Toxicology, 3 (1975) 269-285 @ Elsevier/North-Holland. Amsterdam
in The Netherlands
TOXICOLOGICAL IMPLICATIONS OF PESTICIDES: EFFECTS ON SEEDS OF FOOD PLANTS
and D.K. SALUNKHE**
*Center in Environmental Toxicology, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Term. 37232, and **Department of Nutrition and Food Science, Utah State University, Logan, Ut. 84322 (U.S.A.J (Received (Revision
February 13th, 1974) received August 16th, 1974)
Pesticides are widely used for the protection of economic crops from a variety of noxious pests. The repeated and indiscriminate uses and the extreme stability of certain pesticides have led to their accumulation in plants, animals, soils and sediments, thus effecting widespread contamination of the environment. Soil contaminants are especially serious because they can inhibit or impair the seed germination of our food and feed crops. Seeds can come in close contact with pesticides through processes such as prematurity application, fumigation, seed dressings, and seed treatments. Several reports have indicated the toxic effects of pesticides on seed germination. Possible mechanisms of the toxic action of pesticides during the germination of seeds have been discussed with emphasis on biochemical, histological, and cytological alterations. Bioassay procedures employing seed germination as a simple, feasible, economical, time-saving indicator of toxicity have been described briefly. Attention is then drawn to the possible potential health hazards arising from the presence of pesticidal chemicals in food plants since the toxicological implications of long term exposure to pesticides are often more far-reaching.
Application of pesticides such as fumigants, herbicides and contact insecticides for economical and effective pest control is a common practice. UnAbbreviations: 2,4-D, 2,4-dichlorophenoxyacetic ric acid; DNBP, dinitro-set-butylphenol;
acid; 2,4-DB, 2,4-dichlorophenoxybuty 2,4,5-T, 2,4,5%richlorophenoxyacetic
doubtedly, these chemicals protect food plants from attack by a wide variety of noxious pests affecting agricultural production. The use of organic pesticides (insecticides, herbicides, and fumigants) has been growing annually by 16% and by 1975 pesticide sales at consumer level are expected to equal 3 billion dollars [ 1,2] . The economic value and world-wide importance of pesticides as tools for greater agricultural production are likely to persist. However, their repeated and indiscriminate use can be hazardous as residues accumulate in all segments of the biosphere and ultimately in the resident plants and animals. Presence of pesticidal residues in soil, plants, domesticated animals, fish and various forms of wildlife has been reported in an excellent review by Marth [ 31, and the toxicological implications of the residues in relation to human health have been discussed in a comprehensive review by Durham  . In the past few years distribution of pesticides in the environment -air, water, soils, and biota - has been extensively investigated particularly in respect to the pesticides that may persist in soils for several years [ 51. Much of the persistent residues come from foliage sprays or dusts which miss their target and fall into the soil. It has been estimated that as much as 50% of the sprays applied to foliage may reach the soil in this way  . Even some that is applied to foliage reaches the soil when it is washed or blown off crops or when the plant remains are ploughed into the soil. Moreover, soils can be contaminated by leaching of the chemicals from one place to another. Plants containing translocated pesticide residues may add to soil contamination by decomposing in a soil-water system after the completion of their life cycle. The second main source of pesticide residues in soil is from the large quantities of insecticides applied directly to it to control soil-inhibiting pests. This direct soil application may also include the pesticides employed for seed treatments and seed dressings. In addition to man-made pesticide chemicals, naturally occurring toxic compounds of plant and microbial origin can be deposited either in the soil or in and on the seeds of crop plants. Although a good deal is already known about the persistence, metabolism, and movement of pesticides in soils and the potential translocation of these into the edible parts of crops [ 71 much less is known about the influence of pesticides on toxicity to germinating seeds and seedling of food or feed crops. In the ensuing discussion, an attempt is made to review the available knowledge on the environmental behavior of pesticides and their uses with respect to their damaging effects on seed germination and early development of seedlings especially in the light of the fact that seeds may be the first stage of our economic crop plants to be exposed to the detrimental effects of environmental contamination. Factors affecting effects on seeds
in soil as related
to the toxic
Food crops begin their life with seed germination and seedling growth in soils. Naturally, seeds sowed in the contaminated soil come in contact with the accumulated pesticides and also with toxic compounds occurring in 270
plants that finally persist in soils after the plants complete their life-cycle. Among the economic crops, cereals and legumes are the species of vital importance to man for his living. These species are perennially grown in soils. Therefore, logically the seeds of these crops become the victims of pesticides and also of some naturally occurring plant toxicants present in contaminated soils. It will be worthwhile here to briefly review the numerous factors that affect persistence of pesticides in soils employed for growing food crops. Toxic effects of a given pesticide on seeds will depend on its distribution, persistence and metabolism in the soil. Active form and enough concentration of the pesticide in soil are necessary factors for toxic actions. Although soil is perhaps the largest most important temporary reservoir for the accumulation of pesticide residues [ 81, characteristics of the soil and pesticides exhibit decisive effects on metabolism and persistence of the pesticides that eventually exert adverse effect on seeds. The type of soil treated with a pesticide greatly influences how long residues persist; for instance, heavy clay soils retain pesticides much longer than lighter sandier ones. Pesticides may adsorb to the soil so tightly that they may be non-toxic to either insects or plants. Organic matter content seems to be an important soil factor influencing how long insecticide residues persist in soil. There is evidence that organophosphorus insecticides persist longer in acid soils than in alkaline ones and the amount of mineral ions such as Fe, Al, and Mg present can also affect the adsorption and persistence of the pesticides in soil [ 61. The chemical structure of the pesticide and its resultant intrinsic stability are important factors governing its persistence in soil. The more volatile a pesticide the shorter time it persists in soil and the effectiveness of a soil pesticide tends to be inversely proportional to its water solubility. The larger the dose of pesticide applied to soil the less disappears in terms of the original application in a given time. The persistence of a pesticide in soil depends on its formulation; granules persist longer than emulsions; wettable powders and dusts disappear most readily of all. Thus, the most common and persistent insecticide residues in soil include DDT and dieldrin, and then lindane, chlordane, heptachlor, and aldrin in decreasing order. Organophosphorus insecticides are much short-lived, remaining in the soil for months at most; but, they are more phytotoxic than organochlorine insecticides [ 51. Phytotoxicity of soil-accumulated pesticides is also affected by many other factors such as climatic conditions, presence of other chemicals, and soil fauna and microorganisms. Increased temperatures accelerate the loss of pesticides by volatilization and desorption whereas the presence of other chemicals may increase or decrease the toxicity of a particular pesticidal chemical. On the other hand, the increasing use of pesticides as a means of controlling soil insects has caused concern because of possible cumulative harmful effects on soil fauna and microorganisms that are beneficial to plant life. Way and Scopes  applied organophosphorus insecticides, menazon, phorate, and thionazin to soil as in-row treatments at commercial rates and concluded that these insecticides were most unlikely to do significant harm to soil organisms, even though they may persist for a relatively long time in
some soils. In contrast, Smith and Wenzel [lo] found 40% reduction of nitrifying bacteria from the 50-pound DDT per acre treatment. Other studies have indicated that the soil organisms are more tolerant of the pesticide chemicals than are certain plants, and that dosage rates required to adversely influence the soil organisms are generally far in excess of normal application rates [ 111. At sublethal levels of pesticide residues, soil organisms may decompose these toxic chemicals that would otherwise be harmful to seed germination. On the contrary, very high concentrations of soil residues may inhibit soil population as well as seed germination. Nature of biological response to toxicants in germinating seeds Toxicity implies an injurious effect that is harmful and involves an alteration of structure, function, or response in some living system. Toxicants will generally enter a living system from its environment, and the environment itself may influence the toxicity of a given chemical. For instance, certain insecticides that are toxic to seeds of a certain species when they germinate under one set of environmental conditions may not be toxic under different conditions. The complex metabolic events that occur during seed germination, however, remain the same for all seeds under all acceptable conditions. The events include: the breakdown of certain materials in the seed, the transport of materials from one part of the seed to another, and the synthesis of new materials. Most often toxicants block one or more of these events, and generally by disrupting enzyme activities. Protein synthesis is important to germination and its inhibition by substances such as chloramphenicol forestalls amylase and phosphatase activity, which normally takes place when seeds germinate [ 121. Before it can damage germinating seeds, the toxicant has to reach its site of action. With particular reference to pesticides, an attempt has been made below to briefly describe some of the important factors impairing seed germination. Factors affecting germination process Toxicology is concerned with biological events characterized by distinct dose-response relationships. The first consideration during seed germination is the amount of toxicant to which the seeds are exposed, from whatever source. The second and hardly less important matter is the presence of other substances and environmental conditions that may modify the germination process. A residue of one insecticide in a soil may not inhibit seed germination unless another insecticide is also present (Table I). Thus, the chemical nature of the toxic substance, the amount present, and the surrounding environment have marked effects on the response of a seed. These factors are basic to selective usage of many pesticides, particularly herbicides. Excessive rates of application of most pesticides can produce harmful secondary effects. Lange  reported that a number of factors can affect the advisability of using insecticides as seed treatments. Other workers  observed that prolonged storage increases phytotoxicity to beans and peas from volatile
OF PESTICIDES 120 h
90 87 83 50b 23b
2.87 2.47 2.18 1.02b 1.26b
3.97 3.47 3.10a 2.15b 1.36b
6.72 5.09b 4.00b 1.54b 1.44b
4.51 3.89 3.64a 3.56a 2.47b
3.75 3.07 3.35 3.27 2.94a
4.53 3.46 3.82 2.83a 2.92s
4.28 3.88 3.53 2.86b 3.03a
4.88 4.07 4.18 3.47b 3.38b
0 50 100 200 250
100 90 67b 33b 30b
0 20 40 80 100
100 100 90 90 80b
0 10 20 50 100
100 90 100 93 63b
90 63 60a 50a 23b
4.95 3.75a 4.03 2.60b 2.09b
4.23 2.18b 1.26b 1.04b 0.87b
7.78 6.96 6.45a 4.00b 2.45b
4.48 2.94b 1.75b 1.73b 0.92b
100 87 93 90 90
GS-14254 + Menazon
100 + 250
GS-14254 + Disulfoton
100 + 100
a Significantly b Significantly
at 0.05 level. at 0.01 level [42,71].
mercurial seed dressings. Seed damage can be influenced by different formulations of the same compound. Thus, Duran and Fischer [ 151 found marked differences in injury to seed effected by different proprietary formulations of benzene hexachloride (BHC). “Sticker” chemicals are usually employed for pesticide applications to seeds. These include methyl cellulose, paraffin oil, linseed oil, etc. Most of the pesticides are lipid soluble and as such the stickers may impair seed germination [ 16,171. Often, insecticides and fungicides are applied simultaneously as seed dressings [ 181 . Apart from their protective action, such mixtures increase the possibility of phytotoxicity through additive or synergistic effects. Natural substances derived from microorganisms or plants also can exogenously impair seed germination. Mycotoxins that invade cereal grains and phenolic compounds that inhibit seed germination are well-known examples. It has been recently reported that a mycotoxin, aflatoxin, inhibited hypocotyl elongation of lettuce seeds [ 191.
Soil factors can influence the effects of foreign chemicals on seed germination. Relatively little is known about either mechanisms whereby plants and seeds accumulate pesticides and other toxic chemicals or the internal and external factors that influence them. The genetic constitution of a seed obviously affects tolerance for a foreign chemical as well as its other attributes such as resistance to disease and yield characteristics. For example, a high oil content in a seed can provide a reservoir for methyl bromide and may disrupt seed germination 1201. Wickramasinghe and Fernando [ 211 reported differences in seed-germination among bean varieties following use of endrin as a seed-soak, as did Lange et al.  with treatments of various other pesticides on lima beans. Sclallett and Kurusz [ 231 interpreted their results with germination of barley steeped in 2,4-D as differences in varietal susceptibility. Age and physical condition of the seed are also important to germination response. Roane and Starling  found Ceresan M to be severely phytotoxic to chipped wheat seed, slightly toxic to cracked seed, and nontoxic to sound seed. This suggests that toxicity of at least some chemicals depends upon their crossing various membranes and reaching a specific site. In general, almost any factor having a detrimental influence an seed vigor or quality can increase the likelihood of adverse effects of pesticides or other toxic materials.
Seed germination impaired by pesticide uses Prematurity applications. Various pesticides
are often employed on parent plants while seeds are maturing. That such treatments can influence the subsequent germinability of the seed crop has been well demonstrated. Seeds from cotton plants treated with 2,4-D showed poor germination and surviving seedlings had malformed root tips . Similar observations were made with seeds harvested from cotton plants sprayed with the herbicide dalapon [ 26 ] . Carlson [ 271 treated a beet root seed crop with a series of insecticide mixtures and found low seed viability following the application of a mixture of DDT and disulfoton. Plants and seeds may accumulate indirect residues from absorption, translocation, and subsequent metabolism by plants growing in soils that have been contaminated by pesticides applied to previous crops . Fumigation during storage. Fumigants commonly used to control various insect pests of stored products are basically nonselective in their phytotoxic effects. Methyl bromide, for instance, is also commonly employed as a sterilant in field applications where a complete kill of weed seeds, nematodes, soilborne insects, and disease is sought. According to Richardson [ 291, a number of fumigants tested on seed corn showed wide differences in phytotoxicity. Among the most harmful were acrylonitrile, acrylonitrile-carbon tetrachloride mixture (50 : 50), chloropicrin, and ethylene dibromide. Similarly, bean seedlings displayed various malformations when the seeds had been exposed to the herbicide methyl 2,4-dichlorophenoxyacetate. Seed treatments. Pesticides used to protect seeds and seedlings are applied in the seed hopper or in the row. Any treatment that places the chemical in
intimate contact with the seed or seedling may cause damage. For example, Hanna [ 301 reported that germination and early growth were impaired as a result of such treatment since the chemical remains in contact with the seeds for a longer period of time. Effective insecticidal treatments originated with the development of chlorinated hydrocarbons. Problems in the use of chlorinated hydrocarbon seed dressings have been realized and are well reviewed in detail by several workers [13,31]. Finlayson [ 321 reported that lindane applied as seed treatment to onions was extremely phytotoxic. General symptoms include delay in germination, slight stunting of growth, nonabsorption of cotyledons by certain beans, and reduction in seedling weight. In one case lindane seed-soaks induced mitotic alterations in rye seedlings and various morphological injuries. Although aldrin, dieldrin, and heptachlor have not been as injurious as lindane, they reduced the germination of turnips and rutabaga [ 331. Seed treatment with systemic insecticides has involved seed-soaks or, more recently, impregnated coatings. Many of these compounds are phytotoxic. Soil treatments. Probably all types of herbicides will influence seed germination under certain conditions. Herbicides showing selective pre-emergence activity, however, are more apt to affect seeds than are other types of pesticides because of their specific chemical properties. Therefore, one of the more challenging aspects of many soil-applied herbicides is to offset the effect of the herbicide on seed germination and early development of seedlings of economic crops [ 341 . It is well recognized that, depending on their concentrations, soil residues of many herbicides such as the s-triazine family [35,36] may damage germinating seeds in the soil. Sumitol (GS-14254) is a member of the s-triazine family and appears to be especially phytotoxic and persistent . Even 2,4-D and 2,4-DB, at high rates, reduced or prevented seed germination of several species. DNBP, pentachlorophenol, and diquat, at levels of 10 ppm or less, were found to be phytotoxic to germinating seeds of radish and Sudan grass. Dichlobenil, diquat, and 2,4,5-T were very toxic to germinating cucumber seeds. Several workers [23,30] have reported reduced germination of seeds that were subjected to organochlorine insecticides. Guyer et al.  found that phorate as seed treatment adversely affected wheat germination. Gifford et al.  noted not only reduced wheat germination but also lower seedling survival after phorate treatment. Organophosphates are relatively less persistent than organochlorine insecticides but can remain in soil for several months. They are finding increased use as replacements for persistent chlorinated hydrocarbons, although they appear to be more phytotoxic especially when used as dressings  . Furthermore, the simultaneous presence of various herbicides and insecticides may pose a serious problem since their interactions most commonly result in increased phytotoxicity due to an inhibition of herbicide degradation by insecticides. Many insecticides and fungicides used in seed treatment are applied to soils. Because of seed size or other problems it often is not possible to
provide enough pesticides on the seed to assure protection without causing phytotoxicity. In such cases, various soil applications are attempted. Repeated use of the insecticides and herbicides, therefore, could lead to the build-up of residues in the soil. As already indicated, seed germination can be affected by pesticides. However, effects on germination arise from a complex of factors, often not well understood. Certain elements such as dosage have nearly universal significance and are often readily controllable. Yet, fundamental studies on response of seeds to specific doses of pesticides are not available in many cases. Most other variables are even less well defined. There is a need for better understanding of the problems and for finding ways to cope with them. Mechanism of action of pesticides relative to germination response Suppression of germination and of subsequent seedling growth by a toxicant indicates impairment of some of the biochemical processes of germination. According to Chopra and Nandra , thiometon, an organophosphate insecticide, inhibited germination of sarson seeds (Brassica campestris L.) by limiting the activity of lipase enzyme. The two insecticides, menazon and disulfoton, and a herbicide GS-14254, disrupted mung bean and wheat germination by inhibiting such vital enzymes as ATPase, amylase, and protease . In combination with menazon or disulfoton, GS-14254 severely damaged wheat germination (Table I). However, mung bean germination was not severely impaired. The increased toxicity to wheat germination might have occurred due to the inhibition of detoxification of either of the pesticides at the active site or sites. Kaufman et al.  have observed several such int,eractions for pesticide combinations. Severe injury or death of cotton’ seedlings was reported by Hacskaylo et al.  when systemic phosphate insecticides were applied to soil with the herbicide monuron or diuron. Nash  also observed that combinations of diuron with some phosphate insecticides showed synergistic phytotoxicity to oats and corn. Thus, combining insecticides and herbicides may result in their having severe phytotoxic effects. Very few reports pertain to the effects of pesticides on chemical constituents that are quantitatively formed during the germination process. We found a significant lessening in the formation of reducing sugars and free amino acids in pesticide-treated seeds with a concomitant lower degradation of starch at the end of 3-day germination period as compared to control seeds (Fig. 1). Similar results were reported by Chopra and Nandra [ 411 who observed a decrease in reducing sugars in germinating mustard seeds treated with thiometon. They attributed the decrease to the inhibition of lipase activity in the seeds. Since hydrolytic enzymes such as lipase, amylase, protease, and phosphatase are produced during germination, pesticides may adversely affect their synthesis. According to Penner , barley germination and seedling development were inhibited in culture solutions containing herbicides (amiben or bromoxynil) because of the effect of the herbicide on enzyme development. or synthesis during germination. Inhibition of GA,-
Fig. 1. Effects of menazon, disulfoton, and GS-14254 content of free amino acids , reducing sugars , seeds of mung beans and wheat .
on rate of respiration [ 11, and the and starch  of the germinating
enhanced synthesis of a-amylase by several herbicides has been reported . Again there is a species difference. The inhibition by pesticides of amylase activity or carbohydrate metabolism was especially pronounced in wheat and barley seeds. Mung bean and squash seeds were tolerant to the same concentrations of the pesticides [42,46]. The greater tolerance of mung beans may be attributed to the incomparatively large protein reserves since sulfhydryl groups of proteins have protective effects in biological systems. Alternatively, certain detoxifying enzyme systems, comparable to microsomal enzyme induction in animals, might have been more active in mung beans. Frear and his co-workers  have isolated from cotton plants a mixed-function oxidase enzyme that metabolized 3-(phenyl)-1 -methylurea. Pesticides inhibiting one enzyme may not inhibit another. Young and Varner [ 121 found that inhibitors of amylase and phosphatase syntheses did not have significant effect on proteolytic activity, although de novo synthesis of proteolytic enzymes occurs in the cotyledons during germination. Ashton et al.  demonstrated that herbicides varied widely in their abilities to inhibit increase in proteolytic activity in germinating squash seeds. By
TABLE EFFECT LEUCINE
II OF PESTICIDES INTO PROTEIN
Control Menazon Disulfoton GS-14254 a Significantly b Significantly
ON THE UPTAKE OF GERMINATING
AND INCORPORATION MUNG BEANS
[L -'4 Cl-
Radioactivity Alcohol-soluble (dpm/8 seeds)
96450 82900b 88700b 86200b
53245 31935a 34455a 32280a
at 0.05 at 0.01
level. level [711
Fig. 2. Five-day germinated control and treated mung beans. 1, control; 2, menazon (250 ppm); 3, disulfoton (100 ppm); 4, GS-14254 (100 ppm); 5, alantolactone (100 mg/l); 6, usnic acid (250 mg/l). Note the healthy growth of mung bean seedlings without treatment. Germinating mung beans treated with menazon or disulfoton do not show further growth. Their shoots start thickening and roots are becoming brown due to necrosis. These symptoms are also seen in mung beans treated with GS-14254. In comparison, the natural compounds (alantolactone and usnic acid) are more toxic to seed germination
Fig. 3. Five-day germinated control and treated wheat seeds. 1, control; 2, menazon (250 ppm); 3, disulfoton (100 ppm); 4, (38-14254 (100 ppm); 5, alantolactone (100 ppm); 6, usnic acid (250 ppm). Note the healthy growth of untreated wheat seedlings. Germinating wheat seeds treated with menazon or disulfoton show comparatively poor growth. The shoots and roots appear to be weak. These symptoms are severe in wheat seeds treated with GS-14254. Natural compounds (alantolactone and usnic acid) are included for comparison with pesticides and it is clear that the former are very toxic to germination [ 711.
using labelled amino acid uptake and noting any incorporation into the proteins of germinating seeds, we found that menazon, disulfoton, and GS-14254 not only inhibited uptake of [L-l 4 C] leucine but also significantly inhibited protein synthesis in germinating mung bean seeds (Table II). Litterst et al.  reported that disulfoton affected protein synthesis in HeLa cells. Similarly, Mann and his co-workers  showed the inhibition of protein synthesis by some herbicides. Thus, numerous factors may be involved in the inhibition of germination process by a pesticide treatment. Since the growth of both radicle and plumule in the pesticide-treated seeds was impaired (Figs. 2 and 3), it can be predicted that these pesticides may inhibit cell division as some of the natural compounds do. The phytotoxic effects of these pesticide chemicals on oxidative phosphorylation or on energy-rich compounds required for the synthesis of hydrolytic enzymes may not also be ruled out in the treated seeds. The amounts of menazon (250 ppm) and disulfoton (100 ppm) used in these experiments (Figs. 2 and 3) appear to be excessively large but they represent the range of concentra-
tion that occurs in the drills of commercial in-row or band treatments. Other workers [9,40] have also used similar concentrations. In regard to menazor (250 ppm) only small residues were detected after 2 years and also at 25C ppm it was much less harmful than phorate. In using the high concentrations of these insecticides Way and Scopes  reported, “the work with concen trations as large as 250 ppm of insecticide in soil is not merely of academic interest, because even higher concentrations may occur locally in commercial spot or in-row treatments.” Toxic effects
on cell structures
From the onset of germination, metabolic changes are initiated in seeds from the cellular to the tissue level. Storage reserves are solubilized to smaller units in the cotyledons or endosperm tissue and new cell constituents are synthesized in the growing embryonic tissue. Since the advent of histochemical techniques and electron microscopy, changes in cell constituents and cellular fine structures with respect to cell organelles have been studied during germination by a number of investigators [ 511. Unfortunately, studies on histochemical and fine structural changes during germination of seeds
Fig. 4. Electron micrograph tissue of the control mung mitochondrion; v, vacuole; endoplasmic reticulum and during germination [ 711.
of transverse sections near the embryo of the beans at day 3 of germination (X 14 400). cw, er, endoplasmic reticulum. Note the presence the vacuoles formed due to the depletion of
cotyledonary cell wall; m, of extensive food reserves
treated with pesticides are not extensively documented. Recently, we have reported that menazon, an organophosphate insecticide, inhibited mung bean germination, and that the cotyledons, when examined histochemically, showed abundant starch grains and protein bodies with lesser amounts of nucleic acids as compared to untreated seeds [ 521. In electron microscopic examination, extensive endoplasmic reticulum and ribosomes along with vacuoles and normal mitochondria were seen in control tissues, whereas undigested protein bodies and a disrupted cytoplasmic organization were conspicuous in the menazon-treated mung beans (Figs. 4 and 5). The presence of undigested protein bodies in the pesticide-treated seeds might be the secondary effect of the pesticidal action. Most often pesticides and especially herbicides attack cell membranes and cause disruption of the cytoplasmic organization. Hallam [ 531 observed disruptions in the chloroplast membranes, plasmalemma, and endoplasmic reticulum within 4 h after treatment of bean leaves with 2,4-D. According to another report, a number of herbicides and fungicides disrupted the membrane structures of Chlorella cells and the damage was attributed to the free radical theory [ 541. It is well known that some toxicants are metabolized to free radical forms in living cells and the subsequent attack on membrane lipids by the free radicals
Fig. 5. Electron micrograph of transverse sections near the embryo of the cotyledonary tissue of the menazon-treated mung beans at day 3 of germination (X 14 400). pb, protein body; m, mitochondrion; er, endoplasmic reticulum. Note the presence of undigested large protein bodies and undeveloped endoplasmic reticulum and mitochondria [ 711.
result in the fragmentation of membrane structures. Diquat and paraquat also cause considerable damage to certain membrane systems and a similar mechanism of action was suggested for these herbicides [ 551. Mitochondria are also affected by pesticide treatment. Charnetski et al. [ 561 found multinucleate cells with the presence of intramitochondrial crystals in pea seedlings exposed to lindane and the number of crystals in mitochondria increased with the level of lindane. Other workers [ 57,581 reported that soybean mitochondria were swollen following 2,4-D treatment. In paraquat-treated flax leaves, Dodge [ 551 observed swelling and disruption of mitochondria and tonoplast. He suggested that with tonoplast disruption cell metabolism was irreversibly inhibited and subsequent cell death was expected. Necrotic areas were found in the root apex of pea seedlings treated with lindane [ 561. We have also observed necrosis of root tips of mung bean seedlings treated with various pesticides (Fig. 2). Ashton et al. [ 591 showed that bromacil treatment of oat seedlings resulted in cells with multiple nuclei. This effect was attributed to inhibited cell wall formation. Similar observation on incomplete cell wall formation induced by lindane treatment in pea seedlings was made by Charnetski and his coworkers [ 561 . Thus, several cytological studies have indicated that pesticide treatment may induce the polyploid cells in roots and shoots of crop plants .
Inhibition of germination tal contamination
as a means of detecting
The pesticidal residues in soil can be detected by allowing susceptible seeds to germinate in contaminated soil along with corresponding controls sowed in uncontaminated soil. The concentration of a particular pesticide present in the soil can be determined quantitatively. The dose corresponding to the percentage inhibition of germination by the unknown soil residue can be read off against a dosage-mortality curve constructed from a range of known dosages of pesticides made up freshly in soil. Similar bioassay techniques have been used by several workers [61,62] . In one method wheat seedlings were grown in soil from the treated plots and the systemic action of the insecticide in the seedling leaves was then determined quantitatively using an aphid . Another method was based on mortality of a collembolan placed in soil containing the unknown and known amounts of insecticide . The sensitivity of the methods depends upon the degree of toxicity of the given pesticide. Thus, the use of bioassay in the study of the detection of pesticidal residues is a valuable tool. Though in most cases no qualitative information will be obtained about a particular chemical present, the potential toxic effects on soil population or plants can be determined. In addition to the advantage of a screening test for the presence of toxicants, the use of bioassay procedure is useful as a confirmatory test to results obtained by other analytical procedures.
Significance and toxicological and on seeds It is apparent
of the presence that
of pesticides may adversely
affect seed germination and seedling growth of our economic crops. Furthermore, pesticides present in soil, or those used as seed dressings or applied to the growing cereal crops will be metabolized and varying amounts of the intact pesticide or metabolic products will translocate to maturing grains 1631. For example, a detailed study by Tomizawa and Sato  elucidated malathion and methyl parathion metabolism, translocation, and final concentration of metabolic products in the unripe grains of rice plants. Laws  has reviewed a good deal of information on residues persisting at time of harvest. Examination of certain root and oil crops indicates that some pesticides may accumulate in the root and oil seeds. Presence of pesticides in potatoes, peanuts, and soybeans has been associated with residues in soil  . If such residues are present in seeds of our food plants, they may seriously affect not only germination but may also present a possible health hazard. The fact that the presence of certain pesticides in and on seeds interferes with germination suggests the possibility of using germination as a simple indicator of toxic substances. In addition, the method may serve as a diagnostic tool to determine the biological actions of toxic compounds , as Chlorella has been used for the detection and estimation of toxic mold metabolites . Furthermore, sprouted beans are widely used in oriental foods as a chief source of vitamin C. Malt and brewery industries are essentially based on germination of grains. Use of seeds containing high concentrations of pesticide residues or of other toxicants in the commercial production of sprouts will be detrimental to industry as well as public health. In addition to the inhibition of seed germination of our economic crops, the presence of pesticides in our foods is undesirable since some of these chemicals have been found to be carcinogenic and teratogenic in laboratory animals. Golberg  reported that a variety of organophosphates and organochlorines have carcinogenic and teratogenic effects in mice, whereas common herbicide 2,4,5-T exhibited teratogenic properties. On the other hand, s-triazine derivatives have been found to be carcinogenic [ 701. Although much information is lacking with regard to pesticide residues found in food from crops treated in an approved manner and there is no evidence that these residues cause any ill effects to humans, available evidence indicates that some pesticides do exert damaging effects on seeds of food plants. REFERENCES 1 2 3 4 5 6 5 8 9
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