Prepared by Judith Handlinger

1. Systematic Fish Pathology Part 9. Pathology of the digestive system II. The liver, pancreas, peritoneum & swim bladder Section B: Toxic insults & neoplasia. Prepared by Judith Handlinger With the support of The Fish Health Unit, Animal Health Laboratory, Department Of Primary Industries, Parks, Water and Environment, Tasmania, for Animal Health Australia’s Australian Animal Pathology Standards program(AAPSP)

2. Introduction to Section B. For acknowledgements of funding and photographs see Module 9, Section A. References quoted are listed at the end.

3. Course Outline A. Systematic Fish Pathology 1.Consider the Fish: An evolutionary perspective on comparative anatomy and physiology 2. Pathology of the kidney I – interstitial tissue Part A 3. Pathology of the kidney II – interstitial tissue Part B 4. Pathology of the kidney III – the nephron 5. Pathophysiology of the spleen 6. Fish haematology 7. Fish immunology – evolutionary & practical aspects 8. Pathology of the digestive system I – the oesophagus, stomach, & intestines. 9. Pathology of digestive system II – the liver and pancreas, swim bladder, peritoneum. (Thismodule - split into 4 sections): Section A: General aspects Section B (this presentation): Toxin related pathology and neoplasia. Section C: Pathology of microbial infections (viruses, bacteria & fungi) Section D: Parasitic diseases (protozoa & similar organism, metazoans). 10. Pathology of fish skin 11. Pathology and diseases of circulatory / respiratory system – heart, gills and vessels 12. Pathology of the musculoskeletal system and nervous systems 13. Pathology of gonads and fry

4. Toxin related pathology of liver Section A (general aspects) outlined how widespread but non-uniform liver vacuolation, particularly focal changes, are likely to be pathological rather than physiological or species variations. These are most likely due to hepatotoxins though changes secondary to circulatory failure also occur. This section examines examples of: Acute hepatic necrosis - phenol toxicity Nitrite toxicity Copper toxicity Jelly fish toxin Algal toxins - e.gNodularia; Mycotoxins & discusses the algal related net-pen disease

5. Example 1: Acute toxic hepatopathywith marked zonal necrosis is relatively rare in fish but it is mostly the exposure patterns rather than the pathology or pathogenesis that differ from mammals. This case followed exposure to phenols, due to human error (hopefully rare). There was no indication of anoxic effects, though this fish did show some congestion & pallor of the heart wall. 082143-5

6. Acute toxic hepatopathy continued: Cohort of the above, showing more extensive hepatic necrosis. This fish did not show overt cardiac lesions. 082143-3

7. Example 2: Zone-related liver vacuolation of this small salmon is also due to toxicity – but is probably a secondary effect. The fish had a 24-hour exposure to a tank experiencing on-going mortalities and later found to have high nitrite levels. The vacuolation / cloudy swelling is likely to be due to anoxia resulting from methaemoglobinaemia. 001751-2

8. Nitrite toxicity continued: Similar findings were still present in this Atlantic salmon subjected to the high nitrite tank for 37 days. There was also erythrocyte vacuolation. Erythrophagocytosis in the spleen was much more advanced than the acute case above (see blood & spleen presentations). 001743-3

9. Example 3:Copper toxicity - overview • Copper is as essential element for at least 30 enzymes & glycoproteins and potentially affects a large number of enzyme systems. Circumstances of exposure and adaptation determine which effects reach lethal levels first. It is capable of causing liver degeneration and necrosis in fish, though this is not always seen. • Water (not diet) is often the source for copper toxicity in fish, particularly for acute exposure. Tasmanian Macquarie Harbour cases are typical: high copper load from mine tailings washed down with acid flood waters adding to naturally moderately high levels (i.e. mines are in areas of high mineral content). [There has since been considerable effort to rectify problem tailing areas.] • Copper availability is significantly reduced by organics in water, such as the tannins found in many Tasmanian streams. Copper toxicity is modified by temperature, pH, prior exposure & mucus. • Absorption via the gills increases the opportunity for inactivation of gill enzymes, commonly resulting in failure of ion control being the first major metabolic effect of Cu toxicity. • Toxicity varies greatly between freshwater and seawater, and with water hardness: calcium has a major protective effect & ion control failure is also more rapidly fatal in freshwater: • 96 hour LD50 for fry is about 20 µg/l (ppb) in soft water (hardness 25 mg.L CaCO3), ~100 ppb at hardness of 150 mg/l; & in the order of 2-300 ppb in seawater. • Ionic stress is least at partial salinity that approximates blood sodium levels: some adapted Macquarie harbour salmonids survived 96 hour experimental exposure to tannin-rich water with 1200 ppb added copper, with most deaths at this level occurring after 48 hours, though some fish died earlier. Most survived 300 and 600 ppb. (Hardy-Smith & Handlinger, unpublished DPIPWE report.) • Liver lesions were seen with high exposure at partial salinity (natural & experimental exposure), mostly in rainbow trout. Lesions in other organs are usually minimal. (See also later power-points).

10. Copper toxicity & liver: clinically normal 3 k female rainbow trout (RT) with mild liver changes suspected to be related to copper laden plume from a contaminated river. Hepatocyte vacuolation is the major initial or mild liver change, and a zone-related pattern may be obvious. Mild vacuolation is likely to be reversible, but this is doubtful for the cells showing large confluent vesicles. L-836

11. Acute copper toxicity episode continued (a later submission, RT): Relatively mild lesions of multifocal necrosis associated with elevated gill copper levels. Ballooning of affected cells is characteristic of severe acute copper toxicity at partial salinity in this species (Handlinger & Hardy-Smith, Tas DPIPWE unpublished report). Cell swelling ispresumably due to mitochondrial disruption, as in other animals. L-830

12. Acute copper toxicity continued: More severe and clearly zone-related necrosis in another fish from the same case as previous slide. Note the sparing of portal areas. Generally only cell fragments remain in affected areas. 831

13. Acute copper toxicity continued: Another severely affected fish from the same episode, with slightly earlier lesions still showing the typical ballooned cells. Liver copper levels were not increased. Liver copper may gradually accumulate with age but much of the excess is excreted in bile. (Kamunde et al, 2001). High dietary copper may also cause initial gut lesions. 833

14. http://scyphozoans.tripod.com/ Jellyfish toxicity: The major effects of jellyfish exposure are likely to be local damage to gills and / or skin, but the toxins may also have systemic effects and anumber of jellyfish contain toxins capable of causing liver necrosis. In Tasmania this is mostly seen with Aurelia sp. “moon-jellies” (right), a species with very short tentacles with weak nematocysts (stings) and negligible impact on humans on impact. The toxin is excreted into the mucus that coats small prey. (See also Gills and Skin presentations.) Some jellies produce very extensive necrosis [ e.g. in Chile 2002, with jellyfish following algal blooms in an unusual summer (Pers comm, P. Bustos, Chile). Jellyfish hepatotoxicity is attributed to damage to P-450 enzymes, though other mechanisms may also contribute (reviewed Burnett et al, 1998). This is a typical single necrotic focus, in a small (post-smolt) Atlantic salmon exposed 2 days previously to Aurelia sp mucus. By this time only cell fragments are present within the lesion, but there is little reaction or healing. 841

15. Systemic effects from jelly-fish stings are likely to consist of direct effects on cells absorbing the toxin, and indirect effects via vascular reactions. Observed liver changes vary from focal congestion.... 842

16. 843 (Jellyfish – mild liver changes continued) ... to focal congestion accompanied by focal or irregular vacuolation....

17. 841 • (Jellyfish continued) .....to focal necrosis, generally in the vacuolated areas. • In most cases necrotic foci are small and scattered, but ...

18. Occasionally more extensive confluent areas of necrosis were seen during periods of extensive exposure to Aurelia sp. (Moon jellyfish). The time frame of exposure for this fish is not known. (It is possible that additional factors such as reovirus may have been present, though these lesions were associated with typical acute jelly-fish gill lesions). 844-s2

19. (Jellyfish continued) Liver lesions are not always severe. This moribund salmon showed gill lesions indicative of recent (1-2 days) exposure to jellyfish (almost certainly Aurelia sp.) Liver lesions (predominantly vacuolation) are extensive but mild: the moribund state is almost certainly due to gill & other circulatory lesions. 849

20. (Jellyfish continued.) Records show similar liver lesions after exposure to the bluebottle / Portuguese Man o' War (Physalia physalis - right), but these were rare. A: Apparently older necrotic focus, with reaction to the cell debris. B: The large droplets appear to contain haemoglobin – referable to haemorrhagic gills? A B Jellyfish image from http://en.wikipedia.org/wiki/Portuguese_Man_o%27_War 8BB-4

21. Algal toxicosis Another major source of liver toxins is blooms of algae & cyanobacteria – the plant toxins of the fish world. Toxins of some of the best-studied algae affect gill epithelium. Deaths also occur from anoxia as large blooms collapse. (Not all toxic blooms involve liver). Some toxins are already well-studied in other animals (see right) Others contain toxin in sufficient quantity that toxicity may occur when those algae are a minor component of a mixed bloom. This may prevent identification of which is the toxin-producing species. Algal toxicity case I: Liver necrosis from 6-hour trial exposure to Nodularia spumigena cyanobacteria, run after a major 1993 bloom in Orielton Lagoon (Jones et al, 1994) concerned local fish farmers. Both the organism (1x104ml-1) and extracted nodularin caused liver lesions & peritoneal petechiation in some fish. Findings were similar in trout from the Baltic Sea with major architectural disruption of liver 1-2 after exposure, and partial recovery by 4-8 days (Kankaanpää et al, 2002).

22. Algal or mycotoxin? – hepatocyte globules: Thiscase, in ponds following dirty water during very hot weather (up to 24 oC), was also suspected to be an algal toxicosis, though subsequent history suggested a mycotoxicosis & / or rancidity from feed left in the sun during this period was at least part of the problem. These may all show similar liver pathology. Note the hepatocyte hyaline droplets (A, B), & (in C) renal tubule degeneration. A B C K2 F4 070395

23. Globules case 2: Zone related vacuolation, plus similar globules in an 8 g neomale* salmon, occurring at 12 oC. This was also probably mainly feed related, though the fish also had minor gill and skin lesions suggesting poor water quality. The globule material in this and the preceding case was identified as early ceroid (See section A).. *Neomale : sex reversed by testosterone during early development in order to breed all females. 817

24. Why the suspicion of algae in the high temperature related ceroid case above? Both algal blooms & rancid food are potential consequences of heat-waves. As well as hyaline droplets & other cytoplasmic evidence of liver damage, some of these animals show nuclear changes that have been associated with algae & / or cyanobacteria in other areas. Cohort of the above (same submission): note double nuclei (blue arrow), & apparent nuclear vacuolation (white arrow), probably a pseudo-inclusion due to invagination of the nuclear membrane or an intranuclear glycogen droplet. K2 F6 070395

25. (continued) A later case from the same episode showing multiple hyaline droplets plus similar nuclear changes. By this time renal lesions were minimal. The above nuclear changes, plus marked megalocytosis, can be found with aflatoxins & other toxins (as for mammals), but in fish are also associated with microcytins. L-824 07/0456-1A

26. Net-Pen liver disease (NLD) of salmonids – North American West Coast. Net-pen liver disease is an economically significant salmonid syndrome seen since 1986 in Port Townsend Bay in Washington, USA and later in British Columbia, Canada. Kent, 1990 found summer cumulative mortality 65% in Atlantic salmon, 23% in small Chinook salmon, 13 % in rainbow x steelhead trout (13%), but large farmed chinook, sockeye salmon and Pacific herring, shiner perch and English sole from the area were unaffected. However the disease is seen in 16% mature returning wild chinook salmon. It is characterised initially by hydropic degeneration and necrosis if severe, later by hepatocyte megalocytosis, often with nuclear pseudo-inclusions as shown above and multinucleate forms. There is also evidence of tissue reactions such as perivascular cuffs and ceroid containing melanomacrophages. Mild lesions included individual cell necrosis & bile duct proliferation. Salmon returned to clean water showed liver regeneration and neoplasia. Epidemiology indicated a toxin sourced from the water (not food). Liquid chromatography studies found an inhibitor of protein phosphatase that was chromatographically indistinguishable from microcystin-LR(see next slide) in affected livers, and the syndrome could be reproduced with intraperitoneal injection of microcystin-LR. We have not see this syndrome in Tasmanian salmonids. We do sporadically see cases with mild lesions similar to the above. Such lesions can also be caused in fish by aflatoxins & pyrrolizidine alkaloids, neither of which occur naturally in the aquatic environment (i.e. the source is man-made feed). Pyrrolizidine alkaloid inclusion in fish feeds is unlikely. References: Kent, 1990, Kent et al, 1992; Stephen et al, 1993; Anderson et al, 1993.

27. Cyanobacteria and microcystins. Cyanobacteria (photosynthetic bacteria) inhabit almost every environment and account for ~ 20–30% of Earth's photosynthesis, including ~ half that of the open oceans (http://en.wikipedia.org/wiki/Cyanobacteria), so it is hardly surprising that toxic forms occur. Their toxins include neurotoxins, hepatotoxins (collectively termed microcystins [MC] after the first one isolated from Microcystis aeruginosa, though WHO separates microcystins and nodularins); skin irritants. More than 60 cyanobacterial hepatotoxins have been identified, containing uncommon non-protein amino acids with a common structural complex of 5 (in nodularins) or 7 (in microcystins) peptide linked amino acids. [These contain three ßamino acids (alanine, b-linked erythro-ßmethylaspartic acid, and linked glutamic acid), two variable L-amino acids, R1 and R2, and two unusual amino acids, N-methyldehydroalanine (Mdha) and 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyldeca-4,6-dienoic acid (Adda).]. The most studied common MC is microcystin-LR (MCLR) [structure = cyclo (D-Ala-L-Leu-D-MeAsp-L-Arg-Adda-D-Glu-Mdha], which is both hepatotoxic and carcinogenic. This has been found from multiple cyanobacteria, including M. aeruginosa, but is not the only toxin from these species & several may be produced simultaneously (Chorus I & Bartram J (ed.). 1999). Toxins are contained within the cyanobacteria until released by digestion or collapse of blooms. They are water soluble & unable to penetrate lipid membranes so they affect cells with active uptake. They are also very stable (even after boiling), but slowly degrade with UV light. Acute hepatic necrosis, but not megalocytosis, is ascribed to Microcystis aeruginosa & MCLR in mammals, but the exposure time is generally very short. [I.e. NLD is a more chronic disease.]

28. Microcystins & fish Fish generally are not affected by healthy cyanobacteria blooms, but are susceptible if force fed & from dying blooms. Fish appear as susceptibility to IP injection of disrupted Microcystis aeruginosa as mice: • [Phillips et al, 1985 found 100% deaths 12-36 hr after IP injection of 30-40 mg dry wt. of disrupted cells /kg body wet wt (=LD50 mice this route), with liver necrosis, haemorrhage and fat necrosis round pancreas.] Fish appear less susceptible than mice to extracted microcystin-LR , though this varies with species: • Kotak et al, 1996, found 100% mortality within 10 hours in rainbow trout after IP injection of 1000 µg/kg of microcystin-LR but no mortality to 26 hr with 400 µg/kg (8 times the mouse LD50). Kidney tubule damage also. The slower uptake from slower liver perfusion & low body temperature suspected to increase survival. . • Rabergh et al (1992) found similar survival in Carp (7-day LD50 between 300-550 µg/kg); • Goldfish found to be 30 times less susceptible than mice. (see Chorus & Bartram, 1999, p48). • Most Atlantic salmon survived multiple IP injections of 555 µg/kg, 3 days apart (to 36 days). Anderson et al, 1993. Short-term pathology: tan livers, widespread hepatocellular swelling, followed by multifocal “centrolobular, midzonal or bridging coagulative to liquefactive necrosis, haemorrhage rare”. Longer term findings included megalocytosis & other nuclear abnormalities as seen in Netpen Liver Disease (NLD). [It is likely that similar nuclear abnormalities can also be caused by other insults – see next case.] Liver regeneration seems to vary with species – e.g. basophilic regenerating cells seen as early as 2-3 days in estuarine hardyhead catfish & gulf killifish with hepatic necrosis by 6 hr after IP MC-LR at 45−300 μg/kg, (Fournie & Courtney 2002). Gill damage wasreported during cyanobacteria blooms (pH effect?) – this may have aided toxin absorption.

29. Bioaccumulation of microcystins Cyanobacterial toxins, like toxins from microscopic algae such as diatoms and dinoflagellate, are bio-accumulated by invertebrates. (The source of various forms of so-called Shellfish Poisoning). As invertebrates are a natural fish food, this presents a likely source of cyanobacterial toxins for NLD. Thus keep caged fish well fed and well adapted is the best advice for NLD control. Nodularin (the other group of cyanobacteria toxins) can also bioaccumulate (e.g. in trout liver - Kankaanpää et al, 2002). However as nodularin toxicity trials are usually of short duration it is not known if similar long-term changes (such as nuclear abnormalities) can be inducted. (Similar nuclear abnormalities from other insults are explored further below.)

30. Megalocytosis continued: 9 g Atlantic salmon fry (freshwater, December) with severe bacterial gill disease and marked gill fusion. The scattered fat-type vacuoles almost certainly reflect anoxia. Note the lack of indistinct (glycogen) vacuolation in other cells. Bacterial gill disease is indicative of recent poor water quality / hygiene. A fungal or bacterial toxin thought more likely than microcystins as causing this megalocytosis?. (Also not an acute lesion) 816

31. Megalocytosis continued: The strange case of Lovettia sealii (Tasmanian whitebait), included here for comparison with other megalocytoses. Liver from 4 whitebait. All fish in the population showed bizarre hepatic megalocytosis. Note the pancreas and gut are not affected Fish T1 Fish M2 Consider possible causes of this pathology? T2 Fish M1 Fish T2 Above fish photo from http://www.ifs.tas.gov.au/ifs/IFSDatabaseManager/SpeciesDatabase/tasmanian-whitebait

32. Lovettia sealii megalocytosis continued. • Note pattern of chromatin clumping in some areas. • Electron microscopy was negative for viruses. F1

33. Lovettia sealii hepatic megalocytosis continued. No megalocytosis in other tissues. Other changes included pericardial oedema (left), and a very high level of chloride cell activity in the gill (right, pale cells at white arrow), indicating osmotic regulation effort. M1 Ventricle Liver Auricle F1 Ventricle M2

34. M2 All fish were maturing, and by inference migrating upstream to spawn. Gill & pericardial changes suggest they may not be fully adapted to this osmotic challenge. The one opportunity for examination during brief unpromising study of aquaculture potential suggested this change in all migrating adults. Lovettia sealii hepatic megalocytosis – pathology? Ovary L. sealii is small (~60 mm), endemic to Tasmania, & previously supported a commercial fishery during migration from estuaries to streams for spawning. Most of their life is spent at sea, the transparent fish migrating upstream in spring, then spawn, become almost black & then die. How would you explain the pathology? Is it pathology? Uniformity & life-cycle suggests this could be a programmed change, possibly sacrificing all energy stores for gametes? Any similar findings or studies of single-spawn fish life-cycles would be of interest. Liver Reference: http://www.ifs.tas.gov.au/ifs/IFSDatabaseManager/SpeciesDatabase/tasmanian-whitebait

35. Neoplasia of liver, pancreas (& swim bladder?) Neoplasia has been included with this section as there are many known episodes of toxin-related hepatic neoplasia. Neoplasia of the pancreas is more typically sporadic in nature. I have found no examples of swim-bladder tumours in our records.

36. Toxin related hepatic neoplasia In addition to sporadic tumours of unknown cause, liver tumours have been related to many of the above toxins: both microcystin-LR and aflatoxin are known carcinogens, producing both hepatocyte and biliary tumours. [In the 1960s liver cancer in 90% of 3-year old hatchery rainbow trout was first tracked to aflatoxin from Aspergillus flavus on feed grain – knowledge that has saved countless human lives (Harshbarger, 2004).] Fish neoplasia (liver & / or skin) may also involve xenobiotic toxins (man-made, un-natural), particularly in bottom-dwelling fish such as flounder. Tumour incidence up to 33% has been seen from heavily polluted sites, (e.g. brown bullhead, Black River, Ohio) with implication of polynuclear aromatic hydrocarbons (PAHs), which they metabolise to carcinogenic forms. Transformation can also be protective, for example that by nicotinamide adenine dinucleotide phosphate (NADPH), that detoxifies via one-electron and two-electron biotransformation and conjugation. [In female flounder NADPH is preferentially used for the production of the yolk precursor protein vitellogenin (and therefore less is available for detoxification), resulting in a 3-fold increase of tumours in female compared to male flounder from highly polluted sites.] Despite the above cases, not all oil spills carry a high risk of tumour development: this is much more likely for refined oil, with high levels of PAHs & benzene than the simpler crude oils. Pollution related fish tumour epizootics were initially reported during the 1970-1980s. They appear less common now with efforts to reduce industrial pollution and the banning of the carcinogenic & persistent polychlorinated biphenyls (PCBs) from all but a few well contained applications. References: Baumann & Harshbarger, 1985; Baumann 1998; Koehler & Van Noorden, 2003; Harshbarger, 2004

37. More examples of hepatomas / cholangiomas. Hepatoma Cholangioma hyperplasia/fibrosis. White-sucker Oakville CK, USA 900507-7 cholangioma 900507-7 [Archive slides, source unknown - labeled as per original slides.]

38. Pollution related changes, Lake Bonney, South Australia. By 1970s Lake Bonney was regarded as heavily polluted by discharges from pulp & paper mills (e g the cellulose mill which closed in 1998). 1 Dioxins are perhaps the most concerning pollutants of those commonly found with pulp mill effluents, though a variety of pollutants were present. 1 The related Yarra Pigmy Perch has now disappeared from this lake2 , blamed on pollution as well as habitat loss and predation by introduced species. Archive slide of biliary metaplasia in a Southern Pygmy Perch (Nannoperca australis) from Lake Bonny, suspected to be pollution related. [Slide from archives or Australian Fish Diseases laboratory, original source unknown.] 1. http://www.epa.sa.gov.au/xstd_files/Water/Report/lake_bonney.pdf 2. http://www.environment.gov.au/biodiversity/threatened/publications/recovery/pubs/yarra-pygmy-perch.rtf

39. Virus related neoplasia & liver Virus infections are covered in the next section (Part C), but this is a reminder that retrovirus has been associated with or suspected as a cause or contributor to lymphoid neoplasia, and that liver is a frequent site of secondary tumors. I.e this is not a tumour of hepatocytes, but is nevertheless a relatively common finding in affected populations.

40. Nodular pattern of secondary lymphoid tumour in Atlantic salmon liver. Note the swollen kidney – the primary site.

41. Diffuse pattern of secondary lymphoid tumour in salmon liver.

42. Right: diffuse pattern of secondary lymphoid tumour in salmon liver, compared to the more uniform distribution through kidney interstitia (left). No pathology other than separation of hepatic cords is evident.

43. Another example, with secondary invasion of the pancreas. Note at higher magnification the largely uniform lymphoid cell population separating cells of both fat and acinar tissues.

44. Liver from the same fish as previous slide. Here the invading lymphoid cells include an intravascular mass, with apparent infarction (or at least reduced blood flow), and degeneration of adjacent hepatocytes. Thus tumours of this nature are often benign (though an economic loss through down-grading), but cause occasional acute pathology such as vascular blockage.

45. Neoplasia of the pancreas Pancreatic tumours are relatively rare, generally without known precipitating causes (i.e. typical rare apparently random pattern). Our records show 1 pancreatic acinar cell tumour and 1 presumptive Islet cell tumour in salmonids, from > 2 decades of surveillance.

46. Pancreatic neoplasia case 1 – pancreatic acinar cell tumour in a 3 kg Atlantic salmon. Note focal degeneration (lower right) .. And increased eosinophilic granular cells (ECG) at the margin of the tumour. Normal pancreas

47. Pancreatic neoplasia case 2: Atlantic salmon post-smolt with pancreatic Island cell tumour. The fish was thin, presenting as an apparently unadapted “pinhead”, with the group as a whole showing evidence of recent jelly-fish stress. Note the similarity of tumour cells to normal Islet cells.

48. Islet cell tumour continued, showing the extent of the tumour and nature of neoplastic cells.

49. Islet cell tumour continued: note the tendency to form Islet cell like masses.

50. References – GIT II part B Re copper toxicity: • Kammunde, C N, Grosell, M, Lott, N A, & Wood, C M. 2001. Copper metabolism and gut morphology in rainbow trout (Oncorhynchus mykiss) during chronic sublethal dietary copper exposure. Can. J. Fish. Aquat. Sci. 58: 293-305. Re jellyfish toxins: • Burnett, J.W, D. Weinrish, J.A. Willimson, P.J. Fenner, L.L. lutz and Bloom, D.A. 1998. Autonomic neurotoxicity of jellyfish and marine animal venoms.Clinical Autonomic Research 8:125-130. Re Nodularia toxicity • GJ Jones, SI Blackburn and NS Parker. 1994. A toxic bloom of NodulariaspumigenaMertens in Orielton Lagoon, Tasmania. Australian Journal of Marine and Freshwater Research 45(5): 787 - 800 • HarriKankaanpää, Pekka J. Vuorinen, VesaSipiä and MarjaKeinänen. 2002.Acute effects and bioaccumulation of nodularin in sea trout (Salmotrutta m. trutta L.) exposed orally to Nodulariaspumigena under laboratory conditions. Aquatic Toxicology 61(3-4): 155-168. Re Netpen liver disease • Kent, M. L. (1990). Netpen liver disease (NLD) of salmonid fishes reared in sea water: species susceptibility, recovery, and probable cause. Dis. aquat. Org. 8: 21-28 • Kent, M. L., Andersen, R. J., Dawe, S. C., Williams, D. E.. Le Blanc, M , Taylor, F. J. R. (1992). MicrocystinLR: possibly the cause of netpen liver disease of seawater pen-reared salmon. Am. Fish Soc. Fish Health Sec. Newslett. 20(2): 9-12.