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Radial distribution of thujaplicins and thujic acid in old growth and second growth western redcedar.. 1986

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RADIAL DISTRIBUTION OF THUJAPLICINS AND THUJIC ACID IN OLD GROWTH AND SECOND GROWTH WESTERN REDCEDAR (THUJA PLICATA DONN) by JASON RAY NAULT B . S c , The U n i v e r s i t y o f Winnipeg, 1976 A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF FORESTRY) We a c c e p t t h i s r e p o r t as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d . The U n i v e r s i t y o f B r i t i s h C olumbia A p r i l 13, 1986 ® J a s o n Ray N a u l t , 1986 I n 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 o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e 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 s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f FORESTRY The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date APRIL 14, 1986 i i ABSTRACT R a d i a l c r o s s s e c t i o n s of seven o l d growth c e d a r s and t e n second growth c e d a r s t a k e n a t b r e a s t h e i g h t were c u t i n t o i n c r e m e n t s a v e r a g i n g about 60 y e a r s growth f o r o l d growth t r e e s and 10 y e a r s growth f o r second growth t r e e s . These i n c r e m e n t s were e x t r a c t e d w i t h e t h a n o l : b e n z e n e (1:2). The e x t r a c t i v e s were a n a l y z e d f o r t h u j a p l i c i n c o n t e n t by c o l o r i m e t r y of t h e i r f e r r i c c h e l a t e s , as w e l l as by a new method u t i l i z i n g c a p i l l a r y gas chromatography (GC) of t h e i r m e t h y l a t e d d e r i v a t i v e s . A s t a t i s t i c a l a n a l y s i s o f t h e two methods gave an r ^ v a l u e o f 0.81 and a s l o p e of 0.99. T h u j i c a c i d c o n t e n t s were a l s o d e t e r m i n e d by t h e new GC method. D i s t r i b u t i o n of t h u j a p l i c i n s and t h u j i c a c i d g e n e r a l l y i n c r e a s e d from p i t h t o o u t s i d e heartwood, t h e n d e c r e a s e d i n t h e sapwood. Maximum t h u j a p l i c i n c o n t e n t s were a l s o r e l a t e d t o t h e t r e e age. TABLE OF CONTENTS page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES i v LIST OF FIGURES v LIST OF APPENDICES i x ACKNOWLEDGEMENT x INTRODUCTION 1 LITERATURE REVIEW 2 MATERIALS AND METHODS 7 RESULTS 10 DISCUSSION 12 CONCLUSION 16 LITERATURE CITED 17 i v LIST OF TABLES TABLE 1. GC SAMPLES ANALYZED WITH REPLICATION 21 TABLE 2. COMPARISON OF DATA WITH LITERATURE VALUES...21 LIST OF FIGURES FIGURE 1. SAMPLING PROCEDURE 22 FIGURE 2. RADIAL DISTRIBUTION OF ETHANOL:BENZENE EXTRACTIVES FOR SECOND GROWTH SITE #1 23 FIGURE 3. RADIAL DISTRIBUTION OF ETHANOL BENZENE EXTRACTIVES FOR SECOND GROWTH SITE #2 24 FIGURE 4. RADIAL DISTRIBUTION OF ETHANOL:BENZENE EXTRACTIVES FOR OLD GROWTH TREES 25 FIGURE 5. ETHANOL:BENZENE EXTRACTIVE CONTENT I S AVERAGE RINGS FROM PITH FOR ALL TRES 26 FIGURE 6. COMPARISON OF THUJAPLICIN RESULTS OBTAINED BY COLORIMETRY AND GLC 27 FIGURE 7. THUJAPLICIN CONTENT BY TWO METHODS AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.1 28 FIGURE 8. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.2 28 FIGURE 9. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.3 29 FIGURE 10. THUJAPLICIN CONTENT BY TWO METHODS Y£ AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.4 29 v i FIGURE 11. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.5 30 FIGURE 12. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.1 30 FIGURE 13. THUJAPLICIN CONTENT BY TWO METHODS Y£ AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.2 31 FIGURE 14. THUJAPLICIN CONTENT BY TWO METHODS V_S_ AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.3 31 FIGURE 15. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.4 32 FIGURE 16. THUJAPLICIN CONTENT BY TWO METHODS V_S AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.5 32 FIGURE 17. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #1 33 FIGURE 18. THUJAPLICIN CONTENT BY TWO METHODS Y_S AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #2 33 v i i FIGURE 19. THUJAPLICIN CONTENT BY TWO METHODS V§ AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #3 34 FIGURE 20. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #4 34 FIGURE 21. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #5 35 FIGURE 22. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #6 35 FIGURE 23. THUJAPLICIN CONTENT BY TWO METHODS Y§ N AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #7 36 FIGURE 24. BETA+GAMMA THUJAPLICIN CONTENT V§ AVERAGE RINGS FROM PITH FOR ALL TREES 37 FIGURE 25. BETA+GAMMA THUJAPLICIN CONTENT 1§ LOG (AVERAGE RINGS FROM PITH) FOR ALL TREES...38 FIGURE 26. BETA THUJAPLICIN CONTENT Y£ AVERAGE RINGS FROM PITH FOR ALL TREES 39 FIGURE 27. BETA THUJAPLICIN CONTENT Y£_ LOG (AVERAGE RINGS FROM PITH) FOR ALL TREES...40 FIGURE 28. GAMMA THUJAPLICIN CONTENT Y£ AVERAGE RINGS FROM PITH FOR ALL TREES .41 v i i i FIGURE 29. GAMMA THUJAPLICIN CONTENT V£ LOG (AVERAGE RINGS FROM PITH) FOR ALL TREES...42 FIGURE 30. THUJAPLICIN CONTENT Vg ETHANOL:BENZENE EXTRACTIVE CONTENT FOR ALL TREES 43 FIGURE 31. THUJIC ACID CONTENT V£ ETHANOL:BENZENE EXTRACTIVE CONTENT FOR ALL TREES 44 FIGURE 32. THUJIC ACID CONTENT YS THUJAPLICIN CONTENT FOR ALL TREES 45 FIGURE 33. RADIAL DISTRIBUTION OF THUJIC ACID CONTENT FOR SECOND GROWTH SITE #1 46 FIGURE 34. RADIAL DISTRIBUTION OF THUJIC ACID CONTENT FOR SECOND GROWTH SITE #2 47 FIGURE 35. RADIAL DISTRIBUTION OF THUJIC ACID CONTENT FOR OLD GROWTH TREES 48 FIGURE 36. THUJIC ACID CONTENT I S AVERAGE RINGS FROM PITH FOR ALL TREES 49 FIGURE 37. MULTIPLE ANALYSES OF THUJAPLICIN CONTENT IN SECOND GROWTH TREE #1.1 50 FIGURE 38. MULTIPLE ANALYSES OF THUJAPLICIN CONTENT IN OLD GROWTH TREE #3 51 FIGURE 39. BETA+GAMMA THUJAPLICIN CONTENT I S LOG (AVERAGE RINGS FROM PITH) FOR ALL TREES...52 i x LIST OF APPENDICES APPENDIX 1. PHYSICAL DATA FOR OLD GROWTH TREES 53 APPENDIX 2. OLD GROWTH SAMPLE DATA 53 APPENDIX 3. EXPERIMENTAL DATA FOR OLD GROWTH TREES 55 APPENDIX 4. PHYSICAL DATA FOR SECOND GROWTH TREES 57 APPENDIX 5. SECOND GROWTH SAMPLE DATA 58 APPENDIX 6. EXPERIMENTAL DATA FOR SECOND GROWTH TREES.60 APPENDIX 7. LINEAR REGRESSION STATISTICS FOR GC VS. COLORIMETRIC RESULTS 61 X ACKNOWLEDGEMENT The a u t h o r w i s h e s t o e x p r e s s h i s g r a t i t u d e t o a l l t h o s e p e o p l e who a s s i s t e d w i t h t h i s s t u d y . I n p a r t i c u l a r , a s s i s t a n c e by Dr. J . W. W i l s o n , U.B.C. F a c u l t y of F o r e s t r y , and R e s e a r c h S c i e n t i s t Dr. E. P. Swan, F o r i n t e k Canada Corp. i s a p p r e c i a t e d . I n a d d i t i o n , t h a n k s a r e extended t o Western F o r e s t P r o d u c t s L t d . ( P o r t M c N e i l l ) f o r s u p p l y i n g t h e o l d growth m a t e r i a l . 1 INTRODUCTION Western r e d c e d a r (Thu.ia p l i c a t a Donn) has been l o n g n o t e d f o r t h e n a t u r a l r e s i s t a n c e of i t s heartwood t o decay (11,26). T h i s r e s i s t a n c e has r e s u l t e d i n t h e use of w e s t e r n r e d c e d a r (WRC) i n a p p l i c a t i o n s where t h i s p r o p e r t y i s u s e f u l , e s p e c i a l l y where exposure t o t h e elements i s e x p e c t e d , f o r example i n p o l e s , shakes, s h i n g l e s , s i d i n g and g u t t e r s . T h i s u n i q ue d u r a b i l i t y has been a t t r i b u t e d t o t h e p r e s e n c e of t h u j a p l i c i n s i n t h e heartwood e x t r a c t i v e components. T h u j a p l i c i n s have been shown t o be p o t e n t i n h i b i t o r s of b a c t e r i a l and f u n g a l growth (24,25,26,27,28,30,33,37). WRC i s an i m p o r t a n t Canadian commercial wood. I n t h e y e a r s 1977 t o 1983 B r i t i s h Columbia's l o g p r o d u c t i o n of WRC averaged 7.8 m i l l i o n p e r y e a r ( 9 ) , w h i l e e x p o r t s of WRC s h i n g l e s and shakes a c c o u n t e d f o r $68 m i l l i o n p e r y e a r ( 6 ) . An i n c r e a s i n g s h a r e of our t i m b e r p r o d u c t i o n i s coming from second growth f o r e s t s , and s i l v a c u l t u r a l p r a c t i c e s a r e r e s u l t i n g i n f a s t e r growth of t h e s e s t a n d s . T h i s has r a i s e d c o n c e r n s o v e r t h e q u a l i t y o f p r o d u c t s b e i n g produced from t h i s f a s t - g r o w n wood. W i t h WRC t h e c o n c e r n i s t h a t t h e d u r a b i l i t y f a c t o r i s m a i n t a i n e d i n second growth t r e e s . The purpose of t h i s s t u d y i s t o a n a l y z e t h e r a d i a l d i s t r i b u t i o n o f t h u j a p l i c i n s and t h u j i c a c i d t o d e t e r m i n e i f t h e y a r e r e l a t e d t o t r e e age and how much t h e y v a r y between o l d growth and second growth t r e e s . As w e l l , a new gas c h r o m a t o g r a p h i c method of a n a l y s i s i s compared t o t h e p r e v i o u s c o l o r i m e t r i c method. 2 LITERATURE REVIEW Sowder (30) found t h a t t h e h o t and c o l d water s o l u b l e e x t r a c t i v e s of WRC heartwood and sapwood were t o x i c t o f u n g i , w h i l e e x t r a c t e d wood meal was more s u s c e p t i b l e t o decay t h a n u n e x t r a c t e d . R e n n e r f e l t (24) showed a l p h a , b e t a and gamma t h u j a p l i c i n t o be t o x i c t o decay f u n g i i n low c o n c e n t r a t i o n s , w i t h b e t a b e i n g t h e most t o x i c , f o l l o w e d by gamma, t h e n a l p h a . He a l s o found them t o be more e f f e c t i v e i n decay p r e v e n t i o n t h a n p i n o s y l v i n , t h e most p o t e n t f u n g i c i d e i n h a r d p i n e heartwood. The t h u j a p l i c i n s were of about t h e same e f f e c t i v e n e s s i n decay i n h i b i t i o n as p e n t a c h l o r o p h e n o l . Raa and Goksoyr (23) d i s c o v e r e d t h a t t h u j a p l i c i n s i n h i b i t e d r e s p i r a t i o n i n y e a s t i n t h e i r s t u d y on y e a s t m e t a b o l i c r e a c t i o n s . Rudman (27) s t u d i e d t o x i c i t i e s of a l p h a , b e t a , and gamma t h u j a p l i c i n , and b e t a t h u j a p l i c i n o l and demonstrated a l l t o be p o t e n t i n h i b i t o r s of f u n g a l growth, a l t h o u g h a t low c o n c e n t r a t i o n s (<.1%) t h e y were s t i m u l a n t s t o growth f o r one s p e c i e s of fungus. He a l s o compared t h e i r t o x i c i t y r e l a t i v e t o o t h e r e x t r a c t i v e c h e m i c a l s (28) and found them t o be t h e most t o x i c t o a l l s p e c i e s of f u n g i t e s t e d . T r u s t and Coombs (33) showed b e t a t h u j a p l i c i n t o be a b r o a d spectrum a n t i b a c t e r i a l agent. Coombs and T r u s t (8) found t h a t a n t i b a c t e r i a l a c t i v i t y of b e t a t h u j a p l i c i n was g r e a t l y reduced f o l l o w i n g exposure t o l i g h t , and showed t h a t p h o t o c h e m i c a l d e c o m p o s i t i o n had o c c u r r e d . Anderson and G r i p e n b e r g (1) e l u c i d a t e d t h e s t r u c t u r e of b e t a t h u j a p l i c i n , f o l l o w e d by Erdtman and G r i p e n b e r g ( 1 2 ) , who i s o l a t e d a l p h a and gamma t h u j a p l i c i n and i d e n t i f i e d t h e i r 3 s t r u c t u r e s . The t h u j a p l i c i n s were d i s c o v e r e d t o be u n u s u a l seven member r i n g s which a r e s t r u c t u r a l i s o m e r s . Nozoe e t a l (22) d e s c r i b e d t h e i s o l a t i o n of a l p h a t h u j a p l i c i n from "Hiba-wood". Gardner e t a l (14) i s o l a t e d and i d e n t i f i e d a r e l a t e d s u b s t a n c e , 7-hydroxy-4- i s o p r o p y l t r o p o l o n e o r b e t a t h u j a p l i c i n o l ( which i s b e t a t h u j a p l i c i n w i t h a h y d r o x y l group i n t h e 7 p o s i t i o n ) from e x t r a c t i v e s o f WRC. They r e p o r t e d t h a t t h i s s u b s t a n c e o c c u r r e d i n c o n c e n t r a t i o n s of about one t e n t h t h a t of t h u j a p l i c i n s , and t h a t i t had r e l a t i v e l y low t o x i c i t y t o f u n g i . Z a v a r i n e t a l (38) i s o l a t e d a l p h a t h u j a p l i c i n o l ( 7 - h y d r o x y - 6 - i s o p r o p y l t r o p o l o n e ) from Cupressus pygmaea Lemm. R o f f and W h i t t a k e r (25) found b e t a t h u j a p l i c i n o l t o be as t o x i c t o brown r o t f u n g i as gamma t h u j a p l i c i n and sodium p e n t a c h l o r o p h e n a t e , b u t much l e s s t o x i c t o w h i t e r o t f u n g i t h a n e i t h e r . A u l i n - E r d t m a n (2) p r e s e n t e d u l t r a v i o l e t s p e c t r a f o r t h e t h u j a p l i c i n s , and m o l e c u l a r w e i g h t d e t e r m i n a t i o n s f o r b e t a t h u j a p l i c i n . D o e r i n g and Knox (10) and Cook e t a l (7) d e s c r i b e d methods f o r s y n t h e s i z i n g v a r i o u s t r o p o l o n e s . Gardner e t a l (14) s t u d i e d t h e c o l l e c t i o n of t r o p o l o n e s i n c l u d i n g t h u j a p l i c i n s as copper c h e l a t e s on a copper s c r e e n i n a k i l n d r y i n g WRC lumber. They a l s o d e s c r i b e d t h e f r a c t i o n a t i o n of t h i s t r o p o l o n e m i x t u r e . Gardner and B a r t o n (13) s t u d i e d t h e o c c u r r e n c e of t h u j a p l i c i n s i n t h e steam v o l a t i l e o i l o f WRC, and d i s c u s s e d s e p a r a t i o n of t h e t h u j a p l i c i n s from t h e o i l . B a r t o n and Gardner (3) used acetone e x t r a c t i o n of a l a r g e sample (2.8 kg) of WRC b u t t heartwood t o de t e r m i n e t h e c o n c e n t r a t i o n s o f b e t a and gamma t h u j a p l i c i n and t h u j i c a c i d 4 g r a v i m e t r i c a l l y . They found t h a t t h e wood c o n t a i n e d 0.17 t o 0.35% t h u j a p l i c i n s and 0.11 t o 0.68% t h u j i c a c i d . Maclean and Gardner (17) p r e s e n t e d t h e f i r s t i n s t r u m e n t a l q u a n t i t a t i v e method f o r a n a l y z i n g t h u j a p l i c i n s , i n v o l v i n g c o l o r i m e t r y of t h e f e r r i c c h e l a t e s o f t h e s e compounds. Johnson and C s e r j e s i (15) d e s c r i b e d a g a s - l i q u i d c h r o m a t o g r a p h i c method f o r a n a l y s i s of t r o p o l o n e s and t h u j a p l i c i n i s o m e r s . T h i s method was used (16) t o s t u d y t h e w e a t h e r i n g e f f e c t on t h u j a p l i c i n c o n c e n t r a t i o n s i n WRC shakes. Z a v a r i n and Anderson (35) and Z a v a r i n e t a l (36,37) d e s c r i b e d t h e paper chromatography of t r o p o l o n e s , and d e t e r m i n e d o p t i m a l c o n d i t i o n s f o r a n a l y s i s , and d i s c u s s e d c h a r a c t e r i z a t i o n of t r o p o l o n e s as d i c y c l o h e x y l a m i n e s a l t s . The decay r e s i s t a n c e o f WRC has been found t o be w i d e l y v a r i a b l e . B u c k l a n d (5) s t u d i e d and i d e n t i f i e d WRC decay organisms i n B r i t i s h Columbia, and found t h a t t h e e x t e n t of decay v a r i e d g r e a t l y between o l d and young t r e e s and from s t a n d t o s t a n d . E n g l e r t h and S c h e f f e r (11) s t u d i e d t h e decay r e s i s t a n c e of f o u r w e s t e r n s p e c i e s used as p o l e s , and f o u n d WRC t o be t h e most d u r a b l e . They a l s o showed t h a t t h e decay r e s i s t a n c e v a r i e d w i t h r a d i a l p o s i t i o n and h e i g h t i n t h e p o l e . MacLean and Gardner (18) f o u n d t h e same v a r i a t i o n i n t h e c o n c e n t r a t i o n o f t h u j a p l i c i n s i n WRC heartwoods, r a n g i n g from 0 t o 1.2%. MacLean and Gardner (19) a l s o s t u d i e d t h e c o n c e n t r a t i o n of t h u j a p l i c i n i n WRC " i n c l u d e d sapwood" and found i t t o be t h e same as r e g u l a r sapwood. R o f f e t a l (26) s t u d i e d WRC decay r e s i s t a n c e r e l a t i v e t o s e a s o n i n g and l o g o r i g i n . They found t h a t k i l n d r y i n g d i d not s i g n i f i c a n t l y 5 change decay r e s i s t a n c e , and t h a t decay r e s i s t a n c e v a r i e d w i t h r a d i a l p o s i t i o n . S c h e f f e r (29) s t u d i e d v a r i a t i o n s i n WRC d u r a b i l i t y and f o u n d l a r g e t r e e t o t r e e v a r i a t i o n s , and no r e l a t i o n s h i p s between d u r a b i l i t y and s i t e o r e l e v a t i o n . He a l s o r e p o r t e d t h a t decay r e s i s t a n c e i n c r e a s e d from p i t h t o o u t e r heartwood, t h e n d e c r e a s e d i n t h e sapwood. Swan and J i a n g (31) and Swan e t a l (32) d i s c u s s e d t h e f o r m a t i o n o f heartwood e x t r a c t i v e s i n WRC, ( i n c l u d i n g t h u j a p l i c i n s and t h u j i c a c i d ) and r e v i e w e d t h e i r a n a l y s i s by paper chromatography, t h i n l a y e r chromatography, gas chromatography and c o l o r i m e t r y . They a l s o p roposed a sequence f o r f o r m a t i o n o f l i g n a n s t h r o u g h h y d r o x y l a t i o n r e a c t i o n s i n w h i c h t h u j a p l i c i n s p l a y an i n h i b i t o r y r o l e . T h e i r a n a l y s i s o f a 90 y e a r o l d WRC (31) showed a maximum t h u j a p l i c i n c o n t e n t of 0.34% o c c u r r i n g a t 60 r i n g s from t h e p i t h , but t h e y d i d not a n a l y z e t h e complete r a d i u s . I n t h e i r a n a l y s i s of a 260 y e a r o l d WRC (32) t h e y found a maximum t h u j a p l i c i n c o n t e n t o f 0.42% a t 210 r i n g s from t h e p i t h , w i t h c o n c e n t r a t i o n s i n c r e a s i n g g r a d u a l l y from p i t h t o t h i s maximum, t h e n d e c r e a s i n g r a p i d l y i n t h e sapwood. B a r t o n and MacDonald (4) p r e s e n t e d a comprehensive r e v i e w of WRC c h e m i s t r y t o 1971. N a u l t (20) s t u d i e d t h e r a d i a l d i s t r i b u t i o n of t h u j a p l i c i n s i n second growth WRC by a m o d i f i c a t i o n o f t h e c o l o r i m e t r i c method of Maclean and Gardner ( 1 7 ) . He showed t h a t t h e d i s t r i b u t i o n of t h u j a p l i c i n i n second growth WRC f o l l o w e d t h e same p a t t e r n as i n o l d growth t r e e s , and t h a t comparable maximum l e v e l s were r e a c h e d i n b o t h . 6 R e c e n t l y , N a u l t (21) d e v e l o p e d a c a p i l l a r y g a s - l i q u i d c h r o m a t o g r a p h i c method f o r d e t e r m i n i n g b e t a and gamma t h u j a p l i c i n and t h u j i c a c i d . T h i s i s t h e method used i n t h e p r e s e n t s t u d y . The major r e s e a r c h p e r i o d f o r t h e t h u j a p l i c i n s and r e l a t e d compounds was 1948-63. More r e c e n t work has been by Johnson and C s e r j e s i (15,16), N a u l t (20,21), Swan e t a l (31,32) Coombs and T r u s t (8) and T r u s t and Coombs ( 3 3 ) . 7 MATERIALS AND METHODS B r e a s t h e i g h t samples from s i x f r e s h l y f e l l e d o l d growth WRC were o b t a i n e d from Western F o r e s t P r o d u c t s a t P o r t M c N e i l l , B.C. These t r e e s were c u t from a s t a n d about 11 k i l o m e t e r s west of P o r t M c N e i l l , and ranged from 260 t o 710 y e a r s o f age (Appendix 1 ) . Trees s e l e c t e d were f r e e from o b v i o u s s i g n s of b u t t r o t . A s e v e n t h o l d growth t r e e sample was o b t a i n e d from UBC F o r e s t r y . I t was c u t from a f r e s h l y f e l l e d WRC grown a t t h e UBC r e s e a r c h f o r e s t i n Haney, B.C. T h i s t r e e was 420 y e a r s o l d (Appendix 1 ) , and e x h i b i t e d o b v i o u s s i g n s o f decay i n t h e heartwood. A l l o l d growth samples o b t a i n e d were c u t i n t o s t r i p s a c r o s s t h e d i a m e t e r of t h e t r e e , about 10 cm wide and 5 cm deep. The sapwood was s e p a r a t e d from t h e s e s t r i p s and t h e remainder was c u t i n t o s e c t i o n s c o n s i s t i n g of enough growth r i n g s t o p r o v i d e s u f f i c i e n t m a t e r i a l f o r a n a l y s e s ( F i g u r e 1, Appendix 2 ) . The samples were a i r - d r i e d , t h e n ground t o pass a 50-mesh s c r e e n , w i t h a l l m a t e r i a l saved. The ground samples were e x t r a c t e d i n a s o x h l e t w i t h r e a g e n t grade e t h a n o l : b e n z e n e ( 1 : 2 ) , and t h e e x t r a c t i v e c o n t e n t was d e t e r m i n e d by weight l o s s . The e x t r a c t i v e s from each sample were c o n c e n t r a t e d on a r o t a r y e v a p o r a t o r , t h e n made up t o 25 ml. E t h a n o l : b e n z e n e (1:2) e x t r a c t i v e s from a p r e v i o u s s t u d y by N a u l t (20) r e p r e s e n t i n g r a d i a l b r e a s t h e i g h t samples from t e n second growth WRC from two s i t e s on Vancouver I s l a n d were a l s o 8 i n c l u d e d i n t h e s t u d y (Appendix 4, Appendix 5 ) . These samples and t h e i r e x t r a c t i v e s were p r e p a r e d i n a manner s i m i l a r t o t h a t d e s c r i b e d . However, i n t h e s e samples, heartwood and sapwood were n o t s e p a r a t e d . The outermost sample i n t h e s e r a d i i t h u s c o n t a i n e d heartwood and sapwood mixed i n v a r y i n g r a t i o s . Pure samples of b e t a and gamma t h u j a p l i c i n and t h u j i c a c i d were p r o v i d e d by F o r i n t e k Canada Corp. Diazomethane i n e t h e r was p r e p a r e d from n i t r o s o m e t h y l u r e a and p o t a s s i u m h y d r o x i d e (3 4 ) . P ure s t a n d a r d s were p r e p a r e d by d i s s o l v i n g t h e r e q u i r e d amount o f r e a g e n t i n eth a n o l / b e n z e n e ( 1 : 2 ) . The s t a n d a r d s were m e t h y l a t e d by a d d i t i o n of an e q u a l volume of e t h e r a l diazomethane, t h e n t h e s o l v e n t was e v a p o r a t e d a t 70 *C. The m e t h y l a t e d s t a n d a r d s were t h e n d i s s o l v e d i n eth a n o l / b e n z e n e (1:2) c o n t a i n i n g .00515 g/ml na p h t h a l e n e as an i n t e r n a l s t a n d a r d . A l l e x t r a c t i v e samples, pure s t a n d a r d s , and d e r i v a t i z e d samples were s t o r e d i n a da r k f r e e z e r t o m i n i m i z e d e g r a d a t i o n by h e a t o r l i g h t . A l l samples were a n a l y z e d on a S p e c t r a - P h y s i c s SP7100 gas chromatograph w i t h a d e d i c a t e d d a t a system and c a p i l l a r y c a p a b i l i t y . A 12M QC2/SE30 column w i t h 0.02mm ID and 0.25 micro m e t e r t h i c k c o a t i n g was used, w i t h h e l i u m as t h e c a r r i e r gas. Oven t e m p e r a t u r e was h e l d a t 110 aC f o r 10 min., t h e n r a i s e d t o 200 C a t 10*C/min. D e t e c t i o n was a c c o m p l i s h e d w i t h a flame i o n i z a t i o n d e t e c t o r . Peak a r e a d a t a were c o l l e c t e d on t h e d e d i c a t e d d a t a system, and s t a n d a r d c u r v e s were p r e p a r e d u s i n g 2 m i c r o l i t e r 9 sample i n j e c t i o n s . C a l i b r a t i o n c u r v e s were p r e p a r e d f o r b e t a and gamma t h u j a p l i c i n , and f o r t h u j i c a c i d u t i l i z i n g t h i s t e c h n i q u e . A l l e x t r a c t i v e samples were a n a l y z e d u s i n g t h e same p r o c e d u r e s as f o r t h e s t a n d a r d s . Because m e t h y l a t i o n o f t h u j i c a c i d y i e l d s m e t h y l t h u j a t e , t h e r e p o r t e d c o n c e n t r a t i o n s a r e a c t u a l l y t h e sum of t h e c o n c e n t r a t i o n s o f meth y l t h u j a t e and t h u j i c a c i d . D e t e r m i n a t i o n o f m e t h y l t h u j a t e c o u l d be performed e a s i l y by p r e p a r a t i o n of t h e e t h y l d e r i v a t i v e s , w h i c h would y i e l d e t h y l t h u j a t e from t h u j i c a c i d l e a v i n g m e t h y l t h u j a t e unchanged. A l l samples were a l s o a n a l y z e d c o l o r i m e t r i c a l l y f o r t h u j a p l i c i n c o n t e n t by t h e method of MacLean and Gardner ( 1 7 ) , as m o d i f i e d by N a u l t ( 2 0 ) . 10 RESULTS Seven o l d growth and t e n second growth WRC t r e e s were a n a l y z e d i n r a d i a l i n c r e m e n t s from p i t h t o b a r k (Appendices 3 and 6 ) . The et h a n o l : b e n z e n e (1:2) e x t r a c t i v e c o n t e n t i n a l l t r e e s f o l l o w e d a g e n e r a l p a t t e r n of i n c r e a s i n g from t h e p i t h t o t h e most r e c e n t l y formed heartwood, t h e n d e c r e a s i n g i n t h e sapwood ( F i g u r e s 2-4). A l l o l d growth t r e e s a t t a i n e d much h i g h e r e t h a n o l : b e n z e n e e x t r a c t i v e c o n t e n t s t h a n were e x h i b i t e d i n t h e second growth t r e e s . Maxima ranged from 12.4% t o 22.8% i n t h e o l d growth t r e e s , and from 6.2% t o 9.8% i n t h e second growth t r e e s . E x t r a c t i v e c o n t e n t s i n heartwoods seemed t o be r e l a t e d t o t r e e age ( F i g u r e 5 ) . T h u j a p l i c i n c o n t e n t s as d e t e r m i n e d by t h e c o l o r i m e t r i c method (20) agreed r o u g h l y w i t h t h o s e o b t a i n e d by GLC ( F i g u r e 6,Appendix 7 ) , y i e l d i n g an r ^ v a l u e of 0.81 and a s l o p e of 0.99 f o r GC yj5 c o l o r i m e t r i c r e s u l t s . C o l o r i m e t r i c v a l u e s tended t o be s l i g h t l y h i g h e r f o r second growth t r e e s and s l i g h t l y l o w e r f o r o l d growth t r e e s ( F i g u r e s 7-23). To s i m p l i f y r e p o r t i n g , a l l d a t a i n t h i s paper a r e based on t h e GLC r e s u l t s , w i t h t h u j a p l i c i n c o n t e n t b e i n g t h e sum of b e t a and gamma isomer f r a c t i o n s . T h u j a p l i c i n c o n t e n t i n t h e second growth t r e e s f o l l o w e d t h e p a t t e r n d e s c r i b e d by MacLean and Gardner ( 1 8 ) , t h a t i s , v e r y low c o n t e n t i n t h e i n n e r heartwood, w i t h a r a p i d i n c r e a s e i n t h e l a t e s t formed heartwood ( F i g u r e s 7-16). However, t h e o l d growth t r e e s d i d n o t demonstrate a s i m i l a r p a t t e r n ( F i g u r e s 17-22). The o l d growth t r e e s showed a p p r e c i a b l e t h u j a p l i c i n c l o s e t o t h e p i t h . The maximum l e v e l s a t t a i n e d by o l d growth t r e e s i n t h i s s t u d y were h i g h e r t h a n any r e p o r t e d i n t h e l i t e r a t u r e . T h u j a p l i c i n c o n t e n t s a l s o seemed t o be d i r e c t l y r e l a t e d t o t r e e age, w i t h o l d growth t r e e s h a v i n g a maximum o f 1.8% and second growth t r e e s h a v i n g a maximum o f 0.7%. A p l o t o f t h u j a p l i c i n s vg. r i n g s from p i t h shows t h i s t r e n d ( F i g u r e s 24 and 25). B e t a t h u j a p l i c i n and gamma t h u j a p l i c i n , when a n a l y z e d s e p a r a t e l y , a l s o e x h i b i t e d s i m i l a r t r e n d s ( F i g u r e s 26 t o 29). No r e l a t i o n s h i p s were o b s e r v e d f o r r e l a t i v e amounts o f b e t a and gamma t h u j a p l i c i n and d i s t a n c e from p i t h . T h u j a p l i c i n c o n t e n t s seemed t o be r e l a t e d a l s o t o e x t r a c t i v e c o n t e n t s p r e s e n t i n t h e samples ( F i g u r e 30). T h u j i c a c i d c o n t e n t s ( t h u j i c a c i d p l u s m e t h y l t h u j a t e ) seemed t o be r e l a t e d t o e x t r a c t i v e c o n t e n t s ( F i g u r e 31). No ob v i o u s r e l a t i o n s h i p t o t h u j a p l i c i n c o n t e n t i s seen ( F i g u r e 32), a l t h o u g h t h u j i c a c i d f o l l o w e d t h e same g e n e r a l p a t t e r n as t h u j a p l i c i n , i n c r e a s i n g from t h e p i t h t h r o u g h t h e o u t e r heartwood, t h e n d e c r e a s i n g i n t h e sapwood ( F i g u r e s 33-35). As w e l l , no o b v i o u s r e l a t i o n s h i p i s seen between t h u j i c a c i d and r i n g s from p i t h ( F i g u r e 36). The e x t r a c t i v e s from one second growth and one o l d growth t r e e a n a l y z e d w i t h r e p l i c a t i o n d emonstrate t h e r e p r o d u c i b i l i t y o f t h e GLC method ( T a b l e 1, F i g u r e s 37 and 38). F i g u r e 39 p r e s e n t s a summary o f t h u j a p l i c i n d i s t r i b u t i o n i n a l l 17 t r e e s s t u d i e d . T a b l e 2 p r e s e n t s a comparison o f r e s u l t s from t h i s s t u d y w i t h r e s u l t s o b t a i n e d by o t h e r r e s e a r c h e r s . DISCUSSION Eth a n o l : b e n z e n e e x t r a c t i v e c o n t e n t s i n t h e t r e e s s t u d i e d were g e n e r a l l y i n t h e ranges r e p o r t e d p r e v i o u s l y ( T a b l e 2 ) , a l t h o u g h some o l d growth t r e e s exceeded any l e v e l s p r e v i o u s l y r e p o r t e d . Tree t o t r e e v a r i a t i o n was h i g h , even i n t r e e s from t h e same s t a n d ( F i g u r e s 2 , 3 , 4 ) . A s u r p r i s i n g f i n d i s t h e g e n e r a l i n c r e a s e i n e x t r a c t i v e c o n t e n t w i t h r i n g s from p i t h ( F i g u r e 5 ) . T h i s e x p e r i m e n t i s t h e f i r s t s t u d y of t h u j a p l i c i n s i n WRC u t i l i z i n g c a p i l l a r y GLC. The method i s s e n s i t i v e t o low c o n c e n t r a t i o n s of t h u j a p l i c i n s , and r e q u i r e s no e x t e n s i v e sample p r e p a r a t i o n . The method g i v e s r e p e a t a b l e r e s u l t s , as t h e two t r e e s a n a l y z e d i n t r i p l i c a t e demonstrate ( F i g u r e s 37 and 38). T h i s method i s an improvement o v e r t h e method of Johnson and C s e r j e s i (15) i n t h a t g r e a t e r r e s o l u t i o n of t h e components i s p o s s i b l e due t o t h e use o f a c a p i l l a r y column. As w e l l , t h e d e r i v a t i v e p r e p a r a t i o n u s i n g diazomethane i s e a s i e r and l e s s t i m e consuming t h a n t h e i r method u s i n g s i l y l a t i o n . The d a t a c o l l e c t e d on t h e GC e x h i b i t e d s e v e r a l major peaks w h i c h have not y e t been i d e n t i f i e d . When t h e s e peaks a r e i d e n t i f i e d , and c a l i b r a t i o n c u r v e s p r e p a r e d , more i n f o r m a t i o n on t h e o v e r a l l p a t t e r n o f e x t r a c t i v e d i s t r i b u t i o n w i l l be a v a i l a b l e . The s m a l l volumes of sample r e q u i r e d f o r i n j e c t i o n (2 m i c r o l i t e r s ) and t h e h i g h s e n s i t i v i t y of t h e method s u g g e s t s t h a t by d e c r e a s i n g sample s i z e and e x t r a c t i o n volumes, v e r y s m a l l samples c o u l d be a n a l y z e d . T h i s i s a major advantage o v e r 13 t h e s t a n d a r d c o l o r i m e t r i c method, and c o u l d c o n c e i v a b l y be used t o a n a l y z e i n c r e m e n t c o r e s from s t a n d i n g t r e e s . Concerns have been e x p r e s s e d by t h e f o r e s t p r o d u c t s i n d u s t r y on t h e q u a l i t y of wood from our f u t u r e f o r e s t s , where i n t e n s i v e management w i l l r e s u l t i n h a r v e s t i n g t r e e s a t much younger ages t h a n has been t h e case f o r e x i s t i n g o l d - g r o w t h s t a n d s . T h i s s t u d y s u g g e s t s t h a t l e v e l of t h u j a p l i c i n s i n WRC i s r e l a t e d t o t h e age of t h e t r e e ( F i g u r e s 24 and 25). I n t h e case o f second growth s t a n d s , h a r v e s t i n g a t r e l a t i v e l y young ages may g i v e wood w i t h l o w e r c o n c e n t r a t i o n s of t h e s e compounds. T h i s c o u l d c o n c e i v a b l y y i e l d wood t h a t i s l e s s d u r a b l e t o decay and w e a t h e r i n g ( t r a d i t i o n a l c h a r a c t e r i s t i c s f o r w h i c h WRC wood has been v a l u e d ) . However, t h e h i g h v a r i a b i l i t y i n t h u j a p l i c i n c o n c e n t r a t i o n s i n o l d growth and second growth t r e e s s u g g e s t s t h a t t h e r e i s more i n v o l v e d t h a n s i m p l y t h e age of t h e t r e e . T h i s v a r i a b i l i t y i s c l e a r l y i l l u s t r a t e d by t h e d i f f e r e n c e s between second growth Trees #1.1 ( F i g u r e 7) and #1.2 ( F i g u r e 8 ) . Tree #1.1 has a maximum t h u j a p l i c i n c o n t e n t of 0.323% i n t h e l a s t sample o f pure heartwood, w h i l e Tree #1.2 has a maximum o f o n l y 0.108%, and r e a c h e d t h a t v a l u e a t o n l y 10-15 r i n g s from p i t h . I n t h e o l d growth t r e e s s t u d i e d , t r e e s #5 ( F i g u r e 21) and #7 ( F i g u r e 23) r e p r e s e n t extremes. Tree #5 a t t a i n e d a maximum of 1.77% t h u j a p l i c i n , w h i l e t r e e #7 had a maximum c o n t e n t o f o n l y 0.28%. T h i s agrees w i t h e a r l i e r r e s u l t s p r e s e n t e d by B u c k l a n d (5) and S c h e f f e r ( 2 9 ) , b o t h of whom found g r e a t v a r i a b i l i t y i n WRC wood decay. Perhaps t h e l e v e l o f 14 t h u j a p l i c i n i s a g e n e t i c a l l y c o n t r o l l e d p r o p e r t y , and t r e e s c o u l d be s e l e c t e d f o r p r o p a g a t i o n w i t h t h i s p r o p e r t y i n mind. As s t a t e d , t h e new GC t e c h n i q u e used w i t h n o n - d e s t r u c t i v e i n c r e m e n t c o r e s c o u l d a s s i s t i n such s e l e c t i o n . The maximum l e v e l s o f t h u j a p l i c i n r e p o r t e d h e r e , as up t o 1.8%, exceed any p r e v i o u s l y r e p o r t e d i n t h e l i t e r a t u r e f o r WRC. The p o s s i b i l i t y e x i s t s t h a t p r e v i o u s r e s e a r c h e r s had not p r o t e c t e d t h e i r e x t r a c t i v e samples from l i g h t , and t h a t p h o t o c h e m i c a l d e c o m p o s i t i o n of t h e t h u j a p l i c i n s had o c c u r r e d as d e s c r i b e d by Coombs and T r u s t ( 8 ) . T h i s would r e s u l t i n t h e r e p o r t e d v a l u e s b e i n g l o w e r t h a n t h e a c t u a l v a l u e s . A l s o , t h e c o l o r i m e t r i c method (17) assumes a c o n s t a n t r a t i o of b e t a and gamma t h u j a p l i c i n , w i t h gamma making up 60% of t h e t o t a l t h u j a p l i c i n s . Any d e v i a t i o n from t h i s r a t i o c o u l d change t h e absorbance maximum of t h e sample, and g i v e e r r o n e o u s r e s u l t s . S i n c e t h e samples s t u d i e d v a r i e d from 0% t o 90% gamma t h u j a p l i c i n (Appendices 3 and 6 ) , t h i s a s s u m p t i o n i s shown t o be f a l s e . However, t h e magnitude of t h e e r r o r t h a t t h i s d i f f e r e n c e i n r a t i o s would cause i s not p r e s e n t l y known. As w e l l , t h e p a t t e r n of f a i r l y r a p i d i n c r e a s e of t h u j a p l i c i n c o n c e n t r a t i o n i n t h e heartwood o f o l d growth t r e e s ( F i g u r e s 17 t h r o u g h 23) d i f f e r s from t h e work of MacLean and Gardner (18) and Swan and J i a n g ( 3 1 ) , who fo u n d v e r y low l e v e l s i n t h e o l d e s t heartwood of t h e t r e e s t h e y s t u d i e d . The one o l d growth WRC t r e e w h i c h d i d not have h i g h l e v e l s of t h u j a p l i c i n (Tree #7) was s e l e c t e d f o r s a m p l i n g on t h e b a s i s of e v i d e n t decay. The f a c t t h a t t h e t r e e showed s i g n s of decay 15 and low t h u j a p l i c i n c o n t e n t s u p p o r t s t h e t h e o r y t h a t t h u j a p l i c i n s p l a y a major r o l e i n WRC decay r e s i s t a n c e . Whether th e decay was caused by t h e absence of t h u j a p l i c i n s , o r t h e t h u j a p l i c i n s had been i n a c t i v a t e d by t h e decay organisms remains a q u e s t i o n f o r f u r t h e r r e s e a r c h . A l s o , i t i s noteworthy t h a t t h i s t r e e had r o u g h l y t h e same l e v e l s of t h u j i c a c i d as d i d t h e o t h e r o l d growth t r e e s ( F i g u r e 35). 16 CONCLUSION The new GLC method i s a s e n s i t i v e and c o n v e n i e n t method f o r a n a l y z i n g t h u j a p l i c i n s and a s s o c i a t e d compounds i n WRC e x t r a c t i v e s . The r e s u l t s o b t a i n e d by t h i s method a r e comparable t o t h o s e o b t a i n e d by c o l o r i m e t r i c a n a l y s i s . T h u j a p l i c i n c o n t e n t seemed t o be d i r e c t l y r e l a t e d t o t r e e age, a l t h o u g h t r e e t o t r e e v a r i a b i l i t y was found t o be h i g h . F u r t h e r r e s e a r c h t o det e r m i n e i f t h i s v a r i a b i l t i y i s a g e n e t i c a l l y c o n t r o l l e d p r o p e r t y c o u l d prove i n f o r m a t i v e . N o n - d e s t r u c t i v e i n c r e m e n t c o r e s would f u r n i s h enough m a t e r i a l f o r a n a l y s i s by t h e new GLC method. T h u j i c a c i d was found t o v a r y i n g e n e r a l l y t h e same p a t t e r n as t h u j a p l i c i n s . 17 LITERATURE CITED Anderson, A. B. and J . G r i p e n b e r g . 1948. A n t i b i o t i c s u b s t a n c e s from t h e heartwood o f Thu.ia p l i c a t a D. Don. IV. The c o n s t i t u t i o n o f t h u j a p l i c i n . A c t a . Chem. Scand. 2:644-650. A u l i n - E r d t m a n , G. 1950. S t u d i e s i n t h e t r o p o l o n e s e r i e s . 1. T h u j a p l i c i n s and n o o t k a t i n . A c t a . Chem. Scand. 4:1031-1041. B a r t o n , G. M. and J.A.F. Gardner. 1954. 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On t h e occ u r e n c e of a l p h a t h u j a p l i c i n o l i n t h e heartwood o f Cupressus pygmaea (Lemm.) Sarg. J . Org. Chem. 26:173-176. 21 TABLE 1. GC SAMPLES ANALYZED WITH REPLICATION SAMPLE AVERAGE THUJAPLICINS (% O.D.WOOD) NAME # RINGS TRIAL 1 TRIAL 2 TRIAL 3 AVERAGE FROM BETA GAMMA BETA GAMMA BETA GAMMA BETA GAM PITH SECOND 2.5 0 .03 0 .03 0 .03 0 .03 GROWTH 7.5 0 .02 0 .05 0 .04 0 .04 TREE 12.5 0 .05 0 .06 0 .07 0 .06 # 1.1 17.5 .07 . 13 .04 . 15 .08 . 13 .06 . 14 25 .15 .27 . 15 .25 . 16 .28 .15 .27 35 . 19 .24 . 21 . 28 .25 .29 . 22 . 27 45 .08 . 11 .08 . 13 .09 .21 .08 . 15 OLD 40 .06 .07 .07 .08 .02 .08 .05 .08 GROWTH 125 .08 .24 .04 .27 .08 .36 .07 .29 TREE 210 . 10 .50 .09 .44 .05 .48 .08 .47 # 3 310 . 12 .34 . 12 .33 .09 .31 . 11 .33 410 . 14 . 56 . 14 . 51 . 11 . 58 . 13 .55 475 .22 .38 . 16 . 39 . 15 .38 . 18 .38 558 . 30 .58 . 21 .58 . 18 .59 . 23 .58 610 .02 .04 .33 .04 .25 .03 .20 .04 TABLE 2, COMPARISON OF DATA WITH LITERATURE VALUES COMPONENT SOURCE ETHANOL:BENZENE OF DATA EXTRACTIVES (3) (16) (18) (19) (31) (32) NAULT 13. 8 6.2-22.8 THUJAPLICINS (%) 0.17-0.35 0-0.546 0-1.22 0-0.27 0-0.42 0.01-0.34 0.004-1.8 THUJIC ACID (%) 0.11-0.68 ANALYTICAL METHOD GRAVIMETRIC GC COLORIMETRY COLORIMETRY PAPER CHROM. COLORIMETRY 0.017-1.024 COLORIMETRY AND CAP. GC FIGURE 1. SAMPLING PROCEDURE BREAST HEIGHT RADIAL CROSS SECTION FIGURE 2. RADIAL DISTRIBUTION OF ETHANOL:BENZENE EXTRACTIVES FOR SECOND GROWTH SITE tfl O > 0 0 r ^ c O t / ) < 4 - r o c M (aOOM 3 3H J I 3AII,DVHIXa AH0-N3A0 NO a S S V H ) (%) S3AI10VH1X3 3N3ZN3g:iONVH,L3 FIGURE 3. RADIAL DISTRIBUTION OF ETHANOL BENZENE EXTRACTIVES FOR SECOND GROWTH SITE *2 o o > o o r ^ ( O i n ^ - K ) c > i (aOOM 33H3 3AI1DVHIX3 AHCI-N3A0 NO CI3SVg) (%) S3AII0VH1X3 3N3ZN39:10NVH13 FIGURE 4. RADIAL DISTRIBUTION OF ETHANOL:BENZENE EXTRACTIVES FOR OLD GROWTH TREES (aOOM 3 3 H 3 3AIJ,DVH,LX3 AHCI-N3AO NO a 3 S V 9 ) (%) S 3 A I I 0 V H X X 3 3 N 3 Z N 3 9 • 1 0 N V H I 3 2 4 2 2 H Q § 2 0 & w w a; w > w M > E- H-t U E - < u 03 < E - ttJ X £- W X w w 2 >- W 03 N l Q 2 I W 2 CQ W •• > 3 ° 3 § X E - Q W W < CQ 1 8 1 6 H 1 4 - J 1 2 - J 1 0 H 8 ^ 6 + + + + + + + + + + + + + 2 0 0 400 600 AVERAGE * RINGS FROM PITH HEARTWOOD SAMPLE + HEARTWOOD+SAPWOOD SAMPLE O t-i o a > M < - 3 Q O W t r 1 W t d n W 2 2 O INI m W 2 •n m w o w 2 X h a o w > f c-1 w o o 2 2 >-3 wk cokn 800 CO 00 27 (O Q O e CN>-t m - ' O S W E - « h-l td « > O t-H —o u u < CQ X W CO Z >H C0>-< « < Z o d z o Q W CO < CQ CM d i i — i — i — r co s «o m <t io T i — i — i — i — i — i — r « r ^ o> «o s 1 — 1 — r - . * "1 t 10 N ^ O - — - — - o o o o o o d d p (aOOM 33Hi 3AI10VHIX3 AH0-N3A0 NO (I3SVH) 00 A9 SNIOndVPflHI % FIGURE 7. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #1.1 HEARTWOOD + SAPWOOD AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) 1 1 60 ^COLORIMETRIC 80 FIGURE 8. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE _ RINGS FROM PITH FOR SECOND GROWTH TREE #1.2 © 0.8 8 w w — > o xli < 2 03 t - i E - U X i-t w J p.. >* < 03 » l ac z > o 0.7 - 0.6 - 0.5 " 0.4 - 0.3 - 0.2 - Z °o.i Q W W < 0 HEARTWOOD HEARTWOOD + SAPWOOD I 40 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) — i 1 60 ACOLORIMETRIC 80 0.1 - z o Q w CO < CQ — 0 HEARTWOOD HEARTWOOD + SAPWOOD F I G U R E 9 . T H U J A P L I C I N CONTENT B Y TWO METHODS ¥S A V E R A G E R I N G S FROM P I T H F O R S E C O N D GROWTH T R E E # 1 . 3 i W 0 . 7 W « fa 0.6 — > U0.S CO < Z « O Xo.4 M W •-3 QC 3 O l 33 Z H W >0.2 29 A V E R A G E # R I N G S FROM P I T H • B E T A ( B Y G C ) +GAMMA ( B Y G C ) O T O T A L ( B Y G C ) 1 1 60 ^ C O L O R I M E T R I C 60 F I G U R E 1 0 . T H U J A P L I C I N CONTENT B Y TWO METHODS V £ A V E R A G E R I N G S FROM P I T H F O R S E C O N D GROWTH T R E E # 1 . 4 HEARTWOOD + SAPWOOD 60 60 A V E R A G E # R I N G S FROM P I T H • B E T A ( B Y G C ) + G A M M A ( B Y G C ) O T O T A L ( B Y G C ) A C O L O R I M E T R I C z o a w CO < CQ 0 .1 - HEARTWOOD HEARTWOOD + SAPWOOD FIGURE 11. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE ~ RINGS FROM PITH FOR SECOND GROWTH TREE #1.5 § 0 . 8 S W 0 . 7 w OS fa — > ^ E-» CO < Z K n Eh U * 0 . 4 • W < ! OS n i s z E-< W > 0 . 2 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) — I 1 6 0 ^COLORIMETRIC 6 0 FIGURE 12. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.1 Q 0 . 8 O i _, feo.e H w — > ~ H D . 5 H CO < Z OS < « 0 . 3 - o an z > 0 . 2 O z 00.1 - Q W CO . < o HEARTWOOD HEARTWOOD + SAPWOOD 5 o AVERAGE # RINGS FROM PITH DBETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) 8 0 ^COLORIMETRIC FIGURE 13. THUJAPLICIN CONTENT BY TWO METHODS VS. AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.2 a o £ w w to 0 . B 0 . 7 - 0 . 6 " ^ &-1 0 . 3 - < OS * 0 . 4 H w CO z < 5h § 0 . 3 H I Z > 0 . 2 - O z O 0 . 1 -w CO < CQ HEARTWOOD HEARTWOOD + SAPWOOD AVERAGE * RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) i 60 ^COLORIMETRIC 6 0 FIGURE 14. THUJAPLICIN CONTENT BY TWO METHODS ¥S AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.3 Q 0 . B O £ w OS 0 . 6 -w ~> ~ £ o . 5 H CO < Z OS i - t U X j OS Q I z w >o .2 -o z °ai - Q W CO < 0 0 . 4 - Si •"3 ! 0 . 3 - HEARTWOOD HEARTWOOD + SAPWOOD 6 0 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) ^COLORIMETRIC FIGURE 15. THUJAPLICIN CONTENT BY TWO METHODS V£ AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2 .4 32 § 0 . 8 S WO.7 Cxi « HH U 0 . 5 CO < Z « M E- O X Q . 4 J ^ Q 0 . 3 D I X z E-> W ow*z 0 . 1 - z o D W CO < — o HEARTWOOD HEARTWOOD + SAPWOOD T 4 0 T AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) 1 1 6 0 ^COLORIMETRIC 6 0 FIGURE 16. THUJAPLICIN CONTENT BY TWO METHODS Vg AVERAGE RINGS FROM PITH FOR SECOND GROWTH TREE #2.5 0 . 4 Q 0 B O W W tf TA 0 . 6 W a* I—I w E- 0 . 8 O CQ < Z 05 M E-< O X M W - - < « 0 . 3 o i 3 3 Z * ^ 0 . 2 O O 0 . 1 - a w CO < CQ HEARTWOOD 20 ;HEARTWOOD I-+ SAPWOOD 6 0 AVERAGE # RINGS FROM PITH n BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) aCOLORIMETRIC FIGURE 17, 2 - I - §'•• * 1.8 W •, 7 to «1.6 to 1.5 1.4 w - E h ' ' 3 ° 1 2 2 « 1.1 u x i H O I ' OC 2 0.6 E- to >0.5 °0.4 o " Q0.2 < 0 THUJAPLICIN CONTENT BY TWO METHODS Vg AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #1 3 3 HEARTWOOD ' SAPWOOD — I — 400 — I — 600 800 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) ^COLORIMETRIC FIGURE 18. THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #2 Q 2 gl.» 1.8 H to i 7 to) « 1.6 to 1.5 ~ 2 . 1.4 H 3 CO < 1 , 2 2 « 1.1 O X 1 n W J 0.9 0<><fl. < P 4 ° - 8 D I ' £ 2 0.8 > 0.5 §0.3 H Q 0.2 W 0.1 CO < 0 0 200 H E A R T W O O D ! SAPWOOD 600 A V E R A G E * R I N G S FROM P I T H • B E T A ( B Y GC) +GAMMA ( B Y GC) O T O T A L ( B Y GC) *COLORIMETRIC HEARTWOOD ' SAPWOOD FIGURE 19. THUJAPLICIN CONTENT BY TWO METHODS Y£ AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #3 Ol.9 S 1 . B W 1 . 7 gi .e FE1.5 ^ ^ 1 . 4 - K H 1 . J " u l . 2 CO <, , ox: 1 £j W 0 . 9 a, ><O.B -3 Q 0 . 7 § 2=0 . 6 E - W O . 3 O 0 . 4 Z 0 . 3 ° 0 . 2 § 0 . 1 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) 34 B O O ACOLORIMETRIC FIGURE 20. THUJAPLICIN CONTENT BY TWO METHODS Vg AVERAGE ~ RINGS FROM PITH FOR OLD GROWTH TREE #4 B O O AVERAGE # RINGS FROM PITH DBETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) ACOLORIMETRIC FIGURE 21 THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #5 35 • BETA (BY GC) +GAMMA 200 AVERAGE # RINGS FROM PITH (BY GC) OTOTAL (BY GC) 800 ^COLORIMETRIC FIGURE 22 O 1 fl o 1 , s 3= 1.8 Oh 1.8 1.5 W . . r n O 1.2 CO < Z OS 1.1 O x 1 as zo.e >0.9 °0.4 §0.3 H0.1 CO „ < 0 CQ THUJAPLICIN CONTENT BY TWO METHODS VS AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #6 HEARTWOOD ' SAPWOOD — r -400 — i — 600 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) BOO ^COLORIMETRIC FIGURE 23. THUJAPLICIN CONTENT BY TWO METHODS Vg AVERAGE RINGS FROM PITH FOR OLD GROWTH TREE #7 o o Q 1.5 — > 1 W E - 1 - co < 1 * Z 05 1. M E -O X M W •J o. < OJ°-X20. E- W >0. °0. z o < 0 CQ HEARTWOOD' SAPWOOD T 1 T- , w • I • 1 J 0 200 400 600 800 AVERAGE # RINGS FROM PITH •BETA (BY GC) +GAMMA (BY GC) OTOTAL (BY GC) ^COLORIMETRIC i 1 i r 200 400 AVERAGE # RINGS FROM PITH HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLEO o a ro > W < W W - 3 325 O Q W > w l i—11> 2 O H kp as a w > o ^ s c- ha o »-3 Z O O O 2 " 3 > z s 800 CO -4 1 1 1 1 J 1.2 1.6 2 LOG (AVERAGE # RINGS FROM PITH) HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLEO r 2.4 T 2.8 CO 00 Q O i w w fa ^ > n 2 H o < •-H « J E - DH X < W S >< ac PS H Q I < 2 E - W W > CQ O 2 O Q W CO < CQ 0.7 0.6 - H 0.5 H 0.4 H 0.3 H 0.2 H 0.1 M a r o CO W W n W 2 H O > CO W a o - 9 a E C M 2 "J O O W O 2 > *-3 f 2 t=d co > < > a w T 1 r 200 400 600 AVERAGE # RINGS FROM PITH HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLEO 800 CO CO  200 400 600 AVERAGE # RINGS FROM PITH HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLEO 800 Q O O ^> i—i z e-« u < m 03 - 3 E-t Pu X < W 0 3 « H Q I < 2 2 O Q to CO < 1.4 1.3 H 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 -4 + + 4+ o 0.4 2 0.8 1.2 1.6 LOG (AVERAGE # RINGS FROM PITH) HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLE O + o o 1—*—I"' I l|0 2.4 2.8 DO z I-H o I—I • J < tD X E - 1.8 1.7 ~ 1 . 6 H 11.5 H » 1.4 H e i . 3 h e i . 2 h < Ed CQ E-" 1 1 O ' • 1 < E-" X w 0 . 9 >> § 0 . 8 i O0.6 1 H § 0 . 5 w 0.4 CO 0.2 - | 0.1 0 + 0 + $ + + + + + + + + + + + T T r • f - ~T~ 16 T I 4 8 12 ETHANOL:BENZENE EXTRACTIVES (%) (BASED ON OVEN-DRY EXTRACTIVE FREE WOOD) HEARTWOOD SAMPLE + SAPWOOD CONTAINING SAMPLEO 20 o a CO o W - 3 !x! X - 3 a •-3 f M O O z >-3 Z •-3 O O z •-3 M Z -3 wko t r *-3 ^ 3 Z o tr 1 tx) .. CO W w z tsi 24 •Ik. CO o o g W P=3 to W > t—i EH < Q CS 1-1 H O X ! < W U > • •-• « " 3 Q O I 33 2 EH W > O 2 O Q W CO < 4 8 12 16 ETHANOL:BENZENE EXTRACTIVES (%) (BASED ON OVEN-DRY EXTRACTIVE FREE WOOD) HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLE O r 20 24 0.6 0.8 1 THUJAPLICINS (%) (BASED ON OVEN-DRY EXTRACTIVE FREE WOOD) HEARTWOOD SAMPLE + SAPWOOD CONTAINING SAMPLEO Ot o FIGURE 34. RADIAL DISTRIBUTION OF THUJIC ACID CONTENT FOR SECOND GROWTH SITE #2 c x « - « - 0 ) o o r > > ( O i r ) ^ - r o c N « - o • • — * * • • • • • • • «-•- o o o o o o o o o (dOOM 33H3 3AII0VHIX3 AH0-N3A0 NO (I3SVS) (%) a i o v D i r n H X  i r 200 400 AVERAGE # RINGS FROM PITH HEARTWOOD SAMPLE+ SAPWOOD CONTAINING SAMPLEO 600 0.30 0.28 - _ 0 : 2 6 - 8 0.24 " 0 . 2 2 - \ 2 fa0.20 H w * £ 0 . 1 8 H zolo .16 H I—I £ - t O X 3 W 0 . 1 4 H Pu 3 q 0 . 1 2 H o i H ^ o . i o H o z 0.08 o g O . 0 6 H CQ 0.04 - 0.02 - 0 .00 BETA THUJAPLICIND AVERAGE Ht RINGS FROM PITH AVERAGE BETA^-0- GAMMA THUJAPLICIN+ 0.60 Q O s Eel W 03 td — > as n o CO < 2 03 t-t E-< o x; >• 03 Q I 2 0.50 0.40 H 0.30 H ft 0.20 H 2 o Q w CO < CQ 0.10 H 0.00 BETA THUJAPLICINn 200 400 AVERAGE # RINGS FROM PITH AVERAGE BETA^-^- GAMMA THUJAPLICIN+ 600 AVERAGE GAMMAA-A- FIGURE 39. BETA+GAMMA THUJAPLICIN CONTENT IS LOG (AVERAGE RINGS FROM PITH) FOR ALL TREES 5 2 o o o o o o d o o (CIOOM 3 3 H 3 3AII0VHIX3 AH0-N3A0 NO 0 3 S V 9 ) (%)NiondvrnHi vwwvo + v i s a APPENDIX 1. PHYSICAL DATA FOR OLD GROWTH TREES. SAMPLE SITE PORT M cN E I L L VANCOUVER ISLAND B.C. HANEY, B.C. TREE # DBH (cm) 1 2' 3 4 5 6 7 100. 8 115.6 134.0 135. 6 130.4 116.8 54.2 # RINGS HEARTWOOD/SAPWOOD (AGE) BOUNDARY (RINGS FROM PITH) 287 273 622 604 622 597 710 696 579 554 260 239 420 405 APPENDIX 2. OLD GROWTH SAMPLE DATA TREE # RINGS INCLUDED AVERAGE RINGS RADIUS TO IN SAMPLE FROM PITH LAST RING (cm) 1- 40 20 5.9 41-100 70 17.0 101-160 130 36. 5 161-220 190 41.9 221-250 235 46. 1 251-273 262 48.6 274-287 280 50.4 1-124 62 4.4 125-244 184 16. 6 245-334 289 26.6 335-424 379 36. 6 425-514 469 46. 5 515-574 544 53.1 575-604 589 56.4 604-622 613 58. 6 1- 80 40 3.5 81-170 125 17. 5 171-250 210 33.5 251-370 310 45. 8 371-430 400 52.8 431-520 475 58. 3 521-597 558 64.8 598-622 610 67.3 54 OLD GROWTH SAMPLE DATA TREE # RINGS INCLUDED AVERAGE RINGS RADIUS TO IN SAMPLE FROM PITH LAST RING (cm) 1-100 50 6.7 101-250 175 26.7 251-340 295 35. 2 341-430 385 43.6 431-520 475 50.4 521-610 565 55.8 611-670 640 62.5 671-696 683 65.3 697-710 703 67.7 1-134 67 4.9 135-224 179 14.2 225-284 254 26.5 285-344 314 42.0 345-434 389 54.5 435-494 464 58.5 495-524 509 60. 5 525-554 539 62.5 555-579 566 63.8 1- 40 20 6.1 41-100 70 17. 6 101-160 130 35.9 161-190 175 47.4 191-220 205 52. 7 221-239 230 56. 3 240-260 250 59. 1 1- 30 15 4.8 31-150 90 10. 1 151-240 195 15.1 241-300 270 19. 1 301-360 330 23.1 361-390 375 24.6 391-405 397 25. 9 406-420 412 27. 1 55 APPENDIX 3. EXPERIMENTAL DATA FROM OLD GROWTH TREES (ALL RESULTS % OVEN DRY EXTRACTIVE FREE WOOD) AVE. # EtOH: BETA GAMMA BETA+ GAMMA COLOR. THUJIC RINGS BENZ. THUJA- THUJA- GAMMA /TOT. THUJA- ACID + FROM EXTR. PLICIN PLICIN THUJA- PLICIN METHYL PITH PLICIN THUJATE 20 1.9 .034 .001 .034 .02 . 170 . 198 70 6.5 . 133 .204 .337 .60 .400 .457 130 7.5 . 179 .281 .460 .61 .454 .367 190 9.6 . 194 .349 .543 .64 .465 .494 235 13.2 . 145 .419 . 564 .74 .635 . 586 262 13.0 .135 .520 .655 .79 .702 .573 280 1.7 .033 .067 . 100 .67 . 153 .097 62 4.5 .209 .039 .248 . 16 . 243 .488 184 10. 1 . 220 . 382 .602 .63 . 528 . 527 289 15.2 .135 .732 . 867 .84 .772 . 465 379 13.4 . 134 .718 . 852 . 84 . 794 . 688 469 13.1 .223 .747 .970 .77 .952 1.024 544 14.0 . 134 .409 . 543 . 75 1.079 .671 587 15.6 . 164 .809 .974 . 83 1. 171 .960 615 1.1 .021 .036 .057 .63 . 114 .017 40 3.2 .007 .061 .068 . 90 . 178 . 304 125 5.9 .090 . 288 .377 .76 . 347 .429 210 7.4 .098 .254 .352 .72 .474 . 280 310 11.2 . 381 . 382 . 763 . 50 . 556 . 298 410 13.3 .172 .693 . 865 .80 .763 .388 475 15.0 . 507 .240 . 747 . 32 .728 . 382 558 16. 8 .342 . 578 . 919 . 63 1.039 . 387 610 1.1 .021 .030 .051 . 58 .091 .023 50 7.2 . 119 .201 . 320 . 63 .324 .490 125 13.0 . 218 .488 . 706 . 69 . 662 . 554 295 16.4 . 142 . 548 . 690 .79 .666 .447 385 15.4 . 252 .318 . 570 . 56 .422 . 306 475 19.0 .205 .417 .622 . 67 .658 .320 565 16. 8 . 197 . 181 . 377 .48 . 325 . 238 640 17. 6 .106 .469 . 575 .82 .728 . 401 683 17. 1 . 165 .456 . 621 . 73 .883 .475 703 .6 .007 .008 .015 . 51 .081 .022 56 EXPERIMENTAL DATA FOR OLD GROWTH TREES (ALL RESULTS % OVEN DRY EXTRACTIVE FREE WOOD) AVE. # EtOH: BETA GAMMA BETA+ GAMMA COLOR. THUJIC RINGS BENZ. THUJA- THUJA- GAMMA /TOT. THUJA- ACID + FROM EXTR. PLICIN PLICIN THUJA- PLICIN METHYL PITH PLICIN THUJATE 67 6.3 .024 .000 .024 .00 . 127 .660 179 10.0 . 166 .072 .238 .30 .680 . 920 254 9.8 . 204 . 148 . 352 .42 .872 .793 314 10.0 .219 . 173 .392 .44 .831 .553 389 22.8 . 321 .294 .615 .48 1.000 .794 464 15.7 .466 1.308 1.774 .74 1.216 .868 509 17.0 .502 1.240 1.742 .71 1.186 .797 539 15.8 .270 .437 .707 .62 1.063 .679 566 2.7 .062 .010 .072 . 14 . 134 .071 6 20 2.9 70 6.4 130 7.8 175 11.2 205 12.4 238 12.4 250 2.4 7 15 2.4 90 8.0 195 9.6 270 9.4 330 14.1 375 15.4 383 16.2 412 4.2 .014 .000 . 187 . 146 .347 .260 .469 .372 .587 . 813 .653 .663 .055 .070 .040 .000 .044 .000 .078 .016 .020 .000 .004 .000 . 121 .000 . 201 .077 .030 .002 .014 .00 .333 .44 .607 .43 .840 .44 1.400 .58 1.315 .50 .125 .56 .040 .00 .044 .00 .093 .17 .020 .00 .004 .00 .121 .00 .278 .28 .032 .06 .161 .350 .480 .936 .647 .646 .954 .694 1.131 .603 1.189 .613 .139 .036 .061 .306 .075 .623 .109 .545 .065 .537 .082 .728 .102 .632 .228 .561 .088 .127 APPENDIX 4. PHYSICAL DATA FOR SECOND GROWTH TREES SAMPLE SITE TREE # DBH (cm) # RINGS (AGE) HORNE LAKE, 1.1 46.8 50 VANCOUVER 1.2 44.5 44 ISLAND, B.C. 1.3 37.8 52 1.4 51.6 51 1.5 44.9 63 SHAWNIGAN 2.1 64.6 68 LAKE, 2.2 63.7 52 VANCOUVER 2.3 56.0 71 ISLAND, B.C. 2.4 47.8 42 2.5 52.7 77 HEARTWOOD/SAPWOOD BOUNDARY (RINGS FROM PITH) 44 39 46 46 58 60 46 64 36 72 58 APPENDIX 5. SECOND GROWTH SAMPLE DATA TREE # RINGS INCLUDED AVERAGE RINGS RADIUS TO IN SAMPLE FROM PITH LAST RING (cm) 1- 5 2.5 2.0 6- 10 7.5 4.8 11- 15 12.5 8.3 16- 20 17. 5 11.5 21- 30 25 14. 7 31- 40 35 17.4 41- 50 45 20.3 1- 5 2.5 3.3 6- 10 7.5 5.6 11- 15 12. 5 7.2 16- 20 17.5 9.0 21- 30 25 12.3 31- 44 35 16.2 1- 5 2.5 1.4 6- 10 7.5 3.7 11- 15 12.5 5.8 16- 20 17.5 7.0 21- 30 25 10.4 31- 40 35 14. 5 41- 52 45 19. 2 1- 5 2.5 1.5 6- 10 7.5 3.1 11- 15 12.5 5.2 16- 20 17.5 8.4 21- 30 25 15.9 31- 40 35 20. 1 41- 51 45 23.5 1- 20 10 3.0 21- 25 22. 5 5.2 26- 30 27. 5 8.5 31- 35 32.5 11.1 36- 40 37. 5 12. 6 41- 50 45 16.0 51- 63 55 19.7 59 SECOND GROWTH SAMPLE DATA TREE # RINGS INCLUDED AVERAGE RINGS RADIUS TO IN SAMPLE FROM PITH LAST RING (cm) 1- 25 12.5 3.7 26- 30 27. 5 6.3 31- 35 32.5 8.2 36- 40 37. 5 12.3 41- 45 42.5 14.9 46- 50 47.5 21.1 51- 68 55 30.3 1- 10 5 1.9 11- 15 12.5 5.0 16- 20 17. 5 8.0 21- 30 25 13.1 31- 40 35 20.2 41- 52 45 30.1 1- 25 12. 5 3.4 26- 30 27.5 5.0 31- 35 32. 5 7.5 36- 40 37.5 10.3 41- 45 42. 5 13.3 46- 56 50 17.3 57- 66 60 22. 3 1- 5 2.5 1.3 6- 10 7.5 3.5 11- 15 12.5 6.5 16- 20 17. 5 8.8 21- 30 25 14. 2 31- 42 35 19.2 1- 60 30 10.2 61- 65 62. 5 12. 7 66- 70 67.5 15. 8 71- 77 72. 5 19. 8 60 APPENDIX 6. EXPERIMENTAL DATA FOR SECOND GROWTH TREES (ALL RESULTS % OVEN DRY EXTRACTIVE FREE WOOD) TREE AVE. # EtOH: BETA GAMMA BETA+ GAMMA COLOR. THUJIC # RINGS BENZ. THUJA- THUJA- GAMMA /TOT. THUJA- ACID + FROM EXTR. PLICIN PLICIN THUJA- PLICIN METHYL PITH PLICIN THUJATE 2.5 3.3 .004 .010 .014 . 72 .086 . 128 7.5 3.9 .002 .010 .011 .85 . 112 .310 12. 5 3.6 .013 .044 .058 .77 . 147 . 216 17.5 5.7 .036 . 108 .145 .75 . 197 .331 25 8.0 .055 .225 . 280 .80 .488 .703 35 9.2 .080 .243 .323 .75 .525 .618 45 5.0 .014 .077 .091 .85 . 222 . 246 2.5 3.6 .024 .032 .055 .57 . 109 .312 7.5 5.1 .032 .054 .086 .63 . 148 .477 12.5 7.4 .036 .073 .108 .67 . 180 . 884 17.5 5.4 .034 .047 .081 . 59 . 193 .612 25 5.4 .018 .016 .034 .46 .229 .843 35 6.3 .016 .042 .058 .73 . 191 . 394 2.5 5.2 .016 .005 .021 .25 . 125 . 172 7.5 4.6 .058 .000 .058 .00 . 164 .445 12.5 5.3 .059 .001 .060 .02 . 210 .446 17. 5 7.2 . 108 .003 . I l l .03 . 315 . 600 25 7.4 . 162 .028 .190 . 15 .348 . 650 35 9.8 . 303 .063 . 366 . 17 .481 . 824 45 8.6 . 200 .024 .224 . 11 . 368 .504 2.5 4.8 .021 .014 .035 .40 .078 . 286 7.5 5.0 .042 .028 .070 .40 . 116 . 579 12. 5 4.6 .054 .022 .077 .29 . 108 . 526 17.5 4.6 .044 .016 .060 .27 . 115 .393 25 8.2 . 157 . 107 .264 . 40 . 325 . 772 35 9.7 .292 .233 .525 .44 .544 .989 45 6.1 .095 .060 . 156 .39 . 194 . 361 10 2.8 .022 .000 .022 .00 .030 .069 22.5 3.3 .058 .000 .058 .00 .050 , 157 27. 5 3.8 .089 .023 . 113 . 21 . 115 . 242 32. 5 5.4 . 122 .047 . 169 .28 . 245 .407 37.5 7.0 .226 . 150 .376 .40 .390 .580 45 8.3 . 194 . 154 .348 .44 .511 .661 55 9.3 .242 . 184 .426 .43 .710 .516 61 EXPERIMENTAL DATA FOR SECOND GROWTH TREES (ALL RESULTS % OVEN DRY EXTRACTIVE FREE WOOD) TREE AVE. EtOH: BETA GAMMA BETA+ GAMMA COLOR. THUJIC # RINGS BENZ. THUJA- THUJA- GAMMA /TOT. THUJA- ACID + FROM EXTR. PLICIN PLICIN THUJA- PLICIN METHYL PITH PLICIN THUJATE 2.1 12. 5 3.2 .043 .000 .043 .00 .058 . 215 27.5 4.9 .024 .073 .097 . 75 . 222 .395 32. 5 5.7 .052 . 109 . 161 .68 . 284 . 383 37.5 5.4 .074 . 170 .244 .70 .299 .357 42. 5 9.5 . 125 .341 .467 .73 . 511 .407 50 9.3 .166 .240 .406 .59 .524 .295 60 6.9 . 137 . 299 .436 .69 .397 . 144 2.2 5 3.6 .079 .019 .099 .20 . 137 .205 12. 5 3.9 .094 .040 . 134 . 30 . 128 . 282 17.5 4.3 .152 .034 . 186 . 18 .240 .305 25 6.0 . 206 .044 .249 . 18 . 350 .444 35 6.1 .376 .040 .415 . 10 .466 .290 45 8.2 .315 .000 .315 .00 .622 .232 2.3 12.5 3.7 .027 .006 .033 . 18 .082 . 338 27. 5 4.7 .028 .018 .046 . 39 . 151 . 327 32.5 5.2 .055 .024 .080 . 30 .250 .438 37. 5 6.3 . 189 .016 . 205 .08 . 266 . 614 42.5 7.5 .171 .016 . 188 .09 .389 . 616 50 8.9 . 152 .034 . 186 . 18 . 338 .427 60 8.6 . 300 .027 .327 .08 . 594 . 686 2.4 2.5 4.0 .041 .017 .058 . 29 .084 . 116 7.5 2.4 .007 .000 .007 .05 . 020 .026 12. 5 2.8 .019 .011 .030 . 37 .038 .064 17.5 5.9 .070 .061 . 130 . 46 .238 . 305 25 6.2 .098 .056 . 154 . 36 .299 . 317 35 6.0 . 135 .035 . 169 .20 . 284 . 302 2.5 30 5.2 .054 .046 .099 . 46 . 146 . 392 62.5 6.8 .108 . 105 .213 .49 .355 .440 67. 5 6.3 .046 . I l l . 157 .70 . 336 .444 72.5 6.9 .181 .070 .250 .28 .349 .404 APPENDIX 7. LINEAR REGRESSION STATISTICS FOR GC vs COLORIMETRIC RESULTS SLOPE 994 INTERCEPT CORRELATION COEFFICIENT .062 . 905 819

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