Development of an amperometric flow injection immunoanalysis system for the determination of the herbicide 2,4-dichlorophenoxyacetic acid in water

An amperometric flow injection immunoanalysis (FIIA) system based on an immunoreactor with immobilized biocomponents on a silica surface has been developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). In the antigen coating mode the hapten was immobilized and monoclonal primary antibody against 2,4-D together with alkaline phosphatase (AP)-labelled secondary antibody were used as sensing elements in a titration assay. In the antibody coating mode a biotinylated monoclonal antibody was immobilized on the surface of the immunoreactor and a 2,4-D-AP-conjugate was used for detection. For electrochemical measurements p-aminophenol enzymatically generated from p-aminophenyl phosphate was oxidized at a carbon working electrode at +150 mV versus Ag/AgCl. The system enabled the determination of 2,4-D in drinking water samples in the range from 0.2 to 70 micrograms/l. The whole system was computer controlled with a measuring time of 12 min for one determination.     

Metabolic alterations in hepatocytes promoted by the herbicides paraquat, dinoseb and 2,4-D

The cytotoxic effects of the herbicides paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) on freshly isolated rat hepatocytes were investigated. Paraquat and 2,4-D (1-10 mM) caused a dose and time dependent cell death accompanied by depletion of intracellular glutathione (GSH) and mirroring increase of oxidized glutathione (GSSG). Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000 fold lower than paraquat and 2,4-D), exhibited moderate effects upon the level of GSH and GSSG. These limited effects are at variance with significant effects upon the adenine and pyridine nucleotide contents. ATP and NADH levels are rapidly depleted by herbicide metabolism. This depletion is observed in the millimolar range for paraquat and 2,4-D and in the micromolar range for dinoseb. 2,4-D completely depletes cellular ATP, with subsequent cell death, as detected by LDH leakage. Paraquat rapidly depletes NADH, according to the redox cycling of the herbicide metabolism. The most effective compound is dinoseb since it exerts similar effects as described for paraquat and 2,4-D at concentrations 1000 fold lower. Simultaneously with NADH and ATP depletion, the levels of ADP, AMP and NAD+ increase in hepatocytes incubated in the presence of the herbicides. In contrast to NADH, the time course and extent of ATP depletion and fall in energy charge correlate reasonably with the time of onset and rate of cell death. It is concluded that the herbicides, paraquat and 2,4-D are hepatotoxic and initiate the process of cell death by decreasing cellular GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

A variable response of degrading bacteria to phosphorus added to natural water

The effect of inorganic phosphorus (P) on the degradation of 10 mg l-1 of para-nitrophenol (PNP) or 2,4-dichlorophenoxyacetic acid (2,4-D) by three test bacteria inoculated into Nile water samples was investigated. The response of the organisms to P depended mainly on their affinity for the available P. Thus, Corynebacterium sp. at an initial density of 3.3 x 10(4) cells ml-1 readily degraded 10 mg l-1 of PNP in filter-sterilized Nile water supplemented with 22.8 mg l-1 of P. The same effect was observed when Pseudomonas cepacia was inoculated into Nile water amended with PNP and supplemented with 2.28-22.8 mg l-1 of P. The bacteria grew in Nile water and the final densities were related to the level of the added P. On the other hand, the addition of P, at concentrations ranging from 2.28 to 22.8 mg l-1, to sterile Nile water inoculated with Pseudomonas sp. and amended with 10 mg l-1 of 2,4-D did not stimulate the degradation compared with that obtained with the unsupplemented samples. The affinity of the three strains to P was demonstrated in P-deficient medium amended with PNP or 2,4-D as a sole carbon source. The pH of the medium was adjusted with 0.1 mol l-1 Tris buffer. Pseudomonas sp. at an initial density of 3.3 x 10(4) cells ml-1 degraded 10 mg l-1 of 2,4-D in non-sterile Nile water without added P. A slight enhancement of degradation was observed in water samples amended with a high concentration of P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism mediating basolateral transport of 2,4-dichlorophenoxyacetic acid in rat kidney

The organic anions, p-aminohippurate (PAH) and fluorescein, are transported across the basolateral membrane of the renal proximal tubule in exchange for intracellular alpha-ketoglutarate (alpha KG), a mechanism indirectly coupled to sodium via Na+/alpha KG cotransport. To determine whether this mechanism mediates the basolateral transport of other organic anions, transport of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in rat renal cortical slices and basolateral membrane vesicles. In slices, uptake of 2,4-D increased steadily over time, approaching steady-state tissue/medium ratios of approximately 8 after 60 min. Probenecid, PAH and chlorophenol red inhibited steady-state uptake of 2,4-D. Accumulation of 10 microM 2,4-D was stimulated 2-fold by 60 microM glutarate; other dicarboxylic acids failed to stimulate uptake. In the presence of sodium, the addition of 5 mM LiCl or 2 mM ouabain to the bathing medium abolished glutarate stimulation. Removal of sodium from the bathing medium reversibly inhibited uptake as much as 75%. Furthermore, PAH inhibited 2,4-D uptake by slices in a dose-dependent manner, and increasing the external 2,4-D concentration decreased the inhibitory potency of PAH. In basolateral membrane vesicles, unlabeled 2,4-D inhibited sodium glutarate-coupled uptake of 3H-labeled PAH and 2,4-D in a concentration-dependent manner. Moreover, concentrative uptake of 2,4-D into vesicles could be driven by an outwardly directed gradient of glutarate or alpha KG that was generated by lithium-sensitive Na+/dicarboxylate cotransport or imposed experimentally. An outwardly directed gradient of unlabeled 2,4-D or PAH also stimulated uptake of 2,4-D. Based on these data, basolateral accumulation of 2,4-D by the renal proximal tubule is mediated by 2,4-D/alpha KG exchange, a mechanism energetically coupled to Na+/alpha KG cotransport and shared with PAH.     

Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.     

Medicine in the vocal arts

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the central nervous system (CNS) were studied in rats. Behavioural and neurochemical studies were performed. Results show that acute and oral administration of dimethylamine 2,4-D was able to decrease locomotion and rearing frequencies and to increase immobility duration of rats observed in an open-field test. Treatment of rats with p-chlorophenylalanine (PCPA) was unable to change rat's open-field behaviour; 5-hydroxytryptophan (5-HTP) administration not only increased locomotion and rearing frequencies but also decreased immobility duration. Pretreatment of the rats with PCPA and 5-HTP decreased and increased dimethylamine 2,4-D effects, respectively. The herbicide was not able to change the striatal levels of dopamine and homovanilic acid but decreased the striatal levels of serotonin (5-HT), as observed for the doses of 100 and 200 mg/kg and increased those of 5-hydroxyindoleacetic acid (5-HIAA) as measured after the 200 mg/kg dose treatment. When the levels of serotonin and 5-HIAA were measured at the brain stem level, only those of 5-HIAA were modified, being increased by diethylamine 2,4-D (60; 100 and 200 mg/kg); this increment on 5-HIAA levels was observed even 1 hr after pesticide administration. Further analysis showed that 2,4-D concentrations chromatographically detected both in serum and brain of the intoxicated animals were dose-dependent, being found as early as 1 hr after the smaller dose of the herbicide used (10 mg/kg). The results suggest that diethylamine 2,4-D modify 5-HT functional activity within the CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered behavioral responses in 2,4-dichlorophenoxyacetic acid treated and amphetamine challenged rats

Although the mechanism of 2,4-D neurotoxicity remains unknown the serotonergic system appears to mediate some of the effects of 2,4-D in rats as reported in our previous studies. In the present study we examine the concept that a challenge to a system may overcome compensatory mechanisms and thereby reveal otherwise hidden neurotoxicant-induced damage. We report the behavioral results of 50 or 100 mg/kg 2,4-Dichlorophenoxyacetic acid (2,4-D) acute exposure plus amphetamine challenge of rats. The "Serotonin Syndrome" (SS) involving prominently head movement, piloerection, moist fur, backing, hunching, Straub tail, fore paw tapping of the nose and pivoting, was exhibited by these rats, being females more affected than males. Immobility, social apathy and asymmetry were also observed. All behaviors were not seen in the 2,4-D treated rats. Stereotyped behaviors were observed earlier and were more prolonged in 2,4-D treated and amphetamine challenged rats than in rats treated only with 5 or 10 mg/kg amphetamine. Spiperone blocked all the SS behaviors. In addition, in these rats, rearing and rotation behaviors were showed and were also sex dependent. We also demonstrate that haloperidol, in a non cataleptic dose, induced catelepsy in 2,4-D treated rats. 2,4-D appears to act through serotonergic and dopaminergic mechanisms. The intensity of the response is sex dependent. Our study demonstrates that 2,4-D plus amphetamine induces a Serotonergic Syndrome plus additional Dopaminergic modulation.     

The tfdK gene product facilitates uptake of 2,4-dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4)

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 microM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.     

Development of a displacement immunoassay by exploiting cross-reactivity of a monoclonal antibody

An enzyme-linked immunosorbent assay-based displacement assay was developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Advantage was taken of the cross-reactivity of a monoclonal anti-2,4-D antibody toward 2-methyl-4-chlorophenoxyacetic acid (MCPA). MCPA was conjugated with bovine serum albumin (BSA), immobilized on the surface of a microtiter plate, and saturated with the anti-2,4-D antibody. Due to the low affinity of the antibody toward MCPA (cross-reactivity of approximately 30%), the addition of 2,4-D resulted in a displacement of the antibody. Remaining antibodies were subsequently detected using a peroxidase-labeled goat anti-mouse antibody. The detection limit was as low as 0.1 microgram/liter for 2,4-D, which complies with the European Union Drinking Water Directives. When 2,4-D-BSA was used instead of MCPA-BSA conjugates, no significant displacement of bound antibody was observed.     

Construction of a physiologically based pharmacokinetic model for 2,4-dichlorophenoxyacetic acid dosimetry in the developing rabbit brain

A physiologically based pharmacokinetic (PBPK) model that describes the kinetics of organic anions by using 2,4-dichlorophenoxyacetic (2,4-D) as a representative compound was constructed for the developing rabbit brain at near-term pregnancy (Gestation Day 30). The model consisted of brain, body, and venous and arterial compartments for the mother which were linked to the fetus by a placenta. Maternal brain compartments in the model were brain plasma, cerebrospinal fluid (CSF), and brain tissue including hypothalamus, caudate nucleus, hippocampus, forebrain, brainstem, and cerebellum. The fetus consisted of brain, body, amniotic fluid, and venous and arterial compartments. the maternal body had both a central and a deep compartment; the fetal body had only one compartment. Maternal blood flow to the fetus was modeled as blood flowing to the placenta, where it was equilibrated before it reached the fetus. The brain uptake was membrane-limited by the blood-brain barrier, with saturable clearance from the CSF into the venous blood by the choroid plexus in both fetus and mother. The model was used to compare concentrations of 2,4-D in maternal and fetal brain, maternal and fetal plasma, and amniotic fluid over time with experimental data from pregnant rabbits given 2,4-D intravenously (1, 10, or 40 mg/kg). The model adequately simulated the 2-hr time course of 2,4-D concentrations in both mother and fetus. With continued development, this generic PBPK model should be a useful tool for evaluating the safety of organic acid neurotoxicants in the developing brain.     

Enzyme immunoassay based survey of precipitation and surface water for the presence of atrazine, metolachlor and 2,4-D

The suitability of enzyme immunoassay (EIA) as a method of analysis for 2,4-D, atrazine and metolachlor contamination in water samples was determined by comparing EIA results to gas chromatography (GC) results. The comparison of EIA and GC results yielded a correlation coefficient of 0.92, 0.98 and 0.92 for 2,4-D, atrazine and metolachlor, respectively. EIA was used to monitor seasonal trends in the concentrations of 2,4-D, atrazine and metolachlor in surface water and precipitation throughout the province of Ontario, Canada. 2,4-D was detected in excess of 4 micrograms/L in urban creeks during the period of application. Concentrations of 43 and 9 micrograms/L of atrazine and metolachlor, respectively, were detected during the field application period in surface water samples from the Kintore Creek watershed. The levels of 2,4-D, atrazine and metolachlor detected exceeded the Canadian Water Quality Guidelines for the protection of fresh water aquatic life. Concentrations as high as 445 and 322 ng/L of atrazine and metolachlor, respectively, were detected in precipitation samples collected from 17 locations in Ontario during the herbicide application period. The EIA was shown to be qualitatively and quantitatively comparable to GC analysis.     

Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid

The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate. TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine. In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation. The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA. The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases. Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D. Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate. The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions. On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D. The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events.     

Circadian periodic response of Phaseolus vulgaris l. to 2,4-dichlorophenoxyacetic acid

The susceptibility of bean plants, Phaseolus vulgaris L., cv. Executive, to 2,4-dichlorophentoxyacetic acid (2,4-D) appeared to depend upon the time of application. Oscillations in response to 2,4-D were evident in plants subjected to conditions of alternating light and dark spans, continuous illumination or darkness. The fresh and dry weight of plant material was generally less when 2,4-D was applied to plants near the later portions of the light span.     

2,4-Dichlorophenoxyacetic acid action on in vitro protein synthesis and its relation to polyamines

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the in vitro synthesis of proteins was studied in Chinese hamster ovary cells. A remarkable inhibition of the synthesis of proteins was observed when cells grew for 24 h in presence of 1 mM 2,4-D. This effect was reversed by adding 0.1 mM of the three polyamines (putrescine, spermidine and spermine) to the cultured cells. The mRNA is not altered, indicating that the 2,4-D action is located at a different locus, which may be the ribosomes. From these studies, one can speculate that the alterations in the protein synthesis may be a consequence of the effect of 2,4-D on the polyamine metabolism.     

Degradation of atrazine and 2,4-dichlorophenoxyacetic acid by mycorrhizal fungi at three nitrogen concentrations in vitro

Nine mycorrhizal fungi and free-living saprophytic microorganisms were tested for their ability to degrade two chlorinated aromatic herbicides at two herbicide concentrations and three nitrogen concentrations. Radiolabelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) were used as substrates at concentrations of 1 and 4 mM. After 8 weeks, none of the cultures tested grew at 4 mM 2,4-D. However, when the 2,4-D concentration was reduced to 1 mM, Phanerochaete chrysosporium 1767 had the highest level of 2,4-D mineralization and degradation under all nitrogen conditions. All cultures tested grew at both atrazine concentrations. In all cases, the ericoid mycorrhizal fungus Hymenoscyphus ericae 1318 had the highest level of atrazine carbon incorporated into its tissue. In general, as the nitrogen concentration increased, the total herbicide degradation increased. All of the cultures, except for Rhizopogon vinicolor 7534 and Sclerogaster pacificus 9011, showed increased degradation at 4 mM compared with 1 mM atrazine. The ability to degrade these two herbicides thus appeared to be dependent on the fungus and the herbicide, with no correlation to fungal ecotype (mycorrhizal versus free living).     

2,4-D and alkaloid accumulation in periwinkle cell suspensions

Omission of 2,4-D from culture medium during one subculture of Catharanthus roseus cells, strain C20, resulted in an increased alkaloid accumulation, without effect on growth. Alkaloid accumulation, rather than growth, seemed to be more sensitive to 2,4-D. 2,4-D inhibited alkaloid accumulation essentially during growth phase, but its inhibitory effect during this period was partially reversible. As this reversibility was underlined only during the stationary phase, this suggested that this action could be situated upstream in a terpenoid non-specific pathway. 2,4-D feeding showed that inhibition is weaker and weaker as the alkaloid accumulation period proceeds. Auxin action during this period could take place downstream in specific alkaloid pathways. The lower alkaloid accumulation obtained after loganic acid feeding compared to that obtained with secologanin and loganin could indicate that loganic acid methylation should be one of the 2,4-D target(s).     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D

The responses of three different soil microbial communities to the experimental application of 2,4-dichlorophenoxyacetic acid (2,4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2,4-D, and one had no prior direct application of any herbicide. Dominant 2,4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2,4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2,4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

Interaction of 2,4-dichlorophenoxyacetic acid (2,4-D) with cell and model membranes

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a component of the "agent orange' whose toxicity has been extensively studied without definite conclusions. In order to evaluate its perturbing effect upon cell membranes, 2,4-D was made to interact with human erythrocytes and molecular models. These studies were performed by scanning electron microscopy on red cells, fluorescence spectroscopy on dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles and X-ray diffraction on multilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE). It was observed that 2,4-D induced a pronounced shape change to the erythrocytes. This effect is explained by the herbicide interaction with the outer monolayer of the red cell membrane.     

Phytohormone regulation of isoperoxidases in Catharanthus roseus suspension cultures

Peroxidase (POD) activity was investigated in Catharanthus roseus cell suspensions cultured under different hormonal conditions. Depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) from the culture medium enhanced POD activity in cells and spent medium. Addition of phytohormones, in particular the auxin 2,4-D, reduced POD activity in medium and cellular compartments and enhanced ionically cell-wall bound POD. The differential modulation of POD is due to hormone effects on synthesis and/or accumulation of POD, rather than on the secretion process. Qualitative analysis showed that 2,4-D, but not cytokinins, regulated the synthesis of a basic isoform. The cytokinin treatment seemed to affect acidic rather than basic isoforms. The presence of basic POD is correlated with the capacity of cells to produce indole alkaloids. The major extracellular basic isoperoxidase was purified to homogeneity from culture medium of Catharanthus roseus cell suspensions. The isolated peroxidase is a haem protein with a M(r) of 33,000 and a pI close to 9. The effect of pH on peroxidase activity was studied using guaiacol as substrate and the optimum pH determined at 25 degrees was 6.0. This enzyme acted on guaiacol, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine (o-PD) and pyrogallol, but had no effect on syringaldazine or coniferyl alcohol substrates.     

1H NMR study of dynamics and thermodynamics of acid-alkaline transition in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi)

Molluscicidal activity of the herebicides 2,4-D and Graminol, as well as both extracts and dry powder of the plant Azolla pinnata were evaluated against B. alexandrina snails. It was observed that 2,4-D proved to be the most toxic compound among he tested ones, showing LC90 of 52 ppm after 24 h of exposure. Ethanol extract of Azolla showed the highest molluscicidal activity against the tested snails compared with the other extracts and dry powder (LC90 = 3300 ppm). Ethanol extract at 6600 ppm after 3 h of exposure killed 100% and 19.4% of S. mansoni miracidia and cercariae, respectively. The molluscicidal activity of 2,4-D was not influenced by the presence of Azolla (900 plants/liter) for 7 days, while Graminol effect was significantly reduced. However, the infectivity of S. mansoni miracidia to B. alexandrina snails was not affected by Azolla existence.