Bioguided Fractionation of Local Plants against Matrix Metalloproteinase9 and Its Cytotoxicity against Breast Cancer Cell Models: In Silico and In Vitro Study (Part II).

In our previous work, the partitions (1 mg/mL) of Ageratum conyzoides (AC) aerial parts and Ixora coccinea (IC) leaves showed inhibitions of 94% and 96%, respectively, whereas their fractions showed IC50 43 and 116 µg/mL, respectively, toward Matrix Metalloproteinase9 (MMP9), an enzyme that catalyzes a proteolysis of extracellular matrix. In this present study, we performed IC50 determinations for AC n-hexane, IC n-hexane, and IC ethylacetate partitions, followed by the cytotoxicity study of individual partitions against MDA-MB-231, 4T1, T47D, MCF7, and Vero cell lines. Successive fractionations from AC n-hexane and IC ethylacetate partitions led to the isolation of two compounds, oxytetracycline (OTC) and dioctyl phthalate (DOP). The result showed that AC n-hexane, IC n-hexane, and IC ethylacetate partitions inhibit MMP9 with their respective IC50 as follows: 246.1 µg/mL, 5.66 µg/mL, and 2.75 × 10-2 µg/mL. Toward MDA-MB-231, 4T1, T47D, and MCF7, AC n-hexane demonstrated IC50 2.05, 265, 109.70, and 2.11 µg/mL, respectively, whereas IC ethylacetate showed IC50 1.92, 57.5, 371.5, and 2.01 µg/mL, respectively. The inhibitions toward MMP9 by OTC were indicated by its IC50 18.69 µM, whereas DOP was inactive. A molecular docking study suggested that OTC prefers to bind to PEX9 rather than its catalytic domain. Against 4T1, OTC showed inhibition with IC50 414.20 µM. In conclusion, this study furtherly supports the previous finding that AC and IC are two herbals with potential to be developed as triple-negative anti-breast cancer agents.

In vitro evaluation of cytotoxic effects of di (2-ethylhexyl) phthalate (DEHP) produced by Bacillus velezensis strain RP137 isolated from Persian Gulf.

Phthalates are widely used in polymer science and have potential toxicity related to their chemical structures. However, lots of evidence indicate that phthalate derivatives are undoubtedly produced as secondary metabolites by organisms, including plants, animals, and microorganisms. In the present study, Bacillus velezensis strain RP137 was cultured under optimized conditions. Its biomass was extracted with ethyl acetate with one fraction showing cytotoxic properties. A pure compound was isolated from the active fraction using combined silica gel and LH20 size exclusion column chromatography. Structural evaluation including FT-IR, 1H NMR, 13C NMR, HR-MS and CHN analysis identified the purified compound as di(2-ethylhexyl)phthalate (DEHP) with the formula C24H38O4 and the molecular weight of 389.29 Da. The microorganism-derived (stereospecific) DEHP was strongly reduced the proliferation and induced cytotoxic effects on various eukaryotic cell lines in compare to the synthetic racemic mixture of the compound when assessed by MTT assay. Furthermore, crystal violet assay and morphological changes confirmed the cytotoxic effect of DEHP. Interestingly, non-malignant SV40-immortalized fibroblast cells were less affected by the purified DEHP. Further evaluation on the antibacterial activity of DEHP documented no effect toward Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens even at a high concentration of 100 µM. In conclusion, existence of DEHP as byproduct of microorganism's metabolism can seriously be considered as a warning to human health.

A miniature stainless steel net dumbbell-shaped stir-bar for the extraction of phthalate esters in instant noodle and rice soup samples.

This work reports the development of a very-simple-to-construct stir-bar extraction device so called "a dumbbell-shaped stainless steel stir-bar." The extraction device was assembled from a rolled up stainless steel net filled with an XAD-2 sorbent and a metal rod to allow the use of a magnetic stirrer during extraction. The dumbbell-shaped stainless steel stir-bar was used to extract diethyl phthalate (DEP), dibutyl phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) before analysis by a gas chromatograph equipped with an electron capture detector (GD-ECD). Under the optimal conditions, the developed method provided a good linearity from 10.0 to 1,000.0 ng mL-1 for all three compounds. Limits of detection and limits of quantification were 9.37 ± 0.29 ng mL-1 and 31.22 ± 0.95 ng mL-1 for DEP, 5.73 ± 0.31 ng mL-1 and 19.1 ± 1.0 ng mL-1 for DBP and 3.30 ± 0.06 ng mL-1 and 11.0 ± 0.19 ng mL-1 for DEHP, respectively. Good recoveries in the range of 81.89 ± 0.17 to 109.5 ± 2.0% were achieved when the method was used to extract phthalate esters in five instant noodle and two rice soup samples.

Simultaneous determination of di(2-ethylhexyl) phthalate and diisononylcyclohexane-1,2-dicarboxylate and their monoester metabolites in four labile blood products by liquid chromatography tandem mass spectrometry.

Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that is largely used for PVC blood bags. The migration of DEHP from medical devices into labile blood products (LBP) is a well-known situation. While DEHP has beneficial effects on the storage of red blood cells, it can have toxicological impact due to its potential reprotoxic effects (classified group 1B). Since July 1st, 2015, the French law prohibits the use of tubing made in DEHP-plasticized PVC in paediatric, neonatal and maternity wards. This provision, which could extend in several years more widely to medical devices used for drugs infusion, dialysis, feeding and blood bags, has led manufacturers to replace DEHP to alternative plasticizers such as diisononylcyclohexane-1,2-dicarboxylate (DINCH). In this paper, a liquid chromatography-tandem mass spectrometry (LCMS/MS) method has been developed and validated for the determination of DEHP, DINCH and their corresponding monoester metabolites (MEHP and MINCH) in four labile blood products (LBP): whole blood (WB), red cells concentrate (RCC), plasma and platelet concentrate (PC). Due to strong contamination of blank LBP by DEHP because of its ubiquitous presence in working environment and despite the attention paid to avoid contamination of solvents and glassware, a trap chromatographic column was implemented between the solvent mixing chamber and the injector of the LC system. This set-up permitted to discriminate DEHP present in the sample to DEHP brought by the environmental contamination. In the optimized conditions, all compounds were separated in less than 10 min. The analytes were extracted from LBP samples using a liquid-liquid extraction. After optimization, recoveries were ranged from 47 to 96 %, depending on the analytes and the nature of LBP. Except for DEHP which exhibited RSD values of intermediate precision higher than 20 % at a concentration of 25 nM, all the precision results (repeatability and intermediate precision) were lower than 16 % and trueness values ranged from -16.2-19.8%. Using the validated method, the leachability of DEHP and DINCH from corresponding PVC-blood bags was investigated and the concentrations of their corresponding metabolites, MEHP and MINCH, were determined in whole blood, red cells concentrate, plasma and platelet concentrate.

Diethylhexyl phthalate removal in full scale activated sludge plants: Effect of operational parameters.

Removal of emerging contaminants (ECs) is a serious concern in wastewater industry especially for public acceptance of reclaimed water. Diethylhexyl phthalate (DEHP) is one of the ubiquitous and detectable plasticizers in municipal wastewater across the globe. Water Framework Directive (2000/60/EC) has prioritized it for the establishment of discharge regulations. A cost-effective strategy, especially for developing nations, may be the re-engineering of the existing biological process for the simultaneous removal of ECs and conventional pollutants. Wastewater treatment plants are one of the main sources for DEHP occurrence in surface water. In this study, possible role of activated sludge process operational parameters in effective removal of DEHP was assessed. Principal component analysis of occurrence data showed dissimilarity with the organic and nutrient characteristics of sewage. DEHP concentration in more than half (55%) of treated wastewater samples was more than environmental quality standard value for inland and surface water bodies (1.3 µg/L). At a mixed liquor suspended solid (MLSS) concentration range of 3461-4972 mg/L, overall removal was 23.9 µg/gMLSS.d (92 ±â€¯6%) with biodegraded portion as 22.4 µg/gMLSS.d (85 ±â€¯4%) and sorbed portion of 1.5 µg/gMLSS.d (7 ±â€¯4%). DEHP removal showed an increasing trend at higher oxygen uptake rates (OUR) of sludge with DEHP removal of 8.1  µg DEHP/gMLSS.d (70 ±â€¯6%), in the OUR range of 20-28mgO2/L/h. Increase in overall removal of DEHP showed a positive correlation (r2 = 0.7) with increasing sludge retention time (SRT) and so does the decreasing food to microorganism (F/M) ratio with increasing removal of DEHP (r2 = 0.8). A temperature decrease of 13 °C caused a decrease of 30% in overall removal of DEHP.

Production of di-(2-ethylhexyl) phthalate by Bacillus subtilis AD35: Isolation, purification, characterization and biological activities.

The emergence of extensive antibiotics resistant bacteria increased the demands for finding out new sources of antimicrobial agents. Marine niches were reported to be rich in many competent producers of significant bioactive compounds. On the course of screening program for new antimicrobials, a Bacillus strain was isolated from Alexandria sea shores, Egypt. According to the morphological, cultural and biochemical characteristics, 16S rRNA sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was identified as Bacillus subtilis and designated as B. subtilis AD35. One phthalate derivative namely Di-(2-ethylhexyl) phthalate (DEHP) was purified from the crude extract of B. subtilis AD35 by high performance liquid chromatography (HPLC) and the structural elucidation of this compound was confirmed on the basis of gas chromatography-mass spectroscopy (GC-MS), Fourier infrared spectroscopy (FT-IR) and UV spectrum. The results of MIC of the purified DEHP were as follow: 16 µg/ml (Salmonella typhimurium), 32 µg/ml (Methicillin resistant Staphylococcus aureus, MRSA), 0.25 µg/ml (Listeria monocytogenes), 0.5 µg/ml (Aeromonas hydrophila), 8 µg/ml (Staphylococcus aureus), 4 µg/ml (Staphylococcus epidermidis), 4 µg/ml (Escherichia coli), and 8 µg/ml (Pseudomonas aeruginosa). DEHP produced by B. subtilis AD35 up to a concentration of 2500 µg/ml exhibited no cytotoxic effect against normal Vero cells. In addition, it did not show an antiviral activity against HAV or a significant growth inhibitory effect toward human colorectal adenocarcinoma and human mammary gland adenocarcinoma cell-lines.

Fate of four phthalate plasticizers under various wastewater treatment processes.

The fate of four phthalate plasticizers during wastewater treatment processes at six different wastewater treatment plants (WWTPs) was investigated. Concentrations of benzyl butyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DiNP), and diisodecyl phthalate (DiDP) were determined prior to either aerobic or anaerobic (conventional and advanced) treatment, after treatment, and in final, dewatered solids. Despite their elevated use worldwide, the fate of DiNP and DiDP during wastewater treatment have not been well characterized. DEHP was readily degraded during aerobic treatments while anaerobic digestion resulted in either no significant change in concentrations or an increase in concentration, in the case of more advanced anaerobic processes (thermal hydrolysis pretreatment and a two-phase acid/gas process). Impacts of the various treatment systems on DiNP, DiDP, and BBP concentrations were more varied - anaerobic digestion led to significant decreases, increases, or no significant change for these compounds, depending on the treatment facility, while aerobic treatment was generally effective at degrading the compounds. Additionally, thermal hydrolysis pretreatment of sludge prior to anaerobic digestion resulted in increases in DiNP, DiDP, and BBP concentrations. The predicted environmental concentrations for all four compounds in soils after a single biosolids application were calculated and the risk quotients for DEHP in soils were determined. The estimated toxicity risk for DEHP in soils treated with a single application of sludge from any of the six studied WWTPs is lower than the level of concern for acute and chronic risk, as defined by the US EPA.

Dispersive solid-phase microextraction with graphene oxide based molecularly imprinted polymers for determining bis(2-ethylhexyl) phthalate in environmental water.

A novel graphene oxide-molecularly imprinted polymers (GO-MIPs) was prepared and applied for selective extraction and preconcentration of bis(2-ethylhexyl) phthalate (DEHP) in environmental water samples by using the dispersive solid-phase microextraction (DSPME) method. The GO-MIPs was synthesized via precipitation polymerization using GO, DEHP, methacrylic acid, and ethylene dimethacrylate as supporting materials, template molecules, functional monomer, and cross-linker, respectively. The prepared GO-MIPs were characterized by scanning electron microscope and Fourier transform infrared spectroscopy. The GO-MIPs-DSPME conditions including type and volume of elution solvents, adsorbents amount, initial concentration of DEHP, pH and ionic strength of water samples were investigated. Under optimized conditions, the DEHP was selectively and effectively extracted in real water samples and enrichment factors of over 100-fold were achieved. Good linearity was obtained with correlation coefficients (R2) over 0.999 and the detection limit (S/N=3) was 0.92ngmL-1. The average recoveries of the spiked samples at three concentration levels of DEHP ranged from 82% to 92% with the relative standard deviations less than 6.7%. The results indicated that the proposed GO-MIPs-DSPME extraction protocol combined with HPLC-UV determination could be applied for selective and sensitive analysis of trace DEHP phthalate in environmental water samples.

Survey on plasticizers currently found in PVC toys on the Swiss market: Banned phthalates are only a minor concern.

Plasticizers in toys are a recurring source of criticism and concern, as consumers feel they may endanger the health of their children. Most of the information available in literature concerns the presence or absence of certain phthalic acid ester plasticizers. Very little information can be found in the public domain with respect to the actually used plasticizers at a given time and place. In this paper, we present the plasticizer composition of 118 samples from 88 polyvinyl chloride toys found on the Swiss market in autumn 2015. Bis(2-ethylhexyl) terephthalate (DEHT) was by far the most frequent main plasticizer in the analyzed samples, which is a change when compared to the plasticizers found in toys and child care articles in 2007. Furthermore, the data show that the banned phthalates in toys are only a minor concern. The occurrence, however, is not evenly distributed between importers. If a toy is not designed to be sold on the European market by the manufacturer, it seems to be more likely to contain a banned phthalic acid ester.

Association of diethylhexyl phthalate with obesity-related markers and body mass change from birth to 3†months of age.

BACKGROUND: Several studies have suggested potential links of phthalates to obesity in children and adults. Limited evidence, however, has been available for the relations between diethylhexyl phthalate (DEHP) and obesity-related markers or body mass change in early life. METHODS: 128 healthy pregnant women were recruited and, after delivery, their newborns' first urine and umbilical cord blood samples were collected. We measured urinary levels of two DEHP metabolites, mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP). We also measured the levels of leptin, total cholesterol and triglyceride (TG) in cord serum, and used them along with weight, length, head circumference and ponderal index (PI, 100 g/cm(3)) at birth, as obesity-related markers, and estimated the relations between DEHP metabolites and obesity-related markers using generalised linear models. For the evaluation of body mass increase by early life DEHP exposure, body mass index (BMI) z-score change during 3 months after birth by DEHP metabolites in the first urine samples of the newborns were evaluated using logistic regression. RESULTS: DEHP exposure was associated with decrease of PI and increase of TG (PI, ß=-0.11, p=0.070 and TG, ß=0.14, p=0.027), especially for boys (PI, ß=-0.13, p=0.021; and TG, ß=0.19, p=0.025). Moreover, DEHP exposure was positively associated with body mass increase during 3 months after birth (change of BMI z-scores, OR=4.35, p=0.025). CONCLUSIONS: Our findings suggest that DEHP exposure may affect body mass change in early life through changes of obesity-related markers.

Investigation on removal pathways of Di 2-ethyl hexyl phthalate from synthetic municipal wastewater using a submerged membrane bioreactor.

Highly hydrophobic Di 2-ethyl hexyl phthalate (DEHP) is one of the most prevalent plasticizers in wastewaters. Since its half-life in biological treatment is around 25days, it can be used as an efficiency indicator of wastewater treatment plant for the removal of hydrophobic emerging contaminants. In this study, the performance of submerged membrane bioreactor was monitored to understand the effect of DEHP on the growth of aerobic microorganisms. The data showed that the chemical oxygen demand (COD) and ammonia concentration were detected below 10 and 1.0mg/L, respectively for operating conditions of hydraulic retention time (HRT)=4 and 6hr, sludge retention time (SRT)=140day and sludge concentration between 11.5 and 15.8g volatile solid (VS)/L. The removal efficiency of DEHP under these conditions was higher and ranged between 91% and 98%. Results also showed that the removal efficiency of DEHP in biological treatment depended on the concentration of sludge, as adsorption is the main mechanism of its removal. For the submerged membrane bioreactor, the pore size is the pivotal factor for DEHP removal, since it determines the amount of soluble microbial products coming out of the process. Highly assimilated microorganisms increase the biodegradation rate, as 74% of inlet DEHP was biodegraded; however, the concentration of DEHP inside sludge was beyond the discharge limit. Understanding the fate of DEHP in membrane bioreactor, which is one of the most promising and futuristic treatment process could provide replacement for conventional processes to satisfy the future stricter regulations on emerging contaminants.

Nanostructured copper-coated solid-phase microextraction fiber for gas chromatographic analysis of dibutyl phthalate and diethylhexyl phthalate environmental estrogens.

A novel nanostructured copper-based solid-phase microextraction fiber was developed and applied for determining the two most common types of phthalate environmental estrogens (dibutyl phthalate and diethylhexyl phthalate) in aqueous samples, coupled to gas chromatography with flame ionization detection. The copper film was coated onto a stainless-steel wire via an electroless plating process, which involved a surface activation process to improve the surface properties of the fiber. Several parameters affecting extraction efficiency such as extraction time, extraction temperature, ionic strength, desorption temperature, and desorption time were optimized by a factor-by-factor procedure to obtain the highest extraction efficiency. The as-established method showed wide linear ranges (0.05-250 µg/L). Precision of single fiber repeatability was <7.0%, and fiber-to-fiber repeatability was <10%. Limits of detection were 0.01 µg/L. The proposed method exhibited better or comparable extraction performance compared with commercial and other lab-made fibers, and excellent thermal stability and durability. The proposed method was applied successfully for the determination of model analytes in plastic soaking water.

Simultaneous removal of di-(2-ethylhexyl) phthalate and nitrogen in a laboratory-scale pre-denitrification biofilter system.

This study demonstrated the excellent di-(2-ethylhexyl) phthalate (DEHP) removal performance of a pre-denitrification biofilter system. Experimental results showed that DEHP removal efficiency remained stable while total nitrogen removal efficiency fluctuated with the nitrate recycle ratio changes when the hydraulic loading rate at 1.1m(3)/m(2)h. DEHP removal efficiency increased from 48% to 82% while the hydraulic loading rate increased from 1.1 to 2.2m(3)/m(2)h. DEHP concentration decreased gradually along the wastewater flow direction in the denitrification biofilter and a plug flow model with the reaction order of 5 and the rate constant of 0.54 was obtained. Both the denitrification biofilter and the nitrification biofilter showed similar DEHP removal performance. The overall DEHP removal efficiency of the system was 83.8%, in which biodegradation contributed 72.3%. Biodegradation plays a key role in DEHP removal in the pre-denitrification biofilter system.

[Study on removal of di-(2-ethylhcxyl) phthalate by using of small-scale biological aerated filter].

Ceramsite medium biological aerated filter (BAF) was used to treat the sewage containing di-(2-ethylhcxyl) phthalate (DEHP). The treatment efficiency under different empty bed contact time (EBCT) and temperatures was investigated and the major intermediate products of biodegradation of DEHP were analyzed via GC-MS. Results show that the removal rate of DEHP can be highly achieved at 90.3% under the conditions of 25 degrees C and 8 hours of empty bed contact time. The removal efficiency will be increased after raising temperature or EBCT. The EBCT plays the main role between two influencing factors. The biodegradation of DEHP in BAF is expressed in the first-order kinetics. The major intermediate products of biodegradation of DEHP are made up of mono-2-ethylhexyl phthalate( MEHP), bisethylhexyl phthalate (BEHP), dibutyl phthalate (DBP), dimethyl phthalate (DMP) and phthalic acid (PA). It is supposed that the possible pathway of the biodegradation of di-(2-ethylhcxyl) phthalate is that the long alkyl side-chain of DEHP may be cleavaged into a shorter and more straight one, then PA is formed after cleavaging the two ester bonds, and finally being oxidized into CO2 and H2O.

[Effects of enhanced CO2 fertilization on phytoremediation of DEHP-polluted soil].

Low efficiency of remediation is one of the key issues to be solved in phytoremediation technology. Based on the necessity of reducing CO2 emission in China and the significance of CO2 in plant photosynthesis, this paper studied the effects of enhanced CO2 fertilization on the phytoremediation of polluted soil, selecting the C3 plant mung bean (Vigna radiate) and the C4 plant maize (Zea mays) as test plants for phytoremediation and the DEHP as the target pollutant. DEHP pollution had negative effects on the growth and rhizosphere micro-environment of the two plants. After enhanced CO2 fertilization, the aboveground dry mass of the two plants and the alkaline phosphatase activity in the rhizosphere soils of the two plants increased, the COD activity in the leaves of the two plants decreased, the microbial community in the rhizosphere soils shifted, and the numbers of the microbes with DEHP-tolerance in the rhizosphere soils increased. These changes indicated that enhanced CO2 fertilization could promote the plant growth and the plant tolerance to DEHP stress, and improve the rhizosphere micro-environment. Enhanced CO2 fertilization also increased the DEHP uptake by the two plants, especially their underground parts. All these effects induced the residual DEHP concentration in the rhizospheres of the two plants, especially that of mung bean, decreased obviously, and the phytoremediation efficiency increased. Overall, enhanced CO2 fertilization produced greater effects on C3 plant than on C4 plant. It was suggested that enhanced CO2 fertilization could be a useful measure to enhance the efficiency of phytoremediation.

Determination of phthalic acid diesters in human milk at low ppb levels.

Phthalic acid diesters (PAE) are omnipresent in the human environment and food is a major contributor to the overall human exposure towards these chemicals. Due to developmental effects, PAE infants' exposure via human milk has been subjected to a number of analytical studies. These previous studies, however, revealed that normal laboratory blank values are in the range of or even higher than human milk levels due to the presence of PAE in laboratory environments. In order to provide more reliable data on PAE exposure via human milk, the aim of this study was to develop and validate a robust and sensitive analytical method. This should be capable of removing matrix components efficiently and guarantee limits of quantification in the low ppb range. The method development took into account liquid-liquid extraction and selective pressurised fluid extraction (sPFE) as well as chromatography-based clean-up steps. The final method consisted of a liquid-liquid extraction followed by an automated chromatographic clean-up by an sPFE device. After volume reduction the cleaned extracts were analysed by quadrupole GC/MS. Quantification was based on internal standards. An extensive quality assurance and method test programme demonstrated conservatively determined limits of detection and quantification from 0.3 to 10 ng g⁻¹ in human milk, with recoveries of internal standards from 50% to 101%. Thus, the method allowed the quality-assured detection of di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), di-allyl phthalate (DAP), benzylbutyl phthalate (BBP) and di-cyclohexyl phthalate (DcHP) in 30 human milk samples provided by 30 volunteers from southern Germany. DiBP, DBP and DEHP were the most commonly detected PAE, with median levels of 1.0, 0.6 and 2.3 ng g⁻¹, respectively.

Preparation and characterization of molecularly imprinted polymer for di(2-ethylhexyl) phthalate: application to sample clean-up prior to gas chromatographic determination.

The molecularly imprinted polymer (MIP) selective for di(2-ethylhexyl) phthalate (DEHP) an environmental endocrine disruptor was prepared by suspension polymerization using methacrylamide as functional monomer and N,N'-methylene-bis-acrylamide as cross-linker. The imprinted polymer was employed for solid-phase extraction of DEHP from water samples of environmental importance and characterized by FT-IR and SEM. The adsorption properties of the imprinted polymer were demonstrated by equilibrium rebinding experiments, Pseudo-second-order kinetic model, Sips isotherm and Scatchard analysis. The reusability of MIP was checked for at least six repeated batch adsorption cycles and the results showed almost no deterioration in the adsorption capacity. The competitive recognition studies were performed with DEHP and structurally similar compounds; dimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP). The imprinting factor (IF) of DEHP was found to be 12.86 which was much higher than the imprinting factors (IF) of other phthalates. A method constituted by molecularly imprinted solid-phase extraction (MISPE) with GC-FID was developed for DEHP analysis in water samples under very simple conditions. Sample loading and desorption conditions were also optimized. The MISPE method's linearity ranged from 0.035 to 3.0 µg ml⁻¹ with r² = 0.9998. Intra-assay, interassay precision and accuracy ranged from 0.0168% to 1.017%, 1.130% to 4.799% and 94.98% to 99.35%, respectively. The LOD and LOQ were found to be 0.011 and 0.035 µg ml⁻¹, respectively. Synthesized MIP was employed in MISPE for cleaning up the spiked river water samples prior to gas chromatographic analysis. The river samples were found to contain DEHP in the range of 1.4 × 10⁻³ to 0.349 µg ml⁻¹.

Effects of Potamogeton crispus L. on the fate of phthalic acid esters in an aquatic microcosm.

To study the effects of submerged hydrophytes on the fate of dibutyl phthalate (DBP) and di-2-ethylexyl phthalate (DEHP) in the aquatic environment, a Potamogeton crispus L. (P. crispus) microcosm was constructed. A four-compartment (i.e., water, plant, non-rhizosphere and rhizosphere sediments) level IV fugacity model was established and applied to the simulation experiments in the microcosm. Data obtained from model calculations were in good agreement with those from the experiments. Results of the model calculations showed that the total residues of DBP and DEHP in the microcosm with P. crispus were 39.7% and 19.8% lower than those in the microcosm without P. crispus. The overall biodegradation fluxes of DBP and DEHP in the microcosm with P. crispus increased by 4.7% and 12.3%, respectively, and meanwhile, advective outflow decreased. In the presence of P. crispus, a large fraction of loaded DBP and DEHP (17.7% and 29.0%) transferred to plants, and then to the rhizosphere. 4.8% and 28.0% of loaded DBP and DEHP were removed by biodegradation in P. crispus, and the remaining 12.9% and 1.0% were by biodegradation in rhizosphere sediment which was 3.6% of the total sediment. This finding demonstrates that P. crispus can substantially reduce the accumulation of phthalic acid esters (PAEs) in the experiment system and enhance the removal of PAEs. The enhancement of PAE removal is related to the biodegradation of PAEs in P. crispus, especially for the more hydrophobic DEHP. For the less hydrophobic DBP, biodegradation in the rhizosphere also plays a key role. In addition to nutrient uptake from sediment, transport process between P. crispus and the rhizosphere has also a significant influence on the distribution and fate of PAEs in the aquatic environment.

Antitumour evaluation of di-(2-ethylhexyl) phthalate (DEHP) isolated from Calotropis gigantea L. flower.

The objective of the study is to explore the anticancer activity of di-(2-ethylhexyl) phthalate (DEHP) isolated from Calotropis gigantea flower against Ehrlich ascites carcinoma cells (EAC) in Swiss albino mice. The activity of DEHP was evaluated at doses of 10, 20 and 40 mg kg-1 body mass applied intraperitoneally. DEHP showed a significant decrease in viable cell count (p < 0.05), mass gain (due to tumour burden) and elevated the life span of EAC cell bearing mice. Altered hematological profiles such as RBC, hemoglobin, WBC and differential count were reverted to normal levels in DEHP-treated mice. DEHP also brought back altered biochemical parameters (glucose, cholesterol, triglycerides, blood urea, SALP and SGOT) to normal level. Results of this study indicate that DEHP show potent dose dependent antitumour activity against EAC in vivo.

Taxonomic study and partial characterization of antimicrobial compounds from a moderately halophilic strain of the genus Actinoalloteichus

A moderately halophilic actinomycete strain designated AH97 was isolated from a saline Saharan soil, and selected for its antimicrobial activities against bacteria and fungi. The AH97 strain was identified by morphological, chemotaxonomic and phylogenetic analyses to the genus Actinoalloteichus. Analysis of the 16S rDNA sequence of strain AH97 showed a similarity level ranging between 95.8 percent and 98.4 percent within Actinoalloteichus species, with A. hymeniacidonis the most closely related. The comparison of the physiological characteristics of AH97 with those of known species of Actinoalloteichus showed significant differences. Strain AH97 showed an antibacterial and antifungal activity against broad spectrum of microorganisms known to be human and plant pathogens. The bioactive compounds were extracted from the filtrate culture with n-butanol and purified using thin layer chromatography and high pressure liquid chromatography procedures. Two active products were isolated, one hydrophilic fraction (F1) and another hydrophobic (F2). Ultraviolet-visible, infrared, mass and ÕH and 13C nuclear magnetic resonance spectroscopy studies suggested that these molecules were the dioctyl phthalate (F2) and an aminoglycosidic compound (F1).