Levels of polychlorinated biphenyls and organochlorine pesticides in donkey milk: Correlation with the infection level by intestinal strongyles.

AIM: The study aimed at evaluating the concentration levels of organochlorine pollutants in donkey milk and their modulation on the intestinal strongyle infection. Risk evaluation for consumer health was also investigated. METHODS: We analyzed milk of grazing donkeys living in areas of Southern of Italy affected by organochlorine compounds environmental pollution and parasite infection. The presence of pollutants was assessed through summary statistics; regression analysis of intestinal strongyle on pollutant concentration was performed to investigate the relationship between the two variables. RESULTS: PCB concentrations (mainly non-dioxin-like (ndl)-PCBs) were higher than OCP ones. Mean values of ndl-PCBs across areas ranged from 93.13 to 263.64 ng g-1. In all sample units we detected the six indicator PCBs with the prevalence of the PCB 153, followed by the PCB 28 and the PCB 101. Among the dioxin-like (dl)-PCBs, non-ortho PCB 169, 77 and 126 were assessed in some milk samples; in all areas we detected the mono-ortho PCB 118 and PCB 105. Positive correlation between infection level and six indicator PCBs as well as between the former and HCB, on WW and LW, were observed (at least statistically significant at 5 percent). In some cases, Dl-PCB concentrations emerged as dangerous given the EU maximum residue limit for PCDD/Fs and dl-PCBs. CONCLUSION: Evidence supports the hypothesis of an immunosuppressive role of organochlorine pollutants; risk evaluation reveals the potential health impact of dl-PCB intake, particularly for major donkey milk consumers such as infants, children with cow milk and multiple food intolerance, and elders.

Determination of methotrexate in human urine at trace levels by solid phase extraction and high-performance liquid chromatography/tandem mass spectrometry.

For biological monitoring of hospital personnel occupationally exposed to antineoplastic agents, highly sensitive and specific methods are required. In order to detect trace MTX urinary concentrations, a precise and accurate high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedure, incorporating solid phase extraction, has been developed. Urine samples were purified by solid phase extraction (SPE) on octadecyl bonded, endcapped silica SPE columns. After eluting with methanol, the solvent was evaporated obtaining a 25-fold concentration of the analyte. This procedure was validated by using 7-OHMTX as internal standard. Calibration curves had correlation coefficients always higher than 0.999, and the limit of detection was assessed at 0.2 microg L(-1). High specificity of the HPLC-MS/MS technique assures that no interfering substances are detected rather than the analyte of interest.

Trans,trans-muconic acid, a biological indicator to low levels of environmental benzene: some aspects of its specificity.

BACKGROUND: The specificity of trans,trans-muconic acid (MA) as a biomarker of exposure to low benzene levels and the role of sorbic acid (SA) as a confounding factor were evaluated. MA, a urinary ring-opened metabolite of benzene, has been recently proposed for the biological monitoring of populations exposed to low levels of this chemical. The usual presence of MA in urine of non-occupationally exposed people is generally attributed to benzene world-wide contamination (mainly by smoking habits, urban pollution, and maybe by food contamination). However, the scientific literature reveals that the common food preservative and fungistatic agent SA is converted into MA though in trace amounts. METHODS: Urinary benzene and MA before and after administration of SA were measured in smokers and non-smokers. Benzene dissolved in urine was analyzed injecting a headspace sample in a gas-chromatografic system. Urinary MA was measured by means of a HPLC apparatus. RESULTS: The mean background values of MA were about 60 mg/L (or 50 mg/g creat.); after experimental administration of SA (447 mg), the mean urinary MA concentration became more than 20 times higher. The biotransformation rates of SA into MA after ingestion of 447 mg of SA ranged from 0.05 to 0.51%. The ratio between unmetabolized benzene in the two groups of smokers and non-smokers was significantly different from the ratio between MA in the same two groups. DISCUSSION: Other sources of MA excretion, different from benzene, influence the urinary concentration of the metabolite: only 25% of MA background values can be attributed to benzene. The urinary MA induced by 100 mg of ingested MA is 77% of that expected after an 8-hour benzene exposure to 0.5 ppm (current threshold limit value according to ACGIH). In conclusion, MA is not a sufficiently specific biomarker of low benzene exposure; a significant effect of SA ingestion is predictable.

Matrix interferences in the analysis of benzene in urine.

The analysis of benzene in urine of the general population or of exposed workers can be performed with different methods using the 'purge and trap' or 'solid-phase microextraction' techniques in combination with gas chromatographic analysis and photoionisation or mass spectrometric detection. The published results, however, are deeply conflicting. Differences in sample preparation by different research groups and our own preliminary observations prompted us to investigate pre-analytical and analytical factors potentially capable of modifying the urinary benzene quantification results. Benzene concentrations were measured in 20 urine samples in relation to different conditioning conditions (at 24, 40 and 80 degrees C) and at basic or acid pH. Urinary protein concentrations were measured in the same samples. Urine heating at 80 degrees C yields benzene concentrations on average five times higher than at 24 degrees C. On acidification of urine, the benzene released increases up to 28-fold in comparison to that obtained at uncorrected 'physiological' pH. Despite a widely scattered data distribution, a statistically significant linear correlation was found between 'heat-released' and 'acid-labile' benzene values. There was no correlation between total urinary proteins present in 'physiological' concentrations (between 12 and 110 mg/l) and the different kinds of benzene in urine. Our results could perhaps be explained if it is supposed that part of the benzene in urine is absorbed onto sediment, or bound to specific proteins, or derived from parent molecules and is released with pH modification or heat administration. Our observations may also help to explain why the urinary benzene concentrations reported by different investigators vary considerably even when environmental levels are comparable.

Determination of environmental reference concentration of six PAHs in urban areas (Pavia, Italy).

We propose a sampling strategy, using individual dosimetry to measure the daily inhaled quantity of PAHs in urban air. The method was applied to monitor 56 subjects living in an Italian town (Pavia; 80 000 inhabitants) and the Environmental Reference Concentration (E.R.C.) of six PAHs (classified as 'possible' carcinogenic agents for humans) was determined. The individual environmental samplings took place in two different seasons (winter and summer), for persons living in four different urban areas with different traffic density. Subjects were selected using a specific questionnaire designed to collect information on indoor and indoor+outdoor exposure times. The mean +/- S.D. value of Benzo[a]pyrene [BaP] was 0.37 +/- 0.15 ng m-3 in winter and 0.12 +/- 0.07 ng m-3 in summer. Assuming 18 m3 as the daily inhaled quantity the estimate of the BaP inhaled quantity was 6.66 ng/day in winter and 2.16 ng/day in summer.

[The toxicology and prevention of the risks of occupational exposure to aromatic polycyclic hydrocarbons. II. Toxicology. Exposure assessment. Environmental and biological monitoring]. / Tossicologia e prevenzione dei rischi da esposizione professionale a idrocarburi policiclici aromatici. II. Tossicologia. Valutazione dell'esposizione. Monitoraggio ambientale e biologico.

The evaluation of exposure to polycyclic aromatic hydrocarbons (PAH) should firstly comply with current regulations (D.Lgs. 626/94), that is, identify the compounds and exposed subjects, quantify exposure, adopt preventive measures and health and epidemiological surveillance. Environmental monitoring should take into account the technological cycle and the tasks with higher PAH exposure risk, and the main sources of emissions. In the case of skin contamination, it should be considered the measure of skin PAH by means of sampling or removal techniques; moreover, the determination of urinary hydroxypyrene (1-HP) should be performed. It is mandatory to analyse (Benz[a]) anthracene; Benzo[b]fluroanthene; Benzo[j]fluoranthene; Benzo[k]fluoranthene; Benzo[a]pyrene; Dibenzo[a,h]anthracene, i.e. the PAH marked with the R45-R49 phrase. When 1-HP determination is planned, Pyrene should be added. Biological monitoring has been addressed mainly to hydroxylated metabolites of pyrene and among these 1-HP, the main metabolite of pyrene, although non occupational factors, such as tobacco smoking and consumption of smoked foods are potentially confounding. Urinary mutagenicity tests which are heavily influenced by non occupational factors such as tobacco smoking and diet are not advisable. The determination of DNA and protein adducts is a promising test for evaluation of metabolic active dose but at the moment it is not suitable for routine use in occupational medicine. In order to interpret environmental and biological data, it will be useful to consider appropriate reference values ("limit" "guide", "operative", "maximum admissible") such as 0.1 mg/m3 for total PAH extracted with benzene, 5 micrograms/m3 for the mixture of 15 PAH listed by US NTP, the limits varying from 0.1 to 5 micrograms/m3 for Benzo[a]pyrene, and 2.7-4.4 micrograms/g creat, for 1-HP.

[The toxicology and prevention of the risks of occupational exposure to aromatic polycyclic hydrocarbons. I. Guide lines for the prevention of the risks of occupational exposure to aromatic polycyclic hydrocarbons. Società Italiana Valori di Riferimento and Cattedra di Medicina del Lavoro, Università di Brescia]. / Tossicologia e prevenzione dei rischi da esposizione professionale a idrocarburi policiclici aromatici. I. Linee guida per la prevenzione dei rischi da esposizione professionale a idrocarburi policiclici aromatici. Società Italiana Valori di Riferimento e Cattedra di Medicina del Lavoro, Università di Brescia.

These guidelines mainly deal with prevention of carcinogenic effects following occupational exposure to polycyclic aromatic hydrocarbons (PAH). After some toxicological remarks, the guidelines define a possible method to demonstrate and evaluate occupational exposure to PAH. In particular, it is illustrated the strategy of environmental monitoring and indicated which PAH should be measured, with suggestion about the most appropriate analytical techniques. As regards biological monitoring, the 1-OH-pyreneseems to be currently the most useful indicator since it reflects the recent and global exposure to PAH. The guidelines also give elements to interpret monitoring data, taking into account environmental and biological reference and limit values suggested by some authors, Associations, or current regulations. The most important health effects are carcinogenic and excess risks have been described mainly for lung, bladder and skin cancer in some PAH exposed workers. The studies on cytogenetic effects showed contradictory results. On the basis of such information and current regulations, the guidelines show how to perform health surveillance in preventive and periodical examinations and how to proceed for the information and formation of exposed workers. It is not advisable, on the basis of the current scientific data, to screen asymptomatic PAH exposed workers for early diagnosis of lung or bladder cancer, nor it is opportune the use of tumor markers for health surveillance nor is genetic screening applicable for individual susceptibility evaluation outside research programs.

Environmental and urinary reference values as markers of exposure to hydrocarbons in urban areas.

A study using individual dosimetry to evaluate the daily inhaled dose of sixteen aromatic and aliphatic hydrocarbons in three groups of primary school children, living in three Italian towns with 50,000 inhabitants or less, (Treviglio-Lombardy; Poggibonsi-Tuscany; Valenza-Piedmont) is presented. The simultaneous use of two passive samplers (radial diffusion) for each child, for a 24 h period, determined both the indoor and indoor + outdoor environmental reference concentrations. We measured the urinary levels of benzene, toluene, ethylbenzene and xylenes for each child and hence determined the urinary reference values for BTEX. We also considered the possibility of using benzene in urine as a biomarker of environmental exposure of the general population to this xenobiotic. We evaluated how both the environmental and biological measures were influenced by the presence of smokers in the surveyed children's houses. For the group of children living in Poggibonsi, we considered the influence of the living area and the traffic density on environmental concentrations of benzene (indoor and indoor + outdoor).

[Indoor pollution and biological monitoring of volatile organic compounds (VOC)]. / Indoor pollution e monitoraggio biologico di Composti Organici Volatili (COV).

Indoor air is a complex mixture of chemicals and airborne particles. Volatile Organic Compounds (VOC), a broad class of chemicals including diverse compounds such as Aldehydes, Terpenes, Aromatic and Aliphatic Hydrocarbons and Halogenated Volatile Organics, are an important category of indoor air pollutants. The evaluation of exposure to low doses of Chloroform and Benzene through the measurement of Chloroform and Benzene in urine was performed. Results show that biological monitoring may be helpful in indoor environmental studies in non occupational situations.

Evaluation of occupational exposure to benzene by urinalysis.

Urinary phenol determinations have traditionally been used to monitor high levels of occupational benzene exposure. However, urinary phenol cannot be used to monitor low-level exposures. New biological indexes for exposure to low levels of benzene are thus needed. The aim of this study was to investigate the relations between exposure to benzene (A-benzene, ppm), as measured by personal air sampling, and the excretion of benzene (U-benzene, ng/l), trans,trans-muconic acid (MA, mg/g creatinine), and S-phenylmercapturic acid (PMA, micrograms/g creatinine) in urine. The subjects of the study were 145 workers exposed to benzene in a chemical plant. The geometric mean exposure level was 0.1 ppm (geometric standard deviation = 4.16). After logarithmic transformation of the data the following linear regressions were found: log (U-benzene, ng/l) = 0.681 log (A-benzene ppm) + 4.018; log (MA, mg/g creatinine) = 0.429 log (A-benzen ppm) - 0.304; and log (PMA, micrograms/g creatinine) = 0.712 log (A-benzene ppm) + 1.664. The correlation coefficients were, respectively, 0.66, 0.58, and 0.74. On the basis of the equations it was possible to establish tentative biological limit values corresponding to the respective occupational exposure limit values. In conclusion, the concentrations of benzene, mercapturic acid, and muconic acid in urine proved to be good parameters for monitoring low benzene exposure at the workplace.

[Determination of low levels of urinary trans,trans-muconic acid]. / Il dosaggio dell'acido trans,trans-muconico urinario a basse concentrazioni.

A method is described which allows determination of urinary t,t-muconic acid (MA) by means of HPLC with UV detection even at micrograms/l concentrations. The clean-up procedure of samples involves the use of strong anionic-exchange cartridges (SAX) for solid phase extraction (SPE). In order to improve the reproducibility of the retention time of MA (CV < 1%) and to obtain an adequate separation of MA from interferents, a high performance reverse-phase column (250 x 4.6 [I.D.] mm, 3 microns) is used and a careful control of the temperature (25 degrees C) is made. Also, a column-switching technique is applied to the chromatographic system in order to eliminate the highly retained peaks from the analytical column. The isocratic run is performed at a constant flow rate of 0.7 ml/min; the mobile phase consists of water: methanol: acetic acid (93.5:5.5: 1, v/v) and the UV detector is set at 259 nm. Under these conditions, MA elutes at 21.5 min and a single analysis takes 25 min; the detection limit (at a signal-to-noise ratio of 3) is 3 micrograms/l in urine, when a 200 microliters aliquot of the extract is injected in the analytical apparatus. The recovery of the clean-up procedure is > 90%; both the intra-assay and the inter-assay coefficients of variation are < 4%. The method has been applied to smokers and nonsmokers, subjects occupationally unexposed to benzene; the results showed a statistically significant difference between the two groups. Also, a close correlation was found between urinary excretion of MA measured with this method and environmental concentration of benzene in a population of workers occupationally exposed to low levels of this solvent.

Urinary excretion of unmetabolized benzene as an indicator of benzene exposure.

Benzene concentrations in urine samples (Cu, ng/L) from 110 workers exposed to benzene in chemical plants and gasoline pumps were determined by injecting urine supernate into a gas chromatograph. The urine was saturated with anhydrous N2SO4 to facilitate the passage of benzene in the air over the urine. The solvent was stripped from the urine surface and concentrated on an adsorbent substrate (Carbotrap tube) by means of a suction pump (flow rate 150 ml/m). Wash-up of the head space was achieved by simultaneous intake of filtered air through charcoal. Benzene was thermically desorbed and injected in a column (thermal tube disorder, Supelco; 370 degrees C thermal flash; borosilicate capillary glass column SPB-1, 60 m length, 0.75 mm ID, 1 microns film thickness; GC Dani 8580-FID). Benzene concentrations in the urine from 40 non-exposed subjects (20 smokers > 20 cigarette/d and 20 nonsmokers) were also determined [median value of 790 ng/L (10.17 nmol/L) and 131 ng/L (1.70 nmol/L), respectively]. The 8-h time-weighted exposure intensity (Cl, micrograms/m3) of individual workers was monitored by means of charcoal tubes. The median value for exposure to benzene was 736 micrograms/m3 (9.42 mumol/m3) [geometric standard deviation (GSD) = 2.99; range 64 micrograms/m3 (0.82 mumol/m3) to 13,387 micrograms/m3) (171.30 mumol/m3)]. The following linear correlation was found between benzene concentrations in urine (Cu, ng/L) and benzene concentrations in the breathing zone (Cl, micrograms/m3): log(Cu) = 0.645 x log(Cl) + 1.399 r = .559, n = 110, p < .0001 With exclusion of workers who smoked from the study, the correlation between air benzene concentration and benzene measured in urine was: log(Cu) = 0.872 x log(Cl) + 0.6 r = .763, n = 63, p < .0001 The study results indicate that the urinary level of benzene is an indicator of occupational exposure to benzene.

[The measurement of a benzene metabolite, urinary S-phenylmercapturic acid (S-PMA), in man by HPLC]. / Dosaggio di un metabolita del benzene l'acido S-fenilmercapturico urinario (S-PMA), nell'uomo, mediante HPLC.

Benzene is a widely used solvent, currently present in the industrial environment at concentrations in the order of ppm. A valid method of biological monitoring that is easy to perform is needed for assessing occupational exposures. Benzene is metabolized in the body by microsomal cytochrome P-450 mono-oxygenase system into benzene epoxide. Benzene epoxide is metabolized along three different pathways which end in the excretion of trans, trans muconic acid, S-phenyl-mercapturic (S-PMA) and different phenols. A new method has been developed to evaluate urinary S-PMA of subjects exposed to benzene. Human urine is acidified with HCl to PH 1 and passed through a Sep-Pak C18 cartridge. The cartridges are washed with diluted HCl and a mixture of water/methanol/acetic acid and then eluted with acidified chloroform. The eluate is dried and reconstituted with a buffer phosphate, then passed through an anionic exchange cartridge (SAX) which is washed with diluted buffer and diluted HCl. S-PMA is recovered by eluting with concentrated buffer and is transformed into S-phenyl-cysteine. Finally, S-phenyl-cysteine is detected by HPLC connected with a fluorescence detector (wavelengths: excitation 330 nm, emission 440 nm) after derivatization with o-phthalaldehyde (OPA) and 2-mercapto-ethanol (MCE). The detection limit of the method is about 0.5 micrograms/l, the recovery of S-PMA is 90.0% and the variation coefficient is 3.8%. The method was checked on urine samples of 8 male non-smokers and 10 smokers: median values of 1.3 and 9.2 micrograms/g creatinine respectively of S-PMA were obtained. A further analysis on urine samples of 66 occupationally exposed workers (smokers and non-smokers) revealed a median value of S-PMA of 46.6 micrograms/g creatinine, compared with a median environmental benzene exposure of 1.99 mg/m3. These results suggest that S-PMA can be regarded in the future as a useful indicator for monitoring individual and collective low-level benzene exposure.

[A method for measuring urinary concentrations of benzene. Its use in monitoring of subjects exposed to low levels]. / Un metodo di misura delle concentrazioni urinarie di benzene. Sua utilizzazione per il monitoraggio di soggetti esposti a bassi livelli.

Benzene is a widely diffuse solvent (atmosphere, cigarette smoke, some foods); in the industrial environment benzene is currently present at concentrations of ppm. A valid method of biological monitoring that is easy to perform is needed for assessing occupational and non-occupational exposures. A new method has been developed to evaluate low concentrations of benzene in urine samples by means of a "dynamic" headspace (50 ml of urine in a 120 ml vial). The urine is saturated with anhydrous Na2SO4 in order to support the entrance of benzene in the air over the urine. The solvent is stripped from the urine surface and concentrated on an adsorbent substrate (Carbotrap 100 tube) by means of a suction pump (150 ml/min). A simultaneous intake of filtered air through a charcoal tube allows wash-up of the headspace. Benzene is thermically desorbed and injected in a column (Thermal tube desorber-Supelco; 370 degrees C thermal flash; borosilicate capillary glass column SPB-1 60 m length, 0.75 mm I.D., 1 micron film thickness; G.C. Dani 8580-FID). The detection limit of the method is about 50 ng/l and the variation coefficient is 4.7%. The method was checked on urine samples of 5 non-smokers and 5 smokers: mean values of 135 and 944 ng/l respectively were obtained. A further analysis on urine samples of 60 smokers revealed a significant relationship (p less than 0.001) between urinary benzene concentrations and C0 alveolar concentrations (r = 0.626). A close relationship between benzene exposure levels and urinary concentrations was found in a group of workers exposed to low environmental benzene concentrations (mean value 1200 micrograms/m3) (r = 0.763).

[HPLC determination of low concentrations of benzene]. / Misura in HPLC di benzene a basse concentrazioni.

A laboratory study, using generated atmosphere containing 0.14/23.3 mg/m3 of Benzene, was conducted to adapt an existing industrial hygiene monitoring method for measuring low concentrations of Benzene. This method was developed to determine concentrations of Benzene in the ambient air and around refinery/petrochemical plant. The air sample is collected on a diffusive personal sampler (Zambelli-TK 200) and analyzed by desorption with a mixture of methylene chloride and ethyl acetate followed by quantification using HPLC-Fluorescence detector. The method used did not detect aliphatic or alicyclic hydrocarbons. The HPLC-Fluorescence method is compared with a gas chromatographic method that uses capillary column and flame ionization detection after a collection of air sample on a Carbotrap 100 tube and following thermal desorption.

[Use of gas chromatography with flame ionization (GC-FID) in the measurement of solvents in the urine]. / Impiego della gas cromatografia a ionizzazione di fiamma (GC-FID) per la misura dei solventi tal quali nelle urine.

The urinary concentration of some solvents (acetone, cyclohexane, 1,2 dichloropropane, n-hexane, methyl ethyl ketone, perchloroethylene, styrene, toluene, 1,1,1, trichloroethane) was measured by means of a gas chromatography Hewlett-Packard 5890 supplied with a flame ionization detector (GC-FID, DANI HS 3950). The coefficient of variation of the method was lower than 5%. The sensitivity of the GC-FID was very similar to what of mass spectrometer detector (GC-MSD, HP 5970 A).