Indi an Journal of Experimental Bio logy Vol. 39, May 2001, pp. 453-458

Acute and subchronic toxicity of aflatoxin B 1 to rohu, Labeo rohita (Hamilton)

P K Sahoo, S C Mukherjee, S K Nayak & S Dey

Aquatic Animal Health Division, Central Institute o f Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 75 1 002, India

Fax : 91-0674-465407. Email : cifa@ori.nic.in

Received 13 May /999; revised 12 January 200 I

Pathological alterations in various organs of rohu (L.rohita) fingerlings following acute (0,7.50, 11 .25 and 13.75 mg/kg body weight) and subchronic (0, 1.25 and 2.50 mg/kg body weight) single ip aflatoxin B 1 exposure for I 0 and 90 days, respectively, were investigated. Mortality (dose-dependent) was marked only during acute toxicosis. The changes observed in various organs were dose and time dependent. The acute dose groups revealed toxic changes viz., necrotic and vascular changes in li ver and gi ll lamellae; meningitis, congestion in brain, degeneration and infl ammatory reaction in heart along with degenerative to necrotic changes in kidney tubules and sloughing of the intesti nal mucosa. During subchronic exposure to this tox in, preneoplastic lesions in liver along with changes in spleen, intestine, gi ll and pancreas were recorded. With low doses of aflatoxin, the fish did not reveal any mortality or external signs other than catchexia and increased pigmentation on scales. In composite culture practice of Indian major carps, this could be of economic significance.

Aflatoxins are a family of closely related heterocyclic compounds produced by some strains of the fungi Aspergillus flavus, A. parasiticus and A. nomius. Aflatoxins are best known for their hepatotoxic, cytotoxic, teratogenic, mutagenic, carcinogenic and immunosuppressive properties'. Aflatoxin 8 1 (AFB 1)

and related mycotoxins are of great concern in many tropical countries because they commonly contaminate grains and seeds used as feed ingredients, probably due to their ubiquitous nature and favorable prevai ling climate2

• Aflatoxins have been found in feed stuffs at levels exceeding 2 mg/kg, which greatly exceeds the maximum level of 20 Jlg/kg allowed in commercial feedstuffs by the U. S Food and Drug Administration3

. Although the effects of aflatoxicosis in livestock are well documented and usually indentifiable3

, much less is known about these effects in fish in general and warm water carps in particular. The toxicity of aflatoxins has been documented in different species of fi sh, such as salmonids, channel catfish, common carp, tilapia and channa2

.4- I2 except

in Indian major carps.

Aflatoxins are highly toxic to rainbow trout 13• The

10-day oral LD50 of AFB 1 for rainbow trout is 0.5 mglkg body weight; whereas I 0-day ip LD50 values of AFB 1 for rainbow trout, channel catfish and rohu are 0.81, 11.5 and 12.59 mg/kg of body weight, respectively' 0

· '4

· '5

• The prolong feeding of AFB 1 at a concentration as low as 0.4 mg/kg of diet caused liver neoplasms, hepatic necrosis, and degenerative changes in pancreatic ti ssues and kidneys in rainbow trout6.

Although, rohu is one of the most important cultivable freshwater species of tropical countries, the effects of aflatoxins in this species is still to be explored. The present study has been undertaken with a view to provide information on the pathology of aflatoxicosis in rohu. The objectives are to evaluate the effects of single ip exposure at graded concentrations of AFB 1 for short and prolonged period of observations at the tissue level.

Materials and Methods Experimental conditions-Fourteen 500 I capacity

PVC-lined plastic aquaria equipped with continuous air supply were stocked with 10 rohu fingerlings in each. The basic physico-chemical water parameters viz., dissolved oxygen, pH, alkalinity and total hardness were measured systematically to maintain its optimal level. A commercial pelleted feed (aflatoxin­free) of rohu fingerlings was fed to fish @ 5% of their body weight, once daily through out the experimental period . The water was changed along with waste feed and faecal materials every 24 hr. The water temperature during the experiment was 25°-30°C. The fish were randomly divided into 7 groups for conducting acute and subchronic trials. Each group had 20 fingerlings (weight 35.32 ±4.14 g) maintained in two aquaria.

Aflatoxin preparation and administration schedule-Aflatoxin B 1 (Sigma, USA) was handled with all precautionary measures using necessary protective equipments6

. The aflatoxin was dissolved

454 INDIAN J EXP BIOL, MAY 2001

in chloroform as per the requirement of each group and peanut oil was added to that in required quantity so that each 20 g fish would receive 0.05ml of peanut oil containing needed quantity of aflatoxin. After adding oil, the chloroform was allowed to evaporate. Different levels for acute and subchronic trials were selected based on the following : (a) LD50 value of rohu fingerlings to AFB 1

15; (b) the age and weight

influence of the organism to be tested 6•16

; and (c) the possible rapid metabolism of tropical fi sh to AFB 1

16•

The fish were fed with pe lleted diet for 15 days and then fasted for 24 hr before administration of AFB 1•

All fish were anaesthetized with MS 222 (Sandoz) before injection. The fi sh were injected ip with 7.50, 11.25 and 13.75 mg of AFB/kg of body weight for acu te trial, and 1.25 and 2.50 mg of AFB /kg of body weight for subchroni c trial. The control groups fi sh of both the trials received only peanut oil injecti on without AFB 1• The acute and subchronic trial s were conducted for I 0 and 90 days, respectively.

Mortality and pathology-Per cent mortality in both the trials were recorded. Liver, kidney, spleen, heart, stomach, intestine, pancreas, brain and gill tissues of dead or moribund fish of acute trial; and randomly selected fish (n=5) from subchronic trial during or at the end of the experimental period were visually examined for gross lesions and then removed and placed in 10 % neutral buffered formalin for histological examination. The behavioural changes during the tri als were also recorded. The liver tissues of two moribund fish fro m each acute trial dose groups, and two randomly selected fish of subchronic trial as well as control groups were collected for semithin sectioning immediately after anaesthetization with MS 222. The tissues were sliced, minced ( I mm3

) in chilled fixative (3% glutaraldehyde in phosphate buffer) and fixed in fresh fixative solution for 24 hr at 4°C. They were washed in 3 changes of phosphate buffer solution for 30 min at 4°C, dehydrated in graded series of acetone and infiltrated in araldite resin (CY -21 2, Polysciences Inc, Warrington). Semithin sections (1-2 Jlm) from randomly selected blocks of each liver were cut and stai ned with toluidene blue for 30 S for further light microscopy.

Results Behaviour and external changes-The aflatoxin­

treated fish of acute toxic ity trial reduced their feed intake, showed sluggish movement, lost the equilibrium and had rapid opercular movement 48 hr

of injection, whereas the fi sh of ubchronic tOX ICity trial became cachectic and the scales got darkened.

Mortality and gross pathological changes- In acute dose groups, the mortaiity started after 48 hr of injection. The% mortal ity was do e- related i.e. 20, 30 and 60 % mortalities were marked in 7.50, 11.25 and 13 .75 mg of AFB 1/kg of fish grou ps, respec ti vely. However, there was no mortality in subchron ic as well as control groups till the end of the experiment.

Gross pathological signs- In the acute toxicity trials, some of the moribund fish of 11.25 and 13.75 mg AFB 1 treatmen ts showed haemorrhages in the gills and congest ion at the base of the pectoral fin. On necropsy, the fish revealed hepatomegaly with subcapsular focal congestion, pale ye llow li ver in few cases, cerebral congestion, haemorrhages in the cranial cav ity and serosanguinous fluid in the abdominal cavity in few fish . The lumen of the intestine was filled with whitish mucoid exudates. Mild peritoneal adhesion of the abdominal organs were also marked in some of the fish. There were bloody areas in the livers and abdominal fat.

On the other hand, the fi sh of subchronic toxicity trials revealed pale ye llow, mott led li ver only on necropsy. There was no gross les ion in the fish of control groups o f both the tri als.

Histopathological changes-The lesions observed during acute toxici ty were dose- related and were mostly seen in liver, kidneys, gill s, brain and heart. In liver, the hepatocy tes revealed swelling and individualization to massive necrosis (Fig. I). There was presence of eosinophilic homogenous mass along with few blood cells in the lumen o f hepatic vessel s formin g emboli. Perivascular edema around portal area was marked (Fig . 2). On semithin sections, the cytoplasm of swollen hepatocytes was studded with residual bodies (Fig. 3). The brain revealed generalized congestion and dilatation of meningeal vessels along with infiltration of mononuclear cells (Fig. 4). There was complete olughing of mucosa into lumen of the intestine in most of the cases along with necrotic ti ssue debris (Fig. 5) in comparison to normal mucosa in control fish (Fig. 6). Arteriolar sclerosis and depletion of lymphoid cells were prominent in the spleen (Fig. 7). Disorganization and wavy arrangement of the myofibrils coupled with moderate to marked lymphoid infiltration in myocardium along with rounding of nuclei of myofibrils were pronounced in heart (Fig. 8) in comparison to control which appeared normal (Fig. 9). The gill lamellae showed co mplete necrosis (Fig. I 0)

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Fi g. 1- lndividualization and mass ive necrosis of hepatocytes in liver of acute AFB 1 tox ici ty (H & E X 400); Fig. 2-Perivascular

edema (arrow) along with presence of blood cells in lumen of portal vessel of the li ver of acute AFB 1 tox ic ity (I-1 & E X 200):

Fig. 3-Swollen hepatocytes with packed up residual bodies (arrow) in the cytoplasm -Acute AFB 1 toxici ty (Toluidene blue X 400);

Fig. 4-Generali zed congestion (arrow) of cerebral vessels, dilatation (arrow head) of meningeal vessels and infiltration of mononuclear

ce ll s in the meninges of acute toxic ity ( I-1 & E X 400); Fig . 5- Intestinal lumen of acutely tox ic fi sh fill ed with sloughed up mucosa l

ce ll s and necrotic debris (H & EX 200), Fig. 6 - Normallooking intes tinal mucosa (M) in control fi sh (H & EX 400)

456 INDIAN J EXP BIOL, MAY 2001

and rupture of lamellar tips causing haemorrhages, whereas the gill of control fish had normal filaments and lamellae (Fig. 11 ). The renal tubular epithelia of the kidney revealed necrotic changes and sloughing into lumen.

Alternately, fish of lower dose group in subchronic toxicity trial s did not reveal any marked lesions in any of the organs except liver. Few of the fish of both the subchronic toxicity groups had preneoplastic nodules (40% of the fish examined) to hepatocellular adenoma (10%) in the li ver (Fig. 12). The blood vessels surounding the nodules were dilated. Few of the venules were occluded (Fig. 13). The pancreatic blood vessels were congested along with presence of homogenous mass inside and the acini revealed vacuolative degeneration to necrotic changes. The lamellar hyperplasia and fu sion in the gi ll s were predominant. Submucosal mononuclear reaction in the intestine and lymphocytic depletion along with presence of pigment­laden macrophages in the spleen was also evident.

Discussion In this study, the anorexic change observed in the

fishes used in acute trial may have occurred due to toxicosis. Si mi lar results have also been reported in case of channel catfish 10

• The fi sh of subchronic toxicity trial became cachectic with increased scale pigmentation which have not been observed by earlier workers in any fish species2

·11

. On the other hand , the channel catfi sh and trout exposed to chronic aflatoxin 8 1 remained healthy during the experiment2

'17

, even after being affected with hepatic carcinoma18

. The mortality observed during acute toxicity trial was dose-related and rohu appeared to be more resi stant to AFB, than rainbow trout as indicated by LD50 value'~·' 5 .

Furthermore,the gross lesions obta ined in necropsy from fish of acute trial may be clear indication of toxicosis. Similarly, the acutely toxic channel catfish revealed bloody areas in liver, intestine, stomach. abdominal fat and gonads along with reddish fluid in body cavity' 0

.

Fig. 7-Depletion of lymphoid cells (arrow) in the spleen of acutely intoxicated fish (H & EX 400); Fig. 8-Pericardium and myocardium of control fish showing almost normal appearance of myofibrils (H & EX 200); Fig. 9-Marked lymphocytic infi ltration along with wavy appearance of muscle bundle in the myocardium- Acute AFB 1 toxicity (H & EX 200), Fig. !0-Complete lamell ar necros is in the gill- Acute AFB 1 toxicity (H & EX 200)

SAHOO et a/.: AFLATOXIN 8 1 TOXICITY TO LAB£0 ROHITA 457

Fig. 11 - Normal look ing gill filament s (F) and lamellae (L) of control fi sh (H & E X 400); Fig. 12-Hepatocellular adenoma (A) in the liver during subchronic AFB 1 toxicity (H & E X 400 ), Fig. 13-Semithin section of liver revealing residual bodies studded hepatocytes around the preneoplastic nodu le (N) (Toluidene blue X 400)

On histology, the marked congestion in most of the organs during acute trial are clear indication of toxic effect. The typical changes in the hepatic blood vessels viz .. embolous mass inside lumen, periportal haemorrhages with occluded lumen during acute and

subchronic toxicity indicated the effect of AFB 1 on the endothelial cells of vascular system. Occlusion of the vascular lumen due to aflatoxicosis is not uncommon and has already been reported in cattle 19

.

Epithelial cells in rohu appeared to be highly sensitive to the toxic effects of AFB 1 and most of the lesions were noticed in this cell type. Similar results were also observed in channel catfish 10

. The necrotic changes in liver and intestinal epithelial sloughing of rohu in· case of acute aflatoxicosis marked in our case have also been reported in channel catfish 10

. The changes observed in heart and spleen during acute trial have also been described recently in Catla during acute AFB 1 toxicit/0

• The severity of damage to liver, gill, kidney and heart tissue along with other organs may have resulted in mortality in rohu during acute toxicity .

The development of preneoplastic nodule in li ver during subchronic aflatoxin toxicity in rohu has been described earlier 17

• The clear indication of early development of neoplastic lesions in liver of rohu should serve as a model animal to screen environmental carcinogens. The changes observed in gill and pancreas of rohu during subchronic exposure were more or less similar to that of aflatoxicosis in rainbow trout21

•

Results of the present study indicated that rohu ( L. roll ita) is susceptible to aflatoxicosis and even a single dose of as low as 1.25 mg/kg of body weight could produce significant lesions in the body ti ssues. The presence of this level of AFB 1 toxin in fish feed may be found quite naturally in tropical countries. Although, rohu is comparatively less susceptible than trout and salmonids , due attention should be paid to avoid feed contamination by Aspergillus molds .The typical histopathological changes observed in this study should be regarded as diagnostic aid for aflatoxicosis in fish.

Acknowledgement This work is supported by CSIR and ICAR, New

Delhi.

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Aquaculture, 127 ( 1994) 49. 3 Allocroft R, Atlatoxicosis in farm animals. in Ajlaroxin;

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458 INDIAN J EXP BIOL, MAY 200 1

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8

9

Sinnhuber R 0 & Wales H. Anatoxin Bl . Hepatocarcinigenecity in rainbow trout embryos, Fed Proc, 33 ( 1974) 247. Svobodova Z & Piskac A,The effect of feed with a low content of anatoxin B 1 on the health condi ti on of carp ( Cyprinus cwpio L.). Zivocisna- UVTJZ, 25 ( 1980) 809.

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18 Wood E M & Larson C P, Hepatic carcinoma in rainbow trout, Arch Pat hoi, 7 I ( I 96 I) 471.

I 9 New berne PM . Biological activ ity of the aflatoxins in domestic and laboratory animals. in Trout hepatoma research conference papers edited by J E Hal ver and I A Mitchell (Bureau of sport fi sheries and wildlife, Washington) 1967, 130.

20 Murjani G & Mukherjee S C, Acute afla tox icosis in Cat/a cat/a: An experimen tal study, J Aq:w. 6 ( 1998) 8 1.

2 1 Ashley L M, Hi stopathology of rainbo\\ trout atlatoxicosis. in Troll/ hepatoma research conference papers edi ted by J E Halver and I A Mitchell (Bureau o port fi sheri es and wildlife, Washington) 1967, 103.