Bull Environ Contam Toxicol (2014) 93:532–535 DOI 10.1007/s00128-014-1360-0
Cadmium, Copper, Lead and Zinc Concentrations in Female and Embryonic Pacific Sharpnose Shark (Rhizoprionodon longurio) Tissues M. G. Frı´as-Espericueta • N. G. Cardenas-Nava • J. F. Ma´rquez-Farı´as J. I. Osuna-Lo´pez • M. D. Muy-Rangel • W. Rubio-Carrasco • D. Voltolina
•
Received: 12 March 2014 / Accepted: 13 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract In this work we compared the cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) contents of muscle, liver and placenta of gestating females of the viviparous shark Rhizoprionodon longurio and of muscle, liver and umbilical cord of their respective embryos. The higher values of the essential Cu and Zn were in embryonic or embryo-related tissues (placenta and umbilical cord). Maternal muscle and liver had the highest values of Pb and Cd, respectively. There were significant direct correlations between the Zn and Cd concentrations of placenta and umbilical cord, as well as between maternal muscle and embryonic livers for Pb and Cd, but the relation between these tissues was inverse in the case of Zn. All correlations between the metal content of embryonic tissues and size of the embryos were negative, suggesting an inverse relation between the rate of mother-to-embryo metal transfer and embryonic growth.
M. G. Frı´as-Espericueta J. F. Ma´rquez-Farı´as J. I. Osuna-Lo´pez Facultad de Ciencias del Mar, Universidad Auto´noma de Sinaloa, Paseo Claussen s/n, 82000 Mazatla´n, Sinaloa, Mexico N. G. Cardenas-Nava Programa de Posgrado en Recursos Acua´ticos Facultad de Ciencias del Mar, Universidad Auto´noma de Sinaloa, Mazatla´n, Sinaloa, Mexico M. D. Muy-Rangel W. Rubio-Carrasco CIAD, Unidad Culiaca´n, 80000 Culiaca´n, Sinaloa, Mexico D. Voltolina (&) Laboratorio de Estudios Ambientales UAS-CIBNOR, P.O. Box 1132, 82000 Mazatla´n, Sinaloa, Mexico e-mail: [email protected]
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Keywords Shark Rhizoprionodon longurio Placenta Embryos Metal transfer Metals can be taken up by aquatic organisms through gills or other exposed soft tissues, or because of ingestion of metal-rich diets, and accumulate in the organism’s tissues when the rate of uptake exceeds that of excretion. The most important route of metal uptake of sharks is through their diet (Mathews and Fisher 2009) and, depending on the preys consumed, metals may accumulate to relatively high levels in their tissues (Storelli et al. 2003). Since the reproductive mode of close to 30 % of all shark species is placental viviparity, embryos initially depend for nutrition on the yolk sac. When the yolk reserves are depleted the empty yolk sac implants into the uterine wall to form the yolk sac placenta (Haines et al. 2006), and embryos receive nutrients and metals from the mother through the umbilical cord. Several similarities in placental structure and function between mammals and viviparous sharks seem to indicate convergent evolution of the reproductive and immune systems between these two phylogenetically distant groups (Haines et al. 2006). However, such reproductive physiology has led to the consequence of maternal offloading of anthropogenic pollutants as described for both humans (Gundacker and Hengstschla¨ger 2012; Porpora et al. 2013; Vizcaino et al. 2014) and viviparous sharks (Lyons and Lowe 2013; Mull et al. 2013; Olin et al. 2014). The placental viviparous sharpnose shark (Rhizoprionodon longurio) is a highly migratory species, which appears in the Mexican Pacific coastal waters between November and April, at the height of its reproductive season, when it becomes an important prey for the Mexican Pacific artisanal fishing fleets. Therefore, pregnant females are common in
Bull Environ Contam Toxicol (2014) 93:532–535
the local landings (Ma´rquez-Farı´as et al. 2005). There are several studies on the metal content of shark tissues used for direct or indirect human consumption such as muscle and liver (Endo et al. 2008; Lo´pez et al. 2013) and there is at least one study on this particular species (Hurtado-Banda et al. 2012), but there is no information concerning the mother to embryo metal transfer in placentotrophic sharks. In this work we determined the concentrations and distributions of two essential (Cu and Zn) and two non-essential metals (Cd and Pb) in pregnant R. longurio mothers and in their embryos.
Materials and Methods Pregnant females (n = 15) were obtained between December 2010 and January 2011 from local fishermen of the Mazatla´n (SE Gulf of California) artisanal long line fishing fleet. In the laboratory, the females and the embryos of both uteri were measured (total length, TL) and dissected with a stainless steel knife to obtain liver, placenta and muscle of the females, and muscle, liver and umbilical cord of the embryos. Tissues were stored at -20 °C until processing. Glassware and materials were acid washed (Moody and Lindstrom 1977). Tissues were freeze-dried, homogenized in a Teflon mortar, and triplicate aliquots of known weight (0.5–1 g) were digested at 130 °C in a mod-block unit in 30 mL Teflon vessels with 5 mL of concentrated HNO3 (trace metal grade), transferred to polypropylene vials, diluted to 15 mL with Milli-Q water, and the metal content of each sample was determined by atomic absorption spectrophotometry (Varian Spectra AA) (Ruelas-Inzunza and Pa´ez-Osuna 2007). Blanks were used for accuracy (ranges: Cu = 0–0.013; Zn 0–0.312; Pb = 0.310–0.420; Cd = 0.014–0.33 mg/L, respectively) and for detection limits, which were 0.01 lg/g for Cd, 0.009 lg/g for Pb, 0.09 lg/g for Cu and 0.03 lg/g for Zn. Analysis of certified reference material (DOLT-4, National Research Council Canada) gave recovery values of 91.5 % for Cu, 96.2 % for Cd, 103.4 % for Zn and 105.6 % for Pb (n = 3 in all cases). The mean size and weight of the embryos found in the right and left uterus were similar (Wilkinson’s tests for paired samples p [ 0.05). Therefore, we assumed that they were of similar age and the tissues of embryos obtained in right and left uterus were pooled and analyzed as single samples. The mean content of each metal of the different maternal tissues (including cords and placentas), as well as those of each placenta and of liver and muscle of the respective embryos, were compared with non parametric one-way block ANOVA (Friedman’s) tests because Kolmogorov–Smirnov’s and Bartlett´s tests showed that data were not normal or not homoscedastic. Differences
533
were identified with Dunn’s tests. Possible relations between the metal values found in the tissues of each mother and its respective embryos, and between the metal content and the mean TL of each composite sample of embryos were determined with Spearman’s correlations tests. All tests were with a = 0.05 (Zar 1999).
Results Size at first maturity of R. longurio females is 92.9 ± 0.94 cm TL (Corro-Espinosa et al. 2011). The 15 pregnant females ranged from 96 to 118 cm TL (mean 110.93 ± 5.30 cm), and therefore none could be considered primiparous. They carried a total of 121 embryos (6–10 embryos/female), with a maximum of five embryos/ uterus, all connected to the uterus through placenta and umbilical cord, and ranging in size from 20.0 to 27.8 cm. Their weight ranged from 27.7 to 62.3 g, and the male: female ratio of embryos was 1:1.2. The mean Cu contents were between 5 and 20 times lower than those of Zn, with the exception of the liver of embryos: The orders of concentration of each tissue were different, and the significant differences (p \ 0.05) for Cu were embryonic liver [ embryonic muscle, and of maternal liver and muscle. In the case of Zn, the only significant differences were placenta [ maternal liver and muscle, and the remaining tissues had intermediate values (Table 1). There were also significant differences in the contents of the nonessential Pb and Cd: the Pb content of the mothers’ muscle was significantly higher than those of placenta and cord. All female livers had a Pb content below limit of detection, while the value of Cd was significantly higher (p \ 0.05) than cord and maternal and embryonic muscles (Table 1). There were significant direct correlations between the Zn and Cd contents of cord and placenta, as well as between the Pb and Cd contents of the maternal muscle with the embryonic liver, while the Zn content of the maternal muscle was inversely related to that of the embryonic liver. The correlations between the metal contents of embryonic liver and muscle and between these tissues with their respective placentas were not significant (Table 2). All correlations calculated between the mean TL of the embryos and the metal content of the respective muscle and liver samples were inverse. In the case of the muscle, the only significant value was that of Pb, while in the case of the liver only Cd was not significantly related to TL (Table 3).
Discussion The higher concentrations of Cu and Zn were in embryonic tissues, and in placenta and umbilical cord, whereas Pb was
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Bull Environ Contam Toxicol (2014) 93:532–535
Table 1 Mean metal contents ± standard deviation (lg/g, dry weight) of the tissues of R. longurio females (F) and embryos (E) Cu
Zn
Pb
Cd
v2
59.25
45.49
48.76
49.71
Muscle (F)
1.19 ± 0.24a
16.96 ± 2.29a
4.96 ± 5.88b
0.03 ± 0.03a
Liver (F)
2.43 ± 0.75ab
12.56 ± 4.19a
\LD
1.67 ± 1.19c
Placenta (F)
9.44 ± 1.14bc
45.64 ± 14.36b
0.04 ± 0.07a
0.29 ± 0.27bc
Cord (E)
6.27 ± 4.56bc
29.13 ± 15.23ab
0.24 ± 0.41a
0.14 ± 0.08ab
Muscle (E)
4.10 ± 0.90ab
34.52 ± 10.79ab
2.08 ± 2.03ab
0.08 ± 0.14ab
Liver (E)
46.24 ± 13.89c
23.64 ± 6.65ab
1.43 ± 1.23ab
0.18 ± 0.13abc
\LD = below limit of detection, not used in statistical analysis Equal or common letters indicate lack of significant difference between data in the same column (Friedman tests, p \ 0.05). n = 15 in all cases. v25 values are indicated for the test with each metal Table 2 Spearman’s correlation coefficients between the metal content of placenta, umbilical cord, maternal (Mat) and embryonic (Emb) tissues of R. longurio Tissues
Cu
Placenta-cord
-0.054
Mat liver-emb. muscle
-0.046
0.025
–
0.015
Mat liver-emb. liver
-0.020
0.064
–
0.261
0.193
-0.050
0.351
0.168
-0.754**
0.746**
0.516*
Mat muscle-emb. muscle Mat muscle-emb. liver Placenta-emb. muscle Placenta-emb. liver Cord-emb. muscle
Zn
-0.254
Pb
0.887**
0.407
0.198
-0.293
-0.112
Cd 0.842**
-0.003
0.499
0.157
0.413
0.289
-0.452
-0.167
-0.064
-0.122
Cord-emb. liver
0.286
0.024
0.436
0.095
Emb.muscle-e‘‘mb. liver
0.466
-0.046
0.403
0.509
Probability levels: * \ 0.05, ** \ 0.01 Table 3 Spearman’s correlation coefficients between total length and metal contents of muscle and liver of Rhizoprionodon longurio embryos Cu Muscle Liver
Zn
-0.484 *
-0.582
-0.218 *
-0.564
Pb
Cd
-0.584*
-0.031
-0.646**
-0.382
Probability levels: * \ 0.05, ** \ 0.01
more abundant in the mothers’ muscle and Cd in the mothers’ liver. This could indicate preferential retention of essential metals by the embryos or embryo-related tissues, and explain the negative relation between mothers’ muscles and embryos’ livers in the case of Zn, while the positive correlation between the non essential metal contents of maternal muscle and embryonic livers could be due to unimpeded transfer of metals between mothers and embryos.
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The high content of Pb in the muscle of mothers and embryos, rather than in other tissues was most probably caused by placental Pb transport by diffusion associated to umbilical cord blood flow (Goyer 1990), or because of Pb ionic mimicry of Ca in ionic transport (Evans et al. 2003; Marchetti 2013), although this remains to be confirmed in the case of placental transport in sharks. In the case of Cd, the higher values were in the mothers’ livers, followed by those of the placenta, which could be related to Cd-induced metallothionein synthesis and by retention of this metal by these tissues. Cd retention in the rat placentas was considered by Hazelhoff-Roelfzema et al. (1988) as a protective mechanism for embryos, and Gundacker and Hengstschla¨ger (2012) explained the lower Cd levels in cord than in maternal blood, as due to Cd-induced metallothionein synthesis in human placentas. There are no previous studies on the mother to embryo metal transfer in placentotrophic sharks, but Cd-induced MT synthesis was described in shark male reproductive tissues by Betka and Callard (1999). If this ability was confirmed in other tissues, it might become at least a protective mechanism against this metal. Since the risks of pollutant toxicity to the fetus are greater than for the mother (Leino et al. 2013), this aspect is relevant for the welfare of the species, and should therefore be studied also in sharks. Maternal investment in pup survival after parturition seems to consist in the transfer of reserves to the embryos’ livers, which results in enlarged dimensions and weight of this organ (Hussey et al. 2010). This implies a higher growth rate and a consequent metal dilution in the liver, as compared to whole body tissues, which seems the most likely explanation of these inverse relations, rather than a difference in the rates of mother to embryo and embryo to mother transfers. Acknowledgments Supported by PROFAPI 2011/060, PROMEP 103.5/13/9354 and CONACYT INFRA 2012-01-188029 grants.
Bull Environ Contam Toxicol (2014) 93:532–535
References Betka M, Callard GV (1999) Stage-dependent accumulation of cadmium and induction of metallothionein-like binding activity in the testis of the dogfish shark, Squalus acanthias. Biol Reprod 60:14–22 Corro-Espinosa D, Ma´rquez-Farı´as JF, Muhlia-Melo A (2011) Size at maturity of the Pacific sharpnose shark Rhizoprionodon longurio in the Gulf of California, Mexico. Cienc Mar 37:201–214 Endo T, Hisamichi Y, Haraguchi K, Kato Y, Ohta C, Koga N (2008) Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: relationship between metal concentrations and body length. Mar Pollut Bull 56:1774–1780 Evans TJ, James-Kracke MR, Kleiboeker SB, Casteel SW (2003) Lead enters Rcho-1 trophoblastic cells by calcium transport mechanisms and complexes with cytosolic calcium-binding proteins. Toxicol Appl Pharmacol 186:77–89 Goyer RA (1990) Transplacental transport of lead. Environ Health Perspect 89:101–105 Gundacker C, Hengstschla¨ger M (2012) The role of the placenta in fetal exposure to heavy metals. Wien Med Wochenschr 162:201–206 Haines AN, Flajnik MF, Wourms JP (2006) Histology and immunology of the placenta in the atlantic sharpnose shark, Rhizoprionodon terraenovae. Placenta 27:1114–1123 Hazelhoff-Roelfzema W, Roelofsen AM, Herber RF, Peereboom-Steg JH (1988) Cadmium and zinc concentrations in fetal and maternal rat tissue after parental administration of cadmium during pregnancy. Arch Toxicol 62:285–290 Hurtado-Banda R, Gomez-Alvarez A, Ma´rquez-Farı´as JF, CordobaFigueroa M, Navarro-Garcı´a G, Medina-Jua´rez LA (2012) Total mercury in liver and muscle tissue of two coastal sharks from the northwest of Mexico. Bull Environ Contam Toxicol 88:971–975 Hussey NE, Wintner SP, Dudley SF, Cliff G, Cocks DT, MacNeil MA (2010) Maternal investment and size-specific reproductive output in carcharhinid sharks. J Anim Ecol 79:184–193 Leino O, Kiviranta H, Karjalainen AK, Kronberg-Kippila C, Sinkko H, Larsen EH, Virtanen S, Tuomisto JT (2013) Pollutant concentrations in placenta. Food Chem Toxicol 54:59–69 Lo´pez SA, Abarca NL, Mele´ndez R (2013) Heavy metal concentrations of two highly migratory sharks (Prionace glauca and Isurus oxyrinchus) in the southeastern Pacific waters: comments on public health and conservation. Trop Conserv Sci 6:126–137
535 Lyons K, Lowe CG (2013) Mechanisms of maternal transfer of organochlorine contaminants and mercury in the common thresher shark (Alopias vulpinus). Can J Fish Aquat Sci 70:1667–1672 Marchetti C (2013) Role of calcium channels in heavy metal toxicity. ISRN Toxicol Volume 2013, Article ID 184360. http://dx.doi. org/10.1155/2013/184360 Ma´rquez-Farı´as JF, Corro-Espinosa D, Castillo-Ge´niz JL (2005) Observations on the biology of the Pacific sharpnose shark (Rhizoprionodon longurio, Jordan and Gilbert, 1882), captured in southern Sinaloa Mexico. J N Atl Fish Sci 35:107–114 Mathews T, Fisher NS (2009) Dominance of dietary intake of metals in marine elasmobranch and teleosts fishes. Sci Total Environ 407:5156–5161 Moody JR, Lindstrom RN (1977) Selection and cleaning of plastic containers for storage of trace elements samples. Anal Chem 49:2264–2267 ´ Sullivan JB, Lowe CG Mull CG, Lyons K, Blasius ME, Winkler C, O (2013) Evidence of maternal offloading of organic contaminants in white sharks (Carcharodon carcharias). PLoS One 8:1–8 Olin JA, Beaudry M, Fisk AT, Paterson G (2014) Age-related polychlorinated biphenyl dynamics in immature bull sharks (Carcharhinus leucas). Environ Toxicol Chem 33:35–43 Porpora MG, Lucchini R, Abballe A, Ingelido AM, Valentini S, Fuggetta E, Cardi V, Ticino A, Marra V, Fulgenzi AR, De Felip E (2013) Placental transfer of persistent organic pollutants: a preliminary study on mother-newborn pairs. Int. J. Environ. Res. Public Health 10:699–711 Ruelas-Inzunza JR, Pa´ez-Osuna F (2007) Essential and toxic metals in nine fish species for human consumption from two coastal lagoons in the Eastern Gulf of California. J Environ Sci Health 42A:1411–1416 Storelli MM, Ceci E, Storelli A, Marcotrigiano GO (2003) Polychlorinated biphenyl, heavy metal and methylmercury residues in hammerhead sharks: contaminant status and assessment. Mar Pollut Bull 46:1035–1048 Vizcaino E, Grimalt JO, Ferna´ndez-Somoano A, Tardon A (2014) Transport of persistent organic pollutants across the human placenta. Environ Int 65:107–115 Zar JH (1999) Biostatistical analysis. Prentice Hall, Upper Saddle River
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Cadmium, Copper, Lead and Zinc Concentrations in Female and Embryonic Pacific Sharpnose Shark (Rhizoprionodon longurio) Tissues M. G. Frı´as-Espericueta • N. G. Cardenas-Nava • J. F. Ma´rquez-Farı´as J. I. Osuna-Lo´pez • M. D. Muy-Rangel • W. Rubio-Carrasco • D. Voltolina
•
Received: 12 March 2014 / Accepted: 13 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract In this work we compared the cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) contents of muscle, liver and placenta of gestating females of the viviparous shark Rhizoprionodon longurio and of muscle, liver and umbilical cord of their respective embryos. The higher values of the essential Cu and Zn were in embryonic or embryo-related tissues (placenta and umbilical cord). Maternal muscle and liver had the highest values of Pb and Cd, respectively. There were significant direct correlations between the Zn and Cd concentrations of placenta and umbilical cord, as well as between maternal muscle and embryonic livers for Pb and Cd, but the relation between these tissues was inverse in the case of Zn. All correlations between the metal content of embryonic tissues and size of the embryos were negative, suggesting an inverse relation between the rate of mother-to-embryo metal transfer and embryonic growth.
M. G. Frı´as-Espericueta J. F. Ma´rquez-Farı´as J. I. Osuna-Lo´pez Facultad de Ciencias del Mar, Universidad Auto´noma de Sinaloa, Paseo Claussen s/n, 82000 Mazatla´n, Sinaloa, Mexico N. G. Cardenas-Nava Programa de Posgrado en Recursos Acua´ticos Facultad de Ciencias del Mar, Universidad Auto´noma de Sinaloa, Mazatla´n, Sinaloa, Mexico M. D. Muy-Rangel W. Rubio-Carrasco CIAD, Unidad Culiaca´n, 80000 Culiaca´n, Sinaloa, Mexico D. Voltolina (&) Laboratorio de Estudios Ambientales UAS-CIBNOR, P.O. Box 1132, 82000 Mazatla´n, Sinaloa, Mexico e-mail: [email protected]
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Keywords Shark Rhizoprionodon longurio Placenta Embryos Metal transfer Metals can be taken up by aquatic organisms through gills or other exposed soft tissues, or because of ingestion of metal-rich diets, and accumulate in the organism’s tissues when the rate of uptake exceeds that of excretion. The most important route of metal uptake of sharks is through their diet (Mathews and Fisher 2009) and, depending on the preys consumed, metals may accumulate to relatively high levels in their tissues (Storelli et al. 2003). Since the reproductive mode of close to 30 % of all shark species is placental viviparity, embryos initially depend for nutrition on the yolk sac. When the yolk reserves are depleted the empty yolk sac implants into the uterine wall to form the yolk sac placenta (Haines et al. 2006), and embryos receive nutrients and metals from the mother through the umbilical cord. Several similarities in placental structure and function between mammals and viviparous sharks seem to indicate convergent evolution of the reproductive and immune systems between these two phylogenetically distant groups (Haines et al. 2006). However, such reproductive physiology has led to the consequence of maternal offloading of anthropogenic pollutants as described for both humans (Gundacker and Hengstschla¨ger 2012; Porpora et al. 2013; Vizcaino et al. 2014) and viviparous sharks (Lyons and Lowe 2013; Mull et al. 2013; Olin et al. 2014). The placental viviparous sharpnose shark (Rhizoprionodon longurio) is a highly migratory species, which appears in the Mexican Pacific coastal waters between November and April, at the height of its reproductive season, when it becomes an important prey for the Mexican Pacific artisanal fishing fleets. Therefore, pregnant females are common in
Bull Environ Contam Toxicol (2014) 93:532–535
the local landings (Ma´rquez-Farı´as et al. 2005). There are several studies on the metal content of shark tissues used for direct or indirect human consumption such as muscle and liver (Endo et al. 2008; Lo´pez et al. 2013) and there is at least one study on this particular species (Hurtado-Banda et al. 2012), but there is no information concerning the mother to embryo metal transfer in placentotrophic sharks. In this work we determined the concentrations and distributions of two essential (Cu and Zn) and two non-essential metals (Cd and Pb) in pregnant R. longurio mothers and in their embryos.
Materials and Methods Pregnant females (n = 15) were obtained between December 2010 and January 2011 from local fishermen of the Mazatla´n (SE Gulf of California) artisanal long line fishing fleet. In the laboratory, the females and the embryos of both uteri were measured (total length, TL) and dissected with a stainless steel knife to obtain liver, placenta and muscle of the females, and muscle, liver and umbilical cord of the embryos. Tissues were stored at -20 °C until processing. Glassware and materials were acid washed (Moody and Lindstrom 1977). Tissues were freeze-dried, homogenized in a Teflon mortar, and triplicate aliquots of known weight (0.5–1 g) were digested at 130 °C in a mod-block unit in 30 mL Teflon vessels with 5 mL of concentrated HNO3 (trace metal grade), transferred to polypropylene vials, diluted to 15 mL with Milli-Q water, and the metal content of each sample was determined by atomic absorption spectrophotometry (Varian Spectra AA) (Ruelas-Inzunza and Pa´ez-Osuna 2007). Blanks were used for accuracy (ranges: Cu = 0–0.013; Zn 0–0.312; Pb = 0.310–0.420; Cd = 0.014–0.33 mg/L, respectively) and for detection limits, which were 0.01 lg/g for Cd, 0.009 lg/g for Pb, 0.09 lg/g for Cu and 0.03 lg/g for Zn. Analysis of certified reference material (DOLT-4, National Research Council Canada) gave recovery values of 91.5 % for Cu, 96.2 % for Cd, 103.4 % for Zn and 105.6 % for Pb (n = 3 in all cases). The mean size and weight of the embryos found in the right and left uterus were similar (Wilkinson’s tests for paired samples p [ 0.05). Therefore, we assumed that they were of similar age and the tissues of embryos obtained in right and left uterus were pooled and analyzed as single samples. The mean content of each metal of the different maternal tissues (including cords and placentas), as well as those of each placenta and of liver and muscle of the respective embryos, were compared with non parametric one-way block ANOVA (Friedman’s) tests because Kolmogorov–Smirnov’s and Bartlett´s tests showed that data were not normal or not homoscedastic. Differences
533
were identified with Dunn’s tests. Possible relations between the metal values found in the tissues of each mother and its respective embryos, and between the metal content and the mean TL of each composite sample of embryos were determined with Spearman’s correlations tests. All tests were with a = 0.05 (Zar 1999).
Results Size at first maturity of R. longurio females is 92.9 ± 0.94 cm TL (Corro-Espinosa et al. 2011). The 15 pregnant females ranged from 96 to 118 cm TL (mean 110.93 ± 5.30 cm), and therefore none could be considered primiparous. They carried a total of 121 embryos (6–10 embryos/female), with a maximum of five embryos/ uterus, all connected to the uterus through placenta and umbilical cord, and ranging in size from 20.0 to 27.8 cm. Their weight ranged from 27.7 to 62.3 g, and the male: female ratio of embryos was 1:1.2. The mean Cu contents were between 5 and 20 times lower than those of Zn, with the exception of the liver of embryos: The orders of concentration of each tissue were different, and the significant differences (p \ 0.05) for Cu were embryonic liver [ embryonic muscle, and of maternal liver and muscle. In the case of Zn, the only significant differences were placenta [ maternal liver and muscle, and the remaining tissues had intermediate values (Table 1). There were also significant differences in the contents of the nonessential Pb and Cd: the Pb content of the mothers’ muscle was significantly higher than those of placenta and cord. All female livers had a Pb content below limit of detection, while the value of Cd was significantly higher (p \ 0.05) than cord and maternal and embryonic muscles (Table 1). There were significant direct correlations between the Zn and Cd contents of cord and placenta, as well as between the Pb and Cd contents of the maternal muscle with the embryonic liver, while the Zn content of the maternal muscle was inversely related to that of the embryonic liver. The correlations between the metal contents of embryonic liver and muscle and between these tissues with their respective placentas were not significant (Table 2). All correlations calculated between the mean TL of the embryos and the metal content of the respective muscle and liver samples were inverse. In the case of the muscle, the only significant value was that of Pb, while in the case of the liver only Cd was not significantly related to TL (Table 3).
Discussion The higher concentrations of Cu and Zn were in embryonic tissues, and in placenta and umbilical cord, whereas Pb was
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Bull Environ Contam Toxicol (2014) 93:532–535
Table 1 Mean metal contents ± standard deviation (lg/g, dry weight) of the tissues of R. longurio females (F) and embryos (E) Cu
Zn
Pb
Cd
v2
59.25
45.49
48.76
49.71
Muscle (F)
1.19 ± 0.24a
16.96 ± 2.29a
4.96 ± 5.88b
0.03 ± 0.03a
Liver (F)
2.43 ± 0.75ab
12.56 ± 4.19a
\LD
1.67 ± 1.19c
Placenta (F)
9.44 ± 1.14bc
45.64 ± 14.36b
0.04 ± 0.07a
0.29 ± 0.27bc
Cord (E)
6.27 ± 4.56bc
29.13 ± 15.23ab
0.24 ± 0.41a
0.14 ± 0.08ab
Muscle (E)
4.10 ± 0.90ab
34.52 ± 10.79ab
2.08 ± 2.03ab
0.08 ± 0.14ab
Liver (E)
46.24 ± 13.89c
23.64 ± 6.65ab
1.43 ± 1.23ab
0.18 ± 0.13abc
\LD = below limit of detection, not used in statistical analysis Equal or common letters indicate lack of significant difference between data in the same column (Friedman tests, p \ 0.05). n = 15 in all cases. v25 values are indicated for the test with each metal Table 2 Spearman’s correlation coefficients between the metal content of placenta, umbilical cord, maternal (Mat) and embryonic (Emb) tissues of R. longurio Tissues
Cu
Placenta-cord
-0.054
Mat liver-emb. muscle
-0.046
0.025
–
0.015
Mat liver-emb. liver
-0.020
0.064
–
0.261
0.193
-0.050
0.351
0.168
-0.754**
0.746**
0.516*
Mat muscle-emb. muscle Mat muscle-emb. liver Placenta-emb. muscle Placenta-emb. liver Cord-emb. muscle
Zn
-0.254
Pb
0.887**
0.407
0.198
-0.293
-0.112
Cd 0.842**
-0.003
0.499
0.157
0.413
0.289
-0.452
-0.167
-0.064
-0.122
Cord-emb. liver
0.286
0.024
0.436
0.095
Emb.muscle-e‘‘mb. liver
0.466
-0.046
0.403
0.509
Probability levels: * \ 0.05, ** \ 0.01 Table 3 Spearman’s correlation coefficients between total length and metal contents of muscle and liver of Rhizoprionodon longurio embryos Cu Muscle Liver
Zn
-0.484 *
-0.582
-0.218 *
-0.564
Pb
Cd
-0.584*
-0.031
-0.646**
-0.382
Probability levels: * \ 0.05, ** \ 0.01
more abundant in the mothers’ muscle and Cd in the mothers’ liver. This could indicate preferential retention of essential metals by the embryos or embryo-related tissues, and explain the negative relation between mothers’ muscles and embryos’ livers in the case of Zn, while the positive correlation between the non essential metal contents of maternal muscle and embryonic livers could be due to unimpeded transfer of metals between mothers and embryos.
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The high content of Pb in the muscle of mothers and embryos, rather than in other tissues was most probably caused by placental Pb transport by diffusion associated to umbilical cord blood flow (Goyer 1990), or because of Pb ionic mimicry of Ca in ionic transport (Evans et al. 2003; Marchetti 2013), although this remains to be confirmed in the case of placental transport in sharks. In the case of Cd, the higher values were in the mothers’ livers, followed by those of the placenta, which could be related to Cd-induced metallothionein synthesis and by retention of this metal by these tissues. Cd retention in the rat placentas was considered by Hazelhoff-Roelfzema et al. (1988) as a protective mechanism for embryos, and Gundacker and Hengstschla¨ger (2012) explained the lower Cd levels in cord than in maternal blood, as due to Cd-induced metallothionein synthesis in human placentas. There are no previous studies on the mother to embryo metal transfer in placentotrophic sharks, but Cd-induced MT synthesis was described in shark male reproductive tissues by Betka and Callard (1999). If this ability was confirmed in other tissues, it might become at least a protective mechanism against this metal. Since the risks of pollutant toxicity to the fetus are greater than for the mother (Leino et al. 2013), this aspect is relevant for the welfare of the species, and should therefore be studied also in sharks. Maternal investment in pup survival after parturition seems to consist in the transfer of reserves to the embryos’ livers, which results in enlarged dimensions and weight of this organ (Hussey et al. 2010). This implies a higher growth rate and a consequent metal dilution in the liver, as compared to whole body tissues, which seems the most likely explanation of these inverse relations, rather than a difference in the rates of mother to embryo and embryo to mother transfers. Acknowledgments Supported by PROFAPI 2011/060, PROMEP 103.5/13/9354 and CONACYT INFRA 2012-01-188029 grants.
Bull Environ Contam Toxicol (2014) 93:532–535
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