Introduction

Mercury (Hg) is a toxic trace metal absorbed from the surrounding aquatic environment through biological membranes, being incorporated into fish enzymes and proteins by binding to sulphydryl groups, which leads to its bioaccumulation in tissues and biomagnification through the aquatic food chain1,2. Top pelagic predators of the marine food wed, as tuna, are able to bioaccumulate substantial amounts of Hg, due to its high-performance metabolic rates, which contributes to the enhanced bioaccumulation of Hg3,4.

Bioaccumulation of Hg in fish depends on factors such as fish species, size, diet, position in the trophic chain, and habitat location5,6,7. Species with a relatively long lifespan as tuna, could also have high methylmercury (MeHg) content. According to Burger and Gochfeld (2004) and Médieu et al. (2022), approximately > 89% of the total mercury (THg) present in tuna muscle is in its methylated form. However, THg content in fish consist of a combination of several Hg chemical forms which are absorbed almost 100% in the digestive tract and distributed to various tissues and organs through the blood8,9,10. Therefore, consuming fish and seafood with high concentrations of Hg poses a considerable health risk specially to some vulnerable population groups, such as pregnant women, nursing women, developing children and large fish consumers11,12,13. In adults with a mean body weight of 70 kg, a daily MeHg intake of more than 0.3 mg can lead into chronic Hg poisoning, which represents Hg concentrations of 0.2 mg L−1 in blood and 60 mg kg−1 in hair14. At the cellular level, MeHg inhibit protein synthesis, disrupt microtubules, and can lead to the overproduction of free radicals because of the formation of reactive oxygen species in the kidney, liver, and brain14,15,16,17. Moreover, MeHg can increase intracellular Ca2+ affecting neurotransmitter functions15, and contributing to the signs and symptoms of motor dysfunction, autism spectrum disorders, and Alzheimer’s disease4. It has also been reported, that MeHg intoxication in growing fetus and children can cause loss of IQ, reduced placental and fetal growth, low birth weight and neurodevelopment delays18,19,20,21.

Various techniques have been used to quantify total mercury (THg) in tuna regardless of its chemical form in the sample22, including cold vapor atomic absorption spectroscopy3,6,23,24,25, inductively coupled plasma mass spectrometry7,26, inductively coupled plasma–atomic and optical emission spectroscopy27,28,29,30, cold vapor atomic fluorescence spectrometry30,31, and differential pulse anodic stripping voltammetry32,33.

Regarding fishing and fishery, these are considered the second most important activities34 in Ecuador, becoming one of the major producers and exporters of tuna in Latin America. In 2023, exports reached up to 221.924 tons, principally to the European Union, Latin America, and the United States35, complying with the regulations and standards of quality and health safety of national regulations36. According to the Ministry of Production, Foreign Trade, Investments and Fisheries of Ecuador, the main species of tuna found in Ecuador are yellowfin tuna (Thunnus albacares), bigeye tuna (Thunnus obesus), and skipjack tuna (Katsuwonus pelamis)36,37,38. Most of Ecuadorian canned tuna products can be chunks or flake form in water and different oils such as sunflower or olive39.

Even though, tuna is an important source of protein worldwide40, providing protective benefits to the cardiovascular system, due to its relatively low cholesterol and high omega-3 (n − 3) fatty acids content23,41, it is known to contain high levels of toxic trace metals, as mercury (Hg), cadmium (Cd), and lead (Pb)3,24,42, and other contaminants as polychlorinated biphenyls (PCBs)23. Anthropogenic discharges of these contaminants to the atmosphere, particularly those released from artisanal gold mining, have caused Hg levels to increase, as it is the single largest source of Hg emissions43. In Ecuador, artisanal and small-scale gold mining has been carried out in the country for more than a century, with 29.6 tons of Hg released into the environment44. Additionally, in canned tuna, other factors that influence metal levels are the pH of the canned product, the oxygen concentration in the headspace of the can, storage conditions and coating quality24, which could lead to the release of metal components in the soldered seams of cans45.

According to the European Union Commission Regulation, the maximum permissible limit for Hg in muscle meat of tuna is 1.0 mg kg−1 (fresh weight)46. A maximum acceptable concentration limit in tuna have also been established by the Food and Agricultural Organization (1.2 mg kg−1)47. Likewise, in Ecuador, the Ecuadorian Institute for Standardization has established a maximum acceptable concentration limit in canned tuna of 1.0 mg kg−148.

In Latin American countries, studies of Hg content in canned tuna are still scarce. Few studies carried out in Mexico, Brazil, and Colombia, have reported Hg levels in tuna canned in water and vegetable oil, with values between 0.005 and 1.4725 mg kg−110,24,49,50,51. Among these studies, different techniques have been used to assess Hg content, with results almost below the limits set by national and international regulations. However, most of the studies have not distinguished between different tuna species, origin of canned tuna, and type of muscle, being difficult to compare Hg content with the literature. Moreover, variations in fish size, sampling season, and age of tuna species may also affect Hg content10.

In Ecuador, few studies have examined Hg content in canned tuna25,33,39,52 through atomic absorption spectroscopy and differential pulse anodic stripping voltammetry53. Therefore, in the current study, THg was determined using cold vapor atomic fluorescence spectrometry in samples of canned tuna in water from the most popular brands sold in the Ecuadorian market. Canned tuna in oil was not considered for this study, as several authors have reported no differences between THg concentration in water or oil packaging23,39,41,54.

The results were compared to current national and international regulations and guidelines on permissible Hg limits in food matrixes, as the potential human health risks derived from consumption of canned tuna was assessed.

Materials and methods

Reagents and equipment

Chemicals used were of analytical grade. A 2% solution of tin (II) chloride dehydrate (Sigma Aldrich, Steinheim, Germany, Certified ACS, CAS# 10025-69-1, PubChem CID: 24479) was used as reducing agent, prepared in a 4% (v/v) hydrochloric acid (Fisher Chemical, Ottawa, Canada, ACS certificate, CAS# 7647-01-0, PubChem CID: 313), and a 2% (v/v) HCl solution was also used for the Mercur Plus equipment operation. Argon 99.999% (Linde, Quito, Ecuador, CAS# 7440-37-1, PubChem CID: 23968) was used as the carrier and purge gas. Sample acid digestion was performed using nitric acid (Fisher Chemical, Ottawa, Canada, ACS certificate, CAS# CAS 7697-37-2, PubChem CID: 944), hydrogen peroxide (Fisher Chemical, Ottawa, Canada, Certified ACS, CAS# 7722-84-1, PubChem CID: 784), and perchloric acid (BDH Chemicals).

All mercury standard (9.995 ± 0.056 µg mL−1) (Inorganic Ventures, Virginia, United States) solutions were prepared in high-quality reagent water (resistivity 18.2 MW cm at 25 °C) obtained from a Genie 5 Direct-Pure Water System (Rephile, Shanghai, China).

Certified reference material IAEA-436A (tuna fish flesh homogenate, International Atomic Energy Agency-Marine Environmental Studies Laboratory) was used as quality control.

Mercury measurements were carried out using a Mercur Plus cold vapor coupled to atomic fluorescence spectrometry analyzer (Analytik Jena, Germany). Samples were freeze-dried for 48 h at − 50 °C and 0.150 hPa (Labogene, Bjarkesvej 5, Denmark), and acid digestions were performed with a MARS 6 microwave (CEM Corporation).

Methods

Sampling

From March 2022 to March 2023, sixty cans of tuna were purchased from local supermarkets located in the north and south zones of the Metropolitan District of Quito, Ecuador. Three most representative and leading companies of tuna in Ecuador were selected for the study, with their traditional brands in market55. Brands were coded as A, B, and C according to the price at which they are sold in the market (A < B < C). Labels from brand C indicate that the packaged tuna is Thunnus albacares, brand B is the most consumed in the Ecuadorian market (the label does not indicate the species of packaged tuna), while in brand A only nutritional information is provided. For each brand, twenty cans of tuna packed in water (sample size: 80 g) from different batches were collected and transported to the laboratory, and then stored in a clean dry place until time for preparation.

Sample treatment

Cans were opened and water was drained. Each can tuna meat was homogenized using a blender with a stainless-steel blade. From each can, approximately 20 g was freeze-dried for 48 h and weighed to determine their water content, to report the results as wet weight (w.w.). Dried samples were grinded and placed in plastic bags until digestion procedure.

Acid digestion was performed according to Yánez-Jácome et al.56, weighting 0.1 g of dried sample in high-pressure polytetrafluoroethylene vessels (MARSEasyPrep). 1 mL of HNO3, 1 mL of H2O2, and 1 mL of HClO4 were added to the samples, which were gently mixed. Vials were left open for 10 min before closing them. The microwave was programmed at a temperature of 210 °C, ramp time of 20 min, hold time of 15 min, power of 1400 W, and pressure of 800 psi. After microwave assisted acid digestion, samples were allowed to cool. Subsequently, samples were filtered through filter paper (pore size < 20 µm) into a 50 mL volumetric flask, and 1 mL of HCl was added before filling to volume.

Quantification of total mercury using cold vapor coupled to atomic fluorescence spectrometry

For THg quantification, a Mercur Plus atomic fluorescence spectrometer coupled to a cold vapor generator was used. From a stock solution of Hg+2 (50 μg L−1), calibration curves were diluted at 0.5–5 μg L−1 by the auto-sampler. Linear regression coefficients higher than 0.99 were obtained for calibration plots. The instrumental limits of detection and quantitation were 0.10 μg L−1 and 0.34 μg L−1, respectively.

All digestions and measurements were performed in duplicate for each sample, and the mean concentration was reported.

For quality control, Hg fortifications in tuna samples were prepared at two concentration levels (1 and 4 μg L−1), alternating them per each digestion batch. The mean recovery (mean ± SD) values were between 99% ± 2% (96.1–100.7%) and 91.0% ± 5% (86.5–96.6%), respectively. These results were in good agreement to the recoveries between 60.0 and 115.0% for accuracy established in the Standard Method Performance Requirements for Heavy Metals in a Variety of Foods by the Association of Official Analytical Chemists57.

The accuracy of the analytical procedure was verified by analyzing IAEA-436A certified reference material (tuna fish flesh homogenate) in each sample digestion batch. A mean recovery (mean ± SD) of 103% ± 5% (96−110%) was obtained, within the recovery values according to AOAC57.

Health risk assessment

Human health risk was assessed considering the different THg concentrations (minimum, mean, and maximum) obtained for the three brands of canned tuna. All calculations were performed using Microsoft Office Professional Plus Excel 2016.

According to the Environmental Protection Agency of the United States58, in order to protect human health, THg should be assumed as MeHg in the whole fish sample. Therefore, for the present study, the potential health risk was assessed using the THg concentrations as MeHg in muscle tissue of the canned tuna.

The level of exposure (Ex) for MeHg was calculated using the following equation from the US-EPA (2000):

$$Ex=\frac{Cx \times CR}{BW}$$
(1)

where Cx is the concentration of metal in the edible portion of the samples (mg kg−1), CR is the tuna ingestion rate per day (kg d−1), and BW is the average mean body weight (kg)58. The non-carcinogenic health risk assessment (Rx) through canned tuna consumption was calculated by Eq. (2)59:

$$Rx=\frac{Ex}{RfD}$$
(2)

where Ex is the exposure to the pollutant (mg kg−1 d−1), and RfD is the reference dose of MeHg (1 × 10–4 mg kg−1 d−1)59,60.

Considering the Provisional Tolerable Weekly Intake (PTWI) of 1.6 µg kg−1 Human Body Weight (HBW) for MeHg61, the maximum weekly intake of canned tuna (g weekly) was calculated according to the following equation:

$$Intake \left(g\right)=\frac{PTWI \times HBW}{Cx}$$
(3)

where Cx represents the average MeHg concentration (mg kg−1) in each canned tuna brand measured in this study. Human body weight (HBW) was equal to 70 kg and 15 kg for adult and children, respectively.

Data analysis

To calculate descriptive statistics (mean, standard deviation, range, and recovery), Excel 2016 was used.

THg content between the three different canned tuna brands was assessed using non-parametric methods (Kruskall Wallis) followed by a pairwise Wilcox test. The R software environment for statistical computing and graphics (https://w.w.w.rproject.org/) was used to analyze the data. Chebyshev’s theorem50 was used to evaluate data distribution; the latter was implemented using R software.

Results and discussion

Quantification of total mercury content in samples of canned tuna in water

The highest THg levels (1.98 ± 0.42 mg kg−1) were found in canned tuna of brand B, while the lowest concentration (0.02 ± 0.11 mg kg−1) corresponded to brand A (Table 1). Water content for the studied brands of canned tuna ranged from 66.4 ± 2.5% in brand C to 77.6 ± 2.3% in brand A, which are in good agreement to other humidity percentages in studied brands of canned tuna24.

Table 1 Total mercury levels ± standard deviation (mg kg−1 w.w.) in the three brands of canned tuna analyzed per every year of the studied period.
Full size table

Results from the present study, showed that 96.7% of the analyzed samples were below the threshold limits stablished for THg content. Only one batch of canned tuna from brand B (1.98 mg kg−1), the brand with the intermediate price among the three brands analyzed, exceeded the threshold limits established by the EU (2023), FAO (2019) and NTE INEN (2013), while another sample of the same brand was in the limit of 1.0 mg kg−1 (Table SI_1). Comparing with another research carried out in Ecuador, the average values of our results (0.140.14 ± 0.11, 0.41 ± 0.42, and 0.25 ± 0.22 for brands A, B, and C, respectively) were higher than those reported by Lalangui-López (2017), who showed that the content of THg in canned tuna from the most consumed brands by the Ecuadorian population were lower than 0.5 mg kg−1, with THg values between 0.022 mg kg−1–0.093 mg kg−1. On the other hand, Ormaza-González et al. (2020), reported an average of 0.23 ± 0.14 mg kg−1 of THg content in canned tuna for a study period carried out from 2009 to 2016, where 2572 samples from different brands were analyzed. These results were in agreement with the THg concentrations found in brand C (the brand with the highest price among the three brands analyzed) of our study. In particular, brand A (the brand with the lowest price on the market) had the lowest THg content.

According to the boxplot analysis (Fig. 1), the data distribution for brands B and C was positively skewed, as the mean was greater than the median and mode. For brand B, 75% of the data indicated that the THg content was between 0.22 and 0.63 mg kg−1; for brand A, the content was between 0.06 and 0.37 mg kg−1 and for brand C, between 0.09 and 0.34 mg kg−1. Brand B, had a mean THg value of 0.41 mg kg−1 and was therefore the closest to exceeding the Ecuadorian limit of 0.5 mg kg−1, while brand A had the lowest THg content.

Fig. 1
figure 1

Box plot of the total mercury content (mg kg−1), for the three analyzed brands of canned tuna in water.

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Chebyshev’s theorem was used to determine the confidence intervals and the amount of dispersion for each dataset. Accordingly, the minimum percentage of population values that were within k standard deviations from the mean was calculated using the equation \((1-\frac{1}{{k}^{2}}),\) and the maximum percentage of population values that were beyond k standard deviations was determined with the equation \((\frac{1}{{k}^{2}}),\) where k is the number of standard deviations of interest and must be greater than 1. Table 2 presents the confidence intervals calculated using \(\pi ,\) φ, and e as values for the parameter k, selected for their utility in modeling behaviors and characteristics of important natural and artificial phenomena. When k was assigned the value \(\pi ,\) Chebyshev’s theorem indicated that 89.39% of the data was within 3.1416 standard deviations of the mean; for values of e, 86.47% of the data was within 2.72 standard deviations of the mean, while for the value of Φ, this percentage decreased to 61.80%. Furthermore, the results in Table 2 show that the confidence interval is notably wider for brand A (the brand with the lowest price on the market) data. Given that brand A has a lower THg content compared to the other brands, according to Chebyshev’s theorem, introducing new elements to its data population would result in an 89.39% probability (for k = \(\pi )\) that the majority of these elements would fall within \(\pi\) standard deviations from the mean. Thus, there is a higher level of confidence in attributing a lower THg content to brand A compared to brands B and C.

Table 2 Confidence intervals, according to uncertainties about the distribution of the data, obtained from Chebyshev's theorem.
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Kruskall Wallis test showed differences between THg levels among the three brands under study (p < 0.01), and Wilcox test confirmed that metal content in brand B differs from brand A and C. This difference may be attributed to the two batches which showed the highest THg concentrations. Furthermore, Kruskall Wallis test showed no difference between the two periods of sampling (2022 and 2023) among the three canned tuna brands (p = 0.1298, p = 0.3838, and p = 0.3838, for brands A, B, and C, respectively).

Table 3 compares this study’s results to those published in the literature. In terms of the medium in which the tuna is preserved inside the cans, our results showed similar THg content in comparison to other studies in canned tuna in oil with values between 0.044 to 0.88 mg kg−110,24,49,50,62,63. Burger and Gochfeld (2004) and Ormaza-González et al. (2020) have also found that THg concentration was not associated with the liquid of the cans (water, oil), or if it was drained or undrained.

Table 3 Studies on THg content in canned tuna in water and oil performed in other countries.
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It has been reported three species of tunas, skipjack (Katsuwonus pelamis), yellowfin (Thunnus albacares), and bigeye (Thunnus obesus) account for 93% of all tuna consumed in the world31. Fuentes-Gándara et al. (2018) and Alcalá-Orozco et al. (2017) quantified THg in canned tuna marketed in Colombia in different brands imported from Ecuador. The former showed THg average values of 0.39 mg kg−1 and 0.50 mg kg−1, while the latest indicated contents of 0.36 ± 0.01 mg kg−1, 0.22 ± 0.03 mg kg−1, and below the LOD (< 0.001 mg kg−1). Although cans of brands sold in the Ecuadorian market does not distinguish between different species of tuna, and this information is not usually available to the final consumer.

Other studies have reported similar THg average values in canned tuna in water, 0.362 mg kg−1 in Mexico24, 0.171 mg kg−1 in Brazil56, and 0.332 ± 0.151 mg kg−1 in Spain54, among others as shown in Table 3, which accomplish with the threshold limits for safe consumption. However, few studies have reported samples with THg concentrations higher than 1.0 mg kg−1. Gerstenberger et al. (2010) and Fuentes-Gándara et al. (2018) have presented maximum contents of THg in canned tuna in water in punctual samples (1.666 mg kg−1 and 1.254 mg kg−1; 1.473 mg kg−1, respectively), in agreement to our findings. According to Ruelas-Inzunza et al. (2011), for comparison of Hg levels in canned tuna from different sites, it is highly recommended to use fish of the same species and size, as well as the packing method (water, oil, or sauce).

It has been shown that THg and MeHg content in fish vary widely depending on length, fish species, feeding habits6, fat content39, availability of Hg in their surrounding environment41, and differences in age, growth and metabolic rates, which contributes to the enhanced bioaccumulation of contaminants3,27. High correlations have been found between high Hg levels and larger and older fish species such as yellowfin tuna, skipjack tuna, and bluefin tuna, due to the biomagnification of Hg in the aquatic food web, especially in predatory species64. Additionally, the degree of pollution of the aquatic ecosystem, Hg speciation in water, microbiological, chemical, and physical oceanographic conditions across regions, like the depth of the dissolved oxygen, and the water column, pH, water temperature and transparency, as well as the geographical location and the season where tunas are caught, drives the spatial variability and bioavailability of Hg at the base of the food web7,39,49,65. According to our results, variation of THg content among the brands of canned fish under study, could be attributed to the age, source and size of the fish, similar to the reported by Okyere et al. (2015).

Moreover, Farrel Anthony (2011) reported that skipjack tuna (Katsuwonus pelamis), which is highly popular in terms of fishing and represents 58% of global tuna catch66,67, has a shorter lifespan and therefore accumulates less Hg in its tissues. Thunnus alalunga reaches a maximum weight of approximately 40 kg at 15 years old, while Katsuwonus pelamis has a maximum weight of approximately 30 kg at the same age.

In the Eastern Pacific Ocean (EPO), Ecuador has the largest purse seine fleet of tunas68. Studies have demonstrated that yellowfin tuna from the EPO presented a mean THg concentration of 0.297 ± 0.176, indicating that some combination of trophic status, i.e., feeding behavior, and geographic location could be driving Hg levels in, as they do not migrate across oceans31. Likewise, Ferris and Essigton (2011), showed that yellowfin tuna THg concentrations were slightly higher than those found for bigeye tuna in the eastern equatorial Pacific. However, the eastern Pacific is a region consistently associated with elevated Hg concentrations compared to the central and western regions7,65,69. On the other hand, lower THg levels in skipjack tuna have been found due to a lower trophic position, a shallower vertical habitat that allows access to epipelagic prey with lower Hg concentrations, and a shorter lifespan65.

Human health risk assessment

Table 4 shows the results obtained of the exposure levels, potential non-carcinogenic risk, and recommended weekly intake of fish meat for children and adults, based on mean content of THg found in the three brands of canned tuna in water. According to the FDA/EPA (2022), the highest allowable THg concentration in fish when eating 1 serving per week is 0.46 mg kg−1. Our results showed that adults and children can weekly consume canned tuna of brand A up to 306 g and 63 g, respectively, considering the highest THg content found (Table SI_2). Nevertheless, for brand B and C, 20% and 15% of the samples, respectively, exceeded FDA/EPA reference value (Table SI_1). In view of the high THg levels found in brand B, the recommended amount for consumption for this brand was almost 5 times lower for children and adults (12 g and 57 g, respectively), than for brand A; meanwhile, for brand C, the recommended weekly intake was approximately twice lower (30 g for children and 144 g for adults) in comparison to brand A and B.

Table 4 Exposure levels, potential non-carcinogenic risk, and recommended weekly intake of fish meat for children and adults, based on mean content of MeHg found in the three brands of canned tuna in water.
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According to Ormaza et al. (2022), in Ecuador, canned tuna is consumed once or twice per week, however it does not occur every week. By considering a personal portion of 80 g of canned tuna in water in a single serving per week for an adult, and the half of the content of the can for children (40 g), the Rx calculated for the mean THg content in samples is higher than 1 for brand B. These results suggest that choosing brand B for an intake more than one serving per week can pose a significant potential risk over a lifetime, more specifically for vulnerable populations as pregnant woman with developing fetuses and children. For the other brands, A and C, more tuna can be consumed, however, the non-carcinogenic risks may be greater for children than for adults.

In other countries as Mexico, studies of THg in canned tuna have reported low potential non-carcinogenic risks24,51 nevertheless there is a large difference of consumption among countries. For example, in Mexico, tuna consumption per capita into a daily basis is about 3.92 g day−124, while in Italy, the consumption of canned tuna amounts to 5.71 g day−164. Even though there is not information regarding canned tuna consumption in Ecuador, an intake of 37.0 g day−1 of fish has been estimated70. Thereby risk for frequently canned tuna or other fish species consumption should be a concern71,72,73,74,75,76,77.

Considering the high consumption of canned tuna around the world and the possible consumer health risks due to its consumption, fish species, size, and catch location should be required and regulated on labels. In this regard, Shim et al. (2004), suggested that, because of young children’s susceptibility to Hg’s toxic effects, products with low metal concentrations should be specially labeled as “safe for children”. However, in Ecuador, the labels on this type of product only provide nutritional information.

Conclusions

In the current study, only one lot of canned tuna from brand B (the brand with the intermediate price among the three brands analyzed and the most consumed in Ecuador) showed the highest THg contents (1.98 mg kg−1). For most of the samples from brands A, B, and C, the THg levels were below the threshold limits set by national and international regulations. Variation of THg content among the brands of canned tuna could be attributed to the age, source and size of the fish, since THg in tuna can vary widely depending on several factors such as length, fish species, fat content, degree of pollution of the aquatic ecosystem, geographical location and the season where tunas are caught.

Calculated Rx for MeHg showed that there is no significant potential health risk considering one serving per week of 80 g for adults and 40 g for children; however, the non-carcinogenic risks may be greater for children than for adults when increasing the amount or frequency of canned tuna consumption. Since fish consumption in beneficial for human health, as it provides healthy omega-3 fatty acids, it is important to balance its consumption due to THg content.

Establishing strategies for monitoring canned tuna consumption in Ecuador is highly recommended, in order to protect children and the fetuses of pregnant women, reducing THg exposure. However, in the Ecuadorian market, information regarding tuna species is not always available to the final consumer, being difficult to identify those tuna species with the highest THg content in order to avoid its consumption.