Recovery of 15N Labeled Nitrogen Fertilizer by Fertigated and Drip Irrigated Greenhouse Vegetable Crops

The stable isotope 15N was used to assess the recovery of mineral N fertilizer applied to fertigated and drip-irrigated spring muskmelon and autumn-winter sweet pepper crops grown in greenhouse soil plots. They received 92–96% of mineral N fertilizer as NO3−. 15N-labeled Ca (NO3)2 fertilizer was applied to crops during vegetative growth and fruit production phases. Crops were grown with either conventional management or combined improved N and irrigation management. Improved management for both irrigation and N was based on the combined use of models, to estimate crop requirements, and of monitoring of soil parameters. In sweet pepper, from conventional management, 15N recoveries from the 15N applications made during vegetative growth and fruit production were 66% and 58%, respectively. With improved management in sweet pepper, the corresponding 15N recoveries were 82% and 77%. In muskmelon, 15N recoveries from conventional management from the 15N applications made during vegetative growth and fruit production were 71% and 42%, respectively. With improved management, the corresponding 15N recoveries were 68% and 44%, respectively. The results demonstrated that combined drip irrigation and fertigation systems with frequent irrigation and N fertilizer application can have very high recovery of applied N fertilizer, of 77–82%.


Introduction
With close to 42,000 ha, the greenhouse vegetable production of southeast (SE) Spain is the largest concentration of greenhouses in Europe [1-4]. There are appreciable and expanding areas of similar greenhouses devoted mostly to vegetable production in other Mediterranean Basin countries [1,5]. There is currently a rapid expansion of similar greenhouses in Central America, particularly in Mexico [6]. In China, there are an estimated 4 million ha of greenhouses (Dr. Junjang Yang, Institute of Plant Nutrition and Resources, Beijing, China; personal communication). Two of the most commonly-grown species in the greenhouse-based vegetable production of SE Spain are sweet pepper (Capsicum annum) and melon (Cucumis melo, L.), which are commonly grown in sequence. Pepper with an autumn-winter growing cycle, and melon with a spring growing cycle. Muskmelon is the most commonly-grown type of melon.
Large additions of nitrogen (N) are characteristic of intensive vegetable production systems. Intensive vegetable production is commonly associated with appreciable nitrate (NO 3 − ) leaching

Location and Greenhouses
The work was conducted in two identical plastic greenhouses in the Research Station of Cajamar in El Ejido, Almería, in SE Spain (36 • 48 N, 2 • 43 W). The greenhouses were representative of those used in this region [18,19]. The greenhouses had an asymmetrical, shallow, inverted V-shaped roof, and the structure was of stainless-steel tubes and wires; the cladding was 200 µm-thick colorless low-density polyethylene film. Each greenhouse measured 24 × 18 m. The two greenhouses had an east-west orientation, and were adjacent to one another along the east-west axis. They had passive ventilation and no heating system. Each greenhouse was divided approximately in half along the east-west axis by a 2 m wide concrete path; this work was conducted in the northern half of each greenhouse.
The greenhouses had an artificial soil system known locally as "enarenado" soil, which is commonly used in this vegetable production system [18,19,28]. The soil consisted of a 30 cm layer of imported clay soil, obtained from a local quarry, which was placed over the naturally occurring loam soil; a 10 cm layer of coarse river sand was placed over the imported clay soil as a mulch. The 10 cm sand mulch layer over the soil surface substantially reduces evaporation and weed growth. Generally, in this artificial soil system, roots are mostly concentrated in the imported soil layer. Relevant properties of the soil at the beginning of the study are given in Table 1. All soil depths in Table 1, and referred to subsequently, are relative to the surface of the imported layer of clay soil. This artificial soil system was formed when the greenhouse was constructed in 1995. In June 2003, sheep manure was applied to the surface layer of imported clay soil at a rate of 73 t ha −1 , supplying 1270 kg N ha −1 . The application of manure at these rates, at the formation of the artificial soil system and thereafter every 2-5 years, is common practice in this vegetable production system in response to the low organic matter contents of the sub-soils that are imported, from quarries, into the greenhouses for cropping [18,19].
All plants were grown in north-south aligned rows, with 1 m spacing between rows, and 0.5 m between adjacent plants within rows. All plants were vertically supported using nylon guides.

Crops and Crop Management
Muskmelon (Cucumis melo, L., cv. 'Deneb') type Galia and sweet pepper (Capsicum annum, L., cv. Above-ground drip irrigation was used with one 2.8 L h −1 emitter immediately adjacent to each plant and separated by approximately 5 cm. Complete nutrient solutions were applied by fertigation in all irrigations after the first 2 weeks following transplanting; previously, only water was applied. Examples of the nutrient solutions used for the conventionally managed muskmelon and sweet pepper crops are presented in Supplementary Table S1. All cultural practices (crop pruning and guiding, pollination, fruit harvesting) and pest management operations followed established local practices. Sweet pepper fruit, in both crops, was collected in five harvests conducted at 1-2 week intervals over approximately 2-month period. Muskmelon fruit was harvested at the end of the crop. Irrigation and N management are described subsequently in sub-Section 2.4, and in Tables 2 and 3. Monthly average climatic data inside the greenhouses from January 2005 to January 2007 are presented in Figure 1.

Experimental Design
The working area in the northern half of each greenhouse of 20 rows of 9 plants was divided into four representative plots each of 5 rows with 9 plants per row. All measurements were made with four replications, with one measurement being made in each plot.
In each of the four crops, there were two treatments, being conventional N and irrigation management (CM), or an improved N and irrigation management system (IM). Each treatment was applied to an individual greenhouse. The treatments are fully described in the next sub-section.

Treatments-N and Irrigation Management
For the conventional management treatment, irrigation followed local practices with respect to volume and timing of irrigation. Published [29] and unpublished data (M.D. Fernández, Research Station of Cajamar, El Ejido, Almeria, Spain) relating irrigation volumes to calculated crop evapotranspiration (ETc) were used to characterize conventional irrigation management. Crop evapotranspiration was calculated using the PrHo model developed for vegetable crops in this system [30][31][32]. For conventional management, volumes of applied nutrient solution were in excess of crop evapotranspiration (ETc) until maximum crop coefficient (k c ) values [29] were reached, and then, were similar to ETc [29]. In the improved management systems, irrigation was based on estimated daily ETc [30][31][32] and adjusted to maintain soil matric potential (SMP) at 15 cm depth (measured with tensiometers) between −10 and −40 kPa; the timing of irrigation was in response to SMP. N management in the conventionally managed treatments was based on applying fixed NO 3 − and NH 4 + concentrations in the applied nutrient solutions following the recommendations of local technical advisors. N management in the treatments with improved management was based on a prescriptive-corrective management system [12,14,22,33,34] that was developed with these crops. The prescriptive component developed for N management was based on applying N to match simulated daily crop N uptake that was estimated using the Nup simulation model [22,34]. Nup calculates daily crop N uptake in vegetable crops grown in plastic greenhouses in SE Spain. Inputs are daily values of maximum and minimum air temperatures and the integral of solar radiation, within the greenhouse. It was calibrated and validated for muskmelon and sweet pepper [22,34]. Brief [22] and detailed [34] descriptions are available. The corrective component for N management was based on maintaining the soil solution NO 3 − concentration, in the immediate root zone, within a specified range [12,14,22,34].
The specific N management practices used in each crop are described subsequently.
In the 2005 IM muskmelon crop, the concentration of applied N in the IM treatment was similar to the CM treatment; the 16% reduction in irrigation volume was associated with an 18% reduction in the amount of N applied ( Table 2). In the 2005 IM sweet pepper crop, N management was based on maintaining the soil solution NO 3 − concentration, at 15 cm depth, within the range of 8-12 mM. With the reductions of 17 and 30% in irrigation volume and N concentrations, the amount of applied N was reduced by 42% (Table 3).
In the 2006, IM muskmelon crop, N management was based on estimated crop N uptake, which was simulated using the Nup model [22,34]. With the reductions of 11% and 15% in irrigation volume and applied N concentration, the amount of applied N was reduced by 28% (Table 2). In the 2006 IM sweet pepper crop, N management was based on simulated crop N uptake using the Nup model [22,34] and maintaining soil solution NO 3 − concentration, at 15 cm depth at 8-12 mM. With the reductions of 16 and 24% in irrigation volume and N concentrations, the amount of applied N was reduced by 36% ( Table 3).
The concentrations of the nutrients other than N applied in the complete nutrient solutions followed established local practice. From two weeks after transplanting, the crops received complete nutrient solutions in all irrigations, with 92-96% of N as NO 3 − , and the rest as NH 4 + (Tables 2 and 3) following local practice. Irrigation volumes were measured daily with a volume meter. Samples of applied nutrient solutions were analyzed weekly for NO 3 − and NH 4 + concentrations with an automatic continuous segmented flow analyzer (model SAN++, Skalar Analytical B.V., Breda, The Netherlands).

15 N Applications
In   The vegetative growth phase was defined as being when there was predominantly vegetative growth, and the fruit production phase when there was predominantly fruit growth. The fruit production phase was considered to start when there was the full canopy and the exponential growth of fruit had commenced. In muskmelon, the transition between the vegetative growth and fruit Agronomy 2020, 10, 741 7 of 17 production phases occurred when fruit biomass was approximately 1% of final fruit production. In the sweet pepper crops, it occurred at 12-15% of final fruit production.
For both muskmelon and sweet pepper, the 15  The four individual plants that were directly labeled in each "labeling" of each treatment were in each of the four representative plots of the corresponding treatment. Within each plot, the locations of the 15 N applications were chosen to maximize the distance from any previous 15 N applications.
The 15 N labeled nutrient solutions were prepared firstly as nutrient solutions with a composition of nutrients, other than N, as close as possible to that of the nutrient solutions being applied to the crops. These initial nutrient solutions had 2.0-2.5 mM NO 3 − and no NH 4 + . 15  The 15 N analysis was conducted with elemental analyzer isotope ratio mass spectrometry (EA-IRMS) using an isotope ratio mass spectrometer (IRMS) (Model Europa Scientific 20-20, Sercon Ltd., Crewe, UK) coupled to a Europa Scientific elemental analyzer system (Sercon Ltd., Crewe, UK). Total N content was calculated as the total ion beam area generated by the IRMS. The 15 N analyses were conducted by Iso-Analytical Limited Sandbach, UK. 15 N enrichment was calculated, as 15 N atom% excess by subtracting the 15 N natural abundance values in equivalent materials (e.g., nutrient solution, fruit, leaf, stem, pruned material) that were unlabeled.
The nutrient solutions of 15 N-labeled Ca (NO 3 ) 2 were applied using inverted 1.5 L polyethylene bottles connected by silicon tubing to intra-venous drippers. The inverted bottles were supported on metal stakes and the drippers were positioned 3 cm above the sand layer on the soil surface, directly above where irrigation emitter was normally located. Each day, prior to applying the 15 N-labeled solution, unlabeled nutrient solution was applied through the drip irrigation system, to all plants in each greenhouse except those that were to directly receive the labeled nutrient solutions. For plants that were to be directly labeled, 7 L plastic trays were used to collect the unlabeled nutrient solution. This ensured that the only nutrient solution received during the application period of three consecutive days of labeling was the 15 N-labeled nutrient solution. The applied volumes and the nutrient concentrations of 15 N-labeled nutrient solution were identical to those being applied to the rest of the plants in each greenhouse during the three days labeling periods.
Sheet metal plates 1 m long × 22 cm high were inserted into the soil, to a depth of 22 cm, parallel to and 50 cm from the row of plants being labeled, to limit lateral movement, perpendicular to the rows, and prevent uptake of 15 N by plants in adjacent crop rows. One plate was inserted on each side of each labeled plant, parallel to the crop rows. The center part of each plate was adjacent to the directly labeled plant. plants"; AP), were identified as " 15 N labeled plants". Following labeling, all pruned shoot material from each subsequent pruning of each 15 N labeled plant was collected and oven-dried at 65 • C until constant weight. Similarly, all mature fruit collected from each 15 N labeled plant was collected and oven-dried at 65 • C. At the end of each crop (crop maturity), each labeled plant was cut at the ground level, and separated into leaf, stem and immature fruit. Each of these three components was oven-dried at 65 • C until constant weight.
All dried pruned material collected after labeling was bulked for each plant. Similarly, all dried mature fruit was bulked for each plant. Fallen leaf collected from the soil surface below each plant was collected and bulked. For data analysis, the small amounts of 15 N in fallen leaf were included in that of pruned material. From each labeled plant, both from directly labeled (DLP) and adjacent (AP) plants, there were samples of bulked pruned material, bulked mature fruit, bulked fallen leaf, and the leaf, stem and immature fruit removed at the end of the crop.
For each labeled plant, the bulked pruned material, bulked mature fruit, bulked fallen leaf, and the leaf, stem and immature fruit, removed at the end of each crop, were individually ground with a knife mill (Model SM100 Comfort, Retsch, Germany). A representative sub-sample was then ground with a ball mill (Model MM200 Comfort, Retsch, Germany) until sufficiently fine to pass through a 0.2 mm mesh.

Calculation of 15 N Recovery
For each 15  For each treatment of each crop, four areas of 4 m 2 , each with eight plants were marked to determine fruit production, the mass of pruned material removed during the crop, and final standing biomass (after prior removal of prunings and harvested fruit) at the end of each crop. There was one group of eight plants in each replicate plot. There were several prunings in each crop, one harvest of mature muskmelon fruit and 5-8 harvests of mature sweet pepper fruit. At each fruit harvest, the fruit was separated into commercial and non-commercial fruit using local commercial criteria; fresh and dry weights were determined of both fruit categories. At each pruning, dry matter removed in leaves and stems was determined. Dry matter (DM) content of all crop components was determined by oven drying at 65 • C until constant weight.
At the end of each of crop, the four groups of eight plants in each treatment, were completely removed and separated into leaves, stem and fruit, which were weighed, and the dry matter content determined. Final total shoot dry matter production (final DMP) for each treatment was determined by summing the amount of DM of these sampled plants, the total amount of DM removed in the various prunings, and the total amount of DM harvested as fruit.
Representative samples of leaves, stems, and fruit from the final standing biomass sampling, and of harvested fruit and prunings were ground separately and sequentially with a knife mill and ball mill, as previously described for the equivalent 15 N labeled material. The total N content of each sample was determined using a Dumas-type elemental analyzer system (model EA 3000, EuroVector SpA., Milan, Italy). Total crop N uptake was calculated, as the sum of N in the final standing biomass, harvested fruit and prunings for each replicate group of plants for each treatment.

Statistical Analyses
The following statistical analyses were conducted. Analysis of variance was conducted to examine the differences between treatments in the recovery of 15 N, in the fruit and total dry matter production and in the total N uptake, and standard errors of the means were calculated for all measured results.

Muskmelon
Seventy-one and 68% of 15 N applied, during the vegetative growth phase (VG) in 2005, was recovered by the conventional and improved managed muskmelon crops, respectively (Table 6). Forty-two and 44% of 15 N applied, during the fruit production phase (FP) in 2006, was recovered by the conventional and improved managed muskmelon crops, respectively (Table 6). These results demonstrated high crop recoveries of 15 N applied during vegetative growth, and appreciably lower recoveries of 15 N applied during fruit production. The differences between the conventional and improved management practices applied during the muskmelon crops were insufficient to affect the recovery of 15 N.
In muskmelon, 23-37% of 15 N applied, during vegetative growth, was recovered in the two plants adjacent to the directly-labeled plants ( Table 6). From the 15 N applied during fruit production in muskmelon, only 10-14% of applied 15 N was recovered in adjacent plants (Table 6). From the 15 N application to muskmelon during vegetative growth, 13-37% of the recovered 15 N was in fruit (Table 7), suggesting appreciable remobilization of N within the plants following crop uptake, as at the time of 15 N labeling only 1% of final fruit biomass had formed. Much of the rest of the recovered 15 N from the vegetative growth labeling was in leaves and stem (Table 7). From the 15 N application to muskmelon during fruit production, 47-48% of recovered 15 N was in fruit, and the rest in shoots, mostly in leaves (Table 7). These results demonstrate that approximately 50% of N absorbed during the fruit production phase was used in the production of shoot material.
For the 2005 and 2006 muskmelon crops, total fresh fruit production was 6.8-7.8 kg m −2 and total commercial fruit production was 6.3-7.5 kg m −2 (Table 8). These yields are similar to those for commercial production in the region, and there were no differences between conventional and improved management for either total or marketable fruit production in either the 2005 and 2006 muskmelon crops. Total crop N uptake was 239-266 kg N ha −1 for the 2005 muskmelon crops and 268-283 kg N ha −1 for 2006 muskmelon crops (Table 8). The soil mineral N (NO 3 − -N + NH 4 + -N) content measured at 0-60 cm depth at the beginning and at the end of each treatment of the two muskmelon crops are given in Table 9. There were very high contents of soil mineral N in the 0-60 cm profile throughout all treatments of the 2005 and 2006 muskmelon crops. Nearly all the soil mineral N was in the form of NO 3 − and <5 kg N ha −1 was in the form of NH 4 + -N.  Figure 2a

Sweet Pepper
Total recovery of 15 N applied during vegetative growth was 66% with conventional management and 82% with improved management (Table 10). Total recovery of 15 N applied during the fruit production phase was 58% with conventional management and 77% with improved management

Sweet Pepper
Total recovery of 15 N applied during vegetative growth was 66% with conventional management and 82% with improved management (Table 10). Total recovery of 15 N applied during the fruit production phase was 58% with conventional management and 77% with improved management ( Table 10). The recoveries in the improved management treatments, from the 15 N labeling at both vegetative growth and fruit production, were very high being 77-82%. These recoveries were appreciably higher than the recoveries from the corresponding 15 N applications in the conventionally managed treatments of 58-66%.   In sweet pepper, most of the applied 15 N was recovered in the directly-labeled plants (Table 10). Only 6-8% of applied 15 N during fruit production was recovered in the plants immediately adjacent to the directly labeled plants. From 15 N applied to sweet pepper during vegetative growth, 12-15% of applied 15 N was recovered in adjacent plants.
In sweet pepper, the relative distribution of recovered 15 N within plant components was similar between treatments and 15 N application times (Table 11). Between 44-57% of applied 15 N was recovered in fruit, 25-30% in leaves and 16-26% in stem (Table 11). For the 2005 sweet pepper crop, total fresh fruit production was 6.1-6.8 kg m −2 and total commercial fruit production was 5.5-6.2 kg m −2 (Table 8). For the 2006 sweet pepper crop, total fresh fruit production was 9.8-10.5 kg m −2 and total commercial fruit production was 9.1-9.9 kg m −2 ( Table 8) Table 8).
The soil mineral N (NO 3 − -N + NH 4 + -N) contents measured at 0-60 cm depth at the beginning and at the end of each treatment of the two sweet pepper crops are given in Table 12. There were very  (Table 12).  Figure 3a,b, respectively. Generally, there was an appreciably lower soil solution NO 3 − concentration in the root zone of the crops with improved management compared to those with conventional management.

Discussion
15 N recovery is considered to be a good indicator of N fertilizer efficiency [18]. The sweet pepper crops recovered 58%-82% of 15 N applied during vegetative growth or during fruit production. These are high recovery values, given that N recoveries from mineral fertilizer N applications, in vegetable production, are generally <50% [12,35]. The recovery of 15 N applied to muskmelon during vegetative growth was also relatively high. However, the recovery, by muskmelon, of 15 N applied during fruit production, was notably lower. These relatively low recoveries can be explained by excessive N application during fruit production of muskmelon, as demonstrated by the on-going increases in soil solution NO3 − during the latter part of the melon crops. These data suggest that applied N concentrations during the latter part of melon crops can be appreciably reduced, in relation to what is applied earlier in the crop. This is supported by recent modeling work, in the context of this system, that showed that applied N concentrations, to meet crop N requirements, decline from the vegetative to fruit production phases [20,21,23]. Common commercial management practice in this system is to maintain relatively constant applied N concentrations throughout crops [36].
In the sweet pepper crops, the improved N and irrigation management practices were associated with very high 15 N recoveries of 77%-82% from 15 N applications during both vegetative growth and fruit production. These values suggest that the combination of (i) matching N supply to N demand, (ii) frequent small N applications made every 1-4 days, and (iii) the avoidance of excessive irrigation enabled very high recoveries of applied N. Essentially, N was applied when it was required and to

Discussion
15 N recovery is considered to be a good indicator of N fertilizer efficiency [18]. The sweet pepper crops recovered 58-82% of 15 N applied during vegetative growth or during fruit production. These are high recovery values, given that N recoveries from mineral fertilizer N applications, in vegetable production, are generally <50% [12,35]. The recovery of 15 N applied to muskmelon during vegetative growth was also relatively high. However, the recovery, by muskmelon, of 15 N applied during fruit production, was notably lower. These relatively low recoveries can be explained by excessive N application during fruit production of muskmelon, as demonstrated by the on-going increases in soil solution NO 3 − during the latter part of the melon crops. These data suggest that applied N concentrations during the latter part of melon crops can be appreciably reduced, in relation to what is applied earlier in the crop. This is supported by recent modeling work, in the context of this system, that showed that applied N concentrations, to meet crop N requirements, decline from the vegetative to fruit production phases [20,21,23]. Common commercial management practice in this system is to maintain relatively constant applied N concentrations throughout crops [36].
In the sweet pepper crops, the improved N and irrigation management practices were associated with very high 15 N recoveries of 77-82% from 15 N applications during both vegetative growth and fruit production. These values suggest that the combination of (i) matching N supply to N demand, (ii) frequent small N applications made every 1-4 days, and (iii) the avoidance of excessive irrigation enabled very high recoveries of applied N. Essentially, N was applied when it was required and to where it could be most effectively obtained by the crop. Similarly, high crop recoveries of applied N, averaged over a complete crop, were obtained for optimally managed substrate-grown tomato grown with a similar combined fertigation and drip irrigation system [37]. While the growing media and the frequency of application of nutrient solution are different, it appears that within the drip irrigation bulb in both soil and substrate that precise and frequent N fertilization with optimal management can result in very high recoveries of fertilizer N.
These high 15 N recoveries were obtained in the presence of high soil mineral N contents. A major factor contributing to these high 15 [22,34]. The data of sweet pepper crops with improved N and irrigation management indicate that vegetable crops can obtain nearly of their N requirements from within the drip irrigation bulb, regardless of the amounts of available present outside the drip irrigation bulb. When there is appreciable mineral N present in the upper part of these soil profiles outside the immediate wet bulb, management systems will need to be developed, for drip irrigated and fertigated crops, so that they absorb mineral N from outside the bulb. A notable feature of the results with the sweet pepper crops was the increase in 15 N recovery associated with improved management compared to conventional management. In the sweet pepper crops, the improved management practices resulted in a 36-42% reduction in the total amount of mineral N fertilizer applied. These reductions in applied N were associated with appreciable increases in the 15 N recovery of 16% for the vegetative growth application, and 19% for the fruit production application. The effect of substantial reductions in applied N was also apparent in consistently appreciably lower soil solution [NO 3 − ] in the immediate root zone of the sweet pepper crops with improved management. In the muskmelon crops, the improved management practices did not enhance 15 N recovery compared to conventional management. The reductions in the total amount of fertilizer N of 18 and 28%, in 2005 and 2006, respectively, were apparently insufficient to affect a notable improvement in N efficiency. A factor contributing to the high N recovery of muskmelon during vegetative growth, in the present study, was presumably the very rapid vegetative growth of this crop. The associated high N demand was indicated by the substantial depletion in soil solution [NO 3 − ] in the immediate root zone during this growth phase. The high amounts of soil mineral N in the current study have been observed in other studies conducted in this system [40]. They are consistent with the use of standard nutrient solutions, periodic large manure applications, and that soil mineral N and manure N are generally not considered when developing N management plans [18]. As previously discussed, the 15 N data indicate that the high amounts of soil mineral N had relatively little effect on 15 N recovery.
Optimal N and irrigation management when using fertigation and drip irrigation systems, enable the frequent application of small amounts of N and small volumes of water to accurately meet crop requirements. The current study demonstrated that with optimal N and irrigation management that very high recoveries of applied N fertilizer can be achieved. This greenhouse production system enhances the potential for high recovery of applied mineral because there are no rainfall-related NO 3 − leaching events, and the combination of the sand mulch and the use of NO 3 − based N fertilizers considerably reduces the possibility of NH 3 volatilization loss from these alkaline soils. The use of 15 N-labeled fertilizer demonstrated that N recoveries from N applied were generally high for soil-grown vegetable crops grown in greenhouses using fertigation and drip irrigation. With optimal management practices, recoveries of 77-82% were obtained.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/5/741/s1, Table S1: Muskmelon and sweet pepper. Funding: This research was funded by the "Reduction of nitrate pollution of aquifers from intensive horticultural greenhouse production areas on the Spanish Mediterranean coast" granted by the Spanish Ministry of Education and Science and co-financed by FEDER, grant number AGL2004-07399.