THIN LAYER CHROMATOGRAPHIC STUDIES ON DEPLETION OF SOME HERBICIDES IN TWO SOIL ECOSYSTEMS. A Thesis submitted to the University of Ghana in partial fulfilment for the award of Master of Philosophy in Chemistry. By SAMUEL AFFUL, B.Sc(Hons), Chemistry, (Kumasi). Chemistry Department University of Ghana Legon. 2002 University of Ghana http://ugspace.ug.edu.gh S6?5Tf. 4-/Vf Wfc, C , I ^> 370545 University of Ghana http://ugspace.ug.edu.gh DECLARATION It is hereby declared that this Thesis is the result of the research work undertaken by the author and it has neither wholly nor partially been presented for another degree elsewhere. ........... S. AFFUL (STUDENT) Prof. C.K. AKPABLI Dr. P.O. YEBOAH (SUPERVISOR) (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh THIS THESIS IS DEDICATED TO MY BELOVED WIFE, VICTORIA AND MY DAUGHTERS JOSEPHINE, LORETTA AND LOIS FOR THEIR CONCERN FOR MY WELL BEING. DEDICATION University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDAGEMENT Praise, honour and adoration be to Almighty God for keeping me in his safe hands till now. To him be the glory for the great thing he has done. I wish to express my sincere gratitude to my supervisors, Dr. C. K. Akpabli and Dr. P. O. Yeboah for stimulating my interest in this field of research. Their painstaking direction, inspiration, encouragement and their constructive suggestions and criticisms ensued the reality of this work. My special thanks go to Mr. J. Nortenor of the Soil Science Department, University of Ghana, Legon and Mr. B.Q. Modzinuh of the Chemistry Department, Ghana Atomic Energy Commission for their technical assistance. My thanks also go to Professor J. H. Ephraim, Mr. C.B.J. Semanhyia, Mr. S. A. Dogbe and Mr. D.G. Achel all of the Chemistry Department, Ghana Atomic Energy Commission for their encouragement and moral support, and to my fellow M. Phil colleagues, Mr. Paul Ofosu and Augustus Tawiah, for their useful suggestions. Finally, thanks are due to the entire members of the Chemistry Department, Ghana Atomic Energy Commission and the teaching and non-teaching staff of the Chemistry Department, University of Ghana for their co-operation. University of Ghana http://ugspace.ug.edu.gh CONTENTS DECLARATION......................................................................................................... i DEDICATION............................................................................................................ " ACKNOWLEGEMENT............................................................................................. iii CONTENTS.............................................................................................................. iv ABSTRACT...............................................................................................................ix CHAPTER ONE INTRODUCTION..........................................................1 1.1 History background..................................................................................2 1.2 Background of study................................................................................ 5 1.2.1 Statement of problem............................................................................... 7 1.2.2 Scope and purpose of the work...............................................................8 CHAPTER TWO LITERATURE REVIEW................................................ 9 2.1 Herbicides depletion in soils....................................................................9 2.1.1 Kinetics of depletion of herbicides..........................................................12 2.1.2 Factors influence rate of loss................................................................. 14 2.1.2.1 Concentration..........................................................................................14 2.1.2.2 Herbicide adsorption............................................................................... 15 2.1.2.3 Soil type...................................................................................................15 2.1.2.4 Soil pH..................................................................................................... 16 University of Ghana http://ugspace.ug.edu.gh 2.1.2.5 Soil amendment....................................................................................1' 2.1.2.6 Temperature and moisture..................................................................... 18 2.2 Variability in measurement of herbicide residues..................................19 2.3 Analytical procedure for pesticide residue analysis..............................20 2.3.1 Sampling and sample preparation..........................................................20 2.3.2 Sample extraction................................................................................... 21 2.3.2.1 Pre-analysis.......................................................................................... 21 2.3.2.2 Solvent extraction................................................................................... 22 2.3.2.3 Wet processing technique......................................................................23 2.3.2.4 Extraction of pesticides from vegetables, fruits and soil.......................24 2.3.2.5 Storage of extracts...................................................................................24 2.3.2.6 Concentration of pesticide in stripping solution..................................... 25 2.3.2.6.1 Air evaporation.....................................................................................25 2.3.2.6.2 Concentration using vacuum...............................................................26 2.3.3 Clean up or purification of extract.......................................................... 26 2.3.3.1 Solvent partitioning.............................................................................. 27 2.3.5.2 Acid clean up....................................................................................... 27 2.3.3.3 Column chromatography clean up......................................................... 28 2.3.3.3.1 Solid phase extraction (SPE) clean up method..................................28 2.3.4 Qualitative and quantitative determination of pesticide residue.......... 29 2.3.4.1 Introduction...........................................................................................29 2.3.4.2 Previous work done with TLC............................................................. 30 2.3.4.3 TLC Technique.....................................................................................32 University of Ghana http://ugspace.ug.edu.gh 2 .3.4.3.1 Qualitative analysis(location and identification) using TLC................ 32 2.3.4.3.2 Quantitative determination using TLC.................................................33 2.4 Classification of herbicides.................................................................... 34 2.4.1 Chemical classification..........................................................................34 2.4.2 Classification by phytotoxicity................................................................35 2.4.3 General information on the selected herbicides................................... 37 CHAPTER THREE EXPERIMENTAL METHODS....................................41 3.1 Chemicals and Reagents....................................................................... 41 3.2 Equipment...............................................................................................42 3.3 Sampling field..........................................................................................43 3.3.1 Sampling and sample treatment............................................................ 44 3.4 Physical chemical soil properties analysis.............................................44 3.4.1 Determination of soil moisture................................................................44 3.4.2 Determination of water holding capacity(WHC).................................... 45 3.4.3 Determination particle size.....................................................................45 3.4.4 Determination of soil pH..........................................................................46 3.4.5 Determination of organic carbon and organic matter............................47 3.4.6 Determination of available phosphorus................................................. 47 3.4.7 Determination of total nitrogen............................................................. 48 3.5 Herbicide depletion studies....................................................................49 3.5.1 Preparation of herbicides standards......................................................49 3.5.2 Selection of elution system for analysis of the herbicides.................... 49 University of Ghana http://ugspace.ug.edu.gh 3.5.3 Detection and measurement of herbicides..........................................50 3.5.4 Selection of extraction solvents for the herbicides.............................. 51 3.5.4.1 Procedure for spiking............................................................................51 3.5.4.2 Extraction.............................................................................................. 52 3.5.4.3 Clean up of extracts..............................................................................52 3.5.4.4 TLC analysis of extracts for purity and recovery.................................53 3.5.5 Soil treatment and incubation.............................................................. 53 3.5.5.1 Sub-sampling and extraction procedure..............................................54 3.5.5.2 Thin layer chromatography(TLC) analysis..........................................55 3.5.5.2.1 Quantification of residues.................................................................... 55 3.5.6 Determination of limit of detection (LOD)............................................56 CHAPTER FOUR RESULTS AND DISCUSSION......................................57 4.1 Physico-chemical soil properties.........................................................57 4.2 Selection of elution system for the analysis of the herbicides...........60 4.3 Selection of extraction solvents for the herbicides.............................62 4.4 Thin layer chromatography analysis of extracts................................. 66 4.5 Depletion of the herbicides................................................................. 66 4.6 Kinetics of depletion of the herbicides................................................73 4.7 Detection limits....................................................................................76 CHAPTER FIVE CONCLUSION AND RECOMMENDATION..................79 5.1 Conclusion..........................................................................................79 University of Ghana http://ugspace.ug.edu.gh Recommendation.......................................... 80 REFERENCES...................................................................................81 APPENDIX 1 SAMPLE CALCULATIONS.......................... 94 APPENDIX 2 CALIBRATION CURVES.............................. 97 University of Ghana http://ugspace.ug.edu.gh ABSTRACT Depletion rates of three triazine herbicides, atrazine, simazine and ametryne and two urea base herbicides, diuron and metobromuron, under laboratory conditions have been investigated in soil samples collected from GAEC, a coastal savannah soil, and KNUST, a forest zone soil. Two hundred grammes of the soil samples were treated with herbicides standard solution to generate herbicide-soil concentration of 10 pg/g and incubated at room temperature for 12 weeks. TLC methodology was used to monitor the decline of the herbicides from the soil and the result showed that the decline of the chemicals was comparatively faster in the KNUST soil than the GAEC soil. After two weeks of soil treatment and incubation, atrazine, simazine and metobromuron had depleted more than half of the initial amount applied. In all, the rate of depletion of metobromuron was found to be the highest and at the end of the experiment, it declined to about 2.42 % and 4.38 % of the initial concentration in the KNUST and the GAEC soils respectively. The results obtained, indicated that the kinetics involved in the process of depletion of the herbicides to a higher degree could be described by first order reaction kinetics. The half-lives of the herbicides in the GAEC soil were in the range of 14.8 - 32.2 days and 13.3-31.1 days in the KNUST soil. Soil moisture and organic matter content were found to facilitate the depletion of the chemicals from the soils. Out of the various solvents systems tried for the University of Ghana http://ugspace.ug.edu.gh extraction of the herbicides, acetone, acetonitrile and acetone/hexane mixture (4:1) were found to be efficient for the recovery of the chemicals in the soil ecosystems studied. With the photosynthesis inhibition method used for the detection of the herbicides, the detectability for the unclean extracts was in the range of 0.004 - 0.008 (jg/g and that of the clean-up extracts was in the range of 0.024 - 0.162 pg/g. x University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 HISTORICAL BACKGROUND A weed is essentially any plant grown in a place where it is not wanted[1]. Weeds have been a problem since man began to till the land for food and for other useful purposes. The problem of weeds was even emphasized in the parable of Jesus Christ[2], hence 2000 years ago, they must have been a burden to man. He referred to deleterious effect of weeds in two ways. First, in the parable of the sower, they choked the crop and reduced the yield . Second, in the parable of the tares sown by the enemy, the crop was disturbed and its growth impaired in the process of removing the weed from the crop. Although weeds have challenged man’s effort to survive ever since he started tilling the soil for food, advanced weeds control methods are practiced on limited scale particularly, in the tropics, where hand weeding is still widely carried out. It has been reported by LeRoy Holm[3] that more energy is still expended on the weeding of man’s crop than any other single human task. Before the introduction of advanced methods of weeds control, four measures were adapted to eradicate or limit the spread of weeds. These were manual weeding (by the use of the hand directly or by the use of implements such as 1 University of Ghana http://ugspace.ug.edu.gh hoes and cutlasses), crop rotation, ploughing and various methods of preventing weed seeds from being dispersed. These methods suffer from a basic weakness, in that they are aids to control but they cannot prevent weeds growing with the crops. Apart from this weakness, weeds control by manual weeding through the direct use of the hand, or the use of the farm implements, i.e hoes and cutlasses is time consuming and labour intensive. Advanced methods of weed control or weed eradication involving the use of chemicals have been practised since time immemorial. It is even an established fact that in biblical times, armies sometimes used salts or mixture of brine and ashes to sterilise land that they had conquered, the intention being to make the land uninhabitable by future generations of the enemy. Salts and various industrial by-products such as smelter wastes have also been applied to roadsides and paths to rid them of vegetation hundreds of years ago[4]. Chemicals used to control weeds are called herbicides. Herbicides are one of the main classes of pesticides. They are often employed to kill weeds, sometimes without causing injury to desirable vegetation, for example, to eliminate broad- leaf weeds from lawns without killing the grass. Herbicides, may be considered to have been discovered in 1896 when Bonnet, a French grape grower, observed that the Bordeaux mixture he applied to his vines as protection against downy mildew, turned the leaves of the sinapis arvensis 2 University of Ghana http://ugspace.ug.edu.gh black[5], The weedkiliing properties of sulphates of ammonia, zinc, iron and other metals were soon observed[5]. In the first half of the twentieth century, several inorganic compounds were used as weed killers, principal amongst them being Sodium arsenite Na3As03, Sodium Chlorate NaCI03, and Copper sulphate CuS046]. The latter are only two of a large number of salts formerly used as herbicide sprays whose means of operation is to kill plants by the primitive action of extracting the water from them, and leaving the land to support agriculture. Later milestone in weed control was the introduction of the first organic chemical, 2-methyl-4,6-dinitrophenol in 1932. A few years later, an important discovery was made that chemicals related structurally to plant hormones could be used as selective weedkillers. The use of non-selective and, later selective residual chemicals such as the substituted phenylureas, triazines, and the non-residual chemicals such as paraquat and diquat may be considered as more recent milestones. Inorganic and organometallic herbicides have been phased out because of their persistence in soil. The world consumption level of herbicides between 1896 and 1968 is presented in Table 1 [7], 3 University of Ghana http://ugspace.ug.edu.gh Table 1: Estimated world consumption of Herbicides at consumer level from 1896 to 1968 Area Consumption(million of dollars) North America 550 Latin America 80 Near East, South East, Oceania 80 Japan 70 Western Europe 60 Africa 40 Total 880 It is clear from the Table 1 that more than 50 % of the world consumption of herbicide occurred in North America and the least amount was consumed in Africa within the period being discussed. The situation in Africa today however, shows an improvement with respect to the use of herbicides. Wandiga[8] in the review of pesticide use in Africa reported that between 1986 to 1990 a total of 6830 tonnes of herbicides were imported into Kenya for use. In Ghana the situation is not different, and available information indicates that 21 different kinds of herbicides were imported into the country for agricultural purposes between 1995 and 2000[9], Notably among 4 University of Ghana http://ugspace.ug.edu.gh them were paraquat, atrazine, bromacil, diuron, glyphosate, propanil, ametryne, oxadiazon and alachlor. Table 2 below shows the total amount of herbicides imported into Ghana from 1995 to 2000. Table 2: Quantity of herbicides imported into Ghana from 1995 - 2000[9]. Year Amount imported (in kilogrammes) 1995 88,587 1996 55,414 1997 132,292 1998 224,816 1999 97,584 2000 52,030 1.2 BACKGROUND OF STUDY In recent years, there has been a considerable increase in the use of herbicides in Ghana. This has come about as a result of the intensification of agricultural activities to meet the Country’s food needs and the emphasis on the promotion of non-traditional agriculture products. The use of herbicides makes food production convenient and to some extent easy. This is because a number of them selectively kill the target weeds and leave the cultivated crops/plants intact, thus saving the farmer the problem of 5 University of Ghana http://ugspace.ug.edu.gh having to use farm implements to clear the unwanted weeds. In Ghana, such herbicides as atrazine, diuron, ametryne, glyphosphate,bromacil, paraquat and simazine[10 ] are now being used extensively for the weed control in commercial cultivation of food crops such as rice, maize, pineapple, banana and some vegetables. Despite the immense advantages with the use of herbicides, the associated environmental problem that arises is a matter of concern. After application of the pesticide product on the target pest, the chemical is gradually lost as a result of breakdown, evaporation etc, and the residue is the amount that remains after application. Residues of pesticides in food in general have been a major problem particularly, in the developing countries. When it rains the residues in the field are washed away by flood, run-off and seepage into ground and surface water supplies. As a result of the environmental problem associated with the use of herbicide, it is imperative to embark on a study to have a better understanding of the depletion rate of these chemicals in our environment. In assessing the depletion rates of pesticides in the environment, their behaviour in the soil becomes very important since on application the soil is one of the first points of contact. Depletion of pesticides from treated soil comprises leaching, evaporation and degradation [11 ]. The first two are purely physical phenomena and are governed 6 University of Ghana http://ugspace.ug.edu.gh by the physico-chemical properties of the compound and soil characteristics (i.e. solubility, adsorption capacity, water content etc). The series of chemical conversions, which finally lead to the breakdown of the pesticide, is called degradation [11], Such conversions may proceed on biotic pathway due to light, temperature and pH or chemical composition of the soil. The microorganisms of the soil carry out some of the conversions, either by metabolising the compounds or by catalytic effect of the free enzymes originating from the living organisms. These factors have a complicated interrelation and make it difficult to predict the fate of pesticides in the soil. 1.2.1 STATEMENT OF PROBLEM Depletion of herbicides in the soil has been investigated in other parts of the world. These studies have been advanced to determine the fate of the chemicals in the environment[12]. Although some progress has been made in this field of research, there had been no work done in Ghana on the depletion rates and the lifetime of these herbicides in the Ghanaian soils. Knowledge of depletion of these chemicals in Ghanaian soils would offer a better understanding of their fate and duration in our environment. This would ultimately guide the agronomists and the environmentalists in predicting the amount and nature of residues remaining in the soil after application. 7 University of Ghana http://ugspace.ug.edu.gh 1.2.2 PURPOSE AND SCOPE OF THE WORK In this study, three commonly used triazine herbicides, atrazine. simazine and ametryn, and two substituted urea herbicides, diuron and metobromuron will be studied in a coastal savannah and a forest zone soils with the objectives of ■ determining the depletion rates of the herbicides in the two soil ecosystems for comparative purposes. ■ studying the kinetics involved in the process of depletion of the herbicides for the purpose of assessing their half-lives in the chosen soil ecosystems. ■ determining the physico-chemical soil properties such as moisture content, particle size, soil pH, organic matter etc for the purpose of establishing the relation between these soil properties and herbicide depletion rate. University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 HERBICIDES DEPLETION IN SOILS Work done on pesticides in Ghanaian soils focussed mainly on insecticides such as lindane, endosulfan and propoxur. Very little work has been done on herbicides depletion in Ghanaian soils, particularly, in the area of kinetics of their depletion. Appoh et. a/.[13] studied persistence of lindane in Ghanaian coastal savannah top soil using a radiotracer technique. They reported that dissipation pattern favours a second order kinetics. They further indicated that dissipation from all the soils exhibited a biphase nature with a more rapid dissipation occurring within the first 2 - 6 days followed by relatively, slower phase. Persistence was also observed to be dependent on the organic matter content of the soil. Antwi-Boakye et. al.[ 14] also studied persistence of lindane and endosulfan in two soil ecosystems under laboratory conditions, and established that the degradation pattern of chemicals in both ecosystems was similar. However, after six weeks of incubation, endosulfan had degraded more than lindane. He further indicated that soil moisture facilitated the degradation process. Lowor[15] on the other hand, investigated the fate of propoxur, in cocoa ecosystem using TLC and GC methodology. The investigation established that residues levels of propoxur 9 University of Ghana http://ugspace.ug.edu.gh determined by the two methods were not significantly different. He further indicated that the residue levels of the chemical in the soil decreased rapidly and, by the twenty-first day after application none was detected in the top soil ( 0 - 6 ins). Lowor et. al.[ 16] investigated persistency of atrazine in tropical soils, and concluded that the herbicide is lost from the top soil within five weeks of application. The loss of atrazine was attributed to either degradation or leaching. They further reported that TLC determination of atrazine gave comparable residue data as that of GC. Suess et. a/.[17] studied the degradation of herbicides and herbicides metabolite in different soils and observed that 3.6 to 10.2 % of the applied amount of 1 ppm of atrazine disappeared during an incubation time of 16 weeks, and 77.6 to 77.9 % was adsorbed into the soil complex. No correlation was observed between the degraded amount of atrazine and the amount adsorbed to the soil complex. Espinosa-Gonzalez[1S], investigated the fate and effects of pesticides under tropical field conditions, and showed that atrazine and simazine used for the production of maize, dissipated slowly. He further indicated that the half-life of atrazine in clay soil was of the order of 50 - 60 days. In the study of persistence of some horticultural herbicides in soils at 26 sites throughout United Kingdom, Davison and Clay[19] reported no significant relationship between persistence of simazine and any soil factor, although there was a trend toward lower soil pH. 10 University of Ghana http://ugspace.ug.edu.gh The rate of loss of diuron from soil have also been assessed in different soils in both laboratory test and field trials[20]. No relationship was established between any soil property and the rate of loss. In the laboratory, only 27 % of the variations in the rate of loss could be accounted for by a multiple linear regression with soil properties. The fate and activity of some herbicides in soils was studied by Ogle and Warren[21] who concluded that monuron and diuron decreased progressively from light sandy soil through a silt loam to an organic soil. In the same study, simazine degraded more rapidly in soils with higher organic content. Green et a/[22], also investigated the fate of linuron, monolinuron and metobromuron in water and soil. They observed that metobromuron was soluble in water and is less persistent in soil. In the study of dissipation of herbicides from soil, Kearny et. al.[23] concluded that depletion of the herbicides follows a first order reaction. This implied that at any time, the rate of loss would be proportional to the concentration in the soil. This type of loss is typical of majority of pesticides. Kaufman[24] also investigated the dissipation of certain biodegradable herbicides from soil and reported that a lag phase might occur after initial application in which relatively little pesticide is lost. This is followed by rapid disappearance as a result of microbial metabolism. 11 University of Ghana http://ugspace.ug.edu.gh 2.1.1 Kinetics of Depletion of Herbicides Depletion of some pesticides is characterized by initial lag-period during which little or no change in concentration occurs. This is followed by rapid degradation which appear linear with time, but detailed analysis may show it to follow first order kinetics[25]. Herbicides, with this degradation patterns, include 2,4- dichlorophenoxyacetic acid, dalapam, chloridazon, propham and all compounds of short persistence in soil[26]. Because of the lag-phase, presentation of half- lives for such compounds is not very meaningful. With other compounds there is no lag-phase and the rate of degradation is proportional to concentration so that the result can often be interpreted using first order kinetics, C ^o e '*1 Where C is the concentration after time t, Co is the initial concentration, k is the rate constant, Thus, a plot of the logarithm of concentration against time gives a straight line with slope proportional to the rate constant. Writing ti/2 as the time taken for 50 % depletion, the half-life is T1/2=0.693/k Which is independent of the initial concentration. The half-life concept is a valuable tool in comparing rates of herbicides depletion. Burschel[27] and Freed[28] commented that since the quantity of herbicides in soil is very small in relation to other components, the herbicide concentration is expected to be the rate limiting step, so that first order reaction kinetics should 12 University of Ghana http://ugspace.ug.edu.gh apply. However, since soil is a complex biological and chemical medium, the kinetics of adsorption and desorption might affect rates of loss by controlling the amount of herbicide available for degradation, while the activities of soil microorganisms may vary with time depending on the availability of nutrients and other energy sources. It is therefore not surprising that deviations from simple first order kinetics are observed. Hance and Mckone[29] showed that neither zero-order, half-order, first order nor Michaelis-Menten kinetics described precisely the breakdown of atrazine and linuron in the laboratory. Empirical curve fitting has also been suggested and Hamaker[30] proposed a power-rate equation C = [ C0(1‘n) + (n-1 )kt ] 1/(1'n) Where C is the concentration after time t, Co is the initial concentration, n the apparent order of the reaction and k the rate constant. This equation was used by Kempson-Jones and Hance[31] in the study of kinetics of linuron and metribuzin degradation. In only 10 to 40 separate experiments did the value 1 fall within the 95 % confidence limits of the determined value of n. In the remaining experiments the apparent reaction order was greater than 1 , and in eight of these, it was greater than 4. Hamaker[30] reported similar deviations from first- order reaction kinetics when he used the equation above to calculate apparent order of reaction from previously published degradation data. 13 University of Ghana http://ugspace.ug.edu.gh When degradation is followed for extended periods, many compounds show rates of depletion that are disproportionately slow at lower residual concentrations. Hamaker and Goring[32] suggested a "two compartment” model to explain this. The pesticide is considered divided between available and unavailable fractions, with only the available subject to depletion. Appropriate rate constants control movement into and out of the unavailable pool. Freshly added chemicals are mainly in the available state and the initial rate of degradation is rapid. However, the rate of degradation falls as the pesticide is transferred to the unavailable state and eventually, rate of release from the unavailable pool controls the rate of degradation. Hamaker[33] and Hamaker Goring[32] demonstrated that this model could describe the kinetics of degradation of a number of soil-applied pesticides. There is little doubt that rate equations are valuable for describing pesticide degradation, but it seems unlikely that a universal equation will be found which applies to all compounds and soils. When rate equations are used, their approximate nature must always be kept in mind. 2.1.2 Factors Influencing Rate of Loss 2.1.2.1 Concentration With first order kinetics, rate constant should be independent of initial concentration, but for atrazine, Armstrong et. al.[34] and for simazine, Walker[35] indicated that the rate of loss decreased as initial concentration increased. With 14 University of Ghana http://ugspace.ug.edu.gh some compounds, a lag-phase was demonstrated at increased initial concentrations. Picloram[36] and tri-allate[37] were found to belong to this group. Hance and Mckone[38] suggested that reduced degradation rates at higher initial concentration might result from a limitation in the number of reaction sites in the soil. Hurle[39] suggested that toxic effects on microorganisms or enzymes inhibition might be involved. 2.1.2.2 Herbicide Adsorption Adsorbed herbicides may be degraded more rapidly since the density of microorganisms near colloidal surfaces is greater in the soil[41] Hurle[39] reported increased persistence of atrazine and 2-methyl-4,6- dinitrophenol in soil when straw ash was added. In unamended soils, increasing half-lives with increased adsorption have been reported for simazine, atrazine, ametryne and propazine[42], Moyer et. at. [43] found lower degradation rates for atrazine but not linuron in soil amended with activated charcoal although increased adsorption of the compound was observed. Clay minerals used as carriers may catalyse degradation of pesticide in dust formulation[44], Chloro-s-triazine has been observed to hydrolyse when adsorbed on clay minerals[45]. Armstrong et. a/.[46] suggested that adsorption catalysed hydrolysis, however, later studies by Armstrong and Chesters[47] indicated that adsorption does not necessarily lead to catalyse hydrolysis but specific adsorption bonds are required for this to occur. 15 University of Ghana http://ugspace.ug.edu.gh 2.1.2.3 Soil Type The influence of soil type on herbicide persistence is not well understood. The fact that soil microorganisms are usually involved in degradation means that soil organic matter might be expected to have some effect since microbial activity is often higher in more organic soils. However, adsorption of most herbicides also increases with an increase in soil organic matter and since adsorption reduces the amount of herbicides available in the soil solution, it might provide protection from degradation. Hamaker[33] suggested that an increase in organic matter might increase rates of degradation in mineral soil up to a limiting value, above which the rate of loss would be retarded. Briska et. al.[40] reported that simazine degraded more rapidly in soil with higher organic contents. 2.1.2.4 Soil pH Soil pH may affect depletion directly if the stability of chemical is pH dependent. With simazine, Nearpass [48] found faster depletion in two soils at pH 5.4 and 3.9 than at 6.8 and 7.0. Atrazine depletion was more rapid at pH 5.5 than in the same soil adjusted to pH 7.5[49]. This is consistent with result of Hiltbold and Buchanan [50] working with soil adjusted to pH 5.6 and 7.0. Walker and Thompson[51] found a significant negative correlation between rates of simazine degradation and pH in 18 unamended soils. Hance[52] working with soils adjusted to four different pH in the range of 5.0 to 8.0 reported only slightly increased decomposition rates for atrazine with decreasing pH in one soil, whereas in the other, rates of loss decreased. 16 University of Ghana http://ugspace.ug.edu.gh Corbin and Upchurch[53], investigated the rates of degradation of several herbicides in two organic matter soils adjusted to pH levels in the range of 4.3 to 7.5. They reported maximum degradation of dicamb and 2,4- dichlorophenoxyacetic acid at pH 5.3, and aminotriazole at pH 6.5, but no effect of pH on the rate of loss of diuron or chloramben. These results show that soil pH can influence depletion rates. There is however, very little understanding of the principle involved, and more information is required before general conclusions could be made. 2.1.2.5 Soil Amendments It is generally assumed that the rate of depletion of most herbicides is influenced by soil microbial activity. Since addition of easily degradable organic substrates and mineral nutrients results in a spontaneous increase in microbiological activity, one would expect enhanced herbicide degradation by such treatments. However, this does not always occur. McClure studied accelerated degradation of herbicides in soil and reported that accelerated degradation of minuron, atrazine, dicamba and diuron occurred when microbial nutrient broths were added to the soil[54]. Glucose enhanced disappearance of atrazine[55], ground vetch plant increased atrazine and diuron decomposition[55]. Wolf and Martin[56], reported an enhance decomposition of bromacil and tercil in the soil on addition of maize and bean straw. Hance[57], working on two soils indicated that farm yard manure or straw accelerated atrazine degradation in one soil but not in the other, whereas mineral fertilizers (N, P,K) plus straw accelerated degradation in both soils. 17 University of Ghana http://ugspace.ug.edu.gh 2.1.2.6 Temperature and Moisture Since increasing temperature increases the rates of both non-biological reactions and biological processes, rate of herbicide depletion should be expected to also increase with temperature. Hamaker[30], reviewed the literature for herbicides and other soil-applied pesticides and found that this is generally the case. The dependence of the rate constant, k of a chemical on temperature can be expressed by the Arrhenius equation k = A o e(Ea/RT) Where Ao is a constant, R the gas constant, T the absolute temperature and Ea the activated energy. With first-order reactions, the dependence of half-life on temperature can be expressed by logHi - logH2=Ea/[4.575(1/Ti-1/T2)] where Hi and H2 are half-lives at absolute temperatures T1 and T2 respectively. This equation has been satisfactorily applied to degradation data for a number of soil applied herbicides[58]. Adequate water as well as high temperature is essential for microbiological activity, but in addition, water acts as a solvent and transport agent, a reaction medium for both biological and non-biological processes and a reagent in hydrolytic reactions. There is evidence that herbicides degradation rates are increased under moist soil conditions, which, in most instances probably reflects increased biological activity. 18 University of Ghana http://ugspace.ug.edu.gh In recent years, reports of studies with wide range of compounds have been published[59], and these have shown the expected effect of increase degradation rates with increasing soil moisture up to field capacity. 2.2 VARIABILITY IN MEASUREMENT OF HERBICIDES RESIDUES In laboratory experiments, a well sieved and therefore relatively uniform particle soil samples can be used, the herbicides can be distributed uniformly through the soil and incubation condition can be controlled. In the field, none of these factors can be controlled with precision. Much of the variation in the field compared with laboratory experiment will result from differences in the initial distribution of the herbicides on the soil surface. Some examples of these errors can be taken from data of Fryer and Kirkland[60], Over six years of repeated treated of plots with four different herbicides, initial recoveries of the nominally applied rate varied from 42 to 100 % with 2-metyl-4-chlorophenoxyacetic acid, 37 to 90 % with tri- aliate, 60 to 104 % with simazine and 32 to 144 % with linuron. Similar observation was observed for atrazine. Hence to assess quantitatively the kinetics of degradation under field conditions, detailed measurements of the amount present initially are essential. Fryer and Kirkland[60] also measured the point to point variation in deposition of the spray application. In experiment with linuron, the mean deposition on several 12.9cm2 filter papers placed at random on the soil surface was 94, 65, 73, 81 and 95 % of the theoretical quantity in different tests and the coefficient of variation was 16, 60, 23, 17 and 11 % respectively. Mechanical incorporation of 19 University of Ghana http://ugspace.ug.edu.gh herbicides applied to the soil surface increases this variation[61 ]. Variations in residual concentrations from one point to another within a treated area tend to increase with time, presumably because of variations in soil properties which affect degradation rates[62], 2.3 ANALYTICAL PROCEDURE FOR PESTICIDES RESIDUE ANALYSIS Regardless of the analytical methods used, the procedure for pesticide residue analysis follows the following steps. 1. Sampling and Sample Preparation 2. Sample Extraction 3. Extract Clean-up 4. Determination of Residues 2.3.1 Sampling and Sample Preparation The importance of careful, unbiased and representative sampling in the field cannot be overemphasized. It should always be remembered that chemical residue analyses are usually time- consuming as well as expensive and therefore the minimum sampling to obtain reasonable validity is of paramount importance. Samples collected must have the following characteristics[63], a. Sample must be accurate. The final accuracy of the residue determination is largely dependent on the original field sample. Residue data may be precisely determined but woefully inaccurate due to inadequate field sampling. 20 University of Ghana http://ugspace.ug.edu.gh b. The sample must be valid. A valid sample is the one that is selected in a manner that ensures that each unit of material in the batch being sampled has an equal chance of being selected for the extraction and ultimate test. c. The sample must be representative. A representative sample is not only a random sample but the proportion of each type of the sample material should be identical to that of the gross sample from which it was originally selected. 2.3.2 Sample Extraction 2.3.2.1 Pre-analysis Once a valid, representative field and sub-samples have been selected for eventual residue analysis, the next major problem for the residue chemist is to quantitatively remove the pesticides or its metabolites from the surrounding biological environment. Extraction techniques must be adequate to yield extract which accurately reflect the toxicant residue level. Bann[64] reviewed three basic extraction procedures commonly used for pesticide residue removal. These extraction procedures were represented as follows: a. the whole crop surface rinsed with a suitable solvent. b. maceration of sample with crystalline anhydrous sodium sulphate and extraction with suitable solvent. c. Maceration of the sample in the presence of a suitable solvent or solvent combination. 21 University of Ghana http://ugspace.ug.edu.gh 2.3.2,2 Solvent Extraction The first step in the analysis of pesticide residues is usually separation of the pesticide from the environmental sample by solvent extraction. For efficiency, the solvent must remove the pesticide in a reproducible manner without removing large amount of the co-extractives from the sample. For the past several years, many specialized solvents have been developed for the extraction of pesticides from agricultural samples. The analyst must consider the method of analysis before extraction is begun. For instance to analyse for lindane in a sample using Schechter-Homstein procedure[65], sample should not be extracted with benzene, since benzene is the material to be detected in the final step of analysis. Unless adequate experimental data concerning the solvent purity is known the solvents should be distilled before use. This is especially important in the case of chlorinated solvents such as chloroform, methylene chloride and carbon tetrachloride. These solvents often form phosgene on standing, which not only produce negative analytical results, but may also be hazardous to the analyst. Before the more unstable pesticides were developed, samples were often dried, ground and extracted in a soxhlet or some other type of continuous extractor. However, drying was soon found to cause loss of many of the new organic pesticides. 22 University of Ghana http://ugspace.ug.edu.gh 2.3.2.3 Wet-Processing Technique Residue analysts have found that the wet-processing extraction technique is probably most satisfactory for consistent recovery of pesticides residues[65]. Two satisfactory systems for extracting most pesticides, excluding those that are water-soluble, seem to be extraction with benzene-alcohol mixture, or extraction with chloroform. There are essentially two methods of wet-processing of raw or processed agricultural sample[66]. a. Extraction by rotating or shaking with solvent. Removal of pesticides by shaking or rotating the sample with a single solvent has the major advantage of usually removing the pesticide without the inclusion of large amounts of co-extractives. This method can often be used for soil samples, raw fruits or vegetables crops with surface residue when the pesticide has not been absorbed by the plant tissue. b. Extraction by blending with one or more solvents Extraction of most pesticides can be done best by blending the substrate with the solvents. It has been shown that extraction is most efficient with two solvents. Blending is convenient in warring blender or a large mixing apparatus such as that described by Gunther and Blinn[67], Some workers in the pesticide analysis field recommended that the sample be blended with ethyl or isopropyl alcohol before adding a water-immiscible solvent[68]. In general, the amount of immicible solvent should be 2 ml. per gramme of subtrate. 23 University of Ghana http://ugspace.ug.edu.gh 2.S.2.4 Extraction of Pesticides from Vegetables, Fruits, Forage and Soil Klein et. al.[69] have indicated that co-solvents extraction (extraction with a mixture of two or more solvents) is highly recommended for fresh and frozen leafy vegetables containing considerable quantities of extraneous water. Co­ solvent extraction is not usually necessary or advisable for forage, dried fruits and vegetable, oily crops, nut and shells etc. Precise extraction techniques for the removal of pesticides from the soils cannot always be established because chemical changes affecting adsorptive capacity may be occurring. The use of highly polar solvent such as acetone will yield satisfactory recoveries. Extraction of pesticide with 10 % acetone in hexane has been found to be adequate for the removal of most pesticides without excessive extractive interfering substances[70], 2.3.2.S Storage o f Extracts Patterson and Lehman[71] have shown that stripping or extracted pesticide solutions should be stored under conditions that will permit no change in the pesticide until analysis is performed. If delay is unavoidable, a recovery can be made under the same condition of extraction, and stored along with the sample extracts. Even under optimum conditions, however, prolonged storage of extracts is not advisable, Extract should be stored near 0°C in screw-cap bottles with aluminium liners in the caps. Liners of waxed paper should be avoided, since the wax is usually dissolved by the solvent. Even at low temperatures, pesticide 24 University of Ghana http://ugspace.ug.edu.gh materials may be lost, For example, siven is lost from chloroform extracts at very short time even at low temperatures, however, small volume of added ethanol helps preserve the siven[72]. Extracts should therefore be analysed as soon as possible after extraction unless there is considerable experimental evidence showing that the technical pesticide is stable in the solvent. 2.3.2.6 Concentration of pesticide in stripping or extracting solution After extraction of the pesticide from the sample material, the pesticide is usually at such a low concentration that direct measurement is difficult, so the solution must be concentrated by removal of the solvent. Concentration or the removal may be achieved in several ways, but distillation or evaporation of the solvent are most practical. 2.3.2.6.1 Air evaporation In most cases, evaporation is achieved either by blowing warm air over the sample in a beaker or blowing filtered dry air or nitrogen over a sample held in a warm-water bath. If the sample is being evaporated by stream of air from the laboratory line, it is well to filter the air just before use. A convenient air filter with replaceable cartridge is satisfactory to remove water, oil and rust particle. The temperature of the water bath usually should not be over 50°C, and in some cases must be lower since at higher temperatures most of the pesticides will be lost through evaporation. 25 University of Ghana http://ugspace.ug.edu.gh 2.3.2.6.2 Concentration using vacuum Vacuum can often be used in concentrating the solutions that are sensitive to heat. A Rinco evaporator utilizes the principle of spreading a thin film of solution over a large, rotating surface area and subjecting it to negative pressure. This is a convenient method for vacuum concentration of extracts containing pesticides. 2.3.3 Clean up or purification o f extract Usually one day prior to laboratory analysis the frozen extracts are removed from freezer and allowed to thaw at room temperature, and then purified. In other words, the pesticide of interest must be isolated from the previous environment by suitable solvent. Thus, a clean-up procedure must be devised to quantitatively separate the original applied pesticide from associated interfering materials co-extracted from the original biological environment. Gunther and Blinn[67], Schechter and Hornstein[65] discussed in detail clean-up and isolation of chemical residues from accompanying interfering extractants. The extracted toxicant must be free of most accompanying extractants before precise and valid chemical analysis can be undertaken. Most clean-up procedures are based on a. Chromatographic separation with materials exhibiting a selective adsorption for a compound being determined. 26 University of Ghana http://ugspace.ug.edu.gh b. Chemical removal of interference through oxidation, reduction, saponification or hydrolysis without detrimental effect on the compound itself. c. Physical separation by solvent partition, steam distillation, freezing. 2.3.3.1 Solvent partitioning An example of this is the preferentially solubility of chlorinated pesticides in acetonitrile. Thus, following partitioning of butter fat between petroleum ether and acetonitrile, the pesticides will be concentrated in acetonitrile while the fat is retained in the non polar solvent. 2.3.3.2 Acid clean-up Many pesticides are stable in strong acid medium. Thus, treatment of fats and oil with fuming sulphuric acid will remove the fats while transferring the pesticides to the solvent phase. A typical example is toxaphene. 2.3.5.3 Column chromatography clean-up This is probably the most widely used but least understood clean-up step. Adsorbents normally used are alumina, silica, charcoal, diatomaceous earth, C- 18 and florisil. Solid phase extraction (SPE) is the most widely used column chromatography clean-up. 27 University of Ghana http://ugspace.ug.edu.gh 2.3.3.3.1 Solid phase extraction (SPE) clean-up method Solid phase extraction (SPE) is relatively new technology that is gaining popularity, where low concentrations of analyte can be concentrated from a large sample volume. A typical SPE consists of four major steps: 1 ) conditioning the sorbent beds with solvent to improve the reproducibility of the pesticide retention and to reduce the concentration of any contaminant present. 2) Sorbing the pesticide on the bed, together with undesirable matrix constituents. 3) Rinsing the column with weak solvent to remove undesirable matrix component 4) eluting the pesticide with a sufficiently strong solvent, while leaving the undesirable components on the bed. Useful adsorbents in the extraction of the pesticides include diatomaceous earth, C-18, silica gel and silica supports bonded with ethyl, octyl, octadecyl, cyclohexyl, and cyanopropyl functionalities. SPE is mostly used off-line, the adsorbent being packed in disposable columns or cartridges. 28 University of Ghana http://ugspace.ug.edu.gh 2.3.4 Qualitative and Quantitative determination of pesticide residue 2.3.4.1 Introduction The final step for both qualitative and quantitative determination of pesticide residue usually involved a form of chromatography. The most important chromatographic technique for both qualitative and quantitative analysis is Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). Spectrophotometry can also be used for many pesticides, and calorimetric kits are available for cholinesterase inhibiting insecticides and some fungicides. In this work, TLC was used for the study because it offers opportunity to undertake analysis where there is inaccessibility of instrumental facilities, lack of spare parts and lack of continuous supply of electricity. Thin Layer Chromatography has made a strong impact on analytical chemistry. The popularity of this technique has grown rapidly in the past few years and the technique has been used for the analysis of pesticide residues in soils, plants and vegetables[73], water[74] and urine[75]. Thin layer chromatography is simple and rapid, and is more selective for a greater variety of separations than paper chromatography. The large number of adsorbents are available and the ease of changing conditions, give the chemist a considerable number of parameters which can be varied to obtain a desired separation[76]. Thin layer chromatography has genuine and general utility for pesticides residue analysis. The reasons are, it is applicable to most of the type of analytical 29 University of Ghana http://ugspace.ug.edu.gh problems in which column chromatography, paper chromatography, gas chromatography and electrophoresis can be used, and it is much simpler and faster than these other techniques[76]. 2.3.42 Previous work done with TLC Stammach et. al.[77] chromatographed triazine herbicides, atrazine, atratone and prometryne and determined their Rf values along with those of related compounds which occur in the commercial products. Silica gel G coated plates were used in all cases. For the analysis of atrazine, ethyl acetate/ petroleum ether mixture (3:7) was used as the developing solvents, and in the case of atratone and prometryne, chloroform/absolute ethanol ethyl acetate (90:5:5), and toluene/acetic acid/water (10 :10 :1 ) were used respectively to develop the chromatograms. Henkel and Ebing[78] analysed a group of six triazine herbicides by using a two- step development on air-dried silica gel plates, with chloroform/diisopropyl ether as the developing solvent. Henkel[79] further, reported on the separation of triazine herbicides, using a chloroform/nitromethane solvent in the ratios of 1 :1 and 5:1. The most sensitive reagent for detecting these chemicals was silver nitrate spray using a 0.02-0.1M silver nitrate solution. Other reagents that were used for the detection was Dragendorff reagent and a 0.25% potassium permanganate solution. 30 University of Ghana http://ugspace.ug.edu.gh Abott et. al.[80] chromatographed agroup of eight triazines herbicides in seven solvent systems on silica gel G and on kieselguhr-silica gel (1:1) in a single solvent system. Quantitatively, the compounds were determined by plotting the square root of the spot area against the logarithm of the weight of the material. For quantitative work the spot were visualized by spraying with 0.5 % brilliant green in acetone followed by exposure to bromine vapour. Substituted urea herbicides were analysed by Henkel[81], on air-dried plates of silica gel G with chloroform/ nitromethane (1:1), using a development distance of 10 cm. He reported of the following Rf values, fenuron 0.31, monuron 0.41, diuron 0.53, monochlorlinuron 0.72, linuron 0.79 and neburon 0.77. Golad[82] investigated the thin-layer separation of trifluralin and related compounds by two-dimentional thin-layer chromatography. Using silica gel GF coated plates, chromatograms were developed in the first direction with benezene/1 ,2-dichloroethylene (1 :1 ) and in the second direction with n- hexane/methanol (98:2). Using the natural colour of some of the compounds or the blue absorbing spots under ultraviolet radiation, the sensitivity of detection was 0.5 pg. Bache[83] analysed and detected amiben in tomatoes. Separation was achieved on silica gel G coated plate with benzene/acetic acid (5:1) as developing solvent. With this elution system, he reported Rf value of 0.44. Detection was accomplished by spraying first with 1 % sodium nitrite in 1 M hydrochloric acid 31 University of Ghana http://ugspace.ug.edu.gh followed by a light spray of 0.2 % ethylene diamine dihydrochloride in 2 M hydrochloric acid. Using the extract from the equivalent of 2 g of tomatoes, the method was sensitive to 0.1 ppm. 2.3.4.3 TLC Technique Chromatography can be defined as a technique for the resolution of components of a mixture as a result of differential migration[76]. Thin layer chromatography is analogous to other adsorptive techniques. An adsorbent is spread on a plate and a drop of sample applied. The plate is placed in developing chamber containing a solvent that act as a mobile phase. As the solvent migrates along the plate, it carries the components of the sample mixture along. A continuous adsorption- elution process takes place, and the most mobile compounds travel fastest, causing complex mixtures to be resolved into series of spots. 2.3.4.3.1 Qualitative Analysis (Location and Identification) using TLC The location of spot of a chromatogram is an index to the chemical composition and identification of the compounds separated. The migration is usually expressed as Rf value (relative factor), which is determined by the ratio of distances. The Rf is expressed as Distance of centre of the spot from starting point Distance of solvent front from starting point Variation in temperature, adsorbent batches, moisture, layer thickness and developing chamber saturation may affect the reproducibility of the Rf values. For 32 University of Ghana http://ugspace.ug.edu.gh these reasons it is recommended that a standard quantity of the pesticide under analysis be run concurrently. The variations may then be correlated and represented by the value of RRf(relative retention factor). RRf is given by Distance of centre of sample spot from starting point Distance of centre of standard spot from starting point 2.3.4.3.2 Quantitative Determination using TLC The important quantitative methods using TLC are as follows: 1. Semi-quantitative: A number of standards are spotted on the plate with the samples. After development and visualization, the sample spots are visually compared with the standard spots. The concentration of the sample is taken as that of the standard spot for which the intensities and size are the same. 2. The diameters of standard spots can be taken and plotted against their concentrations to give a calibration curve for which the concentration of the sample can be determined. 3. Colour development on the tic plate, followed by extraction and colorimetry of the coloured material. This involves spraying the plate with a colour developing reagent and treatment to develop the colour. An area around and including the spot is removed, extracted, and the absorbance of the resulting solution is measured at a characteristics wavelength of the compound, and the concentration is then determined from the a calibration curve of standards of known concentration measured at the same wavelength. 33 University of Ghana http://ugspace.ug.edu.gh 2.4 CLASSIFICATION OF HERBICIDES 2.4.1 Chemical Classification There is today an extensive array of herbicides of widely different chemical type which influence the metabolism and hence the growth and behaviour of plants in a number of ways. Two closely related chemicals may also behave quite differently because physical factors such as their adsorption to plant components, volatility, acid or base dissociation differ and these factors may to varying degrees change the apparent activity of the chemical in a plant. In chemical classification, herbicides are divided broadly into inorganic and organic. The organic chemicals are sub-divided into families such as aliphatic and aromatic acids and nitriles, amides, ureas and triazines, where a group is common to a number of herbicides. Inorganic herbicides include chemicals such as ammonium sulphate, ammonium sulphamate, sodium arsenite, sodium tetraborate, calcium cyanamide and others. 34 University of Ghana http://ugspace.ug.edu.gh 2.4.2 Classification by Phytotoxicity In view of the complexity of the system into which herbicides are introduced, it is not surprising to find that their mode of action within plants is not very well understood. For the majority, a number of physiological and morphological effects have been observed, but there is a measure of conjecture in deciding what is direct or indirect effect in relation to herbicidal action. Various authors have attempted to group herbicides by their important biochemical effects in plant. Van Overbeck[84] observed that just two physiological actions account for the herbicidal activity of about 70 named herbicides, and classified herbicides as the hormone weedkillers which produce growth abnormalities and the triazines and substituted ureas which inhibit photosynthesis. King[85] in ‘weed of the world’ classifies herbicides as inhibitors of cell growth, inhibitor of growth and tropic responses, inhibitors of chlorophyll formation and photosynthesis. The third example is that of Moreland[84] who used three biochemical activities, namely, modifications of respiration and mitochondrial electron transport, inhibition of photosynthesis and Hill reaction, and interference with nucleic acid metabolism and protein synthesis to classify herbicides. Inhibition of photosynthesis is the only biochemical effect, which is mentioned in all the three classifications above. One must conclude that there exists no 35 University of Ghana http://ugspace.ug.edu.gh general accepted classification of herbicides based on their physiology. Table 3 lists herbicides that function by photosynthesis Inhibition. Table 3: Herbicides that act by inhibiting photosynthetic electron transport[86] General Grouping Specific Examples Ureas Diuron Linuron Monuron Metobromuron Triazines Ametryne Atrazine Cyanazine Prometryne Simazine Terbutryne Uracils Bromacil Lenacil Terbacil Acylanilides Pentanochlor Propanil 36 University of Ghana http://ugspace.ug.edu.gh 2.4.3 General Information on the selected Herbicides Atrazine Chemical Name: 2-chloro-4-ethylamino-6-isopropyamino-s-triazine Molecular Formula: CsHuCINs Molecular Weight 215.69 Herbicidal activity. Atrazine is pre and post emergence herbicides suitable for general and selective use. Its actual and potential fields of selective application are corn, sorghum, rice, millet, sugar cane, pineapple and fruit trees. Acute oral toxicity. LD50 for mouse 1750 mg/kg, for rat 3080 mg/kg. Rabbit 750 mg/kg. Chronic toxicity. A series of rats fed for 2 years with daily oral amount of 2, 20, and 200 ppm of atrazine was comparable in all respect to the controls Physical properties: Melting point: 173 - 175°C. Solubility in Water at 0°C: 0.0022 % (22 ppm) 27°C: 0.007 % (70 ppm) Chemical properties: It sublimes at higher temperatures and stable in neutral, slightly acidic or basic media. It is hydrolysed to the herbicidally inactive 2- hydroxy-4-ethylamino-6-isopropylamino-s-triazine in acidic or basic media, especially at higher temperatures. Simazine Chemical Name: 2-choro-4,6-bis-ethylamino-s-triazine Molecular Formula: C 7H 12C IN 5 37 University of Ghana http://ugspace.ug.edu.gh Molecular Weight 207.66 Herbicidal activity. Simazine is a pre-emergence herbicide for general and selective use. The actual and potential fields of selective application are corn, vine, small grains, sugar cane, pineapples, tea, coffee, cocoa. Acute oral toxicity. LD50 for mouse, rat, rabbit, chicken - more than 5000 mg/kg. Chronic toxicity. A series of rats fed for 2 years with a daily oral administration of 2, 20 and 200 ppm of simazine 50W were comparable in all respect with the control. Physical properties: Melting point: 225 - 227°C Solubility at 0°C : 0.0002 % (2 ppm) 20°C: 0.0005 % ( 5 ppm) 85°C: 1.2% (12000 ppm) Methanol at 20°C: 0.04 % (400 ppm) Chemical properties: It sublimates at higher temperatures, is stable in neutral, slightly acidic or basic media. It is hydrolysed to the herbicidally inactive 2- hydroxy-4,6-bis-ethylamino-s-triazine in acidic or basic media. Ametryne Chemical Name'. 2-methythio-4-ethylamino-6-isopropylamino-s-triazine Molecular formula: C gH 1 7 N 5S Molecular weight: 221.32 38 University of Ghana http://ugspace.ug.edu.gh Herbicidal activity. Ametryne is pre- and post-emergence herbicide for general and selective use. The potential fields for its selective application include sugar cane, small grains, peanuts and soybean Acute oral toxicity. LD50 for mouse 965 mg/kg, rat 1110 mg/kg. Subchronic toxicity. A series of rats fed for 90 days with a daily oral administration of 20 and 200 mg ametryne 50W was comparable in all behaviour with the control. Physical properties: Melting point: 84 - 86° C Solubility at 20°C: 0.0185 % (185 ppm), in organic acids is high. Chemical properties: It is stable in neutral, slightly acidic or basic media, hydrolyses to the herbicidally inactive 2-hydroxy-4-ethylamino-6-isopropylamino- s-triazine in acidic or basic media. Diuron Chemical Name: 3-(3,4-dichlorophenyl)-1,1 -dimethylurea Molecular Formula: C9H10CI2N2O Mojpeular Weight: 233.1 Herbicidal activity. Diuron is pre-and post- emergence herbicide used for the control of annual weeds in citrus, avocadros, bananas, pineapples, sugar cane. Physical and Chemical Properties: The phenyl alkyl ureas are only sparingly soluble in hydrocarbons. They are stable toward oxidation and hydrolysed under 39 University of Ghana http://ugspace.ug.edu.gh normal conditions, but at elevated temperatures, they can be hydrolysed quantitatively. Much of their analytical chemistry is based on the latter reactions. Melting point: 158 - 159 °C, solubility in water - 42 ppm Metobromuron Chemical Name: 3-(4-bromophenyl)-1 - methoxy-1 -methylurea Molecula Formula: CgHnBrNaC^ Molecular Weight 259.11 Acute oral toxicity. LD50 for rats is 2603 mg/kg Physical and Chemical Properties: The urea is colourless, crystalline solid, and it is more soluble in water than diuron. Solubility in water is 330 ppm at 2Q°C. Melting Point: 95 - 96°C and vapour pressure : 0.40 mPa at 0°C. 40 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE EXPERIMENTAL METHODS 3.1 CHEMICALS AND REAGENTS The chemicals and reagents used in the experiments were obtained from Merck, Darmstadt, Germany and Fluka, Switzerland, unless otherwise stated. The, atrazine, simazine, ametryne, diuron and metobromuron standards were obtained from Dr. Ehrenstorfer, Gmbh. They were of 98 - 99.5 % purity and were used without further purification. Preparation of sodium hexametaphosphate (Calgon). 5 g of sodium hexametaphosphate was dissolved in 100 mL flasks with distilled water and made up to the mark (5 % solution). Preparation of Digestion accelerator. 10 g of potassium sulphate (K2S04), 1 g of hydrated copper sulphate (CuS04.5H20 ) and 0.1 g of selenium (Se) were thoroughly mixed together. Preparation of Mixed indicator. 0.13 g of methyl red was added to 0.066 g of methyl blue and dissolved in 100 mL of 95 % ethanol. Preparation of O-Tolidine + Potassium lodide[OTKI] reagent. 0.5 g of O- tolidine was dissolved in 100 mL of glacial acetic acid, 2 g of Kl was dissolved in10 mL of distilled water, and the two solutions were mixed and diluted to 500 mL with distilled water. 41 University of Ghana http://ugspace.ug.edu.gh Preparation of Borax(Na2B40 7. 10H20) buffer solution. Used to prepare dichlorophenol-indolphenol sodium salt (DCPIP) reagent. 3.325 g of borax were dissolved in 175 mL of distilled water and the solution was added to 75 mL of 0.1 M Hydrochloric acid. Preparation of DCPIP reagent. It was prepared by dissolving 0.1 g of 2,6- dichorphenol-indolphenol sodium salt in 250 mL of the borax buffer. Preparation of the Spraying reagent. 30 g of Panicum maximum leaves and 5 g of sea sand were smashed in a mortar with a pestle. 15 mL of distilled water and 3 mL glycerine were added. These were mixed thoroughly and the liquid squezzed through knapsack into 50 mL flask. 20 mL of this was added to 13 mL of DCPIP reagent to give the spraying reagent. 3.2 EQUIPMENT Auger. This is a metallic implement with a pointed blade at the end, where it is used to dig the soil. 2mm mesh-size sieve. The sieve was used to screen the soil samples to remove stones and other debris. Hydrometer. The hydrometer was used for particle size analysis TOA pH meter HM-30S. The pH meter was used for the measurement of soil pH. Kjeldahl apparatus. This apparatus was used for the determination of total nitrogen in the soil samples. 42 University of Ghana http://ugspace.ug.edu.gh Sp 1800 Spectrophotometer (Unicam). The spectrophotometer was used to measure available phosphorus in the soil samples. 10x20cm and 20x20cm silica gel 60 tic plates, with tic basic set(camag). Also including were application guide, development tank, 5 pL and 10 pL microsyringe with needle(Hamilton) Gallenkamp Flask shaker. The flask shaker was used for the extraction of the herbicides in the soil samples. Forevac diffusion pump. The equipment was used for the vacuum dry of the solid phase extraction (SPE) cartridges after passing the extracts through the SPE columns. 3.3. SAMPLING FIELDS Coastal savannah and forest zone soils obtained from Ghana Atomic Energy Commission(GAEC),and Kwame Nkrumah University of Science and Technology (KNUST) respectively were used for the investigation. Sketch Maps showing the sampling sites are presented in Fig. 1 and Fig. 2 below. At the time of sampling, the savannah field at GAEC was not under cultivation of any crop. However, around the field were pineapple and plantain farms. The field had no history of pesticide application for the past seven years. The field at Kwame Nkrumah University of Science and Technology was under semi-deciduous forest ecological zone. The field was used for the cultivation of yams and cassava in 1998 main farming season. The yams had been harvested 43 University of Ghana http://ugspace.ug.edu.gh Fig. ^ ; A sketch m ap of a section of GAEC showing the Sampling S ite S1 University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh at the time of sampling but there were few cassava plants scattered on the plot. No pesticide was used during cultivation and there was no history of pesticide application on the plot. 3.3.1. SAMPLING AND SAMPLE TREATMENT About 50 m x 50 m-plot size on each field was demarcated for sampling. Soil samples were taken randomly on the demarcated plots with an auger to a depth of 10 cm. Samples were mixed thoroughly and wrapped in an aluminium foil, and then placed in black polythene bags and the polythene taped. In the laboratory, part of the sampled soil was taken a day after sampling for the determination of the soil moisture content. The rest of the soil was wet sieved with 2mm mesh- size sieve aperture to remove stones and other debris, and this was used for the herbicide depletion studies. 3.4 PHYSICAL AND CHEMICAL SOIL PROPERTIES ANALYSIS 3.4.1 Determination of Soil Moisture A known mass of the soil was placed in a previously weighed crucible. The container and its contents were placed in an oven set at 105°C for two hours. It was then cooled in a desiccator. This procedure was repeated until the mass of the crucible and its content became constant and the percentage soil moisture was calculated as 44 University of Ghana http://ugspace.ug.edu.gh mass of soil moisture v 100 mass of soil used 3.4.2. Determination of Water Holding Capacity (WHC) About 100 g of air -dried soil sample was placed in a funnel clogged with cotton wool, with a rubber tube fixed at the stem and a clip attached at the middle of the rubber tube. About 100 mL of distilled water was measured and poured unto the soil in the funnel. It was allowed to soak the soil for 15 minutes. The clip holding the water was opened to allow the water to drain freely into a dried 100 mL measuring cylinder, which had been placed underneath. The amount retained was taken as the difference between the amount drained through and that added and this was used to calculate the percentage water holding capacity. Percentage water holding capacity was calculated as Amount of water retained by soil > 10o Air-dried mass of soil used 3.4.3 Determination of Particle size The particle size analysis was done using the hydrometer method [87], About 40 g of the soil sieved through to the 2 mm sieve was transferred into a one-litre beaker and 200 mL distilled water and 10 mL of 5 % calgon added. The suspension was shaken over-night with a shaker to obtain a uniform dispersion, which was filtered through 45 |im pore size sieve into a graduated litre 45 University of Ghana http://ugspace.ug.edu.gh measuring cylinder. The residue was washed with distilled water until the filtrate leaving the sieve became clear. By doing this, the sieve allowed the clay and silt to pass through leaving the sand behind. The sand portion was then transferred into a previously weighed container, oven dried at 105°C and cooled in desiccator. It was weighed to obtain the amount of the sand in the soil. The filtrate in the graduated cylinder was then stirred with stirring paddle for about one minute. The paddle was removed and the swirling motion was allowed to settle. The hydrometer was carefully immersed and after two minutes, the reading was taken. The reading of the hydrometer estimated the amount of clay. The composition of silt was determined by difference between the amount of soil used for analysis and the sum of composition of sand and clay. 3.4.4 Determination of Soil pH The soil pH was determined using the method of Campbell and Baver[88] in both water and 0.01 M Potassium Chloride solution. About 20 g of the soil was weighed into 100 mL beaker and 20 mL deionised water was added. The suspension was stirred for 30 minutes with a magnetic stirrer and allowed to stand for 15 minutes. The pH of the partly settled suspension was then measured with the pH meter. Another 10 g of the soil sample was weighed into a 100 mL beaker, to which was added 20mL of 0.01 M potassium chloride solution. The suspension was stirred for 30 minutes and allowed to stand for 15 minutes before the pH was measured. 46 University of Ghana http://ugspace.ug.edu.gh 3.4.5 Determination of Organic Carbon and Organic Matter Analysis of organic carbon was based on the method of Walkley and Black[89]. About 1 g of the soil sample was weighed into 250 mL Erlenmeyer flask. 10 mL of 1M K2Cr07 solution was added to the soil in the flask and shaken to disperse the samle. 20 mL of concentrated sulphuric acid was then added, and the content of the flask shaken vigorously for about one minute. The mixture was then allowed to stand for thirty minutes after which, 10 mL distilled water and 10 mL orthophosphoric acid was added. Finally, 2 mL Barium diphenyl amine was added as indicator. The content was titrated against 0.2 M ammonium ferrous sulphate(Fe(NH4)2(S04)2.6H20 ) solution until the colour changed to green. Blank determination was carried out similarly, but without the sample. Triplicate analysis was performed. Percentage organic matter was obtained by multiplying the percentage carbon by 1.724. This is the Van Bemmelen factor. It is used because organic matter contains 58 % carbon. This method is basically the reduction of Cr20 72' by organic compounds and subsequent reduction of the unreacted Cr20 72' by redox titration with Fe2+ 2Cr20 72' + 3C + 16H+ + Fe2+̂ 3C02 + Fe3+ + 8H20 + 4Cr3+ 3.4.6 Determination of Available Phosphorus Available phosphorus was determined using modified Murphy and Riley method[ 90], 2.5g of soil sample was weighed into 100 mL conical flask and 50 mL of 0.5M sodium hydrogen carbonate (NaHC03) of pH 8.5 was added. The University of Ghana http://ugspace.ug.edu.gh suspension was shaken for 30 minutes on a mechanical shaker. After filtration, the phosphorus was determined in the filtrate using spectrophotometer (Unicam Sp 1800 Ultraviolet spectrophotometer) at 712nm. Calibration was done with phosphorus standard of concentration 5 ,10,15 and 20 ppm. 3.4.7 Determination of total Nitrogen This was based on the method of Page, modified by Kalkra and Maynard[91], Two gramme of air-dried soil sample were weighed and placed in a kjedahl flask, 2 mL of distilled water was added to moisten the soil. 2 g-selenium catalyst (digestion accelerator) and 20 mL of concentrated sulphuric acid was also added. The mixture was digested for about 2 hours, transferred to 100 mL volumetric flasks and made up to the mark. 5 mL of the sample solution were pipetted into kjeldahl distillation flask, and 5 mL of 40% sodium hydroxide added. This was distilled, and the distllate collected into 20 mL of 2 % boric acid to which has been added 2 drops of mixed indicator solution. The distillate was then titrated with 0.01 M HCI from light green to a pink colour end point. A duplicate analysis was performed and blank determination was carried out. The nitrogen, which is in the soil sample as sulphate of ammonia, is converted to ammonia by the alkaline solution, sodium hydroxide. This then form a complex with boric acid H3B03. i.e H3B03 + NH3-» NH3H3B03 The ammonium ion is then released upon addition of HCI i.e NH3H3B03 + HCI -» H3B03 + N H / + Cl' 48 University of Ghana http://ugspace.ug.edu.gh 3.5.1 Preparation of the Herbicides Standards To prepare 1 mg/mL (1000ppm) stock solution of the herbicides about 0.1 g of the reference herbicides standard was accurately weighed into 100 mL volumetric flask, except atrazine where 0.05 g was weighed, This was then dissolved with acetone and made up to the mark. These served as the stock solutions of the various herbicides. The stock solutions were diluted ten fold and used as working solutions. 3.5.2 Selection of Elution System for Analysis of the Herbicides Two elution systems were used for the determination of the Rf of the herbicides, i.e. Silica gel-ethyl acetate and Silica gel-dichloromethane systems. Ready made silica gel 60 plates were activated in an oven at 105°C for thirty minutes. Development tank was saturated by the vapour from the developing solvents by using 50 mL of one of the solvents for each determination. Saturation was achieved by lining the walls of the tank with filter paper cut to the size of the tank, and allowing the vapour to soak the tank for about 3 hours with the tank closed. 5 jal of each of the working herbicide solutions was then applied to the sorbent layer of the activated tic plates with 10 pL calibrated microsyringe. The plates were then developed by dipping them into the saturated tank, where the eluent rise by capillary action. The eluent was allowed 3.5 HERBICIDE DEPLETION STUDIES 49 University of Ghana http://ugspace.ug.edu.gh to move more than two-third of the length of the tic plate. It was then taken out of the tank and the eluting layer was allowed to dry and the spots detected as described below. Triplicate determination was carried. The concentrations at which the herbicides were spotted for the determination of Rf is as shown in Table 4. Table 4: Concentrations and Amount of Herbicides applied for the determination of Rf Herbicides Conc.(ng/mL) Amount applied.(^g) Atrazine 55.59 0.278 Simazine 100.70 0.504 Ametryn 103.66 0.518 Diuron 99.78 0.499 Metobromuron 101.58 0.508 3.5.3 Detection and measurement o f Herbicides The O-tolidine + potassium Iodide method(OTKI) and Photosynthesis inhibition method were used. a) For the O-tolidine + potassium Iodide method, developed tic plates were air dried and placed in a tank saturated with chlorine for 30 seconds. The chlorine solution was made by placing a 25 mL beaker containing about 1 - 2 g potassium permanganate at the bottom of a developing tank and adding concentrated HCI. Excess chlorine was removed in a fume hood after which the 50 University of Ghana http://ugspace.ug.edu.gh plates were sprayed with the OTKI reagent. Spots were seen as darkish blue in colour on grey-white background. Spots could stay over-night before disappearing. b) For the Photosynthesis inhibition method, spots were visualized by spraying with the spray reagent, and the plate placed about 20 cm below 60 W ordinary electric bulb for about 2 minutes. Spots were seen as blue in colour in greenish background. Spots disappeared within 1hour. Distances moved by the solvent and chemicals were measured and these were used to calculate the Rf Rf were calculated as Distance of centre of spot from starting point Distance of solvent front from starting point 3.5.4 Selection of Extraction Solvents for the Herbicides Acetone, acetonitrile, hexane, methanol and acetone/hexane(4:1) were investigated for their efficiency in extracting the herbicides from the soil samples spiked with known amount of the herbicide. Extracts were analyzed by tic and the recovery of the various herbicides by each solvent as well as the purity of the soil extracts determined. Each herbicide was analyzed three times. 3.5.4.1 Procedure for spiking About 5 g of the soil sample was accurately weighed into individual extraction flasks. 2 mL of 100 ng/mL of herbicide standard was added and mixed with the 51 University of Ghana http://ugspace.ug.edu.gh soil to generate a concentration of 40 ng/g. In the case of atrazine. 2 mL of 50 |ag/mL was used to yield a concentration of 20 jag/g. The spiked soil in the extraction flask was allowed to stand for 30 minutes. 3.5.4.2 Extraction Extraction was performed by adding 20 mL of each solvent unto the spiked soil and mechanically shaking the mixture on a flask shaker for 2 hours. Filtration was carried out by use of whatman No. 42. filter paper. The residue on the filter paper was washed three times with 3 mL of the solvent and washings were added to the filtrate. The extract was then dried over anhydrous sodium sulphate and filtered. Streams of air were gently blown from a hand-dryer to remove the solvent. The unclean extract was redissolved in 10 mL of acetone and analyzed for recovery and purity. 3.5.4.3 Clean-up of extracts The extraction procedure was repeated, but this time the uncleaned filtrate was clean up as follows. SPE cartridge equipped with C-18 as adsorbent was earlier preconditioned with 2 mL acetone/water(1:9). Filtrate was passed through the preconditioned cartridge. The cartridge and its content were dried for 15 minutes by vacuum pump. The herbicide was then eluted with 10 mL of acetone into 10 mL flask. Eluate was adjusted to the mark with acetone. This is the clean up 52 University of Ghana http://ugspace.ug.edu.gh extract. The clean up extract was also subjected to tic method of analysis. The procedure was repeated for all the solvents. 3.5.4.4 TLC Analysis of Extracts for Purity and Recovery A constant 5 p.1 of each extract was applied to the sorbent layer of the tic plates with a calibrated mircosyringe. The same volume of the standard solution was analyzed concurrently. To spot the standard, 2 mL of the herbicide standard as used to spike the soil samples was pipetted into 10 mL volumetric flask and made up to the mark and 5 [il of the solution used for the analysis. After detection of spots, diameters of the spots of the standards and extracts were measured to estimate concentration. For both the unclean and clean up extracts only one spot was detected, which in each case corresponded to the herbicide analyzed. Triplicate analysis was carried out. Percentage recovery was calculated as Amount recovered x_1Q0 Amount added Amount recovered was calculated as shown in appendix 1( sample calculation 6) 3.5.5 Soil treatment and incubation The method adopted was similar to the one used by G. A. El Zorgani[92], 53 University of Ghana http://ugspace.ug.edu.gh To determine the rate of the herbicide depletion, 200 g of the soil sample was weighed into 500 mL of individual incubation flasks, and treated with 20 mL of 100 ng/mL of the herbicide stock solution to generate herbicide-soil concentration of 10 |_ig/g(10ppm). The soils were thoroughly mixed with the herbicide standard solution by the use of a stirring rod. The soils were then moistened to 50% field capacity by treating the KNUST and GAEC soils in the flasks with 40.land 34.5 mL of distilled water respectively. The soils were then incubated at room temperature (28-30.5°C). Control experiment, without herbicide was also set up. Incubation was done for 12 weeks. 3.5.5.1 Sub-sampling and Extraction procedure Sub-sampling was done at a regular interval of one week after soil treatment. 5 g was of the soil was withdrawn from the incubation flasks into 200 mL extraction flask to determine the rates of the herbicides depletion. To the 5 g of the sub-sampled soil in the extraction flask, 20 mL of acetone/hexane (4:1) was added. This was shaken mechanically on a flask shaker for 2 hours, followed by filtration with Whatman No 42 filter paper. After filtration, the residue was washed 3 times with 3 mL of the solvent. The filtrate was then dried over anhydrous sodium sulphate (Na2S04). The filtrate was concentrated to dryness by gently blowing in streams of air from hand dryer. The residue was then redissolved in 5 mL of acetone and then analyzed by tic. 54 University of Ghana http://ugspace.ug.edu.gh 3.5.5.2 Thin Layer Chromatographic (TLC) Analysis The qualitative and quantitative determination of the extracts was done by thin layer chromatography as described in section 3.5.2 above. 5 pL of the sample extract, extract from control and standard herbicide solution were analyzed concurrenty. Each extract was analyzed two times. Photosynthesis inhibition method as described per section 3.5.3(b) was used for the detection of the spots. The diameters of the spots of the sample extracts were measured, for quantification and the distances moved by the spots of the extracts and the standards herbicide solution was also measured for identification. 3.5.5.2.1 Quantification of residues Calibration curves of average diameters of two replicates measurement of the spots was plotted against the herbicides standard concentration in the range of 1-16 ng as indicated in appendix 2 (graphs 1, 2, 3, 4 and 5) and this was used for quantification of the amount of herbicide in the extracts. The amount extracted (residue level), C was calculated as Concentration in final extract x dilution factor Weight of the sample analyzed. 55 University of Ghana http://ugspace.ug.edu.gh 3.5.6 Determination of Limit of Detection (LOD) Two milli litre of each of atrazine, ametyne, simazine, diuron and metobromuron standard solutions of concentrations 55.59, 103.66, 100.70, 99.78 and 101.59 |jg/mL respectively was each spiked with the 5 g of the soil in the extraction flasks, to generate spiking level of 22.23, 41.46, 40.2839.90 and 40.64 pg/g for atrazine, ametryne, simazine, diuron and metobromuron respectively. Extraction was done with acetone/hexane (4:1) as described per the extraction procedure in section 3.5.5.1 above. Extracts were then subjected to tic method of analysis. To determine the limit of detection, the extracts volume in the range of 10-0.1 (j.L was spotted on the tic plate for analysis, and the least detectable volume noted. Both unclean and clean up extracts were analyzed. Photosynthesis inhibition and O-tolidine+Potassium Iodide methods used for detection. However, for the clean-up extracts only photosynthesis inhibition method was used for detection. Duplicate determination was done. Only the GAEC soil was used for the investigation. Limit of detection was calculated as: Viful) x Level of spiking (ua/g) V2 (ml) x 1000 Vi= least detectable volume, V2= final volume of extract in each case. 56 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS AND DISCUSSION 4.1 PHYSICO-CHEMICAL SOIL PROPERTIES The physical and chemical properties of the soils are presented in Table 5 below. Table 5: Physical and Chemical properties of the soils Properties GAEC soil KNUST soil Soil moisture(%) 1.82 10.24 Water holding capacity(%) 40.1 34.5 Texture Sandy Clay Loam Sandy Loam Soil pH(water) 5.5 7.5 Soil pH(0.01MKCI) 5.2 7.3 Organic carbon(%) 0.633 1.023 Organic matter(%) 1.091 1.764 Available phosphorus(pg/g) 13.06 14.67 Total nitrogen(%) 0.093 0.106 The coastal savannah soil from GAEC had lower % soil moisture but higher % water holding capacity (WHC). The moisture content of 1.82% compared to water holding capacity of 40.1% is an indication that at the time of sampling the soil was quite dry. This was expected since sampling was done in early February. In Ghana the dry season in February is at its peak. However, for the forest zone soil sampling from KNUST, the moisture content of 10.24% compared to water holding capacity of 34.5% indicates that the soil was not too dry at the time of 57 University of Ghana http://ugspace.ug.edu.gh sampling. This observation may be due to the fact that in the forest zone, the land in the dry season is not as bare as it is in the coastal savannah zone. Adequate water is not essential for microbiological activity, but in addition, water acts as a solvent and transporting agent, a reaction medium for both biological and non-biological processes and is a reagent in hydrolytic reactions[107]. The soil pH of the coastal savannah soil was 5.5 in water and 5.2 in 0.01 M potassium chloride solution indicating acidic character, whilst for the forest soil zone the values were 7.5 and 7.3, indicating slightly basic character. Soil pH is affected by the presence of such inorganic species such as hydroxides, bicarbonates which tend to increase pH. The presence of carbon dioxide, oxides of sulphur, nitrogen, the presence of unionised portion of the weakly ionising acid such as carbonic acid lower pH The major nutrients status of the two soils was not too different. Available phosphorus and total nitrogen were only slightly higher in the KNUST soil. However, the organic matter content of the KNUST soil was about 40% higher. This variation was expected since in the forest zone the relative high rainfall pattern promotes decomposition of more organic materials. Soil organic matter might be expected to have some effect on depletion of herbicides since microbial activity is often higher in more organic soils. However, it must be noted that adsorption of most herbicides also increases with increase in soil organic matter and since adsorption reduces the amount of herbicides available in the soil solution, it might provide protection for degradation. 58 University of Ghana http://ugspace.ug.edu.gh Results obtained from the particle size analysis (Figures 3 and 4) gave an indication that the soil particle size is dorminated by sand particularly, the GAEC soil with 61.92 % sand content. The proportions of clay and silt are approximately equal for the GAEC soil, but are quite different for the KNUST soil. □ sand EE! clay □ silt Figure 3: % composition of sand, clay and silt in the GAEC soil. Sand=61.92 %, Clay=20.00 % and Silt=18.08 %. □ sand □ clay □ silt Figure 4: % composition of sand, clay and silt in the KNUST soil. Sand=57.78 %, Clay=12.20 % and Silt=29.63 %. 59 University of Ghana http://ugspace.ug.edu.gh 4.2 SELECTION OF ELUTION SYSTEM FOR THE ANALYSIS OF THE HERBICIDES The Rf values of the pesticides tested with the two elution systems are in agreement with the findings of Lowor et. a/[94], They reported of Rf values of 0.61, 0.61, 0.41 and 0.59 for atrazine, ametryne, diuron and metobromuron respectively at 32±3°C, using silica gel-ethyl acetate elution system. It is obvious from the results that the three triazine herbicides i.e. atrazine, ametryne and simazine, have very close Rf, (Table 6) particularly, atrazine and ametryne under silica gel-etyyl acetate system. Table 6: Rf of the herbicides, using silica gel-ethyl acetate elution system at 30.5±2°C Herbicides Rf values RKmean) SD Atrazine 0.622, 0.620, 0.625 0.622 0.004 Ametryne 0.610, 0.610, 0.617 0.612 0.006 Simazine 0.578, 0.580, 0.588 0.581 0.006 Diuron 0.425, 0.430, 0.432 0.429 0.005 Metobromuron 0.574, 0.580, 0.577 0.577 0.004 SD = Standard deviation This indicates that this elution system could not be very useful for the analysis of these chemicals in a multi-residue procedure involving a mixture of these pesticides. This is because their spots would overlap and resolution would be very difficult. However, for a sample known to contain only one of these 60 University of Ghana http://ugspace.ug.edu.gh chemicals, the system could conveniently be used. In the case of the substituted urea herbicides, i.e. diuron and metobromuron, their Rf values were quite distinct from each other and therefore this elution system is recommended even if these herbicides are administered in a mixture. In the case of silica gel-dichloromethane system, ametryne and diuron, have the same Rf (Table 7). Table 7: Rf of the herbicides, using silica gel-dichloromethane elution system at 30.5+2°C Herbicides Rf values Rf(mean) SD Atrazine 0.064, 0.064, 0.058 0.062 0.006 Ametryne 0.088, 0.085, 0.078 0.083 0.009 Simazine 0.056, 0.048, 0.056 0.053 0.008 Diuron 0.088, 0.088, 0.075 0.083 0.011 Metobromuron 0.258, 0.258, 0.251 0.256 0.006 It is therefore obvious that this elution system could not be very useful to separate these two chemicals if they are in a mixture, as their spots would overlap. It is of interest to note that all the herbicides except metobromuron moved very little from the origin with the silica gel-dichloromethane system. Thus, the Rf values of atrazine, ametrye, simazine and diuron were almost zero. This indicates the high affinity between these chemicals and the stationary phase on one hand and the low affinity between the chemicals and the mobile phase. However, with the silica gel-ethyl acetate system, all the chemicals moved to an 61 University of Ghana http://ugspace.ug.edu.gh appreciable height from the origin. This also means that the stationary phase has less affinity for the chemicals compared to the mobile phase. It therefore appears that perhaps, the silical gel-dichloromethane system might not be suitable for the analysis of these chemicals. For the two systems studied, silica gel-ethyl acetate system appears to be more suitable and was selected. Detection Methods The O-tolidine + potassium iodide was not sensitive to metobromuron as spots appeared very faint. Spots were seen as darkish brown in grey- whitish background. At the concentrations at which the standards were injected, spots could stay overnight before disappearing. With regard to the photosynthesis inhibition method, spots were visualized as blue black in greenish background. Spots stayed for about 45 minutes after detection before disappearing. Comparing the size and intensities of the spots obtained, the photosynthesis inhibition method appeared more sensitive than the O-tolidine + potassium iodide method. 4.3 SELECTION OF EXTRACTION SOLVENTS FOR THE HERBICIDES Recovery of the herbicides investigated sh