not the case for Cr.

Fe shows the least concentrations in all the studied organs, which is not the case for fishes where Fe, constituent of hemoglobin, is very concentrated. Indeed, in bivalves, Fe is involved in the tanning processes of the shell, foot and byssogene gland [START_REF] George | The kinetics of accumulation and excretion of ferric hydrxide in Mytilus edulis (L) and its distribution in the tissue[END_REF][START_REF] Kesavan | Heavy metals in three molluscs and sediments from Vellar Estuary, Southeast coast of India[END_REF].

The hepatopancreas plays a vital role in achieving the metabolism phenomena, and then by crossing the branchial or intestinal walls, metals reach the bloodstream to join this organ, which can explain the importance of the accumulation of the most metals measured in this organ of target metabolism.

Variation of Average Contents of Heavy Metals According to the Size

Figure 3 shows the concentrations of heavy metals measured and detected both in big and small mussels.

The global observation of results concerning the influence of size factor on the accumulation of the studied heavy metals (Fe, Cu, Mn, Co, Cr, Pb and Cd) in Mytilus edulis shows a tendency of concentration in mussels with small size; these results are in concordance with those of the bibliography.

Indeed, many studies have highlighted the relationship metal/size in the mussel. [START_REF] Cossa | Geographical and seasonal variations in the relationship between trace metal content and body weight in Mytilus edulis[END_REF] showed net relationship between the concentrations of different metal contaminants (Cd, Cu, Fe, Mn, Ni, Zn) in the mussel Mytilus edulis and its weight; the small mussels present high concentrations than those measured in large mussels. [START_REF] Morono | The effect of mussel size, temperature, seston volume, food quality and volume specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis)[END_REF] then [START_REF] Morono | Accumulation and transformation of DSP toxins in mussels Mytilus galloprovincialis during a toxic episode caused by Dinophysis acuminate[END_REF] showed that the quantities of metals concentrated in mussels result from their net accumulation (incorporation -excretion) which itself is a function of the size and weight.

According to [START_REF] Casas | Modélisation de la bioaccumulation des métaux (Hg, Cd, Pb, Cu et Zn) chez la moule, Mytilus galloprovincialis, en milieu méditerranéen[END_REF], the metal concentration arises from the quotient of two components: the total metal content in the organism and its weight. Therefore, the changes in the weight of the bioindicator tissues can significantly affect the trace metal concentrations by diluting or simply by concentrating the total mass of the metal. So, the increase of the weight would cause a dilution of trace metals in the organism, which could explain concentrations the high concentrations in small mussels.

Variation between Marine and Estuarine Environment

This study showed that all the measured metals (except the cadmium) revealed in the maritime environment average contents greater or equal to those founded in the estuary environment. This can be explained by the fact that the sea is the final reservoir of all the river draining so the pollutant rates will increase; or may be the human activities (swimming, surfing, zodiac) that start at the Mehdia beach (Kenitra, Morocco) in spring and raise in summer, are behind this noticed increase.

Another hypothesis can be considered to explain this result is the increase of salinity: according to [START_REF] Amiard | Bioaccumulation et nocivité relative de quelques polluants métalliques à l'égard des espèces marines[END_REF] and [START_REF] Kesavan | Heavy metals in three molluscs and sediments from Vellar Estuary, Southeast coast of India[END_REF], natural phenomena of solubilization occurring in the presence of salt water, imply that metals become more available for alive organisms, the forms ionized with chlorides and hydroxides must mainly be considered.

The cadmium bizarrely appears to escape this hypothesis and to be more available in estuary environment than in marine environment. Indeed, studies led by [START_REF] Borchardt | Influence of food quantity on the kinetics of cadmium uptake and loss via food and seawater in Mytilus edulis[END_REF][START_REF] Borchardt | Relationship between carbon and cadmium uptake in Mytilus edulis[END_REF] on the bioaccumulation of cadmium in mussels showed the preponderance of the dissolved way with regard to the particulate way knowing that in the seawater the chloride ions complex strongly the cadmium [START_REF] Baric | Polarography of sea water 1: Ionic state of cadmium and zinc in sea water[END_REF]. However, on the other hand, the study led by [START_REF] Bouquegneau | Localisation biochimique de cadmium chez Murex trunculus en milieu naturel non pollué et après intoxication expérimentale[END_REF] showed that the Murex trunculus mollusc presents a very high contents of cadmium in a bay compared to those collected at sea.

CONCLUSION

Understanding the phenomenon of bioaccumulation of heavy metals in living substance is of extremely complex. This is due to the interpenetration of several parameters which influence the bioaccumulation namely in general: the physico-chemical characteristics of the environment, the chemical properties of the contaminant and the biologic factors of the organism.

In this work, we have tried to contribute to the study of metal contamination in a sensitive point of Sebou's estuary which is the mouth using Mytilus edulis as bioindicator. Obtained results reveal a contamination of the studied area by all the measured metals mainly by the not essential metals: lead, cadmium and chromium.

The comparison of trace metal contents in the estuary environment (Stations 1, 2 et3) and the marine environment (Station 4) revealed a wide availability of Mn, Cu, Co and Cr in this marine environment which may be related to the degree of salinity in the sea.

Also, this study has confirmed the existence of a variation of accumulation of heavy metals according to the size of Mytilus edulis. It showed that the small mussels accumulate more heavy metals than the big, mainly for manganese, chromium and cadmium.

The seven studied trace metals (Mn, Cu, Cr, Pb, Co, Fe and Cd) were detected in the four studied organs (hepatopancreas, gills, gonads and muscle) with variations more or less marked according to the organ: gonads concentrate more Pb, Cd, Cu and Mn; gills accumulate preferentially Cr, Mn, Cu and Fe; hepatopancreas is the preferred site for most metals namely Co, Cu, Fe, Cr and Cd; finally muscle concentrates more Cu and Cd. So, we can deduce that the bioaccumulation is not made in a strictly selective way.

In conclusion, the results of this study show the persistent degradation of the Sebou estuary as a result of industrial, agricultural and domestic discharges, which would increase if no strategy of protection of this environment by concerned authorities, is developed and implemented. So, to reduce the contamination of Sebou estuary, the implementation and the application of regulations on management of releases are needed, as well as the creation of pilot projects of depollution.

Figure 1 :

 1 Figure 1: Approximate locations of sampling sites at the level of Sebou's mouth (Image Source: Google Earth).

Figure 3 :Figure 4 :

 34 Figure 3: Heavy metal contents according to Mytilus edulis size.

ACKNOWLEDGMENTS

The authors thank the superior technicians R. Touhami, Mr. El Hassani and K. Fathi of the Unit of Pollution and Geochemistry of CNESTEN for their technical support.


Inès Galfati
email: ines_galfati@yahoo.fr

Essaïd Bilal
email: bilal@emse.fr

Aïcha Beji Sassi

Hassen Abdallah
email: hassen.abdallah@inrst.rnrt.tn

Ali Zaier

ACCUMULATION OF HEAVY METALS IN NATIVE PLANTS GROWING NEAR THE PHOSPHATE TREATMENT INDUSTRY, TUNISIA

Keywords: Phosphate mud, Heavy metals, Soils, Plants, Phytoremediation, Gafsa-Metlaoui area, Tunisia. Legend Laudry

The enrichment of the phosphates by a humid process can be accompanied with rejects of mud poured in hydrographic network of the region of Gafsa-Metlaoui to Chott El Gharsa. These releases are rich in phosphate and trace elements; the average is around these values: P 2 O 5 : 10.96 %, Cd: 25.83 ppm, Zn: 260.91 ppm, Cr: 387.7 ppm, Cu: 16 ppm, Ni: 26 ppm and Sr: 717.15 ppm. Soils adjoining these rejects present total concentrations in Cd, Zn and Cr higher to the maximal contents tolerated in soils. A two step sequential extraction showed that heavy metals (Cd, Cr, Cu and Ni) were poorly labile (i.e. not soluble in diluted CaCl 2 ), indicating that their leaching under natural conditions is probably very low. However, extraction with DTPA, CaCl 2 and TEA generated significant amounts of metals (mainly Cd and Cu), suggesting that they were potentially mobilizable. However, zinc and strontium are more concentrated in the mobile phase, indicating that the Zn-Sr-solutions are possible in natural conditions. This study evaluates the potential for phytoremediation of 30 species collected from six localities, growing on a contaminated site of the study area. Plants and the associated soil samples were collected and analyzed for total metals concentrations. While total metals concentrations in soils vary from 1 to 36 ppm Cd, 1.6 to 2463 ppm Zn, 8.5 to 442 ppm Cr, 2.8 to 38.5 ppm Cu, 1 to 31.7 ppm Ni and 144.6 to 1310 ppm Sr, while those in the plants ranged from 0.11 to 82 ppm Cd, 0.5 to 567 ppm Zn, 0.04 to 174.7 ppm Cr, 0.95 to 51.73 ppm Cu, 0.17 to 6.9 ppm Ni and 6 ppm to 2858 ppm Sr. None of the plants were suitable for phytoextraction because no hyperaccumulator was identified. However, plants with a high bioconcentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant leaves to roots) have the potential for phytostabilization. The plants most effective in the accumulation of metals in leaves are Malva aegyptiaca (TF=30.7) for Cd, Frankenia thymifolia (TF=8.55) for Zn, Peganum harmala (TF=29.14) for Cu and Citrulus sp (TF=10.42) for Sr. Anthemis stiparum was most suitable for phytostabilization of contaminated sites with Cd (BCF=23.51). Our study showed that native plant species growing on contaminated sites may have the potential for phytoremediation.

INTRODUCTION

In Tunisia natural phosphate is extracted and mineral fertilizers are produced. Gafsa Phosphate Company (CPG) has been active in mining for more than a century. The activity of ore beneficiation and production of various mineral fertilizers is more than half a century old. CPG operates seven open cast quarries and one underground mine. Annual production of merchant phosphate in 2007 reached 8 million tons, placing this country the fifth in the world for phosphate production. After having been exporting all its phosphate rock production during the first fifty years of its activity, Tunisia entered into phosphoric acid and mineral fertilizers production and developed this new activity so that it is now processing more than 80% of its phosphate production.

This industry produces releases under the form of: i) mud discharged into the hydrographical network, and ii) phosphogypsum stored in the form of "Tabia".

These releases are trace elements-rich such as in Cd, Zn, Cr and Sr, can contaminate the environment receptors like air, water, soils and plants. The overall objectives of this research are: i) to determine the concentrations of trace elements (Cd, Zn, Cr, Cu, Ni and Sr) in releases, in soil and in plant biomass growing on the contaminated site; ii) to compare metal concentrations in the leaves, stems and in roots; iii) and to assess the feasibility to use these plants for phytoremediation purpose. Information obtained from this study should provide insight for using native plants to remediate metal-contaminated sites. About a hundred analyze major and trace elements of four samples of mud from the washing units of phosphate and about fifty plants with the soil around the roots were performed. For each plant, leaves, stems and roots were analyzed when they were sampled. To assess the availability of heavy metals in soils around roots, 23 soil samples were performed according to the protocol of [START_REF] Maiz | Evaluation of heavy metal availability in polluted soils by two sequential extraction procedures using factor analysis[END_REF], Damian et al. (2008a,b) and [START_REF] Gazdag | Geochemical background in heavy metals and human health risk assessment at an ore mine site, Gyöngyösoroszi (North Hungary)[END_REF].

MATERIAL AND METHOD

Site description

Centres' ore mines are scattered throughout the Gafsa-Métlaoui Basin (GMB) in Tunisia. For treatment of phosphate ore, ten washing units are operational throughout the region (Fig. 1): four laundries in Métlaoui sector, one Laundry in Moulares sector, one Laundry in Kef Eddour sector, one Laundry in Redeyef sector and three Laundries in the Eastern sector (M'Dhilla and Sehib).

Tunisian Chemical Group (TCG) is located in the M'Dhilla sector, where phosphogypsum releases are stored near the site.

Mud samples were taken from Kef Eddour, Métlaoui, Sehib and M'Dhilla laundries. Plants and soils were collected in December 2006 at the riversides (Fig. 1) that drain wash discharges.

In the M'Dhilla area sampling was made near the processing of phosphoric acid factory. In addition phosphogypsum was taken from TCG Tabia. Four reference samples providing from non polluted area will be used to make comparisons.

Sample preparation and chemical analysis

Mineralogical proportions of mud samples and soils were determined by X-ray diffraction (XRD with a Pan-Analytical brand X' PERT PRO type) carried out at the Laboratory of the Mineral Resources and Environment of the Faculty of Sciences of Tunis. The evaluation of heavy metals availability was performed using a two steps sequential extraction procedure, as proposed by [START_REF] Maiz | Evaluation of heavy metal availability in polluted soils by two sequential extraction procedures using factor analysis[END_REF]. This procedure involved mixing 4 g soil with 20 ml 0.01M CaCl 2 , under agitation for 24 h at room temperature. This first suspension was centrifuged at 3000 t/min during 15 min, and the supernatant was collected for the analysis of the mobile fraction. The residue was then rinsed two times with ultra pure water and resuspended in 20 ml of 0.005M DTPA, 0.01M CaCl 2 and 0.1M TEA, pH 7.3, under continuous agitation for 24 h. After centrifugation at 3000 t/min for 15 minutes, the supernatant was removed and analyzed for the mobilisable fraction. The heavy metals of various extracts were measured by ICP-AES.

Metal accumulation in plant tissues was evaluated from the most abundant species. Plant samples were thoroughly washed in tap water and rinsed three times with distilled water. Samples were then separated into leaves, stems and roots, dried at 40 °C to constant weight and grounded and sieved at 2 mm. Digestion of plant samples was performed using hot nitric concentrated acid, according to [START_REF] Zarcinas | Nitric acid digestion and multielement analysis of plant material by inductively coupled plasma spectrometry[END_REF], [START_REF] Petrescu | Natural actinides studies in conifers grown on uranium mining dumps (The East Carpathians, Romania)[END_REF], 2007), [START_REF] Secu | Lead, zinc and copper in the bioacumulative horizon of soils from Iaşi and the surrounding areas[END_REF]) et Lăcătusu et al. (2009). The plant extracts were analysed by ICP-AES.

RESULTS

Sludge of industry of phosphate

The phosphate laundry factories of Gafsa-Métlaoui Basin reject the muddy fine fraction < 70 µm in the hydrographic network. The mud analyses by XRD show the presence of apatite, calcite, dolomite, quartz, cristobalite and clinoptilolite minerals (Fig. 2A). The clay fraction is represented by the dominant presence of smectite and in lesser amounts by the palygorskite.

The mud chemical composition presents high P 2 O 5 contents, 9.5% in the M'Dhilla laundry and 12.4% in the Métlaoui laundry (Table 1 Phosphogypsum is mainly represented by gypsum and it contains high P 2 O 5 (12 wt%) and some trace elements as Cd (15 ppm), Zn (203 ppm), Cr (33 ppm) and Sr (1325 ppm).

Mineralogy and geochemistry of soil

The mineralogical composition of soil samples, collected around the roots, indicates the presence of quartz, calcite, carbonate fluorapatite, dolomite, opal-CT, gypsum and clinoptilolite (Fig. 2B). The majority of soils collected at M'Dhilla Chott El Gharsa and Selja stations, are very enriched in quartz, with the presence of carbonates (calcite and / or dolomite); there is sometimes traces of gypsum and rare phosphate in some samples (Fig. 2C). Soils sampled at the Métlaoui, Tarfaoui and Sehib stations, are silica-rich minerals as quartz, carbonates; there is also a main amount of carbonate-fluorapatite and the presence of clinoptilolite and opal-CT discharges from washing. The reference samples, taken away from the non polluted sites, are sandy, carbonate-rich and mainly gypsum.

The soils are characterized by low levels of organic matter; the maximum (2%) is recorded in the Métlaoui area (MT1 and MT2 samples). The soil pH is slightly alkaline in the various sampling sites except MD4, MD5 and MD6, taken near the treatment of phosphoric acid factory in M'Dhilla which has an acid pH ranging from 4 and 6 (Fig. 3). The soils of the Gafsa-Métlaoui Basin have quite significant changes; the M'Dhilla and Chott El Gharsa soils are characterized by high levels of SiO 2 above 68% SiO 2 and ratio (Na 2 O+K 2 O)/CaO (0.05 up to 0.1), however, they have low levels of P 2 O 5 , Fe 2 O 3 , MgO, Al 2 O 3 and CaO (Figs 456789) comparatively to Selja, Métlaoui, Sehib and Tarfaoui soils (Figs 10,11).

-The Selja soils are characterized by low P 2 O 5 content of about 1% except Sj1 sample which shows 9%. The CaO contents vary between 7.2 and 9.8 wt% in Sj3 and Sj4 respectively. The highest concentration concerns Sj1 with 20.6 wt% CaO. The SiO 2 contents are the highest (78 and 87 wt%) recorded in the phosphate-poor samples (0.71 wt% and 1.88 wt% P 2 O 5 ). The Al 2 O 3 values vary between 1.13 and 2 wt%; those of Fe 2 O 3 range between 0.6 and 1 wt%. The concentrations of TiO 2 , MgO, Na 2 O and K 2 O are the lowest, showing values below 1 wt%. The trace metal concentrations vary between 2-22 ppm for Cd, 15-122 ppm for Cr, 2-9 ppm for Ni, 7-9 ppm for Cu and 145-665 ppm for Sr; the maximum value in each element is recorded in SJ1 soil phosphate. Zn shows values ranging from 136 up to 582 ppm. -The Métlaoui soils are enriched in phosphates; the P 2 O 5 contents vary between 10.5% (MT1) and 21.5% (MT2) and those of CaO ranged between 26 and 41% in samples MT1 and MT2, respectively. Silica contents are lower than those recorded in the Selja sector, with a maximum of 29% in MT1. The Al 2 O 3 values are interesting, ranging from 1.5% (MT9) and 6% (MT1). The MgO and Fe 2 O 3 concentrations are variable ranging from 1% (MT2) and 2% (MT1). TiO 2 , Na 2 O and K 2 O are characterised by low amounts in the soil collected near the wadi Métlaoui. The Cd, Cr, Zn and Sr high values range between 16-36 ppm, 150 -1299 ppm, 119-443 ppm and 794-1309.9 ppm respectively. Cu and Ni contents are high compared to those analyzed at the Selja respectively, with a maximum of 38.5 and 31 ppm. -The Tarfaoui soils are mainly phosphoric, P 2 O 5 ranging between 8 and 12% except sample T9 (0.78 wt%). CaO also shows values above 25 wt% except sample T9, which is about 14.5 wt%. SiO 2 concentrations range between 26 and 45% in samples TF7 and T2 respectively except T9 sample that shows silica enrichment (63%).

The Al 2 O 3 results range from 2 % (T2) and 5% (TF7). The contents of Fe 2 O 3 present values ranging from 1% (T2) and 2% (T6). MgO shows 1% contents. Trace elements at the Tarfaoui station are high except for the T9 sample, showing low values: 2 ppm (Cd), 74 ppm (Zn), 19 ppm (Cr), 7 ppm (Cu), 4.5 ppm (Ni) and 737 ppm (Sr). The maximum of Cd, Zn, Cr, Cu and Ni are recorded in the TF7 sample with 36, 276, 410, 17.5 and 30 ppm respectively. The highest value of Sr is about 898 ppm (T8).

-At Chott El Gharsa station, soils are depleted in phosphate with 1% approximately, except G2 sample taken from a reject-mound inside the wadi, which contains 10%. However, there is an enrichment of silica with amounts exceeding 70% except for G2 (37 wt% SiO 2 corresponding to the highest content of P2O5). CaO content varies from 6% (G4) to 26% (G2) and those of MgO are about 1 wt%. Al 2 O 3 content ranges between 1 and 3.7 wt% (samples G6'). Fe 2 O 3 shows low values; the maximum is recorded in the G1 sample (Fig. 1) with 1 wt%. The Cd ranges from 1 ppm (G15) to 31 ppm (G2). Zn content ranges from 3 ppm (G16) and 401 ppm (G2), with the exception of G1 and G0 two soil samples showing values above 1200 ppm. Cr shows varying levels with a maximum of 118 ppm (G2). The Cu and Ni values are low with a maximum of 14 ppm. Sr levels range from 157 ppm (G18) and 844 ppm in the G2 sample.

The samples, taken far from non polluted sites by the mud reflect the primary geochemical composition of the soil without phosphoric waste influence; the P2O5 maximum is about 0.3%. Thus, the mud provides a source of P2O5 in the soils sampled near the riversides. Moreover, trace elements show lower values than those found in soils collected near the riversides, except for copper and nickel. Ni and Cu reflect the natural geochemical background of the region; Sr contents remain high.

The comparison of trace elements analysis in soils sampled across the path of the discharge until Chott El Gharsa shows higher values than the normal values of heavy metals in soil reported by [START_REF] Cottenie | Essential and no essential traces elements in the system soil-water-plant[END_REF] and [START_REF] Henin | Les éléments traces dans les sols[END_REF] such as for Cd and Cr, which are about 3 and 25 ppm respectively. The contamination seems to be related to the original releases washing industry of phosphate.

Heavy metal content and availability

Results (Table 2) show that metals (Cr, Ni, Cu and Cd) were virtually insoluble in the first extraction solution (in CaCl 2 ). In fact, the mobile fraction is almost zero (< 0.36%), indicating that the leaching under natural conditions is probably very low. The extraction with DTPA (2 nd extraction) does not allow the release of Cr and Ni. However, it generates an amount of metals (mainly Cd and Cu) suggesting that they are potentially available.

Zinc and strontium are more concentrated in the mobile phase, indicating that the solutions containing these metals exist in natural conditions. These results reveal that the two fractions (mobile and mobilisable) containing acceptable trace elements can be released into the soil solutions. In fact, the heavy metal potential availability can be expressed as follows: Sr > Zn > Cd > Cu > Ni > Cr. [START_REF] Alloway | The accumulation of cadmium by vegetables grown on soils contaminated from a variety of sources[END_REF][START_REF] Secu | Lead, zinc and copper in the bioacumulative horizon of soils from Iaşi and the surrounding areas[END_REF][START_REF] Petrescu | Environmental impact assessment of a uranium mine, east Carpathians, Romania: metal distribution and partitioning of U and Th[END_REF][START_REF] Lăcătuşu | Heavy metals soil pollution state in relation to potential future mining activities in the Roşia Montană area[END_REF]. Plant uptake of heavy metals from soil occurs either passively with the mass flow of water into the roots, or through active transport, and crosses the plasma membrane of root epidermal cells. Under normal growing conditions, plants can potentially accumulate some metal ions in order of magnitude greater than the surrounding medium [START_REF] Kim | Investigation of heavy metal accumulation in Polygonum thunbergii for phytoextraction[END_REF][START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF].

In this study, a total of 30 species was collected from six localities from the site. Total Cd concentrations in plants range from 0.11 to 82 ppm, with the maximum in the leaves of Anthemis stiparum from Chott El Gharsa (Figs 12,13).

The concentrations of Cd are high in different parts of the studied plants (Fig. 12), especially in the leaves. Malva aegyptiaca presents high values in the stems (maximum of 28.87 ppm). Suaeda mollis in different site shows low Cd concentration with maximum in leaves (2.83 ppm). Atriplex inflata in the Métlaoui, Chenopodium chenopodioides, Malva aegyptiaca, Anthemis stiparum, accumulate high levels of Cd in their parts. Cd concentractions in these plants reach phytotoxic levels (Kabata Pandias & Pandias, 1992). In fact, according to [START_REF] Solís-Domínguez | Accumulation and localization of cadmium in Echinochloa polystachya grown within a hydroponic system[END_REF] the normal concentration of Cd in leaf tissue ranges between 0.05-0.2 mg.g -1 , and the excessive or toxic values range from 5-10 up to 30 mg.g -1 .

Figure 12. Variation of Cd in plant species from Selja and Métlaoui plants.

. Zinc concentrations in the plants vary from non-detectable to 567 ppm in leaves of Nerium oleander (Fig. 14). In addition, the leaves of Moricandia arvensis, Malva aegyptiaca, Frankenia thymifolia, Anthemis stiparum and, Chenopodium chenopodioides also enclode significant amounts of Zn (212-360 ppm).

According to Kabata Pendias and Pendias (1992), Zn in these plants is phytotoxic (Table 3). Cr concentrations in different parts of the studied plants are low (Fig. 15). High amounts are recorded in the leaves of Traganum nudatum (15.86 ppm), roots of Salsola vermiculata in the M'Dhilla sector (MD2) with 10.15 ppm. Chromium in roots of Aeluropus littoralis is of 12.19 ppm. Nerium oleander shows the highest values of Cr in the roots (18.8 ppm) and leaves (174.7 ppm).

The Cu contents in the plants ranged from non-detectable to 51.73 ppm in leaves of Nerium oleander (Fig. 16).

Cu concentrations are within the standards except leaves of Peganum harmala (41.48 ppm) and Nerium oleander (51.73 ppm) with values in excess. Ni concentrations in the plants vary from nondetectable to 6.9 ppm (Fig. 17). Like Zn, Cr and Cu, the maximum values are found in the leaves of Nerium oleander. In all studied plants, the Ni concentrations are low. Sr shows high levels ranging from 6 ppm in leaves of Suaeda mollis to 2858 ppm in leaves of Nerium oleander (Fig. 18).

Accumulation and translocation of metals in plants

In this study, none of the plant species reveal metal concentrations higher than 100 ppm of Cd; or higher than 1000 ppm of Cu, Cr, or Ni, or 10000 ppm of Zn in the shoots. No one of them is hyperaccumulator [START_REF] Baker | Terrestrial higher plants which hyperaccumulate metallic elementsa Review of their distribution, ecology and phytochemistry[END_REF]. However, the ability of these plants to tolerate and accumulate heavy metals may be useful for phytostabilization. Both bioconcentration factor (BCF) and translocation factor (TF) can be used to estimate a plant's potential for phytoremediation purpose after [START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF]. A plant's ability to accumulate metals from soils can be estimated using the BCF, which is defined as the ratio of metal concentration in the roots to that in soil [START_REF] Dowdy | Long-tem effects of biosolids application of heavy metal bioavailability in agriculture soils[END_REF][START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF]Gupta & Sinha, 2008). A plant's ability to translocate metals from the roots to the shoots is measured using the TF [START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF][START_REF] Gupta | Phytoextraction capacity of the Chenopodium album L. growing on soil amended with tannery sludge[END_REF]. According to MacFarlane et al. ( 2007), TF is defined as the ratio of concentration of metals in the leaves compared to those in the roots. By comparing BCF and TF, we can compare the ability of different plants in taking up metals from soils and translocating them to the shoots. Plants exhibiting TF and particularly BCF values less than one are unsuitable for phytoextraction [START_REF] Fitz | Arsenic transformation in the soil-rhizosphere-plant system, fundamentals and potential application of phytoremediation[END_REF].

Among the 30 species, Anthemis stiparum, had the highest BCF for Cd with 23.5 (Fig. 19) and a low TF (3.58). The translocation factor for Cd is greater than 1 in most studied plants; the highest value is about 30.7 in Malva aegyptiaca in Selja area. In Gharsa Chott: Plantago albicans, Tribulus terrester, Peganum harmala, Arthrochnemum indicum (in Gharsa and Tarfaoui), and Bassia muricata present BCFs and TFs greater than one.

DISCUSSION AND CONCLUSION

The majority of soils collected around the roots of plants at the Selja, Métlaoui, Sehib and Tarfaoui sites are enriched in phosphates from washing discharges. Samples soils in M'Dhilla and Chott El Gharsa are siliceous with very low levels of phosphate. Chemical analyses reveal that the collected soils at the riversides carrying the washing discharges include high metal concentrations. Comparison with maximum acceptable values [START_REF] Cottenie | Essential and no essential traces elements in the system soil-water-plant[END_REF]& Hennin, 1983) shows that these soils are contaminated mainly with Cd and Cr.

M'Dhilla area is not polluted by mud but by the dust generated by the processing of phosphoric acid.

Atriplex inflata indicates that the bioconcentration factor is great for Sr (1.46), while Citrullus sp. reveals the highest TF (10.42) for this element. Chott El Gharsa which receives all the releases providing from the laundries is located far away from these sources. Phosphate grains because of their high density are deposited along the hydrographic network. Clay minerals become dominant. Both stations show the lowest trace elements (metal) concentrations in the majority of soils but sometimes exceed the standards especially Cd and Cr in some samples.

The mobile fractions were insignificant while the mobilisable fractions, extracted with DTPA, have been a little more interesting (from about 4.3 % of total content for Cd to about 1.5% for Cu). Cr and Ni are not extracted in both phases. Consequently, although these soils contain very high levels of potentially toxic heavy metals (mainly Cd and Cr); contaminants can be considered as quite insoluble and their leaching toward ground water due to rainfalls is probably very low. However, even though metals in soils are poorly leachable, their vertical transfer to above ground vegetation cannot be ruled out [START_REF] Remon | Tolérance et accumulation des métaux lourds par la végétation spontanée des friches métallurgiques: vers de nouvelles méthodes de biodépollution[END_REF].

Indeed it is well known that plants can modify metal solubility [START_REF] Mclaughlin | Soil testing for heavy metals[END_REF][START_REF] Ge | Trace metal speciation and bioavailability in urban soils[END_REF], notably through exudates release and/or modification of soil microbial activity. Consequently, results of sequential extraction procedures are not inevitably correlated with the levels of heavy metals actually accumulated in plants [START_REF] Remon | Tolérance et accumulation des métaux lourds par la végétation spontanée des friches métallurgiques: vers de nouvelles méthodes de biodépollution[END_REF].

In this study, the potential mobilization of trace elements seems to decrease as follows: Sr > Zn > Cd > Cu > Ni > Cr. The absorption of these elements in plants is variable from one species to another, but the majority of plants shows that the accumulation of metals followed this order: Sr > Zn > Cd > Cu > Cr > Ni, indicating that the metal release in the soils correlates with their absorption by the plants.

The main of studied plants show excess values in trace elements and sometimes in the range of phytotoxic levels; this result reveals that the studied plants are tolerant to these elements.

Cadmium is a toxic element for plants [START_REF] Das | Studies on cadmium toxicity in plants: a review[END_REF]Sanita di Toppi & Gabrielli, 1999) and few species can tolerate their high concentrations in the soil. The contents of this element in the studied plants vary between species, but the values are high. Anthemis stiparum and Chenopodium chenopodioides are the two species with the most cadmium accumulation. The main species concentrate cadmium in their leaves but, Malva aegyptiaca accumulates high concentration of this element in the stems.

Zinc, copper and nickel are essential elements for plants [START_REF] Welch | Micronutrient nutrition of plants[END_REF][START_REF] Reid | A case study of cheese associated E. coli O157 outbreaks in Scotland[END_REF][START_REF] Rengel | Role of zinc in plant physiology[END_REF]Marcic, 2005), but they may be toxic in high dose; some plants can tolerate high levels. Zinc, in this work shows normal values with the exception of some species that tolerate high rates. Nerium Oleander, Moricandia arvensis, Malva aegyptiaca, Frankenia thymifolia, Anthemis stiparum and Chenopodium chenopodioides accumulate high concentrations in their leaves. Chromium is a toxic metal for plants; Nerium oleander accumulates high amounts of this element.

Copper is different of chromium by the fact that it is an essential element of the mineral nutrition of plants [START_REF] Punz | The response of roots of herbaceous plant species to heavy metals[END_REF][START_REF] Raskin | Bioconcentration of heavy metal by plants[END_REF]. It is essential for their growth and metabolism [START_REF] Loué | Oligo-éléments en agriculture[END_REF][START_REF] Marschner | Uptake and release of mineral elements by leaves and other aerial plant parts[END_REF][START_REF] Clemens | Molecular mechanisms of plant metal tolerance and homeostasis[END_REF] and in case of failure can cause problems for their growing. Nerium oleander accumulates the maximum value of Cu. The levels of Ni are in the standards. Strontium has the highest levels of analyzed trace elements; Nerium oleander, Salicornia arabica and Zygophylum album contain the most Sr accumulations.

It is noted that Nerium oleander absorbs high levels of Cd, Zn, Cr, Cu and Sr in their leaves.

The process of phytoextraction generally requires the translocation of heavy metals to the easily exploitable, such as leaves. By comparing BCF and TF, we can estimate the ability of different species for the metals, from soil and their translocation to the aerial parts. The species that show values of TF and especially BCF < 1 are inadequate to the phytoextraction [START_REF] Fitz | Arsenic transformation in the soil-rhizosphere-plant system, fundamentals and potential application of phytoremediation[END_REF]. Only those species that show values of BCFs and TFs greater than one, have the potential to be used for the phytoextraction [START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF]. In this present work, many species have BCF and TF greater than one. None of the plants were suitable for the phytoextraction because no hyperaccumulator was identified.

Heavy metal-tolerent species with high BCF and low TF can be used for phytostabilisation of contaminated sites, together with a vegetative cover [START_REF] Yoon | Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site[END_REF]. Examples of such plants in our study included Anthemis stiparum for Cd; Calendula suffruticosa, Plantago albicans, and Halocnemum strobilaceum for Zn and, Hedysarum carnosum, Calendula suffruticosa, Atriplex inflata, Atriplex halimus and Tribulus terrester for Cu. Phytostabilisation can be used to reduce the mobility of metals in contaminated soils [START_REF] Susarla | Phytoremediation: An ecological solution to organic chemical contamination[END_REF]. This process uses the ability of plant roots to change environmental conditions via root exudates. Plants can immobilize heavy metals through absorption and accumulation by roots, adsorption onto roots, or precipitation within rhizosphere. This process reduces the metal mobility and leaching into ground water, and also reduces the metal bioavailability for entry into the food chain.

Among those species collected from the contaminated site, the plants most effective in the translocation of metals are Malva aegyptiaca (TF=30.7) for Cd, Frankenia thymifolia (TF=8.55) for Zn, Peganum harmala (TF=29.14) for Cu and Cittrulus sp. (TF=10.42) for Sr.

The absorption of Cr and Ni by roots and their transfer to the stems and leaves is very low in most plants analyzed.

These species tolerate high levels of trace elements, play an important role for soil remediation at the same time they are toxic to the food chain when consumed as forage.

Figure 1 :

 1 Figure 1: Location of the plants and laundries of Gafsa Phosphates Company (CPG) and the sampling sites in the Gafsa-Métlaoui Basin.

Figure 1 .

 1 Figure 1. Location of sampling sites rounding M'Dhilla laundry and Gafsa Phosphates Company (CPG) plant.

Figure 2 .

 2 Figure 2. XRays Diffractometric diagrams of powder A: mud of Kef Eddour , B: MT1 soil sample of Métlaoui area and C: G1 soil sample of Chott El Gharsa area. CFA: carbonate fluorapatite, D: dolomite, C: calcite, Q: quartz, O-CT: Opal-CT, G: gypsum and Cl: clinoptilolite.

Figure 3 .Figure 4 .

 34 Figure 3. Variation of the pH rate CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure 5 .

 5 Figure 5. Variation of (Na 2 O+K 2 O)/CaO versus CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure 6 .

 6 Figure 6. Variation of Fe 2 O 3 rate CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure 9 .

 9 Figure 9. Variation of Al 2 O 5 rate CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure 8 .Figure10.

 8 Figure 8. Variation of P 2 O 5 rate CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure11.

  Figure11. Variation of Cu and Cr (ppm) VS CaO wt% from the soil of Gafsa-Métlaoui Basin. 1: mud; 2: Selja; 3: Métlaoui; 4: M'Dhilla; 5: Sehib; 6: Tarfaoui; 7: Chott El Gharsa; 8: Soil control and 9: Phosphogypsum.

Figure 13 .

 13 Figure 13. Variation of Cd in plant species from M'Dhilla, Tarfaoui and Chott El Gharsa plants.

Figure 14 .

 14 Figure 14. Variation of Zn in plant species from the plants of Gafsa-Métlaoui Basin

Figure 15 .

 15 Figure 15. Variation of Cr in plant species from the plants of Gafsa-Métlaoui Basin.

Figure 16 .

 16 Figure 16. Variation of Cu in plant species from the Selja and Métlaoui plants.

Figure 17 .

 17 Figure 17. Variation of Cu in plant species from the M'Dhilla, Tarfaoui and Chott El Gharsa plants.

Figure 18 .

 18 Figure 18. Variation of Ni in plant species from the plants of Gafsa-Métlaoui Basin.

Figure 19 .

 19 Figure 19. Variation of Sr in plant species from the Selja and Métlaoui plants.

Figure 20 .

 20 Figure 20. Variation of Sr in plant species from the plants of Gafsa-Métlaoui Basin.

Figure 21 .

 21 Figure 21. Variation of Bio-Concentration Factors (BCF) and Translocation Factors (TF) in plant species from the Gafsa-Métlaoui Basin

Table 1 . Mud chemical composition of laundries; A: Kef Eddour, B: Métlaoui, C: Sehib and D: M'Dhilla and PG: Phosphogypsum composition of Gafsa Phosphates Company plant, LOI*: Loss On Ignition.

 1

	). CaO

Table 2 .

 2 Heavy metal potential availability (expressed in percent of the total metal content) in the studied area. Each value represents the arithmetic mean of the 23 sampling points.

	Metal Mobile (%) Mobilisable (%)
	Cr	0.0023	0.0000
	Ni	0.0000	0.1043
	Cu	0.1509	1.5515
	Cd	0.3502	4.3237
	Zn	3.9046	0.9694
	Sr	10.0091	3.9044

3.4. Metal concentrations in plants

Metal concentrations in plants vary with plant species

ACKNOWLEDGEMENTS

This work was supported by GENERIC department of SPIN division of Ecole Nationale Supérieure des Mines de Saint Etienne, France; The Department of Geology of the Faculty of Sciences, Tunis, University El Manar, and the Laboratory of Geo-power of Power Technology Research Centre. The authors wish to thank Olivier Faure and Esteban Remon, Laboratory of Echophysiology Applied Sciences, Faculty of the Jean Monnet, Saint-Etienne, for their assistance.


Augusto Zanella
email: augusto.zanella@unipd.it

Bernard Jabiol

Jean-François F Ponge

Giacomo Sartori

Rein De Waal

Bas Van Delft

Ulfert Graefe

Nathalie Cools

Klaus Katzensteiner

Herbert Hager

R De

& M Englisch

A European morpho-functional classification of humus forms

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

Introduction 1.Basic concepts

The humus form is the part of the topsoil that is strongly influenced by organic matter and coincides with the sequence of organic and underlying organo-mineral horizons. During the 19 th century, scientists noticed that the type and rate of decomposition of these organic components, as well as the incorporation of organic matter (OM) in mineral horizons, vary according to forest type (review in [START_REF] Jabiol | Quand l'humus est à l'origine de la pédologie. 2. Avant et après Müller: évolution des conceptions sur la description et la typologie "des humus[END_REF]. These observations led [START_REF] Müller | Studier over skovjord, som bidrag til skovdyrkningens teori: om bögemuld og bögemor paa sand og ler[END_REF][START_REF] Müller | Studier over skovjord, som bidrag til skovdyrkningens teori: om muld og mor i egeskove og paa heder[END_REF][START_REF] Müller | Studien über die Natürlichen Humusformen und deren Einwirkung auf Vegetation und Boden: mit Analytischen Belegen von C[END_REF][START_REF] Müller | Recherches sur les formes naturelles de l'humus et leur influence sur la végétation et le sol[END_REF] to define three "humus forms", named Muld (later becoming Mull), Mor and Mullartiger Torf (now Moder), characterized by their climatic, geological and biological conditions of formation in Danish beech forests. From the outset it was evident to Müller that the humus form corresponds to the "expression of life" within the topsoil. Many authors contributed to the development of a classification system of humus forms based on the key role of living components in the topsoil. The most prominent contributions are those of Hesselman (1926), [START_REF] Hartmann | Waldhumusformen[END_REF], [START_REF] Kubiëna | The Soils of Europe[END_REF], [START_REF] Babel | Gliederung und Beschreibung des Humusprofils in mitteleuropäschen Wäldern[END_REF] and [START_REF] Delecour | Les formes d"humus: identification et description[END_REF]. All these concepts still form the basis of modern classifications of humus forms [START_REF] Green | Towards a taxonomic classification of humus forms[END_REF][START_REF] Afes | Référentiel Pédologique 1995[END_REF][START_REF] Baize | A Sound Reference Base for Soils: the "Référentiel Pédologique[END_REF][START_REF] Nestroy | Systematische Gliederung der Boden Österreichs (Österreichische Bodensystematik 2000)[END_REF][START_REF] Zanella | Humus Forestali[END_REF][START_REF] Brunner | Klassifikation der Böden der Schweiz[END_REF][START_REF] Baritz | Humus forms in forests of the northern German lowlands[END_REF][START_REF] Ak Humusformen | Systematik der Humusformen, AK Humusformen der Deutschen Bodenkundlichen[END_REF][START_REF] Zanella | Towards a common humus form classification, a first European approach: few generic top soil references as functional units[END_REF][START_REF] Jabiol | L'Humus sous Toutes ses Formes, 2e édition revue et corrigée[END_REF][START_REF] Van Delft | Field Guide of Humus Forms: Description and Classification of Humus Forms for Ecological Applications[END_REF]AFES, 2009;[START_REF] Jabiol | Typologie des formes d'humus forestières (sous climat tempéré)[END_REF]. Although Canadian (British Columbian) and French classification systems are frequently used in an international context, none of them covers site and climate conditions worldwide, not even all European forest ecosystems. Moreover, the new national classification systems differ according to the parameters used for describing and classifying humus forms as well as for scaling diagnostic parameters [START_REF] Schoeneberger | Field Book for Describing and Sampling Soils, Version 2.0[END_REF][START_REF] Baritz | Humus forms in forests of the northern German lowlands[END_REF]. Similar designations of humus forms often have different contents.

Harmonization purposes

With harmonization purposes in mind, a wide range of European specialists met in Trento (Italy) in 2003 and formed a European Humus Group with the aim of improving the compatibility of established national systems of classification and setting out a unified European reference for humus forms. The present synthesis was elaborated during the course of four plenary field sessions held in Alpine (Trento 2003, Vienna 2004, San Vito di Cadore 2005) and Mediterranean (Cagliari 2007) ecosystems. In these workshops, the place of lesser known terrestrial humus forms such as Tangel and Amphi and that of semi-terrestrial humus forms were discussed and included in a new classification [START_REF] Jabiol | Towards a European classification of terrestrial humus forms[END_REF][START_REF] Zanella | Towards a European humus forms reference base[END_REF]. In the meantime the key of humus forms was also tested by non-specialists in order to improve it and to discard interpretative drawbacks. In the future, the proposed humus form classification will also be included in a worldwide topsoil characterization that is currently being prepared [START_REF] Broll | Topsoil characterization: recommendations for revision and expansion of the FAO-draft (1998) with emphasis on humus forms and biological factors[END_REF]. This paper focuses on the aims and principles of the proposed European classification, details and keys being found in a more complete review available at: http://hal.archives-ouvertes.fr/docs/00/56/17/95/PDF/Humus_Forms_ERB_31_01_2011.pdf

Morpho-functional bases and references for the classification

At the end of the first meeting of the Humus group (Trento, Italy, 2003), after numerous field tests and discussions, the members of the group agreed on a very important "principle of classification": the classification process has to take into consideration only references (objects observable in the field or complexes of objects) which show an evident "morphofunctional" shape or core. This means that: a) each reference must be recognisable thanks to its particular and characteristic morphology; b) this morphology must be easily detectable to the naked eye or with a 5-10X magnifying hand lens; c) the function of the reference within the soil system has to be well expressed, identifiable or reasonably assumed to be possible. The references of the classification are arranged in a nested way: basilar components (from intact leaves/needles until more or less degraded remains, animal droppings, roots, mineral particles…) are arranged in diagnostic horizons (discernable supra-structures of basilar components), themselves arranged in humus forms (evident well structured sets of diagnostic horizons). The detection in the field of the basilar components allows the identification of the diagnostic horizons, and the order and thickness of these horizons in the topsoil allow the humus forms to be classified. The basilar components are presented below, while the diagnostic horizons and humus forms are described in sections 3 and 4.

Recognizable remains vs. humic component

Fresh litter is generally made up of 100% recognizable remains. They correspond to leaves, needles, roots, bark, twig and wood pieces, fragmented or not, whose original organs are recognizable to the naked eye or with a 5-10 X magnifying hand lens. Depending on local climatic and biological conditions, this organic material will be rapidly or slowly transformed into humic component as a consequence of biological activity in the site (Fig. 1a). The humic component is formed by small and non-recognizable organic remains and/or grains of organic or organo-mineral matter, mostly comprised of animal droppings of different sizes. The original plant/animal organs which form the litter and generate smaller particles (free or incorporated in animal droppings) are not recognizable to the naked eye or with a 5-10 X magnifying hand lens.

Micro-, meso-, macroaggregates and mineral component

The humic component often takes the shape of soil aggregates, which are visible to the naked eye or with a magnifying hand lens and are classed in three types of structure, called micro-, meso-and macrostructures (Fig. 2). A level of structure finer than 1 mm cannot be detected by the naked eye (using a 10 X magnifying lens, the limit is 0.1 mm). These very fine granular structures could be comprised of small arthropod or enchytraeid droppings (purely organic or mixed organic and mineral), layering or juxtaposed to mineral particles. Bound mineral particles visible within the humic component and strongly fixed in organo-mineral well-amalgamated aggregates (peds) are categorized as part of the humic component. On the contrary, mineral particles of different sizes, free or very weakly bound to the humic component and visible to the naked eye or with a 5-10 X magnifying hand lens, are considered as other soil parts collectively named mineral component. Above-and below-ground processes are similar. On one side "decomposition" or "weathering", from leaves to molecules or from minerals to elements; on the other side "composition", from mineral elements, organic molecules and water to biological structures (trees, animals…), neoformation clay, humic component, soil sub-units (peds). In aerated soils (terrestrial soils), thick organic horizons form on cold, acid and dry soils; in water saturated soils (semiterrestrial conditions), asphyxia delays biodegradation and the thickness of the organic horizons depends on the length of the saturation period.

Zoogenically vs. non-zoogenically transformed material

Zoogenically transformed material is made of recognizable remains and humic components processed by animals, i.e. leaves, needles and other plant residues more or less degraded by soil animals, mixed with animal droppings. A finely powdered and/or granular structure (less than 1 mm) is typical of the terminal stage of faunal attack in an organic horizon (Fig. 1a). At this last level of biotransformation, the horizon matrix is essentially comprised of organic animal droppings of varying size (droppings of epigeic earthworms, of macroarthropods such as millipedes, woodlice and insect larvae, of microarthropods such as mites and springtails and of enchytraeids dominate). Within organo-mineral horizons, animal activity leads to different types of A horizons, depending on the ability of animals to dig into the mineral soil and to thoroughly mix organic and mineral matter. Zoogenically transformed material may be active (currently inhabited by living animals, freshly transformed, with recent droppings, bite marks or tunnels) or inactive (without living animals or recent signs of animal activity, for 1-2 years or more). Non-zoogenically transformed material is made of recognizable remains and humic components processed by fungi or other non-faunal processes, i.e. leaves, needles and other plant residues more or less fragmented and transformed into fibrous matter by fungi. Recognizable recent animal droppings are absent or not detectable in the mass by the naked eye. Fungal hyphae can be recognized as white, brown, black or yellow strands permeating the organic or organo-mineral substrates. Traces of animal activity (droppings, old bite marks, mucus) may sometimes be detectable but are always marginal. In the last stage of biodegradation of an organic horizon, non-zoogenic substances may essentially be composed of dry, brown plant residues more or less powdered or in tiny fragments. Non-zoogenically transformed material is in any case inactive material that exhibits low biological activity.

Fibric vs. sapric components of histic (peat) horizons

The fibric component of peat is made of non-decomposed or very weakly decomposed hygrophilous plant remains like sphagnum mosses, sedges, rushes, reeds... It is comprised of whole plants, parts of them and/or separate plant organs (leaves, needles, twigs, wood, roots..., Fig. 1b). The sapric component is made of homogeneous dark organic or organomineral matter comprised of well decomposed plant remains pure or partly mixed with mineral particles. Plant structures are not visible to the naked eye or with a 5-10 X magnifying hand lens. Animal droppings are possible in periodically drained horizons and can be abundant in drained peats. [START_REF] Galvan | La forme di humus e la pedofauna. Interpretazione delle interrelazioni e chiavi di riconoscimento[END_REF]. Droppings are divided into three categories, named micro (<=1 mm), meso (1-4 mm) and macro (>4 mm), corresponding to the abovementioned zoogenic A horizons (miA, meA and maA).

Fig. 2. Field classification of droppings of the most common groups of pedofauna (modified from

Diagnostic horizons

A minimum thickness of horizons for description, diagnosis and sampling purposes has been established at 3 mm. Below this threshold, a horizon is considered discontinuous if clearly in patches or absent if indiscernible from other neighbouring horizons. The vagueness of transitions between organic and organo-mineral horizons (or mineral ones, in the absence of an organo-mineral horizon) is an important diagnostic character. Three types of transition have been adopted: very sharp transition within less than 3 mm, sharp transition between 3 and 5 mm and diffuse transition if over more than 5 mm.

Diagnostic horizons of Terrestrial humus forms (aerated topsoils)

Two main types of diagnostic horizons (O for organic and A for organo-mineral) have been distinguished in aerated soils (never, or for few days per year, submerged and/or water-saturated). Roots excluded, following the rate of recognizable remains and humic components, organic horizons of aerated soils have been grouped in three diagnostic horizons, reminiscent of the seminal work of [START_REF] Hesselmann | Studier över barrskogens humustäcke, dess egenskaper och boroende av skogsvården[END_REF], called OL, OF and OH. Suffixes are used to designate specific types of organic horizons then detailed into subtypes, e.g. zoogenic and non-zoogenic OF horizons. At present, the names and suffixes of these organic horizons are not fully in line with IUSS Working Group WRB (2007) or Soil Survey Staff (2010) proposals. However, the following approximated correspondence may be established with the U.S. Soil Taxonomy: OL = Oi; OF = Oe; OH = Oa. A general attempt to harmonize vocabulary and procedures for topsoil classification is in progress [START_REF] Broll | Topsoil characterization: recommendations for revision and expansion of the FAO-draft (1998) with emphasis on humus forms and biological factors[END_REF].

Organic horizons

The OL horizon is characterized by the accumulation of mainly leaves/needles, twigs and woody materials, most of the original plant organs being easily discernible to the naked eye. The humic component amounts to less than 10% by volume; recognizable remains 90% and more, up to 100% in non-decomposed litter (Fig. 3). Suffixes characterize neither fragmented nor transformed/discoloured leaves and/or needles (OLn), and slightly altered, discoloured, bleached, softened up, glued, matted, skeletonized, sometimes only slightly fragmented leaves and/or needles (OLv). The OF horizon is characterized by the accumulation of partly decomposed litter, mainly from transformed leaves/needles, twigs and woody materials, but without any entire plant organ. The proportion of humic component is 10% to 70% by volume (Fig. 3). Depending on humus form, decomposition is mainly accomplished by soil fauna (OFzo) or cellulose-lignin decomposing fungi (OFnoz). Slow decomposition is revealed by a partly decomposed matted layer, permeated by hyphae. The OH horizon is characterized by an accumulation of zoogenically transformed material, i.e. black, grey-brown, brown, reddish-brown well-decomposed litter, mainly comprised of aged animal droppings. A large part of the original structures and materials are not discernible, the humic component amounting to more than 70% by volume (Fig. 3). The OH horizon differs from the OF horizon by a more advanced transformation (fragmentation, humification, …) due to the action of soil organisms. The weight (%) of organic carbon in dry samples of organic horizons without living roots has to be at least 20% by mass (Method: element analyzer, ISO 10694, 1995).

Organo-mineral horizons

The different diagnostic A horizons are identified in the field by observing the soil mass with the naked eye or with a 5-10X magnifying hand lens (Fig. 2), assessing structure and consistence (Soil Survey Division Staff, 1993;FAO, 2006) and measuring the acidity (pHwater) according to ISO 10390 (1995). In the fraction Ø < 2mm of the A horizon, the organic carbon has to be less than 20% by mass following IUSS Working Group WRB (2007). Five diagnostic A horizons may be distinguished: three zoogenic A horizons (biomacrostructured, maA; biomesostructured, meA; biomicrostructured, miA) and two non-zoogenic A horizons (single grain, sgA; massive, msA).

Diagnostic horizons of Semiterrestrial humus forms (waterlogged topsoils)

Histic horizons

Histic horizons are submerged and/or water-saturated for a prolonged period of the year (usually more than 6 months).

Organic horizons

Following the rate of fibric and sapric components, histic organic horizons (H horizons) have been divided in three diagnostic horizons: Hf, Hm and Hs (Fig. 4). Though named differently (Hf = Hi or Oi; Hm = He or Oe; Hs = Ha or Oa or L), these horizons are roughly the same as those used in the main international soil taxonomies (IUSS Working Group WRB, 2007;AFES, 2009;Soil Survey Staff, 2010) for describing peat soils. Carbon content has to be 20% or more (approximately 35-40% organic matter) by weight in dry samples, without living roots (Method: element analyzer, ISO 10694, 1995). The Hf horizon consists almost entirely of practically unchanged plant remains (fibric component ≥ 90%, sapric component < 10% of horizon volume). The Hm horizon consists of half decomposed organic material not fitting the definition of fibric (Hf) or sapric (Hs) horizons (fibric component 10% to 70%, sapric component 90% to 30% by volume). The Hs horizon is in an advanced stage of decomposition (sapric content ≥ 70% of the horizon volume; fibric component less than 30%). Suffixes are used for Hs horizons according to high activity of soil animals (Fig. 4), especially earthworms (Hszo), massive structure with low activity of soil animals (Hsnoz), and high percentage of mineral particles (Hsl). Sapric horizons of brook valley systems and around wells have mostly a higher amount of mineral fraction than those in fens and/or bogs. Although at first sight quite similar, the horizons can differ in structure, pH, nutrient content and base saturation due to differences in water quality, vegetation and soil organisms.

Organo-mineral horizons

Named Aa (from A horizon and anmoor), the histic organo-mineral horizon is mostly built by microorganisms (actinomycetes), dark coloured, with plastic and massive structure, either high or low base-saturated. Earthworms may be abundant during better aerated periods, but the typical structure of their droppings is rapidly destroyed by water immersion and permanence. Carbon content is between 7 and 20% by weight, in dry samples without living roots (Method: element analyser, ISO 10694, 1995).

Fig. 4. Left: Hf, Hm and Hs horizons in graphical definition; right: types of Hs horizons (zoogenic typic and limi; non-zoogenic).

Hydromorphic horizons

Hydromorphic horizons are submerged and/or watersaturated for a non-prolonged period of the year (more than a few days but less than 6 months).

Organic horizons

Formed under non-prolonged water saturation, hydromorphic organic horizons are periodically watersaturated and show the effects of temporary anoxia. On the model of terrestrial horizons, they are named OLg, OFg and OHg: the humic component is less than 10% in volume (roots excluded) in OLg, between 10 and 70% in OFg and more than 70% in OHg. The suffix letter "g" indicates the presence of hydromorphic properties: plant remains become dark, glued together and are often coloured along the venation (more evident than usual) of leaves by black particles of humic component deposited by water during the period of immersion. The humic component is often dark grey or black, massive and plastic, and may be structured in animal droppings during aerated periods.

Organo-mineral horizons

Hydromorphic organo-mineral horizons show evident effects of temporary anoxia such as Fe-mottling and oxidation/reduction colours (orange-red splashes within grey to bluish-grey mass), cover at least 1/3 of the surface of the horizon profile; carbon content is generally less than 7% by weight. All terrestrial A horizons showing hydromorphic properties become hydromorphic organo-mineral horizons. In this case, a letter "g", meaning hydromorphic properties, is added to their corresponding terrestrial code: maAg, meAg, miAg, msAg, sgAg. If carbon content is higher than 7% by weight, similarities with Aa or Hs are possible. However, the structure of maAg or meAg horizons, mostly due to anecic and endogeic earthworms, and although partially destroyed by water, never becomes completely plastic and massive as in the Aa horizon; carbon content of miAg, msAg and sgAg never reaches 20%, which is the case in every kind of Hs horizon.

Humus forms classification

The European classification of humus forms is based on the sequence and morphological characteristics, including evidence of biological activity, of organic and/or organo-mineral horizons observed and described in the field (forests, grasslands, pastures and wetland areas, to the exclusion of croplands). In some cases a few basic chemical data (pH, organic carbon content) are required. A complete set of diagnostic organic and organo-mineral horizons, which are mutually exclusive, is defined. The classification keys use diagnostic horizons and other complementary topsoil or environmental data. The first dichotomy of the classification separates never saturated from saturated (submerged) humus forms (Fig. 5). Within each group of the Terrestrial compartment (Terroforms, Entiforms and Paraforms) and within each group of Hydroforms of the Semiterrestrial compartment (Hydro-, Epihisto-and Histo-forms), the same five "biological types" are identified on a morpho-functional basis: Mull, Amphi, Moder, Tangel and Mor (Fig. 6). Within Histoforms and Epihistoforms, the Tangel biological type is not present, but a characteristic "soil moisture regime" generates the Anmoor biological type. These "biological types", or main references, can be considered as the highest taxonomic levels of the classification (Fig. 6). For Terroforms and Histoforms, the most important and best-known groups of humus forms, a second level of classification has been created. Here, each unit of the main level (Mull, Moder, …) is split in two or more biological sub-types or secondary references (i.e. Eumull, Mesomull; Hemimoder, Dysmoder…).

Terrestrial humus forms

Terrestrial humus forms correspond to the topsoil never or for only a few days per year submerged and/or watersaturated. They are subdivided into Terroforms, Entiforms and Paraforms, the diagnostic criteria of which are briefly summarized in Figures 5 and6.

Terroforms

The five main references (Mull, Moder, Amphi, Mor and Tangel) of Terroforms are identified and described thanks to diagnostic features. Each main reference is in turn subdivided into secondary references according to quantitative differences in the thickness of constitutive horizons (Fig. 7). Note that these names correspond to morpho-functional types that can be found in both environments (Mull, Moder, Mor, Amphi) or not (Anmoor, Tangel) and are at least partly independent of the classification, as a reflection of diagnostic features of biological activity.

Entiforms

Entiforms are subdivided into Lithoforms, Peyroforms and Psammoforms according to the degree of fragmentation of the underlying rock (Fig. 6) and their five main references are characterized, as Terroforms, according to the sequence of diagnostic horizons.

Paraforms

Paraforms are subdivided into Rhizoforms and Lignoforms according to the plant material (roots or wood) which accumulates in the topsoil. As for the preceding units, they are subdivided into Tangel, Amphi, Mull, Moder and Mor secondary references.

Vocabulary notes

In the proposed classification, a terrestrial Mull can be a Terromull (typical form on "normal" well-drained soil), an Entimull (lying directly on a rocky substrate) or a Paramull if subterranean parts of plants or wood dominate it, allowing a large array of poorly identified humus forms, with rarely encountered characters, to be classified. The words Mull, Moder, Mor, Amphi and Tangel without prefixes mean Terromull, Terromoder, Terromor, Terroamphi and Terrotangel, respectively. Note that the word used for every second level of Terroforms is unambiguous: Eumull and Leptoamphi, for instance, can only be Euterromull and Leptoterroamphi respectively; the prefix "terro" is unnecessary and never used for naming these second level units. Lines: diagnostic horizons, superposed as in a real topsoil profile. Columns: second level of classification (examples of Mull: eumull, mesomull, oligomull, dysmull). Mull is considered as central form, divided into sub-forms evolving on calcareous (left) or acid (right) substrates. The order of Mull sub-units could differ from the one marked in the table. In fact, all gradual series from more to less biologically active forms (eu → meso → oligo → dys) are possible as well under calcareous, neutral and siliceous soil/substrate influences.

Semiterrestrial humus forms

The Semiterrestrial humus forms correspond to the topsoil (organic and organo-mineral horizons) submerged and/or water-saturated for more than a few days per year. These conditions of anoxia delay the process of biodegradation and the thickness of organic layers may even increase to several metres. Only the first 40 cm of the topsoil are observed and analyzed in order to classify semiterrestrial humus forms, the underlying part of the profile corresponding to well-detected and described Histosols following methods proposed by the main international soil classifications (FAO, 2006;AFES, 2009;Soil Survey Staff, 2010). Definitions of semiterrestrial Mull, Moder, Amphi, Mor and Tangel differ from terrestrial definitions, the unaltered organic layers acting as parent material, while in terrestrial humus forms parent materials are mostly mineral. A specific Anmoor is also necessary for circumscribing particular conditions of periodic water saturation of the soil. The Semiterrestrial humus forms are subdivided into Histo-, Epihisto-and Hydroforms along a gradient from submerged anoxic soils to drained or infrequently water-saturated ones.

Histoforms

The most typical semiterrestrial forms are the Histoforms. They are submerged and/or water-saturated for a prolonged period of the year (usually more than 6 months). Peat develops at the top of the humus profile due to anaerobic conditions slowing down the biological transformation of organic matter. Selecting the right master diagnostic horizons, a few main references (Histomor, Histomoder, Histoamphi, Histomull and Anmoor) are distinguished in order to separate Histoforms along a gradient of increasing biodegradation rate. A second level of classification of each main reference is shown in Figure 8.

Hydroforms

In Hydroforms organic horizons are rarely submerged and the biotransformation of organic matter is relatively close to aerated conditions, with the same animals and products of their activities. However, organo-mineral horizons always show signs of periodic anoxia. In this first attempt at classifying Hydroforms, five main references (Hydromor, Hydrotangel, Hydromoder, Hydroamphi and Hydromull) have been defined without any second level of classification (Fig. 6). All Hydroforms except Hydromull are often submerged up to the base of the OH horizon and the transition between organic and organo-mineral horizons is often not sharp (> 5 mm); tongues of coloured organic matter can dip into the soil from the top organic horizons. Investigations into Hydrotangel and Hydroamphi forms have never been published. They develop on calcareous soils or basic parent materials, unlike Hydromoder and Hydromor that are formed on acid substrates.

Epihistoforms

Epihistoforms are introduced for classifying initial or transitional semiterrestrial humus forms with organic horizons of both Histoforms and Hydroforms. Selecting the right master diagnostic horizons, a few main references (Epihistomor, Epihistomoder, Epihistoamphi, Epihistomull and Epihistoanmoor) have been distinguished in order to separate Epihistoforms along a gradient of increasing biodegradation (Fig 6).

Vocabulary notes

In the proposed classification, a semiterrestrial Mull can be a Histomull (typical form on "prolonged" water-saturated soil), a Hydromull ("rarely submerged but hydromorphic" topsoil), or an Epihistomull (atypical form, presence of both histo-and hydro-diagnostic horizons). Note that the word Anmoor identifies only semiterrestrial forms and that second level Histoforms are unambiguous: Euanmoor and Limimull, for instance, can only be Euhistoanmoor and Limihistomull respectively; the prefix "histo" is unnecessary for naming these second level units.

Prospects for the use of the European classification of humus forms

Many benefits can be expected from (i) a better characterization of humus forms, in particular for mapping and database purposes, (ii) a common language and vocabulary that could be accepted and used worldwide, (iii) a field instrument for the estimating the effects of global and local changes on soil C pools, (iv) a practical and conceptual tool for planning studies in soil biology, (v) a better knowledge of factors acting at the interface of soil, vegetation and climate. The humus forms contain most of the soil organic carbon, plant and animal remains (entire, fragmented, more or less biodegraded bodies or organs) and organic molecules (exudates, humic acids, proteins,….) within organic and organomineral horizons [START_REF] Batjes | Total carbon and nitrogen in the soils of the world[END_REF]. The humus form is the seat of feed-back interactions, within the topsoil of a given local ecosystem, among roots, animals and associated biodegrader communities [START_REF] Ehrenfeld | Feedback in the plant-soil system[END_REF]. Changing the ecological frame (climate, parent material, human pressure, history…), the system evolves consequently into new biocenoses characterized by adapted humus forms [START_REF] Ponge | Humus forms in terrestrial ecosystems: a framework to biodiversity[END_REF]. The soil as carbon sink will be better understood if the spatial distribution of humus forms is taken into consideration when sampling the area [START_REF] Sartori | Factors controlling alpine humus forms distribution (Venetian Alps, Italy)[END_REF]. It is thus strongly recommended to survey and sample thickness and composition of diagnostic horizons of each different humus form, and to elaborate models of spatial distribution of humus forms [START_REF] Aberegg | Modelling spatial distribution of soil types and characteristics in a high Alpine valley (Val di Sole)[END_REF]. An attempt to extend the system to the microscopic scale is underway and will be published as soon as possible.

(Italy), Jan Sevink (the Netherlands), Marija Grozeva Sokolovska (Bulgaria), Andrea Squartini (Italy), Elena Vendramin (Italy), Roberto Zampedri (Italy), Miglena Zhiyanki (Bulgaria).

Fig. 1 .

 1 Fig. 1. Vocabulary and dynamic formation of an example topsoil in a) Terrestrial and b) Semiterrestrial conditions.Above-and below-ground processes are similar. On one side "decomposition" or "weathering", from leaves to molecules or from minerals to elements; on the other side "composition", from mineral elements, organic molecules and water to biological structures (trees, animals…), neoformation clay, humic component, soil sub-units (peds). In aerated soils (terrestrial soils), thick organic horizons form on cold, acid and dry soils; in water saturated soils (semiterrestrial conditions), asphyxia delays biodegradation and the thickness of the organic horizons depends on the length of the saturation period.

Fig. 5 .

 5 Fig. 5. Main subdivisions of Semiterrestrial and Terrestrial humus forms and their diagnostic horizons.

Fig. 6 .

 6 Fig. 6. The European tree of humus forms classification. The first dichotomy separates Terrestrial humus forms, which are never waterlogged or only for a few days per year, from Semiterrestrial humus forms which are seasonally waterlogged. Each of these main groups is subdivided in three secondary groups, one of them being typical of the main group (Terro for Terrestrial, Histo for Semiterrestrial humus forms), the other two being specialized or atypical forms. Among Terrestrial humus forms, Entiforms are initial forms, subdivided in turn according to substrate, and Paraforms are atypical, subdivided in turn according to the main building agent. Among Semiterrestrial humus forms, Hydroforms are transitional to Terrestrial humus forms and Epihisto are atypical. On the right two circles indicate main humus forms which can be found in both Terrestrial and Semiterrestrial groups.Note that these names correspond to morpho-functional types that can be found in both environments(Mull, Moder, Mor, Amphi) or not (Anmoor, Tangel) and are at least partly independent of the classification, as a reflection of diagnostic features of biological activity.

Fig. 7 .

 7 Fig. 7. Second level classification of Terroforms.Lines: diagnostic horizons, superposed as in a real topsoil profile. Columns: second level of classification(examples of Mull: eumull, mesomull, oligomull, dysmull). Mull is considered as central form, divided into sub-forms evolving on calcareous (left) or acid (right) substrates. The order of Mull sub-units could differ from the one marked in the table. In fact, all gradual series from more to less biologically active forms (eu → meso → oligo → dys) are possible as well under calcareous, neutral and siliceous soil/substrate influences.

Fig

  Fig. 8. Second level classification of Histoforms. Lines: diagnostic horizons, superposed as in a real topsoil profile. Columns: second level classification (examples of Anmoor: Euanmoor, Saprianmoor, Limnianmoor). Note that the classification of Histoforms is based on both quality and thickness of each diagnostic horizon.

Acknowledgements

The authors warmly acknowledge Alain Brêthes (Office National des Forêts, Boigny-sur-Bionne, France), Gabriele Broll (University of Vechta, Germany), Jean-Michel Gobat (University of Neuchatel, Switzerland), Jean-Jacques Brun (CEMAGREF, Saint Martin d'Hères, France), Gerhard Milbert (Geological Survey of North Rhine-Westphalia, Krefeld, Germany), Eckart Kolb (Technische Universität München, Dept. of Ecology/Forest Nutrition and Water Resources, Freising, Germany), Ugo Wolf (University of Florence, Dept. of Agronomy and Land Management, Florence, Italy), Lorenzo Frizzera and Paola Galvan (Centro di Ecologia Alpina, Fondazione Edmund Mach, Viote del Monte Bondone, Italy), Raimo Kolli (Estonian University of Life Sciences, Tartu, Estonia), Rainer Baritz (Federal Institute for Geosciences and Natural Resources, Hannover, Germany), Rolf Kemmers (Alterra, University and Research Centre, Wageningen, the Netherlands ), Andrea Vacca and Gianluca Serra (University of Cagliari, Dept. of Earth Sciences, Cagliari, Italy), Damien Banas (University of Nancy, Vandoeuvre-lès-Nancy, France), Adriano Garlato (ARPAV Servizio Osservatorio Suoli e Rifiuti, Treviso, Italy), Silvia Chersich (University of Pavia, Dept. of Earth Sciences, Pavia, Italy), Emil Klimo (Mendel University of Agriculture and Forestry, Brno, Czech Republic), Roger Langohr (Research Institute for Nature and Forest, Geraardsbergen, Belgium) for their precious help in improving the manuscript. The paper summarizes seven years of exchanges among members of the Humus Group (13 European countries). Their inputs contributed to the success of the synthesis of knowledge about humus forms. Thanks to everyone for their data, ideas, discussions and friendliness. In alphabetical surname order: Anna Andretta (Italy), Michael Aubert (France), Roland Baier (Germany), Rainer Baritz (Germany), Hans-Jörg Brauckmann (Germany), Fabrice Bureau (France), Paolo Carletti (Italy), Nathalie Cassagne (France), Lauric Cécillon (France), Giuseppe Concheri (Italy), Fausto Fontanella (Italy), Hannu Ilvesniemi (Finland), Erik Karltun (Sweden), Tomaz Kralj (Slovenia), Jarmila Lesna (Czech republic), Lars Lundin (Sweden), Erwin Meyer (Austria), Roberto Menardi (Italy), Lucio Montecchio (Italy), Serenella Nardi (Italy), Silvia Obber (Italy), Diego Pizzeghello (Italy), Eva Ritter (Denmark), Mirco Rodeghiero (Italy), Linda Scattolin (Italy), Roberto Secco