In vitro and in vivo impairment of embryo implantation by commonly used fungicide Mancozeb.

The fungicide Mancozeb is an endocrine-disrupting chemical and the mode of action of Mancozeb on embryo implantation is largely unknown. Mancozeb (1 and 3 µg/ml) significantly reduced Jeg-3 trophoblastic spheroids attachment to endometrial epithelial Ishikawa cells. Mancozeb treatment from gestation day (GD) 1 to GD8 or from GD4 to GD8 significantly lowered the number of implantation sites with higher incidence of morphological abnormalities in the reproductive tissues. However, these were not seen in the treatment from GD1 to GD4. Mancozeb at 30 mg/kg BW/d did not alter the expression of p53, COX-2, or PGFS transcripts in the uterus, but down-regulated the PGES transcript and protein. Mancozeb treatment in human endometrial stromal cells did not alter the decidualization response, but the morphological transformation was impaired. Taken together, exposure to Mancozeb affected embryo implantation probably through the modulation of decidualization and to delineate the exact mode of action needs further investigations.

Eco-friendly PEG-based controlled release nano-formulations of Mancozeb: Synthesis and bioefficacy evaluation against phytopathogenic fungi Alternaria solani and Sclerotium rolfsii.

Controlled release (CR) nano-formulations of Mancozeb (manganese-zinc double salt of N,N-bisdithiocarbamic acid), a protective fungicide, have been prepared using laboratory-synthesized poly(ethylene glycols) (PEGs)-based functionalized amphiphilic copolymers without using any surfactants or external additives. The release kinetics of the developed Mancozeb CR formulations were studied and compared with that of commercially available 42% suspension concentrate and 75% wettable powder. Maximum amount of Mancozeb was released on 42nd day for PEG-600 and octyl chain, PEG-1000 and octyl chain, and PEG-600 and hexadecyl chain, on 35th day for PEG-1000 and hexadecyl chain, on 28th day for PEG-1500 and octyl chain, PEG-2000 and octyl chain, PEG-1500 and hexadecyl chain, and PEG-2000 and hexadecyl chain in comparison to both commercial formulations (15th day). The diffusion exponent (n value) of Mancozeb in water ranged from 0.42 to 0.62 in tested formulations. The half-release (t1/2) values ranged from 17.35 to 35.14 days, and the period of optimum availability of Mancozeb ranged from 18.54 to 35.42 days. Further, the in vitro bioefficacy evaluation of developed formulations was done against plant pathogenic fungi Alternaria solani and Sclerotium rolfsii by poison food technique. Effective dose for 50% inhibition in mgL-1 (ED50) values of developed formulations varied from 1.31 to 2.79 mg L-1 for A. solani, and 1.60 to 3.14 mg L-1 for S. rolfsii. The present methodology is simple, economical, and eco-friendly for the development of environment-friendly CR formulations of Mancozeb. These formulations can be used to optimize the release of Mancozeb to achieve disease control for the desired period depending upon the matrix of the polymer used. Importantly, the maximum amount of active ingredient remains available for a reasonable period after application. In addition, the developed CR formulations were found to be suitable for fungicidal applications, allowing use of Mancozeb in lower doses.

Whirling agitated single drop microextraction technique for the simultaneous analysis of Paraquat and Maneb in tissue samples of treated mice.

A new microextraction technique, whirling agitated single drop microextraction, has been proposed for the simultaneous analysis of Paraquat and Maneb in tissue samples before liquid chromatography with tandem mass spectrometry. This technique is based on the idea that the escalatory motion of the sample solution along with the extraction solvent increases the movement of molecules into the extraction solvent. In this technique, a simple handheld rotator was utilized to rapidly agitate the biphasic extraction system for the instantaneous extraction of targeted analytes. After extraction, the extracted phase was directly solidified by cooling in crushed ice and easily collected using a micro-spatula. The method showed good performance by achieving sensitive detection limits at 4.81 ng g(-1) (Paraquat) and 9.12 ng g(-1) (Maneb). Mean recoveries and enrichment factors were obtained >91.21% and up to 114 that ensured the preconcentration capacity of the method. The method precision was verified by evaluating intraday variation (n = 10) ≤4.57 (Paraquat) and ≤4.68 (Maneb) in terms of percent relative standard deviation. Additionally, method efficacy was assured by obtaining very little matrix interferences (≤3.11%). Moreover, the method suitability was also checked with its application on tissue samples of intraperitoneally treated mice with Paraquat and Maneb.

The Effect of Ascorbic Acid on Mancozeb-Induced Toxicity in Rat Thymocytes.

Mancozeb, as a dithiocarbamate fungicide, has been found to exhibit toxicological manifestations in different cells, mainly by generation of free radicals which may alter antioxidant defence systems in cells. The effect of mancozeb on the cells of a primary lymphoid organ has not been studied. In the present study, the effects of mancozeb (0.2, 2 and 5 µg/ml) or mancozeb+ascorbic acid (100 µg/ml), or ascorbic acid alone or control medium alone on the levels of cell viability, apoptosis, intracellular reactive oxygen species production (ROS), mitochondrial membrane potential (MMP) and ATP levels in rat thymocytes were examined in vitro. Cells treated with mancozeb displayed a concentration-dependent increase of hypodiploid cells and ROS production followed by markedly decreased viability of the cells, MMP and ATP levels. Application of ascorbic acid significantly reduced cytotoxicity in cell cultures treated with 0.2 and 2 µg/ml of mancozeb, together with significantly decreased ROS levels and increased MMP and ATP levels. In cells treated with 5 µg/ml of mancozeb, ascorbic acid failed to reduce toxicity while simultaneously increasing the apoptosis rate of thymocytes. These results suggest that ROS plays a significant role in mancozeb-induced toxicity, through alteration of mitochondrial function. Ascorbic acid administration reduced the toxicity rate in cells treated with lower mancozeb concentrations, while it may have the ability to shift cells from necrosis to apoptosis in the presence of highest mancozeb concentrations.

Mancozeb-induced genotoxicity and apoptosis in cultured human lymphocytes.

AIMS: Mancozeb is a dithiocarbamate fungicide known to be genotoxic and induces tumors in rodents at various sites. There is no report in the literature about its genotoxicity in humans. Here, we investigated the association between mancozeb exposure and induction of genotoxic and proapoptotic changes in cultured human lymphocytes (CHLs). MAIN METHODS: Lymphocytes were isolated from peripheral blood of healthy non-smoking donors. Induction of micronuclei and chromosomal aberrations was recorded both by conventional and flow cytometric methods. Annexin-V FITC was used for the differentiation of apoptotic and necrotic cells by flow cytometry. KEY FINDINGS: Mancozeb exposure (0.5, 2 and 5 µg/ml) to CHLs leads to significant induction in the frequency of chromosomal aberrations (CAs) and micronuclei (MN), in a dose-dependent manner. Concomitantly, pro-oxidant potential of mancozeb was also recorded, by increase in the levels of reactive oxygen species (ROS) generation. Our results demonstrated that ROS plays a critical role in the initiation of mancozeb induced apoptosis in CHLs through two ways, primarily through mitochondria-mediated pathway including induction of ROS, decrease in mitochondrial membrane potential (ΔΨm), along with cytochrome c release from mitochondria, and activation of the caspase cascade. The other pathway includes increase in ROS, which resulted in activation of NF-κB, expression of FasL and triggered FasL-dependent pathway, which also involves caspase-8. Therefore, exposure to mancozeb can lead to induction of apoptosis in CHLs through both mechanisms. SIGNIFICANCE: The results of study confirm that mancozeb exposure can induce genotoxicity and apoptosis in CHLs, thus pose a potential risk to exposed human population.

Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats.

Risk assessment is currently based on the no observed adverse effect levels (NOAELs) for single compounds. Humans are exposed to a mixture of chemicals and recent studies in our laboratory have shown that combined exposure to endocrine disrupters can cause adverse effects on male sexual development, even though the doses of the single compounds are below their individual NOAELs for anti-androgenic effects. Consequently, we have initiated a large project where the purpose is to study mixture effects of endocrine disrupting pesticides at low doses. In the initial range-finding mixture studies, rats were gavaged during gestation and lactation with five doses of a mixture of the fungicides procymidone, mancozeb, epoxyconazole, tebuconazole and prochloraz. The mixture ratio was chosen according to the doses of each individual pesticide that produced no observable effects on pregnancy length and pup survival in our laboratory and the dose levels used ranged from 25 to 100% of this mixture. All dose levels caused increased gestation length and dose levels above 25% caused impaired parturition leading to markedly decreased number of live born offspring and high pup perinatal mortality. The sexual differentiation of the pups was affected at 25% and higher as anogenital distance was affected in both male and female offspring at birth and the male offspring exhibited malformations of the genital tubercle, increased nipple retention, and decreased prostate and epididymis weights at pup day 13. The results show that doses of endocrine disrupting pesticides, which appear to induce no effects on gestation length, parturition and pup mortality when judged on their own, induced marked adverse effects on these endpoints in concert with other pesticides. In addition, the sexual differentiation of the offspring was affected. This as well as the predictability of the combination effects based on dose-additivity modelling will be studied further in a large dose-response study.

Yeast adaptation to mancozeb involves the up-regulation of FLR1 under the coordinate control of Yap1, Rpn4, Pdr3, and Yrr1.

FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomyces cerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed.

Temporal effects of paraquat/maneb on microglial activation and dopamine neuronal loss in older rats.

We investigated the effects of combined systemic exposure to the herbicide paraquat (PQ) and the fungicide maneb (MB) in 6-month-old rats, an animal model of Parkinson's disease resulting from environmental toxin exposure. Following two doses of PQ (10 mg/kg) and MB (30 mg/kg), 52% of animals developed fatal lung injury. Examination of the remaining animals showed degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta 6 weeks, but not 4 weeks, following PQ/MB. In contrast, microglial activation was observed at 4 weeks, but had abated by 6 weeks. Compared with our previous findings in younger rats, these results suggest increased susceptibility of older animals to lung and brain toxicity from PQ/MB exposure. Microglial activation preceded, and therefore likely contributed to, DA neurodegeneration. Further, electron microscopy revealed an abnormal appearance of the Golgi apparatus at 4 weeks that was confirmed using double immunostaining for tyrosine hydroxylase and Golgi. This suggests that PQ/MB causes protein processing dysfunction in nigral DA neurons that may be either a direct effect of PQ/MB or the result of microglial activation.

Morphometric analysis of follicular growth and biochemical constituents in albino rats exposed to mancozeb.

Mancozeb, an ethylenebisdithiocarbamate (EBDC), was administered orally at a dose of 700 mg/kg body weight/day to female virgin rats for 5, 10, 20, and 30 days to examine the effect on ovarian follicular development. No significant change occurred in the number of estrous cycles and the duration of proestrus, estrus, and metestrus, but mancozeb treatment for 5 days significantly increased the duration of diestrus. Mancozeb treatment for 10 days significantly increased the number of estrous cycle and the duration of estrus, with a concomitant significant increase in diestrus, but no change in proestrus and metestrus. With mancozeb treatment for 20 and 30 days, the number of estrous cycles and duration of proestrus, estrus, and metestrus were significantly decreased, with a concomitant significant increase in the duration of diestrus. Exposure of rats to mancozeb for 5 days resulted in a significant decrease in stage II and the total number of healthy follicles but no change in atretic follicles. Mancozeb treatment for 10 days resulted in a significant decrease in stages I, II, and IV and in the total number of healthy follicles. A significant increase in atretic follicles was found in rats treated with mancozeb for 20 and 30 days. No significant change occurred in body and organ weights in any group, but the thyroid weight of 20 and 30 days mancozeb-treated rats was significantly increased. The level of protein in the ovary was significantly decreased, but no change was found in the uterus and liver of mancozeb-treated animals. The level of glycogen was significantly decreased in the ovary and the uterus with mancozeb treatment, but not in the liver. With mancozeb treatment, the levels of phospholipids and neutral lipids were significantly increased in the liver but significantly decreased in the uterus. The change in the biochemical constituents of ovary, uterus, and liver was duration dependent. The results of the study thus indicate a marked disruption of the estrous cycle, pathological changes in the gonads, and organ-specific biochemical changes in rats after exposure to mancozeb.

Ethylenethiourea in urine as an indicator of exposure to mancozeb in vineyard workers.

In the present study, the personal exposure to mancozeb and/or ethilenethiourea (ETU) in 13 Italian vineyard workers and in 13 subjects without occupational exposure to pesticides was investigated. With this aim, the level of ETU in urine and the dermal exposure to mancozeb were determined. Baseline urinary ETU results were lower than the analytical limit of detection for all controls (<0.5 microg/g creatinine) and for ten workers (median <0.5, range <0.5-3.4 microg/g creatinine). In workers, urinary ETU was significantly increased at the end of shift (2.5, <0.5-95.2 microg/g creatinine) compared with baseline levels. End-shift urinary ETU was higher in operators using open tractors (n=7) than in those using closed tractors (n=5) (16.2 vs. 2.4 microg/g creatinine), but the difference was not significant (P=0.073). End-shift urinary ETU was positively correlated with dermal exposure to mancozeb determined both over the clothes and on the skin (Spearman's rho=0.770 and 0.702, P=0.009 and 0.024, respectively). Wine consumption positively influenced the excretion of ETU.

Rinsing and management of pesticides' containers.

In order to reduce the effects on the environment, it is necessary to improve the management of pesticides' containers. Usually, users burn or bury empty containers. These methods, even though decreasing must be avoided or even forbidden. Since 1996, empty containers are systematically collected in Belgium and are specifically removed by the firm Phytofar Recover created by the Belgian Federation of pesticides' manufacturers. Since the beginning, the recovery rate (percentage of containers recovered compared with the containers sold) goes on increasing to exceed 85% in 2001. These action and results are a world first (more than 500 tons of empty containers are collected yearly). Once collected, empty containers are subjected to the European Policy about toxic wastes since they contained dangerous products. Their removal must follow a specific removal process by incineration at very high temperature (> 1200 degrees C) with a specific filtration of the smoke. The treatment cost is high and reaches 2 Euros per kg of container. If the container is rinsed and the residue does not exceed 1000, 10,000 or 30,000 mg per kg of container (depending on the dangerousness of product: very toxic, corrosive or toxic), it will be considered as domestic waste and will therefore follow a much more economical energy production process. The study aims at determining the quantities of residue contained in empty containers and the parameters reducing the rinsing efficiency: the formulation (EC, WP, WG), the container's size, packaging's type (plastic container or paper bag), the rinsing technique. Almost 150 tests and analyses of residue have been carried out. A manual rinsing procedure has been set up in order to meet the standards about residue. Rinsing three times with an average volume of water (20 to 30%) allows to reach the lowest residue level. As bags containing powder (WG or WP) container not be rinsed, it is necessary to empty them completely. It is however difficult to reach the 1000 ppm residue limit.

Toxicity of a mancozeb containing fungicide formulation and CU-sulphate to chicken embryos after administration as single compounds or in combination.

Environmental pollution of metal modelled by copper-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air-chamber in a volume of 0.1 ml/egg on day 12 of incubation. The concentration of copper-sulphate was 0.01%. The applied concentration of Dithane M-45 fungicide formulation was 0.2%. Evaluation was done on day 19 of the hatching period. The combined administration of copper-sulphate and the fungicide formulation did not cause a significant reduction in body weight as compared to the control data and the results from individual toxicity study of the test materials. After the combined administration of copper-sulphate and the fungicide formulation the rate of embryomortality was 40%. The incidence of developmental anomalies were sporadic. Light microscopic findings exhibited a degenerative change in the liver tissue of combined administration group. Activities of GPT and GOT enzymes increased markedly in the combined administration group. In summary, it can be established that the interaction of copper-sulphate and an 80% mancozeb containing fungicide formulation (Dithane M-45) caused higher embryomortality with respect to the test of individual toxicity of copper-sulphate and fungicide in our study.

Synergism in mixtures of cymoxanil and mancozeb on Phytophthora infestans in vivo.

The preventive activity of 1:8 mixture of cymoxanil and mancozeb against Phytophthora infestans was higher than that of either the two single ingredients or the other nine mixtures. The synergistic interaction existed (synergy ratio 2.01) between the two at the mixing ratio of 1:8, whereas additive interaction (synergy ratios ranged from 0.73 to 1.34) existed at the mixing ratios ranging from 1:1 to 1:7, from 1:9 to 1:10, 1:8 was the optimal ratio. The preventive activity of 1:8 mixture was higher than the curative and the eradicative. In addition, the eradicative synergism of inhibiting sporangia production on lesions was stronger than the eradicative synergism of inhibiting lesion extension and suppressing infection of sporangia, and than the curative synergism of inhibiting lesion sporulation on detached potato leaflets.

The nigrostriatal dopaminergic system as a preferential target of repeated exposures to combined paraquat and maneb: implications for Parkinson's disease.

Experimental evidence supporting 1,1'-dimethyl-4,4'-bipyridinium [paraquat (PQ)] as a risk factor for Parkinson's disease (PD) is equivocal. Other agricultural chemicals, including dithiocarbamate fungicides such as manganese ethylenebisdithiocarbamate [maneb (MB)], are widely used in the same geographical regions as paraquat and also impact dopamine systems, suggesting that mixtures may be more relevant etiological models. This study therefore proposed that combined PQ and MB exposures would produce greater effects on dopamine (DA) systems than would either compound administered alone. Male C57BL/6 mice were treated twice a week for 6 weeks with intraperitoneal saline, 10 mg/kg paraquat, 30 mg/kg maneb, or their combination (PQ + MB). MB, but not PQ, reduced motor activity immediately after treatment, and this effect was potentiated by combined PQ + MB treatment. As treatments progressed, only the combined PQ + MB group evidenced a failure of motor activity levels to recover within 24 hr. Striatal DA and dihydroxyphenylacetic acid increased 1-3 d and decreased 7 d after injections. Only PQ + MB reduced tyrosine hydroxylase (TH) and DA transporter immunoreactivity and did so in dorsal striatum but not nucleus accumbens. Correspondingly, striatal TH protein levels were decreased only by combined PQ + MB 5 d after injection. Reactive gliosis occurred only in response to combined PQ + MB in dorsal-medial but not ventral striatum. TH immunoreactivity and cell counts were reduced only by PQ + MB and in the substantia nigra but not ventral tegmental area. These synergistic effects of combined PQ + MB, preferentially expressed in the nigrostriatal DA system, suggest that such mixtures could play a role in the etiology of PD.

Assessment of toxicological effects of mancozeb in male rats after chronic exposure.

Mancozeb, an ethylenebisdithiocarbamate fungicide was administered orally to male rats at doses 0, 500, 1000 and 1500 mg/kg/day for 90, 180 and 360 days produced dose dependent signs of poisoning, loss in body weight gain and mortality. However the signs of toxicity and mortality were more pronounced initially at 0-90 days as compared to 90-360 days of treatment period. A significant increase in the relative weight of liver and slight decrease in the kidney weight were observed in animals exposed to mancozeb (1000 and 1500 mg/kg/day) for 180 and 360 days associated with pathomorphological changes in liver, brain and kidney. Mancozeb has produced significant enzymatic changes in the activities of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) throughout the period of study in a dose dependent manner. The alterations in the activity of enzymes associated with pathomorphological changes suggest that the chronic exposure of mancozeb produced significant toxicological effects in rats.

Effect of Ziram, Thiram, and Dithane M-45 on bone marrow cells of mice-assessed by micronucleus test.

Micronucleus test is an extensively used protocol to assess the mutagenicity of environmental chemicals. This was developed by Schmid and his co-workers (Matter and Schmid, 1971; Ledebur and Schmid, 1973). The micronucleus test is simple, quick and as sensitive as the chromosome aberration analysis. It is based on the principle that during anaphase, acentric chromatid and chromosome fragments lag behind, where as centric elements move towards the spindle pole. After telophase both the undamaged chromosomes and the centric fragments give rise to the daughter nuclei. The lagging elements are transferred into one or several secondary nuclei, which are as a rule much smaller than the main nucleus, and therefore called micronucleus (Schmid, 1973). The clastogenic effect of various chemicals is measured by micronucleus test. Erythrocytes are two types, the younger ones are polychromatic erythrocytes (PCE), which stain bluish and the older, the normo chromatic erythrocytes (NCE) which stain reddish. A few hours after the completion of last mitosis the erythroblasts expel their nucleus for unknown reasons and the micronucleus alone remains in the cytoplasm of the Polychromatic erythrocytes, and they are easily recognisable. Erythrocyte micronucleus represents the consequence of chromosomal aberrations induced during preceding mitotic division of erythrocytes (Matter and Grauwiler, 1974).

Carcinogenic activity of a carbamate fungicide, mancozeb on mouse skin.

Mancozeb, a polymeric complex of ethylene bis (dithiocarbamate) manganese with zinc salt is a protective fungicide. In the present study complete carcinogenic activity of mancozeb, has been observed following topical application on dorsal mouse skin. Female Swiss albino mice were exposed to mancozeb at a dose of 100 mg/kg body weight dissolved in 100 microliters dimethyl sulfoxide 3 times per week. Development of tumours was observed after 31 weeks (217 days) of mancozeb application. A high rate of mortality was observed after 54 weeks (378 days) of mancozeb application due to its toxicity and the study was terminated after 60 weeks. On histological examination, these tumours were found mostly to be benign in nature, e.g., squamous cell papillomas and keratoacanthomas.

Acute exposure to maneb alters some behavioral functions in the mouse.

Maneb, an organomanganese fungicide, is largely used in agricultural regions for control of field crop pathologies. Despite its apparent low toxicity, there are reports showing that maneb has harmful effects on peripheral and central nervous systems. In this work the effects of acute administration of maneb were studied on some experimental animal models. Male adult mice were treated with several doses of maneb, IP, and submitted to gross behavioral observation (200-1000 mg/kg) and measurement of locomotor activity, barbiturate-induced sleeping time, isolation-induced aggressiveness, catatonia, climbing behavior and of rota-rod performance (30, 60 and 100 mg/kg). The results showed that maneb has an inhibitory effect on locomotor activity and aggressiveness and increases barbiturate-induced sleeping time and haloperidol-induced catatonia. However, maneb did not affect the apomorphine-induced climbing behavior of animals. These data indicate that maneb has a CNS depressant-like effect, and suggest, at least partially, the involvement of dopaminergic systems in the mediation of this effect.

The behavioral effects of pesticides in male mice.

Male Swiss mice, 25-30 g, were utilized to define some of the behavioral effects of the herbicides Lasso [alachlor 43%; (A)], Basalin [fluchloralin 45%; (F)], Premerge 3 [dinoseb 51%; (D)], and the fungicide Maneb-80 [maneb 80%; (M)]. These compounds were tested for their effects on locomotor activity and for their ability to establish a conditioned taste aversion following oral or dermal exposure. Individual and grouped (N = 5) activity measures were assessed immediately following the dermal administration of the commercially available pesticide formulations. Grouped activity measures were also assessed following the oral administration of the compounds. Total activity was significantly (p less than 0.05) increased over vehicle controls in both grouped and individual subjects by A, F, and D following dermal administration. Grouped activity measures were also increased by A, F, D, and M following the oral administration of the compounds. Similar subjects were tested in a conditioned taste aversion paradigm using a normally preferred 0.3% saccharin solution. Animals were given 30 min access to the saccharin solution followed immediately by the administration of the pesticide or control solution. Twenty-four hours later, animals were given the choice of 2 solutions, one containing water and the other the 0.3% saccharin solution. The percent saccharin consumed and the total fluid intake were calculated for each group (N = 8/group). A, F, and D produced a significant aversion to (N = 8/group) the saccharin following both oral and dermal administration. Oral administration of M, but not dermal exposure, also resulted in a flavor aversion. Total fluid intake, however, was not altered by any of the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)