A number of antinutrients and adventitious toxins may gain entry into fish diets. These components can be integral substances of a feedstuff, they may result from natural contamination, or they may be accidental contaminants derived from human sources. When these materials are present in sufficient concentrations, the effectiveness of the diet may be compromised or the diet may exceed legal restrictions for some substances. This translates into a need for continuous awareness of quality during the selection of ingredients and the processing and storage of the diets.
Numerous substances that occur naturally in plant materials or raw fish preparations from which feedstuffs are derived can affect the performance of fish. These naturally occurring antinutrients include trypsin inhibitors, hemagglutinating agents, phytic acid, gossypol, cyclopropenoic fatty acids, glucosinolates, erucic acid, alkaloids, and thiaminase.
Raw soybeans contain crystalline globular proteins that act as trypsin inhibitors (Mickelsen and Yang, 1966; Liener and Kakade, 1980). These proteins, which form irreversible complexes with trypsin, can be inactivated through heat processing (Ham and Sandstedt, 1944); however, excessive heating reduces the availability of certain amino acids, particularly lysine. The growth inhibition of underheated soybean products for fingerling channel catfish (Robinson et al., 1981) and rainbow trout (Sandholm et al., 1976; Smith, 1977) has been presumed to be due to interference with protein utilization. Several studies, however, with common carp (Viola et al., 1982), channel catfish (Robinson et al., 1985; Wilson and Poe, 1985), and rainbow trout (Ketola, 1975; Rumsey and Ketola, 1975; Dabrowski and Wojno, 1977) have indicated that other antinutritional factors in the soybeans may also be responsible. Robinson et al. (1981) found that dietary soybean products decreased intestinal lipase activity in channel catfish. Research has shown that the sensitivity of various fish species to trypsin inhibitors varies, with salmonids being more sensitive (Sandholm et al., 1976; Smith, 1977) than either channel catfish (Robinson et al., 1985; Wilson and Poe, 1985) or carp (Dabrowski and Kozak, 1979). Channel catfish fed a 35 percent crude protein diet appeared to tolerate soybean meal with much higher trypsin inhibitor activity than fish fed a 25 percent crude protein diet (Wilson and Poe, 1985).
In addition to trypsin inhibitors, soybeans have been found to contain proteins called hemagglutinins, or lectins, that can cause in vitro agglutination of the red blood cells from various species of animals (Jaffe, 1980). Soybean hemagglutinin is readily inactivated by pepsin in the stomach (Mickelsen and Yang, 1966), and therefore would not appear to cause any significant problems for fish with true stomachs.
Approximately 70 percent of the phosphorous in soybean meal and many other feedstuffs of plant origin is in the form of phytate, and its availability to fish is negligible (Ketola, 1985; Ketola, in press). Phytates act as strong chelators and form protein-phytic acid complexes that may reduce the bioavailability of protein (Spinelli et al., 1983) and minerals, such as zinc, manganese, copper, molybdenum, calcium, magnesium, and iron (O'Dell and Savage, 1960; Rackis, 1974; Smith, 1977). Ketola (1975) postulated that this reduction in mineral bioavailability explains in part the need for additional mineral supplementation in soybean-based
diets as compared with those based on fishmeal. Phytates, in conjunction with high concentrations of dietary calcium, caused a zinc deficiency in chinook salmon fed a diet presumed to be adequate in zinc content (Richardson et al., 1985). The addition of 0.5 percent phytic acid to chemically defined diets fed to rainbow trout resulted in a 10 percent reduction in growth and feed conversion, but had no apparent effect on zinc absorption (Spinelli et al., 1983). The conclusion was that the growth retarding effect of phytic acid was related to reduced protein availability. Gatlin and Wilson (1984b) found that the zinc allowance in natural ingredient catfish diets containing about 50 percent of soybean meal should be increased to about five times the normal requirement for growth.
The use of glanded cottonseed meal is limited in fish diets due to its gossypol content. Gossypol is found in the pigment glands of cotton and may account for as much as 2.4 percent of seed weight in certain varieties (Berardi and Goldblatt, 1980). Free gossypol is tolerated at varying amounts by different fish species, but excessive concentrations can depress growth and cause damage to various organ tissues. Gossypol has also been identified as a carcinogen with aflatoxin B in rainbow trout (Sinnhuber et al., 1968a).
Roehm et al. (1967) reported adverse effects of free gossypol on the growth of rainbow trout fed concentrations of 1,000 mg/kg of diet or higher, but not at 250 mg/kg of diet. Herman (1970) found that although growth depression did not occur at concentrations lower than 290 mg/kg of diet, histopathological changes were noted at 95 mg/kg of diet and included thickening of the glomerular basement membrane of the kidney and necrosis and ceroid deposition in the liver. Wood and Yasutake (1956) noted similar histopathology in rainbow trout. These results indicate that the maximum concentration of free gossypol in the diets of salmonids should be restricted to 100 mg/kg of diet or less.
Growth inhibition was observed in fingerling channel catfish fed more than 900 mg of free gossypol/kg of diet (Dorsa et al., 1982), but a portion of the depression was possibly due to a dietary lysine deficiency resulting from the irreversible binding of lysine and gossypol (Wilson et al., 1981; Dorsa et al., 1982). In commercial catfish diets in the United States, 10 to 20 percent cottonseed meal is commonly used. Robinson (1991) reported that the solvent extracted cottonseed meal used in catfish diets, which typically contains 400 to 800 mg of free gossypol/kg, would not provide toxic concentrations of free gossypol when mixed into catfish diets; however, the concentration of available lysine in the diet should be scrutinized when cottonseed meal replaces soybean meal. Blue tilapia have been shown to tolerate free gossypol concentrations as high as 1,800 mg/kg of diet with no apparent growth depression (Robinson et al., 1984).
Cottonseed meal is the primary source of the cyclopropenoic fatty acids (CFAs) (sterculic acid and malvalic acid) in fish diets. CFAs are present in all varieties of cottonseed meal and are not completely removed by the oil extraction process (Mickelsen and Yang, 1966). Dietary CFAs caused lesions, increased glycogen deposition, and elevated saturated fatty acid concentration in the liver in rainbow trout (Roehm et al., 1970; Malevski et al., 1974; Scarpelli et al., 1974; Struthers et al., 1975a,b). The CFAs are powerful carcinogens when fed in combination with aflatoxins for rainbow trout (Lee et al., 1968, 1971; Hendricks et al., 1980) and sockeye salmon (Wales and Sinnhuber, 1972). These compounds also induced hepatomas in the absence of aflatoxins in rainbow trout (Sinnhuber et al., 1976; Hendricks et al., 1980). Dietary CFAs alter the activity of a number of liver enzymes (Taylor et al., 1973; Eisele et al., 1978, 1983), including the inhibition of fatty acid desaturases (Roehm et al., 1970) which may explain the accumulation of saturated fatty acids found in the liver of fish fed CFAs. No conclusive data exist on the effects of dietary CFAs on other fish species.
Glucosinolates are found naturally in oilseed crops such as rapeseed. The compounds are not themselves harmful, but upon enzymatic hydrolysis the products release thiocyanate ion, isothiocyanates, goitrin, and nitrites, all functioning as potent antithyroid agents. Thiocyanate ion inhibits the uptake of iodine by the thyroid while isothiocyanates and nitrites presumably are precursors to thiocyanate ion. The effects of these compounds can be reversed with additional iodine supplementation of the diet. Goitrin is the most potent glucosinolate; it inhibits the ability of the thyroid to bind iodine. The effects of goitrin cannot be reversed with dietary iodine (Tookey et al., 1980).
The glucosinolate content of meal from traditional rapeseed ranges from 3 to 8 percent (Fenwick and Hoggan, 1976; Langer, 1983; van Etten and Tookey, 1983), but selective breeding has led to the development of low glucosinolate varieties of rapeseed, referred to as canola, which provide meal with less than 0.2 mg/g glucosinolates (Higgs et al., 1982, 1983; Hardy and Sullivan, 1983). Yurkowski et al. (1978) showed that feeding rainbow trout traditional rapeseed meal caused thyroid hyperplasia and reduced plasma thyroxine concentration. Heat treatment inactivated the enzyme myrosinase, which hydrolizes the glucosinolates to their toxic by-products, but heating did not eliminate the glucosinolates or allow for growth equal to that of control fish. Extraction of rapeseed meal with water reduced the glucosinolate content and led to improved growth of rainbow trout (Yurkowski et al., 1978; Jones, 1979). Dabrowski and Kozlowska (1981) found that heat treatment of rapeseed
meal did not eliminate all the growth-suppressing factors for common carp.
Higgs et al. (1979) fed diets containing up to 22 percent (low glucosinolate) canola meal to coho salmon and obtained satisfactory growth, but elevated thyroid activity was observed in the fish. They claimed that higher amounts of canola meal could be used in salmon diets if 3,5,3-triiodo-L-thyronine was included in the diet to compensate for the loss of thyroid function (Higgs et al., 1982, 1983). Rainbow trout fed up to 20 percent canola meal showed normal growth rate, but thyroid hyperplasia and increased production of thyroid hormones was induced (Hardy and Sullivan, 1983).
Erucic acid, a constituent of rapeseed oil, is a 22-carbon monounsaturated fatty acid and may constitute from 20 to 55 percent of the oil (Kramer et al., 1975; Slinger, 1977). Diets with erucic acid-containing rapeseed oils were cardiotoxic to rats and caused lipid accumulation followed by necrosis of heart muscle fibers (Slinger, 1977). Unpublished work by Parker and Hendricks (as cited by Hendricks and Bailey [1988]) has shown that inclusion of erucic acid in the diet of coho salmon at 3 to 6 percent led to mortalities and pathologies of the skin, gill, kidneys, and heart. The selective breeding program that developed canola emphasized low erucic acid as well as low glucosinolates. No erucic acid-related pathologies have been associated with the inclusion of rapeseed meal in natural ingredient fish diets (Yurkowski et al., 1978; Higgs et al., 1979; Dabrowski and Kozlowska, 1981; Dabrowski et al., 1981, 1982; Higgs et al., 1982; Hardy and Sullivan, 1983; Higgs et al., 1983).
The pyrrolizidine alkaloids are toxins found in several plant families, but not in any that are commonly used as feedstuffs in fish feeds. Many of these plants are grown in conjunction with soybeans and cotton, and therefore may find their way into fish diets as contaminants of soybean or cottonseed meal. The compounds are metabolized to toxic pyrroles (McLean, 1970) by mixed function oxidases in the liver. Pyrrolizidine alkaloids at 100 mg/kg of diet caused severe growth depression and mortality in rainbow trout (Hendricks et al., 1981); and at concentrations of 2 mg/kg of diet these toxins caused hepatic lesions including necrosis, megalocystis, fiber tissue scarring, and occlusion of the hepatic veins. Liver lesions and mortalities were also noted 6 months after the alkaloids were removed from the diet.
The thiamin-destroying enzyme thiaminase has long been recognized in some raw fish preparations (Green et al., 1941; Wooley, 1941; Wolf, 1942; Deutsch and Hasler, 1943; Lieck and Agren, 1944; Yudkin, 1945; Neilands, 1947). Species that contain thiaminase, as well as those that do not, were reviewed by the National Research Council (1983). The enzyme is found more commonly in freshwater fish than in marine fish. Thiamin is only destroyed after contact with the thiaminase for a period of time; therefore, feeding fresh fish or feeding thiamin in a separate diet from the raw fish will not cause a thiamin deficiency (Camacho, 1974). Heating or ensiling thiaminase-containing raw fish reduces thiaminase activity (Greig and Gnaedinger, 1971; Anglesea and Jackson, 1985).
Some substances may be produced by natural processes in feedstuffs or in the aquatic environment that may affect fish performance. Feedstuffs can become contaminated with mycotoxins, dietary lipids can oxidize, and algal and other marine toxins can be produced in the water and impair proper nutrient uptake or metabolism.
Many fungi grow well on a number of diet ingredients or processed diets under proper temperature and moisture conditions. They produce mycotoxins that are carcinogenic, cytotoxic, or neurotoxic (Eiroa and Nelly, 1979; Lovell, 1991). Feeds contaminated with aflatoxins produced by the mold Aspergillus flavus were a major cause of liver hepatomes in rainbow trout (Wolf and Jackson, 1963; Halver, 1967). Of the different aflatoxins produced by the various strains of Aspergillus , the B1 form was responsible for trout hepatoma (Halver, 1967) and can produce hepatoma at dietary concentrations as low as 0.5 µg/kg (Ashley et al., 1965; Sinnhuber et al., 1965).
The carcinogenic or toxic effects of aflatoxins in fish seem to be species specific. Coho salmon from the Great Lakes are similar to rainbow trout in sensitivity to aflatoxins (Black et al., 1988), but those from sea-run populations (Halver et al., 1967) and brook trout (Wolf and Jackson, 1967) are less sensitive than rainbow trout. Halver (1967) reported the 10-day oral LD50 of aflatoxin B1 for rainbow trout to be 0.5 mg/kg of diet and cited an unpublished study by W. H. Hastings that showed the LD50 for channel catfish was 15 mg/kg of diet. Carp fed diets containing 2 mg/kg of aflatoxin B1 showed no adverse effects. These observations may indicate that warm-water species are less sensitive to aflatoxins than cold-water fish. Jantrarotai and Lovell (1991a) found liver and kidney lesions and a reduction in growth rate and hematocrit but no mortalities in channel catfish fed aflatoxin B1 at 10 mg/kg of diet for 10 weeks. The carcinogenicity
of aflatoxins is affected by other dietary factors such as the presence of cyclopropenoic fatty acids (Lee et al., 1968, 1971; Sinnhuber et al., 1968b) and gossypol (Sinnhuber et al., 1968a) from cottonseed meal and the pesticide dieldrin (Hendricks et al., 1979). Increasing the dietary concentration of protein also increased the carcinogenicity of aflatoxins (Lee et al., 1978; Stott and Sinnhuber, 1978).
Other mycotoxins are produced by molds and fungi that grow on feedstuffs. Friedman and Shibko (1972) identified 27 mycotoxin-producing fungi among 114 species isolated from 20 samples of dried shrimp. The tricothecene toxin, T2, produced by the fungus Fusarium tricintum proved lethal to rainbow trout at a dietary concentration near 6 mg/kg body weight (Marasas et al., 1967). Poston et al. (1983), however, fed rainbow trout T2 at 15 mg/kg of diet and found that the main effects were reduced feed consumption, reduced growth, lower hematocrit, and lower blood hemoglobin. Woodward et al. (1983) showed that rainbow trout had a sensitive taste acuity for vomitoxin produced by Fusarium and reduced their feed intake as the concentration of vomitoxin increased from 1 to 13 µg/g of diet; the fish refused to consume the diet with a vomitoxin concentration of 20 µg/g. Jantrarotai and Lovell (1991b) found that cyclopiazonic acid (CPA), a neurotoxin frequently found in association with aflatoxin, was more toxic to channel catfish than aflatoxins and is more frequently found than aflatoxins in feedstuffs in the southern United States. The minimum dietary concentration that caused a reduction in growth rate was 0.1 mg/kg for CPA as compared with 10 mg/kg for aflatoxin B1.
The existence of toxic marine, estuarine, and freshwater algae has been well documented (Shilo, 1964; Fogg et al., 1973; Taylor and Seliger, 1979; Kungsuwan et al., 1987), and blooms of toxic algae may cause extensive mortality in fish culture facilities (Sparks, 1972; White, 1982; Meriwether et al., 1984; Saunders, 1988). Particular care must be taken to ensure that toxic algal species are not included in diets or nursery ponds of larval fish because the survival of many species depends on direct consumption of phytoplankton. Some mollusks can consume toxic algae and concentrate the toxins in their tissues (Sparks, 1972; White, 1982); therefore, contaminated mollusks must not be included in fish diets. Toxins of certain algae, such as Microcystis aeruginosa, do not seem to affect finfish (Phillips et al., 1985), but the toxins of other algal species, such as Ganyaulax and Gyrodinium spp., are highly toxic (Roberts et al., 1983).
Autoxidation of unsaturated lipids produces a large number of chemical products, including free radicals, peroxides, hydroperoxides, aldehydes, and ketones. These compounds may be toxic to fish or react with other dietary components and reduce their nutritional value (Andrews et al., 1965; Crawford et al., 1966; Roubal and Tappel, 1966; Yamagita et al., 1973; Ko et al., 1975; Forster et al., 1988). The primary effect of feeding diets containing oxidized oils appears to be the interaction of the peroxidative decomposition compounds with vitamin E. Studies with rainbow trout (Sinnhuber et al., 1968b), channel catfish (Murai and Andrews, 1974), common carp (Hashimoto et al., 1966; Watanabe and Hashimoto, 1968; Iijima and Zama, 1979; Hata and Kaneda, 1980), and yellowtail (Ueda and Nagaoka, 1969; Park, 1978) have shown that the pathologies resulting from feeding oxidized oils were similar to those of vitamin E deficiency. Sinnhuber et al. (1968b) and Watanabe and Hashimoto (1968) demonstrated with rainbow trout and common carp that the toxic effects of feeding oxidized lipids could be prevented by increasing the supplement of vitamin E in the diet. Other studies with various fish species have shown that the toxic effects of oxidized oils can be ameliorated by additional vitamin E or a-tocopherol (Hashimoto et al., 1966; Watanabe and Hashimoto, 1968; Murai and Andrews, 1974). Hung and Slinger (1980), however, could show no toxic effects of adding oxidized fish oils to nutritionally complete natural ingredient diets for rainbow trout; the only adverse effect was a slight decrease in liver a-tocopherol level. Adding synthetic or natural antioxidants to feed lipids can prevent or minimize the adverse effects of autooxidized lipids. Antioxidants are discussed in Chapter 2.
Metals may act as both nutrients and toxicants. A detailed discussion of maximum tolerable concentrations of the dietary essential metals is found in a report by the National Research Council (1980). Potential toxicity depends not only on their concentration in the diet but also on the concentration of other minerals, such as calcium and magnesium, in the rearing water (Spear and Pierce, 1978; Waiwood and Beamish, 1978; Carroll et al., 1979). The toxicity of metals can be reduced by other dietary components, such as phytin, which forms a nondigestible organic complex with certain metals. Metal chelators, such as ethylenediaminetetraacetic (EDTA), can reduce the toxicity of cadmium, copper, zinc, lead, and aluminum when added to the diet (Muramoto, 1980; 1981).
The toxicity of mercury to fish depends on its chemical form. Rainbow trout are apparently unable to convert inorganic mercury to the more toxic methyl mercury (Pennacchioni et al., 1976) even though oral doses of inorganic mercury (mercuric chloride) increased tissue total mercury
concentration. Wobeser (1975) fed graded concentrations of methyl mercury chloride to rainbow trout and found that dietary concentrations of mercury up to 24 mg/kg did not cause mortalities, but that fish fed 16 mg/kg or more showed hyperplasia of the gill epithelium and reduced hematocrits. Coho salmon fed dogfishmeal with 2.3 mg total mercury/kg to replace 50 percent of the herring meal in an Oregon moist diet grew as large as control fish and did not accumulate total body mercury concentrations above the U.S. Food and Drug Administration (FDA) tolerance level of 0.5 mg/kg (Spinelli and Mahnken, 1976).
Fish accumulate mercury in muscle tissue and the rate is influenced by dietary form and concentration (Pennacchioni et al., 1976) and fish size (Scott and Armstrong, 1972). The bioaccumulation of mercury is directly correlated with fish size (Friedman and Shibko, 1972). Feedstuffs fed to fish grown for human consumption should be scrutinized for mercury concentration because mercury could possibly accumulate in the fish and exceed the FDA tolerance level. Selenium was found to reduce the toxicity of methyl mercury (Ganther et al., 1972; Friedman et al., 1978) and decrease the rate of mercury bioaccumulation in fish, crayfish, and lake sediment biota (Rudd et al., 1980).
Water-borne cadmium has been shown to be toxic to many fish species (Sangalang and O'Halloran, 1972; Kumada et al., 1973; Clearley and Coleman, 1974; Benoit et al., 1976; Smith et al., 1976). Cadmium absorbed through the gastrointestinal tract (by gastric intubation) was shown to cause liver necrosis and mortality at doses as low as 5 µg/g body weight.
The main source of arsenic in ingredients used in fish feeds comes from marine fishmeal. Reinke et al. (1975) reported arsenic concentrations in tissues of a number of commercial fish species from the North Atlantic ranging from 1.8 to 40 mg/kg; however, most of the arsenic was in the form of an organic complex rather than the highly toxic arsenite. The potential toxicity to fish of feeding diets containing organic arsenic compounds is not known.
The polychlorinated biphenyls (PCBs) are widely used industrially as plasticizers and as heat-transfer, dielectric, and hydraulic fluids. They are poorly biodegraded and accumulate in lipids, and have been found in marine and freshwater organisms from almost all areas of the United States (Addison, 1976; Ito and Konishi, 1980; Peneva, 1980; Brunn et al., 1981; Falandysz and Ganowiak, 1981; Veith et al., 1981). Fish oil and meal represent the primary sources for PCB contamination of fish diets (Hansen et al., 1976). A PCB dosage of 14.5 mg/kg body weight resulted in 100 percent mortality of coho salmon after 260 days (Mayer et al., 1977). Sublethal effects of PCB exposure in fish include liver enlargement, lesions in the liver ultrastructure, inhibition of hepatic aryl hydrocarbon hydroxylase and other hepatic microsomal enzymes (Lidman et al., 1976; Addison et al., 1977, 1978, 1979; Gruger et al., 1977; Shelton et al., 1984), and decreased thyroid activity (Leatherland and Sonstegard, 1978, 1980). These compounds accumulate in fish tissues (Guiney and Peterson, 1980); therefore, prolonged feeding of dietary concentrations below the toxicity level may result in tissue accumulation that would be toxic to the fish or that are above FDA-approved levels (0.2 mg/kg) for human food.
The FDA restrictions on the use and concentration of pesticides in agricultural products used for human or animal consumption make it unlikely that fish feeds will be sufficiently contaminated to cause acute toxicities. Fish are most likely to be exposed to pesticides through accidental contamination of feedstuffs with hazardous quantities of insecticides or rodenticides, or use of water that has been contaminated by these chemicals, such as through aerial spraying. Most pesticides bioaccumulate in fish, therefore, prolonged exposure to small amounts, from the water or the diet, may result in accumulations in the tissue that will affect the health of the fish or the marketability of the product for human food. Ashley (1972) evaluated the toxic effects of chlorinated hydrocarbons in several fishes. Toxicity was greatest in young fish, characterized by dysplasia and sterility of gonads, lethargy, nervous disorders, anorexia, and death. The toxicity to DDT decreased among fish species in the following order: rainbow trout, brown trout, guppy, bluegill, and channel catfish. The insecticide DDT caused inhibition of gill and kidney Na+- and K+-ATPase (Campbell et al., 1974), liver tumors (Halver, 1967), nervous disorders (Bahr and Ball, 1971), and acute toxicity (Buhler et al.,1 969) in various fishes.