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Metal distribution in a lysimeter : experimental methods Dickinson, Anthony C. 1983

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METAL DISTRIBUTION IN A LYSIMETER - EXPERIMENTAL METHODS by ANTHONY C. DICKINSON B.Ap.Sc, U n i v e r s i t y Of B r i t i s h Columbia, 1 980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES Department Of C i v i l E n g i n e e r i n g We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1983 © Anthony C. D i c k i n s o n , 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p e r m i s s i o n fo r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of C i v i l E n g i n e e r i n g The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: J u l y 1983 i i A b s t r a c t North Americans have t r a d i t i o n a l l y used l a n d f i l l s as a cheap method of ref u s e d i s p o s a l . Over the past few years governments have s t a r t e d forming l e g i s l a t i o n to prevent f u r t h e r l a n d f i l l i n g of some i n d u s t r i a l wastes. T h i s r e f l e c t s the r e a l i z a t i o n that metals and o r g a n i c s are not always r e t a i n e d by l a n d f i l l s . The l a c k of i n f o r m a t i o n with res p e c t to metal m o b i l i t y has caused many metal wastes to be barred from l a n d f i l l s . T h i s has c r e a t e d a d i s p o s a l problem f o r commercial and i n d u s t r i a l r e f u s e producers. In 1978 a c o - d i s p o s a l p r o j e c t was i n i t i a t e d at the U n i v e r s i t y of B r i t i s h Columbia to determine the p o t e n t i a l f o r enhanced r e t e n t i o n of metals i n a l a n d f i l l . E l e c t r o p l a t i n g sludge and s e p t i c tank pumpings were added to s i x a i r t i g h t l y s i m e t e r s and the l i q u i d e f f l u e n t was monitored f o r Cd, Cr, Fe, N i , Pb and Zn r e l e a s e s . The study d i d not determine s p e c i f i c metal r e t e n t i o n mechanisms. T h e r e f o r e , the study r e p o r t h e r e i n was i n i t i a t e d to e s t a b l i s h experimental procedures to determine: r e l a t i v e metal m o b i l i t y , the r o l e of b a c t e r i a as metal b i n d i n g agents and the importance of n a t u r a l l i g a n d s as metal b i n d i n g agents. Three separate experiments were conducted u s i n g samples which had been removed from one of the UBC c o - d i s p o s a l study l y s i m e t e r s . A technique f o r removing the samples i n a n i t r o g e n atmosphere was developed to keep the samples a n a e r o b i c . By using anaerobic samples any changes caused by exposure t o a i r were avoided. The f i r s t experiment used a p r o g r e s s i v e e x t r a c t i o n technique to determine the r e l a t i v e m o b i l i t y of Cd, Cr, Cu, Fe, N i , Pb, and Zn. Three samples were t e s t e d u s i n g t h i s technique. The samples were suspensions which had been made by adding d e i o n i z e d d i s t i l l e d water to l y s i m e t e r m a t e r i a l . An untreated sample suspension was used as a c o n t r o l . Another was s o n i c a t e d to rupture c e l l s and fragment p a r t i c l e s . The t h i r d sample was aer a t e d to determine the e f f e c t s of exposure to a i r . By comparing the s o n i c a t e d sample data with the c o n t r o l sample data i t may be p o s s i b l e to estimate the mass of metal bound by b a c t e r i a . A comparison of the aerated sample and c o n t r o l sample data should give an upper bound to the p r o p o r t i o n of the metal mass that w i l l remain f i x e d i n a l y s i m e t e r . The e x t r a c t i o n procedure separated the metals i n t o f i v e d i s t i n c t groups: mobile, e a s i l y complexed, o r g a n i c a l l y bound, s t r o n g l y bound and s t a b l e metals. The second experiment was developed to enumerate the b a c t e r i a i n a sample. A quick and a c c u r a t e enumeration technique was r e q u i r e d so an estimate of the metals a s s o c i a t e d with b a c t e r i a c o u l d be made. For t h i s purpose the f l u o r e s c e n t dye 4,6-diamidino-2-phenylindole was s e l e c t e d t o enhance v i s u a l d e t e c t i o n of b a c t e r i a . Enumeration was u n r e l i a b l e because of the r e l a t i v e l y small q u a n t i t y of b a c t e r i a t h a t were det e c t e d i n the c o m plicated mixture of organic m a t e r i a l s . The t h i r d experiment compared the complexing s t r e n g t h of i v n a t u r a l l i g a n d s with t h a t of EDTA, ethanoic a c i d , g l y c i n e , h i s t i d i n e , 8 - h y d r o x y q u i n o l i n e ( o x i n e ) , NTA and o x a l i c a c i d . S t a b i l i t y c o n s t a n t s f o r the expected m e t a l - c h e l a t e complexes were used to form a g r a d i e n t of complexing agents. I t was not p o s s i b l e to determine the presence or absence of n a t u r a l l i g a n d s from the data c o l l e c t e d . The technique may p r o v i d e a method f o r determining metal s p e c i e s i n complex samples. I t was t h e o r e t i c a l l y p o s s i b l e to determine the mass of each metal s p e c i e s by u s i n g c h e l a t i n g agents which vary i n t h e i r a b i l i t y to complex each metal s p e c i e s . The major l i m i t a t i o n of the theory i s the l a c k of an a c c u r a t e method f o r determining the s t a b i l i t y c o n s t a n t s of complexes which are expected to form. With f u r t h e r work the three experimental methods t e s t e d c o u l d be used to determine the metal m o b i l i t y , the mass of metal a s s o c i a t e d with b a c t e r i a , the s t r e n g t h of n a t u r a l l i g a n d s and the metal s p e c i e s i n an anaerobic l a n d f i l l sample. The p r o g r e s s i v e e x t r a c t i o n technique should be m o d i f i e d to use dry o x i d a t i o n of o r g a n i c s to ensure complete o x i d a t i o n . The c h e l a t i o n experiment r e q u i r e s f u r t h e r t e s t i n g before a c h e l a t e g r a d i e n t can be used to determine metal s p e c i a t i o n and l i g a n d s t r e n g t h . The f l u o r e s c e n t dye technique was the l e a s t s u c c e s s f u l of the three experiments t e s t e d . I t may be p o s s i b l e to improve the technique by u s i n g mithramycin i n s t e a d of 4 , 6 -d i a m i d i n o - 2 - p h e n y l i n d o l e , by experimenting with the c o n c e n t r a t i o n of ethanol r e q u i r e d to f i x b a c t e r i a or by u s i n g a V chemical other than sodium pyrophosphate to break down the c e l l u l o s e f i b e r s i n the sample. Once the three techniques have been s u c c e s s f u l l y used to t e s t l a n d f i l l samples i t w i l l be p o s s i b l e to develop more informed l a n d f i l l management p o l i c i e s . v i Table of Contents A b s t r a c t i i L i s t of Tables v i i i L i s t of F i g u r e s ix I. INTRODUCTION 1 A. OBJECTIVES AND RATIONAL 4 B. METHODOLOGY 5 I I . POTENTIAL METAL REMOVAL MECHANISMS IN LANDFILLS 10 A . GENERAL 10 1. LEACHATE STUDIES 10 2. METALS IN BIOLOGICAL TREATMENT SYSTEMS 16 3. METALS IN SOIL 21 4. NATURAL CHELATORS 25 5. BACTERIA AND METALS 30 B. SUMMARY 32 I I I . EXPERIMENTAL PROCEDURES 35 A. SAMPLING 36 1. SAMPLE SOURCE 36 2. LIQUID SAMPLE COLLECTION 37 3. SOLID SAMPLE COLLECTION .38 4. SAMPLE STORAGE 39 5. SAMPLE TREATMENT 40 6. SAMPLE ANALYSIS 41 B. CHELATION PROCEDURE 43 C. METAL EXTRACTION PROCEDURE 49 D. BACTERIAL COUNTS 54 IV. DATA RESULTS AND DISCUSSION 57 A. CHELATE DATA 57 1. CHELATION DATA SET ONE 60 2. CHELATION DATA SET TWO 62 B. EXTRACTION DATA 67 1. PROCEDURE MODIFICATIONS 69 a. Hydrogen Peroxide 69 b. A c i d - d i g e s t i o n 71 c. C e n t r i f u g i n g 72 2. DATA 7 2 a. Mass R a t i o P l o t s 74 b. B a c t e r i a l Counts 83 c. L i q u i d Sample 86 V. CONCLUSIONS AND RECOMMENDATIONS 88 A. CHELATION 88 B. EXTRACTION 90 C. FUTURE RECOMMENDATIONS 92 REFERENCES 95 GLOSSARY 107 APPENDIX A - - PLOTS OF METAL RELEASED VS TIME 112 APPENDIX B - - CO-ORDINATION CHEMISTRY 114 APPENDIX C - - EXOCELLULAR POLYSACCHARIDES 118 APPENDIX D - - CHELATION PROCEDURE 121 APPENDIX E - - EXTRACTION PROCEDURE 123 APPENDIX F - - SAMPLE EXTRACTION 126 1 . LIQUID SAMPLE 1 26 2. SOLID SAMPLE COLLECTION 128 a . D r i l l i n g 1 30 b. Sample M a n i p u l a t i o n 132 APPENDIX G - - RECOMMENDED DAPI PROCEDURE 136 APPENDIX H - - AVAILABLE METHODS 138 APPENDIX I - - DATA AND CALCULATIONS FOR ESTIMATE OF LIQUID SAMPLE VOLUME 1 42 APPENDIX J - - DATA AND CALCULATIONS FOR ESTIMATE OF SOLID SAMPLE VOLUME 144 APPENDIX K - - DANGEROUS PROPERTIES OF CHEMICALS 145 APPENDIX L - - FLUORESCENT DYES 146 APPENDIX M - - CONCENTRATION VS ABSORBANCY PLOTS FOR CHELATION DATA SET ONE 152 APPENDIX N - - PLOT OF LOG10( STABILITY CONSTANT) VS LIGAND 1 56 APPENDIX 0 - - PERCENT SPIKE VS ABSORBANCY PLOTS FOR CHELATION DATA SET TWO 157 APPENDIX P - - CALCULATIONS FOR ESTIMATES OF COMPLEXED METAL 159 APPENDIX Q - - PSEUDO MASS RATIO CALCULATIONS FOR THE CHELATION EXPERIMENT 161 APPENDIX R - - METAL SPECIES FORMULAE AND DERIVATIONS ...163 APPENDIX S - - CONCENTRATION VS ABSORBANCY PLOTS FOR EXTRACTION DATA 165 APPENDIX T - - MASS RATIO CALCULATIONS FOR THE EXTRACTION EXPERIMENT 170 APPENDIX U - - CONCENTRATION VS ABSORBANCY PLOTS FOR LIQUID DATA 173 APPENDIX V - - REDUCTION POTENTIALS 176 APPENDIX W - - LIST OF SYMBOLS USED 177 v i i i L i s t of Tables I. L i k e l y P r e c i p i t a t e s 19 I I . Chemical And P h y s i c a l S o i l Leaching Parameters ...22 I I I . Metal Ions In S o i l 24 IV. Summary Of S o i l E f f e c t s 25 V. LoglO S t a b i l i t y Constants For Humics - I 27 VI. LoglO S t a b i l i t y Constants For Humics - II 28 V I I . LoglO S t a b i l i t y Constants - PGA 29 V I I I . B i o f l o c - Metal I n t e r a c t i o n s 32 IX. J a r r e l Ash 810 Operating Parameters 42 X. LoglO S t a b i l i t y Constants 44 XI. A p p l i e d And Measured Metals In Lysimeter 45 X I I . C o n c e n t r a t i o n Of Metals In Spike S o l u t i o n s 46 X I I I . S o l u b i l i t y Of Chromium Compounds 46 XIV. Moles Of C h e l a t i n g Agent 49 XV. E x t r a c t i o n Procedure 52 XVI. Expected Complex C o n c e n t r a t i o n s 59 XVII. Pseudo Mass R a t i o s For Data Set One 61 XVIII. Pseudo Mass R a t i o s For Data Set Two 63 XIX. Species In Spike S o l u t i o n s 67 XX. Mass Released From The Lysimeter 87 XXI. Metal C o n c e n t r a t i o n s In L i q u i d Sample 87 XXII. Ligand Groups 115 XXIII. O r b i t a l C o n f i g u r a t i o n s .115 XXIV. Chemical R a d i c a l s Of Charged E x o c e l l u l a r Polymers 118 XXV. C o n c e n t r a t i o n Of Metals In The La s t Leachate Sample 142 XXVI. I n i t i a l Values For C a l c u l a t i o n Of Complex C o n c e n t r a t i o n s 160 XXVII. C a l c u l a t i o n Of Rx 161 XXVIII. C a l c u l a t i o n s Of Xm 162 XXIX. Metal C o n c e n t r a t i o n s In The Leachate Sample 173 XXX. Reduction P o t e n t i a l s 176 ix L i s t of F i g u r e s 1. P l o t Of Metal Release Vs Time 4 2. C r o s s - s e c t i o n Of Lysimeter 37 3. E x t r a c t i o n Funnel 50 4. Flow Chart Of The C h e l a t i o n Procedure 51 5. Flow Chart Of The E x t r a c t i o n Procedure 53 6. Flow Chart Of The F l u o r e s c e n t Dye Technique 56 7. C a l c u l a t i o n s Of Pseudo Mass R a t i o s For Data Set Two ..63 8. L o g 1 0 ( S t a b i l i t y Constant) Vs Ligand 65 9. P l o t s Of Xm Vs Ligand 66 10. Mass R a t i o C a l c u l a t i o n 73 11. E x t r a c t e d Metal Vs E x t r a c t i o n Phase 75 12. Photographs Of S l i d e P r e p a r a t i o n s 85 13. Ligand Bonding S i t e s 116 14. L i q u i d Sample C o l l e c t i o n F l a s k 127 15. Sampling Box 129 16. D r i l l B i t s 131 17. Sample Removal Flow Chart 133 1 8 . Blender L i d 133 1 I . INTRODUCTION North Americans have t r a d i t i o n a l l y used l a n d f i l l s f o r refu s e d i s p o s a l . When re f u s e i s d e f i n e d as an a r b i t r a r y mixture of household, garden, food, commercial and some i n d u s t r i a l wastes i t i s c l e a r that l a r g e volumes of waste have been l a n d f i l l e d . The growing r e a l i z a t i o n t h a t l a n d f i l l s do not r e t a i n some metals and o r g a n i c s has caused governments to form l e g i s l a t i o n r e s t r i c t i n g the types of waste that can be l a n d f i l l e d . T h i s has caused a d i s p o s a l problem f o r i n d u s t r i a l and commercial re f u s e g e n e r a t o r s . When r e s e a r c h e r s f i r s t s t a r t e d s t u d y i n g l a n d f i l l s there was no e f f o r t d i r e c t e d towards metal s t u d i e s because metals were not co n s i d e r e d t o be a problem. When l e a c h a t e s were analyzed f o r metals i t was found that metal contaminants c o u l d be found i n some l e a c h a t e s . Although le a c h a t e cannot be c h e m i c a l l y d e f i n e d i t has a few d i s t i n g u i s h i n g c h a r a c t e r i s t i c s . I t i s a dark c o l o u r e d , unpleasant s m e l l i n g , l i q u i d which d r a i n s from a l a n d f i l l when the l i q u i d h o l d i n g c a p a c i t y of a s i t e i s exceeded. Leachates have been found to vary from a c i d i c to b a s i c s o l u t i o n s depending upon the c o n d i t i o n s and c o n s t i t u e n t s of the l a n d f i l l . The volume of leac h a t e which forms i s a f u n c t i o n of the annual p r e c i p i t a t i o n and i n f i l t r a t i o n r a t e at the s i t e . An a c i d i c l e a c h a t e can be caused by organic a c i d s which are produced as by-products of the orga n i c degradation of r e f u s e . The org a n i c a c i d s mix with the l i q u i d p e r c o l a t i n g through a l a n d f i l l l e a c h i n g metals and a c i d s o l u b l e compounds from the r e f u s e . The 2 net r e s u l t being the g e n e r a t i o n of an obnoxious l i q u i d suspension c o n t a i n i n g d i s s o l v e d and p a r t i c u l a t e matter. T h i s l i q u i d mixture d r a i n s from l a n d f i l l s and i s c a l l e d l e a c h a t e . The metals i n l e a c h a t e can cause environmental contamination and lea c h a t e treatment problems. In 1978, to address the problem of metal contaminants i n l a n d f i l l s , r e s e a r c h e r s at the U n i v e r s i t y of B r i t i s h Columbia s t a r t e d to study the c o - d i s p o s a l of e l e c t r o p l a t i n g sludge with s e p t i c tank pumpings. The study was i n i t i a t e d to t e s t the hypothesis that s e p t i c tank pumpings enhance metal r e t e n t i o n i n a l a n d f i l l . Instead of using a l a n d f i l l to t e s t the r e t e n t i o n h y p o t h e s i s , the UBC group used s i x l a b o r a t o r y models of r e f u s e l e a c h i n g , c a l l e d l y s i m e t e r s . The l i q u i d which d r a i n e d from the l y s i m e t e r s ("leachate") was analyzed f o r i t s metals content using an atomic a b s o r p t i o n spectrometer. The metal c o n c e n t r a t i o n s i n the l y s i m e t e r l e a c h a t e and the volume of l e a c h a t e c o l l e c t e d were used to estimate the mass of metal which had escaped from the l y s i m e t e r s . The l y s i m e t e r s used i n the c o - d i s p o s a l study were c o n s t r u c t e d by packing 300 mm. diameter PVC ( p o l y v i n y l c h l o r i d e ) p i p e s with s o i l and r e f u s e . A l a y e r of pea g r a v e l was p l a c e d at the bottom of the PVC p i p e s . A f t e r p l a c i n g the g r a v e l 15 cm. l a y e r s of r e f u s e were added to the p i p e s . The f i n a l l a y e r was a l a y e r of s o i l . E l e c t r o p l a t i n g s l u d g e s 1 and s e p t i c 1. Most of the metals were i n p r e c i p i t a t e form but some s o l u b l e forms of cadmium were added from a cadmium p l a t i n g bath to boost cadmium l e v e l s . 3 tank pumpings were added to the l y s i m e t e r s d u r i n g packing. Each l y s i m e t e r was s e a l e d from a i r and watered an average of 96 ml./day to simulate a shallow l a n d f i l l r e c e i v i n g roughly 1.0 m. of p r e c i p i t a t i o n per year. The masses of e l e c t r o p l a t i n g sludges and s e p t i c tank pumpings were v a r i e d to e v a l u a t e the enhancement e f f e c t s of s e p t i c tank pumpings. Data were c o l l e c t e d from the c o - d i s p o s a l l y s i m e t e r s over a three year p e r i o d . The c o n c e n t r a t i o n s of Cd, Cr, Fe, N i , Pb, and Zn i n the l y s i m e t e r l e a c h a t e s were determined and p l o t t e d . (An example p l o t i s shown i n F i g u r e 1.) The r a t e of metal r e l e a s e from the l y s i m e t e r s s t a b i l i z e d a f t e r roughly two y e a r s . Mass balance c a l c u l a t i o n s proved t h a t v a r y i n g p r o p o r t i o n s of the e l e c t r o p l a t i n g metals were r e t a i n e d i n the l y s i m e t e r s a f t e r three years of o p e r a t i o n . The study brought to l i g h t the f o l l o w i n g q u e s t i o n s about metal r e t e n t i o n i n l y s i m e t e r s : a) Were the metal p r e c i p i t a t e s i n the e l e c t r o p l a t i n g sludge a s s i m i l a t e d by the l y s i m e t e r contents or had they remained u n a l t e r e d i n the l y s i m e t e r s ? b) How long would the remaining metals stay i n the l y s i m e t e r s ? c) What p r o p o r t i o n of the remaining metals were i n e r t ? Mobile? d) Are any of the metals remaining i n the l y s i m e t e r s a s s o c i a t e d with b a c t e r i a ? e) Are organic c h e l a t i n g agents important metal r e t e n t i o n mechanisms i n a l y s i m e t e r ? No attempt was made to s o l v e q u e s t i o n 'b' above because an a c c e p t a b l e technique f o r p r o j e c t i n g the s t a b i l i t y of the r e s i d u a l metals would r e q u i r e a time study over a p e r i o d of F i g u r e 1 - P l o t Of Metal Release Vs Time 4 T I M E I D f l T S I (X101 ) " 1 G U R E C A D M I U M - C O N C V S . T I M E - C D ! - C . O . r . H . J . K From Atwater et a l . 1981 Note: The r e s t of the p l o t s are i n Appendix A. years. Question 'a' above c o u l d be r e s o l v e d by u s i n g an e l e c t r o n microscope to compare the c r y s t a l s t r u c t u r e of metal p r e c i p i t a t e s i n the e l e c t r o p l a t i n g waste and the l y s i m e t e r samples. T h i s p o s s i b i l i t y was not checked. Questions 'c', 'd' and 'e' cannot be r e s o l v e d u n t i l new experimental techniques are developed. These q u e s t i o n s p r o v i d e d the stimulus f o r the res e a r c h c o n t a i n e d i n t h i s r e p o r t . A. OBJECTIVES AND RATIONAL Methods f o r a n a l y z i n g l a n d f i l l samples are r e q u i r e d f o r informed management and p o l i c y f o r m a t i o n . T h i s study was devi s e d to p r o v i d e a method f o r determining the m o b i l i t y of metals, the importance of b a c t e r i a i n r e t a i n i n g metals, and the s t r e n g t h of n a t u r a l l i g a n d s i n a l a n d f i l l l e a c h a t e . Metal r e t e n t i o n i n l a n d f i l l s i s an important t o p i c because of the c o s t and p o l l u t i o n i m p l i c a t i o n s . With t h i s i n mind the o b j e c t i v e s of t h i s study were: 5 a) To develop an experimental method which c a t e g o r i z e s metals i n a l a n d f i l l sample a c c o r d i n g to t h e i r m o b i l i t y . b) To develop a t e s t to determine the mass of metals a s s o c i a t e d with b a c t e r i a i n a l a n d f i l l sample. c) To develop a technique f o r measuring the s t r e n g t h of n a t u r a l l i g a n d s i n a l a n d f i l l . B. METHODOLOGY A l l three o b j e c t i v e s are r e l a t e d to the d i s t r i b u t i o n of metals i n a l a n d f i l l . A f t e r an i n i t i a l review of the l i t e r a t u r e i t was evident that the samples used f o r t e s t i n g should be anae r o b i c . T h i s would a v o i d any changes i n metal d i s t r i b u t i o n which would be caused by changes i n pH and o x i d a t i o n - r e d u c t i o n p o t e n t i a l . I t i s d i f f i c u l t to o b t a i n anaerobic samples from a l a n d f i l l so a l y s i m e t e r was used f o r a sample source. A l y s i m e t e r which was used i n the UBC c o - d i s p o s a l study was s e l e c t e d f o r t h i s purpose because i t was s e a l e d from a i r and was packed with metal sp i k e d r e f u s e . The l y s i m e t e r contents were s i m i l a r enough to l a n d f i l l r e f u s e that they c o u l d be used to develop experimental methods f o r t e s t i n g l a n d f i l l samples. The refu s e was spik e d with e l e c t r o p l a t i n g wastes so there should be no d i f f i c u l t y d e t e c t i n g metals i n the samples. A l s o , l e a c h i n g data was a v a i l b a l e to i n d i c a t e the mass of metal which had migrated out of the l y s i m e t e r . A l i t e r a t u r e survey was conducted to determine the f i n d i n g s of other l y s i m e t e r s t u d i e s and to i d e n t i f y p o s s i b l e experimental techniques. I t was apparent that l i t t l e has been done to determine which mechanisms cause metal r e t e n t i o n i n l a n d f i l l s or 6 l y s i m e t e r s . Most l y s i m e t e r s t u d i e s attempt to c h a r a c t e r i z e the l e a c h a t e produced when v a r i o u s e x t e r n a l f a c t o r s (eg . p r e c i p i t a t i o n ) or i n t e r n a l f a c t o r s (eg . shredd ing ) are a l t e r e d . The l i t e r a t u r e s e a r c h was broadened to i n c l u d e a e r o b i c or a n a e r o b i c s ludge s t u d i e s and s o i l s t u d i e s which at tempted to i d e n t i f y metal r e t e n t i o n mechanisms. The f o l l o w i n g f a c t o r s were i d e n t i f i e d by t h i s r e s e a r c h e r and the l i t e r a t u r e as p o t e n t i a l meta l r e t e n t i o n mechanisms in a l a n d f i l l or l y s i m e t e r : a) P r e c i p i t a t i o n by h y d r o x y l , c a r b o n a t e , and s u l p h i d e a n i o n s . b) I n s o l u b l e complex f o r m a t i o n . c ) C o n v e r s i o n of meta l s p e c i e s . d) S o r p t i o n to e x t r a c e l l u l a r p o l y s a c c h a r i d e s . e) S o r p t i o n to c e l l w a l l s . f) I n t r a c e l l u l a r u p t a k e . g) S o r p t i o n to s o l i d s . A f t e r c o m p i l i n g a l i s t of p o s s i b l e m e t a l r e t e n t i o n mechanisms, the l i t e r a t u r e was reviewed f o r e x p e r i m e n t a l p r o c e d u r e s which would i s o l a t e meta ls a s s o c i a t e d w i t h one or more of the mechanisms. The p r o c e d u r e s which r e q u i r e d l e s s t ime i n the l a b o r a t o r y and which were s imple to per form were f a v o u r e d . Techniques for sampl ing and t e s t i n g l y s i m e t e r c o n t e n t s were sought . An a n a e r o b i c sampl ing and t e s t i n g procedure was d e v i s e d to a v o i d sample changes caused by exposure to a i r . A l y s i m e t e r from the UBC c o - d i s p o s a l s tudy which had a moderate a d d i t i o n of s e p t i c tank pumpings and a l a r g e a d d i t i o n 7 of e l e c t r o p l a t i n g wastes was s e l e c t e d as a sample source. ( T h i s l y s i m e t e r was l a b e l l e d l y s i m e t e r 'F' i n the UBC c o - d i s p o s a l study.) T h i s study c o n c e n t r a t e d upon the development of a procedure which estimated metal m o b i l i t y because the m o b i l i t y i n d i c a t e s the type of r e t e n t i o n mechanisms. Three experimental procedures were chosen for. t h i s study. One experiment used a s e r i e s of p r o g r e s s i v e e x t r a c t i o n s t o determine the r e l a t i v e m o b i l i t y of metals i n a l y s i m e t e r . A second experiment used a f l u o r e s c e n t dye (4,6-diamidino-2-p h e n y l i n d o l e ) to count b a c t e r i a , so the importance of b a c t e r i a as metal r e t e n t i o n mechanisms, c o u l d be e v a l u a t e d . The t h i r d experiment used a g r a d i e n t of c h e l a t i n g agents to estimate the s t r e n g t h of n a t u r a l c h e l a t i n g agents. The p r o g r e s s i v e e x t r a c t i o n technique was f i r s t developed by Engler (1977) to e x t r a c t metals from r i v e r bottom sediments. The technique c a t e g o r i z e d metals a c c o r d i n g to t h e i r r e l a t i v e m o b i l i t y and allowed a d e t e r m i n a t i o n of the metal mass i n each c a t e g o r y . Samples were ob t a i n e d from a suspension of l y s i m e t e r m a t e r i a l which was made by b l e n d i n g a mixture of d e i o n i z e d d i s t i l l e d water and l y s i m e t e r c o n t e n t s . Three samples of l y s i m e t e r suspension were used. A sample which was not p r e t r e a t e d was used as a c o n t r o l sample. The other two samples were p r e t r e a t e d p r i o r t o the p r o g r e s s i v e e x t r a c t i o n s . The f i r s t pretreatment bubbled a i r through a sample to o x i d i z e the sample and change the pH. A comparison of the c o n t r o l sample and the a e r a t e d sample data should g i v e an estimate of the long term 8 metal s t a b i l i t y . The second pretreatment s o n i c a t e d a sample to rupture c e l l s and break up p a r t i c l e s . A comparison of the c o n t r o l sample and the s o n i c a t i o n sample data c o u l d be used to estimate the i n t r a c e l l u l a r metal uptake. The e x t r a c t i o n procedure should separate the mobile, c a t i o n exchanged, complexed and p r e c i p i t a t e d metals. I t w i l l not d i f f e r e n t i a t e between metals sorbed to c e l l s , e x t r a c e l l u l a r p o l y s a c c h a r i d e s and p a r t i c l e s . S t a b l e i n o r g a n i c complexes may not be separated from p r e c i p i t a t e s but o r g a n i c a l l y bound metals are separated from i n o r g a n i c a l l y bound metals. The second experiment was a technique f o r enumerating b a c t e r i a . Whole c e l l counts c o u l d be made before and a f t e r sample s o n i c a t i o n to allow a q u a n t i t a t i v e d e t e r m i n a t i o n of the metals a s s o c i a t e d with b a c t e r i a . A l i t e r a t u r e search was undertaken to determine the most recent methods f o r cou n t i n g b a c t e r i a . The dye 4,6-diamidino-2-phenylindole (DAPI) was s e l e c t e d f o r t h i s study. I t i s s p e c i f i c f o r b a c t e r i a l DNA and f l u o r e s c e s b r i g h t blue when i r r a d i a t e d with u l t r a v i o l e t l i g h t . Normally b a c t e r i a cannot be observed using an o p t i c a l microscope but the dye enhances v i s u a l i z a t i o n g r e a t l y , to allow o p t i c a l d e t e c t i o n and photography. A f l u o r e s c e n t s t a i n i n g technique u s i n g DAPI was developed, with the he l p of r e s e a r c h e r s at Simon F r a s e r U n i v e r s i t y , to enumerate the b a c t e r i a i n l y s i m e t e r samples. Some method of enhancement was necessary because of the complex nature of the samples and the r e l a t i v e l y small number of b a c t e r i a that were d e t e c t e d . 9 The t h i r d experiment was designed to compare the complexation s t r e n g t h of n a t u r a l l i g a n d s with c h e l a t e s of known complexation s t r e n g t h . S t a b i l i t y c o n s t a n t s of the expected m e t a l - c h e l a t e complexes were used to compare the s t r e n g t h of e t h y l e n e - d i a m i n e - t e t r a - a c e t i c a c i d (EDTA), ethanoic a c i d , g l y c i n e , h i s t i d i n e , 8 - h y d r o x y q u i n o l i n e ( o x i n e ) , n i t r i l o - t r i -a c e t i c a c i d (NTA) and o x a l i c a c i d with n a t u r a l l i g a n d s . The c h e l a t i n g agents were s e l e c t e d to form a g r a d i e n t of complexation s t r e n g t h s ranging from weak agents (eg. a c e t i c a c i d ) to strong ones (eg. EDTA). Examples of anaerobic sampling from l y s i m e t e r s were not found i n the l i t e r a t u r e . T h i s study was assumed to be one of the f i r s t to t r y anaerobic sample removal from a l y s i m e t e r . Anaerobic samples were used f o r t e s t i n g t o more a c c u r a t e l y r e f l e c t c o n d i t i o n s i n a l a n d f i l l . H o p e f u l l y t h i s w i l l h e l p to gain a b e t t e r understanding of the metal r e t e n t i o n mechanisms i n l a n d f i l l s without the d i f f i c u l t y of removing anaerobic samples from them. T h i s study was p o s s i b l e because a three year o l d , a i r t i g h t l y s i m e t e r with metal a d d i t i o n s was a v a i l a b l e f o r t e s t i n g and sampling. 10 I I . POTENTIAL METAL REMOVAL MECHANISMS IN LANDFILLS A. GENERAL The complexity of a l a n d f i l l becomes apparent when one s t a r t s to review l a n d f i l l s t u d i e s . Many f a c e t s of l a n d f i l l s have been s t u d i e d i n an attempt to understand what happens to re f u s e when i t i s i n t e r r e d . Some s t u d i e s have t r i e d to modify the form of the ref u s e and others have v a r i e d e x t e r n a l f a c t o r s i n an attempt to a f f e c t r e f u s e d e g r a d a t i o n . Most l a n d f i l l s t u d i e s analyze the products of a l a n d f i l l ( l e a c h a t e or gas) to monitor the i n t e r n a l workings of a l a n d f i l l . To date no one understands the i n t e r n a l workings of l a n d f i l l s w e l l enough to p r e d i c t the type of leachate which w i l l be produced. With these f a c t s i n mind, s t u d i e s which c o n s i d e r e d metals i n l e a c h a t e , sludge or s o i l were reviewed. 1. LEACHATE STUDIES Many l a n d f i l l s t u d i e s o n l y analyze the le a c h a t e which i s generated. A few simple v a r i a b l e s are known to have an e f f e c t upon the volume and type of l e a c h a t e that escapes a l a n d f i l l . G e n e r a l l y the c o n c e n t r a t i o n of i n o r g a n i c and organic compounds i n l e a c h a t e d e c l i n e s over time a f t e r a peak c o n c e n t r a t i o n has been reached. R a i n f a l l a f f e c t s the l e a c h i n g r a t e and the volume of l e a c h a t e generated. The h y d r a u l i c g r a d i e n t and s o i l c o n d i t i o n s surrounding a s i t e a f f e c t the l e a c h a t e a f t e r i t has been generated. In order to study l e a c h a t e s some re s e a r c h e r s have c o n s t r u c t e d r e f u s e l e a c h i n g models c a l l e d l y s i m e t e r s . Lysimeters are g e n e r a l l y q u i t e small compared to a l a n d f i l l 11 and there i s some c o n t r o v e r s y over t h e i r v a l i d i t y as models of re f u s e l e a c h i n g i n l a n d f i l l s . They are c o n s t r u c t e d by adding a l t e r n a t i n g l a y e r s of s o i l and r e f u s e with s o i l as the f i r s t and l a s t l a y e r . The bottom and s i d e s must be s e a l e d from a i r to si m u l a t e ground c o n d i t i o n s . Most ' r e s e a r c h e r s apply water to sim u l a t e p r e c i p i t a t i o n i n t h e i r area and then study the leac h a t e generated by the l y s i m e t e r s . By b u i l d i n g l y s i m e t e r s , the con t e n t s can be c a t e g o r i z e d and v a r i a b l e s such as compaction or shredding can be c o n t r o l l e d t o determine t h e i r e f f e c t s upon l e a c h a t e g e n e r a t i o n . A l y s i m e t e r study done i n I s r a e l (Raveh and Avnimelech 1979) c h a r a c t e r i z e d the l o c a l wastes which go i n t o l a n d f i l l s . They noted that the average percent food waste i n I s r a e l (50%) was much higher than the average rep o r t e d f o r t y p i c a l U.S. r e f u s e (14%). They estimated the average percentage of food waste, paper p l u s c l o t h , p l a s t i c s , g l a s s , metal cans and le a v e s p l u s wood i n I s r a e l i r e f u s e . Then they used those f i g u r e s to c o n s t r u c t t h e i r l y s i m e t e r s . Raveh and Avnimelech measured the carbon content of l e a c h a t e samples to c h a r a c t e r i z e each l y s i m e t e r . They found that an e x p o n e n t i a l curve c o u l d be f i t t e d to t h e i r data. C = a - b 1 C=rng. carbon/ml. t=time i n seasons (Two seasons/year), a, b=constants The c o n s t a n t s v a r i e d between l y s i m e t e r s due to the d i f f e r e n t r e f u s e d e n s i t i e s and r a i n f a l l r a t e s . They f e l t that the high c o r r e l a t i o n between the data and the ex p o n e n t i a l f u n c t i o n could be used to d e s c r i b e the org a n i c decay i n the l y s i m e t e r . Raveh 1 2 and Avnimelech (1979) found that i n c r e a s i n g the d e n s i t y and shredding the refuse tended t o : slow the r a t e of organic decay, i n c r e a s e the d i s s o l v e d metals, decrease the pH, and i n c r e a s e the volume of water r e t a i n e d . The decay of i r o n and manganese c o n c e n t r a t i o n i n t h e i r l e a c h a t e c o r r e l a t e d with an i n c r e a s e i n pH and a decrease i n o r g a n i c a c i d s . When they analyzed the i r o n data they found that the c o n c e n t r a t i o n s were higher than s o l u b i l i t y data would p r e d i c t (Ksp = 1.1*10" 1°). Raveh and Avnimelech b e l i e v e d that the d i s c r e p a n c y was due to c h e l a t i n g agents i n the l e a c h a t e . They f u r t h e r p o s t u l a t e d that the c h e l a t i n g agents caused the d i s s o l u t i o n and s t a b i l i z a t i o n of other heavy metals and may even e x t r a c t heavy metals from the s o i l surrounding a l a n d f i l l . In another l y s i m e t e r study, Kinman and Walsh (1980) v a r i e d pH, p r e w e t t i n g , i n f i l t r a t i o n , sewage sludge l o a d i n g and i n d u s t r i a l waste l o a d . They measured metal c o n c e n t r a t i o n s i n each l y s i m e t e r by sampling l e a c h a t e from s e v e r a l sample p o r t s , over a f o r t y - s i x month p e r i o d . They compared the metal r e l e a s e s of l y s i m e t e r s with i n d u s t r i a l sludge and sewage sludge loads to c o n t r o l l y s i m e t e r s which had no sludge l o a d s . Cadmium, n i c k e l , chromium and z i n c were r e l e a s e d , from l y s i m e t e r s with i n d u s t r i a l sludges, at the same r a t e or l e s s than the c o n t r o l l y s i m e t e r s . Iron r e l e a s e s were l e s s than c o n t r o l i n a l l cases except the sewage sludge l y s i m e t e r s . The authors were not s p e c i f i c when r e p o r t i n g the l e a d r e l e a s e s . Some of the l y s i m e t e r s with sludge showed an i n c r e a s e d l e a d r e l e a s e with time but i t i s not known i f a l l the sludge l y s i m e t e r s responded s i m i l a r l y . Copper 13 r e l e a s e s were prevented by sewage sludge a d d i t i o n s . In a l l other l y s i m e t e r s copper c o n c e n t r a t i o n s were a f u n c t i o n of sample l o c a t i o n . A study by Atwater et a l . (1981) c o n s i d e r e d changes in l e a c h a t e composition as a f u n c t i o n of time, s e p t i c tank pumpings lo a d and e l e c t r o p l a t i n g waste l o a d . They used l y s i m e t e r s to simulate a shallow l a n d f i l l on an impervious s o i l l a y e r . During three years of l y s i m e t e r o p e r a t i o n , cadmium, chromium, and e s p e c i a l l y l e a d a t t e n u a t i o n were a f f e c t e d by s e p t i c tank pumpings while i r o n , n i c k e l and z i n c were r e l a t i v e l y u n a f f e c t e d . Atwater et a l . s t a t e d that the i n i t i a l s o l u b i l i t y of metals in the e l e c t r o p l a t i n g sludge c o u l d mask the r e t e n t i o n e f f e c t of s e p t i c tank pumpings. They went f u r t h e r p o s t u l a t i n g three p o s s i b l e mechanisms f o r metal a t t e n u a t i o n : i n t r a c e l l u l a r uptake, the formation of i n s o l u b l e s u l p h i d e s and complexation with or g a n i c c h e l a t i n g agents. Pohland and Gould (1980) attempted to understand r e a c t i o n s i n a l a n d f i l l by studying l y s i m e t e r s and e q u i l i b r i a e quations. T h e i r study was i n i t i a t e d so c o n t r o l s t r a t e g i e s f o r management and r e g u l a t i o n of l a n d f i l l s c o u l d be developed. They s t u d i e d s u l p h i d e , hydroxyl and carbonate r e a c t i o n s to determine the f a t e of heavy metals i n a s i t e c o n t a i n i n g m u n i c i p a l and i n d u s t r i a l wastes. Sulphides should dominate the e q u i l i b r i u m r e a c t i o n s because of p r e c i p i t a t e formation with Z n + 2 , P b + 2 , C d + 2 , C u + 2 and to a l e s s e r degree F e + 2 . Chromium should form p a r t i a l l y s o l u b l e h y d r o x y l compounds. At low s u l p h i d e l e v e l s , carbonate p r e c i p i t a t e s of cadmium and l e a d should form while copper w i l l 14 probably form a hydroxy-carbonate compound. Some e f f o r t s have been made to c o n s t r u c t a chemical analogue of leachate to help form t h e o r e t i c a l models of l a n d f i l l p r o c e s s e s . In order to form an analogue, the complexing a b i l i t y , i o n i c s t r e n g t h , pH and r e d u c t i o n - o x i d a t i o n p o t e n t i a l are u s u a l l y modelled. S t a n f o r t h and Stegmann (1979) attempted to s p e c i f y a s y n t h e t i c leachate but they were unable to model b i o l o g i c a l aspects and s o r p t i o n c h a r a c t e r i s t i c s of the p a r t i c u l a t e matter i n l e a c h a t e . However, s y n t h e t i c l e a c h a t e s can supply very g e n e r a l comments about some of the chemistry of l e a c h a t e s . S t a n f o r t h and Stegman (1979) assumed that amine p r o t e i n s and the hydroxyl groups a s s o c i a t e d with humic and f u l v i c a c i d s were the major complexing agents i n l e a c h a t e s . They concluded that the s o l u b i l i t y of metals i n c r e a s e d as the a c t i v i t y and pH of the s o l u t i o n decreased. T h e i s and R i c h t e r (1979) used a computer program to c a l c u l a t e e q u i l i b r i u m and complexation s t a t e s i n ash pond l e a c h a t e s . They a c i d - d i g e s t e d ash pond l e a c h a t e s to determine t o t a l metals, then assumed that Fe, Mn and S i oxides were major s o r b i n g s i n k s . T h e i r computer c a l c u l a t i o n s showed that Cd, Ni and Zn s o l u b i l i t i e s were c o n t r o l l e d by the a d s o r p t i o n c a p a c i t y of Fe and Mn oxides while Cr, Cu and Pb c o n c e n t r a t i o n s were c o n t r o l l e d by p r e c i p i t a t e s o l u b i l i t i e s . When they t e s t e d l e a c h a t e which had passed through s o i l , they found that Zn, Cd and Ni were s t r o n g l y adsorbed by i r o n oxides and to a l e s s e r degree by manganese o x i d e s . Copper p r e c i p i t a t e d out of the lea c h a t e as copper carbonate' (CuC0 3) while l e a d formed the 15 p a r t i a l l y s o l u b l e p r e c i p i t a t e l e a d carbonate (PbC0 3). Chromium formed hydroxyl complexes which are s o l u b l e when the pH > 7.5 (Cr(OH) t t" forms). A l l the metals that they t e s t e d formed weak sulphate complexes. T h e i s and R i c h t e r ' s models were most s t r o n g l y i n f l u e n c e d by pH, sulphate and carbonate c o n c e n t r a t i o n s . A l s o , the r e a c t i o n s with chromium were s t r o n g l y dependent upon the o x i d a t i o n s t a t e . For example, Cr(VI) r e a c t s w i t h d i s s o l v e d s u l p h i d e s and s u l p h y d r y l groups but C r ( l I I ) does not. A l s o , C r ( I I l ) sorbs to s o l i d s and i s o x i d i z e d by Mn oxides (Schroeder and Lee 1975). The pH i s c r i t i c a l as Cr(VI) i s reduced by F e ( l l ) while Fe(OH) 3 sorbs C r ( 1 1 1 ) . When S p o s i t o (1981) was c o n s t r u c t i n g a computer model of heavy metal i n t e r a c t i o n s i n s o l u t i o n , he was unable to obt a i n s t a b i l i t y c onstants f o r f u l v i c a c i d s . The model he used c o n s i d e r e d complexation and s o l u b i l i t y equations to p r e d i c t metal s p e c i e s . As he was more i n t e r e s t e d i n f r e s h water i n t e r a c t i o n s , the r e d u c t i o n - o x i d a t i o n p o t e n t i a l s were not c o n s i d e r e d . C o - o r d i n a t i o n chemistry i n d i c a t e d that the t o x i c i t y of metals i n c r e a s e s as the o x i d a t i o n s t a t e , e l e c t r o n e g a t i v i t y , i o n i c s i z e , p o l a r i z a b i l i t y , and/or a f f i n i t y of metal ions f o r s u l f i d e ions and organic sulphur i n c r e a s e . S p o s i t o (1981) was unable to make many c o n c l u s i o n s due to the l a c k of r e l i a b i l i t y i n computer models. U n t i l a r e l i a b l e method f o r determining the s t a b i l i t y c o n s t a n t s of heterogenous l i g a n d s can be found, models w i l l o n l y serve as guesses. Many leac h a t e s t u d i e s have been undertaken but so f a r none 16 have been able to p r e d i c t metal r e l e a s e s . Some researches have proposed mechanisms which may be r e g u l a t i n g metal r e l e a s e but the l a r g e number of v a r i a b l e s which a f f e c t l e a c h a t e composition have obfuscated the understanding of metal a t t e n t u a t i o n . V a r i a t i o n s i n r e s u l t s have caused the l y s i m e t e r s t u d i e s to be u s e f u l only f o r the s p e c i f i c case that they model. So f a r g e n e r a l i z a t i o n s between s t u d i e s appear c o n t r o v e r s i a l . 2. METALS IN BIOLOGICAL TREATMENT SYSTEMS There i s very l i t t l e i n f o r m a t i o n r e g a r d i n g b a c t e r i a i n a l a n d f i l l . Methane gen e r a t i n g b a c t e r i a , perhaps some sulphur reducing b a c t e r i a and an u n s p e c i f i e d b a c t e r i a l p o p u l a t i o n which generates organic a c i d s are present i n a l a n d f i l l . The anerobic nature of a l a n d f i l l suggests that some s i m i l a r i t i e s may e x i s t between anaerobic sludge b a c t e r i a and l a n d f i l l b a c t e r i a . Anaerobic sludges are r e l a t i v e l y unstudied due to the t e c h n i c a l problems a s s o c i a t e d with anaerobic m i c r o b i o l o g y . T h i s l a c k of i n f o r m a t i o n l e d to a search f o r b a c t e r i a metal i n t e r a c t i o n s i n a e r o b i c sludges. S t u d i e s of a e r o b i c sludge systems have i d e n t i f i e d a number of metal r e t e n t i o n mechanisms which may a l s o e x i s t i n l a n d f i l l s . So the l i t e r a t u r e was reviewed f o r b a c t e r i a - m e t a l i n t e r a c t i o n s i n anaerobic and a e r o b i c sludges. Most of the i n t e r e s t i n heavy metals a s s o c i a t e d with sewage has been s t i m u l a t e d by a need to ensure safe d i s p o s a l mechanisms. Cameron and Koch (1980) found that heavy metals were being removed and s t a b i l i z e d by mixed a e r o b i c treatment of l e a c h a t e . They c o u l d not i d e n t i f y the metal s p e c i e s that were being removed and thus c o u l d not i d e n t i f y s p e c i f i c metal removal 17 mechanisms. Hayes and Theis (1978) separated sludge metals i n t o four c a t e g o r i e s : s o l u b l e , p r e c i p i t a t e , e x t r a c e l l u l a r and i n t r a c e l l u l a r metals. They used a bench s c a l e anaerobic d i g e s t o r to determine the d i s t r i b u t i o n of Cd, C r ( I I I ) , Cu, N i , Pb and Zn (as n i t r a t e s a l t s ) and C r ( I V ) (as dichromate) i n the b i o f l o c . B i o f l o c was c o l l e c t e d f o r a n a l y s i s by removing s e t t l i n g b i o f l o c from the d i g e s t o r s . They used the f i n d i n g s of Cheng et a l . (1975) 2 to separate e x t r a c e l l u l a r metals. P r e c i p i t a t e metals were c o l l e c t e d by a c i d r i n s i n g the biomass with pH=4.0 n i t r i c a c i d . The i n t r a c e l l u l a r metals were c o l l e c t e d by a c i d d i g e s t i n g the biomass. They found a lack of Cd, Cr, Cu, N i , Pb and Zn i n the e x t r a c e l l u l a r category and concluded that t h i s was due to e x t r a c e l l u l a r metal complexes being t r a n s p o r t e d i n t o the c e l l . These f i n d i n g s d i s a g r e e with those of Brown and L e s t e r (1979) who r e p o r t e d that approximately 30% of the metal uptake was due to e x t r a c e l l u l a r polymers i n a c t i v a t e d sludge. Hayes and T h e i s r e p o r t e d s i g n i f i c a n t metal uptake i n the i n t r a c e l l u l a r and i n s o l u b l e p r e c i p i t a t e c a t e g o r i e s . T h i r t y to s i x t y percent of the t o t a l metals i n the b i o f l o c were a s s o c i a t e d with the i n t r a c e l l u l a r c ategory. Metals removed by b a c t e r i a are not s u b j e c t to s o l u b i l i t y e q u i l i b r i a and may be more s t a b l e as i n t r a c e l l u l a r metals than p r e c i p i t a t e s i n the long term. DeWalle et a l . (1979) found metal p r e c i p i t a t e d as 2. Cheng et a l . (1975) found that EDTA had a s t r o n g e r a f f i n i t y f o r heavy metals than e x t r a c e l l u l a r p o l y s a c c h a r i d e s of the c e l l w a l l . 18 s u l p h i d e s , carbonates and hydroxides i n a mixed anaerobic f i l t e r . An e a r l i e r study showed that humic and f u l v i c l i k e a c i d s are present i n an anaerobic f i l t e r and c o u l d c h e l a t e heavy metals. The presence of n a t u r a l c h e l a t o r s h e lps to e x p l a i n why the t o t a l metal c o n c e n t r a t i o n s exceed e s t i m a t e s based on s u l p h i d e and carbonate s o l u b i l i t i e s . A l s o , the data of DeWalle et a l . i n d i c a t e d that s o l u b i l i t y mechanisms were more important than a d s o r p t i o n mechanisms f o r determining suspended and s o l u b l e metal d i s t r i b u t i o n s . O l i v e r and Cosgrove (1974) r e p o r t e d that p r e c i p i t a t i o n and a d s o r p t i o n to b i o f l o c were the dominant removal mechanisms f o r Cd, Cr, Co, Cu, Fe, Pb, Mn, Hg, Ni and Zn i n an a c t i v a t e d sludge system. The removal e f f i c i e n c y was r e l a t e d to the r a t i o of d i s s o l v e d metal to t o t a l metals. Ghosh (1971) r e p o r t e d that s u l p h i d e and carbonate ions are important f o r p r e c i p i t a t i o n of heavy metals. The s u l p h i d e and carbonate ions may p r o t e c t d i g e s t o r organisms from t o x i c l e v e l s of metals. Another f a c t o r may be the i n t e r a c t i o n s of the metals p r e s e n t . Synergism and antagonism c o u l d have important e f f e c t s upon t o x i c i t y and s p e c i a t i o n of metals. Fannin et a l • (1980) r e p o r t e d that the a d d i t i o n of hydroxyl groups to anaerobic d i g e s t o r s w i l l reduce t o x i c i t y due to metals. They were more s p e c i f i c about the e f f e c t s of s u l p h i d e s and r e p o r t e d that Ni and Hg were absorbed. Carbonate ions a f f e c t the s o l u b i l i t y of Zn and to a l e s s e r degree Cd and Pb. Copper s o l u b i l i t y was s t r o n g l y a f f e c t e d by hydroxide i o n s . The carbonate, hydroxide and s u l p h i d e ion l e v e l s have an e f f e c t upon the c o m p e t i t i o n between b a c t e r i a l 19 l i g a n d s and p r e c i p i t a t e s f o r metals. Table I shows some of the p r e c i p i t a t e s that are expected to e x i s t i n an anaerobic d i g e s t o r . Table I - L i k e l y P r e c i p i t a t e s Metal P r e c i p i t a t e Cd Cr Cr(OH) 3 Cu Pb A f t e r Hayes and T heis (1978) If metals are present at high c o n c e n t r a t i o n s they can have i n h i b i t o r y e f f e c t s which may reduce the uptake of metals by b a c t e r i a . An e a r l y study by Barth et a l . (1965) re p o r t e d that b a c t e r i a i n a c t i v a t e d sludge can a c c l i m a t i z e to Cu and Zn but a c c l i m a t i z a t i o n d i d not p r o t e c t the b a c t e r i a from s l u g doses of metals. The treatment e f f i c i e n c y of a c t i v a t e d sludge decreased as the c o n c e n t r a t i o n of Cu, Cr, Ni and Zn i n c r e a s e d . Barth et  a l . found a c o r r e l a t i o n between d i g e s t o r f a i l u r e and s o l u b l e metals. Poon and Bhayani (1971) determined that d i f f e r e n t micro-organisms are a f f e c t e d d i f f e r e n t l y by d i f f e r e n t metals. They compared a pure c u l t u r e of sewage b a c t e r i a to a pure c u l t u r e of fungus and used an enzyme i n h i b i t i o n model (Michaelis-Menten model) to t e s t the e f f e c t of one s p e c i e s of metal ions at a time. T h e i r technique used a l i n e a r i z e d equation (Lineweaver-Burk) and i s l i m i t e d to pure c u l t u r e s and simple s u b s t r a t e s . 20 L e s t e r et a l . (1979) i s o l a t e d three a c t i v a t e d sludge b a c t e r i a l s t r a i n s and t e s t e d the e f f e c t s of Cr, Cu, Pb and Cd on the a c t i v i t y . In a l l cases, the b a c t e r i a showed an i n i t i a l d e c l i n e followed by a recovery a f t e r prolonged exposure to metals. A number of p o s s i b i l i t i e s f o r a c c l i m a t i z a t i o n were c i t e d : the metal s p e c i e s c o u l d be a l t e r e d to a form which p r e c i p i t a t e s , 3 s o l u b l e ammonia-metal complexes c o u l d form, and o r g a n i c l i g a n d s may be r e l e a s e d upon c e l l l y s i s . So the a c c l i m a t i z a t i o n of b a c t e r i a c o u l d cause an i n c r e a s e i n metal uptake or an i n c r e a s e i n metal s o l u b i l i t y ( i f organic l i g a n d s formed s o l u b l e metal complexes). B i o l o g i c a l treatment systems remove heavy metals from sewage. The p r o p o r t i o n of metal which i s removed v a r i e s with the metal s p e c i e s . Metal s p e c i a t i o n a l s o a f f e c t s b a c t e r i a l growth as high c o n c e n t r a t i o n s of some metals w i l l slow down or stop sludge degradation due to b a c t e r i a . Anaerobic b a c t e r i a can use i n t r a c e l l u l a r metal storage to r e t a i n 30% to 60% of the metals measured i n sludge. A e r o b i c b a c t e r i a use e x t r a c e l l u l a r p o l y s a c c h a r i d e s to bind metals and s t o r e s m a l l q u a n t i t i e s of metal w i t h i n the c e l l s . P r e c i p i t a t i o n occurs i n both anaerobic and a e r o b i c systems. The mass of metal which p r e c i p i t a t e s depends upon the carbonate, s u l p h i d e and hydroxyl anions which are a v a i l a b l e . A l s o the c o m p e t i t i o n f o r anions, pH and metal s p e c i e s a f f e c t p r e c i p i t a t e f o r m a t i o n . A l a r g e p r o p o r t i o n of the i n f l u e n t metal which i s 3. Moore et a l . (1961) showed that C r ( V l ) c o u l d be reduced to C r ( I I l ) c a using Cr(OH) 3 to p r e c i p i t a t e . 21 removed from sewage p r e c i p i t a t e s out of s o l u t i o n . Removal mechanisms which were r e p o r t e d to occcur i n anerobic systems i n c l u d e : a) P r e c i p i t a t i o n by h y d r o x y l , carbonate and sulphide anions. b) I n t r a c e l l u l a r Uptake. c) Complex formation. d) Changing the o x i d a t i o n s t a t e of a metal to cause p r e c i p i t a t i o n . e) E x o c e l l u l a r Uptake. Some metal a d s o r p t i o n to c e l l w a l l s may occur but the l i t e r a t u r e was not s p e c i f i c . 3. METALS IN SOIL S o i l i s a component of a l a n d f i l l . As i t i s used to surround and cover r e f u s e , s t u d i e s on the m o b i l i t y of metals i n l a n d f i l l s should c o n s i d e r metal m o b i l i t y i n s o i l s . A l e s i i et  a l . (1980) s t u d i e d the flow r a t e of l e a c h a t e through s o i l . The p a r t i c l e s i z e (or c l a y d i s t r i b u t i o n ) c r i t i c a l l y a f f e c t s a t t e n u a t i o n of l e a c h a t e . R e t e n t i o n of Cd, Cr, Ni and Zn was a f u n c t i o n of i n f l u e n t c o n c e n t r a t i o n . Kirkham (1977) experimented with d i f f e r e n t s o i l types to observe the e f f e c t on metal uptake. G e n e r a l l y , the organic m a t e r i a l s i n s o i l were found to have a v a r y i n g e f f e c t . Kirkham ranked metals a c c o r d i n g to the rate of metal uptake by the s o i l . The uptakes of F e ( I I l ) and C r ( I I I ) were the h i g h e s t . The order of metal uptake r e p o r t e d by Kirkam w a s : F e ( l I I ) and C r ( I I l ) > C u ( l l > N i ( I l ) > Co(II) > F e d I) > M n ( I I ) and Zn(II) > C d ( I I ) . N i c k e l , dichromate and c h r o m i u m d l l ) were bound s t r o n g l y to s e r p e n t i n e s o i l , while only 22 chromium uptake i n c r e a s e d i n a l k a l i n e s o i l s . Kirkham i n d i c a t e d that the predominant mechanism f o r cadmium uptake was v i a c a t i o n exhange. F u l l e r and A l e s i i (1979) o u t l i n e d a l i s t of chemical and p h y s i c a l parameters u s u a l l y a s s o c i a t e d with the m i g r a t i o n of heavy metals i n s o i l . Some of these parameters are l i s t e d i n Table I I . Table II - Chemical And P h y s i c a l S o i l Leaching Parameters Chemical Parameters P h y s i c a l Parameters Surface area of S o i l p art i c l e s Clay content pH P h y s i c a l shape Lime content Depth of s o i l Q u a n tity of hydrous oxides of Fe, Mn, and A l S t r a t i f i c a t o n of s o i l S o l u b l e s a l t s content Compaction of s o i l Organic matter content Water i n f i l t r a t i o n C a t i o n exchange c a p a c i t y Frequency of cementations and a c c r e t i o n s H y d r a u l i c c o n d u c t i v i t y The c l a y content and pH e f f e c t the m o b i l i t y of a l l metals. A drop i n pH w i l l i n c r e a s e the c o n c e n t r a t i o n of metals i n s o l u t i o n and an i n c r e a s e i n c l a y content decreases the rate of flow through the s o i l . The metals Cu, Fe, Mn and Zn are the most s e n s i t i v e to c l a y content, f r e e i r o n oxides and the t o t a l s o i l content of Co, Cr, Mn, Ni and Zn. There i s a complex r e l a t i o n s h i p between a d s o r p t i o n s i t e s , l i g a n d s and competing 23 ions i n the s o i l . When G r i f f i n et a l . (1976) were studying metal a t t e n u a t i o n due to c l a y , they found t h a t Fe and Mn were e l u t e d from the s o i l . The e l u t i o n c o u l d have been due to c a t i o n exchange or r e d u c t i o n of the Fe and Mn i n c l a y to a more s o l u b l e s p e c i e s . They r e p o r t e d that Cd, Hg, Pb and Zn were attenuated more than the other metals s t u d i e d . The a t t e n u a t i o n of metals by c l a y was a f u n c t i o n of c a t i o n exchange c a p a c i t y , i n i t i a l c a t i o n s i n the system, l e a c h a t e composition and leachate pH. Above pH=6, p r e c i p i t a t i o n i s the dominant metal removal mechanism (F r o s t and G r i f f i n 1977). Another study by G r i f f i n et  a l . (1977) found that the c a t i o n i c s p e c i e s of Cd, Cu, C r ( l l l ) , Hg, Pb and Zn were i n c r e a s i n g l y r e t a i n e d with r i s i n g pH while the a n i o n i c s p e c i e s C r ( V I ) , As and Se were d e c r e a s i n g l y r e t a i n e d . Table III l i s t s some f u r t h e r comments by G r i f f i n et  a l . with respect to s p e c i f i c metal i o n s . T i r c h et a l . (1979) used ammonium a c e t a t e to estimate the c a t i o n exchange c a p a c i t y of s o i l . The degree of p r e c i p i t a t i o n and the pH had a s i g n i f i c a n t e f f e c t on the c a t i o n exchange c a p a c i t y . They ranked three ions based on t h e i r success at competing f o r ammonium ac e t a t e s i t e s [Cu > Cd > Ca]. Wollan and Beckett (1979) t r i e d to c h a r a c t e r i z e s l u d g e - s o i l i n t e r a c t i o n s by the r a t i o of e x t r a c t a b l e metal to t o t a l metal. Twelve months a f t e r the sludge had been added, Cu, Ni and Zn r a t i o s were independent of the sludge type ( f o r the two sludges that they t e s t e d ) . T h e i r EDTA e x t r a c t i o n s ( f o r Cu and Zn) and a c e t i c a c i d e x t r a c t i o n s ( f o r Ni and Zn) were s e n s i t i v e to experimental c o n d i t i o n s . They found t h a t added heavy metals were more 2 4 Table III - Metal Ions In S o i l Metal Ion Comments Pb Not e f f e c t i v e i n competition with i r o n f o r c a t i o n exchange s i t e s . When the pH > 6 hyd r o x y l complexes are adsorbed by c l a y and lead carbonate p r e c i p i t a t e s form. Cu, Cd, Zn Form hyd r o l y z e d ions i n c r e a s i n g the t o t a l metals i n s o l u t i o n as the pH r i s e s . Cu Competes s u c c e s s f u l l y f o r c a t i o n exchange s i t e s . C r + 6 Very c o m p e t i t i v e f o r a n i o n i c exchange s i t e s . H C r C " Is adsorbed by s o i l . C r + 3 Forms h y d r o l y z e d ions i n a c i d and forms a p r e c i p i t a t e when the pH > 5. Competes f o r c a t i o n exchange s i t e s . r e a d i l y e x t r a c t e d than n a t i v e heavy metals. Metals are r e t a i n e d by d i f f e r e n t s o i l s to v a r y i n g degrees. Some of the metals are bound, but s t i l l a v a i l a b l e to p l a n t s , and some are very mobile. The c a t i o n exchange c a p a c i t y , c l a y c o n t e n t , pH and rate of water a p p l i c a t i o n appear to be the most important v a r i a b l e s a f f e c t i n g l e a c h a t e m o b i l i t y . Table IV summarizes the e f f e c t s on metals of v a r i o u s s o i l p r o p e r t i e s . There are many other v a r i a b l e s which a f f e c t metal i n t e r a c t i o n s i n s o i l but u n t i l the metal s p e c i e s i n leac h a t e can be determined, p r e d i c t i o n of le a c h a t e p r o p e r t i e s w i l l be hampered. 25 Table IV - Summary Of S o i l E f f e c t s Property Metals Most E f f e c t e d I n f l u e n t c o n c e n t r a t i o n Cd, Cr, Ni and Zn C a t i o n exchange c a p a c i t y Cu> Cd> Ca Bound by S o i l Cr, Ni Adsorbed by Clay Cu, Fe, Mn and Zn Adsorbed by Iron Oxides Cu, Fe, Mn and Zn E f f e c t e d by t o t a l content of Co, Cr, Mn, Ni and Zn Cu, Fe, Mn and Zn Attenuated by Clay Cd, Hg, Pb and Zn 4. NATURAL CHELATORS Evidence presented by Knox and Jones (1979) and Raveh and Avnimelech (1979) i n d i c a t e d the e x i s t e n c e of n a t u r a l c h e l a t i n g agents i n l y s i m e t e r l e a c h a t e s . Other s t u d i e s have shown the e x i s t e n c e of n a t u r a l c h e l a t i n g agents i n s o i l , peat and lakewater. Estimates of the s t a b i l i t y and c a p a c i t y of n a t u r a l l i g a n d s would h e l p to estimate metal m o b i l i t y i n a l a n d f i l l or l y s i m e t e r . S o i l , sludge and le a c h a t e s t u d i e s which c o n s i d e r n a t u r a l l i g a n d s have been reviewed i n an attempt to determine the v a r i a b l e s which a f f e c t complex formation i n the n a t u r a l environment. ( D e t a i l s of c o - o r d i n a t i o n chemistry are co n t a i n e d i n Appendix B.) Knox and Jones (1979) i n v e s t i g a t e d the complexation of Cd i n l y s i m e t e r l e a c h a t e . They t r i e d t o e v a l u a t e the e f f e c t s of pH and metal co m p e t i t i o n but were unable to maintain anaerobic 26 c o n d i t i o n s . T h e i r r e s u l t s i n d i c a t e that one to one l i g a n d - m e t a l complexes were predominant. By studying the amount of metal complexed at v a r y i n g pH, they concluded that c a r b o x y l i c , p h e n o l i c , h y d r o x y l , s u l f h y d r y l and n i t r o g e n groups were the most l i k e l y l i g a n d types. As they were unable to i d e n t i f y s p e c i f i c l i g a n d s they d i d not o b t a i n s t a b i l i t y c o n s t a n t s . P r e c i p i t a t e d i r o n f l o e s absorbed Cd and other metal c a t i o n s but the r e a c t i o n s were pH dependent. Cheng et a l . avoided the problem of i d e n t i f y i n g l i g a n d s i n sludge by determining an apparent s t a b i l i t y c o nstant. They separated f r e e ions from complexed ions and found that complex formation tended to i n c r e a s e with pH u n t i l hydroxyl complexes s t a r t e d to form. EDTA, NTA, o x a l a t e and g l y c i n e ( i n order of d e c r e a s i n g strength) were stronger c h e l a t i n g agents than sewage sludge. T h e i r ranking f o r sludge uptake of Pb > Cu > Cd > Ni does not agree with the general order of s t a b i l i t i e s determined by I r v i n g and W i l l i a m s (Cu > Ni > Cd). Cheng et a l . found t h a t the I r v i n g - W i l l i a m s order depended upon the l i g a n d s a v a i l a b l e and the pH. Mantoura and R i l e y (1975) used a g e l that achieved 92% and 72% r e c o v e r i e s f o r humic a c i d and f u l v i c a c i d r e s p e c t i v e l y . The g e l was assumed to separate f r e e ions from complexed i o n s . Mantoura and R i l e y used a Scatchard p l o t to c a l c u l a t e s t a b i l i t y c o n s t a n t s f o r complexes of C u ( I I ) , N i ( I l ) and Zn(II) with humic and f u l v i c a c i d s e x t r a c t e d from s o i l , peat, and lakewater. Table V shows s t a b i l i t y c o n s t a n t s f o r a few humic-metal complexes. 27 Table V - LoglO S t a b i l i t y Constants For Humics - I LogK C u + 2 Ni + 2 Zn + 2 S o i l 3.3 3.1 2.4 Peat 7.2-6.5 4.3-5.6 4.8 Lakewater 8.1-8.8 5.2 5.2 MacCarthy (1977) developed a t h e o r e t i c a l method for c a l c u l a t i n g s t a b i l i t y c o n s t a n t s f o r s o i l samples assuming Schubert c o n d i t i o n s ( [ l i g a n d ] >> [ m e t a l ] ) . MacCarthy's method esti m a t e s an o v e r a l l average s t a b i l i t y constant f o r a s o l u t i o n under given temperature, pH, and i o n i c s t r e n g t h . He estimated the c o n c e n t r a t i o n of f r e e and s o i l complexed metals by using a g e l t o complex f r e e ions and by v a r y i n g c o n c e n t r a t i o n s of a known l i g a n d . In a q u a l i t a t i v e study T r u i t t and Weber (1979) found that f u l v i c a c i d enhanced the removal of C u ( l l ) , C d ( I I ) , and Z n ( l l ) i n wastewater d u r i n g alum c o a g u l a t i o n . They ranked f i v e metals a c c o r d i n g to the r e l a t i v e s t r e n g t h of the f u l v i c acid-metal complex. (Pb, Hg > Cu > Cd, Zn). In a f u r t h e r study by Mantoura et a l . (1978), i t was reported that the t r a d i t i o n a l assumptions used f o r s p e c i a t i o n m o d e l l i n g are not v a l i d f o r high i o n i c s t r e n g t h s o l u t i o n s . They assumed that the humic a c i d -metal complexes were roughly one to one. A l s o , mixed l i g a n d T o m p l e x e s , p o l y n u c l e a r complexes and interphase r e a c t i o n s were not s i g n i f i c a n t . Although they used the Davies equation they concluded that humic-metal complexes were very s i g n i f i c a n t i n f - a s h water. Copper, c a l c i u m , and magnesium and to a l e s s e r degree mercuric c h l o r i d e s , complexed the humics i n sea water. 28 The model used by Mantoura et a l . was very s e n s i t i v e to pH and the assumed s t a b i l i t y c o n s t a n t s . The s t a b i l i t y c o nstants that they were able to determine f o r humic-metal complexes are shown in Table VI. Table VI - LoglO S t a b i l i t y Constants For Humics - II C a + 2 Ni + 2 Cu + 2 Zn + 2 C d + 2 Mg+ 2 Peat 3.65 4.98 7.85 4.83 4.57 3.81 Lakes 3.95 5.14 9.83 5.14 4.57 4.00 Seawater 4. 12 5.51 9.71 5.31 4.69 3.98 A f t e r Mantoura et a l . (1978). Stoveland et a l . (1979) found that NTA i n sewage formed s o l u b l e metal complexes. They a l s o added sodium t r i p o l y p h o s p h a t e to sewage and found that i t formed metal complexes which p r e c i p i t a t e d . T h i s i n d i c a t e s that s o l u b l e metal ions or complexes are a v a i l a b l e and that the s o l u b l e complexes are r e l a t i v e l y weak. J e l l i n e k and Sangal (1971) used a number of n a t u r a l p o l y e l e c t r o l y t e s to determine t h e i r e f f e c t i v e n e s s at metal complexation i n p o l l u t e d water. None of the p o l y e l e c t r o l y t e s ( p o l y m e t h a c r y l i c a c i d - from bovine albumin and g e l a t i n ; a l g i n i c a c i d - from g e l a t i n ; p e c t i n ; o x i d i z e d s t a r c h from g e l a t i n ; and p o l y g a l a c t u r o n i c a c i d (PGA)) complexed a l l of the metals t e s t e d (Cd, Cu, N i , Zn and C r ( V l ) ) . They found that PGA was the most e f f e c t i v e c h e l a t i n g agent as i t formed complexes with a l l the metals except C r ( V l ) . Estimates of the 29 PGA-metal complex s t a b i l i t i e s are shown i n Table V I I . T h e i r r e s u l t s i n d i c a t e that more than one l i g a n d i s r e s p o n s i b l e f o r n a t u r a l complexation. Table VII - LoglO S t a b i l i t y Constants - PGA Metal Logl 0 Removal E f f i c i e n c y C u + 2 1.7 98% C d + 2 0.59 90% Ni + 2 0.21 70% Zn + 2 0.34 78% C r * 3 75% Note: Data was c o l l e c t e d at 20°C By J e l l i n e k and Sangal (1972). In a study of c h e l a t i o n by NTA and EDTA from anthropogenic sources, Zn, Cd, and Cu c h e l a t i o n were favoured (Raspor et a l . 1981). As long as Ca and Mg c o n c e n t r a t i o n s were not four orders of magnitiude g r e a t e r than the metal c o n c e n t r a t i o n s , the metals were c h e l a t e d . The presence of humic m a t e r i a l s only a f f e c t e d Cu(II) and Hg(II) c o n c e n t r a t i o n s . Measurement of c h e l a t i n g c a p a c i t y has been most s u c c e s s f u l i n s o i l s because a g e l has been d i s c o v e r e d which i s o l a t e s the humic and f u l v i c a c i d s . A major element in the d e t e r m i n a t i o n of the c h e l a t i n g c a p a c i t y i n l e a c h a t e s , s o i l s and water i s the experimental technique being employed. To date r e s e a r c h e r s have been unable to measure c h e l a t i n g c a p a c i t y d i r e c t l y but a number of o b s e r v a t i o n s can be made regarding the f a c t o r s which i n f l u e n c e c h e l a t e c a p a c i t y . The f o l l o w i n g f a c t o r s have been 30 shown to be s i g n i f i c a n t i n the c o m p e t i t i o n of metals f o r complexing l i g a n d s . a) Metal s p e c i e s . b) pH. c) Temperature. d) Exposure to a i r . e) A v a i l a b l e l i g a n d s (eg. f u l v i c a c i d s , OH r a d i c a l ) . f ) A v a i l a b l e r a d i c a l s f o r p r e c i p i t a t e formation (eg, s u l p h i d e s ) . g) A v a i l a b l e sorbing s i n k s (eg. i r o n and manganese o x i d e s ) . h) Clay content of s o i l i n l a n d f i l l . i ) I o n i c s t r e n g t h of l e a c h a t e . The s t a b i l i t y c onstants ' r e p o r t e d by Mantoura et a l . (1978) are the most comprehensive a v a i l a b l e f o r humic-metal complexes i n s o i l s and water. T h e i r f i n d i n g s and those of others i n d i c a t e t h a t Pb* 2, Hg* 2, Cu* 2, N i * 2 , Z n + 2 and then Cd* 2 are most l i k e l y to form n a t u r a l complexes i n s o i l s and water. 5. BACTERIA AND METALS Most s t u d i e s of b a c t e r i a i n sludge, l e a c h a t e or sewage f a l l i n t o two c a t e g o r i e s . They are e i t h e r s t u d i e s of a few s p e c i f i c b a c t e r i a known to e x i s t i n the environment or s t u d i e s of u n s p e c i f i e d b a c t e r i a l masses. B a c t e r i a d e f i n i t e l y play some r o l e i n l a n d f i l l s but the extent of the r o l e with respect to metal r e t e n t i o n i s d i f f i c u l t to determine. There i s evidence sugges t i n g that b a c t e r i a can b i n d heavy metals i n e x t r a c e l l u l a r or e x t r a c e l l u l a r p o l y s a c c a r i d e s . C o n s i d e r a b l e s p e c u l a t i o n 31 e x i s t s r e g a r d i n g the s o r b i n g c a p a b i l i t i e s of b a c t e r i a . G u t h r i e et a l . (1977) i s o l a t e d n a t u r a l l y o c c u r r i n g b a c t e r i a and used pure s t r a i n s to determine metal uptake. Hatch and Menawat (1979) i s o l a t e d a pure b a c t e r i a s t r a i n and a pure fungus s t r a i n . They i s o l a t e d the e x t r a c e l l u l a r p o l y s a c c h a r i d e s a s s o c i a t e d with a b a c t e r i a ( S p h a e r o t i l u s natans) and a fungus ( L e p t o n i t u s ) . Both micro-organisms occur i n sludges and p o l l u t e d waters and were found to bind metal s a l t s . When the p r o t e i n - p o l y s a c c h a r i d e - l i p i d complex that forms the e x t r a c e l l u l a r p o l y s a c c h a r i d e s was analyzed by a scanning e l e c t r o n microscope and X-ray c r y s t a l l o g r a p h y , a uniform d e p o s i t of i r o n was found. The i r o n was d i s t r i b u t e d throughout the c e l l mass as a f i n e e x t r a c e l l u l a r p r e c i p i t a t e . Hatch and Menawat were able to grow S. natans i n s u l p h a t e s of Fe, Cu, Co, Cd and Mg. T h e i r data showed that the b a c t e r i a reached a metals s a t u r a t i o n p o i n t . An e x t e n s i v e l i t e r a t u r e review by Brown and L e s t e r (1979) rep o r t e d t h a t the b i o f l o c , which forms i n an a c t i v a t e d sludge p r o c e s s , sorbs a l a r g e q u a n t i t y of metals. A l s o , more metal was sorbed by the v i a b l e sludge than the non-a c t i v e sludge. They o u t l i n e d four p o s s i b l e mechanisms f o r metal removal by b i o f l o c and four types of metal t r a n s f o r m a t i o n by b a c t e r i a t hat c o u l d cause an i n c r e a s e i n metal s o r p t i o n . Table VIII l i s t s the removal and t r a n s f o r m a t i o n mechanisms presented by L e s t e r and Brown. The l e a s t l i k e l y mechanism i s the v o l a t i l i z a t i o n of metals to the atmosphere as t h e r e are no mass balance s t u d i e s to support t h i s theory. A l l of the other mechanisms have been 32 Table VIII - B i o f l o c - Metal I n t e r a c t i o n s Removal Mechanisms Metal Transformations P h y s i c a l t r a p p i n g of p r e c i p i t a t e s Formation of organo-m e t a l l i c complexes in f l o e B i n d i n g of s o l u b l e metal to e x o c e l l o l a r p o l y s a c c h a r i d e s Metal s u b s t i t u t i o n Accumulation of s o l u b l e metals i n c e l l s B i o m e t h y l a t i o n V o l a t i l i z a t i o n of metal to atmosphere Changing the metal valency shown to occur to some degree. E x t r a c e l l u l a r p o l y s a c c h a r i d e s c o u l d bind s o l u b l e metals by s o r p t i o n and/or c a t i o n exchange. I t has been shown by many re s e a r c h e r s that e x t r a c e l l u l a r p o l y s a c c h a r i d e s r e t a i n metals. F u r t h e r d e t a i l s of e x t r a c e l l u l a r p o l y s a c c h a r i d e s metal uptake mechanisms are shown i n Appendix C. G e n e r a l l y e x t r a c e l l u l a r p o l y s a c c h a r i d e s r e t a i n metals by a d s o r p t i o n , chemical bonding, a b s o r p t i o n , complexation or by p r o v i d i n g s i t e s f o r p r e c i p i t a t i o n . Both anaerobes and aerobes are capable of manufacturing e x t r a c e l l u l a r p o l y s a c c h a r i d e s . A l s o i t may be p o s s i b l e f o r some b a c t e r i a to o x i d i z e e x t r a c e l l u l a r p o l y s a c c h a r i d e s to form s t a b l e end products. B. SUMMARY Leachate s t u d i e s show that metals are r e t a i n e d to varying degrees i n l y s i m e t e r s . Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e to e x p l a i n why some metals are r e t a i n e d longer than o t h e r s . A l s o , l i t t l e has been done to estimate the long term s t a b i l i t y of metals i n a l y s i m e t e r . The metal s p e c i e s seems to be an 33 important f a c t o r f o r metal r e t e n t i o n and s t a b i l i t y but there are few s t u d i e s of the metal s p e c i e s i n l a n d f i l l s or l y s i m e t e r s . I t i s most l i k e l y t h a t m u l t i p l e f a c t o r s are r e s p o n s i b l e f o r the r e t e n t i o n of each metal i n a l y s i m e t e r or l a n d f i l l . The degree of metal f i x a t i o n by b a c t e r i a i n a l a n d f i l l i s not documented. Stud i e s of a e r o b i c and anaerobic sewage treatment systems show that b a c t e r i a can sorb and perhaps complex metals. B a c t e r i a probably immobilize metals i n t h e i r surrounding environment by one or more of the f o l l o w i n g methods: a) I n t r a c e l l u l a r uptake. b) S o r p t i o n to c e l l w a l l . c) Bonding with e x t r a c e l l u l a r p o l y s a c c h a r i d e s . d) Entrapment i n e x t r a c e l l u l a r p o l y s a c c h a r i d e s . e) Conversion of metal to a s p e c i e s which complexes or p r e c i p i t a t e s . Three other metal removal mechanisms c o u l d occur i n a l y s i m e t e r or l a n d f i l l : s o r p t i o n , p r e c i p i t a t i o n and complexation. P r e c i p i t a t e s of s u l p h i d e s , carbonates and hydroxides are expected to form. (A l i s t of expected p r e c i p i t a t e s i s shown in Table I.) Complexation occurs between or g a n i c components of s o i l and metals. Organic components other than humic and f u l v i c a c i d s may complex metals. Both p r e c i p i t a t i o n and complexation are a f f e c t e d by pH, metal s p e c i e s and c o m p e t i t i o n f o r r e a c t i v e r a d i c a l s . S o r p t i o n by s o i l s may a l s o be a s i g n i f i c a n t metal removal mechanism. The most important v a r i a b l e s a f f e c t i n g s o r p t i o n by s o i l s are the c l a y content, c a t i o n exchange c a p a c i t y , pH and 34 r a t e of water a p p l i c a t i o n . I f i r o n or manganese oxides are present, they can adsorb some metal s p e c i e s . 35 I I I . EXPERIMENTAL PROCEDURES A c h e l a t i o n exper iment , an e x t r a c t i o n pro cedure and a f l u o r e s c e n t dye t echn ique were used to determine the meta l m o b i l i t y , b a c t e r i a l numbers and the r e l a t i v e s t r e n g t h of n a t u r a l l i g a n d s in a l y s i m e t e r . The f i r s t experiment was a p r o g r e s s i v e e x t r a c t i o n t e c h n i q u e which c a t e g o r i z e d meta ls a c c o r d i n g to t h e i r m o b i l i t y . Three samples were t e s t e d wi th t h i s p r o c e d u r e : a s o n i c a t e d sample , an a e r a t e d sample and an u n t r e a t e d sample used as a c o n t r o l . The mass of meta l s e x t r a c t e d i n each c a t e g o r y was d i v i d e d by the dry weight of each sample for comparison of the three sample t y p e s . S o n i c a t i o n sho u ld r u p t u r e c e l l s r e l e a s i n g i n t r a c e l l u l a r meta l s and a e r a t i o n shou ld cause a l l m e t a l s , which are not s t a b l e when exposed to a i r , ( i e . F e ( I l ) w i l l be c o n v e r t e d to F e ( l I I ) ) to be r e l e a s e d . By u s i n g a second experiment to determine b a c t e r i a l numbers i t may be p o s s i b l e to c o r r e l a t e some meta l r e l e a s e s w i t h the d e s t r u c t i o n of v i a b l e b a c t e r i a . For these purposes the f l u o r e s c e n t dye 4 , 6 - d i a m i d i n o -2 - p h e n y l i n d o l e was t e s t e d . The t h i r d experiment used a g r a d i e n t of c h e l a t i n g agents to compare the s t r e n g t h of n a t u r a l l i g a n d s w i t h l i g a n d s of known complex ing a b i l i t y . Thus , an e s t i m a t e of the s t a b i l i t y c o n s t a n t s f o r m e t a l - n a t u r a l l i g a n d complexes c o u l d be made. A l l t h r e e exper iments r e q u i r e d suspended samples which had not been exposed to a i r . To a c h i e v e t h i s , samples were removed i n a n i t r o g e n atmosphere from one of the l y s i m e t e r s used i n the UBC c o - d i s p o s a l s t u d y . A f t e r remova l , the samples were b lended w i t h d e i o n i z e d d i s t i l l e d water to form a s l u r r y . A s h o r t d i s c u s s i o n of the a n a e r o b i c sampl ing method i s i n c l u d e d i n 36 t h i s chapter and f u r t h e r d e t a i l s are provided i n Appendix F. A. SAMPLING 1. SAMPLE SOURCE A l y s i m e t e r which had e l e c t r o p l a t i n g wastes added to i t was s e l e c t e d f o r sampling. The e l e c t r o p l a t i n g wastes boost metal c o n c e n t r a t i o n s i n the l y s i m e t e r s i m p l i f y i n g metal d e t e c t i o n requirements. One l y s i m e t e r from the c o - d i s p o s a l study conducted by Atwater et a l . (1981), was used as the sample source. P l o t s of metal r e l e a s e vs time f o r the l y s i m e t e r i n d i c a t e that the r a t e of metal r e l e a s e had s t a b i l i z e d . ( P l o t s of metal r e l e a s e vs time f o r the l y s i m e t e r are reproduced i n Appendix A.) The age of the l y s i m e t e r may have helped to reduce c o m p l i c a t i o n s which c o u l d a r i s e i n data i n t e r p r e t a t i o n . F i g u r e 2 i s a c r o s s - s e c t i o n view of the l y s i m e t e r . I t shows the i n t e r n a l l a y e r s and the l o c a t i o n s of the sampling h o l e s . The f i r s t hole was 15 cm. from the bottom of the l y s i m e t e r . T h i s sample s i t e was estimated to c o i n c i d e with the top of the f i r s t l i f t . A l l but the top l i f t were composed of a 15 cm. l a y e r of r e f u s e compacted at 220 p s i . The f i n a l l i f t was a 15 cm. l a y e r of s o i l . A second hole was made 45 cm. from the bottom of the l y s i m e t e r and should be near the top of the t h i r d l i f t . These l o c a t i o n s were chosen because e l e c t r o p l a t i n g wastes were added to the top of the f i r s t and t h i r d l i f t s . 37 F i g u r e 2 - C r o s s - s e c t i o n Of Lysimeter Water Input nd D r i l l e t D r i l l e To Leachate Collection A l l refuse was hydraulically packed at a maximum pressure of 220 psi. A l l dimensions are in cm. After Atwater et a l . 1981 2. LIQUID SAMPLE COLLECTION Before d r i l l i n g a hole i n the l y s i m e t e r a leac h a t e sample was c o l l e c t e d from the bottom of the l y s i m e t e r . T h i s sample was taken to ensure that no changes had occured i n the l y s i m e t e r s i n c e the l a s t metal a n a l y s i s of the c o - d i s p o s a l study. A n i t r o g e n atmosphere was maintained i n the leac h a t e c o l l e c t i o n f l a s k to prevent p r e c i p i t a t e formation. A l l of the samples 38 c o l l e c t e d were analyzed using a J a r r e l Ash 810 atomic a b s o r p t i o n spectrophotometer. An estimate of the l i q u i d volume r e q u i r e d f o r a n a l y s i s was based on data from the UBC c o - d i s p o s a l study. The data and c a l c u l a t i o n s are shown i n Appendix I. The l e a c h a t e was allowed to d r a i n by g r a v i t y i n t o a f l a s k f i l l e d w ith n i t r o g e n gas. The n i t r o g e n gas p r e s s u r e was kept s l i g h t l y above atmospheric p r e s s u r e by submerging the gas e x i t tube 5 mm. below the s u r f a c e of a beaker of water. T h i s prevented an a i r leak i n the f l a s k i f the gas flow f a i l e d . 3. SOLID SAMPLE COLLECTION S o l i d samples were c o l l e c t e d i n a n i t r o g e n atmosphere to a v o i d pH or metal s p e c i a t i o n changes. A pressure of 4 cm. of water above atmospheric was used to prevent contamination by a i r . A l s o , a n i t r o g e n atmosphere may h e l p to keep true anaerobic b a c t e r i a v i a b l e u n t i l they are f i x e d . A sampling chamber, made of l u c i t e , was c o n s t r u c t e d to f i t a g a i n s t the si d e of the l y s i m e t e r . (A d e t a i l e d diagram of the chamber i s shown i n Appendix F.) The chamber was designed to allow sampling to occur i n a n i t r o g e n atmosphere. Nitrogen gas was s u p p l i e d from a 100 l b . c y l i n d e r with a r e g u l a t o r to maintain a steady n i t r o g e n p r e s s u r e . D e t a i l s of the sampling procedure are c o n t a i n e d i n a step by step procedure l i s t e d i n Appendix F. A f t e r a hole was d r i l l e d i n t o the s i d e of the l y s i m e t e r , two d r i l l b i t s were used to remove samples. One was a s t e e l tube with a band saw blade welded onto the end. The other was a bar with two tungsten c a r b i d e t e e t h and a high speed t w i s t d r i l l welded to i t (Sketches of the d r i l l b i t s are shown i n Appendix 39 F ) . Both b i t s tended to tear p i e c e s of sample from the l y s i m e t e r r e s u l t i n g i n a shredded sample. A s t a i n l e s s s t e e l tube or blade was used to pic k r e f u s e p i e c e s from the l y s i m e t e r . The p i e c e s were p i l e d on a paper towel which was used to funnel the r e f u s e i n t o a 4 l i t r e nalgene c o n t a i n e r . When s u f f i c i e n t sample had been c o l l e c t e d , the nalgene c o n t a i n e r was s e a l e d using a t h i n f i l m of p l a s t i c ( ' P a r a f i l m 1 ) • . To c a l c u l a t e the approximate volume of sample that was r e q u i r e d , i t was assumed that the metals i n the l y s i m e t e r were uni f o r m l y d i s t r i b u t e d . The masses of metals i n the l y s i m e t e r were estimated by s u b t r a c t i n g the masses of metals leached from the masses a p p l i e d , to the l y s i m e t e r . T h i s should give a low estimate of the metals c o n t a i n e d i n the l y s i m e t e r as the masses of metals i n the r e f u s e are not i n c l u d e d . The c a l c u l a t i o n s are shown i n Appendix J . Roughly 1200 ml. of s o l i d sample are r e q u i r e d f o r the three experiments. 4. SAMPLE STORAGE A l l the c o n t a i n e r s used to s t o r e samples were a c i d washed with a 30% n i t r i c a c i d s o l u t i o n then r i n s e d with d e i o n i z e d d i s t i l l e d water. Each p i p e t t e , f u n n e l , s p a t u l a or other implement that c o n t a c t e d the sample was a c i d washed and r i n s e d with d e i o n i z e d d i s t i l l e d water. Samples which were not i n use were se a l e d , using p a r a f i l m , and s t o r e d at 4°C, away from l i g h t . 4. The manufacturers of p a r a f i l m c l a i m that i t prevents gas d i f f u s i o n . 40 Large c o n t a i n e r s were s e a l e d using p a r a f i l m which was taped to make sure i t stayed i n p l a c e . The small 50 ml. Erlenmyer f l a s k s used f o r b a c t e r i a l samples were s e a l e d with a p l a s t i c stopper. B a c t e r i a l samples were f i x e d with ethanol or 5% formaldehyde s o l u t i o n s to prevent b a c t e r i a l growth. 5. SAMPLE TREATMENT Before the s o l i d samples were t e s t e d , the s o l i d s were suspended i n d e i o n i z e d d i s t i l l e d water. A Waring blender was used to homogenize the s o l i d s . Large p i e c e s of metal, p o r c e l a i n and wood were removed from the sample t o a v o i d damaging the b l e n d e r . S o l i d s and d e i o n i z e d d i s t i l l e d water were added to the blender to make a t o t a l volume of 600 ml. i n the blender. I n i t i a l l y a l l the b l e n d i n g was conducted o u t s i d e the anaerobic chamber. So a l u c i t e top with a neoprene s e a l and e l a s t i c s , to h o l d the top down, was used to keep samples anaerobic (A diagram i s shown i n Appendix F ) . When s o l i d s from the second hole were blended, a l l the b l e n d i n g was conducted i n s i d e the chamber using the l u c i t e top to r e t a i n s p l a s h e s . The f i r s t s o l i d s sample was roughly 1.2 L. and the second was 0.9 L. before d i l u t i o n . The f i r s t sample was blended and d i l u t e d to 10 L. The second sample was d i v i d e d i n t o three samples. Each t h i r d was d i l u t e d to 0.9 L. and blended j u s t p r i o r to use. T h i s was more convenient as s m a l l e r volumes were manipulated. A mass balance was used to determine the dry mass o f l y s i m e t e r s o l i d s being t e s t e d i n each sample. 41 6. SAMPLE ANALYSIS Gas samples were taken from the bench top anaerobic chamber, the anaerobic sampling box and the l i q u i d c o l l e c t i o n f l a s k . A F i s h e r Hamilton gas p a r t i t i o n e r was used with a Hewlett Packard s t r i p c h a r t r e c o r d e r to determine oxygen l e v e l s . In a l l cases f i v e minutes of f l u s h i n g with n i t r o g e n reduced oxygen to l e s s than 0.5% of the atmosphere. So the v a r i o u s anaerobic chambers were f l u s h e d f o r f i v e minutes before a sample was exposed to the n i t r o g e n atmosphere. A J a r r e l Ash 810 atomic a b s o r p t i o n spectrophotometer l i n k e d to a Hewlett Packard 7127A s t r i p - c h a r t r e c o r d e r was used f o r metals a n a l y s i s . Other a n a l y s i s methods were c o n s i d e r e d and they are d i s c u s s e d i n Appendix H. The advantages of the atomic a b s o r p t i o n spectrometer over the other methods were: o p e r a t i n g s i m p l i c i t y , minimal sample p r e p a r a t i o n , a v a i l a b i l i t y and r e p e a t a b i l i t y . The d e s i r e d accuracy was obtained u s i n g the parameters l i s t e d i n Table IX. P r i o r t o a n a l y s i s w i t h the J a r r e l Ash 810 atomic a b s o r p t i o n spectrophotometer, a l l samples were a c i d d i g e s t e d using c o n c e n t r a t e d n i t r i c a c i d as recommended by S. L i p t a k of the UBC Environmental E n g i n e e r i n g Laboratory. Each sample was evaporated to dryness then the r e s i d u e was r e d i s s o l v e d using, c o n c e n t r a t e d n i t r i c a c i d . The mixture was d i l u t e d with d e i o n i z e d d i s t i l l e d water and f i l t e r e d through a Whatman number 541 f i l t e r b e fore d i l u t i n g up to a f i n a l volume of 100 ml. Stock s o l u t i o n s s u p p l i e d by F i s h e r Chemicals were used to c a l i b r a t e the atomic a b s o r p t i o n spectrometer readings. 42 To a v o i d i n a c c u r a c i e s due to o l d s o l u t i o n s , a l l the stock s o l u t i o n s were compared to f r e s h l y made t e s t s o l u t i o n s . Table IX - J a r r e l Ash 810 Operating Parameters Parameter Cd Cr Cu Fe Ni Pb Zn Lamp Current 6 10 10 10 10 7.5 7.5 i n ma. Flame Type Oxid Redu Oxid Redu Oxid Oxid Oxid Absorbing 2289 3579 3247 2484 2320 2833 2138 Wavelength (A) Non-absorbing 2268 3520 2961 2316 2825 2127 Wavelength (A) S l i t Width (A) 4 4 4 2 2 4 4 Channel A-B A-B A-B A A-B A-B A-B C o n f i g u r a t i o n An attempt to measure pH and o x i d a t i o n - r e d u c t i o n p o t e n t i a l (orp) was made to c h a r a c t e r i z e the i n s i t u c o n d i t i o n s of l y s i m e t e r F. U n f o r t u n a t e l y the orp probe s t a r t e d to respond e r r a t i c a l l y d u r i n g warmup. Another orp probe was not a v a i l a b l e at that time so an orp measurement was not made. The pH measurements were obtained by surrounding the t i p of the probe with the l y s i m e t e r c o n t e n t s . T h i s was done to ensure c o n t a c t with the probe t i p because there was no f r e e l i q u i d i n the l y s i m e t e r . The pH probe was p l a c e d three times to ensure c o n s i s t e n c y . A l l the pH readings and c a l i b r a t i o n s were conducted i n a n i t r o g e n atmosphere to a v o i d exposing the l y s i m e t e r contents to a i r . 43 B. CHELATION PROCEDURE A c h e l a t i o n experiment was d e v i s e d to t e s t the e x i s t e n c e and s t r e n g t h of n a t u r a l l i g a n d s i n a l y s i m e t e r . The experiment compared the metal masses complexed by seven c h e l a t i n g agents (EDTA, ethanoic a c i d , g l y c i n e , h i s t i d i n e , 8 - h y d r o x y q u i n o l i n e ( o x i n e ) , NTA and o x a l i c a c i d ) to the masses that they should t h e o r e t i c a l l y complex. T h i s i s accomplished by p l o t t i n g the mass of metal complexed vs the l i g a n d used f o r each complex. A separate curve i s generated f o r each metal c o n s i d e r e d . A t h e o r e t i c a l curve can be generated by p l o t t i n g the mass of metal t h a t would be expected to complex with each l i g a n d knowing the i n i t i a l metal c o n c e n t r a t i o n , the i n i t i a l l i g a n d c o n c e n t r a t i o n and the s t a b i l i t y constant f o r each complex. If the l i g a n d s are arranged i n order from weakest to s t r o n g e s t a c h e l a t e g r a d i e n t i s e s t a b l i s h e d . By c o r r e c t i n g the measured mass of metal f o r c h e l a t i o n e f f i c i e n c y the measured data can be compared to the t h e o r e t i c a l data. Any d i f f e r e n c e s between the t h e o r e t i c a l and measured curves should i n d i c a t e the presence of n a t u r a l l i g a n d s . The p o i n t where the t h e o r e t i c a l and measured curves d i v e r g e r e p r e s e n t s the upper l i m i t of n a t u r a l l i g a n d complex s t r e n g t h . It i s p o s s i b l e to f u r t h e r r e f i n e the experiment by s e l e c t i n g l i g a n d s to provide a s e r i e s of smaller increments f o r the c h e l a t e g r a d i e n t . The s t a b i l i t y c o n s t a n t s , that were a v a i l a b l e , f o r the metals t e s t e d are shown in Table X. Two d i f f e r e n t experimental techniques were used to determine the mass of metal complexed. The f i r s t was c a l l e d the Table X - LoglO S t a b i l i t y Constants 44 Ion Cu + 2 Ni + 2 Zn + 2 Pb + 2 C d + 2 F e + 3 F e + 2 Mn* 2 Cr + 3 Cr + 2 Mg+ 2 C a + 2 A l + 3 EDTA 18.92 18.36 16.26 18.32 16.9 25 14 14 23 13 11.0 11.0 16.7 1 2 5 4 61 Etha 2.23 1 .43 1 .57 2.68 1 .93 2.63 1 .82 1 .4 1 .8 1 .28 1.12 Glyc 8.6 6.4 5.9 5.11 6.0 10.0 4.3 3.9 8.62 1 0* H i s t 10.21 8.5 6.4 96 65 0 85 24* 8-Hy 13.49 1 1 .44 9.96 10.61 9.43 1 3.69 8.77 7.3 13.3 13.29 4.35* NTA 1 1 1 1 10 1 1 10 15 8.84 8.6 13.5 5.46 6.46 9.5 5 54 44 47 0 91 Oxal 4.85 5. 16 44 32 2 39 05 97 34 2.39* 1 .66 Note: * - i n d i c a t e s a s t a b i l i t y constant f o r two Ligands per metal. A f t e r S i l l e n 1971. method of standard comparisons and the second was c a l l e d the method of standard a d d i t i o n s . Both techniques r e q u i r e d the use of s pike s o l u t i o n s or standard s o l u t i o n s . The standards were made up by mixing atomic a b s o r p t i o n stock s o l u t i o n s i n molar p r o p o r t i o n s t h a t were s i m i l a r to those expected i n the samples. Table XI l i s t s the metal c o n c e n t r a t i o n s that were used to estimate the moles of c h e l a t i n g agents that would be r e q u i r e d . Table XII l i s t s the c o n c e n t r a t i o n s of each metal i n the s p i k e s o l u t i o n s . The method of standard comparisons assumes that the e x t r a c t i o n e f f i c i e n c y of each c h e l a t i n g agent i s the same f o r the sample and the standards. A l s o , the metal s p e c i e s in the standards and sample are assumed to be the same. T h i s l a s t assumption was found t o be i n v a l i d a f t e r the f i r s t set of c h e l a t i o n data was c o l l e c t e d . I t was d i s c o v e r e d that a p r e c i p i t a t e was forming i n the standards r e n d e r i n g them u s e l e s s . 45 Table XI - A p p l i e d And Measured Metals In Lysimeter Parameter Cd Cr Cu Fe Ni Pb Zn gm/mole 112.4 52.0 63.5 55.9 58.7 207.2 65.4 Net input In mM 4.78 1 53 1 4000 13.8 180 2400 uM/ml of con t e n t s 0.096 3.06 279 0.276 3.6 48.0 Measured Va1ue s in uM metal/gm dry wt Sample A-raw B-blended C-blended 0.4 0.35 0.035 1 .8 0.535 0.205 0.59 0. 178 1 .33 59 24.6 82 0.032 0. 175 0. 1 43 1 .37 2.11 3.0 4.73 5.1 5 9.83 Notes: 1. Net tank v a l u e s from Atwater et a l . (1981). 2. A--14.2 gms. of l y s i m e t e r m a t e r i a l d i g e s t e d i n a c i d . Sample was taken near a l a y e r of e l e c t r o p l a t i n g waste. 14.2x0.4 = 5.68 gms. dry weight. 3. B--258.4 gms. of blended sample. Sample was taken near a l a y e r of e l e c t r o p l a t i n g waste. 258.4x0.013 = 3.4 gms. dry weight. 4. C—70.6 gms. of blended sample. Sample was taken near a l a y e r of s e p t i c tank waste. 70.6x0.069 = 4.87 gms. dry weight. 5. Six d i f f e r e n t moisture content d e t e r m i n a t i o n s were taken f o r three l y s i m e t e r samples: 0.394,0.364,0.439 g i v i n g an average = 0.40 6. Tank Volume = 50 1. Diameter = 30 cm. Height = 70 cm. Volume = 7T70(30) 2/4 = 50000 cm 3 = 50 1. The g r a v e l l a y e r (15 cm.) and the compressed s o i l cover (10 cm.) were s u b t r a c t e d from the height of the l y s i m e t e r contents (95 cm.) f o r the height e s t i m a t e . Table XIII l i s t s the s o l u b i l i t y of v a r i o u s chromium compounds which c o u l d be p r e c i p i t a t i n g i n the standards. A f t e r some t e s t i n g i t was determined that l e a d chromate (PbCrO,) had 46 Table XII - C o n c e n t r a t i o n Of Metals In Spike S o l u t i o n s Cd Cr Cu Fe Ni Pb Zn DS1 uM/ 50 ml. Mix 1 0.44 4.81 1 .34 206 1 .45 4.83 36.7 Mix 2 0.04 0.48 0.13 20.6 0.14 0.48 3.8 Spike 3 0.44 3.85 — 35.8 Mix 4 0.89 9.62 3.15 358 3.41 9.65 76.5 Mix 5 0.18 1 .92 0.63 71.6 0.68 1 .93 15.3 DS2 uM/ Spike 100 2.5 16.9 5.67 358 27.3 8.88 67.3 50 1 .25 8.4 2.83 1 79 13.6 4.44 33.6 10 0.25 1 .69 0.57 35.8 2.7 0.89 6.7 Notes: 1. DS1 = C h e l a t i o n data set one 2. DS2 = C h e l a t i o n data set two 3. Mix 1,2,3,4 or 5 = Standards which were c h e l a t e d . 4. Spike 3 = T h i s i s a spike which was added to a sample. 5. Spike 10, 50 or 100 = These are spike s o l u t i o n s which were added to samples f o r data set two. Table XIII - S o l u b i l i t y Of Chromium Compounds Metal M(Cr 20«) M ( C r 2 0 7 ) M(CrO«) Cd i Cu i vs i Fe i s Pb d 5.8X10" 6 Zn vs i A f t e r Weast (1971 ). Note: CrO, and C r 2 0 7 should be i n e q u i l i b r i u m i n the spike s o l u t i o n , with a m a j o r i t y of the chromium i n the CrOfl form. Where: i = i n s o l u b l e d=decomposes s=soluble vs=very s o l u b l e 47 p r e c i p i t a t e d i n the s p i k e s o l u t i o n s . Instead of u s i n g the atomic a b s o r p t i o n spectroscopy stock s o l u t i o n f o r chromium s p i k e s , a chromium c h l o r i d e ( C r C l 3 ) s o l u t i o n was used. The second technique (method of standard a d d i t i o n s ) was used to a v o i d the problems of c h o s i n g standards i n the same c o n c e n t r a t i o n range as the samples. Spike s o l u t i o n s were added to three samples and a f o u r t h sample had no spike a d d i t i o n . The atomic a b s o r p t i o n spectrometer readings were p l o t t e d vs the percent s p i k e added to the sample (The p l o t s are shown i n Appendix 0 ) . The c o n c e n t r a t i o n of each metal i n the spike s o l u t i o n was known (Values are l i s t e d i n Table XII) so i t i s t h e o r e t i c a l l y p o s s i b l e to determine the c o n c e n t r a t i o n of complexed metal i n a sample. There i s an u n d e r l y i n g assumption with t h i s technique, t h a t the complex formation and e x t r a c t i o n e f f i c i e n c y i s the same f o r the s p i k e s o l u t i o n s and the samples. The organic s o l v e n t m e t h y l - i s o - b u t y l ketone 5 (MIBK) was used to e x t r a c t a l l the complexes. MIBK has a lower d e n s i t y than water so i t forms a l a y e r above water. The MIBK and sample mixture were w e l l mixed by shaking and then l e f t to s i t o v e r n i g h t . That a l l o w s the MIBK enough time to d i s s o l v e a p o r t i o n of the metal complexes and to form a l a y e r above the water based sample. In t h i s study i t was necessary to c e n t r i f u g e samples to remove the s o l i d s because they tended to f l o a t at the MIBK-water i n t e r f a c e making s e p a r a t i o n of the two 5. Care should be taken when using MIBK because i t i s very v o l a t i l e . I t i s a l s o a s k i n and i n h a l a t i o n i r r i t a n t . 48 l a y e r s i m p o s s i b l e . The r e s u l t i n g supernatant contained MIBK and a water s o l u t i o n which was r e a d i l y separated. Each c h e l a t e was allowed to react independently so there was one sample per c h e l a t e and seven samples per sample s e t . The number of moles of each c h e l a t e was determined a f t e r c o n s i d e r i n g the s t r e n g t h of the c h e l a t e , the number of bindin g s i t e s per molecule of c h e l a t e and the t o t a l moles of metal expected i n a sample. The l i g a n d s were added so that they would be a f a c t o r of f i v e i n excess of the expected metals. T h i s should allow other p o t e n t i a l complexes (eg. c a l c i u m complexes) to form without causing a l i g a n d shortage. Table XIV l i s t s the moles of c h e l a t i n g agent added to the samples. A l l the c h e l a t i o n r e a c t i o n s were co n t a i n e d i n e x t r a c t i o n f u n n e l s as they are s p e c i f i c a l l y designed f o r c h e l a t i o n e x t r a c t i o n s . F i g u r e 3 shows a diagram of an e x t r a c t i o n f u n n e l . The va l v e at the bottom was used to g r a v i t y d r a i n the water l a y e r from the f u n n e l . P l a s t i c stoppers were used to help keep the samples anaerobic u n t i l the c h e l a t i n g agents were added. U n f o r t u n a t e l y there was i n s u f f i c i e n t c l e a r a n c e i n the anaerobic chamber to allow p i p e t t i n g of the c h e l a t i n g agents. A f t e r the c h e l a t i n g agents were added, the e x t r a c t i o n f u n n e l s were shaken and allowed to s i t o v e r n i g h t . MIBK was added the next day and the samples were mixed and l e f t overnight a g a i n . Then the whole sample was t r a n s f e r r e d to l i g h t p o l y e t h y l e n e b o t t l e s f o r c e n t r i f u g i n g (Nalagene c e n t r i f u g e b o t t l e s were a t t a c k e d by MIBK). A f t e r c e n t r i f u g i n g , the 49 Table XIV - Moles Of C h e l a t i n g Agent EDTA Eth Gly H i s t Hyd NTA Oxa B i n d i n g S i t e s / L igand 4 1 1 1 1 3 2 [Ligand] mM/1 95.4 1715 857 171 .2 85.42 425.5 856.3 mM Bond S i t e s / m l 0.38 1.7 0.86 0.17 0.085 1 .28 1 .7 mM Added to DS1 7.6 11.9 6.0 3.0 6.0 9.0 1 1 .9 mM Added to DS2 4.77 17.15 8.57 6.85 5.98 6.38 8.56 Note: 1. DS1 used 120 ml. samples with a 0.03 s o l i d s content = 3.6 gm. dry weight. 2. DS2 used 50 ml. samples with a 0.0363 s o l i d s content f o r Blk and 50% samples = 1.8 gms dry weight. Samples 10% and 100% had a 0.0307 s o l i d s content = 1.5 gms dry weight. 3. DS1 r e q u i r e d 2.5 mM. of c h e l a t i n g agent. DS2 r e q u i r e d 1.5 mM. of c h e l a t i n g agent. supernatant was decanted i n t o the e x t r a c t i o n f u n n e l s . Then the water l a y e r was removed. The MIBK l a y e r was d r a i n e d through a Whatman number 541 f i l t e r and c o l l e c t e d f o r a n a l y s i s with the atomic a b s o r p t i o n spectrometer. A flow c h a r t of the c h e l a t i o n procedure i s shown i n F i g u r e 4. C. METAL EXTRACTION PROCEDURE A s u c c e s s i v e e x t r a c t i o n technique was used to determine the m o b i l i t y of metals. Samples were e x t r a c t e d using a s l i g h t l y m o d i f i e d v e r s i o n of a procedure developed by Engler (1977). The metals were grouped i n t o s i x c a t e g o r i e s : f r e e , r e a d i l y e x t r a c t e d , c a t i o n exchangeable, o r g a n i c a l l y bound, moderately e x t r a c t a b l e and r e s i d u a l . E n g l e r ' s procedure was m o d i f i e d by F i a u r e 3 - E x t r a c t i o n Funnel 5 0 P L A N VIEW P R O F I L E u s i n g a c e n t r i f u g e to separate supernatants i n s t e a d of a f i l t r a t i o n system. T h i s m o d i f i c a t i o n helped to maintain anaerobic samples. The e x t r a c t i o n procedure used three sample types, a l l of which had been suspended and homogenized to allow wet chemistry m a n i p u l a t i o n s . One sample was a e r a t e d before e x t r a c t i o n so an estimate of the " s t a b l e " metal mass c o u l d be made. Another sample was s o n i c a t e d before e x t r a c t i o n to determine the metal mass r e t a i n e d by organisms. A t h i r d sample was not aerated or s o n i c a t e d before e x t r a c t i o n . T h i s sample was used as a c o n t r o l f o r comparison with the a e r a t e d and s o n i c a t e d samples. The samples were l a b e l l e d by using the sample type and e x t r a c t i o n phase to form sample numbers. There were s i x e x t r a c t i o n phases numbered 1 through 6 which were p r e f i x e d by FOX, SON or B L K F i g u r e 4 - Flow Chart Cf The C h e l a t i o n Procedure 51 D i l u t e 300 ml. of blended sample and d i l u t e with d e i o n i z e d d i s t i l l e d water to 900 ml. I Put 50 ml. of sample i n t o each e x t r a c t i o n f l a s k . Add s p i k e s o l u t i o n . I Add c h e l a t i n g agent. Shake w e l l I Add m e t h y l - i s o - b u t y l ketone. I C e n t r i f u g e and pour supernatant i n t o e x t r a c t i o n f u n n e l s . Then allow the r e s i d u e to d r a i n i n t o f u n n e l s . I Shake the e x t r a c t i o n f u n n e l s w e l l an J. leave o v e r n i g h t . I D r a i n o f f the lower l a y e r of l i q u i d and d i s c a r d . ( T h i s i s the water l a y e r . ) D r a i n the remainder of the l i q u i d through a f i l t e r i n t o the sample c o n t a i n e r s . ( T h i s i s the MIBK l a y e r . ) I Analyze. which r e f e r r e d to a e r a t e d , s o n i c a t e d or c o n t r o l samples r e s p e c t i v e l y . An i n i t i a l sample of the blended l y s i m e t e r mixture was taken before commencing with the e x t r a c t i o n procedure. These samples were l a b e l l e d IIKO, SON0 and FOX0. Each phase of the e x t r a c t i o n procedure used the res i d u e from the p r e v i o u s phase f o r an e x t r a c t i o n sample. The samples were d i l u t e d u s i n g d e i o n i z e d d i s t i l l e d water and then allowed to re a c t with the e x t r a c t i n g agent. The only exceptions occured i n phases four and s i x where a sample d i g e s t i o n proceded the a d d i t i o n of an e x t r a c t i n g agent. A f t e r adding the e x t r a c t i o n agents the samples were c e n t r i f u g e d and the supernatant was 52 c o l l e c t e d . The re s i d u e was kept f o r the next phase of e x t r a c t i o n and the supernatant was kept as the sample f o r that phase. The phases are d e s c r i b e d i n Table XV and a flow c h a r t of the e x t r a c t i o n procedure i s shown i n F i g u r e 5. Table XV - E x t r a c t i o n Procedure Phase Treatment E x t r a c t s I n t e r s t i t i a l Water D i l u t e and C e n t r i f u g e Pour o f f supernatant Free Water S o l u b l e metals Exchangeable Add Ammonium Acetate E a s i l y E x t r a c t e d Metals E a s i l y Reducible Add Hydroxylamine Hydrochlor ide Manganese Oxides Metals with Manganese Oxides Organic Dige s t with Hydrogen Peroxide*** and E x t r a c t with Ammonium Acetate D i g e s t s Organics Metals r e l e a s e d by d e s t r u c t i o n of or g a n i c s Moderately Reduc i b l e Add Sodium c i t r a t e Sodium d i t h i o n i t e E x t r a c t s Iron Oxides Metals with Iron Oxides R e s i d u a l A c i d D i g e s t E x t r a c t s metals from r e s i d u e 'Fixed' metals *** See the d i s c u s s i o n i n the s e c t i o n t i t l e d 'Procedure M o d i f i c a t i o n s ' on page 68. The supernatant from each phase and the r e s i d u e from the l a s t phase were a c i d d i g e s t e d using c o n c e n t r a t e d n i t r i c a c i d . A f t e r d i g e s t i o n , the samples were f i l t e r e d through a Whatman number 541 f i l t e r and made up to volume u s i n g d e i o n i z e d d i s t i l l e d water. Phase 4, 5 and 6 samples were made up to 250 F i g u r e 5 - Flow Chart Of The E x t r a c t i o n Procedure S p l i t roughly 900 ml. of sample i n t o 3-300 ml. groups I D i l u t e a l l 3 samples up t o 900 ml. w i t h d e i o n i z e d d i s t i l l e d water I Put one sample i n t o a 4000 watt s o n i c a t i o n bath f o r 30 min. Bubble a i r through another f o r 30hr. Do not t r e a t the t h i r d sample. | Remove a subsample from each sample f o r : i ) A b a c t e r i a l sample, i i ) moisture content sample, i i i ) An i n i t i a l metals sample. (Subsamples BLK0,SON0,FOX0) I Remove supernatant and d i l u t e sample to 900 ml. with d e i o n i z e d d i s t i l l e d water. (Subsamples BLK1,SON 1,FOX 1) Add 19.3 gm. ammonium a c e t a t e t o each sample and shake w e l l . I C e n t r i f u g e each sample. I Remove supernatant and d i l u t e sample t o 900 ml. w i t h d e i o n i z e d d i s t i l l e d water. (Subsamples BLK2,SON2,FOX2) Add 2.61 gms. of hydroxylamine h y d r o c h l o r i d e t o each sample and C e n t r i f u g e I Remove supernatant and d i l u t e sample t o 1000 ml. with d e i o n i z e d d i s t i l l e d water'. (Subsamples BLK3,SON3,FOX3) Add 150 ml. of hydrogen^peroxide t o 1000 ml. of sample. Add 9.65 gms. ammonium a c e t a t e t o 500 ml. of sample and C e n t r i f u g e Remove supernatant and d i l u t e sample t o 300 ml. w i t h d e i o n i z e d d i s t i l l e d water. (Subsamples BLK4,SON4,FOX4) I Add 10 gm. sodium c i t r a t e and 20 gms. sodium d i t h i o n i t e to each sample. C e n t r i f u g e . I Remove supernatant (Subsamples BLK5,SON5,F0X5) The r e s i d u e forms Subsamples BLK6,SON6,FOX6 * Note that t h i s step should be r e p l a c e d by dry o x i d a t i o n i n an oven at 500°C. 54 ml. and the f i l t e r s were r i n s e d with ammonium a c e t a t e to improve metal r e c o v e r y . Samples from a l l other phases were made up to 100 ml. and the f i l t e r s were only r i n s e d with d e i o n i z e d d i s t i l l e d water. D. BACTERIAL COUNTS To determine the mass of metals a s s o c i a t e d with b a c t e r i a , techniques f o r counting b a c t e r i a l numbers were i n v e s t i g a t e d . A f l u o r e s c e n t dye s t a i n i n g technique which employed the dye 4,6-di a m i d i n o - 2 - p h e n y l i n d o l e was s e l e c t e d f o r t h i s study. Many re s e a r c h e r s r e p o r t e d t h e i r success at co u n t i n g small numbers of b a c t e r i a with an o p t i c a l microscope (Hobbie 1977, Daley and Hobbie 1979, Ka p u s c i n s k i and Skoczylas 1977, S a l a r i and Ward 1979, Cowell and Franks 1980, P o r t e r and F e i g 1980, Alan and M i l l e r 1980, Coleman 1980, Coleman et a l . 1981). (Further d e t a i l s of the i n f o r m a t i o n a v a i l a b l e i n the l i t e r a t u r e i s c o n t a i n e d i n Appendix L ) . None of the a r t i c l e s reviewed r e p o r t e d the use of 4,6-diamidino-2-phenylindole f o r co u n t i n g b a c t e r i a i n complex organic samples l i k e l e a c h a t e . A f t e r some experimentation and per s o n a l communications with Iqubal V e l g i (SFU), a procedure was formulated. A d e t a i l e d l i s t of the experimental steps i s co n t a i n e d i n Appendix G. B a c t e r i a l samples were taken from the s o n i c a t e d a e r a t e d and blank samples. Each sample was weighed and the moisture content was determined. A l l the b a c t e r i a l samples were p l a c e d i n a c i d washed, d e i o n i z e d d i s t i l l e d water r i n s e d , 50 ml. Erlenmyer f l a s k s and d i l u t e d to 40 ml. with d e i o n i z e d d i s t i l l e d water. A f t e r d i l u t i o n 5 ml. of formaldehyde s o l u t i o n was added to f i x 55 the b a c t e r i a . A l a t e r m o d i f i c a t i o n r e p l a c e d formaldehyde with 5 ml. of 70% ethanol s o l u t i o n . The samples were s t o r e d i n a dark room at 4°C u n t i l the counts were to be taken. To o b t a i n counts, sodium pyrophosphate was added to the f i x e d b a c t e r i a l samples, before s o n i c a t i o n , t o break up the s u b s t r a t e . A f t e r s o n i c a t i o n , the sample was f i l t e r e d through a 25 mm., diameter, 0.02 urn. Nucleopore f i l t e r . The f i l t e r s were s t a i n e d with i r g a l a n black before use, to reduce background luminescence under 'the microscope. When 6 ml. of prepared sample had been f i l t e r e d , the f i l t e r s were p l a c e d on a microscope s l i d e . One drop of c a r g y l e B o i l was added before a cover s l i p was pl a c e d over the f i l t e r . The s l i d e s were observed using a Xenon lamp with a 350 380 nm. e x c i t a t i o n f i l t e r . A drop of c a r g y l e B o i l was used f o r immersion o i l and a 100X m a g n i f i c a t i o n was used. Six f i e l d s of view were observed on each s l i d e and ten s l i d e s were made of each sample. According to T r o l l d e n i e r (1973), that should give a 5% p r o b a b i l i t y that the r e a l v alue was more than 10% d i f f e r e n t from the counts obtained.' A flow c h a r t of the dye procedure i s shown i n F i g u r e 6. F i g u r e 6 - Flow Chart Of The F l u o r e s c e n t Dye Technique Add 5 ml. of 70% e t h a n o l to 5 ml. of s o l i d s i n a 50 ml. s t e r i l i z e d erlenmeyer f l a s k . I D i l u t e mixture with f i l t e r e d d e i o n i z e d d i s t i l l e d water to 50 ml. I D i l u t e 1 ml. of mixture to 10 ml. with f i l t e r e d d e i o n i z e d d i s t i l l e d water and mix w e l l . I Add 5 ml. of 0.01% f i l t e r e d sodium pyrrophosphate to 2 ml. of d i l u t e d mixture. I Incubate mixture at room temperature f o r 30 min. and mix every f i v e min. I S o n i c a t e pyrrophosphate mixture f o r 5 min. I Add 0.2 ml. of 0.1 M DAPI s o l u t i o n and mix w e l l . I A f t e r 15 min. f i l t e r through a 25 mm. diameter, 0.02 urn. Nucleopore f i l t e r s t a i n e d with i r g a l a n b l ack . (Use 8 mm. Hg vacuum to f i l t e r . ) I Put f i l t e r on a microscope s l i d e . I Add a drop of c a r g y l e B immersion o i l and cover with a cover s l i p . I Observe s l i d e at 100X u s i n g a 340-370 nm. e x c i t a t i o n beam. 1 Count 4-6 f i e l d s of view on 10 or more s l i d e s . 5 7 IV. DATA RESULTS AND DISCUSSION Three separate experiments were conducted f o r t h i s study: a c h e l a t i o n experiment; an e x t r a c t i o n experiment; and a b a c t e r i a l s t a i n i n g experiment. The e x t r a c t i o n and b a c t e r i a l s t a i n i n g experiments were to be used together to determine the mass of metals r e t a i n e d by b a c t e r i a . They were t e s t e d s e p a r a t e l y to e v a l u a t e t h e i r f e a s i b i l i t y . None of the experiments had been p r e v i o u s l y used to t e s t l y s i m e t e r samples. So a p r e l i m i n a r y sample, taken from the f i r s t sampling hole, was used to t e s t the s a f e t y and w o r k a b i l i t y of the experimental procedures. A. CHELATE DATA The c h e l a t i o n experiment was designed to determine the presence and s t r e n g t h of n a t u r a l l i g a n d s i n l y s i m e t e r or l a n d f i l l samples. The experiment used seven complexing agents ( l i g a n d s ) t o form complexes with metals i n l y s i m e t e r sample suspensions. One l i g a n d was added to each sample and the complexes were e x t r a c t e d i n m e t h y l - i s o - b u t y l ketone (MIBK). The MIBK e x t r a c t s were analyzed to determine the number of moles of complex that had formed. These measured v a l u e s were then to be compared with c a l c u l a t e d v a l u e s . The complex c o n c e n t r a t i o n s (ML) were c a l c u l a t e d knowing the i n i t i a l l i g a n d c o n c e n t r a t i o n (LO), the s t a b i l i t y constant (Ks) of each complex and the approximate number of moles of metal i n i t i a l l y i n the samples (MO). Any d i f f e r e n c e s between measured and c a l c u l a t e d complex m o l a r i t y t h a t c o u l d not be accounted f o r by the recovery e f f i c i e n c y were a t t r i b u t e d to the e f f e c t s of 58 n a t u r a l l i g a n d s and u n a v a i l a b l e m e t a l s . 6 Metals which were not complexed by the l i g a n d s with the h i g h e s t s t a b i l i t y c o nstants were c o n s i d e r e d u n a v a i l a b l e f o r complexation. To c a l c u l a t e the expected m o l a r i t y of l i g a n d s i n the MIBK e x t r a c t s the f o l l o w i n g assumptions were made: a) The metals r e a c t e d i n 1:1 p r o p o r t i o n s with the l i g a n d s . b) An excess q u a n t i t y of l i g a n d was added. c) The metal c o n c e n t r a t i o n s measured i n the e x t r a c t i o n experiment c o u l d be used to estimate the i n i t i a l metal c o n c e n t r a t i o n s of the samples. d) A l l the metal e x t r a c t e d i n the MIBK had formed a complex. These assumptions r e s u l t e d i n the formula: Ks(MO-ML)(LO-ML) = ML T h i s formula i s d e r i v e d i n Appendix P. I t has only one unknown so the complex c o n c e n t r a t i o n can be determined. Table XVI shows the complex c o n c e n t r a t i o n s expected f o r a 1.8 gm. sample (dry weight at 104°C). ( T h i s corresponded to roughly 50 gms. of the wet c h e l a t i o n samples used f o r the second data s e t . ) A l i g a n d g r a d i e n t was e s t a b l i s h e d t o f a c i l i t a t e data i n t e r p r e t a t i o n . Each l i g a n d was put i n order a c c o r d i n g to i t s a b i l i t y to form complexes with the metals t e s t e d . The o r d e r i n g 6. T h i s study d e f i n e d the recovery e f f e c i e n c y (Re) as the moles of metal that were complexed, d i v i d e d by the moles of metal i n i t i a l l y p r e s e n t . The recovery e f f i c i e n c y combines the e x t r a c t i o n e f f i c i e n c y of MIBK with the complexation e f f i c i e n c y of a l i g a n d . 59 Table XVI - Expected Complex Co n c e n t r a t i o n s uM of Metal Complexed/ 1.8 gm of Dry Lysimeter Sample EDTA Eth Gly H i s t Hyd NTA Oxa C d + 2 0.72 0.43 0.72 0.72 0.72 0.72 0.67 Cr + 3 3.24 3.24 3.24 3.24 3.24 3.24 C u + 2 2.34 1 .74 2.34 2.34 2.34 2.34 2.34 Fe + 3 1 48 130 1 48 146 1 48 1 48 148 F e + 2 1 48 78 147 1 48 148 1 48 134 Ni + 2 0.32 0.1 0.32 0.32 0.32 0.32 0.32 Pb + 2 5.37 4.79 5.36 5.37 5.37 5.37 5.09 Zn* 2 17.8 6.9 17.8 17.8 17.8 17.8 17.1 The data i s c a l c u l a t e d f o r c h e l a t i o n data set two. The formulae are shown i n Appendix P. used was (from s m a l l e s t to l a r g e s t s t a b i l i t y c o n s t a n t ) : ethanoic a c i d , o x a l i c a c i d , g l y c i n e , h i s t i d i n e , NTA, EDTA and 8-h y d r o x y q u i n o l i n e . T h i s o r d e r i n g used maximum s t a b i l i t y c o n s t a n t s because some l i g a n d s (eg. 8-hydroxyquinoline) form much stronger complexes when two l i g a n d s or more are a s s o c i a t e d with a c o o r d i n a t i n g metal. When the measured and c a l c u l a t e d data are compared using a l i g a n d g r a d i e n t i t should be p o s s i b l e to determine the e f f e c t s of n a t u r a l l i g a n d s . The d i f f e r e n c e s between the measured and c a l c u l a t e d data at the hig h s t a b i l i t y constant end of the g r a d i e n t should be due to u n a v a i l a b l e metals. I f t h i s i s e s t a b l i s h e d as a base value a new c a l c u l a t e d metals c o n c e n t r a t i o n can be determined by s u b t r a c t i n g the base c o n c e n t r a t i o n from each of the c a l c u l a t e d c o n c e n t r a t i o n s . Then any d i f f e r e n c e s between the measured and newly c a l c u l a t e d 60 c o n c e n t r a t i o n s should be due to n a t u r a l l i g a n d s a l o n e . A sharp decrease i n metal complexed by n a t u r a l l i g a n d s would i n d i c a t e that the bulk s t a b i l i t y constant of the n a t u r a l l i g a n d s i s l e s s than the s t a b i l i t y constant of the added l i g a n d . In t h i s way an upper bound to the bulk s t a b i l i t y constant of n a t u r a l l i g a n d s can be e s t a b l i s h e d f o r each metal. 1. CHELATION DATA SET ONE I n i t i a l l y a method of standard comparisons was used to determine complex c o n c e n t r a t i o n s and e x t r a c t i o n e f f i c i e n c i e s . T h i s technique d i d not work w e l l because the metal s p e c i e s i n the standards were not the same as those i n the samples and the chromium standard formed a p r e c i p i t a t e when mixed with the l e a d standard. So the mixed metal standards were u s e l e s s . The problems with the standard comparison technique gave r i s e to two changes: the use of C r C l 3 d i s s o l v e d i n 0.05% n i t r i c a c i d f o r a chromium standard and the use of the method of standard a d d i t i o n s . The q u a n t i t y of l i g a n d was a l s o i n c r e a s e d f o r the second data set because only Cr, Cu, Fe and Zn were d e t e c t e d i n the MIBK e x t r a c t s . The raw data i s shown in Appendix M. Metal c o n c e n t r a t i o n s c o u l d not be determined f o r the standard comparison data because a p r e c i p i t a t e formed i n the standard s o l u t i o n s and the p r e c i p i t a t e was not observed u n t i l the standards were prepared f o r the second data s e t . The l a c k of c o n c e n t r a t i o n data prevented the d e t e r m i n a t i o n of recovery e f f i c i e n c i e s . T h i s study was not of s u f f i c i e n t d u r a t i o n to permit a repeat of the standard comparison data. 61 A pseudo mass r a t i o (Rx) value was c a l c u l a t e d f o r each metal to p r o v i d e a c l e a r example of the c a l c u l a t i o n s that would be r e q u i r e d to compare c h e l a t e s . I t may be p o s s i b l e at a future date to use the Rx values f o r a comparison with other s t u d i e s . To c a l c u l a t e the pseudo mass r a t i o s the absorbancy values r e g i s t e r e d by the atomic a b s o r p t i o n spectrometer were assumed to be d i r e c t l y p r o p o r t i o n a l to the metal c o n c e n t r a t i o n s . T h i s assumption c o u l d not be v e r i f i e d without r e p e a t i n g the c h e l a t i o n procedure. The pseudo mass r a t i o i s the r a t i o of absorbancy to c o r r e c t e d sample mass. The c o r r e c t e d sample mass represented the dry weight of sample d i v i d e d by the volume of MIBK that was recovered. To t r a n s l a t e the Rx v a l u e s to mass r a t i o s the recovery e f f i c i e n c y (Re), and the c o n c e n t r a t i o n c o n v e r s i o n f a c t o r 7 must be known. More s p e c i f i c d e t a i l s f o r c a l c u l a t i n g the Rx v a l u e s , shown i n Table XVII, are c o n t a i n e d i n Appendix Q. Table XVII - Pseudo Mass R a t i o s For Data Set One Rx Values EDTA Eth Gly H i s t * Hyd NTA Oxa Cr 2.65 2.35 2.60 1 .63 4.03 6.42 1 .43 Cu 0 .81 0.68 0.59 0.61 >6.3 0.34 2.1 Fe 0.75 1 .07 4.10 1 .37 >8 1.14 <0.53 Zn >8 0.15 0.35 0.14 1 .48 0.19 0.65 * S p i l t roughly 7 ml. of sample. See Appendix Q f o r pseudo mass r a t i o c a l c u l a t i o n s . 7. The c o n c e n t r a t i o n c o n v e r s i o n f a c t o r i s the f a c t o r i s the m u l t i p l i e r used to convert absorbancy values to c o n c e n t r a t i o n s . 62 2. CHELATION DATA SET TWO The second c h e l a t i o n data set was c o l l e c t e d using the method of standard a d d i t i o n s . Absorbancy v a l u e s , from the a n a l y s i s of MIBK e x t r a c t s , were p l o t t e d on absorbancy vs percent s p i k e graphs. The data was expected to form a smooth curve so the metal c o n c e n t r a t i o n s and recovery e f f i c i e n c i e s c o u l d be determined. U n f o r t u n a t e l y t h i s was not the case. As the p l o t s i n Appendix 0 show, none of the data formed a smooth curve. T h i s may be due to v a r i a t i o n s i n the samples or to problems with the standards. To demonstrate the r e q u i r e d c a l c u l a t i o n s and to f i l t e r out the e f f e c t s of d i f f e r e n t sample s i z e s a pseudo mass r a t i o (Xm) was c a l c u l a t e d f o r the second data s e t . The method of c a l c u l a t i o n f o r the Xm v a l u e s d i f f e r e d from the Rx values i n two ways but the assumptions were the same. The Xm value d i d not c o r r e c t f o r f l u c t u a t i o n s i n the volume of MIBK that was recovered and the Xm v a l u e s were normalized by d i v i d i n g the wet weights of the samples by f i f t y . The Xm v a l u e s represented pseudo mass r a t i o s f o r a 1.8 gm. sample ( a f t e r d r y i n g at 104°C). An example Xm c a l c u l a t i o n i s shown i n F i g u r e 7 and f u r t h e r d e t a i l s of the c a l c u l a t i o n s are c o n t a i n e d i n Appendix Q. Table XVIII shows the c a l c u l a t e d Xm v a l u e s . The 10, 50 and 100 numbers correspond to samples with a 10%, 50% and 100% spike a d d i t i o n . The Xm v a l u e s f o r a given metal and c h e l a t o r complex c? r. be compared to the other complexes of the same metal i f the recovery e f f i c i e n c i e s are assumed to be s i m i l a r . 63 F i g u r e 7 - C a l c u l a t i o n s Of Pseudo Mass R a t i o s For Data Set Two RR = Mm/WP RR = CmVi/WP RR/Vi = Cm/WP Cm = xAm RR/Vi = xAm/WP x = a constant which i s d i f f e r e n t f o r each metal. M u l t i p l y both s i d e s of the equation by 50 gms. to c a l i b r a t e the samples to a 50 gms. wet mass sample. RR50/xVi = Am50/WP = Xm Le t : f2 = 50/WP Xm = Am(f2) Note: Appendix W c o n t a i n s a l i s t of symbols. Table XVIII - Pseudo Mass R a t i o s For Data Set Two Xm Values EDTA Eth Gly H i s t Hyd NTA Oxa Cr Blk 7.21 4.42 2.9 1 .7 8.0 5.1 0.6 10 10.9 17.1 7.3 7.7 22 8.1 3.2 50 6.0 6.5 4.8 5.0 7.7 5.5 1 .2 1 00 10 1 1 8.3 8.6 10 8.3 2.4 Cu Blk 2.3 0.8 1 .3 1.5 49 1 .3 1 .5 10 2.0 3.0 1 .2 1 .3 84 1 .5 2.0 50 2.3 1.8 1.5 0.9 5. 1 2.5 1.8 100 2.4 2.7 1 .4 1 .5 3.1 2.3 1 .9 Fe Blk 20 0.17 5.8 6.6 310 1 .0 13 10 21 9.9 3.5 4.8 210 1 .4 6.4 50 29 1.3 2.4 9.8 92 49 25 100 19 1 .5 3.6 4.3 27 1 3 10 Zn Blk 17 3.0 8.6 1 1 0.3 0.9 10 17 2.4 0.7 1.8 2.6 1 .4 1.2 50 20 0.29 1 . 1 2.1 0.45 33 5.0 100 18 0.84 0.27 2.0 0.76 1 4 6.2 Note: Zn has a n o n - l i n e a r response i f the Measured c o n c e n t r a t i o n exceeds 12 mg/1. S i m i l a r l y f o r Fe > 60 mg/1. See Appendix Q f o r pseudo mass r a t i o c a l c u l a t i o n s . 6 4 Again, only Cr, Cu, Fe and Zn were d e t e c t e d i n MIBK e x t r a c t s . E i t h e r Cd, Ni and Pb were u n a v a i l a b l e f o r complexation or the c h e l a t i o n e x t r a c t i o n e f f i c i e n c y was very poor f o r these metals. I t i s u n l i k e l y t h at a l i g a n d shortage occured to prevent Cd, Ni or Pb complexation. Iron was the most abundant metal i n the l y s i m e t e r and there should have been enough l i g a n d to c h e l a t e over s i x times the a v a i l a b l e i r o n mass. A l s o , the s t a b i l i t y c o n s t a n t s of Cd, Ni and Pb are comparable with those of Zn so even an abundant metal l i k e Ca would not cause a l i g a n d shortage. Calcium would need to be a v a i l a b l e at c o n c e n t r a t i o n s four orders of magnitude g r e a t e r than n i c k e l to s u c c e s s f u l l y compete f o r l i g a n d s . (Note: L o g ( S t a b i l i t y constant)=6.5 f o r Ca-NTA and 11.5 f o r Ni-NTA). The Xm data cannot be used to prove the e x i s t e n c e of n a t u r a l l i g a n d s because the metal c o n c e n t r a t i o n s are unknown. T h i s a l s o prevented the c a l c u l a t i o n of the sample recovery e f f i c i e n c i e s . Instead the data was used to demonstrate how the predominant metal s p e c i e s i n a sample c o u l d be i d e n t i f i e d . For t h i s purpose the recovery e f f i c i e n c i e s were assumed to be s i m i l a r f o r a l l the complexes of a given metal. Then the Xm data were p l o t t e d to form histograms. These histograms were for comparison with the s t a b i l i t y constant vs l i g a n d histogram shown in F i g u r e 8. The l i g a n d s were ordered so the s t a b i l i t y constant i n c r e a s e d toward the r i g h t f o r most metals. The same l i g a n d o r d e r i n g was used f o r the p l o t shown in F i g u r e 9. If the p a t t e r n of r e l a t i v e magnitudes i s the same f o r a given metal i n the Xm histogram and the Ks histogram then the metals were 6 5 assumed to be the same s p e c i e s . In t h i s manner the histograms could be used to determine metal s p e c i e s . F i g u r e 8 - L o g l 0 ( S t a b i l i t y Constant) Vs Ligand Log W(Ks) vs Ligand b o Cr(JII) O E B C-u(IJ) Maximum Ks E23 Ft(IlI) EZ2 FB(II) EZ2 Zrx(II) t -T-J"T Eth \ \ N Cly Hist Ligand NT A i • i 1 i' 1 EDTA Hyd The r e l a t i v e o r d e r i n g of the Xm magnitudes changed as more spike was added to the samples. The Xm histogram i n d i c a t e s that C r ( I I I ) , F e ( I I ) and Zn(II) are present i n the blank. Copper i s probably present as C u ( l ) i n the blank, which would e x p l a i n why the order d i f f e r s from the s t a b i l i t y constant order f o r C u ( I I ) . more spike was added the o r d e r i n g a l t e r e d to i n d i c a t e C r ( I I I ) , C u ( I l ) , F e ( I I I ) and Z n ( l l ) . (Only the blank and 100% spike data were shown i n the histograms as they represented the extremes.) T h i s corresponded with the s p e c i e s known to predominate i n the spike s o l u t i o n s . Table XIX l i s t s the metal s p e c i e s i n the spike s o l u t i o n s and those expected i n the F i g u r e 9 - P l o t s Of Xm Vs Ligand 66 LogtO(Xm) vs Ligand LogW(Xm) vs Ligand Cly j T i » f NTA IDTA Hyd Ligand Log tO(Xm) vs Ligand LogtO(Xm) vs Ligand )Cu-tlk I ru~roo Ulllll Ftk Ox Cty M\»t ffTA £DTA Hyd samples. Before "he c h e l a t i o n experiment can be used to determine the s t r e n g t h of n a t u r a l l i g a n d s or metal s p e c i a t i o n , some improvements are r e q u i r e d . F i r s t , the spike s o l u t i o n s must be reduced. T h i s c o u l d be accomplished by bubbling n i t r o g e n gas through the spi k e and by using the reduced forms of i r o n and copper f o r the spike s o l u t i o n . Secondly, the e x t r a c t i o n e f f i c i e n c y of MIBK must be determined (method of standard comparisons) f o r a l l the metals under c o n s i d e r a t i o n . T h i r d l y an excess supply of l i g a n d s i s e s s e n t i a l i f the e f f e c t s of r e a c t i o n Table XIX - Species In Spike S o l u t i o n s 6 7 Cd Cr Cu Fe Ni Pb Zn Data Set 1 D i l u t e d i n Spec i e s metal DN C d + 2 KCrO„ W Cr + 3 CrO«- 2 ( CuO DN Cu + 2 ;uCr0 4 F e C l 3 DN Fe * 3 metal DN Ni * 2 metal DN PbCrO„ ZnCrO„ ZnO DN Zn + 2 Data Set 2 D i l u t e d i n Species metal DN Cd + 3 C r C l 3 DN Cr + 3 CuO DN Cu + 2 FeCl 3 DN Fe + 3 metal DN Ni + 2 metal DN ZnO DN Zn + 2 Species i n Sample C d + 2 C r + 3 Cu + F e + 2 Ni + 2 Zn + 2 DN = D i l u t e N i t r i c A c i d (0.05%) W = Water time and s o l u t i o n a c t i v i t y , upon c h e l a t i o n e f f i c i e n c y , are to be avoided. The formulae r e q u i r e d f o r determining metal s p e c i a t i o n are shown in Appendix R. Values f o r the s t a b i l i t y c o n s t a n t s , i n i t i a l l i g a n d m o l a r i t y , i n i t i a l metal m o l a r i t y and complex m o l a r i t y must be determined to s o l v e the equations f o r the m o l a r i t y of each metal s p e c i e s . B. EXTRACTION DATA Four s e t s of e x t r a c t i o n data were c o l l e c t e d . The f i r s t data set was a p r e l i m i n a r y run to see how w e l l the procedure worked. A l l the other s e t s were run s i m u l t a n e o u s l y . One set was s o n i c a t e d , another was o x i d i z e d with a i r , and the t h i r d was l e f t u n t r e a t e d as a blank f o r comparison. Major improvements a f t e r the f i r s t data set were the i n c r e a s e d number of moisture content samples and a b e t t e r mass balance. Two d i f f e r e n t sample pretreatments were s e l e c t e d to h e l p d i f f e r e n t i a t e between b a c t e r i a l e f f e c t s and chemical r e a c t i o n s . 68 The f i r s t pretreatment method s o n i c a t e d a sample f o r 1/2 h r . 8 By using a ' f l u o r e s c e n t dye to enumerate the b a c t e r i a before and a f t e r s o n i c a t i o n the a c t u a l percentage of b a c t e r i a which were s o l u b i l i z e d c o u l d be determined. Then a comparison between the metals e x t r a c t e d from the s o n i c a t e d and blank samples may i n d i c a t e the mass of metals r e l e a s e d by ruptured b a c t e r i a . U n f o r t u n a t e l y the b a c t e r i a l s t a i n i n g method d i d not work w e l l so the b a c t e r i a were not enumerated. Without b a c t e r i a l counts a c o r r e l a t i o n between metal r e l e a s e s and s o n i c a t i o n cannot be made because the p a r t i c l e fragmentation caused by s o n i c a t i o n may r e l e a s e metals. The b a c t e r i a should be enumerated before and a f t e r a e r a t i o n to determine i f the number of whole c e l l s changes a f t e r the sample has been a e r a t e d . 9 A r e d u c t i o n i n the number of c e l l s c o u l d i n d i c a t e the presence of anaerobic b a c t e r i a . If a c o r r e l a t i o n can be e s t a b l i s h e d between metal r e l e a s e and a drop i n whole c e l l numbers i t would be p o s s i b l e to estimate the mass of metal h e l d by anaerobes. The second pretreatment method aerated a sample f o r 36 hours. A e r a t i o n should d e s t r o y anaerobes, i n c r e a s e the pH and i n c r e a s e the o x i d a t i o n - r e d u c t i o n p o t e n t i a l . I t was hoped that 36 hours would provide enough time for complete mixing of the 8. Slade and V e t t e r (1956) repo r t e d that a f t e r 1/2 hr. Of s o n i c a t i o n only 10% of the b a c t e r i a were v i a b l e and that 50% of the c e l l s were s o l u b i l i z e d . 9. Prolonged exposure to a i r may cause a p o p u l a t i o n of aerobes to grow. 69 sample and a i r without a l l o w i n g new p o p u l a t i o n s of aerobes to propagate. The b a c t e r i a should be enumerated before and a f t e r a e r a t i o n so metal r e l e a s e s can be c o r r e l a t e d to changes in b a c t e r i a numbers. A e r a t i o n w i l l probably o x i d i z e organic and i n o r g a n i c m a t e r i a l s i n the samples. Only r e l a t i v e l y s t a b l e compounds and newly formed p r e c i p i t a t e s should remain a f t e r a e r a t i o n . The s t a b l e compounds l e f t i n the r e s i d u a l phase of the e x t r a c t i o n procedure should provide an upper l i m i t to the mass of metals which w i l l remain i n the l y s i m e t e r . Only gradual l e a c h i n g of low s o l u b i l i t y compounds should r e l e a s e more metals. The e x t r a c t i o n procedure was a s l i g h t l y m o d i f i e d v e r s i o n of the procedure developed by En g l e r (1977). I t produced s i x subsamples and a seventh subsample was taken f o r a t o t a l metals a n a l y s i s . Each step of the procedure used the r e s i d u e of the p r e v i o u s s t e p f o r e x t r a c t i o n . A f t e r removing the l i q u i d phase by c e n t r i f u g i n g , the r e s i d u e was ready f o r the next s t e p . Other methods were c o n s i d e r e d and a summary of the l i t e r a t u r e reviewed i s c o n t a i n e d i n Appendix H. Table XV shows the order and c a t e g o r i e s of the e x t r a c t i o n procedure developed by E n g l e r . 1. PROCEDURE MODIFICATIONS a. Hydrogen Peroxide There were two major problems encountered i n the procedure used. One was the hydrogen peroxide d i g e s t i o n and the other was a c i d - d i g e s t i o n of samples with a high c o n c e n t r a t i o n of suspended s o l i d s . The hydrogen peroxide d i g e s t recommended by En g l e r et  a l . (1977) r e q u i r e s h e a t i n g of a sample u n t i l a l l the hydrogen 70 peroxide i s used up. T h i s i s extremely dangerous because the hydrogen peroxide can become con c e n t r a t e d . When hydrogen peroxide i s conc e n t r a t e d beyond 90%, i t i s flammable and e x p l o s i v e (See Appendix K f o r d e t a i l s of h e a l t h hazards). A hydrogen peroxide d i g e s t can be accomplished by adding d e i o n i z e d d i s t i l l e d water d u r i n g h e a t i n g to maintain the l i q u i d volume and keep the hydrogen peroxide d i l u t e . With t h i s m o d i f i c a t i o n a v i s u a l check does not i n d i c a t e when the hydrogen peroxide i s used up. To ensure that the r e a c t i o n was complete the s o l u t i o n was heated f o r at l e a s t one hour a f t e r the l a s t v i s i b l e r e a c t i o n of hydrogen peroxide with the o r g a n i c s ( i e . A f t e r the foaming stopped). Another m o d i f i c a t i o n was the slow a d d i t i o n of hydrogen per o x i d e . A l l the samples had high l e v e l s of or g a n i c s which r e s u l t e d i n the formation of foam when hydrogen peroxide was added. I t was necessary to use a mechanical s t i r r e r to keep the foam mixed and to add the hydrogen peroxide 10 ml. at a time. A f t e r a l l the hydrogen peroxide had been added and allowed to r e a c t , the sample was slowly heated. The whole hydrogen peroxide d i g e s t took four to f i v e hours t o perform and i t i s unknown how complete the d i g e s t i o n was. (Note: the sample d i d not foam when the l a s t 10 ml. of hydrogen peroxide was added which may i n d i c a t e that a l l the o r g a n i c s were o x i d i z e d . ) In the f u t u r e , i t would be s a f e r t o use d r y - a s h i n g f o r o x i d i z i n g samples with such h i g h c o n c e n t r a t i o n s of org a n i c m a t e r i a l s . T h i s would ensure complete o x i d i z a t i o n of o r g a n i c s but would cause the r e l e a s e of s t r o n g l y sorbed water a s s o c i a t e d with 71 m i n e r a l c o l l o i d s and the r e l e a s e of hydroxyl groups, b. A c i d - d i g e s t i o n A c i d - d i g e s t i o n of samples with high suspended s o l i d s can only be achieved by using more d i l u t e samples and l a r g e r d i g e s t i o n c o n t a i n e r s . I f the suspended s o l i d s are too high, the sample tends to 'bump'. (The whole sample heaves i n s t e a d of s m a l l bubbles r i s i n g to the s u r f a c e of the sample.) The bumping can cause the beaker to v i b r a t e . A l s o , bumping tends to s p i t sample out of the d i g e s t i o n c o n t a i n e r . Another d i f f i c u l t y encountered when a c i d - d i g e s t i n g samples with h i g h suspended s o l i d s was d i s s o l v i n g the r e s i d u e l e f t a f t e r d i g e s t i n g samples. A l l of the sub-samples numbered 0, 4, 5, and 6 d i d not r e d i s s o l v e p r o p e r l y . 1 0 Sub-samples 4, 5, and 6 were d i l u t e d to 250 ml. i n s t e a d of 100 ml. and the f i l t e r cakes were washed with ammonium a c e t a t e to minimize metal l o s s e s . An a n a l y s i s of the f i l t e r cake to determine the mass of metals l o s t was not made. A comparison of the t o t a l metals e x t r a c t e d and the sum of the metals e x t r a c t e d from each subsample i n d i c a t e d incomplete recovery of metals. Subsample four d i d not c o n t a i n l a r g e c o n c e n t r a t i o n s of metals so the d i g e s t e d sample cannot be d i l u t e d very much. The other three subsamples, zero, f i v e and s i x c o u l d a l l be d i l u t e d by a f a c t o r of 100 f o r a n a l y s i s , which circumvented the r e d i s s o l u t i o n problem. 10. The '0' r e p r e s e n t e d the i n i t i a l samples l a b e l l e d BLK0, SON0 and FOX0. The numbers 1,2,3,4,5 or 6 i n d i c a t e the number of e x t r a c t i o n s performed on the sample. 72 c. C e n t r i f u g i n g A major d i f f e r e n c e between the procedure used and the one re p o r t e d by Engler et a l . was the use of a c e n t r i f u g e . When the i n i t i a l data set was c o l l e c t e d , the f i r s t phase was f i l t e r e d . To a v o i d l o s i n g metals adsorbed to the f i l t e r , the f i l t e r cakes were r i n s e d with a c i d i f i e d d e i o n i z e d d i s t i l l e d water. The f i l t e r s were very q u i c k l y clogged so vacuum was a p p l i e d . A l l the f i l t e r i n g had to be done i n a n i t r o g e n atmosphere to keep the system a n a e r o b i c . When a l l the d i f f i c u l t i e s with f i l t e r i n g were compared to the s i m p l i c i t y of c e n t r i f u g i n g , a l l steps i n the procedure which r e q u i r e d f i l t r a t i o n , except those f o l l o w i n g a c i d - d i g e s t i o n , were r e p l a c e d with c e n t r i f u g i n g . C e n t r i f u g i n g and f i l t r a t i o n recover f r e e water so the s o l u t i o n s c o l l e c t e d should be comparable. C e n t r i f u g i n g does not c o l l e c t f i n e suspended s o l i d s as ef f i c i e n t l y as f i l t r a t i o n . The f i n e suspended s o l i d s w i l l be i n the supernatant of the f i r s t e x t r a c t i o n phase but very l i t t l e metal was de t e c t e d i n the supernatant of the f i r s t phase. So c e n t r i f u g i n g was co n s i d e r e d adequate f o r the e x t r a c t i o n experiment. 2. DATA Each sub-sample was ana l y z e d on a J a r r e l Ash 810 atomic a b s o r p t i o n spectrophotometer f o r Cd, Cr, Cu, Fe, N i , Pb, and Zn. Every sub-sample was compared to a minimum of three s i n g l e s p e c i e s standard s o l u t i o n s . Then a graph of c o n c e n t r a t i o n vs absorbancy was generated. The c o n c e n t r a t i o n of metal i n the 73 sub-sample was i n t e r p o l a t e d from the c o n c e n t r a t i o n vs absorbancy graphs contained i n Appendix S. A f t e r determining the c o n c e n t r a t i o n i n each sub-sample, the moisture content of each sub-sample was found. Then a mass r a t i o (RR= the mass of metal /the dry weight of l y s i m e t e r c o n t e n t s ) , was c a l c u l a t e d so the data c o u l d be compared. The mass r a t i o values were p l o t t e d vs the e x t r a c t i o n phase to show any trends which may occur. An example c a l c u l a t i o n of a mass r a t i o i s shown i n F i g u r e 10. F i g u r e 10 - Mass R a t i o C a l c u l a t i o n C i = metal c o n c e n t r a t i o n i n sample V i = volume of sample Mm = metal mass i n sample Mm = C i V i W = weight of sample P = s o l i d s f r a c t i o n RR = Mm/WP So f o r sample BLKO (An i n i t i a l c o n t r o l sample): C i = 1.75 mg/1 of Cd V i = 100 ml. = 0.1L. Mm = 0.175 mg. of Cd W = 70.6 P = 0.069 AW = atomic weight of the metal = 112.4 gm/mole = 112.4 ug/umole f o r Cd RR = 0.175/70.6x0.069 = 0.36 ug/dry gram sample OR RR = 36/112.4 = 0.32 uM/ gms. of dry sample Note: Appendix W c o n t a i n s a l i s t of symbols. I n i t i a l metal c o n c e n t r a t i o n s were determined f o r each sample s e t . The sum of the metal mass r a t i o s e x t r a c t e d i n each e x t r a c t i o n phase should correspond to the t o t a l mass r a t i o of a 74 raw sample. Only the sums of cadmium and copper e x t r a c t e d from the c o n t r o l sample were w i t h i n f i v e percent of the t o t a l of each metal e x t r a c t e d . There are two reasons f o r the f i n d i n g . The f i r s t reason was the small s i z e of the c o n t r o l sample which was r e a d i l y d i s s o l v e d a f t e r a c i d - d i g e s t i o n . A second reason was the probable l o s s of metal on the f i l t e r cake coupled with some p r e c i p i t a t e formation a f t e r a c i d d i g e s t i o n . T h i s was e s p e c i a l l y t rue f o r samples from phases 0, 4 r 5 and 6. The three i n i t i a l samples (BLKO, SON0, and FOX0) should have roughly the same metals l e v e l s because each sample was removed from the same bulk sample. Appendix T c o n t a i n s the f i g u r e s used to c a l c u l a t e the mass r a t i o at each phase of e x t r a c t i o n and the c a l c u l a t e d mass r a t i o v a l u e s . F i g u r e 11 shows histograms of the log(mass r a t i o ) vs the e x t r a c t i o n phase f o r each metal. In a l l seven histograms the metal c o n c e n t r a t i o n s are expressed as a r a t i o of the metal mass over the dry weight of sample, a. Mass R a t i o P l o t s The s o n i c a t e d , a e r a t e d and blank data, f o r each metal, are shown i n the histograms i n F i g u r e 11. A l l seven histograms have the e x t r a c t i o n phase p l o t t e d on the x - a x i s . Phase one corresponds to metals which were washed from the sample when d e i o n i z e d d i s t i l l e d water was added. T h i s w i l l i n c lude any metals r e l e a s e d by c e l l s which ruptured due to the osmotic pressure c r e a t e d by adding s a l t f r e e water. The metals r e l e a s e d i n t h i s phase are s o l u b l e and mobile. Ammonium acetate e x t r a c t e d phase two metals which are e a s i l y complexed metals. These metals are weakly h e l d i n the l y s i m e t e r and correspond to 75 F i g u r e 11 - E x t r a c t e d Metal Vs E x t r a c t i o n Phase 7 6 7 7 78 Extraction Phase vs. Mass Ratio S i extraction Phase metals which would be h e l d by s o i l in a c a t i o n exchange r e a c t i o n . Hydroxylamine h y d r o c h l o r i d e was used to e x t r a c t phase three metals. Metals i n t h i s phase are e a s i l y exchanged so they would be bound by s o i l i f they escaped a l a n d f i l l . Phase four used hydrogen peroxide to o x i d i z e o r g a n i c m a t e r i a l s . O x i d a t i o n c o u l d r e s u l t i n any combination of the f o l l o w i n g circumstances: a) C e l l l y s i s r e l e a s i n g metal complexing agents. b) C e l l l y s i s p r o v i d i n g new s i t e s f o r metal s o r b t i o n . c) C e l l l y s i s r e l e a s i n g bound, complexed, p r e c i p i t a t e d or s o l u b l e metals. d) Release of p r e c i p i t a t e s trapped by o r g a n i c s . e) S t a b i l i z a t i o n of o r g a n o - m e t a l l i c complexes. f) Formation of new metal p r e c i p i t a t e s with s t a b l e o x i d a t i o n p r o d u c t s . The s t r o n g o x i d i z i n g c a p a b i l i t y of hydrogen peroxide should prevent the formation of any o r g a n o - m e t a l l i c combinations but new p r e c i p i t a t e s or the r e l e a s e of p r e c i p i t a t e s c o u l d r e s u l t in metals which w i l l not be e x t r a c t e d i n phase f o u r . Phase f i v e 79 used sodium c i t r a t e and sodium d i t h i o n i t e to e x t r a c t i r o n o x i d e s . Sodium c i t r a t e and sodium d i t h i o n i t e are very strong i r o n complexing agents so only very s t a b l e compounds should remain i n the r e s i d u a l phase (phase s i x ) . Metals a s s o c i a t e d with phases one, two or three w i l l p robably l e a c h from a l y s i m e t e r . Most of the other metals should remain f a i r l y s t a b l e as long as: a) The reducing c o n d i t i o n s are maintained. b) The b a c t e r i a l p o p u l a t i o n remains the same. c) Anaerobic c o n d i t i o n s are maintained. An increase i n the pH w i l l cause many of the metals to form p r e c i p i t a t e s r e n d e r i n g them immobile. I f a pH change a l t e r s the b a c t e r i a l p o p u l a t i o n , metals may be r e l e a s e d . I t i s not known i f the metals a s s o c i a t e d with o r g a n i c s are h e l d by b a c t e r i a or humic compounds. By comparing the s o n i c a t e d samples to the c o n t r o l samples, one c o u l d i n f e r what p r o p o r t i o n of the metals are h e l d by b a c t e r i a . T h i s assumes that s o n i c a t i o n only d e s t r o y e d b a c t e r i a and d i d not break up anything e l s e which may h o l d metals. Metals a s s o c i a t e d with b a c t e r i a can be h e l d by a number of methods. a) Adsorbed i n s i d e the c e l l . b) P r e c i p i t a t e d i n s i d e the c e l l . c) Sorbed to the c e l l w a l l . d) Bound to e x t r a c e l l u l a r p o l y s a c c h a r i d e s . e) P r e c i p i t a t e d i n e x t r a c e l l u l a r p o l y s a c c h a r i d e s . Some general comments can be made with respect to the 80 histograms. In the i r o n and z i n c histograms the three samples responded s i m i l a r l y . T h i s i n d i c a t e s that very s i m i l a r r e t e n t i o n mechanisms are o p e r a t i n g f o r both metals or that some of the a p p l i e d p r e c i p i t a t e s never d i s s o l v e d i n the l y s i m e t e r . If these metals were r e t a i n e d then t h e i r predominance i n the phase f i v e e x t r a c t s i n d i c a t e s that c o - p r e c i p i t a t i o n i s the probable removal mechanism. Cadmium was the most mobile of a l l the metals i n a l l three circumstances and was the only metal which was added to the l y s i m e t e r i n a s o l u b l e f o r m . 1 1 Thus, any cadmium r e t e n t i o n i s e n t i r e l y due to the contents of the l y s i m e t e r . Chromium was predominant i n the phase four e x t r a c t s while l e a d c o n s i s t e n t l y had high masses in the r e s i d u a l phase. The shape of the copper curve was the same f o r a l l three sample types. I t showed a peak at phases four and s i x . Very l i t t l e copper was a s s o c i a t e d with phase f i v e i n d i c a t i n g d i f f e r e n t r e t e n t i o n mechanisms from a l l the other metals. I t i s important to r e a l i z e that the l a c k of mobile metals i s probably due to the age of the l y s i m e t e r which was sampled. Any n a t u r a l complexes which remain would be i n p r e c i p i t a t e form. Metals h e l d by organic l i g a n d s should be r e l e a s e d by a e r a t i o n or hydrogen peroxide o x i d a t i o n . Complexes which are s t a b i l i z e d by oration or o x i d a t i o n should be e x t r a c t e d i n phase f i v e or s i x . The mobile metals in the samples i n d i c a t e that some Cd, Cu and Ni r e t e n t i o n mechanisms form moderately s o l u b l e compounds. 11. E x t r a cadmium was added as l i q u i d from a cadmium p l a t i n g bath to boost cadmium l e v e l s . T h i s was done because there was very l i t t l e cadmium in the e l e c t r o p l a t i n g wastes. 81 Otherwise the h i g h l y mobile metals that were e x t r a c t e d should have been leached from the l y s i m e t e r a l r e a d y . So copper, n i c k e l and cadmium were r e t a i n e d i n m i l d l y exchangeable and s o l u b l e forms. These metals were more s t a b l e a f t e r s o n i c a t i o n . T h i s e f f e c t c o u l d be due to s o p t i o n or the formation of compounds a f t e r c e l l l y s i s . The l a r g e mass of cadmium which i s mobile a f t e r a e r a t i o n i n d i c a t e s that cadmium compounds are very s u s c e p t a b l e to oxygen and /or pH i n c r e a s e s . Cadmium and n i c k e l are probably sorbed to o r g a n i c s as shown by the in c r e a s e i n phase four metals a f t e r s o n i c a t i o n . S o n i c a t i o n causes the d i s r u p t i o n of c e l l s and the fragmentation of s o l i d p a r t i c l e s . C e l l l y s i s c o u l d r e l e a s e organic compounds which w i l l complex metals and /or c e l l fragments c o u l d sorb metals. P a r t i c l e fragmentation j u s t i n c r e a s e s the exposed s u r f a c e a r e a . T h i s should i n c r e a s e the a v a i l a b i l i t y of metals f o r c a t i o n exchange and in c r e a s e the s i t e s f o r metal s o r p t i o n . If c e l l l y s i s causes metals to be complexed, the i n s o l u b l e compounds w i l l show up i n phase four while the s o l u b l e complexes should be recovered i n phase one. The fragmentation of p a r t i c l e s should cause: more metals to be recovered i n phases two and three i f metals are weakly sorbed or in c r e a s e d recovery i n phases f i v e and s i x i f metals are s t r o n g l y sorbed. S o n i c a t i o n data i n d i c a t e s that organic complexes of cadmium, chromium and copper form. A l l the metals except copper are s t r o n g l y sorbed by i r o n compounds e x t r a c t e d i n phase f i v e . 82 A l l the metals are s t r o n g l y sorbed by m i n e r a l s present i n the r e s i d u e ( e s p e c i a l l y copper and lead) or they form s t a b l e p r e c i p i t a t e s . The r e s i d u a l phase of the aerated sample i n d i c a t e s the mass of metal which i s s t a b l e a f t e r : exposure to a i r and a pH i n c r e a s e . These metals w i l l probably be r e t a i n e d i n a l y s i m e t e r f o r many ye a r s . Some l e a c h i n g w i l l occur but i t may not be measureable. If they are s l i g h t l y s o l u b l e they would leach out of the l y s i m e t e r e v e n t u a l l y . Roughly 40% of the l e a d , 20% of the i r o n , 15% of the chromium, 15% of the copper, 15% of the n i c k e l and 10% of the z i n c i s f i x e d (phase s i x a e r a t e d m e t a l s ) . I t i s p o s s i b l e that a e r a t i o n and the corres p o n d i n g pH increase caused some of the metals to form p r e c i p i t a t e s . So the esti m a t e s provide an upper l i m i t f o r the unleachable metal mass. The l a r g e r e l e a s e of cadmium i n phase one i n d i c a t e s that cadmium i s r e a d i l y complexed by o r g a n i c s and/or a e r a t i o n d e s t r o y s cadmium compounds i n a l y s i m e t e r . Metals a s s o c i a t e d with o r g a n i c s other than b a c t e r i a f a l l i n t o two c a t e g o r i e s : mobile or f i x e d compounds. A l l the f i x e d compounds should remain s t a b l e u n t i l the pH, o x i d a t i o n - r e d u c t i o n p o t e n t i a l or anaerobic c o n d i t i o n s are changed. I f they are i n p r e c i p i t a t e form, they w i l l maintain an e q u i l i b r i u m with the surrounding water f i l m , so they w i l l s lowly l e a c h from the system. If the metals are f i x e d to n o n - b a c t e r i a l o r g a n i c s by s o r p t i o n , bonding or entrapment, they w i l l remain f i x e d u n t i l the o r g a n i c s are degraded. In e i t h e r case, metals are probably 83 r e l e a s e d slowly over a very long time i n t e r v a l . G e n e r a l l y , the e x t r a c t i o n procedure q u a n t i f i e s the metals i n a l a n d f i l l or l y s i m e t e r . I t i s a l s o p o s s i b l e to determine the r e l a t i v e m o b i l i t y of v a r i o u s metals and hence p r e d i c t the metals t h a t would be expected to l e a c h from a l y s i m e t e r or l a n d f i l l . A major problem a s s o c i a t e d with extending the procedure to a n a l y s i s of l a n d f i l l m a t e r i a l i s the removal of an anaerobic sample. There may a l s o be some problems when a c i d d i g e s t i n g the e x t r a c t from phases one, two and three because of the bumping e f f e c t d u r i n g volume r e d u c t i o n . (Dry o x i d a t i o n at 500°C may be necessary.) b. B a c t e r i a l Counts To f u r t h e r r e f i n e the e x t r a c t i o n procedure an experiment was d e v i s e d to determine the e f f e c t s of b a c t e r i a upon metal m o b i l i t y . The experiment r e q u i r e d two samples, one with b a c t e r i a and another with b a c t e r i a which were d i s r u p t e d by s o n i c a t i o n . Each sample would be e x t r a c t e d u s i n g the e x t r a c t i o n procedure. D i f f e r e n c e s i n metal m o b i l i t y c o u l d then be a t t r i b u t e d to the e f f e c t s of b a c t e r i a . To q u a n t i f y the r e s u l t s the number of b a c t e r i a / gram of sample must be known. Th i s r e q u i r e d a b a c t e r i a l enumeration technique so a method us i n g an cjpif l u o r e s c e n t dye was s e l e c t e d . The dye 4,6-diamidino-2-p h e n y l i n d o l e (DAPI) was chosen f o r the experiment as i t i s very s n e c i f i c f o r b a c t e r i a . The s t a i n i n g technique used was developed by Iqubal V e l g i at Simon F r a s e r U n i v e r s i t y f o r a Masters t h e s i s i n B i o l o g y . 84 The samples were found to have at l e a s t two types of b a c t e r i a shape: rod and c o c c i . I n i t i a l l y only small areas of a s l i d e p r e p a r a t i o n c o u l d be counted due to a blue haze which obscured most of the s l i d e s . A f t e r some experimentation, r e s e a r c h e r s at SFU found that most of the background m a t e r i a l c o u l d be broken up by adding pyrophosphate to the sample. Pyrophosphate treatment coupled with s o n i c a t i o n broke up a l l the background luminescence that was p r e v i o u s l y observed. One drawback with the pyrophosphate i s that i t forms a p r e c i p i t a t e with potassium, c a l c i u m , and magnesium. These p r e c i p i t a t e s are probably what confuse c o u n t i n g as the s l i d e p r e p a r a t i o n s look l i k e a f i e l d of g r a v e l . I t i s very d i f f i c u l t to d i s c e r n a few b r i g h t blue specs r e p r e s e n t i n g b a c t e r i a among the very many specs of d u l l blue to white ' g r a v e l ' . In f a c t , i t was not p o s s i b l e to o b t a i n c o n s i s t e n t counts. The low c o n c e n t r a t i o n of b a c t e r i a i n the sample prevented f u r t h e r d i l u t i o n to make counts e a s i e r . So i t was not p o s s i b l e to use the technique as planned. Other methods are a v a i l a b l e 1 2 f o r determining b i o l o g i c a l mass or even e s t i m a t i n g b a c t e r i a l numbers but they c o u l d not give an accurate and quick estimate of the number of whole b a c t e r i a l c e l l s . The most e f f e c t i v e DAPI procedure t r i e d i s o u t l i n e d i n Appendix G. F i g u r e 12 shows photographs of s l i d e s under a microscope at 100X power. The f i r s t photograph shows a s l i d e of a l y s i m e t e r sample. No 12. See C o s t e r t o n (1979); Mara (1974); S t r i c k l a n d and Parsons (1972). A l s o Appendix L c o n t a i n s a s p e c i f i c d i s c u s s i o n of dye s t a i n i n g techniques. F i g u r e 12 - Photographs Of S l i d e P r e p a r a t i o n s 85 A photograph of a s l i d e prepared by Iqubal V e l g i to show b a c t e r i a (The b r i g h t blue dots are b a c t e r i a ) . 86 b a c t e r i a can be d i s c e r n e d due to the haze. The second s l i d e i s an example of Iqubal V e l g i ' s showing b a c t e r i a . The very small b r i g h t blue dots are b a c t e r i a , c. L i q u i d Sample A l y s i m e t e r l e a c h a t e sample was c o l l e c t e d under anaerobic c o n d i t i o n s . Data from the UBC c o - d i s p o s a l study (Atwater et a l . 1981) was used with a t o t a l metals a n a l y s i s of the l y s i m e t e r l e a c h a t e to determine the mass of metal which had leached from the l y s i m e t e r before the f i r s t s o l i d s sample was removed. The mass r e l e a s e d from the tank i s shown in Table XX and a sample c a l c u l a t i o n i s shown below the t a b l e . Table XXI shows the r e s u l t s of the l i q u i d a n a l y s i s . The data corresponds with the trends e s t a b l i s h e d i n the c o - d i s p o s a l r e p o r t by Atwater et a l . (1981). An a n a l y s i s of the l i q u i d was performed to ensure that none of the metals had suddenly leached out of the system. There was an i r o n r e l e a s e a f t e r the f i r s t hole was made in the l y s i m e t e r . The i r o n r e l e a s e cannot be r e a d i l y e x p l a i n e d and seems to be a temporary occurance. There was an a i r leak due to problems with the plug that was used to s e a l the h o l e . The a i r leak would cause a s h i f t i n pH and o x i d a t i o n - r e d u c t i o n p o t e n t i a l which should cause i r o n to p r e c i p i t a t e . The observed i n c r e a s e of i r o n i n the leachate must be due to the r e l e a s e of complexed i r o n . The f a u l t y plug was subsequently r e p l a c e d by a l a y e r of p a r a f i l m , a l l o w i n g the the system to go anaerobic again. 87 Table XX - Mass Released From The Lysimeter Metal Mass Released (gms.) Cd 0.033 Cr 0.347 Fe 62.6 Ni 0.59 Pb 0.009 Zn 10.0 Ca 36.9 Mg 7.38 Use lea c h a t e a n a l y s i s to determine the c o n c e n t r a t i o n of metal i n a sample = C i Measure the t o t a l volume of l e a c h a t e c o l l e c t e d from the l y s i m e t e r = V i So f o r a time i n t e r v a l T1 to T2: At time T1: C1 = metal c o n c e n t r a t i o n i n sample taken at T1 VI = t o t a l volume of l e a c h a t e generated M1 = cummulative mass to time T1 At time T2: C2 = metal c o n c e n t r a t i o n i n sample taken at T2 V2 = t o t a l volume of lea c h a t e generated t h e r e f o r e M2 = (C2+C1/2) x (V2-V1) + M1 M2 = mass r e l e a s e d over time T1 to T2. Table XXI - Metal C o n c e n t r a t i o n s In L i q u i d Sample Metal ug/1 uM/1 Cd 3 0.03 Cr 73 1 .4 Cu 46 0.72 Fe 2290 41 Ni 990 1 7 Pb 10 0.05 Zn 2810 43 Note: Cd and Pb were at the l i m i t s of d e t e c t i o n . A t o t a l of 356 ml. were t e s t e d . 88 V. CONCLUSIONS AND RECOMMENDATIONS The experimental methods developed i n t h i s study c o u l d be used to i n c r e a s e the understanding of metal d i s t r i b u t i o n i n a l a n d f i l l . I t i s so d i f f i c u l t to remove anaerobic samples from a l a n d f i l l t h a t the p r e l i m i n a r y t e s t s conducted i n t h i s study used samples from a l y s i m e t e r . Three d i f f e r e n t experiments were conducted to determine metal m o b i l i t y , n a t u r a l l i g a n d s t r e n g t h and b a c t e r i a l numbers. A. CHELATION Two methods, standard comparisons and standard a d d i t i o n s , were used to develop a t e s t f o r the s t r e n g t h and presence of n a t u r a l l i g a n d s . The experimentation gave r i s e to the f o l l o w i n g c o n c l u s i o n s : a) Both the standard comparison method and standard a d d i t i o n s method should be used. b) The standards a d d i t i o n s used upset the metal e q u i l i b r i u m i n the samples. c) The volume of MIBK recovered should be measured f o r each sample although t h i s i n c r e a s e s MIBK l o s s e s . d) C e n t r i f u g i n g the MIBK-sample suspension mixture does work but may be improved with higher c e n t r i f u g a l f o r c e s . e) The use of C r C l 3 f o r a chromium standard d i d prevent v i s i b l e p r e c i p i t a t i o n i n the standard s o l u t i o n s . f) The lack of Cd, Ni and Pb i n MIBK e x t r a c t s was due to low i n t e r a c t i o n s with other metals or a low recovery e f f i c i e n c y f o r these metals. g) The s i m i l a r responses of Fe and Zn i n d i c a t e d s i m i l a r c h e l a t i o n b e h a v i o r . h) A grad i e n t of complex s t r e n g t h s can be e s t a b l i s h e d to determine metal s p e c i a t i o n . 8 9 i ) A comparison of the pseudo mass r a t i o s and s t a b i l i t y c o n stants of each metal i n d i c a t e d the presence of C r ( I I I ) , C u ( l ) , F e ( I l ) and Zn(II) i n the sample. The comparison i n d i c a t e d C r ( I I l ) , C u ( I I ) , F e ( l l l ) and Zn(II) i n the standards. Both a n a l y s i s methods should be used so the recovery e f f i c i e n c y can be determined f o r the sample. A method was not found f o r breaking the recovery e f f i c i e n c y i n t o i t s two components, namely c h e l a t i o n e f f i c i e n c y and MIBK e x t r a c t i o n e f f i c i e n c y . The method of standard a d d i t i o n s can be used to determine the recovery e f f i c i e n c y f o r the sample+standard. The recovery e f f i c i e n c y f o r the standards can be determined using the method of standard comparisons. Any d i f f e r e n c e s between the two methods can be a t t r i b u t e d t o a d i f f e r e n t recovery e f f i c i e n c y f o r metals i n the sample. The l a c k of Cd, Ni and Pb i n the MIBK e x t r a c t s may be due to a low recovery e f f i c i e n c y f o r these metals. There was i n s u f f i c i e n t time a v a i l b l e to rerun the standard comparison experiment using C r C l 3 so the recovery e f f i c i e n c i e s are unknown. I t i s a l s o p o s s i b l e that a r e a c t i o n between these metals and the non-reduced standards occured which prevented t h e i r c h e l a t i o n . A g r a d i e n t of s t a b i l i t y c o nstants can be e s t a b l i s h e d f o r each metal s p e c i e s . A comparison of the order of s t a b i l i t y constant g r a d i e n t s and pseudo mass r a t i o s can then determine the s p e c i e s present i n the sample i f the f o l l o w i n g are t r u e : a) The s t a b i l i t y c o n s t a n t s of the added l i g a n d s exceed those of the n a t u r a l l i g a n d s . b) The recovery e f f i c i e n c i e s can be determined f o r each metal i n the sample. 9 0 c) The c o n c e n t r a t i o n of the complex that i s recovered i s dependent upon the magnitude of the s t a b i l i t y constant of a complex. d) If the recovery e f f i c i e n c i e s are e q u i v a l e n t then a higher s t a b i l i t y constant w i l l r e s u l t in a g r e a t e r c o n c e n t r a t i o n of complex i n the MIBK e x t r a c t . e) There i s a d i f f e r e n t g r a d i e n t o r d e r i n g f o r each s p e c i e s of a metal. The presence and s t r e n g t h of n a t u r a l c h e l a t i n g agents can only be determined i f the complex c o n c e n t r a t i o n s and recovery e f f i c i e n c i e s are measured. A p o s s i b l e s p i n o f f of the technique may be the a b i l i t y to determine the metal s p e c i e s and t h e i r c o n c e n t r a t i o n s . The s p e c i e s d e t e r m i n a t i o n w i l l only be a f f e c t e d by n a t u r a l l i g a n d s i f they form very strong complexes. To determine the c o n c e n t r a t i o n of each s p e c i e s the formulae i n Appendix R can be used. The complex c o n c e n t r a t i o n s used fo r the c a l c u l a t i o n should be those due to the s t r o n g e s t complexing agents. T h i s w i l l a v o i d e r r o r s caused by the presence of n a t u r a l l i g a n d s . B. EXTRACTION A s e r i e s of e x t r a c t i o n s were performed to determine the r e l a t i v e m o b i l i t y of metals i n four samples. The metals i n the samples were separated a c c o r d i n g to t h e i r ease of e x t r a c t i o n . One sample was s o n i c a t e d to separate b a c t e r i a l and n o n - b a c t e r i a l metals. Another sample was aerated to d i s c o v e r the f r a c t i o n of metals which would be m o b i l i z e d i n an o x i d i z i n g environment. The a e r a t e d and s o n i c a t e d samples were compared to untreated samples (blanks or c o n t r o l s ) . The samples were c o l l e c t e d near a 91 l a y e r of e l e c t r o p l a t i n g waste. The p r o g r e s s i v e e x t r a c t i o n experiment was s u c c e s s f u l l y employed to c a t e g o r i z e the m o b i l i t y of metals i n a l y s i m e t e r sample. S o n i c a t i o n and a e r a t i o n of two d i f f e r e n t samples d i d p r o v i d e more in f o r m a t i o n about metal r e t e n t i o n . U n f o r t u n a t e l y the mass of metal a s s o c i a t e d with b a c t e r i a c o u l d not be determined because the b a c t e r i a l enumeration technique d i d not work w e l l enough. A l l the e x t r a c t i o n data was converted to a mass r a t i o value (moles of m e t a l / mass of l y s i m e t e r suspension a f t e r d r y i n g at 104°C) to f a c i l i t a t e comparison with other s t u d i e s . A f t e r a n a l y z i n g the data the f o l l o w i n g c o n c l u s i o n s were made: a) A f t e r threee years of l y s i m e t e r o p e r a t i o n most of the mobile metals had a l r e a d y been leached out of the l y s i m e t e r . b) Over 10% of the cadmium and n i c k e l was mobile because they formed p a r t i a l l y s o l u b l e p r e c i p i t a t e s or a chemical was slowly r e l e a s e d which formed s o l u b l e compounds with these two metals. c) The l a r g e metal r e l e a s e s caused by a e r a t i o n c o n f i r m the b e l i e f that metal r e t e n t i o n mechanisms are s e n s i t i v e to pH and o x i d a t i o n - r e d u c t i o n p o t e n t i a l changes. d) The smaller p r o p o r t i o n of metals i n phases two and three e x t r a c t s i n d i c a t e s that c a t i o n exchange i s not a major r e t e n t i o n mechanism i n the l y s i m e t e r t e s t e d . e) S o n i c a t i o n caused more Cd and Ni to be r e t a i n e d by o r g a n i c s i n d i c a t i n g that c e l l chemicals may bind these metals. f) A e r a t i o n m o b i l i z e d a l l the metals except n i c k e l i n d i c a t i n g the presence of very s t a b l e n i c k e l compounds. g) Most of the chromium was e x t r a c t e d i n phases 4,5 and 92 6 i n d i c a t i n g the presence of f a i r l y s t a b l e Cr compounds. h) A maximum of 40% of the l e a d , 20% of the i r o n , 15% of the chromium, 15% of the n i c k e l , 10% of the copper and 1% of the cadmium i s s t a b l e a f t e r a e r a t i o n . C. FUTURE RECOMMENDATIONS The c h e l a t i o n experiment t e s t e d i n t h i s study c o u l d be developed f u r t h e r . Future attempts to measure the s t r e n g t h of n a t u r a l l i g a n d s should i n c o r p o r a t e the f o l l o w i n g : a) Determine s t a b i l i t y c onstants f o r a l l the s p e c i e s under c o n s i d e r a t i o n . b) Use reduced spike s o l u t i o n s which have had n i t r o g e n gas bubbled through them. c) E x t r a c t a sample u s i n g only the s o l v e n t to check for metals which are s o l u b l e i n the s o l v e n t . d) Prove whether metal s p e c i e s can be q u a n t i f i e d . e) Try to determine s t a b i l i t y c o n s t a n t s f o r n a t u r a l l i g a n d s by comparison and/ or MacCarthy's method (1977). f) Determine why Cd, Ni and Pb were not d e t e c t e d . The c h e l a t i o n g r a d i e n t d i d not produce good data i n t h i s study because of problems with the spike s o l u t i o n s but the technique o f f e r s an o p p o r t u n i t y to measure s p e c i e s c o n c e n t r a t i o n and n a t u r a l l i g a n d s t r e n g t h s . P r e v i o u s l y the s p e c i e s c o u l d not Le determined i n most cases and n a t u r a l l i g a n d s t r e n g t h s could only be estimated f o r a few metals. So f u r t h e r work should be conducted to t e s t the f e a s i b i l i t y of the technique f o r metal s p e c i a t i o n measurements. A standard without c h e l a t i n g agents should be e x t r a c t e d 93 using MIBK. I f the mass of metal e x t r a c t e d i s comparable to the complexed metal e x t r a c t e d another s o l v e n t should be used. This e x t r a c t i o n was overlooked i n t h i s study. F u r t h e r e f f o r t s to determine b a c t e r i a l numbers should be d i r e c t e d a t : a) Avo i d i n g d i l u t i o n of samples which may cause c e l l l y s i s due to osmotic p r e s s u r e s . b) T e s t i n g the s l i g h t l y more s p e c i f i c b a c t e r i a l s t a i n c a l l e d mithramycin. c) Experimenting with the ethanol f i x a t i o n to determine the optimal c o n c e n t r a t i o n f o r f i x i n g b a c t e r i a from a l y s i m e t e r . The d e t e r m i n a t i o n of b a c t e r i a l numbers must be s u c c e s s f u l before metal r e t e n t i o n due to b a c t e r i a can be e v a l u a t e d . The e x t r a c t i o n procedure demonstrated the success of c h e l a t i o n without s o l v e n t s . As the e x t r a c t i o n data c o l l e c t e d i n t h i s study was used to t e s t the technqiue, f u r t h e r work should be done t o : a) Compare anaerobic l a n d f i l l samples to l y s i m e t e r samples to determine the v a l i d i t y of using l y s i m e t e r s as ref u s e l e a c h i n g models. b) Compare d i f f e r e n t l a n d f i l l s . c) Compare d i f f e r e n t sample l o c a t i o n s i n a l a n d f i l l . d) Replace wet o x i d a t i o n of o r g a n i c s with dry o x i d a t i o n by oven d r y i n g samples at 500°C to ensure complete o x i d a t i o n of the o r g a n i c s . e) Conduct a time study of a l a n d f i l l to determine the metal m o b i l i t y changes over time. f) Try d i f f e r e n t a e r a t i o n c o n t a c t times t o check the s e n s i t i v i t y of samples and to determine the conta c t time r e q u i r e d f o r maximum metal r e l e a s e . 94 P r o g r e s s i v e e x t r a c t i o n s t u d i e s of anaerobic l a n d f i l l samples w i l l h e lp to determine the v a l i d i t y of l y s i m e t e r s t u d i e s . A l s o , time s t u d i e s should be conducted so estimates of metal l e a c h i n g can be made. L a s t l y a comparison of the metal d i s t r i b u t i o n at d i f f e r e n t l a n d f i l l l o c a t i o n s w i l l help to understand the metal l e a c h i n g p r o c e s s . 95 REFERENCES 1 A l b e r t , A. The A c r i d i n e s . Edward Ar n o l d L t d . London. 1966. 2 A l e s i i , B.A., et a l . E f f e c t of Leachate Flow Rate on Metal M i g r a t i o n Through S o i l . J o u r n a l of  Environmental Q u a l i t y . V ol.9, No.1, pp.119-126, 1980. 3 A l l a n , R.A., and J . J . M i l l e r . I n f l u e n c e of S-adenosylmethionine on DAPI-induced F l u o r s c e n c e of Polyphosphate i n the Yeast Vacuole. Canadian  J o u r n a l of M i c r o b i o l o g y . Vol.26, pp.912-920, 1980. 4 Atwater, J.W., et a l . C o d i s p o s a l of E l e c t r o p l a t i n g and A r s e n i c Wastes with Refuse and S e p t i c Tank  Pumpings.—Draft Report. UBC Dept. C i v i l E n g i n e e r i n g , 1981. 5 Barth, E.F., et a l . Summary Report on the E f f e c t s of Heavy Metals on the B i o l o g i c a l Treatment Processes. J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.37, No.1, pp.86-96, January 1 965. 6 B a t l e y , G.E., and T.M. F l o r e n c e . A Novel scheme f o r the C l a s s i f i c a t i o n of Heavy Metals Species i n N a t u r a l Waters. A n a l y t i c a l L e t t e r s . V o l.9, No.4, pp.379-388, 1976. 7 B a t l e y , G.E., and D. Gardner. Sampling and Storage of N a t u r a l Waters For Trace Metal A n a l y s i s . Water  Research. Vol.11, pp.745-756, 1977. 8 B e l l , C F . P r i n c i p l e s and A p p l i c a t i o n s of Metal C h e l a t i o n . Oxford U n i v e r s i t y . Oxford. 1977. y Bergman, S.C., et a l . The Use of Zonal C e n t r i f u g a t i o n i n D e l i n e a t i n g Trace Element D i s t r i b u t i o n s i n Sewage Sludges from Dayton, Ohio, Area. J o u r n a l of  Environmental Q u a l i t y . Vol.8, No.3, pp.416-422, 1979. 96 Brown, M.J., and J.N. L e s t e r . Metal Removal i n A c t i v a t e d Sludge : The Role of B a c t e r i a l E x t r a c e l l u l a r Polymers. Water Research. Vol.13, pp.817-837, 1979. Buck, J.D. The P l a t e Count i n Aquatic M i c r o b i o l o g y . Native Aquatic B a c t e r i a : Enumeration, A c t i v i t y ,  and Ecology. C o s t e r t o n , J . M. and C o l w e l l , R. R. eds. ASTM, pp.19-28, 1979. Budde, W.L., and J.W. E i c h e l b e r g e r . Organics A n a l y s i s  Using Gas Chromatography Mass Spectrometry. Ann Arbor. Michigan. 1979. Cameron, R.D., and F.A. Koch. Trace Metals and Anaerobic D i g e s t i o n of Leachate. J o u r n a l Water P o l l u t i o n  C o n t r o l F e d e r a t i o n . Vol.52, No.2, pp.282-292, February 1980. Carrondo, M.J.T., et a l . Comparison of a Rapid Flameless Atomic A b s o r p t i o n Procedure f o r the A n a l y s i s of the M e t a l l i c Content of Sewages and Sewage E f f l u e n t s with Flame Atomic A b s o r p t i o n Methods. Science of the T o t a l Environment. Vol.12, pp.1 -12, 1979. Chao, T.T. S e l e c t i v e D i s s o l u t i o n of Manganese Oxide From S o i l s and Sediments with A c i d i f i e d Hydroxylamine H y d r o c h l o r i d e . S o i l Science S o c i e t y of America.  Proceedings. Vol.36, pp.764-768, 1972. Chen, K.Y., et a l . Trace Metals i n Wastewater E f f l u e n t s . J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.46, No.12, pp.2663-2676, December 1974. Cheng, M.H., et a l . Sludge. F e d e r a t i o n . T975~: Heavy Metals Uptake by A c t i v a t e d J o u r n a l Water P o l l u t i o n C o n t r o l Vol.47, No.2, pp.362-376, February Chian, E.S., and F.B. DeWalle. C h a r a c t e r i z a t i o n of S o l u b l e Organic Matter i n Leachate. Environmental Science and Technology J o u r n a l . Vol.11, No.2, pp.158-163, February 1977. Coleman, A.W., M.J. Maguire, and J.R. Coleman. Mithramycin- and 4'-6-Diamidino-2-Phenylindole(DAPI)- DNA S t a i n i n g f o r Fluorescence M i c r o s p e c t r o p h o t o m e t r i c Measurement of DNA i n N u c l e i , P l a s t i d s , and V i r u s P a r t i c l e s . The  J o u r n a l of H i s t o c h e m i s t r y and Cytochemistry. Vol.29, No.8, pp.959-968, 1981. 97 20 Coleman, A.W._ Enhanced D e t e c t i o n of B a c t e r i a i n N a t u r a l Environments by Fluorochrome S t a i n i n g of DNA. Limnology and Oceanography. Vol.25. No 5* pp.948-951. 1980. ' 21 Corpe, W.A. M e t a l - B i n d i n g P r o p e r t i e s of Surface M a t e r i a l s from Marine B a c t e r i a . Developments i n I n d u s t r i a l  M i c r o b i o l o g y . Garamond/Pridemark Press. L~. H T U n d e r k o f l e r ed. Baltimore,Maryland. Vol.16, pp.249-255, 1975. 22 C o s t e r t o n , J.W., and G.G. Geesey. Which P o p u l a t i o n s of Aquatic B a c t e r i a Should We Enumerate. Native  Aquatic B a c t e r i a : Enumeration, A c t i v i t y , and  Ecology. C o s t e r t o n , J . M. and C o l w e l l , R. R. eds. ASTM, pp.7-18, 1979. 23 Cowell, J.K., and L.M. Franks. A Rapid Method f o r Accurate DNA Measurements i n S i n g l e C e l l s In S i t u Using a Simple M i c r o f l u o r i m e t e r and Hoechst 33258 as a Q u a n t i t a t i v e Fluorochrome. The J o u r n a l of  H i s t o c h e m i s t r y and Cytochemistry. Vol.28, No.3, pp.206-210, 1980. 24 Daley, R.J. D i r e c t E p i f l u o r e s c e n c e Enumeration of Native Aquatic B a c t e r i a : Uses, L i m i t a t i o n s , and Comparative Accuracy. Native Aquatic B a c t e r i a :  Enumeration, A c t i v i t y , and Ecology. C o s t e r t o n , J . M. and C o l w e l l , R. R. eds. ASTM, pp.29-45, 1979. 25 Daley, R.J., and J.E. Hobbie. D i r e c t Counts of Aquatic B a c t e r i a by a M o d i f i e d E p i f l u o r e s c e n c e Technique. Limnology and Oceanography. Vol.20, pp.875-882, 1975. 26 Dean J.A., ed. Langes Handbook of Chemistry --12 e d i t i o n . McGraw-Hill. New York. 1979. 27 DeWalle, F.B., S.K. Chian, and J . Brush. Heavy Metal Removal With Completely Mixed Anaerobic F i l t e r . J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.51, No.1, pp.22-36, January 1979. 28 E n g l e r , R.M, et a l . A P r a c t i c a l S e l e c t i v e E x t r a c t i o n Procedure f o r Sediment C h a r a c t e r i z a t i o n . Chemistry of Marine Sediments. T.F. Yen ed. Ann Arbor Science P u b l i s h e r s . Ann Arbor, Mich. 1977. 98 29 Fannin, K.F., et a l . Anaerobic P r o c e s s e s . ( L i t e r a t u r e Review") J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.52, No.6, pp.1182-1195, 1980. 30 F l o r e n c e , T.M. Trace Metal Species i n Fresh Waters. Water Research. Vol.11, pp.681-687, 1977. 31 F l o r e n c e , T.M., and G.E. B a t l e y . Determination of the Chemical Forms of Trace Metals i n N a t u r a l Waters, With S p e c i a l Reference t o Copper, Lead, Cadmium and Z i n c . T a l a n t a Mini-Review. Vol.24, pp.151-158, 1977. 32 F r a n c i s , C.W., et a l . The U t i l i t y of E x t r a c t i o n Proceedures and T o x i c i t y T e s t i n g With S o l i d Wastes. D i s p o s a l of Hazardous Waste. Proceedings of the Annual Research Symposium (6th) Held at Chicago, I l l i n o i s on March 17-20, pp.39-45, 1980. 33 F r a n c i s c o , D.E. , R.A. Mah, and A.C. Rabin. A c r i d i n e O r a n g e - e p i f l u o r e s c e n c e Technique f o r Counting B a c t e r i a i n N a t u r a l Waters. T r a n s a c t i o n s of the  American M i c r o s c i e n c e S o c i e t y . Vol.92, No.3, pp.416-421, J u l y 1973. 34 F r o s t , R.R., and R.A. G r i f f i n . E f f e c t of pH on Ad s o r p t i o n of Copper, Z i n c , and Cadmium From L a n d f i l l Leachate by Clay M i n e r a l s . J o u r n a l of  Environmental Science and H e a l t h : Part A. VO1.A12, No.4&5, pp.139-156, 1977. 35 F u l l e r , W.H., and B.A. A l e s i i . Behavior of M u n i c i p a l S o l i d Waste Leachates. I I . In S o i l . J o u r n a l of  Environmental Science and H e a l t h : Part A. Vol.AM, No.7, pp.559-592, 1979. 36 F u r r , A.K. Elemental A n a l y s i s of P r o t e i n - C o n t a i n i n g Food M a t e r i a l s From V a r i o u s Sources. J o u r n a l of Food  Science. Vol.39, pp.887-891, 1974. 37 Gabb, M.H., and W.E. Latchem. A Handbook of Laboratory S o l u t i o n s . Chemical P u b l i s h i n g Co. Inc. New York, 1968. 38 Ghosh, S. Anaerobic Processes. J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.44, No.6, pp.948-959, June 1972. 99 Goldberg, M.C., L. Delong, and L. Kahn. Continuous E x t r a c t i o n of Organic M a t e r i a l s from Water. Environmental Science and Technology J o u r n a l . Vol.5, No.2, pp.161-163, 1971. Gould, M.S. And E . J . G e n e t e l l i . The E f f e c t of M e t h y l a t i o n and Hydrogen Ion C o n c e n t r a t i o n on Heavy Metal B i n d i n g by A n a e r o b i c a l l y Digested Sludges. Water Research. Vol.12, pp.889-892, 1978. G r i f f i n , . R.A., et a l M . . •= . A t t e n u a t i o n of P o l l u t a n t s i n M u n i c i p a l L a n d f i l l Leachate by Clay M i n e r a l s . rl^r, " H e a v y - m e t a l A d s o r p t i o n . Environmental  Geology Notes. Vol.79, 1977. ~ - £ -G r i f f i n , R . A . , et a l , A t t e n u a t i o n of P o l l u t a n t s M u n i c i p a l L a n d f i l l Leachate by Passage Through Environmental Science and Technology V o 1 - 1 0 ' No.13, pp.1262-1268, December Clay. J o u r n a l 1976. G u t h r i e , R.K., et a l . A q u a t i c B a c t e r i a l P o p u l a t i o n s and Heavy Metals I I . I n f l u e n c e of Chemical Content of Aquatic Environments on B a c t e r i a l Uptake of Chemical Elements. Water Research. Vol.11, pp.643-646, 1977. Hatch, T.H., and A. Menawat. B i o l o g i c a l Removal and Recovery of Trace Heavy M e t a l s . Biotechnology  and B i o e n g i n e e r i n g Symposium. Vol.8, pp.191-203, 1979. Hawley, G.G., Condensed Chemical D i e t i o n a r y . - - 8 e d i t i o n . Van Nostrand R e i n h o l d Co. New York. 1971. Hayes, T.C., and T.L. T h e i s . The D i s t r i b u t i o n of Heavy Metals i n Anaerobic D i g e s t i o n . J o u r n a l Water  P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.50, No.1, pp.61-72, 1978. H i n e s l y , T.D., et a l . E f f e c t s of Annual and Accumulative A p p l i c a t i o n s of Sewage Sludge on A s s i m i l a t i o n of Zinc and Cadmium by Corn (Zea mays L . ) . Environmental Science and Technology J o u r n a l . Vol.11, No.2, pp.182-187, February 1977. Hobbie, 100 Hoffmann, M.R., et a l . C h a r a c t e r i z a t i o n of S o l u b l e and C o l l o i d a l - P h a s e Metal Complexes i n Ri v e r Water by U l t r a f i l t r a t i o n . A Mass-Balance Approach. Environmental Science and Technology J o u r n a l . V o l . 1 5 , No.6, pp.655-661, June 1981. Holmgren, G.G.S. A Rapid C i t r a t e - D i t h i o n i t e E x t r a c t a b l e Iron Procedure. S o i l Science S o c i e t y of America.  Proceedings. Vol.31, pp.210-211, 1967. Jackson, M.L. S o i l Chemical A n a l y s i s . P r e n t i c e - H a l l Inc. Englewood C l i f f s , N.J. 1958. Jeanes, A. A p p l i c a t i o n s of E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e - p o l y e l e c t r o l y t e s : Review of L i t e r a t u r e , I n c l u d i n g Patents. J o u r n a l of  Polymer Science: Polymer Symposia. I on  C o n t a i n i n g Polymers. No.45, pp.209-227, 1974. Jeanes, A. E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s : new H y d r o c o l l o i d s Having Both Fundamental and P r a c t i c a l Import. Polymer Science and Technology . Vol.2, pp.227-242, Aug 1972. J e l l i n e k , H.H., and S.P. Sangal. Complexation of Metal Ions With N a t u r a l P o l y e l e c t r o l y t e s [Removal and Recovery of Metal Ions From P o l l u t e d Waters]. Water Research. Vol.6, pp.305-314, 1972. Jones, J.G. Some Obs e r v a t i o n s on D i r e c t Counts of Freshwater B a c t e r i a Obtained with a Fluorescence Microscope. Limnology and Oceanography. Vol.19, pp.540-543, 1974. K a p u s c i n s k i , J . , and B. of DNA with indole.2HC1 or i n d o l e . A n a l y t i c a l pp.3775-379y, October, 1980. Sk o c z y l a s . F l u o r e s c e n t Complexes DAPI ^ 4', 6-diamidine-2-phenyl DCI 4', 6-dicarboxyamide-2-phenyl L e t t e r s . V ol.5, No.10, K a p u s c i n s k i , j . , and F l u o r i m e t r i c B. S k o c z y l a s . Method f o r Simple and Rapid A n a l y t i c a l B i o c h . ^ ^ r y — V o l ° N A " i c r o a s s a y . 1977, pp.252-257, Kmman, R . N . ; a d J j Walsh. Leachate from M u n i c i p a l and I n d u s t r i a l Waste L a n d f i l l S i m u l a t o r s . D i s p o s a l of Hazardous Waste. Proceedings of the Annual Research Symposium (6th) Held at Chicago, I l l i n o i s on March 17-20, pp.203-222, 1980. 101 Kirkham, M.B. Organic Matter and Heavy Metal Uptake. Compost Sc i e n c e . Vol.18, No.1, pp.18-21, Jan-Feb 1977. Klimek, J . , and D.F. O l l i s . E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s : K i n e t i c s of Pseudomonas sp., Azotobacter v i n e l a n d i i , and Aureobasidium p u l l u l a n s Batch Fermentations. Biotechnology and  B i o e n q i n e e r i n g . Vol.22, No.11, pp.2321-2342, 1 980. Knox, K., and P.H. Jones. Complexation C h a r a c t e r i s t i c s of S a n i t a r y L a n d f i l l Leachates. Water Research. Vol.13 pp.839-846, 1979. Leemann, U., and F. Ruch. S e l e c t i v e E x c i t a t i o n of Mithramycin or DAPI Fl u o r e s c e n c e on Double-S t a i n e d C e l l N u c l e i and Chromosomes. H i s t o c h e m i s t r y . Vol.58, pp.329-334, 1978. L e s t e r , J.N., et a l . The I n f l u e n c e of Heavy Metals on a Mixed B a c t e r i a l P o p u l a t i o n of Sewage O r i g i n i n the Chemostat. Water Research. Vol.13 No.11, pp.1055-1063, 1979. L e s t e r , J.N., et a l . Rapid Flameless Atomic Absorption A n a l y s i s of the M e t a l l i c Content of Sewage Sludges. I. Lead, Cadmium and Copper. Science  of the T o t a l Environment. V o l . 8 , pp.153-158, 1 977. L i n g l e , J.W., and E.R. Hermann. Mercury i n Anaerobic Sludge D i g e s t i o n . J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.47, No.3, pp.466-471, March 1975. MacCarthy, P. An I n t e r p r e t a t i o n of S t a b i l i t y Constants f o r S o i l Organic Matter-Metal Ion Complexes Under Schubert C o n d i t i o n s . J o u r n a l of Environmental  Science and H e a l t h : Part A. Vol.A12, No.1&2, pp.43-59, 1977. Mantoura, R.F.C., A. Dickson, and J.P. R i l e y . The Complexation of Metals with Humic M a t e r i a l s i n N a t u r a l Waters. E s t u a r i n e and C o a s t a l Marine  S c i e n c e . V o l . 6 , pp.387-408, 1978. Mantoura, R.F.C., and J.P. R i l e y . The Use of Gel F i l t r a t i o n In The Study of Metal B i n d i n g by Humic A c i d s and Re l a t e d Compounds. A n a l y t i c a Chimica  A c t a . Vol.78, pp.193-200, 1975. 102 69 Mantoura, R.F.C., and J.P. R i l e y . The A n a l y t i c a l C o n c e n t r a t i o n of Humic Substances from Nat u r a l Waters. A n a l y t i c a Chimica A c t a . Vol.76, pp.97-106, 1975. 70 Mara, D.D. B a c t e r i o l o g y f o r S a n i t a r y Engineers. C h u r c h i l l L i v i n g s t o n e , London, 1974. 71 McFadden, W.H. Techniques of Combined Gas Chromatography /Mass Spectrometry. A p p l i c a t i o n s i n Organic  A n a l y s i s . John Wiley and Sons. New York. 1973. 72 Moore, W.A., et a l . E f f e c t s of Chromium on the A c t i v a t e d Sludge Process. J o u r n a l Water P o l l u t i o n C o n t r o l  F e d e r a t i o n . Vol.33, No.1, pp.54-72, January 1961 . 73 Mosey, F.E. The T o x i c i t y of Cadmium to Anaerobic D i g e s t i o n : I t s M o d i f i c a t i o n by Inorganic Anions. Water P o l l u t i o n C o n t r o l (G.B.). Vol.70, No.5, pp.584-598, 1971. 74 Naimski, P., A. B i e r z y n s k i , and F. Magdalena. Q u a n t i t a t i v e F l u o r e s c e n t A n a l y s i s of D i f f e r e n t Conformational Forms of DNA Bound to the Dye, 4' , 6-Diamidine-2-phenylindole, and Separated by Gel E l e t r o p h o r e s i s . A n a l y t i c a l B i o c h e m i s t r y . Vol.106, pp.471-475, 1980. 75 Neufel d , R.D., and E.R. Hermann. Heavy Metal Removal by Acc l i m a t e d A c t i v a t e d Sludge. J o u r n a l Water  P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.47, No.2, pp.310-329, February 1975. 76 Obayashi, A.W., and A.F. Gaudy J r . Aero b i c D i g e s t i o n of E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s . J o u r n a l  Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.47, No.7, pp.1584-1594, J u l y 1973. 77 O l i v e r , B.G., and E.G. Cosgrove. The E f f i c i e n c y of Heavy Metal Removal by a Conventional A c t i v a t e d Sludge Treatment P l a n t . Water Research. Vol.8 pp.869-874, 1974. 78 Pace, G.W., and R.C. R i g h e l a t o . Production of E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s . Advances i n Biochemical E n g i n e e r i n g . F i e c h t e r , A. Ed. S p r i n g e r - V e r l a g New York, Vol.15, pp.41-88, 1979. 103 79 P a t r i c k , F.M., and M.W. L o u t i t . The Uptake of Heavy Metals by E p i p h y t i c B a c t e r i a on Alisma Plant Ago-a q u a t i c a . Water Research. Vol.11, pp.699-703, 1977. 80 81 82 P a t r i c k , F.M., and M.W. L o u t i t . Passage of Metals i n E f f l u e n t s , Through B a c t e r i a to Higher Organisms. Water Research. Vol.10, pp.333-335, A p r i l 1976. Pohland, F.G., and J.P. Gould. S t a b i l i z a t i o n of M u n i c i p a l L a n d f i l l s C o n t a i n i n g I n d u s t r i a l Wastes. D i s p o s a l of Hazardous Waste. Proceedings of the Annual (6th) Held at Chicago, 242-253, 1980. Research Symposium I l l i n o i s on March 17-20, pp. Poon, C.P.C., and K.H. Bhayani. Metal T o x i c i t y to Sewage Organisms. ASCE J o u r n a l of S a n i t a r y E n g i n e e r i n g  D i v i s i o n . Vol.97, No.SA2, pp.161-169, A p r i l 1971 . 83 P o r t e r , K.G., and Y.S. F e i g . The use of DAPI f o r I d e n t i f y i n g and Counting Aquatic M i c r o f l o r a . Limnology and Oceanography. Vol.25, No.5, pp.943-948, 1980. 84 Powell, D.A. P o l y s a c c h a r i d e S t r u c t u r e and I n t e r a c t i o n s . M i c r o b i a l P o l y s a c c h a r i d e s and P o l y s a c c h a r a s e s . Berkeley, R. C. W. ; Gooday, G. W. and Ellwood, D. C. eds. Academic Press, New York, pp.150-153, 1979. 85 Raspor, B., H.W. Nurnberg, P. V a l e n t a , and M. B r a n i c a . Voltammetric S t u d i e s on the S t a b i l i t y of the Zn(II) C h e l a t e s With NTA and EDTA and the K i n e t i c s of T h e i r Formation i n Lake O n t a r i o Water. Limnology and Oceanography. Vol.26, No.1, pp.54-66, 1981. 86 Raveh, A., and Y. Avnimelech. Leaching of P o l l u t a n t s From S a n i t a r y L a n d f i l l Models. J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.51, No.11, pp.2705-2716, 1979. 87 Roberson, B.X., and J.H. Schwab. S t u d i e s on P r e p a r a t i o n of B a c t e r i a l C e l l Walls and C r i t e r i a of Homogeneity. Biochemica et B i o p h y s i c a . Vol.44, pp.436-444, 1960. 1 04 88 S a l a r i , S.H., and M.E. Ward. E a r l y D e t e c t i o n of Chlamydia trachomatis Using F l u o r e s c e n t , DNA Binding Dyes. J o u r n a l of C l i n i c a l Pathology. Vol.32, pp.1155-1162, 1979. 89 Sanderson, W.W., and G.B. C e r e s i a . Continuous E x t r a c t i o n of C h l o r i n a t e d Aromatic P e s t i c i d e s i n P a r t s per B i l l i o n . J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . Vol.37, No.8, pp.1177-1179, 1965. 90 Sandford, P.A. E x o c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s . Advances in Carbohydrate Chemistry and  B i o c h e m i s t r y . Tipson, R. S. and Horton, D. Eds. Academic Press, New York, Vol.36, pp.266-292, 1979. 91 Sax, N.I. Dangerous P r o p e r t i e s of I n d u s t r i a l M a t e r i a l s . R e i n h o l d P u b l i s h i n g Co. New York. 1958. 92 Schomaker, N.B., and B.L. Rittenhouse. Current Research on Land D i s p o s a l of Hazardous Wastes. D i s p o s a l  of Hazardous Waste. Proceedings of the Annual Research Symposium (6th) Held at Chicago, I l l i n o i s on March 17-20, pp.1-14, 1980. 93 Schroeder, D.C, and F.G. Lee. P o t e n t i a l Transformations of Chromium in N a t u r a l Waters. Water, A i r , a n d  S o i l P o l l u t i o n . Vol.4, pp.355-365, 1975. 94 S i l l e n , L.G., S t a b i l i t y Constants Supplement No.1. S p e c i a l P u b l i c a t i o n 25. The Chemical S o c i e t y London. Alden Press, Oxford. 1971. 95 Slade, H.D., and J.K. V e t t e r . S t u d i e s on S t r e p t o c c u s Pyogenes I -- Observations on the M i c r o s c o p i a l and B i o l o g i c a l Aspects of the D i s i n t e g r a t i o n and S o l u b i l i z a t i o n of a Type 6 S t r a i n by Sonic O s c i l l a t i o n . J o u r n a l of B a c t e r i o l o g y . Vol.71, pp.236-243, 1956. 96 Sharon, N., and R.W. J e a n l o z . A Procedure f o r the P r e p a r a t i o n of Gram-quantities of B a c t e r i a l C e l l W a l l s . E x p e r i e n t a . Vol.20, pp.253-254, 1964. 97 S p o s i t o , G. Trace Metals i n Contaminated Waters. Environmental Science and Technology J o u r n a l . Vol.15 No.4, pp.396-403, A p r i l 1981. 105 98 99 S r i v a s t a v a , S.K., et a l . Gels i n the Research. V o l S t u d i e s on the Use of Inorganic Removal of Heavy Metals. Water ,14 No.2, pp. 1 13-1 15, 1980. Srna, R.F., et a l . Copper Complexation C a p a c i t y of Marine Water Samples from Southern C a l i f o r n i a . Environmental Science and Technology J o u r n a l . Vol.14, No.12, pp.1482-1486, December 1980. 100 a l S t a n f o r t h , R., et M u n i c i p a l P o l l u t i o n C o n t r o l Development of L a n d f i l l Leachate. _ F e d e r a t i o n . V o l a S y n t h e t i c J o u r n a l Water 51 No. 7 pp.1965-1975, 1979. 101 Stoveland,, S., J.N. L e s t e r , R. Perry. The Influence of N i t r i l o t r i a c e t i c A c i d on Heavy Metal T r a n s f e r i n the A c t i v a t e d Sludge Process — I.At Constant Loading. Water Research. Vol.13, pp.949-965, 1979. 102 S t r i c k l a n d , J.D.H. and T.R. Parsons. A P r a c t i c a l Handbook of Seawater A n a l y s i s . B u l l e t i n Research Board of Canada, Ottawa, 167. F i s h e r i e s O n t a r i o . 1972. 103 T h e i s , T.L., and R.O. R i c h t e r . Chemical S p e c i a t i o n of Heavy Metals i n Power Plant Ash Pond Leachate. Environmental Science and Technology J o u r n a l . Vol.13, No.2, pp.219-224, February 1979. 104 T i r s c h , F.S., et a l . Copper and Cadmium Reactions With S o i l s i n Land A p p l i c a t i o n s . J o u r n a l Water P o l l u t i o n C o n t r o l F e d e r a t i o n . pp.2649-2660, 1979. Vol.51, No.11 105 T r o l l d e n i e r , G. The use of Fluorescence Microscopy f o r Counting S o i l Micro-organisms. Modern Methods in  the Study of M i c r o b i a l Ecology. Rosswall, Thomas ed. (Proceedings of a Symposium h e l d at the A g r i c u l t u r a l C o l l e g e , Uppsala Sweden. 1972. ) p.53-59, 1973. June 19-23 106 T r u i t t , R.E., and J.H. Weber. I n f l u e n c e of F u l v i c A c i d on the Removal of Trace C o n c e n t r a t i o n s of Cadmium(Il), C o p p e r ( I I ) , and Z i n c ( I I ) From Water by Alum C o a g u l a t i o n . Water Research. Vol.13, No.12, pp.1171-1177, 1979. 107 Watson, S.W., et a l . Determination of B a c t e r i a l Number and Biomass i n the Marine Environment. A p p l i e d  and Environmental M i c r o b i o l o g y . Vol.33, No.4, pp.940-946, A p r i l 1977. 106 108 Weast, R.C, ed. Handbook of Chemistry and P h y s i c s . Chemical Rubber Co. 52 and 62 e d i t i o n s . C l e v e l a n d , Ohio. 1971, 1981. 109 Wollan, E., and P.H.T. Beckett. Changes i n the E x t r a c t a b i l i t y of Heavy Metals on the I n t e r a c t i o n of Sewage Sludge with S o i l . Envi ronmental  P o l l u t i o n J o u r n a l . Vol.20, No.3, pp.215-230, 1979. 1 07 GLOSSARY -A-Absorb - To h o l d w i t h i n a volume. A c t i v i t y - C o n c e n t r a t i o n x a c t i v i t y c o e f f i c i e n t ( f i ) - the a c t i v i t y i s a f u n c t i o n of the number of ions i n a s o l u t i o n . L o g l O ( f i ) = -0.5 Z i 2 ( U ) 0 ' 5 /(1 + ( U ) 0 ' 5 ) Where: Ci= m o l a r i t y U= i o n i c s t r e n g t h Zi= charge Adsorb - To h o l d on the s u r f a c e . Antagonism - The a b i l i t y of a c a t i o n to decrease the t o x i c i t y of another c a t i o n . -B-B i o f l o c - A z o o g l e a l mass of c e l l s j o i n e d by s l i g h t l y s o l u b l e e x t r a c e l l u l a r p o l y s a c c h a r i d e s which form polymer f i b r i l s . The f l o e forms i n a c t i v a t e d sludge systems and s e t t l e s out of s o l u t i o n i n a c l a r i f i e r . R e fers to a l l of the l o c a l b i o l o g i c a l ecosystems. -C-Exchange C a p a c i t y - CEC. The number of moles of a d s o r b t i o n s i t e s that are a v a i l a b l e i n a s o i l f o r c a t i o n exchange. C a t i o n exchange occurs when a f r e e c a t i o n i s more s t a b l y bound to the s o i l than the c a t i o n that i s a l r e a d y bound by the s o i l . C h e l a t i n g agent - A compound which i s c o o r d i n a t e d by a c h e l a t o r . A c h e l a t i n g agent p r o v i d e s a supply of l i g a n d s f o r a c h e l a t e to c o o r d i n a t e with. C h e l a t i o n - When a c h e l a t o r bonds c o v a l e n t l y with a l i g a n d to form one or more r i n g s c h e l a t i o n has occured. C h e l a t o r - U s u a l l y a metal which i s j o i n e d by c o v a l e n t bonds to two or more other atoms. The atoms can be s u p p l i e d by one or more molecules c a l l e d l i g a n d s . C o d i s p o s a l Report (UBC) - Atwater et a l . (1981) s t u d i e d the i n t e r a c t i o n of e l e c t r o p l a t i n g wastes with s e p t i c tank wastes i n a l y s i m e t e r . C o l l o i d a l P a r t i c l e s - G e n e r a l l y i n the 2.5-12.0 nm p a r t i c l e s i z e range. The a c t u a l range used by an author may vary a c c o r d i n g to the f i l t e r s that were a v a i l a b l e . B i o t a -Cat ion 108 Complex - A complex forms when a l i g a n d molecule binds to a c h e l a t o r . -D-Dentate - l i t e r a l l y means t o o t h . b i d e n t a t e - two t e e t h - i n c o - o r d i n a t i o n chemistry i t r e f e r s to a l i g a n d which p r o v i d e s two l o c a t i o n s f o r a c h e l a t o r to bond t o . DAPI - 4', 6 d i a m i d i n o - 2 - p h e n y l i n d o l e . A strong base and a f l u o r e s c e n t dye. Davies Equation - L o g f i = 0 . 5 1 Z 2 ( ( U ) 0 ' 5 /1+(U) 0' 5 - 0.3U) Z=Charge on the ion U=Ionic s t r e n g t h f i = A c t i v i t y D i a l y s i s - The use of d i f f e r e n t i a l c o n c e n t r a t i o n s to d r i v e molecules a c r o s s a membrane and hence separate small molecules from l a r g e molecules. Small molecules d i f f u s e a c r o s s the membrane more r a p i d l y . -E-E l e c t r o p h o r e s i s - The a p p l i c a t i o n of a p o t e n t i a l d i f f e r e n c e to a s o l u t i o n to separate p o s i t i v e and negative s p e c i e s . E l u t e - to wash out. -F-F u l v i c a c i d - that f r a c t i o n of the organic matter i n s o i l that remains i n s o l u t i o n a f t e r e x t r a c t i o n with a l k a l i and p r e c i p i t a t i o n by a c i d . -G-Gram-negative - b a c t e r i a t h a t f a i l the gram-staining t e s t i n d i c a t e s a two l a y e r c e l l w a l l - more r e s i s t a n t to environmental s t r e s s e s . Gram-positive - gram-staining i s s u c c e s s f u l with t h i s type of b a c t e r i a - i n d i c a t e s a s i n g l e l a y e r c e l l w a l l - l e s s r e s i s t a n t to environmental s t r e s s e s . Gram- s t a i n i n g t e s t - uses c r y s t a l v i o l e t to s t a i n b a c t e r i a - p o s i t i v e r e s u l t s s t a i n purple - negative r e s u l t s do not s t a i n with gram s t a i n . 109 -H-Humic a c i d - the p o r t i o n of the organic matter i n s o i l that p r e c i p i t a t e s when a c i d i s added to an a l k a l i e x t r a c t i o n of the s o i l . Humic substances - substances from the organic f r a c t i o n of s o i l . Humin - the f r a c t i o n that i s l e f t i n the s o i l a f t e r a c i d and base e x t r a c t i o n . Humus - dark organic f r a c t i o n of s o i l formed by decaying substances. - I -Ion-exchange - separates p o s i t i v e , negative, and n e u t r a l s p e c i e s . I o n i c s t r e n g t h - U = 0.5 (Ci x Z i 2 ) " 0 ' 5 I r v i n g - W i l l i a m s - e s t a b l i s h e d an order f o r the s t r e n g t h of complexes. I r v i n g and W i l l i a m s r e p o r t e d the f o l l o w i n g o r d e r : Mg < Ca < Cd, Mn < Co < Zn, Ni < Cu <Hg. [Nature V o l 162 p746 1948 and J of Chem Soc V o l 3 p3l92 1953] -L-L a n d f i l l - g e n e r a l l y a s i t e where re f u s e from households and i n d u s t r y i s i n t e r r e d . M a t e r i a l s which are brought to the s i t e are dumped on the s u r f a c e or i n an excavated area which i s predetermined. Then the r e f u s e i s covered with s i x inches or more s o i l . The s i t e i s b u i l t on u n t i l there i s no more s o i l a v a i l a b l e to cover the r e f u s e . To be a s a n i t a r y l a n d f i l l the s i t e must be i s o l a t e d from the surrounding s o i l . The refuse must be covered by s o i l w i t h i n 24 h r s . of dumping and the s i t e should be d r a i n e d to c o l l e c t any l e a c h a t e which may be generated. The whole s i t e should be above the l o c a l water t a b l e . O p t i m a l l y , the m a t e r i a l s being dumped at the s i t e can be r e s t r i c t e d . Leachate - A dark c o l o u r e d , unpleasant s m e l l i n g , l i q u i d which d r a i n s from a l a n d f i l l when the l i q u i d h o l d i n g c a p a c i t y of a s i t e has been exceeded. The volume of l e a c h a t e which forms i s a f u n c t i o n of the ground water i n f i l t r a t i o n and the annual p r e c i p i t a t i o n at the s i t e . Leachates can be a c i d i c , b a s i c or n e u t r a l . The a c i d i c nature of l e a c h a t e can be caused by organic a c i d s 110 which are produced as b y - p r o d u c t s of the organ ic d e g r a d a t i o n of r e f u s e . These a c i d s mix w i t h the water • seep ing through a l a n d f i l l . They can l e a c h metals and a c i d s o l u b l e compounds i n the r e f u s e . The net r e s u l t i s the g e n e r a t i o n of an obnoxious l i q u i d suspens ion c o n t a i n i n g d i s s o l v e d and p a r t i c u l a t e m a t t e r . T h i s l i q u i d i s c a l l e d l e a c h a t e . The l i q u i d d r a i n e d from the l y s i m e t e r was r e f e r r e d to as l y s i m e t e r l e a c h a t e because i t was s i m i l a r to l a n d f i l l l e a c h a t e . L i g a n d - a compound or molecule which p r o v i d e s one or more bonding s i t e s f o r a c h e l a t o r . (Note: two s i t e s = b i d e n t a t e to s i x s i t e s = hexadentate) L i n e w e a v e r - B u r k E q u a t i o n - 1/V = Km/VmS + 1/Vm Where: V= mg/1 0 2 Uptake /gm. of b i o s o l i d c u l t u r e / m i n . Vm= V f o r an unhindered s u b s t r a t e S= s u b s t r a t e c o n c e n t r a t i o n i n gm. COD/gm. b i o l o g i c a l s o l i d Km= M i c h a e l i s Menten c o n s t a n t L y s i m e t e r - l a b o r a t o r y s i z e model of a s a n i t a r y l a n d f i l l . In t h i s case a 30 cm. d iameter PVC tube w i t h a l t e r n a t i n g l a y e r s of s o i l and r e f u s e packed under a 220 p s i p r e s s u r e . L y s i s - the r u p t u r i n g of the c e l l w a l l of a whole c e l l . - M -M i c h a e l i s - M e n t e n Model - a model of enzyme i n h i b i t i o n . - 0 -O x i d a t i o n - Occurs when an ion donates e l e c t r o n s . -P-Phenol s - a c l a s s of compounds w i t h , one or more hydroxy groups a t t a c h e d to a benzene r i n g . - R -R e d u c t i o n - Occurs when an ion ga in s e l e c t r o n s r e d u c i n g i t ' s p o s i t i v e c h a r g e . Recovery e f f i c i e n c y - D e f i n e d as the r a t i o of meta l d e t e c t e d in MIBK e x t r a c t / the i n i t i a l c o n c e n t r a t i o n in the sample 111 to be c h e l a t e d . Note that t h i s i s the combination of the MIBK e x t r a c t i o n e f f i c i e n c y and the c h e l a t i o n e x t r a c t i o n e f f i c i e n c y . -S-Scatchard- p l o t - a p l o t of Vav/ Mf vs Vav . The p l o t d e s c r i b e s the equation Vav/Mf = Ki ( n i - Vav) Where: Vav = Moles of metal bound / Moles of Humic a c i d Mf = c o n c e n t r a t i o n of f r e e metal ion ni = Number of b i n d i n g s i t e s / molecule of Humic a c i d (Note: T h i s i s the Vav a x i s i n t e r c e p t ) Ki = S t a b i l i t y constant (Slope of p l o t ) . Schubert c o n d i t i o n s - occur i n c o - o r d i n a t i o n chemistry when the c o n c e n t r a t i o n of l i g a n d s i s very much gr e a t e r than the c o n c e n t r a t i o n of the c h e l a t o r . (eg [ l i g a n d ] >> [metal]) Sorb - to h o l d by a d s o r p t i o n or a b s o r p t i o n . S t a b i l i t y constant - when i t r e f e r s to a c h e l a t i o n r e a c t i o n , i t i s of the form: K = [MnLm] / [M] * [L] where M i s g e n e r a l l y the c h e l a t o r metal, K i s the s t a b i l i t y constant and L i s the l i g a n d molecule and [] i n d i c a t e s c o n c e n t r a t i o n . The e q u i l i b r i u m equation would be: xM + yL = MmLn Synergism - when two or more c a t i o n s have a g r e a t e r e f f e c t together than the a d d i t i o n of t h e i r independent e f f e c t s . -U-U l t r a f i l t r a t i o n - Using a very f i n e f i l t e r made of p l a s t i c to f i l t e r out molecules over a s p e c i f i e d s i z e . U s u a l l y r e q u i r e s an a p p l i e d pressure to overcome the l a r g e head l o s s . APPENDIX A - - PLOTS OF METAL RELEASED VS TIME 1 13 R J GURE N I C K E L - CONC VS. '.'IF - CD! - C D C " . J . K 1981 . M G U R E Z I N C - CONC VS TIM£ - CDI - C.C C - J K 1 1 4 APPENDIX B - - CO-ORDINATION CHEMISTRY When a metal bonds with a r a d i c a l to form one or more co v a l e n t bonds, i t has been complexed. C o - o r d i n a t i o n chemistry i s the study of complex formation. A r a d i c a l which donates one or more e l e c t r o n p a i r s to form a complex i s c a l l e d a l i g a n d . When the atoms of a l i g a n d and a metal form a r i n g , the complex i s c a l l e d a c h e l a t e and the l i g a n d i s c a l l e d a c h e l a t o r or a c h e l a t i n g agent. A l l the l i g a n d s i n t h i s study are c h e l a t o r s except ethanoic a c i d . Each of the metals analyzed i n t h i s study (Cd, Cr, Cu, Fe, N i , Pb, and Zn) can c o - o r d i n a t e up to s i x co v a l e n t bonds. In g e n e r a l a l i g a n d t hat has 'x' donor atoms w i l l form 'x-1' c h e l a t e r i n g s ( B e l l 1977 and Dean 1979). The l a r g e r the value of 'x' the more s t a b l e the complex w i l l tend to be. Some lig a n d - m e t a l complexes w i l l d i s p l a y enhanced s t a b i l i t y due to a p a r t i c u l a r l y good f i t . Table XXII l i s t s some b a s i c and a c i d i c l i g a n d groups. The b a s i c l i g a n d s i n t e r a c t with a metal by donating a lone p a i r of e l e c t r o n s to form a co v a l e n t bond. A c i d i c groups l o s e a proton (H + ) before donating an e l e c t r o n p a i r to form a c o v a l e n t bond. F i g u r e 13 shows the bonding s i t e s f o r the seven l i g a n d s used. When a complex forms, the c o n s t i t u e n t molecules form a r i n g . The s t a b i l i t y of a complex decreases as the number of members i n the r i n g , the bulkyness or the asymmetry of the core ion i n c r e a s e s . Core symmetry i s a f u n c t i o n of the o r b i t a l s which are occ u p i e d . Table XXIII i l l u s t r a t e s the o r b i t a l 1 15 Table XXII - Ligand Groups Basic I jigands A c i d i c Ligar ids -NH2 -NH -N= =0 =N-0H -OH - s -Amino Imino T e r t i a r y A c y c l i c or Hetero-c y c l i c N Carbonyl Oxime A l c o h o l T h i o e t h e r -COOH -S0 3H -PO(OH) -OH =N-OH -SH C a r b o x y l i c Sulphonic Phosphoric E n o l i c Phenolic Oxime T h i o e n o l i c T h i o p h e n o l i c Table XXIII - O r b i t a l C o n f i g u r a t i o n s Ion 1s 2s 2p 3s 3p 4S 3d 4p 5s 4d 5p 6s 4f 5d 6p Cr 2 2 6 2 6 1 5 C r + 2 2 2 6 2 6 4 C r + 3 2 2 6 2 6 3 C r + 6 2 2 6 2 6 Fe 2 2 6 2 6 2 6 F e + 2 2 2 6 2 6 6 Fe + 3 2 2 6 2 6 5 Ni 2 2 6 2 6 2 8 Ni + 2 2 2 6 2 6 8 Cu 2 2 6 2 6 1 10 Cu + 2 2 6 2 6 1 0 C U + 2 2 2 6 2 6 9 Zn 2 2 6 2 6 2 10 Z n + 2 2 2 6 2 6 10 Cd 2 2 6 2 6 2 10 6 2 10 C d + 2 2 2 6 2 6 2 10 6 10 Pb 2 2 6 2 6 2 10 6 2 10 6 2 1 4 10 2 P b + 2 2 2 6 2 6 2 1 0 6 2 10 6 2 14 10 c o n f i g u r a t i o n s f o r Cd, Cr, Cu, Fe, N i , Pb, and Zn. S t a b i l i t y c onstants are used to n u m e r i c a l l y i n d i c a t e the s t r e n g t h or s t a b i l i t y of a given complex. The more p o s i t i v e the ^ l a b i l i t y constant the g r e a t e r the s t a b i l i t y and the gr e a t e r the l i k e l i h o o d t h a t the complex w i l l form. The s t a b i l i t y constant 'Kn' i s d e f i n e d by the f o l l o w i n g F i g u r e 13 - Ligand Bonding S i t e s A c i d i c Ligand =COOH2 0-Q P _ 0 X =C X H =C * to H-1 N-l Bas i c L i g a n d -NH2 M ,H -N -N* Note: That carbon and n i t r o g e n bonds are shown. *- marks bonding l o c a t i o n s C = carbon ; N = n i t r o g e n ; 0 = oxygen; H = hydrogen LIGAND MOLECULAR MODEL (Molecular Formulae) EDTA NH 2 CH=COOH2 C 1 0 H 1 6 0 B N 2 I / H 2 O O C = C H - C - C - C H = C O O H 2 N H 2 \ : H = C O O H 2 oooooooooooooooooooooooooooooooooooooooooooooooooooooo E t h a n i c A c i d CH 2=COOH 2 C 2 H n 0 2 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO G l y c i n e NH2-CH=COOH2 C 2H 50 2N oooooooooooooooooooooooooooooooooooooooooooooooooooooo H i s t i d i n e H2OOC CH 2-C = CH C 6 H 9 N 3 0 2 \ / • I \ CH=C HN N . 2 CH oooooooooooooooooooooooooocooooooooooooooooooooooooooo N - \ H 8-Hydroxyquinoline CH CH C 9H 7ON / ' HC C^ I II HC ,C /,CH \ / \ /, oooooooooooooooooooooooooooooooooooooooooooooooooooooo NTA CH2-COOH C 6H 9N0 6 \ HOOC-N-COOH CH 2 v_n 2 oooooooooooooooooooooooooooooooooooooooooooooooooooooo O x a l i c A c i d HOOC=COOH C 2H 20, oooooooooooooooooooooooooooooooooooooooooooooooooooooo formula: Kn = [MLn]*([M]*[MLn-1])- 1 Where: M=metal ion 1 1 7 L=ligand n=number of l i g a n d s complexed The bulk s t a b i l i t y constant 'Bn' i s the product of the Kn. Bn = [MLn]*([M]*[L] J " 1 = n K i 118 APPENDIX C - - EXOCELLULAR POLYSACCHARIDES Corpe (1975) separated the s u r f a c e a c t i v e p o l y m e r s ( e x t r a c e l l u l a r p o l y s a c c h a r i d e s ) produced by primary f i l m forming b a c t e r i a i n s a l t water. He found s u b s t a n t i a l q u a n t i t i e s of metals bound to c e l l envelopes and e x t r a c e l l u l a r p o l y s a c c h a r i d e s . The b a c t e r i a were capable of forming i n s o l u b l e p r e c i p i t a t e s with s a l t s of Fe, Cu, and Pb. N e u t r a l polymer s o l u t i o n s formed n o n - d i f f u s i b l e complexes with Co, N i , Zn, Mn, and Ca. Table XXIV l i s t s the r a d i c a l s r e s p o n s i b l e f o r the charge on e x t r a c e l l u l a r p o l y s a c c h a r i d e s . Jeanes (1972) observed Table XXIV - Chemical R a d i c a l s Of Charged E x o c e l l u l a r Polymers Spec i e s R a d i c a l Causing Charge N e u t r a l P o l y s a c c h a r i d e s P o l y a n i o n i c P o l y s a c c h a r i d e s P o l y c a t i o n i c P o l y s a c c h a r i d e s N e u t r a l or N - s u b s t i t u t e d amino sugars Uronic a c i d c o n s t i t u e n t s or a c i d i c s u b s t i t u t e s U n s u b s t i t u t e d amino sugars that u n c h a r a c t e r i z e d b a c t e r i a l p o l y s a c c h a r i d e s were r e s p o n s i b l e f o r the uptake of uranium and other o r e s . E x t r a c e l l u l a r p o l y s a c c h a r i d e s can be produced by some s t r a i n s of waste water, p l a n t and s o i l b a c t e r i a . Some aerobic and anaerobic b a c t e r i a can generate e x t r a c e l l u l a r p o l y s a c c h a r i d e s . Obayashi and Gaudy (1973) s t u d i e d two waste water, two s o i l , and one p l a n t b a c t e r i a . They found that b a c t e r i a can o x i d i z e e x t r a c e l l u l a r p o l y s a c c h a r i d e s and form a 119 s t a b l e end product. E x t r a c e l l u l a r p o l y s a c c h a r i d e s can be n e u t r a l , p o l y a n i o n i c or p o l y c a t i o n i c (Jeanes 1974). When e x t r a c e l l u l a r p o l y s a c c h a r i d e s c o n t a i n metal ions they tend to be h y d r o p h i l l i c and form c o l l o i d a l d i s p e r s i o n s , i n s o l u t i o n . Sandford (1979) suggested three c a t e g o r i e s of polymers a s s o c i a t e d with b a c t e r i a . They are i n t r a c e l l u l a r , c e l l w a l l , and e x t r a c e l l u l a r polymers. The e x t r a c e l l u l a r p o l y s a c c h a r i d e s are e i t h e r loose or c o v a l e n t l y bonded to c e l l s . Klimek and O l l i s (1980) r e p o r t e d that p r o d u c t i o n of p o l y s a c c h a r i d e s may be growth r e l a t e d , wholly non-growth r e l a t e d or mixed. They were abl e to use rate equations to model production of e x t r a c e l l u l a r p o l y s a c c h a r i d e s . Pace and R i g h e l a t o (1980) s t u d i e d the s y n t h e s i s of e x t r a c e l l u l a r p o l y s a c c h a r i d e s . Some of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s are sy n t h e s i z e d o u t s i d e the c e l l w a l l but most are s y n t h e s i z e d i n the c e l l w a l l . The molecular weight of e x t r a c e l l u l a r p o l y s a c c h a r i d e s appears to be h i g h l y v a r i a b l e . Powell (1979) t h e o r i z e d that the e x t r a c e l l u l a r p o l y s a c c h a r i d e s e x i s t near the c e l l w a l l to allow c e l l s to i n t e r a c t with t h e i r environment. I t may be p o s s i b l e f o r the e x t r a c e l l u l a r p o l y s a c c h a r i d e s t o adopt a c o n f i g u r a t i o n that would allow complexation to occur . Many micro-organisms are capable of producing e x t r a c e l l u l a r p o l y s a c c h a r i d e s . I t i s not known i f they can form complexes but they do r e t a i n metals. A n a l y s i s of some e x t r a c e l l u l a r p o l y s a c c h a r i d e s has shown t h a t p r e c i p i t a t e s can form i n them. There i s a l s o a p o s s i b i l i t y that they can form i o n i c bonds with metal i o n s . U s u a l l y e x t r a c e l l u l a r p o l y s a c c h a r i d e s are 120 p o l y a n i o n i c a l l y and n e u t r a l l y charged. Most important i s the d i s c o v e r y of a s t a b l e end product a f t e r they were o x i d i z e d by le a c h a t e b a c t e r i a . T h i s may mean that metals bound by e x t r a c e l l u l a r p o l y s a c c h a r i d e s can be f i x e d . 121 APPENDIX D - - CHELATION PROCEDURE A. A c i d wash seven e x t r a c t i o n funnels (at l e a s t 250 ml. s i z e ) , one 100 ml. graduated c y l i n d e r and one f u n n e l . Then r i n s e them with d e i o n i z e d d i s t i l l e d water. B. Weigh the fu n n e l s , a d e i o n i z e d d i s t i l l e d water wash b o t t l e and the sample. C. P l a c e the f u n n e l s , a wash b o t t l e , p a r a f i l m , scotch tape, the graduated c y l i n d e r , and the blended sample i n t o the glove box. F l u s h the glove box with n i t r o g e n and r a i s e the pre s s u r e u n t i l i t i s 2 cm. of water above atmospheric. Note: the tops of the e x t r a c t i o n funnels should be removed to allow the a i r i n s i d e to be f l u s h e d out. D. Shake the sample c o n t a i n e r to mix the sample. Pour 50 ml. of sample i n t o the graduated c y l i n d e r . Then pour contents of c y l i n d e r i n t o an empty f u n n e l . Repeat s i x times so a l l the fu n n e l s are f u l l . E. Replace the e x t r a c t i o n funnel t o p s . Seal the sample c o n t a i n e r with p a r a f i l m and tape. F. Turn o f f the n i t r o g e n gas. Remove the sample c o n t a i n e r and e x t r a c t i o n f u n n e l s . G. Weigh the sample c o n t a i n e r and each e x t r a c t i o n f u n n e l . Put the sample c o n t a i n e r back i n the c o l d room i f sample removal i s f i n i s h e d . H. Using a vo l u m e t r i c p i p e t t e , add the c a l c u l a t e d volume of spike s o l u t i o n to each f u n n e l . Shake each funnel and leave them two hours to allow e q u i l i b r i u m to be approached. I. Add the c a l c u l a t e d volume of c h e l a t i n g agent to each sample. Shake each funnel and leave o v e r n i g h t . J . Add 50 ml. of MIBK to each e x t r a c t i o n f u n n e l . Shake w e l l . Note: a pressure b u i l d - u p w i l l occur so the pressure must be r e l i e v e d . I nvert the f l a s k and open the stopcock to do t h i s . Leave f u n n e l s o v e r n i g h t . K. Pour the contents of each e x t r a c t i o n funnel i n t o a 250 ml. a c i d washed l i g h t p o l y e t h y l e n e c o n t a i n e r . C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. f o r ten minutes. L. Pour the supernatant i n t o the corresponding f u n n e l . Allow the l i g h t p o l y e t h y l e n e b o t t l e s to d r a i n f o r 15 122 minutes. M. Shake each f l a s k then leave them to s i t o v e r n i g h t . N. D r a i n o f f the water (the bottom l a y e r ) . 0. D r a i n the MIBK l a y e r i n t o a Whatman number 541 f i l t e r and c o l l e c t the f i l t r a t e f o r a n a l y s i s with a J a r r e l Ash 810 atomic a b s o r p t i o n spectrometer. 1 23 APPENDIX E - - EXTRACTION PROCEDURE For Each Sample: A. Weigh a one l i t r e f l a s k . Add roughly 300 ml. of blended sample and weigh the f l a s k . Make the a d d i t i o n i n the glove box under n i t r o g e n atmosphere. Seal the f l a s k using p a r a f i l m and tape. B. Place the sample i n the glove box and d i l u t e with d e i o n i z e d d i s t i l l e d water to roughly 900 ml. Weigh the f l a s k . C. Under a n i t r o g e n atmosphere, remove a moisture content sample, b a c t e r i a l count sample, and a sample f o r an i n i t i a l metals d e t e r m i n a t i o n (BLKO, SON0, or FOX0). D. Do one of the f o l l o w i n g to each sample: a) Place sample i n a M e t t l e r E l e c t o n i c s 5.5 g a l . s o n i c a t i o n bath f o r h a l f an hour at 20 KHz and 4000 peak watts. b) Bubble a i r through sample f o r 30 hours. Before a e r a t i n g the sample, bubble the a i r through an a c i d t r a p to remove p a r t i c l e s , metals, and water. c) T h i s sample r e c e i v e s no treatment as i t i s the c o n t r o l . E. Take a b a c t e r i a l count sample. F. I n s i d e the glove box, pour sample i n t o four 250 ml. c e n t r i f u g e b o t t l e s . Then weigh the c e n t r i f u g e b o t t l e s . G. C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. Remove the supernatant f o r a n a l y s i s (BLK1, SON1, or FOX1). H. I n s i d e the glove box add 9.635 gms. of ammonium a c e t a t e to each c e n t r i f u g e b o t t l e . Then d i l u t e the samples with d e i o n i z e d d i s t i l l e d water u n t i l each c e n t r i f u g e b o t t l e c o n t a i n s roughly 250 ml. Shake w e l l and leave o v e r n i g h t . I. Weigh the c e n t r i f u g e b o t t l e s and balance the b o t t l e s i n p a i r s by adding d e i o n i z e d d i s t i l l e d water. J . C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. and remove supernatant f o r a n a l y s i s . (BLK2, SON2, FOX2). K. I n s i d e the glove box add 0.435 gms. of hydroxylamine h y d r o c h l o r i d e to each c e n t r i f u g e b o t t l e . Then d i l u t e with d e i o n i z e d d i s t i l l e d water u n t i l each c e n t r i f u g e 1 24 b o t t l e c o n t a i n s roughly 250 ml. Shake w e l l and leave o v e r n i g h t . L. Weigh the c e n t r i f u g e b o t t l e s and balance the b o t t l e s i n p a i r s by adding d e i o n i z e d d i s t i l l e d water. M. C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. and remove supernatant f o r a n a l y s i s . (BLK3, SON3, FOX3). N. Weigh out a moisture content sample and weigh the r e s i d u e . 0. Put the res i d u e i n a two l i t r e beaker and d i l u t e with d e i o n i z e d d i s t i l l e d water to roughly one l i t r e . P. Add hydrogen peroxide 10 ml. at a time u n t i l 150 ml. of hydrogen peroxide has been added to the sample. S t i r c o n t i n u o u s l y with a mechanical s t i r r e r . Only add more hydrogen peroxide when the foaming has subsided.**** Q. Pour some of the hydrogen peroxide d i g e s t e d sample i n t o two c e n t r i f u g e b o t t l e s and add 9.635 gms. of ammonium a c e t a t e to each b o t t l e . R. C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. and pour o f f the supernatant f o r a n a l y s i s . (BLK4, SON4, FOX4). S. D i l u t e the r e s i d u e with d e i o n i z e d d i s t i l l e d water. Add 5.0 gms. of sodium d i t h i o n i t e and 12.5 gms. of sodium c i t r a t e t o each of the two c e n t r i f u g e b o t t l e s . T. C e n t r i f u g e at a r e l a t i v e c e n t r i f u g a l f o r c e of 1600 g. and pour o f f the supernatant f o r a n a l y s i s . (BLK5, SON5, FOX5). U. Weigh the r e s i d u e and take a moisture content sample. V. A c i d - d i g e s t the residue then f i l t e r through a Whatman number 541 f i l t e r . (BLK6, SON6, FOX6). W. Rinse the f i l t e r cake with ammonium a c e t a t e . X. Add a c i d i f i e d d e i o n i z e d d i s t i l l e d water (0.5% n i t r i c a c i d ) to make sample up to 250 ml. NOTE: A l l the sub-samples f o r a n a l y s i s were a c i d -d i g e s t e d and f i l t e r e d through a Whatman number 541 f i l t e r . The f i l t e r cake was r i n s e d with ammonium a c e t a t e (Sub-samples 4, 5, and 6) or a c i d i f i e d d e i o n i z e d d i s t i l l e d water (Sub-samples 0, 1, 2, and 3). Then sub-samples 4, 5, and 6 were d i l u t e d up to 1 25 250 ml. and sub-samples 0, 1, 2, and 3 were d i l u t e d up to 100 ml. ( A c i d i f i e d d e i o n i z e d d i s t i l l e d water was used to d i l u t e the sub-samples) Sub-samples 0, 1, 2, and 3 were made up to 100 ml. and the f i l t e r cakes were r i n s e d with a c i d i f i e d d e i o n i z e d d i s t i l l e d water. **** An a l t e r n a t e method f o r o x i d i z i n g o r g a n i c s which avoids the use of hydrogen peroxide : Oven-dry the sub-samples at 500°C, then d i s s o l v e the residue i n d e i o n i z e d d i s t i l l e d water. Add ammonium a c e t a t e to e x t r a c t the metals r e l e a s e d by the o x i d a t i o n of the o r g a n i c s . Dry-ashing tends to vapourize the v o l a t i l e o r g a n i c s and some l e a d . I f v o l a t i l e o r g a n i c s and l e a d form a s i g n i f i c a n t p o r t i o n of a sub-sample, the use of hydrogen peroxide may be advantageous. Otherwise dry-ashing i s recommended as i t i s a s a f e r method f o r o x i d i z i n g o r g a n i c s . 126 APPENDIX F - - SAMPLE EXTRACTION I t i s very c l e a r that samples need to be kept at i n - s i t u c o n d i t i o n s i f one wishes to measure metal s p e c i a t i o n . The most d i f f i c u l t i n - s i t u c o n d i t i o n to maintain, i s an anaerobic environment. In order to r e t r i e v e s o l i d and l i q u i d samples under anaerobic c o n d i t i o n s , s p e c i a l designs were r e q u i r e d . An anaerobic environment i s r e q u i r e d before the pH can be measured i n - s i t u . 1. LIQUID SAMPLE To ensure that enough sample was c o l l e c t e d to be a b l e to measure cadmium, 400 ml. of l i q u i d ( l e a c h a t e ) was c o l l e c t e d under an anaerobic atmosphere, from the bottom of a l y s i m e t e r . An anaerobic atmosphere was c r e a t e d by a l l o w i n g the l e a c h a t e to d r a i n from the l y s i m e t e r i n t o a one l i t r e erlenmeyer f l a s k which had a n i t r o g e n atmosphere. The whole f l a s k was acid-washed to prevent contamination. The n i t r o g e n pressure was 2 cm. of water above atmosphere. A very small pressure was used to prevent a i r e n t e r i n g the f l a s k but a l s o to a v o i d f l u s h i n g the l y s i m e t e r with n i t r o g e n . (Nitrogen was s u p p l i e d by a 100 l b . c y l i n d e r ) . The p u r i t y of the n i t r o g e n atmosphere was checked by u s i n g a F i s h e r Hamilton gas p a r t i t i o n e r and a Hewlett Packard I n t e g r a t o r Model 3380 A. Oxygen was l e s s than 0.5% of the atmosphere a f t e r f i v e minutes of f l u s h i n g with n i t r o g e n . I t took over twenty-four hours to c o l l e c t 400 ml. of sample. So a flow r e s t r i c t i o n was used, to a v o i d d r a i n i n g the n i t r o g e n c y l i n d e r too q u i c k l y . F i g u r e 14 1 27 shows a s i d e view of the l i q u i d sample c o l l e c t i o n f l a s k , The F i g u r e 14 - L i q u i d Sample C o l l e c t i o n F l a s k N £ I n p u t L e a c h a t e I n p u t N2 O u t p u t R u b b e r S t o p p e r 1 L . E r l e n m y e r F l a s k N o t e : T h e N2 i n p u t t u b e i s l o n g e r t o m i n i m i z e s h o r t - c i r c u i t i n g T h e N2 i n p u t was m a i n t a i n e d a t a p r e s s u r e 2 mm. a b o v e l o c a l a t m o s p h e r i c . cork i n the top had three tubes, one f o r the l e a c h a t e to e n t e r , one f o r the n i t r o g e n gas to enter, and one f o r the gas to e x i t . To prevent s h o r t c i r c u i t i n g of the n i t r o g e n gas flow, the input tube was l o c a t e d near the bottom. A water t r a p was put on the n i t r o g e n gas o u t l e t to prevent oxygen d i f f u s i n g i n t o the system. A f t e r the sample was c o l l e c t e d i t was co n c e n t r a t e d to 100 ml. before p r e p a r a t i o n f o r a n a l y s i s with a J a r r e l Ash 810 atomic a b s o r p t i o n spectrometer. 128 2. SOLID SAMPLE COLLECTION C o l l e c t i o n of a s o l i d sample below the s u r f a c e of a sealed l y s i m e t e r presented two problems. One was c u t t i n g a hole under anaerobic c o n d i t i o n s and the other was removing the sample under anaerobic c o n d i t i o n s . A sampling box was d e v i s e d to meet these needs. F i g u r e 15 shows a s i d e view and an end view of the sampling box. The box was made of p l e x i g l a s s . I t had a removable end p l a t e which had a hole i n the c e n t r e . A hole was r e q u i r e d to allow a s t e e l rod or a wire i n t o the box. There were three gas p o r t s on the bottom of the box. One was to measure gas pressu r e , one was a n i t r o g e n gas i n l e t , and the t h i r d was a n i t r o g e n gas o u t l e t . The o u t l e t went to a water t r a p . At the f r o n t of the box there was a removable end pie c e with a curve to f i t the c u r v a t u r e of the l y s i m e t e r . A s t r i p of neoprene foam was glued to the edge of the curved p i e c e to he l p form an a i r t i g h t s e a l . Two tags, one on e i t h e r s i d e of the box, were used to a t t a c h a loop of rope to the box. The loop was passed around the l y s i m e t e r and t i g h t e n e d to h e l p form a t i g h t s e a l . A stand was made to support the weight of the box. There were two glo v e s , a t t a c h e d to a c o l l a r by hose clamps, on each s i d e of the box. They were f l e x i b l e g i v i n g an ope r a t o r , o u t s i d e the box, the a b i l i t y to manipulate items i n s i d e the box. 129 F i g u r e 15 - Sampling Box Add f o a m t o f o r m a s e a l "7 16.5 cm. T o f a s t e n b o x t o l y s i m e t e r A l l p o r t s 16mm QD. N £ p o r t s f o r : • I n p u t • O u t p u t • P r e s s u r e g a u g e S u p p o r t : c o l l a r C o l l a r f o r g l o v e I I '— 0 .6 mm P l e x i g l a s s -i—r H o l e f o r d r i l l s s h a f t H o l e f o r w i r e s Zcj c m . -?.9 c m . -P R O F I L E 45 cm. BOTTOM VIEW END VIEW 130 a. D r i l l i n g To remove a sample from the box a 8.9 cm. wood d r i l l b i t was used to cut the w a l l of the l y s i m e t e r (The l y s i m e t e r w a l l was made of PVC). A v a r i a b l e speed, 1.9 cm. chuck, e l e c t r i c d r i l l was used f o r a l l the d r i l l i n g . The power c o r d entered the box v i a the hole i n the back p l a t e . A s p l i t cork was used to form a s e a l with the w ire. Once a hole was cut i n the s i d e of the l y s i m e t e r , a p i e c e of p a r a f i l m was taped to the s i d e of the l y s i m e t e r to form an a i r t i g h t s e a l . ( I n i t i a l l y a wooden cork was used but i t d i d not form a good s e a l . ) A f t e r c u t t i n g the i n i t i a l h o l e , the i n - s i t u pH was measured. An attempt to measure the o x i d a t i o n - r e d u c t i o n p o t e n t i a l was made but the instrument d i d not f u n c t i o n properly.. The temperature was a l s o measured before r e s e a l i n g the l y s i m e t e r . Once the i n - s i t u measurements were made, the anaerobic box was set up f o r d r i l l i n g . To d r i l l out the sample, two b i t types were t e s t e d . The f i r s t b i t was a 7.6 cm. diameter pipe with a band saw blade welded to the l e a d i n g edge. I t was hoped that the b i t would remove a core sample. The mixture of m a t e r i a l s i n the l y s i m e t e r proved too much f o r a band saw blade. A f t e r c u t t i n g through one t i n can, the b i t was too d u l l f o r use. So, another b i t was d e v i s e d . F i g u r e 16 shows the two b i t s . The second b i t used two tungsten c a r b i d e t e e t h to cut and a h i g h speed t w i s t d r i l l to c e n t r e the b i t . (The t w i s t d r i l l broke when i t was a q u a r t e r of an i n c h longer than the tungsten 131 F i g u r e 1 6 - D r i l l B i t s Bandsaw blade Nut to f i t onto threaded d r i l l shaft 4 . 5 mm CORING BIT High speed twist d r i l l bit 1 > 1 /— Tungsten carbide teeth N — N u t to f i t onto d r i l l shaft MULCHING BIT c a r b i d e t e e t h ) . W i t h t h e s e c o n d b i t , a l o n g e r b i t l i f e a n d a h i g h e r d r i l l i n g r a t e w e r e a c h i e v e d . One o f t h e f e a t u r e s o f t h e s e c o n d b i t was t h a t i t m u l c h e d t h e s a m p l e . I n o r d e r t o a p p l y some p r e s s u r e on t h e d r i l l b i t , a r o d , t h r e e f e e t l o n g , was u s e d . A n e o p r e n e r i n g was i n s e r t e d i n a c o l l a r o v e r t h e h o l e i n t h e b a c k p l a t e o f t h e a n a e r o b i c b o x . T h e r i n g h e l p e d p r o v i d e a l o w f r i c t i o n b e a r i n g s u r f a c e a n d f o r m e d a s e a l a r o u n d t h e s t e e l r o d . I t was p o s s i b l e t o l e a v e a f o u r l i t r e s a m p l e c o n t a i n e r i n t h e s a m p l e b o x w h i l e d r i l l i n g b e c a u s e t h e d r i l l c o u l d be a t t a c h e d t o t h e e n d o f t h e r o d . A l s o , t h e d r i l l c o u l d be h e l d p r o p e r l y o u t s i d e t h e b o x . (A l a r g e t o r q u e c o u l d be g e n e r a t e d by t h e b i t j a m m i n g s o i t was 1 32 important to have a f i r m g r i p on the d r i l l ) . To f i l l the sample c o n t a i n e r , a bent p i e c e of one inch pipe was used. The bend gave some p r y i n g c a p a b i l i t y . There was not much room to manipulate the pipe due to the l a r g e c o l l a r (3.8 cm. c o l l a r ) used to a t t a c h the gloves to the sid e of the sample box. Once the sample was scooped i n t o the sample box, the top was s e a l e d using p a r a f i l m and f r e e z e r tape. The p a r a f i l m was s t r e t c h e d to h e l p form a t i g h t s e a l . I n i t i a l l y , the c o n t a i n e r top was screwed i n p l a c e but i t tended to break the s e a l formed by the p a r a f i l m . Once the sample was r e t r i e v e d , the l y s i m e t e r and the sample c o n t a i n e r were s e a l e d . A flow c h a r t of the sample removal i s shown i n F i g u r e 17. b. Sample M a n i p u l a t i o n A glove box was used to manipulate samples a f t e r removal. The glove box had been purchased p r e v i o u s l y f o r another experiment. I t was made of heavy p l a s t i c and supported by s t e e l rods. A double lock p l a s t i c z i p p e r was used to maintain an a i r -t i g h t s e a l . The top of the box was t r a n s l u c e n t to allow m a n i p u l a t i o n s . There was one gas i n l e t and one gas o u t l e t p o r t . O r i g i n a l l y , the box had a s i d e chamber but the z i p p e r f o r t h i s s e c t i o n was no longer a i r - t i g h t . A tube from the gas o u t l e t went to a water t r a p and a U-tube f o r measuring gas p r e s s u r e . When the sample was removed from the l y s i m e t e r , i t was p a r t i a l l y broken-up by the d r i l l b i t . For the experimental work, a Waring blender was used to break down the sample even f u r t h e r . A s p e c i a l top was made out of l u c i t e to form an a i r -t i g h t s e a l at the top of the blender jug. F i g u r e 18 shows a F i g u r e 17 - Sample Removal Flow Chart A t t a c h sample box P r e s s u r i z e with N 2 D r i l l h o l e s e a l and p l a c e pH probe and thermometer i n box I A t t a c h and p r e s s u r i z e box Measure pH and temperature Se a l l y s i m e t e r and d e p r e s s u r i z e box At t a c h d r i l l b i t t o the end of the rod Put a sample c o n t a i n e r and removal t o o l i n box At t a c h and p r e s s u r i z e box D r i l l i n t o l y s i m e t e r c o n t e n t s Dig, pry and scrape l y s i m e t e r contents i n t o sample c o n t a i n e r Seal l y s i m e t e r and s e a l sample c o n t a i n e r I D e p r e s s u r i z e and d i s c o n n e c t the box Put the sample ^container i n t o a dark, cold ( 4°C) room F i g u r e 18 - Blender L i d S l o t s f o r e l a s t i c s U s e f o a m s t r i p s t o f o r m a s e a l A N 2 I n p u t — ' r N2 O u t p u t -s' ^ BOTTOM VIEW P R O F I L E 134 s i d e view and bottom view of the l i d . There were three gas p o r t s on the l i d . One gas port was an i n t a k e , another an o u t l e t , and the other port was f o r measuring the p r e s s u r e . A neoprene foam was used to form a s e a l between the j a r top and the l u c i t e l i d . The l i d was h e l d i n p l a c e by four p i e c e s of rubber, one from each corner. I n i t i a l l y , the blending was done o u t s i d e the glove box. That method only allowed b l e n d i n g of 600 ml. of sample and d i l u t i o n water. Blending o u t s i d e the glove box r e q u i r e d more time and n i t r o g e n gas although i t d i d allow more working room. There was a problem with the sample b l o c k i n g the gas o u t l e t , so most of the b l e n d i n g was done i n s i d e the glove box. To a v o i d shearing the b a c t e r i a , a two minute maximum ble n d i n g time was used. A f t e r b l e n d i n g , the sample was p l a c e d i n four l i t r e nalagene sample c o n t a i n e r s . The c o n t a i n e r s were a c i d washed and s e a l e d with p a r a f i l m and f r e e z e r tape. Large p i e c e s of wood, p o r c e l a i n , and cans were removed from the sample to a v o i d damaging the b l e n d e r . A l i s t of the step by step sample e x t r a c t i o n procedure i s i n c l u d e d below. A. Place the d r i l l , d r i l l b i t , p a r a f i l m , and s c o t c h tape i n the sampling box. B. Move the sample box i n t o p o s i t i o n and c i n c h the curved end a g a i n s t the s i d e of the l y s i m e t e r using a p i e c e of rope. C. F l u s h the sample box with n i t r o g e n gas and p r e s s u r i z e to roughly 2 cm. of water above atmospheric p r e s s u r e . 135 D. D r i l l a 8.9 cm. hole i n t o the s i d e of the l y s i m e t e r . E. S e a l the l y s i m e t e r and turn o f f the n i t r o g e n gas. F. Remove the d r i l l , d r i l l b i t , and p i e c e of l y s i m e t e r from the sample box. Replace them wit h : a p i e c e of p a r a f i l m , s c o t c h tape, a thermometer, a pH probe, b u f f e r s o l u t i o n s , a wash b o t t l e , and a o x i d a t i o n -r e d u c t i o n p o t e n t i a l probe. G. Repeat steps 'B' and ' C . H. Remove the l y s i m e t e r s e a l to measure the temperature, pH, and o x i d a t i o n - r e d u c t i o n p o t e n t i a l . Place a new s e a l on the l y s i m e t e r . I. Remove the contents of the sample box. Place i n the box: p a r a f i l m , s c o t c h tape, a d i g g i n g t o o l , a sample c o n t a i n e r , and a d r i l l b i t . Note: i t i s e a s i e r to a t t a c h the b i t t o the d r i l l i n g s h a f t p r i o r to p o s i t i o n i n g the sampling box. J . Repeat steps 'B' and ' C . K. Remove roughly 1.2 l i t r e of sample and pl a c e i n the sample c o n t a i n e r . Seal the l y s i m e t e r and sample c o n t a i n e r u s i n g p a r a f i l m . Use f r e e z e r tape to hold p a r a f i l m to the sample c o n t a i n e r . L. Turn o f f the n i t r o g e n gas and remove the sample c o n t a i n e r . M. Put the sample c o n t a i n e r i n a dark c o l d room (4°C.) u n t i l use. 1 36 APPENDIX G - - RECOMMENDED DAPI PROCEDURE 1. Add roughly 5 ml. of s o l i d s to an a c i d washed f l a s k . 2. F i l t e r a l l s o l u t i o n s through a 25 mm. diameter, 0.02 um. Nucleopore f i l t e r . 3. Add roughly 5 ml. of 70% et h a n o l s o l u t i o n . Then d i l u t e up to 50 ml. with d e i o n i z e d d i s t i l l e d water. 4. Store at 4°C u n t i l time of s l i d e p r e p a r a t i o n . 5. Remove 1 ml. of mixture and d i l u t e up to 10 ml. Then shake v i g o r o u s l y . 6. Remove 2 ml. of mixture and add 4 ml. of 0.03M sodium pyrrophosphate ( f i l t e r e d ) . 7. Allow mixture to incubate at room temperature f o r 30 minutes shaking v i g o r o u s l y every 5 minutes. 8. Son i c a t e with an u l t r a s o n i c probe (120 W) f o r 60 sec. 9. D i l u t e up to 6 ml. with d e i o n i z e d d i s t i l l e d water and shake v i g o r o u s l y . Add 0.2 ml of 0.1 M. DAPI s o l u t i o n . Leave 15 minutes shaking every 5 minutes. 10. F i l t e r mix through a 0.22 um. nucleopore f i l t e r , which has been soaked i n i r g a l a n black f o r a minimum of 8 h r s . and r i n s e d i n d e i o n i z e d d i s t i l l e d water, u s i n g 8 mm. of Hg vacuum u n t i l the f i l t e r i s dry. 11. P l a c e f i l t e r on a s l i d e and add 1 drop of Cargyle B immersion o i l . 12. Allow o i l to spread before p l a c i n g cover s l i p on top. 13. P l a c e a drop of C a r g i l l e B immersion o i l on the cover s l i p and observe the s l i d e under a 340-370 nm. e x c i t a t i o n beam at 100X power. 1 37 14. Count 4-6 f i e l d s of view i n 10 or more p r e p a r a t i o n s . NOTE: I t i s very important to f i l t e r a l l s o l u t i o n s and s t e r i l i z e the sample c o n t a i n e r s . R e s i d u a l n i t r i c a c i d , from a c i d washing c o n t a i n e r s , should be washed o f f by r i n s i n g with d e i o n i z e d d i s t i l l e d water. 138 APPENDIX H - - AVAILABLE METHODS Many experiments have been d e v i s e d which separated metals i n t o groups a c c o r d i n g to t h e i r p h y s i c a l p r o p e r t i e s . So f a r no one has managed to determine which s p e c i e s are i n those groupings. C a t i o n exchange g e l s , r e s i n s , d i a l y s i s , u l t r a f i l t r a t i o n , e l e c t r o p h o r e s i s and c e n t r i f u g i n g are some of the methods used to separate metals from sludges, s o i l and l e a c h a t e s . F l o r e n c e and B a t l e y (1977) looked at f i v e general methods f o r a n a l y s i s of metals i n f r e s h water. They s t a t e d that s e p a r a t i o n techniques c o u l d only segregate metals a c c o r d i n g to t h e i r chemical or p h y s i c a l c h a r a c t e r i s t i c s . Examples of s e p a r a t i o n methods are d i a l y s i s , c e n t r i f u g i n g , e l e c t r o p h o r e s i s , ion exchange and u l t r a f i l t r a t i o n . A l l of these techniques probably a l t e r the balance of the metal s p e c i e s i n a sample. S e p a r a t i o n methods t y p i c a l l y have contamination problems e s p e c i a l l y from rubber o - r i n g s which can add Zn and Cd to samples. A number of p o t e n t i a l methods were c o n s i d e r e d . S p e c i f i c e l e c t r o d e s and a n o i d i c s t r i p v oltametry have c h a r a c t e r i s t i c a l l y poor d e t e c t i o n l i m i t s (except i n the case of copper where 1 ppm. can be detected) and long warm-up times. The technique of p o l a r o g r a p h i c h a l f wave p o t e n t i a l s h i f t s i s s t i l l i n the experimental stages but has been used s u c c e s s f u l l y f o r s t a b i l i t y constant d e t e r m i n a t i o n s i n s y n t h e t i c s o l u t i o n s . F l o r e n c e and B a t l e y r e p o r t e d that c h e l a t i o n e x t r a c t i o n methods tend to underestimate the percent metal a s s o c i a t e d with the o r g a n i c phase. So they developed a procedure which separated metals a c c o r d i n g to p h y s i c a l p r o p e r t i e s ( B a t l e y and Florence 139 1976 and F l o r e n c e 1976). B a t l e y and F l o r e n c e used a n o i d i c s t r i p voltametry t i t r a t i o n s f o r metal a n a l y s i s of each category they had separated. They warned that i n o r g a n i c c o l l o i d s ( i e . hydrated i r o n or manganese oxides, c l a y s i l i c a and sulphates) and organic c o l l o i d s ( i e . humics, animal d e b r i s and p l a n t d e b r i s ) i n n a t u r a l waters have a s o r b i n g c a p a c i t y which may g i v e r i s e to a low estimate of the metal c o n c e n t r a t i o n . T h e i r r e s u l t s f o r Cd, Cu, Pb and Zn s p e c i a t i o n i n n a t u r a l water and sea water were u n a f f e c t e d by storage at 4°C. They were a b l e to separate metal s p e c i e s i n t o groups but were unable to i d e n t i f y the mass of a given s p e c i e s w i t h i n the p h y s i c a l l y separated groups. Bergman et a l . (1979) used zonal c e n t i f u g a t i o n to separate d e n s i t y f r a c t i o n s . The technique does not separate metal s p e c i e s but the data obtained i n d i c a t e d at l e a s t two methods of metals uptake i n sewage sludges. They found metals i n the low and high d e n s i t y ranges. Most of the metals were found i n a narrow d e n s i t y range which was probably a s s o c i a t e d with o r g a n i c m a t e r i a l s . S r i v a s t a v a et a l . (1980) used a g e l (chromium f e r r o c y a n i d e ) f o r s e p a r a t i n g metals from s o l u t i o n s with a h i g h a c i d i t y and s a l t c o n t e n t . They r e p o r t e d that the g e l d i d not recover a l l metals but s p e c i f i c g e l s may one day be a good a n a l y s i s t o o l . Chen et a l . (1974) used f i l t r a t i o n and c e n t r i f u g a t i o n to separate the p a r t i c u l a t e f r a c t i o n from sewage e f f l u e n t . A 0.22 um. f i l t e r removed 99% of the metals and a 0.8 um. f i l t e r removed 97% of the metals. The optimum metal and sludge 1 40 s e p a r a t i o n was achieved by c e n t r i f u g i n g at 225 g's. ( r e l a t i v e c e n t r i f u g a l f o r c e ) f o r ten minutes. In order to compare samples Chen et a l . c a l c u l a t e d the mass of metals per dry weight mass of p a r t i c u l a t e . Chian and DeWalle (1977) used membrane u l t r a f i l t r a t i o n , g e l permeation, gas chromatography, and s p e c i f i c organic a n a l y s i s to separate o r g a n i c c a t e g o r i e s in l e a c h a t e . By c o l l e c t i n g samples under anaerobic c o n d i t i o n s , they prevented c o l l o i d a l i r o n hydroxide f o r m a t i o n . They used the membranes to separate weight f r a c t i o n s and then analyzed the f r a c t i o n s . The l a r g e s t organic f r a c t i o n c o n t a i n e d f r e e v o l a t i l e f a t t y a c i d s and the next l a r g e s t organic f r a c t i o n c o ntained ' f u l v i c - l i k e ' o r g a n i c s which were high i n c a r b o x y l and aromatic hydroxyl groups. The s m a l l e s t o r g a n i c f r a c t i o n c o n t a i n e d high molecular weight humic carbohydrate complexes with h y d r o x l y z a b l e amino a c i d s . T h e i r r e s u l t s were s i m i l a r f o r d i f f e r e n t l e a c h a t e s . Hoffman et a l • (1981) used an u l t r a f i l t r a t i o n cascade and a mass balance to c h a r a c t e r i z e c o l l o i d a l metal complexes i n r i v e r water. They found that Cd, Fe, and Mn ions were a s s o c i a t e d with oxides, hydroxides or s u r f a c e adsorbing p a r t i c l e s . T h e i r data i n d i c a t e d that Cu, Cd, and Pb would probably be complexed with o r g a n i c s . Engler et a l . (1977) developed an e x t r a c t i o n procedure f o r c h a r a c t e r i z i n g metals i n sediment. The procedure used ammonium a c e t a t e , hydroxylamine h y d r o c h l o r i d e , hydrogen pero x i d e , sodium c i t r a t e t o form an e x t r a c t i o n g r a d i e n t . Engler used a combination of e x t r a c t i o n methods which had been developed f o r e x t r a c t i n g metals from s o i l . Chao (1972) reported 141 that hydroxylamine h y d r o c h l o r i d e s e l e c t i v e l y d i s s o l v e d manganese oxides, l e a v i n g i r o n oxides i n the r e s i d u e . Holmgren (1967) determined the optimum c o n c e n t r a t i o n s of sodium d i t h i o n a t e and sodium c i t r a t e f o r d i s s o l v i n g e x t r a c t a b l e i r o n . Jackson (1958) o u t l i n e d an experimental procedure f o r using ammonium acetate to determine exchangeable m e t a l l i c c a t i o n s p e c i e s . He a l s o o u t l i n e d a technique f o r o x i d i z i n g organic matter with hydrogen per o x i d e . The low temperature o x i d a t i o n ( l e s s than 100°C) prevents the r e l e a s e of hydroxyl and s t r o n g l y sorbed water a s s o c i a t e d with mineral c o l l o i d s . 142 APPENDIX I - - DATA AND CALCULATIONS FOR ESTIMATE OF LIQUID SAMPLE VOLUME Samples were prepared f o r atomic a b s o r p t i o n spectrometer a n a l y s i s by a c i d d i g e s t i n g a bulk sample and eva p o r a t i n g the sample to dryness. Then the r e s i d u e was d i s s o l v e d i n a c i d and d i l u t e d . A f t e r f i l t e r i n g the sample through a Whatman number 541 f i l t e r the sample was d i l u t e d up to a volume of 50 ml. A 50 ml. sample was r e q u i r e d f o r atomic a b s o r p t i o n spectrometer a n a l y s i s of seven metals. The l i s t below shows the metal c o n c e n t r a t i o n s i n the most r e c e n t l y t e s t e d l y s i m e t e r leachate sample. The d e t e c t i o n l i m i t s f o r each metal are a l s o shown. The c o n c e n t r a t i o n v a l u e s were obtained from the UBC c o - d i s p o s a l study (Atwater et a l . 1981) Table XXV - C o n c e n t r a t i o n Of Metals In The Las t Leachate Sample Leachate D e t e c t i o n L i m i t (mg/1) (mg/1) Cd 0.03 0.05 Cr 0.25 0.05 Fe 83 0.05 Pb 0.09 0.5 Ni 0. 14 0.05 Zn 9.5 0.05 The s m a l l e s t metal c o n c e n t r a t i o n was Cd so i t was used as the l i m i t i n g metal f o r the c a l c u l a t i o n s . To ensure that Cd c o u l d be det e c t e d a sample c o n t a i n i n g f i v e times the cadmium d e t e c t i o n l i m i t should be c o l l e c t e d . Sample volume*[detection l i m i t ] = minimum mass 143 0.05 x 0.05 = 0.0025 mg Minimum mass/[concentration 0.0025 / 0.03 = 0.083 L 0.083 x 5 = 0.412 L So a volume of 400 ml. was volume. i n l e a c h a t e ] = minimum volume c o l l e c t e d f o r the l i q u i d sample 1 44 APPENDIX J - - DATA AND CALCULATIONS FOR ESTIMATE OF SOLID SAMPLE VOLUME The f o l l o w i n g c a l c u l a t i o n s were based on c o n c e n t r a t i o n data from the Atwater et a l . study (1981). A l l the c a l c u l a t i o n s were f o r Cd as i t was a v a i l a b l e i n the s m a l l e s t q u a n t i t i e s . A sample volume of 0.05 L. was assumed f o r atomic a b s o r p t i o n spectrometer samples. Minimum moles of Cd f o r d e t e c t i o n = 0.05x0.05 = 0.0025 mg. Mass d e s i r e d = 5 x 0.0025 = 0.0125 mg. Net mass i n Tank F = 0.57 - 0.033 = 0.537 gms. Volume of Tank F = 70 x ( 3 0 ) 2 x 0.25 x i = 50 L. Note: the a c t u a l height i s 95 cm. but there are 15 cm. of t o p s o i l and at l e a s t 5 cm. of dead space (top and bottom) which do not c o n t a i n metals. So 25 cm. were s u b t r a c t e d from the a c t u a l h e i g h t . C o n c e n t r a t i o n of Cd i n Tank F = 0.537/50 = 10 mg/1. To allow f o r 28 e x t r a c t i o n samples and 35 c h e l a t i o n samples (63 t o t a l ) : 65 x 0.0125 = 0.8125 mg. 0.81 / 10 = 0.08 L of s o l i d sample are r e q u i r e d . T h i s c a l c u l a t e s the compressed volume of s o l i d s that would be r e q u i r e d . The mulching of the sample d u r i n g d r i l l i n g i n c r e a s e s the sample volume so the sample s i z e must be based on the s i z e of the c a v i t y c r e a t e d by sampling. 1 45 APPENDIX K - - DANGEROUS PROPERTIES OF CHEMICALS Ammonium Acetate — No hazards l i s t e d . E t h a n i c A c i d ( A c e t i c a c i d ) — I r r i t a n t , c a u s t i c , flammable i f exposed to flame. Dangerous with HN0 3. Can burn, penetrates s k i n e a s i l y c a using d e r m i t i t i s and u l c e r s . I r r i t a t e s mucous membranes of the nose. Ethanol -- Ra p i d l y o x i d i z e s , i r r i t a t e s eyes and nose i f exposed to 5000-10000 ppm. May cause drowsiness i f exposure i s prolonged over an hour. G l y c i n e — No hazards l i s t e d . Hydroxylamine h y d r o c h l o r i d e -- Toxic c h l o r i d e fumes on contact with a c i d , dangerous when heated. Hydrogen peroxide — T i s s u e i r r i t a n t , can burn at 35% c o n c e n t r a t i o n . Eyes are very s e n s i t i v e to c o n t a c t . If h i g h l y c o n c e n t r a t e d i t i s e x p l o s i v e . L - H i s t i d i n e -- No hazards l i s t e d . 8-hydroxyquinoline — A c r i d fumes when heated, s l i g h t f i r e hazard. A l s o c a l l e d 8 - q u i n o l i n o l . M e t h y l - i s o - b u t y l ketone -- Dangerous when exposed to heat or flame. Skin i r r i t a n t and i n h a l a t i o n i r r i t a n t . N i t r i l o - t r i - a c e t i c a c i d -- No hazard l i s t e d . N i t r i c A c i d — Moderate f i r e hazard, fumes are h i g h l y i r r i t a n t to eyes and nose. Pyrrophosphate — No hazard l i s t e d . O x a l i c A c i d -- C o r r o s i v e , vapour i r r i t a t e s eyes and nose. . Prolonged exposure t o vapour has severe s i d e e f f e c t s . Sodium c i t r a t e — No hazard l i s t e d . Sodium d i t h i o n i t e — No hazard l i s t e d . NOTE: Hazard l i s t i n g s are from Dangerous P r o p e r t i e s of I n d u s t r i a l M a t e r i a l s . — N. Sax 1958. 146 APPENDIX L - - FLUORESCENT DYES S c i e n t i s t s around the world have been u s i n g f l u o r e s c e n t dyes as a i d s f o r d e t e c t i n g m i c r o s c o p i c bodies f o r the past three decades . A c r i d i n e orange (AO) has been one of the more popular dyes and was used u n t i l r e c e n t l y . S t a i n i n g techniques have now been developed, using 4', 6 d i a m i d i n o - 2 - p h e n y l i n d o l e (DAPI), and mithramycin, which provided g r e a t e r enhancement and s p e c i f i t y than AO. When AO techniques were f i r s t being developed i t was o r g i n a l l y b e l i e v e d 1 that l i v i n g c e l l s f l u o r e s c e d green while dead c e l l s f l u o r e s c e d red. B u c h e r e r 2 l a t e r showed that the c o l o u r d i s t i n c t i o n was caused by an excess of AO glowing red. The plasma of l i v i n g gram p o s i t i v e c e l l s tended to have a higher s o r p a t i v e c a p a c i t y than gram negative c e l l s so they glowed red even when a l i v e . T h i s d i d not stop s c i e n t i s t s from u s i n g AO. F r a n c i s c o et a l • (1973) showed that the counts obtained u s i n g AO compared f a v o u r a b l y with a P e t r o f f - H a u s e r Counting chamber (PHC chamber). The PHC chamber has a d e t e c t i o n l i m i t of 10 7 b a c t e r i a / m l . So i t cannot be used f o r b a c t e r i a l counts i n n a t u r a l waters. F r a n c i s c o recommended that AO be used f o r t o t a l whole c e l l counts i n f r e s h water. To ensure c o n s i s t e n c y , F r a n c i s c o p r e f i l t e r e d a l l s o l u t i o n s and used AO from one manufacturer. (AO v a r i e d between manufacturers) 1. The o r i g i n a l a r t i c l e was by S. Strugger i n F l u r e s z e n z m i k r o s k o p i e and M i k r o b i o l o g i e (1949). I t was r e f e r e n c e d i n the a r t i c l e by T r o l l d e n i e r . 2. The a r t i c l e by Bucherer (1966) i s w r i t t e n i n German and was r e f e r e n c e d by T r o l l d e n i e r . 147 T r o l l d e n i e r (1973) attempted to extend the use of AO to b a c t e r i a l counts i n s o i l s . By adding sodium metaphosphate he was a b l e to d i s p e r s e the s o i l p a r t i c l e s which gave higher b a c t e r i a l counts and a random b a c t e r i a l d i s t r i b u t i o n . T r o l l d e n i e r found that 4 to 6 f i e l d s of view/ p r e p a r a t i o n and 10 p r e p a r a t i o n s r e s u l t e d i n a 5% p r o b a b i l i t y t h a t the r e a l value was more than 10% d i f f e r e n t from the value o b t a i n e d . T r o l l d e n i e r obtained a h i g h c o r r e l a t i o n between p l a t e counts and f l u o r e s c e n t counts. He used AO to enumerate b a c t e r i a i n s o i l . As long as the s o i l p a r t i c l e s are w e l l d i s p e r s e d , a c c u r a t e counts c o u l d be made, otherwise AO i s n o n - s p e c i f i c f o r i n - s i t u s o i l micro-organisms. AO i s not u s e f u l f o r a complex mixture u n l e s s i t i s s p e c i f i c f o r the b a c t e r i a i n the m a t e r i a l under study or the m a t e r i a l and b a c t e r i a can be d i s p e r s e d . The c o n c l u s i o n s of T r o l l d e n i e r and others caused r e s e a r c h e r s to look f o r a l t e r n a t e dyes and b e t t e r techniques. Jones (1974) compared AO to f l u o r e s c e i n i s o t h i o c y n a t e (FITC) and euc h r y s i n e 2GNX (E-2GNX). Jones concluded t h a t E-2GNX was c o n s i s t e n t l y b e t t e r than AO but that the c o n c l u s i o n was dependent upon the experimental method used. Daley (1975) came to a s i m i l a r c o n c l u s i o n ; the r e s u l t s were s e n s i t i v e to sample type, c o n t a c t time, c o n c e n t r a t i o n of fluorochrome, operator, lamp type, f i l t e r s i z e , f i x a t i o n , and procedure. AO and E-2GNX both i n c r e a s e the r e s o l u t i o n but the r e s u l t s o b tained using the experimental technique of F r a n c i s c o et a l . (1973) or Zimmerman ai'id M e y e r - R e i l 3 , were not reproduceable f o r c e l l counts i n 148 n a t u r a l waters. Daley recommended using a sample which giv e s 15 to 30 b a c t e r i a per g r a t i c u l e f o r co u n t i n g , the use of 2-5% formaldehyde s o l u t i o n f o r f i x a t i o n and a black f i l t e r background. Some re s e a r c h e r s compared AO to other counting methods. Watson (1977) compared AO with a t r a n s m i s s i o n e l e c t r o n microscope (TEM), a L i p o p o l y s a c c h a r i d e method, and an ATP de t e r m i n a t i o n . AO and TEM are d i r e c t count methods and are able to d i s c r i m i n a t e between p r o c a r y o t e and eucarote c e l l s . The other techniques only estimate c e l l mass. So AO or other f l u o r e s c e n t dye techniques are the q u i c k e s t and cheapest methods fo r o b t a i n i n g b a c t e r i a l counts i n n a t u r a l waters. Hobbie et a l . (1977) o u t l i n e d g e n e r a l p r i n c i p l e s f o r d i r e c t count techniques: a) A l l b a c t e r i a must be r e t a i n e d by the f i l t e r . b) A l l b a c t e r i a must be v i s i b l e at the f i l t e r s u r f a c e . c) S t a i n i n g and o p t i c s must produce a high c o n t r a s t between b a c t e r i a and background. Hobbie was abl e to improve the background by s t a i n i n g 0.22 urn. nucleopore f i l t e r s with i r g a l a n black and by using a C a r g i l l e type B immersion o i l . His technique agreed w e l l with the l i m a l u s l y s a t e , scanning e l e c t r o n microscope, and TEM methods. In f u r t h e r work by Daley (1979), he found AO to be b e t t e r than LPS, FITC, and E-2GNX f o r e s t i m a t i n g t o t a l numbers of aq u a t i c b a c t e r i a i n n a t u r a l waters. He found that aggregation of c e l l s 3. T h i s a r t i c l e was r e f e r r e d to by Daley (1979) and i s found i n the j o u r n a l K i e t e r Meeresforschungen Vol.30, pp.24-27, 1974. 1 49 c o u l d not be avoided but that a minimum of 6 ml. of sample should be f i l t e r e d g i v i n g 5-25 c e l l s per f i e l d f o r best r e s u l t s . A c o n t r o l should be run because a f i l t e r e d blank c o u l d s t i l l have 1-2% of t y p i c a l sample counts. Costerton and Geesey (1979) found that molecular oxygen decomposes aldehydes to form a c i d s which may decompose c e l l s . So they recommended a 5% a l k a l i n e b u f f e r e d argon and purged g l u t a l d e h y d e f o r sample f i x a t i o n . While Hobbie, Daley, and others t r i e d to p e r f e c t AO methods, other r e s e a r c h e r s were developing a technique f o r 4', 6 d i a m i d i n o - 2 - p h e n y l i n d o l e (DAPI). In 1975, i t was shown th a t the DAPI-DNA complex i s f l u o r e s c e n t * . K a p u s c i n s k i and S k o c z y l a s (1977) developed a f l u o r o m e t r i c microassay technique which was s e n s i t i v e to 5*10" 1 0 gm DNA/ml. T h i s was much b e t t e r than the 5*10" 7 gm DNA/ml. p r e v i o u s l y achieved with mithramycin. T h e i r technique was not a f f e c t e d by RNA, n u c l e o t i d e s , h i s t o n e s , urea or pH (5.0 < pH < 10.0). The f l u o r e s c e n t i n t e n s i t y was found to decrease with i n c r e a s e d i o n i c s t r e n g t h . Leeman and Ruch (1978) and K a p u s c i n s k i and Skoczylas (1980) experimented to determine how DAPI was bound to DNA. Both mithramycin and DAPI r e q u i r e d at l e a s t two b i n d i n g s i t e s . Each dye had a s p e c i f i c dye-DNA s i t e and one g e n e r a l s i t e that would accept e i t h e r dye. The DNA-DAPI complex was found to be very s t a b l e and formed a r i g i d bond. Leeman and Ruch found that DAPI-DNA banding appeared sharper a f t e r r e f r i g e r a t i o n f o r a few 4. T h i s f i n d i n g i s c r e d i t e d to W.C. Russel et a l . (Nature(London) Vol.253, pp.461-462, 1975.) and Williamson and Fennel (Methods i n C e l l B i o l o g y . D.M. P r e s c o t t ed. Vol.12, pp.335-351. Academic P r e s s , New York.) i n an a r t i c l e by K a p u s c i n s k i and S k o c z y l a s (1977). 1 50 days. K a p u s c i n s k i and Skoczylas found that f l u o r e s c e n t i n t e n s i t y i n c r e a s e d as v i s c o s i t y , s o l i d s , SDS c o n c e n t r a t i o n ( a n i o n i c detergent) i n c r e a s e d . Low c o n c e n t r a t i o n s of NaCl and s u p e r f i c i a l t e n s i o n s had no e f f e c t on f l u o r e s c e n c e . In a 1979 paper, S a l a r i and Ward (1979) compared DAPI with hoechst 33258 and a giesma s t a i n . They found that DAPI and hoechst 33258 had g r e a t e r s e n s i t i v i t y and was e a s i e r to use. They used a phosphate b u f f e r e d s a l i n e s o l u t i o n t o wash out b a c t e r i a , yeast, white c e l l s , e p i t h e l i a , and other DNA c o n t a i n i n g d e b r i s from the f i x e d c e l l s they looked a t . Cowell and Franks (1980) found that pretreatment with RNase (1 mg. /ml. f o r 20 minutes at 37°C) removed the general c y t o p l a s m i c s t a i n i n g without a f f e c t i n g the n u c l e a r s t a i n i n g i n t e n s i t y . They found that methanol i n a c e t i c a c i d (3:1) as a f i x a t i v e gave 20% higher f l u o r e s c e n c e than formaldehyde and methanol i n a c e t i c a c i d (10:85:5). Naimski et a l . (1980) found t h a t DAPI bound p r o p o r t i o n a l l y to DNA i n the 0.032-0.320 ug. DNA range. There was some dependence upon the q u a n t i t y of deoxyadenylate-thymidylate (AT n u c l e o t i d e s ) r e g i o n s . Porter and F e i g (1980) used DAPI f o r counting and i d e n t i f y i n g shapes of n a t u r a l a q u a t i c m i c r o f l o r a . They found DAPI f a r s u p e r i o r to AO because of in c r e a s e d v i s u a l i z a t i o n , i n c r e a s e d storage times (up to 24 weeks), and i n c r e a s e d s t a b i l i t y of the DAPI-DNA complex under the microscope. A l l a n and M i l l e r (1980) compared mithramycin with DAPI f o r s t a i n i n g yeast n u c l e i and found that r ; t h r a m y c i n s t a i n e d more c o n s i s t e n t l y . Coleman (1980) compared D A P I w i t h mithramycin f o r s t a i n i n g i n n a t u r a l environments. She 151 found that both dyes c o u l d be used i n the pH 4 to 8 range and 5-30 min. of exposure to the dye ensured p e n e t r a t i o n . Although DAPI f l u o r e s c e s b r i g h t e r than mithramycin, i t binds with some m u c o c y s t - t r i c h o c y s t m a t e r i a l i n f l a g e l l a t e s , polyphosphate g r a n u l e s , and very s l i g h t l y to b a c t e r i a l slime t r a i l s . Both dyes gave uniform and r e p e a t a b l e r e s u l t s . She found that a pH=4.4 M c l l v a i n e b u f f e r s o l u t i o n reduced the background f l u o r e s c e n c e and w a l l b i n d i n g . A l s o , a 3:1 or 70% ethanol f i x a t i o n procedure was more c o n s i s t e n t than using formaldehyde. Coleman et a l . (1981) p i c k e d DAPI i n p r e f e r e n c e to ethidium bromide, AO, hoechst 33258, feugen r e a c t i o n , and chromomycin A-3 because of i t ' s g r e a t e r s e n s i t i v i t y and slow f a d i n g . C o n s i d e r i n g the p u b l i s h e d m a t e r i a l s , some experimentation and p e r s o n a l commmunications with Iqubal V e l g i (SFU), the procedure o u t l i n e d i n Appendix G was compiled. 152 APPENDIX M - - CONCENTRATION VS ABSORBANCY PLOTS FOR CHELATION DATA SET ONE Chelate Test — Cone, -vs Abs. — Cd o—o CdSTD r • CADATA Concentration — ma/1 Chelate Test Cone, v s Abs. — C r 153 Chelate Test Cone, vs Abs. — Cr2 Chelate Test Cone, -vs Abs. Cu Chelate Test — Cone, vs Abs. — Cu2 Concentration —— mg/1 154 - i — i £50.0 Concentration — mj/l Chelate Test — Cone, -vs Abs. — Ni Concentration — mg/l Chelate Test — Cone, vs Abs. Pb Chelate Test — Cone, vs Abs. — Pb2 T — r — i — i — i — i — r — i — i — i — r — i — i — i — i — r CS U> /_5 JtO 4 5 »J> i s 4J> Concentration — mg/1 Chelate Test Cone, vs Abs. Zn o <D ZnSTD * ZnData ~l 1 1 1 1 1 1 1 r £.4 3.6 4M 6.0 Tji 6.4 Concentration — mg/l Chelate Test — Cone, vs Abs. — Zn2 i 1 1 1 r — i 1 r 30.0 40.0 SO.O tOO TOO Concentration — mg/l ~r — i 1 1 — ~r — i 1 40.0 tO.O 100.0 156 APPENDIX N - - PLOT OF LOG10( STABILITY CONSTANT) VS LIGAND Log 10(Ks) vs Ligand Fth Cly Hist NTA EDTA Hyd Ligand 157 A P P E N D I X 0 - - P E R C E N T S P I K E V S A B S O R B A N C Y P L O T S F O R C H E L A T I O N D A T A S E T TWO Chelates — Cr — Abs. vs Spike Absorbancy Chelates — C u — Abs. vs Spike Absorbancy Chelates — Hydroxy — Abs. vs Spike 158 Chelates — Fe — Abs. vs Spike Abtorbancy (XIO' ) Chelates -- Zn — Abs. vs Spike Absorbancy 159 APPENDIX P - - CALCULATIONS FOR ESTIMATES OF COMPLEXED METAL Assume: 1) E x t r a c t i o n e f f i c i e n c y = 1 f o r each complex 2) Schubert c o n d i t i o n s occur ( [ l i g a n d ] > > [ m e t a l ] ) . Equations: 1) ZIMnLm = ZEBnm * Mn * (L) 2) LEMnLm = MO - EMn 3) LEMnLm = LO - L 4) ZEMnLm = ML If values f o r Bnm can be determined e x p e r i m e n t a l l y then equations 1-4 can be s o l v e d f o r n=1,2 and m=1,2...6. In s p e c i a l cases the equations can be s o l v e d when n=3 (the t h i r d s p e c i e s i s a s o l i d ) . The valu e s of MO and ML can be determined e x p e r i m e n t a l l y and the l i g a n d c o n c e n t r a t i o n LO i s c o n t r o l l e d . Table XVI was c a l c u l a t e d assuming: 1) 1:1 l i g a n d - m e t a l complexes (m=1). 2) Only one s p e c i e s i s present i n s i g n i f i c a n t c o n c e n t r a t i o n s (n=1). The second assumption may not be v a l i d f o r i r o n , chromium or copper. S t a b i l i t y c o nstants were not a v a i l a b l e f o r the Cu(I) s p e c i e s so ML was not c a l c u l a t e d i n t h i s case. I f n=2 the value of ML cannot be determined without experimental data. For n=m=1 equations 1-4 become: 1) M1L1 = B11 * M1 * L 2) M1L1 = MO -M1 3) M1L1 = LO - L 4) M1L1 = ML The terms M1L1, ML, L and M1 are unknowns and there are four equations so a s o l u t i o n can be found. The equations reduce t o : ML = K * (MO-ML) * (LO-ML) OR OM0L0-ML (LO+K " 1 +M0) +ML 2 ML can be s o l v e d using the s o l u t i o n f o r a q u a d r a t i c e a u a t i o n . The i n i t i a l v alues used f o r Table XVI are shown i n Taole XXV and the B11 values were taken from Table X . Note that the equation f o r ML changes i f there are 1:2 and 1:1 complexes. ML= B12*(MO-ML)*(LO-ML) 2 A t r i n o m i a l i n ML. 1 60 Table XXVI - I n i t i a l Values For C a l c u l a t i o n Of Complex Concentrat ions I n i t i a l Values Cd Cr Cu Fe Ni Pb Zn Maximum [Metal] uM/dry gm. 0.4 1.8 1.3 82 0.18 3.0 9.8 Log10(Cone.) DS1-3.6 gm. (moles) DS2-1.8 gm. (moles) -5.84 -6.14 -5. 19 -5.49 -5.63 -5.63 -3.53 -3.83 -6.19 -6.49 -4.97 -5.27 -4.45 -4.75 LO Values DS2 4.77 17.15 8.57 6.85 5.98 6.38 8.56 161 APPENDIX Q - - PSEUDO MASS RATIO CALCULATIONS FOR THE CHELATION EXPERIMENT Table XXVII - C a l c u l a t i o n Of Rx Data Set One — DS1 I f : Vs= volume of MIBK added t o sample before r i n s e — i n ml. Vr= volume of MIBK r i n s e — i n ml. Mm= measured metals — gm. Cm= c o n c e n t r a t i o n of metals i n sample — mg/l. P= f r a c t i o n of s o l i d s W= mass of sample — gm. RR= mass r a t i o — mass of metals/mass of sample dry weight x = a d i f f e r e n t constant f o r each metal f1 = mass c o r r e c t i o n f a c t o r Am = absorbancy u n i t s Then: RR = Mm/WP Mm = Cm(Vs+Vr) RR =Cm(Vs+Vr)/WP L e t : f1 = (Vs+Vr)/WP Mm = xAm Then: RR/x = Rx = Am(fl) EDTA Eth Gly H i s t Hyd NTA Oxa w 121 122.5 113.7 122. 1 1 16.8 84.3 117.7 Vs 30.5 29 34.5 23* 29.5 35.5 32 f 1 6.97 6.63 8.15 6.83 7.05 11.2 7.43 * - S p i l t roughly 7 ml. of sample. 162 Table XXVIII - C a l c u l a t i o n s Of Xm Data Set Two — DS2 RR = Mm/WP RR = CmVs/WP RR/Vs = Cm/WP Cm = xAm RR/Vs = xAm/WP x = a constant which i s d i f f e r e n t f o r each metal. M u l t i p l y both s i d e s of the equation by 50 gms. to c a l i b r a t e the samples to a 50 gms. wet mass sample. RR50/xVs = Am50/WP = Xm L e t : f2 = 50/WP Xm = Am(f2) EDTA Eth Gly H i s t Hyd NTA Oxa Blk W f 2 39.5 35 41.4 33.5 41.1 33.8 41 .7 33.3 37.4 37. 1 39.9 34.8 39.2 35.4 10% w f 2 47.0 34.3 50.7 31 .8 43.6 37.0 46.4 34.8 51 .3 31.4 51 .3 31 .4 49.6 32.5 50% W f2 52.2 26.6 48.6 28.6 51 .4 27.0 47. 1 29.5 49.3 28.2 51 .5 27.0 51 .0 27.2 100% W f 2 42.5 38.0 38.2 42.2 41.1 39.2 40.6 39.7 40.5 39.8 39.6 40.7 42.9 37.6 Note: P = 0.036 f o r Blk and 50% P = 0.031 f o r 10% and 100% 163 APPENDIX R - - METAL SPECIES FORMULAE AND DERIVATIONS Assume: a) Shubert c o n d i t i o n s LO>>MO and L>>Mn. b) Values f o r En, Bnm, MA and MO can be determined e x p e r i m e n t a l l y . c) The value of LO i s known. d) The a c t i v i t y = c o n c e n t r a t i o n . e) A maximum of three metal s p e c i e s can occur i n s o l u t i o n s . M3=1 i n d i c a t e s that the t h i r d s p e c i e s i s a s o l i d . Then: 1) ZIMnLm = ML 2) IIMnLm = ZZBnm*Mn*(L) 3) ZIMnLm = MO - ZMn 4) LO = L + ZZMnLm 5) LIMnLm*Enm = MA 6) IZMnLm = ZEBnm*Mn*Enm*(L) A b b r e v i a t i o n s : Zn = IBnm*Enm*L Sn = IBnm*L S o l v i n g f o r L the f o l l o w i n g equation i s ob t a i n e d : (MA-Z3)*(S1-S2)-MO*(Z2*S1-Z1*S2)+S3*(Z1-Z2) = = (L-L0)*(Z1-Z2-S1*Z2+S2*Z1) The equation can be s o l v e d f o r any m but values of m g r e a t e r than one r e q u i r e a polynomial s o l u t i o n . In the s p e c i a l case where m=1 ( i e . EDTA) a q u a d r a t i c s o l u t i o n can be found. 164 Values f o r Bnm, MO and MA should be determined e x p e r i m e n t a l l y . Values f o r LO are c o n t r o l l e d i n the l i g a n d a d d i t i o n s but they should be much l a r g e r than the expected metal c o n c e n t r a t i o n s f o r optimal r e s u l t s . 165 A P P E N D I X S - - C O N C E N T R A T I O N V S A B S O R B A N C Y P L O T S F O R E X T R A C T I O N D A T A Extraction — Cd — Cone, vs Abs. Cone, (mg/l) Extraction — Cd — Cone, vs Abs. Cone, (mg/l) Extraction — Cd — Cone, vs Abs. * ID e o Cd4 * * dCd4 * 0*1/25 e CS -•° s o o tn Xi / e i " 0 X 3 / 1 0 / s / , r T - 1 1 1 1 Cone. (mg/l) 166 Extraction — Cr — Cone vs Abs < • Cr2 • • dCrl x x dCr2 Extraction — Cr — Cone vs Abs 2 4 -Cr4 * dCr3 sane orb' 0,0- SOM5/ Extraction — Cu — Cone, vs Abs. 5J ? 5 -CVLI dCu> Cone, (mg/l) . E x t r a c t i o n — C u — Cone, vs Abs. Cone. (mg/I) Extraction — C u — Cone, vs Abs. Cone, (mg/l) 1 E x t r a c t i o n — P b — Cone, vs Abs. Cone. (mj/1) Extraction — P b — Cone, vs Abs. Cone, (mg/l) Extraction — P b — Cone, vs Abs. 8 o -o e Pb4 * * dPb4 e -»- *1-o <>-r . 0 So o o4——, , , , . —• . 1 1—1 1 — - 1 1 1 1 1 1 1 4 — i 1 1 i 1 i i 1 1 1 r - i 1 1.0 1-0 *• *.o so so 7-0 TJ> Cone. (mg/1) 169 Extraction Ni — Cone, vs Abs. ft e -O O •9 -o ftif -r NiZ • oLVi/ x dNiZ '• _  K <*>»} , , , _ ~ i 1 T ^ T " — i 1 1 1 1 1 1 1 1 1 1 1 1 i 0J> 0.1 1.6 H4 tJt 4.0 4 J 3.S « « 7Jt Cone. (mg/l) Extraction — Zn — Cone, vs Abs. ZnZ dZnZ OtSAK Cone, (mg/l) Extraction — Zn — Cone, vs Abs. O O Zn1 + + dZnl 4 0 . 0 — i 1 1 — (6.0 1 0 . 0 Cont. (mg/l) I I 1 4 . 0 —I *eo 170 APPENDIX T - - MASS RATIO CALCULATIONS FOR THE EXTRACTION EXPERIMENT The data l i s t e d i n matrix form below was o b t a i n e d from the graphs i n Appendix S. The values are l i s t e d i n mg/1. Cd Cr Cu Fe N i Pb Zn SON 0 1 . 160 9. 940 65.000 2700. 000 2. 690 122. 000 388. 000 SON 1 0. 630 0. 390 10.600 69. 000 0. 810 3. 920 47. 000 SON 2 0. 960 0. 510 28.900 740. 000 0. 700 8. 200 37. 000 SON 3 0. 970 0. 800 10.900 320. 000 0. 370 1 1 . 000 55. 000 SON 4 0. 300 7. 350 13.900 68. 000 0. 370 3. 160 25. 500 SON 5 0. 380 l 1 . 100 1.610 3600. 000 1. 660 21 . 900 238. 000 SON 6 0. 330 4. B10 28.000 2200. 000 0. 720 24. 800 87. ,000 BLK 0 1. 750 10. 000 65.000 4000. 000 6. 900 146. 000 480. 000 BLK l 1. 360 0. 090 1 1.100 74. 000 5. 700 1 . 800 56. ,600 BLK 2 2. 170 0. 210 29.500 48, ,000 4. 940 5. 500 41 . ,800 BLK 3 0. 960 0.200 9.700 76. .000 1. 850 8. 000 37. ,000 BLK 4 0. ,490 3. ,690 8.20C 940. .000 0. ,340 26. 400 65. ,800 BLK 5 0. ,040 3. ,310 1 .460 2300, ,000 0, ,720 7. 400 266, ,000 BLK 6 0. .690 9. ,220 42.000 3800, .000 2. ,480 146, ,000 236, ,000 FOX 0 1. ,330 3. ,510 53.000 2500, .000 1, ,540 126, ,000 396, .000 FOX 1 177, ,500 1 , 660 395.000 1630, .000 4 , 900 134. ,000 547, .000 FOX 2 43. .400 0. ,160 43.000 45, .000 1 , 660 6. ,700 88. .600 FOX 3 14. .000 0. .330 7.900 230, .000 0, ,620 5. .500 46 .400 FOX 4 4. .290 21 .800 81 .000 390, .000 0, ,715 5, ,900 50 .000 FOX 5 1, .460 4 , 670 4.790 '41 00 .000 1, .740 15, .000 586 .000 FOX 6 0. .690 5, .210 30.000 1400 .000 0, .710 34. .500 105 .000 The program on the next page reads a l l the a e r a t e d , blank or s o n i c a t e d data and c a l c u l a t e s the mass r a t i o s , % of t o t a l mass r a t i o and cummulative % of t o t a l m a s s r a t i o . T h i s data i s outputed as x and y c o o r d i n a t e s f o r a p l o t t i n g program. /COMPILE C THE PURPOSE OF THIS FILE IS TO CALCULATE AN OUTPUT FILE FOR MASS RATIO C VS PHASE PLOTS. THE MASS RATIO VALUES FOR BLK AND FOX DATA ARE CALCULATED C USING THE SAME PROGRAM. ALL OCCURANCES OF SON ARE CHANGED TO BLK OR FOX C WHEN THE OTHER DATA IS CALCULATED. A DATA FILE MUST BE CONCATENATED WITH C THE END OF THIS FILE. c •••*•*•**••••• T H i S SECTION INPUTS THE DATA **»******»»•*••• REAL SON(7.7),SONM(7.7),C(7),V.B(7),SUM(7).SONM2(7,7) READ.((SON(I,J).J=1,7).1-1,7),(C(JJ).JJ-1.7) C J-METAL NUMBER :1-CD. 2=CR. 3-CU. 4-FE, 5-NI. 6-PB, 7«2N C I'PHASE NUMBER C V-VOLUME OF AA SAMPLE C C= DRY WEIGHT FACTOR c ••••*»•••••»•••» THIS SECTION ADJUSTS THE DATA FOR SAMPLE VOLUME *••**•*** DO 20 I"1,7 V- 0. 1 DO 10 J-1.7 SUM(J)» 0 IF(I.GE.5) V-0.25 SONM(I.J)-SON(I,d)*V/C(I) 10 CONTINUE 20 CONTINUE c *».*...*....,.»» THIS SECTION PRINTS THE INPUT DATA IN MATRIX FORM ******* PRINT 50 50 F0RMAT(T13,'Cd',T23.'Cr',T33,'Cu',T43.'Fe',T53.'NI' c ,T63,'Pb',T73.'Zn',/./) DO 25 1-1. 7 M-I-1 25 PRINT 100.M,(SON(I,J).J=1,7) 100 FORMAT(T2,'SON'.12.T8,6(F9.3,1X),F9.3,/) c *•»••••*.•** THIS SECTION OUTPUTS X AND Y CO-ORDINATES FOR PLOTTING ****** DO 33 7 B(J)-0 DO 33 I«1, 7 MMM-I-1 33 PRINT 33O,MMM,S0NM(I,J) 330 F0RMAT(I3,F12.6) DO 44 1*2. 7 DO 44 J*1, 7 B(J ) *B(J ) + SONM(I.J) 44 CONTINUE DO 35 J-1.7 DO 35 I»1 . 7 SONM(I.J)- S0NM(I,J)*1O0/B(J) MM-I-1 IF(I.LE.2) GOTO 34 S0NM2(I,J)-S0NM2(1-1.J)+SONM(I.J) GOTO 37 34 CONTINUE S0NM2(I.J)"SONM(I,J) 37 CONTINUE 35 PRINT 200,MM,SONM(I,J),MM,S0NM2(I,J) 200 F0RMAT(I3.F12.6.I3.F12.G) STOP END /DATA ILE 172 The data l i s t e d in matrix form below was obtained from the program on the previous page. A l l valu e s are mass r a t i o s of metal mass over dry mass of sample ( a f t e r d r y i n g at 104°C). Cd Cr Cu Fe NI PD Zn BLK 0 0.035714 0.204082 1 .326530 81 .632640 0.140816 2.979592 9.795918 BLK 1 0.004345 0.000288 0.035463 0.236422 0.018211 0.005751 0.180831 BLK 2 O.OOB037 0.000778 0.109259 0. 177778 0.018296 0.020370 0. 1548 15 BLK 3 0.003556 0.000741 0.035926 0.281482 0.006852 0.029630 0.137037 BLK 4 0.0O5104 0.038437 0.085417 9.791666 0.00354 2 0.275000 0.6854 17 BLK 5 0.000714 0.059107 0.026071 41.07 1420 0.012857 0.132143 A 750000 BLK 6 0.015682 0.209545 0.954545 86.363630 0.056364 3.318181 b.363636 Cd Cr Cu Fe NI Pb Zn SON 0 0.013395 0. 114781 0.750577 31 . 177820 0.031062 1.408776 4.480370 SON 1 O.0O222S 0.001378 0.037456 0.243816 0.002862 0.013852 0.166078 SON 2 0.003429 0.001821 0.103214 2.642858 0.002500 0.029286 0.132143 SON 3 0.003469 0.0O287B 0.039209 1 . 151078 0.001331 0.039568 0.197842 SON 4 0.0207 18 0.507597 0.959945 4.696133 0.025552 0.218232 1.761049 SON S 0.027143 0.792857 0.115000 257.142800 0.118571 1.564285 17.000000 SON 6 0.025000 0.364394 2.121211 166.666600 0.054545 1.878788 6.590908 Cd Cr Cu Fe NI Pb Zn AER 0 0.011176 0.029496 0.445378 21.008400 0.012941 1.058824 3.327731 AER 1 0.393570 0.003681 0.875831 3.614190 0.010865 0.297118 1.212860 AER 2 0.143234 0.000528 0.141914 0.148515 0.005479 0.022112 0.292409 AER 3 0.056000 0.001320 0.031600 0.920000 0.002480 0.0220O0 0 185600 AER 4 0.045445 0.230932 0.858051 4.131354 0.007574 0.062500 0.529661 AER 5 0.017299 0.055332 0.056754 48.578170 0.020616 I -.77725 6.943126 AER 6 0.008756 0.066117 0.380711 17.766490 0.009010 0.437817 1 332487 1 73 APPENDIX U - - CONCENTRATION VS ABSORBANCY PLOTS FOR LIQUID DATA The cadmium data was at d e t e c t i o n l i m i t s so the data was not used. For a l l the other metals t e s t e d the c o n c e n t r a t i o n s are r e p o r t e d below. A1 was a sample of the supernatant a f t e r c e n t r i f u g i n g the l e a c h a t e sample. The t o t a l volume of the sample used was 356 ml. S2 was a mixture of supernatant and res i d u e w hile S3 was mainly r e s i d u e . (S1+S2+S3)*0.05/0.356 = mg./I c o n c e n t r a t i o n of Each metal i n the Leachate. Table XXIX - Metal C o n c e n t r a t i o n s In The Leachate Sample Metal S1 S2 S3 ug/1 Cr 0.21 0.16 0.13 70 Cu 0.11 0.10 0.06 38 Fe 7.6 3.9 4.0 2200 Ni 1.9 3.5 3.5 1200 Pb 0.08 0.09 0.09 37 Zn 10.5 5.0 4.9 2900 Concentration vs Absorbancy — Leachate Sample — Cd Concentration (mg/l) Concentration vs Absorbancy — Leachate Sample • T " — 1 Concentration (mg/l) * Concentration vs Absorbancy — Leachate Sample — Concentration (mg/l) Concentration vs Absorbancy — Leachate Sample Concentration (mg/l) Concentration vs Absorbancy — Leachate Sample — C o n c e n t r a t i o n (mg/l) Concentration vs Absorbancy — Leachate Sample -Concentration (mg/l) A P P E N D I X V - - REDUCTION POTENTIALS Table XXX - Reduction P o t e n t i a l s Equation Reduction P o t e n t i a l In V o l t s + e" = C r * 2 + 3e- = Cr + 2e" = Cr 7 - - 14H* + Ge-Cd* 2 + 2e- = Cd Cr* 3 C r + 3 C r + 2 C r 2 0 7 " 2 + + 6e- = 2Cr* 3 + 7H 20 C r 0 2 " + 2H 20 + 3e- = Cr + 40H" CrO«" 2 + 4H 20 + 3e" = Cr(OH) 3 + 50H" Cr(OH) 3 + 3e" = Cr + 30H" HCrO«- + 7H* + 3e" = C r + 3 + 4H 20 Cu + + e" = Cu + e- = Cu* + 2e" = Cu + 2e" = Fe + 3e" = Fe + e" = F e * 2 + 2e" = Ni 4H* + 4e" = 2H 20  Cu* 2 Cu* 2 F e * 2 Fe* 3 Fe* 3 N i * 2 0 2 + 2H 20 + 4e" = 40H" Pb* 2 + 2e" = Pb S + 2H* + 2e" = H 2S(aqu) Zn* 2 + 2e" = Zn -0.4026 -0.41 -0.74 -0.557 + 1 .33 -1 .2 -0.12 -1 .3 + 1 . 195 +0.522 +0.158 +0.3402 -0.4092 -0.036 +0.770 -0.23 + 1 .229 +0.401 -0. 1263 + 0.141 -0.7628 A f t e r Weast (1971) Note: A p o s i t i v e p o t e n t i a l i n d i c a t e s t h a t the r e a c t i o n w i l l proceed to the r i g h t . 177 APPENDIX W - - LIST OF SYMBOLS USED AO = A c r i d i n e Orange ASV = a n o i d i c s t r i p v oltametry AW = atomic weight Bnm = bulk s t a b i l i t y constant C i = metal c o n c e n t r a t i o n i n sample EDTA = e t h y l e n e - d i a m i n e - t e t r a - a c e t i c a c i d Enm = e x t r a c t i o n e f f i c i e n c y of l i g a n d m when complexed with metal s p e c i e s n. DAPI = 4,6-diamidino-2-phenylindole Ks = s t a b i l i t y constant LO = moles of l i g a n d added m = number of l i g a n d s complexed MA = measured moles of complexed metal ML = measured moles of complexed metal i f the e x t r a c t i o n e f f i c i e n c y i s 100% Mm = metal mass i n sample Mn = moles of metal s p e c i e s n MnLm = moles of complex with s p e c i e s n and m l i g a n d s MO = i n i t i a l moles of a metal n = metal s p e c i e s number NTA = n i t r i l o - t r i - a c e t i c a c i d P = s o l i d s f r a c t i o n determined by oven d r y i n g at 104°C PGA = p o l y g a l a c t u r o n i c a c i d RR = mass r a t i o Rx = pseudo mass r a t i o f o r c h e l a t i o n data set one V i = MIBK volume recovered 178 W = weight of sample Xm = pseudo mass r a t i o f o r c h e l a t i o n data set two 

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