Article pubs.acs.org/JAFC
Effect of Intestinal Tapeworm Clestobothrium crassiceps on Concentrations of Toxic Elements and Selenium in European Hake Merluccius merluccius from the Gulf of Lion (Northwestern Mediterranean Sea) Jordi Torres,*,†,‡ Catarina Eira,§ Jordi Miquel,†,‡ Dolors Ferrer-Maza,∥ Eulàlia Delgado,∥ and Margarida Casadevall∥ †
Department of Sanitary Microbiology and Parasitology, University of Barcelona, Avinguda de Joan XXIII, 08028 Barcelona, Spain Institut de Recerca de la Biodiversitat, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain § Centro de Estudos do Ambiente e do Mar and Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal ∥ Department of Environmental Sciences, University of Girona, E-17071 Girona, Spain ‡
ABSTRACT: The capacity for heavy metal bioaccumulation by some fish parasites has been demonstrated, and their contribution to decreasing metal concentrations in tissues of parasitized fish has been hypothesized. The present study evaluated the effect of the cestode Clestobothrium crassiceps on the accumulation of trace elements in 30 European hake, Merluccius merluccius, in Spain (half of them infested by C. crassiceps). Tissue samples from all M. merluccius and specimens of C. crassiceps from the infected hakes were collected and stored until element analysis by inductively coupled plasma mass spectrometry (ICP-MS). Arsenic, mercury, and selenium were generally present in lower levels in the cestode than in all hake tissues. The mean value of the muscular Se:Hg molar ratio in the infested subsample was higher than that in hakes without cestodes. Values indicate that the edible part of infested hakes presents a lower amount of Cd and Pb in relation to noninfested hakes. KEYWORDS: heavy metals, helminth parasite/host interaction, Clestobothrium crassiceps, Merluccius merluccius, Mediterranean Sea
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some protection against mercury exposure.14,15 It has been argued that a selenium: mercury molar ratio >1 is protective against potentially adverse mercury effects.14,16,17 The capacity of some helminth parasites of fish to bioaccumulate diverse toxic elements (usually heavy metals) has been widely demonstrated by several studies performed in both fresh and estuarine waters, but very scarce information is currently available in marine ecosystems.18−21 It seems that some helminth parasites and heavy metal pollution might exert antagonistic reactions over the hosts.22 However, the potential influence of parasites has never been taken into account when evaluating toxic element concentrations in edible fish or when evaluating a specific toxic element estimated daily intake (EDI). Adult M. merluccius specimens are carnivorous fish feeding on small fish and some cephalopods, therefore occupying a higher level of the food chain. In the coastal area of the Gulf of Lion (northwest Mediterranean), the European hake is a species of economic importance, with an annual average consumption of about 4−7 kg/person depending on age and gender.23 Increasing toxic element levels have been reported in hakes from the present study area.3,4 Also in this area, hakes are frequently parasitized by the cestode Clestobothrium crassiceps (Rudolphi, 1819).24 The objective of the present study was to evaluate the effect of C. crassiceps on the concentration of As, Cd, Hg, Pb, and
INTRODUCTION Marine waters contain dissolved toxic metals that arise from many sources, usually as a result of anthropogenic sources, atmospheric releases, and industrial activities.1,2 The European hake (Merluccius merluccius, Linnaeus, 1758), as well as other predator fish species, may bioaccumulate high levels of diverse potentially toxic elements such as Cd, Pb, and Hg. Therefore, fish consumption contributes decisively to the dietary human intake of toxic elements.3,4 Due to the potential harmful effects of these toxic elements on humans, some restrictions have been recommended concerning the consumption of some marine species, and maximum levels of Cd, Pb and Hg in foodstuffs have been regulated.5,6 More recently, the need for reassessing the provisional tolerable weekly intake (PTWI) of inorganic arsenic (15 μg/kg body mass) has been pinpointed by the Joint FAO/WHO Expert Committee on Food Additives.7,8 A notable number of surveys have been recently carried out in different countries determining the concentrations of toxic elements (mainly concerning mercury) in several edible marine fishes, including M. merluccius, with estimates of human exposure via fish ingestion.3,4,9 It has long been known that mercury is an element of special concern that may be several orders of magnitude higher in fish tissues than in the water column.10,11 Biomethylation of mercury occurs in the sediment, allowing for food chain biomagnification, and levels of methylmercury may be sufficiently high in some fish to cause adverse health effects in people that frequently consume fish.12,13 However, fish are also relatively rich in selenium, an element necessary for selenoenzyme functions, which has long been known to offer © XXXX American Chemical Society
Received: August 7, 2015 Revised: September 29, 2015 Accepted: October 4, 2015
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DOI: 10.1021/acs.jafc.5b03886 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Data Analysis. Data were log (x +1) transformed, and whenever normal distributions were obtained, analysis of variance (ANOVA; posthoc Turkey’s test) and unpaired t-tests (two-tailed) were used to compare element concentrations and Se:Hg molar ratios. Otherwise, Kruskal−Wallis (posthoc Dunn’s test) and Mann−Whitney tests were used in Prism 5. Relationships between trace element levels in cestodes and their hosts, and between trace elements and the HSI and Se:Hg ratios, were performed by linear regression analysis in JMP 9 (SAS Institute). A minimum significance level P < 0.05 was applied in all tests. All results were expressed in micrograms of element per gram of tissue (parts per million, ppm) wet weight. Bioaccumulation factors (BF) were determined as the ratio of element concentration in the parasite to that in different host tissues [BF = C(parasite)/C(host tissue)].28 Molar concentrations of Se and Hg were obtained by dividing concentrations by each respective molecular weight (200.59 for Hg and 78.9 for Se).17
Se in different tissues of the European hake (M. merluccius) from the Gulf of Lion, which in turn will allow us to quantify the influence of C. crassiceps on daily intake of the assessed toxic elements by European hake consumers.
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MATERIALS AND METHODS
Study Area and Sampling. The European hake is a carnivorous demersal edible fish, which is widely distributed throughout the Mediterranean Sea and the Atlantic coast of Europe and western North Africa, and that lives mainly at 200−500 m depths in ca. 5 °C waters. It is a species of great economic importance, highly appreciated and quite abundantly consumed worldwide. Some international organisms have warned about its decreasing abundance and have proposed measures for its recovery.25−27 Samples of M. merluccius were collected from January 2010 to March 2012 from commercial trawling and longline catches in the Gulf of Lion (northwest Mediterranean Sea), between the Cap de Creus (Spain) and the Marseille coast (France), at depths ranging from 45 to 510 m. Samples were obtained at the port of Roses (Figure 1), which is one of the most important fishing harbors in the region.
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RESULTS Previous data for the same study area revealed a C. crassiceps prevalence of 76.98% in European hakes.24 The following results refer to a group of 30 hakes, half of which were infested by the referred cestode. Limits of detection (LOD) were less than 0.1 ng·mL−1 for all elements. Recovery rates always ranged from 90% to 110%. All information concerning element concentrations detected in liver, kidney, and muscle of M. merluccius and in its parasite C. crassiceps, as well as the molar ratio of selenium to mercury (Se:Hg), are presented in Table 1. The mean value of the muscular Se:Hg molar ratio in the infested subsample was higher (2.6) than that of the nonparasitized hakes (2.0) (t test, t = 2.412, P = 0.0227). Whereas the highest values for Cd and Hg were found in liver in both infested and noninfested hakes, the highest values for Pb and Se were detected in kidney and the highest arsenic concentrations were found in hake muscle (Table 1). Considering the element levels detected in C. crassiceps and in the tissues of the respective host subsample (Table 1), it was possible to detect that arsenic, mercury, and selenium were generally present in lower levels in the cestode than in all hake tissues. In fact, the arsenic concentration in cestodes is significantly lower (Kruskal−Wallis, H = 35.99, P < 0.0001) than in kidney and muscle (Dunn’s, both p < 0.0001) and liver (p < 0.01) of infested hakes. The Hg concentrations in cestodes (Kruskal−Wallis, H = 16.63, P = 0.0008) are significantly lower than in kidney (Dunn’s, p < 0.05) and liver (Dunn’s, p < 0.01) of infested hakes, and the same was found for Se concentrations in cestodes (Kruskal−Wallis, H = 45.98, P < 0.00010) compared with kidney (Dunn’s, p < 0.0001) and liver (Dunn’s, p < 0.05) of infested hakes. With respect to bioaccumulation factors, the highest value (BF = 98.9) was obtained for cadmium concentrations in C. crassiceps with respect to muscle (Table 1). In fact, the Cd concentration in cestodes is significantly higher than in muscle of infested hakes (Kruskal−Wallis, H = 50.70, P < 0.0001; Dunn’s, p < 0.0001). Even though the cadmium BF of C. crassiceps with respect to kidney was 3.2, this value is not statistically significant. As for the Pb concentration in cestodes, the obtained value was significantly higher than the Pb concentration in muscle from hakes with cestodes (ANOVA, F = 59.71, P < 0.0001; Tukey, p < 0.0001). In fact, the lead BF of C. crassiceps in relation to hake muscle was 12.1 (Table 1). Current values indicate that the edible part of infested hakes presents a lower amount of Cd and Pb in relation to noninfested ones (less 28.0% for Cd and 29.0% for Pb; see Table 1).
Figure 1. Sampling area: Gulf of Lion (northwest Mediterranean Sea) between the Cap de Creus (Spain) and the Marseille coast (France). Hakes were dissected with stainless steel instruments and Milli-Q water. All digestive tracts were removed and scanned for helminths using a stereomicroscope. To perform the current study, 30 specimens of M. merluccius (15 hakes presented C. crassiceps and another 15 individuals were cestode-free) were selected for analysis. Samples of kidney, liver, and muscle from all 30 hakes and specimens of C. crassiceps from the 15 infected hakes were collected and stored in glass vials and frozen (−20 °C) until processing for trace element analysis. Almost all hake specimens were females with a mean length of 506 mm (416−567 mm) and a mean weight of 902 g (506−1307 g). The hepatosomatic indices (HSI) of the same hakes were reported in a previous study and those infested by C. crassiceps presented higher HSI than uninfested hakes.24 Analytical Procedure. Samples were weighted (100−200 mg wet weight) and digested in Teflon vessels with HNO3 (2 mL) and H2O2 (1 mL) (Merck, Suprapure) at 90 °C in an oven and left overnight. All materials used in the digestion process were thoroughly acid-rinsed. After digestion, samples were diluted with 30 mL of Milli-Q water. Total concentrations of As, Cd, Pb, Hg, and Se were quantified by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer Elan 6000). The analytical procedure was checked against standard reference material dogfish (Squalus acanthias) liver (DOLT-3) and muscle (DORM-2) (National Research Council, Canada). Several analytical blanks were prepared and analyzed along with samples in order to determine the detection limits. The analytical process was ́ performed at the CCiTUB, Centres Cientifics i Tecnològics de la Universitat de Barcelona. B
DOI: 10.1021/acs.jafc.5b03886 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Cd
0.25 (±0.2) 0.1−0.9
172.1 (±108.5) 47.9−388.3
7.3 (±2.7) 3.5−12.4
NI
3.2
