Published online 4 January 2008
Published in J Environ Qual 37:182-185 (2008)
DOI: 10.2134/jeq2007.0329
© 2008 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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Isotopic Discrimination as a Tool for Organic Farming Certification in Sweet Pepper
Francisco M. del Amora,*,
Joaquín Navarroa and
Pedro M. Apariciob
a Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, 30150 La Alberca, Murcia, Spain
b Instituto de Agrobiotecnología, UPNA.CSIC.Gobierno de Navarra, 31006 Pamplona, Spain
* Corresponding author (franciscom.delamor{at}carm.es).
Received for publication June 20, 2007.
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ABSTRACT
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Organic farming is a form of agriculture that excludes the use of synthetic fertilizers, pesticides, and genetically modified organisms. These fertilizers have been traditionally overused in conventional farming to avoid lost revenue, but this often not does not take into account the potential contamination of aquifers and river due to nitrate leaching. Transition to organic farming practices could provide an instrument to reduce contamination and increase potential income. It is difficult to determine to what extent those fertilizers could have been used within a complete traceability of the production process. In this experiment, we evaluated the use of 15N/14N isotopic discrimination in sweet pepper plants to test the hypothesis that synthetic fertilizers significantly reduce 15N/14N compared with exclusively organic practices. Therefore, three common types of organic manures (sheep, hen, or horse) were applied at a rate of 8 kg m–2 with or without synthetic fertilizer amendments under fully controlled environmental and irrigation conditions. Results indicate that (i) use of synthetic fertilizers significantly reduced 15/14N2vsN2atm compared with treatments that only received water; (ii) with respect to the plant organs, old leaves and fruits were more sensitive to the synthetic fertilizer additions with reductions in 15/14N2vsN2atm of 24.1 and 27.8%, respectively; and (iii) independently of the organic manure used, no additional fertilization (synthetic or organic) is required before 106 days after transplanting at that dosage because plant fresh weight was not reduced.
Abbreviations: DAT, days after transplanting • OM, organic matter • TN, total nitrogen
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INTRODUCTION
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THERE is increasing concern about food quality and safety. Consumers are interested in organic products because they are considered to be safer for health due to the absence of pesticide residues and because they are produced in a more environmentally compatible manner (Brandt and Mølgaard, 2001). According to the USA and European Union regulations on organic farming, organic products are subject to controls by an accreditation and certification system. In particular, they must be recorded at each step of production, thereby ensuring the complete traceability at all stages of production, processing, and marketing. In addition to the biological control for pest and diseases, soil fertilization in organic farming is only possible with organic fertilizers and amendments such as composted or uncomposted cattle manure or other organic waste, whereas in conventional production systems, synthetic fertilizers are used (Rapisarda et al., 2005). However, in managing organic farming, it could be difficult to establish a correct fertilization because of differing compositions of the manure used. Furthermore, we should take into account net mineralization of soil organic N under different field conditions, which arises from the effects of temperature and moisture supply on the N-cycle processes (Mulvaney et al., 2001). Thus, in organic farming, it is difficult to determine the availability of this N to the crop during its cycle, and plant growth could be reduced if appropriate monitoring techniques are not applied (del Amor, 2006). To overcome these problems while maintaining high yields, farmers could use synthetic fertilizers to avoid lost revenue.
Conventional farming, especially in intensive horticultural crop production, is the main source of nitrate contamination to aquifers and rivers. Transition to a more sustainable production systems is therefore required especially in the nitrate-vulnerable zones, and the extra income gained through selling surplus (premium prices for certified organic) could be an attractive incentive to reduce contamination. However, managing organic farming could be more difficult than conventional farming, and systems to control and monitor N inputs or N leaching for each farm are costly and often not possible. Therefore, we evaluated a technique to determine the use of chemical fertilizers in horticultural crops with two goals: (i) to identify the use of chemical fertilizers and therefore the increased potential contamination via N leaching and (ii) to develop a tool to detect fraud in organic farming production and thus to help genuine organic farmers, consumers, and certification policies.
The hypothesis of use of N-isotopic composition is as follows: The N in synthetic N fertilizers is derived from atmospheric N2 by high-temperature catalytic reaction with hydrogen to form ammonia, which is subsequently oxidized to nitrate. This process results in little change in the original atmospheric N isotope ratio. Therefore, ammonia and nitrate derived from such a fertilizer source has a 15N value close to 0
, the value of atmospheric N2 (Shearer et al., 1974; Vitoria et al., 2004). By contrast, nitrate derived from sewage sources can have values greater than +20, signifying a 15N enrichment of 2% over the original 15N/14N ratio in starting N2. This is significantly different from the fertilizer N isotopic value (Jeffrey et al., 2002).
We tested this hypothesis about the use of isotopic discrimination in crops to detect addition of chemical N-fertilizers in sweet pepper plant with three common manures, with or without the addition of chemical fertilizers. If a significant reduction in 15N/14N in any plant organ could be observed, then this reduction would provide us with a tool to identify nonorganic procedures and to reduce contamination through the use of certified organic production practices.
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Materials and Methods
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Plant Material and Growth Conditions
Sweet pepper plants, cv. Cierva, were transplanted from a commercial nursery on 18 Jan. 2006. The greenhouse was divided in three independent zones corresponding with the three manure treatments. Manure was applied at preplant. During the crop cycle, half of the plants in each manure zone received only water, and the other plants received chemical fertilizers as commonly used in conventional cultivation. Thus, six treatments were studied corresponding to three manure types and two drip-irrigation regimens (only water or addition of chemical fertilizers) per each type of manure.
Manure (8 kg m–2) was applied for each treatment. The manures had the following composition in dry weight (%): Sheep manure: organic matter (OM), 73.8; total N (TN), 2.01; C/N, 21.13; P2O5, 1.04; K2O, 3.78; CaO, 5.86; MgO, 1.52. Horse manure: OM, 70.3; TN, 1.77; C/N, 23.04; P2O5, 1.5; K2O, 3.08; CaO, 7.18; MgO, 0.87. Hen manure: OM, 57.4; TN, 2.41; C/N, 13.82; P2O5, 3.24; K2O, 3.57; CaO, 16.54; MgO, 1.52. Chemical fertilizers were applied with the following composition in the nutrient solution in mmol L–1: 4.2 NO3–; 1.7 H2PO4–; 3.4 SO42–; 3.8 Ca2+; 1.2 K+; 2.0 Mg2+. All treatments received the same amount of irrigation (water or fertigation). Each treatment had four rows with 78 plants each. The rows at both ends were not controlled and were considered as border plants; thus, each treatment had four blocks with 16 plants per block in the central two rows. All sampled plants or fruits were surrounded by plants of the same treatment.
Plant Biomass and Gas Exchange Parameters
At 106 days after transplanting (DAT), plants were harvested for plant biomass, CO2 assimilation, and isotopic discrimination. Plants were harvested and separated into young and old leaves, stems, roots (only the upper part due to the difficulty in extracting all the organs), and mature fruit. Plants organs were dried for at least 72 h at 65°C.
Net CO2 assimilation and leaf transpiration were determined with a LI-6400 (Li-COR Biosciences, Lincoln, NE). Light intensity (photosynthetically active radiation) was fixed at saturated light conditions (1500 µmol m2 s–1 photon flux density) and 370 mg L–1 CO2 inside the leaf chamber. Four measurements were made per treatment on the youngest and fully expanded leaves.
Isotopic Determination
From each sample, 5 to 8 mg of powdered plant material was packed in tin capsules and analyzed by isotope ratio mass spectrometry for the 15N/14N (Continuous Flow Isotope Ratio Mass Spectrometer-CF-IRMS, Thermo-Quest Delta plus). Abundance of 15N is expressed by the ratio 15N/14N. Natural 15N abundance in the atmosphere, the largest N reserve in the world, is at the level of 0.0036765 and remains constant around the world (Ehleringer and Cerling, 2002). Natural 15N abundance in samples is normally related to 15N abundance in the atmosphere by the following expression (Robinson 2001):
Several physical, chemical, and biological processes and reactions present different affinities for 15N or 14N isotopes. Due to this discrimination, products resulting from these reactions are usually enriched or impoverished in 15N in comparison with the original. For batch calibration in the isotope ratio analysis, plant materials, previously calibrated against standard material of known isotope composition, were used as working standards.
Statistical Analyses
Analysis of variance was performed on main effects (manure type, fertilization post-transplant, and organ) with Statgraphics Plus 5.1. When the interaction between organs and fertilization was found (P 
0.05), mean values for each organ were separated by LSD at P 0.05.
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Results and Discussion
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Sheep manure significantly increased (P 0.05) shoot biomass compared with hen or horse manure (Table 1
). No significant effect was found due to the irrigation treatments (only water or with chemical fertilizers amendment). At later stages of crop production (164 DAT), only water application reduced growth in all manures (data not shown). Net CO2 assimilation rate and transpiration were not affected by the addition of chemical fertilizers. However, transpiration was higher with sheep manure rather than hen or horse manure.
Organic carbon additions from farm manures affect soil physicochemical and biophysical properties; therefore, manures can increase soil porosity and plant-available water capacity, which results in better soil structural conditions (Bot and Benites, 2005). Therefore, these soil conditions and characteristics provided by manures may affect the leaf transpiration. Sheep manure had higher OM than horse or hen manure and could improve soil water capacity better than hen or horse manures. Additionally, plants with sheep manure had higher fresh weight; thus, not only OM but also textural and microbiological proprieties of this manure could be implicated in this response.
This result indicates that proper manure management in sweet pepper is more important than chemical fertilization. This result agrees with other findings where animal manure is a valuable resource in crop nutrition, but its efficient management is a greater challenge than when using mineral fertilizers (Thomsen, 2005; del Amor, 2006).
Taking into account the main effects (manure, fertilization, and plant organ) on the isotopic composition of sweet pepper (Fig. 1
), our results show that there is no significant effect (P 0.05) of the type of manure; however, an important reduction in 
15/14N2vsN2atm was observed related to the fertilization type (water or with chemical fertilizer amendment). Thus, when organic manure was irrigated with chemical fertilizers, 15/14N2vsN2atm was significantly reduced from 11.16 to 8.72 (21.8%) with respect to the addition of water alone. When all plant organs were analyzed, roots and fruits of sweet pepper plants showed in general a lower 15/14N2vsN2atm than young or old leaves or stems. Choi et al. (2003) proposed the possibility of using the 15N of an organic product as a tool for verifying the organic or inorganic nature of N fertilizers used in organic farming. For this to happen, two requirements need to be satisfied: (i) The 15N of organic and inorganic fertilizers should be largely different, and (ii) plant 15N should reflect the 15N of received fertilizers. Evans (2001) asserted that whole-plant and leaf N isotope composition is determined by the isotope ratio of the external N source and by physiological mechanisms within the plant. Whole-plant isotope composition can reflect that of the N source when plant demand exceeds N supply. Recent studies have reported that N-isotope studies can provide unique information on features such as nutrient sources, nutrient cycling, mixing regime, water column stability, and sediment provenance (Naseeb et al., 2006).
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Fig. 1. Isotopic composition according to (A) different organic manures, (B) irrigation (water or with additional chemical fertilizers), and (C) different plant organs of sweet pepper plants. Vertical bars indicate LSD values (P 0.05) for treatment comparisons.
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Any biological processes implicated in the synthesis and transformation of organic compounds could show a different affinity for 15N and for 14N. Thus, the 15/14N of the resulting compounds could be altered (Evans 2001). Organic fertilizers of animal origin show the highest 15/14N, followed by fertilizers of plant origin. This is similar to the 15/14N of nonfertilized soil. This could be due to the fact that most of the N present in nonfertilized soils comes from the decomposition of vegetable residues. On the other hand, the 15/14N of synthetic fertilizers is similar to atmospheric 15/14N. Our results clearly show this fact and that the addition of synthetic fertilizers to any of the studied manures significantly reduced 15/14N2vsN2atm. No significant differences were found between organic manures; this fact proves the wide range of this technique in organic systems where different types of animal manures are applied.
The ANOVA considering the fertilization type effect on 15/14N2vsN2atm in each plant organ (Table 2
) showed the highest significance values for old leaves (p = 0.0001) and fruits (p = 0.0005) rather than young leaves (p = 0.003) or stem (p = 0.004), and it was not significant for roots. This is especially relevant for monitoring organic crops in the field and for organic certification of fruits.
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Table 2. Isotopic composition of different plant organs for plants irrigated with water or with chemical fertilizers management.
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Conclusions
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Agencies for organic farming certification require techniques to verify the organic nature of the N fertilizers applied to crops. Results show that significant differences have been found between organic and not fully organic practices. Thus, this study demonstrates that with N-isotopic techniques it is possible to discriminate the use of chemical fertilizers in the organic production of sweet peppers with respect to strictly nonorganic crops. Further studies including the effects of different soils characteristics, climate, and biotic or abiotic stress could be useful in determining the proper interval of 15N/14N ratio to exclude nonorganic fertilization practices.
The transition to organic farming could significantly reduce nitrate contamination in this crop. Additionally, it has been found that regardless of the organic manure used, no additional fertilization (synthetic or organic) is required before 106 DAT because plant fresh weight was not reduced. This conclusion is important for conventional farmers who apply manure pre-planting for biofumigation with solarization because the use of methyl bromide (a gas that has been used as a structural fumigant to control a wide variety of pests in the soil) is banned. Thus, the requirement for biofumigation in conventional farming makes unnecessary the use of synthetic fertilizers during a significant period of the crop, avoiding nitrate contamination.
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ACKNOWLEDGMENTS
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The analyses of d15/14N were performed by "Servicio Apoyo Investigación" of Universidad Pública de Navarra. This research was funded by Fundación Seneca de la Región de Murcia under the project 554/PI/04. We thank D.J. Walker for English revision of the manuscript and G. Ortuño (IMIDA) and A.J. García (CDTA-El Mirador) for valuable assistance with crop management and measurements.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.
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