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Persistent organic pollutants and histological lesions in Mayan catfish Ariopsis assimilis from the Bay of Chetumal, Mexico
Marine Pollution Bulletin 48 (2004) 263–269 www.elsevier.com/locate/marpolbul
Persistent organic pollutants and histological lesions in Mayan catfish Ariopsis assimilis from the Bay of Chetumal, Mexico a E. Nore~ na-Barroso a, R. Sim a-Alvarez , G. Gold-Bouchot a
a,b,*
, O. Zapata-Perez
a
Centro de Investigaci on y Estudios Avanzados del I.P.N. Unidad M erida, Km 6 Antigua Carretera a Progreso A.P. 73 Cordemex, 97310 M erida, Yucat an, Mexico b Centro EPOMEX, Av. Agustin Melgar S/N, Campeche, Mexico
Abstract Livers of catfish (Ariopsis assimilis) from the Bay of Chetumal were analyzed for organochlorine compounds and hydrocarbons as part of a study to diagnose the environmental health of the Bay after a catfish mass mortality that occurred in 1996. The presence of histological lesions in several organs of the fish as result of chemical exposure was also evaluated. The concentrations of organic pollutants found in the Bay may be considered high if compared to the levels reported for sites affected by chemical pollution. High prevalences of cellular alteration histopathologies were found in liver, including hepatic tumors. The presence of some lesions may be related statistically to environmental pollution in the Bay, specially with chlorinated compounds. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Organochlorines; Hydrocarbons; Catfish; A. assimilis; Histopathology
Between May and October 1996, several thousand Mayan catfish (Ariopsis assimilis) died in the Bay of Chetumal, a coastal system located on the border between Mexico and Belize. A few years before, the presence of organochlorine pesticides in sediments was reported (Ortiz-Hern andez et al., 1997). In addition, preliminary studies in the liver of a few catfish made during the fish kill showed high hydrocarbon and pesticide content, as well as histological lesions in gills and liver, probably related to the presence of organic pollutants (Vidal et al., 1996). Given the situation mentioned before, the aim of this study was to take part in an integrated environmental evaluation of the Bay after the fish kill, through the determination of hydrocarbon and chlorinated compound levels in the liver of catfish that survived the mass mortality event, and the histological analysis of several fish organs to determine possible relationships between chemical pollution and biological effects in catfish. As part of this integrated study, concentrations of organic * Corresponding author. Address: Centro de Investigaci on y Estudios Avanzados del I.P.N. Unidad Merida, Km 6 Antigua Carretera a Progreso A.P. 73 Cordemex, 97310 Merida, Yucatan, Mexico. Tel.: +52-99-812960x530; fax: +52-99-812923. E-mail address: [email protected] (G. Gold-Bouchot).
0025-326X/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2003.08.001
pollutants in sediments of the Bay were also determined (Nore~ na-Barroso et al., 1998) and sediment toxicity was tested (Zapata-Perez et al., 2000). The information generated can be useful in conservation programs for the Bay, which has been declared a refuge for the West Indies manatee (Trichechus manatus). Seventy-six catfish (A. assimilis) were collected at five stations along the northern part of the Bay of Chetumal in October 1996 (Fig. 1). Livers were removed and sectioned for chemical analysis; a portion was preserved in liquid nitrogen and transported to the laboratory. Sections of liver, kidney, spleen and gills of each fish were fixed in 10% formalin solution buffered with sodium phosphate for histological analysis. Fish otoliths were removed for age determination, and sex recorded. Concentrations of hydrocarbons and chlorinated organic compounds were determined according to procedures described in Sericano et al. (1990). Freeze-dried liver tissue was extracted with hexane and methylene chloride. Extracts were fractionated in an alumina:silica column. Lipids were removed from the aromatic fractions by size exclusion chromatography. Organic compounds were determined by gas chromatography using a Hewlett Packard 5890 Series II gas chromatograph equipped with a 30 m 0.25 mm HP-5 column. Hydrocarbons were quantified with a flame ionization
264
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
Fig. 1. The Bay of Chetumal and localization of the sites where catfish were collected. Numbers in parenthesis indicate the number of fish collected at each site.
Table 1 Organochlorine pesticides (ng g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
IQ range
1,2,4,5-TCB 1,2,3,4-TCB Pentachlorobenzene HCB Pentachloroanisole a-HCH b-HCH c-HCH Heptachlor Heptachlor epoxide a-Chlordane c-Chlordane Trans-nonachlor Cis-nonachlor Aldrin Dieldrin Endrin Endosulfan II 2,40 -DDE 4,40 -DDE 4,40 -DDD 4,40 -DDT Mirex
25.08 63.30 126.21 52.74 78.57 43.67 26.96 46.42 48.04 31.62 42.59 64.21 32.96 35.64 277.60 32.91 40.04 26.57 9.73 315.30 427.95 84.89 72.04
0.00 0.00 2.97 5.99 1.97 0.00 0.00 6.53 6.40 0.01 5.38 3.72 2.49 0.00 0.00 4.63 3.18 0.00 0.00 17.14 1.18 3.18 0.00
4.46 5.42 12.27 11.55 7.32 7.70 7.47 13.45 14.75 0.28 8.43 8.05 9.02 3.33 14.54 8.71 12.32 3.73 0.00 25.78 9.38 11.09 7.62
Total pesticides
795.80
143.65
125.00
detector and chlorinated pesticides and PCBs with an electron capture detector. Quality assurance of the analytical procedures included addition of internal standards and analysis of a procedural blank for each set of
samples. For histology, tissues were dehydrated and embedded in paraffin. Tissue sections (0.5 lm) were stained with haematoxylin and eosin, and periodic acid and Schiff.
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
265
Table 2 Polychlorinated biphenyls (ng g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
IQ range
PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB
112.27 30.50 551.83 28.28 41.90 22.10 540.71 7.53 33.20 25.55 30.66 29.24 52.95 88.99 1603.16 32.99 33.58 28.69 19.18 46.26
2.18 4.07 5.88 0.00 0.00 1.44 0.90 0.00 4.19 0.00 0.00 0.00 4.02 5.77 28.64 0.60 4.28 0.00 0.11 3.25
12.67 7.66 9.43 2.62 3.13 5.30 8.95 1.84 7.14 3.66 5.94 2.26 8.38 11.08 2.99 5.81 9.93 4.43 4.08 6.78
1646.72
83.84
99.86
8 18 28 29 44 52 66 87 101 110 118 128 138 153 170 180 187 195 200 206
Total PCBs
Table 3 Polynuclear aromatic hydrocarbons (lg g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
Naphthalene 1-Methylnaphthalene 1,2,4-Triethylbenzene 1,3,5-Triethylbenzene Acenaphthylene Acenaphthene Fluorene Phenanthrene 1-Methylphenanthrene 2-Methylphenanthrene Anthracene Fluoranthene Pyrene 1-Methylpyrene Benzo[a]anthracene Chrysene Benzo[a]pyrene Perylene
1.43 4.52 0.16 0.63 2.31 1.27 3.22 3.22 38.18 31.46 3.34 132.55 5.16 16.89 114.74 4.80 752.19 356.94
0.00 0.59 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.77 0.15 1.69 0.00 0.28 0.97 0.00 30.31 0.00
0.00 1.11 0.00 0.00 0.71 0.35 0.71 1.15 0.62 1.44 0.70 8.24 1.47 1.32 4.69 0.00 88.18 18.61
Total PAHs
762.24
77.06
103.49
Catfish total length ranged from 24.5 to 42.0 cm, and age from 2 to 6 years. Most fish were 4 and 5 years old. In relation to sex, 58 organisms were females, five males (for 13 fish, sex was not recorded). Twenty-three individual pesticides were identified in livers (Table 1). DDT and its metabolites (28.7 ± 42.4 ng g1 ), and total P chlordanes ( heptachlors, chlordanes and nonachlors; 26.2 ± 29.2 ng g1 ) had greatest median concentrations. Total organochlorine pesticides ranged from 39.1 to 795.8 ng g1 with a median value of 143.7 ± 125.0 ng g1 .
IQ range
On the other hand, twenty different PCB congeners were found in the samples analyzed (Table 2). PCBs were grouped according to the number of chlorine atoms in their structure, with highest median concentrations for trichlorobiphenyls (triCBs) and hexachlorobiphenyls (hexaCBs), 14.5 ± 19.0 and 13.1 ± 17.8 ng g1 , respectively. The minimum value of total PCBs was 10.9 ng g1 and the maximum 1646.7 ng g1 , with a median concentration of 83.8 ± 99.9 ng g1 . Highly chlorinated congeners such as hexaCBs are known to magnify in
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Fig. 2. Variation of organic compounds content in catfish (Ariopsis assimilis) from Bay of Chetumal in relation to sex and age of the organisms.
food chains and to accumulate in higher organisms (Erickson, 1997). Published studies dealing with the determination of persistent organic pollutant (POPs) levels in fish from Mexican coastal lagoons are scarce, and all determined contaminants were in muscle, so their results are not comparable with ours. McCain et al. (1996) evaluated chlorinated compounds in liver of hardhead catfish Arius felis (a species closely related to A. assimilis) from highly polluted sites in Tampa P Bay, FL. These authors found mean chlordane levels ( chlordane and transnonachlor) similar to the mean detected in the Bay of Chetumal (35.6 ± 14.9 and 14.28 ± 16.31 ng g1 dry-wt, respectively), but mean values of total DDTs (228.9 ± 214.0 ng g1 dry-wt) and PCBs (760 ng g1 drywt) for Tampa Bay were much higher than those we
found (54.06 ± 88.80 and 139.15 ± 210.17 ng g1 , respectively). Regarding PAHs, 18 individual compounds were identified in livers. Concentration values are given in Table 3, with benzo[a]pyrene standing out with the highest median (30.3 ± 88.2 lg g1 ). Total PAHs ranged from 6.6 to 762.2 lg g1 , with a median of 77.1 ± 103.5 lg g1 . Predominance of high molecular (five rings) PAHs was observed with a concentration of 41.5 ± 81.7 lg g1 . Also, there was a clear predominance of parent compounds over alkyl derivatives. The same pattern was observed in sediments, where it was considered as evidence of a pyrogenic source of these compounds in the Bay (Nore~ na-Barroso et al., 1998). Variation of persistent organic pollutant levels in relation to the age and sex of the fish analyzed is shown in
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
Fig. 2. Pesticides, PCBs and PAHs follow the same trend. In females, the concentration of chemicals decrease with age. In males the tendency is inverse, with highest concentrations found in 5-year-old fish. However, this variation should be carefully considered since only a few organisms ages two and six were found. The same can be said about differences related to sex, considering that most fish analyzed were females. Organic compound accumulation in fish depends on lipid content, age, sex and diet of the organisms (Elskus et al., 1994). It has been reported that lipid content in fish increases in relation to age and size; however, there are other factors that should be considered such as metabolic rate, reproductive status, season of the year and activity pattern of the organism (Weatherley and Gill, 1987). Lack of information on the basic biology of A. assimilis makes it difficult to explain our results. Since age data are not available for all 76 catfish analyzed, and considering that catfish age is highly correlated to total length (Spearman’s R ¼ 0:9460; p < 0:05), non-parametric Spearman’s R correlation coefficients were calculated to evaluate the relationships between total length and the levels of the different organic compounds found in liver. This provides indirect evidence of any association between chemicals and age. Total length was negatively correlated with several pesticides such as a-HCH (R ¼ 0:2343; p < 0:05), heptachlor (R ¼ 0:3373; p < 0:05), aldrin (R ¼ 0:2893; p < 0:05) and dieldrin (R ¼ 0:2715; p < 0:05). Moreover, length was also negatively correlated with groups of PCBs having highest median concentrations: triCBs (R ¼ 0:2285; p < 0:05) and hexaCBs (R ¼ 0:2954; p < 0:05). No significant correlations were observed between PAHs and total length, except a positive one with 1,3,5-triethylbenzene (R ¼ 0:2369; p < 0:05). A multivariate analysis of variance indicated significant differences in organic pollutant content among sites (Wilks’ k ¼ 0:70; p < 0:05). A post-hoc Tukey (HSD) test showed differences in liver pesticide and PAH content at El Ramonal with those from other sampling stations, but none for PCB content between sites. On the other hand, even though the major sources of pollutants are located on the west coast of the Bay, higher bioaccumulation of organic chemicals took place in fish from the east coast. This pattern corresponds to that observed in sediments, and may be related to an accumulation of these compounds in sediments, depending on grain size and organic matter content (Nore~ na-Barroso et al., 1998). Most catfish analyzed (98.7%) had at least one histological lesion in liver, kidney and gills. The prevalence of the different lesions observed in each organ is given in Table 4. A high prevalence of liver damage involving cellular degeneration and granuloma (60.5%), as well as an increase in the number of hepatocytes and hyperaemia (69.7%) were found. Cellular alteration foci can be
267
Table 4 Prevalence of histological lesions observed in catfish Ariopsis assimilis from Bay of Chetumal, Mexico Lesion
Number of fish
%
Liver Hyperaemia Lymphocyte infilt Granuloma Pycnosis Tumors
53 50 46 11 6
69.74 65.79 60.53 14.47 7.89
Kidney Glomerulonefritis Calcium deposits Granuloma
58 45 2
76.32 59.21 2.63
Gills Hypertrophy Hyperplasia Edema Telangiectasis
74 60 52 39
97.37 78.95 68.42 51.32
Spleen Hemosiderosis
59
77.63
considered preneoplastic lesions or precursors in hepatic neoplasms histogenesis (Hawkins et al., 1990). The prevalences of hepatic lesions involving cellular alteration registered in this study are similar to those reported by McCain et al. (1988) for highly polluted urban zones of the USA west coast (10–36%). Among hepatic lesions, the presence of granulomas and tumors (7.9%) stand out in severity. Tumor prevalence is within the same range (2–18%) reported by Malins et al. (1988) for several sites in Puget Sound, and it is higher than the 2.2% found in a closely related catfish species (A. felis) from Tampa Bay (McCain et al., 1996). In addition, hemosiderosis in the spleen was detected in 77.6% of the fish analyzed. Hemosiderosis usually occurs as a result of excessive or accelerated breakdown of erythrocytes in vertebrates and the break down product, hemosiderin (a pigment that contains iron), can be found in hematopoietic tissue and spleen (Thiyagarajah et al., 1998). Hemosiderosis suggests fish anemia due to erythrocyte destruction (Estrada and Garcıa, 1991). Liver, spleen and kidney hemosiderosis in fish has been associated with the presence of organic pollutants in the environment (Thiyagarajah et al., 1998). Significant logistic correlations between histological lesions in catfish and organic contaminants in liver are given in Table 5. Chlorinated compounds show a stronger effect on the presence of lesions, since they were significantly related to a higher number of histological lesions than PAHs. This might be due to the fact that biliary PAH metabolites were not measured. PAHs in fish are biotransformed in the liver and readily excreted to biliary products (Varanasi et al., 1989). Chlorinated pesticides were associated with the presence of histopathologies in
268
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
Table 5 Relationship between organic compounds and histological lesions in catfish Ariopsis assimilis from Bay of Chetumal, Mexico Lesion Pesticides Hyperaemia (L) Pycnosis (L) Tumors (L) Hyperplasia (G) Ca deposits (K) Hemosiderosis (S) PCBs Granuloma (L) Pycnosis (L) Edema (G) Telangiectasis (G) Glomerulonefritis (K) PAHs Hypertrophy (G) Hyperplasia (G) Glomerulonefritis (K)
Compound
p-Value
Pentachlorobenzene Endosulfan II Dieldrin Dieldrin Endosulfan II Mirex DDTs
0.0399 0.0149 0.0011 0.0144 0.0299 0.0089 0.0092
HexaCBs TetraCBs TetraCBs TetraCBs HeptaCBs NonaCBs Total PCBs
0.0418 0.0181 0.0242 0.0186 0.0494 0.0035 0.0083
4-Ring PAHs 2-Ring PAHs 4-Ring PAHs
0.0466 0.0406 0.0381
Explained deviance (%) 4.62 16.94 14.28 6.02 6.66 8.39 4.06 8.89 5.36 7.73 13.02
21.41 5.36 5.17
Logistic regression by steps (n ¼ 76). L ¼ liver; G ¼ gills; K ¼ kidney; S ¼ spleen. * Significant correlations.
all organs examined, especially Endosulfan II, which is highly toxic to fish (Miles and Pfeuffer, 1997), and Dieldrin, related to hepatic cellular alteration and neoplastic damage. Total DDTs (found in elevated levels in liver and sediments) showed a significant correlation with spleen hemosiderosis. In relation to PCBs, tetrachlorobiphenyls influenced the appearance of histological damage in liver as well as in gills. Hexachlorobiphenyls (having the second highest concentration in liver) are associated with hepatic granulomas, while PAHs may be related to hepatic hyperaemia and gill hyperplasia. The degree of association between contaminants and lesion prevalences, and explained variance, reported here are similar to those reported in the review by Malins et al. (1988). Association between fish histopathologies and the presence of organic pollutants should be carefully studied given the histological lesions observed. Relatively high concentrations of pollutants were found in Mayan catfish, and some of these pollutants, particularly chlorinated compounds such as pesticides and polychlorobiphenyls, are associated with the presence of histological lesions. Also, high levels of PAHs in fish livers deserve more consideration. The fact that most catfish analyzed had at least one lesion in liver, kidney and gills, suggests that fish in the Bay are in bad condition. Given that fish were collected several months after the mass mortality event the relationship between the mortality and these results cannot be assessed.
Acknowledgements We acknowledge Pablo Ivan Caballero (CETMar No. 10) for his support for sample collection and experience on the study area. We also thank Claudia Leonce for her help analyzing otoliths and to Gregory Arjona for his help with histological samples. References Elskus, A.A., Stegeman, J.J., Gooch, J.W., Black, D.E., Pruell, R.J., 1994. Polychlorinated biphenyl congener distributions in winter flounder as related to gender, spawning site, and congener metabolism. Environmental Science and Technology 28, 401–407. Erickson, M.D., 1997. Analytical Chemistry of PCB’s, second ed. CRC Press, Boca Raton, FL. p. 667. Estrada, R.R., Garcıa, P.B., 1991. Atlas de Histopatologıa. Progreso, Mexico. Hawkins, W.E., Walker, W.W., Overstreet, R.M., Lytle, J.S., Lytle, T.E., 1990. Carcinogenetic effects of some polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures. Science of Total Environment 94, 155–167. Malins, D.C., McCain, B.B., Landahl, J.T., Myers, M.S., Krahn, M.M., Brown, D.W., Chan, S.L., Roubal, W.T., 1988. Neoplastic and other diseases in fish in relation to toxic chemicals: an overview. Aquatic Toxicology 11, 43–67. McCain, B.B., Brown, D.W., Krahn, M.M., Myers, M.S., Clark, R.C., Chan, S., Malins, D.C., 1988. Marine pollution problems, North American West Coast. Aquatic Toxicology 11, 143–162. McCain, B.B., Brown, D.W., Horn, T., Myers, M.S., Pierce, S.M., Collier, T.K., Stein, J.E., Chan, S., Varanasi, U., 1996. Chemical
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269 contaminant exposure and effect in four fish species from Tampa Bay, Florida. Estuaries 19, 86–104. Miles, C.J., Pfeuffer, R.J., 1997. Pesticides in canals of South Florida. Archives of Environmental Contamination and Toxicology 32, 337–345. Nore~ na-Barroso, E., Zapata-Perez, O., Ceja-Moreno, V., Gold-Bouchot, G., 1998. Hydrocarbon and organochlorine residue concentrations in sediments from Bay of Chetumal, Mexico. Bulletin of Environmental Contamination and Toxicology 61, 80–87. Ortiz-Hern andez, M.C., Saenz, R., Zavala, A., 1997. Plaguicidas organoclorados. In: Estudio Emergente sobre la Mortandad de Bagres en la Bahia de Chetumal. Informe Tecnico, Secretarıa de Infraestructura, Medio Ambiente y Pesca, Gobierno del Estado Quintana Roo, El Colegio de la Frontera Sur Unidad Chetumal, Chetumal, Q. Roo, Mexico. Sericano, J.L., Atlas, E.L., Wade, T.L., Brooks, J.M., 1990. NOAA’s status and trends mussel watch program: chlorinated pesticides and PCBs in oyters (Crassostrea virginica) and sediments from the Gulf of Mexico, 1986–1987. Marine Environmental Research 29, 161– 203.
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Thiyagarajah, A., Harley, W.R., Abdelghani, A., 1998. Hepatic hemosiderosis in buffalo fish (Ictiobus spp.). Marine Environmental Research 46 (1–5), 203–207. Varanasi, U., Stein, J.E., Nishimoto, M., Varanasi, U., 1989. Biotransformation and disposition of polycyclic aromatic hydrocarbons in fish. In: Varanasi, U. (Ed.), Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. CRC Press, Boca Raton, FL, pp. 93–149. Vidal, M.V.M., Sima, R., Gold, G., Zapata, O., 1996. Informe parcial del estudio sobre la mortalidad del bagre (Arius felis) de la Bahıa de Chetumal, Quintana Roo, Informe Tecnico, CINVESTAV IPN, Unidad Merida, Merida, Yucatan, Mexico. Weatherley, A.H., Gill, H.S., 1987. The Biology of Fish Growth. Academic Press, Great Britain. Zapata-Perez, O., Sima-Alvarez, R., Nore~ na-Barroso, E., G€ uemes, J., Gold-Bouchot, G., Ortega, A., Albores-Medina, A., 2000. Toxicity of sediments from Bahıa de Chetumal, Mexico, as assessed by hepatic EROD induction and histology in Nile tilapia Oreochromis niloticus. Marine Environmental Research 50, 385– 391.
Persistent organic pollutants and histological lesions in Mayan catfish Ariopsis assimilis from the Bay of Chetumal, Mexico a E. Nore~ na-Barroso a, R. Sim a-Alvarez , G. Gold-Bouchot a
a,b,*
, O. Zapata-Perez
a
Centro de Investigaci on y Estudios Avanzados del I.P.N. Unidad M erida, Km 6 Antigua Carretera a Progreso A.P. 73 Cordemex, 97310 M erida, Yucat an, Mexico b Centro EPOMEX, Av. Agustin Melgar S/N, Campeche, Mexico
Abstract Livers of catfish (Ariopsis assimilis) from the Bay of Chetumal were analyzed for organochlorine compounds and hydrocarbons as part of a study to diagnose the environmental health of the Bay after a catfish mass mortality that occurred in 1996. The presence of histological lesions in several organs of the fish as result of chemical exposure was also evaluated. The concentrations of organic pollutants found in the Bay may be considered high if compared to the levels reported for sites affected by chemical pollution. High prevalences of cellular alteration histopathologies were found in liver, including hepatic tumors. The presence of some lesions may be related statistically to environmental pollution in the Bay, specially with chlorinated compounds. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Organochlorines; Hydrocarbons; Catfish; A. assimilis; Histopathology
Between May and October 1996, several thousand Mayan catfish (Ariopsis assimilis) died in the Bay of Chetumal, a coastal system located on the border between Mexico and Belize. A few years before, the presence of organochlorine pesticides in sediments was reported (Ortiz-Hern andez et al., 1997). In addition, preliminary studies in the liver of a few catfish made during the fish kill showed high hydrocarbon and pesticide content, as well as histological lesions in gills and liver, probably related to the presence of organic pollutants (Vidal et al., 1996). Given the situation mentioned before, the aim of this study was to take part in an integrated environmental evaluation of the Bay after the fish kill, through the determination of hydrocarbon and chlorinated compound levels in the liver of catfish that survived the mass mortality event, and the histological analysis of several fish organs to determine possible relationships between chemical pollution and biological effects in catfish. As part of this integrated study, concentrations of organic * Corresponding author. Address: Centro de Investigaci on y Estudios Avanzados del I.P.N. Unidad Merida, Km 6 Antigua Carretera a Progreso A.P. 73 Cordemex, 97310 Merida, Yucatan, Mexico. Tel.: +52-99-812960x530; fax: +52-99-812923. E-mail address: [email protected] (G. Gold-Bouchot).
0025-326X/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2003.08.001
pollutants in sediments of the Bay were also determined (Nore~ na-Barroso et al., 1998) and sediment toxicity was tested (Zapata-Perez et al., 2000). The information generated can be useful in conservation programs for the Bay, which has been declared a refuge for the West Indies manatee (Trichechus manatus). Seventy-six catfish (A. assimilis) were collected at five stations along the northern part of the Bay of Chetumal in October 1996 (Fig. 1). Livers were removed and sectioned for chemical analysis; a portion was preserved in liquid nitrogen and transported to the laboratory. Sections of liver, kidney, spleen and gills of each fish were fixed in 10% formalin solution buffered with sodium phosphate for histological analysis. Fish otoliths were removed for age determination, and sex recorded. Concentrations of hydrocarbons and chlorinated organic compounds were determined according to procedures described in Sericano et al. (1990). Freeze-dried liver tissue was extracted with hexane and methylene chloride. Extracts were fractionated in an alumina:silica column. Lipids were removed from the aromatic fractions by size exclusion chromatography. Organic compounds were determined by gas chromatography using a Hewlett Packard 5890 Series II gas chromatograph equipped with a 30 m 0.25 mm HP-5 column. Hydrocarbons were quantified with a flame ionization
264
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
Fig. 1. The Bay of Chetumal and localization of the sites where catfish were collected. Numbers in parenthesis indicate the number of fish collected at each site.
Table 1 Organochlorine pesticides (ng g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
IQ range
1,2,4,5-TCB 1,2,3,4-TCB Pentachlorobenzene HCB Pentachloroanisole a-HCH b-HCH c-HCH Heptachlor Heptachlor epoxide a-Chlordane c-Chlordane Trans-nonachlor Cis-nonachlor Aldrin Dieldrin Endrin Endosulfan II 2,40 -DDE 4,40 -DDE 4,40 -DDD 4,40 -DDT Mirex
25.08 63.30 126.21 52.74 78.57 43.67 26.96 46.42 48.04 31.62 42.59 64.21 32.96 35.64 277.60 32.91 40.04 26.57 9.73 315.30 427.95 84.89 72.04
0.00 0.00 2.97 5.99 1.97 0.00 0.00 6.53 6.40 0.01 5.38 3.72 2.49 0.00 0.00 4.63 3.18 0.00 0.00 17.14 1.18 3.18 0.00
4.46 5.42 12.27 11.55 7.32 7.70 7.47 13.45 14.75 0.28 8.43 8.05 9.02 3.33 14.54 8.71 12.32 3.73 0.00 25.78 9.38 11.09 7.62
Total pesticides
795.80
143.65
125.00
detector and chlorinated pesticides and PCBs with an electron capture detector. Quality assurance of the analytical procedures included addition of internal standards and analysis of a procedural blank for each set of
samples. For histology, tissues were dehydrated and embedded in paraffin. Tissue sections (0.5 lm) were stained with haematoxylin and eosin, and periodic acid and Schiff.
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
265
Table 2 Polychlorinated biphenyls (ng g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
IQ range
PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB
112.27 30.50 551.83 28.28 41.90 22.10 540.71 7.53 33.20 25.55 30.66 29.24 52.95 88.99 1603.16 32.99 33.58 28.69 19.18 46.26
2.18 4.07 5.88 0.00 0.00 1.44 0.90 0.00 4.19 0.00 0.00 0.00 4.02 5.77 28.64 0.60 4.28 0.00 0.11 3.25
12.67 7.66 9.43 2.62 3.13 5.30 8.95 1.84 7.14 3.66 5.94 2.26 8.38 11.08 2.99 5.81 9.93 4.43 4.08 6.78
1646.72
83.84
99.86
8 18 28 29 44 52 66 87 101 110 118 128 138 153 170 180 187 195 200 206
Total PCBs
Table 3 Polynuclear aromatic hydrocarbons (lg g1 dry-wt) in catfish Ariopsis assimilis liver from Bay of Chetumal, Mexico Compounds
Max
Median
Naphthalene 1-Methylnaphthalene 1,2,4-Triethylbenzene 1,3,5-Triethylbenzene Acenaphthylene Acenaphthene Fluorene Phenanthrene 1-Methylphenanthrene 2-Methylphenanthrene Anthracene Fluoranthene Pyrene 1-Methylpyrene Benzo[a]anthracene Chrysene Benzo[a]pyrene Perylene
1.43 4.52 0.16 0.63 2.31 1.27 3.22 3.22 38.18 31.46 3.34 132.55 5.16 16.89 114.74 4.80 752.19 356.94
0.00 0.59 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.77 0.15 1.69 0.00 0.28 0.97 0.00 30.31 0.00
0.00 1.11 0.00 0.00 0.71 0.35 0.71 1.15 0.62 1.44 0.70 8.24 1.47 1.32 4.69 0.00 88.18 18.61
Total PAHs
762.24
77.06
103.49
Catfish total length ranged from 24.5 to 42.0 cm, and age from 2 to 6 years. Most fish were 4 and 5 years old. In relation to sex, 58 organisms were females, five males (for 13 fish, sex was not recorded). Twenty-three individual pesticides were identified in livers (Table 1). DDT and its metabolites (28.7 ± 42.4 ng g1 ), and total P chlordanes ( heptachlors, chlordanes and nonachlors; 26.2 ± 29.2 ng g1 ) had greatest median concentrations. Total organochlorine pesticides ranged from 39.1 to 795.8 ng g1 with a median value of 143.7 ± 125.0 ng g1 .
IQ range
On the other hand, twenty different PCB congeners were found in the samples analyzed (Table 2). PCBs were grouped according to the number of chlorine atoms in their structure, with highest median concentrations for trichlorobiphenyls (triCBs) and hexachlorobiphenyls (hexaCBs), 14.5 ± 19.0 and 13.1 ± 17.8 ng g1 , respectively. The minimum value of total PCBs was 10.9 ng g1 and the maximum 1646.7 ng g1 , with a median concentration of 83.8 ± 99.9 ng g1 . Highly chlorinated congeners such as hexaCBs are known to magnify in
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Fig. 2. Variation of organic compounds content in catfish (Ariopsis assimilis) from Bay of Chetumal in relation to sex and age of the organisms.
food chains and to accumulate in higher organisms (Erickson, 1997). Published studies dealing with the determination of persistent organic pollutant (POPs) levels in fish from Mexican coastal lagoons are scarce, and all determined contaminants were in muscle, so their results are not comparable with ours. McCain et al. (1996) evaluated chlorinated compounds in liver of hardhead catfish Arius felis (a species closely related to A. assimilis) from highly polluted sites in Tampa P Bay, FL. These authors found mean chlordane levels ( chlordane and transnonachlor) similar to the mean detected in the Bay of Chetumal (35.6 ± 14.9 and 14.28 ± 16.31 ng g1 dry-wt, respectively), but mean values of total DDTs (228.9 ± 214.0 ng g1 dry-wt) and PCBs (760 ng g1 drywt) for Tampa Bay were much higher than those we
found (54.06 ± 88.80 and 139.15 ± 210.17 ng g1 , respectively). Regarding PAHs, 18 individual compounds were identified in livers. Concentration values are given in Table 3, with benzo[a]pyrene standing out with the highest median (30.3 ± 88.2 lg g1 ). Total PAHs ranged from 6.6 to 762.2 lg g1 , with a median of 77.1 ± 103.5 lg g1 . Predominance of high molecular (five rings) PAHs was observed with a concentration of 41.5 ± 81.7 lg g1 . Also, there was a clear predominance of parent compounds over alkyl derivatives. The same pattern was observed in sediments, where it was considered as evidence of a pyrogenic source of these compounds in the Bay (Nore~ na-Barroso et al., 1998). Variation of persistent organic pollutant levels in relation to the age and sex of the fish analyzed is shown in
E. Nore~na-Barroso et al. / Marine Pollution Bulletin 48 (2004) 263–269
Fig. 2. Pesticides, PCBs and PAHs follow the same trend. In females, the concentration of chemicals decrease with age. In males the tendency is inverse, with highest concentrations found in 5-year-old fish. However, this variation should be carefully considered since only a few organisms ages two and six were found. The same can be said about differences related to sex, considering that most fish analyzed were females. Organic compound accumulation in fish depends on lipid content, age, sex and diet of the organisms (Elskus et al., 1994). It has been reported that lipid content in fish increases in relation to age and size; however, there are other factors that should be considered such as metabolic rate, reproductive status, season of the year and activity pattern of the organism (Weatherley and Gill, 1987). Lack of information on the basic biology of A. assimilis makes it difficult to explain our results. Since age data are not available for all 76 catfish analyzed, and considering that catfish age is highly correlated to total length (Spearman’s R ¼ 0:9460; p < 0:05), non-parametric Spearman’s R correlation coefficients were calculated to evaluate the relationships between total length and the levels of the different organic compounds found in liver. This provides indirect evidence of any association between chemicals and age. Total length was negatively correlated with several pesticides such as a-HCH (R ¼ 0:2343; p < 0:05), heptachlor (R ¼ 0:3373; p < 0:05), aldrin (R ¼ 0:2893; p < 0:05) and dieldrin (R ¼ 0:2715; p < 0:05). Moreover, length was also negatively correlated with groups of PCBs having highest median concentrations: triCBs (R ¼ 0:2285; p < 0:05) and hexaCBs (R ¼ 0:2954; p < 0:05). No significant correlations were observed between PAHs and total length, except a positive one with 1,3,5-triethylbenzene (R ¼ 0:2369; p < 0:05). A multivariate analysis of variance indicated significant differences in organic pollutant content among sites (Wilks’ k ¼ 0:70; p < 0:05). A post-hoc Tukey (HSD) test showed differences in liver pesticide and PAH content at El Ramonal with those from other sampling stations, but none for PCB content between sites. On the other hand, even though the major sources of pollutants are located on the west coast of the Bay, higher bioaccumulation of organic chemicals took place in fish from the east coast. This pattern corresponds to that observed in sediments, and may be related to an accumulation of these compounds in sediments, depending on grain size and organic matter content (Nore~ na-Barroso et al., 1998). Most catfish analyzed (98.7%) had at least one histological lesion in liver, kidney and gills. The prevalence of the different lesions observed in each organ is given in Table 4. A high prevalence of liver damage involving cellular degeneration and granuloma (60.5%), as well as an increase in the number of hepatocytes and hyperaemia (69.7%) were found. Cellular alteration foci can be
267
Table 4 Prevalence of histological lesions observed in catfish Ariopsis assimilis from Bay of Chetumal, Mexico Lesion
Number of fish
%
Liver Hyperaemia Lymphocyte infilt Granuloma Pycnosis Tumors
53 50 46 11 6
69.74 65.79 60.53 14.47 7.89
Kidney Glomerulonefritis Calcium deposits Granuloma
58 45 2
76.32 59.21 2.63
Gills Hypertrophy Hyperplasia Edema Telangiectasis
74 60 52 39
97.37 78.95 68.42 51.32
Spleen Hemosiderosis
59
77.63
considered preneoplastic lesions or precursors in hepatic neoplasms histogenesis (Hawkins et al., 1990). The prevalences of hepatic lesions involving cellular alteration registered in this study are similar to those reported by McCain et al. (1988) for highly polluted urban zones of the USA west coast (10–36%). Among hepatic lesions, the presence of granulomas and tumors (7.9%) stand out in severity. Tumor prevalence is within the same range (2–18%) reported by Malins et al. (1988) for several sites in Puget Sound, and it is higher than the 2.2% found in a closely related catfish species (A. felis) from Tampa Bay (McCain et al., 1996). In addition, hemosiderosis in the spleen was detected in 77.6% of the fish analyzed. Hemosiderosis usually occurs as a result of excessive or accelerated breakdown of erythrocytes in vertebrates and the break down product, hemosiderin (a pigment that contains iron), can be found in hematopoietic tissue and spleen (Thiyagarajah et al., 1998). Hemosiderosis suggests fish anemia due to erythrocyte destruction (Estrada and Garcıa, 1991). Liver, spleen and kidney hemosiderosis in fish has been associated with the presence of organic pollutants in the environment (Thiyagarajah et al., 1998). Significant logistic correlations between histological lesions in catfish and organic contaminants in liver are given in Table 5. Chlorinated compounds show a stronger effect on the presence of lesions, since they were significantly related to a higher number of histological lesions than PAHs. This might be due to the fact that biliary PAH metabolites were not measured. PAHs in fish are biotransformed in the liver and readily excreted to biliary products (Varanasi et al., 1989). Chlorinated pesticides were associated with the presence of histopathologies in
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Table 5 Relationship between organic compounds and histological lesions in catfish Ariopsis assimilis from Bay of Chetumal, Mexico Lesion Pesticides Hyperaemia (L) Pycnosis (L) Tumors (L) Hyperplasia (G) Ca deposits (K) Hemosiderosis (S) PCBs Granuloma (L) Pycnosis (L) Edema (G) Telangiectasis (G) Glomerulonefritis (K) PAHs Hypertrophy (G) Hyperplasia (G) Glomerulonefritis (K)
Compound
p-Value
Pentachlorobenzene Endosulfan II Dieldrin Dieldrin Endosulfan II Mirex DDTs
0.0399 0.0149 0.0011 0.0144 0.0299 0.0089 0.0092
HexaCBs TetraCBs TetraCBs TetraCBs HeptaCBs NonaCBs Total PCBs
0.0418 0.0181 0.0242 0.0186 0.0494 0.0035 0.0083
4-Ring PAHs 2-Ring PAHs 4-Ring PAHs
0.0466 0.0406 0.0381
Explained deviance (%) 4.62 16.94 14.28 6.02 6.66 8.39 4.06 8.89 5.36 7.73 13.02
21.41 5.36 5.17
Logistic regression by steps (n ¼ 76). L ¼ liver; G ¼ gills; K ¼ kidney; S ¼ spleen. * Significant correlations.
all organs examined, especially Endosulfan II, which is highly toxic to fish (Miles and Pfeuffer, 1997), and Dieldrin, related to hepatic cellular alteration and neoplastic damage. Total DDTs (found in elevated levels in liver and sediments) showed a significant correlation with spleen hemosiderosis. In relation to PCBs, tetrachlorobiphenyls influenced the appearance of histological damage in liver as well as in gills. Hexachlorobiphenyls (having the second highest concentration in liver) are associated with hepatic granulomas, while PAHs may be related to hepatic hyperaemia and gill hyperplasia. The degree of association between contaminants and lesion prevalences, and explained variance, reported here are similar to those reported in the review by Malins et al. (1988). Association between fish histopathologies and the presence of organic pollutants should be carefully studied given the histological lesions observed. Relatively high concentrations of pollutants were found in Mayan catfish, and some of these pollutants, particularly chlorinated compounds such as pesticides and polychlorobiphenyls, are associated with the presence of histological lesions. Also, high levels of PAHs in fish livers deserve more consideration. The fact that most catfish analyzed had at least one lesion in liver, kidney and gills, suggests that fish in the Bay are in bad condition. Given that fish were collected several months after the mass mortality event the relationship between the mortality and these results cannot be assessed.
Acknowledgements We acknowledge Pablo Ivan Caballero (CETMar No. 10) for his support for sample collection and experience on the study area. We also thank Claudia Leonce for her help analyzing otoliths and to Gregory Arjona for his help with histological samples. References Elskus, A.A., Stegeman, J.J., Gooch, J.W., Black, D.E., Pruell, R.J., 1994. Polychlorinated biphenyl congener distributions in winter flounder as related to gender, spawning site, and congener metabolism. Environmental Science and Technology 28, 401–407. Erickson, M.D., 1997. Analytical Chemistry of PCB’s, second ed. CRC Press, Boca Raton, FL. p. 667. Estrada, R.R., Garcıa, P.B., 1991. Atlas de Histopatologıa. Progreso, Mexico. Hawkins, W.E., Walker, W.W., Overstreet, R.M., Lytle, J.S., Lytle, T.E., 1990. Carcinogenetic effects of some polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures. Science of Total Environment 94, 155–167. Malins, D.C., McCain, B.B., Landahl, J.T., Myers, M.S., Krahn, M.M., Brown, D.W., Chan, S.L., Roubal, W.T., 1988. Neoplastic and other diseases in fish in relation to toxic chemicals: an overview. Aquatic Toxicology 11, 43–67. McCain, B.B., Brown, D.W., Krahn, M.M., Myers, M.S., Clark, R.C., Chan, S., Malins, D.C., 1988. Marine pollution problems, North American West Coast. Aquatic Toxicology 11, 143–162. McCain, B.B., Brown, D.W., Horn, T., Myers, M.S., Pierce, S.M., Collier, T.K., Stein, J.E., Chan, S., Varanasi, U., 1996. Chemical
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