The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

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).     

Combining supercritical fluid extraction of soil herbicides with enzyme immunoassay analysis

Supercritical fluid extraction (SFE) of soil herbicides followed by enzyme immunoassay analysis (EIA) is explained in a step-by-step process. Extracted herbicides, include 2,4-D, simazine, atrazine, and alachlor. The herbicide, trifluralin was not successfully analyzed by EIA because of crossreacting metabolites. Problems with SFE, including uneven packing of cells, leaks, uneven flow and clogging, can largely be eliminated as the method parameters are optimized. It was necessary to add modifiers including methanol or acetone to the SF CO₂ to increase the solubility of the analytes. Detection limits of 2.5 ng/g soil for atrazine and alachlor and 15 ng/g soil for simazine and 2,4-D without concentration of the sample were achieved. Recoveries above 80% and relative standard deviations (RSDs) less than 15% for 2,4-D simazine, atrazine and alachlor were achieved. Atrazine and alachlor recoveries were above 90% with RSDs below 10%. Forty soil samples could be extracted and analyzed in an 8-h day.     

DNA interaction with human serum albumin studied by affinity capillary electrophoresis and FTIR spectroscopy

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.     

Interaction of taxol with human serum albumin

Taxol (paclitaxel) is an anticancer drug, which interacts with microtuble proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures (Nogales et al., Nature 375 (1995) 424-427). This study was designed to examine the interaction of taxol with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 0.0001-0.1 mM, and HSA (fatty acid free) concentration of 2% w/v. Gel electrophoresis, absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second-derivative resolution enhancement were used to determine the drug binding mode, binding constant and the protein secondary structure in the presence of taxol in aqueous solution. Spectroscopic evidence showed that taxol-protein interaction results into two types of drug-HSA complexes with overall binding constant of K=1.43 x 10(4) M(-1). The molar ratios of complexes were of taxol/HSA 30/1 (30 mM taxol) and 90/1 (90 mM taxol) with the complex ratios of 1.9 and 3.4 drug molecules per HSA molecule, respectively. The taxol binding results in major protein secondary structural changes from that of the alpha-helix 55 to 45% and beta-sheet 22 to 26%, beta-anti 12 to 15% and turn 11 to 16%, in the taxol-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of taxol in aqueous solution.     

Translocation and Complex Formation of Picloram and 2,4-D in Rape and Sunflower

Uptake, translocation and complex formation of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of rape (Brassica napus L. cv. Nilla) and sunflower (Helianthus annuus L. var. uniflorus) were studied. Sunflower is susceptible both to 2,4-D and picloram, while rape is susceptible to 2,4-D but more tolerant to picloram. The uptake of the herbicides through the leaves was almost complete in both species. Translocation of 2,4-D into the roots took place more readily than that of picloram. In sunflower about 50 per cent of the applied 2,4-D was extruded through the roots into the nutrient solution after 9 days. In the picloram-treated sunflower most of the activity was found in the aerial parts, while in picloram-treated rape most of the activity still occurred in the treated leaf after 9 days. No activity at all was found in the roots or in the nutrient solution of the picloram-treated rape seedlings. While the major part of 2,4-D always was found in the state of free herbicide, a large fraction of picloram was rapidly bound into water-soluble complexes. This binding was especially pronounced in rape. Separation by paper chromatography showed that different radioactive compounds were formed. Most of these could be hydrolyzed, thereby releasing free herbicide. The results support the hypotheses that complex formation could counteract herbicide translocation and toxicity of auxin herbicides.     

Human serum albumin complexes with chlorophyll and chlorophyllin

Porphyrins and their metal derivatives are strong protein binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA and protein. The presence of several high-affinity binding sites on human serum albumin (HSA) makes it possible target for many organic and inorganic molecules. Chlorophyll a and chlorophyllin (a food-grade derivative of chlorophyll), the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis and interact with protein and DNA in many ways. This study was designed to examine the interaction of HSA with chlorophyll (Chl) and chlorophyllin (Chln) in aqueous solution at physiological conditions. Fourier transform infrared, UV-visible, and CD spectroscopic methods were used to determine the pigment binding mode, the binding constant, and the effects of porphyrin complexation on protein secondary structure. Spectroscopic results showed that chlorophyll and chlorophyllin are located along the polypeptide chains with no specific interaction. Stronger protein association was observed for Chl than for Chln, with overall binding constants of K(Chl) = 2.9 x 10(4)M(-1) and K(Chln) = 7.0 x 10(3)M(-1). The protein conformation was altered (infrared data) with reduction of alpha-helix from 55% (free HSA) to 41-40% and increase of beta-structure from 22% (free HSA) to 29-35% in the pigment-protein complexes. Using the CDSSTR program (CD data) also showed major reduction of alpha-helix from 66% (free HSA) to 58 and 55% upon complexation with Chl and Chln, respectively.     

The effect of the glyphosate, 2,4-D, atrazine e nicosulfuron herbicides upon the Edaphic collembola (Arthropoda: Ellipura) in a no tillage system

The use of herbicides is a common and intensive practice in no tillage systems. The herbicides can influence, directly or indirectly, the population of edaphic arthropods. Collembola is a group that functions as a bio-indicator of soil conditions. The degree of abundance and diversity of Collembola provides the level of soil disturbance provoked by agricultural practices. This experiment was designed to compare the influence of herbicides on the population fluctuation of Collembola in a no-till soil preparation system. The work was conducted in a non irrigated no-till area at the Núcleo Experimental de Ciências Agrárias of the Universidade Federal de Mato Grosso do Sul (UFMS), Campus de Dourados, in soil planted with corn as a surface covering, during the period of December, 2002 to December, 2003. The data were analyzed according to a completely randomized model, in a split plot design. The plots received four types of herbicides: glyphosate, atrazine, 2,4-D and nicosulfuron. A fifth plot did not receive any herbicide (control), for a total of five treatment types. The sub plots were represented by their collection times (10, 20, 30 and 40 days after the herbicide applications). Both the type of herbicide and the time of data sampling influenced the Collembola population fluctuaction. The treatments with atrazine and 2,4-D caused the most reduction of the population of Collembola, depending on the time of application.     

Co-induction of a glutathione-S-transferase, a glutathione transporter and an ABC transporter in maize by xenobiotics

Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by four herbicides (2,4-D, atrazine, metolachlor and primisulfuron) and a herbicide safener (dichlormid) on the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect on gene expression varies with both chemicals and genes. The expression of ZmGST27 and ZmMRP1 was up-regulated by all five compounds, whereas that of ZmGT1 was increased by atrazine, metolachlor, primisulfuron and dichlormid, but not by 2,4-D. For all chemicals, the inducing effect was first detected on ZmGST27. The finding that ZmGT1 is activated alongside ZmGST27 and ZmMRP1 suggests that glutathione transporters are an important component in the xenobiotic detoxification system of plants.     

Translocation and Complex Formation of Root-applied 2,4-D and Picloram in Susceptible and Tolerant Species

Translocation and complex formation of ¹⁴C-labelled 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) in seedlings of sunflower (Helianthus annuus L. var. uniflorus), rape (Brassica napus L. cv. Nilla), wheat (Triticum aestivum L. cv. Starke), and Norway spruce (Picea abies (L.) H. Karst.) were studied. The herbicides were absorbed through the roots from the nutrient solution, Picloram was well translocated to the shoots of the four species; while the acropetal translocation of 2,4-D was small except in rape. In 2,4-D-susceptible sunflower and rape and in picloramsusceptible sunflower and spruce the herbicides were recovered mainly in the uncomplexed form. In 2,4-D tolerant wheat and spruce most of the absorbed 2,4-D was converted into water-soluble or TCA-insoluble complexes. In picloram-tolerant wheat and in relatively picloram-tolerant rape, the absorbed picloram was also converted into complexes recovered predominantly in the water-soluble fraction. Most of the complexes released free herbicides by hydrolyzing in NaOH or HCI. The results further support the hypothesis that complex formation counteracts herbicide toxicity.     

Translocation and Complex Formation of Picloram and 2,4-D in Wheat Seedlings

Uptake, translocation and metabolism of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.     

Effect of herbicide residues in a sandy loam on the growth,nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterraneum

The herbicides 2,4-D, amitrole, atrazine, diclofop-methyl, diquat, paraquat and trifiluralin were applied at rates of 0, 2, 5 and 10 μg ai. g⁻¹ to a sandy loam soil and allowed to degrade for 120 days. After this period, subterranean clover seedlings were transplanted into treated soil and the effect of herbicide residues on plant growth, number of nodules formed and nitrogenase activity was investigated. At all rates of atrazine and chlorsulfuron, and at all rates of amitrole in excess of 2 mg ai g⁻¹ of soil, sufficient herbicide remained to be lethal to the seedlings. When amitrole was applied at the rate of 2 mg ai g⁻¹ of soil, plant growth, nodulation and nitrogenase activity of plants were reduced. Residues of diquat reduced all plant parameters studied while, residues of 2,4-D reduced plant growth and nodule formation, but plant nitrogenase activity was unaffected. Residues of trifluralin had no effect on plant growth parameters but the number of nodules formed per plant was reduced. Residues of paraquat and diclofop-methyl had no effect on any of the plant parameters studied.     

Response of multiple herbicide resistant strain of diazotrophic cyanobacterium, Anabaena variabilis, exposed to atrazine and DCMU

Effect of two photosynthetic inhibitor herbicides, atrazine (both purified and formulated) and [3-(3,4-dichlorophenyl)-1,1-dimethyl urea] (DCMU), on the growth, macromolecular contents, heterocyst frequency, photosynthetic O2 evolution and dark O2 uptake of wild type and multiple herbicide resistant (MHR) strain of diazotrophic cyanobacterium A. variabilis was studied. Cyanobacterial strains showed gradual inhibition in growth with increasing dosage of herbicides. Both wild type and MHR strain tolerated < 6.0 mg L(-1) of atrazine (purified), < 2.0 mg L(-1) of atrazine (formulated) and < 0.4 mg L(-1) of DCMU indicating similar level of herbicide tolerance. Atrazine (pure) (8.0 mg L(-1)) and 4.0 mg L(-1) of atrazine (formulated) were growth inhibitory concentrations (lethal) for both wild type and MHR strain indicating formulated atrazine was more toxic than the purified form. Comparatively lower concentrations of DCMU were found to be lethal for wild type and MHR strain, respectively. Thus, between the two herbicides tested DCMU was more growth toxic than atrazine. At sublethal dosages of herbicides, photosynthetic O2 evolution showed highest inhibition followed by chlorophyll a, phycobhiliproteins and heterocyst differentiation as compared to carotenoid, protein and respiratory O2 uptake.     

Impact of Polyacrylamide Treatment on Sorptive Dynamics and Degradation of 2,4-D and Atrazine in Agricultural Soil

High-molecular-weight, anionic polyacrylamide (PAM) is added to irrigation water to reduce soil erosion during furrow irrigation of crops. The chemical nature of PAM, together with the observation that the polymer can be biotransformed by soil bacteria, led us to question the impact of PAM treatment on the fate of coapplied agrochemicals. The herbicides, atrazine (nonionic) and 2,4-D (anionic), were tested for pesticide sorption, desorption, and degradation in PAM-treated and untreated soils. Sorption of atrazine and 2,4-D in soil was unaffected by PAMtreatment, as was atrazine desorption. However, 2,4-D desorbedmore readily from the PAM-treated soil than from untreated soil. With respect to pesticide degradation, mineralization of the 2,4-D aromatic ring was not impacted by PAM treatment, but decarboxylation of the 2,4-D carboxylic acid side chain was significantly reduced in the PAM-treated soil. Limited mineralization (7 to 10%) of atrazine was observed in both soils. However, in PAM-treated soils atrazine conversion to ¹⁴CO₂ and bound residue components was significantly reduced, and there was an increase in the level of methanol extractable metabolites. These results may indicate that PAM application can alter the environmental fate of some pesticides in soils, especially under the high dose treatment conditions examined in this study.     

Herbicides for Establishing Switchgrass in the Central and Northern Great Plains

Weed interference limits switchgrass (Panicum virgatum L.) establishment from seed. Our objectives were to determine the effect of selected post-plant, preemergence herbicides on stand establishment and subsequent biomass yields of adapted upland switchgrass cultivars grown in three environments in the Central and Northern Great Plains. A separate experiment was conducted in eastern Nebraska to determine if there were any differences among switchgrass ecotypes for herbicide tolerance to the optimal herbicide combination. Herbicides applied immediately after planting were different concentrations of atrazine [Aatrex 4L®; 6-chloro-N-ethyl-N′-(1-methylethyl)-1,3,5-triazine-2,4-diamine], quinclorac (Paramount®; 3,7-Dichloro-8-quinolinecarboxylic acid), atrazine+quinclorac, imazapic {Plateau®; 2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylic acid}, and quinclorac+imazapic. Herbicide efficacy was determined by measuring stand frequency of occurrence and biomass yield the year after establishment. The application of quinclorac plus atrazine resulted in acceptable stands and high biomass yields. Imazapic often reduced switchgrass stands in comparison to the nontreated control and is not recommended for switchgrass establishment. In the multi-state trials, the herbicide by cultivar interaction was not significant for stands or biomass yields, indicating that the effects of herbicides on switchgrass stands and biomass yields were consistent over the upland cultivars used in the trials. No differences were detected among switchgrass lowland and upland ecotypes for tolerance to atrazine and quinclorac. Quinclorac, which provides effective control of grassy weeds, and herbicides such as atrazine which provide good broadleaf weed control are an excellent herbicide combination for establishing switchgrass for biomass production in the Great Plains and the Midwest.     

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Polyamine analogues bind human serum albumin

Polyamine analogues show antitumor activity in experimental models, and their ability to alter activity of cytotoxic chemotherapeutic agents in breast cancer is well documented. Association of polyamines with nucleic acids and protein is included in their mechanism of action. The aim of this study was to examine the interaction of human serum albumin (HSA) with several polyamine analogues, such as 1,11-diamino-4,8-diazaundecane (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333), in aqueous solution at physiological conditions using a constant protein concentration and various polyamine contents (microM to mM). FTIR, UV-visible, and CD spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind nonspecifically (H-bonding) via polypeptide polar groups with binding constants of K333 = 9.30 x 10(3) M(-1), KBE-333 = 5.63 x 10(2) M(-1), and KBE-3333 = 3.66 x 10(2) M(-1). The protein secondary structure showed major alterations with a reduction of alpha-helix from 55% (free protein) to 43-50% and an increase of beta-sheet from 17% (free protein) to 29-36% in the 333, BE-333, and BE-3333 complexes, indicating partial protein unfolding upon polyamine interaction. HSA structure was less perturbed by polyamine analogues compared to those of the biogenic polyamines.     

Influence of Auxin-Like Herbicides on Tobacco Mosaic Virus Multiplication

Tobacco (Nicotiana tabacum L., cv. Samsun) leaf discs inoculated with tobacco mosaic virus (TMV) were treated with auxin-like herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), 3-amino-1,2,4-triazol (Amitrol) and 6-chloro-2-ethylamino-4-isopropylamino-1,3,5-triazine (Atrazin). All herbicides in the concentration of 10^–7 M enhanced the virus content (MCPA to 227.4 %, Amitrol to 218.1 % and Atrazin to 257.3 % of values found in TMV-infected, herbicide untreated discs). The 2,4-D alone did not affect the activity of the glucose-6-phosphate dehydrogenase and ribonucleases, but the 2,4-D treatment together with TMV infection raised their activities twice as high as in the untreated control discs. Polyacrylamide gel electrophoresis of acidic extracellular proteins washed from leaf discs treated with 2,4-D did not prove the induction of PR-proteins.     

Bioconjugation of CdTe quantum dot for the detection of 2,4-dichlorophenoxyacetic acid by competitive fluoroimmunoassay based biosensor

Quantum dots (QD) are semiconductor fluorescent nanoparticles, which can be made use of for environmental monitoring with high sensitivity. In view of the alarming levels of pesticides and herbicides being used in agriculture practices, there is a need for their rapid, sensitive and specific detection in food and environmental samples, as pesticides and herbicides are harmful to living beings even at trace levels. Present study was carried out to develop a reliable and rapid method for analysis and detection of 2,4-D (herbicide) using cadmium telluride quantum dot nanoparticle (CdTe QD). Fluoroimmunoassay based on the fluorescent property of quantum dot was used along with immunoassay to detect 2,4-D. CdTe capped with mercaptopropionic acid, was conjugated using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS) to alkaline phosphatase (ALP) which was in turn conjugated to 2,4-D molecule. Anti 2,4-D-IgG antibodies were immobilized in an immunoreactor column using Sepharose CL-4B as an inert matrix. The detection of 2,4-D was carried out by fluoroimmunoassay-based biosensor using competitive binding between conjugated 2,4-D-ALP-CdTe and free 2,4-D with immobilized anti 2,4-D antibodies in an immunoreactor column. It was possible to detect 2,4-D upto 250pgmL(-1). Present study also emphasizes on the resonance energy transfer between ALP and CdTe QD as a result of bioconjugation, which can be used for future biosensor development based on quantum dot-biomolecular interactions.     

Herbicide Persistence in Seawater Simulation Experiments

Herbicides are detected year-round in marine waters, including those of the World Heritage listed Great Barrier Reef (GBR). The few previous studies that have investigated herbicide persistence in seawater generally reported half-lives in the order of months, and several studies were too short to detect significant degradation. Here we investigated the persistence of eight herbicides commonly detected in the GBR or its catchments in standard OECD simulation flask experiments, but with the aim to mimic natural conditions similar to those found on the GBR (i.e., relatively low herbicide concentrations, typical temperatures, light and microbial communities). Very little degradation was recorded over the standard 60 d period (Experiment 1) so a second experiment was extended to 365 d. Half-lives of PSII herbicides ametryn, atrazine, diuron, hexazinone and tebuthiuron were consistently greater than a year, indicating high persistence. The detection of atrazine and diuron metabolites and longer persistence in mercuric chloride-treated seawater confirmed that biodegradation contributed to the breakdown of herbicides. The shortest half-life recorded was 88 d for growth-regulating herbicide 2,4-D at 31°C in the dark, while the fatty acid-inhibitor metolachlor exhibited a minimum half-life of 281 d. The presence of moderate light and elevated temperatures affected the persistence of most of the herbicides; however, the scale and direction of the differences were not predictable and were likely due to changes in microbial community composition. The persistence estimates here represent some of the first appropriate data for application in risk assessments for herbicide exposure in tropical marine systems. The long persistence of herbicides identified in the present study helps explain detection of herbicides in nearshore waters of the GBR year round. Little degradation of these herbicides would be expected during the wet season with runoff and associated flood plumes transporting a high proportion of the original herbicide from rivers into the GBR lagoon.