Investigating links between polychlorinated biphenyl (PCB) exposure and thymic involution and thymic cysts in harbour porpoises (Phocoena phocoena)

Marine Pollution Bulletin 64 (2012) 2168–2176

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Investigating links between polychlorinated biphenyl (PCB) exposure and thymic involution and thymic cysts in harbour porpoises (Phocoena phocoena) Xinli Yap a,1, Rob Deaville a, Matthew W. Perkins a, Rod Penrose b, Robin J. Law c, Paul D. Jepson a,⇑ a

Institute of Zoology, Zoological Society of London, Regent’s Park, NW1 4RY London, UK Marine Environmental Monitoring, Penwalk, Llechryd, Cardigan SA43 2PS, UK c The Centre for Environment, Fisheries and Aquaculture Science, CEFAS Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK b

a r t i c l e

i n f o

Keywords: Polychlorinated biphenyls Thymus Thymic involution Thymic cyst Harbour porpoise Phocoena phocoena

a b s t r a c t The associations between polychlorinated biphenyls (PCBs) exposure and involution of lymphoid tissue and development of epithelial-lined cysts in the thymus of UK-stranded harbour porpoises (Phocoena phocoena) (n = 170) were tested. Percentage of thymic lymphoid tissue (%TLT) was histologically quantified. Multiple regression analyses (n = 169) demonstrated significant positive correlation between %TLT and nutritional status (p < 0.001) and significant negative association between %TLT and onset of sexual maturity (p < 0.001). However, in a subgroup of porpoises with total PCB levels above a proposed threshold of toxicity (>17 mg/kg lipid weight) (n = 109), the negative association between %TLT (as dependent P variable) and summed blubber concentrations of 25 chlorobiphenyl congeners ( 25CBs) remained significant (p < 0.01) along with nutritional status (p < 0.001) and onset of sexual maturity (p < 0.001). These results suggest PCB-induced immuno suppression may be occurring in harbour porpoises in UK waters but only at concentrations that exceed proposed toxicity thresholds for marine mammals. In contrast, development of thymic cysts appears predominantly age-related. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The harbour porpoise (Phocoena phocoena) is distributed globally in the northern hemisphere, mainly confined to shelf waters (Hammond et al., 2008; Reid et al., 2003). The species is the most common marine mammal occurring in United Kingdom (UK) waters, with the majority of the population in the south-western North Sea (SCANS-II, 2008). Harbour porpoise populations seem to have declined or been eliminated in some areas, particularly the eastern Channel, Black Sea and Baltic Sea (Kuiken et al., 1994; Reid et al., 2003). Factors thought to have caused the decline are entanglement in fishing nets, changes in food supply, habitat destruction due to increased shipping activity and noise (Evans, 1991; Kuiken et al., 1994). Studies have also suggested that pollutants such as polychlorinated biphenyls (PCBs) may have contributed to population decline (Aguilar and Borrell, 1994). PCBs are planar halogenated aromatic hydrocarbons (PHAHs), including the coplanar PCBs, totalling 209 individual congeners (Loganathan and Kannan, 1994; Mullins et al., 1984). Their toxicity to wildlife resulted in bans on their production and use in most industrialised countries in the late 1970s or early 1980s ⇑ Corresponding author. Tel.: +44 207 449 6691; fax: +44 207 7586 2870. E-mail address: [email protected] (P.D. Jepson). Present address: Wildlife Reserves Singapore, 80 Mandai Lake Road, Singapore 729826, Singapore. 1

0025-326X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2012.07.038

(Loganathan and Kannan, 1994) but they remain persistent due to their stability and are bioaccumulative due to their lipophilic nature (Mullins et al., 1984). Marine mammals bioaccumulate persistent organochlorine compounds throughout their lifespan as they feed at high trophic levels (Tanabe et al., 1994; Pauly et al., 1998). Studies have shown that PCBs can have toxic effects on the reproductive (Murphy et al., 2010; Pierce et al., 2008; Reijnders, 1986; Schwacke et al., 2002) and immune systems (De Swart et al., 1996; Hall et al., 2006). In studies of UK harbour porpoises, PCB concentrations were found to be associated with infectious disease mortality (Jepson et al., 1999, 2005). High PCB levels have also been associated with the cetacean morbillivirus epizootic in striped dolphins (Stenella coeruleoalba) in the Mediterranean between 1990 and 1992 (Aguilar and Borrell, 1994). Experimental evidence has also shown that PCBs cause immuno suppression and increase mortality in rodents and marine mammals (De Swart et al., 1996; Imanishi et al., 1980; Lahvis et al., 1995; Loveren et al., 2000; Mos et al., 2006; Ross et al., 1996a, 1996b, 1997). Harbour seals experimentally fed with relatively highly contaminated Baltic herring compared to Atlantic herring exhibited impaired in vitro and in vivo immune function (De Swart et al., 1996; Loveren et al., 2000; Ross et al., 1996a). The thymus is essential for the differentiation and maturation of T-cell lymphocytes of the immune system (Cowan, 1994; Park et al., 2008). Few papers have been published on the pathologies and involution (loss of thymic lymphoid tissue) of the cetacean thymus (Beineke et al., 2007; Clark et al., 2005; Cowan, 1994;

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Wünschmann et al., 1999) and fewer still published on the effects of PCBs on the cetacean thymus (Beineke et al., 2005). However, it is known that it has a typical mammalian organisation, involutes at puberty (Cowan and Smith, 1999), albeit more slowly than in humans (Clark et al., 2005; Wünschmann et al., 1999). Microcyst formation, which may be due to cystic dilatation of Hassall’s corpuscles or condensation of the thymic epithelial reticulum, are occasionally observed in humans and can be observed macroscopically in healthy adult harbour porpoises (Wünschmann et al., 1999) and bottlenose dolphins (Cowan, 1994) rendering it a possibly age-related process. However, thymic macrocysts following accelerated thymic atrophy are frequently observed in animals with chronic diseases (Wünschmann et al., 1999). In a study of 61 harbour porpoises stranded in the German parts of the North and Baltic seas, an association was found between PCBs and thymic involution, albeit notwithstanding the effects of confounding factors (Beineke et al., 2005). This paper intends to examine the potential effects of PCBs on an organ of the immune system, the thymus, in the harbour porpoise. The main objective of this study was to test the hypothesis that thymic involution and thymic cyst growth is associated with elevated PCB levels in harbour porpoises by utilising a larger dataset than was examined previously (Jepson, 2003). For this purpose, multiple regression analyses were made between PCB concentration and percentage of thymic lymphoid tissue and presence of thymic cyst. Lymphoid organs are affected by a variety of factors and so the potential confounding effects of sexual maturity, sex and nutritional status were factored into the analyses. Toxicological experiments conducted on a range of aquatic mammalian species including seals, otters and minks have indicated that total PCBs might have adverse toxic effects (including immuno suppression) above a threshold level of total PCB greater than 17 mg/kg lipid weight in blubber (Kannan et al., 2000). Total PCBs (as Aroclor 1254) was calculated in this study using the formula [sumICES7 CB congeners⁄3] (for full explanation see Jepson et al., 2005) and two subsets of porpoises with total PCB levels above and below 17 mg/ kg lipid weight were used to statistically test for associations between PCB exposure and indices of lymphoid tissue abundance and thymic cyst presence in animals with high and low blubber PCBs concentrations.

2. Materials and methods 2.1. Source of animals and tissue samples The 170 harbour porpoises (80 females and 90 males) included in this study were stranded or bycaught in commercial fishing gear between November 1991 and November 2004 around England and Wales (Fig. 1). Animals were included on the basis of carcass condition and availability of blubber concentrations of a standard suite P of 25 chlorobiphenyl congeners ( 25CBs) and a thymic tissue sample preserved in 10% neutral buffered formalin. A total of 160 fresh carcasses were examined after being stored between 0 °C and ambient temperature. Ten carcasses were frozen at 20 °C until a postmortem examination was carried out. Thymic tissues for histological examination were embedded in paraffin, cut at 2– 6 lm, mounted on slides and stained with hematoxylin and eosin (H&E). Wherever possible a cause of death was determined based of established pathological and other criteria (Jepson, 2006). For statistical analyses, the causes of death were grouped into three categories consisting of death due to acute physical trauma (mainly by-catches); death due to infectious disease(s) and a third category of ‘‘other causes of death’’ comprising mainly starvation or cases where the most probable cause of death could not be established.

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Fig. 1. Distribution of the 170 harbour porpoises used in this study around England and Wales.

Age was determined for 162 individuals by analysing the number of growth layer groups in decalcified tooth histologic sections, a method generally adopted by marine mammalogists (Cowan, 1994; Lockyer, 1995a). Where age determination using the above method was not possible in juvenile porpoises, age was estimated using body length (<105 cm = 0 years; 105–120 cm = 1 years; 121– 130 cm = 2 years, and >130 cm = 3 years) (Jepson et al., 2005; Lockyer, 1995b). Sexual maturity was determined by the examination of gonads and histological detection of spermatogenesis in testes (Jepson et al., 2005). Both mean blubber thickness (taken from three sections: dorsal, lateral and ventral) and the residuals of the best-fit regression line between body weight and body length have been commonly used as indices for nutritional status (Jepson, 2003; Jepson et al., 2005; Kuiken et al., 1994; Pierce et al., 2008). However, diseased animals lose mass from other body components apart from blubber (Kuiken et al., 1994) and there are also marked seasonal variations in mean blubber thickness in porpoises (Fig. 2A) due to thermoregulatory function (Jepson et al., 2005; Pierce et al., 2008). In this study, the nutritional status of each individual was derived using its residual from the best-fit regression line between the natural logarithm values of body weight and body length (relative body weight). Since there was a strong correlation between relative body weight and mean blubber thickness (n = 168, p < 0.001, r2 = 0.30) (Fig. 2B), and since relative body weight had been suggested to be a better nutritional status indicator (Kuiken et al., 1994), only relative body weight was used in this study.Descriptive details for each individual can be found in the Appendix.

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Fig. 2. (A) Seasonal changes of mean blubber thickness in the 170 porpoises used in this study. (B) Correlation of mean blubber thickness (mm) and nutritional status (relative bodyweight) (n = 168, p < 0.001, r2 = 0.30).

2.2. Determination of PCB concentration Only freshly dead and slightly decomposed individuals were used as blubber organochlorine concentrations can change during decomposition (Borrell and Aguilar, 1990; Law, 1994). The blubber concentrations of 25 individual chlorobiphenyl (CB) congeners (mg/kg wet weight) were determined using internationally standardised methodology routinely used in the Centre for Environment, Fisheries and Aquaculture Science Laboratory (Allchin et al., 1989; Law, 1994). A brief description of the analytical method, analytical quality control procedures undertaken and identities of the 25 CB congeners are given elsewhere (Ballschmiter and Zell, 1980; Law et al., 2010). Using the proportion of hexane extractable lipid in individual blubber samples, the sum of the concentrations of the 25 CB congeners (R25CBs mg/kg wet weight) was then converted to a lipid weight basis (mg/kg lipid weight). The conversion factor [Total PCB concentration (as Aroclor 1254) = 3.0  sum of ICES7 congeners (lipid weight)] was used to determine total PCB concentration. The conversion was obtained through the regression of seven International Council for the Exploration of the Sea (ICES) congeners (CB28, CB52, CB101, CB118, CB138, CB153, and CB180) (ICES, 1986), studied using fish of six species (cod, dab, flounder, lemon sole, plaice and whiting), and the formulation, Aroclor 1254 (based on the Aroclor 1254 envelope as determined in the same samples) (Jepson et al., 2005). This conversion from congener to ‘‘total PCBs’’ allows for direct comparisons with older toxicological studies that determined PCB concentrations on a commercial formulation (rather than individual CB chlorobiphenyl congener) basis. It also permits a comparison between data in this study with the proposed ‘‘total PCBs’’ threshold (as Aroclor 1254) of 17 mg/kg lipid weight in marine mammal blubber (Kannan et al., 2000). 2.3. Measurement of percentage thymic lymphoid tissue (%TLT) All thymic sections were examined ‘‘blind’’ without prior knowledge of the individual’s age, nutritional status or PCB exposure. A binocular Leica Diaplan light microscope was used with a 10 eyepiece (fitted with a 10 mm by 10 mm graticule) and 2.5 objective (= 25 total magnification) to assess the relative proportions of lymphoid tissue and interstitial tissue (Fig. 3). To determine %TLT, four areas of the thymic section were chosen at random to be read. The %TLT was calculated as the mean of the four readings. If thymic cysts were present, areas of the thymus without

Fig. 3. Usage of a graticule to measure the percentage of thymic lymphoid tissue (25 magnification).

cysts were selected for %TLT calculation. Each section was then examined at high power magnification (100) for the presence and morphology of thymic cysts. Scores were given for each of the sections: Absence of cysts = zero; Presence of cyst(s) less than 1.0 mm in diameter (microcysts) = one; Presence of cyst(s) greater than 1.0 mm in diameter (macrocysts) = two. If both microcysts and macrocysts were present in a section, a score of two was given.

2.4. Statistical analysis For statistical purposes, the natural logarithm for all continuously distributed data (except %TLT and mean blubber thickness) had to be used to establish a normal distribution. A normal distribution for %TLT was obtained using the equation ‘‘arcsine (square root (%TLT/100))’’. Since age data were highly skewed with a majority of neonates and juveniles (n = 121 out of 170), sexual maturity was used to represent different age groups. With %TLT as the dependent variable, one-way analysis of variance (ANOVA) and linear regression were used to test its association with categorical independent variables (sexual maturity, sex and presence of thymic cyst) and continuously distributed variables (nutritional P 25CBs (mg/kg lipid)) respectively (Sokal and Rohlf, status and 1995; SYSTAT, 1998). Multiple regression models were then used to separate the confounding effect of any independent variable that

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was found to have a significant association with %TLT (Sokal and Rohlf, 1995; SYSTAT, 1998). For greater statistical power, the animals with cyst score ‘‘1’’ and ‘‘2’’ were merged and the association with absence/presence of thymic cyst was investigated using Pearson Chi-squared test for categorical variables (sexual maturity and sex) and binary logistic regression for continuously distributed variables (nutritional P status and 25CBs (mg/kg lipid) (Sokal and Rohlf, 1995; SYSTAT, 1998). Multiple logistic regression models were then used to test the effect of covariates on the presence of thymic cyst (Sokal and Rohlf, 1995; SYSTAT, 1998). To investigate the proposed threshold for PCB toxicity (Kannan et al., 2000), porpoises were divided into two subsets: above and below 17 mg/kg total PCB. The sample size for each statistical analysis varied since not all the parameters were established for each porpoise. Finally, a one-way analysis of variance was used to test for associations between %TLT and cause of death categories (trauma; infectious disease; others) with %TLT as the dependent variable.

3. Results 3.1. Morphology of the thymus As described in other studies, thymus analysed were typically mammalian with many lobules, each with a distinct cortex and medulla (Fig. 4B). Hassall’s corpuscles were identified in a few porpoises, mainly in juveniles (Fig. 4A).

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3.2. Histological characterisation of thymic involution In this study, thymic involution was determined in 170 harbour porpoises by the shrinkage and irregularity of the lobules, loss of distinction between cortex and medulla, loss of cortical lymphoid tissue and increased interlobular connective (adipose) tissue. The %TLT measured in all porpoises ranged from 0% to 99.5% (Mean = 59.5%, Median = 65.2%) (Fig. 4B–D) (see Appendix for details).

3.3. Histological characterisation of thymic cysts Of all the 170 porpoises studied, 129 porpoises had no detectable cysts and a total of 41 porpoises had cysts. Of these, 15 had microcysts (<1.0 mm diameter) and 26 had macrocysts (>1.0 mm diameter). Microcysts usually did not occur singly and were lined by a layer of squamous or columnar epithelium, with eosinophilic stained colloid-like contents (Fig. 5A). Macrocysts were irregularly shaped with a single layer of flattened epithelial cells filled with eosinophlic contents stained weaker than microcysts (Fig. 5B). It was possible to find both microcysts and macrocysts in an individual (Fig. 5C). In a polycystic example, macrocysts seemed to join and form a bigger macrocyst (Fig. 5D). Age range for animals with no detectable thymic cysts was zero to 15 years. Of these animals, 16 out of 129 were adults, with four above 6 years. Porpoises with microcysts ranged from 0 to 8 years, with eight adults. Only one out of 26 porpoises which had macrocysts was not an adult (range = 4–21 years). Seven out of

Fig. 4. (A) SW2004/165. Characteristic eosinophilic stained concentrically lamellated Hassall’s corpuscle in the medulla of a juvenile porpoise (arrow). (100 magnification) (H & E). (B) SW1996/126. Thymus in a 0 year old porpoise with abundant thymic lymphoid tissue (98.5%). Note the distinct cortex (C) and medulla (M), and little interstitial tissue. (200 magnification) (H & E). (C) SW2002/250. Thymus in an adult porpoise with partial involution of thymic lymphoid tissue (55.0%). Microcysts present but not in view. Note the increased interstitial tissue and shrinkage of thymic lobules. (200 magnification) (H & E). (D) SW2002/19. Highly atrophied thymus in an adult porpoise with little thymic lymphoid tissue (15.5%). Macrocysts present but not in view. Note the extensive interstitial tissue, degeneration of thymic lobules and loss of distinction between cortex and medulla. (200 magnification) (H & E).

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Fig. 5. (A) SW2003/385. Weakly stained eosinophilic microcyst found in an adult porpoise (arrow). Cyst is lined with a single layer of squamous epithelium. (100 magnification) (H&E). (B) SW2003/385. Microcyst found in the same individual as in Fig. 5A (arrow). Note that the material in the cyst has a weaker eosinophilic stain than the microcyst. (400 magnification) (H & E). (C) SW2002/151. Highly atrophied polycystic thymus with both microcysts (Mi) and macrocysts (Ma) (%TLT = 0.005). (200 magnification) (H & E). (D) SW2002/19. Polycystic thymus with several macrocyst joined together to form a bigger macrocyst. (200 magnification) (H & E).

121 sexually immature porpoises had cyst (microcysts = 6, macrocysts = 1), and 16 out of 49 sexually mature animals had no cysts. Four out of five adults above 15 years had macrocysts. Only one out of 41 animals aged zero had cysts (microcysts). 3.4. Statistical analyses of %TLT The %TLT was measured for 170 porpoises including sexually immature females (n = 60), sexually immature males (n = 61), sexually mature females (n = 20) and sexually mature males (n = 29). The results of the statistical analyses for single associations between %TLT and the respective independent variables (sexual maturity, sex, presence of thymic cyst, nutritional status and P 25CBs (mg/kg lipid weight)) are shown in Table 1. Sexual maturity was significantly associated with %TLT with decreased %TLT in sexually mature animals (Fig. 6A). Although statistical analyses could not be performed with age as a normally distributed variable, a decreasing trend of %TLT with age can be seen in Fig. 6B.

Also, %TLT was positively correlated with nutritional status (Fig. 6C). There was a negative correlation between %TLT and P 25CBs (Fig. 6D). A strong association was found between presence of thymic cyst and %TLT. Although males had a lower average %TLT compared to females, this result was not significant. P To test for the significance of %TLT and 25CBs after accounting for confounding factors, multiple linear regressions using the significant independent variables in the univariate analyses (nutriP tional status, sexual maturity and 25CBs) were performed (Table 2). After controlling for confounding effects, nutritional status and sexual maturity remained strongly associated with %TLT but P 25CBs was no longer independently correlated with %TLT. Subset of porpoises with total PCBs > 17 mg/kg lipid weight was then selected. This threshold was suggested by Kannan et al. (2000) as the level that adverse health effects could be detected in a range of experimental toxicological studies. In this subset, %TLT was positively correlated with nutritional status (Fig. 6C), negatively P correlated with 25CBs (Fig. 6D) and had a strong association

Table 1 Results of the analyses between percentage thymic lymphoid tissue (%TLT) and the independent variables showna. Factor

n

b (standard error)

b

One-way analysis of variance (ANOVA) between %TLT and independent variables Sexual maturity 170 Sex 170 Presence of thymic cyst 170 Linear regression analyses between %TLT and independent variables Nutritional status 169 0.65 (0.15) P 25CBs 170 0.09 (0.03)

0.32 0.23

F-ratio

p

F1,168 = 40.8 F1,168 = 0.71 F2,168 = 48.9

<0.001⁄⁄⁄ >0.40 <0.001⁄⁄⁄

F1,167 = 19.3 F1,168 = 9.50

<0.001⁄⁄⁄ <0.01⁄⁄

a Percentage thymic lymphoid tissue (%) is the dependent variable in the model. Factor refers to the independent variable in each test. Analyses were performed using SYSTAT (1998).

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Fig. 6. (A) Mean thymic lymphoid tissue (%) in porpoises of different sexual maturity (n = 170). (B) Thymic lymphoid tissue (%) against age (years) (n = 162). (C) Thymic lymphoid tissue (%) against nutritional status (relative body weight) (n = 169). (D) Thymic lymphoid tissue (%) against sum of 25 CBs (mg/kg lipid weight) (n = 170). (E) Thymic lymphoid tissue (%) of different thymic cyst scores (n = 170). (F) Mean age of different thymic cyst scores (n = 162).

with sexual maturity and presence of thymic cyst. As in the analysis using all 170 porpoises, there was no association with sex. Testing for harbour porpoises with total PCBs < 17 mg/kg lipid weight, %TLT was correlated with nutritional status (Fig. 6C) but not with P 25CBs (Fig. 6D). There remained a strong association with sexual maturity and presence of thymic cyst. Again, there was no association with sex. Multiple linear regressions with the dependent variable, %TLT, and the independent variables (nutritional status, sexual maturity P and 25CBs) were performed for both subsets above and below 17 mg/kg lipid weight of total PCB. As seen in Table 2., after selection of porpoises with total PCB > 17 mg/kg lipid weight, nutritional status and sexual maturity remained strongly associated P with %TLT. Interestingly, 25CBs was then also strongly and independently correlated with %TLT even after correcting for

confounding effects. However, with the subset for total P PCB < 17 mg/kg lipid weight, 25CBs was not independently associated with %TLT. Blubber PCBs (sum25CBs) levels were significantly higher in adult males (mean = 32.3 mg/kg lipid; n = 29) compared to adult females (mean = 8.12 mg/kg lipid; n = 20) (F1,47 = 41.3 p < 0.001) but there was no significant difference in %TLT between adult males and adult females (F1,47 = 0.763; p > 0.35). Finally, a one way analysis of variance with %TLT as the dependent variable and three categories of cause of death (physical trauma; infectious disease; ‘‘others’’) (n = 170) showed that %TLT was significantly different in all three cause of death categories (overall model: F2,167 = 43.4; p < 0.001). In this analysis mean %TLT was greatest in the ‘‘healthy’’ animals that died of acute physical trauma (mean = 73.5%; n = 92) and lowest in those animals that died of

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Table 2 Summary of multiple linear regression analyses with percentage thymic lymphoid tissue as the dependent variablea. Dependent variable

Independent predictors

b (standard error)

p

Thymic lymphoid tissue (%) (All data, n = 169)

Nutritional status Sexual maturity P 25CBs

0.66 (0.14) 0.35 (0.05) 0.04 (0.03)

0.33 0.45 0.102

<0.001⁄⁄⁄ <0.001⁄⁄⁄ >0.10 <0.001⁄⁄⁄

Nutritional status Sexual maturity P 25CBs

0.60 (0.17) 0.29 (0.06) 0.15 (0.05)

0.28 0.38 0.26

=0.001⁄⁄⁄ <0.001⁄⁄⁄ <0.01⁄⁄ <0.001⁄⁄⁄

Nutritional status Sexual maturity P 25CBs

0.65 (0.22) 0.35 (0.09) 0.01 (0.09)

0.35 0.47 0.02

<0.01⁄⁄ <0.001⁄⁄⁄ >0.85 <0.001⁄⁄⁄

b

Overall model: R2 = 0.33 Thymic lymphoid tissue (%) (subset of porpoises with total PCB > 17 mg/kg lipid weight, n = 109)

Overall model: R2 = 0.38 Thymic lymphoid tissue (%) (subset of porpoises with total PCB < 17 mg/kg lipid weight, n = 60)

Overall model: R2 = 0.29

a Percentage thymic lymphoid tissue (%) is the dependent variable in the model. Independent predictors are the factors and covariates included in each multiple regression model. Analyses were performed using SYSTAT (1998).

infectious disease (mean = 33.2%; n = 45). The porpoises that died of ‘‘other’’ causes of death had a mean %TLT of 56.5% (n = 33) and comprised mainly starvation cases or animals where the cause of death could not be established. 3.5. Statistical analyses of thymic cysts As discussed earlier, %TLT was highly associated with presence of thymic cysts, and seems to decrease with increasing thymic cyst score with a high degree of statistical significance (Fig. 6E). Pearson chi-squared tests and binary logistic regression were performed using presence of thymic cyst as the dependent variable and sexual P maturity, sex, nutritional status and 25CBs as the independent variables (Table 3). Univariate analyses showed that sexual maturity was significantly associated with presence of thymic cyst, and graphically, an increased mean age was seen for increasing thymic cyst score (Fig. 6F). Sex and nutritional status did not have an association with presence of thymic cyst. From the dataset, the oldest P animal (15 years) with no cyst had a 25CBs of 2.80 and the only P animal at 0 years with cysts (microcyst) had a 25CBs of 8.30. Although presence of thymic cyst seems to be associated with P 25CBs, this result was not significant. Performing a multiple logistic regression analysis with sexual maturity and %TLT as the independent variables and presence of thymic cyst as the dependent variable, both sexual maturity and %TLT remained independently correlated with presence of thymic cyst. Selecting for porpoises above the proposed threshold of total PCB > 17 mg/kg lipid weight, univariate analyses showed that presence of thymic cyst was significantly and positively associated P with sexual maturity and 25CBs but not with sex and nutritional status. The association between presence of thymic cyst and P 25CBs was no longer significant when a multiple logistic regression was conducted using sexual maturity as the confounding factor. However, sexual maturity remained strongly associated with presence of thymic cyst.

4. Discussion This is one of only a few studies to test for potential associations between PCBs and thymic involution and the development of thymic cysts in the harbour porpoise (Beineke et al., 2005; Jepson, 2003). The results of this study show a statistically significant assoP ciation between 25CBs and thymic involution in harbour porpoises above a total PCB threshold of 17 mg/kg lipid weight (Kannan et al., 2000) that was not confounded by any other measured variables including sexual maturity, sex and nutritional status. However, when porpoises of all PCB levels were analysed, the significant association with thymic involution in the univariate analysis was lost when other confounding factors were considered. PCB level of porpoises below the 17 mg/kg lipid weight threshold did not show any significant association with %TLT in the univariate analysis. Another study utilising a much smaller dataset (n = 61) by Beineke et al. (2005), found thymic involution to be associated with PCB level only at the univariate level when porpoises of all PCB concentrations were evaluated. However, Beineke et al. (2005) used only a semi-quantitative approach to rank the degree of thymic atrophy. The results obtained in this study were highly consistent with the proposed total PCBs toxicity threshold of 17 mg/kg total PCBs lipid weight for marine mammal blubber by Kannan et al. (2000) and suggest that above that threshold, PCBs may have an immuno toxic effect on the thymus. After selecting for animals above the proposed threshold, the association between PCBs and thymic involution was even stronger, despite the reduced sample size, and remained highly significant after accounting for the strong potentially confounding factors of onset of sexual maturity and nutritional status. This is consistent with experimental PCB-induced thymic involution in rodents and chicken embryos (Goff et al., 2005; Safe et al., 1985; Silkworth and Antrim, 1985) and impairment of natural killer cell activity, T-lymphocyte function and other changes in immune cellular activity in harbour seals

Table 3 Results of the analyses between presence of thymic cyst and the independent variables showna. Factor

n

v2

Wald v2

b (standard error)

eBb

Pearson chi-squared analyses between presence of thymic cyst and independent variables Sexual maturity 170 v21 = 70.3 Sex 170 v21 = 0.06 Binary logistic regression between presence of thymic cyst and independent variables Nutritional status 169 0.45 P 25CBs 170 2.95 a b

p <0.001⁄⁄⁄ =0.80

0.70 (1.05) 0.37 (0.21)

Presence of thymic cyst (yes/no) is the dependent variable in the model. Factor refers to the independent variable in each test. eB = Odds ratio. Analyses were performed using SYSTAT (1998).

2.02 0.69

>0.50 >0.05

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fed PCB-contaminated Baltic herring (Ross et al., 1996a). One-way analysis of variance also showed a highly statistically significant association between %TLT and cause of death category. The highest mean %TLT was in the ‘‘healthy’’ animals that died of acute physical trauma and the lowest mean %TLT in the porpoises that died of infectious diseases. These findings are therefore consistent with blubber PCB levels causing generalised immuno suppression in a dose-dependent manner and resulting in mortality due to a range of infectious diseases as has been suggested in other studies of UKstranded harbour porpoises (Jepson et al., 2005; Hall et al., 2006). Elevated blubber PCB concentrations could be the reason why harbour porpoises from North Sea, Baltic Sea and Norwegian waters show higher levels of thymic atrophy compared to porpoises from Icelandic waters (Beineke et al., 2005) and may pose a serious concern for the individual health and ongoing population viability of harbour porpoises UK and other even more highly PCB-contaminated areas like the Baltic Sea. A strong positive correlation was found between thymic involution (loss of thymic lymphoid tissue) and sexual maturity in all statistical tests. This correlation was expected since several studies have found that the cetacean thymus, like the human thymus (Park et al., 2008), increase to a maximum size in juveniles, then involutes (loses lymphoid tissue) with age (Clark et al., 2005; Cowan and Smith, 1999; Wünschmann et al., 1999). However, studies suggest that thymic involution with age is slow in the harbour porpoise as it was observed in most healthy adults (Clark et al., 2005; Wünschmann et al., 1999). In this study, the thymus was found in the two of the oldest animals (20 and 21 years old), albeit both thymuses were highly atrophied (%TLT = 4.0 and %TLT = 0.50 respectively). Because age values were highly skewed in the dataset, a categorical predictor, sexual maturity, had to be used. Using a continuous variable such as age could increase the statistical power of the analyses so a larger dataset with more adult samples could ideally be used in the future. Thymic involution was strongly and negatively correlated with nutritional status in all statistical analyses and corresponds with other cetacean studies (Beineke et al., 2005; Cowan, 1994). The body condition of the animal is very important when assessing the thymus, because the thymus can undergo involution in chronically stressed animals (Cowan, 1994). Nutritional status of the animal can also affect metabolism. When porpoises undergo starvation, fat and contaminants from the blubber are mobilized and can travel into the blood stream to cause toxicity in other tissues or be excreted (Kuiken et al., 1994). Males had a lower average %TLT than females, although this result was not statistically significant in any age group. Adult males had significantly higher blubber PCB levels than adult females but there was no significant difference in PCB levels between male and female juveniles. The lower PCB levels in adult females were mainly due to maternal offloading of lipophilic pollutants like PCBs to calves during gestation and lactation (Aguilar and Borrell, 1994; Borrell et al., 1995). The first calf is thought to receive the highest amount of PCBs from maternal offloading (Aguilar and Borrell, 1994; Borrell et al., 1995) with up to 80% of the mother’s organochlorine burden transferred within the first two months of lactaction (Cockcroft et al., 1989). Maternal offloading of PCBs to calves may account for the high PCB levels and loss of thymic lymphoid tissue in some neonate porpoises found in this and other studies (Beineke et al., 2005; Jepson et al., 1999, 2005). An extended study with a larger dataset could be undertaken in order to test for differences in %TLT among sexually immature males/females and sexually mature females/males and for any association with PCB concentrations. The morphology of the microcysts and macrocysts is consistent with those observed in other studies of cetacean thymuses (Cowan, 1994; Wünschmann et al., 1999). Thymic cysts were not correlated

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with 25CBs when all porpoises were analysed but results were significant in the univariate analysis with the subset of porpoises above the threshold (total PCB > 17 mg/kg lipid weight). However, this significance was lost when a multiple regression was conducted with sexual maturity as the confounding factor. The %TLT remained highly associated with presence of thymic cysts after accounting for sexual maturity. These results support other studies which report the development and enlargement of cetacean thymic cysts to be correlated with atrophy of thymic lymphoid tissue and age (Cowan, 1994; Wünschmann et al., 1999). If high PCB levels do cause thymic involution, it is theoretically possible that PCBs may have a secondary (indirect) causal effect on thymic cyst formation.

5. Conclusion This study supports the hypothesis that elevated PCB levels in harbour porpoises leads to thymic involution (loss of thymic lymphoid tissue), possibly contributing to immuno suppression and increased disease susceptibility. This association was only found in porpoises above the blubber ‘‘total PCBs’’ toxicity threshold (>17 mg/kg lipid weight in blubber) proposed by Kannan et al. (2000). Thymic cyst growth appears mainly age-related, although, if PCBs do act as thymotoxic contaminant, they could have a secondary role as a factor in cyst development. These results support a wider body of research demonstrating links between chemical pollutant exposure and potential health effects. Further investigations of the potential health effects of PCB exposure in marine mammals are needed in UK and other waters. Acknowledgements This research was conducted under the aegis of the UK Cetacean Strandings Investigation Programme (CSIP), which is funded by the Department for Environment, Food and Rural Affairs (Defra) and the Devolved Administrations in Scotland and Wales. Contaminant analyses were funded by Defra. The authors would also like to thank Thijs Kuiken, Vic Simpson, Nick Davison, John Baker, James Kirkwood and Stella Turk who conducted and facilitated some of the necropsies carried out in this study.

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