Liesbeth Temme

Stefanie Marie Vandevijvere
email: stefanie.vandevijvere@wiv-isp.

Christine Vinkx

Inge Huybrechts

Leo Goeyens

Herman Van Oyen

Elisabeth Hm Temme

Leo

Goeyens

Average daily nitrate and nitrite intake in the Belgian population older than 15 years

Keywords: Nitrate, nitrite, food consumption survey, exposure assessment, ADI, Belgium

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

NUSSER method. The mean usual daily intake of nitrate was 1.38 mg/kg bodyweight (bw)/day and the usual daily intake at the 97.5 percentile was 2.76 mg/kg bw/day. Exposure of the Belgian population to nitrate at mean intake corresponded to 38% of the ADI (while 76% at the 97.5 percentile). For the average consumer half of the intake was derived from vegetables (especially lettuce) and 20% from water and water-based drinks. Average daily intake of nitrate and nitrite from cheese and meat products was low (respectively 0.2% and 6% of ADI at average intake). Scenario analyses with higher consumption of vegetables or higher nitrate concentration in tap water showed a significant higher intake of nitrate. Whether this is beneficial or harmful must be further assessed. The aim of this study was to assess the dietary intake of nitrate and nitrite in Belgium. The 20 nitrate content of processed vegetables, cheeses and meat products was analyzed. These data 21 were completed by data from non-targeted official control and from literature. In addition, the 22 nitrite content of meat products was measured. Concentration data for nitrate and nitrite were 23 linked to food consumption data of the Belgian Food Consumption Survey. This study 24 included 3245 respondents, aged 15 years and older. Food intakes were estimated by a 25 repeated 24-h recall using EPIC-SOFT. Only respondents with two completed 24-hour recalls 26 (n=3083) were included in the analysis. For the intake assessment, average concentration data 27 and individual consumption data were combined. Usual intake of nitrate/nitrite was calculated 28 using the NUSSER method. The mean usual daily intake of nitrate was 1.38 mg/kg 29 bodyweight (bw)/day and the usual daily intake at the 97.5 percentile was 2.76 mg/kg bw/day. 30 Exposure of the Belgian population to nitrate at mean intake corresponded to 38% of the ADI 31 (while 76% at the 97.5 percentile). For the average consumer half of the intake was derived 32 from vegetables (especially lettuce) and 20% from water and water-based drinks. Average 33 daily intake of nitrate and nitrite from cheese and meat products was low (respectively 0.2% 34 and 6% of ADI at average intake). Scenario analyses with higher consumption of vegetables 35 or higher nitrate concentration in tap water showed a significant higher intake of nitrate. 36 Whether this is beneficial or harmful must be further assessed. 37 Recently the role of nitrate and nitrite as healthful dietary components has been reconsidered 39 [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF][START_REF] Lundberg | Cardioprotective effects of vegetables: is nitrate the answer?[END_REF][START_REF] Lundberg | Cardiovascular prevention by dietary nitrate and nitrite[END_REF]Lundberg et al. 2009;Lundberg et 40 al. 2011;Lundberg and Weitzberg 2009;[START_REF] Lundberg | The biological role of nitrate and nitrite: the times they are a-changin[END_REF]. Nitrate and nitrite 41 rich food sources may play a physiological role in vascular and immune function. Higher 42 intakes are hypothesized to be associated with lower blood pressure and a better 43 cardiovascular function [START_REF] Lundberg | Cardioprotective effects of vegetables: is nitrate the answer?[END_REF][START_REF] Lundberg | Cardiovascular prevention by dietary nitrate and nitrite[END_REF][START_REF] Lundberg | Roles of dietary inorganic nitrate in cardiovascular health and disease[END_REF]. 44 Till now the risk of nitrate and nitrite has been emphasized. Nitrate toxicity is, for adults, 45 thought to be related to the in vivo conversion to nitrite after ingestion [START_REF] Hartman | Review: putative mutagens and carcinogens in foods. I. Nitrate/nitrite ingestion and gastric cancer mortality[END_REF]. Nitrite 46 reacts with amines at the stomach pH to form nitrosamines that are known as carcinogenic 47 compounds (Office fédéral de la santé publique Division science des aliments 2000). The EC 48 Scientific Committee on Food (SCF) considered in their independent expert committee in 49 1995 the safety of nitrate and recommended an Acceptable Daily Intake (ADI) of 3.65 mg per 50 kg bodyweight per day (equivalent to 220mg per day for an adult of 60 kg) (JECFA Food 51 Additives Series 50, Nitrate and Nitrite). The ADI for nitrite is 0.07 mg per kg bodyweight 52 per day, which is equivalent to 4.2 mg per day for an individual weighing 60 kg. 53

Nitrate and nitrite are allowed as preservative by European Authorities (European Parliament 54 and Council Directive No 95/2/EC of 20 February 1995). 55

It is worthwhile to assess the exposure of the Belgian population to nitrate and nitrite and its 56 major sources, both because of possible beneficial as well as harmful aspects. 57

The nitrate intake by the Belgian consumer was estimated previously in 1994 (Dejonckheere 58 et al. 1994). Additive intake was not taken into consideration in that study. At that moment 59 actual food intake data were not available, since no national dietary survey had been carried 60 out. Consumption data were estimated from household purchase data. 61 The objective of the present study was to re-estimate the nitrate and nitrite intake via the diet 62 in Belgium by use of actual concentrations and individual food consumption data. To 63 complement the existing data on occurrence of nitrate in unprepared vegetables and fruits, 64 obtained from Dejonckheere et al [START_REF] Dejonckheere | Nitrate in food commodities of vegetable origin and the total diet in Belgium (1992-1993)[END_REF], analyses were performed in 65 processed vegetables and fruits (canned, jarred or deep frozen, as available on the market). 66

For some products, for example spinach, including the processing in intake assessment is 67 especially important because of the higher consumption of frozen compared to fresh spinach 68 in Belgium. To our knowledge the effects of processing methods on nitrate concentrations in 69 vegetables available on the Belgium market have not been evaluated before. In addition, food 70 groups (cheese, meat products) were analyzed in which nitrate or nitrite is added as a food 71 additive. 72

73

METHODS

74

Study design 75

For the analysis of nitrate in foods, the focus was on fruits and vegetables, potato (products), 76 cheese and processed meat. Food items for nitrate or nitrite analyses were chosen on the basis 77 of data on nitrate concentrations present in foods available in Belgium: from literature, from 78 the Belgian Federal Agency for the Safety of the Food Chain or available product 79 information. Already existing data on nitrate concentrations in fresh vegetables and fruits 80 from Belgium were used from Dejonckheere et al [START_REF] Dejonckheere | Nitrate in food commodities of vegetable origin and the total diet in Belgium (1992-1993)[END_REF], as well as more 81 recent data of fresh vegetables from non-targeted official control programmes. In case both 82 consulted sources contained data for a certain vegetable or fruit, the most recent data were 83 used. In Additional analyses were performed that could add to these already existing data. The focus 87 was on the nitrate content of differently processed vegetables and fruits and nitrate used as 88 additive in cheese and processed meats. The average nitrate content of bottled water, as 89 communicated by the industry to the Federal Public Service of Health (2.6 mg/L; average of 90 positive concentrations), was used for mineral and source waters. For other drinks based on 91 water (coffee, tea, soup), the nitrate concentration was assumed to be 21 mg/l, which is the 92 average nitrate concentration as communicated by official distributors of tap water in Brussels 93 and Wallonia. For the analysis of nitrite, focus was on the group of processed meats because 94 of the high allowed levels (residual concentration of 50-175 mg/kg) compared to its presence 95 in other food items (European Parliament and Council Directive No 95/2/EC of 20 February 96 1995). 97

Afterwards, the nitrate and nitrite concentrations of the different foods were linked to 98 corresponding foods recorded in the individual food consumption survey after applying 99 correction factors for peeling, washing, and home cooking as done by Dejonckheere 100 [START_REF] Dejonckheere | Nitrate in food commodities of vegetable origin and the total diet in Belgium (1992-1993)[END_REF]. The effect of processing was taken into account for vegetables 101 using the corrected concentration value whenever the processing or conservation mode of the 102 food was registered in the food consumption survey. The utilized factors can be found in 103 analyzed for nitrate and 15 cheese and 43 processed meat pooled samples for nitrite. A pooled 118 sample contained up to 16 different samples, each from a different trade mark and/or a 119 different kind of food within the food group. After sampling and before treatment of samples 120 they were stored in the fridge or in the freezer (-18°C or less) depending on the preservation 121 mode at sampling. If the analyses were not conducted on the same day, the sample was stored 122 deep frozen (-18°C or less) for a maximum time of 1 month. The stability of nitrate during 123 this type of storage has been demonstrated elsewhere [START_REF] Chung | Changes in nitrate and nitrite content of four vegetables during storage at refrigerated and ambient temperatures[END_REF]. In case of fresh 124 products, the edible part was analyzed (eg. celeriac was peeled). 125

Nitrate and nitrite quantification method 126

Individual samples from the same kind of foods (type and preservation mode) but from 127 Grade System coupled after a Millipore Elix 100 deionised water production system). 10g of 136 the pooled homogenized sample was mixed at about 15 000rpm in an Ultra-Turrax T25 (IKA, 137

different
Germany) with 100ml of a 6.55mmol l -1 Borax (Fluka, Switzerland) buffer heated at 60°C just 138 before extraction. The Ultraturax was rinsed 2 times with a total of 40ml extraction solution 139 which is then added to the sample extract. 2ml of a 15% potassium hexacyanoferrate (II) 140 (Merck, Germany) and 2ml of 30% zinc sulfate (Merck, Germany) are added to the extract 141 which is shaken after each addition. After cooling, the extract is brought up to 200. The signal (peak high, in this case) for a particular compound in an unknown sample was 156 compared with the one of the same compound in a standard solution of known concentration. 157

To prove the linearity of the detection system, a minimum of 7 different standard 158 concentrations were used in the range 0.1 to 200mg l -1 of NO 3 -or NO 2 -. The calibration 159 solutions were prepared from purified water with resistivity lower then 18.2MΩ cm -1 and 160 NaNO 3 (Riedel-deHaën, Switzerland) and NaNO 2 (Riedel-deHaën, Switzerland) reagent 161 grade or 1000mg l -1 solutions. Those solutions were prepared on each series of analysis. The 162 relationship between signal and concentration was linear with a first order polynomial 163 equation between 0.1 and 50mg l -1 and also between 10 and 200mg l -1 . A second order 164 polynomial regression can also be used over the all range. The purity, accuracy and stability 165 (after 18 days) of the standards were checked with a 1/20 dilution of ready certified standard 166 solutions for ion chromatography 1 000mg l -1 (Fluka, Switzerland) in NO 3 -or NO 2 -ions. The 167 recovered amounts were 97.7% nitrate and 101.8% nitrite. Each series of analysis was 168 composed of a calibration curve, solvent blank, sample blank, control sample, samples and 169 control standard. 170 171

The method precision was validated by injecting 9 replicates of the same sample over 3 day's 172 of work. This was done at 3 different concentrations chosen in regards of the expected 173 concentration in the type of food targeted. There were 3 different matrices representing meat 174 products, vegetables and cheese (chopped pork meat, tomatoes and Emmental). For nitrate, 175 the precision was greater in meat and vegetable products (less than 5% variation for the 176 highest concentrations tested) than in cheese where very high variation was found at 177 concentration near the quantification limit (nearly 90%). In the case of nitrite, the precision 178 was lower than for nitrate between 25 and 50mg l -1 (maximum variation result of 36% but 179 once again, for concentrations near the quantification limit, the variation observed (in 180 vegetable) was higher (66%). 181 The quantification limit was 5.0mg kg -1 for nitrate and 6.7mg kg -1 for nitrite. A selectivity test 182 of the method has been carried out and demonstrated that the separation of nitrate and nitrite 183 was achieved from phosphate, chloride, ascorbic acid and citric acid that can also be present 184 in the sample in plus minus high quantities. 185 186

Food consumption data 187

The food consumption data were obtained from the Belgian Food Consumption Survey (FCS) 188 2004[START_REF] Vriese | The Belgian food consumption survey: aims, design and methods[END_REF]. The target population covered all Belgian inhabitants of 15 189 years or older with no upper limit of age. The sample was stratified by province and included 190 3245 participants randomly selected from the National Register. 191 192 Information on dietary intake was collected by a repeated non-consecutive 24h recall in 193 combination with a food frequency questionnaire. The respondents reported the quantity of all 194 foods and beverages consumed during the preceding day. Quantification of foods and recipes 195 was performed using photographs, standard units or ml/g. In order to get information on the 196 within-person variation, two non-consecutive 24h recalls per respondent were collected. The 197 24h recall was carried out using the standardized EPIC-SOFT program (Slimani and Valsta 198 2002). 199 200

Statistical analyses 201

Only respondents with two complete 24-h recall interviews were included in the analyses (n= 202 3 083; 1 546 men and 1 537 women). The individual intake of nitrate and nitrite from a 203 certain food product was estimated by: 204 where y i is the intake of nitrate/nitrite by individual i from a particular food (in mg per kg 206 bodyweight and per interview day), x i is the consumption of a certain food by individual i 207 (kg), c i is the concentration of nitrate/nitrite in that food (mg per kg; when nitrate 208 concentrations were below the limit of detection (LOD), 0 mg kg -1 concentration was 209 applied) [START_REF] Kroes | Assessment of intake from the diet[END_REF]

Vegetables and fruits 229

The highest nitrate concentrations were found for lettuce, some other leafy vegetables and 230 celery, with large differences depending on the method of processing. The highest nitrate 231 concentrations for all vegetables studied (except for peas and carrots) were found in fresh 232 vegetables followed by frozen vegetables and canned vegetables or vegetables in glass 233 respectively. Nitrate concentration in peas in glass or can was 3.8 times higher than in fresh 234 peas while nitrate was not detected in frozen peas. Nitrate concentration in fresh carrots was 235 higher than in carrots in glass or can and frozen carrots. Nitrate concentrations in frozen 236 vegetables were on average 43% (19%-62%) and in canned or glass packed vegetables 69% 237 (54%-91%) lower than in fresh vegetables. 238

In the previous intake calculations of nitrate performed by Dejonckheere et al (Dejonckheere 239 et al., 1994), only vegetables were considered, for which data were available for the 3 240 different conservation methods (fresh, frozen and canned or glass). 241 242In order to estimate the variation in concentrations obtained in pooled samples, the analysis 243 was done within samples of the same processing method for some vegetables, namely endive, 244 beans with pods and celery. Nitrate (NO 3 -) concentrations in frozen endive (n=2) were 245 1264±119 mg/kg (CV of 9.4%), in frozen endive with added cream (n=9) 758±95 mg/kg (CV 246 of 13%) and in white celery, in can or glass (n=5) 757±173 mg/kg (CV of 23%). 247 The largest variations were observed for frozen beans (n=12) with a nitrate concentration of 248 429±250 mg/kg (CV of 59%) and beans in glass jar (n=10) 264±79 mg/kg (CV of 30%). The 249 average concentrations measured in these additional analyses, however, were similar to the 250 pooled sample analyses as reported in table 1, except for white celery. 251 252

Nitrate and nitrite additive use in cheese and meat products 253

Some cheeses were analyzed and the concentrations found in pooled samples are shown in 254 table 2. Some cheeses were not pooled because they are special foods ("appellations") of 255 controlled origin. The average concentration of 9 pooled Gouda half-and old cheeses was 256 about the same as the average concentration of 11 young ones. This was an unexpected result 257 because half and old Gouda are more dry than the young one, so the concentration was 258 expected to be higher. More investigations should be done on individual analysis basis. 259 Meat products were analyzed for nitrate after pooling of individual samples. The results are 260 given in table 2. Due to the huge number of meat products available on the market, it was 261 decided in some cases to make more than one pooled sample in order to represent major types 262 of processed meat products. Not more than 15 individual samples were pooled per sample. 263

264

The standard deviation of the mean nitrate concentration found in samples of the same meat 265 product (samples within a pooled sample) varied from 11% of the mean concentration in the 266 case of creamy pie to up to 81% of the mean concentration in the case of raw meat sausages. 267

Those results suggested that depending on the manufacturer the concentration can vary 268 substantially. The obtained results of nitrite concentrations in meat products are also given in 269 table 2. The measured concentrations were between 5 and 34 mg kg -1 . 270 This result was unexpected because of the relatively high allowed residual concentrations (50-271 175 mg/kg). Even for bacon, the concentration was very low (5 mg kg -1 ). 272

Nitrate intake 273

The average age of the population was 47±19 yr: 46±18 yr for men and 48±19 yr for women. 274

The average self-reported weight of the population was (71±14) kg; (78±13) kg for men and 275 (65±12) kg for women (De [START_REF] Vriese | De Belgische Voedselconsumptiepeiling 1 -2004 : Rapport[END_REF]) 276

277

The estimated average usual daily intake of selected foods (g/day) is given in table 3. The 278 combination of individual consumption data and average concentration data for the different 279 food items regrouped in categories is given in table 4. The total estimated average daily intake 280 of nitrate from these foods was 1.38 mg/kg bw/day (or 96 mg/day) when beverages (non-281 alcoholic and soup) were included and 0.885 mg/kg bw/day (or 61 mg/day) on average when 282 only potatoes, vegetables and fruits were included. When all foods were considered, half of 283 the mean usual nitrate intake was from vegetables and 20% from non-alcoholic beverages. 284

Potatoes and soups (soups were supposed to consist of 30% vegetable mix and 70% tap water, 285 resulting in a calculated nitrate concentration for soups of 105 mg/kg) contributed each for 286 around 10% to the daily nitrate intake. Of the vegetables, lettuce was the main contributor to 287 total nitrate intake, both because of its high nitrate concentration and high consumption in the 288 Belgian population. Carrots had a relatively lower nitrate concentration but a high 289 consumption; on the other hand spinach has a high nitrate content but was consumed only by 290 a small part of the population. The category 'other (mixtures) of vegetables' consisted of 291 vegetables, which were not specified by the respondent, during the 24-h recalls, or mixtures 292 of vegetables. 293 the tap water used to brew coffee and tea. The estimated daily food additive intake of nitrate, 296 from meat products and cheese, was 1.16 mg (or 0.013 mg/kg bw/day), which was only 0.9% 297 of the total average daily intake. The ADI was not exceeded even in the high percentiles of 298 intake, although it should be noted that average and not maximum concentrations of nitrate 299 were used in the different foods At total average intake, the intake represented 38% of ADI 300 and at the 97.5 percentile of intake it corresponded to 76% of ADI. 301 302

Nitrite intake from processed meat 303

Table 5 shows the estimated usual food additive intake of nitrite from meat products, which 304 was on average 0.24mg/day or 0.003mg/kg bw/day, representing 6% of the ADI. Although 305 boiled ham had a nitrite concentration near to the LOD, because of the high consumption of 306 this meat product, it was the main source of intake (24%). 307 308 DISCUSSION 309

Nitrate concentrations of foods 310

Concentrations of nitrate were the highest for fresh rucola (rocket salad), lamb's lettuce, 311 lettuce, radish, celery, spinach and endive (Table 1). Nitrate concentrations of most vegetables 312 analyzed during this study fall within the wide range of values reported before by 313 Dejonckheere et al, 1996) However, other vegetables contained higher nitrate concentrations 314 (e.g. turnip) and for others no concentrations have been reported so far (rhubarb, courgette) 315 [START_REF] Belitz | Food Chemistry[END_REF][START_REF] Chung | Survey of nitrate and nitrite contents of vegetables grown in Korea[END_REF][START_REF] Penttilä | Estimation of food additive and pesticide intakes by means of a stepwise method[END_REF][START_REF] Petersen | Nitrate and nitrite in vegetables on the Danish market: content and intake[END_REF]316 Tamme et al. 2006). 317 Concentrations of nitrate in vegetables vary, in addition, according to the processing method 318 with the highest concentrations in fresh, then frozen and the lowest in vegetables preserved in 319 can or glass. This was confirmed in this study, except for peas and carrots. The nitrate content 320 in processed carrots (frozen, canned or jarred) was lower than in fresh carrots, but the level in 321 frozen carrots was not higher than the level in canned or jarred carrots. A likely explanation is 322 that nitrate is lost during blanching of frozen vegetables and into the water in the jar, as far as 323 the nitrate content in the vegetable is higher than in water. For vegetables with low nitrate 324 content such as peas and carrots, a transfer of nitrate to the vegetables might be possible from 325 the added water used for preparation (if the nitrate concentration in water is higher than that in 326 the vegetables). Variation of concentrations even within the same vegetable and processing 327 method is high; within the group of processed vegetables studied, the highest variation of 328 58% was noticed for frozen beans with pods. This spread may be explained by different 329 varieties, growing and processing conditions. Food additive nitrate concentrations of cheese 330 and meat products are low compared to the possible contamination observed in vegetables 331 and tap water. 332

Nitrate intake 333

In the current intake assessment, using individual consumption data and average 334 concentration data, the nitrate intake was estimated to be 96 mg per day or 1.38 mg/kg bw/day 335 (38% of the ADI). The intake assessment took into account potatoes, vegetables and fruits, 336 processed meat, cheese, water and drinks based on water. Half of the intake was from 337 vegetables, of which 20% from lettuce. When comparing these figures with previous ones, 338 different aspects have to be taken into account, like the type of food consumption data and the 339 food groups included in the analyses. 340 Comparing our results with the results of the previous Belgian intake estimate (Dejonckheere 342 et al., 1994), a higher intake of 2.11 mg/kg bw/day was recorded in the previous Belgian 343 study. The data used in the latter study were derived from household budget surveys and food 344 balance sheets for Belgium. These types of food consumption data are collected at group level 345 with no information on the actual amounts consumed by individuals, including waste, 346 resulting thus in a higher estimate of the daily intake. In addition, dietary patterns might have 347 changed during the past years. In the previous study for instance, the intake of vegetables and 348 potatoes was 202 and 250g per person per day respectively [START_REF] Dejonckheere | Nitrate in food commodities of vegetable origin and the total diet in Belgium (1992-1993)[END_REF], while 349 the consumed quantities currently measured were only 132 and 98 g per person per day, 350 respectively. 351

The recommended intake of vegetables (Vlaams Instituut voor Gezondheidspromotie 2007) is 352 much higher than the amounts actually consumed. In a scenario where vegetable intake would 353 comply with the recommended amount of 350g/day instead of 132g/day, the mean intake of 354 nitrate would be 2.49mg/kg bw/day (68% of ADI) instead of 1.38mg/kg bw/day. 355

As the ADI of nitrate will not be exceeded by complying with the recommendation for 356 vegetable consumption, the beneficial effects of increasing vegetable intake outweigh the 357 possible adverse effects. In the current food consumption survey only 1% of the respondents 358 reached the recommendations for vegetables [START_REF] Vandevijvere | The gap between food-based dietary guidelines and usual food consumption in Belgium, 2004[END_REF]). In addition, certain 359 nutritional habits, such as changing especially in the winter season from green house head 360 lettuce to iceberg lettuce with lower nitrate concentration could reduce intake. Among 361 beneficial effects might be the effects of nitrate and nitrite themselves. Recent prospective 362 epidemiologic studies have shown that green leafy vegetables are among the foods most 363 protective against coronary heart disease and ischemic stroke risk [START_REF] Joshipura | Fruit and vegetable intake in relation to risk of ischemic stroke[END_REF]. The 364 authors hypothesize that: the content of inorganic nitrate in certain vegetables and fruit can 365 provide a physiologic substrate for reduction to nitrite, nitric oxide, and other metabolic 366 products (NOx) that produce vasodilatation, decrease blood pressure, and support 367 cardiovascular function [START_REF] Mcknight | Dietary nitrate in man: friend or foe?[END_REF].These studies call into question the rationale 368 for recommendations to limit nitrate and nitrite intake from plant foods. EFSA's 369 Contaminants Panel (CONTAM) has assessed the risks and benefits to consumers from 370 nitrates in vegetables and concluded that the beneficial effects of eating vegetables and fruit 371 outweigh potential risk to human health from exposure to nitrate through vegetables. 372

373

In studies with similar individual dietary assessments as in the current study (24h recall, food 374 records), intake is largely influenced by whether water and water based drinks are taken into 375 account. Taking into account only potatoes, vegetables and fruits and some drinks, results are 376 similar to a Finnish study that reported an intake of 55mg/day (or 0.92mg/kg bw/day) 377 [START_REF] Penttilä | Nitrate, nitrite, and N-nitroso compounds in Finnish foods and the estimation of the dietary intakes[END_REF]) . Penttilä et al [START_REF] Penttilä | Nitrate, nitrite, and N-nitroso compounds in Finnish foods and the estimation of the dietary intakes[END_REF] probably underestimated the intake 378 of nitrate by drinks. Calculating back the intake of coffee and soft drinks is on average 379 50ml/day. In the present study the intake of coffee was 270ml/day. Another important 380 difference is that the nitrate content of water was estimated to be 10mg/kg whereas in the 381 current study an average content of 21mg/L was used. Individual food consumption data 382 (dietary history) estimated a total intake of 77mg/day from foods for Finnish adults (Dich et 383 al. 1996). Nitrate from drinking water was not taken into account. Of this estimate 90% was 384 derived from vegetables (including potatoes). Our results are very similar; the average 385 estimated nitrate intake is 76 mg when the non alcoholic beverages are not taken into account, 386 60% of this intake is from vegetables. 387

388

In a recent French study among adults [START_REF] Menard | Assessment of dietary exposure of nitrate and nitrite in France[END_REF]) the average nitrate intake was 3.7 389 mg per kg bw/day or 40% of the ADI.. The major contributors were, vegetables (24% of 390 ADI), potatoes (5% of ADI), and water (5% of ADI). Intake of nitrite was 0.06 mg per kg 391 bw/day (33-67% of the ADI), with major contributions of additive food vectors. Another 392 dietary assessment method was used (7-day food record) than in our study. 393 394 To estimate the intake from water and water based drinks it was assumed that the 395 concentration of nitrate in water was on average 21mg/L. This average is based on 396 concentrations communicated for tap water in the Wallonia and Brussels regions. For 397 different food groups (soft drinks, juices reconstituted or not) it was assumed that they 398 consisted for 100% of drinking water, which might be an overestimation. A maximal level of 399 50mg/kg is allowed for drinking water. In case of this upper level, intakes will be higher 400 (average of 1.76mg/kg bw/day instead of 1.38mg/kg bw/day or 48% of ADI). Drinking water 401 in Belgium, however, rarely exceeds this limit although regional differences might exist (van 402 Grinsven et al. 2006). 403 404 In a combined situation of both an assumed median vegetable intake which equals the 405 recommended vegetable intake and water with maximum nitrate concentrations, the average 406 nitrate intake will be 2.59 mg/kg bw/day (75.7% of ADI) instead of 1.38 mg/kg bw/day. It is 407 thus clear that the recommendation for vegetable consumption is safe with regard to nitrate 408 intake. In such unlikely scenario in which all consumers increase their vegetable intake with 409 the same percentage as the median consumer would do to reach the recommended vegetable 410 intake, and in which all tap water (also used to make coffee, tea, to reconstitute fruit juice and 411 to make soft drinks) reaches the maximum nitrate level, almost 25% of the adult population in 412 Belgium would exceed the acceptable daily intake. Whether this is beneficial or harmful must 413 be further assessed. Intake of the additive nitrite from processed meats was 0.24mg/day. This is only 6% of the 418 ADI. For nitrite the main issue is the use as additive. Further evaluation must be focused on 419 more vulnerable groups such as children, with possibly high consumption of certain products 420 like ham sausages. 421 422

Methodological issues 423

In this study, the intake of nitrate and nitrite in the adult Belgian was estimated as accurately 424 as possible. The nitrate concentration data for potatoes, vegetables and fruits took into 425 account, if available, waste and preparation method. Consumption data are reported as 426 consumed. In many cases, however, information on correction factors is not available, 427 resulting in an overestimation of the nitrate concentration. 428

429

In only a very limited number of foods analyzed, only some cheeses, nitrate concentrations 430 were below the limit of detection and in these cases a concentration of 0 mg/kg was applied. 431 This could have lead to an underestimation of the intake but the foods concerned were not 432 consumed at a high level and not frequently. Additionally, the mean concentration values 433 were used in the intake assessment; no maximum concentrations. 434

The variation in concentration among the different variety or brands of one particular food 435 could expose some consumers to higher nitrate concentrations in time than the mean 436 concentration value if brands or varieties are not regularly varied and if consumers choose 437 regularly those ones with the higher nitrate concentrations. A common problem in dietary assessment in general [START_REF] Zhang | Sex ratio of total energy intake in adults: an analysis of dietary surveys[END_REF]Zhang et al. 2000) 440 and also in the present study is the possibility of underreporting of foods. It was estimated, 441 based on energy requirements, that 12% of the respondents under recorded their real food 442 intake. This might affect nitrate intake assessment. On the other hand large efforts were made 443 in this study to include a representative sample of the Belgian population older than 15 years 444 of age by choosing respondents among all categories of age, regions and social classes of 445 Belgium. In addition, this study is reliable because it takes into account the most important 446 foods consumed in Belgium and food concentrations were reported not only in the case of an 447 additive usage but also when it could be assumed to be present on a natural way 448 ("contaminant") in the food. 449

CONCLUSION 450

Usual exposure to nitrate in Belgian adults was on average 96 mg per day or 1.38 mg/kg 451 bw/day and represented 38% of the ADI. Half of the intake was derived from vegetables 452 (especially lettuce) and 20% of water and drinks based on water. Daily intake of the food 453 additives nitrate (0.2% of ADI) and nitrite (6% of ADI) from cheese and meat products was 454 low. Scenario analyses showed the possible effect of a higher vegetable intake and higher 455 nitrate content of water on estimated nitrate intake.

  0ml with 142 MilliQ water. The extract was filtered through a 0.22µm PVDF syringe filter after 143 centrifugation at 7 000rpm. 144 145 The final extract was injected into an High Performance Liquid Chromatography (HPLC) 146 system formed of a 232XL injector (Gilson, USA), a 9010 HPLC pump (Varian inc., 147 Australia), a column oven (Alltech, USA), a UV detector model 785A (Applied Biosystem -148 PerkinElmer Inc., USA) operated at 220nm and a signal acquisition system formed of a 149 Star800 interface and Galaxy version 1.7 (Varian Inc, Australia). Further calibration and 150 quality assurance calculations were done in Microsoft Excel version 10 SP3. The analytes 151were injected isocratically at 1.0ml min -1 on a Lichrospher100 RP18 250mm length, 4.6mm 152 internal diameter and 5µm particles thickness maintained at 35°C. The eluent was constituted 153 by a mix of tetrabutylammonium sulfate 50mmol l -1 (Fluka, Switzerland), purified water and 154 methanol Lichrosolve grade (Merck, Germany) in 10/68/22 proportion. 155

  little to nitrate intake. Of the drinks almost half of the nitrate is from 295

  Table 1 it can be found for each vegetable or fruit which source for concentration 84 There seemed to be no important evolution in time in concentrations of nitrate in vegetables. 86

		F o r
		P
		e
		e r
		R
		e
		v i e
		w
		O n l
		y
	85	data has been used.
		5

Table 1

 1

	104	.
	105
	106
	107
	108

Nitrate and nitrite concentration of selected foods 109

	110	Food sampling
	111	Efforts were made to ensure representative concentration data. Samples of cheese and meat
	112	products, however, were targeted towards products for which nitrate or nitrite use was known
	113	from the label. Sampling was performed from January till March 2006 in the 5 most
	114	frequented supermarket chains (6 for the frozen products).
	115	Different seasons were not taken into account because it is impossible to determine the time
	116	of harvest. Moreover nitrate or nitrite addition to meat or cheese is not a seasonal practice. A
	117	total of 49 vegetable and fruit, 15 cheese and 43 processed meat pooled samples were

  and bw i is the self-reported body weight of individual i (kg). To 210 estimate the total intake of nitrate/nitrite per group of foods and/or per day, individual daily 211 intakes of the contaminant from different foods were summed. The concentration in a food is 212 either a concentration in a pooled sample, or an average concentration from different 213 individual and/or pooled samples. 214215The usual nitrate intake distribution was estimated with the Nusser method(Nusser et al. 216 1996) using the C-side program(Iowa State University 1996). Several statistical methods are 217 available to estimate usual intake distributions with the correct mean, variance and skewness. 218

	225	RESULTS
	226	Nitrate concentrations of (conserved) foods
	227	The nitrate concentrations in fruits and vegetables used in the exposure assessment are given
	228	in table 1, for cheese and meat products in table 2.
		F o r
		P
		e
		e r
		R
	219	These statistical procedures adjust for within-person or day-to-day variability. The Nusser e
	220 221 222	method eliminates the within-individual variance and additionally transforms the data to obtain approximately normally distributed data. The method is suitable to estimate usual v i e intake distributions in a population both for normally and non-normally distributed foods and w
	223 224	nutrients.	O n l
			y
		11

Table 1 .

 1 Concentrations of nitrate ([NO 3 -] (mg/kg) in selected vegetables, potatoes and fruit. Non-targeted official control data from 2004-2005 received from the Belgian Federal Agency for Safety of the Food Chain, unpublished data, personal communication. All analyses performed by the Scientific Institute of Public Health (2006).

	456
	457
	458
	459	ACKNOWLEDGEMENTS
	460	The study was funded by the Federal Public Service of Health, Food Chain Safety and
	461	Environment.

a

Table 4 .

 4 Estimated habitual nitrate intake (mg/kg bodyweight/day) in the Belgian population older than 15 years Nitrate (mg/kg bodyweight/day) % of ADI Nitrate expressed as nitrate ion, the acceptable daily intake (ADI) for nitrate is 3,65 mg/kg bw/day The habitual dietary intakes are weighted and adjusted for the age and sex distribution of the Belgian population 2004 and adjusted for interview day and season. Total number of consumption days is 6166 * Habitual intake could not be estimated by the Nusser method because of too few replicate intakes.

	Mean	SD	P50	P97,5

32 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

VEGETABLES

The average dietary intakes are weighted and adjusted for the age and sex distribution of the Belgian population 2004 and adjusted for interview day and season. The acceptable daily intake for nitrite is 0,07 mg/kg bw/day * The habitual intake could not be estimated by the Nusser method because of too few replicate intakes.


A M Soto

# C Sonnenschein

P A Miquel

On physicalism and downward causation in developmental and cancer biology

The dominant position in Philosophy of Science contends that downward causation is an illusion. Instead, we argue that downward causation doesn't introduce vicious circles either in physics or in biology. We also question the metaphysical claim that "physical facts fix all the facts." Downward causation does not imply any contradiction if we reject the assumption of the completeness and the causal closure of the physical world that this assertion contains. We provide an argument for rejecting this assumption. Furthermore, this allows us to reconsider the concept of diachronic emergence.

Introduction

Physicalism and antireductionism in biochemistry and in molecular biology are topics of renewed current interest. Rosenberg and Kaplan attempted to reconcile them (Philosophy of Science (2005). For this purpose they developed an argument whereby they reject the downward causation by undermining one of the epistemological pillars of biology: i.e., the very distinction in Mayr's view (1961), between the question "how?" that has a meaning in biochemistry as well as in the physical sciences, and the question "how come and why" that has sense only in evolutionary biology. This duality introduces a gap between physics and biology. Rosenberg and Kaplan attempt to close this gap by trying to force a biological concept, that of natural selection, into the realm of physics. To do so, they claim that the principle of natural selection is an underived law about physical systems; in other words, it is already present in the field of physics and chemistry. According to the authors, the difference between physical and biological objects is not due to fitness, a notion similar to entropy, but rather due to reproduction.

In order to be swayed to Rosenberg and Kaplan's position, one should acknowledge that fitness is a concept that belongs to physics. And yet, in their view, natural selection is also a supervenient property like entropy in statistical mechanics. However, this doesn't involve any ontological implication, because it doesn't give rise to irreducible levels of properties and entities; it just reveals the limits of our knowledge instruments. Thus, even if they believe that biological theories are not reducible to physical ones in an epistemic sense, this assertion doesn't lead to any "implicit or explicit commitment to metaphysical or ontological differences" between physics and biology. Like [START_REF] Kim | Supervenience and Mind[END_REF], they defend a "vindicating physicalism" based on the physical determination thesis ("physical facts fix all the facts") and on the ontological principle of completeness and of causal closure of the physical world. Consequently, they reject downward causation because it would not be compatible with physicalism 's commitment (2005).

We will argue to the contrary that downward causation does not introduce vicious circles either in physics or in biology. Downward causation does not imply any contradiction once we reject Rosenberg and Kaplan's assumption concerning the completeness and the causal closure of the physical world. Such an ontological claim introduces the God's Eye fallacy in ones' metaphysical vision of Nature [START_REF] Putnam | Realism as a Human Face[END_REF]. By rejecting this claim we are allowed to reconsider the concept of diachronic emergence.

1-Fitness as a physical property

Rosenberg and Kaplan claim that the argument of "substrate-neutrality" of fitness doesn't prove that the principle of natural selection (PNS) is an "underived" law of biology. According to their view, the law of entropy in macroscopic thermodynamics is also substrate neutral, and yet it can be reduced to the growth of the logarithm of the complexion number up to the state of equilibrium in a closed system where all accessible microstates are equiprobable. Therefore, they ponder whether a similar reduction could be made for fitness.

Let us explore the argument of substrate neutrality (Dennett, 1996: 50-51). In Dennett's view, substrate neutrality is linked to the idea of an algorithm as a mechanical procedure, an idea coming from Turing, Gödel and Von Neumann. We acknowledge the significance of this notion in the construction of classical molecular biology that derives in turn from the concept of microcode in Schrödinger's view. However, Dennett uses it in the context of evolutionary biology. If he is right, natural selection acts as a mindless and mechanical meta-engine that modifies biological engines, which in turn generate the growth of complexity and adaptability. Yet, adaptability is not an equilibrium state that can be predicted. It is, instead, the result of an instantiation of a mindless algorithm under rules of blind design, which makes sense only retrospectively because it is a product of evolution. Dennett proposed this concept of "accumulation of design" in contrast to the vision of a creative order that produces novelty from nothing that he attributes to Gould. In Dennett's view, natural selection would be a "crane" and not a "skyhook 1 ".

According to Dennett, natural selection adds logical constraints to the physical ones. Thus, a distinction must be made between the physical structure in which the algorithm is implemented on the one hand, and the function that it fulfils, on the other. And here is where Dennett introduces the principle of the substrate neutrality (PSN), founded on the idea that "the power of the procedure is due to its logical structure" which can be realized in very different substrates [START_REF] Dennett | Darwin's Dangerous Idea[END_REF].

Rosenberg and Kaplan are cautious about this definition of the PSN. According to their views, there cannot be any real "biological kinds", any real difference of level between physics and biology, even if one accepts that "biology relies on a theory that is not reducible to a more fundamental physical theory" [START_REF] Rosenberg | How to reconcile Physicalism and Antireductionism about Biology[END_REF]. This could be the case, only if all natural facts (chemical, biological) are fixed as physical ones. Given that this is true, the distinction between physics, biology and psychology is only semantic due to human "conceptual incommensurabilities or epistemic limitations," and would have no ontological implications. This point is also emphasized by Kim (1999 2 ).

According to Rosenberg and Kaplan, entropy seems to be also a "supervenient" property, and yet it can be reduced to statistical disorder 3 . The fact that temperature is not varying at the equilibrium state is founded on the ergodicity hypothesis 4 : thus, the system will continually (eternally?) instantiate all these states in compatibility with its temperature. In this case, one must presume, first, that such a system is closed; second, that a gas is composed "of concrete constituents with mass and velocity"; and third, that such a hypothesis is true "in the limit of infinite time (2005)". Rosenberg and Kaplan know that this sentence has no physical sense. But for them, idealisations are just showing the semantic imperfections of theories as instruments. They have no ontological meaning. Mechanical facts fix thermodynamic facts.

1 "The skyhook concept is perhaps a descendant of the deus ex machina of ancient Greek dramaturgy: when second-rate playwrights found their plots leading their heroes into inescapable difficulties they were often tempted to crank down a god into the scene, like Superman, to save the situation supernaturally" (Dennett, 1996, 74). 2 "That is, we interpret the hierarchical levels as levels of concepts and descriptions, or levels within our representational apparatus, rather than levels of properties and phenomena in the world". 3 Normally, "substrate neutrality" and "supervenience" are not compatible with a strong reductionism, since supervenient properties cannot be deduced form physical ones: "no actual derivation may be effected, owing to the famous "many-one" relation between the physical supervenience base and the biological properties to which the PNS adverts" [START_REF] Rosenberg | How to reconcile Physicalism and Antireductionism about Biology[END_REF]). 4 A system of molecules will assume in a long period of time all conceivable microstates that are compatible with the conservation of energy.

Why cannot this also be the case with natural selection? Of course, this PNS needs replicative entities that are present in a non-replicative realm. It becomes difficult to understand how PNS can have a meaning before the existence of replicative entities. Yet the authors pursue the idea, already used by Cairn [START_REF] Cairns-Smith | Genetic takeover and the mineral origin of life[END_REF], to explain the transition between the prebiotic and the biotic world. Under certain chemical conditions, we can observe that more stable molecules can be produced which have a greater chance to be preserved and to lead to a more stable macrostructure. One could imagine connections between such structures and the origin of replication, and that a specific equilibrium could be reached between unstable replicative entities and stable non-replicative molecules. Accordingly, the PNS could be applied to stable and unstable molecules in an abiotic world, as it happens with replicative biological entities. Thus, for Rosenberg and Kaplan, chemical facts would fix biological facts.

Rosenberg and Kaplan also conclude that downward causation is incompatible with their physicalist credo. On this point, their view regarding the causal agency of supracellular structures differs from that of P. Kitcher. In Kitcher's view (1984) the formation of a gap among cells would affect the density gradient of a molecular gene product during wing morphogenesis that would be responsible for the formation or malformation of the wing. According to Rosenberg and Kaplan, the supracellular geometrical structure is a supervenient property affecting the genes, and fixed by the evolution of a chemical milieu controlled by the PNS. Thus, the downward causation effect would be a product of this "wholly physical process."

Their view assumes that natural selection is a "physical law" on the same footing as that of entropy: "(x)(y)(E)[If x is fitter than y in environment E at generation n, then probably there is some future generation n' after which x has more descendants than y] [START_REF] Rosenberg | How to reconcile Physicalism and Antireductionism about Biology[END_REF]." We disagree with this view.

To begin with, can PNS be expressed as an algebraic formula of the kind the authors propose? If we follow their reasoning, one may assume that the change in a genetic pool between a generation n and a future generation n' is predictable by a formal analysis. This was precisely Fisher's credo in Genetical Theory of Natural Selection [START_REF] Fisher | The Genetical Theory of Natural Selection[END_REF]. Fisher accepted the assumption of gene additivity, and thought that the value of fitness depended solely on a change in a gene frequency. However, in 1932, Sewall Wright claimed that such an assumption was unrealistic, since genes and alleles are interacting by epistasis, pleiotropy and dominance (S. [START_REF] Wright | The Roles of Mutation, Inbreeding, Crossbreeding, and Selection[END_REF]. He insisted on the division of a genetic population in demes far away from panmictic equilibrium, and on the role of the sampling effect in each deme. He concluded that in a "shifting balanced theory of evolution", the change in fitness value could be illustrated by adaptive landscapes. In this case, fitness was not predictable from a formal analysis of gene frequency. For [START_REF] Wright | The Roles of Mutation, Inbreeding, Crossbreeding, and Selection[END_REF][START_REF] Wright | Evolution and the Genetics of Population[END_REF][START_REF] Wright | Genic and Organismic Selection[END_REF], in evolutionary biology, what comes first is creativity and novelty, not prediction. Moreover, scientific explanations in population genetics are not fully quantitative.

Second, if the PNS would be a basic law of physics or of chemistry, it should be operational at the lower-level systems of molecules, but this does not imply automatically, as Rosenberg and Kaplan assume, that it is also operational "at each level of organisation of matter" (2005).

Third, seventy years after Wright's observation, the unpredictability problem remains unsolved, concerning the applicability of the PNS. For instance, in Kauffman's simulations on Boolean networks, even when initial conditions and logical laws are chosen randomly, the reproduction of the same computations with the same initial conditions produce the same results. Then, it is possible to give an "epistatic" value K to the fitness contribution of a given gene by other genes. His NK model is an idealisation of an organism constituted by N genes. When a specific value is assigned to K, the evolution of a multipeaked fitness landscape can be simulated. However, the properties that emerge from the use of such computations are generic features, in other words, they are not described by the initial conditions. In order to predict an outcome, one must first reproduce a situation, and not the contrary. That is probably why Kauffman uses the concept of emergence. Natural selection cannot be understood as a law by which a prediction can directly be made. Natural selection acts from the outside, like "an invisible hand" (Kauffman, 2000, 204) on a system which is not completely defined by its internal constraints (such as the value of the connectivity parameter K, the number N of Boolean automata symbolising the genes, the iteration mode, etc). From this perspective, the PNS would be a "contextual open property". It could not be characterised as a fundamental law of physics, because it is neither a property of the elements composing a system (the automata, for instance) or the relations between these elements, nor a property of a system determined as a whole (Bunge, 2003, 13). Such Boolean networks have specific generic properties that could occur without assuming that external forces are acting to give each microelement a fitness value. On the contrary, in this model, such a system is a part of a much more complex system that is not self-similar5 . Kauffman developed this explanation of the properties of a system by integrating it into a more complex one. He tried to simulate coevolution [START_REF] Kauffman | Investigations[END_REF].

Finally, Rosenberg and Kaplan conclude, that PNS is "a basic law of chemistry", and not of physics, because "there is no scope in microphysics for differences in "fitness" along the lines that molecules exemplify" (2005). Thus, if they introduce a new gap between physics and chemistry in order to reduce biological systems to chemical ones, they provide an explanation that is hardly solving the problem. They are just regressing from the first level at which the problem was posed, to another one.

2-Physicalism and diachronic emergence

"Not only is reification of worldviews a highly theoretical move of doubtful internal coherence, it stems from a radical misconception of the human condition" [START_REF] Van Fraassen | The Manifest Image and the Scientific Image[END_REF].

Rosenberg and Kaplan's vision of the PNS is based on the physical determination thesis that they emphasize ("physical facts fix all the facts"). This thesis is founded on two principles more explicitly developed by [START_REF] Kim | Supervenience and Mind[END_REF][START_REF] Kim | Philosophy of Mind[END_REF][START_REF] Kim | Making sense of Emergence[END_REF]: the causal closure and the completeness of the physical world. Rosenberg and Kaplan claim "that this thesis enjoys near universal acceptance" (2005) in philosophy of biology. Yet, we will argue that this thesis must be challenged in order to understand the very difference between real emergent biological phenomena and a very misleading application of the concept of supervenience in the field of evolutionary biology.

A-First, the physical determination thesis ("the physical facts fix all the facts") is a metaphysical claim, a "Weltanschauung". In other words, this is not a simple scientific assertion concerning a set of events that a scientist isolates, defined by kinds that facts instantiate and by laws that explain them. This proposition implies that the domain in which physical facts are ("all natural facts") is completely intelligible, even if we cannot describe them completely. This we can call the completeness of the physical world (CPW): "the idea that real connections exist and the idea that the world is intelligible and controllable are arguably equivalent ideas" (Kim, 1993: 53). We argue that this principle is based on a hidden logical fallacy.

From the CPW perspective, Kim jumps from the level of a finite system to the level of the world. How is it possible to make this jump? Obviously this cannot be done on science alone! And the answer is historically well known: one needs a Demon. Demons are everywhere in physics: Laplace, Maxwell, Boltzmann implicitly or explicitly used them. A physicist has a human eye, which symbolizes our epistemic limitation and the status of our scientific theories as instruments. Concerning the entire world, a physicist-as a physicist-can say nothing. Consequently, in order to become a physicalist -to reduce the real world itself to a set of physical events-the physicist needs a God's-Eye View [START_REF] Putnam | Realism as a Human Face[END_REF] 6 . He must employ an objective and impersonal entity, like the Laplacian demon. Yet, how can we accept the help of a supernatural entity on the one hand, if on the other we want to reduce the real world to a set of physical events?

One could counter that we can use the "physical determination thesis" without introducing a Laplacian Demon. We will answer, that it is impossible to justify "that physical facts fix all the facts" without to jump, "as if we were outside our own skins7 ", from the level of the "facts" to the level of the world ("all the facts"). Furthermore, no connexion can be introduced between the idea of "unobservable entities", like atoms or electrons, and the idea of "the intelligibility of the world". We can try to give a limited representation of the electron with the help of experimental tools. This cannot be the case with respect to the "intelligibility of the world". Thus, we reject the CPW. Nobody is able to give a complete description of the world.

B-Rosenberg and Kaplan's thesis also implies that we can identify causal relations between natural facts that are physical ones. Physical facts fix the facts, meaning that biological or psychological facts cannot have any causal action in Nature. This means that the physical domain is not only intelligible, but also "causally closed". As conceptualised by Kim: "thus no causal chain involving a physical event will ever cross the boundary of the physical into the non-physical: if x is a physical event and y is a cause or effect of x, then y, too, must be a physical event" (1993: 147). In short, every physical event would have a physical cause: let's call it the Causal Closure Principle (CCP).

The physical determination thesis also means that the causal power of higher-level properties is reducible to the causal power of the base-level properties. Kim calls this the Causal Inheritance Principle (CIP, [START_REF] Kim | Supervenience and Mind[END_REF][START_REF] Kim | Making sense of Emergence[END_REF][START_REF] Perovic | The Limitation of Kim's Reductive Physicalism[END_REF]. Accordingly, even if one accepts either synchronic or diachronic emergence, one must reject the principle of "the causal efficiency of the emergents" [START_REF] Kim | Making sense of Emergence[END_REF]. Emergent properties could not have causal powers of their own. If causes would work simultaneously at the basal and at the higher levels, they would produce an overdetermination of the effect. If the instantiation of a property M of an object X would cause the instantiation of another property M*, and if M would depend on a physical property P by which M is realized, or implemented, it would mean that, either M* depends on another physical property P*, having no ontological efficiency, or M* would modify a microproperty P in P*, which would violate the CCP.

In this view, "emergence" could be accepted as an epistemic property involving unpredictability from lower levels as long as it is connected with mereological supervenience, like it seems to be the case for Rosenberg and Kaplan. But emergence could not be an ontological property, involving qualitative novelty [START_REF] Bunge | Emergence and Convergence[END_REF]. By mereological supervenience, one means that "…systems with an identical total microstructural property have all over properties in common. Equivalently, all properties of a physical system supervene on, or are determined by, its microstructural property" [START_REF] Kim | Making sense of Emergence[END_REF]. This view is compatible with the idea that there is a "complete microstructural description" [START_REF] Kim | Making sense of Emergence[END_REF] of the system, and that the upper-level conditions are not "resulting" from the basal-conditions. Macrostructural properties could be said to be unpredictable and in a certain way unexplainable. The relation between M and P is not univocal. M is depending on P, or on P*, or on P**. Yet with the same physical systems, one would always have the same supervenient macroproperties. Thus, physical facts would seem to fix the facts.

Bunge has discussed all the difficulties related to the use of this concept of supervenience (2003: 14). We will instead focus on the fact that the CCP and the CIP are founded on the CPW. Given the incomplete description of the physical world, it would be impossible to assert that all physical events have a physical cause. This clears the way for a new conception of diachronic emergence compatible with downward causation.

C-In our view, a scientific theory in classical physics (not in non-linear dynamics or in thermodynamics of open systems far from equilibrium) is nothing but "an interpretation" of a finite system (even the universe is one), characterised by a certain class of theoretical models concerning the spatial coexistence and the temporal existence of physical events [START_REF] Duhem | Mechanical induction of twist in the Drosophila foregut/stomodeal primordium[END_REF][START_REF] Van Fraassen | Laws and Symmetry[END_REF][START_REF] Longo | Mathématiques et Sciences de la Nature[END_REF]. We would add that even in quantum mechanics, a class of theoretical models is specified by its invariants or internal constraints, which are always finite and measurable. That's why the structure of such systems is not depending on time [START_REF] Van Fraassen | Laws and Symmetry[END_REF].

The determinism principle establishes that "the same causes produce the same effects": at T1 of a specific system (like the solar system) one can isolate initial conditions, laws and invariants, and based on them predict a final state at T2. This is not true of biological systems. Biological systems are not only finite, because they are not defined only by internal conditions or constraints, but they are open systems. A living organism has a metabolism, biochemical cycles, and it is sensitive to natural selection. External constraints are always operating on them. This has been called a "contextually non separable system" [START_REF] Perovic | The Limitation of Kim's Reductive Physicalism[END_REF]. By non separability, Perovic assumes that a contextual open property N "is not instantiated in each individual constituent but rather in the context of the system's (individual) constituents" [START_REF] Perovic | The Limitation of Kim's Reductive Physicalism[END_REF]).

Yet "non separability" is not only a property of the higher level that must be distinguished from those specifying the basal-level. We assume that the internal constraints defining a system are always disturbed by external ones, so that in order to understand what is going on in the system, we must jump simultaneously to multiple levels on which this system is integrated [START_REF] Stengers | Cosmopolitiques VI[END_REF]. For instance, the cell, as a whole is integrated in a more complex system, the tissue, the organism, in which it will not act, as if it were placed in an in vitro culture. Let us take for example a muscle cell in the heart. The components of this system are proteins that channel the ions, (calcium, potassium), and they carry currents that changes the cell voltage. In turn the cell voltage changes the ion channels (Noble, 2006: 57). The components alter the behaviour of the heart and the heart alters the behaviour of the components, yet both components and the heart are integrated in a higher multicellular structure, the organism. This means that the working of such systems is never defined by initial internal constraints. D-When dealing with open systems, new systemic properties emerge at T2 and they can modify the initial properties. Thus what was described at T1 was not the essence of the system. In other words, when one states that the biological facts at T1 cause physical facts at T2, and that they compete with the explanation of these facts as purely physical ones, we are making a mere idealisation. At T2, the system is not the same as the one at T1, because it has new properties that were not there at T1. Therefore, a system's description of natural events is not a complete description of what this system does. By diachronic emergence we mean the fact that in specific natural or formal systems the initial relations and properties of elements cannot teach us how they would be applied as the system evolves. Thus, the historical way by which a system of natural events operates is not a consequence of its description. It acts and it produces novelty (novel qualities and novel structures) in the real world, which leads to the conclusion that emergence has an ontological meaning [START_REF] Bunge | Emergence and Convergence[END_REF]. It is not a simple epistemic property.

Consequently, we reject Rosenberg and Kaplan's argument against antireductionism because it introduces a conceptual confusion between emergence and supervenience. They note that, if physical facts at T1 fix the physical facts at T2, even if physical facts are biological facts at T1, it adds (ontologically) nothing to physical facts. They argue that, if this would not be true, contradictions would be inevitable. Our simple answer is: physical facts in a physical system (A) at T1 fix the physical facts at T2 in our representation of the system at T1. Nevertheless, our representation can never be a complete description of the system. This description can change by the mere fact that the system is working between T1 and T2. At T2, the system is historically changed, without any contradiction, since the real biological emergent properties were not present in our description, at T1. They are a result of the system's activity. Dobhzansky's assertion that "nothing in biology makes sense except in the light of evolution" (1973) substantiates the view that diachronic emergence is a central property of biological systems. It suggests that predictions can only be achieved by means of reproductions of specific classes of phenomena. Diachronic emergence explains that irreversibility is a fundamental property of all evolutionary processes in biology. This irreversibility does not preclude that evolution is the result of computations represented by Boolean Networks. Admittedly, such computations are dynamic process by which new systemic properties emerge.

This brings about how computational sciences could contribute to the understanding of the concept of emergence. Let us recall an old and interesting example, i.e., Von Neumann's well-known model of reproductive automata (1961) that deals with the concept of complication.

3-Self-reproduction and self-replication

In order to model reproduction we must understand how complication "becomes selfsupporting and even increasing" [START_REF] Neumann | The General and Logical Theory of Automata[END_REF]. Degenerative complication is defined by the fact that an automaton A that has the ability to produce B needs a complete description of B plus "various arrangements in A that see to it that this description is interpreted and that the constructive operations that it calls are carried out." Then "a degenerative tendency must be expected" [START_REF] Neumann | The General and Logical Theory of Automata[END_REF]. For instance, in the well known system of three automata (A, B, C), A constructs an automaton which is described by an instruction (i). B makes a copy of the instruction (i) that is furnished to it, and C combines A and B operations. This is illustrated below:

A iA A C AiA B iA iA
Let us take the aggregate D denoted by A+B+C. The instruction to reproduce D and to make a copy of iD must be inserted in A and in B, respectively, so that:

A iD D C (D,iD) = E

B iD iD

Von Neumann concluded that E is "clearly self-reproductive" without any vicious circle and "according to the rules of logic" and the complication of such a construction becomes "selfsupporting" and may even increase. The D system acquires the new systemic function of selfreproduction by the fact that iD is inserted in the aggregate (A, B, C), thus becoming E. This transformation results from the fact that the description of D is different in this system from what is constructed with this description. E is a result of this construction and not an element of this description. E emerges in the sense of diachronic emergence. We can reproduce the same construction in a deterministic way and without mystery.

Let us now return to the problem of self-replication in experimental biology that is at the centre of Rosenberg and Kaplan's paper. They deal with the problem of knowing how selfreplication can be a result of catalytic activities of several molecules and not a fundamental property of a certain class of molecules. This means that auto-replication can be constructed in a reproductive way. Consider the example emphasized by [START_REF] Kauffman | Investigations[END_REF] and experimented by [START_REF] Sievers | Self-replication of complementary nucleotide-based oligomers[END_REF] of a single stranded DNA hexamer that is replicated by a collective catalytic system. The hexamer A does not contain the genetic information to produce a copy of itself. Instead, the two triplets of A have the power to catalyse the ligation of B while the two triplets of B catalyse the ligation of A. Thus, reproduction is the result of this collective catalytic operation:

CGGCGG CCG+ CCG CCGCCG CCGCCG CGG+CGG CGGCGG
In the classical molecular model, a fragment of DNA codes for the synthesis of proteins by which DNA is replicated, thus falling in the well-known vicious circle whereby genes are translated into proteins with the help of proteins. In this novel way of thinking the circle becomes virtuous. In it, we have properties (stability or instability of molecules, auto and cross catalysis, etc.) and objects (DNA or RNA as ribozymes). Systemic properties, like selfreplication, result from what is constructed by the initial properties and objects. Therefore, the products cannot be elements of the initial description of the process. The first step is accomplished in order to understand how prebiotic genes can be inserted in a biological system.

4-The entanglement enigma

Assuming that systemic properties result from what is constructed from these properties and objects is valid, it implies that in the scientific methodology, one must take into account the action of downward causation. Let's come back to the origin of the concept in Kim's analysis.

In our previous example (see Section 2c), the cell voltage is a systemic property. It depends on base properties by which it is realized (electric charged ions). Yet, Kim rejects the "causal efficacy" of systemic properties. According to Kim, emergent properties have no causal power of their own. Either the systemic property E is identified with the disjunction of its realizers (Q1 vs Q2 vs Q3…), or it is a "second order functional expression" by which we cannot generate new properties, "but only new ways of indifferently picking out, or grouping, first order properties" [START_REF] Kim | Making sense of Emergence[END_REF]. However, in our example the cell voltage is a systemic property that changes the protein channel (which would be the definition of downward causation). This contradicts Kim because it leads to the idea that a macroproperty M of a system can add to the set of properties Pi of its elements a new property Pj, and this violates the CCP and the CPW.

According to our view, to the contrary, downward causation is obviously possible since the natural world is not closed or completely described by any Demon. As we can say without any contradiction that ions are changing the cell voltage (upward); we can add that the change in cell voltage (systemic property) modifies the protein channels (downward), since both effects are not simultaneous, or were not simultaneous at the beginning. Kim neglects the fact that time is acting, and that this action of time is not an epistemic property. It is, instead, an ontological one. Let's consider Kim's celadon vase that is dropped from the window of his office (1999). Even, if we conclude that the vase, by virtue of its mass, has a set of causal powers that none of its micro-constituents have, the mass of this object is a resultant property. However, the electric voltage emerging from the activity of the ions is not a distributive property and it acts downward on the protein channel. Why is it so different from the celadon example? Our assumption is that time is acting on the system of elements considered as a whole modifying its internal structure, and thus the relation and the properties of its elements. This brings us to Noble's concept of "middle out", which is nothing but a heuristic tool for exploring downward causation in complex systems (Noble, 2003). A priori, one cannot distinguish, what is on the top and what is down, what is the system and what are its elements.

In order to identify the elements, we need to understand first how the system has been modified by its history. They are no "elements per se", in the sense that they do not exist in isolation. They are "the elements" in a system modified by its history. That's why, on each level of explanation, we find strange entanglements in biology: genes are coding for the synthesis of proteins by proteins; the basal membrane is producing the architecture of tissues, yet the stroma and the epithelial cells are producing the basal membrane, etc (see section below).

We will argue that it is not downward causation what leads to these forms of reciprocal causation and to the entanglement enigma. We propose, on the contrary, that this enigma can be solved by the use of the concept of downward causation. Let us consider some biological examples in order to understand this point.

a-Tissue interactions

Since the introduction of the cell theory, most biologists readily accept that the cell is the unit of life. However, in multicellular organisms, single cells do not have an existence independent of the whole organism. These organisms and their cells are ontogenetically linked. Rather, a zygote-a cell resulting from the union of a female and a male gamete-divides, producing more cells, which are organized in a three-dimensional pattern. Both association patterns and cell types change as tissues and organs are formed. Hence, we consider multicellular organisms as complex systems in which the relations among parts are contextual and interdependent. From the beginning of ontogeny, each cell undergoes "differentiation8 " under the influence of neighboring cells. This reciprocity makes it difficult to establish detailed cause and effect relationships. In addition, molecules are being sent from one cell to its surroundings. Such a molecule residing outside cells is no more a part of a cell. These extracellular molecules act on the immediate neighbors, or are carried into distant places by the bloodstream, where they modify the activity of yet other cells, which in turn produce molecules that react with other cells, and so on. These facts reveal that these hierarchical levels are entangled, at times precluding experimentally isolated cells from revealing their role in situ in the originating organism (Soto & Sonnenschein, 2004).

Considering that cells constitute the "low level" parts of the higher level "tissue" or organism may not be always adequate. A tissue results from a long series of interactions during which cells move around in relation to one another and change in the process. By the time the tissue is formed, the "parts" that we identify in them are no longer the parts that interacted in their formation. The cellular components now present did not pre-exist the tissue itself-they are interacting in a particular way that is reciprocal. When we artificially separate the components of the tissue, for instance the cells forming epithelium9 and its subjacent stroma 10 , cells cease to perform the functions they executed when together in their proper three-dimensional arrangement. However, when recombined, oftentimes they form a tissue similar to the one from which they originated. Parenthetically, this is an oversimplification because neither the parenchyma nor the stroma exists in isolation from one another. For example, recombining stroma and parenchyma 11 (usually epithelium) from different organs has provided evidence about the inductive role of the stroma over the epithelium, as well as some indications that the epithelium possesses some degree of cellular identity that is not influenced by the stroma [START_REF] Sakakura | Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme[END_REF]. This is illustrated by recombining fetal mammary gland stroma with salivary gland epithelium which results in a tissue that resembles the mammary gland regarding ductal structure. The recombination of salivary gland stroma with mammary epithelium results in a tissue that resembles a salivary gland. However, the epithelial cells in each case preserve their specific cellular response, i.e., mammary epithelial cells respond to the hormone prolactin by secreting milk proteins regardless of the provenance of the stroma.

As researchers tend to think that the important part of a tissue is its parenchyma, descriptions of changes occurring in the stroma are rare. However, the basement membrane that is interposed between the epithelium and the stroma is made "in collaboration" by the stroma and epithelial cells. Remarkably, some of those basement membrane proteins 12 are produced by both cell types, while others are selectively produced. Nonetheless, the kinetics of production of each protein is different when the epithelium and the stroma are cultured in isolation, indicating that one cell type influences the output of the other cell type; that is, they influence each other reciprocally [START_REF] Smola | Dynamics of Basement Membrane Formation by Keratinocyte-Fibroblast Interactions. In: Organotypic Skin Culture[END_REF]. The secreted proteins interact producing a highly organized structure. The epithelial cells form hemidesmosomes 13 , attaching themselves to the basement membrane. Notably, when epithelial cells are placed in a plain plastic culture dish, they arrange themselves, in a way that does not reflect the pattern of the epithelium of origin. If instead, they are placed in a similar dish coated with basement membrane proteins they associate and recover the original three-dimensional architecture of the epithelium of origin. They attach to each other forming either sheets or ducts. Moreover, the individual cell shapes also change, as does the intracellular placement of their organelles 14 . How can causation be studied here? Is the tissue causing the formation of a basement membrane? And then, is the basement membrane "causing" the normal architecture of the epithelium-and thus the tissue? This suggests downward causation. Tooth development offers another example of reciprocal causation. The mandibular epithelium "causes" the underlying mesenchyme15 to condense (i.e. it appears more cell-dense at the light microscope). If the mandibular epithelium is now isolated and recombined with mesenchyme from other organs, it induces the generation of tooth structures. Soon thereafter this "inductive" potential of the mandibular epithelium is lost. If the condensed mesenchyme from underneath the mandibular epithelium is now recombined with epithelia from other parts of the body, the recombinant generates tooth structures [START_REF] Gilbert | Developmental Biology[END_REF]. Moreover, many gene products involved in early stages of tooth development, mammary gland development and in hair follicle development are similar. Hence, in order to understand how form is generated it may be more productive to concentrate on the higher-level phenomena (cell movement, cell proliferation, cell death) than exclusively in patterns of gene expression.

Thus, in the context of multicellular organisms, single cells are ontogenically linked. Epithelial cells and stromal cells jointly produce the basement membrane, while at the same time this membrane is producing the architecture of the tissue (stroma and epithelium). In this context, it is impossible to isolate gene or cell activity from tissue interactions. In order to understand the problem, one may start at the cell level, or at the level of tissue interactions; however, one will be forced to eventually move up and down levels of organization since multiple levels are participating in the phenomenon being explored. A complete explanation is out of reach because levels are entangled. In this context a useful heuristic tool would be to overcome the bottom-up and the top-down explanation by adopting the middle out approach proposed by [START_REF] Noble | The Music of Life[END_REF]. Since our ontological approach deals with open systems, all levels of organisation can be the starting point for a simulation. They can be many middles now, since the centre is everywhere and the circumference nowhere. Instead of explaining the higher level by the lower one, we can look for the lower level "by the filter of the higher level " (2006: 72).

This reciprocal causality is also central to the understanding of cancer. The cancer phenotype is not fixed from within the cancer cells [START_REF] Sonnenschein | The Society of Cells[END_REF]. Cells from the inner cell mass of blastocysts (early embryos) grow as carcinomas when transplanted under the kidney capsule of a host. When these cells are transplanted back into normal blastocysts, they end up as normal cells in practically all the tissues of the mice that developed from those blastocysts [START_REF] Mintz | Normal genetically mosaic mice produced from malignant teratocarcinoma cells[END_REF]. Similar normalization phenomena have been observed when liver carcinoma cells are transplanted into a normal liver [START_REF] Coleman | Regulation of the differentiation of diploid and aneuploid rat liver epithelial (stem-like) cells by the liver microenvironment[END_REF], when mammary carcinoma cells are transplanted into normal mammary gland stroma [START_REF] Maffini | Stromal regulation of neoplastic development: Age-dependent normalization of neoplastic mammary cells by mammary stroma[END_REF] or when melanoma cells are placed into zebrafish embryos [START_REF] Hendrix | Reprogramming metastatic tumour cells with embryonic microenvironments[END_REF]. However, the ability of the host organ to normalize cancer cells is influenced by developmental factors. For example, the ability of the liver to "normalize" liver cancer cells is more robust in young animals than in older ones, while the ability of the mammary stroma to normalize mammary carcinoma cells is enhanced by the age of the recipient and it is further enhanced if the animals have undergone pregnancy and nursed their offspring [START_REF] Maffini | Stromal regulation of neoplastic development: Age-dependent normalization of neoplastic mammary cells by mammary stroma[END_REF].

The ontological stance of researchers is a major determinant of the models proposed to study a phenomenon. For decades, reductionists have interpreted the phenomenon of chemicallyinduced mammary cancer as due to a direct interaction between the carcinogen and the DNA of the "founder" epithelial cell of the resulting carcinoma. This stance overlooked two facts, namely, that the carcinogen was injected into a live animal, where all cells are exposed, and that the carcinogen interacts with multiple macromolecules, in addition to DNA. By overlooking these facts, they exclusively searched for mutations in the DNA of the epithelial cells that became neoplastic. Only recently experiments were conducted to address whether the target of the carcinogen resided in the epithelium or in the stroma of the mammary gland. Using a theory-neutral experimental strategy, Maffini et al observed that the recombination of stroma exposed to a carcinogen with normal epithelial cells resulted in neoplasms (Maffini et ai, 2004). The reverse combination did not. This observation suggests that the stroma, rather than individual cells in the epithelium, is the target of the carcinogen. These results also point to the contextuality of the neoplastic phenotype.

But where is this reciprocal causality coming from? The context of multicellular organisms is a product of history (evolution and ontogeny). We refuse all a priori views that predicate the possibility to see all different contexts, or all possible worlds, "instead of studying cases of emergence in the real world" (Bunge, 2003: 14). We are arguing then, that this contextdependence is an effect of diachronic emergence, and not the contrary.

Let us now look at a well-identified system of chemical objects that by interacting add new properties to this system. We take into account the system's history. Therefore new properties are acting and have the power to modify the system without any contradiction, or any infinite regression, since we don't need to assume that they could be described at the beginning. The new definition of the system must be done at another time, and it acts downward and not arbitrarily on the initial elements of the system. Let's relate a representative example from ontogeny in order to illustrate this point.

b-An example in morphogenesis

During embryogenesis the generation of form (morphogenesis) is mediated by cellular processes such as the direction and number of cell divisions, changes in cell shape, cell movement, cell growth, cell death and changes in the composition of the cell membrane and extracellular matrix [START_REF] Gilbert | Developmental Biology[END_REF]. The reductionist view proposes that each of these cellular processes is generated by the differential expression of developmental genes. We consider instead, that in addition to this upward causation, cellular and tissue events occurring before the expression of a particular set of genes takes place may act downwardly modifying the expression of these genes at a later time (diachronic emergence). During gastrulation 16 of the Drosophila embryo 17 , the mesoderm 18 invaginates, i.e., it moves from the surface to the interior of the embryo, and the germ band 19 extends (T1). This movement generates mechanical force that results in compression of the stomodeal cells (the anterior part of the alimentary canal; future mouth). As a result of this compression, the stomodeal cells express the gene twist (T2). This is a crucial event in the formation of the anterior gut. Mutant embryos that fail to undergo germ band extension also fail to express twist in the stomodeal cells. When these embryos are subjected to gentle compression, twist is expressed in the 16 Gastrulation: after fertilization a series of rapid mitotic divisions (cleavage) result in the blastula. Next, the process of gastrulation takes place, whereby cells change positions with respect to each other; as the result of this process the germ layers (ectoderm, mesoderm and endoderm) are formed. 17 Drosophila: a genus of small dipteran flies that include several (as D. melanogaster) that are extensively used in genetic research (modified from: http://www.nlm.nih.gov/medlineplus/mplusdictionary.html).

18 Mesoderm: the middle of the three primary germ layers of an embryo that is the source especially of bone, muscle, connective tissue, and dermis; broadly speaking, tissue derived from this germ layer (modified from http://www.nlm.nih.gov/medlineplus/mplusdictionary.html). 19 Germ band: a collection of cells along the ventral midline that includes all the cells that will form the trunk of the embryo (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?indexed=google&rid=dbio.section.1959).

stomodeal cells and development continues. Furthermore, ablation of dorsal cells in normal embryos relieves compression during germ band extension; as a result, twist expression in the stomodeal cells fails to occur [START_REF] Duhem | Mechanical induction of twist in the Drosophila foregut/stomodeal primordium[END_REF]. This causal chain, from molecular events resulting in germ band extension to physical stress inducing the next molecular event (twist induction in stomodeal cells) represents an emergent event. In other words, a moving group of cells produces compression on another group of cells that respond to this force by expressing a gene product, twist, promoting in this way a downward causal chain. Reductionists may argue that this is not a truly downward cause, because an operator could also apply compression and generate the same outcome; however, the fact remains that during the process of organogenesis, the only option the embryo has to instantiate compression is by creating a three-dimensional structure that generates this force and hence, this outcome. From our perspective, it is clear that mechanical forces, in an abiotic system produce mechanical effects that are predictable. In a biological system, they may acquire novel properties, such as that of inducing gene expression, which cannot be predicted from our knowledge of the physical world. This novel property appears in a "tissue-world" whereby a mass of moving cells is needed to generate this force.

Conclusion

If we admit that a physical property Pj can be at the same time physical and biological as a result of the phylogenetic and ontogenetic history of organisms, all potential vicious circles are avoided. We have illustrated the problem with examples taken from organismal biology, specifically, embryonic development and carcinogenesis. When acting in a biological system such as an embryo during morphogenesis, a mechanical force acquires the new property of inducing the expression of a particular gene (Pj). A simple chemical molecule, such as a carcinogen, has the new property to disrupt the architecture of a tissue and to initiate neoplastic development. In the context of new systemic properties, basic properties are changing. However, this new type of constraint is not fixed at the beginning. This is the meaning of downward causation (see section 4).

After a certain period of time t, a new macroproperty M is emerging in a system of elements characterized by a set of physical properties Pi. Time is acting, not on a part of the system, but on the system conceived "as a whole". This action is real and has an ontological meaning.

Thus the system, having M at t, causes the new property Pj (this is our definition of downward causation). Note that Kim is curiously not in disagreement with this conception of "reflexive downward causation" based on the use of diachronic emergence (1999). Yet, it is more compelling when one rejects the CCP, the CPW and the physical determination thesis. Thus, we deal with ontological emergence, in the sense that the action of time explains how the new property M is emerging. It is not only a question of prediction. It is not due to our limited cognitive capacities.

Downward causation makes philosophical sense only when it correlates with diachronic emergence. It requires that the system in which it appears be time-dependent. That is, the system is a process, not a thing. Downward causation leads to a vicious circle only in the context of synchronic emergence [START_REF] Kim | Making sense of Emergence[END_REF]. This is so because if systemic properties depend on basic properties; the former cannot complete or modify the basic properties simultaneously without contradiction.

This explains why, in order to understand what is going on in the system after a period of time t, we must jump simultaneously to multiple levels on which this system is integrated (see section 2C). Genes are coding for the synthesis of proteins by the proteins. Proteins carry current that change the cell voltage. In turn the cell voltage changes the ion channels. Such a conclusion legitimates the heuristic tool proposed by Noble and by which both reductionist and holist approaches are rejected in the fields of developmental biology and phyisiology. "Middle out" is neither a bottom-up reconstruction, nor a top-down synthesis (see also Bunge 2003: 46-47). This new design allows us to understand how "the orchestra of life" works, and "how large it is" [START_REF] Noble | The Music of Life[END_REF].

Our analysis also has evolutionary relevance. Diachronic changes can be simulated and natural selection can be used in this simulation [START_REF] Kauffman | Investigations[END_REF]. Yet this simulation will not explain how natural selection as a biological or a chemical property is emerging itself in the world, as Rosenberg and Kaplan propose. Moreover, Kaplan and Rosenberg's assertion by which natural selection is understood as a natural law is not obvious.

There is a difference between explanation and computation. Kauffman's automata have a fitness value in NK models, where N= automata and K= interactions. NK networks have little to do with classical models of maximisation in population genetics. What these systems of automata are doing is not explained by the description of their elements. New constraints are emerging by the process of computation that simulates evolution. Thus, we all have to deal again with the old French aphorism: "le temps est ce qui se fait, et même ce qui fait que tout se fait. 20 "

The origin of this idea is in[START_REF] Feibleman | Theory of integrative levels[END_REF], see also[START_REF] Stengers | Cosmopolitiques VI[END_REF].

"The same notion of a God'-Eye View"… the same epistemic ideal of achieving a view from an "Archimedean point" -a point from which we can survey observers as if they were not ourselves, survey them as if we were, so to speak, outside of our own skins -is involved"(Putnam, 1990: 17).

(Putnam, 1990: 17).

Differentiation : the sum of the developmental processes whereby from a single cell, the zygote the organism attains the diverse adult structures (cells, tissues and organs) and functions.

Epithelium: an avascular tissue that covers a free surface or lines-up a tube or cavity in an animal body and serves especially to enclose and protect the other parts of the body, produces secretions and excretions, and functions in assimilation (modified from: http://www.nlm.nih.gov/medlineplus/mplusdictionary.html).

Mesenchyme: loosely organized mesodermal embryonic tissue that give rise to connective tissues. (modified from: http://www.nlm.nih.gov/medlineplus/mplusdictionary.html).

Acknowledgements:

We thank Professor Ulises Moulines (University of Munich) and Doctor Slobodan Perovic (Carleton University) for their critical reading and helpful comments concerning an earlier draft of this manuscript.