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High levels of polychlorinated biphenyls in tissues of Atlantic turtles stranded in the Canary Islands, Spain
Chemosphere 74 (2009) 473–478
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Chemosphere j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / ch e m o s p h e r e
High levels of polychlorinated biphenyls in tissues of Atlantic turtles stranded in the Canary Islands, Spain J. Orós a,*, O.M. González-Díaz b, P. Monagas a a b
Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontana s/n, 35413 Arucas (Las Palmas), Spain Department of Chemistry, University of Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Spain
a r t i c l e
i n f o
Article history: Received 18 March 2008 Received in revised form 24 August 2008 Accepted 25 August 2008 Available online 4 December 2008 Keywords: Polychlorinated biphenyls Sea turtles Caretta caretta Chelonia mydas Dermochelys coriacea Atlantic Ocean
a b s t r a c t Polychlorinated biphenyls (PCBs 28, 31, 52, 101, 138, 153, 180, and 209) were measured in tissue samples (liver and fat) from 30 loggerhead turtles Caretta caretta, 1 green turtle Chelonia mydas, and 1 leatherback Dermochelys coriac ea stranded on the coasts of the Canary Islands, trying to establish a possible relation between PCB con centrations and the lesions and causes of death. Tissues from these turtles contained higher levels of PCBs than those reported in turtles from other geographical regions. )PCB concentrations (1980 ± 5320 ng g¡1 wet wt.) in the liver of loggerheads were higher than in the adipose tissue (450 ± 1700 ng g¡1 wet wt.). Concentrations of PCB 209 in the liver (1200 ± 3120 ng g¡1 wet wt.) of loggerheads and in the liver (530 ng g¡1 wet wt.) and adipose tissue (500 ng g¡1 wet wt.) of the leatherback were remarkable. Frequencies of detection of PCB 209 in the liver (15.5%) and adipose tissue (31%) were also remarkable. Cachexia was detected in 7 turtles (22%) and septicemia was diagnosed in 10 turtles (31%). Statistically, a positive correlation was detected between )PCBs concentration and cachexia. Poor physic al condition, cachexia and/or septicaemia could explain the high levels of PCBs and tissue distribution. However, no histological lesions exclusively attributed to the acute effects of PCBs were described. The most prevalent histologic al lesions were ulcerative and purulent oesopha gitis, purulent dermatitis, necrotizing enterit is, and granulomatous pneumonia. The bacteria most frequently isolated were Escherichia coli, Staphylococcus sp., and Aeromonas sp. Although immunosupression as a result of PCBs pollution has been described previously, other factors in this study, such as incidental fishing, nutritional status, and exposition to different micro-organisms, make it dif ficult to establish a clear association between PCB concentrations and causes of death. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Two families and seven species of sea turtles are currently recognised (Pritchard, 1997) and included in the red list of the World Conservation Union (IUCN, 2007). The family Dermochelyi dae includes only the leatherback (Dermochelys coriacea). The fam ily Cheloniidae includes the green turtle (Chelonia mydas), logger head (Caretta caretta), hawksbill (Eretmochelys imbricata), Kemp’s ridley (Lepidochelys kempi), olive ridley (Lepidochelys olivacea), and flatback turtles (Natator depressa). The most common species in the Canary Islands is the loggerhead turtle (Mateo et al., 1997). However, evidence of a decline in the population of turtles in the Canary Islands has been reported (López-Jurado and González, 1983; Blanco and González, 1992). Diseases and causes of mortality among turtles stranded in the Canary Islands have been previously reported (Orós et al., 2004, 2005). However, data available for baseline levels of contaminants
* Corresponding author. Tel.: +34 928451089; fax: +34 928457431. E-mail address: jo[email protected]pgc.es (J. Orós). 0045-6535/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.08.048
and effects on the turtle populations of the Canary Islands are scarce (Torrent et al., 2004). Polychlorinated biphenyls (PCBs) have a particular significance because of their undesirable effects on environmental quality and animal health (Ahlborg et al., 1994). PCBs were manufactured from the 1930s to the 1970s for several industrial applications, such as liquid coolants for electrical transformers or as softeners in the production of plastics and as components of hydraulic fluids and lubricating oils. PCBs are able to bioaccumulate through the food chain and their effects have been reported on the immune, endo crine, and reproductive systems of different animal species (Fox, 2001). Although much was reported to date on PCBs concentra tions in large predators, few studies have been dedicated to turtles. These studies have been focused on turtles from Long Island (Lake et al., 1994), Virginia (Rybitski et al., 1995), Scotland (Mckenzie et al., 1999), the Hawaiian Islands (Miao et al., 2001), the Baja Cal ifornia Peninsula (Gardner et al., 2003) and North Carolina (Kel ler et al., 2004, 2006). Studies focused on the Mediterranean Sea have a particular significance because of their number (Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Perugini et al., 2006; Storelli et al., 2007).
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The aim of this study was to evaluate the presence and pat terns of eight PCB congeners (28, 31, 52, 101, 138, 153, 180, and 209) in tissue samples (liver and fat) from 32 turtles stranded on the coasts of the Canary Islands between August 2002 and Novem ber 2005. We also tried to determine a possible relation between the PCB concentrations and the lesions and causes of death using the Spearman’s rho correlation method to calculate the correlation between )PCB concentrations in both tissues and physic al condi tions such as cachexia and septicaemia.
was obtained, bacteria were identified based on the biochemical profile (API 20 E, API 20 NE, and API 20 STAPH, BioMérieux).
2. Materials and methods
2.4. PCBs analysis
2.1. Turtles
Polychlorinated biphenyls (IUPAC Nos. 28, 31, 52, 101, 138, 153, 180, and 209) were analysed according to the method described by Tanabe et al. (1994). The validity of analytical methods was con firmed with Standard Reference Materials (CARP-2: ground whole carp, Cyprinus carpio) obtained from the National Research Council of Canada. Precision and accuracy are reported in Table 2. Briefly, aliquots (4–7 g) of the homogen ized samples were ground with anhydrous sodium sulphate in a mortar, and extracted using Soxhlet apparatus for 6 h with 300 mL of diethyl ether:hex ane (3:1) solvent mixture. Extracts were concentrated in volume to 10 mL in Kuderna-Danish, and the aliquots (2 mL) were transferred to a glass column packed with 20 g of Florisil and dried by passing through nitrogen gas. Organochlorines adsorbed on Florisil were eluted with 150 mL of 20% hexane-washed water in acetonitrile and transferred to a separatory funnel containing 600 mL of hex ane-washed water and 100 mL of hexane. After partitioning, the hexane layer was concentrated, cleaned up with sulphuric acid, and passed through a 12 g Florisil-packed glass column for sepa ration. Final determination of PCBs was carried out using a Varian 3600 gas chromatograph fitted with an electron capture detec tor (GC-ECD). In all the analyses a fused-silica capillary column Supelco (length = 30 m, inner diameter 0.53 mm and film thick ness 0.50 lm) was used. The column oven temperature was pro grammed from 60 to 160 °C, held for 10 min, and then increased to 260 °C at a rate of 2 °C/min and held for 20 min. Injector and detec tor temperatures were set at 260 and 280 °C, respectively. Nitrogen was used as a carrier gas at 63.3 mL/min. The PCB patron used as an internal standard was the PCB-Mix 12, Iso-octane (Lab. Dr. Ehren storfer). Quantification interval used for PCBs was from 1 ng g¡1 (instrumental detection limit) to 50 000 ng g¡1 (optimum linear limit). Concentrations of PCBs, means of four measurements, are presented as ng g¡1 on a wet weight basis.
Between August 2002 and November 2005, 32 turtles that got stranded on the coasts of four islands belonging to the Canary Islands [Gran Canaria (n = 25; 78.1%), Tenerife (n = 3; 9.4%), Fuerteventura (n = 2; 6.2%), and El Hierro (n = 2; 6.2%)] were sub mitted for necropsy to the Veterinary Faculty, University of Las Pal mas de Gran Canaria (ULPGC). Some of them had been previously submitted to the Tafira Wildlife Rehabilit ation Center (TWRC) for health evaluation, medical management, and possible rehabilita tion. Species identifications were made according to Frick (1996). Turtles belonging to three different species were examined: 30 loggerheads, C. caretta (93.7%), 1 green turtle, C. mydas (3.1%), and 1 leatherback, D. coriacea (3.1%). Species, sex, age group, and bio metrics data are shown in Table 1. Age group was determined on the basis of straight carapace length (SCL) (Bjorndal et al., 2001; Seminoff et al., 2004) and sexual maturity (estimated from the appearance of their gonads). 2.2. Pathological and microbiologic al studies Necropsies were performed at the Veterinary Faculty (ULPGC) within the first 12 h after death. The gross postmortem examina tions were carried out using the procedures previously described (Wolke and George, 1981; Orós and Torrent, 2001). Gross lesions were recorded and tissue samples from all major organs were fixed in 10% neutral buffered formalin, embedded in paraf fi n, sectioned at 5 lm for light microscopy and stained with haematoxylin and eosin. Special stains used on selected cases included Gram stain for bacteria, Ziehl-Neelsen stain for acid-fast organisms, periodic acid-Schiff stain for protozoa and fungal hyphae, Grocott’s methe namine silver nitrate for fungi, and von Kossa stain for calcium (Bancroft and Stevens, 1996). For the microbiologic al studies, sam ples were taken from gross lesions and cultured on a variety of selective and non-selective media, including blood agar (Oxoid), MacConkey agar (Oxoid), Baird Parker agar (Oxoid) for staphylo cocci, and Sabouraud Dextrose agar (Oxoid) for fungi and yeasts. All cultures were incubated at 25 °C aerobically. Once a pure growth
2.3. Samples collection for PCB analysis Tissues samples (liver and coelomic fat) were collected during necropsy. After collection, the samples were wrapped in an alu minium foil and stored at ¡20 °C until analysis. Prior to analysis, tissue samples were homogen ized using a commercial blender.
2.5. Statistical analysis Mann–Whitney U test was conducted to determine whether the difference in the levels of PCBs were related to the tissues. Spearman’s rho correlation method was used to calculate the
Table 1 Species, sex, age group and biometrics data (mean ± standard deviation) of the tur tles analysed
Table 2 Precision and accuracy of analytical methods obtained using a certified ground whole carp Cyprinus carpio (CARP-2)a
Species
Sex
Age group
PCBs
Caretta caretta (n = 30)
Female (n = 27; 90%) Male (n = 3; 10%) Female
Pelagic juvenile 11.5 ± 8.3 (n = 12; 40%) Juvenile (n = 18; 60%)
41 ± 11.7
Juvenile
21
52
Female
Adult
231.5
nm
Chelonia mydas (n = 1) Dermochelys coriac ea (n = 1)
SCL: straight carapace length. n: number of turtles. nm: not measured.
Weight (kg)
SCL (cm)
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180 PCB 209
CARP-2 Certified
Foundb
34 ± 4.0 138 ± 43 145 ± 48 103 ± 30 105 ± 22 53.3 ± 13.0 4.6 ± 2.0
31 ± 3.1 119.7 ± 13.9 150.1 ± 24.1 99.5 ± 18.7 116 ± 16.6 59.2 ± 10.7 4.8 ± 3.6
a
The concentrations are given in ng g¡1 wet wt.
b
Number de replicates is 4.
J. Orós et al. / Chemosphere 74 (2009) 473–478
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c orrelation between )PCB concentrations in both tissues and physic al conditions such as cachexia and septicaemia. According to the necropsy reports, values of 0, 1, and 2 were given to tur tles with absence of cachexia, mild cachexia, and severe cachexia, respectively. Values of 0, 1, and 2 were also given to turtles with absence of infections, infection in one organ, and severe septicae mia.
Table 3 PCBs concentrations (range, arithmetic mean ± standard deviation) (ng g¡1 wet wt.) in liver and fat of the three species of sea turtles analysed.
3. Results
PCB 28,31
3.1. Gross pathology, histopathology, and microbiology
Caretta caretta n = 30
)PCB
PCB 52 PCB 101
The pathological study revealed that 23 turtles (72%) died from lesions associated with human activities such as boat-strike inju ries (n = 4; 12.5%), entanglement in derelict fishing nets (n = 7; 22%), and ingestion of hooks and monofilam ent lines (n = 12; 37.5%). Only 9 turtles (28%) died from spontaneous diseases, including pulmonary emphysema, ulcerat ive keratoconjunctivitis, purulent adenitis of the salt glands and purulent cloacitis. Cachexia was detected in 7 turtles (22%). Septicemia was diagnosed in 10 turtles (31%). The histological lesions observed in the loggerheads included severe necrotic hepatitis (n = 4; 13%), severe multifocal granulom atous hepatitis (n = 4; 13%), severe ulcerat ive and purulent oesoph agitis (n = 10; 33%), mild purulent gastritis (n = 2; 6.7%), mild to severe necrotizing enteritis (n = 5; 16.7%), severe purulent cloacitis (n = 1; 3%), mild to severe granulomatous pneumonia (n = 5; 16.7%), severe pulmonary emphysema (n = 1; 3%), severe necrotic spleni tis (n = 1; 3%), severe ulcerative and purulent keratoconjunctivitis (n = 3; 10%), severe purulent adenitis of the salt glands (n = 4; 13%) and severe purulent dermatitis (n = 9; 30%). Bacteria isolated from the tissue lesions included Escherichia coli (n = 5; 16.7%), Proteus sp. (n = 3; 10%), Vibrio alginolyticus (n = 2; 6.7%), Pseudomonas sp. (n = 2; 6.7%), Streptococcus sp. (n = 2; 6.7%), Citrobacter freundii (n = 2; 6.7%), Aeromonas sp. (n = 3; 10%), Staphylococcus sp. (n = 4; 13%), and Kleb siella sp. (n = 2; 6.7%). Histological lesions observed in the specim en of C. mydas included severe multifocal granulomatous nephritis associated to Candida guillermondi infection and severe oedema of the lamina propia and intestinal submucosa. Histological lesions observed in the specimen of D. coriac ea included severe multifocal necrotic nephritis, severe granulomatous pneumonia and severe necrotiz ing enteritis, and all these lesions were associated with Serratia marcescens infection. 3.2. PCBs PCB concentrations measured in the two tissues of the three species of turtles analysed are shown in Table 3. Loggerheads showed the highest hepatic )PCB (IUPAC Nos. 28, 52, 101, 138, 153, 180) mean concentration (1980 ± 5320 ng g¡1 wet wt.), followed by the only specimen of D. coriacea analysed (445 ng g¡1 wet wt.). )PCB concentration in the liver of the only specim en of C. mydas analysed was lower (116 ng g¡1 wet wt.) than those detected in loggerheads and leath erback turtles. Loggerheads also showed the highest )PCB mean concentration in the adipose tissue (450 ± 1700 ng g¡1 wet wt.). Loggerheads showed the highest hepatic PCB 209 mean con centration (1200 ± 3120 ng g¡1 wet wt.), followed by the only specimen of D. coriacea analysed (530 ng g¡1 wet wt.). However, the leatherback showed the highest PCB 209 concentration in the adipose tissue (500 ng g¡1 wet wt.). Six loggerhead turtles had individual PCB concentrations higher than 2000 ng g¡1, and seven turtles showed individual PCB concentrations higher than 900 ng g¡1. All these turtles showed severe septicaemia and/or severe cachexia.
PCB 138 PCB 153 PCB 180 PCB 209
Chelonia mydas n = 1
Liver
Fat
BDL – 34900 1980 ± 5320 BDL – 590 42 ± 131 BDL – 1000 98 ± 262 BDL – 670 32 ± 130 BDL – 13300 456 ± 2426 BDL – 15440 915 ± 3180 BDL – 3890 440 ± 1016 BDL – 14900 1200 ± 3120
BDL – 9800 450 ± 1700 BDL – 38 3.8 ± 9 BDL – 56 5.5 ± 12.5 BDL – 84.5 6 ± 19 BDL – 2040 83 ± 370.5 BDL – 2600 133 ± 477 BDL – 5006 217 ± 907 BDL – 218 15 ± 45.5
Dermochelys coriacea n = 1
Liver
Fat
Liver
Fat
116
144
445
77
BDL
5.7
71.5
9.7
BDL
7.3
8.7
11.4
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
58
251
56
116
73
114
BDL
BDL
BDL
530
500
BDL: below detection limit.
)PCB = sum of PCBs nos. 28, 52, 101, 138, 153, and 180.
As revealed by statistical analysis, turtles showed significant higher PCBs 28,31, PCB 153, PCB 209 and )PCBs concentrations in the liver than in the adipose tissue. However, no significant dif ferences between concentrations in both tissues were detected for PCB 138 and PCB 180. The congener most frequently detected in the liver was PCB 180 (46.8%), followed by PCB 153 (31.2%), PCBs 28, 31 (25%), PCB 52 (25%), PCB 209 (15.6%), PCB 138 (15.6%), and PCB 101 (12.5%). The congeners most frequently detected in the adipose tissue were PCB 180 (50%) and PCB 153 (50%), followed by PCBs 28,31 (34.4%), PCB 52 (34.4%), PCB 209 (31.2%), PCB 138 (28.1%), and PCB 101 (18.7%). PCB profiles in the C. caretta tissues were dominated by the higher chlorinated congeners (Fig. 1). Hexachlorobiphenyls (PCBs 138 and 153) were predominant and accounted for 43.6% of the total PCBs, followed by decachlorobiphenyls (PCB 209) (33%) and heptachlorobiphenyls (PCB 180) (18%). Tetrachlorobiphenyls (PCB 52) accounted for 3% of the total PCBs, and trichlorobiphenyls (PCBs 28 and 31) accounted for 1.3% of the total residue. Concern ing individual congeners, the most abundant was PCB 209 (33%) followed by PCB 153 (29%), PCB 180 (18%), and PCB 138 (15%). The isomer pattern of PCBs in C. caretta was different in the liver and the fat. In the liver, the most abundant was PCB 209 (37.6%), fol lowed by PCB 153 (29%), PCB 138 (14%) and PCB 180 (14%). In the fat, the most abundant was PCB 180 (47%), followed by PCB 153 (28.6%), PCB 138 (18%) and PCB 209 (3%).
3Cl
4Cl
5Cl
6Cl
7Cl
10Cl
C. caretta (liver) C. caretta (fat) C. mydas (liver) C. mydas (fat) D. coriacea (liver) D. coriacea (fat) 0%
20%
40%
60%
80%
100%
Fig. 1. Fingerprints of liver and fat of the three species of sea turtles analysed.
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Regarding PCB profiles in the only specimen of C. mydas ana lysed, the most abundant was PCB 180 (73%) followed by PCB 153 (22%) and PCB52 (3%). Although PCB 180 was the most abundant in both tissues, this congener was the only PCB detected in the liver of this turtle. Regarding PCB profiles in the only specimen of D. coriacea ana lysed, the most abundant was PCB 209 (66%) followed by PCB 153 (20%) and PCB 180 (7%). The isomer pattern of PCBs in this tur tle was slightly different in the liver and the fat. In the liver, the most abundant was PCB 209 (54%) followed by PCB 153 (26%) and PCB 180 (12%). In the fat, the most abundant was PCB 209 (87%), followed by PCB 153 (10%), and PCB 52 (2%). Hepatic )PCBs concentrations were positively correlated with cachexia (r = 0.250, P = 0.04). No significant correlation was observed between )PCBs concentrations and septicaemia. 4. Discussion The present study represents the first data of PCB levels for any turtle species from the Canary Islands. The turtle tissues analysed in the current investigation contained high levels of PCBs com pared to concentrations reported in turtles collected in other loca tions around the world (Table 4) (McKim and Johnson, 1983; Lake et al., 1994; Rybitski et al., 1995; Mckenzie et al., 1999; Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Gardner et al., 2003; Keller et al., 2004; Perugini et al., 2006; Storelli et al., 2007). The high degree of varia tion of concentrations of PCBs detected in our study may be attributed to the different exposure of
i ndividual turtles to pollutants and to the physical condition of the animals. Sea turtles visit different areas with different degree of contamination during migration, resulting in differences in expo sure for each animal (Ziswiler, 1986). According to recent studies, loggerhead turtles stranded on the coasts of the Canary Islands come from two different migration routes: from the Western Atlantic arriving to the Canary Islands by the Gulf Stream (PérezJiménez, 1997), and from Cape Verde (Dellinger, 2006). In addition, turtles included in this study varied widely in physical condition, ranging from turtles that had been killed incidentally through human interaction to turtles that showed severe emaciation and septicaemia after prolonged illness. Six loggerhead turtles had individual PCB concentrations higher than the quantification limit of 2000 ng g¡1 established by the Food and Drug Administration (FDA). In addition, seven turtles showed individual PCB concentrations higher than 900 ng g¡1. All these turtles showed severe septicaemia and/or severe cachexia. Statistically, a positive correlation was detected between )PCBs concentration and cachexia. Although sea turtles are long-living high trophic organisms that would be expected to contain high concentrations of PCBs (Lake et al., 1994), we think the poor physical condition of these animals could be the reason for these high PCB levels. It has been described that in cetaceans many diseases affect metabolic centres and thus the capacity to metabolise or excrete pollutants may be affected, questioning whether PCB concentrations in the tissues of these specimens are representative of normal conditions (Aguilar et al., 1999).
Table 4 PCB concentrations (average concentration or range, ng g¡1 wet wt.) in hepatic and adipose tissue samples from sea turtles reported by other authors Species
Locality
Tissue (n) )PCB
PCB 118
PCB 138
PCB 153
PCB 180
Reference
L. kempi
Long Island Virginia and North Carolina
98.3 148.9 BDL-59.5
114.94 195 BDL-65.8
170.04 272.5 2.04–152
53.62 102.7 BDL-49.4
Lake et al. (1994)
C. caretta
Liver (22) 512.6 Fat (13) 863 Liver (18) 7.46–514 Fat (20) Liver (3) Fat (3) Liver (4)
55.4–1730 158–608 90.4–904 50–102
<0.5–<1.6
<0.5–<1.6
<0.5–3.4
4.55–193 14.8–18.7 19.4–91.9 3.3–8.7
3.77–236 26.1–35.6 38.9–156 12–24
12.1–406 43.1–259 90.4–283 13–29
BDL-148 10.3–19.5 19.1–64 7.3–18
Fat (3) Liver (1) Liver (2) Fat (2) Liver (9)
775–893 159 3.1–3.7 47–178 <1.1–77
<4.8–<8.0 <0.9 <0.6–<0.8 <4.7–<7.2 <0.7–<3.4
8.6–37 15 2.2–3.9 12 0.7–4.3
8–32 16 <0.6–<0.8 5.8–<7.2 0.9–7.4
54–71 12 <0.6–2.8 <7.2–8.3 <0.7–4.8
132–169 20 <0.6–0.8 <7.2–25 <1.1–11
229–261 24 1.3–1.5 7.8–46 <1.1–13
73–154 12 <0.6–<0.8 <7.2–24 <1.1–7.4
<2.2–<3.7
13.25
5.7–33
<2.2–27
<2.2–28
<2.2–3.2
<2.2–13
L. kempi C. caretta
Mediterranean Sea
C. caretta Scotland D. coriacea Scotland C. mydas
Mediterranean Sea
C. caretta
Adriatic Sea
Fat (3) Liver (11)
C. caretta
Adriatic Sea
Liver (4)
39–261 3.22–6.54A,a 0.21–0.76A,b 119
C. mydas
Baja California peninsula
Fat (4) Liver (7)
334 BDL-44.7
Fat (7) Liver (1) Fat (1) Liver (1) Fat (1) Fat (44) Liver (11)
BDL-49.5 58.1 18.4 41 BDL 2010 ± 2960B 83 7.6–247.3
L. olivacea C. caretta C. caretta C. caretta
North Carolina Adriatic Sea
Fat (11) C. caretta
Adriatic Sea
459.7 2.9–1472.1 Liver (19) 6.85–297.49
BDL: below detection limit. A Value expressed as mg kg¡1 lipid weight. B Value expressed as ng g¡1 lipid weight. a Young turtles. b Adult turtles.
PCB 28
PCB 52
PCB 101
4.68–11.10
14.25–42.82
17.84–45.50
868–28.83
9.55–36.97
23.78–103.70 31.15–134.46 19.60–78.10
Rybitski et al. (1995)
Mckenzie et al. (1999)
Storelli and Mar cotrigiano (2000) Corsolini et al. (2000) Gardner et al. (2003)
11 1.1–26.5 62.4 0.33–150.8
26.7 2.2–92.9 24.5 2.5–75.7 115.8 115 0.6–294.2 0.6–369.2
Keller et al. (2004) Perugini et al. (2006)
Storelli et al. (2007)
J. Orós et al. / Chemosphere 74 (2009) 473–478
ng/g wet wt
2000 1500 1000
Fat
500
Liver
0
C. caretta
C. mydas
D. coriacea
ng/g wet wt
2000 1500 1000
Fat
500
Liver
0
C. caretta
C. mydas
D. coriacea
Fig. 2. )PCB (IUPAC nos. 28, 52, 101, 138, 153, 180) mean concentrations (a) and PCB 209 mean concentrations (b) in the hepatic and adipose tissue samples of the three species of sea turtles analysed.
According to other authors, the pattern of tissue distribution of PCBs reported in turtles and other marine species is as follows: adipose tissue > liver > kidney > muscle (Tanab e et al., 1983; Marti neau et al., 1987; Colborn and Clement, 1992; Rybitski et al., 1995; Mckenzie et al., 1999). This is attributed to the highest total lipid content and triglycerides for the adipose tissues and the strong correlations between these and their burden of lipophilic pollu tants (Cockcroft et al., 1989). However, we detected higher )PCBs concentrations in the liver than in the adipose tissue (Fig. 2). Thir teen turtles showed higher PCB levels in the liver than in the fat. All these turtles showed severe cachexia and/or septicaemia. Accord ing to different studies, animals with abundant fat deposits can accumulate and tolerate higher concentrations of toxic chemicals because lipophilic pollutants are stored in the adipose tissue and are less available to target organs and receptors. In contrast, ema ciated animals which have mobilised their lipid stores may be more susceptible to toxic effects as a result of remobilisation of the pollutants, resulting in higher concentrations of PCBs in the remaining tissues such as the liver (Bernhoft and Skaare, 1994). Thus, the cachexia observed in our turtles may indicate a previous remobilis ation of the fat and the pollutants, and could explain the high concentration of PCBs in the liver. In addition, the septicae mic condition reported in these turtles could be caused by a possi ble inmunosupression as one of the most distinguished effects of organochlorine compounds. In our study, PCB profiles in the C. caretta tissues were domi nated by the higher chlorinated congeners (Fig. 1). Hexaclorobi phenyls and heptaclorobiphenyls are predomin ant in turtles and marine mammals (Muir et al., 1988; Rybitski et al., 1995; Colborn and Smolen, 1996; Alam and Brim, 2000; Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Miao et al., 2001; Perugini et al., 2006; Storelli et al., 2007). However, Gardner et al. (2003) reported in C. mydas a profile domin ated by lower-chlorinated congeners. Differences in PCB patterns in turtles may be attributed to dif ferences in the congener compositions of environmental media among regions, dietary differences or differences in the abilities of the various species and populations to metabolize PCBs (Gardner et al., 2003). In our study, the concentrations and frequencies of detection of PCB 209 were remarkable. Usually, published reports do not
477
include this congener. Gardner et al. (2003) detected very low concentrations of PCB 209 following a decreasing order in muscle, liver and adipose tissue of turtles. Storelli et al. (2007) reported in a study on 19 loggerhead turtles that PCB 209 accounted only for 0.1–1.6% of total PCBs. One of our objectives was to establish a possible relationship between PCBs concentrations and the causes of death of each tur tle. It was dif ficult to assess the impact of organochhorine com pounds in the turtles of our study because the most frequent causes of death (72%) were attributed to fishing activity. In addi tion, few toxicological studies have been published regarding organochlorines in reptiles and no safe concentrations have been established. It is remarkable that almost all turtles with severe septicaemia showed very high levels of PCBs. Immunosupression as result of PCBs pollution has been previously described (Keller et al., 2004, 2006). However, in our study the presence of other factors different to PCBs concentrations, such as incidental fish ing, nutritional status, and exposition to different micro-organ isms, make it dif ficult to establish a clear association between PCB concentrations and causes of death. According to the histopathol ogical study no lesions exclusively attributed to acute effects of PCBs were described. All the histological lesions were attributed to bacterial and/or fungal infections mainly associated with gross lesions caused by fishing activity. However, chronic effects of PCBs are much dif ficult to determine. While there was an insuf ficient number of samples to assess differences among species, qualitative evaluation could indicate the potential effects of poor physical condition differences among individuals of C. caretta and subsequent poor metabolic perfor mance of detoxification systems. In addition, the detection of high levels of PCBs in these turtles makes it necessary to continue to monitor the levels of these chemicals in turtles from the Canary Islands to contribute to the protection of these currently endan gered species. Acknowledgements The authors would like to thank members of the Consejería de Medio Ambiente, Cabildo de Gran Canaria, for providing the turtles. This investigation was partially supported by the national project I + D REN2000-1753 MAR. References Aguilar, A., Borrel, A., Pastor, T., 1999. Biologic al factors affecting variability of persistent pollutant levels in cetaceans. J. Cetacean Res. Manage. 1, 83–116. Alam, S.K., Brim, M.S., 2000. Organochlorine, PCB, PAH, and metal concentrations in eggs of loggerhead sea turtles (Caretta caretta) from northwest Florida, USA. J. Environ. Sci. Health 35, 705–724. Ahlborg, U.G., Becking, G.C., Birnbaum, L.S., Brouwer, a., Derks, H.J.G.M., Feeley, M., Golor, G., Hanberg, A., Larsen, J.C., Liem, A.K.D., Safe, S.H., Schlatter, C., Waern, F., Younes, M., Yrjänheikki, E., 1994. Toxic equivalency factors for dioxin-like PCBs: report on WHO-ECEH and IPCS consultation, December 1993. Chemosphere 28, 1049–1067. Bancroft, J.D., Stevens, A., 1996. Theory and Practice of Histological Techniques, fourth ed. Churchill Livingstone, New York. Bernhoft, A., Skaare, J.U., 1994. Levels of selected individual polychlorinated biphe nyls in different tissues of harbour seals (Phoca vitulina) from the southern coast of Norway. Environ. Pollut. 86, 99–107. Bjorndal, K.A., Bolten, A.B., Koike, B., Schroeder, B.A., Shaver, D.I., Teas, W.G., W itzell, W.N., 2001. Somatic growth function for immature loggerhead sea turtles, Caretta caretta, in southeastern US waters – Statistical Data included.. Fish. Bull. 99, 240–246. Blanco, J.C., González, J.L., 1992. Libro rojo de los vertebrados españoles. Ministerio de Agricultura, Pesca y Alimentación. Colección Técnica ICONA, Madrid. Cockcroft, V.G., De Kock, A.C., Lord, D.A., Ross, G.J.B., 1989. Organochlor ines in bot tlenose dolphins (Tursiops truncatus) from the east coast of South Africa. S. Afr. J. Mar. Sci. 8, 207–217. Colborn, T., Clement, C., 1992. Chemically Induced Alterations in Sexual and Func tional Development: The Wildlife/human Connection. Princeton Scientific Pub lishing, Princenton, New Jersey.
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Colborn, T., Smolen, M.J., 1996. Epidemiol ogical analysis of persistent organo chlorine contaminants in cetaceans. Rev. Environ. Contam. Toxicol. 146, 91–172. Corsolini, S., Aurigi, S., Focardi, S., 2000. Presence of polychlorobiphenyls (PCBs) and coplanar congeners in the tissues of the Mediterranean loggerhead turtle Caretta caretta. Mar. Pollut. Bull. 40, 952–960. Dellinger, T., 2006. Vida no mar alto: os juvenis pelágicos das tartarugas marinhas no Atlântico. In: Proceedings IX Congreso Luso-Español de Herpetología. San Sebastián, Spain, pp. 27–28. Fox, G.A., 2001. Wildlife as sentinels of human health effects in the Great Lakes-St. Lawrence Basin. Environ. Health Perspect. 109, 853–861. Frick, M.G., 1996. A Guide for the Identification of Stranded Dead Turtles: The East ern United States and the Gulf of Mexico. Savanah Science Museum Special Publication. Gardner, S.C., Pier, M.D., Wesselman, R., Juárez, J.A., 2003. Organochlorine con taminants in sea turtles from the Eastern Pacific. Mar. Pollut. Bull. 46, 1082– 1089. I.U.C.N./S.S.C., 2007. 2007 I.U.C.N. Red List of threatened Species. (accessed 02.12.07). Keller, J.M., Kucklick, J.R., Harms, C.A., McClellan-Green, P.D., 2004. Organochlorine contaminants in sea turtles: correlations between whole blood and fat. Environ. Toxicol. Chem. 23, 726–738. Keller, J.M., McClellan-Green, P., Kucklick, J.R., Keil, D.E., Peden-Adams, M.M., 2006. Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments. Envi ron. Health Perspect. 114, 70–76. Lake, J.L., Haebler, R., McKinney, R., Lake, C.A., Sadove, S.S., 1994. PCBs and other chlorinated organic contaminants in tissues of juvenile Kemp’s ridley turtles (Lepidochelys kempi). Mar. Environ. Res. 38, 313–327. López-Jurado, L.F., González, S., 1983. Las tortugas en Canarias. Aguayro 147, 29– 31. Martineau, D., Beland, P., Desjardins, C., Legace, A., 1987. Levels of organochlo rine chemicals in tissues of beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Queb ec, Canada. Arch Environ. Contam. Toxicol. 16, 137–147. Mateo, J.A., Andreu, A.C., López-Jurado, L.F., 1997. Las tortugas marinas de la Penín sula Ibérica, Baleares, Azores, Madeira y Canarias: Introducción. In: Pleguezue los, J.M. (Ed.), Distribución y Biogeografía de los Anfibios y Reptiles en España y Portugal. Universidad de Granada Editorial, Granada, pp. 433–434. Mckenzie, C., Godley, B.J., Furness, R.W., Wells, D.E., 1999. Concentrations and pat terns of organochlorine contamin ants in marine turtles from Mediterranean and Atlantic waters. Mar. Environ. Res. 47, 117–135. McKim Jr., J.M., Johnson, K.L., 1983. Polychlorinated biphenyls and p,p’ -DDE in loggerhead and green postyearling Atlantic sea turtles. Bull. Environ. Contam. Toxicol. 31, 53–60.
Miao, X., Balazs, G.H., Murakawa, S.K.K., Li, Q., 2001. Congener-specific profile and toxicity assessment of PCBs in green turtles (Chelonia mydas) from the Hawaiian Islands. Sci. Total Environ. 281, 247–253. Muir, D.C.G., Norstrom, R.J., Simon, M., 1988. Organochlorine contaminants in Arc tic marine food chain: accumulation of specific polychlorinated biphenyls and chlordane-related compounds. Environ. Sci. Technol. 22, 1071–1079. Orós, J., Calabuig, P., Déniz, S., 2004. Digestive pathology of sea turtles stranded in the Canary Islands between 1993 and 2001. Vet. Rec. 155, 169–174. Orós, J., Torrent, A., 2001. Manual de necropsia de tortugas marinas. Ediciones del Cabildo de Gran Canaria, Las Palmas de G. C. Orós, J., Torrent, A., Calabuig, P., Déniz, S., 2005. Diseases and causes of mortal ity among sea turtles stranded in the Canary Islands Spain (1998–2001). Dis. Aquat. Org. 63, 13–24. Pérez-Jiménez, A., 1997. Caretta caretta (Linnaeus, 1758). In: Pleguez uelos, J.M. (Ed.), Distribución y Biogeografía de los Anfibio s y Reptiles en España y Portugal. Uni versidad de Granada, Granada, Spain, pp. 435–437. Perugini, M., Giammarino, A., Olivieri, V., Guccione, S., Lai, O.R., Amorena, M., 2006. Polychlorinated biphenyls and organochlorine pesticida levels in tissues of Caretta caretta from the Adriatic Sea. Dis. Aquat. Org. 71, 155–161. Pritchard, P.C.H., 1997. Evolution, phylogeny, and current status. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, Florida, pp. 1–28. Rybitski, M.J., Hale, R.C., Musick, J.A., 1995. Distribution of organochlorine pollu tants in Atlantic sea turtles. Copeia 2, 379–390. Seminoff, J.A., Resendiz, A., Resendiz, B., Nichols, W.J., 2004. Ocurrence of logger head sea turtles (Caretta caretta) in the Gulf of California, Mexico: evidence of life- history variation in the Pacific Ocean. Herpetol. Rev. 35, 24–27. Storelli, M.M., Marcotrigiano, G.O., 2000. Chlorobiphenyls, HCB, and organochlo rine pesticides in some tissues of Caretta caretta (Linnaeus) specimens beached along the Adriatic Sea, Italy. Bull. Environ. Contam. Toxicol. 64, 481–488. Storelli, M.M., Barone, G., Marcotrigiano, G.O., 2007. Polychlorinated biphenyls and other chlorinated organic contaminants in the tissues of Mediterranean logger head turtle Caretta caretta. Sci. Total Environ. 373, 456–463. Tanabe, S., Hidaka, H., Tatsukawa, R., 1983. PCBs and chlorinated pesticides in Ant arctic atmosphere and hydrosphere. Chemosphere 12, 277–288. Tanabe, S., Sung, J.K., Choi, D.Y., Baba, N., Kiyota, M., Tatsukawa, R., 1994. Persis tent organochlorine residues in northern fur seal from the Pacific coast of Japan since 1971. Environ. Pollut. 85, 305–314. Torrent, A., González-Díaz, O.M., Monag as, P., Orós, J., 2004. Tissue distribution of metals in loggerhead turtles (Caretta caretta) stranded in the Canary Islands Spain. Mar. Pollut. Bull. 49, 854–874. Wolke, R.E., George, A., 1981. Sea turtle necropsy manual, In: NOAA Technical Mem orandum NMFS-SEFC-24, National Oceanic and Atmospheric Administration, Kingston, RI. Ziswiler, V., 1986. Clase Reptiles Reptilia. In: Omega (Ed.), Zoología especial Verte brados Tomo II: Amniotas. Barcelona, Spain, pp. 23–72.
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Chemosphere j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / ch e m o s p h e r e
High levels of polychlorinated biphenyls in tissues of Atlantic turtles stranded in the Canary Islands, Spain J. Orós a,*, O.M. González-Díaz b, P. Monagas a a b
Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontana s/n, 35413 Arucas (Las Palmas), Spain Department of Chemistry, University of Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Spain
a r t i c l e
i n f o
Article history: Received 18 March 2008 Received in revised form 24 August 2008 Accepted 25 August 2008 Available online 4 December 2008 Keywords: Polychlorinated biphenyls Sea turtles Caretta caretta Chelonia mydas Dermochelys coriacea Atlantic Ocean
a b s t r a c t Polychlorinated biphenyls (PCBs 28, 31, 52, 101, 138, 153, 180, and 209) were measured in tissue samples (liver and fat) from 30 loggerhead turtles Caretta caretta, 1 green turtle Chelonia mydas, and 1 leatherback Dermochelys coriac ea stranded on the coasts of the Canary Islands, trying to establish a possible relation between PCB con centrations and the lesions and causes of death. Tissues from these turtles contained higher levels of PCBs than those reported in turtles from other geographical regions. )PCB concentrations (1980 ± 5320 ng g¡1 wet wt.) in the liver of loggerheads were higher than in the adipose tissue (450 ± 1700 ng g¡1 wet wt.). Concentrations of PCB 209 in the liver (1200 ± 3120 ng g¡1 wet wt.) of loggerheads and in the liver (530 ng g¡1 wet wt.) and adipose tissue (500 ng g¡1 wet wt.) of the leatherback were remarkable. Frequencies of detection of PCB 209 in the liver (15.5%) and adipose tissue (31%) were also remarkable. Cachexia was detected in 7 turtles (22%) and septicemia was diagnosed in 10 turtles (31%). Statistically, a positive correlation was detected between )PCBs concentration and cachexia. Poor physic al condition, cachexia and/or septicaemia could explain the high levels of PCBs and tissue distribution. However, no histological lesions exclusively attributed to the acute effects of PCBs were described. The most prevalent histologic al lesions were ulcerative and purulent oesopha gitis, purulent dermatitis, necrotizing enterit is, and granulomatous pneumonia. The bacteria most frequently isolated were Escherichia coli, Staphylococcus sp., and Aeromonas sp. Although immunosupression as a result of PCBs pollution has been described previously, other factors in this study, such as incidental fishing, nutritional status, and exposition to different micro-organisms, make it dif ficult to establish a clear association between PCB concentrations and causes of death. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Two families and seven species of sea turtles are currently recognised (Pritchard, 1997) and included in the red list of the World Conservation Union (IUCN, 2007). The family Dermochelyi dae includes only the leatherback (Dermochelys coriacea). The fam ily Cheloniidae includes the green turtle (Chelonia mydas), logger head (Caretta caretta), hawksbill (Eretmochelys imbricata), Kemp’s ridley (Lepidochelys kempi), olive ridley (Lepidochelys olivacea), and flatback turtles (Natator depressa). The most common species in the Canary Islands is the loggerhead turtle (Mateo et al., 1997). However, evidence of a decline in the population of turtles in the Canary Islands has been reported (López-Jurado and González, 1983; Blanco and González, 1992). Diseases and causes of mortality among turtles stranded in the Canary Islands have been previously reported (Orós et al., 2004, 2005). However, data available for baseline levels of contaminants
* Corresponding author. Tel.: +34 928451089; fax: +34 928457431. E-mail address: jo[email protected]pgc.es (J. Orós). 0045-6535/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.08.048
and effects on the turtle populations of the Canary Islands are scarce (Torrent et al., 2004). Polychlorinated biphenyls (PCBs) have a particular significance because of their undesirable effects on environmental quality and animal health (Ahlborg et al., 1994). PCBs were manufactured from the 1930s to the 1970s for several industrial applications, such as liquid coolants for electrical transformers or as softeners in the production of plastics and as components of hydraulic fluids and lubricating oils. PCBs are able to bioaccumulate through the food chain and their effects have been reported on the immune, endo crine, and reproductive systems of different animal species (Fox, 2001). Although much was reported to date on PCBs concentra tions in large predators, few studies have been dedicated to turtles. These studies have been focused on turtles from Long Island (Lake et al., 1994), Virginia (Rybitski et al., 1995), Scotland (Mckenzie et al., 1999), the Hawaiian Islands (Miao et al., 2001), the Baja Cal ifornia Peninsula (Gardner et al., 2003) and North Carolina (Kel ler et al., 2004, 2006). Studies focused on the Mediterranean Sea have a particular significance because of their number (Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Perugini et al., 2006; Storelli et al., 2007).
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The aim of this study was to evaluate the presence and pat terns of eight PCB congeners (28, 31, 52, 101, 138, 153, 180, and 209) in tissue samples (liver and fat) from 32 turtles stranded on the coasts of the Canary Islands between August 2002 and Novem ber 2005. We also tried to determine a possible relation between the PCB concentrations and the lesions and causes of death using the Spearman’s rho correlation method to calculate the correlation between )PCB concentrations in both tissues and physic al condi tions such as cachexia and septicaemia.
was obtained, bacteria were identified based on the biochemical profile (API 20 E, API 20 NE, and API 20 STAPH, BioMérieux).
2. Materials and methods
2.4. PCBs analysis
2.1. Turtles
Polychlorinated biphenyls (IUPAC Nos. 28, 31, 52, 101, 138, 153, 180, and 209) were analysed according to the method described by Tanabe et al. (1994). The validity of analytical methods was con firmed with Standard Reference Materials (CARP-2: ground whole carp, Cyprinus carpio) obtained from the National Research Council of Canada. Precision and accuracy are reported in Table 2. Briefly, aliquots (4–7 g) of the homogen ized samples were ground with anhydrous sodium sulphate in a mortar, and extracted using Soxhlet apparatus for 6 h with 300 mL of diethyl ether:hex ane (3:1) solvent mixture. Extracts were concentrated in volume to 10 mL in Kuderna-Danish, and the aliquots (2 mL) were transferred to a glass column packed with 20 g of Florisil and dried by passing through nitrogen gas. Organochlorines adsorbed on Florisil were eluted with 150 mL of 20% hexane-washed water in acetonitrile and transferred to a separatory funnel containing 600 mL of hex ane-washed water and 100 mL of hexane. After partitioning, the hexane layer was concentrated, cleaned up with sulphuric acid, and passed through a 12 g Florisil-packed glass column for sepa ration. Final determination of PCBs was carried out using a Varian 3600 gas chromatograph fitted with an electron capture detec tor (GC-ECD). In all the analyses a fused-silica capillary column Supelco (length = 30 m, inner diameter 0.53 mm and film thick ness 0.50 lm) was used. The column oven temperature was pro grammed from 60 to 160 °C, held for 10 min, and then increased to 260 °C at a rate of 2 °C/min and held for 20 min. Injector and detec tor temperatures were set at 260 and 280 °C, respectively. Nitrogen was used as a carrier gas at 63.3 mL/min. The PCB patron used as an internal standard was the PCB-Mix 12, Iso-octane (Lab. Dr. Ehren storfer). Quantification interval used for PCBs was from 1 ng g¡1 (instrumental detection limit) to 50 000 ng g¡1 (optimum linear limit). Concentrations of PCBs, means of four measurements, are presented as ng g¡1 on a wet weight basis.
Between August 2002 and November 2005, 32 turtles that got stranded on the coasts of four islands belonging to the Canary Islands [Gran Canaria (n = 25; 78.1%), Tenerife (n = 3; 9.4%), Fuerteventura (n = 2; 6.2%), and El Hierro (n = 2; 6.2%)] were sub mitted for necropsy to the Veterinary Faculty, University of Las Pal mas de Gran Canaria (ULPGC). Some of them had been previously submitted to the Tafira Wildlife Rehabilit ation Center (TWRC) for health evaluation, medical management, and possible rehabilita tion. Species identifications were made according to Frick (1996). Turtles belonging to three different species were examined: 30 loggerheads, C. caretta (93.7%), 1 green turtle, C. mydas (3.1%), and 1 leatherback, D. coriacea (3.1%). Species, sex, age group, and bio metrics data are shown in Table 1. Age group was determined on the basis of straight carapace length (SCL) (Bjorndal et al., 2001; Seminoff et al., 2004) and sexual maturity (estimated from the appearance of their gonads). 2.2. Pathological and microbiologic al studies Necropsies were performed at the Veterinary Faculty (ULPGC) within the first 12 h after death. The gross postmortem examina tions were carried out using the procedures previously described (Wolke and George, 1981; Orós and Torrent, 2001). Gross lesions were recorded and tissue samples from all major organs were fixed in 10% neutral buffered formalin, embedded in paraf fi n, sectioned at 5 lm for light microscopy and stained with haematoxylin and eosin. Special stains used on selected cases included Gram stain for bacteria, Ziehl-Neelsen stain for acid-fast organisms, periodic acid-Schiff stain for protozoa and fungal hyphae, Grocott’s methe namine silver nitrate for fungi, and von Kossa stain for calcium (Bancroft and Stevens, 1996). For the microbiologic al studies, sam ples were taken from gross lesions and cultured on a variety of selective and non-selective media, including blood agar (Oxoid), MacConkey agar (Oxoid), Baird Parker agar (Oxoid) for staphylo cocci, and Sabouraud Dextrose agar (Oxoid) for fungi and yeasts. All cultures were incubated at 25 °C aerobically. Once a pure growth
2.3. Samples collection for PCB analysis Tissues samples (liver and coelomic fat) were collected during necropsy. After collection, the samples were wrapped in an alu minium foil and stored at ¡20 °C until analysis. Prior to analysis, tissue samples were homogen ized using a commercial blender.
2.5. Statistical analysis Mann–Whitney U test was conducted to determine whether the difference in the levels of PCBs were related to the tissues. Spearman’s rho correlation method was used to calculate the
Table 1 Species, sex, age group and biometrics data (mean ± standard deviation) of the tur tles analysed
Table 2 Precision and accuracy of analytical methods obtained using a certified ground whole carp Cyprinus carpio (CARP-2)a
Species
Sex
Age group
PCBs
Caretta caretta (n = 30)
Female (n = 27; 90%) Male (n = 3; 10%) Female
Pelagic juvenile 11.5 ± 8.3 (n = 12; 40%) Juvenile (n = 18; 60%)
41 ± 11.7
Juvenile
21
52
Female
Adult
231.5
nm
Chelonia mydas (n = 1) Dermochelys coriac ea (n = 1)
SCL: straight carapace length. n: number of turtles. nm: not measured.
Weight (kg)
SCL (cm)
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180 PCB 209
CARP-2 Certified
Foundb
34 ± 4.0 138 ± 43 145 ± 48 103 ± 30 105 ± 22 53.3 ± 13.0 4.6 ± 2.0
31 ± 3.1 119.7 ± 13.9 150.1 ± 24.1 99.5 ± 18.7 116 ± 16.6 59.2 ± 10.7 4.8 ± 3.6
a
The concentrations are given in ng g¡1 wet wt.
b
Number de replicates is 4.
J. Orós et al. / Chemosphere 74 (2009) 473–478
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c orrelation between )PCB concentrations in both tissues and physic al conditions such as cachexia and septicaemia. According to the necropsy reports, values of 0, 1, and 2 were given to tur tles with absence of cachexia, mild cachexia, and severe cachexia, respectively. Values of 0, 1, and 2 were also given to turtles with absence of infections, infection in one organ, and severe septicae mia.
Table 3 PCBs concentrations (range, arithmetic mean ± standard deviation) (ng g¡1 wet wt.) in liver and fat of the three species of sea turtles analysed.
3. Results
PCB 28,31
3.1. Gross pathology, histopathology, and microbiology
Caretta caretta n = 30
)PCB
PCB 52 PCB 101
The pathological study revealed that 23 turtles (72%) died from lesions associated with human activities such as boat-strike inju ries (n = 4; 12.5%), entanglement in derelict fishing nets (n = 7; 22%), and ingestion of hooks and monofilam ent lines (n = 12; 37.5%). Only 9 turtles (28%) died from spontaneous diseases, including pulmonary emphysema, ulcerat ive keratoconjunctivitis, purulent adenitis of the salt glands and purulent cloacitis. Cachexia was detected in 7 turtles (22%). Septicemia was diagnosed in 10 turtles (31%). The histological lesions observed in the loggerheads included severe necrotic hepatitis (n = 4; 13%), severe multifocal granulom atous hepatitis (n = 4; 13%), severe ulcerat ive and purulent oesoph agitis (n = 10; 33%), mild purulent gastritis (n = 2; 6.7%), mild to severe necrotizing enteritis (n = 5; 16.7%), severe purulent cloacitis (n = 1; 3%), mild to severe granulomatous pneumonia (n = 5; 16.7%), severe pulmonary emphysema (n = 1; 3%), severe necrotic spleni tis (n = 1; 3%), severe ulcerative and purulent keratoconjunctivitis (n = 3; 10%), severe purulent adenitis of the salt glands (n = 4; 13%) and severe purulent dermatitis (n = 9; 30%). Bacteria isolated from the tissue lesions included Escherichia coli (n = 5; 16.7%), Proteus sp. (n = 3; 10%), Vibrio alginolyticus (n = 2; 6.7%), Pseudomonas sp. (n = 2; 6.7%), Streptococcus sp. (n = 2; 6.7%), Citrobacter freundii (n = 2; 6.7%), Aeromonas sp. (n = 3; 10%), Staphylococcus sp. (n = 4; 13%), and Kleb siella sp. (n = 2; 6.7%). Histological lesions observed in the specim en of C. mydas included severe multifocal granulomatous nephritis associated to Candida guillermondi infection and severe oedema of the lamina propia and intestinal submucosa. Histological lesions observed in the specimen of D. coriac ea included severe multifocal necrotic nephritis, severe granulomatous pneumonia and severe necrotiz ing enteritis, and all these lesions were associated with Serratia marcescens infection. 3.2. PCBs PCB concentrations measured in the two tissues of the three species of turtles analysed are shown in Table 3. Loggerheads showed the highest hepatic )PCB (IUPAC Nos. 28, 52, 101, 138, 153, 180) mean concentration (1980 ± 5320 ng g¡1 wet wt.), followed by the only specimen of D. coriacea analysed (445 ng g¡1 wet wt.). )PCB concentration in the liver of the only specim en of C. mydas analysed was lower (116 ng g¡1 wet wt.) than those detected in loggerheads and leath erback turtles. Loggerheads also showed the highest )PCB mean concentration in the adipose tissue (450 ± 1700 ng g¡1 wet wt.). Loggerheads showed the highest hepatic PCB 209 mean con centration (1200 ± 3120 ng g¡1 wet wt.), followed by the only specimen of D. coriacea analysed (530 ng g¡1 wet wt.). However, the leatherback showed the highest PCB 209 concentration in the adipose tissue (500 ng g¡1 wet wt.). Six loggerhead turtles had individual PCB concentrations higher than 2000 ng g¡1, and seven turtles showed individual PCB concentrations higher than 900 ng g¡1. All these turtles showed severe septicaemia and/or severe cachexia.
PCB 138 PCB 153 PCB 180 PCB 209
Chelonia mydas n = 1
Liver
Fat
BDL – 34900 1980 ± 5320 BDL – 590 42 ± 131 BDL – 1000 98 ± 262 BDL – 670 32 ± 130 BDL – 13300 456 ± 2426 BDL – 15440 915 ± 3180 BDL – 3890 440 ± 1016 BDL – 14900 1200 ± 3120
BDL – 9800 450 ± 1700 BDL – 38 3.8 ± 9 BDL – 56 5.5 ± 12.5 BDL – 84.5 6 ± 19 BDL – 2040 83 ± 370.5 BDL – 2600 133 ± 477 BDL – 5006 217 ± 907 BDL – 218 15 ± 45.5
Dermochelys coriacea n = 1
Liver
Fat
Liver
Fat
116
144
445
77
BDL
5.7
71.5
9.7
BDL
7.3
8.7
11.4
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
58
251
56
116
73
114
BDL
BDL
BDL
530
500
BDL: below detection limit.
)PCB = sum of PCBs nos. 28, 52, 101, 138, 153, and 180.
As revealed by statistical analysis, turtles showed significant higher PCBs 28,31, PCB 153, PCB 209 and )PCBs concentrations in the liver than in the adipose tissue. However, no significant dif ferences between concentrations in both tissues were detected for PCB 138 and PCB 180. The congener most frequently detected in the liver was PCB 180 (46.8%), followed by PCB 153 (31.2%), PCBs 28, 31 (25%), PCB 52 (25%), PCB 209 (15.6%), PCB 138 (15.6%), and PCB 101 (12.5%). The congeners most frequently detected in the adipose tissue were PCB 180 (50%) and PCB 153 (50%), followed by PCBs 28,31 (34.4%), PCB 52 (34.4%), PCB 209 (31.2%), PCB 138 (28.1%), and PCB 101 (18.7%). PCB profiles in the C. caretta tissues were dominated by the higher chlorinated congeners (Fig. 1). Hexachlorobiphenyls (PCBs 138 and 153) were predominant and accounted for 43.6% of the total PCBs, followed by decachlorobiphenyls (PCB 209) (33%) and heptachlorobiphenyls (PCB 180) (18%). Tetrachlorobiphenyls (PCB 52) accounted for 3% of the total PCBs, and trichlorobiphenyls (PCBs 28 and 31) accounted for 1.3% of the total residue. Concern ing individual congeners, the most abundant was PCB 209 (33%) followed by PCB 153 (29%), PCB 180 (18%), and PCB 138 (15%). The isomer pattern of PCBs in C. caretta was different in the liver and the fat. In the liver, the most abundant was PCB 209 (37.6%), fol lowed by PCB 153 (29%), PCB 138 (14%) and PCB 180 (14%). In the fat, the most abundant was PCB 180 (47%), followed by PCB 153 (28.6%), PCB 138 (18%) and PCB 209 (3%).
3Cl
4Cl
5Cl
6Cl
7Cl
10Cl
C. caretta (liver) C. caretta (fat) C. mydas (liver) C. mydas (fat) D. coriacea (liver) D. coriacea (fat) 0%
20%
40%
60%
80%
100%
Fig. 1. Fingerprints of liver and fat of the three species of sea turtles analysed.
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Regarding PCB profiles in the only specimen of C. mydas ana lysed, the most abundant was PCB 180 (73%) followed by PCB 153 (22%) and PCB52 (3%). Although PCB 180 was the most abundant in both tissues, this congener was the only PCB detected in the liver of this turtle. Regarding PCB profiles in the only specimen of D. coriacea ana lysed, the most abundant was PCB 209 (66%) followed by PCB 153 (20%) and PCB 180 (7%). The isomer pattern of PCBs in this tur tle was slightly different in the liver and the fat. In the liver, the most abundant was PCB 209 (54%) followed by PCB 153 (26%) and PCB 180 (12%). In the fat, the most abundant was PCB 209 (87%), followed by PCB 153 (10%), and PCB 52 (2%). Hepatic )PCBs concentrations were positively correlated with cachexia (r = 0.250, P = 0.04). No significant correlation was observed between )PCBs concentrations and septicaemia. 4. Discussion The present study represents the first data of PCB levels for any turtle species from the Canary Islands. The turtle tissues analysed in the current investigation contained high levels of PCBs com pared to concentrations reported in turtles collected in other loca tions around the world (Table 4) (McKim and Johnson, 1983; Lake et al., 1994; Rybitski et al., 1995; Mckenzie et al., 1999; Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Gardner et al., 2003; Keller et al., 2004; Perugini et al., 2006; Storelli et al., 2007). The high degree of varia tion of concentrations of PCBs detected in our study may be attributed to the different exposure of
i ndividual turtles to pollutants and to the physical condition of the animals. Sea turtles visit different areas with different degree of contamination during migration, resulting in differences in expo sure for each animal (Ziswiler, 1986). According to recent studies, loggerhead turtles stranded on the coasts of the Canary Islands come from two different migration routes: from the Western Atlantic arriving to the Canary Islands by the Gulf Stream (PérezJiménez, 1997), and from Cape Verde (Dellinger, 2006). In addition, turtles included in this study varied widely in physical condition, ranging from turtles that had been killed incidentally through human interaction to turtles that showed severe emaciation and septicaemia after prolonged illness. Six loggerhead turtles had individual PCB concentrations higher than the quantification limit of 2000 ng g¡1 established by the Food and Drug Administration (FDA). In addition, seven turtles showed individual PCB concentrations higher than 900 ng g¡1. All these turtles showed severe septicaemia and/or severe cachexia. Statistically, a positive correlation was detected between )PCBs concentration and cachexia. Although sea turtles are long-living high trophic organisms that would be expected to contain high concentrations of PCBs (Lake et al., 1994), we think the poor physical condition of these animals could be the reason for these high PCB levels. It has been described that in cetaceans many diseases affect metabolic centres and thus the capacity to metabolise or excrete pollutants may be affected, questioning whether PCB concentrations in the tissues of these specimens are representative of normal conditions (Aguilar et al., 1999).
Table 4 PCB concentrations (average concentration or range, ng g¡1 wet wt.) in hepatic and adipose tissue samples from sea turtles reported by other authors Species
Locality
Tissue (n) )PCB
PCB 118
PCB 138
PCB 153
PCB 180
Reference
L. kempi
Long Island Virginia and North Carolina
98.3 148.9 BDL-59.5
114.94 195 BDL-65.8
170.04 272.5 2.04–152
53.62 102.7 BDL-49.4
Lake et al. (1994)
C. caretta
Liver (22) 512.6 Fat (13) 863 Liver (18) 7.46–514 Fat (20) Liver (3) Fat (3) Liver (4)
55.4–1730 158–608 90.4–904 50–102
<0.5–<1.6
<0.5–<1.6
<0.5–3.4
4.55–193 14.8–18.7 19.4–91.9 3.3–8.7
3.77–236 26.1–35.6 38.9–156 12–24
12.1–406 43.1–259 90.4–283 13–29
BDL-148 10.3–19.5 19.1–64 7.3–18
Fat (3) Liver (1) Liver (2) Fat (2) Liver (9)
775–893 159 3.1–3.7 47–178 <1.1–77
<4.8–<8.0 <0.9 <0.6–<0.8 <4.7–<7.2 <0.7–<3.4
8.6–37 15 2.2–3.9 12 0.7–4.3
8–32 16 <0.6–<0.8 5.8–<7.2 0.9–7.4
54–71 12 <0.6–2.8 <7.2–8.3 <0.7–4.8
132–169 20 <0.6–0.8 <7.2–25 <1.1–11
229–261 24 1.3–1.5 7.8–46 <1.1–13
73–154 12 <0.6–<0.8 <7.2–24 <1.1–7.4
<2.2–<3.7
13.25
5.7–33
<2.2–27
<2.2–28
<2.2–3.2
<2.2–13
L. kempi C. caretta
Mediterranean Sea
C. caretta Scotland D. coriacea Scotland C. mydas
Mediterranean Sea
C. caretta
Adriatic Sea
Fat (3) Liver (11)
C. caretta
Adriatic Sea
Liver (4)
39–261 3.22–6.54A,a 0.21–0.76A,b 119
C. mydas
Baja California peninsula
Fat (4) Liver (7)
334 BDL-44.7
Fat (7) Liver (1) Fat (1) Liver (1) Fat (1) Fat (44) Liver (11)
BDL-49.5 58.1 18.4 41 BDL 2010 ± 2960B 83 7.6–247.3
L. olivacea C. caretta C. caretta C. caretta
North Carolina Adriatic Sea
Fat (11) C. caretta
Adriatic Sea
459.7 2.9–1472.1 Liver (19) 6.85–297.49
BDL: below detection limit. A Value expressed as mg kg¡1 lipid weight. B Value expressed as ng g¡1 lipid weight. a Young turtles. b Adult turtles.
PCB 28
PCB 52
PCB 101
4.68–11.10
14.25–42.82
17.84–45.50
868–28.83
9.55–36.97
23.78–103.70 31.15–134.46 19.60–78.10
Rybitski et al. (1995)
Mckenzie et al. (1999)
Storelli and Mar cotrigiano (2000) Corsolini et al. (2000) Gardner et al. (2003)
11 1.1–26.5 62.4 0.33–150.8
26.7 2.2–92.9 24.5 2.5–75.7 115.8 115 0.6–294.2 0.6–369.2
Keller et al. (2004) Perugini et al. (2006)
Storelli et al. (2007)
J. Orós et al. / Chemosphere 74 (2009) 473–478
ng/g wet wt
2000 1500 1000
Fat
500
Liver
0
C. caretta
C. mydas
D. coriacea
ng/g wet wt
2000 1500 1000
Fat
500
Liver
0
C. caretta
C. mydas
D. coriacea
Fig. 2. )PCB (IUPAC nos. 28, 52, 101, 138, 153, 180) mean concentrations (a) and PCB 209 mean concentrations (b) in the hepatic and adipose tissue samples of the three species of sea turtles analysed.
According to other authors, the pattern of tissue distribution of PCBs reported in turtles and other marine species is as follows: adipose tissue > liver > kidney > muscle (Tanab e et al., 1983; Marti neau et al., 1987; Colborn and Clement, 1992; Rybitski et al., 1995; Mckenzie et al., 1999). This is attributed to the highest total lipid content and triglycerides for the adipose tissues and the strong correlations between these and their burden of lipophilic pollu tants (Cockcroft et al., 1989). However, we detected higher )PCBs concentrations in the liver than in the adipose tissue (Fig. 2). Thir teen turtles showed higher PCB levels in the liver than in the fat. All these turtles showed severe cachexia and/or septicaemia. Accord ing to different studies, animals with abundant fat deposits can accumulate and tolerate higher concentrations of toxic chemicals because lipophilic pollutants are stored in the adipose tissue and are less available to target organs and receptors. In contrast, ema ciated animals which have mobilised their lipid stores may be more susceptible to toxic effects as a result of remobilisation of the pollutants, resulting in higher concentrations of PCBs in the remaining tissues such as the liver (Bernhoft and Skaare, 1994). Thus, the cachexia observed in our turtles may indicate a previous remobilis ation of the fat and the pollutants, and could explain the high concentration of PCBs in the liver. In addition, the septicae mic condition reported in these turtles could be caused by a possi ble inmunosupression as one of the most distinguished effects of organochlorine compounds. In our study, PCB profiles in the C. caretta tissues were domi nated by the higher chlorinated congeners (Fig. 1). Hexaclorobi phenyls and heptaclorobiphenyls are predomin ant in turtles and marine mammals (Muir et al., 1988; Rybitski et al., 1995; Colborn and Smolen, 1996; Alam and Brim, 2000; Corsolini et al., 2000; Storelli and Marcotrigiano, 2000; Miao et al., 2001; Perugini et al., 2006; Storelli et al., 2007). However, Gardner et al. (2003) reported in C. mydas a profile domin ated by lower-chlorinated congeners. Differences in PCB patterns in turtles may be attributed to dif ferences in the congener compositions of environmental media among regions, dietary differences or differences in the abilities of the various species and populations to metabolize PCBs (Gardner et al., 2003). In our study, the concentrations and frequencies of detection of PCB 209 were remarkable. Usually, published reports do not
477
include this congener. Gardner et al. (2003) detected very low concentrations of PCB 209 following a decreasing order in muscle, liver and adipose tissue of turtles. Storelli et al. (2007) reported in a study on 19 loggerhead turtles that PCB 209 accounted only for 0.1–1.6% of total PCBs. One of our objectives was to establish a possible relationship between PCBs concentrations and the causes of death of each tur tle. It was dif ficult to assess the impact of organochhorine com pounds in the turtles of our study because the most frequent causes of death (72%) were attributed to fishing activity. In addi tion, few toxicological studies have been published regarding organochlorines in reptiles and no safe concentrations have been established. It is remarkable that almost all turtles with severe septicaemia showed very high levels of PCBs. Immunosupression as result of PCBs pollution has been previously described (Keller et al., 2004, 2006). However, in our study the presence of other factors different to PCBs concentrations, such as incidental fish ing, nutritional status, and exposition to different micro-organ isms, make it dif ficult to establish a clear association between PCB concentrations and causes of death. According to the histopathol ogical study no lesions exclusively attributed to acute effects of PCBs were described. All the histological lesions were attributed to bacterial and/or fungal infections mainly associated with gross lesions caused by fishing activity. However, chronic effects of PCBs are much dif ficult to determine. While there was an insuf ficient number of samples to assess differences among species, qualitative evaluation could indicate the potential effects of poor physical condition differences among individuals of C. caretta and subsequent poor metabolic perfor mance of detoxification systems. In addition, the detection of high levels of PCBs in these turtles makes it necessary to continue to monitor the levels of these chemicals in turtles from the Canary Islands to contribute to the protection of these currently endan gered species. Acknowledgements The authors would like to thank members of the Consejería de Medio Ambiente, Cabildo de Gran Canaria, for providing the turtles. This investigation was partially supported by the national project I + D REN2000-1753 MAR. References Aguilar, A., Borrel, A., Pastor, T., 1999. Biologic al factors affecting variability of persistent pollutant levels in cetaceans. J. Cetacean Res. Manage. 1, 83–116. Alam, S.K., Brim, M.S., 2000. Organochlorine, PCB, PAH, and metal concentrations in eggs of loggerhead sea turtles (Caretta caretta) from northwest Florida, USA. J. Environ. Sci. Health 35, 705–724. Ahlborg, U.G., Becking, G.C., Birnbaum, L.S., Brouwer, a., Derks, H.J.G.M., Feeley, M., Golor, G., Hanberg, A., Larsen, J.C., Liem, A.K.D., Safe, S.H., Schlatter, C., Waern, F., Younes, M., Yrjänheikki, E., 1994. Toxic equivalency factors for dioxin-like PCBs: report on WHO-ECEH and IPCS consultation, December 1993. Chemosphere 28, 1049–1067. Bancroft, J.D., Stevens, A., 1996. Theory and Practice of Histological Techniques, fourth ed. Churchill Livingstone, New York. Bernhoft, A., Skaare, J.U., 1994. Levels of selected individual polychlorinated biphe nyls in different tissues of harbour seals (Phoca vitulina) from the southern coast of Norway. Environ. Pollut. 86, 99–107. Bjorndal, K.A., Bolten, A.B., Koike, B., Schroeder, B.A., Shaver, D.I., Teas, W.G., W itzell, W.N., 2001. Somatic growth function for immature loggerhead sea turtles, Caretta caretta, in southeastern US waters – Statistical Data included.. Fish. Bull. 99, 240–246. Blanco, J.C., González, J.L., 1992. Libro rojo de los vertebrados españoles. Ministerio de Agricultura, Pesca y Alimentación. Colección Técnica ICONA, Madrid. Cockcroft, V.G., De Kock, A.C., Lord, D.A., Ross, G.J.B., 1989. Organochlor ines in bot tlenose dolphins (Tursiops truncatus) from the east coast of South Africa. S. Afr. J. Mar. Sci. 8, 207–217. Colborn, T., Clement, C., 1992. Chemically Induced Alterations in Sexual and Func tional Development: The Wildlife/human Connection. Princeton Scientific Pub lishing, Princenton, New Jersey.
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