INTRODUCTION

Trace metals reach coastal regions through continental runoff and atmospheric transport by natural and anthropogenic causes (^{Abdel-Ghani 2015}). Nevertheless, they can accumulate at high concentrations in both the aquatic environment and marine organisms due to sewage disposal from human activities such as mining, livestock, poultry, agriculture, and aquaculture. Hazardous pollutants in wastes from anthropogenic activities are discharged into local irrigation systems and from there to estuaries and coastal ecosystems (^{Rainbow 2002}).

Occupying the second trophic level of aquatic ecosystems, sessile filter-feeding bivalve mollusks inevitably accumulate high levels of metals in their soft tissues, representing a danger for human consumption. Marine bivalves feed selectively, filtering large volumes of seawater through their gills (^{Silvester et al. 2005}) with which they trap suspended materials and sediments, phytoplankton, fecal pellets, detritus, and high molecular weight substances (^{Liu and Deng 2007}) that are eventually ingested. Thus, metals associated with the aqueous phase, food, and sediments accumulate in the shells (^{Cravo et al. 2002}) and soft tissues of these organisms (^{Góngora-Gómez et al. 2018}).

Due to their sensitivity and rapid response to pollutants, bivalves such as mussels and oysters (^{Bray et al. 2015}) are used as biomonitors of metal contamination in aquatic systems (^{Kanthai et al. 2014}), the bioavailability of contaminants, and the degree of pollution. To a lesser extent, clams also serve as sentinel organisms for marine eco-toxicological tests to ensure coastal water quality with respect to metals. For instance, ^{Bendell (2009}) performed cadmium analysis on oysters, mussels, clams, and scallops sampled from commercial and natural sources on the west coast of British Columbia, Canada, and ^{Kehrig et al. (2006)} reported the levels of methyl and total mercury in mussels, oysters, and clams from two estuaries in Rio de Janeiro, Brazil. In both studies, mussels and oysters showed higher metal accumulation than clams and scallops. The latter two are part of the sedentary non-sessile benthic fauna for which metal accumulation levels strongly depend on the metals associated with particulates that settle in bottom sediments (^{Inengite et al. 2010}) and on the specific bioavailability of different trace metals (^{Ferreira et al. 2004}).

Megapitaria squalida, commonly known as the squalid callista clam, represents one of the most important bivalve species on the southeastern coast of the Gulf of California, Mexico (^{Arellano-Martínez et al. 2006}). This clam, which is naturally distributed from Baja California to Peru, digs in sandy clay sediments in the intertidal zone to depths of approximately 160 m (^{Coan and Valentich-Scott 2012}). It is a popular raw seafood for human consumption with wild banks located in coastal lagoon systems in the southeastern Gulf of California, where intense anthropogenic activities also occur. Thus, these areas are under permanent pressure and the trace metal burdens in M. squalida, consequently, represent a potential risk for human health.

Due to the significance of copper (Cu), cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), arsenic (As), zinc (Zn), and mercury (Hg) to public health (^{WHO 1996}), their high degree of toxicity (^{ATSDR 2006}), and their routine association with human activities around the sample sites (^{Gómez-Arroyo et al. 2013}), the levels of these metals were assessed both in sediments and in the soft tissues of M. squalida from two locations on the southeastern coast of the Gulf of California during 13 consecutive months. The levels were evaluated based on national and international permissible levels for human consumption.

MATERIALS AND METHODS

RESULTS

Metal levels in the sediments and clams from Altata and Agiabampo varied spatially (Table I). Zn had the highest monthly and mean values in both sediments (46.55 and 31.07 µg/g dw for Altata and Agiabampo, respectively) and clams (51.39 and 46.81 µg/g dw for Altata and Agiabampo, respectively). The CV values for metals fluctuated from 9.67 % for Cu in sediments to 212.50 % for Cr in clams at Altata Bay and from 6.51 % for Zn in clams to 200 % for Cd in sediments at Agiabampo Bay.

With the exception of Cd and Hg in sediments, the ranking of metal concentrations presented the same order that in sediments (Zn > Pb > Cr > Ni > Cu > As > Cd > Hg at Altata and Zn > Pb > Cr > Ni > Cu > As > Hg > Cd at Agiabampo) and clams (Zn > Cu > Pb > As > Ni > Cd > Hg > Cr at Altata and Zn > Cu > Pb > As > Ni > Cd > Hg > Cr at Agiabampo). Only the Zn levels in sediments and clams from Altata were similar (p = 0.08, Table II).

The concentrations of Cu, Ni, and Zn were significantly different (p ≤ 0.05) between sediments from both sites and between clams from the two bays (Table III).

The metal rankings for sediments and clams from both sample sites indicate that Zn was the most abundant element with a concentration ranging from 31.07 µg/g dw for Altata sediments to 51.39 µg/g dw in Altata clams.

None of the metals studied surpassed the permissible limits in sediments (^{CCME 2003}; ^{SEMARNAT 2007}) from both sites; however, some metal contents in clams did exceed permissible limits. Cd and Pb contents in clams from Altata (2.49 and 5.68 µg/g dw, respectively) and Agiabampo (2.38 and 5.54 µg/g dw, respectively) were higher than permitted by Mexican regulations (2 and 1 µg/g dw, respectively (^{SSA 1995}, ²⁰¹¹).

At Altata, Cd and Pb were significantly correlated (r = 0.55) in clams and correlations were found between Ni and Cr, Pb and Cr, Pb and Ni, Zn and Cr, and Zn and Pb (r = 0.63-0.93) in sediments. For Agiabampo, significant correlations were obtained for Cd/Cr (r = 0.73), Pb/Ni (r = 0.76), and Zn/Cd (r = 0.72) in clams, and for Ni/Cr (r = 0.65), Ni/Cd (r = 0.65), and Pb/Cd (r = 0.67) in sediments.

Overall, the BSAF for Altata and Agiabampo presented the following order: Cd > As > Hg > Cu > Zn > Pb > Ni > Cr and Cd > As > Hg > Cu > Zn > Ni > Pb > Cr, respectively (Table IV). The clam soft tissues exhibited the highest levels of absorption capacity for Cd (10.75 and 7.07), As (5.42 and 5.25), Hg (4.83 and 5.04), Cu (1.44 and 1.74), and Zn (1.15 and 1.62) for Altata and Agiabampo, respectively.

DISCUSSION

Due to the ecological and commercial importance, as well as the high consumption demand for different mollusk species in the study region, several biomonitoring studies focusing on heavy metal accumulation in a variety of bivalve species have been conducted and provide comparative data. Wild and cultivated oysters, pen shells, and clams have been used as biological monitors for heavy metals. For instance, ^{Frías-Espericueta et al. (2008)} obtained higher Cu (21-73.2 µg/g dw) and Cd (1.8-7.2 µg/g dw) levels, but lower Pb (2.6-8.7 µg/g dw) burdens in a wild bank of the pleasure oyster (Crassostrea corteziensis) from the Altata bay. The Zn (1-1.66 µg/g dw), Cr (0.4-1 µg/g dw), Ni (0.33-2.3 µg/g dw), Pb (0.66-2 µg/g dw), and As (0.2-0.57 µg/g dw) concentrations reported by ^{Góngora-Gómez et al. (2018)} in the muscle of the pen shell (Atrina maura) cultivated at a farm located between the two sample sites were lower than the levels we obtained for Zn (2.44-5.16 µg/g dw), Cr (3.51-9.76 µg/g dw), Ni (2.49-8.55 µg/g dw), Pb (6.68-14.45 µg/g dw), and As (0.4-1.82 µg/g dw). ^{Muñoz-Sevilla et al. (2017}) registered variations of Cu (10.2-26.6 µg/g dw), Cd (0.8-3.8 µg/g dw), Pb (1.3-3.6 µg/g dw), Zn (57.2-219.4 µg/g dw), and Hg (0.02-0.11 µg/g dw) in Pacific oysters (C. gigas) cultivated near the Agiabampo bay. These variations can be attributed to differences in human activities around the study areas, latitude, environmental conditions, and the bivalve species studied.

Different baseline metal orders in sediments have been reported by ^{Widmeyer and Bendell-Young (2008)} in British Columbia, Canada (Cu > Zn, Pb > Cd), ^{Tarique et al. (2012)} (Pb > Cu > Cd > Hg) in the Kuwait bay, and ^{Jonathan et al. (2017)} (Zn > Cr > Ni > Cu > Pb > As > Cd > Hg) in the southwestern Gulf of California. In the case of Altata and Agiabampo, metal concentrations in sediments were affected by a number of factors, including sewage effluents from industry, urban sewage disposal, agricultural activities, and aquaculture farms close to the sample sites (^{Páez-Osuna et al. 2003}, ^{Hernández-Cornejo et al. 2005}, ^{Muñoz-Sevilla et al. 2017}); also, by the relationship between grain size and texture and metal adsorption that in this case favors degradation of organic matter by tidal mixing (^{Forrest and Creese 2006}), as well as the natural erosion of the rocks, dissolution processes, and water runoff from upstream of the estuaries (^{Murray and Busty 2015}). The external and natural inputs at each specific locality may help to understand the differences among the aforementioned results.

The metal bioaccumulation orders observed in M. squalida from both sites differed from those reported by ^{Tarique et al. (2012)} (Zn > Cu > Cr > Ni > Hg > Cd > Pb) and ^{Mohammad et al. (2017)} (Zn > Pb > Cu > Cd) for the clams Amiantis umbonella in the Kuwait bay and Donax semistriatus in the Mediterranean Sea, respectively. ^{Góngora-Gómez et al. (2017)} analyzed the metal concentrations in oysters (C. gigas) cultivated in a lagoon system between Altata and Agiabampo, registering the following rank order of accumulation: Zn > Cu > Cr > Cd > Ni > Pb > As > Hg, which is similar to those obtained by ^{Osuna-Martínez et al. (2011)} (Zn > Cu > Cd > Pb) and ^{Vázquez-Boucard et al. (2014)} (Zn > Cd > Pb) for the same oyster species in the studied region.

Several biotic factors affect metal bioaccumulation in bivalve mollusks. Some trace metals like Zn, Na, and Cu are essential for tissue formation, metabolic physiology, cellular metabolism, and nutrient synthesis and metabolism (^{Barile 2008}), but levels exceeding normal requirements can impair growth, reproduction, and general development. ^{Jara-Marini et al. (2013)} stated that Cu levels in adult oysters decreased during the post-spawning event, whilst ^{Lango-Reynoso et al. (2010)} registered variations in Cd accumulation during different reproductive stages. ^{Páez-Osuna et al. (1995)} concluded that concentrations of some metals vary seasonally with reproductive events and gonadal development after finding higher levels of Cu and Zn at the end of the reproductive cycle in oysters. These findings do not coincide with the results obtained in this study, since M. squalida from both sites did not show a normal seasonal pattern for bioaccumulation of the metals examined. On the other hand, no tools for evaluating clam reproductive phases were employed in this study, as this was not the goal of our research. In fact, no reports on the relationship between metal accumulation and reproduction of the squalid callista are available so far.

As with other bivalves, M. squalida is a popular raw dietary item not only in coastal areas of the Gulf of California, but also in cities where there is a high demand for this resource (^{Amezcua-Castro et al. 2015}). However, only a few studies have been conducted on heavy metals in the squalid callista along the coasts of the Gulf of California. ^{Méndez et al. (2006)} analyzed Pb, Ni, Cd, Mn, Zn, Cu, and Fe levels in M. squalida from an apparently contamination-free site in the southwestern Gulf of California, and concluded that this clam should be consumed before the rainy season due to Pb and Cd accumulation. ^{Cantú-Medellín et al. (2009)} stated that some antioxidant defense mechanisms in M. squalida changed in response to the bioaccumulation degree of some metals, such as Cd, Pb, and Fe. Through morphometry and condition index, ^{Yee-Duarte et al. (2017)} concluded that health of the squalid callista clam in Santa Rosalía, Baja California Sur, evidenced negative physiological effects, possibly caused by contamination from metals produced by the local mining activity. ^{Romo-Piñera et al. (2018)} evaluated the total Hg concentration in the squalid callista, finding relatively low values of Hg (0.06-0.09 µg/g dw) in soft tissues that nonetheless represent a potential risk for human health.

These works were carried out in wild squalid callista populations from the southwestern Gulf of California. Only ^{Frías-Espericueta et al. (2008)} recorded metal contents (Cd, Cu, Pb, and Zn) in oysters, mussels, and clams from the Altata-Pabellones lagoon system (southeastern Gulf of California) and found differences in metal bioaccumulation during the rainy and dry seasons, and between the different bivalve species. They concluded that the inner mangrove swamp species (C. corteziensis and Mytella strigata) bioaccumulated higher levels of Cd and Cu than M. squalida, which inhabits areas under marine influence. This confirms the strong influence of habitat on metal bioaccumulation in different bivalve species, as well as the need for more research related to heavy metal accumulation in M. squalida from natural banks at the southeastern coast of the Gulf of California.

Since limits for Zn, Cu, Cr, and Ni in soft tissue of clams have not yet been set in Mexico, the concentrations of these metals were compared with standards established in other countries. Zn (51.39 µg/g dw) in M. squalida from Altata and Hg (0.32 µg/g dw) in clams from Agiabampo surpassed the permissible limits established by the Ministry of Agriculture, Fisheries and Food (50 and 0.3 µg/g dw, respectively) in the UK (^{Sally et al. 1996}). Cu, Cd, and Zn burdens were lower than those reported by ^{Frías-Espericueta et al. (2008)} for M. squalida from Altata. However, Ni was lower than the level obtained by ^{Méndez et al. (2006)} in a wild population of squalid callista clam on the southwestern coast of the Gulf of California. Recently, ^{Romo-Piñera et al. (2018)} reported Hg at 0.05-0.09 µg/g dw in soft tissues of M. squalida from La Paz bay, Baja California Sur, Mexico, which is lower than the Hg levels in both bays examined in the present study. The presence of high levels of Cd and Pb in clams from both places is associated to phosphate fertilizers used in agriculture (^{Jara-Marini et al. 2013}) and aquaculture and industrial influxes from the region (^{Luoma and Rainbow 2005}), respectively.

A strong association has been reported between particular groups of metals, suggesting a possible common source (^{García-Rico et al. 2001}, ^{Jonathan et al. 2017}, ^{Muñoz-Sevilla et al. 2017}). It is reported that in approximately 200 000 ha that surround the two studied regions, the agriculture of different grains and vegetables, such as corn, tomato, and beans, is practiced; thus, enormous amounts of agrochemicals containing Cd, Ni, and Zn are used in fertilizers (^{Páez-Osuna et al. 1993}), insecticides, fungicides, and herbicides (^{Gómez-Arroyo et al. 2013}). These heavy metals are eventually leached from the soil and transported to the coastal lagoon systems inhabited by these clams (^{Páez-Osuna and Osuna-Martínez 2015}). This was confirmed by the correlations found in the sediments between Ni/Cr, Pb/Cr, Pb/Ni, Zn/Cr, and Zn/Pb at Altata, between Ni/Cr, Ni/Cd, and Pb/Cd at Agiabampo, in the clam soft tissue between Cd/Pb at Altata, and between Cd/Cr, Pb/Ni, and Zn/Cd at Agiabampo.

Since both sites are connected to irrigation districts, they receive agrochemicals not only from agriculture and livestock industries, but also from shrimp aquaculture ponds (^{Hernández-Cornejo et al. 2005}), resulting in high contamination influxes to the estuaries in the study area. There are reports for decades ago that the lagoon systems near our sample sites is receiving tons of inorganic nitrogen, inorganic phosphorous, fungicides, pesticides, and herbicides from anthropogenic activities highlighting that their influxes would increase over time (^{Escobedo-Urías 2010}, ^{Gómez-Arroyo et al. 2013}). The presence of Pb and Cr relationships in sediments and clams reflects the impact of untreated sewage discharges from the different (domestic, aquaculture, and agriculture) activities (^{Wong et al. 2007}) and industrial complexes (sugar mills) in the region (^{Gaxiola 2003}).

The BSAF orders in our study reflect the high absorption capacity of Cd, As, and Hg; the moderate absorption capacity of Cu and Zn, and the low absorption capacity of Pb, Ni, and Cr in the soft tissues of clams. ^{Soto-Jiménez et al. (2001)} mentioned that the degree of metal availability from sediments is partially due to the exchangeable carbonates and organic phases made available to the organisms by water currents. Cd had the highest biosediment accumulation factor values at both sites, but the Cu and Zn concentrations in sediments were lower, suggesting a higher rate of accumulation of these metals by M. squalida. Pb, Ni, and Cr were generally lower in the clam soft tissues than in sediments from both sites, suggesting that the clams’ capacity to regulate these metals was not surpassed by the metal levels in sediment, as observed by ^{Mohammad et al. (2017)} in the clam D. semistriatus. For the rest of the metals evaluated, factors such as metal concentration in water and clam metabolism (^{Páez-Osuna et al. 1995}), among other internal and external factors, may partially explain the BSAF reported here.

CONCLUSIONS

All metals assessed were found in sediment and M. squalida soft tissue from both sites, confirming the strong influence from the various anthropogenic activities present around both studied bays. Domestic sludges and sewage waste from industry, agriculture, and aquaculture practices impacted the metal concentration patterns in sediments and M. squalida. Similar to the conclusions reported by ^{Góngora-Gómez et al. (2018)} for the pen shell A. maura, Cd and Pb burdens in the squalid callista clam exceeded the permissible limits for human consumption established by Mexican standards, posing a human health risk. Therefore, a continuous monitoring system is highly recommended and obligatory for the healthy production and consumption of this shellfish species.