Survey of Some Selected Metals and Tannin in Awash Wine, Ethiopia

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Chemistry Thesis and Dissertations

2019-12-04 Survey of Some Selected Metals and Tannin in Awash Wine, Ethiopia

BIRHANU, HUNDE http://hdl.handle.net/123456789/10020 Downloaded from DSpace Repository, DSpace Institution's institutional repository

BAHIR DAR UNIVERSITY

OFFICE OF GRADUATE STUDIES

DEPARTMENT OF CHEMISTRY

Survey of Some Selected Metals and Tannin in Awash Wine, Ethiopia

BY: BIRHANU HUNDE

SEPTEMBER, 2019

SURVEY OF SOME SELECTED METALS AND TANNIN IN AWASHWINE, ETHIOPIA

ATHESIS SUBMITTED TO THE COLLEGE OF SCIENCE, POST GRADUATE PROGRAME, DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY

BY: BIRHANU HUNDE SUFA

ADVISOR: TIHITINNA ASMELLASH (PhD)

SEBTEMBER, 2019

BAHIR DAR UNIVERSITY

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LETTER OF APPROVAL

The thesis entitled “SURVEY OF SOME SELECTED METALS AND TANNIN IN AWASH WINE, ETHIOPIA “by Birhanu Hunde is approved for degree of Master in Chemistry.

Board of Examiners

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Name of External Examiner Signature Date

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Name of Internal Examiner Signature Date

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Name of Internal Examiner Signature Date

DECLARATION

This is to certify that entitled SURVEY OF SOME SELECTED METALS AND TANNIN IN AWASH WINE, Ethiopia submitted in partial fulfillment of the requirements for degree of Master of Science in chemistry to the graduate program of college of science, Bahir Dar University is authentic work conducted by Birhanu Hunde under the supervision of Tihitinna Asmellash (PhD).

Birhanu Hunde ______

Student Signature Date

Tihitinna Asmellash (PhD) ______

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DEDICATION

This thesis is dedicated to my all thing Beza Woldeyohannis, my brother Ribera Hunde and my deepest friend Dange Guta for their everlasting love, support and guides me in all my life.

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ACKNOWLEDGEMENTS

I would like to take this opportunity to gratefully thank the almighty God for letting me to finish this thesis work. Next I would like to thank my Advisor, Tihitinna Asmellash (PhD) for all here patience, guidance and support throughout my thesis research work. It is a great concession and honor for me of being advised by here.

I gratefully express indebtedness to Chemistry department for permission ICP – OES to read sample for conducting my studies. My great thank is also to Chemistry laboratory assistances for their professional and material support throughout my thesis research work.

I gratefully acknowledge Bahir Dar University, School of Graduate Studies, and Chemistry Department for the financial and Equipment support. Finally I wish to thank my parents and friends for all their help, support and guidance throughout my studies. At the last but not the least, I would like to thank Oromia Education Bureau for sponsoring

ABSTRACT

Wine is an alcoholic beverage which contains inorganic as well as organic components. The quality of wine depends on the concentration of these components. In this study, after digestion of wine sample and preparation of standard KMnO4, the selected metals and tannins were determined in the six Awash brands of Ethiopian wines using ICP – OES and Titrimetric method respectively. The result indicates, all Awash wine brands were rich by macro metals (K, Na, Ca, Mg). From all trace metals, Fe was the most abundant in both Gebeta Red and Gebeta White, but least in Axumite Red. Pb was not detected in three Awash brands (Gouder red, Axumite Red and Kemila white), but Gebeta red, Gebeta white and Awash Tekishino were contain Pb. Cd was not detected in all six Awash wines. The level of Cr in Gouder red was below detection limit, but Kemila white has the highest content of Cr than all brands.

Awash wine brand also contain tannin in the range of 0.021-0.032% with mean value of 0.0451%. The one way ANOVA for most metals in Awash brand were significant difference with (p <0.05), Na was significantly different in all wine brands and some metals were not significantly different in each brands with (p> 0.05). Comparing the level of tannin in Red wines with white wines, it was significantly different (p< 0.05), but not significant with another some red wines. This indicates that, the level of tannin in red wine was more concentrated than in white wines.

Keywords: Metals, Tannin, wine, ICP–OES and Titrimetric Method.

TABLE OF CONTENTS

Contents Page

LETTER OF APPROVAL...... i

DECLARATION...... ii

DEDICATION...... iii

ACKNOWLEDGEMENTS ...... iv

ABSTRACT ...... v

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

ABBREVIATIONS AND ACRONYMS ...... xiii

CHAPTER 1: INTRODUCTION ...... 1

1.1. Background of the Study ...... 1

1.2. Statement of the Problem ...... 2

1.3. Objective of the Study ...... 3 1.3.1. General Objectives ...... 3 1.3.2. Specific Objective ...... 3

1.4. Scope of the Study...... 4

1.5. Significance of the Study ...... 4

CHAPTER 2: LITRATURE REVIEW ...... 5

2.1. History of Wine ...... 5

2.2. Quality of Wine ...... 6

2.3. Classification of Wine ...... 7

2.4. Chemistry of Wine ...... 9

2.5. Components of Wine ...... 9 2.5.1. Essential Macro Metals ...... 9 2.5.2. Essential Micro Metals ...... 11 2.5.3. Toxic Metals ...... 13 2.5.4. Tannin ...... 15

2.6. Review on Determination of Metals and Tannins in Wine ...... 18

CHAPTER 3: MATERIALS AND METHEDS ...... 22

3.1. Study Area ...... 22

3.2. Chemicals and Reagents...... 22

3.3. Instruments and Apparatus ...... 22

3.4. Sampling and Sample Preparation ...... 23 3.4.1. Digestion of the Wine Sample ...... 23

3.5. Determination of the Metals in Wine Samples by ICP-OES ...... 25

3.6. Determination of Tannin ...... 25

3.7. Instrumental Calibration ...... 26

3.8 Method Validation...... 31 3.8.1. Repeatability ...... 31 3.8.2. Limit of Detection (LOD), Limit of Quantification (LOQ) and Instrumental Detection Limit (IDL) ...... 31 3.8. 3. Recovery Test ...... 32

3.9. Statistical analysis ...... 34

CHAPTER 4: RESULTS AND DISCUSSION ...... 35

4.1. Levels of Metals in Wine Samples ...... 35 4.1.1. The level of Major metals (K, Na, Ca, and Mg) in each wine Samples ...... 37 4.1.2. Level of Minor Metals (Fe, Cu, Zn, Mn, Co and Ni) ...... 41 4.1.3. The Levels of Toxic Metals (Cr, Pb and Cd) ...... 46 4.1.4. Comparisons of the Levels of Metals in Each Ethiopian Wines ...... 48

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4.1.4.1. Gebata Red Wine ...... 48 4.1.4.2. Gebata White Wine ...... 48 4.1.4.3. Kemila White Wine ...... 49 4.1.4.4. Axumite Red Wine ...... 49 4.1.4. 5. Awash Tekishino Red Wine ...... 49 4.1.4.6. Gouder Red Wine ...... 49 4.1.5. Comparison of the Level of Metals in Ethiopian Wines with another World Data ...... 51 4.1.6. Comparison of the Level of Metals with OIV ...... 56

4.2. Determination of Tannins...... 57 4.2.1. Comparison of the level of Tannins with each Sample of wines ...... 59 4.2.2. Comparison of the Level of Tannins with World Wide of Wines ...... 59

CHAPTER 5: CONCLUSION AND RECOMMENDATION ...... 61

REFERENCE ...... 63

APPENDEX A: Lists of Table ...... 72

APPENDEX B: Lists of Figure and graphs ...... 80

APPENDEX C:Analysis of Variances (ANOV) at 95% confidence interval Multiple Comparisons ...... 86

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LIST OF TABLES

Table 1: Optimized volume ratio, temperature and duration of time for digestion of sample ...... 24 Table 2: Concentration of intermediate Standards (mg/L), Concentration of Working Standard (mg/L) and Correlation coefficient of calibration carve ...... 27 Table 3: The MDL, LOD, LOQ, IDL, % Recovery and Correlation Coefficients of calibration curve (R2) were given as the following table...... 33 Table 4: Level of metals in Awash brand of wine ...... 36 Table 5: Level of K, Na, Ca and Mg in Awash brand wine ...... 37 Table 6: Level of Fe, Cu, Zn, Mn, Ni and Co in Awash brand wine ...... 41 Table 7: Level Cr, Co and Pb in Awash brand wine ...... 46 Table 8: Average Values of some metals in Ethiopian Wines...... 52 Table 9: Comparison of the level of metals in wines of different Countries ...... 53 Table 10: Comparison of the level of metals in wines of different Countries with OVI .. 56 Table 11: The amount of Tannins in six Awash brand of Wine sample ...... 58

LIST OF FIGURES

Figure 1: Instrumentation (ICP – OES) ...... 21 Figure 2: Preparation of sample ...... 23 Figure 3: The digested sample ready for ICP – OES ...... 24 Figure 4: when titration was conducted ...... 26 Figure 5: Plot of calibration curve for the studied metals (a-m)...... 31 Figure 6: comparison of major metals (K, Na, Ca and Mg) in Ethiopian Wines ...... 40 Figure 7: comparison of trace metals (Fe, Cu, Zn and Mn) in Ethiopian Wines...... 45 Figure 8: The comparisons of both trace and heavy metals in Ethiopian wine brand ...... 48 Figure 9: The Comparisons of Essential and Non essential metals in Ethiopian wines ... 50 Figure 10: The percent Tannin in Ethiopian Wine brand ...... 59

ABBREVIATIONS AND ACRONYMS

AAS Atomic Absorption Spectroscopy AES Atomic Emission Spectrometry ANOVA Analysis of Variance CV –AAS Cyclic Voltammetry Atomic Absorption Spectroscopy FAAS Flame Atomic Absorption Spectroscopy

FSO2 Free sulfur dioxide GA Gallic Acid GFAAS Graphite Furnace Atomic Absorption Spectrometry HGAAS Hydride Generation Atomic Absorption Spectroscopy HPLC High performance liquid chromatography ICP – AES Inductively coupled Plasma Atomic Emission Spectrometry ICP – OES Inductively coupled Plasma Optical Emission Spectrometry ICP – MS Inductively coupled Plasma mass spectrometry IDL Instrument Detection Limit LOD Limit of Detection LOQ Limit of Quantification NAA Neutron activated analysis OIV International Organization Vine RSD Relative Standard Deviation SD Standard Deviation TCA Tri chloroanisole

T SO2 Total Sulfur dioxide

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CHAPTER 1: INTRODUCTION

1.1. Background of the Study

Traditional alcoholic beverages are indigenous to a particular area and are prepared by the local people using old age techniques from locally available raw materials [1]; accordingly, different countries, including Ethiopia have various indigenous alcoholic beverages [2]. The most familiar traditional fermented beverages are: high alcoholic beers such as Tella , low alcoholic beers such as Keribo, Buqri, Shameta, Borde and wine such as Tej made from honey. Similarly, the most popular distilled beverage is Areke [3].

Wine is one of the most common alcoholic beverages consumed in all over the world. Wine is also widely produced and consumed in Ethiopia. Awash Winery is the first commercial winery in Ethiopia, established in 1943. The wineries produce a range of well-made wines, Including Gouder Red, Dukem Red and Awash Tekishino, Axumite Red, Gebeta Red and Gebeta white wine [3].

Minerals (essential and non-essential metals), Acids, additive chemical (like SO2) and Phenolic compounds (tannin) - are the main components of wine. Tannin belongs to a class of phenolic compounds and comes from grape skins and seeds; it is mostly found in red wines and also can be found in some white wines. Tannins are a collective name for colorless but bitter flavonoids. Tannins can be divided into two groups namely the hydrolysable tannins and condensed tannins [4]. Hydrolysable tannins are easily oxidized and thereby decreasing the oxygen availability for the reaction. It also has ability to inhibit the growth of several wood decaying fungi [5]. Whereas condensed tannins does not significantly affect the chemical age and color intensity of the wine but external addition of these tannins increase total phenols of the tannin [6, 7]

The content of metals in wine can be attributed to the natural sources, the atmospheric deposition of airborne particulate matter on grapes and transfer of metals from the soil via the roots to the grapes and finally to wine, also contamination during the winemaking process is the source of metals in wine. [9, 8]. Metals were the largest

1 part of the total metal content in wine. It is connected with the maturity of the grapes, their variety, the type of soil in the vineyard, and the climatic conditions during their growth [11]. The contribution of metals of a secondary origin is associated with external impurities that reach wine during growth of grapes or at different stages in winemaking (from harvesting to bottling). During the growth of grapes in a vineyard, contaminations can be classified as geogenic (originating in the soil), from protection and growing practices, or from environmental pollution [10, 12].The levels of certain elements in wine are of great concern because of harm full in un recommended amount.

1.2. Statement of the Problem

Wine was the most common alcohol consumed in Ethiopia, it contains organic and in organic substances or metals and tannin. Knowing the content of metals in wine is very important. The alcoholic beverages of wine constitute more than 12 % of the consumption of beverages in general. Then, metals are an important source of several metal ions. Therefore monitoring of certain elements in wines, gain an special attention due to their toxic effect on the human body in case of excessive intake (above the recommended amount) [12 - 16]. The Oxidized wines lose the brightness of wine, both in color and in flavor; contamination usually from real cork [17-20]. Excess Mineral precipitates wines, [21]. The dosage of tannin in wine results the dosage of tannins in wine arise problems like blood pressure, slow blood clotting, heart diseases, brain damage after stroke, etc and low level make bitter test [18].Therefore, to overcome these problems the content of mineral and tannin in wine must be determined, by conducting research.

1.3. Objective of the Study

1.3.1. General Objectives

 To determine the level of tannins and some selected metals in different brand of Awash wine.

1.3.2. Specific Objective

 To determine levels of metals in six in Awash brand wines  To determine tannin content in six in Awash brand wines  To compare the level of metals and Tannins in Ethiopian wine the reported before.

1.4. Scope of the Study

Previously (2008) by Woldemariam and Chandra one work was done on metal analysis of some type of Awash wine, other than this no work was done on metal analysis and tannin content of all Awash winery products. Therefore, the scope of this research is concerned on the determination of Tannins and some selected metals in Ethiopian Awash wine, and also compare and contrast the various wine, based on the content of tannins and metals which determine the quality of wine

1.5. Significance of the Study

This research is expected to give the following benefits:

 Giving information for the consumers and concerned bodies about the level of tannin and some selected metals studied in wine.  It also helps cultivator to improve their production methods if there is excess level in the metals studied.  Help for further more investigation

CHAPTER 2: LITRATURE REVIEW

2.1. History of Wine

Grape and wines are well known in Ethiopia which is primarily imported through Red sea port of Adulis, and Ethiopia starts brewery traditional fermented beverage from local available raw materials [22].

In Ethiopia, the production of wine can be traced to the early centuries of the first millennium A.D. [23]. The historian Richard Pankhurst observed that early references to Axumite wine can be found in one of the stele erected by the 4th century ruler Ezana. Axumite viticulture is also attested to by carvings on the base of the great 3rd century obelisk at Axum [23]. The traditional honey winetej has also long been widely popular. Contemporary viticulture in Ethiopia dates to 1956, to the establishment of the Awash Winery by entrepreneur Mulugeta Tesfakiros. As of 2014 the Awash winery had an annual output of 10 million bottles, most of which is consumed locally [24]. In 2014 the French beverage corporation Castel began producing wines of a number of varieties on a 120-hectare estate near Ziway in the Ethiopian Rift Valley. As of 2013 annual production was 3 million bottles, approximately half of which was exported, mainly to China [24].

Climatic and geographic conditions in the Rift Valley make it well suited to viticulture. Annual rainfall measures about 650 mm, temperatures average 25 degrees Celsius per year and the region enjoys sandy soils, which are ideal conditions for wine production. Furthermore, due to Ethiopia’s proximity to the equator, it is possible to make two harvests per year [24]. The western slopes of the Ethiopian highlands also enjoy an inland Mediterranean climate, also conducive to wine-growing.

2.2. Quality of Wine

A wine’s quality is not absolute: how great a wine is or isn’t depends on who is doing the judging. The combined opinion of a group of trained, experienced palates (also known as wine experts) is usually considered a definitive judgment of a wine’s quality. The standards of performance that wine experts use to judge wine quality include the following [25, 26].

Balance: The relationship of four components -sweetness, acidity, tannin, and alcohol - to one another. A wine is balanced when nothing sticks out as you taste it, like harsh tannin or too much sweetness. Most wines are balanced to most people [25, 26].

Length: Used to describe a wine that gives an impression of going all the way on the palate you can taste it across the full length of your tongue - rather than stopping short halfway. Many wines today are very up front on the palate -they make a big impression as soon as you taste them- but they don’t go the distance in your mouth. They are short. Generally, high alcohol or excess tannin is to blame. Length is a sure sign of high quality.

Depth: This is another subjective unmeasurable attribute of a high quality wine. We say a wine has depth when it seems to have a dimension of verticality that is. It does not taste flat and one-dimensional in the mouth. A “flat” wine can never be great [25, 26].

Complexity: There’s nothing wrong with a simple, straightforward wine, especially if we enjoy it. But a wine that keeps revealing different things about itself, always showing you a new flavor or impression a wine that has complexity is usually considered better quality. Some experts use the term complexity specifically to indicate that a wine has a multiplicity of aromas and flavors, while others use it in a more holistic (but less precise) sense, to refer to the total impression a wine gives you.[25, 26].

Finish: The impression a wine leaves in the back of your mouth and in your throat after you have swallowed it is its finish or aftertaste. In a good wine, you can still perceive the wine’s flavors such as fruitiness or spiciness at that point. Some wines may finish hot, because of high alcohol, or bitter, because of tannin both shortcomings. Or a wine may have nothing much at all to say for itself after you swallow.

Typicity: In order to judge whether a wine is true to its type, you have to know how that type is supposed to taste. So you have to know the textbook characteristics of wines made from the major grape varieties and wines of the world’s classic wine regions. For example, the Cabernet Sauvignon grape typically has an aroma and flavor of blackcurrants and the French white wine called Pouilly-Fumé typically has slight gunflint aroma content [25].

2.3. Classification of Wine

The most common classifications of wine are done by; place of origin (or appellation), vinification methods and style, sweetness, taste, quality, vintage or varietal (which describes from what variety of grapes was selected wine been made [25, 26].

One of the first classifications of wine was by their place of origin (or appellation), for example Bordeaux, Rioja, Mosel and Chianti all described the place of wine's origin and sometimes style in which were created. These classifications also specified exactly which grapes were used for making each wine, and the process of their fermentation. Example, Champagne wine made from grapes grown in Champagne region of France [27].

Regional wine classification: were introduced to monitor the state and authenticity of wines created at a specific location (estates and vineyards) in the world. The first of those laws was introduced in 1855s "Bordeaux Wine Official Classification" which catalogued all best wines created in their Bordeaux region. Some of the most notable regional specifications include Classification of Saint-Émilion wine of Bordeaux (updated every 10 years), Classification of Graves wine of Bordeaux, Classified estates of Provence and Grand cru of Burgundy and Alsace. [26].

Classification by vinification: refers to how the wines are made, and they separate all wines into three major categories: table wines, sparkling wines and fortified wines. Table wines (also called natural wines) are mostly consumed with food, and they serve as a compliment to the meal. Sparkling wines are mostly dedicated for consumption at celebrations, and fortified wines are used before or after the meal (or in cooking as an ingredient).

Taste classification: describes the character of wine as dry (not sweet, containing 2-3% of sugar and about 10% of alcohol), semidry, semisweet (5-6% sugar, 13-14% alcohol) and sweet (often called dessert wines, 14-16% sugar and 16% alcohol).

Vintage classification: regulates wines that are made from grapes that were grown in specific year which is accordingly dated on its packaging [25, 26].

Varietal classification: regulates the origin of dominant grapes in wine. As wines may not be entirely made from one type of grapes, various countries enforce different amounts of grapes before naming that wine "varietal". United States asks for at least 75% particular grape before allowing it to be labeled varietal, but European Union has a minimum of 85% and it has strict laws about naming wines with multiple grape types.

Color classification: There are four main types of wine, also based on their color. Red, White, Rose or blush and Champagne, There are many subtypes within these groups, and different areas of the world produce varying varieties due to soil conditions and climates [28].

Red Wine: Wine makers make red wine from red grapes, but this is not why the wine is red. During the fermentation process of red wine, the skins of the grapes are left on and this is what causes the striking red color of red wine. Some popular red wines include Cabernet Sauvignon, Merlot, Montepulciano, Nebbiolo, Pinot Noir, Sangiovese, Syrah, Tempranillo and Zinfandel. Serve red wine with red meats such as beef.

White Wine: Wine makers usually make white wine from white grapes, although they may make it from red grapes as well. During the fermentation process, white wine grapes are separated from their skins so that no color comes over from the skin, leaving the wine white in color. Expensive white wines are frequently aged in oak barrels while less expensive white wines are soaked in oak chips instead. Some popular white wines include Chardonnay, Gewurztraminer, Pinot Grigio or Pinot Gris, Riesling, Sauvignon Blanc and Semillon. Serve white wine with fish and poultry [25, 28].

Rose or Blush Wine: Wine makers make rose or blush wine from red grapes but the grape skins are only left on for a short time. This causes the wine to turn a pinkish color instead

8 of the deep red of red wines. Popular rose or blush wines are white merlot and white zinfandel. Despite the name white, these are actually rose or blush wines. Serve rose or blush wine with fish or poultry [26, 28].

Champagne: Champagne is a bubbly form of wine that was originally produced in France. Champagne making requires the fermentation process to be interrupted and then restarted. This traps carbon dioxide particles in the bottle. Growers use three kinds of grapes, white Chardonnay, red Pinot Noir and red Pinot Meunier. Champagne can range from very dry (Brut) to dry (Sec) to semi-sweet (Demi-Sec). Serve champagne on special occasions such as holidays or anniversaries [25, 26].

2.4. Chemistry of Wine

Wine has a complex composition of water, alcohols (ethanol and methanol), and a great variety of inorganic and organic compounds such as glycerin, sugars ,glucose, fructose, tartaric, malic, citric, lactic, acetic acid) and volatile acids, flavor compounds (esters, aldehydes, terpenes), phenolic compounds (anthocyanins, tannins), vitamins, minerals (anions and cations) and amino acids [29 and30]. Its composition is influenced from many factors such as grape variety, soil composition and viticultural applications such as fertilizer or pesticide applications, climate, winemaking practices including yeast culture, aging, storage, quality and hygiene of vinery facilities [31].

2.5. Components of Wine

2.5.1. Essential Macro Metals

Metals find their way into alcoholic beverages at different stages and from various sources including the raw materials, type of brewing process, equipment bottling aging/storage and adulteration [32]. The contents of metals in alcoholic beverages vary within a broad range [33]. Vehicle exhaust fumes or other emissions to air, water and soil contain and environment pollution including industrial and urban sewage and air born particulate matter and substances added during brewing such as hops, acids, bases, silica gel, dilution water, flavoring agents, additives, and stabilizers are potential sources of metal ions in the brewing process [34]. 9

Calcium (Ca)

Calcium is important for healthy bones and teeth; helps muscles relax and contract; important in nerve functioning, blood clotting, blood pressure regulation, immune system health. If there is deficiency of Ca, it increases the risk of bone loss. Source; Milk and milk products; canned fish with bones (salmon, sardines); fortified tofu and fortified soy beverage; greens (broccoli, mustard greens); legumes [35],also it seen in wine by different levels that is exceed 60 mg/L for red wines and 80 mg/L for white wines. Knowing the concentration of calcium in wine will give an understanding of the risk of calcium instability. If a wine has calcium levels close to or above those levels, then other factors that may increase pH, such as malolactic fermentation and blending, can also increase the likelihood of calcium L-tartrate precipitation [36].

Potassium (K)

Potassium needed for proper fluid balance, nerve transmission, and muscle contraction, Source; Meats, milk, fresh fruits and vegetables, whole grains, legumes [35] and also wine. Much of the potassium in wine comes from the soil where grape wines are grown. In fact, many grape farmers add potassium to the soil because it can produce a larger crop of wine grapes. A 5-ounce glass Red wine also Merlot contains 187 mg of potassium and white wine and also chardonnay contains 104mg of potassium. Also sweet desert wine contains 135mg of potassium. The daily potassium limit for most healthy adults is 4,700 milligrams per day [10].

Sodium (Na)

Sodium needed for proper fluid balance, for nerve transmission, muscle contraction and the major source of Na is Table salt, soy sauce; large amounts in processed foods; small amounts in milk, breads, vegetables and unprocessed meats [35], Sodium is also found indifferent wines by different levels.4-Ounces of domestic red wine typically contain about 12 mg of sodium, while domestic whites have about 19mg in the same serving. The daily sodium limit for most healthy adults is 2,300 milligrams per day [37].

Magnesium (Mg)

Magnesium is a co-factor for many coenzymes, also affects metabolism of K and Ca and Found in bones; needed for making protein, muscle contraction, nerve transmission, immune system health. Its deficiency is related to high blood pressure, pregnancy problems, and poor heart function. Food sources include vegetables, especially dark green, leafy ones [37]. Also, Wine appears to have a mean Mg content of about 114mg/L [38].

2.5.2. Essential Micro Metals

Iron (Fe)

Iron is a part of hemoglobin, found in red blood cells that carries oxygen in the body; needed for energy metabolism. Its deficiency causes anemia. In addition to Organ and grain source [35], Iron content in wines is an important parameter controlling their quality and stability. The OIV rules do not define limits on the concentration of iron in wines. A major problem that appears in wines is their instability to iron concentrations greater than 10 mg/L. At concentrations greater than 10 mg/L, Fe(III) creates an insoluble suspensions with tannin and phosphates which are known as hazes or “casses” In the present study iron content in wines varied from 0.5 to 7.9 mg/L (ICP- AES data) and 0.4-8.8 mg/L (NAA data). The maximum values were obtained from Cabernet, Pinot Noir and Malbec wines. In these wines the concentration of iron was higher than 5 mg/L [39].

Copper (Cu)

Copper the metal copper is found in old pennies, high-quality plumbing, and electrical wires. In our bodies, copper helps mop up dangerous "free radicals," highly reactive chemicals that have been linked to an increased risk of cancer and heart disease. It also used many enzymes; needed for iron metabolism. Food source, obster, crabs, beans, and nuts, Legumes, nuts and seeds, whole grains, organ meats, drinking water [35].

Manganese (Mn)

Manganese is a part of many enzymes. In our cells, it helps break down fats, carbohydrates, and proteins to convert food into energy. It helps to give Sacajawea dollar coins their shiny, golden luster and amethysts their purple hue. Manganese is present in whole grains especially plant foods and cereal products [35]. Manganese also present in wine naturally. A wine may exceed this arbitrary limit of 2mg/l despite manganese never having been added to the wine [2].

Cobalt (Co)

Cobalt forms the core of vitamin B12 and is important in the body for making red blood cells. It mixed with other metals; cobalt is used in jet engines. It also gives a brilliant blue color to pottery, glass, and tiles. This metal is found in meat, dairy products, and leafy green vegetables [35]. The estimated safe and that required dietary intake of Cr (III) is 0.05 – 0.20mg/l pare day. Chromium is also found in wine that may originated from natural source (soil and grape), Environmental contamination, Fertilizer, Pesticide, Industrial processing and the containers [40].

Zinc (Zn)

Zinc is a part of many enzymes; needed for making protein and genetic material; has a function in (a range of household products, from batteries to cosmetics), taste perception, wound healing, normal fetal development, production of sperm, normal growth and sexual maturation, immune system health, (important for controlling gene activity and regulating hormones.) It is Food source, Meats, fish, poultry, leavened whole grains, vegetables [39]. Also there is Zinc in wines with recommended (by OIV) concentration of 0.2 – 1.3mg/l, that originates from soil, fungicides, insecticides, and vinification equipment. Low zinc concentrations in wines play a vital role during fermentation, whereas high concentrations negatively influence organoleptic properties [39].

Nickel (Ni)

Nickel is widely found in the environmental and released from natural sources such as volcanic emission, soil dust, forest fire, vegetation and sea salt, while it its anthropogenic sources include combustion of diesel, fuel oil and coal, metallurgical operations, electroplating, alloys batteries, coins spark plugs and etc [41] and it is also found in wine as concentrations ranged from 5.4 to 87.9 μg/L in red wine, from 7.5 -74.5 μg/L in white wine, from 19.5 to 24.6 μg/L in rose wine, and from 8.9 to 26.9 μg/L in Champagne in grapes ranged from 4.2 to 94.0 μg/kg. Ni contamination is from, stainless steel storage tanks and its bottles during the storage of wines. Total Ni daily dietary intake for French population from wine consumption was calculated to be 4.37 μg/day/. The daily contribution of Ni from wine in dietary intake may be as high as 7% [42].

Molybdenum (Mo) is Part of some enzymes. Source of Mo: Legumes, breads and grains; leafy greens; leafy, green vegetables, milk, liver. Other trace nutrients known to be essential in tiny amounts include silicon, and vanadium [35].

2.5.3. Toxic Metals

Chromium (Cr- III) is works closely with insulin to regulate blood sugar (glucose) levels, Food source, unrefined foods, especially liver, brewer's yeast, whole grains, nuts, cheeses[35].Also chromium is the essential trace metal found in human body that involved in Lipid metabolism. However, in excessive intake, particularly Cr (VI), it considered to be high toxic. The estimated safe and that required dietary intake of Cr (III) is 0.05 – 0.20mg/l pare day. Chromium is also found in wine that may originated from natural source (soil and grape), Environmental contamination, Fertilizer, Pesticide, Industrial processing and the containers [40].

Metal toxicity or metal poisoning is the toxic effect of certain metals in certain forms and doses on life. Some metals are toxic when they form poisonous soluble compounds. Certain metals have no biological role, i.e. are not essential minerals, or are toxic when in a certain form [43]. In the case of lead, any measurable amount may have negative health effects [44]. Often heavy metals are thought as synonymous, but lighter metals may also

13 be toxic in certain circumstances. Not all heavy metals are particularly toxic, and some are essential, such as iron. The definition may also include trace elements when in abnormally high doses maybe toxic. An option for treatment of metal poisoning may be chelation therapy, which is a technique which involves the administration of chelation agents to remove metals from the body.

Toxic metals sometimes imitate the action of an essential element in the body, interfering with the metabolic process resulting in illness. Many metals, particularly heavy metals are toxic, but some heavy metals are essential, and some, such as bismuth, have a low toxicity. Most often the definition of toxic metals includes at least Cadmium, Manganese, Lead, Mercury and the radioactive metals [45].

Metalloids (arsenic, polonium) may be included in the definition. Radioactive metals have both radiological toxicity and chemical toxicity. Metals in an oxidation state abnormal to the body may also become toxic: Chromium (III) is an essential trace element, but Chromium (VI) is a carcinogen [45].

Toxicity is a function of solubility. Insoluble compounds as well as the metallic forms often exhibit negligible toxicity. The toxicity of any metal depends on its ligands. In some cases, organometallic forms, such as methyl mercury and tetraethyl lead, can be extremely toxic. In other cases, organometallic derivatives are less toxic such as the cobalt ocenium cation [45].

Decontamination for toxic metals is different from organic toxins: because toxic metals are elements, they cannot be destroyed. Toxic metals may be made insoluble or collected, possibly by the aid of chelating agents, or through bioremediation. Alternatively, they can be diluted into a sufficiently large reservoir, such as the sea, because immediate toxicity is a function of concentration rather than amount [45].

Toxic metals can bio accumulates in the body and in the food chain [45]. Therefore, a common characteristic of toxic metals is the chronic nature of their toxicity. This is particularly notable with radioactive heavy metals such as radium, which imitates calcium to the point of being incorporated into human bone, although similar health

14 implications are found in lead or mercury poisoning. The exceptions to this are Barium and Aluminum, which can be removed efficiently by the kidneys [45].

2.5.4. Tannin

Tannin belongs to a class of compounds called phenols and comes from grape skins and seeds; it is mostly found in red wines but can be found in some white wines. It plays an important a role in the aging of wine; therefore high-tannic red wines such as Cabernet, Sauvignon and Nebbiolo are those that can be aged longest. An easy way to understand the effect of tannin is to think of a cup of hot tea in which the tea bag has steeped for too long; the tea will have a very strong, harsh, almost bitter (tannic) flavor that can only be softened by the addition of milk. This same concept applies to wine, that is why cheese and wine is a classic pairing (the protein in cheese neutralizes or balances the tannins in wine) [46].

Tannins are a collective name for colorless but bitter flavonoids. Tannins can be divided into two groups namely the hydrolysable tannins and condensed tannins [4]. Hydrolysable tannins are easily oxidized and thereby decreasing the oxygen availability for the reaction. It also has ability to inhibit the growth of several wood decaying fungi [5]. Whereas condensed tannins does not significantly affect the chemical age and color intensity of the wine but external addition of these tannins increase total phenols of the tannin [6].

Tannins are also known to precipitate protein, inhibit digestive enzyme and affect the utilization of vitamin and minerals [17]. Due to this anti carcinogenic effect of tannins, this work was carried out to know how much percent of tannins are present in various wine samples. Also the legal limit of Tannins in wine accepted in g/l of Gallic acid is; for Red wine not exceeded 3g/l GA and for white wine not exceeded 0.8g/l GA [7]. Sulfur Dioxide is one of the most common additives in wine, you probably refer to Sulfur Dioxide (SO₂) as “Sulfites” Acting as an antioxidant and antibiotic, Sulfur Dioxide is commonly used to preserve the grapes, stabilize the wine and prevent oxidation during the winemaking

15 process. It is also commonly used to help sanitize barrels and other winemaking equipment. [46]

The components of wine provide clues about where the wine was grown and how it was made. They also affect qualities such as taste and mouthfeel, therefore knowing the percent (the amount of parameter of wine in wine is important in order to balance the quality of wine, health care (example the dosage of tannins arise the problems like slow blood clotting, increases blood pressure, increases the serum lipid level, heart and diabetes diseases, brain damage after stroke, makes shortness of breath, makes the tongue and the moth dry out, etc.) [10]. And the appropriate level of sulfur dioxide in wine is used to inhibit or kill unwanted yeast and bacteria and to protect wine from oxidation. Also the dosage of sulfur dioxide in wine, loses the color, loses the test. Generally, juice and wine bacteria are more sensitive to the effect of sulfur dioxide that are yeasts, Acetic acid bacteria vary in their sensitivity to sulfur dioxide, Lactic acid bacteria (LAB) vary in their sensitivity to sulfur dioxide and in the cause of Leuconostoc oenos, levels as low as

10mg/L may be lethal [47]. The legal limit for total SO2 varies from one country to another; 250 ppm [for Red wine 150ppm and for white wine 200ppm] is a commonly accepted value [48].

Although there is no legal limit on the amount of free SO2, levels from 20–40 ppm safeguard the wine without affecting its taste. If the level is below 10 ppm in a white wine it is in danger of going bad [16]. Sulfur dioxide found in the form of free sulfur 2- dioxide that is found in the forms H2SO3, HSO3 and SO3 . Combined sulfur dioxide is found in all other forms, the sugar and other carbon compounds present in wine are able to act as sulfur dioxide binding. SO2 can also bind with phenolic compounds of red wine. Total sulfur dioxide is the sum of the free sulfur dioxide and the combined sulfur dioxide. Also the legal limit of Tannins accepted in g/l of Gallic acid is; for Red wine not exceeded 3g/l GA and for white wine not exceeded 0.8g/l GA [48].

Acids are the most important element in the pulp other than water and sugar, as a grape ripens its sugar content increases and its acid content decreases; the challenge is to harvest precisely when optimal balance is struck. The primary acids in wines ( Malic acid

16 and Tartaric acid) and the other acids in wine ( acetic acid, ascorbic acid, butyric acid citric acid, lactic acid and sorbic acid) are used for maintain chemical stability of wine, influence test and color [28].

Acid balances alcohol and sweetness and sometimes adds a crisp, refreshing sensational may cause our mouth to pucker (like if we were biting into a lemon wedge)Many wines undergo malolactic fermentation, which transforms, hard malic acid into softer lactic acid Grapes grown in cooler regions tend to have higher levels of acidity[28].

Alcohol• is produced during fermentation when yeasts come in contact with the natural grape sugar in the grape pulp. High-alcohol wines are full-bodied with a richer mouthfeel [28].

Alcohol generally has a sweet flavor. A wine with high levels of alcohol sometimes gives-off a hot, burning sensation that you can smell and taste. High levels of alcohol indicate that the grapes were very ripe at harvest [28].

Sugar comes from ripe grapes (although some grape varieties naturally contain more sugar than others). It is mostly converted into alcohol during fermentation. Any remaining sugar is called “residual sugar” [26]

A wine with high levels of residual sugar generally tastes sweet, has a richer mouthfeel and fuller body Grapes grown in warmer climates tend to get riper and contain more sugar Wines with no apparent sweetness (or low levels of sugar) are referred to as “dry” also water is the largest elements of wine that pressed from the grape’s pulp, constitutes the single largest element of wine.

2.6. Review on Determination of Metals and Tannins in Wine

Many of the components or the parameters of wines, which affect its quality, are determined by a number of analytical methods [49]. In 2017 (Justyna Płotka-Wasylkaand et al), suggested the determination of metals in different types of food and beverages samples has drawn significant attention due to several reasons with the most important one being the nutritional and toxic effects of these elements or their compounds. The knowledge of certain elements content in wines/fruit wines is of special interest due to their toxicity in case of excessive intake and also the effect they seem to have on the organoleptic properties of wine [45].

In 2006 (Moutounet and et al), was extracted the level of phenolic compound in wine fruit, by lash release technique [50]. The phenolic compounds are major non-volatile components in wines, which are widely recognized as an essential component of wine quality. They provide color [51] to wine. The phenolic composition of the finished wines depends on the grape and winemaking practices. Polyphenols are unstable compounds, with their reactions starting as soon as the grape is crushed or pressed, continuing throughout winemaking and aging. These reactions may involve interaction or binding with aroma compounds, thus influencing aroma release and perception [52].

White wines are essentially composed of phenols found in flesh of the grapes such as gallic acid, hydroxynammic acid esters, catechin, epicatechin, gallocatechin gallate, and procyanidin and catechin gallate. The content varies and normally found in few mg/L units. On the other hand, red wines are mainly composed of several closely related chemical groups: flavonol-3 group (catechin), flavane (3, 4) diol group (leucocyanidin), flavonol-3 (quercetin), anthocyanins and tannins, in addition to those phenolic compounds found in white wines [53, 54].

Among the phenolic compounds, tannins are of particular importance in red wine. Tannins, the most abundant class of phenolics in grapes contribute to perceived astringency. Astringency is an important attribute in wine influencing consumer acceptability. It is often described as the drying, roughing and puckering feeling [55] attributed to the precipitation of a non-covalent complex between salivary-muco proteins 18 and proline-rich proteins and tannins in the mouth [56 ,57]. The cross-linking of mucoproteins results in its precipitation and consequently, reduction in lubrication and increased friction in the mouth [58] while the proline-rich proteins bind favorably with tannin [59]

The sensory properties of tannins are thought to be dependent upon their structure, molecular size and concentration. Tannins may be bitter and/or astringent. Bitterness is restricted to small molecules with particular structural features enabling them to enter the receptor and activate the signal transduction process, whereas astringency depends on the number of protein interaction sites in the molecule [60]. The low molecular weight flavanol are both bitter and astringent [61]. Tannins become gradually less bitter but more astringent as the molecular weight increases to about 10 units. The perception of astringency as also observed to significantly increase as tannin concentration increased, with a tendency to mask perceived bitterness [62].

Sugars: (Glucose and fructose) account for the sweet taste of some wines with recognition thresholds equivalent to 16 g/L and 9.5 g/L, respectively [53]. Dry wines generally contain less than 1.5 g/L of these residual sugars dominated by fructose, thus are not perceived as sweet. In contrast, ice wines, which are typically produced from Vidal and Riesling grape varieties contain 140 to 280 g/L range of sugar in the finished wines and are sweet and aromatic [63].

Polysaccharides present in wine originate from the primary cell walls of grapes (arabinogalactan-proteins and RG-I and II) and microorganisms such as yeasts (mainly mannoproteins (MP) used in winemaking [64]

The influence of other components such as ethanol, acids, and tannins on the perception of sweetness has been accounted previously. The sweet sensation has a mitigating effect on perceived sourness and bitterness. In addition, sugar concentration was also reported to increase the volatility of aromatic compounds [26]. At the level of sugar in model white wines increased (80 to 250 g/L), approximate equal to that of ice wine, both the perceived density and viscosity increased [65].

Proteins: Protein nitrogen in wine is only about 2% of the total nitrogen content. Almost all amino acids are included in proteins. Most abundant amino acids are alanine, aspartic acid, glutamic acid, glycine, serine and threonine (each between 9-17% of the total amino acids). In general, there is an occasional increase in protein content in wine compared to its must content that is attributable to the yeast during and after fermentation [88]. However, recent investigation revealed that the majority of the polypeptides present in wine originated from grape pulp [66].

The proteins in wine are also beneficial to winemakers, particularly those involved in the production of sparkling and champagne wines owing to their role in foam formation and foam stability [67].

In 2014 (Henock Woldemichael et al), studied on Effects of Variety and Fermentation Time on the Quality of Rice Wine accordingly, the physicochemical analysis of wine prepared from different rice varieties clearly indicated that there was significant difference in the rice varieties with respect to pH. Alcohol is an important parameter to measure wine quality [68].

In one study, the volatile compounds, isoamyl acetate, ethyl hexanoate, n-hexanol and β- ionone were found to have lower activity coefficient in the artificial wine composed of organic acids (0.4% tartaric acid, 0.3% malic acid and 0.01% acetic acid), salts (0.0025% magnesium sulfate and 0.01% potassium sulfate) and 10% ethanol than in water [69]. In another study, decreases of 30-35% in the partition coefficient of volatile compounds were found when ethanol concentration was increased from 5 to 80 mL/L in a model wine (10 g/L glycerol, 1 g/L potassium at pH 3.2)[70].

Generally many analytical methods are used to determine the level of metals in wines sample. This are like AAS(Atomic Absorption Spectroscopy), FAAS (Flame Atomic Absorption Spectroscopy), GFAAS (Graphite Furnace Atomic Absorption Spectroscopy), CV AAS (Cold Vapor Atomic Absorption Spectroscopy), HGAAS (Hydride Generation Atomic Absorption Spectroscopy), NAA (Neutron Activation Analysis), ICP – MS (Inductively Coupled Plasma Mass Spectroscopy) and ICP–OES (Inductively Coupled Plasma – Optical Emissions Spectroscopy) which shown in the 20 following figure 1, is the most important for determination of the concentration of 60 metals in ppm with in (1minute) by low cost than ICP – MS which is very expensive[49].Therefore in this study the determination or Analysis of metals were carried out using ICP – OES techniques, because of its availability and multi metal analysis. Also the titrimetric method was preferred for determine the level of tannin in wine, because of its availability and simplicity

Figure 1: Instrumentation (ICP – OES)

CHAPTER 3: MATERIALS AND METHEDS

3.1. Study Area

The wine samples were collected from Bahir Dar City at different super markets

3.2. Chemicals and Reagents

Chemicals that used in the analysis were all analytical grades.HNO3 65% , (N43725-UNI-

CHEM) and H2O2 30%, (Dallul pharmaceutical PIC, AA.) Were used for the digestion of wine samples, Stock standard solutions containing 1000 mg/L, in 2% HNO 3 , of the metals (K, Mg, Ca, Na, Fe, Zn, Mn, Cu, Cr, Co, Ni, Cd, and Pb) were used for preparation of calibration standards and in the spiking experiments. Working standard solutions were obtained by suitable dilution from stock solution, Potassium permanganate

KMnO4 99.5%, (ALPHA CHEMIKAL, INDIA), Indigo caramine 10%, (BDH Chemical Ldt, Pool England) and activated charcoal 99.9%, (BLULUX LABRATORIES (P) limited, FARIDABAD 121005, India), H2SO4 98%, (Labo Chemic PUC, Ltd, India) were purchased and Distilled water was used to dilution, for preparation of solution and rinsing of apparatus prior to analysis.

3.3. Instruments and Apparatus

A refrigerator (Samsung Hitachi, Japan) was used to keep the wine samples cool till the analysis. 50 ml, 250 mL round bottomed, hot plate to digest the wine samples. Inductively coupled plasma optical Emission spectroscopy (ICP OES) equipped with deuterium ark background corrector was used for analysis of the analyte metals (K, Mg, Ca, Na, Fe, Zn, Mn, Cu, Cr, Co, Ni, Cd, and Pb) using Argon lamb. A micropipette (100 -1000 μl) used for volume measurement of reagents and standards, Conical flask, beakers, burette with standing Iron, water bath to heat the sample, Filtered paper (Whatman NO. 42), electronic balance, Stirrer, different size of measuring cylinders.

3.4. Sampling and Sample Preparation

According to Lazos and Alexakis [71], the samples were prepared as the following figure 2. For this study, six bottled Awash wine brands (Axumite Red Wine, Gouder Red Wine, Awash Tekishino Wine, and Kemila White Wine, Gebeta Red wine and Gebeta white wine) were selected. Two bottles for each brand were collected randomly from six supermarkets at different sites of Bahir Dar city, (to make sample representative). By taking 100 mL from each bottle of the same brand six bulk samples (200 mL each) were prepared. The bulk samples were shaken slightly to ensure mixing.

Figure 2: Sample of Awash wine brands studied

3.4.1. Digestion of the Wine Sample

From the bulk samples (200 mL), 10 mL wine samples were digested to decompose the organic substances and make clear solution in doublet according to previously reported work [72]. The digestion procedure was optimized by taking the amount of

H2O2 and HNO3added to the sample, the digestion time the maximum temperature required, and the color and clearness of the digest in to consideration.

The procedures which consumed least time at fixed temperature for the digestion, giving clear solution with no precipitate were considered as the optimum procedure. Based on these parameters, different optimization results for wine samples were obtained. Smaller time than the optimum parameters resulted in incomplete digestion of samples which were indicated by colored and unclear digest with precipitates. The optimized digestion procedures are indicated in Table 1.

The cooled digest was filtered and transferred quantitatively to 50 mL volumetric flask and diluted to the volume with distilled water. The digest was being kept in the refrigerator until the analysis by ICP-OES. Two Blank samples (used to calibrate the performance of instrument), were prepared the same way as sample digestion.

Figure 3: The digested sample ready for ICP – OES Table 1: Optimized duration of time for digestion of sample

Sample Ratio of Temp Time consumed to form clear solution (in minutes) volume Reagents in . (in and remark (in color formation) (in ml) (7ml) Oc) H2O2 HNO3 50 65 70 90

10 6 1 220 Yellowish Yellowis Yellowish Yellowish clear h clear clear clear

10 5 2 220 Yellowish Yellowis Nearly Clear with clear h clear clear no ppt

10 4 3 220 Yellowish Yellowis Clear with - clear h clear no ppt

10 3 4 220 Clear with - - - no ppt

3.5. Determination of the Metals in Wine Samples by ICP-OES

ICP – OES is one of the most popular analytical instrument and mainly used for determination of the level of almost all elements of the periodic table in sample. The technique has a wide dynamic concentration range and can measure a trace to high concentration. Also it has a better detection limits, free from chemical matrix effect, and multi element capability [73].

In this study metals were determined by ICP using an Argon lamb. Potassium (K) was out of the range of the prepared standard solution, this may caused due to the contamination, lack of good calibration of instrumentation. Therefore further diluting the digested wine samples were conducted, to bring the concentration within the linear range of the calibration. Stock standard solutions containing 1000 mg/L, in 2% HNO3, of the metals Na, K, Ca, Mg, Mn, Fe, Cu, Zn, Ni, Co, Pb, Cd, and Cr were used for the preparation of calibration standards and in the spiking experiments. For the preparation of calibration curve, a series of six working standards were prepared for each metal.

3.6. Determination of Tannin

A 0.004 M KMnO4 solution was prepared by dissolving 0.158 g of KMnO4 in 100 mL of distilled water and then it was diluted up to 250 mL with distilled water. The indicator indigo caramine (0.5%) was prepared by dissolving 0.5 g of indigo caramine in 60 mL of warm distilled water. The solution was cooled to room temperature, 4 mL of concentrated sulfuric acid was added and then it was diluted up to 100 mL with distilled water. This solution was filtered by Whatman filter paper number 42.

According to Mulani et al [74], the determination of tannin was conducted. 5 mL of wine sample was pipetted out in a conical flask and 10 mL of distilled water was added. This solution was heated on water bath until volume was reduced to 5 mL. To this conical

25 flask, 10 mL of distilled water and 2 mL of 0.5% indigo caramine indicator was added.

The above solution was titrated with a 0.004 M KMnO4 solution until the end point

(golden yellow color) was observed. The consumed volume of KMnO4 was recorded as back titration reading (A). Blank titration was carried out with 25 mL of wine sample and 1 g of charcoal, stirring thoroughly and it was kept aside for 15 minutes and then followed by filtration. 5 mL of above decolorized wine sample was pipetted out in conical flask and continued the procedure as applied for back titration. The consumed volume of KMnO4 was recorded as blank titration reading (B).

Then the amount of potassium permanganate used in oxidation of tannins is calculated as

A- B=C mL. The standard tannin solution for which 1 mL of 0.004 M KMnO4 = 0.0832 mg of tannin. Therefore, percent of tannins in wine was =0.01664×C.

A B

A= Titrating the sample B= after titration Golden Yellow observed (end point)

Figure 4: when titration was conducted

3.7. Instrumental Calibration

The technique consists of plotting the instrument responses against the series of standard working concentration [75].By convection the calibration on graph is plotted with the concentration of the standard on X –axis and the reading of the instrument response (Intensity) on the Y – axis. So the suitable working range for standards must be selected. The proper way of constructing the graph is essentially the performance of a regression analysis, i.e. the statistical establishment of a linear relationship between concentration of the analyte and intensity read by ICP – OES. This regression analysis (y = a + bx), 26 intensity Y, and concentration X, yields correlation r as measure for the fit of the point to a straight line (by means of least square) [75]. The r, correlation with > 0.99, indicates that the strong relationship between intensity and concentration. In linear calibration curve, y = a + bx, a; is the intercept and zero is the expected value, but because of interaction, interference, noise, contamination and other bias this is seldom the case; measures for the sensitivity of the procedure , steeper the slope the more sensitive procedure (the strong response instrument) [75].

The quality of result obtained using ICP-OES is affected by the calibration and the process of preparing standard solution. The instrument was calibrated using six working standard that relates standard concentration, correlation coefficient values of calibration carve (graph) for each elements as the following fig 5(a-m).

Table 2: Concentration of intermediate Standards (mg/L), Concentration of Working Standard (mg/L) and Correlation coefficient of calibration carve

Metals Concentration of intermediate Concentration of Working Standards (mg/L) Standard(mg/L) K 75 0.01, 15, 30, 45, 60, 75 Na 75 0.01, 15, 30, 45, 60, 75 Ca 75 0.01, 15, 30, 45, 60, 75 Mg 75 0.01, 15, 30, 45, 60, 75 Cu 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Zn 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Mn 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Ni 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Fe 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Co 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Cr 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Cd 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Pb 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05

40 POTASIUM (K) CALCIUM (Ca) y = 351665.04426 +449851.17607 * X 0.3 30 Y =2428.95415+3844.7831*x R = 0.99924 R = 0.99523 0.2 20

0.1 10

Intensity Intensity (M)

Intensity (M) Intensity 0 0.0

0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 concentration (mg/l) Concentration (mg/L)

(a) (b)

0.6 SODIUM (Na) MAGNESIUM (Mg) Y =-7859.78825 + 8295.1725 * X 100 Y = 879992.11141 +1.32223E6 *x R = 0.9984 R = 0.9965 0.4

Intensity Intensity (M) 0.2

Intensity (M) Intensity

0 0.0

0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Concentration (mg/L) Concentration (mg/L)

(c ) (d)

ZINC (Zn) 0.4 4 Y = 2987.09267 + 74673.48 * X COPPER (Cu) R = 0.99988 3 Y = -24261.51929 + 715469.24286X 0.3 R = 0.99986

2 0.2

Intensity (M) Intensity Intensity (M) Intensity 0.1

0 0.0 0.0 1.5 3.0 4.5 6.0 0.0 1.5 3.0 4.5 6.0 Concentration (mg/L) Concentration (mg/L)

(e) (f)

IRON (Fe) Y = 5956.2809 + 372545.33429 * x 0.8 NICKEL (Ni) 2.0 R = 0.99993 Y = 1572.34486 + 149778.53143 * X R = 0.99995 1.5 0.6

1.0 0.4

Intensity (M) Intensity Intensity (M) Intensity 0.5 0.2

0.0 0.0

0.0 1.5 3.0 4.5 6.0 0.0 1.5 3.0 4.5 6.0 Concentration (mg/L) Concentration (mg/L)

(g) (h)

20 MANGANESE (Mn) 3 Y = -73993.64529 + 3.80703E6 * X CHROMIUM (Cr) Y = 13849.93976 + 476969.58571 * X 15 R = 0.9999 2 R = 0.99964

10 1

Intensity (M) Intensity Intensity(M) 5

0 0

0 2 4 6 0 2 4 6 Concentration (mg/L) Concentration (mg/L)

(i) (j)

LEAD (Pb) 0.10 Y = 732.74329 + 19856.27714 * X CADMIUM (Cd) R = 0.99985 Y = 5032.478 + 294363.0*X 1.4 R = 0.999

0.05 0.7

Intensity (M)

0.00 (M) Intensity 0.0

0.0 1.5 3.0 4.5 6.0 0.0 1.5 3.0 4.5 6.0 Concentration (mg/L) Concentration (mg/L)

(k) (l)

COBALT (Co) 0.8 Y = 1586.1803 + 166156.58*X R =0.9998

0.4

Intensity(M) 0.0

0 2 4 6 Concentration (mg/L))

(m)

Figure 5: Plot of calibration curve (a-m); K, ca, Na, Mg, Zn, Cu, Fe, Ni, Mn, Cr, Pb and

Cd respectively), Emission intensity Vs concentration for the studied metals

3.8 Method Validation

In this study, the validation methods used were Repeatability, Limit of detection, Limit of quantification and the Present Recovery that were listed below.

3.8.1. Repeatability It is a term used when independent test results are obtained with the same method on identical test items in the same laboratory by the same operator using the same equipment within short intervals of time.

In this paper, it is the repeatability that was determined in terms of precision (% relative standard deviation (% RSD, which is calculated as the ratio of standard deviation of sample to the mean concentration of the sample multiplied by 100) [75].

3.8.2. Limit of Detection (LOD), Limit of Quantification (LOQ) and Instrumental Detection Limit (IDL)

LOD is the lowest concentration of analyte that can be detected and reliably distinguished from zero (or the noise level of the system), but not necessarily quantified; the concentration at which a measured value is larger than the uncertainty associated with it.

LOD can be expressed in response units and is taken typically as three times the noise level for techniques or three times the standard deviation of the sample blank.[MDL or LOD = 3 times (SD of blank reagent)][76].

퐿푂퐷 = 3푆퐷

LOD (Limit of detection is lowest concentration of the measured can be detected at specific level of confidence and it is the concentration at which we can decide whether an element is present or not, but it do not reported [76] Limit of Quantification (LOQ) is lowest concentration at which the performance of a method or a measurement system is acceptable for specific use and this concentration is reported. It is calculated as LOQ = 10 times (SD of blank reagent) [76]

퐿푂푄 = 10푆퐷

The standard deviation of the blank samples, for each element was calculated from the measured blank samples. Most common protocol for estimation of LOD and LOQ including the ISO – standards assume that the instrument output at low concentrations can result in negative readings concentrations [77]. Instrumental Detection Limits do not establish the limits, but used to compare the calculated IDLs with LOQ. It can be calculated as the mean of Blank sample plus three times standard deviation of the blank reagent [78].

3.8. 3. Recovery Test

Recovery shows that, the accuracy of the analytical procedure ,by spiking a suitable known amount of the analyte metals into a test portion of the sample having a known concentration of the analyte.( the value of % Recovery of the studied metals were given in the following table 3)

푠푝푖푘푒푑 푠푎푚푝푙푒 − 푢푛 푠푝푖푘푒푑 % 푅푒푐표푣푒푟푦 = 푥100 푎푑푑푒푑 푐표푛푐푒푛푡푟푎푡푖표푛

Table 3: Recovery test for the studied metals Metals Concentration in Added amount Concentration in % Recovery un spiked (mg/L in (mg/L) spiked (mg/L) K 62.95 3 65.8 95.00 Na 19.83 3 22.72 96.33 Ca 9.3 3 12.35 101.67 Mg 11.08 3 13.97 96.33 Cu 0.056 0.5 0.571 103.00 Zn 0.14 1.5 1.489 89.93 Mn 0.146 1.5 1.451 87.00 Ni 0.065 0.5 0.578 102.60 Fe 0.52 1.5 1.976 97.07 Co -0.0011 0.25 0.222 89.24 Cr -0.012 0.25 0.221 93.20 Cd -0.015 0.25 0.218 93.20 Pb -0.171 0.25 0.054 90.00

Table 4: The MDL, LOD, LOQ, IDL, and Correlation Coefficients of calibration curve (R2) were given as the following table.

Metals Wave MDL LOQ IDL Correlation Length coefficient of calibration curve (R2) K 766.490 0.0441 0.147 0.9952 Na 589.592 0.5484 1.828 0.0690 0.9984 Ca 317.933 0.5131 1.710 0.01 0.9992 Mg 285.213 0.1627 0.542 0.0016 0.9965 Cu 327.393 0.0443 0.148 0.0097 0.9998 Zn 206.200 0.0009 0.003 0.0059 0.9998 Mn 257.610 0.0571 0.190 0.0014 0.9999 Ni 231.604 0.0003 0.001 0.0150 0.9999 Fe 238.204 0.0811 0.270 0.0046 0.9999 Co 228.616 0.0003 0.001 0.0070 0.9999 Cr 267.716 0.0003 0.001 0.0071 0.9996 Cd 228.802 0.003 0.01 0.0027 0.9998 Pb 220.353 0.0009 0.003 0.042 0.9998

3.9. Statistical analysis

All data obtained from the experiment were subjected to one-way Analysis of Variance (ANOVA) using IMB SPSS statistical Version 20 software to compare mean results between wine sample by least significance difference test at (p<0.05) significance level.

CHAPTER 4: RESULTS AND DISCUSSION

4.1. Levels of Metals in Wine Samples

The concentrations of metals were determined by ICP - OES in the wine samples after digested and diluted with deionizer water. The results are summarized in table 5. Although most metals were in quantifiable level, Cd in all sample was not detected, Cr in Gouder Red and Pb in three samples of Wine were below detection limit.

Table 3: Level of metals in Awash brand of wine

Awash brand wines Metal Gebata Red wine Gebata White wine Kemila white wine Axumite red wine Awash Tekishino Gouder Red Wine Red Wine Concentration Concentration Concentration Concentration Concentration Concentration (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) K 906.9±2.64 661.5±2.31 699.1±0.504 881.2±1.33 163.1±0.08 281.97±0.14

Na 49.75±0.41 126.49±0.157 90.2±0.036 139.05±0.33 79.45±0.15 64.15±0.053

Ca 74.35±0.94 49 ±0.286 62.3±0.075 69.9±0.045 36.29±0.0314 35.925±0.0177 Mg 55.425±0.44 55.425 ±0.44 54.95±0.079 65.3±0.116 14.935±0.0184 22.535±0.0056 Cu 1.5±0.0015 0.25±0.001 0.225±0.0001 0.3±0.0005 0.555±0.111 0.23±0.0005 Zn 2.35±0.177 1.14±0.0016 1.2±0.00047 3.2±0.0005 1.2±0.00047 1.03±0.0005 Mn 1.545±0.006 1.63±0.0192 1.565±0.004 1.15±0.0008 0.205±0.041 0.32±0.064 Ni 0.611±0.077 0.25±0.002 0.2±0.00047 5.15±0.074 0.25±0.0001 0.16±0.0028 Fe 4.095±0.0164 3.7±0.025 2.6±0.0017 0.03±0.0038 2.46±0.0013 1.8±0.00047 Co 0.035±0.001 0.045±0.001 0.0015±0.0005 0.03±0.0001 0.025±0.0005 0.00335±0.0005 Cr 0.0825±0.0075 0.065±0.001 1.565±0.004 0.03±0.0014 0.105 ND Cd ND ND ND ND ND ND Pb 0.45±0.002 0.25±0.027 ND ND 0.05±0.0022 ND

4.1.1. The level of Major metals (K, Na, Ca, and Mg) in each wine Samples

The summary of mean concentrations for K, Na, Ca, and Mg in different types of wine presented in Table 5 and Figure 6, The ANOVA (IBM SPSS, Statistics version 20) results displayed in table 6

Table 4: Level of K, Na, Ca and Mg in Awash brand wine

Awash brand wines Gebata Red Gebata White Kemila white Axumite red Awash Gouder Red wine wine wine wine Tekishino Red Wine Wine

Concentration Concentration Concentration Concentration Concentration Concentration

Metal (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

K 906.9±2.64 661.5±2.31Ns 699.1±0.504 Ns 881.2±1.33 163.1±0.08 281.97±0.14

Na 49.75±0.41 126.49±0.157 90.2±0.036 139.05±0.33 79.45±0.15 64.15±0.053

Ca 74.35±0.94 49 ±0.286 62.3±0.075 69.9±0.045 36.29±0.0314Ns 35.925±0.018 Ns Mg 55.425±0.44Ns 55.425 ±0.44Ns 54.95±0.079Ns 65.3±0.116 14.935±0.0184 22.535±0.006

NS- show that Non significant at (p>0.05),

Potassium: the K availability is in excess, in association with organic acids, K changes qualitative factors of wine [79]. The level of potassium in wine is affected by grapes variety, soil and climatic conditions, time of harvest and other variables such as the temperature of fermentation and storage, the pH. So its levels are different according to the type of wine and their geographical origin [80].

The six wines of Awash brand [Gebeta White (GEW), Gebeta Red (GER), Kemila White (KW), Axumite Red (AXR), Awash Tekishino Red (ATR) and Gouder Red (GR)] contains the main portion of the four major elements (K, Na, Ca, and Mg), with the average value of concentration in mg/L: K (598.98), Na (91.52), Ca (54.63) and Mg (44.76). Compared to other wine, Gebeta red showed the highest content followed by Axumite ,Kemila ,Gebeta white ,Gouder red and Awash tekesheno respectively. 37

Therefore, Potassium is the highest and Sodium is the second concentrated metals in Ethiopian Awash brand wines, from all of the four major metals.

The range of potassium in these wines had similar interval to that reported for Ethiopian, Czech, Italy and German. However, the level of potassium in this study had lower level of K compared to Polish wines [80] this indicates that, Ethiopian grape and soils are rich in Potassium.

Comparing the mean of all the six brands for their Potassium (K) contents at the 95% confident level, were significantly different (P < 0.05). But the comparison of K content in between Gebeta white – Kemila white and Gebeta Red – Axumite Red were not significant different (P > 0.05). This showed that, the K content of Gebeta red was significantly higher than Axumite Red. Also the K level of Awash Tekishino was significantly lowest level.

Sodium: The concentrations of Na in these wines ranged from 49.75 to 139 mg/L (Table 6). The highest mean concentration of Na was observed in Axumite red wine followed by Gebata White wine. The OIV acceptable limit of Na in wine is set at 60 mg/L [81] which indicate that, except Gebata Red wine all wine in this study show higher level of sodium. In Ethiopia, Na concentrations of 24– 25 mg/L were reported in wine [79] Sodium concentrations of 0.0053- 3.82 mg/L have been reported for Polish wines. The concentrations of Na in this study are differing with the wine data reported in the literature. Therefore, further analysis with other instrument like flame photometry and atomic absorption might be need before concluding the results.

The Na concentration of all wine brands have significantly different mean value (P < 0.05), which indicated the content of Na in all wine brand significantly different. Na content in Axumite Red is significantly higher and least in Gebeta Red.

Calcium: The mean concentrations of Ca in the wine varied from 35.93 to 74.35 mg/L (Table 5). The highest mean level of Ca was observed in Gebata Red wine and lowest in Gouder Red Wine. Woldemariam and Chandravanshi [80] reported Ca levels ranging from 58.1 -79.2 mg/L in Ethiopian wine and 26.22 to 98.02 mg/L concentration have been reported for Turkey wine [79]. The calcium content in present study agrees with 38 previously reported work. The One way ANOVA (IBM SPSS Statistics Version 20) indicated the existence of significantly different of the Calcium (Ca) level in Ethiopian wine brand (P < 0.05). But the comparison of Ca content Awash Tekishino and Gouder Red were not significantly different (P > 0.05), which indicated that, the level of Ca in Awash Tekishino and Gouder Red have similar amount. The concentration of Ca in Gebeta Red was significantly higher, but least in Gouder Red.

Magnesium: Magnesium content in wines correlates with the natural Mg content of grape berries. Magnesium deficiency in organisms may lead to serious biochemical and symptomatic changes. This element is involved in more than 300 essential metabolic reactions [82].Mg level in the study range from 14.935 to 65.3 mg/L (Table 6) the concentration were similar to those found in other types of wine in the literature [79,80,83]. The concentrations of Mg in these wine types varied considerably (P < 0.05). But the comparison of the Mg level in between Gebeta white – Kemila white and Gebeta white – Gebeta Red were not significant different (P >0.05).

1000 POTASIUM (k) 900 k SODIUM (Na) 800 150 700 600 Na 500 100 400 300 50 200

100 Concentration(mg/l)

0 0 concentration(mg/L) AWASH BRAND WINES AWASH BRAND WINES

Magnsium(Mg) Calcium (Ca) 80 70 70 Ca Mg 60 60 50 50 40 40 30 20 30 10 20 concentration(mg/L) 0 10

concentration(mg/L) 0

AWASH BRAND WINES AWASH BRAND WINES

1000 k K, Na, Ca and Mg Na 800 Ca 600 Mg

400

concentration(mg/L) 200

AWASH BRNAD WINES

Figure 6: comparison of major metals (K, Na, Ca and Mg) in Ethiopian Awash Wines 40

4.1.2. Level of Minor Metals (Fe, Cu, Zn, Mn, Co and Ni)

There are certain metals which are very important for humans and other animals, because they play an essential role in diverse processes. The fermentation process is influenced by Fe, Cu, Zn and Mn; these metals are also the part of important anti-oxidant process that prevent spoilage in wine [84]. The comparison of the four metals in the wines analyzed is shown in a Table 7 and Figure 7

Table 5: Level of Fe, Cu, Zn, Mn, Ni and Co in Awash brand wine

Awash brand wines

Gebata Red Gebata White Kemila white Axumite red Awash Gouder Red Wine wine wine wine wine Tekishino Red Wine

Concentration Concentration Concentration Concentration Concentration Concentration

Metal (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Cu 1.5±0.0015 0.25±0.001 0.225±0.0001Ns 0.3±0.0005 0.555±0.111 0.23±0.0005Ns Zn 2.35±0.177 1.14±0.0016* Ns 1.2±0.00047 Ns 3.2±0.0005 1.2±0.00047 Ns 1.03±0.0005* Ns Mn 1.545±0.006Ns 1.63±0.0192Ns 1.565±0.004Ns 1.15±0.0008 0.205±0.041 0.32± 0.064 Ni 0.611±0.077 0.25±0.002 0.2±0.00047 5.15±0.074 0.25±0.0001 0.16±0.0028 Fe 4.095±0.0164 3.7±0.025 2.6±0.0017 Ns 0.03±0.0038 2.46±0.0013Ns 1.8±0.00047 Co 0.035±0.001 Ns 0.045±0.001 0.0015±0.0005*Ns 0.03±0.0001Ns 0.025±0.0005 Ns 0.00335±0.0005*Ns

NS- show that Non significant at (p>0.05),

Copper: is a component of more than 30 enzymes in the human body. Copper is a major and much-needed component for human, it can also become toxic to living cells if present in higher concentrations [85].Previous work reported the concentration of Cu, Fe and Zn in deferent selected wine samples produced in different country. In the Ethiopian Awash brand wines, the concentration of Copper is from 0.2 -1.5 mg/L. Gebeta red has more level of Cu with 1.5mg/L, and Kemila wine with the lowest level of 0.2 mg/L and the rest Gebeta White, Gouder Red, Axumite Red and Awash Tekishino contain almost the same level of Cu. Their order was, Gebeta Red (1.5 mg/L), Awash Tekishino (0.56 mg/L), Axumite Red (0.3 mg/L), Gebeta white (0.25 mg/L), Gouder Red (0.23 mg/L) and

Kemila whit (0.225 mg/L) (Table 7). So copper in Gebeta red is slightly higher than recommended value that set by OIV [81] which is 1mg/L. The presence of copper (Cu) is known to impact on the development of wine in bottle by influencing the oxidative or reductive evolution of the wine, and/or its turbidity [86]. Therefore, the measurement of concentration of Cu is important to maintain wine quality. One way ANOVA result for Copper (Cu) indicated that, the mean values of the six wine brands were significantly different (P < 0.05) however, the comparison of Cu level in between Kemila white and Gouder Red showed no significantly different (P > 0.05). In Gebeta Red, the level of Cu was significantly the highest.

Iron: The iron content in wines originates from either grapes or contamination during the production process, transportation and/ or storage when the wine comes in contact with iron containing alloys. Iron can accelerate oxidation and at low concentrations this may be a desired effect which can help the wine develop new sensory characteristics. However, Iron can also form complexes with tannins and phosphates, which leads to haze formation, known as “iron casse”. [85]. Iron concentration in wine may vary because of conditions includes the stage of maturity of the grape, the soil type, soil pH and chemical residues in the soil, climate changes. According to Danijela et al [87] the concentrations of Fe vary from 2.93 to 36.2 mg/L. In the present study, the concentration of Iron range from 0.03 to 4.095 mg/L and it is the most abundant trace metal in Awash brand (Table 7). Gebeta red has highest level of Fe with 4.095 mg/L, and Axumite red wine with the least level of 0.03 mg/L. The decreasing orders of their concentration were Gebeta Red (4.095 ppm), Gebeta white (3.7 ppm), Kemila white (2.6 ppm), Awash Tekishino (2.46), Gouder Red (1.8 ppm) and Axumite Red (0.03). Generally the concentration of Fe in Ethiopian wine is in the range of the legal limit set by OIV. The mean of Iron (Fe) values statistically, analysis for their significance difference, which was significantly different in all six wine brands (P < 0.05), but no significant different in between Awash Tekishino and Kemila White (P > 0.05). Thus there was significantly equal level of Fe in both wines. The level of Fe in Gebeta white was significantly the highest, but it was the least in Axumite wine.

Zinc: Zinc, which is a trace element naturally found in soil, plays a key role in the growth of plants. The values by OIV range from 0.2 to 1.3 mg/l, low zinc concentrations in wines play a vital role during fermentation, whereas high concentrations negatively influence organoleptic properties [39].M.Banovic et al [88] reported that, the level of Zn in Croatian wine 0.266 – 2.434 mg/L. In this study, the concentration of Zinc in Awsh brand Wine was found in the range of 1.03 mg/L to 3.2 mg/L (Table 6), the highest level of Zn found in Axumite wine, but lowest level of Zn is in Gouder Red. The order of Zn in all six wines were, Zn in Axumite (3.2 mg/L) > Gebeta Red (2.35 mg/L) > Kemila and Awash Tekishino (1.2 mg/L) > Gebeta White (1.24 mg/L) > Gouder (1.03 mg/L). Zn is the second most abundant trace metals in Ethiopian wine next to Fe.

The mean values of Zinc (Zn) comparing in between Gebeta White and Awash Tekishino, Kemila white, Gouder Red were not significant different (P > 0.05). Therefore, the level of Zn in all wine brands, except in Gebeta Red and Axumite Red, were significantly equal. Axumite Red has significantly highest level of Zn

Manganese: is one of trace metal that used in function of enzyme, different researchers were reports the level of Mn in wines of different countries, R. Stobbaerts, et al, [89] determined the value of Mn in Europe wines by using Electro thermal Atomic Absorption Spectroscopy (ET-AAS) and the reported value was from 0.5 to7.3 mg/L, In the present study the Ethiopian wine contain Mn in range of 0.205 – 1.63 mg/L (Table 6) which is similar with the previous value (1.04 mg/L and 1.88 mg/L ) reported by Woldemariam and Chandravanshi [80] , Gebeta white has more Mn level than all six Awash brand wines, and Gouder Red wine were the lest content of Mn. Thus, Mn in Gebeta White (1.63 ppm) >Kemila white (1.565 ppm) Gebeta Red (1.545 ppm)>Axumite Red (1.15 ppm) > Gouder Red (Awash Tekishino (0.205 ppm), Mn is the firs most abundant trace metal in Ethiopian white Wine.

Nickel: Total Ni daily dietary intake for the daily contribution of Ni from wine in dietary intake may be as high as 7%. It was reported that Ni in wine ranged from 5.4 to 87.9 μg/L in red wine, from 7.5 -74.5 μg/L in white wine, from 19.5 to 24.6 μg/L in rose wine, and from 8.9 to 26.9 μg/L in Champagne in grapes ranged from 4.2 to 94.0 μg/kg. Ni

43 contamination is from, stainless steel storage tanks and its bottles during the storage of wines. [42].

In this study Ni concentration range from 0.16 to 5.15 mg/L, Axumite wine has the highest level of Ni and Gebeta Red found the second with 0.16 mg/L. So Axumite Red wine may stay along time in maceration storage that leads contact with the barrel made from stainless still. Ni in Gebeta white and Awash Tekishino contain equal level which is ranked the third with (0.25 mg/L) and Kemila white and Gouder Red is the fourth and the last with 0.2 and 0.16mg/L respectively. The two wine brand (Gebeta Red wine and Gebeta White wine) show higher value compared to previous work reported by [80] which was 0.18 – 0.2 mg/L.

Cobalt: Cobalt is beneficial for human, because of it is part of Vitamin B12 which is essential to maintain human healthy. The average human Adult requires 0.0001 mg/day of Co. The high levels of Cobalt in wine can results heart, kidney and Liver problems [90].

Ethiopian wine (Awash brand) has some level of Cobalt that pronounced in Gebeta white (0.045 mg/L) and some in Gebeta red and Axumite white with 0.035 and 0.03 mg/L respectively.

The mean concentrations of Ni, and Co in most wine brands were statistically not significant (P > 0.05).

Iron (Fe) Zinc (Zn) 4.5 3.5 3 4 Zn 3.5 Fe 2.5 3 2 2.5 2 1.5

1.5 1 concentration(mg/L)

concentration(mg/L) 1 0.5 0.5 0 0

AWASH BRAND WINES AWASH BRAND WINES

Manganese (Mn) 2 Copper (Cu) 2

1.5 Cu 1.5 Mn 1 1

0.5 0.5

concentration(mg/L) concentration (mg/L) 0 0

AWASH BRAND WINES AWASH BRAND WINES

4.5 4.5 Cu Gb w 4 4 Zn Gb R 3.5 3.5 3 Mn Aw TK 3 2.5 Fe Ax R 2.5 2 Km W 1.5 2 Gu R

1 1.5 Concentration(mg/L)

0.5 (mg/L) concentration 1 0 0.5 Gb w Gb R Aw Ax R Km Gu R TK W 0 Fe Zn Mn Cu Awash brand wine Trace metals

Figure 7: comparison of trace metals (Fe, Cu, Zn and Mn) in Ethiopian Wines.

4.1.3. The Levels of Toxic Metals (Cr, Pb and Cd)

Increasing heavy Metals in wine may result from long maceration time, long contact time with metal containing equipment [91]. Similar to other metals for example, high levels of Cobalt in wine can results heart, kidney and Liver problems [92], others also have effect either in low or in dosage. The comparison of the three toxic metals in the analyzed wines is shown in Figure 8 and Table 8

Table 6: Level Cr, Co and Pb in Awash brand wine

Awash brand wines

Gebata Red wine Gebata White Kemila Axumite red Awash Tekishino Gouder Red wine white wine wine Red Wine Wine

Concentration Concentration Concentratio Concentration Concentration Concentratio

Metal (mg/L) (mg/L) n (mg/L) (mg/L) (mg/L) n (mg/L)

Cr 0.0825±0.0075 0.065±0.001 1.565±0.004 0.03±0.0014 0.105 ND Cd ND ND ND ND ND ND

Pb 0.45±0.002 0.25±0.027 ND ND 0.05±0.0022 ND

Chromium: The average human Adult requires 0.005 mg/day of Chromium [93]. Chromium is one of the essential trace metal in human body and use in metabolism of glucose and some lipids (cholesterol), But the excessive intake of Cr, particularly Cr(VI) is toxic and arise pathogenesis disease such as lung and gastro intestinal cancer [94]. The study by Pieter et al [94] reported that, the level of different wine sample of France contains 0.008 – 0.019 mg/L.

In this study, chromium was not detected in Gouder red, but concentrated in Kemila White with level of 1.565 mg/L and least in Axumite Red wine. Cr found to be in the range of ND – 1.565 mg/L. Kemila white wine has more Cr than Co, but Gouder Red has equal concentration of Cr and Co, which is ND (Table 8).

Cadmium: According to The OIV the level of Cd in wine is 0.01 mg/L. But in Ethiopian wine the level of Cd is below detection limit and not detected in six examined wine.

Lead: There are a number of sources of Pb in wine, such as Soil composition, fertilizers, Industrial emission, the winery equipments [95], and is toxic metal even in small amount. The level of Pb results various health effects, such as hematological, nervous, renal and reproductive system problems. According to the International Organization of Vine the recommended level of Pb in wine is 0.15 mg/l [95]

The concentration of Pb in this study was found in the range ND – 0.45 mg/L (Table 8) and was not detected in Axumite, Kemila and Gouder Red wines. Pb is more concentrated in Gebeta red and the second leveled in Gebeta white 0.45 mg/L and 0.25 mg/L respectively and this value are above the OIV. This may results due to the growth/ the origins of the raw material for wine production, or water used for wine production or contamination. Value higher than the limit set by OIV Where also reported previously in Ethiopian wine [80] This result indicate the wine producers should be more cautious about the content of this metal in their wines. The level of Pb in Awash Tekishino (0.05 mg/L) is below the value of OIV, Gebeta Red wine has more Pb than Cr, but Awash Tekishino contains less Pb than Cr (Table 8).

While Lead show significant difference (P < 0.05) in Ethiopian wine brand, but not detected in Axumite Red, Kemila white and Gouder Red whereas Co not significantly different (P > 0.05) in Ethiopian wine brands. Cd and Cr were below detection limit in most wine brands.

5 Gb w Gb R Aw TK 4 Ax R Km W 3

Gu R concen. (mg/L) 2

0 Ni Co Cr Cd Pb

Trace and heavy metals

Figure 8: The comparisons of both trace and heavy metals in Ethiopian wine brand

4.1.4. Comparisons of the Levels of Metals in Each Ethiopian Wines

4.1.4.1. Gebata Red Wine

In Awash brand of Gebeta Red wine, K is the first ranked and Ca, Mg, Na were the second, third and the fourth respectively. Oppositely Co is the least ranked and Cd is not detected in all six Awash brand wines. The metals in Gebeta red wine was in the order :K > (906.9) > Ca(74.35) > Mg(55.425) > Na(49.75) > Fe(4.095) > Zn(2.35) > Mn(1.545) > Cu(1.5) > Ni (0.611) > Pb (0.45) > Cr(0.0825) > Co(0.035).

4.1.4.2. Gebata White Wine

Gebeta white wine contains more concentrated K and Na, Ca, Mg were followed, but Co is the least concentrated, Cd is not detected. Na is the second ranking in this wine which is ranked as K(661.5) > Na(126.49) > Mg(55.425) > Ca(49.0) > Fe(3.5) > Mn(1.63) > Zn(1.14) > Cu = Ni = Pb (0.25) > Cr(0.065) > Co (0.045) in mg/L.

4.1.4.3. Kemila White Wine

In this study, Kemila white wine has the same ranked of essential (major) elements as Gebeta White, but K and Ca more concentrated in Kemila than Gebeta White. Cobalt is the least amount in Kemila white and Cd and Pb were not detected in Kemila white. The decreasing order of metals in Kemila white wine in mg/L is give as;

K(699.1)>Na(90.2)>Ca(62.3)>Mg(54.95)>Fe(2.6)>Mn(1.565)=Cr(1.565)>Zn(1.2)>Cu(0. 225)>Ni(0.2)>Co(0.0015)

4.1.4.4. Axumite Red Wine

From the six Awash brand wine, Ni was the more concentrated in Axumite Red wine than the left wines. But Fe, Co and Cr were the least and like in Kemila white wine, Cd and Pb are not detected. The order of metals that examined in this study were distributed in Axumite Red measured by mg/L is;

K(881.2)>Na (139.05)>Ca(69.9)>Mg(65.3)>Ni(5.15)> Zn(3.2)> Mn(1.15)>Cu(0.3)>Fe(0.03) = Co(0.03) = Cr(0.03)

4.1.4. 5. Awash Tekishino Red Wine

Awash Tekishino Red Wine has higher level of K, than another metal present in wine, but less as compared with other Awash brand wines. Least level of lead as well found in this wine. Cd is not detected in Awash Tekishino Red Wine. The level of metals in Awash Tekishino Wine was decreased from K to Pb in mg/L

K(163.1)>Na(79.45)>Ca(36.29)>Mg(14.935)>Fe(2.46)>Zn(1.2)>Cu(0.555)>Ni(0.25)> Mn (0.205)> Cr(0.105)> Co(0.025)> Pb (0.005).

4.1.4.6. Gouder Red Wine

Pb and Cr were not detected in Gouder Red wine, the 10 metals detected was ranked as: K(281.97) > Na(64.15) > Ca(35.925) > Mg(22.535) > Fe(1.8) > Zn(1.03) > Mn(0.32) > Cu(0.23) > Ni(0.16) > Co(0.00335) in mg/L. Cobalt was found in very small amounts.

1000

900 GW 800 GR 700 AW T 600 Ax R 500 Km W GDR

400 concn.(mg/L) 300 200 100 0 k Na Ca Mg Cu Zn Mn Ni Fe Co Cr Cd Pb Metals Figure 9: The Comparisons of some metals in Ethiopian wines

Generally in the studied wine, K- is the highest and the first ranked, but Co, except in Awash Tekishino wine, the least and the last ranked. In all wines except in Axumite Red wine, Fe is the fifth most abundant metal in wines. In all cause Cd was not detected. All of the Ethiopian six wines of Awash brand were more concentrated with K than other essential and non-essential metals, K is more concentrated in Gebeta Red and the least in Awash Tekishino. K in Gebeta Red (906.9) > in Axumite (881.2) > Kemila (699.1) > Gebeta white (661.5) > Gouder Red (281.97) >Awash Tekishino (163.1) that all concentrations are expressed in (mg/L).

Sodium is the second Ranked in Ethiopian wine next to K, with different levels and it is the highest abundant in Axumite Red and the least occurred in Gebeta Red. Thus Na in Axumite Red (139.05) > Gebeta white (126.49) > Kemila white (90.2) > Awash Tekishino (79.45) > Gouder Red (64.15) > Gebeta Red (49.75)

Calcium also the third most abundant metal that is first ranked in Gebeta Red and second, third, fourth, fifth and sixth in Axumite, Kemila White, Gebeta white, Awsh Tekishino and in Gouder Red respectively.

Magnesium also the fourth ranked in Ethiopian wine and more concentrated in Axumite Red than other wines, the least in Awash Tekishino. 50

From trace essential metals Fe, Zn, Mn, and Cu placed first to fourth ranked in Ethiopian Awash Brand wines. These metals were more concentrated in Gebeta Red, Axumite Red, Kemila white and Gebeta Red also least concentrated in Axumite Red, Gouder Red, Gouder Red and Kemila white respectively.

Cadmium was not detected in all wines, but lead was detected only in three wines of Awash brand. Cobalt and Cr were found in Ethiopian wines in very small levels. Cr not detected in Gouder Red.

4.1.5. Comparison of the Level of Metals in Ethiopian Wines with another World Data

The average values of metals in Ethiopian Awash brand wines may differs from the other levels of metals in wine reported in different literatures. The present study, reported that, the average values and the mean values of the different metals found in Ethiopian brand wines (Gebeta Red, Gebeta White, Axumite Red, Gouder Red, Kemila White and Awash Tekishino), as shown in the table 9. Most of these values were related with another values studied in different countries with different analytical methods.

The level of K in Ethiopian wines (Awash brand) with mean value of (599) mg/L and found with range from (163.1- 906.6) mg/L, which is the larger interval comparing with data reported earlier in table 8 bellow. Whereas wine of polish reported in the range of 97 – 3250 mg/L which was the widest interval than most of published data.

Magnesium was reported, the lowest essential metal in Ethiopian awash brand wine with mean Values 44.76 mg/L and found in the range of 14.7 – 65.3 mg/L as compared with wines of America, Italy, Czech, Greek that were found in the rand of 100 – 245 mg/L, 30-153 mg/L, 7.8 – 138 and 82.5-122.5 mg/L respectively, in table 9.

The average value of Sodium level in Ethiopian wines 91.52 mg/L and found with the range of (49.75 – 139.05 mg/L). It is the second most abundant major metals next to K. This value was higher than some level of sodium reported in review literature. The level of Na reported in Spanish, German and French wines were 0.0053-3.80 mg/L, 6-25 mg/L and 7.7-14.6 mg/L respectively which are lower than the Ethiopian wine content, But

51 wines of America, Italy has the wider range of Na level 7-106 and 3.4 -200 mg/L respectively, this may because of the origin of grapes, and in some country additive of Na salts are the source of level of Na in wine.

The mean level of Ca in present study were reported 54.63mg/L that found in the range of 35.9 – 74.35 mg/L, is comparable with that of Greek ranged from 14-47.5 mg/L and America ranged from 17-94 mg/L, but it was lower range than Ca in wine of German (58-200 mg/L), Polis (32-137 mg/L), Italy (30 -151 mg/L) and wine of French (65-161 mg/L) Also the level of Ca reported in Ethiopian wine by Woldemariam and et al [80], was found (28 – 37mg/L) that was nearly in the same range comparing with the present study

Table 7: The range Values of some metals in Ethiopian Wines

Metals Range value (mg/L) Ranking K 906.9 – 163.1 1 Na 49.75 – 139.05 2 Ca 35.9 – 74.35 3 Mg 14.9 – 65.3 4 Cu 0.225 – 1.5 9 Zn 1.03 – 3.2 6 Mn 0.32 – 1.63 8 Ni 0.16 – 5.15 7 Fe 0.03 – 4.095 5 Co 0.0015 – 0.045 12 Cr 0.03 – 1.565 10 Cd - - Pb 0.05 – 0.45 11

Table 8: Comparison of the level of metals in Awash wine brand with wine of different Countries reported previous

Metals and Concentration (mg/L) Ref.

Wine of; K Na Mg Ca Fe Zn Cu Co Ni Cd Pb Cr Mn

-5 -5 97- 3250 0.0053- 42.7-161 32-137 0.558- 0.0075- 2x10 - 0.00002– 2.3x10 - 0.006- 0.0037– 0.33-9.22 [81] 3.82 2.775 2.34 0.0068 0.245 0.0025 0.0349 0.095 [96]

Polish

493-3056 2-110 7.8-138 40-100 0.9-5.2 0.012 –6.827 0.019– 5.5x10-5- 0.032– 0.28-3.26 [80] 0.03 0.0033

Czech 0.037

750-1500 3.4-200 53-115 30-151 1.35-27.8 0.135-4.8 0.001 –1.34 0.003- 0.015- 0.0012- 0.01-0.35 0.02-0.05 0.67-2.5

Italy 0.009 0.21 0.0016 [79]

480-1860 6-25 56-105 58-200 0.4-4.2 0.3-1.5 0.02–0.71 0.004- - - 0.005 [80]

German

462-1147 7-106 100-245 17-94 1.2-6.6 0.75-3.60 0.05–0.58 - -

America

955-2089 5.5-150 82.5- 14-47.5 0.7-7.3 0.05-8.9 0.2 –1.65 ND-0.62 122.5

Greek

265-426 7.7- 55-96 65-161 0.81– 2.51 0.44– 0.74 ND-0.48 0.004– 0.006- 0.63-0.96 14.6 0.011 0.09

French

693a -766 24 58 - 79 28-37 1.4-3.16 1.82-2.7 0.5-1.5 ND-0.091 0.18-0.25 ND 0.14-0.31 ND-0.091 1.04-1.88 [80]

Ethiopi

281-906.9 49.75– 14.9 – 35.9 – 0.03– 1.03–3.2 0.225-1.5 0.0015– 0.16-5.15 ND 0.05 – 0.45 0.03 – 0.32 – Pres.st 139.05 65.3 74.35 4.095 0.045 1.565 1.63 udy

Ethiopia

In the present study, the level of Fe in Ethiopian wine of Awash brand found in the range of 0.03 – 4.095mg/L with mean value of 2.448ppmwhich is less than the maximum permitted levels that set by OIV, also the concentration of Fe in Ethiopian wine that reported earlier was below the OIV. Fe was the first most abundant from the trace essential metals. Also it is concentrated in wine of America(1.2-6.6mg/L), Czech (0.9-5.2 mg/L) and the highest concentrated in wine of Italy found in the range of (1.35- 27.8mg/L).The lower concentration of Fe merit for metabolism and fermentation processes, The level of Fe in wine increase due to Geographical/Environmental factors or device used for production of wines.

From trace metals Zn is the second most abundant in Ethiopian wines, which is ranging from 1.03-3.2ppm with mean value of 1.687mg/L and more concentrated than, Zn in wine of German and French that were reported in published data 0.3-1.5 and 0.44 – 0.74 mg/L respectively. But the concentration of Zn in wine of Greek (0.05-8.9mg/L) was, more concentrated than Ethiopian wines. The level of Zn, Cu, Pb, Co and Mn were report in Awash brand wine before this study were nearly the same, but Co were not detected in all wines.

Cu was found in Ethiopian Awash brand ranged from 0.225 – 1.5mg/L with average value of 0.51 and below legal limits set by International Organization Vine and Wine. The wine of German and French contain less concentration of Cu than Ethiopian, with having 0.02 – 0.71 and ND – 0.48 ppm respectively, but the level of Cu in wine of Czech is higher range which was (0.012 – 6.827 mg/L) than most reported levels in America (0.05 – 0.58mg/L), Italy(0.001 – 1.34mg/L) and including Ethiopian. Most of Ethiopian wines contain less level of Cu comparing with OIV value. Soil and grapes type, contamination during cultivation and harvesting and using Cu based chemicals for pesticide, herbicide and increasing wine productivity were factor for increasing of Cu level in wines.

Manganese and Nickel also one of micro and trace metals found in wines. Ethiopian wine (Awash brand wine) contain 0.32 – 1.36 mg/L, 0.16 -5.15 mg/L with mean values of 1.069 and 1.104 mg/L respectively. These metals were more concentrated in Ethiopian

54 wine than Cu. The level of Mn in most of Ethiopian wines is less than in many countries reported in review literature. Wines of German (0.28 -3.26 mg/L), USA (0.81 -4.08 mg/L), Italy (0.67 – 2.5 mg/L) contain higher concentration of Mn than Ethiopian wine. But the wine of French reported (0.63 – 0.96) was less level of Mn than Ethiopian wines. Polish wine more concentrated with 0.33-9.22 mg/L).

The concentration of Ni in Ethiopian wine that reported mean value 1.104 ppm and found in the range of (0.16 – 5.15 mg/L)was more concentrated than wines of Polish (0.00002– 0.245 mg/L and Italy (0.015 – 0.21 mg/L).

However, the concentration of Cobalt detected in Ethiopian wines in very small level (0.005 -0.045 mg/L) with mean value of 0.023 mg/L, it is larger than the reported levels in table above. The level of Cobalt in wines of German (0.004 – 0.005 mg/L), Italy (0.003 – 0.009 mg/L) and French (0.004 – 0.011mg/L) which were smaller range than the Ethiopian wines

Heavy metals also found in Ethiopian wines (Awash brand) in smaller amount. Cd was not detected in all six Ethiopian wines, similar to that reported before this study. but the concentration of Cd reported in wine of Czech, Polish and German in the range of 0.0012 – 0.0016 mg/L, 2.3x10-5 – 0.0025 mg/L and 5.5x10-5 – 0.0033 mg/L respectively.

Pb was detected only in three Ethiopian wines that found in the range of (0.05 – 0.45 mg/L) with mean value of 0.25 mg/L. Thus some of Ethiopian wines have concentrated Pb above the permitted value. The level of Pb reported in published data with very smaller values, a wine of Polish has 0.004 – 0.011mg/L of Pb and in Italian wine 0.01- 0.35mg/L of Pb was observed.

Chromium is found in Ethiopian wines in the range of 0.03 – 1.565 mg/L and 0.37mg/L average value. In review literature reported, the level of Cr wines French wines found in different range. Red wine (0.007 - 0.09 mg/L), Rose wine (0.0073 – 0.0147 mg/L), White wine (0.0066 – 0.0439) and Champagne wine (0.0105–0.036 mg/L)[140], the reported level of Cr in wine of Polish (0.0037–0.095 mg/L), Italy (0.02 -0.05 mg/L) and Czech (0.032–0.037 mg/L) were lower than Ethiopian wines. The high level of Cr may caused

55 due to contamination during maceration or containing wine with equipment that contain chromium alloy, or the soil that affected by chemicals containing Cr.

The level of Cr, Zn and Fe in Ethiopian wine reported by this study were, some higher than Awash wine that reported before, but the concentration of Pb, Mn, Cd and Co were nearly the same level.

The concentration of trace (Cu, Fe, and Zn) and heavy metals were decreased in wine due to the precipitation and yeast consumes metals during fermentation process and during clarification of wine. And also the level of trace (Cu, Fe, and Zn) and heavy metals increased due to the long maceration time and long contact time with metal containing equipment [82].

4.1.6. Comparison of the Level of Metals with OIV

Table 9: Comparison of the level of metals in wines of different Countries with OVI

Element OIV Concentration of metals (mg/L) in the; Australia Germany Italy Poland Ethiopia Cd 0.01 0.05 0.01 0.03 ND Cu 1 5 5 10 0.51 Pb 0.15 0.2 0.3 0.3 0.3 0.25 Zn 5 5 5 5 1.687 Fe 10 2.448 Mn 0.43 1.069 Na 60 91.52 K 200- 599 2000 Ref. [85],[81], [96] and [80] Present study

The levels of most metals in Ethiopian wines were in the range of legal limit that set by OIV. The concentration of Cu in Gebeta Red (1.5 mg/L) which is greater than OIV and the level of Pb in Gebeta white (0.25 mg/L) and in Gebeta red (0.45 mg/L) were greater than the recommended, amount. The Concentration of Zn and Fe were less than the maximum permitted (OIV) in all Ethiopian six wines. (Level of Fe in Gebeta Red (4.095 mg/L), Gebeta white (3.5mg/L), Awash Tekishino (2.46mg/L), Axumite Red (0.03mg/L), Kemila white (2.6 mg/L), Gouder Red (1.8 mg/L) and level of Zn in (Gebeta Red (2.35 mg/L), Gebeta white (1.14 mg/L), Awash Tekishino (1.2 mg/L), Axumite Red (3.2 mg/L), Kemila white (1.2 mg/L), Gouder Red (1.03 mg/L). Thus there was no risk regarding to Fe and Cu in Ethiopian six Awash brand wines.

Except in Gouder wine (0.32 mg/L) and Awash Tekishino(0.205 mg/L), the concentration of Mn in all (Kemila white 1.565 mg/L, Gebeta Red 1.545 mg/L, Gebeta white 1.63mg/L and Axumite Red 1.15mg/L) greater than the legal limit set by OIV.

The level of Na in Ethiopian wines was greater than the maximum limit, except in Gebeta red with 49.75 mg/L.

4.2. Determination of Tannins

The amount of Tannins in the in Ethiopian Awash brand wine, determined by titration method, which it based on the volume of titrant (KMnO4) and the analyte sample which contain Indigo carmine indicator that used to remove the pigment (color).The back and blank titration used to determine the amount of KMnO4 oxidized[74].

The amount of potassium magnate used in oxidation of tannins can be calculated as Back titration –Blank titration =C mL. The standard tannin solution for which 1 mL of 0.004 M

KMnO4 = 0.0832 mg of tannin. Therefore, percent of tannins in wine=0.01664×C [74].Accordingly, the following data was obtained from experimental procedure

Table 10: The amount of Tannins in six Awash brand of Wine sample

Amount of KMnO used Samples 4 in oxidizing of Tannin % Tannin in Wine= Mean SD RSD (A - B = C) 0.01664C 2.767 0.058 0.021 Kemila white 0.6 0.1 0.167 2.168 0.0361 9.6 0.2 0.021 Axumite Red 6.767 0.153 0.023 2.833 0.0472 4.3 0.265 0.062 Gouder Red 1.233 0.208 0.169 3.067 0.0510 3.067 0.1151 0.038 Gebeta White 0.8 0.265 0.331 2.267 0.0377 12.867 0.153 0.012 Gebeta Red 10.1667 0.208 0.021 2.7 0.045 2.2 0.1 0.046 Awash Tekishino 0.267 0.058 0.217 1.933 0.0322

4.2.1. Comparison of the level of Tannins with each Sample of wines

%Tannin 0.06

0.05

0.04 KM W

0.03 AX R

%Tannin G R 0.02 Ge W 0.01 Ge R Aw T

G R G

AX R AX

Ge R Ge

Aw T Aw

Ge W Ge KM W KM Wine Brand

Figure 10: The percent Tannin in Ethiopian Wine brand

The level of Tannin content in Gouder red was the highest were as Awash Tekishino was the least content.

The percent Tannin content in Gouder Red (0.0503) > Axumite Red (0.04715) > Gebeta Red (0.04493) >Gebeta White (0.03772) ≈ Awash Tekishino (0.03217) Kemila White (0.03605)

4.2.2. Comparison of the Level of Tannins with World Wide of Wines

Ethiopian wines contain tannin mean value of 0.0415% with the range of 0.032 – 0.051%. Mulani .K et al, [98] reported the level of tannin in Indian wine found in the range of (0.016 – 0.25%), which is wider range than Ethiopian wine. This may happen by the different sources of tannins (Skin, seed, Stems, the newer oak barrel, amount of tannin added, Climate condition). So one of these affects the Ethiopian wine that make lower Tannin level than that of Indian wine

The level of Tannin was high in Gebeta red wine, Axumite Red wine like K, Fe and Ni, But les in Kemila white and Awash Tekishino. 59

The one way analysis of variance (ANOVA) , IBM SPSS Statistics Version 20 , attached to appendix, for tannin in Kemila white, it was significant different with red wines (Gouder red, Axumite Red and Gebeta Red), but not significant in with gebeta White (P > 0.05). Also Tannin in Gouder Red was significant different with white wines (P< 0.05), but not significant with some reds.

This implies that, the level of tannin in Red wines not the same distribution with of white wines. The concentration of tannin in Red wine is greater than that of in white wine of Awash brand.

CHAPTER 5: CONCLUSION AND RECOMMENDATION

There are a number of wine parameters like metals, Tannins, Sulfur dioxides, Flavonoids, Poly Phenols, that affects the quality of wines (test, Color, Mouth feel), health care.

This study emphasized on the analysis of selected metals and Tannins in Ethiopian Awash brand wines, by using ICP – OES and Titrimetric method respectively, that follows the principal procedure. The analysis was done by first collecting the sample randomly from different super markets in Bahir Dar city. Then digestion followed by the optimization condition that necessary for quality control parameter.

The validation of whole analytical procedure was checked by recovery test that expressed by percentage recovery. The % recovery was obtained in the range of 100±15. This shows that the analytical procedure followed was valid for determination of metals in Ethiopian Awash brand.

Also titrimetric method was used to determine the percent of Tannin content Ethiopian wine, that followed by the care procedure, based on the titrating the titrant (KMnO4) with analyte until the end point /color change appeared.

In Ethiopian wine, it was reported that, the %Tannin found in the range 0.032 – 0.051%.with mean value of 0.0415%. Gouder Red contains more level of Tannin than Gebeta white and Kemila white. The %Tannin concentrated in red wine than in White wines.

It was reported that Ethiopian wine brand contain K in highest level and followed by Na that was found in the range of 35.9 – 139.05 mg/L with average value of 91.52 mg/L, which was above the maximum amount permitted that set by OIV, but in Gebeta Red (49.75 mg/L) that is below the maximum recommended and that is significant difference with p > 0.05

Toxic metal (Cd) was not detected in all six Ethiopian wine. Pb was detected in three wines (Gebeta Red, Gebeta White and Awash Tekishino) in the range of 0.05 – 0.45 mg/L with average value of 0.25 mg/L. This value was in small extent larger than the

International Organization for vine. Pb was not detected in Axumite Red, Kemila White and Gouder Red).

Fe was the first most abundant from micro metals, that found in the range of 0.03 – 4.095 mg/L with mean value of 2.448 mg/L. this value was below recommended maximum value (OIV).

Generally Essential and Non –essential metals (K, Na, Ca, Mg, Fe, Zn, Ni, Mn, Co, Cr, Cd and Pb) and Tannins were contributes for the daily nutritional requirement in recommended amount.

Also Tannin in Red wines was significant different with white wines of Awash brand P < 0.05), but not significant with some reds, which indicates that the level of tannin in red wine was more concentrated than white wines.

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APPENDEX A: Lists of Table

Sam. Sample Ratio of Time consumed to form clear solution (in

volume Reagents in c) minutes) and remark (in color formation) O (in ml) (7ml)

H2O2 HNO3 50 65 70 90

Tem (in Tem Wine 10 2 5 220 Yellowish Clear with - - clear no ppt 10 3 4 220 Clear with - - - no ppt

10 4 3 220 Yellowish Yellowish Clear - clear clear with no ppt 10 5 2 220 Yellowish Yellowish Nearly Clear with clear clear clear no ppt 10 6 1 220 Yellowish Yellowish Yellowi Yellowish clear clear sh clear clear 10 1 6 220 Yellowish Yellowish Yellowi Yellowish clear clear sh clear clear

Table 1: Optimized volume ratio, temperature and duration of time for digestion of sample

Table 2: Concentration of intermediate Standards (mg/L), Concentration of Working Standard (mg/L) and Correlation coefficient of calibration carve Metals Concentration of Concentration of Working intermediate Standards Standard(mg/L) (mg/L) K 75 0.01, 15, 30, 45, 60, 75 Na 75 0.01, 15, 30, 45, 60, 75 Ca 75 0.01, 15, 30, 45, 60, 75 Mg 75 0.01, 15, 30, 45, 60, 75 Cu 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Zn 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Mn 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Ni 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Fe 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Co 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Cr 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Cd 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05 Pb 5.05 0.05, 1.05, 2.05, 3.05, 4.05, 5.05

Table 3:MDL, IDL, % Recovery,and LOD of the instruments and Correlation coefficient of calibration curve Metals Wave MDL LOQ IDL % Recovery Correlation Length coefficient of calibration curve K 766.490 0.0441 0.441 95 0.99523 Na 589.592 0.5484 0.267 0.0690 96 0.9984 Ca 317.933 0.5131 0.216 0.01 101.1 0.99924 Mg 285.213 0.1627 1.152 0.0016 96.4 0.9965 Cu 327.393 0.0443 0.003 0.0097 103 0.99986 Zn 206.200 0.0009 0.009 0.0059 89.96 0.99988 Mn 257.610 0.0571 0.57 0.0014 87 0.9999 Ni 231.604 0.0003 0.003 0.0150 102.5 0.9999 Fe 238.204 0.0811 0.021 0.0046 97 0.99993 Co 228.616 0.0003 0.003 0.0070 89 0.9999 Cr 267.716 0.0003 0.003 0.0071 98 0.99964 Cd 228.802 0.003 0.03 0.0027 95 0.99986 Pb 220.353 0.0003 0.0009 0.009 95 0.99985

Table 6: Level of metals in Awash brand of wine

Awash brand wines Metal Gebata Red wine Gebata White wine Kemila white wine Axumite red wine Awash Tekishino Gouder Red Red Wine Wine Concentration Concentration Concentration Concentration Concentration Concentration (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) K 906.9±2.64 661.5±2.31 699.1±0.504 881.2±1.33 163.1±0.08 281.97±0.14

Na 49.75±0.41 126.49±0.157 90.2±0.036 139.05±0.33 79.45±0.15 64.15±0.053

Table 7:The range Values of metals studied in Ethiopian Wines

Table 8: Comparison of the level of metals in wines of different Countries

Metals and Concentration (mg/L) Ref.

Wine of; K Na Mg Ca Fe Zn Cu Co Ni Cd Pb Cr Mn [81]

97- 0.0053- 42.7-161 32-137 0.558- 0.0075- 2x10-5- 0.00002 2.3x10-5- 0.006- 0.0037– 0.33- [83] 3250 3.82 2.775 2.34 0.0068 –0.245 0.0025 0.0349 0.095 9.22

Polish

493- 2-110 7.8-138 40-100 0.9-5.2 0.012 –6.827 0.019– 5.5x10-5- 0.032– 0.28- [80] 3056 0.03 0.0033 3.26

Czech 0.037

750- 3.4-200 53-115 30-151 1.35-27.8 0.135-4.8 0.001 –1.34 0.003- 0.015- 0.0012- 0.01-0.35 0.02-0.05 0.67-2.5

Italy 1500 0.009 0.21 0.0016

480- 6-25 56-105 58-200 0.4-4.2 0.3-1.5 0.02–0.71 0.004- - - 1860 0.005

German

462- 7-106 100-245 17-94 1.2-6.6 0.75-3.60 0.05–0.58 - - 1147

America

955- 5.5-150 82.5- 14-47.5 0.7-7.3 0.05-8.9 0.2 –1.65 ND-0.62 2089 122.5

Greek

265- 7.7- 55-96 65-161 0.81– 2.51 0.44– 0.74 ND-0.48 0.004– 0.006- 0.63- 426 14.6 0.011 0.09 0.96

French

693- 24 58 - 79 28-37 1.4-3.16 1.82-2.7 0.5-1.5 ND-0.091 0.18- ND 0.14-0.31 ND-0.091 1.04- [80] 766 0.25 1.88

Ethiopia

163.1- 49.75– 14.9 – 35.9 – 0.03– 1.03–3.2 0.225-1.5 0.0015– 0.16- ND 0.05 – 0.45 0.03 – 0.32 – present 906.9 139.05 65.3 74.35 4.095 0.045 5.15 1.565 1.63 study

Ethiopia 77

Table 9: The accepted level of metals content (mg/l) in wine set by OIV

Elemen OIV Concentration of metals (mg/L) in the; ts Australia Germany Italy Poland Ethiopia Cd 0.01 0.05 0.01 0.03 ND Cu 1 5 5 10 0.51 Pb 0.15 0.2 0.3 0.3 0.3 0.25 Zn 5 5 5 5 1.687 Fe 10 2.448 Mn 0.43 1.069 Na 60 91.52 K 200-2000 599 Ref. [85]&[81] [83] and [80] Present study

Table 10: The amount of Tannins in six Awash brand of Wine sample

Volume of KMnO4 Amount of

Samples Consumed (ml) KMnO4 used in oxidizing of % Tannin in Trial Titration Tannin (A - B = Wine= Trial 1 Trial 2 3 Type Mean SD RSD C) 0.01664C 2.8 2.8 2.7 Back (A) 2.766667 0.057735 0.02087 Kemila white 0.6 0.7 0.5 Blank (B) 0.6 0.1 0.1667 2.166666667 0.036053333 9.8 9.4 9.6 Back (A) 9.6 0.2 0.02083 Axumite Red 6.8 6.6 6.9 Blank (B) 6.766667 0.1527525 0.022574 2.833333333 0.047146667 4.2 4.6 4.1 Back (A) 4.3 0.2645751 0.06153 Gouder Red 1 1.4 1.3 Blank (B) 1.233333 0.2081666 0.1688 3.066666667 0.051029333 Gebeta 3 3.2 3 Back (A) 3.066667 0.1154701 0.0376533 White 1.1 0.7 0.6 Blank (B) 0.8 0.2645751 0.330712 2.266666667 0.037717333 13 12.9 12.7 Back (A) 12.86667 0.1527525 0.011872 Gebeta Red 10.1 10.4 10 Blank (B) 10.16667 0.2081666 0.02046 2.7 0.044928 Awash 2.1 2.2 2.3 Back (A) 2.2 0.1 0.04545 Tekishino 0.3 0.2 0.3 Blank (B) 0.266667 0.057735 0.2165064 1.933333333 0.032170667

APPENDEX B: Lists of Figure and graphs

40 CALCIUM (Ca) y = 351665.04426 +449851.17607 * X 30 R = 0.99924

Intensity Intensity (M)

0 10 20 30 40 50 60 70 80 concentration (mg/l)

Figure 5: graph of calibration curve [similar straight line for the rest metals]

1000 K, Na, Ca and Mg 900 800 700 600 500 k 400 Na

concentration(mg/L) 300 Ca 200 Mg 100 0

AWASH BRNAD WINES

Figure 6: comparison of major metals (K, Na, Ca and Mg) in Ethiopian Wines.

Cu, Zn, Mn and Fe) Cu 4.5

4 Zn

3.5 Mn

3 Fe 2.5

1.5 concentration(mg/L) 1

0.5

AWASH BRAND WINES

Figure 7: comparison of trace metals (Fe, Cu, Zn and Mn) in Ethiopian Wines

Ni 3 Co 2 Cr

Concentration(mg/L) Cd 1 Pb

0 GW GR AW T Ax R Km W GDR Wine brands

Figure 8: The comparisons of both trace and heavy metals in Ethiopian wine brand

1000 GW GR 900 AW T 800 Ax R Km W 700 GDR 600

500

400 concentration(mg/L) 300

200

100

0 k Na Ca Mg Cu Zn Mn Ni Fe Co Cr Cd Pb Wine brand

Figure 9: Comparisons of Essential and Non essential metals in Ethiopian wines 82

%Tannin 0.06

0.05

0.04 KM W

0.03 AX R

G R %Tannin 0.02 Ge W 0.01 Ge R Aw T

G G R

AX R AX

Ge R Ge

Aw T Aw

Ge W Ge KM W KM Wine Brand Figure 10: The percent Tannin in Ethiopian Wine brand

Figure 1: ICP – OES instrumentation

Figure 2: Preparation of Sample

Figure3: digested sample ready for ICP – OES

Titrating the sample Golden Yellow (end point)

Figure4: when titration was conducted

APPENDEX C:Analysis of Variances (ANOV) at 95% confidence interval Multiple Comparisons

LSD Dependent Variable (I) Wine Type (J) Wine Type Mean Difference Std. Error Sig. 95% Confidence Interval (I-J) Lower Bound Upper Bound Gebeta red 3.7700000* .1689492 .000 3.415051 4.124949 awash Tekishino 9.4116667* .2069196 .000 8.976945 9.846389 Gebeta white Axumite red -2.5150000* .2069196 .000 -2.949722 -2.080278 Kemila white 7.2583333* .2069196 .000 6.823611 7.693055 Gouder red 12.4716667* .2069196 .000 12.036945 12.906389 Gebeta white -3.7700000* .1689492 .000 -4.124949 -3.415051 awash Tekishino 5.6416667* .2069196 .000 5.206945 6.076389 Gebeta red Axumite red -6.2850000* .2069196 .000 -6.719722 -5.850278 Kemila white 3.4883333* .2069196 .000 3.053611 3.923055 Gouder red 8.7016667* .2069196 .000 8.266945 9.136389 Gebeta white -9.4116667* .2069196 .000 -9.846389 -8.976945 Gebeta red -5.6416667* .2069196 .000 -6.076389 -5.206945 awash Tekishino Axumite red -11.9266667* .2389302 .000 -12.428640 -11.424693 Kemila white -2.1533333* .2389302 .000 -2.655307 -1.651360 Gouder red 3.0600000* .2389302 .000 2.558026 3.561974 Sodium Gebeta white 2.5150000* .2069196 .000 2.080278 2.949722 Gebeta red 6.2850000* .2069196 .000 5.850278 6.719722 Axumite red awash Tekishino 11.9266667* .2389302 .000 11.424693 12.428640 kemila white 9.7733333* .2389302 .000 9.271360 10.275307 Gouder red 14.9866667* .2389302 .000 14.484693 15.488640 Gebeta white -7.2583333* .2069196 .000 -7.693055 -6.823611 Gebeta red -3.4883333* .2069196 .000 -3.923055 -3.053611 Kemila white awash Tekishino 2.1533333* .2389302 .000 1.651360 2.655307 Axumite red -9.7733333* .2389302 .000 -10.275307 -9.271360 Gouder red 5.2133333* .2389302 .000 4.711360 5.715307 Gebeta white -12.4716667* .2069196 .000 -12.906389 -12.036945 Gebeta red -8.7016667* .2069196 .000 -9.136389 -8.266945 Gouder red awash Tekishino -3.0600000* .2389302 .000 -3.561974 -2.558026 Axumite red -14.9866667* .2389302 .000 -15.488640 -14.484693 kemila white -5.2133333* .2389302 .000 -5.715307 -4.711360

Gebeta red -21.0400000* 1.2164495 .000 -23.595666 -18.484334 awash Tekishino 49.8433333* 1.4898403 .000 46.713295 52.973372 Gebeta white Axumite red -21.9666667* 1.4898403 .000 -25.096705 -18.836628 kemila white -2.7566667 1.4898403 .081 -5.886705 .373372 Gouder red 37.9533333* 1.4898403 .000 34.823295 41.083372 Gebeta white 21.0400000* 1.2164495 .000 18.484334 23.595666 awash Tekishino 70.8833333* 1.4898403 .000 67.753295 74.013372 Gebeta red Axumite red -.9266667 1.4898403 .542 -4.056705 2.203372 kemila white 18.2833333* 1.4898403 .000 15.153295 21.413372 Gouder red 58.9933333* 1.4898403 .000 55.863295 62.123372 Gebeta white -49.8433333* 1.4898403 .000 -52.973372 -46.713295 Gebeta red -70.8833333* 1.4898403 .000 -74.013372 -67.753295 awash Tekishino Axumite red -71.8100000* 1.7203194 .000 -75.424257 -68.195743 kemila white -52.6000000* 1.7203194 .000 -56.214257 -48.985743 Gouder red -11.8900000* 1.7203194 .000 -15.504257 -8.275743 K Gebeta white 21.9666667* 1.4898403 .000 18.836628 25.096705 Gebeta red .9266667 1.4898403 .542 -2.203372 4.056705 Axumite red awash Tekishino 71.8100000* 1.7203194 .000 68.195743 75.424257 kemila white 19.2100000* 1.7203194 .000 15.595743 22.824257 Gouder red 59.9200000* 1.7203194 .000 56.305743 63.534257 Gebeta white 2.7566667 1.4898403 .081 -.373372 5.886705 Gebeta red -18.2833333* 1.4898403 .000 -21.413372 -15.153295 kemila white awash Tekishino 52.6000000* 1.7203194 .000 48.985743 56.214257 Axumite red -19.2100000* 1.7203194 .000 -22.824257 -15.595743 Gouder red 40.7100000* 1.7203194 .000 37.095743 44.324257 Gebeta white -37.9533333* 1.4898403 .000 -41.083372 -34.823295 Gebeta red -58.9933333* 1.4898403 .000 -62.123372 -55.863295 Gouder red awash Tekishino 11.8900000* 1.7203194 .000 8.275743 15.504257 Axumite red -59.9200000* 1.7203194 .000 -63.534257 -56.305743 kemila white -40.7100000* 1.7203194 .000 -44.324257 -37.095743

Gebeta red -3.7850000* .2090866 .000 -4.224275 -3.345725 awash Tekishino 8.0980000* .2560778 .000 7.560001 8.635999 Gebeta white Axumite red -1.9716667* .2560778 .000 -2.509666 -1.433667 kemila white .0950000 .2560778 .715 -.442999 .632999 Gouder red 6.5783333* .2560778 .000 6.040334 7.116333 Gebeta white 3.7850000* .2090866 .000 3.345725 4.224275 awash Tekishino 11.8830000* .2560778 .000 11.345001 12.420999 Gebeta red Axumite red 1.8133333* .2560778 .000 1.275334 2.351333 kemila white 3.8800000* .2560778 .000 3.342001 4.417999 Gouder red 10.3633333* .2560778 .000 9.825334 10.901333 Gebeta white -8.0980000* .2560778 .000 -8.635999 -7.560001 Gebeta red -11.8830000* .2560778 .000 -12.420999 -11.345001 awash Tekishino Axumite red -10.0696667* .2956931 .000 -10.690895 -9.448438 kemila white -8.0030000* .2956931 .000 -8.624228 -7.381772 Gouder red -1.5196667* .2956931 .000 -2.140895 -.898438 Mg Gebeta white 1.9716667* .2560778 .000 1.433667 2.509666 Gebeta red -1.8133333* .2560778 .000 -2.351333 -1.275334 Axumite red awash Tekishino 10.0696667* .2956931 .000 9.448438 10.690895 kemila white 2.0666667* .2956931 .000 1.445438 2.687895 Gouder red 8.5500000* .2956931 .000 7.928772 9.171228 Gebeta white -.0950000 .2560778 .715 -.632999 .442999 Gebeta red -3.8800000* .2560778 .000 -4.417999 -3.342001 kemila white awash Tekishino 8.0030000* .2956931 .000 7.381772 8.624228 Axumite red -2.0666667* .2956931 .000 -2.687895 -1.445438 Gouder red 6.4833333* .2956931 .000 5.862105 7.104562 Gebeta white -6.5783333* .2560778 .000 -7.116333 -6.040334 Gebeta red -10.3633333* .2560778 .000 -10.901333 -9.825334 Gouder red awash Tekishino 1.5196667* .2956931 .000 .898438 2.140895 Axumite red -8.5500000* .2956931 .000 -9.171228 -7.928772 kemila white -6.4833333* .2956931 .000 -7.104562 -5.862105

Gebeta red -.1445000 .3281917 .665 -.834005 .545005 awash Tekishino 2.5446667* .4019511 .000 1.700199 3.389135 Gebeta white Axumite red -4.1740000* .4019511 .000 -5.018468 -3.329532 kemila white -2.6573333* .4019511 .000 -3.501801 -1.812865 Gouder red 2.6176667* .4019511 .000 1.773199 3.462135 Gebeta white .1445000 .3281917 .665 -.545005 .834005 awash Tekishino 2.6891667* .4019511 .000 1.844699 3.533635 Gebeta red Axumite red -4.0295000* .4019511 .000 -4.873968 -3.185032 kemila white -2.5128333* .4019511 .000 -3.357301 -1.668365 Gouder red 2.7621667* .4019511 .000 1.917699 3.606635 Gebeta white -2.5446667* .4019511 .000 -3.389135 -1.700199 Gebeta red -2.6891667* .4019511 .000 -3.533635 -1.844699 awash Tekishino Axumite red -6.7186667* .4641332 .000 -7.693774 -5.743559 kemila white -5.2020000* .4641332 .000 -6.177108 -4.226892 Gouder red .0730000 .4641332 .877 -.902108 1.048108 Ca Gebeta white 4.1740000* .4019511 .000 3.329532 5.018468 Gebeta red 4.0295000* .4019511 .000 3.185032 4.873968 Axumite red awash Tekishino 6.7186667* .4641332 .000 5.743559 7.693774 kemila white 1.5166667* .4641332 .004 .541559 2.491774 Gouder red 6.7916667* .4641332 .000 5.816559 7.766774 Gebeta white 2.6573333* .4019511 .000 1.812865 3.501801 Gebeta red 2.5128333* .4019511 .000 1.668365 3.357301 kemila white awash Tekishino 5.2020000* .4641332 .000 4.226892 6.177108 Axumite red -1.5166667* .4641332 .004 -2.491774 -.541559 Gouder red 5.2750000* .4641332 .000 4.299892 6.250108 Gebeta white -2.6176667* .4019511 .000 -3.462135 -1.773199 Gebeta red -2.7621667* .4019511 .000 -3.606635 -1.917699 Gouder red awash Tekishino -.0730000 .4641332 .877 -1.048108 .902108 Axumite red -6.7916667* .4641332 .000 -7.766774 -5.816559 kemila white -5.2750000* .4641332 .000 -6.250108 -4.299892

Gebeta red .0025000* .0006211 .001 .001195 .003805 awash Tekishino -.0610000* .0007607 .000 -.062598 -.059402 Gebeta white Axumite red -.0123333* .0007607 .000 -.013932 -.010735 kemila white .0050000* .0007607 .000 .003402 .006598 Gouder red .0043333* .0007607 .000 .002735 .005932 Gebeta white -.0025000* .0006211 .001 -.003805 -.001195 awash Tekishino -.0635000* .0007607 .000 -.065098 -.061902 Gebeta red Axumite red -.0148333* .0007607 .000 -.016432 -.013235 kemila white .0025000* .0007607 .004 .000902 .004098 Gouder red .0018333* .0007607 .027 .000235 .003432 Gebeta white .0610000* .0007607 .000 .059402 .062598 Gebeta red .0635000* .0007607 .000 .061902 .065098 awash Tekishino Axumite red .0486667* .0008784 .000 .046821 .050512 kemila white .0660000* .0008784 .000 .064155 .067845 Gouder red .0653333* .0008784 .000 .063488 .067179 Cu Gebeta white .0123333* .0007607 .000 .010735 .013932 Gebeta red .0148333* .0007607 .000 .013235 .016432 Axumite red awash Tekishino -.0486667* .0008784 .000 -.050512 -.046821 kemila white .0173333* .0008784 .000 .015488 .019179 Gouder red .0166667* .0008784 .000 .014821 .018512 Gebeta white -.0050000* .0007607 .000 -.006598 -.003402 Gebeta red -.0025000* .0007607 .004 -.004098 -.000902 kemila white awash Tekishino -.0660000* .0008784 .000 -.067845 -.064155 Axumite red -.0173333* .0008784 .000 -.019179 -.015488 Gouder red -.0006667 .0008784 .458 -.002512 .001179 Gebeta white -.0043333* .0007607 .000 -.005932 -.002735 Gebeta red -.0018333* .0007607 .027 -.003432 -.000235 Gouder red awash Tekishino -.0653333* .0008784 .000 -.067179 -.063488 Axumite red -.0166667* .0008784 .000 -.018512 -.014821 kemila white .0006667 .0008784 .458 -.001179 .002512

Gebeta red -.0823333* .0098989 .000 -.103130 -.061537 awash Tekishino .2446667* .0121236 .000 .219196 .270137 Gebeta white Axumite red -.2900000* .0121236 .000 -.315471 -.264529 kemila white .2176667* .0121236 .000 .192196 .243137 Gouder red .3750000* .0121236 .000 .349529 .400471 Gebeta white .0823333* .0098989 .000 .061537 .103130 awash Tekishino .3270000* .0121236 .000 .301529 .352471 Gebeta red Axumite red -.2076667* .0121236 .000 -.233137 -.182196 kemila white .3000000* .0121236 .000 .274529 .325471 Gouder red .4573333* .0121236 .000 .431863 .482804 Gebeta white -.2446667* .0121236 .000 -.270137 -.219196 Gebeta red -.3270000* .0121236 .000 -.352471 -.301529 awash Tekishino Axumite red -.5346667* .0139991 .000 -.564078 -.505256 kemila white -.0270000 .0139991 .070 -.056411 .002411 Gouder red .1303333* .0139991 .000 .100922 .159744 Fe Gebeta white .2900000* .0121236 .000 .264529 .315471 Gebeta red .2076667* .0121236 .000 .182196 .233137 Axumite red awash Tekishino .5346667* .0139991 .000 .505256 .564078 kemila white .5076667* .0139991 .000 .478256 .537078 Gouder red .6650000* .0139991 .000 .635589 .694411 Gebeta white -.2176667* .0121236 .000 -.243137 -.192196 Gebeta red -.3000000* .0121236 .000 -.325471 -.274529 kemila white awash Tekishino .0270000 .0139991 .070 -.002411 .056411 Axumite red -.5076667* .0139991 .000 -.537078 -.478256 Gouder red .1573333* .0139991 .000 .127922 .186744 Gebeta white -.3750000* .0121236 .000 -.400471 -.349529 Gebeta red -.4573333* .0121236 .000 -.482804 -.431863 Gouder red awash Tekishino -.1303333* .0139991 .000 -.159744 -.100922 Axumite red -.6650000* .0139991 .000 -.694411 -.635589 kemila white -.1573333* .0139991 .000 -.186744 -.127922

Gebeta red .0173333* .0067605 .020 .003130 .031537 awash Tekishino .2853333* .0082798 .000 .267938 .302729 Gebeta white Axumite red .0983333* .0082798 .000 .080938 .115729 kemila white .0130000 .0082798 .134 -.004395 .030395 Gouder red .2623333* .0082798 .000 .244938 .279729 Gebeta white -.0173333* .0067605 .020 -.031537 -.003130 awash Tekishino .2680000* .0082798 .000 .250605 .285395 Gebeta red Axumite red .0810000* .0082798 .000 .063605 .098395 kemila white -.0043333 .0082798 .607 -.021729 .013062 Gouder red .2450000* .0082798 .000 .227605 .262395 Gebeta white -.2853333* .0082798 .000 -.302729 -.267938 Gebeta red -.2680000* .0082798 .000 -.285395 -.250605 awash Tekishino Axumite red -.1870000* .0095607 .000 -.207086 -.166914 kemila white -.2723333* .0095607 .000 -.292420 -.252247 Gouder red -.0230000* .0095607 .027 -.043086 -.002914 Mn Gebeta white -.0983333* .0082798 .000 -.115729 -.080938 Gebeta red -.0810000* .0082798 .000 -.098395 -.063605 Axumite red awash Tekishino .1870000* .0095607 .000 .166914 .207086 kemila white -.0853333* .0095607 .000 -.105420 -.065247 Gouder red .1640000* .0095607 .000 .143914 .184086 Gebeta white -.0130000 .0082798 .134 -.030395 .004395 Gebeta red .0043333 .0082798 .607 -.013062 .021729 kemila white awash Tekishino .2723333* .0095607 .000 .252247 .292420 Axumite red .0853333* .0095607 .000 .065247 .105420 Gouder red .2493333* .0095607 .000 .229247 .269420 Gebeta white -.2623333* .0082798 .000 -.279729 -.244938 Gebeta red -.2450000* .0082798 .000 -.262395 -.227605 Gouder red awash Tekishino .0230000* .0095607 .027 .002914 .043086 Axumite red -.1640000* .0095607 .000 -.184086 -.143914 kemila white -.2493333* .0095607 .000 -.269420 -.229247

Gebeta red -.0035000 .0025441 .186 -.008845 .001845 awash Tekishino -.0080000* .0031158 .019 -.014546 -.001454 Gebeta white Axumite red .0070000* .0031158 .037 .000454 .013546 kemila white .0110000* .0031158 .002 .004454 .017546 Gouder red .0150000* .0031158 .000 .008454 .021546 Gebeta white .0035000 .0025441 .186 -.001845 .008845 awash Tekishino -.0045000 .0031158 .166 -.011046 .002046 Gebeta red Axumite red .0105000* .0031158 .003 .003954 .017046 kemila white .0145000* .0031158 .000 .007954 .021046 Gouder red .0185000* .0031158 .000 .011954 .025046 Gebeta white .0080000* .0031158 .019 .001454 .014546 Gebeta red .0045000 .0031158 .166 -.002046 .011046 awash Tekishino Axumite red .0150000* .0035978 .001 .007441 .022559 kemila white .0190000* .0035978 .000 .011441 .026559 Gouder red .0230000* .0035978 .000 .015441 .030559 Cr Gebeta white -.0070000* .0031158 .037 -.013546 -.000454 Gebeta red -.0105000* .0031158 .003 -.017046 -.003954 Axumite red awash Tekishino -.0150000* .0035978 .001 -.022559 -.007441 kemila white .0040000 .0035978 .281 -.003559 .011559 Gouder red .0080000* .0035978 .039 .000441 .015559 Gebeta white -.0110000* .0031158 .002 -.017546 -.004454 Gebeta red -.0145000* .0031158 .000 -.021046 -.007954 kemila white awash Tekishino -.0190000* .0035978 .000 -.026559 -.011441 Axumite red -.0040000 .0035978 .281 -.011559 .003559 Gouder red .0040000 .0035978 .281 -.003559 .011559 Gebeta white -.0150000* .0031158 .000 -.021546 -.008454 Gebeta red -.0185000* .0031158 .000 -.025046 -.011954 Gouder red awash Tekishino -.0230000* .0035978 .000 -.030559 -.015441 Axumite red -.0080000* .0035978 .039 -.015559 -.000441 kemila white -.0040000 .0035978 .281 -.011559 .003559

Gebeta red .0020000* .0005092 .001 .000930 .003070 awash Tekishino .0036667* .0006236 .000 .002357 .004977 Gebeta white Axumite red .0030000* .0006236 .000 .001690 .004310 kemila white .0086667* .0006236 .000 .007357 .009977 Gouder red .0083333* .0006236 .000 .007023 .009643 Gebeta white -.0020000* .0005092 .001 -.003070 -.000930 awash Tekishino .0016667* .0006236 .016 .000357 .002977 Gebeta red Axumite red .0010000 .0006236 .126 -.000310 .002310 kemila white .0066667* .0006236 .000 .005357 .007977 Gouder red .0063333* .0006236 .000 .005023 .007643 Gebeta white -.0036667* .0006236 .000 -.004977 -.002357 Gebeta red -.0016667* .0006236 .016 -.002977 -.000357 awash Tekishino Axumite red -.0006667 .0007201 .367 -.002180 .000846 kemila white .0050000* .0007201 .000 .003487 .006513 Gouder red .0046667* .0007201 .000 .003154 .006180 Co Gebeta white -.0030000* .0006236 .000 -.004310 -.001690 Gebeta red -.0010000 .0006236 .126 -.002310 .000310 Axumite red awash Tekishino .0006667 .0007201 .367 -.000846 .002180 kemila white .0056667* .0007201 .000 .004154 .007180 Gouder red .0053333* .0007201 .000 .003820 .006846 Gebeta white -.0086667* .0006236 .000 -.009977 -.007357 Gebeta red -.0066667* .0006236 .000 -.007977 -.005357 kemila white awash Tekishino -.0050000* .0007201 .000 -.006513 -.003487 Axumite red -.0056667* .0007201 .000 -.007180 -.004154 Gouder red -.0003333 .0007201 .649 -.001846 .001180 Gebeta white -.0083333* .0006236 .000 -.009643 -.007023 Gebeta red -.0063333* .0006236 .000 -.007643 -.005023 Gouder red awash Tekishino -.0046667* .0007201 .000 -.006180 -.003154 Axumite red -.0053333* .0007201 .000 -.006846 -.003820 kemila white .0003333 .0007201 .649 -.001180 .001846

Gebeta red -.0721667* .0257407 .012 -.126246 -.018087 awash Tekishino 0E-7 .0315258 1.000 -.066233 .066233 Gebeta white Axumite red -.0240000 .0315258 .456 -.090233 .042233 kemila white .0083333 .0315258 .795 -.057900 .074567 Gouder red .0180000 .0315258 .575 -.048233 .084233 Gebeta white .0721667* .0257407 .012 .018087 .126246 awash Tekishino .0721667* .0315258 .034 .005933 .138400 Gebeta red Axumite red .0481667 .0315258 .144 -.018067 .114400 kemila white .0805000* .0315258 .020 .014267 .146733 Gouder red .0901667* .0315258 .010 .023933 .156400 Gebeta white 0E-7 .0315258 1.000 -.066233 .066233 Gebeta red -.0721667* .0315258 .034 -.138400 -.005933 awash Tekishino Axumite red -.0240000 .0364028 .518 -.100480 .052480 kemila white .0083333 .0364028 .822 -.068146 .084813 Gouder red .0180000 .0364028 .627 -.058480 .094480 Ni Gebeta white .0240000 .0315258 .456 -.042233 .090233 Gebeta red -.0481667 .0315258 .144 -.114400 .018067 Axumite red awash Tekishino .0240000 .0364028 .518 -.052480 .100480 kemila white .0323333 .0364028 .386 -.044146 .108813 Gouder red .0420000 .0364028 .264 -.034480 .118480 Gebeta white -.0083333 .0315258 .795 -.074567 .057900 Gebeta red -.0805000* .0315258 .020 -.146733 -.014267 kemila white awash Tekishino -.0083333 .0364028 .822 -.084813 .068146 Axumite red -.0323333 .0364028 .386 -.108813 .044146 Gouder red .0096667 .0364028 .794 -.066813 .086146 Gebeta white -.0180000 .0315258 .575 -.084233 .048233 Gebeta red -.0901667* .0315258 .010 -.156400 -.023933 awash Tekishino -.0180000 .0364028 .627 -.094480 .058480 Axumite red -.0420000 .0364028 .264 -.118480 .034480 Gouder red kemila white -.0096667 .0364028 .794 -.086146 .066813

Gebeta red .0023333* .0002833 .000 .001738 .002928 awash Tekishino .0018333* .0003469 .000 .001104 .002562 Gebeta white Axumite red .0035000* .0003469 .000 .002771 .004229 kemila white .0051667* .0003469 .000 .004438 .005896 Gouder red .0088333* .0003469 .000 .008104 .009562 Gebeta white -.0023333* .0002833 .000 -.002928 -.001738 awash Tekishino -.0005000 .0003469 .167 -.001229 .000229 Gebeta red Axumite red .0011667* .0003469 .003 .000438 .001896 kemila white .0028333* .0003469 .000 .002104 .003562 Gouder red .0065000* .0003469 .000 .005771 .007229 Gebeta white -.0018333* .0003469 .000 -.002562 -.001104 Gebeta red .0005000 .0003469 .167 -.000229 .001229 awash Tekishino Axumite red .0016667* .0004006 .001 .000825 .002508 kemila white .0033333* .0004006 .000 .002492 .004175 Gouder red .0070000* .0004006 .000 .006158 .007842 Cd Gebeta white -.0035000* .0003469 .000 -.004229 -.002771 Gebeta red -.0011667* .0003469 .003 -.001896 -.000438 Axumite red awash Tekishino -.0016667* .0004006 .001 -.002508 -.000825 kemila white .0016667* .0004006 .001 .000825 .002508 Gouder red .0053333* .0004006 .000 .004492 .006175 Gebeta white -.0051667* .0003469 .000 -.005896 -.004438 Gebeta red -.0028333* .0003469 .000 -.003562 -.002104 kemila white awash Tekishino -.0033333* .0004006 .000 -.004175 -.002492 Axumite red -.0016667* .0004006 .001 -.002508 -.000825 Gouder red .0036667* .0004006 .000 .002825 .004508 Gebeta white -.0088333* .0003469 .000 -.009562 -.008104 Gebeta red -.0065000* .0003469 .000 -.007229 -.005771 Gouder red awash Tekishino -.0070000* .0004006 .000 -.007842 -.006158 Axumite red -.0053333* .0004006 .000 -.006175 -.004492 kemila white -.0036667* .0004006 .000 -.004508 -.002825

Gebeta red -.1850000* .0588382 .006 -.308614 -.061386 awash Tekishino .0448333 .0720618 .542 -.106563 .196229 Gebeta white Axumite red -.3521667* .0720618 .000 -.503563 -.200771 kemila white .0468333 .0720618 .524 -.104563 .198229 Gouder red .0815000 .0720618 .273 -.069896 .232896 Gebeta white .1850000* .0588382 .006 .061386 .308614 awash Tekishino .2298333* .0720618 .005 .078437 .381229 Gebeta red Axumite red -.1671667* .0720618 .032 -.318563 -.015771 kemila white .2318333* .0720618 .005 .080437 .383229 Gouder red .2665000* .0720618 .002 .115104 .417896 Gebeta white -.0448333 .0720618 .542 -.196229 .106563 Gebeta red -.2298333* .0720618 .005 -.381229 -.078437 awash Tekishino Axumite red -.3970000* .0832098 .000 -.571817 -.222183 kemila white .0020000 .0832098 .981 -.172817 .176817 Gouder red .0366667 .0832098 .665 -.138151 .211484 Zn Gebeta white .3521667* .0720618 .000 .200771 .503563 Gebeta red .1671667* .0720618 .032 .015771 .318563 Axumite red awash Tekishino .3970000* .0832098 .000 .222183 .571817 kemila white .3990000* .0832098 .000 .224183 .573817 Gouder red .4336667* .0832098 .000 .258849 .608484 Gebeta white -.0468333 .0720618 .524 -.198229 .104563 Gebeta red -.2318333* .0720618 .005 -.383229 -.080437 kemila white awash Tekishino -.0020000 .0832098 .981 -.176817 .172817 Axumite red -.3990000* .0832098 .000 -.573817 -.224183 Gouder red .0346667 .0832098 .682 -.140151 .209484 Gebeta white -.0815000 .0720618 .273 -.232896 .069896 gebeta red -.2665000* .0720618 .002 -.417896 -.115104 Gouder red awash Tekishino -.0366667 .0832098 .665 -.211484 .138151 Axumite red -.4336667* .0832098 .000 -.608484 -.258849 kemila white -.0346667 .0832098 .682 -.209484 .140151

Gebeta red -.0401667* .0099003 .001 -.060966 -.019367 awash Tekishino .0433333* .0121253 .002 .017859 .068808 Gebeta white Axumite red .0726667* .0121253 .000 .047192 .098141 kemila white .1000000* .0121253 .000 .074526 .125474 Gouder red .1370000* .0121253 .000 .111526 .162474 Gebeta white .0401667* .0099003 .001 .019367 .060966 awash Tekishino .0835000* .0121253 .000 .058026 .108974 Gebeta red Axumite red .1128333* .0121253 .000 .087359 .138308 kemila white .1401667* .0121253 .000 .114692 .165641 Gouder red .1771667* .0121253 .000 .151692 .202641 Gebeta white -.0433333* .0121253 .002 -.068808 -.017859 Gebeta red -.0835000* .0121253 .000 -.108974 -.058026 awash Tekishino Axumite red .0293333 .0140011 .051 -.000082 .058749 kemila white .0566667* .0140011 .001 .027251 .086082 Gouder red .0936667* .0140011 .000 .064251 .123082 PB Gebeta white -.0726667* .0121253 .000 -.098141 -.047192 Gebeta red -.1128333* .0121253 .000 -.138308 -.087359 Axumite red awash Tekishino -.0293333 .0140011 .051 -.058749 .000082 kemila white .0273333 .0140011 .067 -.002082 .056749 Gouder red .0643333* .0140011 .000 .034918 .093749 Gebeta white -.1000000* .0121253 .000 -.125474 -.074526 Gebeta red -.1401667* .0121253 .000 -.165641 -.114692 kemila white awash Tekishino -.0566667* .0140011 .001 -.086082 -.027251 Axumite red -.0273333 .0140011 .067 -.056749 .002082 Gouder red .0370000* .0140011 .017 .007585 .066415 Gebeta white -.1370000* .0121253 .000 -.162474 -.111526 Gebeta red -.1771667* .0121253 .000 -.202641 -.151692 Gouder red awash Tekishino -.0936667* .0140011 .000 -.123082 -.064251 Axumite red -.0643333* .0140011 .000 -.093749 -.034918 kemila white -.0370000* .0140011 .017 -.066415 -.007585 *. The mean difference is significant at the 0.05 level.

Multiple Samples Test Paired Differences t df Sig. (2- Mean Std. Std. Error 95% Confidence Interval of the tailed) Deviation Mean Difference Lower Upper Axumite Red - 6.50000000* .41952354 .17126977 -6.94026295 -6.05973705 -37.952 5 .000

Gouder Red -1.08333333* .53072278 .21666667 -1.64029273 -.52637394 -5.000 5 .004 Kemila Gebeta White -.25000000 .18708287 .07637626 -.44633143 -.05366857 3.273 5 .052 white Gebeta Red -9.83333333* .30767949 .12560962 -10.15622315 -9.51044351 -78.285 5 .000 Awash .45000000* .18708287 .07637626 .25366857 .64633143 5.892 5 .002 Tekishino Gouder Red -5.41666667 .36009258 .14700718 5.03877268 5.79456065 36.846 5 .061

Gebeta White 6.25000000* .41352146 .16881943 5.81603584 6.68396416 37.022 5 .000 Axumite Gebeta Red -3.33333333* .27325202 .11155467 -3.62009374 -3.04657292 -29.881 5 .050 Red Awash Tannin 6.95000000* .52440442 .21408721 6.39967131 7.50032869 32.463 5 .000 Tekishino

Gebeta White -.83333333* .53541261 .21858128 .27145225 1.39521441 3.812 5 .000

Gouder Gebeta Red -8.75000000 .28809721 .11761519 9.05233948 -8.44766052 -74.395 5 .052 Red Awash 1.53333333* .66833126 .27284509 .83196269 2.23470397 5.620 5 .002 Tekishino Gebeta Red -9.58333333* .34302575 .14003968 9.94331678 -9.22334988 -68.433 5 .05 Gebeta Awash White .70000000* .26076810 .10645813 .42634067 .97365933 6.575 5 .001 Tekishino Gebeta Awash 10.28333333* .47923550 .19564707 9.78040652 10.78626015 52.561 5 .000 Red Tekishino *. The mean difference is significant at the 0.05 level.