Environ. Sci. Technol. 2005, 39, 3933-3938
Investigations of the Potential Influence of Environmental Contaminants on the Thymus and Spleen of Harbor Porpoises (Phocoena phocoena) A N D R E A S B E I N E K E , * ,† U R S U L A S I E B E R T , ‡ MICHAEL MCLACHLAN,§ REGINA BRUHN,| KRISTINA THRON,⊥ KLAUS FAILING,¶ GUNDI MU ¨ LLER,† AND W O L F G A N G B A U M G A¨ R T N E R † Institut fu ¨ r Pathologie, Tiera¨rztliche Hochschule Hannover, 30559 Hannover, Germany, Forschungs- und Technologiezentrum Westku ¨ ste, Universita¨t Kiel, 25761 Bu ¨ sum, Germany, Institute of Applied Environmental Research, Stockholm University, 10691 Stockholm, Sweden, GKSS Research Centre, 21502 Geesthacht, Germany, Institut fu ¨ r Ostseeforschung, Universita¨t Rostock, 18119 Rostock, Germany, and Institut fu ¨ r Veterina¨r-Physiologie, Justus-Liebig-Universita¨t, 35392 Giessen, Germany
Harbor porpoises from the German North and Baltic Seas exhibit a higher incidence of bacterial infections compared to whales from less polluted arctic waters. The potential adverse effect of environmental contaminants such as polychlorinated biphenyls (PCBs) and heavy metals on the immune system and the health status of marine mammals is still discussed controversially. The aim of the present study was to investigate the possible influence of PCB, polybrominated diphenyl ether (PBDE), toxaphene, (p,p′dichlorodiphenyl)trichlorethane (DDT), and (p,p′-dichlorodiphenyl)dichlorethene (DDE) on the immune system of harbor porpoises. Lymphoid organs are influenced by a variety of factors, and therefore special emphasis was given to separating the confounding effect of age, health status, nutritional state, geographical location, and sex from the effect of contaminant levels upon thymus and spleen. Contaminant analysis and detailed pathological examinations were conducted on 61 by-caught and stranded whales from the North and Baltic Seas and Icelandic and Norwegian waters. Stranded harbor porpoises were more severely diseased than by-caught animals. Thymic atrophy and splenic depletion were significantly correlated to increased PCB and PBDE levels. However, lymphoid depletion was also associated with emaciation and an impaired health status. The present report supports the hypothesis of a contaminant-induced immunosuppression, possibly contributing to disease susceptibility in harbor porpoises. However, further studies are needed to determine if * Corresponding author phone: +49-511-953-8620; fax: +49-511953-8675; e-mail:
[email protected]. † Tiera ¨ rztliche Hochschule Hannover. ‡ Universita ¨ t Kiel. § Stockholm University. | GKSS Research Centre. ⊥ Universita ¨ t Rostock. ¶ Justus-Liebig-Universita ¨ t. 10.1021/es048709j CCC: $30.25 Published on Web 04/22/2005
2005 American Chemical Society
lymphoid depletion is primarily contaminant-induced or secondary to disease and emaciation in this cetacean species.
Introduction The harbor porpoise (Phocoena phocoena) is the only resident cetacean species in the German North and Baltic Seas (1). There is growing concern about the adverse effects of persistent environmental contaminants on this and other marine mammal species (2, 3). High concentration of polychlorinated biphenyls (PCBs) and pesticides, such as (p,p′-dichlorodiphenyl)trichlorethane (DDT) and (p,p′dichlorodiphenyl)dichlorethene (DDE) are present in harbor porpoises from the North and Baltic Seas (4, 5). Furthermore, pinniped and cetacean populations around the world including the harbor porpoise are contaminated with polybrominated diphenyl ethers (PBDEs), a group of chemicals used as flame retardants (6). Marine mammals are at the end of the aquatic food chain where they accumulate persistent organic contaminants and heavy metals throughout their lifespan. The high trophic feeding level and the limited capacity to metabolize xenobiotics observed in different cetacean species are suspected to predispose the harbor porpoise to chronic intoxication (7, 8). There is a current debate about the potential adverse effect of environmental contaminants such as PCBs and heavy metals on the immune system and therefore on the health status of marine mammals. (3, 4, 9, 10). Harbor porpoises from waters around Germany, Great Britain, Belgium, and France exhibit a higher incidence of infectious diseases compared to animals from less contaminated arctic water (11-14). Thus, an inhibitory contaminant-induced effect on the pulmonary or systemic immune system is suspected to contribute to pneumonia in these whales (15). Furthermore, PCB-induced impairment of the immune system is suspected to play a contributing role to the devastating morbillivirus epizootics in different pinniped and cetacean species worldwide (16-18). Inhibited cellular immune response has been demonstrated in freeranging bottlenose dolphins (Tursiops truncatus) associated with increased PCB and DDT levels. Furthermore, harbor seals, experimentally fed with PCB-contaminated fish, show an impaired cellular and humoral immune response (1820). The aim of the present study is to investigate the possible influence of environmental contaminants on the immune system of the harbor porpoise. Because lymphoid organs are influenced by a variety of factors, special emphasis is given to separate the confounding effect of age, health status, nutritional state, geographical location, and sex from the effect of contaminant levels upon the thymus and the spleen.
Experimental Section Necropsy was performed on 61 harbor porpoises (36 female [?] and 25 male animals [/]; North and Baltic Seas, n ) 30; Icelandic waters, n ) 12; Norwegian waters, n ) 19) as described elsewhere (10). Fifty of these animals were bycaught, while 11 were stranded whales. The age was determined by counting the dental growth layers (age range, 0.1-12 years). Samples for histopathological, microbiological and contaminant analysis were collected and stored in 10% formalin or at -20 °C, respectively, until used (Supporting Information, Table SI-1). Gross and Histopathological Examination. All organ systems of 61 animals were examined macroscopically, and samples of all organs and pathological lesions were taken. VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Samples were fixed in 10% formalin, embedded in paraffin wax, cut into 5 µm thick sections, and stained with hematoxylin and eosin (HE). The health status was evaluated on the basis of the severity of the main pathological findings and/or the nutritional state and graded into very good, good, moderate, and poor (21). The nutritional state was judged from blubber thickness and muscle condition. Accordingly, investigated animals were divided into four categories: very good, good, moderate, and emaciated (13). Bacteriology. Samples of lung, liver, kidney, spleen, intestine, and mesenteric lymph node of 56 out of 61 harbor porpoises were submitted for routine bacteriological examination. Additionally, pathological lesions suspected to contain bacterial infections were examined. Selective media for the isolation of Vibrio and Brucella species, Erysipelothrix rhusiopathiae, and fungi were used (13). Scoring of Thymus and Spleen. The degree of depletion in thymus and spleen was evaluated semiquantitatively using HE-stained slide sections as follows: group I ) normal tissue architecture and group II ) mild, group III ) moderate, and group IV ) severe depletion. In the normal spleen numerous areas with primary and secondary follicles could be distinguished. Mild depletion was characterized by reduction of the size of the white pulp and a loss of follicles, whereas a marked reduction of size of the white pulp and a loss of follicles were indicative of moderate depletion. In severely depleted spleens, the white pulp was no longer recognizable. For grading thymic alterations, cellularity and distinction of cortical and medullary borders as well as the amount of interlobular connective tissue were the most important parameters (22). Toxicological Analysis. Between 2 and 10 g of subcutaneous blubber sampled behind the dorsal fin were mixed with a similar quantity of sodium sulfate and Soxhlet extracted for 6 h in a 1:1 mixture of n-hexane and dichloromethane. The extractable lipid was determined gravimetrically in an aliquot of the extract. Separate aliquots of the extract were taken for PCB, DDT, DDE, toxaphene, and PBDE analysis. Internal standard was added (13C12-labeled p,p′-DDE, and 13 C12-labeled PCB congeners 28, 52, 101, 138, 153, and 180 to the PCB + DDT + DDE aliquot; toxaphene congener Parlar 58 to the toxaphene aliquot; PBDE congener 85 to the PBDE aliquot), and the extracts were purified as described elsewhere (4, 23). PCB, DDT, and DDE were analyzed using GC/MS-MS (EI), toxaphene was analyzed using GC/MS (NCI), and PBDE was analyzed using GC/MS (EI). The sum of the dominant congeners of the chemical mixtures was used in the statistical evaluation: IUPAC Nos. 99, 149, 153, 138, 180, and 187 for PCB; Parlar Nos. 26, 40, 42, 44, and 50 for toxaphene; and IUPAC Nos. 47, 99, 100, 153, and 154 for PBDE. Samples of 59 harbor porpoises were available for the analysis of PCB, DDT, and DDE levels. Furthermore, concentrations of PBDE and toxaphene were quantified in the blubber of 52 and 53 harbor porpoises, respectively. All contaminant concentrations were normalized to the extractable lipid content. Statistical Analysis. The statistical analysis of the data was performed with the statistical program package BMDP/ Dynamic, Release 7.0 (24). The distribution of xenobiotic concentration was skewed to the right; therefore, data of these variables were logarithmically transformed before performing statistical analysis. The relationships between lymphoid depletion (thymus, spleen) and contaminant levels were described in two steps. As a crude measurement of association, the rank correlation coefficient of Spearman (rs) was used. Subsequently, polychotomous stepwise logistic regression for ordinal dependent variables was applied to separate the confounding effect of age, health status, geographical location, and sex from the effect of contaminant levels. First the analysis was done with the complete model including all main effects of contaminant 3934
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levels, age, sex, geographical location, and health status. In a subsequent step the most insignificant influential variable was removed from the model. At each step the p-values and the adjusted odds ratio (OR) were calculated. The used level of significance was p e 0.05 (3).
Results Pathological Findings. Complete postmortem examinations were performed on 61 harbor porpoises. The respiratory system was most frequently affected. In total, 42 harbor porpoises (68.9%) showed suppurative or granulomatous bronchopneumonias associated with acute congestion and alveolar edema. Most cases of bronchopneumonia were related to verminous infestation. Thirteen animals (21.3%) showed pulmonary congestion and alveolar edema without inflammatory lesions of the lung. The remaining whales exhibited no pulmonary lesions. Lymphoplasmacytic to granulomatous cholangitis and/ or pericholangitis (inflammation of the bile duct and/or surrounding tissue), mostly due to trematode infestation, was found in 29 harbor porpoises (47%). Two animals showed a mixed cellular and lymphoplasmacytic hepatitis, respectively. Hepatic lipidosis was present in 14 animals (23%), while two animals exhibited single-cell necrosis of hepatocytes. Portal to periportal fibrosis and biliary hyperplasia, associated with other lesions (inflammatory or degenerative), were present in 12 cases (19.7%). Lymphoplasmacytic to granulomatous pancreatitis was found in seven harbor porpoises (11.5%). Pancreatic interstitial fibrosis was present in two cases. Focal to multifocal ulcerative gastritis, associated with nematode parasites, was observed in 23 whales (37.7%). One animal showed mucosal hyperkeratosis in this gastric compartment. Two animals had a focal granulomatous and multifocal ulcerative esophagitis, respectively. In addition, a case of acute diffuse catarrhal enteritis and a case of ulcerative transmural enteritis were found. Individual intestinal crypt abscesses were present in one whale. Single cases of suppurative necrotizing nephritis, chronic lymphocytic uretheritis, subcapsular renal hemorrhages, and renal tubular epithelial lipidosis were observed. Lymphoplasmacytic to necrotizing vaginitis was present in four harbor porpoises. In addition, a case of chronic suppurative endometritis (inflammation of the uterus) and lymphohistiocytic mastitis (inflammation of the mammary gland) was described. Subcutaneous hemorrhages were found in 14 animals (23%). Two animals showed a disseminated ulcerative and pyogranulomatous necrotizing dermatitis. Multiple cerebral hemorrhages were found in seven cases (11.5%). A focal nonsuppurative panencephalitis of unknown cause was present in one harbor porpoise. No morbillivirus antigen was detected in the brain by immunohistochemistry (25). Uni- or bilateral hyphema (hemorrhage in the anterior chamber of the eye) was present in three cases. One whale showed a bilateral focal ulcerative keratitis and unilateral hypopyon (suppurative exudate in the anterior chambers of the eye). In addition, bilateral perforating corneal ulcers were observed in one harbor porpoise. Single cases of myocardial hemorrhages with myofiber necrosis, periaortal hemorrhages, and suppurative myocarditis with chronic fibrinous suppurative epicarditis were found. In addition, a diffuse fibrinous pericarditis was present in one whale (26). Health Status. The heath status was determined in 61 harbor porpoises, on the basis of the severity of main pathological lesions. A very good health status was found in four animals (6.6%) with no pulmonary or other lesions. One of these animals
TABLE 1. Rank Correlation Analysis between Thymic Atrophy and Splenic Depletion, Respectively, and Contaminant Levels, Nutritional State, Health Status, and Age of Harbor Porpoises thymic atrophy no. of investigated animals total PCB DDE DDT toxaphene PBDE nutritional state health status age
59 59 59 54 53 61 61 59
by-caught 49 49 49 45 44 50 50 48
rsa total 0.31 0.15 0.17 0.11 0.45 0.42 0.38 -0.08
splenic depletion significance (p-value)
by-caught
total
0.19 0.11 0.19 0.11 0.31 0.04 0.06 -0.01
0.02b 0.27 0.19 0.44 0.01b 0.01b 0.01b 0.55
by-caught 0.19 0.44 0.19 0.49 0.04b 0.76 0.65 0.93
no. of investigated animals total 50 50 50 47 46 52 50 50
by-caught 41 41 41 38 37 42 42 40
significance (p-value)
rsa total 0.36 0.10 0.06 0.02 0.42 0.37 0.51 -0.15
by-caught
total
by-caught
0.28 0.11 0.15 0.04 0.35 0.04 0.28 0.02
0.01b
0.07 0.48 0.36 0.82 0.03b 0.81 0.07 0.89
0.51 0.70 0.90 0.01b 0.01b 0.01b 0.29
a r ) rank correlation coefficient of Spearman. b These values are significant; total ) total data set including stranded and by-caught harbor s porpoises; by-caught ) data set including only by-caught harbor porpoises.
exhibited a very good and three whales a good nutritional state. Twenty-two animals (36.1%) exhibited a good health status, characterized by mild pneumonic lesions or mild to moderate pancreatic or hepatic lesions, respectively. All of these animals exhibited a good nutritional state. A moderate health status was described in 23 animals (37.7%) with moderate pneumonic lesions. One harbor porpoise showed a very good, 16 a good, and six a moderate nutritional state. A poor health status was found in 12 cetaceans (19.7%), characterized by severe pneumonic lesions (six animals), severe disseminated suppurative necrotizing dermatitis (two animals), severe diffuse fibrinous peritonitis (one animal), and severe diffuse fibrinous suppurative polyserositis with suppurative myocarditis (one animal). One of these animals showed a very good, two a good, two a moderate, and five an emaciated nutritional state. Additionally, two harbor porpoises with severe emaciation of unknown cause were included to this group (Supporting Information, Table SI-1). Main pathological findings are given in Supporting Information, Table SI-2. Three out of 50 by-caught whales exhibited a very good health status (6.0%), 22 a good (44.0%), 21 a moderate (42.0%), and four a poor health status (8.0%). In comparison, one out of 11 stranded harbor porpoise showed a very good (9.1%), two a moderate (18.2%), and eight a poor health status (72.7%; Supporting Information, Table SI-1). Microbiological Results. Microbiological examination was performed on tissue samples of 56 harbor porpoises. No bacterial growth was found in 19 cases, while 33 whales showed various bacterial organisms without any associated pathological lesions. Pathogenic bacteria associated with pathological lesions were found in four cases. Staphylococcus aureus was isolated from a whale with fibrinous suppurative polyserositis and suppurative myocarditis. Clostridium perfingens was associated with ulcerative transmural enteritis in one case. Streptococcus spp. was isolated from an animal with pyogranulomatous necrotizing dermatitis. Furthermore, β-hemolytic Escherichia coli was cultured in a case of fibrinous suppurative pleuropneumonia (Supporting Information, Table SI-2). Histological Changes in Thymus and Spleen. The degree of thymic atrophy was evaluated in 61 harbor porpoises. An intact thymus was present in 22 harbor porpoises (group I, 36.1%). Mild, moderate, and severe thymic atrophy was found in 18 (group II, 29.5%), 13 (group III, 21.3%), and eight cases (group IV, 13.1%), respectively (Supporting Information, Table SI-1). The spleens of 52 harbor porpoises were available for histological grading. Normal splenic architecture was found in 10 animals (group I, 19.2%). Mild, moderate, and severe splenic depletion was found in 29 (group II, 55.8%), 10 (group
III, 19.2%), and three animals (group IV, 5.8%), respectively (Supporting Information, Table SI-1). Contaminant Levels. Harbor porpoises showed a median PCB concentration of 3400 ng/(g of lipid) (?, median ) 2890 ng/(g of lipid); /, median ) 5033 ng/(g of lipid)). Median concentrations of DDE and DDT were 983 ng/(g of lipid) (?, median ) 969 ng/(g of lipid); /, median ) 1200 ng/(g of lipid)) and 270 ng/(g of lipid) (?, median ) 229 ng/(g of lipid); /, median ) 333 ng/(g of lipid), respectively. Median toxaphene concentration of investigated whales was 965 ng/ (g of lipid) (?, median ) 1060 ng/(g of lipid); /, median ) 824 ng/(g of lipid)). PBDE showed a median concentration of 138 ng/(g of lipid) (?, median ) 98 ng/(g of lipid); /, median ) 161 ng/(g of lipid)). Correlation between Lymphoid Depletion and Contaminant Levels. Contaminant levels are given as Supporting Information in Table SI-1. Thymic atrophy and splenic depletion were associated with increasing levels of contaminants. Statistical analysis by the rank correlation coefficient of Spearman of the total data set, including by-caught and stranded whales, showed highly significant correlations between thymic atrophy as well as splenic depletion and elevated levels of PCB and PBDE. Concentrations of DDE, DDT, and toxaphene were not significantly correlated with thymic atrophy or splenic deletion. Impairment of the nutritional state and health status were significantly related to thymic atrophy and splenic depletion, but age was not (Table 1, Figure 1). Statistical analysis of by-caught animals showed a significant correlation between thymic atrophy as well as splenic depletion and elevated PBDE levels. Other confounding factors showed no significant correlation with changes in thymus and spleen. A barely not significant correlation (p ) 0.07) was found between splenic depletion and elevated PCB concentrations as well as an impaired health status, respectively (Table 1). Polychotomous stepwise logistic regression of the total data set, including by-caught and stranded whales, was applied to separate the confounding effect of age, health status, geographical location, and sex from the effect of contaminant levels upon the thymus and spleen. High PBDE levels and a poor health status were positively correlated with lymphoid depletion of the thymus (odd ratios, 5.6 and 3.7), while the animals age was negatively associated with thymic atrophy (odds ratio, 0.2). Applying all confounding factors and contaminants to the statistical model, no association between thymic atrophy and geographical location, sex, toxaphene, PCB, DDE, and DDT was found. Considering these confounding factors and contaminant levels, splenic depletion was only associated with a poor health status (odd ratio, 4.2), while PCB, PBDE, toxaphene, DDE, and DDT levels did not correlate with lymphoid VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Rank correlation between thymic atrophy and splenic depletion, respectively, and contaminant levels in the blubber of by-caught and stranded harbor porpoises present as box and whisker plot. Box and whisker plot displays values as minimum, maximum, median, and the lower and upper quartile: I ) no, II ) mild, III ) moderate, IV ) severe thymic atrophy and splenic depletion, respectively. p ) p-value; rs ) rank correlation coefficient of Spearman.
TABLE 2. Polychotomous Stepwise Logistic Regressiona thymic atrophy
PCB DDE DDT toxaphene PBDE sex geog location health status age
splenic depletion
p-value total/ by-caught
adjusted odds ratio total/ by-caught
p-value total/ by-caught
adjusted odds ratio total/ by-caught
0.93/0.74 0.83/0.57 0.31/0.82 0.80/0.99 0.02/0.77 0.58/0.11 0.56/0.01 0.01/0.98 0.01/0.63
-/-/-/-/5.6/-/-/1.5 3.7/0.2/-
0.72/0.97 0.72/0.94 0.72/0.90 0.38/0.34 0.87/0.97 0.18/0.03 0.12/0.22 0.01/0.01 0.47/0.21
-/-/-/-/-/-/15.9 -/4.2/6.7 -/-
a Association between thymic atrophy and splenic depletion, respectively, and contaminant levels, sex, geographical location, health status, and age of harbor porpoises. “-” ) effect was removed from the model; total ) total data set including stranded and by-caught harbor porpoises; by-caught ) data set including only by-caught harbor porpoises.
depletion. In addition, no significant relationships were found between splenic changes and the age, geographical location, and sex of the animals (Table 2). Statistical analysis of bycaught animals showed a correlation between thymic atrophy and geographical location. More whales from the North and Baltic Seas as well as from Norwegian waters exhibit thymic atrophy compared to whales from Icelandic waters (odd ratio, 1.5). Health status, nutritional state, and age as well as contaminant levels did not correlate with thymic atrophy. Splenic depletion was associated with the health status and sex of the animals (odd ratios, 6.7 and 15.9). Referring to this, splenic depletion was associated with an impaired health status. Furthermore, depleted spleens were more present in male harbor porpoises. Other confounding factors and contaminant levels showed no correlation with splenic depletion using polychotomous stepwise logistic regression analysis (Table 2).
Discussion The aim of the present study was to investigate the possible influence of environmental contamination upon lymphoid 3936
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organs of the harbor porpoise. The analyzed compounds are found in high concentrations in this cetacean species and are suspected to be toxic to marine mammals (4, 6). Thymic atrophy in harbor porpoises was characterized by cortical lymphoid depletion, loss of the corticomedullary junction, and increased interlobular connective tissue, as described in humans (27). Physiologic thymic involution in cetaceans begins after sexual maturity with 3 years and progresses slowly, as observed in harbor porpoises and bottlenose dolphins (21, 28). In the present study, thymic atrophy was predominantly found in diseased and contaminated juvenile animals (
