Effect of combined radioactive and chemical (heavy metals,herbicides) pollution on the yield of cytogenetic disruptions in the intercalary meristem of spring barley

The yield of aberrant cells and their loading with lesions of intercalary meristem of spring barley in conditions of the combined action of 137Cs, heavy metals and 2.4-D herbicide was studied. At simultaneous insertion of 137Cs and Cd into the soil, the type of non-linearity of the yield of aberrant cells was determined by radioactive pollution: in a range of low concentrations the synergistic effects took place, in a range of higher ones the antagonistic effects did. In the presence of cadmium, the severity of radiation induced damage to aberrant cells was significantly less. The interaction in 137Cs-Pb pair had an antagonistic character. The yield of aberrant cells was determined mainly by lead, whereas the radioactive pollution played the main role in loading of the aberrant cells with lesions. At the combined pollution with 137Cs and other contaminants, the antagonistic effects were more pronounced with Pb than with Cd, and further increased with the herbicide, both in the test on frequency of aberrant cells and the test on the loading of aberrant cells with lesions. In case of combination of the chemical agents the non-linear effects in test on loading of aberrant cells with lesions were mainly synergistic, whereas in the test on frequency of aberrant cells they were antagonistic. Only Pb-Cd pair and only in the test on loading of aberrant cells with lesions no significant non-linear effects were found. The results show high rate of the induction of synergistic and antagonistic effects in conditions of the combined action of low concentrations of the investigated agents on plants. These effects substantially determined the yield of cytogenetic disturbances in the studied range.     

Effect of separate radioactive and chemical pollutants on the yield of cytogenetic disruptions in the intercalary meristem in spring barley

Dependencies of the yield of cytogenetic disturbances within intercalary meristem cells of spring barley on the soil level of 137Cs (1.48-14.8 MBq/m2), Cd, Pb (2-50 and 30-300 mg per kilogram of the soil) and 2.4-D herbicide (1 or 2 kg/ha) had a non-linear character in the studied range. At low concentrations the yield of cytogenetic disturbances grew faster than at higher ones. Concentrations of lead in soil (at the level of maximum concentration limit) and doses of 2.4-D recommended for agricultural use resulted in the increase in the rate of aberrant cells. The observed rate of cytogenetic disturbances was comparable with the effect of the maximum studied level or the radioactive soil pollution. The heaviest damage to aberrant cells was found in the presence of 137Cs.     

Rhizobium leguminosarum bv. viciae 3841 Adapts to 2,4-Dichlorophenoxyacetic Acid with “Auxin-Like” Morphological Changes,Cell Envelope Remodeling and Upregulation of Central Metabolic Pathways

There is a growing need to characterize the effects of environmental stressors at the molecular level on model organisms with the ever increasing number and variety of anthropogenic chemical pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we determined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.     

A simple solid phase assay for the detection of 2,4-D in soil

Contaminated soils are usually characterized using chemical analyses. However, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. Thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. Although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies have been carried out in slurries or soil extracts rather than soil itself. The following study presents the development of a simple solid-phase bioassay for the direct detection of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) in soil using Ralstonia eutropha JMP 134-32, a luxCDABE-based 2,4-D whole cell bioreporter. The bioreporter was spotted onto glass microfibre filter discs that allowed its retrieval and analysis after exposure to 2,4-D amended soils. These disc-fixed cells responded in a concentration dependent manner to 2,4-D in solution (0-25 mg/L) and in spiked soil (0-50 mg/kg). The influence of environmental factors on bioavailability was demonstrated in soil with a low moisture content which prevented 2,4-D-induced bioluminescence but which did not affect bioluminescence from already induced cells. This rapid and low cost bioassay provides a proof of concept demonstrating that retrievable disk-fixed cells can be induced in soil, thus providing a measure of solid-phase bioavailability. This method overcomes some of the limitations associated with the inoculation and monitoring of bioreporters directly in soil. Additionally, this simple system should be amenable to use with other bioreporters.     

Effects of 2,4-D on mature and juvenile plants of water hyacinth (Eichornia crassipes (Mart.) Solms)

The effects of various concentrations of the herbicide 2,4-D were investigated on two vegetative forms of water hyacinth including juvenile and mature forms. The results obtained suggest that efficient chemical control of water hyacinth may be best applied at the early stages of vegetative development of the water hyacinth, i.e. at the juvenile phase. This would require the use of 2,4-D at a concentration of 60 000 ppm (2.2 kg/ ha) which equals to half the rate of 2,4-D normally used for control of water hyacinth in the Sudan. The lower rate of 2,4-D not only means a considerable reduction in quantity and cost of the consumed herbicide but also a substantial minimisation in the level at which pollution may build up in the White Nile of the Sudan.     

The problem of the transgeneration phenomenon of genome instability in sick children of different age groups after the accident at the Chernobyl Nuclear Power Plant

A complex genetic examination of children which belong to two cohorts and their parents were carried out. The first cohort included children and constantly living on territories contaminated with radionuclides (Novozybkov district, Bryansk region). They were subdivided in groups according to the ontogenetic age periods of development of their parents at the time of the Chernobyl accident. In the children born in 1986-1995 the level of aberrant genomes is significantly higher as compared to the control (p < 0.001). In children born in 1998-2002 the differences are insignificant (p > 0.05). The frequency of aberrant genomes had a tendency to decrease with the period of time between the birth date of a child and the moment of the accident. Analysis of the results of cytogenetic investigation for the same living on territories with different densities of radioactive contamination (zone I-- 627-688 kBq/m2, 137Cs and zone II-- 135-402 kBq/m2, 137Cs) revealed insignificant differences in the spectrum and average frequencies of chromosome aberrations. The second cohort included children born in 1987-1991 and 1993-2002 from irradiated fathers (Chernobyl clean-up workers) and unirradiated mothers living on territories without radionuclide contamination. These children also displayed increased frequencies of aberrant genomes as compared to the control (p < 0.001). The analysis of the dynamics years of birth of cytogenetic disturbances in the same cohorts of children showed the average frequencies of aberrant genomes remain higher than the control level. In most of the children of both cohorts the repair synthesis of genome DNA by gamma- and UV-radiation is reduced as compared to one in the children from the control group.     

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.     

Micronucleus frequency in Gomel (Belarus) children affected and not affected by thyroid cancer.

Cytogenetic monitoring was carried out on a group of children from Gomel (Belarus), one of the areas most severely affected by radioactive contamination following the accident at the Chernobyl nuclear power plant, in 1986. We performed the micronucleus test (MN) in binucleated lymphocytes of 42 children (mean age: 11+/-2.34 years), 16 of whom were affected by thyroid gland tumor. Thirty healthy children living in Pisa (mean age: 14.96+/-2.17 years) were enrolled in the study as controls. Thyroid tumor affected children living in Gomel showed a statistically significant increase in the frequency of micronucleated cells as compared with the healthy children from Pisa. Moreover, a significant correlation was found between MN frequency and both the presence of tumor and higher 137Cs contamination. In addition, higher 137Cs contamination was more frequently observed in tumor affected children. These results suggest that the increased MN frequency is attributable more to 137Cs contamination rather than to the presence of the tumor itself.     

Testicular steroidogenesis is not altered by 137 cesium Chernobyl fallout,following in utero or post-natal chronic exposure

The testis is especially sensitive to pollutants, including radionuclides. Following the Chernobyl nuclear power plant accident, several of these radionuclides were emitted and spread in the environment. Subsequently, children presented some disruptions of the endocrine system. To determine whether these disruptions were due to 137 cesium (¹³⁷Cs) exposure, the effects of chronic contamination with low doses of ¹³⁷Cs in utero or from birth on testicular steroidogenesis in rats were studied. Contamination was continued for 9 months. No modification was observed in circulating level of hormones (17β-estradiol, testosterone, follicle-stimulating hormone, luteinizing hormone) following in utero or post-natal contamination. Expression of several genes involved in testicular steroidogenesis was affected (cyp19a1, fxr, sf-1), without modification of protein expression or activity. Our results suggest that growing organisms may be affected at the molecular level by ¹³⁷Cs contamination at this post-accidental dose.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Clastogenicity of pentachlorophenol, 2,4-D and butachlor evaluated by Allium root tip test

The meristematic mitotic cells of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. For assessing genotoxicity of pentachlorophenol (PCP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-chloro-2,6-diethyl-N-(butoxymethyl) acetanilide (butachlor), 50% effective concentration (EC(50)), c-mitosis, stickiness, chromosome breaks and mitotic index (MI) were used as endpoints of genotoxicity. EC(50) values for PCP and butachlor are 0.73 and 5.13 ppm, respectively. 2,4-D evidently induced morphological changes at higher concentrations. Some changes like crochet hooks, c-tumours and broken roots were unique to 2,4-D at 5-20 ppm. No such abnormalities were found in PCP and butachlor treated groups, however, root deteriorated and degenerated at higher concentrations (<3 ppm) in PCP. MI in 2,4-D showed a low average of 14.32% followed by PCP (19.53%), while in butachlor it was recorded 71.6%, which is near to the control value. All chemicals induced chromosome aberrations at statistically significant level. The highest chromosome aberration frequency (11.90%) was recorded in PCP at 3 ppm. Large number of c-mitotic anaphases indicated that butachlor acts as potent spindle inhibitor, whereas, breaks, bridges, stickiness and laggards were most frequently found in PCP showing that it is a potent clastogen.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Uptake and development of phytotoxicity following exposure to vapour of the herbicide C 2,4-D butyl by tomato and lettuce plants

Tomato and lettuce plants were exposed to vapour of the herbicide ¹⁴C 2,4-D butyl at concentrations in the range of 1–660 pg/l for 6, 24 or 72 hr. The relationship between herbicide uptake, measured as amount of radiolabel in the plant, and vapour concentration was linear and independent of the duration of exposure for both species. The rate of uptake of herbicide vapour at any one concentration by tomato was about twice that of lettuce. Immediately after exposure, the leaves of lettuce plants contained similar amounts of herbicide residue, whereas leaves at the base of tomato plants usually contained the least herbicide, and those at the apex the most. In terms of leaf area, both species had highest residue contents in the apical leaves. Different exposure periods did not affect the amounts of herbicide residue in the apical leaves or the leaf below, indicating that herbicide present in the apex is largely due to uptake and not to translocation. Both species developed visible symptoms of phytotoxicity following exposure, the severity being directly related to the amount of herbicide vapour received. Leaf contents of herbicide residue in tomato plants 40 days after exposure were related to the total plant content measured immediately after exposure; the leaf below the apex at the time of exposure retained about 17% of the herbicide originally present. Shoot dry weight, 40 days after exposure, was reduced by 10% following uptake of about 250 ng/plant in tomato, whereas for lettuce, about twice this amount of 2,4-D butyl was required to cause noticeable weight reduction. Doses of this magnitude are of the order of 10⁻⁵ the amount used in field application, and so it is clear that 2,4-D vapour is a potential hazard to tomato and lettuce crops.     

The analysis of coefficient of 137Cs transfer from soil into potato in relationship with 137Cs content in soil and plant mineral feed

The soil-potato transfer factor for 137Cs (TF) was estimated by using results of 137Cs activity concentration measurements in 214 samples of soil and potato taken at fields with various level of contamination with 137Cs. The relationships between the coefficient TF and soil characteristics (acidity pH (KCl), content of K2O, P2O5, CaO and MgO in soil) and soil contamination with 137Cs have been analysed. The results show that the TF values tend to decrease with increasing concentration of 137Cs, K2O, P2O5, and CaO in considered sod-podsolic sandyloam soil. The regression function describing the TF dependence of 137Cs, K2O, P2O5, and CaO content in soil has been derived.     

Simultaneous assessment of the effects of an herbicide on the triad: rhizobacterial community, an herbicide degrading soil bacterium and their plant host

Aims

This work addresses the relevant effects that one single compound, used as model herbicide, provokes on the activity/survival of a suitable herbicide degrading model bacterium and on a plant that hosts this bacterium and its bacterial rhizospheric community.

Methods

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), on Acacia caven hosting the 2,4-D degrading bacterium Cupriavidus pinatubonensis JMP134, and its rhizospheric microbiota, were simultaneously addressed in plant soil microcosms, and followed by culture dependent and independent procedures, herbicide removal tests, bioprotection assays and use of encapsulated bacterial cells.

Results

The herbicide provokes deleterious effects on the plant, which are significantly diminished by the presence of the plant associated C. pinatubonensis, especially with encapsulated cells. This improvement correlated with increased 2,4-D degradation rates. The herbicide significantly changes the structure of the A. caven bacterial rhizospheric community; and it also diminishes the preference of C. pinatubonensis for the A. caven rhizosphere compared with the surrounding bulk soil.

Conclusions

The addition of an herbicide to soil triggers a complex, although more or less predictable, suite of effects on rhizobacterial communities, herbicide degrading bacteria and their plant hosts that should be taken into account in fundamental studies and design of bio(phyto)remediation procedures.     

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mol photons/m²s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.This revised version was published online in November 2004 with corrections to Volume 48.     

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.     

Influence of herbicide, 2,4-dichlorophenoxy acetic acid, on haemocyte DNA of in vivo treated mussel

The influence of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) on haemocyte DNA of in vivo treated mussels Mytilus galloprovincialis has been investigated by flow cytometry and epifluorescence microscopy. Haemocyte proliferation and atypical flow cytometric DNA histograms were observed in mussels treated with 20 and 100 μg/g of 2,4-D. The stimulation of proliferation by 2,4-D was also obvious by DNA labelling with BrdU followed by FITC conjugated anti-BrdU MoAb visualised by epifluorescence microscopy. An apoptotic sub-G₁ peak resulted in mussels that were exposed to higher doses of herbicide at 100 and 500 μg/g as well as subpopulation could be detected by flow cytometric analysis. In these experiments morphological changes characteristic for apoptotic cells were looked for by fluorescence microscopy. A low percentage of cells in S as well as in G₂M phase indicating G1 arrest were detected in haemocytes from these mussels that had survived 4 days of 20 μg/g 2,4-D exposure. In addition, sister-chromatid exchanges (SCE) could be seen with the immunolabelling BrdU method. Thus, in vivo treatment and the subsequent uptake of 2,4-D causes serious genetic consequences and raises concerns regarding the potential overall fitness and health effects in mussel populations.     

Assessing eco-toxicological effects of industrial 2,4-D acid iso-octylester herbicide on rat pancreas and liver

Abstract

We studied the eco-toxic and carcinogenic effects of a commonly used 2,4-D acid iso-octylester herbicide on rat liver and pancreas. The rats in Group 1 were fed a standard feed and the rats in Group 2 were fed with standard feed to which was added 200 mg/kg/day 2,4-D acid iso-octylester for 16 weeks. Azaserine, 30 mg/kg/body weight, was injected into rats of Groups 3 and 4 to investigate the effects of 2,4-D acid iso-octylester on the development of neoplasms. After feeding the rats with neoplasms in Group 4 with food including 200 mg/kg/day 2,4-D acid iso-octylester for 16 weeks, an autopsy was carried out on all animals. We found that 2,4-D acid iso-octylester caused the formation of atypical cell foci (ACF) in the pancreata and livers of rats. ACF that were formed experimentally by exposure to azaserine had increased diameter, volume and number of atypical cell foci/mm² and mm³ after exposure to 2,4-D acid iso-octylester. Our observations indicated that this herbicide potentially is a cancer initiator.