Xjenza Vol. 8 - 2003 - Needs Cover

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Xjenza 2003; Vol. 8

Brief Research Report THE DETERMINATION OF TRACE NUTRIENT METALS IN CATTLE FEEDS* Sonia J. Vella, A. Meilak and George Peplow† Department of Chemistry, University of Malta, Msida, Malta. www: http://home.um.edu.mt/chemistry

Trace metals are of great importance in animal nutrition.

The dietary elements essential for animals include Cr, 1 Mn, Fe, Co, Cu, Zn and Mo . Most of the trace nutrient elements needed by livestock are supplied through the daily intake of animal feed. According to the guideline requirements specified by the Agricultural Research 2 Council , cattle diets are considered adequate if they contain at least 10 mgCu/kg, 50 mgZn/kg and 40 mgMn/kg. Toxicity symptoms are manifested when the concentration of Cu and Zn in the feed exceeds 115mg and 5000mg/kg3 respectively.

The choice of a suitable digestion technique in multielement analysis is generally based on the need for a method that is adequate for the determination of the required elements. Previous work4 has demonstrated that the nitric-perchloric acid mixture is relatively trouble-free and particularly suitable for recovery of trace elements.

As reported by other workers5, flame (FAAS) may be employed for the determination of metals including Cu, Zn and Mn when their concentration is high enough in preference to graphite furnace atomic absorption spectrometry (GFAAS) since it is more rapid and less prone to interferences. AAS techniques typically involve the use of a calibration procedure for standardisation.

The use of standard additions or normal concentration calibration depends on the complexity and nature of the sample matrix.

The objective of the present study was the establishment of a model analytical method for determining the trace nutrient elements Cu, Mn and Zn in cattle feeds and mineral mix supplements. The selection of these metals was instigated by their nutritional importance, their adverse effects at deficient and toxic levels, as well as the intriguing methods of analyses available for trace

amounts. The determination of Cu, Zn and Mn was carried out on samples of compound pellet cattle feeds (F), and mineral-mix supplements of two types: dairy (D) and calf-starter (CLF-ST). The supplements contain minerals and trace metals in relatively high concentrations. The specified composition6 of the feeds was 15 – 30 mgCu/kg, 50 – 100 mgZn/kg and 60 – 100 mgMn/kg. The mineral-mix supplements were specified6 to contain 1000 – 1500 mgCu/kg, 3000 – 5000mgZn/kg and 3000 – 6000mgMn/kg.

Sample group A was digested by the Kjeldahl method, tested for Cu and Mn by GFAAS, and for Zn by FAAS. Group B was digested by the digestion tube block method, and tested for Cu, Mn and Zn using the same techniques as in group A. Group C was digested by the digestion tube block, followed by determination of the metals by FAAS. A control sample, consisting of maize and barley, (M + B), was included with sample groups B and C, and spiked with known quantities of Cu, Zn and Mn stock solutions to determine the percentage recovery following sample treatment.

A standard reference material (bovine liver) was also used to evaluate the accuracy of the technique.

The results obtained in this investigation showed that concentration of trace nutrient metals in cattle feeds was 25.1±2.2mgCu/kg, 103.9±1.8mgMn/kg and 76.5±5.3mgZn/kg. The mineral mix supplements were found to have trace metals concentrations of 1191±106mgCu/kg, 4659±180 mgMn/kg, and 3300±190mgZn/kg. All results compared well with specified ranges. The comparison of digestion method showed that the digestion tube block method was more precise and more adequate for complete dissolution of the samples than the Kjeldahl digestion. The digestion tube

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Paper presented at the First National Chemistry Symposium, Malta, February 2002.

†

Corresponding Author. Tel: +356 2340-2276, E-mail: george.peplow@um.edu.mt

2 block method was a relatively faster technique of sample preparation, enabling the simultaneous digestion of up to 40 samples at a time and accurate temperature control. In all sample groups, the % recovery was satisfactory and higher than 94 %.

The determination of Cu and Mn in feeds and supplements by FAAS gave results that were more accurate and precise. Compared to GFAAS, FAAS also offered the possibility of working in higher concentration ranges without the need for serial dilutions. The method of extended linear range was found to be very convenient for the determination of the metals in mineral supplements at a higher concentration range. The results of this study showed that there was no significant difference between the method of normal calibration and the method of standard additions. Based on the results obtained, the recommended procedure for the routine determination of Cu, Mn and Zn in compound dairy feeds and supplements is tube digestion followed by FAAS using the normal calibration technique.

References: [1] E. J. Underwood, ‘Trace Elements in Human and Animal Nutrition’, 3rd Ed., (1971), Academic Press.

[2] National Research Council Committee on Animal Nutrition, Nutrient Requirements of Beef Cattle, 6th Ed., (1984), NAP, USA, p. 22-23.

[3] R. Puls, ‘Mineral Levels in Animal Health’, (1988), Sherpa International.

[4] T.T. Gorsusch, Analyst, (1955), 84, 135.

[5] J. Sneddon & K.S. Farah, Simultaneous Determination of Copper, Iron, Manganese and Zinc in Bovine Liver and Estuarine Sediment using Flame Atomic Absorption Spectrometry with Background Correction, Analytical Letters, (1993) 26 (4), 709 – 719.

[6] C. Fenech, Personal Communication, (1993).

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Xjenza 2003; Vol. 8

Brief Research Report INFLUENCE OF SERVICE STATIONS ON AIR QUALITY* Marion Zammit STMicroelectronics, Industry Rd., Kirkop, Malta.

Alfred J. Vella† Department of Chemistry, University of Malta, Msida, Malta. www: http://home.um.edu.mt/chemistry

Air pollution problems have been caused throughout the ages by the use of fuel. Benzene, toluene, xylenes and methyl-tert-butyl ether (MTBE) are volatile substances found in gasoline, which through evaporation can contaminate our environment leading to adverse effects on human health and other ecosystems. Service stations are a point source of such organic substances emissions.

In Malta there are 48 petrol stations and 26 kerbside stations, while in Gozo there are 7 petrol stations and 3 kerbside stations (at time of study). Most of these are situated on main roads surrounded by domestic dwellings.

In this study, air quality in the vicinity of service stations was investigated in order to determine whether these fuel marketing outlets are producing a significantly higher risk to people in their area. Benzene, toluene, xylene and MTBE concentrations in air at points proximate and distal from service stations were used as indicators of air quality. This study also included an investigation of variation in wind direction and speed between street sites and the Luqa Meteorological Station.

Analysis was performed following methodologies validated by the National Institute for Occupational Safety and Health in the US (NIOSH) and OSHA Analytical Laboratory [1-4]. Volatile organic compounds (VOCs) in air were adsorbed onto activated charcoal and subsequently desorbed into carbon disulfide. Analysis of the resultant solution was carried out by GC-FID. Due to co-elution of MTBE and 2,3-dimethylbutane (also found in gasoline), concentrations of MTBE in air had to be

quoted as MTBE equivalents (eq.). Both active and passive samplers were used in this study.

Concentrations of VOCs in air detected in active samplers showed that wind direction and height of vents of the underground storage tanks at the service station are important determinants of pollutant concentrations in the vicinity of service stations. Passive samplers showed a clear trend of significantly higher concentrations in the vicinity of the service stations compared with the situation 300 - 400 m away. In one case passive samplers exposed during the same period detected 491 µg m-3 of MTBE eq., 34 µg m-3 of benzene, 97 µg m-3 toluene, 55 µg m-3 p- + m-xylene and 17 µg m-3 of o-xylene in the vicinity of a service station compare to 18 µg m-3 benzene, 38 µg m-3 toluene and 29 µg m-3 p- + m-xylene, but no MTBE eq. and o-xylene at a point 400 m away from the service station.

Measurements of wind direction and speed showed that topography and structures in a given area have a strong bearing on the micrometeorology at a specific site and therefore field studies should be performed when carrying out air pollution measurements.

This study shows that concentrations of VOCs in Malta air may reach worrying levels both in our streets and particularly so in the vicinity of service stations. It is also evident that service stations are probably causing a significantly higher risk to people living in the area of the service station.

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Paper presented at the First National Chemistry Symposium, Malta, February 2002.

†

Corresponding Author. Tel: +356 2340-2290, E-mail: alfred.j.vella@um.edu.mt

4 An intensive monitoring campaign, investigating air quality for VOCs should be carried out on a national scale, while authorities responsible for fuel marketing should seriously consider the adoption of technologies which mitigate the impact of service stations on air quality.

References: [1] Elskamp, C. J. (1980) Method No. 12 Benzene; Organic Methods Evaluation Branch, OSHA Analytical Laboratory: Utah.

[2] NIOSH (1994) Method No. 1501 - Aromatic Hydrocarbons; National Institute for Occupational Safety and Health.

[3] NIOSH (1994) Method No. 1615 - MTBE; National Institute for Occupational Safety and Health.

[4] Lodge, J.P. (1988) Methods of Air Sampling and Analysis; Lewis: Florida, p. 678-685.

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Xjenza 2003; Vol. 8

Brief Research Report ETHANOLIC FRACTIONATION OF DILUTE GELATIN SOLUTIONS * Alfred Aquilina and Emmanuel Sinagra Department of Chemistry, University of Malta, Msida, Malta. www: http://home.um.edu.mt/chemistry

Claude Farrugia† Chemistry Department, Junior College, University of Malta, Msida MSD06, Malta www: http://staff.um.edu.mt/cfar2

Gelatin is a heterogeneous protein with a broad molecular weight profile (MWP). Addition of a non-solvent to gelatin solutions causes progressive desolvation and aggregation of the polymer. Modification of the net charge of the protein, by adjusting the solution pH to values ranging about the iso-electric point (IEP), influences the degree of interaction between the different molecular weight fractions, and hence the response of the protein [1]. The objective of this work was to determine the response of gelatins of different bloom strengths, and hence with different MWP’s, to the non-solvent ethanol at different pH’s.

Unbuffered gelatin solutions were prepared by heating aqueous suspensions of undissolved gelatin to 40°C with stirring for 20 minutes. The pH was adjusted to 3, 5, 7, 9 or 11. The gelatin solutions were then incubated at 20°C, 39°C or 56°C for 1.5 hours and mixed with ethanol/water mixtures that had been similarly incubated such that the final solutions contained 0.2% w/w gelatin and ethanol concentrations from 40 to 75% w/w. The threecomponent systems were incubated for a further 20 minutes and the turbidity of the solutions measured by percent transmittance using a Shimadzu 160 UV/Vis spectrophotometer operated at 600nm. The data obtained was subjected to nonlinear regression analysis and the parameter V50 (the ethanol concentration at the % transmittance midway between the initial and final values) was used to monitor the effects of the experimental conditions on the phase behaviour of gelatin in solution, lower V50 values being indicative of a greater sensitivity to desolvation.

The behaviour of the gelatin solutions was observed to be highly dependent on the solution pH. Gelatin solutions adjusted to pH 3 and 11 were insensitive to the desolvating effect of ethanol, while solutions adjusted to pH 5, 7 and 9 exhibited increased turbidity with increasing ethanol concentration, with the solutions adjusted to pH 5 being the most sensitive. In terms of the DLVO theory, gelatin solutions incubated at extremes of pH carry a net charge that gives rise to intermolecular repulsive forces and to a double layer around the gelatin molecules, which provided an energy barrier inhibiting aggregation. On the other hand, the proximity of pH 5 to the IEP of B-type gelatins ensured that the gelatin molecules in solution carried a reduced net charge. Thus, the electrical double layer surrounding each molecule was not efficient in inhibiting aggregation, and precipitation resulted. The V50 values of B225 type gelatin solutions were sensitive to both changes in temperature (F=16.9, p<0.05) and pH (F = 49.1, p<0.01), while those of B75 type gelatins were sensitive to changes in pH (F=10.0, p<0.05) but not in temperature (F=1.59, p>0.05).

Earlier studies have shown that factors altering the MWP of gelatin in solution affect the phase behaviour of gelatin solutions in the presence of a desolvating agent such as ethanol [1]. Thus, increasing temperature causes a shift in the MWP to lower molecular weights, accounting for the above observations. Lower bloom strength gelatins already have a MWP that is shifted towards lower

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Paper presented at the First National Chemistry Symposium, Malta, February 2002.

†

Corresponding Author. 9985-8107, e-mail: claude.farrugia@um.edu.mt

6 molecular weights [2], accounting for the lack of temperature effects with B75 gelatin.

References: [1] C.A. Farrugia and M.J. Groves, J. Pharm.

Pharmacol., 51 (1999) 643.

[2] C.A. Farrugia et al., Pharm. Pharmacol. Commun., 4 (1998) 1.

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Xjenza 2003; Vol. 8

Brief Research Report THE BINDING OF CATIONIC SURFACTANTS TO GELATIN * Claudette Mifsud, Emmanuel Sinagraâ&#x20AC; , S. Vassallo and Mark A. Scerri Department of Chemistry, University of Malta, Msida, Malta. www: http://home.um.edu.mt/chemistry

The interaction between gelatin and anionic surfactants is an important phenomenon and has been reported in a number of articles [1]. The study of the interaction between gelatin and cationic surfactants has been paid less attention in comparison, however, it has been concluded that the interaction is weaker than that between the biopolymer and anionic surfactants[2]. The objective of this work was to investigate the effect of (i) variation of surfactant head-group type and chain length and (ii) variation of pH on the binding of cationic surfactants and gelatin.

their shorter-chained analogues, these surfactants showed ctc values below their cmc. The values of the ctc were also found to decrease with increasing pH. Through these studies, we were able to conclude that the order of binding strength (BS) is likely to be: Greatest BS :

dodecylammonium Chloride

Medium BS :

hexadecyltrimethylammonium bromide, hexadecylpyridinium chloride dodecyltrimethlammonium bromide, dodecylpyridinium chloride

Lowest BS :

The binding studies were performed using either equilibrium dialysis or a purposely-prepared surfactant selective electrode. The gelatin used was of the B type with a bloom stength of 225 and an isoelectric point (IEP) of 4.9.

Dodecyltrimethlammonium bromide was found to bind cooperatively to gelatin above the IEP of the polymer.

The cooperativity threshold concentration (ctc) was found to match the critical micelle concentration (cmc) of the surfactant. Below this value very little surfactant adhered to the gelatin. The amount of surfactant bound to the gelatin increased on increasing the pH of the solution.

Similarly the amount of dodecylpyridinium chloride binding to the gelatin was found to be negligible below the cmc of the surfactant. Dodecylammonium chloride was found to bind cooperatively with the gelatin even at pH values below the IEP of the polymer. Furthermore the ctc was detected below the cmc of the surfactant and its value was observed to decrease with increasing pH.

References: [1] Knox, W.J.; Parshall, T.O. J. Colloid Interface Sci. 1970, 33, 16, Tavernier, B.; J. Colloid Interface Sci. 1983, 93, 419, Whitesides, T.H.; Miller, D.D. Langmuir 1994, 10, 2899.

[2] Fruhner, H.; Kretzschmar, G. Colloid Polym. Sci. 1989, 267, 839, Henriquez, M.; Abuin, E.; Lissi, E. Colloid Polym. Sci. 1993, 271, 960.

The binding of hexadecyltrimethylammonium bromide and hexadecylpyridinium chloride with gelatin was also found to be cooperative above the IEP of gelatin. Unlike *

Paper presented at the First National Chemistry Symposium, Malta, February 2002.

â&#x20AC;

Corresponding Author. 2340-2396, e-mail: emmanuel.sinagra@um.edu.mt

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Xjenza 2003; Vol. 8

Brief Research Report PRELIMINARY EVIDENCE FOR IN VITRO METHYLATION OF TRIBUTYLTIN IN A MARINE SEDIMENT* Alfred J. Vella† and Jean Pierre Tabone Adami Department of Chemistry, University of Malta, Msida, Malta. www: http://home.um.edu.mt/chemistry

Organotin compounds, especially tributyltin (TBT), are found in sea-water and sediment samples from the principal commercial harbours in Malta. TBT, and to a lesser extend its degradation products, i.e. dibutyltin and monobutyltin, have been shown to produce harmful effects in a variety of marine biota. Recent reports from our laboratory on the occurrence of methylbutyltins in marine sediments and seawater suggest that these compounds are formed in the environment by the methylation of both TBT and that of its degradation products, to give MenBu(4-n)Sn for which n = 1, 2 and 3 respectively.

(maximum concentration 6.0 µgSn L-1). The minimum conversion yield of TBT into MeBu3Sn was estimated to be 0.3%. MeBu3Sn has a significantly lower affinity for sediment than TBT and, therefore, is more mobile in the marine environment, possibly also migrating into the atmosphere to generate a hitherto unsuspected flux of organotin compounds into that phase.

We investigated the possibility of inducing methylation of TBT in seawater-sediment mixtures in experiments carried out in vitro using environmental materials collected from a yacht marina in Msida, Malta. Three water-sediment mixtures, which were shown to contain TBT, dibutyltin and monobutyltin but no other organotins, were spiked with tributyltin chloride (90 mg in 100 mL sea-water/100 ml sediment); to one mixture was added sodium acetate and to another methanol, to act as possible additional carbon sources, and all mixtures were allowed to stand at 25°C in stoppered clear-glass bottles in diffused light for a maximum of 315 days.

Speciation and quantification of organotins was performed using aqueous phase boroethylation with simultaneous solvent extraction followed by gas chromatography with flame photometric detection. The atmosphere inside the bottles quickly became reducing with abundant presence of H2S, and after an induction period of about 112 days, and only in the reaction mixture containing methanol, methyltributyltin (MeBu3Sn) was observed in both sediment (maximum concentration 0.87 µgSn g-1) and overlying water *

Paper presented at the First National Chemistry Symposium, Malta, February 2002.

†

Corresponding Author. 2340-2290, e-mail: alfred.j.vella@um.edu.mt