RESEARCH ARTICLE

 

Evaluation of pesticide residues and heavy metals in common food tubers from Nigeria

 

 

Kingsley O. Omeje^I; Benjamin O. Ezema^{II, III, IV}; Sabinus O.O. Eze^I

^IDepartment of Biochemistry, University of Nigeria, Nsukka, Nigeria
^IIThe Biochemistry Unit, Department of Science Laboratory Technology, University of Nigeria, Nsukka, Nigeria
^IIIAston Institute of Materials Research, Aston University, Birmingham, UK
^IVEnergy and Bioproducts Research Institute, Aston University, Birmingham, UK

Correspondence

 

 

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ABSTRACT

Pesticide residues and heavy metal content of cassava, yam, cocoyam, potato, water yam and carrot were evaluated by gas chromatography-mass spectrometry and atomic absorption spectroscopy. The detected pesticide residues in the samples were 2,4-dichlorophenoxyacetic acid, glyphosate, hexachlorobenzene (HCB), dichlorobiphenyl, aldrin, endosulfan, profenofos, g-chlordane, carbofuran, biphenyl, heptachlor, lindane and t-Nonachlor. The concentration of HCB ranged between 0.0799 ± 0.06 mg/kg and 0.1596 ± 0.00 mg/kg, which was greater than the permitted maximum limit of 0.5 mg/kg established by the US Environmental Protection Agency. The concentration of aldrin and profenofos detected was lower than the predetermined maximum allowed limits. Endosulfan concentrations in cocoyam (0.2500 mg/kg) and potato (0.3265 mg/kg) were higher than the limits allowed by the Canadian Department of Industrial Research. The heavy metals detected in these samples include cobalt, nickel, lead, manganese, chromium, arsenic and mercury in at least one of the samples evaluated. There was not much difference between the concentration of cobalt in yam (0.036 mg/kg) and the maximum allowed concentration (0.043 mg/kg). Lead was detected in potatoes and carrots but was below detectable concentration in cassava, yam, cocoyam and water yam. Similarly, cocoyam was found to have a significant mercury content (0.658 mg/kg), but mercury content was below detectable concentrations in cassava, yam and water yam.
SIGNIFICANCE:
Heavy metal pollutants and pesticide residues can impair human health, and their presence in food can cause various illnesses and health issues. It is important to prevent exposure to these contaminants and ensure that food is safe by identifying and monitoring them. Farmers may provide consumers with more assurance that their products are safe by identifying and monitoring pesticide residues and heavy metal contamination in these food crops. Overall, it is crucial to find and monitor pesticide residues and heavy metal contamination in food to safeguard customer confidence, ensure legal compliance and preserve human health.

Keywords: tubers, pesticide residue analysis, heavy metal analysis, gas chromatography-mass spectrometry, atomic absorption spectroscopy, maximum permissible limit

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Introduction

Pesticides are chemicals that are used to control pests which are harmful to humans, plants and the environment.¹ In parallel, pesticide residues are described as substances that are found in foods for consumption by humans or other animals, and are chemical derivatives considered to be toxic to living organisms.² Similar to pesticide residues, the entrance of heavy metals into the food chain is their major route into the human system, which could cause autoimmune disorders and inhibit the functions of some biochemical processes.³ Heavy metal toxicity has been reported regularly in recent times, with some deaths attributed to it. Other effects of heavy metals on humans include cancers, high blood pressure and gene mutation.^2,4,5 Uncontrolled disposal of household and electronic waste, animal dung and abandoned metallic parts are some of the environmental sources of heavy metals.⁶ Environmental pollution is a serious problem in today's modern world, with pesticides and heavy metal pollution being the most prevalent due to their ability to contaminate air and water.⁷

Cassava, yam, cocoyam, potato, water yam and carrot are commonly cultivated in Nigeria due to their multiple usage and nutritional values. These tubers are tropical crops consumed by about 2 billion people and are the major sources of carbohydrates providing energy for the roughly 700 million residents of tropical and subtropical regions.⁸ The production of these products and their conversion into goods derived from food is expanding, and farmers profit significantly from their market.⁸ Their high post-harvest losses, due to contamination by external and internal hazardous substances (such as mycotoxins, heavy metals and insecticides), reduce economic value and income. These roots/tubers have a crude fat content on the fresh weight of 0.1-0.5% and 1-3% on the dry weight, of which 80% is starch. The carbohydrate content of cassava is larger than that of potatoes.⁸ Cassava is a potent source of energy despite being deficient in lipids, minerals and proteins.⁹ On the other hand, yam provides energy in the range of 80-120 kcal/100 g.¹⁰ Vitamin A is produced from beta-carotene, and it is present in adequate amounts in potatoes.¹¹ These tubers' essential amino acid composition is higher than that recommended by the Food and Agriculture Organization (FAO) for daily protein intake and greater than that of soybean protein.¹² These tubers may become polluted during cultivation and food processing and also contain certain endogenous antinutrients.

Currently, there are common applications of several agrochemicals in the cultivation of these crops to ward off pests. All the grown food crops are highly vulnerable to various insect attacks, especially on the farm or in the cultivation area, which has a detrimental impact on both the financial and dietary elements of product quality. Pesticides must be used to control pest infestations, which helps to improve the quality of crops and their production on farms.¹³ Consequently, these tubers may get contaminated, and the risks associated with consuming these roots can be divided into two groups: those related to potentially harmful substances present in the crop itself, and those related to processing and product development. However, there are some significant concerns regarding food safety and security. A lot of research still needs to be carried out concerning the level of heavy metal and pesticide pollution, especially that seen in Nigerian food crops like the tubers that are commonly consumed, such as cassava, yam and cocoyam^14,15

The amount of residues from pesticides and heavy metals in tuber-derived food products varies based on the location of the growing area and the system of farming. In Nigeria, pesticide residues detected in some tubers (cassava and yam) were HCB (0.0247), endosulfan (0.0340 and 0.090) and aldrin (0.0000 and 0.0937) mg/kg, respectively.¹⁵ The concentration of isopropylamine in yam was 0.2165 ± 0.00 mg/kg and in cassava was 0.1649 ± 0.00 mg/kg, while the concentration of t-Nonachlor in yam was 0.1093 ± 0.00 mg/kg and in cassava was 0.0006 ± 0.00 mg/kg, as reported by Omeje et al.¹⁵ Adeyeye and Osibanjo¹⁶ detected high concentrations of organochlorine residues in yam (aldrin = ~5.0 μg/kg; dieldrin = ~24.0 μg/kg and p,p'-DDE = ~13.0 μg/kg) and cassava (aldrin = ~6.0 μg/kg; dieldrin = ~31.0 μg/ kg and p,p-DDE = ~21.0 μg/kg) in their study. Heavy metals have been found in tubers like potatoes, yams and cassava in previous studies. The amount of cadmium (Cd) found in yam was reported to be 0.11 mg/kg, and lead (Pb) and nickel (Ni) were also detected.¹⁷ In addition, 0.21 mg/ kg of Pb, 42 mg/kg of copper (Cu), 24 mg/kg of zinc (Zn), 18 mg/kg of manganese (Mn) and 12 mg/kg of Ni were reported by Wilberforce and Nwabue¹⁸. Arsenic (As) concentration in cassava was detected to be 0.017 mg/kg.¹⁵ According to Onianwa et al.¹⁹, the range reported for Ni concentrations in tubers is 0.93-1.79 mg/kg. Akinyele and Shokunbi²⁰ detected Mn in yams (~4.42 mg/kg), and Orisakwe et al.²¹ reported Pb (~0.33 mg/kg), Cd (~0.10 mg/kg) and Ni (~0.30 mg/kg) in cassava.

To enrich the existing body of information, we assessed the presence and concentration of common food pesticide residues and heavy metal contaminants in essential and commonly consumed tuber crops cultivated in Nigeria using gas chromatography-mass spectrometry (GC-MS) and atomic absorption spectroscopy.

 

Materials and methods

Chemicals and materials

The chemicals and reagents used were of analytical quality and included chloroform, perchloric acid, sodium sulfate, concentrated sulfuric acid (Sigma-Aldrich), n-hexane (Loba Chemie, India) and concentrated nitric acid, anhydrous sodium sulfate, methanol (Sigma-Aldrich) and benzene. The pesticide standards (purity > 95%) were obtained from Restek (Sigma-Aldrich, USA). With concentrations ranging from 50 ng/mL to 200 ng/mL, stock standard solutions of 47 organochlorine pesticides (OCPs), organophosphorus pesticides (OPPs) and other pesticides were made in ethyl acetate and stored at 4 °C in a dark location until analysis. Pesticides are present in spiking solutions in amounts of 10-50 ng/L. The internal standard was aldrin solution (Sigma-Aldrich) in acetone at a concentration of 50 ng/L.

Samples

In April 2022, fresh tubers weighing 500 g each (cassava, yam, cocoyam, potato, water yam and carrot) were purchased from Nsukka open market situated in Enugu State (6°51'24" N and 7°23'45" E) in southeast Nigeria, and transported to the laboratory of the Department of Biochemistry, University of Nigeria, Nsukka. They were processed and stored at 4 °C for further analysis.

Pesticide residue analysis

The pesticide residues were determined with the help of a GC analysis and prepared following the AOAC method²², with minor modifications. Ten grams (10 g) of the homogenised sample was mixed with 60 g of anhydrous sodium sulfate in an agate mortar to absorb moisture. The homogenate was transferred into a 500 mL beaker, and the extraction was carried out with 300 mL of n-hexane for 24 h. The obtained crude extract was concentrated using a rotary vacuum evaporator at 40 °C to dryness. The sample residue (1 mL) was measured into 50 mL of chloroform transferred to a 100 mL volumetric flask and diluted. Most of the chloroform was evaporated at room temperature before adding 1 mL of the solvent mixture (20% benzene and 55% methanol). The mixture was sealed and heated at 40 °C using a water bath for 10 min. After heating, the organic sample was extracted with n-hexane and water in a proportion of 1:1. The mixture was shaken vigorously for 2 min, and n-hexane phase was transferred onto a small test tube for injection into a Buck 530 Gas Chromatograph (GC) equipped with an on-column, automatic injector, electron capture detector and an HP 88 capillary column (100 mm X 0.25 film thickness) (Agilent Technologies, Santa Clara, cA, USA), with injector and detector temperatures of 180 °C and 300 °C, respectively. Overall, the GC enabled the identification of pesticide residues, which were recorded in mg/kg, as the results emerged.

Heavy metal analysis

The heavy metal analysis (Co, Ni, Pb, Mn, Cr, As, Hg and Cd) was performed using a Varian aA240 Atomic Absorption Spectrophotometer (AAS; Varian Inc., Palo Alto, CA, USA) equipped with an acetylene air flame, adapting the protocol described by Quarcoo and Adotey²³, with slight modifications. The pyrolytic-coated graphite tubes of the AAS were equipped with platform instrument settings and furnace programs that helped to ascertain the peak signals. A known concentration of the sample (~2 g) was put into a digestion flask, along with 20 mL of acid mixture (which consisted of 650 mL concentrated HNO₃; 80 mL perchloric acid; 20 mL concentrated H₂SO₄), and subsequently heated until a clear digest was obtained. The digest was diluted with distilled water to the 100 mL mark. The acid level samples as they came along were monitored by a pH meter. The digestate was quantified, assayed for heavy metals using a Varian AA240 Spectrophotometer, and reported in mg/kg. The reference standards (Fluka Analytical, Sigma-Aldrich Chemie GmbH, Switzerland) for the detected element, blanks and their duplicates were digested using conditions consistent with those of the samples.

Statistical analysis

The emergent heavy metal and pesticide residue data were obtained from triplicate determinations of different samples from a given food crop batch. A one-way analysis of variance (ANOVA), using SPSS for Windows (version 16, SPSS Inc., Chicago, IL, USA), was used to establish differences in heavy metals/pesticide residues across the studied food crop samples. Data are expressed as mean ± standard error (SE). A simple f-test was used to compare the heavy metals/ pesticide residue concentration data and the established/referenced maximum permissible limits (MPLs). The probability level was set at p < 0.05 (95% confidence level).

 

Results

The concentrations of nickel (Ni), chromium (Cr), cobalt (Co), arsenic (As), manganese (Mn), cadmium (Cd), lead (Pb) and mercury (Hg) in the tubers are shown in Table 1.

All the heavy metals evaluated were present in the samples, except for Cd, which was below the detectable concentration in all the samples studied (Table 1). Among the samples, the maximum level of Ni (0.012 ± 0.00 mg/kg) was found in cassava. The concentration of Ni was 0.009 ± 0.00 mg/kg in cocoyam, 0.006 ± 0.00 in yam, 0.007 ± 0.00 in water yam, 0.001 ± 0.00 in carrot and 0.006 ± 0.00 in potato (Table 1).

Co, one of the common heavy metals in the environment, was among those evaluated. The maximum Co concentration of 0.036 ± 0.00 mg/kg was detected in yam. A Co concentration of 0.026 ± 0.00 mg/kg was detected in cocoyam, 0.016 ± 0.00 mg/kg in water yam, 0.011 ± 0.00 mg/kg in cassava, 0.010 ± 0.00 mg/kg in carrot and 0.002 ± 0.00 mg/ kg in potato. According to Leyssens et al.²⁴, Co is an essential constituent of nature, which is released during many anthropogenic activities and is a cofactor of vitamin B₁₂.

Cr was also detected in all the samples. Cr concentrations of 0.093 ± 0.00 mg/kg and 0.083 ± 0.00 mg/kg were found in cocoyam and cassava, respectively. Potato and water yam had Cr concentrations of 0.073 ± 0.00 mg/kg and 0.078 ± 0.00 mg/kg, respectively. The lowest level of Cr was detected in carrots (0.010 ± 0.00 mg/kg). Similarly, cobalt was detected in all the samples, with the highest concentration (0.036 ± 0.00 mg/kg) found in yam. Co was detected in the potato sample, although at the lowest concentration (0.00 ± 0.00 mg/kg) when compared to those of the other samples.

Similarly, As was found in the samples in various amounts. The amounts found in cassava, yam and cocoyam were 0.045 mg/kg, 0.010 mg/ kg and 0.049 mg/kg, respectively. Also, 0.056 ± 0.00 mg/kg, 0.037 ± 0.00 mg/kg and 0.019 ± 0.00 mg/kg As were detected in samples of potato, water yam and carrot, respectively. Potatoes had the highest concentration of As discovered, whereas yam had the lowest concentration (0.010-0.00 mg/kg) (Table 1). The presence of Mn, Cd and Pb was evaluated in the samples. High concentrations of Mn (0.838 ± 0.00 and 0.750 ± 0.00 mg/kg) were detected in water yam and potato. Cassava contained 0.138 ± 0.00 mg/kg of Mn, as shown in Table 1.

Lead was not detected in cassava, yam, cocoyam or water yam. However, higher concentrations of 0.032 mg/kg and 0.028 mg/kg were detected in cassava and carrots, respectively. The World Health Organization (WHO) limit for Mn is not yet established.¹⁵ Mn serves as a cofactor for some enzymes but could cause neurological disorders when above >5 mg/dm³.^5,25

The concentrations of Pb were 0.032 ± 0.00 and 0.028 ± 0.00 mg/kg in cassava and carrot, which were below FAO/WHO established MPLs (10 ± 0.00 mg/kg). The concentration of Pb in cassava, yam, cocoyam and water yam was below the detectable range, as shown in Table 1. The concentration of mercury (Hg) was lowest in potato (0.153 ± 0.00 mg/kg) and highest in cocoyam (0.658 ± 0.02 mg/kg), but below the detectable range in cassava, yam and water yam.

Metallic mercury exposure has been reported to cause lung damage.²⁶ The concentration of Hg in the cocoyam (0.658 ± 0.00 mg/kg) was greater than the 0.5 mg/kg FAO/WHO maximum acceptable limits.²⁷ However, potatoes showed a Hg concentration below the MPL.²⁷

Table 2 shows the concentrations of pesticide residues present in the tubers studied. 2,4-dichlorophenoxyacetic acid, dichlorobiphenyl, HCB, endosulfan, aldrin, profenofos, carbofuran, lindane, g-chlordane, dichlorvos (DDVP), heptachlor, glyphosate, t-Nonachlor and biphenyl were the different pesticide residues detected in the six tubers studied. Aldrin was observed in all the samples studied; the highest amounts were detected in potato (0.1161 ± 0.00 μg/m³) and cocoyam (0.0779 ± 0.000μg/m³), followed by carrot (0.0711 ± 0.00μg/m³), cassava (0.0617 ± 0.00 μg m³), water yam (0.0580 ± 0.00 μg/m³) and then yam (0.0004 ± 0.00 i g/m³). Similarly, DDVP was found in every sample studied, with the maximum concentration observed in cassava (0.5208 ± 0.00), potato (0.3635 ± 0.05 μg/m³), followed by carrot (0.3632 ± 0.06), yam (0.1334 ± 0.04) i g/m³, cocoyam (0.0683 ± 0.00 i g/m³) and then water yam (0.0562 ± 0.00μg/m³). Endosulfan (0.2500 ± 0.01 μg/m³), lindane (0.0914 ± 0.01 μg/m³), g-chlordane (0.0000 ± 0.120 μg/m³), biphenyl (0.9228 ± 0.00 i g/m³), 2,4-dichloro phenoxy acetic acid (0.1127 ± 0.00 μg/m³), HCB (0.1018 ± 0.00 μg/m³), profenofos (0.2138 ± 0.00 μg/m³), glyphosate (0.1876 ± 0.00 μg/m³) and t-Nonachlor (0.1084 ± 0.001 i g/m³) were detected only in cocoyam. Furthermore, cassava contained 0.0431 ± 0.00 i g/m³ 2,4-dichlorophenoxyacetic acid, 0.1596 ± 0.00 μg/m³ HCB, 0.1693 ± 0.00 μg/m³ p'p'-DDD, 0.1476 ± 0.00 μg/m³ profenofos and 0.0988 ± 0.00 μg/m³ glyphosate. Also, 0.1285 ± 0.00, 0.799 ± 0.00, 0.0732 ± 0.00, 0.0000 ± 0.00 and 0.1098 ± 0.00 ng/ m³ were the concentrations of 2,4-dichlorophenoxyacetic acid, HCB, p'p'-DDD, profenofos and glyphosate detected in yam, as shown in Table 2. For potato, 2,4-dichloro and p'p'-DDD were not detected, HCB (0.1415 ± 0.00 μg/m³), profenofos (0.3130 ± 0.00 μg/m³), glyphosate (0.3130 ± 0.00 μg/m³), endosulfan (0.3265 ± 0.08 μg/m³), biphenyl (0.7418 ± 0.00 μg/m³) and dichlorobiphenyl (0.1653 ± 0.02 μg/m³) were detected. Lindane, g-chlordane, t-Nonachlor, carbofuran and heptachlor were also not detected in the sample, as shown in Table 2. Subsequently, 2,4-dichlorophenoxyacetic acid, HCB, p' p'-DDD, profenofos and glyphosate were detected in water yam as 0.0527 ± 0.00, 0.0000 ± 0.00, 0.0002 ± 0.00, 0.0000 ± 0.00 and 0.0000 ± 0.00 μg/m³, respectively. Biphenyl and carbofuran concentrations were 0.2472 ± 0.00 and 0.1256 ± 0.05 i g/m³, respectively. Some pesticide residues found in potatoes include 2,4-dichloro (0.0957 ± 0.00 i g/m³), HCB (0.0000 ± 0.00 μg/m³), p'p'-DDD (0.1603 ± 0.00 μg/m³), profenofos (0.0011 ± 0.00 μg/ m³), glyphosate (0.1039 ± 0.00 μg/m³), lindane (0.0015 ± 0.00 μg/m³), biphenyl (1.1842 ± 0.00 μg/m³) and heptachlor (0.0867 ± 0.00 μg/m³). The chemical abstract service (CAS) numbers of some of the pesticide residues tested are listed in Supplementary table 1.

 

Discussion

Currently, due to the advances in crop production and cultivated food crops, there is an increase in the pollution of heavy metals and chemical residues. Thus, there is a need for continuous evaluation of their presence and concentration to aid in mitigating or preventing any public health issues that could occur as a result.

Several heavy metals, including nickel, lead, cobalt, arsenic, manganese, chromium, cadmium and mercury, were detected in the six samples (cassava, yam, cocoyam, potato, water yam and carrot) using atomic absorption spectroscopy and efficient techniques. The levels of Co in yam did not differ significantly from one another (0.036 ± 0.00 mg/ kg) and from the MPL (0.043 mg/kg) established by the US Food and Nutrition Board (2004).

Although Ni was present at varying concentrations in the samples, its concentration was below the MpLs (100 μg/L) as stipulated by the US Environmental Protection Agency and, thus, may not pose any serious health challenges to consumers, such as skin allergies and lung cancer, which are signs of Ni toxicity manifestation. A higher concentration of Ni residue (0.93-0.179 mg/kg) in tubers has been reported by Onianwa et al.¹⁹. Ni occurs naturally as part of different mineral complexes, with its deficiency in the human system causing retardation of intra-uterine development and reduced iron reabsorption.²⁰ The highest Co intake in humans occurs through diet. Co has been detected in okra, as reported by Orisakwe et al.²¹

One of the major sources of As in the environment is arsenic-rich fertilisers³⁶, which are released when applied. Previous researchers have shown high heavy metal accumulation in leafy vegetables.³¹ The MPL for Cd is 0.3 mg/kg as set by the FAO/WHO (2006). It has been detected in different food materials such as cassava, yam¹⁵ and cereals. Onianwa et al.¹⁹ reported Cd concentrations of 0.03-0.28 mg/kg in tubers. Also, Commission Regulation (EC) No 1881/2006⁴⁶ reported the presence of Cd in rice. Due to its slow excretory rate, high Cd levels threaten human health and could damage the kidneys and liver.⁴⁶

Pb is made available to the environment through lead-containing pipes, combustion of leaded gasoline and the use of lead-based paint.³⁰ Lead is known to affect the cardiovascular, nervous, skeletal, muscular and immune systems, and causes gastrointestinal symptoms and organ damage when ingested or from prolonged exposure.³⁰ The maximum exposure permissible limits of some pesticide residues established by international regulators are shown in Table 3.

The concentration of HCB was greater than the MPL (0.002 mg/m³) reported by OSHA, the US government's workplace safety and health authority. The concentrations of aldrin and profenofos were below the MPL, as reported by FAO/WHO. The pesticide residual amounts in the tests were lower than the permitted exposure level specified by international organisations such as WHO/FAO, the US Environmental Protection Agency and CDIR. However, care should be taken when consuming these food tubers, as prolonged consumption could lead to their bioaccumulation in the body of organisms.

A similar study by Lien et al.⁴⁷ reported high concentrations of organochlorine residues in yam. Some pesticide residues are highly persistent in the environment.⁴⁷ Oyinloye et al.⁴⁸ also detected different concentrations of aldrin, carbofuran, endosulfan and profenofos in T. occidentalis. There are many risks associated with pesticide residue exposure, including effects on human health; thus, concentrations of these residues in food samples should be monitored regularly.

Increased d-glutaric acid metabolism and allergic reactions/skin rashes have both been linked to aldrin and lindane.¹⁵ Aldrin, heptachlor, endosulfan and dieldrin were some of the pesticide residues that were detected by Njoku et al.⁴⁹ Jayaraj et al.⁵⁰ reported some adverse effects of aldrin, heptachlor and lindane, including neurotoxic effects. This could be why the Nigerian government prohibits their use.

 

Conclusions

Using AAS and GC-MS, we assessed the levels of heavy metals and pesticide residues in regularly grown tuber crops in Nigeria. In all the samples, 13 different pesticide residues were found. In every sample examined, the Cd concentration was below the threshold for detection.

Similarly, Pb and Hg were below the detectable concentration in cassava, yam and cocoyam. Other heavy metals present were lower than the MPLs established by standard organisations. Aldrin and dichlorvos (DDVP) were present in all the samples, with cassava having the highest concentration. Almost all the pesticide residues were detected in cocoyam, potato and carrot. Thus, there should be continuous monitoring of these staple foods to ensure their consumption does not predispose the consumer to heavy metal toxicities, as continuous consumption could potentially threaten people's health.

 

Competing interests

We have no competing interests to declare.

 

Authors' contributions

K.O.O.: Conceptualisation and resources, project administration, writing -original draft, supervision. B.O.E.: Formal analysis, investigation, writing - revision and editing, data curation. S.O.O.: Conceptualisation and resources, supervision, writing - revision and editing, validation. All the authors read and approved the final version of the manuscript.

 

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Correspondence:
Benjamin Ezema
Email: ezema.onyebuchi@unn.edu.ng

Received: 17 Apr. 2023
Revised: 28 Oct. 2023
Accepted: 06 Nov. 2023
Published: 27 Mar. 2024

 

 

Editor: Priscilla Baker
Funding: None

 

 

Supplementary Data

The supplementary data is available in pdf: [Supplementary data]