Trish J Lavery
email: trish.lavery@flinders.edu.au

Catherine M Kemper

Ken Sanderson

Christopher G Schultz

Peter Coyle

James G Mitchell

Laurent Seuront

Heavy metal toxicity of kidney and bone tissues in South Australian adult bottlenose dolphins (Tursiops aduncus)

Keywords: Heavy Metals, Toxicity, Mammals, Dolphins, Metallothionein, Kidney, Bone, Cadmium, Mercury, Lead, Zinc 1

Metallothioneins (MT) concentration, renal damage, and bone malformations were investigated in 38 adult Tursiops aduncus carcasses to determine any associations with cadmium, copper, zinc, mercury, lead and selenium. Significantly higher concentrations of cadmium, copper, and zinc in the liver were observed in dolphins showing evidence of more advanced renal damage. No significant differences in metal or selenium concentrations in the liver were observed between groups differing in level of bone malformations. Some dolphins displayed evidence of toxicity and knowledge of metal toxicity pathways were used to elucidate the cause of these abnormalities. Two dolphins had high metal burdens, high MT concentrations, renal damage, and evidence of bone malformations, indicating possible severe and prolonged metal toxicity. One dolphin showed evidence of renal damage, but the lack of any other symptoms suggests that this was unlikely to be caused by metal toxicity. We recommend examining a range of metal toxicity symptoms simultaneously to aid in distinguishing metal toxicity from unrelated aetiologies.

INTRODUCTION

Metal concentrations have been recorded in over 60 species of cetaceans [START_REF] O'shea | Environmental contaminants and marine mammals[END_REF] but few studies have explored the relationship between metals and toxicity. Metal toxicity causes irregular metallothioneins (MT) protein synthesis, renal damage, and disruption of bone structure in humans and wildlife [START_REF] Beiglbock | Environmental cadmium induces histopathological changes in kidneys of roe deer[END_REF][START_REF] Jarup | Cadmium overload and toxicity[END_REF][START_REF] Sato | Recent studies on metallothionein: Protection against toxicity of heavy metals and oxygen free radicals[END_REF]. Studies of metal toxicity in cetaceans, particularly beachstranded carcasses, are hindered by difficulties arising from examining tissues that have undergone post-mortem autolysis. Since these are usually the only specimens available, it is important that methods of measuring metal toxicity in decomposed carcasses are developed to allow determination of dolphin health, even in degraded specimens.

Metallothioneins (MT) are metal-binding proteins that preferentially bind to Cd (cadmium) and Zn (zinc), reducing the bioavailability of these elements [START_REF] Sato | Recent studies on metallothionein: Protection against toxicity of heavy metals and oxygen free radicals[END_REF][START_REF] Das | Cu, Cd and Hg binding to metallothioneins in harbour porpoises Phocoena phocoena from the southern North Sea[END_REF] and providing an early indicator of cellular responses to metal toxicity [START_REF] Petering | Metal-binding proteins and peptides for the detection of heavy metals in aquatic organisms[END_REF]. Although MT are often correlated with liver and kidney metal burdens, few attempts have been made to correlate them with markers of heavy metal toxicity in cetaceans. This is an absolute prerequisite to support the use of MT overexpression as a bioindicator of metal toxicity.

Metals are toxic to mammalian renal cells [START_REF] Wang | Cytopathology induced by mercuric chloride and methylmercury in cultured renal cells of Atlantic spotted dolphin (Stenella plagiodon)[END_REF], causing damage that leads to leakage of phosphates, calcium, glycogen and proteins (proteinuria) from the kidney [START_REF] Loghman-Adham | Renal effects of environmental and occupational lead exposure[END_REF]. Histological indicators of proteinuria have been used as a preliminary measure of renal pathology in Tursiops aduncus in South Australia [START_REF] Long | Cadmium accumulation and toxicity in the bottlenose dolphin, the common dolphin, and some dolphin prey species in South Australia[END_REF] but may have been impacted by post-mortem autolysis. In contrast, metal-induced swelling of the Bowman's capsule, and protein leakage within an intact capsule, are not affected by post-mortem decomposition [START_REF] Beiglbock | Environmental cadmium induces histopathological changes in kidneys of roe deer[END_REF].

While MT and renal structure swelling may determine toxicity in mildly degraded specimens, bone structure is resistant to post-mortem degradation. Loss of bone mineral structure occurs in cases of Cd, Pb (lead), and Hg (mercury) toxicity in humans [START_REF] Escribano | Effect of lead on bone development and bone mass: A morphometric, densitometric, and histomorphometric study in growing rats[END_REF][START_REF] Jarup | Cadmium overload and toxicity[END_REF][START_REF] Suzuki | Both mercury and cadmium directly influence calcium homeostasis resulting from suppression of scale bone cells: The scale is a good model for the evaluation of heavy metals in bone metabolism[END_REF] while increased and decreased bone mineral density have been observed in response to Zn and Se (selenium) excess and deficiency respectively [START_REF] Turan | A biomechanical and spectroscopic study of bone from rats with selenium deficiency and toxicity[END_REF][START_REF] Yun | Effect of zinc on bone metabolism in fetal mouse limb culture[END_REF]. Loss of bone density is a clinical endpoint marker of chronic metal toxicity and indicates accompanying sub-clinical behavioural and immunological deficits [START_REF] Staessen | Environmental exposure to cadmium, forearm bone density and risk of fractures: prospective population study[END_REF] which are difficult to examine directly in free-living cetaceans.

The causes of elevated MT, renal damage, and loss of bone density and complexity are probably interrelated. MT induced by metal toxicants form a large metal-MT complex which leads to damage of renal structures. This results in leakage of calcium, phosphate and proteins from the kidney, hindering bone remodelling and leading to a loss of bone density (bone mineral density) and complexity (bone histomorphometry) [START_REF] Alfven | Cadmium and lead in blood in relation to low bone mineral density and tubular proteinuria[END_REF]. Thus, dolphins displaying metal-induced bone malformations should show symptoms of elevated MT synthesis and damage of renal structures, assuming these markers are reflecting metal toxicity and not extraneous influences (e.g., post-mortem cellular degradation, unrelated parasitism, unrelated disease, normal mammalian ageing process). Consistency between MT, renal damage and bone structure should provide a valuable tool for helping to distinguish metal-induced toxicity from other aetiologies.

South Australian adult Indo-Pacific bottlenose dolphins, Tursiops aduncus, have been shown previously to have high concentrations of Cd, Hg, and Se in the liver, moderate concentrations of Pb, Cu, and Zn in the liver and moderate concentrations of Cd and Pb in bone compared to dolphins elsewhere [START_REF] Lavery | Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988 -2004[END_REF]. This study aims to explore any links between MT, renal damage, bone density and structure, and liver tissue concentrations of Cd, Hg, Pb, Zn, Cu and Se in adult bottlenose dolphins.

MATERIALS AND METHODS

Necropsy procedures and sample selection

Necropsy procedures and determination of Se and metal concentrations by Inductively

Coupled Plasma Atomic Emission Spectrometry in 71 T. aduncus from South Australia are detailed elsewhere [START_REF] Lavery | Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988 -2004[END_REF]. All dolphins were collected by the South Australian Museum (SAMA) and given a museum identification number. Some dolphins had multiple analyses conducted on one tissue, and the mean metal or Se value was used in these cases. Since animal age impacted metal accumulation in these dolphins, only a subset of 38 sexually and / or physically mature adults of tooth category 3 and above (see [START_REF] Kemper | Dolphin interactions with tuna feedlots at Port Lincoln, South Australia and recommendations for minimising entanglements[END_REF], for details of dolphin development compared to tooth category)

were examined in the present study. This sample of 38 adult dolphins consisted of 17 male and 21 female adult dolphins, 16 of which had kidney samples with intact nuclei and glomeruli available for histopathological examination [START_REF] Geraci | Marine Mammals Ashore: A field guide for strandings[END_REF] decomposition code 2 and 3). Kidney (N = 15) and liver (N = 14) samples from a subsample of these animals were analysed for MT. Bone density was measured in four vertebrae of 30 animals and the histomorphometry of costal rib tips examined from 15 animals.

Metallothionein quantification

Soft tissues were collected following procedures that minimise cross contamination [START_REF] Geraci | Marine Mammals Ashore: A field guide for strandings[END_REF]. Subsampling of frozen (-20 o C) tissue entailed collecting approximately 0.5 g of liver and kidney for MT analysis. MT was quantified using the modified cadmium-haemoglobin affinity assay [START_REF] Eaton | Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues[END_REF]. Briefly, this methodology requires adding radioactively labelled 109 Cd to the tissue supernatant, allowing it to bind to MT present. A 'Cobra Auto-gamma' counter (Packard Company)

was used to determine the Cd content of the resultant supernatant, which allows for determination of the equivalent MT concentration, expressed as nmol of Cd bound g -1 wet weight.

Histological examination

Histology was carried out on 16 formalin-fixed kidney tissues: 15 samples were previously frozen (-20 o C) and one fixed without prior freezing (SAMA: M21243).

Haematoxylin and eosin (H&E) preparations were carried out on each of the 16 kidney samples using standard histological methodology [START_REF] Brancroft | Theory and practice of histological techniques[END_REF]). An

Olympus BH-2 light microscope was used with 20× magnification. Digital microscope images of 60 glomeruli and Bowman's capsules observed were captured, and software (Motic Images Plus, Version 2.0) was used to measure the area of 20 randomly selected glomerulus and Bowman's capsules from each slide. The area of the space between the glomeruli and Bowman's capsule, and the presence of proteins within each Bowman's capsule was recorded.

2.4

Quantification of bone density and structure Bone strength depends on the amount of bone and the organisational structure of the trabeculae [START_REF] Kleerekoper | The role of three-dimensional trabecular microstructure in the pathogenesis of vertebral compression fractures[END_REF], so two measures of bone density were obtained;

Dual energy X-ray Absorptiometry (DXA) and histomorphometry. DXA measures the total amount of bone (cortical and trabecular bone), but skeletal metal stores can bias results [START_REF] Puzas | Lead in the skeleton interferes with bone mineral density measurements[END_REF]. Histomorphometry directly quantifies the 3-dimensional micro-architectural structure and organisation of bone and is not biased by metal burden. 1.

Statistical analyses

Our primary aim was to determine if metal concentrations were associated with markers of toxicity. To examine the influence of metal concentrations on kidney structures a hierarchical cluster analysis (data not shown) was employed which sorted 14 individuals into two groups based on their concentrations of MT and their levels of renal toxicity.

Between groups linkage was used to designate groups based on all available information and squared Euclidian distances were used to derive proximity measurements. Values were transformed using Z scores due to variables being measured on different scales.

Once groups were designated, an independent samples t-test was then used to determine any significant differences in log transformed metal concentrations, dolphin length, or blubber thickness between the two groups. This process was then repeated, using cluster analysis to sort bone structure variables of 15 individuals into two groups, and a t-test to determine differences in log transformed metal concentrations, dolphin length, and blubber thickness between the groups. All analyses were conducted using SPSS version 14.0.

Qualitative analyses

Our second aim was to examine the datasets for outlier animals with extreme levels of toxicity which could be examined to shed light on the aetiology of the observed toxicity.

Three outlier animals were clearly identifiable from scatter plots (data not shown) and examined. We assume here that metal toxicity aetiology will be highlighted by consistency along the metal toxicity pathway (high metal concentrations, high MT concentrations, advanced renal damage and advanced bone malformations). We assume one of these indicators alone suggests an aetiology that is unrelated to metal toxicity.

RESULTS

High levels of variation among individuals were observed in all toxicity markers (Table 2) and qualitative differences among animals were observed for histological indicators (Figure 1) and costal rib histomorphometry (Figure 2).

Renal damage

Cluster analysis classified individuals into two groups based on differences in their measured renal parameters (renal structure swelling, protein leakage, and MT concentrations). Renal Group 1 had low scores for indicators of renal damage, whereas

Renal Group 2 was characterised by high levels of renal damage, with advanced renal structure (glomerulus, Bowman's capsule, and the space between glomerulus and Bowman's capsule) swelling, higher levels of protein leakage, and higher MT (liver and kidney) concentrations (Table 3). Renal Group 2 had significantly higher levels of Cd (p = 0.045), Cu (p = 0.047) and Zn (p = 0.047) than Renal Group 1. No significant difference in dolphin length or blubber thickness existed between the groups (p > 0.05).

Bone malformations

Classifications based on bone parameters were less clear than those based on renal parameters. Bone Group 1 was defined by lower spinal bone mineral density whereas Bone Group 2 had lower levels of rib bone trabecular organisation (Table 4). Metal concentrations, animal length, and blubber thickness were not significantly different between the groups (p > 0.05). A 2.3 m male (SAMA: M21243) from Gulf St Vincent had the highest recorded glomerulus and Bowman's capsule areas (Table 2) but this was not consistent with other measured parameters. Values for tissue metal burdens and other markers of toxicity were very close to the mean and there were no other signs of toxicity in this dolphin.

Consistency among toxicity markers

DISCUSSION

Renal damage

Concentrations of Cu and Cd in the liver were significantly higher in the cluster of animals defined by high levels of renal damage. Cadmium is a known nephrotoxicant [START_REF] Wang | Cytopathology induced by mercuric chloride and methylmercury in cultured renal cells of Atlantic spotted dolphin (Stenella plagiodon)[END_REF]. Exposure to Cd is known to alter Cu metabolism [START_REF] Peraza | Effects of micronutrients on metal toxicity[END_REF] and the close association between Cd, Cu and renal damage in the present study raises the possibility that alteration of Cu metabolism may be a mechanism for the observed damage. The relationship between MT and renal damage provides support for the use of MT overexpression as a suitable biomarker of Cd-induced renal damage in cetaceans.

The cluster defined by high levels of renal damage also had significantly higher levels of hepatic Zn. Zinc is an essential metal but it can be toxic in high concentrations [START_REF] Walsh | Zinc: Health effects and research priorities for the 1990s[END_REF]. Large anthropogenic quantities of Zn (> 30,000 kg) are released into the marine environment annually in Spencer Gulf, South Australia (NEPC, 2003), and in the long term could result in unusually high Zn accumulation in dolphin tissues and toxic impacts. The significance of renal structure swelling has not been studied in cetaceans specifically; however, laboratory mammal and human studies have found an increase in annual mortality risk of 40 -80% in human patients with metal-induced renal pathology [START_REF] Iwata | Follow up study of renal tubular dysfunction and mortality among residents of an area polluted with cadmium[END_REF][START_REF] Nakagawa | Urinary B 2microglobulin and mortality in a cadmium polluted area[END_REF][START_REF] Nishijo | Relationship between urinary cadmium and mortality among inhabitants living in a cadmium polluted area in Japan[END_REF].

Bone malformations

The bone demineralising effects of environmental contaminants are well documented.

East Greenland polar bear and Baltic grey seal populations show evidence of bone mineral disruption associated with high body burdens of organochlorine contaminants [START_REF] Lind | Bone mineral density in male Baltic grey seal[END_REF][START_REF] Sonne | Is bone mineral composition disrupted by organochlorines in East Greenland polar bears (Ursus maritimus)?[END_REF]. While laboratory studies suggest that metals also influence bone structure and mineralisation [START_REF] Suzuki | Both mercury and cadmium directly influence calcium homeostasis resulting from suppression of scale bone cells: The scale is a good model for the evaluation of heavy metals in bone metabolism[END_REF], associations between metals and bone in wildlife studies are less readily observed [START_REF] Sonne-Hansen | Cadmium toxicity to ringed seals (Phoca hispida) -an epidemiological study of possible cadmium induced nephropathy and osteodystrophy in ringed seals (Phoca hispida) from Qaanaaq in Northwest Greenland[END_REF] although see [START_REF] Beiglbock | Environmental cadmium induces histopathological changes in kidneys of roe deer[END_REF] Rather than sorting individuals based on high or low levels of bone damage, cluster analysis sorted dolphins into groups based on different types of bone organisation. Bone

Group 1 was characterised by low levels of spinal bone density and Bone Group 2 were characterised by low levels of rib bone trabecular organisation. Perhaps due to this method of clustering, and despite the known impacts of metals on bone, there were no significant differences in metal concentrations between groups, suggesting metals may be exerting no influence on bone structure in this study (although see sections 4.3 below).

Alternatively, it is known that Pb preferentially accumulates in the skeleton, leading to overestimates of bone mineral density and complexity [START_REF] Puzas | Lead in the skeleton interferes with bone mineral density measurements[END_REF]. Thus, animals with high bone mineral density scores here may be biased by bone metal content.

Interpretation of these results is hindered by the fact that measurements were taken in different bones. Histomorphometry was measured in peripheral rib bones while bone mineral density was measured in the axial spinal skeleton. The axial skeleton is more sensitive to the effects of toxicants than the peripheral skeleton [START_REF] Laan | Differential effects of glucocorticoids on cortical appendicular and cortical vertebral bone mineral content[END_REF].

Future increases in microtomograph scanning size may allow for quantification of spinal histomorphometry and would aid in clarifying the relationship between metals, bone density, and bone organisation.

Consistency among toxicity markers

Three dolphins had evidence of toxicity and these animals were examined more closely to reveal possible aetiologies. A 2.3 m male (SAMA: M21243) from Gulf St Vincent had high levels of renal structure swelling but no other markers of toxicity suggesting this animal was probably not suffering from metal toxicity, but from an undetermined renal condition.

A female dolphin from Spencer Gulf (SAMA: M20877) had high Cd and Cu concentrations in the liver, MT overexpression, renal damage and extreme bone malformations. While difficult to prove, the consistency amongst toxicity markers may suggest this dolphin was suffering from severe metal toxicity. This animal had a tooth category of five, indicating that it should be both physically and sexually mature [START_REF] Kemper | A study of the life history parameters of dolphins and seals entangled in tuna farms near Port Lincoln, and comparisons with information from other South Australian dolphin carcasses[END_REF]. However, examination of ovaries and skeleton revealed that this animal was physically and sexually immature. It is possible, therefore, that the high levels of metal toxicants have delayed sexual and physical maturity in this animal.

However, due to the non-experimental nature of these observations, it is impossible to rule out the presence of an extraneous influence that either caused the observed pathology, or rendered this dolphin more susceptible to the effects of metal toxicity.

A 2.1 m male dolphin (SAMA: M20877) from upper Spencer Gulf with high Zn tissue concentrations showed decreased bone complexity, high concentrations of liver MT, and renal structure swelling. This dolphin had a tooth category of 5 and was sexually and physically mature [START_REF] Kemper | A study of the life history parameters of dolphins and seals entangled in tuna farms near Port Lincoln, and comparisons with information from other South Australian dolphin carcasses[END_REF]. The consistency between toxicity markers suggests this dolphin may have been suffering from metal toxicity. Zinc toxicity in humans occurs above a threshold level of 465 mg kg -1 dry weight [START_REF] Anon | National irrigation water quality program[END_REF], which is well below the dry weight Zn concentration of 999 mg kg -1 recorded in this animal.

The relationship between metals, toxicity of the kidney, and bone malformations, has been demonstrated in experimental and natural studies of humans and wildlife [START_REF] Staessen | Environmental exposure to cadmium, forearm bone density and risk of fractures: prospective population study[END_REF][START_REF] Alfven | Cadmium and lead in blood in relation to low bone mineral density and tubular proteinuria[END_REF][START_REF] Jarup | Low level cadmium exposure, renal and bone effects -the OSCAR study[END_REF]. A study of 100 ringed seals (Phoca hispida) in Greenland, however, found no relationship between cadmium, degree of bone mineralisation, and nephropathy [START_REF] Sonne-Hansen | Cadmium toxicity to ringed seals (Phoca hispida) -an epidemiological study of possible cadmium induced nephropathy and osteodystrophy in ringed seals (Phoca hispida) from Qaanaaq in Northwest Greenland[END_REF] and the authors concluded that high dietary intakes of vitamin D and calcium (among other minerals) in these ringed seals may protect seals from cadmium toxicity. However, the sample of ringed seals consisted mainly of animals younger than 10 years. Cadmium-induced renal damage typically occurs only after 10 years exposure [START_REF] Friberg | Cadmium and health: A toxicological and epidemiological appraisal[END_REF] and the effect of Cd on bone is more pronounced in older bone [START_REF] Alfven | Cadmium and lead in blood in relation to low bone mineral density and tubular proteinuria[END_REF]. While accurate ages of the bottlenose dolphins examined here are unknown, all are tooth category 3 and above, and would be older than 10 years [START_REF] Kemper | Dolphin interactions with tuna feedlots at Port Lincoln, South Australia and recommendations for minimising entanglements[END_REF].

Experimental studies of metal toxicity in dolphins are undesirable, necessitating the use of natural experiments to infer relationships between health and toxicants. Natural experiments must contend with a lack of experimental control and the presence of confounding factors (e.g., reproductive status, nutritive condition etc). Results obtained from natural studies must be viewed cautiously but can provide important information that can guide future research despite the absence of controlled experimental data. The influence of age on metal accumulation [START_REF] Lavery | Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988 -2004[END_REF] and bone mineral density [START_REF] Butti | Age estimation in the Mediterranean bottlenose dolphin Tursiops truncatus (Montagu 1821) by bone density of thoracic limb[END_REF] have been minimised in the present study by including only adult animals but not eliminated as knowledge of the accurate ages of each dolphin included in this study is not known.

There was a large hiatus between the highest Cd levels (M22410, 98.58 mg kg -1 ) which seem to be associated with toxicity, and the second highest (M21231, 22 mg kg -1 ) which showed no association with toxicity. Thus, toxicity thresholds for T. aduncus could not be determined. Future discoveries of such intermediate Cd tissue burdens in South Australian T. aduncus may allow for the establishment of a toxicity threshold which will aid in interpreting the relevance of metal concentrations already determined throughout the world (see O'Shea, 1999, for a review).

CONCLUSION

Higher metal concentrations were found in a group of animals suffering from renal damage. No differences in metal concentrations were found between groups sorted on the basis of bone density and organisation. Knowledge of the toxicological pathway of metals can aid in distinguishing symptoms of metal toxicity from other, unrelated aetiologies. While only adult dolphins were examined here, the influence of fine scale changes in dolphin age and undetected pathologies represent a methodological limitation in carcasses studies such as this. Extraneous influences are difficult to determine or overcome but we suggest that, by examining a range of metal toxicity symptoms concurrently, future studies can better allow for determination of whether observed health deficits are related to metal toxicity, or an unrelated aetiology.

6.

Figure Captions.

Table 1 Description of trabecular microarchitectural parameters acquired by histomorphometric analyses. Metal and Se concentrations in liver and bone are a subsample of those measured during a previous study [START_REF] Lavery | Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988 -2004[END_REF]. * = minimum or maximum values recorded from M22410; ‡ = minimum or maximum values recorded from M20877; § = maximum values recorded from M21243.

  DXA scans were performed on a GE Lunar Prodigy Vision Dual X-ray Absorptiometry, GE Lunar Madison, Wisconsin. Four caudal vertebrae from each of 30 animals were analysed to obtain measurements of bone mineral density, BMD (g cm -2 ), and bone mineral content, BMC (g). Caudal vertebrae were selected on the basis of being the most posterior four caudal vertebrae with small transverse processes. Small animal software (version 8.10.027) on standard settings was used to examine a maximum area of 25 × 25 cm. Vertebrae were laid longitudinally to best simulate the longitudinal stress placed on the caudal region of the spine by powerful dorsal and ventral muscles associated with swimming. Bones were laid on a 1 cm thick Lucite tissue equivalent block and scanned with parameters set at 76 kilovolts and 150 A. Pixel size was 1.05 × 0.6 mm. BMD and BMC of the vertebrae and posterior process (but not the transverse processes) were determined. Histomorphometry was performed using a Skyscan 1072 X-ray Microtomograph on the largest costal rib available from each of 15 animals. A piece 2 cm long was sampled from the largest end of the bone and placed in the X-ray Microtomograph with the cut facing down. Pixel size was 18.95 m while the X-ray parameters were set at 16× magnification, 100 kilovolts and 98 A. A ring artefact correction was used and a beam hardening correction of 100% was set. Cone reconstruction software was used to reconstruct the 3-dimensional structure of the bone from 1,000 separate 2-dimensional cross section images. Bone density parameters from the 3-dimensional reconstructions were calculated using CTan software (Skyscan). Density parameters calculated by CTan are shown in Table

  Three dolphins showed extreme values in one or more of the measured markers (MT, liver histology, bone density or bone organisation). A 2 m female dolphin (SAMA: M22410) from lower Spencer Gulf showed consistency among the measured parameters. This dolphin had the lowest recorded spinal bone mineral density, highest tissue concentrations of Cd and Cu (Table2), moderately high levels of Zn in the liver (209.35 mg kg -1 ), the highest concentration of liver MT, the largest area between Bowman's capsule and glomeruli, the highest percentage of protein leakage within the Bowman's capsule (Table2), the second highest Bowman's capsule area (19477 m 2 ) and third highest glomerulus area (11703 m 2 ) recorded. Examination of the skeleton revealed serious malformations in the lumbar-caudal region. The last lumbar vertebra had no transverse processes on the right side. The first caudal vertebra had no transverse processes at all, while the second and third caudal vertebrae were missing transverse processes on the left side. Neural spines were thickened on the second, third and fourth caudal vertebrae.A 2.1 m male dolphin (SAMA: M20877) from upper Spencer Gulf had the lowest recorded bone complexity values (BV/TV, Tb.Pf, SMI) (Table2) and also showed consistency among measured parameters. This male dolphin had the second highest recorded scores for concentrations of liver MT (876 nmol of Cd bound g -1 wet weight), glomerulus area (11922 m 2 ), and area of space between Bowman's capsule and glomerulus (6675 m 2 ). It had the third largest Bowman's capsule area (18597 m 2 ), and second highest recorded liver Zn burden (270 mg kg -1 ).

(

Table 3

 3 Copper, cadmium, lead, mercury, selenium and zinc concentrations in the liver, cadmium and lead concentrations in the bone, and renal parameters measured in two groups of adult bottlenose dolphins (Tursiops aduncus) differentiated by cluster analysis on the basis of renal damage.

Table 4

 4

		Copper, cadmium, lead, mercury, selenium and zinc concentrations in the
	liver, cadmium and lead concentrations in the bone, and bone parameters measured in
	two groups of adult bottlenose dolphins (Tursiops aduncus) in two groups differentiated
	on the basis of bone structure.
	Figure 1	Examples of renal parameters measured in adult bottlenose dolphin
	(Tursiops aduncus) carcasses collected from South Australia. A: no space between
	glomeruli and surrounding Bowman's capsule. B: enlarged space between the glomeruli
	and surrounding Bowman's capsule. C: enlarged space between glomeruli and
	Bowman's capsule with no proteins enclosed. D: glomeruli with protein leakage inside
	the Bowman's capsule.

Table 2

 2

	Measurement	N	Minimum	Maximum	Mean	SD
	Liver Cu (mg kg -1 wet weight)	21	8.35	36.21*	19.56	7.76
	Liver Cd (mg kg -1 wet weight)	30	0.26	98.58*	8.64	18.17
	Liver Pb (mg kg -1 wet weight)	30	0.04	14.15	0.68	2.55
	Liver Hg (mg kg -1 wet weight)	30	66	2651	932	677
	Liver Se (mg kg -1 wet weight)	30	33	1188	355	263
	Liver Zn (mg kg -1 wet weight)	30	34	347	97	73
	Bone Pb (mg kg -1 wet weight)	35	0.29	16.00	2.84	3.10
	Bone Cd (mg kg -1 wet weight)	15	0.005	0.33	0.05	0.08
	Glomerulus (µm 2 )	16	3067	14880 §	6856	3349
	Bowman's capsule (µm 2 )	16	4756	20873 §	10862	5020
	Space between glomerulus and	16	1690	7774*	4006	1829
	Bowman's capsule (µm 2 )
	Bowman's capsules with proteins (%) 16	10	95*	43	28
	Liver MT (nmol of Cd bound g -1 wet	14	2	1347*	219	394
	weight)
	Kidney MT (nmol of Cd bound g -1 wet	15	7	193	67	61
	weight)
	Bone Mineral Density (g cm -2 )	30	1.21*	1.80	1.53	0.16
	Bone Mineral Content (g)	30	15.58	42.17	31.45	5.76
	Bone volume / tissue volume (%)	15	23.29 ‡	38.85	30.97	4.00
	Bone surface / bone volume (mm -1 )	15	0.33	0.44	0.37	0.04
	Bone surface / tissue volume (mm -1 )	15	0.09	0.13	0.11	0.02
	Trabecular pattern factor (mm -1 )	15	-0.09	0.02 ‡	-0.05	0.03
	Structural model index	15	-0.38	0.96 ‡	0.17	0.37
	Trabecular thickness (mm)	15	7.81	9.57	8.95	0.52
	Trabecular number (mm -1 )	15	0.03	0.04	0.03	0.01
	Trabecular separation (mm)	15	21.59	30.74	25.97	3.30

Table 4

 4

	Measurement	Group 1			Group 2
		Low bone BMD, BMC	Low bone organisation
		N	Mean	SD	N	Mean	SD
	Liver Cu	9	19.41	8.39	4	16.31	5.33
	Liver Cd	9	17.87	31.39	5	3.35	2.90
	Liver Pb	9	0.23	0.25	5	0.29	0.13
	Liver Hg	9	731.8	531.6	5	1082	706
	Liver Se	9	258	175	5	481	263
	Liver Zn	9	79	58	5	97	97
	Bone Pb	10	2.76	2.91	5	2.91	2.05
	Bone Cd	0			0
	BMD	10	1.45	0.14	5	1.59	0.10
	BMC	10	28.70	4.02	5	34.54	2.37
	BV/TV	10	32.59	3.45	5	27.72	3.07
	BS/BV	10	0.37	0.03	5	0.36	0.04
	BS/TV	10	0.12	0.01	5	0.10	0.004
	Tb.Pf	10	-0.07	0.01	5	-0.01	0.02
	SMI	10	0.09	0.34	5	0.33	0.41
	Tb.Th	10	8.85	0.56	5	9.16	0.38
	Tb.N	10	0.04	0.003	5	0.03	0.002
	Tb.Sp	10	24.29	2.61	5	29.33	1.27

Stockin. Anonymous reviewers greatly improved the quality of earlier manuscripts. We are greatly indebted to all these people.

ACKNOWLEDGEMENTS Funding was provided by Flinders University, University of Adelaide, and Environmental Protection Agency, Adelaide. Carcass collection, management, and necropsies were carried out by staff and volunteers from the South Australian Museum, including Lynette Queale, Bob Hamilton-Bruce, David Stemmer, Ross Goble, Sue Gibbs, Martine Long and members of the Dolphin Trauma Group. Officers of the National Parks and Wildlife SA and Department of Primary Industries Fisheries are thanked for collecting many of the carcasses. Martine Long and Nicole Butterfield were instrumental in laying the groundwork for this project by their preliminary work on metal burdens in South Australian marine mammals. Sam Gaylard was extremely helpful in ensuring that the results of previous researchers were available for inclusion in this study. Ben Roudnew provided invaluable assistance with collecting and preparing samples. Helpful advice and assistance was given by Mike Bossley, Michelle Lewis, Robert Reid, Roger Byard, Nick Fazzalari, Helen Beard, Ian Parkinson, John Stirling, John Edwards, David Duncan, Peter Self, Brian Woodward, Alan McLennan, Kylie Lange, Nardi Cribb and Karen


Baodong Wang
email: wangbd@fio.org.cn

Hydromorphological Mechanisms Leading to Hypoxia off the Changjiang Estuary

Keywords: Hypoxia, Dissolved oxygen, Oxygen depletion, Hydrography, Morphology, Bottom topography, The Changjiang (Yangtze

Based on the seasonal surveying data and long-term data, the annual changes in the geographical locations, occurrence frequency, affected areas and the minimum oxygen level as well as the formation mechanism of the summer hypoxia off the Changjiang estuary are summarized and discussed in this paper. The historical data indicates that there were episodes of hypoxia in the past 50 years but not every year, and the event of summer hypoxia could be traced back to as early as late 1950s off the Changjiang estuary. The minimum oxygen levels in the hypoxia zone did not show any decline in the past 50 years, but all the events with large size of affected-area (>5000km 2 ) were observed after the late 1990s, suggesting an enlarging trend. The author argues that the development of summer hypoxia off the Changjiang estuary was related not only to stratification and input of suspended particulate matter, but also to the inflow of Taiwan Warm Current Water as well as the bottom topography.

Introduction

Because of their geomorphology and circulation patterns, some marine systems have a greater tendency to develop hypoxic conditions. The basic features of a system that make it prone to hypoxia are low physical energy (tidal, currents, or wind) and large freshwater input [START_REF] Diaz | Overview of hypoxia around the world[END_REF]. These features combine to form stratified or stable water masses near the bottom that become hypoxic when they are isolated from reoxygenation with surface waters.

Hypoxia and anoxia have been widely observed in many estuarine and coastal regions over the last several decades, of which the most well-known areas for hypoxia/anoxia are the Gulf of Mexico, Chesapeake Bay, the North Sea, Tokyo Bay, the Black Sea, and the Baltic Sea [START_REF] Diaz | Overview of hypoxia around the world[END_REF]Rabalais, Turner andWiseman, 2001, 2002). Hypoxic zones also occur in coastal waters of China Seas, such as the summer hypoxia off the estuaries of Changjiang and Pearl River (e.g., [START_REF] Chen | Hypoxia in the East China Sea: One of the largest coastal low-oxygen areas in the world[END_REF][START_REF] Wei | Summer hypoxia adjacent to the Changjiang Estuary[END_REF][START_REF] Dai | Oxygen depletion in the upper reach of the Pearl River estuary during a winter drought[END_REF][START_REF] Li | Oxygen depletion off the Changjiang (Yangtze River) Estuary[END_REF][START_REF] Yin | Temporal and spatial distribution of dissolved oxygen[END_REF].

Previous studies have obtained some preliminary understanding about the formation mechanism of the summer hypoxia off the Changjiang estuary; however, these findings were based on the data of some single cruises [START_REF] Chen | Hypoxia in the East China Sea: One of the largest coastal low-oxygen areas in the world[END_REF][START_REF] Li | Oxygen depletion off the Changjiang (Yangtze River) Estuary[END_REF][START_REF] Wei | Summer hypoxia adjacent to the Changjiang Estuary[END_REF]. The annual changes in the geographical locations, the frequency, the affected areas, the minimum oxygen levels, and the regional specific characteristics of summer hypoxia development in this area still remain unknown.

Based on the seasonal surveying data and the long-term data on summer hypoxia off the Changjiang estuary, the author attempted to answer the above questions and identify the regional specific hydromorphological mechanisms leading to the development of summer hypoxia off the Changjiang estuary.

Materials and Methods

The source of field data used in this paper is the same as that of [START_REF] Wang | Nutrient conditions in the Yellow Sea and the East China Sea[END_REF]. Since this paper focuses on the chemical hydrography in areas adjacent to the Changjiang estuary, ten cross-shelf transects covering the shelves of the Yellow Sea (YS) and East China Sea (ECS) were selected from the original database. The data collected from November 1997, May 1998, August 1998and January 1999 cruises were designed to represent the autumn, spring, summer and winter conditions, respectively. The station locations, sampling resolution and analytical methods of temperature and salinity were described in detail elsewhere [START_REF] Wang | Nutrient conditions in the Yellow Sea and the East China Sea[END_REF]. Dissolved oxygen was measured using Winkler titration method. The precision in the measurements of temperature, salinity and dissolved oxygen were  0.005C,  0.005,  0.06 mg L -1 , respectively. Historical records of summer hypoxia were also compiled for comparison.

Results

In the winter, circulation in areas adjacent to the Changjiang estuary is characterized by northward extension of the Taiwan Warm Current (TWC) and southward flow of coastal currents along the banks [START_REF] Guan | Patterns and structures of the currents in Bohai, Huanghai and East China Seas[END_REF]. Bottom temperature distribution showed that the saline warm water of TWC flowed northward along 123E from the south of ECS and turned northwestward at 31N into the trough off the Changjiang estuary, while the cold, less saline coastal water extended southeastward in a tongue-like shape from the west of YS to the northern ECS (Fig. 1a). The shapes of the isotherms were quite similar to isobaths (Fig. 1a).

Similar distributions were also found for salinity (not shown) and dissolved oxygen (Fig. 1b). Water with DO <8 mg L -1 from the TWC occupied the trough off the Changjiang estuary, while >8 mg L -1 coastal water occupied the coastal areas as well as the middle shelf of the northern ECS (Fig. 1b). The vertical profiles of T, S and DO showed that the water column was homogeneous and the TWC water in the trough was very distinct with high temperature (>15C) and salinity (>34.2) and lower oxygen level (<7.8 mg L -1 ) (Fig. 2a, b &c).

With the southerly monsoon predominant in spring, the coastal current in ECS moved northward along the coast with increasing width and velocity, while the coastal current in YS extended southeastward into the northern ECS with less intensity than that in winter [START_REF] Guan | Patterns and structures of the currents in Bohai, Huanghai and East China Seas[END_REF]. The TWC at all levels below the sea surface moved north-northeastward along isobaths offshore with intensity stronger than that in winter [START_REF] Guan | Patterns and structures of the currents in Bohai, Huanghai and East China Seas[END_REF]. The bottom salinity distribution indicated that the saline frontal water of TWC extended far reaching into the trough off the Changjiang estuary, which could reach 31.5N as shown by isohaline of 33 psu (Fig. 1c). The oxygen level of the bottom water of TWC was lower than that in the winter.

An area with oxygen level less than 5.0 mg L -1 was found in the bottom water inside the trough and there were sharp gradients with surrounding waters (Fig. 1d).

Solar radiation and river runoffs (mainly from the Changjiang River) made a strengthened stratification in the spring in the northern ECS shelf. In the area of the trough, there were a strong halocline, a weak thermocline and lower dissolved oxygen at the depth of 10 to 30 m (Fig. 2d2f). Below the halocline and the thermocline lied the deep water of TWC inside the trough, which was characterized by low temperature (<19.0C), high salinity (>33.5) and low oxygen level (<5.0 mg L -1 ). In the middle shelf of the northern ECS, there was a stronger thermocline at the depth of 10 to 20 m and below the thermocline lied a cold water (<13C) with low salinity (<33.0) and high oxygen starting level (> 8.0 mg L -1 ) (Fig. 2d2f).

In the summer, the circulation pattern and the composition of water masses in the studied area were similar to that in the spring. However, the forces of the coastal current in ECS and TWC were enhanced due to higher runoffs and dominance of southerly wind [START_REF] Guan | Patterns and structures of the currents in Bohai, Huanghai and East China Seas[END_REF]. The surface of the northern ECS and south of YS was occupied by Changjiang Diluted Water with low salinity (< 31.0) and high temperature (see Fig. 2 in [START_REF] Wang | Nutrient conditions in the Yellow Sea and the East China Sea[END_REF]. The bottom water from TWC with low temperature (<21.0C) and high salinity (>33.0) occupied the middle shelf as well as the trough off the Changjiang estuary, and its frontal water could reach 32N (as shown by the 33.0 isohaline) (Fig. 3a). The oxygen level of the bottom water of TWC was even lower than that in the spring. A closed area with oxygen level less than 3.0 mg L -1 was found in bottom water inside the trough as well as on its right slope (Fig. 3b) and there were sharp gradients both in temperature and oxygen levels with its eastern and northern surrounding waters (Fig. 3a &b).

Stronger solar radiation and higher river runoffs during the summer made a stronger stratification than that in the spring on the ECS shelf. In the inner shelf of the northern ECS, there were a very strong thermocline, a halocline and a spring layer of dissolved oxygen at the depth of 5 to 30 m (Fig. 2g~2i). The upper water in the northern ECS shelf was occupied by Changjiang Diluted Water with salinity less than 31.0 (Fig. 2h) and the lower layer water was the deep water of TWC with high salinity (>34.0), low temperature (<19C) and low oxygen level (<4.0 mg L -1 ). The In the autumn, the circulation pattern and intensities of the currents were similar to that in the winter. The extending range of the bottom frontal water of TWC was almost the same as that in the winter (as shown by 33.0 isohaline, Fig. 3c). The oxygen levels in the bottom waters inside the deep trough as well as on the middle shelf of ECS were less than 5.0 mg L -1 (Fig. 3d). The vertical profiles showed that the water column was nearly vertically uniform on the northern ECS shelf except inside the deep trough, where there still existed a halocline and a spring layer of dissolved oxygen at the lower layer, which was the remnant deep water of TWC (Fig. 2j~2l).

Discussion

The locations of hypoxia zone off the Changjiang estuary

Variation of the minimum oxygen levels and the affected areas of the hypoxia zone

A historical picture of oxygen conditions off the Changjiang estuary, as shown in Table 1, indicates that there were episodes of hypoxia in the past 50 years but not every year. The overall probability of occurrence of summer hypoxia was 60% in the past 50 years, but up to nearly 90% after the 1990s, demonstrating an increasing trend. In terms of the duration of the hypoxic events, the available monthly surveying data conducted in 1959 revealed that hypoxic events occurred only in August, suggesting that the duration of the hypoxic events was probably rather short.

The minimum oxygen concentrations observed in the area off the Changjiang estuary were different in different years, but there was no obvious trend of decline or increase. The size of the hypoxia zone also differed greatly in different years.

However, all the events of hypoxia with affected area greater than 5000 km 2 occurred after the end of the 1990s, indicating that the extent of summer hypoxia became more severe in recent years, possibly due to the increasing input of anthropogenic nutrients to this area [START_REF] Wang | Cultural eutrophication in the Yangtze River plume: history and perspective[END_REF].

The formation mechanism of the hypoxia zone off the Changjiang estuary

Based on the analysis of the seasonal distributions of temperature, salinity and dissolved oxygen, the formation mechanism of hypoxia zone off the Changjiang estuary may be described as the following: in the winter, the frontal water of TWC with high temperature and salinity and low oxygen level occupied the whole water column in the area of the trough off the Changjiang estuary; in the spring, the combination of higher runoff and stronger solar radiation made a strong stratification in the northern ECS shelf, especially in the area of the trough, resulting in decreased oxygen level at the lower layer through decomposition of organic matter; in the summer, the combination of the highest runoff and the strongest solar radiation made the strongest stratification in the northern ECS shelf, especially in the area of the trough, resulting in the continuous decrease in the oxygen level at the lower layer through decomposition of organic matters and finally the hypoxia zone formation inside the trough; and in the autumn, water column stratification is broken down was by strong vertical advection, the bottom water is reoxygenated, and the hypoxia zone disappears.

Previous studies emphasized two principal factors, i.e., water column stratification and decomposition of organic matter in the bottom water, leading to the development of hypoxia off the Changjiang estuary [START_REF] Chen | Hypoxia in the East China Sea: One of the largest coastal low-oxygen areas in the world[END_REF][START_REF] Li | Oxygen depletion off the Changjiang (Yangtze River) Estuary[END_REF][START_REF] Wei | Summer hypoxia adjacent to the Changjiang Estuary[END_REF]. In this paper, the author argues that there are also some other factors controlling the development of summer hypoxia off the Changjiang estuary.

First, the inflow of the TWC water with high salinity and lower temperature (during spring and summer) provides an essential prerequisite for the formation of water column stratification in the area of the trough during the spring and the summer and it also provides the bottom water which is originally low in oxygen level.

Secondly, the bottom topography might play a key role in the processes of the development of summer hypoxia off the Changjiang estuary. The deep trough just off the estuary isolated the bottom water inside the trough from exchange with surrounding oxygen-rich waters through advection, making the bottom water inside the trough to be rather stable, so that the hypoxia could develop and persist inside the trough. This is why almost all of the centers of hypoxia zones were located inside the trough in the past 50 years. As a matter of fact, the development of most of the well-known hypoxia or anoxia zones in the world oceans has something to do with bottom topography, such as the hypoxic zone in the deep channel of the Chesapeake Bay [START_REF] Hagy | Hypoxia in Chesapeake Bay, 1950-2001: long-term change in relation to nutrient loading and river flow[END_REF], and the hypoxic zone in the V-shaped deep trough of the central Baltic Sea [START_REF] Conley | Hypoxia in the Baltic Sea and basin-scale changes in phosphorus biogeochemistry[END_REF], as well as the anoxic zone in the deep basin with steep slope in the Black Sea [START_REF] Neretin | A sulfur budget for the Black Sea anoxic zone[END_REF].

Moreover, owing to its special bottom topography and hydrographic condition, the deep trough is favorable for the sedimentation of fine sediments from the Changjiang River [START_REF] Milliman | Transport and deposition of river sediment in the Changjiang estuary and adjacent continental shelf[END_REF][START_REF] Shi | Bottom fine sediment boundary layer and transport processes at the mouth of the Changjiang Estuary, China[END_REF]. This could be supported by the existence of a closed area of muddy sediments inside the trough [START_REF] Hu | Upwelling and sedimentation dynamics I. The role of upwelling in sedimentation in the Yellow Sea and East China Sea[END_REF]. Additionally, upwelling in this area is favorable for the resuspension of fine sediments [START_REF] Hu | Upwelling and sedimentation dynamics I. The role of upwelling in sedimentation in the Yellow Sea and East China Sea[END_REF][START_REF] Wang | Chemical hydrography of the coastal upwelling in the East China Sea[END_REF]Zhao, 1993). All these processes accelerate the reduction of oxygen level through decomposition of organic matter in the bottom water which can lead to the development of hypoxia off the Changjiang estuary. C h a n g ji a n g R . C h a n g ji a n g R .

China

China China

Figure 1

 1 Figure 1

Figure 2 remnant

 2 Figure 2

Figure 4

 4 Figure4shows the distribution of the locations of the centers of hypoxia zones

  Figure 3

  Figure 4

Fig. 1

 1 Figure captions:

  Figure1

  Figure 3

Table 1

 1 Historical record of the locations of the minimum values of dissolved oxygen concentrations and affected areas of the hypoxia zone off the Changjiang estuary

		Location	Min. of	Affected
	Time	longitude	latitude	DO Conc.	area	Reference
				(mg L -1 )	(km 2 )
	Aug.,1959	12245	3115	0.34	1000	Office of Integrated Oceanographic
						Survey of China ,1961
	Aug.,1976-85	12300	3100	≥0.80	?	Zhang, 1990
	Aug.,1981	12300	3050	2.0	-	Limeburner, Beardsley and Zhao,1983
	Aug.,1982	12230	3115	2.85	-	Chen, Chen and Yang, 1988
	Aug.,1985	12230	3130	2.29	-	Ren, 1992
	Aug.,1988	12300	3050	1.96	<200	Tian, Hu and Martin, 1993
	Aug.,1998	12400	3210	1.44	600	Wang and Wang, 2007
	Aug.,1999	12259	3051	1.0	13700	Li et al., 2002
	Aug., 2002	12229	3200	1.73	<500	Shi, Lu, Zhang and Wang, 2006
		12300	3100	1.99
	Jun., 2003	12250	3050	1.0	5000	Xu, 2005
	Aug., 2003	12330	3130	2.0	-	Chen et al., 2007
	Sept., 2003	12256	3049	0.8	20000	Wei et al., 2007
		12245	3155	<1.5
	Aug., 2004	12240	3115	>2.0	-	Pei, 2007
	Sept., 2006	12306	3009	1.96	<300	Zou, Yang, Liu and Shi, 2007

in the Pearl River Estuary and adjacent coastal waters. Continental Shelf Research 24, 1935-1948. Zhang, Z., 1990. Maximum and Zou, J., Yang, G., Liu, J., Shi, X., 2007. Features in distribution of dissolved oxygen in the Changjiang estuary and adjacent coastal waters. Advances in Marine Science (in press) (in Chinese with English abstract).


M-F Van Bressem

Marcos César De Oliveira Santos

Júlia Emi De Faria Oshima

Skin diseases in Guiana dolphins (Sotalia guianensis) from the Paraná estuary, Brazil: a possible indicator of a compromised marine environment

Keywords: diseases, lobomycosis-like disease, nodules, skin, pollution, Sotalia guianensis, Lagamar estuary, coastal waters, Brazil

We report on the presence of lobomycosis-like disease (LLD) and nodular skin disease (NSD) in a community of Guiana dolphins (Sotalia guianensis) inhabiting the biologically and chemically contaminated Paraná estuary (Brazil) and on their absence in the community living in the cleaner Cananéia estuary. Prevalence rates of LLD and NSD were 3.9% and 12.6%, respectively, in 103 photo-identified (PI) dolphins from the Paraná estuary in the period 2006-2007. Adults and calves were affected. Lobomycosis-like lesions may be extensive and form large plaques. Skin nodules were sometimes ulcerated and associated with cutaneous traumas suggesting that traumatic injuries may play a role in the pathogenesis of this condition. In two adult dolphins, NSD evoked the beginning of LLD. In 1996-2007 none of the 200 PI Cananéia S. guianensis had LLD or NSD, a highly significant difference. Interestingly, these dolphins were reported to harbour relatively low concentrations of organochlorines. LLD and NSD are possibly indicators of environmental changes.

INTRODUCTION

Caused by the fungal pathogen known as Lacazia loboi (Taborda et al., 1999) (syn. Loboa loboi; [START_REF] Caldwell | Lobomycosis as a disease of the Atlantic bottle-nosed dolphin (Tursiops truncatus Montagu, 1821)[END_REF], lobomycosis (or lacaziosis) naturally affects humans, bottlenose dolphins (Tursiops truncatus) and Guiana dolphins (Sotalia guianensis) (de [START_REF] De Vries | A case of lobo's diseases in the dolphin Sotalia guianensis[END_REF][START_REF] Caldwell | Lobomycosis as a disease of the Atlantic bottle-nosed dolphin (Tursiops truncatus Montagu, 1821)[END_REF]Simões-Lopes et al., 1993;[START_REF] Reif | Lobomycosis in Atlantic bottlenose dolphins from the Indian River Lagoon, Florida[END_REF][START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF]. The natural reservoir of L. loboi is unknown but soil and vegetation seem to be likely sources of infection for humans [START_REF] Honda | Lobomycosis[END_REF]. Lobomycosis in dolphins is characterized by grayish, whitish to slightly pink, verrucous lesions, often in pronounced relief that may ulcerate [START_REF] Migaki | Lobo's disease in an Atlantic bottlenosed dolphin[END_REF]. T. truncatus from the Indian River Lagoon (IRL), Florida, affected by cutaneous lobomycosis, were found to have significant impairment in adaptative immunity possibly related to chronic exposure to environmental stressors [START_REF] Reif | Immune dysfunction in Atlantic bottlenose dolphins (Tursiops truncatus) with lobomycosis[END_REF]. Variation in salinity and water temperature may also play a role in the infection [START_REF] Reif | Lobomycosis in Atlantic bottlenose dolphins from the Indian River Lagoon, Florida[END_REF]. Lobomycosis and lobomycosis-like disease (LLD), a disease very similar to lobomycosis but for which a histological diagnostic is missing, have been reported in inshore dolphins from several South American countries with the first case reported in a S. guianensis caught in the estuary of the Surinam River in 1971 (de [START_REF] De Vries | A case of lobo's diseases in the dolphin Sotalia guianensis[END_REF]Simões-Lopes et al., 1993;[START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF]. Photo-identification was shown to be a useful tool to detect and monitor these diseases [START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF][START_REF] Murdoch | Lobomycosis in Bottlenose Dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida: Estimation of Prevalence, Temporal Trends, and Spatial Distribution[END_REF].

Two communities of S. guianensis inhabit the Lagamar estuary (Figure 1 (e.g. Santos and Rosso, 2007;2008;Santos et al., 2000;2001;2002;2003) Area in 1984(Schaeffer-Novelli et al., 1990). It is, however, severely impacted by anthropogenic factors. A channel dug at the end of the 1880s to connect its northern limit to the Ribeira do Iguape River has caused dramatic changes in its fauna composition and structure (Tommasi, 1985). Though prohibited by law in Brazil since 1985, chlorinated hydrocarbons were vastly used till the end of the 1990s and still contaminate the estuary [START_REF] Ferreira | Alguns dados sobre a poluição por pesticidas clorados na região lagunar estuarina de Cananéia[END_REF][START_REF] Mattos | Resíduos de pesticidas organoclorados e bifenilos policlorados em sedimentos e algas de Santos e Cananéia[END_REF]. In the southern portion of the estuary, Paranaguá harbour, Brazil's second most important port, was built in 1935. Oil spills are common in this area as well as shipwrecks inside and outside local estuarine waters [START_REF] Wiczorek | Mapeamento de sensibilidade a derramamentos de petróleo do Parque Estadual da Ilha do Cardoso-PEIC e áreas do entorno[END_REF]. As top predators, small cetaceans are exposed to, and affected by, persistent organic pollutants (POP) including polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and related compounds [START_REF] Ross | The immunotoxicity of environmental contaminants to marine wildlife: a review[END_REF][START_REF] Ross | The role of immunotoxic environmental contaminants in facilitating the emergence of infectious diseases in marine mammals[END_REF]).

Here we report on the presence of LLD and possibly related nodular skin disease (NSD) in S. guianensis from the Paraná estuary and on their absence in the community inhabiting the Cananéia area.

MATERIAL AND METHODS

1. Image and data collection

Photo-identification boat-based (15 and 30hp motorized vessels) surveys were conducted from May 1996 to August 2007 in the Cananéia estuary and from April 2006 to September 2007 in the northern portion of the Paraná state. The survey path followed a zig-zag pattern to maximize chances of encountering dolphins. Surveys were conducted in good sea conditions (Beaufort Scale = 0-2). A 35mm reflex camera using printed films and with a 300mm zoom lens was used till May 2004 and subsequently replaced by digital cameras with 300 and 400mm lenses. Photographs were taken at distances ranging from two to 10 m with shutter speeds ranging between 1/500s to 1/2000s.

Dolphins

All dolphins were individually identified from natural marks present on the dorsal fin and body [START_REF] Würsig | The photographic determination of group size, composition, and stability of coastal porpoises (Tursiops truncatus)[END_REF][START_REF] Würsig | Methods of photo-identification for small cetaceans[END_REF]. About 100,000 and 26,000 pictures of dorsal fins were examined for the Cananéia and Paraná dolphins, respectively. On most occasions, all individuals from a group including calves were photographed. Females were identified on the basis of an accompanying calf. Calves are defined as individuals whose body length is one-third or less than that of other dolphins in the area and that usually remain close to their mothers in the infant position [START_REF] Mann | Behavioral development of wild bottlenose dolphin newborns[END_REF][START_REF] Mann | Female reproductive success in bottlenose dolphins (Tursiops sp.): life history, habitat, provisioning, and group-size effects[END_REF]. Adults are large, robust dolphins.

Nodular skin disease and lobomycosis-like disease

Nodular skin disease is characterized by circumscribed, raised, grey, orange or reddish, sometimes ulcerated, skin lumps. Lobomycosis-like disease consists in raised, gray-white to slightly pink, verrucous, at times ulcerated, proliferating lesions that may form large plaques, and grossly most closely resemble those caused by L. loboi in T. truncatus.

The lesions were counted and their broadest size estimated on the basis of a mean dorsal fin base length of 23 cm in adults (Santos et al., unpublished data). They were further classified as small (≤ 10 mm), medium (>10 mm and < 30 mm) and large (≥ 30 mm). Severity of the disease was scored as follows: 1= one small to medium-sized lesion, 2= two to seven, small to medium-sized, localized nodules, 3= five or more, small to large lesions affecting more than one body areas, 4= more than 10 lesions, aggregating, affecting several body areas and covering approximately 10% or more of the visible body surface.

RESULTS

In the Paraná estuary we observed LLD and NSD in 18 (17.4%) of the 103 dolphins photo-identified in 2006-2007. Four (3.9%) dolphins including a calf, an adult female and two adults of unknown sex had LLD with severity scores ranging from 2 to 4 (Table 1). In two adults, including the female, the lesions were extensive and proliferating and affected the back, flanks and tailstock (Figure 2a).

Individual lesions ranged from an approximate 5 to 50 mm in their broadest dimension (Table 1).

When congregating the sores formed large plaques in adult female 'J' (approximately 355 x 25 mm, 283 x 16 mm and 119 x 08 mm) and in adult 'L' of unknown sex (about 180 x 80 mm-the sizes of the two other visible plaques could not be measured). In the calf the disease was apparently restricted to the back and dorsal fin (Figure 2b). The elevated white lumps measured around 8 till 20 mm in their broadest dimension. All dolphins were seen in Guaraqueçaba bay (25S 16' 14.2", 48W 21' 06.1' ' , Figure 1) and surrounding areas in February-August 2007. On the only picture available, the calf's mother had a small white and round mark on her right side that was not considered to be LLD or NSD but may represent a very early stage of these diseases (Figure 2b). NSD was seen in 14 (12.6%) dolphins including 13 adults and a large calf with severity scores ranging from 1 to 3 (Table 1). The nodules were seen on the flanks, back and tailstock. They ranged from an estimated 5 to 45 mm in their broadest dimension, were occasionally ulcerated (Figure 3a, Table 1) and were sometimes associated with skin traumas (Figure 3b) suggesting that wounds may play a role in the pathogenesis of this disease. In two adult dolphins of unknown sex, the lumps evoked the beginning of LLD: though still circumscribed they were grouped on a small area behind the dorsal fin and had a tendency to form plaques (Figure 4). However, these lesions were only sighted once and their evolution is unknown. Nine of the 13 adults with lumps were seen in Guaraqueçaba bay and surroundings. The mother of the calf with the reddish lump did not have visible skin lesions but only one picture was available. None of the 200 Cananéia dolphins had LLD or NSD in the period 1996-2007, a highly significant (χ 2 = 37.32, df= 1, P= 1 -09 ) difference.

DISCUSSION

This photo-identification study reports for the first time on the presence of LLD and NSD in a community of S. guianensis inhabiting the Paraná estuary, Brazil. It is also the first time that LLD is observed in a calf. The epidemiological status of LLD and NSD in S. guianensis from the Lagamar estuary is unknown. The presence of extensive lobomycosis-like lesions in two adults, and minor lesions in a third, suggests that the disease has been present for at least some months in this community. Prevalence of LLD was 3.9% in 103 dolphins photo-identified in 2006-2007. In inshore T. truncatus from South America, prevalence levels of this syndrome varied from 1.6% (Gulf of Guayaquil, Ecuador) to 20% (Tramandaí estuary, Brazil) in the period [START_REF] Würsig | Methods of photo-identification for small cetaceans[END_REF]-2007[START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF]. The role of L. loboi as the aetiological agent of LLD was confirmed by histology in a dead T. truncatus from the Tramandaí estuary, Brazil, visually diagnosed as positive when still alive [START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF][START_REF] Moreno | Mycotic dermatitis in common bottlenose dolphins (Tursiops truncatus) from southern Brazil, with a confirmed record of lobomycosis disease[END_REF]. Further studies will examine whether this fungus is also the causal agent of LLD in S. guianensis from the Paraná estuary.

Though the 'nodular skin disease' observed in the Paraná dolphins suggests an early form of LLD, it may represent another disorder. Besides, L. loboi at least four other pathogens are known to cause skin nodules in small cetaceans. Streptococcus iniae, primarily a bacteria of fishes, triggered 'golf ball disease', a condition characterised by multiple subcutaneous abscesses in botos (Inia geoffrensis) kept in captivity in the US [START_REF] Bonar | A third report of "golf ball disease" in an Amazon River dolphin (Inia geoffrensis) associated with Streptococcus iniae[END_REF]. Lumps resembling golf ball disease were seen in I. geoffrensis from the Mamirauá Sustainable Development Reserve, Brazil, but the aetiological agent of these lesions is unknown [START_REF] Da Silva | Skin disease and lesions of the boto Inia geoffrensis in the Central Amazon[END_REF]. There is no other report of this syndrome in free-ranging river dolphins, and the organism has not been reported in fishes from Brazil [START_REF] Agnew | Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination[END_REF]). An uncharacterised papillomavirus (PV) caused cutaneous warts in a harbour porpoise (Phocoena phocoena) stranded in Germany in December 1993 [START_REF] Van Bressem | Cutaneous papillomavirus infection in a harbour porpoise (Phocoena phocoena) from the North Sea[END_REF]. Nevertheless, the warts had a different aspect from the lumps observed during this study, the PV disease did not seem to be very contagious and has not been reported in South American cetaceans. Fusarium spp. hyphae caused raised, firm, erythematous, 2 to 5 mm cutaneous nodules that were most prominent on the head, trunk, and the caudal portion of an Atlantic whitesided dolphin (Lagenorhynchus acutus) and a pygmy sperm whale (Kogia breviceps) stranded along the northeast coast of the USA in 1991 [START_REF] Frasca | Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals[END_REF]. Fusarium spp. are widespread in South America causing diseases in plants and animals [START_REF] Ortoneda | Fusarium oxysporum as a multihost model for the genetic dissection of fungal virulence in plants and mammals[END_REF][START_REF] Guilhermetti | Fusarium spp. as agents of onychomycosis in immunocompetent hosts[END_REF]. A fungus identified as Trichophyton spp. was seen in conical, ulcerated, 16-36mm nodules on the trunk of an Atlantic T. truncatus kept in captivity in Japan (Hoshina and Sugiura 1956). The role of these pathogens in the aetiology of the cutaneous nodules in S. guianensis should be further explored. If NSD represents indeed an early form of LLD, prevalence of this syndrome would reach 17.4% in S. guianensis from the Paraná estuary.

Although more than 100,000 photographs of S. guianensis inhabiting the Cananéia estuary were analysed over a period of 10 years, LLD and NSD were never seen in this community. Interestingly, these dolphins were reported to harbour relatively low concentrations of organochlorines [START_REF] Yogui | Chlorinated pesticides and polychlorinated biphenyls in marine tucuxi dolphins (Sotalia fluviatilis) from the Cananéia estuary, southeastern Brazil[END_REF]. On the other hand, prevalence of LLD and possibly related NSD was relatively high (17.4%) in the individuals from the Paraná estuary. A large port, small villages with up to 2,000 inhabitants and an illegal shrimp farm that operated for a short time in 2006-2007 (Santos, pers. (Santos, pers. observations) likely biologically and chemically contaminated this area. Aquaculture, especially shrimp and salmon farms, in South America is known to heavily use prophylactic antibiotics that may alter the normal skin fauna and create an appropriate environment for fungal invasion [START_REF] Frasca | Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals[END_REF][START_REF] Cabello | Antibioticos y acuicultura en Chile: consecuencias para la salud humana y animal[END_REF][START_REF] Cabello | Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment[END_REF]. Water ballast from the numerous ships transporting grain could bring alien micro-organisms [START_REF] Ruiz | Global spread of microorganisms by ships[END_REF][START_REF] Drake | Potential microbial bioinvasions via ships' ballast water,sediment, and biofilm[END_REF] including L. loboi into the Paraná estuary. Interestingly all inshore T. truncatus populations from South America affected by LLD inhabited heavily polluted waters surrounding large ports and cities and, in the case of Guayaquil, also harbouring intense shrimp farming activities [START_REF] Van Bressem | A preliminary overview of skin and skeletal diseases and traumata in small cetaceans from South American waters[END_REF]. Environmental changes may favour the dissemination of the L. loboi and promote its introduction in naïve populations and communities.

CONCLUSIONS

The high prevalence of LLD and NSD in the S. guiannensis from the Paraná estuary and their absence in the well-studied community of the Cananéia estuary is striking. These diseases may be indicators of environmental changes. Their aetiology, pathogenesis, epidemiology, evolution and impact on survival of coastal dolphins should be further explored.

ACKNOWLEDGEMENTS

Long

TABLE 1 10. FIGURES

  ), Brazil. One dwells in the coastal waters surrounding the island of Cananéia (25 o 03'S; 47 o 55'W) and the other lives along the northern coast of the Paraná state (25 o 22'S; 48 o 25'W). The first one has been studied since 1996

  while photo-identification surveys of the second one started in April 2006. In the Cananéia and Paraná areas 200 and 103 dolphins were photo-identified, respectively. Approximately 180 km long, the Lagamar estuary is located between the states of São Paulo and Paraná and is in close contact with the Southwest Atlantic Ocean. It is surrounded by large mangrove forests and was designated a National Protected

  Figure 1. Map of the Lagamar estuary, Brazil, including the Cananéia and Paraná estuaries

Figure 3a .

 3a Figure 3a. Ulcerated nodules on the back of adult S. guianensis 'C'.

Figure 3b .

 3b Figure 3b. Reddish nodule on tailstock and lesions associated with cutaneous traumas on the back of

Figure 4 .

 4 Figure 4. Nodules evoking the beginning of LLD on the back of adult S. guianensis 'O'.

Table 1 . Details of Guiana dophins (Sotalia guianensis) from Guaraqueçaba and adjacent areas affected by lobomycosis-like disease (LLD) and nodular skin disease (NSD) Dolphin Sex Age class Date NSD LLD Nber Severity Description

 1 size reddish nodule on tail stock, two in relief lesions associated with skin traumas on left flank.Specimens are ordered by disease and code. Abbreviations are: indet= indeterminate, nber= number.

	J	female	adult	8 Aug 07 No Yes several	4	dark gray and brownish, at times ulcerated, small to large nodules forming plaques on flanks and back.
	L	indet	adult	8 Feb 07	No Yes several	4	extensive, small to large, nodular lesions forming plaques on dorsal fin, back and tailstock.
	G	indet	calf	8 Jul 06	No Yes	3	2	two whitish, small to medium, verrucous lesions on back behind dorsal and one on dorsal fin.
	V	indet	adult	2 Aug 07 No Yes	2	2	small to medium lesions on the back, apparently associated with a skin trauma.
	A	indet	adult	30 Jul 06 Yes No	5	3	in relief small to large lesions on dorsal fin and left flank.
	B	female	adult	30 Jul 06 Yes No	2	2	small to medium nodules on back and left flank.
	C	indet	adult	8 Jul 06 Yes No	3	2	ulcerated, medium-sized nodules on the back below dorsal.
	D	indet	adult	8 Jul 06 Yes No	3	2	small to large, grouped nodules on left flank.
	E	indet	adult	8 Jul 06	Yes No	2	2	a small nodule on the back and a medium on tailstock.
	F	female	adult	8 Jul 06	Yes No	1	1	medium-size, ulcerated nodule on left flank.
	H	indet	calf	8 Aug 07 Yes No	1	1	heart shaped, medium, reddish nodule on the back before dorsal fin.
	M one medium-N indet adult 8 Feb 07 Yes No 1-3 2 indet adult 8 Jan 07 Yes No ~ 7 2 small to medium nodules associated with scars and evoking the beginning of LLD on the back behind dorsal fin
	O	indet	adult	31 Jul 06 Yes No	5	2	medium, whitish nodules evoking the beginning of LLD on the back behind dorsal fin.
	P	indet	adult	8 Aug 07 Yes No	1	1	reddish, medium-size nodule on right flank.
	Q	indet	adult	8 Aug 07 Yes No	5	2	medium-size, whitish nodules on the left flank and back behind dorsal fin.
	R	female	adult	8 Aug 07 Yes No	1	1	medium-size nodule on the back behind head.
	S	indet	adult	2 Aug 07 Yes No	1	1	small nodule on the back behind dorsal.

Table Figure 1

 Figure

Estudos e Projetos (FINEP), Ministério da Ciência e Tecnologia (MCT), Programa de Recursos Humanos da ANP para o setor de petróleo e gás -PRH-ANP/MCT. Logistical support was provided by the Instituto Oceanográfico da Universidade de São Paulo (Cananéia research station) and the Núcleo Pró-Ação de Guaraqueçaba da Pontifícia Universidade Católica do Paraná. Ednilson da Silva and Eduardo Pacífico helped in fieldwork surveys. The work of M-F Van Bressem was funded by the WDCS and CSI. We kindly thank two anonymous reviewers for their constructing comments on the manuscript.

Taborda, P.R., Taborda, V.A., McGinnins, M.R., 1999. Lacazia loboi gen. Nov., comb., the etiologic agent of Lobomycosis. Journal of Clinical Microbiology, 37, 2031-2033. Tommasi, L.R., 1985 Projeto Valo Grande. Boletim do Instituto Oceanográfico, S. Paulo 3: 27-31.