Denis E Corpet

Red meat and colon cancer: Should we become vegetarians, or can we make meat safer?

Keywords: Red meat, processed meat, safer meat, colon cancer, epidemiology, heme iron

The effect of meat consumption on cancer risk is a controversial issue. However, recent meta-analyses show that high consumers of cured meats and red meat are at increased risk of colorectal cancer. This increase is significant but modest (20-30%). Current WCRF-AICR recommendations are to eat no more than 500g per week of red meat, and to avoid processed meat. Moreover, our studies show that beef meat and cured pork meat promote colon carcinogenesis in rats. The major promoter in meat is heme iron, via N-nitrosation or fat peroxidation. Dietary additives can suppress the toxic effects of heme iron. For instance, promotion of colon carcinogenesis in rats by cooked, nitrite-treated and oxidized high-heme cured meat was suppressed by dietary calcium and by α-tocopherol, and a study in volunteers supported these protective effects in humans. These additives, and others still under study, could provide an acceptable way to prevent colorectal cancer.

Introduction

Is it safe to eat meat? The news media reports that meat causes cancer, each time a new scientific study is published. Is the causal link truly demonstrated, or is it only a speculative assumption? Anyway, current recommendations take this risk in account: To reduce the risk of cancer, the 2007 report of the World Cancer Research Fund makes the recommendation to limit the consumption of red meat and to avoid processed meat intake (World Cancer Research Fund & American Institute for Cancer Research, 2007). Based on this report, the French National Cancer Institute recommends: "Limit intake of red meat to less than 500 g per week. Limit intake of cured meats, especially high fat or very salty ones. Those who eat cured meat should choose it less often and reduce portion size." [START_REF] Inca | Nutrition et Prévention des Cancers: des connaissances scientifiques aux recommandations[END_REF]. If these recommendations were adhered to, cancer incidence may be reduced, but farmers and meat industry would suffer important economical problems, while the impact of meat on the risk of cancer is a controversial topic [START_REF] Demeyer | The World Cancer Research Fund report 2007: A challenge for the meat processing industry[END_REF][START_REF] Parnaud | Colorectal cancer: controversial role of meat consumption[END_REF]Santarelli, Pierre, & Corpet, 2008). Although meat intake is not the only risk factor for colorectal cancer, the aim of this article is to focus on meat, to review epidemiological and experimental data and to report recent rodent studies pointing to possible solutions.

Colorectal cancer: Epidemiological studies

1.1-Correlation studies, case-control studies, cohort studies. Correlation between cancer mortality and diet is remarkably strong at the international level: colorectal cancer is frequent in Western countries were red meat is frequently consumed; in contrast, this type of cancer is rare in less affluent countries where meat intake is low (S. [START_REF] Bingham | Diet and cancer -The European prospective investigation into cancer and nutrition[END_REF]. However, correlation is not causation, and it is clear that many other lifestyle factors are different in affluent and poor countries. The hypothesis that red meat favors cancer must be tested at the individual level. Nearly one hundred publications report a link between meat intake and colorectal cancer risk, most of them being retrospective case-control studies, some of them prospective cohort studies. In a retrospective study, people are asked on their past diet, and the answers of hundreds of cancer patients are compared to those of noncancer paired controls. However, the estimation of foods consumed years before is inaccurate, and cancer changes memories, which biases case-control comparison. In addition, results can change depending of the chosen controls, which casts doubts on retrospective studies conclusion. Cohort studies are much longer and more expensive, but they avoid these limitations: thousands to million of healthy people are questioned on their current diet and lifestyle. The cohort is followed for ten to twenty years, and occurring diseases are registered. The statistical link between current diseases and past food intake can then be searched for. One case-control study out of three, and one cohort study out of five, shows a significant link between colorectal cancer risk and red meat or processed meat intake [START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF].

1.2-Major meta-analyses on meat and cancer

In order to estimate the risk associated with meat intake, all of these studies were gathered in two major meta-analyses, whose major results are reported below [START_REF] Larsson | Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies[END_REF][START_REF] Norat | Meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies[END_REF]. A metaanalysis is a statistical approach that gathers all data from published epidemiological studies, after exclusion of poor quality studies. Theoretically, the global result is equivalent to a single large study including all the subjects of the original studies. Due to the very high number of included subjects, even relative risks that are not far from one may be significant. In addition it enables the study of sub-groups that were too small to be analyzed in the original studies.

Norat's meta-analysis gathers 23 cohort and case-control studies, selected out of 48 studies by using pre-established quality criteria [START_REF] Norat | Meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies[END_REF]. Larsson's meta-analysis gathers 18 prospective studies selected out of 23, aggregating more than one million subjects [START_REF] Larsson | Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies[END_REF]. Both meta-analyses are rather independent from each other, because subjects included in Norat's study make only 15% of Larsson's one. The WCRF-AICR 2007 report also describes a meta-analysis based on original studies already included in Larsson's study, and whose results are very close to Larsson's ones. These three meta-analyses bring global and consistent conclusions for different types of meat: total meat intake, red meat, processed meat, and poultry meat. "Red meat" and "processed meat" definitions are tricky points, since it does not mean the same thing in all studies. In most publications, "red meat" gathers beef, veal, mutton, pork and offal, and "processed meat" (equivalent: deli meat) gathers cooked, dried, smoked, or cured meat and offals from any animal, but mostly pork. Some studies make a distinction between fresh and processed meat.

1.3-Major results of meta-analyses

Major results of Norat's and Larsson's metaanalyses were reported as follows: -Consumption of total meat (all types of meat) is not associated with colorectal cancer risk.

-Consumption of "red meat" is associated with a moderate risk increase: = Norat reports a relative risk (RR) of colorectal cancer of 1.35 for the quartile of people eating the highest amount of red meat (including processed meat). It means that cancer risk increased by 35% compared with the quartile eating the least red meat. The 95% confidence interval (95%CI) is 1.21-1.51 and does not include "one", telling the increase is significant. After exclusion of processed meat, the risk associated with eating 120 g/d fresh red meat was still significant (+19%) [START_REF] Norat | Meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies[END_REF].

= Larsson reports a colorectal cancer RR of 1.28 (95%CI 1.15-1.42) for the high red meat eaters (including processed meat). Intake of fresh red meat (excluding processed meat) was reported in nine studies out of 15, and the associated RR is 1.22 (significant). The risk associated with eating 120 g/d of red meat is +28%. Larsson's analysis is less detailed than Norat's: dose-effect was not calculated specifically for fresh red meat, and some points remain obscure (e.g., meat categories) [START_REF] Larsson | Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies[END_REF].

= The WCRF 2007 report gives a summary effect estimate RR of 1.29 (95%CI 1.04-1.60) for 100 g/day from only three selected original studies (World Cancer Research Fund & American Institute for Cancer Research, 2007).

-Consumption of processed meat (mostly deli or cured meat) is associated with colorectal cancer risk: highest vs. lowest category RR are 1.31 (95%CI 1.13-1.51) and 1.20 in those two studies respectively [START_REF] Larsson | Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies[END_REF][START_REF] Norat | Meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies[END_REF]. The WCRF-AICR reports a summary effect estimate of 1.21 (95%CI 1.04-1.42) for 50 g/day (World Cancer Research Fund & American Institute for Cancer Research, 2007). Per gram of meat eaten, cured meat appears to be ten times more efficient to promote cancer than fresh red meat in Norat's study, and twice more in Larsson's.

-Consumption of "white meat", mostly poultry, is not associated with cancer risk (RR = 1.01; 95%CI 0.90-1.13), and a high intake of fish brings a significant protection (RR = 0.85; 95%CI 0.75-0.98) (highest vs. lowest category).

-The method of meat cooking and the doneness, and the human subjects' genetic polymorphism were not taken in account in the above reported studies, although many epidemiological studies address these questions. However, carcinogen chemicals are produced in meat when it is heated above 100°C or when it is cooked on an open flame (e.g., barbecue, see below). These carcinogens can be metabolized slowly in someone and fast in another one. The difference is due to genetic variations in p450 and N-acetyltransferase, key detoxifying enzymes that help to eliminate carcinogens [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF]. The RR values given above thus represent the mean effect of meat, whatever the cooking, on the whole population, whatever the phenotype.

1.4-Consequences and reliability of metaanalyses conclusions

The risk fraction attributable to current levels of red meat intake in various countries was computed by Norat, under the hypothesis that there is a causal link between meat and cancer. The calculation suggests for instance that 25% of colorectal cancers are attributable to the average of 168g of red meat that people are eating daily in Argentina. According to Norat's estimation, the excess risk would almost be zero when people eat less than 70g red meat per week. Elio Riboli provided a recent estimate of the preventability of colorectal cancer (World Cancer Research Fund, 2010). According to his calculation, if USA citizen were eating red meat and processed meat less than once a week, colorectal cancer risk would be decreased by 5 and 12% respectively (World Cancer Research Fund & American Institute for Cancer Research, 2009). WCRF and AICR recommendations are to limit fresh red meat intake to less than 500 g/week in meat eaters, and to avoid processed meat (0 g per week). However, the choice of these thresholds is not clearly substantiated in the report (World Cancer Research Fund & American Institute for Cancer Research, 2007).

In conclusion, these meta-analyses consistently show that red meat and processed meat consumption is significantly associated with a moderate increase in colorectal cancer risk (a relative risk lower than two is considered as "moderate").

Large prospective studies published after 2006 clearly confirmed these conclusions, notably the 500,000 subjects AARP cohort (A. J. [START_REF] Cross | A large prospective study of meat consumption and colorectal cancer risk: an investigation of potential mechanisms underlying this association[END_REF][START_REF] Cross | A prospective study of red and processed meat intake in relation to cancer risk[END_REF]. The excess risk associated with red or processed meat intake was significant in both studies, and the hazard ratios (HR) values were 1.16-1.20 and 1.24 respectively, for the fifth quintile of meat intake compared with the first quintile. It is not surprising that most studies published before 2006 did not show a significant risk, because a small size study cannot show significance when the RR is close to one: these "negative" studies thus do not contradict the general pattern. Meat intake is not the only lifestyle factor that modulates colorectal cancer. According to the WCRF report, the following factors convincingly or probably decrease risk of colorectal cancer: physical activity, foods containing dietary fiber, garlic, milk and calcium; the following factors convincingly or probably increase risk: red and processed meat, alcoholic drinks, body and abdominal fatness, and adult attained height (World Cancer Research Fund & American Institute for Cancer Research, 2007). Cigarette smoking also increases the risk, but was beyond the WCRF report scope. Table 1 shows that the magnitude of red meat effect on colorectal cancer is similar to that of other factors (fatness, alcohol, and smoking).

In addition, few review articles provide criticisms on the above cited studies, attempting to show that the link between meat and cancer is insignificant, but they failed to convince the author of the present review [START_REF] Alexander | Red meat and colorectal cancer: a critical summary of prospective epidemiologic studies[END_REF][START_REF] Mcafee | Red meat consumption: an overview of the risks and benefits[END_REF][START_REF] Truswell | Meat consumption and cancer of the large bowel[END_REF]. To quote Demeyer et al.: "Although criticisms of the inaccurate definition of processed meats and the insufficient accounting for the large variability in composition of meat products have been expressed, it is clear that this problem urges proper action by the meat and nutrition research community and the meat industry" [START_REF] Demeyer | The World Cancer Research Fund report 2007: A challenge for the meat processing industry[END_REF].

Cohort studies are observations: they cannot fully avoid confounding factors. Thus a metaanalysis of cohort studies cannot demonstrate that a food is the cause of a cancer. Only a direct experiment can prove that a cause produces an effect. Indeed, many experimental studies have been done on meat-fed rodents. Do they support the meat-cancer link, and can they explain it? We will briefly review below the mechanistic hypotheses and the animal studies on the meat and cancer link.

Meat and colorectal cancer: Mechanistic Hypotheses

Several mechanistic hypotheses could explain how red meat and processed meat can increase colorectal cancer risk. Pro-cancer factors in red meat might be excess fat, excess protein, excess iron, or heat-induced mutagens. These factors may also act in processed meat, plus salt and nitrite added during the curing process. Other mechanisms might also play a role, but have not yet been investigated thoroughly. Dietary fat increases bile acids secretion inside the gut, and they act as aggressive surfactants for the mucosa thus increasing cell loss and proliferation [START_REF] Bruce | Recent hypotheses for the origin of colon cancer[END_REF]. In addition, fatty diets favor obesity which in turn increases insulin resistance and associated changes in blood values (high glucose, free fatty acids, insulin and IGF1): these circulating factors increase proliferation and decrease apoptosis (= cell suicide) of precancerous cells, thus promoting tumor growth [START_REF] Calle | Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms[END_REF]. Excess protein is fermented in the large bowel yielding amines, phenols and H 2 S that are toxic to the mucosa [START_REF] Visek | Dietary Protein and Cancer[END_REF]. Iron induces production of genotoxic free radicals in the colonic stream [START_REF] Nelson | Iron and colorectal cancer risk: human studies[END_REF] and endogenous N-nitrosated compounds such as carcinogenic Nnitrosamines (S. A. [START_REF] Bingham | Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer?[END_REF]. Last, cooking meat at a high temperature or on an open flame (e.g., grilling, frying or barbecuing) produces heterocyclic amines and polycyclic aromatic hydrocarbons, which are potent carcinogens [START_REF] Sugimura | Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish[END_REF].

However none of those hypotheses seems able, as such, to explain the link between meat intake and cancer risk. For instance, intervention studies in human volunteers do not show any change in intestinal tumor incidence with low-fat diet, suggesting fat is not a major promoter [START_REF] Beresford | Low-fat dietary pattern and risk of colorectal cancer: the Women's Health Initiative Randomized Controlled Dietary Modification Trial[END_REF]. In addition, a recent meta-analysis gathering 1.5 million subjects shows that animal fat intake is not a risk factor for cancer [START_REF] Alexander | Meta-analysis of animal fat or animal protein intake and colorectal cancer[END_REF]. The fermentation products from dietary proteins do not promote colon carcinogenesis in rodents [START_REF] Corpet | Colonic protein fermentation and promotion of colon carcinogenesis by thermolyzed casein[END_REF]. In several studies, inorganic iron failed to promote colorectal carcinogenesis, but Ilsley et al. showed in mice that a diet overloaded with ferric (FeIII) citrate increased tumor size, without promoting preneoplastic lesions or the incidence of colon adenoma. The oxidative status of iron in the gut was not determined [START_REF] Ilsley | Dietary iron promotes azoxymethane-induced colon tumors in mice[END_REF]. Carcinogenic doses of heterocyclic amines in rodents are more than 10000 times higher than levels found in human foods. Grilled and fried chicken contain much more heterocyclic amines than beef meat, but intake of poultry is not related to cancer risk [START_REF] Heddle | A test of the mutagenicity of cooked meats in vivo[END_REF]. It is however likely that all heterocyclic amines have not the same carcinogenic potency (beef ones seems more potent in humans than chicken ones), and that some individuals are more susceptible, due to genetic polymorphisms or intestinal microbiote. For instance, smokers with fast N-acetyltransferase are more susceptible to cancer promotion by well done meat than those with a slow N-acetyltransferase [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF]. Also the intestinal microbiote adapts to meat intake and heterocyclic amines might be more genotoxic in individuals that consume high amounts of meats [START_REF] Kassie | Effect of intestinal microfloras from vegetarians and meat eaters on the genotoxicity of 2-amino-3-methylimidazo 4,5-f quinoline, a carcinogenic heterocyclic amine[END_REF].

However, most studies of meat and phenotypes interactions are deceiving and the general picture is not convincing. Last, cereals, not meat, are the major source of polycyclic aromatic hydrocarbons [START_REF] Phillips | Polycyclic aromatic hydrocarbons in the diet[END_REF]. It is however probable that heat-induced mutagens found on the surface of well-done beef meat can cause colon cancer in people with genetic predisposition. Salt (sodium chloride) and sodium nitrite do not promote colon carcinogenesis in rodents, and salt intake is not associated with CRC risk (but with gastric cancer risk, see below). However, sodium chloride could enhance fat oxidation in meat, increasing the TBARs level and slightly reducing the pool of antioxidant enzymes [START_REF] Gheisari | Chloride salt type/ionic strength and refrigeration effects on antioxidant enzymes and lipid oxidation in cattle, camel and chicken meat[END_REF]. Since none of the above cited hypotheses seem satisfactory, we will review here the animal studies on meat and cancer, and report recent studies from our laboratory, and related studies in Omaha, Nebraska and in Cambridge, UK.

Meat and colorectal cancer: Cancer studies in rodents

Before 2004 twelve rodent studies investigated the effect of a meat-based diet, but none could show the promoting effect of meat on tumorigenesis in rats or mice. In contrast, and very surprisingly, meat diets appear to protect rats and mice against chemically induced carcinogenesis. Below is given a brief summary of those twelve studies that have been reviewed elsewhere [START_REF] Parnaud | Colorectal cancer: controversial role of meat consumption[END_REF]: -Diets that are very high in fat or in protein usually promote carcinogenesis in rats, whatever the fat or protein source, and meat is not "worse" than soy or casein [START_REF] Reddy | Effect of a diet with high levels of protein and fat on colon carcinogenesis in F344 rats treated with 1,2-dimethylhydrazine[END_REF]. Rats given a high-beef meat diet (50%, low fat) have the same number of tumors than casein-fed rats [START_REF] Lai | Non-promoting effects of iron from beef in the rat colon carcinogenesis model[END_REF]. Raw and grilled beef meat diet (20%) do not change tumor incidence in rats compared with a soy-protein diet [START_REF] Clinton | 1,2-dimethylhydrazine induced intestinal cancer in rats fed beef or soybean protein[END_REF]. Kangaroo meat diet (23%) results in the same tumor incidence than casein or soy protein diets in rats [START_REF] Mcintosh | Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats[END_REF].

-Surprisingly, a diet with 60% cooked beef meat significantly protects rats against carcinogenesis compared with a casein control diet [START_REF] Pence | Non-promoting effects of lean beef in the rat colon carcinogenesis model[END_REF]. Compared with a casein-based diet, well done cooked meat (60% of diet, with 35% moisture and a high load of heterocyclic amines) reduces colon cancer risk in rats, in a high-fat context. By contrast in a low-fat context, welldone meat increases cancer risk [START_REF] Pence | Feeding of a well-cooked beef diet containing a high heterocyclic amine content enhances colon and stomach carcinogenesis in 1,2-dimethylhydrazine-treated rats[END_REF]. Mice given a highbeef meat diet (46%) have fewer tumors than casein fed mice [START_REF] Nutter | BALB/c mice fed milk or beef protein: differences in response to 1,2dimethylhydrazine carcinogenesis[END_REF]. Grilled beef meat or bacon diets (30 and 60%), do not increase the number or the size of carcinogen-induced aberrant crypt foci (ACF, preneoplastic lesions) in rats, but bacon diet reduces the ACF size [START_REF] Parnaud | Effect of meat (beef, chicken, and bacon) on rat colon carcinogenesis[END_REF]. Min mice are mutated on the Apc gene and develop many intestinal tumors. Female Min mice given beef meat have less tumors than control Min mice given a no-meat diet [START_REF] Kettunen | Intestinal immune responses in wild-type and apc(min/+) mouse, a model for colon cancer[END_REF].

-Three studies seem to contrast with the above cited ones, but a careful look at the methods reveals meat was not responsible for the tumor promotion: Rats given a humanized diet containing 25% beef meat have more colon cancer than rats on a rodent chow. However, the rodent chow contained much more fibers and less fat than the humanized diet [START_REF] Alink | Effect of heat processing and of vegetables and fruit in human diets on 1,2dimethylhydrazine-induced colon carcinogenesis in rats[END_REF]. A small increase in jejunum polyp number was reported in Min mice given a 24% beef meat diet, but the effect was not significant, and the meat diet contained five times more fat than the control diet [START_REF] Mutanen | Beef induces and rye bran prevents the formation of intestinal polyps in apc(min) mice: relation to betacatenin and PKC isozymes[END_REF]. Last, compared with a whey protein diet, a kangaroo meat diet increases the number of ACF in rats, but whey proteins have known chemopreventive properties and may not be a "neutral" control diet [START_REF] Belobrajdic | Whey proteins protect more than red meat against azoxymethane induced ACF in wistar rats[END_REF].

The discrepancy between epidemiology and animal studies is a paradox: Epidemiology suggests red meat promotes cancer while meat diets show no effect or protection on rodents. Could this discrepancy be explained, and resolved? The next paragraph reports the most likely hypothesis that can, according to the author, explain the effect of meat on cancer and resolve the above-cited paradox.

The heme iron hypothesis: fat peroxidation and N-nitroso pathways

We reasoned that red meat would contain a toxic compound absent in white meat. This toxic compound would be either inactive in rodents or inhibited by rodent diet. Based on works of Van der Meer [START_REF] Sesink | Red meat and colon cancer: the cytotoxic and hyperproliferative effects of dietary heme[END_REF], and of Sawa [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF] we thus speculated that heme iron would be a major player in cancer promotion, explaining why red meat, but not white meat, is associated with cancer risk. This hypothesis is supported by a meta-analysis of epidemiological studies that shows a suggestive association between dietary heme and risk of colon cancer [START_REF] Bastide | Heme Iron from Meat and Risk of Colorectal Cancer: A Meta-analysis and a Review of the Mechanisms Involved[END_REF]. We also speculated that calcium would bind heme iron and suppress its toxicity. This would explain why no animal study published before 2004 and using the high-calcium standard AIN76 diet could show red meat promotion (AIN, 1977).

Our team brought the first demonstration that beef meat added to a low-calcium diet promotes early stages of colon carcinogenesis in chemically-initiated rats. We also demonstrated a dose-response relationship between heme iron and promotion: Tumor number was higher in black pudding-fed (blood sausage) rats than in beef meat fed rats. Tumor promotion was identical in beef meat-fed rats and in rats given a heme-equivalent diet with hemoglobin, but not in rats given the same level of inorganic iron (ferric citrate). In contrast, chicken breast meat did not promote carcinogenesis as it contains little heme iron [START_REF] Pierre | Beef meat and blood sausage promote the formation of azoxymethane-induced mucin-depleted foci and aberrant crypt foci in rat colons[END_REF]. Our hypothesis on heme iron, calcium and cancer was thus demonstrated experimentally. We then wanted to explore the mechanism(s) by which heme iron can promote cancer, and we now think that two independent pathways may link heme and cancer: The fat peroxidation pathway and the N-nitroso pathway that are presented on Fig. 1 (reprinted from [START_REF] Bastide | Heme Iron from Meat and Risk of Colorectal Cancer: A Meta-analysis and a Review of the Mechanisms Involved[END_REF]).

-We think that the fat peroxidation pathway mainly explains tumor promotion by fresh red meat. Our studies consistently show that carcinogenesis promotion by dietary heme iron is associated with the urinary excretion of a fat peroxidation biomarker, called 1,4dihydroxynonane mercapturic acid (DHN-MA) (F. [START_REF] Pierre | Beef meat and blood sausage promote the formation of azoxymethane-induced mucin-depleted foci and aberrant crypt foci in rat colons[END_REF]. DHN-MA excretion also increases in the urine of volunteers that are given black pudding, a heme iron loaded blood sausage [START_REF] Pierre | New marker of colon cancer risk associated with heme intake: 1,4-dihydroxynonane mercapturic Acid[END_REF]. In feces also, high-heme iron diets consistently increase the level of TBARs, an overall measure of aldehyde molecules due to fat oxidation. The oxidation of polyunsaturated fatty acids by hemoglobin leads to peroxyl radicals formation in refined vegetable oils [START_REF] Sawa | Lipid peroxyl radicals from oxidized oils and heme-iron: implication of a high-fat diet in colon carcinogenesis[END_REF]. The main aldehyde molecules are malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) [START_REF] Marnett | Oxyradicals and DNA damage[END_REF]. MDA is toxic and binds DNA, forming mutagenic adducts. 4-HNE induces apoptosis and kills normal cells, but not precancerous cells that are mutated on the Apc gene, because they resist to apoptosis induction [START_REF] Pierre | Apc mutation induces resistance of colonic cells to lipoperoxide-triggered apoptosis induced by faecal water from haem-fed rats[END_REF]. The selective cytotoxicity of 4-HNE explains tumor promotion by a selection process, like the selection of resistant bacteria by an antibiotic [START_REF] Corpet | Colon tumor promotion: is it a selection process? effects of cholate, phytate, and food restriction in rats on proliferation and apoptosis in normal and aberrant crypts[END_REF].

-We think the N-nitroso pathway mainly explains that nitrite-cured meat favors cancer. Feces from rats and mice fed bacon-or hot-dogbased diets contains 5-20 times more N-nitrosocompounds than feces from control rodents fed a casein-based diet [START_REF] Mirvish | N-nitroso compounds in the gastrointestinal tract of rats and in the feces of mice with induced colitis or fed hot dogs or beef[END_REF][START_REF] Parnaud | Endogenous N-nitroso compounds, and their precursors, present in bacon, do not initiate or promote aberrant crypt foci in the colon of rats[END_REF]. Our studies show that cured-meat promotion of carcinogenesis in rats is associated with a high level of fecal apparent total nitroso-compounds [START_REF] Santarelli | Meat processing and colon carcinogenesis: cooked, nitrite-treated, and oxidized high-heme cured meat promotes mucin-depleted foci in rats[END_REF]. This pathway is not limited to cured-meat, since a diet high in fresh red meat (600g/d compared with 60g/d) induces a 3-fold increase in fecal nitroso-compounds [START_REF] Bingham | Does increased endogenous formation of N-nitroso compounds in the human colon explain the association between red meat and colon cancer?[END_REF]. This endogenous production of nitroso-compounds is specifically caused by the intake of heme iron in fresh red beef meat [START_REF] Cross | Haem, not protein or inorganic iron, is responsible for endogenous intestinal n-nitrosation arising from red meat[END_REF]. Pork meat contains less heme iron than beef meat, but nitrite favors the endogenous production of nitroso-compounds in volunteers given cured-meat [START_REF] Joosen | Effect of processed and red meat on endogenous nitrosation and DNA damage[END_REF]. The nature of the nitroso-compounds formed in the gut is not fully known [START_REF] Zhou | Partial purification from hot dogs of N-nitroso compound precursors and their mutagenicity after nitrosation[END_REF]. Most assays indeed gather Fe-nitrosyl heme, Snitroso-thiols with N-nitroso-compounds, and the resulting value is called ATNC for "apparent total nitroso-compounds" [START_REF] Kuhnle | Diet-induced endogenous formation of nitroso compounds in the GI tract[END_REF]. The main part of ATNC in volunteers given red meat is made of Fe-nitrosyl-heme, but those given cured-meat had 2-3 times more "true" Nnitroso-compounds than fresh meat eaters [START_REF] Joosen | Effect of processed and red meat on endogenous nitrosation and DNA damage[END_REF]. Several N-nitrosocompounds are known carcinogen in rodents, and they can alkylate DNA. In volunteers, the red meat associated endogenous NOC formation has been correlated with the formation of the Nnitroso-specific DNA adduct, O6carboxymethylguanine (O6-CMG) in vivo [START_REF] Lewin | Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk[END_REF].

-A third pathway may also explain the effect of red meat: a direct effect of heme on colonic cells. This mechanism has received limited support from studies on cancer cells in vitro. They show that hemin induces DNA damage in human cells of colonic origin [START_REF] Glei | Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes[END_REF], via hydrogen peroxide produced by hemeoxygenase, which can be inhibited in vitro by Zn-protoporphyrin [START_REF] Ishikawa | Heme induces DNA damage and hyperproliferation of colonic epithelial cells via hydrogen peroxide produced by heme oxygenase: a possible mechanism of heme-induced colon cancer[END_REF].

Making safer meat

We then reasoned that knowing the toxicity of heme iron and its pathways to toxicity, we may find ways to suppress the toxicity. As reported above, we knew from van der Meer's publications that heme iron is trapped by calcium phosphate and by chlorophyll [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF]. Van der Meer and colleagues speculated that heme, a planar hydrophobic molecule with polar side chains (like unconjugated bilirubin and bile salts) would bind with calcium ions incorporated in a crystal, by alignment between anionic groups and calcium [START_REF] Sesink | Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium[END_REF][START_REF] Van Der Veere | Rapid association of unconjugated bilirubin with amorphous calcium phosphate[END_REF]. They also speculated that chlorophyll and heme that both are planar hydrophobic porphyrins can stack together in the hydrophobic phase of the luminal contents (de Vogel, Jonker-Termont, [START_REF] De Vogel | Natural chlorophyll but not chlorophyllin prevents heme-induced cytotoxic and hyperproliferative effects in rat colon[END_REF]. We thus designed an experiment showing that promotion of carcinogenesis in the colon of rats by hemin, a chlorinated chemical form of free heme iron, is fully suppressed by dietary calcium [START_REF] Pierre | Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats[END_REF]. We also showed that calcium carbonate suppresses promotion by beef meat [START_REF] Pierre | Beef meat promotion of dimethylhydrazine-induced colorectal carcinogenesis biomarkers is suppressed by dietary calcium[END_REF] and is more efficient than calcium phosphate, without side-effects [START_REF] Allam | Calcium carbonate suppresses haem toxicity markers without calcium phosphate side effects on colon carcinogenesis[END_REF]. However, although it is non-toxic and shows potent and consistent protection, calcium has two drawbacks: (i) it modifies meat Callow's structure and makes it hard and dry; and (ii) it binds heme iron and thus reduces its absorption. In Europe, iron deficiency is one of the main nutritional deficiency disorders affecting large fractions of the population, particularly menstruating and pregnant women. We thus looked for other way to prevent meat promotion without blocking heme iron, by suppressing the fat peroxidation pathway or the N-nitroso pathway.

Peroxidation and nitrosation may be reduced by adding antioxidant or antinitrosant additives to meat. In addition, peroxidation is prevented by removing oxygen, and nitrosation is prevented by removing nitrite from meat or from the gastrointestinal tract. In a study on chemicallyinitiated rats, cured pork meat without sodium nitrite, or packaged to prevent oxidation, does not promote carcinogenesis, in contrast to nitritecured meat exposed to open air for five days in a refrigerator [START_REF] Santarelli | Meat processing and colon carcinogenesis: cooked, nitrite-treated, and oxidized high-heme cured meat promotes mucin-depleted foci in rats[END_REF]. Freezedried cooked ham (with nitrite) purchased in a shop also promotes carcinogenesis in rats [START_REF] Pierre | Freeze-dried ham promotes azoxymethane-induced mucindepleted foci and aberrant crypt foci in rat colon[END_REF], because freeze-drying boosts fat peroxidation [START_REF] Gasc | 4-hydroxynonenal in foodstuffs: heme concentration, fatty acid composition and freeze-drying are determining factors[END_REF]. Adding antioxidant butylated hydroxyanisole with rutin, or oxidation-resistant olive oil, to a hemin-loaded diet fully prevents the promoting effect of hemin, a proxi for meat heme iron (F. [START_REF] Pierre | Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats[END_REF]. In rats, cured meat increased the number of precancerous lesions in the gut, and fecal lipoperoxidation (TBARs). When added as a food additive to the curing solution, α-tocopherol (vitamin E) fully normalized the preneoplastic lesions per colon, and reduced fecal TBARS in cured meat-fed rats. Similarly, TBARS significantly increased in stools of volunteers given cured meat compared to the meat-free period. Calcium supplements or αtocopherol addition fully normalized fecal TBARS in volunteers given cured meat (Santarelli, manuscript in preparation).

We thus have demonstrated in animal studies that red meat and processed meat can promote colon carcinogenesis. As reported above, we provide several ways to prevent this toxic effect by changing the diet, the process, or additives: -Diet change: Calcium carbonate supplements bind heme iron and suppress carcinogenesis promotion in rats, and associated peroxidation biomarkers in rats and volunteers. We suggest that dairy products would produce the same effect. Other way to change diet is to reduce meat intake, following WCRF recommendations.

-Process changes: Preventing the oxidation of fat during meat processing storage with an anaerobic packaging reduces ham-induced promotion. Also, omission of nitrite in curing solution suppressed ham-induced promotion. However, it will not be easy to get rid of nitrite.

-Additives: α-tocopherol added to the curingsolution suppresses cured-meat promotion in rats, and associated biomarkers in human volunteers (unpublished results). Our team is still working on this issue, looking for natural antioxidant and/or anti-nitrosant agents that might be added to meat, notably plant polyphenols. Twelve molecules or extracts from fruits, leaves or rhizome have already been tested in short-term in vivo studies with biochemical endpoints. We are currently testing the most promising chemopreventive agents in a long term carcinogenesis study.

Meat intake and other cancers

Meat consumption appears to increase modestly the risk of colorectal cancer, and thus to be a minor cause of cancer in Western countries. Could meat intake increase also the risk of other cancers, particularly the frequent breast and prostate cancers? Several cohort studies show that cured meat particularly boosts the risk of gastric cancer, likely because of salt and nitrite, but this cancer is rare in affluent countries. For instance in the EPIC study, total meat intake is associated with a RR of stomach cancer of 3.5 (95%CI 2-6) [START_REF] Gonzalez | Meat intake and risk of stomach and esophageal adenocarcinoma within the European Prospective Investigation Into Cancer and Nutrition (EPIC)[END_REF]. However, the WCRF-AICR report concluded the risk was not convincing nor probable but limitedsuggestive. The link seems much weaker with breast and prostate cancers, and did show up neither in a breast cancer meta-analysis [START_REF] Missmer | Meat and dairy food consumption and breast cancer: a pooled analysis of cohort studies[END_REF], nor in the very large European EPIC study of half a million persons. In an American study of similar size, elevated risks (ranging from +20% to +60%) were evident for oesophageal, colorectal, liver, and lung cancer (but neither breast nor prostate) (A. J. [START_REF] Cross | A prospective study of red and processed meat intake in relation to cancer risk[END_REF].

Discussion and conclusion

The above reported observation studies clearly show that consumers of processed meat (mainly cured pork) and of red meat (mainly beef) have a modest increase in their risk to develop a colorectal cancer. Our experimental studies in rats suggest the effect is not due to confounding factors, but comes from true toxic factor(s) in red and processed meat. From an individual perspective, +25% risk is a rather small increase. Let us assume that one person out of 20 have a colorectal cancer, this figure would increase from 1.0 to 1.2 out of 20 in the most "carnivorous" fraction of the population. In contrast, the risk increase seems large from a public health perspective. Let us assume that one hundred people in France are told each day they have colorectal cancer. The excess risk associated with a daily steak, +25%, would now translate to an extra 25 people each day with cancer, which is not acceptable! One may think that the global risk had been estimated mostly from American data and would not apply to other parts of the world, particularly Europe. But Larsson's meta-analysis specifically addressed this question, and her data show that the risk increase per gram of meat consumption is not different in Europe and in the USA [START_REF] Larsson | Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies[END_REF]. In addition, results from meat intake surveys do not show large differences between meat intake in Europe and North-America. For instance, the French INCA2 survey shows that red meat and processed meat intake are 370g and 270g per week respectively in France [START_REF] Volatier | La place de la viande et des produits à base de viande comme aliments-vecteurs dans les apports nutritionnels de la population française[END_REF]. Distribution data show that a quarter of the French adult population (39% men and 13% women) eats more red meat than the recommended 500g, and a quarter eats more than 50g/d processed meat (we do not know how much these two populations overlap). Thus, at least in France, the cancer burden due to fresh meat consumption should be roughly equivalent to the burden due to processed meat.

Our experimental studies in rats provide direct evidence that red meat and processed meat can increase colon carcinogenesis. They also strongly support the hypothesis that heme iron is the major cause of cancer promotion by red meat. Based on works by other researchers, our results add some evidence to two pathways linking dietary heme iron and cancer promotion. Lastly, we are suggesting several ways to prevent the toxic effect of meat, either by increasing the calcium load of the meal, by changing the meat processing, or by choosing new additives. Full demonstration of mechanisms and of chemopreventive substances has not yet been given, but we expect that these studies will lead to a reduction of the risk of colorectal cancer without losing the nutritional benefit and the pleasure of eating meat. 1. b-Percent change = 100 times the absolute value of (RR-1) c-Summary estimates of Relative Risk were extracted from the WCRF-AICR 2007 report, except value for smoking, not reported in the report, and extracted from a recent meta-analysis [START_REF] Botteri | Smoking and colorectal cancer: a meta-analysis[END_REF]. d-Significance: * the 95% confidence interval excludes 1.00; NS, non significant; MS, marginally significant (1.00 is the upper value of the confidence interval) e-Abdominal fatness measured by the Waist-to-Hip ratio, and body fatness by the Body Mass Index. [START_REF] Bastide | Heme Iron from Meat and Risk of Colorectal Cancer: A Meta-analysis and a Review of the Mechanisms Involved[END_REF]. Heme iron catalyzes nitrosation and fat peroxidation. End products are N-nitroso compounds (NOCs), malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE). These pathways explain, at least in part, the promoting effect of red and cured meat on colorectal cancer. The catalytic effects of heme iron can be inhibited by trapping heme with calcium carbonate or chlorophyll. The endogenous formation of NOCs is inhibited by vitamin C and E. Ongoing studies suggest that specific polyphenols can inhibit fat peroxidation and/or nitrosation.

Figure 1 Fig. 1 .

 11 Figure 1

Table 1 : Summary estimates of relative risk on colorectal cancer, from cohort studies meta-analysis

 1 (World Cancer Research Fund & American Institute for Cancer Research, 2007). -C, convincing; P, probable. Factors with limited/suggestive evidence are not reported in Table

		Evidence	Percent	Summary	Signif.
	Factor	strength a	change b	RR c	d	per
	Abdominal fatness e	C	30	1.30	*	0.1 W-to-H
	Red meat	C	29	1.29	*	100 g/d
	Garlic	P	27	0.73	*	high vs. low
	Alcohol	C	27	1.27	*	30 g/d
	Smoking c	C	25	1.25	* ever vs. never
	Processed meat	C	21	1.21	*	50 g/d
	Body fatness e	C	15	1.15	*	5 kg/m 2
	Dietary fiber	P	10	0.90	*	10 g/d
	Adult attained height	C	9	1.09	*	5 cm
	Milk	P	6	0.94	NS	serving/d
	Calcium	P	2	0.98	MS	200 mg/d

a

Acknowledgments

Many thanks are due to Fabrice Pierre for his help in writing this review, to Conner Middelmann-Whitney for editing the title, to Nadia Bastide for composing the figure, and to the four anonymous Reviewers who suggested many improvements. D.E. Corpet's team researches are supported by French INRA, DGER, and ANR projects HemeCancer and SecuriViande.


Amelène El-Mufleh
email: amelene.el-mufleh@lcpc.fr

Véronique Béatrice Bechet
email: beatrice.bechet@lcpc.fr

B Béchet

V Ruban

Characterisation and modelling of the interactions between pollutants and solid matrix in mixed contaminated urban soils

Keywords: Urban water, contaminants, risk assessment, organic and inorganic pollutants, characterisation, modelling

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

To control build-up area's extension and to fulfil people wishes in terms of life's quality improvement, soil preservation became a national and European priority. For the local authorities, the aim is to limit primeval sites and manage the potentially contaminated area.

In urban and suburban environments, the degradation of soils and water resources may result from craft or industrial activities, the re-use of industrial by-products in civil engineering, water management practices … The authorities are highly concerned with the impact of soil pollutions on human health, and waiting forward management solutions for contaminated areas. Currently, the available tools used in urban management project are not taken into account the specificities of urban soils in terms of structural and biogeochemical complexity. Improvements of these tools require a multidisciplinary approach.

This study is developed in the framework of a research project (POLESUR) on soil and water pollutions in urban areas.

The aim of this study is to address part of this complexity through characterisation of the mixed pollution within specific urban soils, and through the modelling of the interactions between pollutants (trace metals and organic compounds) and solid matrix. The review of the literature of urban soils indicates that most of the studies focus on the characterisation and treatment of industrial soils and stormwater drainage. The most frequently studied sites are heavily contaminated industrial sites and retention/infiltration ponds.

Those studies describe retention process of the pollutants in the soil as the ions exchanges, the surface complexation, the precipitation or the hydrophobic interactions " WEBER et al. (1991)".

Studies also define the factors influencing the retention of the pollutants " DURIN et al. (2005)", such as pH, oxido-reduction conditions or ionic force. " CLOZEL et al. (2005)" describe the trace metals mobility and put forward the solid phases which bear the pollutants.

The originality in this study lies in the consideration of the mixed pollution in soil and in the qualitative but also quantitative approach of the pollution repartition.

The first objective of this study is to develop a methodology to study the mixed pollutant distribution on solid phases applied to specific urban soils, such as stormwater sediment and former industrial site. The second objective is to study the adsorption/desorption process.

The study is organized into steps: selection of specific urban sites, to define a methodology, to establish a method for physical and chemical characterisations, including microscopy observations to localized pollutants in the solid phases, and to implement batch and column experiments in order to model the pollutant release.

This paper presents the results of preliminary sampling of waters and sediments in infiltration basins, the advancement state of the experimental sites selection and proposes a methodology for the study of the mixed pollutants speciation.

MATERIAL & METHODS

Criteria for the choice of sampling sites

The investigation phase focuses on two types of sites, retention/infiltration basins and former craft and/or industrial areas containing both organic and inorganic pollutants.

In large cities, such as Nantes or Lyon, these kinds of sites are common. About 250 infiltration structures have been inventoried in Nantes or in Lyon areas.

Criteria for the choice of retention/infiltration basins are as follows: basins receiving highway runoff waters with a high traffic; size around or greater than 1 ha; containing old and non redisturbed sediments and a secure access.

The industrial site (or former craft), should have an important and well identified waste storage or rejection surface, pollutants should be detected in groundwater, the hydrogeology of the site have to be identified and easy to understand and the most important point is to have the authorization of the exploiting.

Analytical methods

Trace metal analysis in surface water

Surface water were analysed for total trace metals (NF EN ISO 11885). 100 mL of sampling water were acidified by 0.5 of the site have to be identified and easy to understand and the most important point is to have the mL of pure HNO3 (nitric acid), sonificated during 15 minutes and kept in contact with the acid during 165 minutes. Then, the solution was filtered using a 0.45 m Whatman (n°40) filter. The analysis of major and trace elements was performed using an ICP-OES (Inductively Coupled Plasma -Optical Emission Spectrometer) (Varian 720-ES). The quantification limit for As, Cd, Cr, Cu, Ni, Pb and Zn are respectively 6, 0.5, 5, 2, 10, 10 and 2µg/L. In case of concentration under the limit of quantification, the sample was analyzed using an ICP-MS (Inductively Coupled Plasma -Mass Spectrometer) (Varian 820-MS). The ICP-MS limit of quantification for water is ranging from 0.02 g/L to 0.3 g/L depending on the element (Table 1).

Trace metals analysis in sediment

Trace metals were extracted by an acid attack from the sediments collected in infiltration basins (NF X 31-147). 0.5g of sediment is sieved to 2 mm to remove gravel and roots, and calcinated during 3 hours at 450°C. Then, after the addition of 10 mL of HF (hydrofluoric acid) and 3 mL of HClO4 (perchloric acid), the sample was left to boil dry on a hotplate at 160°C. Once dry, the residue is taken up by 1mL of HNO3 (nitric acid) at room temperature during 30 min, then 10mL of ultrapure water is added and heated at 60°C for 1h. The residue was transferred into a 100mL flask and filled up with ultrapure water to the mark. This sample was filtered by a 0.45 m Whatman (n°40) filter and analyzed using an ICP-MS (Varian 820-MS) for major and trace elements.

Organic pollutant analysis in sediments

The analyses of total hydrocarbons, PAHs and PCBs were conducted on sediment samples (at IDAC Nantes laboratory). PAHs and PCBs were analyzed using a gas chromatography coupled to a mass-spectrometer (GC/MS (/MS)) according to the XP X33-012 norm. For the total hydrocarbons, the ISO 11046 (X31-410) norm is used and analysis is performed with an Infrared spectrophotometer.

Methodology for studying pollutant speciation

The following methodology is available for both kinds of sites: retention-infiltration basins and industrial areas.

The soil can be regarded as a complex solid matrix, consisting of mineral and organic phases. To locate pollutants in these different phases of the soil, there are two main types of methods: physical methods (or direct), which consist in a non-destructive separation of the soil phases, depending on their physical properties (size, density ...); and chemical methods (or indirect) where the pollutant is taken out of it structural context, by chemical attacks, analyses are indirect insofar as they are done in the solution "CORNU et al. ( 2000)".

Physical fractionation

The use of physical fractionation decreases the heterogeneity of the soil, thus chemical analysis of pollutants can be made for each fraction and so indicates the repartition of those pollutants on the soil different phases. One of the fractionation usually studied is the densimetry (fractionation by density of soil phases). " DOELSCH et al (2006)" proposed an original density fractionation, based on a simple preparation, which allows the identification of pollutants in the different density fraction. In addition to quantify the pollutants, this non destructive method allows the identification of the phase structure by using X-ray diffraction, Nuclear magnetic resonance spectroscopy… The main problem encountered is the organomineral aggregation which changes the density of the organic and the mineral phases of the agglomerate. 1). The fractions chosen are those likely to be affected by variations in environmental conditions. The extractant used in the four steps differ from one author to another, but a standard method from the BCR (Bureau Communautaire de Référence) is extensively used (Figure 1).

Metals analysis ICP/MS

Chemical fractionation

Exchangeable fraction

Reducible fraction

Oxidiseable fraction

Residual fraction

Soil or sediment sample

Residue

Residue

Residue

Supernatant is recovered

Step 1 CH 3 COOH

Step 2 NH 2 OH, HCl

Step 3

H 2 O 2 + CH 3 COONH 4
Step 4 HF/HClO 4 + HNO 3

Characterisation and modelling of the interactions between pollutants and solid matrix in mixed contaminated urban soils -Amelène EL MUFLEH

/ 10

The chemical fractionation suggested by "DOICK et al ( 2005)" depends on the chemical proprieties (mainly the solubility). It is a non destructive method which allows the fractionation of humic substances (fulvic acid, humic acid and humin) and also of the mineral phase (Figure 2). Both organic and trace metals remain associated to the different fractions. The original solvent used is the MIBK (Methyl IsoButyl Ketone).

Dry soil sample

Mineral fraction

Fulvic acid

Humic acid in aqueous phase

Acidify to pH<2 and centrifuge The foreseen methodology

Humic acid

In order to localise pollutants in the different phases of the soil, the previous methods (chemical extraction and fractionation) will be used. Indeed, the chemical extractions procedure will inform us on the mobility of trace metals in the operationally defined fractions. It could be interesting to adapt these methods to the organic compounds. Furthermore, it will be useful to assemble the physical and chemical fractionations, i.e. to carry out a density fractionation to the mineral fraction resulting from the MIBK fractionation procedure. Then quantification of both the organic and inorganic pollution in all phases will be possible. This by trying to minimize the organomineral aggregations could also be reduced in the density fractionation.

Otherwise, in each case, to follow the separation (in fractionation methods) or the solubilisation (in sequential extractions), we will associate analytical techniques as X-ray diffraction, Scanning or Transmission Electronic Microscopy, Infrared spectrophotometer or X-ray Photoelectron Spectroscopy.

RESULTS AND DISCUSSION

Infiltration basin selection

A first set of experimental sites was selected along the ring-road of Nantes (west of France). Five retention-infiltration basins receiving highway runoff waters were selected and a preliminary sampling campaign was performed in December 2008 and January 2009 to collect surface water and sediments from a central zone of the basins: along A11 highway (four basins) and next to Nantes ring-road (Cheviré basin) (Figure 3). This last basin has been studied by LCPC since 1991.

Figure 3: Location of the five infiltration basins and the landfill in Nantes area

Sites from the A 11 highway

These basins are located in the east of Nantes (Figure 3), a few kilometers from each other, and are named as follows (from the farther west to the most easterly), Gesvres, Boisbonne, Erdreau and Jouhandière. All have been in use since the opening of the A11 in 1992 and had never been dredged since this date.

Gesvres is on the border between the A11 highway and the Nantes ring-road. It carries an average daily flow of 80 000 vehicles. The overflow of the basin discharges into a stream named Gesvres.

For Boisbonne, Erdreau and Jouhandière, the drained runoff waters only come from the A11 highway. The flow of vehicles is an average of 27 000 per day.

Cheviré basin

This basin is located in the south west of Nantes (Figure 3), it receives runoff waters from the south part of the Cheviré bridge which is a section of the Nantes ring-road. Opened in 1991, the Cheviré bridge now carries an average daily flow of 90 000 vehicles. The drainage surface area

Prairie de Mauve' landfill

Cheviré

Gesvres

Boisbonne Erdreau

Jouhandière

LCPC

Characterisation and modelling of the interactions between pollutants and solid matrix in mixed contaminated urban soils -Amelène EL MUFLEH 7 / 10 is around 1.9 ha. Runoff waters are collected in gulleys, driven through an aluminium gulley and then trough a concrete pipe to the basin. The surface of the basin is 0.78 ha and its depth is about 1.5 m. The runoff waters are collected and infiltrated via a single flow path from the entrance to the overflow of the basin to the Loire river. The basin had not been dredged since 1991, expect in a small area close to the main inlet. This results in the settlement of a sedimentary layer whose thickness varies from 5 to 30 cm depending on the location in the basin, laying on a sandy subsoil.

Analytical results

Table 1 summarizes the results of trace metals in surface water for each site. The trace metal concentrations are as follows, As (from 5.10 to 20.52 g/L), Cd (from 0.01 to 2.8 g/L), Cr (from 0.30 to 8.6 g/L), Cu (from 1.03 to 296 g/L), Ni (from 0.92 to 12.4 g/L), Pb (from 0.28 to 80 g/L) and Zn (from 6.10 to 1946 g/L). Cheviré's trace metal concentrations, expect for arsenic, are from 2 to 300 times higher than concentrations for the other sites. For arsenic, the values are almost in the same order of concentration. We can also notice that cadmium values for the A11 sites are close the limit of quantification thus negligible. The results of trace metals in sediments for each site are presented in table 2. The trace metal concentration are as follows, As (from 28 to 174 mg/kg), Cd (from 0.3 to 1.3 mg/kg), Cr (from 57 to 97 mg/kg), Cu (from 31 to 411 mg/kg), Ni (from 32 to 76 mg/kg), Pb (from 33 to 271mg/kg) and Zn (from 125 to 1655 mg/kg). We can notice again that Cheviré's values for copper, lead and zinc are in average respectively 13, 8 and 13 times higher than the other for the extreme values. Nevertheless, for the A11 basins, arsenic concentrations seem to be higher and require intervention. Nickel, chromium and cadmium concentrations were in the same range. Trace metals in surface water and in sediments both presented higher concentrations for Cheviré basin than for A11 basins (expect for As). This also applied to Gesvres, which have roughly the same traffic. The geochemical background and the highway maps were consulted. The main difference in the construction between Cheviré and A11 basins lies in their pits (in which the runoff water transits to the basin). In fact, for the A11 basins all the pits are grassy whereas Cheviré pits are all concrete. Consequently, for A11 sites, the hypothesis is that the pollutants infiltrate before the entrance in the basins. Furthermore, according to the geochemical background, the high arsenic concentration is due to the geochemical background of Nantes region (ranging from 20 to 500 mg/kg in road basin sediments) and can't be due to anthropogenic sources.

On the basis of trace metal concentrations, we decided to carry out the organic pollutant analyses only for Gesvres and Boisbonne sites. Some results are available for Cheviré basin "Durand et al.

2004". (2) Dutch standard applied to a soil containing 25% clay and 10% organic matter * Abbreviations: Benzo(a)anthracene (Ba-ANT), Benzo(a)pyrene (Ba-PYR), Benzo(b)fluoranthene (Bb-FLA), Benzo(ghi)perylene (Bghi-PYR), Benzo(k) fluoranthene, Chrysene (CHY), Fluoranthene (FLA), Indeno(1,2,3cd)pyrene(Icd-PYR), Phenanthrene (PHE), Pyrene (PYR) and total hydrocarbons (Hc(tot)).

The three sites are contaminated by hydrocarbons (including PAHs). Gesvres and Boisbonne have a greater variety of PAHs even if they are less concentrated. As for trace metals, the Cheviré concentrations of fluoranthene, phenanthrene, pyrene and the total hydrocarbons are 5 to 10 times higher than the others, the results for Boisbonne are higher than Gesvres, although this was not always the case previously for trace metals.

Site choice

According to the pollutant results, Cheviré and Boisbonne are obviously the selected sites for the following methodology, even if Boisbonne doesn't show high concentration compared to Cheviré.

Industrial sites selection

The BRGM (the French Geological Survey) identified several sites in the Nantes region, based on results of BASIAS and BASOL (DRIRE) data bases. A visit of the landfill of "Prairie de Mauve" near Nantes (Figure 3) has been organized, but the heterogeneity of the site was such that a pre-study should be done before the selection. This pre-study consists in searching a delimited area in the landfill in which the heterogeneity is lower and the hydrogeology easier to understand and will be performed by the BRGM. In addition, negotiations with an industrial operator are in progress for another site. According to the BRGM' census, this site is interesting in terms of mixed pollution.

Cheviré basin previous results

On Cheviré basin, pollutants and sediments speciation have been studied by "DURAND ( 2003)". These speciation consisted in the characterisation of the sediment regarding the mineral and organic phases with several analytical techniques, and in following the pollutant mobility with extraction method. The XRD composite sample results show a fairly uniform pattern, with crystalline phases that are mostly primary silicates (albite, clinochlore, microcline, muscovite and quartz). These results are confirmed by the Scanning Electron Microscopy (SEM) coupled to an Energy Dispersive Xray (EDX) spectroscopy. The organic phases have been studied with the IHSS (International Humic Substance Society) protocol, which separates humic substances themselves identified with NMR (Nuclear magnetic resonance spectroscopy), Infrared spectroscopy or GC-MS (Gas chromatography-mass spectrometry). Humin represents 57.6% of total organic matter, fulvic acid 4.5%, humic acid 18% and lipids 19.9%. It should be noted that in this protocol, it seems that the humin fraction contains mineral phases. The mobility of trace metals has been followed within the IHSS protocol for the organic matter and with sequential extractions for the different sediment phases. The following observations have been noted: Cd, Pb and Zn are mainly in fulvic acids fraction, Ni and Cr are mainly in humin fraction, Cu in both humic acid and fulvic acid fractions, furthermore, Cd and Zn are located in the exchangeable fraction (of the sequential extractions), Ni and Cr are in the residual fraction and Cu and Pb are bond to the organic fraction.

Our new methodology should go beyond these conclusions. In fact, we should be able to localise more precisely both organic pollutants and trace metals in all the soil phases by proceeding to the MIBK extraction with the densimetric fractionation. The obtained results will be compared to sequential extractions for the trace metals. Additionally, we will observe and characterise the soil phases with several technical's analysis as XPS (X-ray Photoelectron Spectroscopy), μ-XRF (Xray Fluorescence micro-analyzers) which should give more information on pollutant speciation.

CONCLUSIONS

For the site research, two retention/infiltration basins have been selected (Cheviré and Boisbonne), however, investigation continues, we are looking for an industrial site and other retention-infiltration basins containing both organic and inorganic pollutants. In fact, the study of several sites will be useful to compare and validate our results. The new methodology will be applied on the Cheviré and Boisbonne sediments; it will be extended to the future sites. Finally it could be very interesting to establish a correlation between a given pollutant and the abundance of a particular phase. This would allow the development of simple indicators based on soil type obtained with simple analytic techniques which would have been validated by more complicated techniques. Furthermore, these results should give information for the choice of the future modelling parameters.

8th

  World Wide Workshop for Young Environmental Scientists WWW-YES 2009: Urban waters: resource or risks? (2-5 June 2009)

Figure 1 :

 1 Figure 1: Sequential extraction procedure, according to "JAMALI et al. (2008)"

Figure 2 :

 2 Figure 2: MIBK fractionation procedure, according to "DOICK et al (2005)"

8th

  World Wide Workshop for Young Environmental Scientists WWW-YES 2009: Urban waters: resource or risks? (2-5 June 2009)

  Results from Durand's thesis(2003).

8th

  World Wide Workshop for Young Environmental Scientists WWW-YES 2009: Urban waters: resource or risks? (2-5 June 2009) 10 / 10

  Three types of methods were presented: simple, kinetic and sequential extractions. These methods have been used for speciation of trace metals.o Simple extractions help to differentiate labile or "available" compartment, from non mobile compartment. Indeed, this simple method is commonly used in soil science in particular to predict the availability of the trace metals for plants. Four types of extraction solutions are mainly used: deionized water (often to simulate natural conditions of soil), saline solutions (mainly CaCl 2 and BaCl 2 ), diluted acids and organic complex as EDTA or DTPA. Phases extracted by the extraction solution containing the most mobile metals, are potentially the most extractable. o More complex techniques allow the differentiation of three compartments, the labile, the slightly mobile and immobile (or not extractable). Kinetic extractions are based on simple extractions of trace metals at different contact times by a single extractant (salt or EDTA complexes in most of cases). o Sequential extractions were developed in the early 1980s. Initially for trace metals speciation in sediments, they have been adapted to soils. The principle is to submit a sample, in order, to a series of increasing strength reagents to solubilise different phases (or fractions) of soil. The various metals associated with these phases or fractions are solubilised at the same time and quantified. Patterns of extractions contained usually four steps (Figure

8th World Wide Workshop for Young Environmental Scientists WWW-YES 2009: Urban waters: resource or risks? (2-5 June 2009) 4 / 10

Chemical extractions

Table 1 : Analytical results for trace metals in surface water for each site

 1

	(μg/L)		As	Cd	Cr	Cu	Ni	Pb	Zn
	LQ (1)		0.02	0.02	0.02	0.02	0.05	0.2	0.3
	Gesvres		20.52	0.01	0.70	3.39	3.32	0.63	12.55
	Boisbonne		7.36	0.05	0.32	2.06	10.67	0.39	34.19
	Erdreau		7.92	0.06	0.30	1.98	2.89	0.45	6.10
	Jouhandière		5.10	0.02	0.39	1.03	0.92	0.28	10.76
	Cheviré		14.1	2.8	8.6	296	12.4	80	1946
	Dutch standard	(2)	50	5	50	1000	50	50	5000
	(1) Limit of quantification for the Varian 820-MS ICP/MS
	(2) Threshold at which a soil is considered contaminated (according to the Dutch standard)

Table 2 : Analytical results for trace metals in sediments for each site

 2

	(mg/kg)	As	Cd	Cr	Cu	Ni	Pb	Zn
	Gesvres	174	1	72	85	41	83	327
	Boisbonne	93	1.3	59	70	76	44	410
	Erdreau	71	0.3	64	31	38	33	125
	Jouhandière	40	0.5	57	51	32	46	465
	Cheviré	28	1	97	411	37	271	1655
	Polluted soil (1)	29	0.8	100	36	35	85	140
	Intervention's
	threshold (2)	55	12	380	190	210	530	720

(1)

Value at which a soil is considered contaminated (according to the Dutch standard)

(2)

Value at which intervention is necessary (according to the Dutch standard)

Table 3 ,

 3 presented the concentrations of 10 PAHs from the 16 PAHs recognized as proprietary pollutants by the US-EPA (US Environmental Protection Agency) and the total hydrocarbon values. The other PAHs and the PCBs haven't significant values.

Table 3 : Analytical results for PAHs and Hc(tot) in sediments for the following basins

 3

	μm/kg	Ba-ANT*	Ba-PYR*	Bb-FLA*	Bghi-PL*	Bk-FLA* CHY* FLA*	Icd-PYR* PHE* PYR*	Hc(tot)* mg/kg
	Gesvres	34	51	75	118	51	71	93	75	54	98	246
	Boisbonne	59	74	103	133	74	108	128	96	83	135	997
	Cheviré (1)	-	-	-	-	-	-	670	-	313 1223	7000
	Dutch
	standard (2)

11-WWW-YES-2009-France-El-Mufleh-paper.doc

AKNOWNLEDGES

This work benefits from the financial support of the "Région des Pays de la Loire" in the framework of the regional research project POLESUR "Soil and water pollution in urban areas" (2008)(2009)(2010)(2011).

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