Kinetics of the metabolic effects, distribution spaces and lipid-bilayer affinities of the organo-chlorinated herbicides 2,4-D and picloram in the liver.

Tordon® is the commercial name of a mixture of two organo-chlorinated herbicides, 2,4-D and picloram. Both compounds affect energy transduction in isolated mitochondria and the present study aimed at characterizing the actions of these two compounds on liver metabolism and their cellular distribution in the isolated perfused rat liver. 2,4-D, but not picloram, increased glycolysis in the range from 10 to 400 µM. The redox potential of the cytosolic NAD+-NADH couple was also increased by 2,4-D. Both compounds inhibited lactate gluconeogenesis. Inhibitions by 2,4-D and picloram were incomplete, reaching maximally 46% and 23%, respectively. Both compounds diminished the cellular ATP levels. No synergism between the actions of 2,4-D and picloram was detected. Biotransformations of 2,4-D and picloram were slow, but their distributions occurred at high rates and were concentrative. Molecular dynamics simulations revealed that 2,4-D presented low affinity for the hydrophobic lipid bilayers, the opposite occurring with picloram. Inhibition of energy metabolism is possibly a relevant component of the toxicity of 2,4-D and of the commercial product Tordon®. Furthermore, the interactions of 2,4-D with the membrane lipid bilayer can be highly destructive and might equally be related to its cellular toxicity at high concentrations.

Persistence of auxinic herbicides applied on pasture and toxicity for succeeding crops.

The aim of this work was to determine the persistence of auxinic herbicides applied on tropical pasture and toxicity for succeeding crops. The herbicides were applied in an area of dystrophic red‒yellow latosol with pasture infested of weeds. At 40, 80, and 280 days after application of herbicide, the soil samples were collected at depths of 0 to 20 cm. Soil with residues of 2,4-D, 2,4-D + picloram, triclopyr, and a soil without herbicide application were analyzed with six replicates. Seven crops were cultivated in these soils: cucumber (Cucumis sativus L.), velvet bean [Mucuna pruriens (L.) DC.], pigeon pea [Cajanus cajan (L.) Millsp.], alfalfa (Medicago sativa L.), lablab bean [Lablab purpureus (L.) Sweet], corn (Zea mays L.), and sorghum [Sorghum bicolor (L.) Moench]. The plants of cucumber, pigeon pea, and alfalfa were the most susceptible to the auxinic herbicide residues. However, the lablab bean was the only one among the dicot evaluated that showed tolerance to the 2,4-D + picloram residual when cultivated in soils at 280 days after application of herbicide. Corn and sorghum showed lower chlorophyll content in soils with 2,4-D + picloram residual up to 80 days after application of herbicide.

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

Toxicity of neurons treated with herbicides and neuroprotection by mitochondria-targeted antioxidant SS31.

The purpose of this study was to determine the neurotoxicity of two commonly used herbicides: picloram and triclopyr and the neuroprotective effects of the mitochondria-targeted antioxidant, SS31. Using mouse neuroblastoma (N2a) cells and primary neurons from C57BL/6 mice, we investigated the toxicity of these herbicides, and protective effects of SS1 peptide against picloram and triclopyr toxicity. We measured total RNA content, cell viability and mRNA expression of peroxiredoxins, neuroprotective genes, mitochondrial-encoded electron transport chain (ETC) genes in N2a cells treated with herbicides and SS31. Using primary neurons from C57BL/6 mice, neuronal survival was studied in neurons treated with herbicides, in neurons pretreated with SS31 plus treated with herbicides, neurons treated with SS31 alone, and untreated neurons. Significantly decreased total RNA content, and cell viability in N2a cells treated with picloram and triclopyr were found compared to untreated N2a cells. Decreased mRNA expression of neuroprotective genes, and ETC genes in cells treated with herbicides was found compared to untreated cells. Decreased mRNA expression of peroxiredoxins 1-6 in N2a cells treated with picloram was found, suggesting that picloram affects the antioxidant enzymes in N2a cells. Immunofluorescence analysis of primary neurons revealed that decreased neuronal branching and degenerating neurons in neurons treated with picloram and triclopyr. However, neurons pretreated with SS31 prevented degenerative process caused by herbicides. Based on these results, we propose that herbicides--picloram and triclopyr appear to damage neurons, and the SS31 peptide appears to protect neurons from herbicide toxicity.

An ecological risk assessment of the acute and chronic toxicity of the herbicide picloram to the threatened bull trout (Salvelinus confluentus) and the rainbow trout (Onchorhyncus mykiss).

We conducted acute and chronic toxicity studies of the effects of picloram acid on the threatened bull trout (Salvelinus confluentus) and the standard coldwater surrogate rainbow trout (Oncorhynchus mykiss). Juvenile fish were chronically exposed for 30 days in a proportional flow-through diluter to measured concentrations of 0, 0.30, 0.60, 1.18, 2.37, and 4.75 mg/L picloram. No mortality of either species was observed at the highest concentration. Bull trout were twofold more sensitive to picloram (30-day maximum acceptable toxic concentration of 0.80 mg/L) compared to rainbow trout (30-day maximum acceptable toxic concentration of 1.67 mg/L) based on the endpoint of growth. Picloram was acutely toxic to rainbow trout at 36 mg/L (96-h ALC50). The acute:chronic ratio for rainbow trout exposed to picloram was 22. The chronic toxicity of picloram was compared to modeled and measured environmental exposure concentrations (EECs) using a four-tiered system. The Tier 1, worst-case exposure estimate, based on a direct application of the current maximum use rate (1.1 kg/ha picloram) to a standardized aquatic ecosystem (water body of 1-ha area and 1-m depth), resulted in an EEC of 0.73 mg/L picloram and chronic risk quotients of 0.91 and 0.44 for bull trout and rainbow trout, respectively. Higher-tiered exposure estimates reduced chronic risk quotients 10-fold. Results of this study indicate that picloram, if properly applied according to the manufacturer's label, poses little risk to the threatened bull trout or rainbow trout in northwestern rangeland environments on either an acute or a chronic basis.

Using accelerated life testing procedures to compare the relative sensitivity of rainbow trout and the federally listed threatened bull trout to three commonly used rangeland herbicides (picloram, 2,4-D, and clopyralid).

We conducted 96-h static acute toxicity studies to evaluate the relative sensitivity of juveniles of the threatened bull trout (Salvelinus confluentus) and the standard cold-water surrogate rainbow trout (Onchorhyncus mykiss) to three rangeland herbicides commonly used for controlling invasive weeds in the northwestern United States. Relative species sensitivity was compared using three procedures: standard acute toxicity testing, fractional estimates of lethal concentrations, and accelerated life testing chronic estimation procedures. The acutely lethal concentrations (ALC) resulting in 50% mortality at 96 h (96-h ALC50s) were determined using linear regression and indicated that the three herbicides were toxic in the order of picloram acid > 2,4-D acid > clopyralid acid. The 96-h ALC50 values for rainbow trout were as follows: picloram, 41 mg/L; 2.4-D, 707 mg/L; and clopyralid, 700 mg/L. The 96-h ALC50 values for bull trout were as follows: picloram, 24 mg/L; 2.4-D, 398 mg/L; and clopyralid, 802 mg/L. Fractional estimates of safe concentrations, based on 5% of the 96-h ALC50, were conservative (overestimated toxicity) of regression-derived 96-h ALC5 values by an order of magnitude. Accelerated life testing procedures were used to estimate chronic lethal concentrations (CLC) resulting in 1% mortality at 30 d (30-d CLC1) for the three herbicides: picloram (1 mg/L rainbow trout, 5 mg/L bull trout), 2,4-D (56 mg/L rainbow trout, 84 mg/L bull trout), and clopyralid (477 mg/L rainbow trout; 552 mg/L bull trout). Collectively, the results indicated that the standard surrogate rainbow trout is similar in sensitivity to bull trout. Accelerated life testing procedures provided cost-effective, statistically defensible methods for estimating safe chronic concentrations (30-d CLC1s) of herbicides from acute toxicity data because they use statistical models based on the entire mortality:concentration:time data matrix.

A toxicity view of the pesticide picloram when immobilized onto a silica gel surface.

The pesticide picloram (4-amino-3,5,6-trichloropicolinic acid) was anchored onto silica gel to yield a new surface. Isothermal microcalorimetry was applied to study the toxic effects caused to microbial activity of a typical Brazilian agricultural soil by application of free and immobilized picloram. The activity of the microorganisms in 1.50 g of soil sample was stimulated by addition of 6.0 mg of glucose plus 6.0 mg of ammonium sulfate under 34.8% controlled humidity at 298.15+/-0.02 K. The activity was recorded through power-time curves for increasing amounts of the active principle, varying from zero to 10.00 microg g(-1). The increasing amounts of picloram, either free or immobilized, caused a decrease of the original thermal effect. The calorimetric data showed that the anchored pesticide presented a much lower toxic effect than the free picloram on the microbial activity.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

Toxic effect caused on microflora of soil by pesticide picloram application.

The toxic effect of the herbicide picloram on the microbial population of a typical Brazilian red Latosol soil was studied in a series of microcalorimetric experiments. The activity of the soil was stimulated by the addition of 6.0 mg of glucose and 6.0 mg of ammonium sulfate, under 34.8% controlled moisture, to a 1.50 mg soil sample, at 298.15 +/- 0.02 K. The net thermal effect due to the addition of the picloram to the soil was determined by interpreting the power-time curves, which were recorded on the microcalorimeter. The total thermal effect evolved by the microorganisms was affected by the increasing doses of herbicide, and varied from 0 to 10.00 micrograms g-1. An increase in picloram exposure caused a decrease of the original thermal effect, reaching a null value above 20.89 micrograms of herbicide per gram of soil. The decreases of the thermal effect evolved by microorganisms and the increase of the lag phase period are associated with the death of the microbial population. The effects caused by picloram application in this typical Brazilian soil resulted in a strong effect on the soil microbial communities.

Effects of a herbicide formulation, Tordon 75D, and its individual components on the oxidative functions of mitochondria.

This investigation evaluates the toxicity of a herbicide formulation, as well as testing its active and other components (other components comprise all components of Tordon 75D excluding the active components: i.e. the solvents, triisopropanolamine and diethyleneglycol monoethyl ether, a silicone defoamer and a proprietary surfactant, polyglycol 26-2). The results showed that Tordon 75D (a mixture of the triisopropanolamine salts of 2,4-dichlorophenoxy acetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) and its other components) impaired the oxidative functions of submitochondrial particles (SMPs). The effective concentrations that caused 50% inhibition of SMP activity (EC50s) for Tordon 75D were in the low micromolar range for 2,4-D and picloram in the presence of the other components, while in the absence of the other components exposure to 136 times higher concentrations of the triisopropanolamine forms of 2,4-D and picloram administered as a mixture were required to inhibit the oxidative functions of SMPs. Tordon 75D also significantly decreased the respiratory control ratio of intact rat liver mitochondria. The results show that the toxic effects of Tordon 75D on SMPs (at the EC50) and intact rat liver mitochondria were not due to any additive or synergistic actions of a mixture of its active and other components, but rather were caused solely by the proprietary surfactant. Since mitochondria are responsible for over 90% of the energy production in all eukaryotic organisms, the use of the SMP assay provides a convenient in vitro assay for evaluating cellular toxicity and can be regarded as an informative screening assay when designing chemical products which contain mixtures of chemicals.

Tradescantia-micronucleus and -stamen hair mutation assays on genotoxicity of the gaseous and liquid forms of pesticides.

The clastogenic and mutagenic effects of the insecticide Dimethoate (Cygon-2E), herbicides Atrazine, Simazine (Princep), Dicamba (Banvel D) and Picloram (Tordon) were studied using the Tradescantia-micronucleus (Trad-MCN) and Tradescantia-stamen hair mutation (Trad-SHM) assays. In clone 4430, dimethoate fumes both significantly increased the pink mutation events and reduced the number of stamen hairs per filament with increasing dosages. The pink mutation events were elevated by the liquid treatment with Picloram at 100 ppm concentration. The result of Trad-MCN test on Dimethoate fumes was not significantly different between the control and treated groups. The herbicide Atrazine showed positive effects at 10-50 ppm dose (liquid) and signs of overdose at 100 and 500 ppm concentrations. Simazine was mildly positive in elevating the MCN frequencies in the dose range of 5 to 200 ppm (liquid doses). Both Dicamba and Picloram induced a dosage-related increase in MCN frequencies in the Trad-MCN tests using Tradescantia clone 03. However, in higher dosages (200 ppm or higher), there were signs of overdose, reduction of MCN frequencies and physical damage of the leaves and buds of plant cuttings.

Effect of roundup and tordon 202C herbicides on antibody production in mice.

Female CD-1 mice were exposed to Tordon 202C (2,4-dichlorophenoxyacetic acid [2,4-D] and picloram) or Roundup (glyphosate) in drinking water for 26 d at concentrations ranging from 0 to 0.42% or from 0 to 1.05%, respectively. The mice were inoculated with sheep red blood cells to produce a T-lymphocyte, macrophage dependent antibody response on day 21 of the herbicide exposure period. Tordon 202C dosing reduced weight gain and water consumption at the 0.42% level of exposure. Roundup exposure did not alter weight gain or water consumption. Antibody production was unaffected by Roundup dosing, suggesting that Roundup is unlikely to cause immune dysfunction under normal application conditions. In contrast, all levels of Tordon 202C exposure reduced antibody production by as much as 45%. The immunosuppressive activity of Tordon 202C was associated with levels more than 12 x the normal application level, although it was not determined which component of the formulation was responsible for the immunosuppression effect. The presence of immune alteration subsequent to exposure to Tordon 202C at levels marginally above the normal application levels suggests that chronic exposure to Tordon 202C in the environment has the potential to alter immune function.

Effect of Tordon 2,4-D 64/240 triethanolamine BR on the energy metabolism of rat liver mitochondria.

Tordon herbicide, which is a mixture of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D), depresses the phosphorylation efficiency of the rat liver mitochondria, as inferred from the decrease of the respiratory control coefficient and the ADP/O ratios when NAD(+)-dependent substrates were used; NADH oxidase and NADH cytochrome c reductase were also inhibited, without any effect on the other enzymatic complexes of the respiratory chain. Tordon (66.2 nmol picloram + 270 nmol 2,4-D mg-1 protein) affected the amplitude of swelling induced by glutamate, succinate, (N,N,N',N'-tetramethyl-p-phenyldiamine + sodium ascorbate and ATP. These results characterize an interaction of Tordon with complex I of the respiratory chain and also a partial collapse of the proton motive force of the mitochondrial inner membrane without affecting its elasticity.

Biochemical and toxicological studies on the mixtures of three commonly-used herbicides in mice.

Herbicidal-mixtures have not been adequately studied in biological systems. Therefore, mixtures of three commonly-used herbicides were evaluated. Alachlor (AL), atrazine (AT), picloram (PI), AL+AT, AT+PI, PI+AL and AL+AT+PI, at 10 ppm (in drinking water) of each herbicide, were provided to mice for 30, as well as for 90, days ad libitum; these herbicides and mixtures, at 100 mg/kg (in corn oil) of each herbicide, were also given to additional groups of mice by oral intubation daily for 21 days. In the 30-day test, the spleen/body weight ratios on Day 31 with respect to the control were increased in the AT (53%) and AL+AT (44%) groups. Decreases in the body weights were noted in the treated groups after Day 31 during the 90-day test. Serum glutamic-pyruvic transaminase (SGPT), glutamic-oxalacetic transaminase (SGOT) and alkaline phosphatase activities with AL+AT+PI were elevated by 36-92% on Day 91; SGPT (34%) and SGOT (73%) activities were increased with AL. During the 21-day oral intubation study, the mouse body weights in the mixture groups were generally lower than the control (p < or = 0.05). The liver/body weight ratios were elevated in all groups (16-38%); the spleen/body weight ratio increased with PI (50%). The kidney/body weight ratios were high with PI+AL and AL+AT+PI (p < or = 0.05). Excluding AT, the pentobarbital-induced sleep was less (51-77%) in the herbicide-treated groups. Also, necrosis of individual and small groups of hepatocytes was noted with the mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 2,4-dichlorophenoxyacetic acid and picloram on biotransformation, peroxisomal and serum enzyme activities in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) exposed to a mixture of picloram and 2,4-dichlorophenoxyacetic acid (2,4-D) for 10 days displayed increased activities of hepatic ethoxyresorufin O-deethylase (EROD), decreased serum chloride concentrations and decreased liver/body weight ratios. These changes were not observed in fish exposed to either compound alone. Neither compound, nor their mixture, increased hepatic peroxisomal catalase or lauroyl CoA oxidase activities. The results of this study indicate that 10-day exposure of 2,4-D or picloram does not induce peroxisomal enzymes in channel catfish; however, exposure to a mixture of 2,4-D and picloram may cause physiological effects to catfish not observed with either compound alone.

The effects of Tordon 202c exposure on urethan-induced lung adenoma formation in female CD-1 mice.

Female CD-1 mice were exposed to Tordon 202c, a herbicide containing 2,4-dichlorophenoxyacetic acid and picloram, in the drinking water for 15 w at concentrations ranging from 0 to 0.3% of the product formulation. After 3 w of the 15-w treatment period, the mice received 1.5 mg/g urethan ip. Pulmonary adenoma production was evaluated 12 w later. Tordon 202c exposure produced a dose-dependent increase in tumor number, but had no effect on tumor size. Urethan-induced sleeping times which reflected the rate of urethan metabolism or excretion were altered, but a specific dose-related effect which could be correlated with tumor production was not observed. This suggests that Tordon 202c exposure influences adenoma formation by immunological mechanisms rather than by causing indirect effects on urethan metabolism or excretion.

Chronic toxicity and oncogenicity of picloram in Fischer 344 rats.

The chronic toxicity and oncogenicity of the herbicide picloram was studied in male and female Fischer 344 rats administered 0, 20, 60, or 200 mg/kg.d technical-grade picloram via their feed for 2 yr. A comprehensive set of in-life and clinical pathology parameters was measured and an extensive list of tissues was examined grossly and by light microscopy from control and treatment groups of animals. The primary treatment-related effect observed in the study was hepatocellular swelling and altered tinctorial properties in the central regions of the liver lobules of both sexes of rats ingesting 60 or 200 mg/kg.d picloram. Males were more affected than females. Increases in liver weights accompanied these changes in both sexes of rats ingesting the high dose level of picloram. All other histopathologic lesions observed were typical of those that normally occur in aged Fischer 344 rats. There were no treatment-related increases in the incidence of any particular tumor type or in total tumors. No treatment-related effects were observed in rats ingesting 20 mg/kg.d of the test material.

Effects of preconceptional and gestational exposure to Tordon 202c on fetal growth and development in CD-1 mice.

Female CD-1 mice were exposed to Tordon 202c (a picloram and 2,4-D combination herbicide) in the drinking water at concentrations of 0.21, 0.42, and 0.84% for 60 days prior to mating with untreated males. One-half of the pregnant females subsequently continued treatment throughout gestation while the remaining females were maintained on distilled water. Fetal weight, crown-rump length, placental weight, and maternal gestational weight gain were reduced in a dose-dependent manner following combined preconceptional and gestational exposure. The incidence of malformed fetuses (cleft palate, renal agenesis, hydronephrosis, unilateral testicular agenesis, and umbilical hernia) and fetuses with variants (especially incomplete ossification of the skeleton) were increased in a dose-dependent manner following combined exposure. Increased maternal mortality and decreased preconception weight gain were observed in the highest-dosage group. Relative maternal liver weight was increased in a dose-dependent manner. The results suggest that combined preconceptional and gestational exposure to Tordon 202c is required for teratogenesis and fetal growth depression. Preconceptional exposure alone is not effective in increasing the risk for embryotoxicity.