Location

This study was conducted in the department of Magdalena, Colombia, at the geographic coordinates 10°46'00" N and I 74°8'00" W. The natural vegetat10n is classified as tropical 3001 dry forest, and the climate is warm and dry, with a mean annual temperature of 27°C and relative humidity of 82% (^{Piraneque, Aguirre, & Reis, 2018}). Plant material and soil samples were collected in 20 georeferenced agricultural plots distributed in the Sierra Nevada of Santa Marta (zone 1) and the Banana Zone (zone 2) (Figure 1).

Source: Authors

Figure 1 Locat10n of the agricultural plots and sampling zones where Cd content in the cocoa tissues and soils cultivated with cocoa was identified. 

Soils characteristics

Soils in zone 1 were classified as Entic Haplustolls originating from granodiorite, quartz diorite, and granite. These are deep, well-drained soils with loam to sandy-loam texture; they are moderately acidic to neutral (pH 6.3 to 7.7) with moderate to high natural fertility due to low organic matter content and cation exchange capacity. These soils contain kaolinite (50%), micas (5-30%), montmorillonite (30-50%) and, vermiculite (5-30%) in the clay fraction and quartz (515%), feldspar (5-15%), and interstratified trace minerals in the sand fraction (^{Aguirre, Piraneque, & Vasquez, 2015}; ^{IGAC, 2009}).

Soils in zone 2 were classified as Typic Ustorthens originating from gneiss and schists. These are relatively deep, well-drained soils with sandy texture; they are neutral to slightly acidic with moderate natural fertility due to medium organic matter content and high cation exchange capacity. These soils contain non crystalline material, kaolinite (40%), gypsite (30-50%) and, vermiculite (5-30%) in the clay fraction and quartz (22-26%), feldspar (36-38%), and hornblende (20-22%) in the sand fraction (^{Aguirre, Piraneque, & Vasquez, 2015}; ^{IGAC, 2009}).

Plant material, sampling and establishment

Different cocoa varieties are grown in small cultivated plots, but the product is recognized in the International market for its aroma and provides economic sustenance for indigenous people and farmers. The plots have the added value of proximity to the seaport, which is a determinant of cocoa export. Using a transect, 20 agricultural plots were selected with productive plants older than eight years from which cocoa leaves (healthy and functional from the central portion of the branches in the middle third of the crown) and fruits were collected. The plant material was transported to the Soil Laboratory of the University of Magdalena, were it was washed with distilled water and dried at room temperature; the beans were separated from the fruit or pod (shell), and the material was dried at 70°C for 48 hours, ground, packed and labeled.

Soil sampling and determinations

Soil samples (1 kg) were randomly collected for analysis from the 20 cocoa farms at two depths (0-10 and 10-20 cm) per plot. The samples were air-dried, then, were sieved through a 2-mm mesh, packed in plastic bags, and tightly sealed (Figure 2).

Source: Authors

Figure 2 Cocoa pods and soil samples were collected in cocoa-producing areas in northern Colombia to determine the heavy metal content.  

The tissue and soil samples were taken to the laboratory of the International Center for Tropical Agriculture (CIAT) in Palmira for analysis, where the respective determinations were made per methods described in Table 1.

Table 1 Laboratory methods used to determine the heavy metal content in cocoa tissue samples from cocoa-producing areas in northern Colombia. 

Source: Authors

Statistical analysis

The parameters obtained from the chemical determinations (plant tissue and soil) were subjected to analysis of variance and Tukey's post hoc test (p < 0.05). The results that differed were subjected to Pearson's correlation analysis between the heavy metal content in the plant tissue and soil properties. Statistical analyses were conducted using R software v. 3.02 (R Development Core Team 2015, available at www.r-project.org).

Heavy metals in plant tissues

The mean Cd, Ni, Pb, Cr, Ca, Mg and K content in the beans, shells and leaves of T. cacao grown in the study area are shown in Appendix 1. The beans from zone 1 had a mean Cd content of 0.5I mg kg^-1, whereas the beans from zone 2 had a mean of 0.66 mg kg^-1. These values exceeded the maximum permissible limit (0.5 mg kg^-1) reported by ^{García and Dorronsoro (2002)}. The beans from plot 3 of zone I had a Cd content of 0.96 mg kg^-1, and those from plots 17, 11 and 15 of zone 2 had Cd contents of 1.25 mg kg^-1, I.08 mg kg^-1 and 0.86 mg kg^-1, respectively. These values are considered unsafe for direct consumption. In contrast, plots 6 and 16 had the lowest Cd contents, with 0.21 mg kg^-1 and 0.15 mg kg^-1, respectively (Figure 3).

Source: Authors Legend:_Reference level from ^{Garcia and Dorronsoro (2002)}

Figure 3 Total Cd content in cocoa beans from cocoa-producing areas.  

The mean Cd content in the leaves did not differ significantly across the analyzed samples, with means of 0.43 mg kg^-1 in zone 1 and 0.41 mg kg^-1 in zone 2. The highest Cd content was reported in plot 6, at 0.81 mg kg^-1, and in plot 11, at 1.24 mg kg^-1.

The Cd content in the shells was heterogeneous, but without significant differences. The mean values were 0.50 mg kg^-1 in zone 1 and 0.40 mg kg^-1 in zone 2. These values were lower than those reported by ^{INIAP-PROMSA (2003)} in Ecuador, where a mean of I.08 mg kg^-1 Cd was recorded in the shells.

The values reported for the shells and leaves generally did not exceed the permissible limits: however, more Cd had accumulated in the beans, with 0.51 and 0.67 mg kg for zones 1 and 2, respectively, demonstrating Cd mobility in the plant. ^{Miranda et al. (2008)} state that the Cd present in leaf tissue is linked to specific physiological stages and is involved in diverse metabolic pathways.

Heavy metals in soils

Table 2 shows the soil analysis results, in which the pH ranged from 5.7 to 6.07, with no significant differences among sites or between zones. The Fe and Mn levels were optimal (10 to 50 mg kg^-1), and the Zn level was adequate in some sites but deficient in others. The Ca+² content ranged from intermediate (3.0 cmol+ kg^-1) to high (≥6.0 cmol+ kg^-1), the Mg content was intermediate (1.5 to 1,7 cmol+ kg^-1), and K was deficient (< 0.2 cmol+ kg^-1). For this reason, the Ca/K and (Ca+Mg)/K cation ratios were analyzed, which confirmed that K deficiency may be induced. The percentage of organic matter was low. Regarding the total metal concentrations in the soil, no evidence of Cd, Pb, Ni or Cr contamination was found. Concentrations were not reported from some sites because the values were lower than the method's limit of quantification (< LOQ).

Table 2 Results of the soil analysis by cocoa-producing zone in northern Colombia. 

Depth: 1 = 0-10 cm, 2 = 10-20 cm; Cd, Pb, Ni and Cr concentrations were determined by inductively coupled plasma mass spectrometry at the CIAT Laboratory

Source: Authors

The mean heavy metal content in the analyzed soils did not exceed the maximum concentrations established by ^{García and Dorronsoro (2002)}. In zone 1, the Cd concentration ranged from 0.19 mg kg^-1 at 10 cm deep to 0.02 mg kg^-1 at 20 cm deep. A similar pattern was observed in zone 2, consistent with the studies of ^{Mite, Carrillo, & Durango (2010)} and ^{Gramlich et al. (2018)}, who reported surface Cd accumulation. Heavy metal adsorption is strongly conditioned by pH (and thus its solubility) and the organic matter content, clay, oxides and hydroxides in the soil (^{Olivares-Rieumont et al., 2013}).

Bioavailability is associated with soil concentration and plant species. Pearson correlation analysis between Cd, Pb, Ni and Cr content in the soil with foliar variables (bean, leaf and shell content) indicated an association between the heavy metal concentrations in the soil and foliar contents (Table 3).

Table 3 Pearson correlation between the Cd, Pb, Ni and Cr content available in the soil and their relationship to other nutrients. 

Significant correlation (P < 0.05); s - soil, p - plant tissue. s - soil, f - aerial organs

Source: Authors

The soil Cd content correlated positively with the plant Cd content as well as with Ca concentrations in the soil. However, it was inversely proportional to the plant's Ca content, while the plant's Cd content was inversely proportional to the Ca and K uptake.

Association between variables

The results showed that the soil Ni concentration was affected by that of soil Cr and is inversely proportional to the foliar Ca content as well as the pH. Although Cr poses no toxicological risk, it was positively correlated with the Ni content in the soil and is affected by the pH. Ni and Cr in the soil were inversely correlated with the pH, indicating that this parameter directly affects their solubility.

Figure 4 shows the association between the studied variables regarding the heavy metal concentrations in the cocoa crop in the cocoa-producing areas of northern Colombia. Larger balls indicate greater association, blue represents positive associations, and red represents inverse relationships, which confirm the previous analysis.

Source: Authors

Figure 4 Associations between the variables measured in the soil and aerial organs of cocoa plants grown in northern Colombia. Blue circle = positive associations, and red circle = inverse relationships.  

Heavy metals in plant tissues

^{Chávez et al. (2015)} reported Cd values of 0.02 to 3 mg kg^-1 in cocoa beans in southern Ecuador. ^{Gramlich et al. (2018)} recorded Cd levels higher than 1.1 mg kg^-1 in beans from Honduras. ^{Huamaní-Yupanqui et al. (2012)} reported a mean Cd content of 1.55 mg kg-^I in cocoa beans from the province of Leoncio Prado in Peru, and ^{Martínez and Palacio (2010)} found Cd content ranging from 4 to 7 mg kg^-1 in San Vicente de Chucurí, Santander department, Colombia. These concentrations were higher than those found in the present study area.

Nevertheless, the results of the Cd analysis for the cocoa beans of some plots exceeded the maximum value cited by ^{García and Dorronsoro (2002)} and therefore, the value allowed by ^{Lanza, Churión, Liendo, & López (2016)} and the EU. This finding suggests the need to further investigate Cd's origin to build a distribution map and monitor concentrations in the beans by zone.

The lowest leaf Cd content was observed in plot 17, at 0.20 mg kg^-1, but in this plot, the Cd content of the beans was relatively high, indicating Cd mobility in the plant. This finding agrees with the report by ^{Caunii et al. (2015)}, who cautioned about Cd mobility in leaves and considered the maximum tolerable Cd content to be 0.5 mg kg^-1.

Finally, the values reported for the shells and leaves generally did not exceed the permissible limits: however, more Cd had accumulated in the beans, with 0.51 and 0.67 mg kg^-I for zones 1 and 2, respectively, demonstrating Cd mobility in the plant. ^{Miranda et al. (2008)} state that the Cd present in leaf tissue is linked to specific physiological stages and is involved in diverse metabolic pathways. Likewise, ^{Pernía, De Sousa, Reyes, & Castrillo (2008)} reported that Cd uptake by the roots reflects its concentration and availability in the soil.

Heavy metals in soils

Regarding the total metal concentrations in the soil, no evidence of Cd, Pb, Ni or Cr contamination was found. Concentrations were not reported from some sites because the values were lower than the method's limit of quantificat10n (< LOQ). Thus, the mean heavy metal content in the analyzed soils did not exceed the maximum concentrat10ns established by ^{García and Dorronsoro (2002)}. In zone I, the Cd concentrat10n ranged from 0.19 mg kg^-1 at 10 cm deep to 0.02 mg kg^-1 at 20 cm deep. A similar pattern was observed in zone 2, consistent with the studies of ^{Mite, Carrillo, & Durango (2010)} and ^{Gramlich et al. (2018)}, who reported surface Cd accumulation and that Heavy metal adsorption is strongly conditioned by pH (and thus its solubility) and the organic matter content, clay, oxides, and hydroxides in the soil (^{Olivares-Rieumont et al,, 2013}).

Although ^{Roberts (2014)} reported that applied phosphorus fertilizers (phosphate rock) are a source of Cd contamination in agricultural land and constitute more than 50% of the Cd that enters into the soil (this hypothesis is not tested here). However, given its extensive use, phosphate rock may be a possible contamination route for Cd in this area, further studies with more focus on fertilizers, are suggested in the north of Colombia.

The solubility and availability of metals in soils is affected by the interactions between pH, cation exchange capacity (CEC), precipitation, complexation and chelation. In this regard, ^{Silveira et al. (2003)} reported that when metals adsorb to soil particles, their concentration in the solution is reduced; thus, the metals cannot be quantified therefore, soils with high CECs have more exchange sites available in the colloidal fraction for adsorption and possible immobilization. Likewise, ^{Contreras, Herrera and Izquierdo (2005)} identified that soil Ca is found both as free Ca and as carbonate and calcium phosphate; therefore, Cd can be controlled by alkalization and precipitated.

Further research is required to assess the risk of Cd soil contamination, and regular monitoring of heavy metals should be carried out to evaluate the existence of any potential threat to food safety. For this, it is necessary to advance in new detection techniques such as the use of two-dimensional electrical resistivity tomography described by ^{Bravo and Benavides (2020)} or application of organic amendments like biochar for reducing the soil Cd movement and Cd plant uptake reported by ^{Cui et al. (2011)}, which promotes the soil quality, soil health, as well as food security.

Association between variables

Regarding the Ca and Cd association, ^{Cui et al (2011)} and ^{Solti et al. (2011)} mentioned that high Ca concentrations in soils can reduce Cd uptake because ionic competition occurs between these elements, thus reducing their uptake. Likewise, ^{Solti et al. (2011)} reported that Cd entering the roots directly competes with Ca, K, Fe and Cu because the same transporter proteins carry out Cd absorption. ^{Huamani-Yupanqui et al. (2012)} explained that for Cd to precipitate, it must have affinity for carbonates or phosphates, even if pH is slightly acidic.

The solubility and availability of metals in soils is affected by the interactions between pH, cation exchange capacity (CEC), precipitation, complexation and chelation. Soils with high CECs have more exchange sites available in the colloidal fraction for adsorption and possible immobilization. Likewise, as the soil Ca is found either in free form or as carbonate and calcium phosphate, therefore, Cd can be controlled by alkalization and precipitated (^{Olivares-Rieumont et al., 2013}).

Carbonate presence in the study soils was not analyzed, but the study area's geological characteristics and proximity to the sea indicate that carbonate may have been present; thus, insoluble Cu, Zn and Cd compounds can form and be adsorbed by carbonates, and Ca and Cd in the soil may remain adhered and undetectable this is an essential issue for future research. So, the fact that soil Ni and Cr correlated inversely with pH, is consistent with that reported by ^{Chavez et al. (2015)}, who reported that low redox potential increases heavy metal solubility, and with the Agency for Toxic Substances & Disease Registry (^{ATSRD, 2012}), which maintains that Ni is immobile in alkaline soils and precipitates in the form of carbonates and insoluble phosphates. This is important to select new areas for the cocoa crop.

The foliar calcium content was associated with most of the variables studied and was inversely proportional to Cd, Ni, Pb and Cr uptake. If Ca uptake is increased, cocoa metal uptake may decrease. Although the calcium concentration in the soil is in the intermediate range, increasing it would be beneficial without neglecting the cationic relationships. Further research should focus on the application of soluble Ca effect on cocoa Cd uptake growing in northern Colombia.