Microtitration of Free Fatty Acids in Oil and Biodiesel Samples Using Absorbance and/or Fluorescence of Pyranine

Free fatty acids (FFA) are measured before alkaline transesterification of oil and when testing quality of the produced biodiesel. Titration of FFA is usually monitored on a potentiometer (EN 14104 and ASTM D 664 methods) or employing the indicator phenolphthalein (AOCS Cd 3d-63). Both procedures have some disadvantages including relatively large sample masses (2–20?g), elaborate maintenance of the electrode, subjective observation of the color transition, etc. Here we describe a microtitration method based on absorbance/fluorescence of pyranine (aqueous pK 7.3). All reactants were dissolved in a medium with universal solubility, which allowed accurate optical measurements. FFA standards and test samples (5–150?mg) were titrated on standard equipment, and the sigmoid titration curves were produced. The upper bend of each sigmoid corresponded to neutralization of FFA. Fluorescence of pyranine had better “signal to noise” characteristics than absorbance when working with heavily pigmented oil samples. Blind examination of different experimental mixtures (FFA?=?0.15–40?%) revealed a close correspondence between the pyranine method and two other established procedures (EN 14104 and a variant of AOCS Cd 3d-63).     

Comparison of four analytical methods for the determination of peroxide value in oxidized soybean oils

Previous work in our laboratory demonstrated that soybean oil oxidation, expressed as PV, can be determined using NIR transmission spectroscopy as an alternative to the official AOCS iodometric titration method. In the present study, a comparison of four peroxide analytical methods was conducted using oxidized soybean oil. The methods included the official AOCS iodometric titration, the newly developed NIR method, the PeroxySafe^™ kit, and a ferrous xylenol orange (FOX) method, the latter two being colorimetric methods based on oxidation of iron. Five different commercially available soybean oils were exposed to fluorescent light to obtain PV levels of 0–20 meq/kg; periodic sampling was done to ensure having representative samples throughout the designated range. A total of 46 oil samples were analyzed. Statistical analysis of the data showed that the correlation coefficient (r) and standard deviation of differences (SDD) between the standard titration and NIR methods were r=0.991, SDD=0.72 meq/kg; between titration and the PeroxySafe^™ kit were r=0.993, SDD=0.56 meq/kg; and between the standard titration and FOX method were r=0.975, SDD=2.3 meq/kg. The high correlations between the titration, NIR, and PeroxySafe^™ kit data indicated that these methods were equivalent.     

A Visual Titration Method for the Determination of the Acid Number of Oils and Fats: a Green Alternative

A visual and green titrimetric method is proposed for the determination of the acid number of oils and fats. The sample is dissolved in a water–ethanol mixture (1:1 v/v) and titrated with a 0.02?mol L^?1 aqueous NaOH solution. Phenolphthalein is used as acid-basic indicator. Oils of canola, sunflower, linseed, castor, corn and soy as well as swine lard, a total of twelve real samples, were analyzed. The results obtained were compared with those from the application of the AOCS Cd 3d-63 method, through a statistical linear regression procedure at the 95?% confidence level and no evidence for systematic differences between the two sets of results was observed. The mean RSD (relative standard deviation ) for the proposed procedure is 4.8?%, whereas that of the official method was found to be 5.4?%.     

Evaluation of SafTestTM methods for monitoring frying oil quality

The feasibility of applying methods developed by Safety Associates, Inc., to monitor oil degradation products, including malondialdehydes (AldeSafe^TM), FFA (FASafe^TM), and peroxides (PeroxySafe^TM), in fresh and heat-abused deep-fat frying oil was evaluated. Based on performance qualification studies, the AldeSafe method was the most suitable SafTest^TM assay for monitoring the quality of frying oil because of its high accuracy, precision, linearity, and reproducibility, and low detection/quantitation limits. A strong correlation (r=0.924) between the AldeSafe method and its counterpart, AOCS Official Method Cd 19-90, also supported the suitability of the SafTest method for monitoring oil quality. Moreover, the FASafe method had a moderately strong relationship with AOCS Official Method Ca 5a-40 (r=0.761). Our studies suggest that this test can be applied for monitoring frying oil; however, certain method performance limitations must be considered for routine analysis purposes. In contrast, the PeroxySafe method probably should not be used to monitor heat-abused oil without further development because of high variability, low accuracy, and low correlation (r=0.062) with the AOCS Official Method Cd 8-53 assay.     

Rapid Method for the Determination of Moisture Content in Biodiesel Using FTIR Spectroscopy

A new, rapid, and direct method was developed for the determination of moisture content in biodiesel produced from various types of oils using Fourier transform infrared (FTIR) spectroscopy with an attenuated total reflectance (ATR) element. Samples of biodiesels used in this study were produced using sludge palm oil (SPO). The calibration set was prepared by spiking double-distilled water into dried biodiesel samples in ratios (w/w) between 0 and 10% moisture. Absorbance values from the wavelength regions 3,700–3,075 and 1,700–1,500 cm⁻¹, and the partial least square (PLS) regression method were used to derive a FTIR spectroscopic calibration model for moisture content in biodiesel samples. The coefficient of determinations (R ²) for the models was computed by comparing the results obtained from FTIR spectroscopy against the values of the moisture concentrations (%) determined using the American Oil Chemists’ Society (AOCS) oven method Ca 2d-25. Same comparison was done using International Union of Pure and Applied Chemistry (IUPAC) distillation method 2.602. R ² was 0.9793 and 0.9700 using AOCS and IUPAC methods, respectively. The standard error (SE) of calibration was 1.84. The calibration model was cross validated within the same set of samples, and the standard deviation (SD) of the difference for repeatability (SDDr) and accuracy (SDDa) of the FTIR method was determined. With its speed and ease of data manipulation, FTIR spectroscopy is a useful alternative method to other methods for rapid and routine determination of moisture content in biodiesel for quality control.     

Collaborative test of determination of iodine value in fish oils. 2. Carbon tetrachloride or cyclohexane/acetic acid as solvents

Nine laboratories participated in a collaborative test to determine the iodine value (IV) of eight samples of fish oil (four with IV<150; four with IV>150) with either carbon tetrachloride (AOCS Official Method Cd 1–25) or cyclohexane/acetic acid (AOCS Recommended Practice Cd 1d-92) as solvent and 1 h of reaction time. Laboratories received coded duplicate samples (hidden duplicates) and carried out duplicate determinations on each oil by each method (open duplicates). Replacing carbon tetrachloride with cyclohexane/acetic acid resulted in similar mean values for both low- and high-IV oils and similar estimates of repeatability and reproducibility. The repeatability standard deviation (s _r ), based on hidden duplicates, with carbon tetrachloride and cyclohexane/acetic acid were 1.71 and 1.55, respectively. The corresponding reproducibility standard deviations were 1.81 and 1.98.     

Synthesis,characterization, and application of a new chelating resin functionalized with dithiooxamide

Chloromethylated polystyrene‐divinylbenzene has been functionalized with dithiooxamide. The resulting chelating resin (DTOA) has been characterized by elemental analyses, infrared spectroscopy, thermogravimetric analysis, and metal ion sorption capacities. It has been used for the preconcentration and separation of Cu(II), Zn(II), Cd(II), and Pb(II) prior to their determination by FAAS. Parameters such as the amount of the resin, effect of pH, equilibration rate, sorption and desorption of metal ions, and effect of diverse ions have been studied. The maximum sorption capacities found are 0.97, 0.12, 0.08, and 0.12 mmol g^?1 for Cu(II), Zn(II), Cd(II), and Pb(II) at pH 6.0, 5.5, 1.0, and 5.5, respectively. The preconcentration factors are 100, 100, 50, and 50 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. Recoveries of the metal ions were 96 ± 5, 97 ± 6, 96 ± 5, and 96 ± 5 at 95% confidence level, whereas the limits of detection are 2.0, 1.3, 2.5, and 25.0 μg L^?1 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The calibration curves were linear up to 12 μg mL^?1 (R² = 1.000), 2 μg mL^?1 (R² = 0.998), 2 μg ml^?1 (R² = 1.000), and 5 μg mL^?1 (R² = 0.979) for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The reliability of the method has been tested by analyzing certified samples. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2281–2285, 2007     

A rapid method for the analysis of hydrogenated fats by GC with IR detection

A rapid method for analysis of trans and cis FA in hydrogenated fats has been developed. The method is based on a single anlaysis by CG with IR detection. Multivariate partial least squares regression is applied on the IR spectra to predict the number of cis and trans double bonds. For each chain length the method provides information about the amount of the saturated FA, the amount of trans monoenes, the amount of cis monoenes, the amount of PUFA, and the average number of cis and trans double bonds in PUFA. The method has been validated by summing the values to a total trans value and total unsaturation. These sum values were compared with total trans unsaturation, as determined by AOCS method Cd 14-95, and iodine value, as determined by AOCS Cd 1d-92.     

Determination of the oxidative stability of fats and oils: Comparison between the active oxygen method (AOCS Cd 12-57) and the rancimat method

Oxidative stability is an important parameter in the characterization of fats and oils. The determination of this parameter with the Active Oxygen Method (AOM; AOCS Method Cd 12-57) is both very costly and labor intensive, owing to the repeated peroxide value determinations involved. The alternative rancimat method is based on the conductometric determination of volatile degradation products and features automatic plotting of the conductivity against time. The evaluation is performed graphically after completion of the experiment. The labor required for this method is considerably less as it is not necessary to perform titrations at regular intervals. In the determination of the peroxide values of six samples at three temperatures, ca 151 mixed solvent and chemicals valued at SFr. 400 (ca $180 US) were consumed. The induction times (t _I ) determined with both methods using six different fats and oils show a good correlation (slope 1.005, correlation coefficient 0.987). The rancimat method thus yields results equivalent to the AOCS Method Cd 12–57, but offers a real alternative for the determination of oxidative stabilities owing to the appreciable saving in labor.     

Application of a Novel,Heated, Nine-Reflection ATR Crystal and a Portable FTIR Spectrometer to the Rapid Determination of Total <Emphasis Type="Italic">Trans</Emphasis> Fat

Since trans fat labeling requirements became mandatory in the US and many other countries, there has been a need for rapid and accurate analytical methodologies that can facilitate compliance with the various regulations. The determination of total trans fatty acids by mid-infrared (IR) spectroscopy is a widely used procedure that was standardized and validated as AOCS Official Method Cd 14e-09 (negative second derivative infrared spectroscopic method for the rapid (5 min) determination of total isolated trans fat) in 2009. The C–H out-of-plane deformation mid-IR band observed at 966 cm⁻¹ is uniquely characteristic of isolated (non-conjugated) double bonds with trans configuration. AOCS Official Method Cd 14e-09, the most recent attenuated total reflection-Fourier transform IR (ATR-FTIR) official method, entails the measurement of the height of the negative second derivative of the trans absorption band. In the present study, the performance of a novel, portable FTIR system equipped with a heated 9-bounce diamond ATR crystal was evaluated and compared to that of a conventional benchtop single-bounce ATR-FTIR spectrometer. The introduction of the 9-bounce diamond ATR crystal resulted in the lowering of the limit of quantification of trans fat, as a percentage of the total fat, from approximately 2 to 0.34%. The data collected from accurately weighed gravimetric standards and 28 unknown test samples ranging in trans fat content from about 0.5 to 54%, as a percentage of the total fat, indicated that this IR official method and the use of the new 9-bounce portable ATR-FTIR instrumentation could lead to a five-fold enhancement in sensitivity relative to single-bounce systems. Implementing these changes would facilitate regulatory compliance and verification of fat and oil samples for trans fat content in the US and other countries, since all of the published regulations (e.g., “0 g trans fat per serving”) have levels of trans fat, as percentage of total fat, that exceed 0.34%.     

A fully robotic method for the determination of acid values in olive oil without titration

A method for the determination of acid values (AV) in olive oil without titration is proposed, based on pH measurements on an oil sample emulsion in a suitable reagent and fully automated using a robotic station. The robot performs the weighing of the sample, adds the reagent, emulsifies both, and measures the pH of the emulsion. The data are acquired and treated by the computer. After optimization, two versions of the measurement step (interpolation within a calibration curve and addition of standard) are developed and applied to 51 oil samples with AV values that also have been determined by the standard method based on potentiometric titration. Both versions are statistically compared with the standard method, and no significant differences are found. Excellent correlation (R>0.99) between all three methods demonstrates the usefulness of the fully automated approach that works at a sample rate of 15 sample h⁻¹ with relative standard deviations lower than 2.5%.     

Evaluation of the dielectric properties of grease containing copolymers and ester

Poly(1-octadecene-co-maleic anhydride) bis behanate ester, poly(1-octadecene-co-maleic anhydride) bis stearate ester, and ethylene glycol bis stearate ester were prepared and their structures were confirmed by IR spectroscopy. Wax gel was prepared from base oil blend (base lube oil grade 260/290, transformer oil) and microcrystalline wax in ratio 2.3 : 1, respectively, in the presence of antioxidant and anticorrosion additives in ratio of 0.1–2%. Atactic polypropylene was added in mixture with the above copolymers and ester to the wax gel in different proportions. Dielectric constant, dielectric loss, and volume resistivity at frequency range of 1–10³ kHz 35°C have shown better results than that of wax gel alone. The grease which includes poly(1-octadecene-co-maleic anhydride) bis behanate ester in mixture with atactic polypropylene recorded the best results. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Preparation,Crystal Structure,Thermal Decomposition,and Explosive Properties of [Cd(en)(N3)2]n

A new multi‐ligand coordination polymer of cadmium(II) ethylenediamine azide, [Cd(en)(N₃)₂]_n (en=ethylenediamine), was synthesized and characterized by using elemental analysis and FT‐IR spectrum. Its crystal structure was determined by means of X‐ray single crystal diffraction. The obtained results show that this crystal belongs to monoclinic, P2₁/n space group, a=0.6548(1) nm, b=1.0170(2) nm, c=1.2246(2) nm, β=90.23(1)°, V=0.8156(2) nm³, D_c=2.090 g⋅cm⁻³, Z=4, R₁=0.024, wR₂ (I>2σ(I))=0.0416 and S=0.998. The Cd(II) ion is six‐coordinated with four azido ligands by μ‐1, 1 azido bridges, and two ethylenediamine molecules which serve as bidentate ligands through the nitrogen atoms. The thermal decomposition mechanism of the title complex was studied by using differential scanning calorimetry (DSC) and thermogravimetry‐differential thermogravimetry (TG–DTG) techniques. Under nitrogen atmosphere with a heating rate of 10 K⋅min⁻¹, the thermal decomposition of the complex contains two main successive exothermic processes between 519 and 701 K in the DSC curve, and the final decomposed residue at 725 K is Cd. The non‐isothermal kinetics parameters were calculated by using the Kissinger's method, Ozawa–Doyle's method, pervasive integration method, and differential method, respectively. The sensitivity properties of [Cd(en)(N₃)₂]_n were also determined with standard methods.     

Synthesis of polyphenylmethylsiloxanes and their enhancement on tribological properties of titanium complex grease

Polyphenylmethylsiloxanes (PPMSs) with the phenyl/methyl mole ratios of 0.25:1–0.75:1 were facilely synthesized using phenylmethyldichlorosilane, diphenyldichlorosilane, dimethyldichlorosilane, and hexamethyldisiloxane as the raw materials with the hydrolysis-condensation method. The molecular weights (M _w) of the as-synthesized PPMSs ranged from 1248.7 to 27 014.0. The maximum molecular weight was obtained at the phenyl/methyl ratio of 0.65:1. Titanium complex greases were prepared by using titanium carboxylate as the thickener and PPMS/mineral oil (KN4010) as the mixed base oil. When the weight percentage of PPMS (phenyl/methyl mole ratio of 0.45:1) in the mixed base oil was 20%, the dropping point of the resultant titanium complex grease increased by 21 °C as compared with the sole mineral oil-based titanium complex grease. The friction coefficient and the wear scar dimension were 0.54 and 0.69 times those of the sole mineral oil-based titanium complex grease, respectively. According to thermogravimetric analysis–differential scanning calorimetry analysis, the working temperature of the titanium complex grease could reach 240 °C. The addition of PPMS in the titanium complex grease obviously improved its thermal stability and tribological properties. The present work developed a new category of titanium complex grease using PPMS and mineral oil as the mixed base oil. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47168.     

High Performance Liquid Chromatography–Size Exclusion Chromatography for Rapid Analysis of Total Polar Compounds in Used Frying Oils

Analysis of used frying oil samples by high performance liquid chromatography–size exclusion chromatography (HPLC–SEC or HPSEC) was compared to AOCS Official Method Cd 20-91 (silica gel column chromatography) for the purpose of developing a rapid analysis of total polar compounds (TPC). In a direct comparison of the two analytical methods using four different sets of used frying oils (21 total oil samples) ranging from fresh to discard quality (4.3 to 35.4% TPC by column chromatography), the weight percent total polar compounds (%TPC) determined by HPLC–SEC averaged 0.71% higher than the values by silica gel column chromatography. Reproducibility of the HPLC–SEC method of s _r = 0.30 and RSD_r% = 1.22 compares to the variability of s _r = 0.29 and RSD_r = 1.3 for samples of approximately the same %TPC, reported in AOCS Method Cd 20-91. Because the rapid method does not separate pure (non-polar) triacylglycerol (TAG) and polar, oxidized TAG (OX-TAG), a high concentration of OX-TAG will quantitatively affect the results. This places practical limits on the types of studies to which the method may be applied if a separate analysis for the OX-TAG is not performed. Advantages of the HPLC–SEC method include the following. It uses about 75% less solvent than standard column chromatography methods for determination of %TPC. This HPLC–SEC method is very similar to AOCS Official Method Cd 22-91, and thus, also separates and quantifies polymerized triacylglycerols. The HPLC–SEC method determines both TAG polymer concentration and %TPC of used frying oils in about 1 h.     

Quick method for estimating free gossypol in cottonseed,meats, collets,and extracted meals

A method for estimating free gossypol (FG) has been developed that decreases sample-determination time from over 2 h to about 25 min per sample. With auto pipetters and bottle-to dispensers, six sample determinations can be completed in approximately 50 min. The method consisted of adding water and acetone separately to a fixed sample weight, mixing, filtering, diluting with 65% acetone, and reading absorbance on a spectrophotometer. Absorbance was plotted against the official American Oil Chemists’ Society’s FG method for samples that contained FG between 0.02 and 0.9%. Quadratic least squares regression for 31 samples had a correlation coefficient ofr ²=0.986 and a standard error of estimated FG of 0.032%. Presented in part at the AOCS Annual Meeting, Anaheim, California, April 1993.     

Comparison of attenuated total reflection infrared spectroscopy to capillary gas chromatography for trans fatty acid determination

Gas chromatography (GC) has been a standard analytical tool in lipid chemistry. The rapid attenuated total reflection (ATR) infrared (IR) American Oil Chemists’ Society (AOCS) Recommended Practice (Cd 14d-97) was compared to the capillary GC AOCS Recommended Practice (Ce 1f-97) that was optimized to accurately determine total trans fatty acids on highly polar stationary phases. This comparative evaluation was validated in an independent laboratory. These procedures were used to quantitate the total trans fatty acid levels in partially hydrogenated vegetable oils, measured as neat (without solvent) triacylglycerols (TAG) by ATR and as fatty acid methyl ester (FAME) derivatives by capillary GC. Unlike FAME, TAG determination by ATR required no derivatization, but samples had to be melted prior to measurement. Five blind replicates for each of three accuracy standards and three test samples were analyzed by each technique. The GC and ATR determinations were in good agreement. Accuracy was generally high. The ratios of ATR mean trans values (reported as percentage of total TAG) to the true values (based on the amount of trielaidin added gravimetrically) were 0.89, 0.98, and 1.02 for accuracy standards having about 1, 10, and 40% trans levels. The corresponding GC values, determined as percentage of total FAME, were 0.98, 0.99 and 1.04. The ratios of mean trans values determined by these techniques were ATR/GC 0.85, 1.04, and 1.01 for test samples having trans levels of about 0.7, 8, and 38%, respectively. The optimized GC procedure also minimzed the expected low bias in trans values due to GC peak overlap found with the GC Official Method Ce 1c-89. Satisfactory repeatability and reproducibility were obtained by both ATR and GC.     

An International Collaborative Study on Trypsin Inhibitor Assay for Legumes,Cereals, and Related Products

For determining trypsin inhibitor activity (TIA) in soy products, the American Oil Chemists' Society (AOCS) Method Ba 12-75 has been used. It measures differences in absorbance at 410 nm of bovine trypsin activity toward a synthetic substrate (Nα-benzoyl-DL-arginine-p-nitroanilide) in the absence and presence of an inhibitor. Recently, a significantly improved method was developed (JAOCS, 2019, 96:635–645), featuring 5 mL of total assay volume, enzyme-last sequence, and single inhibitor level in duplicate. It is proposed as the AOCS Method Ba 12a-2020. As a part of the AOCS method approval process, a collaborative study involving 12 international laboratories was conducted to evaluate the performance of the proposed method. The study involved measuring TIA in 10 selected test samples plus a blind duplicate. They included soybeans, pulses, cereals, and their processed products (flours, concentrates, and isolates). After rigorous statistical treatment of the data, only three outliers were removed from the data of two samples. Repeatability relative standard deviations (RSDr) for the 11 samples ranged from 0.99% to 5.52%. Reproducibility RSD (RSD_R) ranged from 7.07% to 22.92%, with seven samples having RSD_R around 10% or less. The remaining four samples had very low TIA, and their RSD_R values ranged from 13.34% to 22.92%. The study has demonstrated reliable performance of the proposed AOCS method. Several collaborators carried out additional experiments addressing some aspects of the method, leading to further refinements. The proposed method is undergoing evaluation by the AOCS Uniform Methods Committee for adoption as an Official Method for measuring TIA in various legume and grain products.     

Regulatory Infrared Spectroscopic Method for the Rapid Determination of Total Isolated Trans Fat: A Collaborative Study

Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, collaborating scientists in ten different laboratories measured (in duplicate) the total trans fat content of ten fat or oil test samples, two of which were blind duplicates. The procedure used entailed measuring the height of the negative second derivative of the IR absorption band at 966 cm^?1. This absorption is attributed to the C?CH deformation vibration that is characteristic of isolated (non-conjugated) double bonds with the trans configuration. The precision of ATR-FTIR results in this international collaborative study was satisfactory and led to the approval of this validated procedure as official method AOCS Cd 14e-09 in late 2009. This official method is also suitable for analysis of total isolated trans fat and oil products containing, or supplemented with, trans conjugated linoleic acid (CLA) isomers. Although this method does not require derivatization of the oil or fat test materials, as required for GC, fats and oils in foods must be extracted with organic solvents before analysis. This method is also rapid (5 min) and does not require any weighing or quantitative dilution of unknown neat fat or oil test samples in any solvent. The AOCS Cd 14e-09 method is suitable for determination of test samples with zero trans fat, which is defined according to the US labeling regulations as 0.5 g trans fat per serving or 1.8% trans fat, as a percentage of total fat.     

Polycyclic Aromatic Hydrocarbons,Heavy Metals,and Genotoxicity of the Suburban Soils from Guangzhou,China

During the past few decades, urban and suburban developments have grown at unprecedented rates and extents with unknown consequences for ecosystem function. The problem of soil pollution as a result of the accelerating development of Guangzhou in China is becoming great concerns. In the present study, gas chromatograph coupled mass spectrometry (GC-MS), inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) were employed to determine the 16 US Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs) and the heavy metals (As, Cr, Cu, Pb, Cd, Hg, and Se) of soils collected from suburban areas of Guangzhou. The genotoxicity of these soils was screened with micronucleus (MN) assay in Vicia faba root cells. The concentrations of the pollutants in the soils were (dried weight): ΣPAHs (230.6–1263 ng·g^?1), As (2282.6–36064 μg·kg^?1), Cr (7109–64699 μg·kg^?1), Cu (7047–56388 μg·kg^?1), Pb (9675.9–93739 μg·kg^?1), Cd (68.5–847.3 μg·kg^?1), Hg (85.4–549.2 μg·kg^?1), and Se (219.2–968 μg·kg^?1), which fell in the moderately polluted range. However, six out of nine soil-exposed groups had a significant increases of MN frequencies observed in the V. faba root cells compared with the negative group (P < 0.05, P < 0.01), indicating that they had potential genotoxic risks. Bringing together the chemical analyses with the biological effects observed in this study, the genotoxic response could at a certain degree be explained by both the soil PAHs and heavy metals. Our results suggested that apart from chemical analysis, bioassays like the MN assay of V. faba root cells should also be included in a battery of tests to assess the eco-environmental risks of urban and/or urbanization in the developing areas on the soils.