From production to consumption: tracing C, N, and S dynamics in Brazilian agroecosystems using stable isotopes

Da produção ao consumo: traçando a dinâmica de C, N e S nos agroecossistemas brasileiros com uso de isótopos estáveis

Abstract

Brazilian scientists have played a pioneering role in developing and applying stable isotope methodologies, in terms of natural abundance and enriched levels, to trace carbon and nutrient flows in terrestrial ecosystems, including natural and agroecosystems. Significant contributions have been made in the areas of biological N₂ fixation, carbon dynamics in soil, synthesis and evaluation of labeled fertilizers, and food science. These contributions have originated from several decentralized units of Embrapa, from research institutions, and from federal or state universities. In order to capitalize the existing Brazilian expertise, it is necessary to provide, at an institutional level, analytical facilities for stable isotope research, aiming to strengthen national capacity and to maintain the international competitiveness of the research.

Index terms:
biological N₂ fixation; fertilizer use efficiency; food science; labeled fertilizer; soil carbon; stable isotopes

Resumo

Pesquisadores brasileiros têm tido um papel pioneiro no desenvolvimento e na aplicação das metodologias de isótopos estáveis, em termos de abundância natural e de níveis enriquecidos, para traçar os fluxos de carbono e nutrientes em ecossistemas terrestres, que incluem os naturais e os agroecossistemas. Contribuições significativas têm sido feitas nas áreas de fixação biológica de N₂, de dinâmica de carbono nos solos, de síntese e avaliação de fertilizantes marcados, e da tecnologia dos alimentos. Essas contribuições têm se originado de diversas unidades decentralizadas da Embrapa, de institutos de pesquisa e de universidades federais ou estaduais. Para capitalizar a experiência brasileira existente, é necessária a provisão, nas instituições, de instalações analíticas para pesquisa com isótopos estáveis, a fim de fortalecer a capacidade nacional e manter a competitividade internacional das pesquisas.

Termos para indexação:
fixação biológica de N₂; eficiência de uso de fertilizantes; tecnologia de alimentos; fertilizante marcado; carbono do solo; isótopos estáveis

Introduction

The Brazilian climate is predominantly tropical and subtropical, in marked contrast to the temperate or colder regions of Europe and North America. Therefore, the soils within the country are, in general, more highly weathered and support a greater variety of both intensive and extensive agricultural enterprises. Despite this, Brazilian soils share a similar set of problems encountered in their northern hemisphere counterparts, namely loss of soil organic matter and declining fertility under arable cropping. Stable isotope tracers have played a crucial role in gaining insight into carbon, nitrogen, and sulfur cycling in agroecosystems, including the estimation of fertilizer use efficiency and the contribution of biological N₂ fixation to the N nutrition of legumes and C₄ grasses. In the globalization era of agricultural products of plant and animal origin, the public is demanding accountability and authenticity of the external description on food and beverage packaging. In this case, stable isotopes are being used to detect adulteration and to verify geographic origin and mode of production, such as organic vs. conventional systems.

The objective of this review was to highlight contributions that Brazilian science has made and continues to make to the global efforts to trace carbon and nutrient flows in terrestrial ecosystems; synthesize and evaluate the efficiency of labeled fertilizers; and authenticate foods and beverages. It is an overview of the significant accomplishments, seen through a small window or aperçu, from the perspective of an outsider, who, nevertheless, has been fortunate to be involved in Brazilian stable isotope research and review since 1979.

This review addresses key examples, mainly but not exclusively, from agroecosystems, but does not attempt to make an extensive coverage of all relevant Brazilian literature. Recognition has been given to published papers in which the research was conducted and analytical services were provided in the country, irrespectively of the national or international affiliations of the scientists involved. Although considerable expertise exists in Brazil, this review does not cover aquatic environments (except for one example of fish production regime), microbial and plant ecology, plant and animal physiology, and pure and applied sciences, such as entomology, zoology, hydrology, geochemistry, paleontology, and related disciplines.

Absolute (x) and relative (δ) values of isotopic abundance

The lighter elements of interest in biological sciences, that is, H, C, N, O, and S, have naturally occurring stable isotopes: two, in the case of H, C, and N; three, in that of O; or four, in that of S (Table 1). The absolute abundance of each isotope, expressed as mole fraction, is given in Table 1. These values are equivalent to the atom fraction of an element, x(ⁱE), which is defined as the amount of a specified atom (isotope) of an element divided by the total amount of atoms of the element within the mixture (Coplen, 2011). Therefore, for an element with two stable isotopes, such as N, the absolute value is expressed by the equation:

Table 1
Isotopic composition of the important elements in biological sciences. Adapted from Berglund & Wieser (2011).

• ⁽¹⁾Superscript is mass number. ⁽²⁾Best measurement from a single terrestrial source. ⁽³⁾Terrestrial materials.

If a sample is artificially enriched in an isotope, the excess over natural abundance is expressed as an excess atom fraction, x, which is the difference between the mole fraction of an isotope of an element in a substance and that of a reference (Coplen, 2011). For N, for instance, the isotopic enrichment is expressed by the equation:

in which the reference is air, which has a ¹⁵N mole fraction equal to 0.003 663.

The range of natural variations in isotopic abundances, expressed as mole fraction, in terrestrial materials is also given in Table 1. However, it is more common to express small variations in natural abundance as relative (δ) rather than absolute (x) values. The relative value is the isotope ratio (R) of a sample, relative to the isotope ratio of the international standard for the element, as in the equation: δ(ⁱE) = (R_sample/R_standard) - 1.

For N, R is the ratio of ¹⁵N/¹⁴N. The international standard is air, with an R value equal to 0.003 676 47 and, by definition, a δ¹⁵N or x^E(¹⁵N) value equal to zero (Chalk et al., 2015b). In order to avoid fractions, δ values have traditionally been expressed in per mil (‰), by multiplying the right hand side of the equation of the isotope ratio by 1,000. Values of δ¹⁵N can be negative or positive depending on whether the sample isotope ratio is, respectively, less than or greater than the isotope ratio of air.

A full description of the source, availability, and properties of primary and secondary international reference materials for isotope-ratio analysis is given in Brand et al. (2014).

Biological N₂ fixation (BNF)

Short-term measurements of the ability of legumes or actinorhizal plants to fix atmospheric N₂ can be performed by exposure of the whole plant or root system to an atmosphere containing enriched ¹⁵N₂ (De-Polli et al., 1977). Although the method is direct, it poses many practical difficulties in relation to the containment of the labeled gas and to the maintenance of plants in an enclosure, which is why it is seldom attempted.

Indirect methods, commonly used to estimate legume symbiotic dependence, such as the proportional contribution of biologically-fixed N₂ to the N nutrition of the legume, involve paired treatments of a legume and a non-legume reference plant. The latter is required to estimate the ratio of labeled to unlabeled N derived from the soil by the N₂-fixing plant.

The ¹⁵N enrichment (E) method involves the addition of a fertilizer or organic material artificially enriched in ¹⁵N to the soil in the paired plots. The proportional dependence (P_atm) of the legume on biologically-fixed N is estimated by isotope dilution, using the following equation: P_atm = 1 - (x^E(¹⁵N)_legume/x^E(¹⁵N)_{reference plant}).

The E methodology was reviewed by Chalk & Ladha (1999).

The ¹⁵N natural abundance (NA) method is based on the same principle, but isotopic discrimination, i.e., the B value, during N₂ fixation must be additionally measured, usually by growing the legume in an N-free medium. Symbiotic dependence is estimated according to the equation: P_atm = [(δ(¹⁵N)_{reference plant} - δ(¹⁵N)_legume)/δ(¹⁵N)_{reference plant} - B] .

The NA methodology was reviewed by Unkovich et al. (2008).

Endophytic BNF

Brazilian and visiting scientists working with Dr. Johanna Döbereiner, at Embrapa Agrobiologia, were the first to recognize the importance of diazotrophic bacteria in endophytic associations with tropical C₄ Gramineae. The first isotope dilution field measurement to quantify endophytic BNF was also carried out at Embrapa Agrobiologia with Paspalum notatum Flugge (Boddey et al., 1983). Since these pioneering studies, endophytic BNF has been quantified by both the E and NA methods in several C₄ forage grasses and in sugarcane (Saccharum officinarum L.) (Table 2). This research has unequivocally shown the importance of endophytic BNF to the N nutrition of C₄ plants. For example, in a four-year study, Urquiaga et al. (2012) found that P_atm values varied from 43 to 61% among nine varieties of sugarcane.

Table 2
Brazilian studies of Embrapa Agrobiologia, using ¹⁵N-based methodologies to quantify endophytic N₂ fixation in C₄ forages and in sugarcane (Saccharum officinarum).

• ⁽¹⁾Bahia grass, Paspalum notatum; Guinea grass, Panicum maximum; signal grass, Urochloa brizantha, U. decumbens, U. humidicola, U. ruziziensis, and U. arrecta; elephant grass, Pennisetum purpureum; and sugarcane, Saccharum officinarum. ⁽²⁾E, ¹⁵N enrichment; and NA, ¹⁵N natural abundance. ⁽³⁾P_atm, proportional dependence of the legume on biologically-fixed N.

Legume BNF

In Brazil, symbiotic dependence has been estimated for grain and forage legumes in agroecosystems, using the E and NA methods, and for native leguminous shrubs and trees at undisturbed sites in the Cerrado and Caatinga biomes, using the NA method. Some examples are given in Table 3.

Table 3
Brazilian field studies using 15N dilution to quantify symbiotic N2 fixation in legumes⁽¹⁾.

• ⁽¹⁾
  Common bean, Phaseolus vulgaris; soybean, Glycine max; and groundnut, Arachis hypogaea. ⁽²⁾Wheat, Triticum aestivum; Nn, non-nodulating; Weeds, Digitaria horizontalis, Sorghum arundinaceum, and Cenchrus echinatus; C₄ grasses, Urochloa brizantha, U. arrecta, and Panicum maximum; Weeds, Sorghum sp., Zea mays, Cynodon dactylon, Cyperus rotundus, and Sida glaziovii; and Nnlegume, the non-nodulating legumes Bauhinia cheilanta and Caesalpinia pyramidalis. ⁽³⁾E, ¹⁵N enrichment; and NA, ¹⁵N natural abundance. ⁽⁴⁾P_atm, proportional dependence of the legume on biologically-fixed N. ⁽⁵⁾B value was assumed to be zero (Ramos et al., 2001), -1.3‰ (Teixeira et al., 2006). ⁽⁶⁾Stem tissue, low range is dry season, and high range is wet season.

Hungria & Vargas (2000) suggested that high temperature, water stress, and soil acidity were the main abiotic constraints to BNF in tropical grain legumes in the country. However, this conclusion was based on an examination of the literature in which qualitative data, such as nodulation parameters or assays on acetylene reduction activity, were reported instead of quantitative data on legume symbiotic dependence. Isotope-based methodologies have played an important role in identifying both biotic and abiotic constraints to legume performance in Brazil. For example, the E methodology was used to identify the critical growth stages of common bean (Phaseolus vulgaris L.) during which water stress had a large negative impact on yield and BNF. Similarly, by reviewing the published studies in which the E methodology was used, Chalk et al. (2010) were able to identify temperature, water, salinity, sodicity, acidity, and mineral nutrition as major abiotic factors affecting legume BNF globally.

Moreover, biotic factors - such as the absence of indigenous rhizobia or arbuscular mycorrhizal fungi (Ibijbijen et al., 1996) - and legume genotype can also affect legume symbiotic dependence. A comprehensive review of this subject was published by Chalk et al. (2006).

BNF dynamics

Apart from the quantification of legume BNF, a suite of indirect ¹⁵N methodologies has been developed to estimate the transfer of BNF or legume N (LN) to companion non-legume species, as reviewed by Chalk et al. (2014b). Other ¹⁵N-based methods have been developed to quantify the transfer of LN or BNF to an array of crops in sequential rotations. A frequently used method involves the application of a ¹⁵N-labeled crop residue obtained from a particular source to unlabeled plots in a subsequent experiment, which might be separated in space and time from the original source. Primo et al. (2014), for example, measured cumulative recoveries of ¹⁵N-labeled Gliricidia sepium (Jacq.) Kunth biomass in a pot experiment, in three subsequent crops of corn (Zea mays L.), cotton (Gossypium hirsutum L.), or cowpea [Vigna unguiculata (L.) Walp.], obtaining values of 28, 35, and 41%, respectively. In an attempt to overcome the obvious limitations of such an approach, an in situ method based on ¹⁵N natural abundance was proposed by Perin et al. (2006), who estimated that corn following sunn hemp (Crotalaria juncea L.) green manure derived 15% of its grain N content from N₂ fixed by the preceding sunn hemp crop.

Organic carbon dynamics in soil

For carbon, the relative isotopic abundance is expressed by the isotope ratio equation, in which R is the ratio of ¹³C/¹²C. The international standard, in this case, is Vienna Pee Dee Belemnite (VPDB), which has a δ value of -0.030 031.

It is well known that ¹³C isotopic fractionation occurs during photosynthesis, so that plant organic C is naturally depleted in ¹³C, when compared with atmospheric CO₂ (δ¹³CO₂ = -8‰). Different CO₂ assimilation pathways between C₃ (the Calvin cycle), C₄ (the Hatch-Slack pathway), and C_CAM (Crassulacean acid metabolism) plants result in markedly different bulk δ¹³C signatures of photosynthetic plants, mainly due to diffusion and enzymatic effects. The ranges in the δ¹³C values for C₃, C₄, and C_CAM plants are -22 to -30‰, -9 to -13‰, and -10 to -20‰, respectively.

In undisturbed natural ecosystems, the δ¹³C signature of surface soil organic matter is similar to that of the native vegetation from which it was derived. Many of the soils used in agriculture were originally under C₃ forest and, therefore, had very negative δ¹³C values. With the introduction of sugarcane and tropical forage grasses, such as C₄ species (Table 2), the above-ground C input into the ecosystem had a much higher δ¹³C signature, and, over time, the δ¹³C value of the soil organic matter became less negative (Vitorello et al., 1989; Moraes et al., 1996; Pinheiro et al., 2010) (Table 4).

Table 4
δ¹³C signatures of Brazilian soils under forest and after a number of years of C₄ pasture or sugarcane (Saccharum officinarum) established on cleared forest.

• ⁽¹⁾Equation of proportional N recovery per plant.

An innovative methodology to estimate the proportions of soil C from the original and introduced vegetation is based on the shift in the δ¹³C signature of soil C and was developed by Cerri et al. (1985) at Centro de Energia Nuclear na Agricultura (Cena), the center for nuclear energy in agriculture of Universidade de São Paulo (USP). The proportion of organic C in a soil layer derived from the introduced C₄ vegetation (P_C4soil) was expressed by the following equation: P_C4soil = δ(¹³C)_C4soil - δ(¹³C)_C3soil)/(δ(¹³C)_C4plant - δ(¹³C)_C3soil)], in which δ(¹³C)_C4soil is the δ¹³C value of a soil layer under C₄ vegetation; δ(¹³C)_C3soil is the δ¹³C value of a soil layer under the original C₃ vegetation; and δ(¹³C)_C4plant is the δ¹³C value of the C₄ vegetation. P_C4soil increased with increasing years under C₄ pasture or sugarcane (Table 4), and Moraes et al. (1996) reported that the original C₃ signal in the 0-10-cm soil layer had disappeared after 81 years under C₄ pasture.

The concentrations of soil organic C and δ¹³C values are not constant with soil depth (Sisti et al., 2004). Organic C concentrations generally decrease with soil depth. However, in C₃ forest, soil δ¹³C values increased with depth, although, in soil under C₄ pasture or sugarcane, these values also decreased with depth due to the decreasing effect of the C₄ pasture with depth (Vitorello et al., 1989; Moraes et al., 1996; Pinheiro et al., 2010).

Management factors can affect the dynamics of C when the system is undergoing interchange between C₃- and C₄-dominant vegetation. For instance, the tillage system (zero vs. conventional) under different long-term crop rotations was shown to affect the decay of the original C₃-derived soil organic C to a depth of 100 cm (Sisti et al., 2004). Balieiro et al. (2008) showed that the stocks of soil C and δ¹³C signatures to 10-cm depth were affected by the reintroduction of two C₃-forest species, Eucalyptus grandis W.Hill and Pseudosamanea guachapele Harms, the latter being a N₂-fixing tree. The site had formerly supported C₄Panicum maximum Jacq. for ten years. The rate of replacement of soil C by new forest C after five years was much faster under a mixed plantation of trees rather than under pure plantations of either species (Balieiro et al., 2008).

There is an increasing interest worldwide in the use of soil additives, such as biochars, due to their potential to sequester soil C. In a 30-day laboratory incubation study with three biochars derived from C₃ oilseed residues added to a sandy soil containing C_{4 carbon}, Rittle et al. (2015) found that respired CO₂ came principally from the biochars and that these decelerated the mineralization of the indigenous C (negative priming effect). Therefore, there may be both direct and indirect effects of biochar addition on C sequestration.

Synthesis and evaluation of labeled fertilizers

Scientists at Cena of USP have been involved for many years in the synthesis of labeled fertilizers using ion exchange chromatography (Bendassolli et al., 1997). They have also developed new methods for the isotope-ratio analysis of labeled plants and soils (Carneiro et al., 2008). Some examples of labeled fertilizers synthesized at Cena are given in Table 5. The isotopic enrichment of the product is largely determined either by the isotopic enrichment of the source materials or by the degree of isotopic fractionation during ion exchange chromatography.

Table 5
Synthesis of labeled fertilizers at Centro de Energia Nuclear na Agricultura of Universidade de São Paulo, as well as fertilizer use efficiency.

• ⁽¹⁾Rice, Oryza sativa; sunn hemp, Crotalaria juncea; sugarcane, Saccharum officinarum; and coffee, Coffea arabica. ⁽²⁾Recovery in plant tops, in which data in parentheses represent growth periods in days.

By using isotopically-labeled fertilizers, it is possible to determine the recovery in the plant-soil system. The proportion of ¹⁵N-enriched fertilizer recovered by a plant, for example, can be calculated according to the equation: proportional N recovery_plant = (mass N_plant× x^E(¹⁵N)_plant/mass N_fertilizer× x^E(¹⁵N)_fertilizer). in which x^E(¹⁵N)_plant/x^E(¹⁵N)_fertilizer is the fraction of plant N derived from the fertilizer. The same procedure is used to determine the recovery of fertilizer in the soil, and fertilizer loss from the plant-soil system can be calculated by mass balance.

Many studies have been undertaken with different crops and strategies of N fertilizer application, in order to estimate N fertilizer efficiency. Recovery by annual crops seldom exceeds 60%, and the cumulative residual value of the N fertilizer in two subsequent crops is usually <10%. Some examples of recoveries of ¹⁵N- and ³⁴S-labeled fertilizers in plant tops are given in Table 5.

In addition to studies with labeled synthetic (industrially manufactured) N fertilizers, such as urea and ammonium-based forms, efficiency studies have also been performed with labeled organic sources, including composts (Chalk et al., 2013) and animal excreta (Primo et al., 2014), or with labeled slow- or controlled-release products (Chalk et al., 2015a). It is also possible to trace the dynamics of N from source (fertilizer) to product (food) in agroecosystems using ¹⁵N natural abundance (Chalk et al., 2014a).

Food and beverage authentication

In the globalization era of agricultural products of plant and animal origin, the public is demanding authenticity and accountability in food and beverage labelling. Stable isotopes are playing an expanding role in detecting adulteration and in verifying geographic origin and production regime. Some examples of research carried out in Brazil are summarized in Table 6, of which several will be outlined in some detail.

Table 6
The use of stable isotope δ values to authenticate Brazilian foods and beverages.

• ⁽¹⁾The geographic origin of the seized samples of the illicit drug marijuana has also been determined by stable isotope (¹³C, ¹⁵N) analysis (Shibuya et al., 2006). ⁽²⁾Cachara, Pseudoplatystoma fasciatum.

Adulteration

Marked differences are observed in the δ¹³C signatures between sugars derived from C₃ and C₄ plants. This makes it possible to detect the presence of C₄ sugar or C₄ ethanol in a product ostensibly derived from a C₃ source. Therefore, it allows identifying, for instance, the sources of ethanol in Brazilian brandy (Pissinatto et al., 1999) or sparkling wine (Martinelli et al., 2003), or whether C₄ sugar has been added to freshly-squeezed orange juice (Pupin et al., 1998) or to honey (Padovan et al., 2003).

Honey is produced from the nectar collected by bees from flowering plants, mainly from C₃ plants, and, to a lesser extent, from C₄ and C_CAM plants. The δ¹³C signature of the protein extract of honey will reflect the dominant source from which the honey was derived, e.g. -22 to -33‰ for C₃ plants, -10 to -20 for C₄ plants, and -11 to -13.5 for C_CAM plants (Padovan et al., 2003). Adulteration of pure honey with cheap C₄ sugars, such as cane or corn syrup, has become an international problem. It can be detected by δ¹³C analysis of the bulk honey and of the protein extracted from the honey, although the latter is not affected by the adulteration. A difference of 1 δ indicates 7% of C₄ sugar added, which is the limit tolerated worldwide (Padovan et al., 2003).

Geographic origin

The most useful indicators of the geographic origin or provenance of terrestrial plants are the δ¹⁸O or δ²H values of stem water. At a given geographic location, the relationship between δ¹⁸O and δ²H of meteoric water, i.e., water from precipitation, is linear, referred to as the meteoric water line. δ¹⁸O is defined by the isotope ratio equation, in which R is the ratio of ¹⁸O/¹⁶O. The primary international standard, in this case, is Vienna Standard Mean Ocean Water (VSMOW), which, by definition, has δ¹⁸O and δ²H values of zero (Brand et al., 2014).

The isotopic composition of meteoric water is affected by physical phenomena such as condensation and evaporation, with temperature being the main variable that affects isotopic fractionation. The δ¹⁸O and δ²H composition of meteoric water follows a predictable geographic pattern that is related to latitude, altitude, amount of precipitation, and distance from the ocean. The annual mean δ¹⁸O in meteoric water ranges from +2 to -2‰, in equatorial regions, to as low as -22‰ in the north-polar region. It should be noted that there is no isotopic fractionation during water uptake by terrestrial plants and, therefore, stem water has the same signature as the water source, regarding access to deep vs. shallow water.

The three main wine producing regions in the state of Rio Grande do Sul could be differentiated on the basis of δ¹⁸O signatures of water in the wines (Table 7) (Dutra et al., 2011), but not in the wine ethanol. These authors speculated that the variation in δ¹⁸O among regions may be due to differences in local meteorological conditions, such as rainfall and temperature, but that altitude and distance to the ocean may also play a role. A more intensive data analysis of site-specific and regional climate and geography is necessary to separate these variables.

Table 7
δ¹⁸O in water and δ¹³C in ethanol in wines from three regions of the state of Rio Grande do Sul, Brazil (Dutra et al., 2011)⁽¹⁾.

• ⁽¹⁾
  Means followed by equal letters do not differ by Tukey’s test, at 5% probability.

Geographic origin may also be differentiated by differences in δ¹³C signatures. For example, a survey of δ¹³C signatures of Big Mac hamburger patties from outlets in Brazil and Great Britain showed extreme median values of -11.1 and -25.4‰, respectively, indicating the predominant C₄ (tropical grasses) or C₃ (temperate species) diet of the beef cattle (Martinelli et al., 2011).

Both δ¹³C and δ¹⁵N signatures have also been successfully used to differentiate the regional production of the illegal drug marijuana (Cannabis sativa L.) in Brazil (Shibuya et al., 2006). From the known producing areas of the states of Pernambuco and Bahia (dry region) and of Mato Grosso do Sul and Pará (wet region), mean δ¹⁵N values of the samples seized from the dry region varied from +1 to +2‰, whereas those from the wet region were higher, varying from +5 to +6.8‰. The mean δ¹³C values for the dry region were of -26 to -26.5‰, differing from those of the wet region, which varied from -29.2 to -30.2‰. This information provides the basis for the allocation of police resources to intercept shipments from the source to the largest population of consumers in the city of São Paulo, in the state of São Paulo.

Production regime

The δ¹³C signature of the feces or body tissues of domestic animals is a robust indicator of diet composition. For instance, in another study, the ¹³C natural abundance of cattle manure was an effective predictor of the proportion of legume in a diet consisting of legume (Desmodium ovalifolium Wall.) + grass [Urochloa dictyoneura (Fig. & de Not.) Veldkamp (Syn. Brachiaria dictyoneura )] forage (Macedo et al., 2010). Colleta et al. (2012) were able to separate barn vs. free-range chicken according to their diet, in which the latter had more C₄ in their diet and higher δ¹⁵N values, which the authors attributed to the ingestion of animal protein.

The presence of poultry offal meal, i.e., a product derived from the entrails and internal organs of poultry, in broiler diets is a potential threat to the large Brazilian export market in chicken meat due to concerns regarding contamination with Salmonella or Escherichia coli . Both δ¹³C and δ¹⁵N have shown promise under controlled experimental conditions as a diagnostic tool to identify the presence of offal in broiler diets (Oliveira et al., 2010), but, by themselves, cannot be used as a conclusive proof. Despite this, δ¹³C values showed clear differences between a species of farmed and wild freshwater fish (Cachara) from the Alta Floresta region in the Amazon (Sant'Ana et al., 2010). This was observed both in the wet and dry seasons, with mean values varying from -23.6 to -24‰ for farmed fish and from -29.1 to -30‰ for wild fish.

Furthermore, δ¹³C composition is also a good index of the ingredients used in the brewing process of Brazilian beers (Mardegan et al., 2013). Large breweries use a relatively high proportion of C₄ corn resulting in a mean δ¹³C value of -20‰, whereas small artisanal (boutique) breweries use a higher proportion of C₃ cereals, resulting in a mean δ¹³C value of -25‰. These differences are driven by the need of small brewers to market a product distinctive in quality from mass-produced beer, and also by the need to use fewer ingredients of lower cost, more rapid fermentation, and higher alcohol content, which corn provides.

The use of stable isotopes to differentiate organic vs. conventional plant and animal products was reviewed by Inácio & Chalk (2015) and Inácio et al. (2015a), respectively. In a study of conventionally and organically produced lettuce (Lactuca sativa L.) in the state of Rio de Janeiro, Inácio et al. (2015b) concluded that the δ¹⁵N signatures of lettuce were an indicator of the principal N inputs (BNF, organic or manufactured fertilizer) instead of a definitive indicator of the production regime.

Brazilian institutions involved in stable isotope applications in agroecosystems

The precise measurement of isotopic abundance of the lighter elements requires an isotope-ratio mass spectrometer (IRMS). Computer-controlled, automated sample processing and analysis is a feature of modern instruments, which are equipped with multiple inlet and collector systems, allowing for the simultaneous measurement of several ions differing in mass-to-charge ratio. IRMS is a mature but somewhat expensive technology, requiring a relatively large initial capital investment and on-going technical support and maintenance costs.

Several Brazilian research institutions have been involved in stable isotope research for many years, and their increasing number testifies to the importance of this technology. The known Brazilian institutions involved in stable isotope research are identified in Table 8.

Table 8
Brazilian institutions involved in stable isotope research in terrestrial and agroecosystems⁽¹⁾.

• ⁽¹⁾
  While endeavoring to be comprehensive, some institutions may have been inadvertently overlooked. ⁽²⁾IRMS, isotope-ratio mass spectrometer.

Several educational institutions, including Cena of USP, or institutions affiliated with Universities, such as, Embrapa Agrobiologia and Universidade Federal Rural do Rio de Janeiro, have strong post-graduate research training and teaching programs in stable isotope applications. Therefore, Brazilian scientists are well equipped to participate not only in national programs but also in international ones involving stable isotope applications in soil, plant, food, and animal sciences. Among the opportunities for Brazilian scientists, are possible contributions to the research programs of the Division of Nuclear Techniques in Food and Agriculture of the Joint Food and Agriculture Organization of the United Nations/International Atomic Energy Agency (FAO/IAEA) and to the technical cooperation programs of IAEA, either as project participants or as institutional employees. The programs in food and agriculture cover the areas of plant breeding and genetics, soil and water management and crop nutrition, insect pest control, animal production and health, and food and environmental protection (Iaea, 2015). Participation in such programs raises the international profile of the important work being undertaken in Brazil.

Conclusions

Brazilian scientists have made significant original contributions, through stable isotope tracing, for better understanding of carbon and nutrient cycling in agroecosystems, as well as in food and beverage authentication. There is an increasing awareness of the importance of stable isotope research in many branches of science, for which IRMS is an essential piece of equipment that is best provided at institutional level. The less desirable and often impractical alternative is to rely on expensive fee-for-service laboratories or to collaborate with a sister institution that has IRMS facilities. In this sense, one of the main impediments to a more innovative research in nutrient cycling in natural and agroecosystems in Brazil is the obstacle created by the current paucity of IRMS facilities in the national institutions.

New or emerging technologies in the agricultural sector will require the application of stable isotope techniques to follow the dynamics of C, N, and S in the soil-plant-animal-atmosphere continuum. For example, for the introduction of new legume species or cultivars, as well as of new crop rotations, including legumes or new inoculants, it will be necessary to assess the contribution of BNF to N balance in agroecosystems. Similarly, the efficacy of new slow- or controlled-release fertilizers or chemical inhibitors will need to be evaluated using stable isotope techniques. The effect of innovative agronomic practices, such as the use of biochar, will require the strategic application of stable isotopes to verify the sequestration of C, N, and S. The alternative use of radioactive tracers (e.g. ¹⁴C and ³⁵S) is generally precluded due to restrictions imposed by environmental and safety issues, whereas the half-life of ¹³N of 9.97 min is too short to be of practical value. Stable isotopes have a role in characterizing negative interactions between agriculture and the environment, such as methane emissions from ruminants and rice paddies, and also fertilizer-induced emissions of nitrous oxide. Scientists in Brazil are in a unique position to assess innovative technologies to enhance resource-use efficiency across a wide range of soils and climatic conditions, extending from the humid tropics to temperate regions.

Acknowledgements

To Embrapa colleagues, for constructive comments on the manuscript; to Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj), for financial support (grant No. 102.515/2009, 101.466/2014); and to University of Melbourne, for assistance with travel expenses.

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Publication Dates

• Publication in this collection
  Sept 2016

History

• Received
  24 July 2015
• Accepted
  29 Feb 2016