Cytotoxicity, morphological and ultrastructural effects induced by the neonicotinoid pesticide, imidacloprid, using a rat Leydig cell line (LC-540).

Imidacloprid is a systemic neonicotinoid insecticide widely used to combat agricultural pests and flea infestations in dogs and cats. Despite its low toxicity to mammals, imidacloprid is reported to cause male reproductive toxicity. This study evaluated the cytotoxic effects of 75-800 µM imidacloprid on a rat Leydig cell line (LC-540). The effect of exposure to 300, 400, and 500 µM imidacloprid on selected cytoskeletal proteins, mitochondrial morphology, lysosomal acidity, and ultrastructure were investigated. Cell viability was markedly reduced after 48 and 72 h of exposure to higher imidacloprid concentrations. The immunocytochemical analysis revealed that the cytoskeletal filaments exhibited disorganization, disruption, and perinuclear aggregation in treated LC-540 cells. Ultrastructurally, cytoplasmic vacuoles, autophagic vacuoles, lysosomes, and mitochondrial damage were detected. Changes in the mitochondrial morphology and lysosomes induced by imidacloprid were confirmed. The cytotoxicity of imidacloprid observed in LC-540 cells might be due to its mitochondrial damage and cytoskeletal protein disruption.

What is in a name? Scientific name changes of potentially poisonous plants and fungi in South Africa.

Changes over the past five decades in the scientific names of some potentially poisonous plants and toxigenic fungi in South Africa are briefly reviewed. Some of the reasons why taxonomists change names are highlighted. In recent years, DNA sequencing data have contributed considerably towards establishing phylogenetic relationships among plants, often resulting in changes in generic circumscription and, consequently, the names of species. Philosophical differences between the phylogenetic and the evolutionary schools of plant classification are briefly explained as these may manifest as different classifications for the same group of plants. Although choice of classification remains the prerogative of the end-user of plant names, in this review, the classifications for plants currently adopted by the South African National Biodiversity Institute (SANBI) in its online database, Plants of Southern Africa (POSA), were followed. Noteworthy generic changes include Pachystigma to Vangueria, Homeria to Moraea, and Urginia to Drimia. Following much controversy, the species native to southern Africa that were formerly treated as Acacia are now classified in either Vachellia or Senegalia, with the genus name Acacia being retained for the mainly Australian members of the group, the latter commonly known as wattles. Former southern African members of Acacia implicated in poisoning include Vachellia erioloba(camel thorn), Vachellia sieberiana var. woodii (paperbark thorn), and Senegalia caffra (common hook thorn).

Putative neuromycotoxicoses in an adult male following ingestion of moldy walnuts.

A tremorgenic syndrome occurs in dogs following ingestion of moldy walnuts, and Penicillium crustosum has been implicated as the offending fungus. This is the first report of suspected moldy walnut toxicosis in man. An adult male ingested approximately eight fungal-infected walnut kernels and after 12 h experienced tremors, generalized pain, incoordination, confusion, anxiety, and diaphoresis. Following symptomatic and supportive treatment at a local hospital, the man made an uneventful recovery. A batch of walnuts (approximately 20) was submitted for mycological culturing and identification as well as for mycotoxin analysis. Penicillium crustosum Thom was the most abundant fungus present on walnut samples, often occurring as monocultures on isolation plates. Identifications were confirmed with DNA sequences. The kernels and shells of the moldy walnuts as well as P. crustosum isolates plated on yeast extract sucrose (YES) and Czapek yeast autolysate (CYA) agars and incubated in the dark at 25 °C for 7 days were screened for tremorgenic mycotoxins and known P. crustosum metabolites using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. A relatively low penitrem A concentration of only 1.9 ng/g was detected on the walnut kernels when compared to roquefortine C concentrations of 21.7 µg/g. A similar result was obtained from P. crustosum isolates cultured on YES and CYA, with penitrem A concentrations much lower (0.6-6.4 µg per g mycelium/agar) compared to roquefortine C concentrations (172-1225 µg/g). The authors surmised that besides penitrem A, roquefortine C might also play an additive or synergistic role in intoxication of man.

Multimycotoxin analysis of South African Aspergillus clavatus isolates.

Aspergillus clavatus poisoning is a neuromycotoxicosis of ruminants that occurs sporadically across the world after ingestion of infected feedstuffs. Although various toxic metabolites are synthesized by the fungus, it is not clear which specific or group of mycotoxins induces the syndrome. A. clavatus isolates were deposited in the culture collection of the Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council during incidences of livestock poisoning (1988-2016). Six isolates were still viable and these plus three other South African isolates that were also previously deposited in the collection were positively identified as A. clavatus based on morphology and ß-tubulin sequence data. The cultures were screened for multiple mycotoxins using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Twelve A. clavatus metabolites were detected. The concentrations of the tremorgenic mycotoxins (i.e., tryptoquivaline A and its related metabolites deoxytryptoquivaline A and deoxynortryptoquivaline) were higher than patulin and cytochalasin E. Livestock owners should not feed A. clavatus-infected material to ruminants as all the South African A. clavatus isolates synthesized the same compounds when cultured under similar conditions.

Lead ingestion as a potential contributing factor to the decline in vulture populations in southern Africa.

Vulture populations in southern Africa have been on the decline for years, which unlike the Asian vulture crisis, has no one specific cause. Reasons attributable are deliberate and secondary poisonings, drowning, power line injuries, electrocutions, traditional medicine ("muti" trade) and calcium deficiencies. However, lead toxicity as a potential causal factor is hardly mentioned. The potential for lead toxicity needs to be considered as substantial game hunting occurs in the region with little regulatory control on bullet types. In this study, we determined the whole blood lead concentrations of captive and wild vulture populations in South Africa and Namibia (n=185). Results were compared to previous published ranges indicative of background exposure (<10µg/dL), non-toxic point exposure based upon the range established from captive birds and subclinical exposure. In general, whole blood lead concentrations were higher for wild African White-backed vultures (Gyps africanus)(AWBV) than Cape vultures (G. coprotheres)(CGV) at 15.54±12.63µg/dL vs 12.53±8.88µg/dL (non-significantly different), while in the Bearded vultures (Gypaetus barbatus) no indication of exposure was evident. Very similar exposures resulted irrespective of the birds being in captivity or under wild, free-roaming conditions. A proportion of wild birds did, however, appear to be exposed to another source of lead than purely environmental (±12% and 30.6% for AWBV and CGV respectively). One bird, which had a whole blood concentration of 100µg/dL, died soon after capture. To find the relationship between whole blood lead concentration and likely exposure factors, birds were compared by their rural/urban location, vicinity to mines and surrounding soil lead concentrations. With no relationship being present for the latter factors, we believe that this is evidence that the portion of southern African vultures being exposed to unknown source of lead, which we suggest arises from leaded ammunition remaining from hunting.

The pathology of acute Nolletia gariepina poisoning of cattle.

Toxicity in cattle by the shrub Nolletia gariepina was induced experimentally by intraruminal administration of 3 g/kg dried, milled plant material as a single dose. The animals had to be starved for 24 hours before dosing, as dosing on a full rumen did not induce any signs of toxicity during 5 days of observation and clinical pathology monitoring. Clinical signs were not specific and varied according to the duration (acute versus subacute) of the toxicological process. Clinical pathological parameters indicated renal and to a lesser extent hepatic damage, with raised serum concentrations of urea, creatinine, aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT). Increased urinary sodium and potassium concentration and GGT activity, as well as proteinuria, were evident. Histological and electron microscopic examinations revealed acute renal tubular epithelial cell degeneration and necrosis, especially of the proximal convoluted tubules. Mild hepatocellular degeneration was also noticeable.

A fluorescent investigation of subcellular damage in H9c2 cells caused by pavetamine, a novel polyamine.

Gousiekte, which can be translated literally as "quick disease", is one of the six most important plant toxicoses that affect livestock in South Africa. It is a plant-induced cardiomyopathy of domestic ruminants characterised by the sudden death of animals within a period of 4-8weeks after the initial ingestion of the toxic plant. The main ultrastructural change in sheep hearts is degradation of myofibres. In this study, fluorescent probes were used to investigate subcellular changes induced by pavetamine, the toxic compound that causes gousiekte, in H9c2 cells. The sarcoplasmic reticula (SR) and mitochondria showed abnormalities that were not present in the control cells. The lysosomes of treated cells were more abundant and enlarged than those of the control cells. There was increased activity of cytosolic hexosaminidase and acid phosphatase, indicating increased lysosomal membrane permeability. Lysosomes play an important role in both necrosis and apoptosis. The degradation of the myofibres may be a consequence of the increased lysosomal membrane permeability. Pavetamine was also found to cause alterations in the organisation of F-actin. F-actin in the nucleus is a transcription regulator and can therefore influence protein synthesis. Actin filament organisation also regulates the cardiac L-type Ca(2+) channels. Fluorescent staining demonstrated that pavetamine may damage a number of organelles, all of which can influence the proper functioning of the heart.

Damage to some contractile and cytoskeleton proteins of the sarcomere in rat neonatal cardiomyocytes after exposure to pavetamine.

Pavetamine, a cationic polyamine, is a cardiotoxin that affects ruminants. The animals die of heart failure after a period of four to eight weeks following ingestion of the plants that contain pavetamine. This immunofluorescent study was undertaken in rat neonatal cardiomyocytes (RNCM) to label some of the contractile and cytoskeleton proteins after exposure to pavetamine for 48 h. Myosin and titin were degraded in the RNCM treated with pavetamine and the morphology of alpha-actin was altered, when compared to the untreated cells, while those of beta-tubulin seemed to be unaffected. F-actin was degraded, or even absent, in some of the treated cells. On an ultrastructural level, the sarcomeres were disorganized or disengaged from the Z-lines. Thus, all three contractile proteins of the rat heart were affected by pavetamine treatment, as well as the F-actin of the cytoskeleton. It is possible that these proteins are being degraded by proteases like the calpains and/or cathepsins. The consequence of pavetamine exposure is literally a "broken heart".

Cytotoxicity and ultrastructural changes in H9c2(2-1) cells treated with pavetamine, a novel polyamine.

Intake of pavetamine, a novel polyamine, synthesized by certain rubiaceous plants, is the cause of gousiekte ("Quick disease") in ruminants. The disease is characterized by a latent period of 4-8 weeks, followed by heart failure. The aim of this study was to firstly investigate the cytotoxicity in H9c2(2-1) cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and LDH (lactate dehydrogenase) release assays. Maximum cell death occurred after pavetamine exposure of cells for 72h at a concentration of 200muM (55%+/-9.84), as measured by the MTT assay. LDH release was only observed after 72h exposure to pavetamine. Secondly, the ultrastructural changes induced by pavetamine in H9c2(2-1) cells were investigated. Changes in the mitochondria and sarcoplasmic reticula were observed. The nucleus was not affected during the first 48h exposure of cells to pavetamine and no chromatin condensation occurred. However, after 72h exposure to pavetamine, the nucleus became fragmented and membrane blebbing occurred. It was concluded that the ultimate cell death of H9c2(2-1) cells treated with pavetamine, was through necrosis and not apoptosis. Thirdly, the effect of pavetamine on the mitochondrial membrane potential (DeltaPsi) was evaluated by using the JC-1 (5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) and TMRM (tetramethylrhodamine methyl ester perchlorate) probes. Pavetamine treatment led to significant hyperpolarization of the mitochondrial membrane potential. Cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly, indicating that the MPTP (mitochondrial permeability transition pore) plays no role in the cytotoxicity of pavetamine.

Potential plant poisonings in dogs and cats in southern Africa.

Plant poisoning occurs less commonly in dogs and cats than in herbivorous livestock, but numerous cases have been documented worldwide, most of them caused by common and internationally widely cultivated ornamental garden and house plants. Few cases of poisoning of cats and dogs have been reported in southern Africa, but many of the plants that have caused poisoning in these species elsewhere are widely available in the subregion and are briefly reviewed in terms of toxic principles, toxicity, species affected, clinical signs, and prognosis. The list includes Melia azedarach (syringa), Brunfelsia spp. (yesterday, today and tomorrow), Datura stramonium (jimsonweed, stinkblaar), a wide variety of lilies and lily-like plants, cycads, plants that contain soluble oxalates, plants containing cardiac glycosides and other cardiotoxins and euphorbias (Euphorbia pulcherrima, E. tirucalli). Poisoning by plant products such as macadamia nuts, onions and garlic, grapes and raisins, cannabis (marijuana, dagga) or hashish and castor oil seed or seedcake is also discussed. Many of the poisonings are not usually fatal, but others frequently result in death unless rapid action is taken by the owner and the veterinarian, underlining the importance of awareness of the poisonous potential of a number of familiar plants.

Putative Aspergillus niger-induced oxalate nephrosis in sheep.

A sheep farmer provided a maize-based brewer's grain (mieliemaroek) and bales of Eragrostis curvula hay to ewes and their lambs, kept on zero-grazing in pens. The 'mieliemaroek' was visibly mouldy. After 14 days in the feedlot, clinical signs, including generalised weakness, ataxia of the hind limbs, tremors and recumbency, were noticed. Six ewes died within a period of 7 days. A post mortem examination was performed on 1 ewe. The carcass appeared to be cachectic with mild effusions into the body cavities; mild lung congestion and pallor of the kidneys were observed. Microscopical evaluation revealed nephrosis and birefringent oxalate crystals in the renal tubules when viewed under polarised light. A provisional diagnosis of oxalate nephrosis with subsequent kidney failure was made. Amongst other fungi, Aspergillus niger was isolated from 'mieliemaroek' samples submitted for fungal culture and identification. As A. niger is known to synthesise oxalates, a qualitative screen to detect oxalic acid in the mieliemaroek and purified A. niger isolates was performed using high-performance liquid chromatography (HPLC). Oxalic acid was detected, which supported a diagnosis of soluble oxalate-induced nephropathy.

Molasses as a possible cause of an "endocrine disruptive syndrome" in calves.

During the mid 1990s a potentially serious, chronic syndrome was reported in well-managed beef and dairy herds from unrelated parts of South Africa. Farmers reported that it manifested as various combinations of decreased production, decreased weaning masses, apparent immune breakdown in previously immunocompetent animals, increased reproductive disorders, various mineral imbalances in non-deficient areas and goitre, noticeable as enlarged thyroid glands. The farmers associated this syndrome with certain batches of sugar cane molasses and molasses-based products. The syndrome was reminiscent of an "endocrine disruptive syndrome". The objective of this study was to evaluate the suspected endocrine disruptive effect of molasses included in cattle feed. Using existing in vitro assays, four batches of molasses syrup were screened for possible inclusion in a calf feeding trial. Two batches were selected for the trial. Thirty-two, 4- to 6-week-old, weaned Holstein bull calves were included in the single phase, three treatment, parallel design experiment. In two of the groups of calves, two different batches of molasses were included in their rations respectively. The control group was fed a ration to which no molasses was added, but which was balanced for energy and mineral content. The mass gain of the calves was recorded over the 6-month study period. The calves were clinically examined every week and clinical pathology parameters, immune responses and endocrine effects were regularly evaluated. Even though endocrine disrupting effects were detected with the in vitro screening assays, these could not be reproduced in the calves in the experiment. The two batches of molasses utilized in the calf feeding trial did not induce major differences in any of the parameters measured, with the exception of a lower mass gain in one of the molasses-fed groups (Group 1), which tended towards significance. The results of the study indicate that the two batches of molasses had no endocrine disruptive or immunosuppressive effects in calves.

Evaluation of activated charcoal as treatment for Yellow tulp (Moraea pallida) poisoning in cattle.

The efficacy of activated charcoal as a treatment for cattle (n = 57) poisoned by Yellow tulp (Moraea pallida) was investigated. Treatment with activated charcoal resulted in full recovery, irrespective of the degree of posterior paresis, provided that this clinical sign did not develop within the first 12 hours after initial exposure to Yellow tulp-infested grazing. For instance, despite treatment, 1 of 7 cattle succumbed after manifesting mild posterior paresis 6 to 8 h after initial exposure and 3 of 3 treated cattle died after developing severe posterior paresis within 6 to 12 h.

Poisonous plants of veterinary and human importance in southern Africa.

Southern Africa is inherently rich in flora, where the habitat and climatic conditions range from arid environments to lush, sub-tropical greenery. Needless to say, with such diversity in plant life there are numerous indigenous poisonous plants, and when naturalised exotic species and toxic garden varieties are added the list of potential poisonous plants increases. The economically important poisonous plants affecting livestock and other plant poisonings of veterinary significance are briefly reviewed. In addition, a synopsis of the more common plant poisonings in humans is presented. Many of the plants mentioned in this review are also used ethnobotanically for treatment of disease in humans and animals and it is essential to be mindful of their toxic potential.

Provisional clinical chemistry parameters in the African sharptooth catfish (Clarias gariepinus).

Pollution affects aquatic systems worldwide and there is an urgent need for efficient monitoring. Fish are generally sensitive to their environment and are thus considered to be valuable bioindicator species. The African Sharptooth catfish (Clarias gariepinus) is particularly important in this respect because of its very wide distribution. In order to use C. gariepinus as a bioindicator species its baseline clinical chemistry must be defined. Existing data are scarce, and the objective of this work was therefore to establish clinical chemistry parameters for C. gariepinus. Blood was collected from male and female catfish and a number of clinical chemistry parameters were determined. Plasma protein values, but particularly those of plasma albumin, were found to be very low, approximately half the value for dogs, but similar to the values in Channel catfish (Ictalurus punctatus). Plasma urea values in Sharptooth catfish were found to be much lower than in dogs, but only marginally lower than in Channel catfish. Plasma creatinine in Sharptooth catfish, however, was only a quarter of that of dogs and one third of that found in Channel catfish. These findings may have implications for using urea and/or creatinine as an index of renal glomerular filtration, as is done in mammals. Plasma enzyme activity ranges were much lower in Sharptooth catfish than in dogs, particularly for alkaline phosphatase (ALP) and alanine aminotransferase (ALT). By comparison, Channel catfish have an even lower ALT activity range but an ALP range that is very similar to dogs. The implications for using these enzymes as markers for liver disease are not clear from these data, as factors such as plasma half-life and tissue distribution remain to be determined. The very low plasma thyroxine (T4) levels have important implications for laboratory personnel, who will have to set up calibration and standardisation adaptations for the methods that are generally designed for human samples. Although the sample size was too small for reliable comparisons, it appeared that there was little difference in the parameters measured between male and female fish. The values obtained are a useful starting point for using C. gariepinus as a bioindicator species.

Establishment and validation of primary hepatocytes of the African sharptooth catfish (Clarias gariepinus).

In vitro systems such as primary cells and continuous cell lines are gaining momentum in ecotoxicological studies. Cytotoxicity tests with fish cells as well as tests using specific endpoints such as CYP1A induction are valuable in the toxicity assessment of environmental samples. The main objective of this study was to establish and validate the use of primary hepatocytes from the African sharptooth catfish (Clarias gariepinus) as an in vitro toxicity monitoring system. The successful isolation of primary hepatocytes from the sharptooth catfish was achieved using an in situ perfusion method. The primary hepatocytes responded to CYP1A induction, while a continuous Chinese hamster ovary (CHO-K1) cell line showed no activity when exposed to various concentrations of benzo[a]pyrene (B[a]P) (p<0.0001). Cytotoxicity, as measured by the methyl thiazol tetrazolium (MTT) assay, was not observed following a 72 h exposure of the primary hepatocytes and the CHO-K1 cell line to different B[a]P concentrations. However, the hepatocytes were damaged at higher B[a]P concentrations (>10(-6)M) as shown by transmission electron microscopy. This cytotoxicity effect was also confirmed by the trypan blue exclusion assay (TD(50) of 10(-6)M). Differences in the results between the MTT and trypan blue exclusion assays are probably due to mitochondria that are still metabolically active, causing the tetrazolium salt to be dehydrogenated. The internal architecture of normal primary hepatocytes included large quantities of rough endoplasmic reticulum (often in close proximity to the nucleus), mitochondria, aggregates and scattered glycogen, a few lipid droplets and spherical nuclei with distinct nucleoli. The primary catfish hepatocyte cell culture system, expressing CYP1A when exposed to B[a]P, could be used as a biomarker for aromatic hydrocarbon pollutants in aquatic ecosystems of southern and East Africa.

A potential krimpsiekte vaccine.

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.

The toxicity of Senecio inaequidens DC.

This study was designed to confirm the toxicity of a plant implicated in an outbreak of poisoning of stock in Frankfort, Free State Province, South Africa. Cows died acutely after being introduced into a camp, where an abundant, green shrublet was noted to be heavily grazed. This plant was subsequently identified as Senecio inaequidens DC. (Asteraceae) by the South African National Biodiversity Institute (SANBI). Extraction and chemical analyses for pyrrolizidine alkaloids (PAs) in Senecio inaequidens revealed the presence of 4 different compounds, namely retrorsine and senecionine (known to be hepatotoxic) and 2 unidentified compounds. The average total PA (free base plus N-oxide) concentration in plant parts of S. inaequidens collected at Frankfort during the outbreak was 0.81%, compared with the total alkaloid content in the dried, milled S. inaequidens plant material, collected 7 weeks after the outbreak, of only 0.18%. Male Sprague-Dawley rats (n = 4), aged 8-9 weeks, were dosed per os. Each rat received a different dose of the crude Senecio inaequidens extract, ranging from 0.049 mg/g body weight (b.w.) to 0.25 mg/g b.w. No clinical signs were observed in the rat receiving the lowest dose. Rats receiving higher doses showed depression, an unsteady gait, pilo-erection and jaundice, which was particularly noticeable in the ears. Clinical chemistry evaluation revealed an increase in the activities of ALP (except Rat 4), AST and GGT in all animals. Total serum bilirubin, creatinine and urea concentrations were also elevated. All rats had low serum globulin concentrations with an A/G ratio above 1.2. Post mortem examination of the rats revealed marked hepatic lesions. Histopathologically, these changes were characterised by necrosis (variable in extent) of the centrilobular and midzonal hepatocytes (but sparing the portal hepatocytes), with extensive haemorrhage and congestion. Proliferation of the bile ducts, fibrosis and oedema were also present. Ultrastructural changes in affected rats were characterised by margination of chromatin, the presence of numerous autolysosomes in necrotic hepatocytes, intramitochondrial woolly inclusions and changes in the endoplasmic reticulum. A sheep, also dosed with the crude extract, failed to exhibit clinical signs, clinical chemistry aberrations or macroscopic lesions; however, examination of the liver of this sheep revealed histopathological and ultrastructural changes similar, though milder, to those displayed by the rats. Pyrrolizidine alkaloids were extracted from the liver and kidneys of the rats and the sheep. In the case of the sheep, retrorsine was also detected in the lungs, urine and bile.

Chronic vanadium poisoning in calves and its treatment with calcium disodium ethylenediaminetetraacetate.

Sixteen Friesland heifer calves aged between 96 and 157 days were removed from a dairy farm that had been polluted with vanadium and randomly allocated into two equal groups (n = 8). The objective of the trial was to determine whether calcium disodium ethylenediaminetetraacetate (CaNa(2)EDTA) could be used as a treatment for cattle running in environments high in background vanadium. The treatment group received 80 mg CaNa(2)EDTA per kg body weight intraperitonealy (i.p.) twice a week over a 10-week period. The control group received normal saline i.p. over the same period. During the trial calves were exposed to a daily intake of vanadium in the form of contaminated tef hay derived from the farm of origin. In addition, the total mixed ration was spiked with a further 20 mg V(2)O(5)/kg feed to compensate for possible on-farm inhalation exposure. A stochastic model was used to estimate daily intake of vanadium as a distribution function. The model estimated that the daily intake of vanadium varied between an absolute minimum of 33 mg/day to an absolute maximum of 124 mg/day. The average intake of vanadium was 71.8 mg per day per calf. Various chemical pathology parameters were measured throughout the trial as well as urine excretion rates of vanadium and lymphocyte stimulation counts. All calves were slaughtered and necropsied in cohorts of 4-6 animals at monthly intervals after completion of the trial and withdrawal of vanadium from the ration. Tissue concentrations of vanadium were determined and necropsy findings were noted. The study found that CaNa(2)EDTA appears to enhance the excretion of vanadium in calves, but could not prove that the treatment had a protective effect against vanadium exposure. Calves were able to tolerate the prolonged treatment with CaNa(2)EDTA without side-effects.