Cadmium (Cd) distribution and soil-plant relationship in cacao farms in Costa Rica

The current cadmium (Cd) regulations in chocolate threaten the cacao supply chain in several Latin American countries. The factors contributing to Cd accumulation in cacao beans have been poorly studied in Central America. The objective of this research was to identify the location of Cd hotspots as well as soil properties and management practices influencing the Cd concentration in cacao beans. A survey was carried out and soil, leaf, and beans were sampled from 150 farms in the three principal cacao-producing regions in Costa Rica. Total soil Cd concentration ranged from <0.1 to 1.05 (average 0.22 mg kg−1) which is typical of uncontaminated soils. Bean Cd concentration ranged from 0.12 to 3.23 (average 0.56 mg kg−1) and 22% of the samples exceeded the selected threshold of 0.80 mg kg−1, located mostly in the Huetar Caribe and Huetar Norte regions. Variability in bean Cd concentration was better explained by total soil Cd and soil organic carbon (SOC) (R2 = 0.62, p < 0.05). In addition, bean Cd concentration was affected by leaf nutrient content and management practices. Leaf Zn and P were positively correlated with bean Cd while K and Mn were negatively correlated (p < 0.05). Farm altitude and orchard age were also negatively correlated with bean Cd. Overall, this study shows that bean Cd contamination does not reach the extent observed in other Latin American countries such as Ecuador, Colombia, or Honduras. Nevertheless, research is needed in hotspot areas to assess the feasibility of potential mitigation strategies, particularly the use of mineral or organic soil amendments, which may allow better for planning in existing plantations or the expansion into new cacao-growing areas in the country.


Introduction
Cacao (Theobroma cacao) is the key ingredient for chocolate manufacturing and various other widely consumed products.Consumption of dark chocolate can have multiple health benefits, but the presence of high cadmium (Cd) concentrations can lead to detrimental health effects (Smolders & Mertens, 2013;Vanderschueren et al., 2021).Due to the risk of Cd exposure in the European population, stringent regulations regarding the maximum Cd concentration in chocolate are now in place.The regulation focuses on 1 3 Vol:. ( 1234567890) the Cd content in the final product and not the raw cacao beans (European Food Safety Authority, 2012).
High Cd concentrations in cacao beans have been predominantly reported in cacao originating from Latin American countries (Bertoldi et al., 2016;Vanderschueren et al., 2019;Vanderschueren et al., 2021).However, the perception that the majority of Latin American cacao-producing areas have high Cd content is inaccurate and threatens the sustainability of the emerging cacao sectors in producing countries, such as Costa Rica.
Countries in Latin America, such as Ecuador (Argüello et al., 2019;Barraza et al., 2017;Chavez et al., 2015), Colombia (Bravo et al., 2021), Perú (Arévalo-Gardini et al., 2017;Thomas et al., 2023;Zug et al., 2019), and others (Gramlich et al., 2018), have already conducted soil/plant surveys to identify high soil Cd areas and the corresponding soil or agronomic factors aggravating the issue.The presence of Cd hotspots suggests that pollution is localized and highly variable within locations; thus, generalizations at a wider scale should be avoided (Vanderschueren et al., 2021).
Nevertheless, the belief persists that cacao produced in Latin America contains Cd levels that will not meet EU regulations, particularly in the absence of available information.Costa Rica is a marginal cacao producer worldwide, with an approximate area of 3200 hectares (MAG, 2018).In 2018, Costa Rica produced 550 Mg of dry cacao beans, ranking 17 th and 5 th among cacao producers in Latin and Central America, respectively (FAO, 2019).Approximately 79% of the cacao production in this country is managed by small-scale (<2 ha) low-income farmers (SEPSA, 2017).
In Costa Rica, soils with Cd concentration below 3 mg kg −1 are classified as "non polluted" according to national regulation (Ministerio de Salud, 2014).However, no supporting information, such as the country's average soil Cd concentration, has been published to justify the selection of this threshold.Furcal-Beriguete and Torres-Morales (2020) conducted a small survey (n=40) to determine the Cd concentrations in soils collected from cacao farms in Huetar Norte region.They reported soil Cd concentrations lower than the limit of detection (LoD) of the analytical procedure (LoD = 1 mg kg −1 ).It is worth mentioning that this LoD is much higher than total soil Cd reported elsewhere (Vanderschueren et al., 2021), which could potentially lead to erroneous conclusions.In contrast, samples collected in the Brunca region showed soil Cd concentrations of up to 4 mg kg −1 .These data highlight the variability of Cd concentrations within the country, emphasizing the need for more detailed information.
Furthermore, it is essential to determine the factors that influence bean Cd accumulation, such as soil properties, management practices, or climatic conditions.Several studies have demonstrated that Cd availability in soils is influenced by total soil Cd concentrations, soil pH, and soil organic carbon (SOC) (Smolders & Mertens, 2013).These factors can vary depending on soil and environmental conditions, consequently affecting bean Cd concentrations.A recent meta-analysis showed that pH is a crucial soil characteristic affecting bean Cd levels in different countries (Vanderschueren et al., 2021).
In contrast, in some surveys, when a proxy for available soil Cd is measured, soil pH is not the only factor influencing bean Cd levels.A recent review also concluded that total soil Cd, pH, and leaf Cd concentrations were the best predictors for bean Cd concentrations.The authors also reported that SOC and pH had relatively smaller effects on bean Cd (Wade et al., 2022).
Within this context, the objectives of this study were to (i) identify the geospatial distribution of high Cd concentrations in cacao beans in the three main producing regions, and (ii) determine the soil and agronomic factors affecting Cd uptake in cacao beans.This information will help in locating the contaminated areas, providing support for mitigation strategies, and exploring the potential for expansion of the cacao-producing regions in Costa Rica while minimizing the risk of elevated Cd concentrations in cacao beans.

Study area and sample collection
Costa Rica, situated in Central America, is a tropical country with approximately 3200 hectares of cacao spread across nearly 1400 farms.Cacao is mostly cultivated in the Huetar Norte, Huetar Caribe, and Brunca regions, with cacao farms scattered throughout 18 cantons.The average annual temperature in Vol.: (0123456789) these zones varies from 16 to 32 °C and the average precipitation ranges between 1800 and 4000 mm (Table 1, IMN, 2016).Cacao farms in Costa Rica are located over a range of altitudes from 4 to 1000 m.
In this survey, paired soil and plant samples were collected from 150 cacao farms, representing 10% of the total (Fig. 1).The farms were distributed across the 17 cantons to ensure representativeness.The main criteria for selecting farms included a minimum area (> 0.5 ha), availability of ripe cacao pods, and accessibility for sampling.The coordinates of each farm were recorded using a GPS (Garmin Montana 680).
One composite soil sample was collected per farm.Composite soil samples resulted from mixing 8 to 10 subsamples collected from the 0-10 cm deep soil layer around the base of cacao trees (approximately 1m from the trunk, a maximum of 5 trees).Soil subsamples were homogenized in a plastic bucket.Approximately 500 g of homogenized soil was placed in a labelled plastic bag for transportation to the laboratory.
For each of the trees mentioned above, a total of 20 leaves in developmental stage I2 (Greathouse, 1971) were collected.Additionally, between three to five ripe and healthy cacao pods were harvested from the same trees from which soil samples were taken.During the sampling process, farmers responded to a questionnaire regarding basic agronomic practices, such as fertilization, orchard age, and the type of management (organic or conventional).

Sample preparation and analyses
Soil samples were air dried for 72 h.The air-dried soils were ground and sieved through a stainlesssteel mesh (<2mm).Leaf samples were washed with deionized water to remove any adhered particles.Washed leaf samples were then oven-dried at 65 °C for 48 h.The dried leaf samples were crushed to a fine powder using a ceramic mortar and pestle.Cacao pods were opened and the beans were extracted.Mucilage tissue was not removed from the beans.Cacao beans were then oven-dried at 65 °C for 72 h.The completely dry beans were crushed using a ceramic mortar and a pestle before further analyses.
Soil pH was measured in a 1:2.5 soil to deionized water ratio.Soil organic matter (SOM) was determined using the Walkley and Black method (1938) through wet combustion with potassium dichromate.Organic carbon (OC) was estimated by Van Bemmelen factor dividing the % SOM by 1.724 (Heaton et al., 2016).Exchangeable aluminum (Al-Exc.) was determined in a 1:10 soil to solution (1M KCl) extract ratio.The soil suspension was shaken for 10 min.Afterwards, the suspension was centrifuged at 3000 rpm for 15 min.Supernatants were collected, and Al-Exc was determined by titration with 10 mL NaOH solution.
Soil subsamples (500 mg) were digested at 200 °C in a microwave digestion system (CEM MARS 6, North Carolina, USA) using an acid mixture: 2 mL HNO 3 69%, 1.25 mL HCl 37%, 0.25 mL HF 40%, 1.25 mL H 3 BO 3 5%, and 0.25 mL HClO 4 70% for 45 min and two consecutive cycles.For leaf and beans, 500 mg of the sample and an acid mixture consisting of: 2.5 mL of 69% HNO 3 and 2.5 mL of 37% HCl were used in the microwave at 200 °C for 45 min in two cycles.The elemental concentrations (including Cd) were determined by an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES 5800 Agilent, USA).The ICP-OES was calibrated using five levels of cadmium standard concentrations.
The Method Detection Limit (MDL) was 0.10 mg kg −1 for soils and 0.20 mg kg −1 for leaves and beans.

Statistical analyses
All continuous variables (except pH) were log-transformed to meet statistical assumptions.Stepwise regression analyses (backwards) were performed to identify significant variables affecting bean Cd concentration.The variable selection was based on the maximization of adjusted R 2 and minimization of the Bayesian Information Criteria (BIC).Multiple regression analysis using the Least Square Model (LSM) was conducted with the selected variables.Statistical significance was set at P ≤ 0.05.The Shapiro-Wilk test was employed on the regression residuals to assess the assumption of normality.The Variation Inflation Factor (VIF) was used to test the hypothesis of no multicollinearity, and variables with VIF values higher than permissible limits were excluded.The VIF values were calculated using the formula VIF = 1/(1 − R 2 ) (Mason & Perreault, 1991;O'brien, 2007).All data analyses were carried out using the JMP Pro Version 16.1 software (SAS Institute INC, 2021).

Soil properties
Descriptive statistics for soil parameters (including total soil Cd) are displayed in Table 2. Total soil Cd ranged from < 0.1 to 1.05 mg kg −1 (average 0.22 mg kg −1 ) whereas soil pH ranged from 4.50 to 7.49 and SOC from 0.56 to 13.11 g 100 g −1 .Seventy-five percent of the samples had an acidity less than 0.95 cmol(+)/L and only 5% of soil samples surpassed a pH of 6.51, primarily located in the Huetar Norte and Caribe regions.Furthermore, only 18 samples presented SOC values higher than 6 g 100 g −1 , with 61% of these samples found in the Huetar Norte region and the remainder in the Brunca region.
Total soil Cd concentration was highly correlated with bean Cd (Table 3), whereas other variables such as acidity and pH showed weak correlations with either bean or soil Cd concentration.
The relationship between soil properties, soil, and bean Cd has been reported in several local studies (Adams et al., 2004;Chavez et al., 2015;Gramlich et al., 2018) and in a recent metanalysis (Vanderschueren et al., 2021).As expected, soil Cd is a major factor affecting bean or leaf Cd accumulation.Additionally, Argüello et al. (2019) reported that bean or leaf Cd displays a negative correlation with pH.The increase in negative charges in the solid soil phase in high-pH soils promotes Cd sorption and reduces its availability for plants (Zug et al., 2019).However, soil is a complex matrix, and other factors such as cation exchange capacity, Fe/Mn oxides and hydroxides, acidity saturation, and clay content could also play a role in the sorption/desorption of soil Cd and its subsequent plant uptake (Ur Rahman et al., 2021).

Cacao leaf and bean elemental concentration
Leaf Cd concentration ranged from <0.10 to 8.3 mg kg −1 , with geometric and arithmetic means of 0.52 and 0.98 mg Cd kg −1 , respectively (Table 4).On average, leaf Cd was a factor of 1.6 times higher than bean Cd.Zinc and Mn-leaf concentrations ranged from 27 to 266 and 46 to 1447 mg kg −1 , respectively.
Geometric and arithmetic mean bean Cd concentrations were 0.41 and 0.56 mg Cd kg −1 , respectively, with 75% of the samples showing concentrations of less than 0.69 mg kg −1 (Table 2).The Zn and Mn-bean concentrations ranged from 34 to 243 and 2 to 156 mg kg −1 , respectively.Except P, the concentration of all the other elements analyzed (including Cd) was higher in leaves than the beans.This trend was also reported in Gramlich et al. (2018).Average Zn and Mn concentrations in leaves are considered optimal and slightly above optimal, respectively, from a nutritional standpoint (Fageria et al., 2002).
The correlation analysis between bean Cd and leaf elements showed a strong and positive correlation between bean and leaf Cd, as well as a moderate and positive correlation with P (p < 0.01), whereas other variables showed weaker correlations.Relationships solely between leaf element concentrations were moderate (r < 0.48).The strongest correlation was found between P and Cd (Table 5).
Similar to this study, Gramlich et al. (2018) found weak correlations (r < 0.20) between elements in leaves and they also reported a positive correlation between Zn and Cd leaf concentration.Argüello et al. (2019) also observed a strong relationship between bean Cd and soil properties; therefore, soil variables were included for modeling bean Cd concentration.

Geospatial distribution of soil and bean Cd
The geospatial distribution of cadmium is depicted in Fig. 2. In 33 samples (22% of the total), bean Cd concentration surpassed the selected threshold of 0.80 mg kg −1 .These samples were distributed as follows in the three studied areas: Huetar Norte (30%) > Huetar Caribe (20%) > and Brunca (17%).
Elevated bean Cd could be attributed to factors such as soil formation, artisanal mining, or the cumulative impact of high fertilization rates, all of which may contribute to increased Cd input into soils.P fertilization has been identified as one of the exogenous causes of Cd contamination in agricultural soils in other countries (Kabatia-Pendias, 2010;Jiao et al., 2012).However, high rates of P fertilizers are not commonly applied in cacao systems, rendering the effect of fertilizer application negligible.In contrast, it is more likely that the weathering of Cd-enriched soil minerals is the primary contributor to excessive bean Cd levels (Vanderschueren et al., 2021).Soil Cd concentration displayed the highest variability in the Brunca region (Coefficient of Variation [CV] = 77%), followed by Huetar Norte (CV = 68%) and Caribe (CV = 60%) regions; similar results were reported by Bravo et al. (2021) in Colombia.Bean Cd concentration was highly variable, particularly in the Brunca and Caribe regions (CV >100%), with lower variability in Huetar Norte (CV = 68%).High bean Cd variability was also reported by Argüello et al. (2019) in Ecuador, at both the farm and regional scales.These findings underscore the potential of some areas to combine cacao bean with high and low Cd concentrations to meet trade requirements.
Other studies have reported that the cacao tree is capable of taking up and accumulating greater Cd concentrations compared to other plants, i.e., the translocation factor (ratio between soil and bean Cd) is greater than 1.This capacity of the cacao tree can be enhanced by an increased soil-available Cd and the recycling of Cd through litter decomposition (Gramlich et al., 2018;Kwawukume et al., 2014).The authors have suggested that the cacao tree should be considered a moderate Cd accumulator (Vanderschueren et al., 2021).This suggestion aligns with the data found in our study, where the transfer factor ranged from 0.58 to 11 (average 2.79), similar to the ranges of 0.13-12.5 reported in Ecuador by Argüello et al. (2019), 0.50-16.3calculated in Honduras by Gramlich et al. (2018), and 0.26-7.84 reported in Ecuador by Barraza et al. (2017).

Relationship between soil and plant factors affecting bean Cd
A stepwise (backward) regression analysis was performed to determine the soil and plant factors affecting Cd accumulation in cacao beans.A total of 4 soil properties, 9 leaf and bean variables, and 3 agronomic management practices were included.The regression models were grouped according to (i) soil Cd and soil properties, (ii) leaf variables, (iii) soil properties + leaf variables, and (iv) soil properties + agronomic practices (Table 6).
The variability of bean Cd concentration could be elucidated by soil properties (Table 6), which is consistent with results from other studies (Argüello et al., 2019;Chavez et al., 2015;Gramlich et al., 2018).Total soil Cd alone explained ~58%, whereas the addition of SOC enhanced the regression model by 3% (Eq.1).In contrast, pH and acidity had no statistical effect; therefore, total soil Cd and SOC were the most significant variables and together explaining up to 62% of the bean Cd variability.
Along with total soil Cd, SOC is one of the soil properties affecting available soil Cd (Argüello et al., 2019;Smolders & Mertens, 2013).Model 1 calculated that doubling SOC decreased Cd concentration (1) log 10 (Bean Cd) = 0.36 + 0.82 × log 10 Soil Cd T − 0.25 × log 10 (%SOC) in cacao beans by a factor of 1.18 which is lower than the reduction computed by Argüello et al. (2019).A higher percentage of SOC and organic acids promotes the formation of chemical bonds between Cd and the carboxylic and phenolic groups, thus increasing the immobilization of the metal in soils (Bravo et al., 2014;Khan et al., 2018).
In model 2, Zn and P leaf concentrations (Table 6) showed a positive effect on bean Cd explaining up to 9.13 and 6.39%, respectively.The positive effect between Zn and Cd found in this study coincides with the results reported by Arévalo-Gardini et al. (2017) in cacao and by Tkalec et al. (2014) in tobacco, that could be explained by the shared uptake pathway for these elements in plants (Tang et al., 2014).Although some authors have reported antagonistic effects between Zn and Cd (Gramlich et al., 2018;Kabatia-Pendias, 2010), this effect is likely to be concentration dependent.
In contrast, K and Mn showed a negative effect on bean Cd.The K effect on Cd is not totally understood.The addition of K in sunflower plants reduced Cd uptake without affecting plant biomass (Samet et al., 2017).In rice, Liu et al. (2013) also determined that shoots and roots of K-deficient seedlings had higher Cd concentrations compared to plants with optimal K concentration, which could explain the negative effect found in model 2 (Table 6).
It has been shown that storage of Cd in root vacuoles is one of the main mechanisms to overcome the effects of Cd toxicity and possible translocation to shoots, seeds, and other tissues as well as a development of normal cells (Kopittke et al., 2013;Martinoia et al., 2012;Vanderschueren et al., 2021).
In terms of higher adjusted R 2 (0.74), the best-fitting model was computed using total soil Cd and leaf-P.The VIF of the predictors in this model remained below the permissible values.Limited information exists regarding the influence of P concentration on Cd in cacao beans.This nutrient is involved in protein metabolism (Zhao et al., 2019) and recently, Blommaert et al. (2022) found that cacao nibs had 5-fold higher P/Cd ratio than other tissues and approximately 85% of the P content in cacao nibs was in the form of phytate.This finding may explain the positive relation between Cd-P found in model 3.
In model 4, total soil Cd explained most of the bean Cd variability whereas agronomic practices had little effect (<6%).Orchard age and altitude showed a negative correlation with bean Cd concentrations.Similar results were reported by Gramlich et al. (2018) where alluvial soils contained higher Cd levels than volcanic soils.
In this context, the weathering of parental material can increase Cd accumulation in topsoil.Wen et al. (2020) found that Cd concentrations in sedimentary rocks were higher (average 1.17 mg kg −1 ) than in igneous rocks (average 0.22 mg kg −1 ).Oliva et al. (2020) reported that altitude explained soil Cd and its relationship with Cd accumulation in cacao beans.Using machine learning, Li et al. (2021) found that soil Cd concentration decreases with increasing altitude, which could be attributed to soil-forming factors such as parent material, topography, and climate.Furthermore, these authors noted that as altitude increased, soil pH and SOC better explained the variability in soil Cd.These studies support the findings in model 4 and our abovementioned statement that, for the majority of high Cd areas, soil genesis is the key factor explaining Cd levels.
In our study, approximately 60% of the farmers did not apply any fertilizer and relied solely on the decomposition of litter and crop residues as a source of nutrients.The cycling of Cd in the decomposed biomass could increase soil Cd levels and, consequently, lead to translocation to the beans (Barraza et al., 2019).However, more research is needed to determine the potential input of Cd from litter and the nature of the soil contamination in cacao areas.

Conclusion
Elevated Cd concentration in cacao beans can threaten the sustainability of cocoa production in Latin American countries due to cadmium regulations enforced by different agencies.This has motivated research to study the extent of Cd-contaminated areas and understand the soil properties related to plant uptake.In this study, 22% of the beans from sampled farms exceeded a selected threshold of 0.80 mg Cd kg −1 , which may restrict their sale to regulated markets such as the European Union.These high concentrations can be related to soil factors such as total Cd and SOC.
Although the average soil and bean Cd concentration reported in this study are lower than those reported in other countries, conducting laboratory analysis remains crucial to avoid setting up new plantations in areas with high Cd concentrations.The statistical models presented in our study can be useful for avoiding Cd-contaminated lands as well as to propose mitigation strategies in current cacao farms.According to the soil modeling, increasing SOC could ameliorate the uptake of Cd.Furthermore, application of Mn is recommended.In our study, soil pH was not statistically related to bean Cd, even though other researchers suggest that increasing soil pH is the most suitable Cd management practice in cacao.These potential mitigation strategies should be studied in more comprehensive multi-year field trials.
and the Caribbean Cocoa 2030-2050" with grant ATN/RF-17235-RG.The opinions expressed in this publication are exclusively those of the authors and do not necessarily reflect the point of view of FONTAGRO, its Board of Directors, or the countries it represents.

Fig. 1
Fig. 1 Distribution of farms sampled in the three major cacao-producing regions in Costa Rica

Table 1
Temperature, rainfall, number, and area of cacao farms in the main producing regions of Costa Rica

Table 4
Descriptive statistics of selected elements in cacao leaves and beans n=101 and 138 for leaves and beans, respectivelyLeaf BeanCd mg kg −1 K % Zn mg kg −1 Mn mg kg −1 P % Cd mg kg −1 K % Zn mg kg −1 Mn mg kg −1 P %

Table 5
Correlation matrix of elements in leaves and bean Cd in cacao plants.All variables were log 10 transformed ** p< 0.01; * p< 0.05 Vol.: (0123456789)