Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

Responses of Hyalella azteca and Ceridaphnia dubia to reservoir sediments following Chelated Copper Herbicide Applications

In response to nuisance growths of algae and vascular plants, such as dioecious hydrilla ( Hydrilla verticillata L.f. Royle), copper formulations have been applied in lakes and reservoirs for a number of years. Concerns have arisen regarding the long-term consequences of copper applications and those concerns have appropriately focused on sediment residues. In this study, we evaluated the toxicity of sediments from treated (for a decade) and untreated areas in Lake Murray, South Carolina and estimated the capacity of those sediments to bind additional copper. Two sentinel aquatic invertebrates, Hyalella azteca Saussure and Ceriodaphnia dubia Richard, were used to measure residual toxicity of treated and untreated sediments from the field and after laboratory amendments. (PDF has 5 pages.)

Herbicide evaluation for the control of wild taro

Wild taro (Colocasia esculenta (L.) Schott), is an exotic, emergent perennial that has established in many shallow-water wetlands throughout the southern United States. Although wild taro is a cultivated crop in many tropical and subtropical areas of the world, its invasion in riverine and lacustrine wetlands in the U.S. has resulted in the loss of habitat for native plant species. Once established, wild taro forms dense, monotypic stands that reduce the diversity of native vegetation, as has occurred in Louisiana, Florida, and Texas (Akridge and Fonteyn 1981, Simberloff et al. 1997). Akridge and Fonteyn (1981) reported that although wild taro is considered naturalized in south-central Texas, its present dominance along the San Marcos River has altered the native vegetational structure and dynamics of this river system. The objective of this study was to evaluate the efficacy of four aquatic herbicides for control of wild taro.

The toxicity of four insecticides and a herbicide under tropical conditions

The toxicities of four insecticides and a herbicide to Tilapia macrochir were tested in the laboratory. The 24 hour LC50's were estimated as follows: Endrin 20% ,0.008 ppm; Lindane 5% granules, 4.6 ppmm; Synexa 50 (HCH) 50%), 5.6 ppm; Synex 25 (HCH 25%), 14.8 ppm; TOK herbicide (Nitrofen), 100% survival for 24 hours at 100 ppm. These estimates agree with results obtained by other workers elsewhere in the world. The laboratory determination of toxicity is important in estimating the direct effects of poisonous substances on fish, but other indirect effects may result from their use. These should be investigated in the field.

Effects of atrazine herbicide on physiological, biochemical and histological biomarkers of developmental stages of larvae and fry in Caspian kutum, Rutilus frisii kutum

To investigation of the toxic effects of atrazine on newly hatched larvae and releasing age fry of the Caspian Kutum, Rutilus frisii kutum, the 96h LC50 was determined as 18.53 ppm and 24.95 ppm, respectively. Newly hatched larvae were exposed to three sublethal concentrations of atrazine (1/2LC50, 1/4LC50 and 1/8LC50) for 7 days. Different histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed larvae. Fry’s were exposed to one sublethal concentration of atrazine (1/2LC50) for four days, and like the larvae’s, many histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed fry’s, too. Also, measurements of the body ions: Na+, K+, Ca2+, Mg2+ and Cl- in atrazine exposed larvae and fry’s compare to control groups showed that atrazine is changed the body ions composition. No significant differences were found in length growth rate, weight growth rate and the condition factor of the atrazine exposed larvae and fry. Immunohistochemical localization of the Na+, K+-ATPase in integumentary and gill ionocytes, showed no differences in dispersion pattern of the ionocytes in atrazine exposed larvae and fry, compare to control group. Measuring the dimensions of the ionocytes and counting the ionocytes showed that atrazine is affecting on ionocytes by mild increasing in size and mild decreasing in number. Ultrastructural studies, using SEM and TEM, showed that atrazine have significant effects on cellular and subcellular properties. It caused necrosis in surface of the pavement cells in branchial epithelium, necrosis in endoplasmic reticulum of the ionocytes and changed the shape of the mitochondria in these cells. Results showed that sublethal concentrations of atrazine were very toxic to larvae and fry of the Rutilus frisii kutum, and at these levels can made some serious histopathological alterations in their tissues. Related to the severe histopathological alterations in osmoregulatory organs, like gill, kidney and digestive system, and the alterations in the body ion composition, it could be concluded that atrazine could interfere with the osmoregulation process of the Rutilus frisii kutum at the early stages of the life history.

Electrochemical determination of the herbicide bentazone using a carbon nanotube b-Cyclodextrin modified electrode

An electrochemical sensor has been developed for the determination of the herbicide bentazone, based on a GC electrode modified by a combination of multiwalled carbon nanotubes (MWCNT) with b-cyclodextrin (b-CD) incorporated in a polyaniline film. The results indicate that the b-CD/MWCNT modified GC electrode exhibits efficient electrocatalytic oxidation of bentazone with high sensitivity and stability. A cyclic voltammetric method to determine bentazone in phosphate buffer solution at pH 6.0, was developed, without any previous extraction, clean-up, or derivatization steps, in the range of 10–80 mmolL 1, with a detection limit of 1.6 mmolL 1 in water. The results were compared with those obtained by an established HPLC technique. No statistically significant differences being found between both methods.

Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes

Mecoprop-p [(R)-2-(4-chloro-2-methylphenoxy) propanoic acid) is widely used in agriculture and poses an environmental concern because of its susceptibility to leach from soil to water. We investigated the effect of soil depth on mecoprop-p biodegradation and its relationship with the number and diversity of tfdA related genes, which are the most widely known genes involved in degradation of the phenoxyalkanoic acid group of herbicides by bacteria. Mecoprop-p half-life (DT50) was approximately 12 days in soil sampled from <30 cm depth, and increased progressively with soil depth, reaching over 84 days at 70–80 cm. In sub-soil there was a lag period of between 23 and 34 days prior to a phase of rapid degradation. No lag phase occurred in top-soil samples prior to the onset of degradation. The maximum degradation rate was the same in top-soil and sub-soil samples. Although diverse tfdAα and tfdA genes were present prior to mecoprop-p degradation, real time PCR revealed that degradation was associated with proliferation of tfdA genes. The number of tfdA genes and the most probable number of mecoprop-p degrading organisms in soil prior to mecoprop-p addition were below the limit of quantification and detection respectively. Melting curves from the real time PCR analysis showed that prior to mecoprop-p degradation both class I and class III tfdA genes were present in top- and sub-soil samples. However at all soil depths only tfdA class III genes proliferated during degradation. Denaturing gradient gel electrophoresis confirmed that class III tfdA genes were associated with mecoprop-p degradation. Degradation was not associated with the induction of novel tfdA genes in top- or sub-soil samples, and there were no apparent differences in tfdA gene diversity with soil depth prior to or following degradation.

Estimating the spatial scale of herbicide and soil interactions by nested sampling, hierarchical analysis of variance and residual maximum likelihood

An unbalanced nested sampling design was used to investigate the spatial scale of soil and herbicide interactions at the field scale. A hierarchical analysis of variance based on residual maximum likelihood (REML) was used to analyse the data and provide a first estimate of the variogram. Soil samples were taken at 108 locations at a range of separating distances in a 9 ha field to explore small and medium scale spatial variation. Soil organic matter content, pH, particle size distribution, microbial biomass and the degradation and sorption of the herbicide, isoproturon, were determined for each soil sample. A large proportion of the spatial variation in isoproturon degradation and sorption occurred at sampling intervals less than 60 m, however, the sampling design did not resolve the variation present at scales greater than this. A sampling interval of 20-25 m should ensure that the main spatial structures are identified for isoproturon degradation rate and sorption without too great a loss of information in this field.