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Selected metals in earthworms, lettuce and soil amended with sewage sludge Kenney, Elizabeth Anne 1983

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SELECTED METALS IN EARTHWORMS, LETTUCE AND SOIL AMENDED WITH SEWAGE SLUDGE by ELIZABETH ANNE KENNEY (nee HAYES) B.Sc. hons . , Dalhousie U n i v e r s i t y , 1976 •> A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of S o i l Science) We accept th i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1983 © E l i z a b e t h Anne Kenney, 1983 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 a d v a n c e d d e g r e e 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 a g r e e 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 a g r e e 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 r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t 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 . D e p a r t m e n t o f The 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 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Ear thworms ( L u m b r i c u s r u b e l l u s , A p o r r e c t o d e a s p p . and O c t o l a s i o n  cyaneum) were k e p t i n s o i l t r e a t e d w i t h s i x a p p l i c a t i o n r a t e s o f m i l o r g a n i t e . A f t e r t e n d a y s , the e a r t h w o r m s , t h e i r f a e c e s and the s o i l were a n a l y z e d f o r cadmium, c o p p e r , l e a d and z i n c . Cadmium and Zn were c o n c e n t r a t e d i n the worm t i s s u e o f a l l t h r e e t a x a , o v e r s o i l l e v e l s , w h e r e a s Cu and Pb were n o t . Cadmium c o n c e n t r a t i o n s i n t h e body t i s s u e i n c r e a s e d w i t h i n c r e a s i n g s o i l C d , u n t i l s o i l c o n c e n t r a t i o n s r e a c h e d 7 ug /gm, a f t e r w h i c h the body t i s s u e c o n c e n t r a t i o n s l e v e l l e d o u t . The body t i s s u e Cd and Zn c o n c e n t r a t i o n s e x c e e d e d the c o n c e n t r a t i o n o f t h e s e m e t a l s i n t h e f a e c e s . The f a e c e s had h i g h e r Cu and Pb c o n c e n t r a t i o n s t h a n t h e body t i s s u e . The ear thworms a p p e a r t o be c a p a b l e o f a c c u m u l a t i n g Cd and r e g u l a t i n g body t i s s u e c o n c e n t r a t i o n s o f C u , Pb and Z n . T h e r e were d i f f e r e n c e s among e a r t h w o r m t a x a i n t h e m e t a l c o n c e n t r a t i o n s i n b o t h body t i s s u e s and f a e c e s . A p o r r e c t o d e a s p p . had t h e l o w e s t body t i s s u e Cd c o n c e n t r a t i o n s , as w e l l a s , had l o w e r f a e c a l Cd c o n c e n t r a t i o n s t h a n L . r u b e l l u s . Body t i s s u e Cd c o n c e n t r a t i o n s f o r _L. r u b e l l u s and (). cyaneum were not d i f f e r e n t . Copper and Pb c o n c e n t r a t i o n s i n the body t i s s u e o f 0. cyaneum were g r e a t e r t h a n t h o s e f o r the o t h e r two t a x a , w h i c h d i d no t d i f f e r s i g n i f i c a n t l y i n t h e i r Cu and Pb c o n c e n t r a t i o n s . L u m b r i c u s r u b e l l u s had h i g h e r Cu l e v e l s i n i t s f a e c e s t h a n d i d A p o r r e c t o d e a s p p . , w h e r e a s t h e r e were no d i f f e r e n c e s i n the f a e c a l Pb c o n c e n t r a t i o n s f o r t he t h r e e t a x a . Z i n c c o n c e n t r a t i o n s - i i i -were d i f f e rent i n the body t i ssues of a l l three taxa and was highest i n the Aporrectodea spp. The l a t t e r taxon also had the lowest faeca l Zn concentrat ions . Earthworms might be useful b i o l o g i c a l monitors of Cd p o l l u t i o n , but not for Cu, Pb and Zn, i n s o i l s r ece iv ing sewage sludge. In the experiment on the poss ib le e f fects of mi lorgani te and earthworms on growth and on mtal uptake by l e t t u c e , the add i t ion of the sewage sludge resul ted i n a s i g n i f i c a n t increase i n plant y i e l d . The cadmium and z inc concentrations i n lea f and root t i s sue were s i g n i -f i c a n t l y increased by milorganite addi t ions and n i c k e l concentrations were decreased. Copper concentrations were unaffected by the sludge. Mean lead concentrations were higher i n the mi lorgani te treatments. However, the v a r i a b i l i t y was great and there was not s i g n i f i c a n t d i f f e rences . The add i t ion of mi lorgani te to the s o i l re su l ted i n increased concentrations of diethylene triamine penta ace t i c ac id (DTPA) extractable metals. At an a p p l i c a t i o n rate of 20 gm mi lorgani te /kg s o i l , DTPA extractable Cu, Ni and Zn were s i g n i f i c a n t l y corre la ted with l e t tuce lea f concentrations for these 3 metals. However, at the higher mi lorgani te a p p l i c a t i o n rate of 50 gm/kg, only DTPA extractable Zn was s i g n i f i c a n t l y corre la ted with plant t i s sue concentrat ions . The add i t ion of earthworms to the s o i l d id not a f fec t l e t tuce y i e l d s or the concentrations of DTPA extractable metals. Lead concentrations i n the roots were s i g n i f i c a n t l y lower when earthworms were present i n the s o i l . Earthworms did not a f fect the concentrations of Cd, Cu, Ni and Zn i n le t tuce t i s s u e s . - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES i x LIST OF PLATES x ACKNOWLEDGEMENTS x i i General Introduction 1 Sewage Sludge Application to Land - A B r i e f Overview References 9 PART 1: Selected Metal Levels i n Three Taxa of Earthworms (Lumbricidae) Kept i n S o i l Contaminated by Milorganite 13 1. Introduction and Literature Review 14 2. Materials and Methods 19 2.1 Sample Preparation and Chemical Analysis 19 2.2 S t a t i s t i c a l Analysis 29 3. Results 34 3.1 Metal Concentrations i n Milorganite, S o i l , Earthworms and Faeces 34 3.2 Cadmium 51 3.3 Copper 56 3.4 Lead 63 3.5 Zinc 68 4. General Results and Discussion 75 4.1 S o i l Metal Concentrations 75 4.2 S o i l Metal Concentrations - Comparisons with Other Studies 77 4.3 Body Weights 79 4.4 Earthworms as B i o l o g i c a l Indicators of Metal Contamination 85 5. Conclusions 91 6. References 93 TABLE OF CONTENTS Page P A R T 2: The Uptake of Cadmium, Copper, Lead, Ni c k e l , and Zinc by Lettuce (Lactuca sativa L.) Grown i n S o i l Amended with Milorganite and i n the Presence of Earthworms 99 1. Introduction and Literature Review 100 2. Materials and Methods 104 2.1 Experimental Design and Preparation 104 2.2 Sample Preparation and Chemical Analysis 105 2.3 S t a t i s t i c a l Analysis 108 3. Results 109 3.1 Survival and A c t i v i t y of the Earthworms 109 3.2 Y i e l d of Lettuce Tissue 109 3.3 Metal Concentrations i n Milorganite 109 3.4 Metal E x t r a c t a b i l i t y and Correlations with Lettuce Metal Content 115 3.5 Metal Concentrations i n Lettuce Tissues 119 3.6 Metal Concentrations i n the S o i l 121 3.7 S o i l Properties 122 4. Discussion • 124 4.1 Y i e l d 124 4.2 Extractable Metals 126 4.3 Correlations Between Extractable Metals and Lettuce Metal Concentrations 127 4.4 Lettuce Cadmium Concentrations 130 4.5 Lettuce Copper Concentrations 133 4.6 Lettuce Nickel Concentrations 135 4.7 Lettuce Lead Concentrations 137 4.8 Lettuce Zinc Concentrations 138 4.9 E f f e c t s of Earthworms on Lettuce Metal Concentration 139 5. General Discussion 141 6. Conclusions 144 7. References 146 - v i -LIST OF TABLES - PART 1 Page Table 1: Diagnostic characteristics of four earthworm species 22 Table 2: Instrument parameters for HGA 26 Table 3: Analysis of N.B.S. Orchard Leaf Standard 27 Table 4: Analysis of N.B.S. Liver Standard 27 Table 5: Metal concentration (ug/gm dry weight) in milorganite 34 Table 6: Metal concentrations (ug/gm dry weight) in bulk Ap sample 34 Table 7: Selected chemical and physical properties of Ap samples 35 Table 8: Cadmium concentration (ug/gm dry weight) in s o i l , body tissue and faeces of three earthworm taxa from six milorganite application rates 36 Table 9: Copper concentrations (ug/gm dry weight) in s o i l , body tissue and faeces of three earthworm taxa from six milorganite application rates 37 Table 10: Lead concentrations (ug/gm dry weight) in s o i l , body tissue and faeces of three earthworm taxa from six milorganite application rates 38 Table 11: Zinc concentrations (ug/gm dry weight) in s o i l , body tissue and faeces of three earthworm taxa from six milorganite application rates 39 Table 12: ANOVA Table for cadmium in earthworm body tissue (5% probability level) 43 Table 13: ANOVA Table for cadmium in earthworm faeces (5% probability level) 43 Table 14: ANOVA Table for copper in earthworm body tissue (5% probability level) 44 Table 15: ANOVA Table for copper in earthworm body faeces (5% probability level) 44 Table 16: ANOVA Table for lead in earthworm body tissue (5% probability level) 45 - v i i -Page Table 17: ANOVA Table for lead i n earthworm faeces (5% p r o b a b i l i t y l e v e l ) 45 Table 18: ANOVA Table for z inc i n earthworm body t i s sue (5% p r o b a b i l i t y l eve l ) 46 Table 19: ANOVA Table for z inc i n earthworm faeces (5% p r o b a b i l i t y l e v e l ) 46 Table 20: S i g n i f i c a n t groupings (5% p r o b a b i l i t y l e v e l ) of mi lorgani te a p p l i c a t i o n rates (p.g/kg) for Cd concentrations i n 3 earthworm taxa boddy t i s sue and faeces 47 Table 21: S i g n i f i c a n t groupings (5% p r o b a b i l i t y l e v e l ) of mi lorgani te a p p l i c a t i o n rates (|j,g/kg) for Cu concentrations i n 3 earthworm taxa boddy t i s sue and faeces 48 Table 22: S i g n i f i c a n t groupings (5% p r o b a b i l i t y l e v e l ) of mi lorgani te a p p l i c a t i o n rates (|ag/kg) for Pb concentrations i n 3 earthworm taxa boddy t i s sue and faeces 49 Table 23: S i g n i f i c a n t groupings (5% p r o b a b i l i t y l eve l ) of mi lorganite a p p l i c a t i o n rates (p,g/kg) for Zn concentrations i n 3 earthworm taxa boddy t i s sue and faeces 50 Table 24: Proposed Ontario gu ide l ines for maximum metal concentrations i n a g r i c u l t u r a l s o i l s and maximum metal addi t ions i n sewage sludges 76 2 Table 25: Regression Equations and r values r e l a t i n g body dry weights (mg) and element contents (\xg) for Lumbricus rubel lus for s ix mi lorgani te t rea tment . . . 80 2 Table 26: Regression Equations and r values r e l a t i n g body dry weights (mg) and element contents (p.g) for Aporrectodea spp. for s ix mi lorgani te t r e a t m e n t . . . . 81 2 Table 27: Regression Equations and r values r e l a t i n g body dry weights (mg) and element contents (p.g) for Octo las ion cyaneum for s ix mi lorgani te t rea tment . . . 82 Table 28: S o i l metal concentrations and concentrat ion factors of Cd, Cu, Pb, Zn, for three taxa of earthworms 87 - v i i i -LIST OF TABLES - PART 2 Page Table 1: Fresh and dry weight ( 8 0 ° C ) and standard dev ia t ion fo r l e t tuce lea f t i s sue harvested a f ter 2 months exposure to treatment 113 Table 2: T o t a l cadmium, copper, l ead , n i c k e l and z i n c concentrat ions (p-g/gm) i n l e t tuce l ea f t i s sue grown i n s o i l amended with 20 gm mi lorgan i te /kg s o i l , the mi lorgani te and s o i l 113 Table 3: T o t a l and extractable s o i l cadmium, copper, l ead , n i c k e l and z inc concentrations ([ig/gni ± 1 s td . dev.) extractable metals also expressed as percentage of t o t a l s o i l metal concentrat ion 114 Table 4: C o r r e l a t i o n c o e f f i c i e n t s between d i f f e ren t s o i l extractants and metal l eve l s i n l e t tuce l e a f s , grown i n s o i l amended with 20 gm mi lorgan i te /kg s o i l 114 Table 5: DTPA extractable metals (ng/gm dry wt ± 1 S.D) i n s o i l a f ter 2 months exposure to treatments 116 Table 6: C o r r e l a t i o n Coe f f i c i en t s (r values) for metal concentrat ion i n l e t tuce leaves , t o t a l s o i l metal concentrat ion and DTPA extractable metals , with and without mi lorgani te added to the s o i l . 116 Table 7: Metal Concentrations (|ig/gm dry wt ± 1 S .D.) i n le t tuce leaves a f ter 2 months exposure to treatment 118 Table 8: Metal concentrations (jig/gm dry wt. ± 1 S .D.) i n le t tuce roots a f ter 2 months exposure to treatment. 118 Table 9: T o t a l metal concentrations ((ig/gm dry wt. ± 1 S .D. ) i n s o i l a f ter 2 months exposure to treatments 120 Table 10: Selected s o i l proper t ie s of the bulk Ap and of an Ap hor izon from the Cresent S o i l Series 123 - i x -LIST OF FIGURES - PART 1 Page Figure 1: E f f e c t s of sewage sludge a p p l i c a t i o n on cadmium l e v e l s i n tissue and faeces of mature adults of Lumbricus rubellus 40 Figure 2: E f f e c t s of sewage sludge a p p l i c a t i o n on copper le v e l s i n tissue and faeces of mature adults of Lumbricus rubellus 41 Figure 3: E f f e c t s of sewage sludge a p p l i c a t i o n on cadmium lev e l s i n tissue and faeces of mature adults of Lumbricus rubellus 42 - x -LIST OF PLATES - PART 1 Page P la te 1: C l i t e l l a t e specimen of Lumbricus rube l lu s 20 Plate 2: C l i t e l l a t e specimen of Octo la s ion cyaneum 20 Plate 3: Non c l i t e l l a t e specimen of Aporrectodea spp 21 - x i -LIST OF PLATES - PART 2 Page P l a t e 1: A e s t i v a t i n g e a r t h w o r m s . The t a x o n , on the l e f t i s A p o r r e c t o d e a s p p . and O c t o l a s i o n cyaneum i s on the r i g h t . There i s some e v i d e n c e o f b u r r o w i n g a c t i v i t y t o the l e f t and above t h e O c t o l a s i o n cyaneum i n d i v i d u a l 110 P l a t e 2: An example of the s o i l f r om t r e a t m e n t w i t h e a r t h w o r m s , t o i l l u s t r a t e t he b u r r o w i n g a c t i v i t y . . I l l P l a t e 3: An example o f t h e s o i l f r o m t r e a t m e n t w i t h no ea r thworms 112 - x i i -ACKNOWLEDGEMENTS This study was conducted under the supervision of Dr. A. Carter. The other members of the committee were Dr. L.M. Lavkulich, Dr. K. H a l l and Dr. CA. Rowles. I wish to thank the following: Mr. P. Kenney and Ms. S. Norman aided i n the c o l l e c t i n of earthworms and s o i l s . Dr. C. Paul, for data and assistance with determinations for the approximate f i e l d capacity and bulk density for the experimental containers. Technical assistance was provided by Mr. B. van Spindler, Ms. J. Lansiquot and Ms. T.D. Nyuynen. The Department of Animal Science permitted the author to use t h e i r block digestor. Dr. T. Guthrie provided information on a n a l y t i c a l procedures, as did Dr. A. Carter. Mr. B. Carson supplied a n a l y t i c a l data for milorganite. Dr. D. Moon assis t e d i n c o l l e c t i n g p r o f i l e descriptions and s o i l samples. Dr. D.P. Schwert i d e n t i f i e d the earthworm species used i n t h i s study. Help i n s t a t i s t i c a l analysis was provided by Drs. M. Greig, J. H a l l , H. Schreier, and M. Sandheim. Dr. T.A. Black provided the hygrothermograph and advice on photoperiods for l e t t u c e . Dr. A. Bomke provided the lettuce seeds and advice on supplemental nutrients. Photographs were taken by Mr. B. von Spindler and reproduced by Mr. W. Mc Diarmid. Drafting was done by Ms. S. Brightwell and the rough draft was typed by Ms. J . Melzer. Mr. R. Reynolds and Mr. H. Reynolds of Westham Island, Delta, B.C. allowed the author to c o l l e c t s o i l and earthworms from t h e i r farmland. - x i i i -This study was financed by the Natura l Appl ied and Hea l th Sciences Grant Committee of the U n i v e r s i t y of B r i t i s h Columbia, Nat ional Sciences and Engineering Research Counci l of Canada (67-6177) and A g r i c u l t u r e Canada (65-0360) grants . - 1 -1. GENERAL INTRODUCTION Sewage Sludge A p p l i c a t i o n to Land - A B r i e f Overview Sewage sludge i s the by-product produced by municipal wastewater 5 treatment. In Canada, approximately 10 tonnes of dry so l id s were produced i n 1973 and the projected production by 1985 i s 10 6 tonnes dry so l id s per annum (Webber, 1979). Disposa l prac t i ce s have inc luded i n c i n e r a t i o n , ocean dumping, f resh water dumping, l a n d f i l l i n g and storage lagoons, as w e l l as, a p p l i c a t i o n to land for d i sposa l purposes, cropland improvement and for reclamation of dis turbed lands such as mine s p o i l s . In 1970, the Environmental Pro tec t ion Service was e s tab l i shed and i s responsible for federa l p o l l u t i o n c o n t r o l l e g i s l a t i o n . The major f edera l acts are the Environmental Contaminants A c t , Clean A i r A c t , Ocean Dumping Contro l A c t , F i s h e r i e s Act and Canada Water A c t , as w e l l as s i m i l a r p r o v i n c i a l l e g i s l a t i o n (Environment Canada, 1981; Sanderson et a l . 1973). There i s s i m i l a r l e g i s l a t i o n i n the United States (Parr e t . a l . , 1978). The main thrust of th i s l e g i s l a t i o n i s to encourage m u n i c i p a l i t i e s to consider applying sewage sludge to land e i t h e r for d i sposa l or use i n a g r i c u l t u r e . The add i t ion of sewage sludge to a g r i c u l t u r a l lands i s not new. For 300 years , beginning i n 1559, sewage was spread on a g r i c u l t u r a l lands i n Prus s i a . (Bates, 1972; Dowdy et a l . 1976). Sewage farms were s tar ted i n England's London area during the 1860's and a g r i c u l t u r a l land i s s t i l l being used for sludge d i sposa l today (Thompson and Dickens, 1979). About 25% of a l l sludge produced i n the U .K . i s appl ied to farmland as l i q u i d digested sludge. (Coker, 1979). Since 1897, - 2 -Melbourne, A u s t r a l i a has spread sewage sludge on land which i s used p r i m a r i l y for l i v e s t o c k grazing (Seabrook, 1975). In the U .S . about 20% of municipal sludges are spread on land (Parr et a l . 1978). Coote et a l . (1981) reviewed the Canadian use of sewage on l and . In B r i t i s h Columbia's Okanagan sewage sludge i s spread on a g r i c u l t u r a l and parkland. Several major p r a i r i e c i t i e s a lso spread sludge on farmland. Ontar io disposes of 34% of the sludge produced onto farmland. Both Quebec and the A t l a n t i c Region do not p rac t i ce land a p p l i c a t i o n of sewage sludge. The a d d i t i o n of sewage sludge to a g r i c u l t u r a l land may have severa l b e n e f i c i a l e f f e c t s , as w e l l as, harmful e f fect s on s o i l chemical p r o p e r t i e s . Sludges contain varying amounts of n i t rogen , phosphorus and micronutr ients and genera l ly low leve l s of potassium and have been used to promote crop growth (Bates, 1972; Chawla et a l . 1874; Coker, 1979; Dowdy et a l . , 1976, Parr et a l . , 1978; Stewart and Webber, 1976). Eps te in et a l . (1976) ind ica te that sewage sludges may contain from 2-8% ni t rogen and 1 -4 °C phosphorus. Coker (1979) suggested that the n i t rogen a v a i l a b i l i t y i n sewage sludges could be estimated as the amount of ammonium - N present plus 1/6 of the organic - N present. Both Damprey (1979) and Coker (1979) suggested that the n i t rogen and phosphorus content of sludges are su i t ab le for grasses and cerea l crops. The ef fects of sludge addi t ion on s o i l pH i s v a r i a b l e . Eps te in et a l . (1976) reported decreases whereas Coker (1979) ind ica ted a r i s e i n s o i l pH. This may r e f l e c t d i f f e r e n t processes u t i l i z e d i n sludge s t a b i l i z a t i o n . The a d d i t i o n of lime i s only one of several means of - 3 -s t a b i l i z i n g sewage (Bates, 1972, Dowdy et a l , 1976). Eps te in et a l . (1976) report an increase i n s o i l ca t ion exchange capacity when sludge was added to the s o i l . Various sa l t s are used i n wastewater treatment and when sludge i s added to the s o i l , there i s an increase i n s o i l s a l i n i t y and e l e c t r i c a l c o n d u c t i v i t y . Excess s a l t s can be harmful to plant growth and reduce germination while excess sodium can also be harmful to s o i l s t ruc ture . (Dowdy et a l . , 1976; Eps te in et a l . , 1976; Stewart and Webber, 1976). The add i t ion of sewage sludge to s o i l genera l ly improves the p h y s i c a l p r o p e r t i e s . The organic matter i n sewage sludge i s r e l a t i v e l y s table as the e a s i l y decomposed mater ia l i s destroyed during process ing (Coker, 1979). Webber (1978) and Gupta et a l (1977) reported decreases i n s o i l bulk densi ty when sludge was added. Sludge addi t ions can increase the percentage of water stable aggregates present i n the s o i l . (Eps te in , 1975; Webber, 1978). Water re tent ion i n s o i l s treated with sludge us sua l ly i s increased . (Coker, 1979; E p s t e i n , 1976; Gupta et a l , 1977), although Webber (1978) and Eps te in (1978) reported an increase i n 6 2 water holding capacity only at the 1.5 x 10 N/m (15 bars) and no increase at the lower pressures . Sewage sludges d i f f e r from other organic supplements added to s o i l i n that they general ly contain varying amounts of metals . The add i t ion of large amounts of metals to s o i l i s a p o t e n t i a l hazard because many of the metals can be tox i c to plants at c e r t a i n concentrat ions , and once taken up by plants the metals enter the food chain and can threaten the hea l th of l i v e s t o c k and humans. Long term - 4 -a p p l i c a t i o n of sludge containing metals may re su l t i n a long term s o i l contamination and render the s o i l non productive for crop production (Bates, 1972; CAST, 1976; Dowdy et a l . , 1976; Parr et a l . , 1978). The movement of metals i n the s o i l p r o f i l e a f ter sludge a p p l i c a t i o n has been reviewed by Page (1974) and Will iams et a l . , (1980). In genera l , a large proport ion of the metals present i n sewage sludges occur i n s table and nonmobile organic forms. Meta l s , i n c l u d i n g Cd, Cu, N i , Pb and Zn, tend to remain i n the surface and only a small percentage (< 7%) of the added metals i n sewage sludges w i l l be found below the plow l a y e r . Wil l iams et a l . (1980) reported than Zn, Cu, Pb and Cd concentrat ions decreased with depth a f ter sewage sludge was incorporated in to the surface 20 cm and that for a l l 4 metals the concentrations from sludge t reated s o i l s and contro l s were equal at 25 to 30 cm, or 5 to 10 cm below the zone of sludge a d d i t i o n . Most researchers refer to the metals as heavy metals. However, the term "heavy metal" has been used i n c o n s i s t e n t l y i n the l i t e r a t u r e . Heavy metals are those metals which have a densi ty greater than 5,000 kg/m 3 yet i n the l i t e r a t u r e concerning metal p o l l u t i o n , elements that are ne i ther metals or heavy have been included i n th i s group (Hughes et a l . , 1980). The metals of most concern are z i n c , copper and n i c k e l which can be phytotoxic and cadmium which can accumulate i n plant t i s sue without the plant e x h i b i t i n g tox ic symptoms and become p o t e n t i a l hea l th hazards to l i v e s t o c k and humans. (Cast, 1976; Dowdy et a l , 1976; Stewart and Webber, 1976). P lants (and also animals) grown i n s o i l s amended with sewage - 5 -sludge may e i ther accumulate, concentrate or not r e t a i n metals i n t h e i r t i s s u e s . When the concentrat ion r a t i o (the r a t i o of metal concentrat ion i n the organism to the metal concentrat ion i n the s o i l ) does not d i f f e r from zero , no re tent ion of the metal by the organism has occurred. Accumulation occurs when the plant (or animal) t i s sue contains i n c r e a s i n g l y higher amounts of a metal as a re su l t of being exposed to one l e v e l of the metal i n the environment over a per iod of time, or at one given time the t i s sues contain increa s ing l eve l s of metals i n propor t ion to l eve l s i n the environment. Concentrat ion of a given metal occurs when the concentrat ion r a t i o i s greater than uni ty (Hartenstein et a l . 1980, Hughes et a l . , 1980; Van Hook, 1974). The chemical composition of sludge var ie s widely from c i t y to c i t y and with the time of day, and depends on the type of i n d u s t r i a l waste, treatment processes, storm dra ins , using the treatment p l a n t . (Bates, 1972; Dowdy et a l . 1976). Metal concentrations i n sewage sludges from various centres have been publ i shed . In Canada, Chawla et a l . (1974) review metal content i n severa l Ontario sludges and Koch et a l . (1977) present data on the composition of sludges from the Greater Vancouver Region. Page (1974) reviewed metal concentrations i n sludges from a number of American c i t i e s , as did Sommers et a l . (1976), Horvath and Koshut (1981) and Sommers (1977). Information for B r i t i s h and Welsh sludges has been summarized by Berrow and Webber (1972). A l l these reviews emphasize the v a r i a b i l i t y i n metal concentrations i n d i f f e ren t s ludges. - 6 -Sommers (1977) ind ica te s that p r i o r to applying sludges to land, the composition i n c l u d i n g metal concentrat ion should be known. Various countr ies have proposed guide l ines to regulate the amount of metals which can be added to the s o i l i n sewage sludge i n an attempt to prevent metal concentrations from reaching tox ic l e v e l s . (Freedman and Hutchinson, 1981). Not only have m u n i c i p a l i t i e s spread sludge on land as a means of d i sposa l but some c i t i e s have packaged sludge and marketed i t as a f e r t i l i z e r . In B r i t a i n , sewage was sold as manure during the 1800's. Since the turn of the century, H a l i f a x , England has been packaging processed sludge and s e l l i n g i t as a f e r t i l i z e r under the name "Organi fax" . Other processing plants i n B r i t a i n also s e l l processed sludge under a v a r i e t y of trade names (Wood, 1979). In 1927, M i l o r g a n i t e , a heat-dr ied ac t iva ted sludge from Milwaukee, Wisconsin was the f i r s t sludge to be sold i n the United States as a f e r t i l i z e r (Anderson, 1959). Earthworms have a lso been added to s o i l to increase crop growth. (Bar ley , 1961 and Ghabbour, 1966) and i n New Zealand the add i t ion of earthworms to pastures i s a recommended procedure for increa s ing pasture product ion . ( S t o c k d i l l , 1966). Edwards and Lof ty (1977) and Hughes et a l . (1980) reviewed the uptake of metals by earthworms. In genera l , Cd and Zn are concentrated by earthworms and th i s poses a threat to organisms feeding on the earthworms. A l s o , when the earthworms d i e , the metals reta ined by the earthworms may become ava i l ab le for plant uptake. I re land (1975) - 7 -reported that decaying earthworms contained much more ava i l ab l e Pb and Zn than d id the s o i l or earthworm cast s . Kirkman (1979) reported that earthworms increased the growth of wheat i n s o i l s both with and without sewage sludge as w e l l as increased Mn uptake by wheat. The present study cons i s t s of two par t s . The object ives of the f i r s t part were: 1. To determine i f earthworms belonging to three taxa would accumulate cadmium, copper, lead and z inc i n response to increa s ing app l i ca t ions of mi lorgani te to the s o i l i n which the earthworms were kept. 2. To determine i f there was a taxon d i f ference i n metal uptake by earthworms. 3. To determine i f metal concentrat ion i n the earthworms was dependent upon body weight. 4. To determine i f earthworms are p o t e n t i a l l y useful as b i o l o g i c a l monitors of metal l eve l s i n the s o i l . Thi s experiment was conducted i n the c o n t r o l l e d environment of a growth chamber. This study d i f f e r s from other studies i n v o l v i n g metal uptake by earthworms kept i n sludge amended s o i l s i n that s ix a p p l i c a t i o n rates of sludge are used to test for accumulation by the worms i n response to increas ing metal loading of the s o i l . The second par t ' s ob ject ives were: 1. To determine i f the cadmium, copper, l ead , n i c k e l , and z i n c concentrations i n lea f l e t tuce would be increased when mi lorgani te was added to the s o i l i n the presence and absence - 8 -of earthworms. 2. To determine i f mi lorgani te and earthworms increased l e t tuce y i e l d s . This experiment was conducted i n a greenhouse. M i l o r g a n i t e was used because i t was r e a d i l y ava i l ab le from gardening suppl iers and was reported to have high concentrations of cadmium, copper, n i c k e l , lead and z i n c . (John and van Laerhoven, 1976). This experiment d i f f e r s from the only other experiment t e s t ing the inf luence of earthworms on metal uptake by plants grown i n sludge amended s o i l (Kirkman, 1979) i n that severa l taxa of f i e l d earthworms were u t i l i z e d rather than the manure worm (E i sen ia foet ida) which l i v e s i n compost heaps and i n s o i l s only with high organic matter contents (Reynolds, 1977). - 9 -REFERENCES Anderson, M . S . , 1959. F e r t i l i z i n g C h a r a c t e r i s t i c s of Sewage Sludge. Sewage and I n d u s t r i a l Wastes. 31: 678-682. Bar ley , K . P . 1961. The abundance of earthworms i n a g r i c u l t u r a l land and t h e i r poss ib le s i gn i f i c ance l n a g r i c u l t u r e . Advan. Agron. 13: 249-268. Bates, T . E . , 1972. Land A p p l i c a t i o n of Sewage Sludge, report on pro jec t 71-4-1. Canada-Ontario Agreement, Res. Rept. No. 1., Env. Canada/Ont. Min . of E n v i r . Ottawa/Toronto, 212 pp. Berrow, M . L . , and J . Webber, 1972. Trace Elements i n Sewage Sludges. J . S c i . Fd . A g r i c . 23, 93-100. Canada Water A c t . 1969-70. Revised Statues of Canada 1970 1st Supplement. Chap. 5. Queen's P r i n t e r of Canada, Ottawa. CAST, 1976. A p p l i c a t i o n of Sewage Sludge to Cropland: A p p r a i s a l of P o t e n t i a l Hazards of Heavy Metals to Plants and Animals, USA Counci l for A g r i c u l t u r a l Science and Technology, Report No. 64, EPA-430/9-76-013. Washington. 63 pp. Chawla, V . K . , J . P . Stephenson, and D. L i u , 1974. Biochemical C h a r a c t e r i s t i c s of Digested Chemical Sewage Sludges. In Proceedings Sludge Handling and Disposa l Seminar, Conference Proceedings No. 2. Env. Canada/Ont. Min . of Env. Ottawa/Toronto pp 63-94. Clean A i r Act of 1971. 1972. Statutes of Canada, 1970-71-72. v o l . 1. Chap. 47. Queen's P r i n t e r of Canada, Ottawa. Coker, E . G . 1979. The U t i l i z a t i o n of L i q u i d Digested Sludge. In U t i l i s a t i o n of Sewage Sludge on Land. Papers and Proceedings of a Water Research Centre Conference, Oxford, England, 1978. pp. 110-129. Coote, D . R . , J . Dumanski, and J . F . Ramsey, 1981. An Assessment of the Regradation of A g r i c u l t u r e Lands i n Canada. L . R . R . I . Cont r ibu t ion No. 118. Research Branch, A g r i c u l t u r e Canada, Ottawa. Dampney, T . D . 1979. Sewage Sludge - the farmer's view i n U t i l i s a t i o n of Sewage Sludge on Land. Papers and Proceedings of a Water Research Centre Conference, Oxford, England, 1978, pp. 164-170. Dowdy, R . H . , R . E . Larson, and E . E p s t e i n , 1976. Sewage Sludge and Ef f luent Use i n A g r i c u l t u r e . In Land A p p l i c a t i o n of Waste M a t e r i a l s , S o i l Conserv. Soc. Am., Ankeny, Iowa, pp. 138-153. Edwards, C A . and J .R . L o f t y . 1977. Biology of Earthworms. Chapman and H a l l . London, 2nd ed. 333 pp. - 10 -Environment Canada, 1981. The Environmental Pro tec t ion Service i n A l b e r t a , Saskatchewan, Manitoba, and the Northwest T e r r i t o r i e s . E p s t e i n , E . , 1975. Ef fect of Sewage Sludge on Some S o i l P h y s i c a l P r o p e r t i e s . J . Env i ron . Qual i ty , 4, 139-142. E p s t e i n , E . , J . M . T a y l o r , and R . L . Chaney, 1976. E f fec t s of Sewage Sludge and Sludge Compost Appl ied to S o i l on some S o i l Phys i ca l and Chemical P r o p e r t i e s . J . Env i ron . Qual i ty . 5, 422-426. F i s h e r i e s Act of 1969-70, 1970. Revised Statutes of Canada 1970. 1st Supplement. Chap. 17. Queen's P r i n t e r , Ottawa. Freedman, B . , and T . C . Hutchinson, 1981. Sources of Metal and Elemental Contamination of T e r r e s t r i a l Environments. In E f f e c t of Heavy Meta l P o l l u t i o n on Plants Vo l 2 Metals i n the Environment. Lepp, N.W. ed. App l i ed Science Pub l i sher s , London, pp. 35-94. Ghabbour, S . I . 1966. Earthworms i n A g r i c u l t u r e : A modern eva lua t ion . Rev. E c o l . B i o l . S o l . I l l : 259-271. Gupta, S . C , R .H. Dowdy, and W.E. Larson. 1977. Hydraul ic and Thermal Propert ies of a Sandy S o i l as Influenced by Incorporat ion of Sewage Sludge. S o i l S c i . Soc. Am. J . 41: 601-605. Har tens te in , R . , E . F . Neuhcuser, and J . C o l l i e r . 1980. Accumulation of hevy metals i n the earthworm E i s e n i a f o e t i d a . J . Env i ron . Qual. 9: 23-26. Horvath, D . J . , and R .A. Koshat. 1981. Proport ions of Several Elements Found i n Sewage Ef f luent and Sludge from Several M u n i c i p a l i t i e s i n West V i r g i n i a . J . Env i ron . Qual. 10: 491-497. Hughes, M . K . , N.W. Lepp and D.A. Phipps . 1980. A e r i a l Heavy Meta l P o l l u t i o n and T e r r e s t r i a l Ecosystems. Advan. E c o l . Res. 11: 217-327. I r e l a n d , M.P. 1975. The E f fec t of the Earthworm Dendrabaena rubida on the S o l u b i l i t y of Lead, Zinc and Calcium i n Heavy Metal Contaminated S o i l i n Wales. J . S o i l S c i . 26: 313-318. John, M . K . , and C J . van Laerhoven, 1976. E f fec t s of Sewage Sludge Composit ion, A p p l i c a t i o n Rate and Lime Regime of Plant A v a i l a b i l i t y of Heavy Meta l s . J . Env i ron . Qual. 5, 246-251. Kirkham, M.B. 1979. A v a i l a b i l i t y to Wheat of Elements i n Sludge Treated S o i l with Earthworms. In : Proc . Conf. U t i l i z a t i o n of S o i l Organisms i n Sludge Management, (R. Hartenste in ( e d . ) ) . Syracuse, N.Y. 15-17 June 1978. State U n i v e r s i t y of New York , Syracuse, pp. 103-121. Koch, F . H . , K . J . H a l l , and I. Y e s a k i , 1977. Toxic Substances i n ' the Westwaters from a' Met ropo l i t an Area , Westwater Research Cent re ,Technica l Report No. 12 U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 107 pp. - 11 -Ocean Dumping Contro l Act of 1975, 1976. Statutes of Canada, 1974-75-76. V o l . 1 Chap. 55, Queen's P r i n t e r for Canada, Ottawa. Page, A . L . , 1974. Fate and E f fec t s of Trace Elements i n Sewage Sludge When Appl ied to A g r i c u l t u r a l Lands, A L i t e r a t u r e Review Study. EPA-670/2-74-005. C i n c i n n a t i , Ohio. Pa r r . J . F . , G.B. Wilson and E . E p s t e i n , 1978. Current Research on Composting of Sewage Sludge from U t i l i z a t i o n of S o i l Organisms i n Sludge Management, Conference Proceedings, Syracuse, New York, Har tens te in , R(ed). pp 59-72. Reynolds, J .W. 1977. The Earthworms (Lumbricidae and Sparganophilidae) of Onta r io . Miscel laneous P u b l i c a t i o n s , Royal Ontario Museum, Toronto. 141 pp. Sanderson, E . , J . E . Brydon, and J . Monteith 1973. Environmental Impl icat ions of M u l t i j u r i s d i c t i o n a l Problems i n Waste Management. In Proceedings of the In te rna t iona l Conference on Land for Waste Management. Ottawa, pp. 317-326. Seabrook, B . L . 1975. Land A p p l i c a t i o n of Wastewater i n A u s t r a l i a , The Werribee Farm System. E . P . A . Washington 27 pp. Sommers, L . E . 1977. Chemical Composition of Sewage Sludges and Ana lys i s of t h e i r P o t e n t i a l Use as F e r t i l i z e r s . J . Env i ron . Qual. 6: 225-232. Sommers, L . E . D.W. Nelson, and K . J . Yos t . 1976. V a r i a b l e Nature of Chemical Composition of Sewage Sludges. J . Env i ron . Qual. 5: 303-306. Stewart, B.A. and L .R . Webber, 1976. Considerat ion of S o i l s for Accept ing Wastes. In Land A p p l i c a t i o n of Waste M a t e r i a l s , S o i l Conserv. Soc. Am. Ankeny, Iowa. pp. 8-47. S t o c k d i l l , S .M. J . 1966. Earthworms a Must for Maximum Product ion . N . Z . J . A g r i c . 119: 61-67. Thompson, L . H . and W.J . Dickens, 1979. The Land U t i l i z a t i o n of Sludges form Urban Works from U t i l i s a t i o n of Sewage Sludge on Land, Papers and Proceedings of a Water Research Centre Conference, Oxford, England, pp. 19-32. Van Hook, R . I . 1974. Cadmium, Lead, and Zinc D i s t r i b u t i o n s between Earthworms and S o i l s : Po tent i a l s for B i o l o g i c a l Accumulation. B u l l . Env i ron . T o x i c o l . 12: 509-512. Webber, L . R . , 1978. Incorporat ion of Nonsegregated, Non composted S o l i d Waste and S o i l P h y s i c a l P r o p e r t i e s . J . Env i ron . Qual. 7, 397-400. - 12 -Webber, M.D. 1979. Phosphate F e r t i l i z e r and Sewage Sludge Use on A g r i c u l t u r a l Land. The P o t e n t i a l for Cadmium Uptake by Crops. E n v i r o n . Canada Report No. EPS 4-WP-79-2. pp 30. W i l l i a m s , D . E . , J . Vlamis , A . H . Pukite and J . E . Corey. 1980. Trace Element Accumulation, Movement, and D i s t r i b u t i o n i n the S o i l P r o f i l e from Massive App l i ca t ions of Sewage Sludge. S o i l S c i . 129: 119-132. - 13 -PART 1 Selected Metal Levels in Three Taxa of Earthworms (Lumbricidae) Kept in Soil Contaminated by Milorganite. - 14 -1 INTRODUCTION AND LITERATURE REVIEW Earthworms are an important component of the s o i l fauna, p a r t i c u l a r l y i n temperate c l i m a t i c reg ions . T h e i r feeding and burrowing a c t i v i t i e s have been shown to increase s o i l i n f i l t r a b i l i t y , s o i l p o r o s i t y , s o i l aggregate s t a b i l i t y and the mixing and t r a n s l o c a t i o n of s o i l cons t i tuent s . Not a l l of the Lumbricidae af fect the s o i l to the same degree, as some species are deep burrowers, while others are surface dwellers ( S a t c h e l l , 1967; Edwards and L o f t y , 1977). Earthworms are a food source for many organisms e s p e c i a l l y a large number of b i r d species and moles, as w e l l as, many invertebrates (Edwards and L o f t y , 1977; Gish and Chri s tensen, 1973). I re land (1977) demonstrated that toads could accumulate lead from t h e i r d i e t , which consis ted of earthworms with high t i s sue concentrat ions of l ead . The burrowing a c t i v i t i e s of earthworms have been shown to inf luence the r e d i s t r i b u t i o n of elements i n the s o i l . In s o i l cores 109 topped with l i t t e r spiked with Cd, v e r t i c a l migrat ion of cadmium was greater i n cores wi th earthworms, Lumbricus t e r r e s t r i s and Octolas ion lacteum, than i n cores with no earthworms present . The two species have d i f f e r e n t feeding habits and the former taxon also i s a deeper burrower than the l a t t e r species . Lumbricus t e r r e s t r i s was more e f f ec t ive i n the r e d i s t r i b u t i o n of cadmium (Oak Ridge Nat iona l Laboratory, 1974). Re ichle et a l . (1971) and Cross ley and co-workers (1971) demonstrated that Octo las ion lacteum and Lumbricus t e r r e s t r i s were both important i n 137 organic matter turnover, and i n r e d i s t r i b u t i o n of Cs i n forested s o i l systems i n the southern U.S . Earthworms were shown to be important i n - 15 -the r e d i s t r i b u t i o n of 6 0 C o i n the s o i l (Peredelsky et a l 1957). Various species of earthworms from both uncontaminated and contaminated s o i l s have been found to accumulate, and i n some cases, concentrate ce r t a in metals found i n the s o i l into t h e i r t i s sues (see reviews by Edwards and L o f t y , 1977; Hughes et a l , 1980). The contaminated s i t e s inc luded : (1) urban and r u r a l s o i l s i n the v i c i n i t y of extens ive ly t r a v e l l e d highways; (2) s o i l s to which sewage sludges with high l eve l s of metals were added; (3) s o i l s contaminated by mine t a i l i n g s ; (4) s o i l s located downwind from lead - z inc smelters . The general f indings were that Cd and Zn concentrations i n earthworm t i s sue exceeded the concentrations of these metals i n the s o i l . Cadmium concentrations i n earthworms tended to increase with s o i l concentrat ions i n d i c a t i n g Cd accumulation, whereas Zn concentrations tended to be regulated . Lead and copper tended not to be concentrated or accumulated by earthworms. Only earthworms sampled from s o i l s contaminated by lead-zinc mine spo i l s were found to concentrate Pb above s o i l concentrations ( I r e l a n d , 1975, 1979; Ire land and Richards , 1977). Van Rhee (1963, 1975, 1977) found decreased earthworm populat ions i n orchard s o i l s where copper fungicides such as copper sulphate had been used and i n pasture s o i l s where pig s l u r r y containing high amounts of copper had been app l i ed . High concentrations of copper were found to reduce earthworm r e p r o d u c t i v i t y capacity as w e l l as body growth. Copper l e v e l s i n a mixed species sample of earthworms showed increas ing l eve l s of copper i n the body t i s sue with increas ing s o i l l eve l s although the l e v e l s i n the earthworms did not exceed that of the s o i l . - 16 -I re land and hi s coworkers c a r r i e d out a number of studies to determine the metal content i n earthworms, p r i m a r i l y the ac id to le rant spec ies , Dendrobaena rub ida , c o l l e c t e d from s o i l s with low pH's ranging from 3.6 to 5 .1 . I re land and Wooton (1976) ind ica ted that the lead concentrat ions i n the t i s sues of I), rubida were seasonal ly var i ab le and were s i g n i f i c a n t l y greater i n the winter . They a lso found that Zn concentrat ion i n D. rubida were s i g n i f i c a n t l y lower i n November for 2 of the 3 s i t e s s tudied . Accumulation of a metal by earthworms occurs when the earthworm t i s sue contains inc rea s ing ly higher amounts of a metal as a r e su l t of being exposed to one l e v e l of the metal i n the environment over a per iod of time, or at one given time the t i s sues contain increa s ing l eve l s of metal i n proport ion to l eve l s i n the enviornment. Concentrat ion of a given metal occurs when the r a t i o of metal concentrat ion i n the earthworms to the concentrat ion i n the s o i l i s greater than uni ty (Hartenstein et a l . 1980; Van Hook, 1974). Ash and Lee (1980), Andersen (1979, 1980), I re land and Richards (1977) and Ire land (1979) reported s i g n i f i c a n t d i f ferences i n metal uptake among d i f f e rent earthworm species . I re land and Richards (1977) suggested that species d i f ferences i n body t i s sue concentrations may be a re su l t of d i f f e rent metal tolerances between species , whereas I re land and Richards (1981) ind ica ted that d i f ferences i n feeding habits may account for d i f f e rent metal concentrations among d i f f e rent earthworm species . Carter et a l . (1980) c o l l e c t e d L . rubel lus c l i t e l l a t e i n d i v i d u a l s - 17 -and j u v e n i l e n o n - c l l t e l l a t e specimens from an a g r i c u l t u r a l f i e l d . The n o n - c l i t e l l a t e specimens had lower body t i s sue Cd concentrations than d id the c l i t e l l a t e specimens. Zinc regula t ion was reported by Bryan and Hummerstone (1973) for the marine polychaete , Nereis d i v e r s i c o l o r . Z inc regu la t ion i n earthworms has been reported by Ire land and Wooton (1976), I re land (1979), I re land (1976) and Ire land and Richards (1977), as w e l l as, Ash and Lee (1980). Earthworms also regulate copper ( Ire land and Richards 1981). Faeces are the major excre t ing s i t e s for lead and z inc i n the earthworm species Dendrobaena rubida ( I re land , 1976). Van Hook (1974), Helmke et a l . (1979), Czarnowska and Jopkiewicz (1978) and Carter et a l . (1980) have suggested that earthworms are p o t e n t i a l l y useful b i o l o g i c a l monitors of metal l eve l s i n the s o i l . A b i o l o g i c a l monitor organism i s a member of the b io t a indigenous to the study area and accumulates po l lu tant s from the s o i l or ingested food i n i t s t i s sue . The organism acts as an i n d i c a t o r i n that the l e v e l s of a po l lu tant i n i t s t i s sue are taken to be an index of the degree of po l lu tan t contamination at the sampling s i t e ( P h i l l i p s , 1978). The object ives of th i s study were as fo l lows : 1. To determine i f earthworms would accumulate the metals i n proport ion to the l e v e l s i n the s o i l s and, there fore , p o t e n t i a l l y be useful b i o l o g i c a l monitors of metal l eve l s i n the s o i l . 2. To determine i f there were d i f ferences among three taxa of earthworms: Lumbricus r u b e l l u s , Aporrectodea spp. and - 18 -Octolasion cyaneum, i n the metal concentrations of t h e i r body tis s u e s . 3. To determine i f the concentrations of metals i n the body tissue was dependent upon the body weight of the earthworms. - 19 -2 MATERIALS AND METHODS 2.1 Sample Preparation and Chemical Analysis Bulk s o i l samples were c o l l e c t e d from the Ap hor izon of a Crescent-Westham Is land s o i l complex (Luttmerding, 1980) located on Mr. Hugh and Bob Reynolds farms on Westham Is land i n the Fraser Del ta of B r i t i s h Columbia. ( S o i l p r o f i l e descr ip t ions are given i n Luttmerding, 1981a,b). The s o i l was a i r - d r i e d , crushed with a wooden r o l l e r and passed through a 6 mm screen. The s o i l (2760 grams) was throughly mixed wi th 0 ,5 ,10,25,50 or 100 grams of M i l o r g a n i t e per ki logram of s o i l on an a i r - d r i e d bas i s . These a p p l i c a t i o n rates are equivalent to 0 ,11,25,22.50,56.25,112.50,225.0 metric tonnes of mi lorgani te per hectare . The s o i l - m i l o r g a n i t e mixture was packed (bulk density of 1200 kg/m ) in to p l a s t i c flower pots (15 cm x 17 cm). M i l o r g a n i t e i s a heat dr ied ac t iva ted sewage sludge sold by the Milwaukee Sewerage Commission. The sludge i s h ighly contaminated and was used as the source of metals; cadmium, copper, lead and z i n c . A f t e r f i l l i n g the pot s , they were watered with 1000 mis of d i s t i l l e d water (to approximate f i e l d capacity) weighed and the weights recorded. Four Lumbricus  rube l lus Hoffmeister , four Aporrectodea spp. ( e i ther Aporrectodea  trapezoides Duges, Aporrectodea tuberculata E i s e n , or a combination of these two species) and two Octo las ion cyaneum Savigny were placed on top of the s o i l - m i l o r g a n i t e mixture of each pot and allowed to burrow. Only c l i t e l l a t e adult earthworms were used. (See Plates 1-3 and Table 1 for de scr ip t ions of these earthworms). Earthworms were obtained by digging on Westham Is land near the f i e l d where the s o i l was obtained. Plate 2. C l l t e l l a t e specimen of Octolasion cyaneum. - 21 -Table 1 Diagnostic Characteristics of Four Earthworn Species (Adapted from Reynolds, 1977) Length (mm) Diameter (mn) Number of Segments Praatomlum 1st Dorsal Pore Clltellum Tuberculua Pubertatls Setal Pairing Genital Tumescenes Pigmentation L u n b r l c u s r u b e l l u s ( r e d marsh worm) Octotaslon cyaneum (woodland blue worm) A p o r r e c t o d e a t u b e r c u l a t a 50-150 (usually 60 mm) 65-180 A p o r r e c t o d e a  t r a p e t o l d e a 90-150 80-140 4-6 7-8 70-120 140-158 tanyloblc 5/6-8/9 xxvl, xxvli-xxxl, xxxll eplloblc 11/12 or 12/13 xxlx-xxxlv xxvlll xxxu xxx—xxxlll 3-7 93-169 eplloblc generally 130 12/13 xxvll, xxvl11-xxxlll , xxxlv Closely v l l l - x l l (less frequently on x) xx-xxl11, xxvl-xxxvl Closely paired setae of x .xv l l l , anteriorly widely paired posterloly 4-8 146-196 eplloblc 11/12 or 12/13 xxvli-xxxlv xxx.xxxi- closely xxxlll,xxxlv xxxl-xxxlll closely xlx.xx.xxi fre-quently bn white genital tumescences absent ln xxxl and xxxl l l , present ln xxx, xxxll and xxxlv and fre-quently ln xxvl Genital tumescences Including a and b setae only ln lx-xi ,xxxl i -xxxlv, often ln xxvlll and occasionally ln the region of xxvl-xxlx Ruddy brown or red violet lrrldescent dorsally, pale yellow ventrally Blue-gray with (usually) li lac blue dorsal line, last 4-5 segments yellow, anterior segments pink, clltellum red-orange unplgmented almost white or greyish or sometimes with light pigmentation on the dorsum variable, often lighter behind the clltellum until near, the hind end, the deeper brownish-reddish - 23 -The tops and bottoms of the pots were covered with nylon mesh i n order to prevent the earthworms escaping. The pots were placed i n a P e r c i v a l growth chamber set at a constant-temperature of 15 °C and 12 hr photoperiod using a mix of incandescent and f luorescent l i g h t . Because of evaporat ion, d i s t i l l e d water was added d a i l y to br ing the s o i l - m i l o r g a n i t e mixture back to the i n i t i a l weight. The experiment was run for ten days. The s i x treatments were r e p l i c a t e d f ive t imes. The pots were set up i n a repeated measure des ign. In a repeated measures experiment, more than one measurement i s made on the same experimental uni t (F i sher and MacDonald, 1978; Lee, 1975; and Winer, 1971). In t h i s experiment a flowerpot i s an experimental u n i t , and there are three taxa of earthworms sampled from each u n i t . The earthworms are the repeated measure. The pots were set up as i n a randomized block design. Af ter ten days, the surv iv ing earthworms were recovered, r insed with d i s t i l l e d water, placed on moist ashless f i l t e r paper (Whatman #40) i n p e t r i dishes and placed i n an incubator at 15 °C for four days to a l low the earthworms to void t h e i r gut contents . During the four days the f i l t e r papers were changed d a i l y so as to prevent poss ib le coprophagy. Where po s s ib l e , faeces from two i n d i v i d u a l s of each of Lumbricus r u b e l l u s , Aporrectodea spp. and Octo la s ion cyaneum, from each pot were c o l l e c t e d and saved for a n a l y s i s . A l l the surv iv ing earthworms were k i l l e d and stored i n a deep freezer u n t i l analysed. The s o i l - m i l o r g a n i t e mixtures were a i r - d r i e d , crushed with a wooden r o l l e r and passed through a 2 mm s t a in l e s s s t e e l s i eve . Three r e p l i c a t e s for each s o i l mi lorgani te mixture were prepared and analyzed - 24 -for Cd, Cu, Pb and Zn. Samples weighing 0.5 gm were placed i n acid-washed glass t a l l f o r m 100 ml beakers and digested with 9 mis concentrated HN03 and 3 mis concentrated HCI on a Lindberg SB type H-2 hot p late on se t t ing 4 (medium set t ing) for at least 1/2 hour. The beakers were covered with ac id washed watch g lasses . 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 Whatman #42 f i l t e r paper and brought to 25 ml volume with d i s t i l l e d water (van Loon and Lichwa, 1973). Ana lys i s for Cd, Cu, Pb and Zn were done using a Perk in Elmer Model 306 atomic adsorpt ion spectrophotometer (AAS) equipped wi th background cor rec t ion and an a i r -acety lene flame. Wavelengths used were: 288.8 nm for Cd, 324.7 nm for Cu, 283.3 nm for Pb and 213.9 nm for Zn. The same procedure was also used to determine Cd, Cu, Pb and Zn concentrations i n the bulk Ap sample and the mi lo rgan i te . The earthworms were placed i n acid washed glass v i a l s and p a r t i a l l y f r eeze-dr i ed . The worms and the faeces were then dr ied at 8 0 ° C for 3 days and then weighed on a Cahn 25 automatic e lec t roba lance . The sample preparat ion method for earthworm t i s sue and faeces depended on sample s i z e . Earthworms less than 150 mg dry weight and faeces less than 100 mg dry weight were placed i n ac id washed Kimax glass v i a l s (70 x 20 mm O.D.) and digested at 1 5 0 ° C and a block d ige s te r . One m i l l i l i t e r of concentrated reagent grade n i t r i c ac id was added to the v i a l and then evaporated to dryness. This was repeated twice . Then 1 ml of 30% reagent grade hydrogen peroxide was added to the v i a l and evaporated to dryness. This was repeated once. Next, 5 mis of 0.16 N HNO3 were added to each v i a l . This procedure was adapted - 25 -by T. Guthrie (personal communication) from Koirtyohann et a l . (1976). Earthworms heavier than 150 mg dry weight and faeces samples heavier than 100 mg dry weight were placed i n ac id washed 100 ml t a l l f o r m beakers and digested on a hotplate at medium se t t ing with 9 mis concentrated reagent grade HNO3 and 3 mis concentrated reagent grade HCI. Beakers were covered with ac id washed watch glasses . Af te r d ige s t ing for at leas t 1/2 hour, or u n t i l the volume was reduced by one h a l f , the samples were removed from the hotplate and allowed to c o o l . F i v e mis deionized water were then added and the samples f i l t e r e d through Whatman #42 f i l t e r paper into ac id washed 25 ml volumetric f l a s k s . The samples were brought to volume using deionized water. The samples were then t ransferred to ac id washed 50 ml p l a s t i c bot t le s and stored i n a r e f r i g e r a t o r u n t i l analyses . A l l glassware and p l a s t i c bot t l e s used for preparing earthworm and faeces samples were ac id washed with 8 N HN03 and r insed with deionized water. Cadmium, Cu, and Zn l eve l s i n the earthworm t i s sue and Cu and Zn l e v e l s i n the faeces were determined using the atomic absorption spectrophotometer as described previous ly for the s o i l - m i l o r g a n i t e mixtures . Cadmium and lead l eve l s i n the faeces and lead l eve l s i n the earthworm t i s sues were determined using a Perkin Elmer model 306 AAS equipped with the Perkin Elmer Hollow Graphite Analyzer (HGA) model 2100 and a deuterium arc background c o r r e c t o r . Twenty m i c r o l i t e r i n j e c t i o n s were made from each sample with an Eppendorf m i c r o l i t e r p i p e t . These i n j e c t i o n s were repeated two or three times for each sample. Argon was used as the c a r r i e r gas. Furnace operating parameters, wavelengths and - 26 -s l i t se t t ings are given i n Table 2. The re su l t s were recorded using a Perk in Elmer model 56 s t r i p chart recorder set at 10 mv with a speed of 120 mm min. Peak heights were used for quant i t a t ive purposes. Table 2 Instrument Parameters for HGA Dry Ash Atomize element s l i t number Gas mode HGA 2100 se t t ings Cd 229.3 nm 3 normal 20 Sec 10 Sec 10 Sec at 9 0 ° C at 3 0 0 ° C at 2 0 0 0 ° C Ni 232.3 nm 3 normal 20 Sec 10 Sec 10 Sec at 9 0 ° C at 3 0 0 ° C at 2 0 0 0 ° C Pb 387.7 nm 3 normal 20 Sec 10 Sec 10 Sec at 9 0 ° C at 3 0 0 ° C at 2 0 0 0 ° C The same methods were used by Carter et a l . (1980,1983) to determine metal concentrations i n earthworm samples. Along with the earthworm samples, Nat iona l Bureau of Standards orchard leaves , bovine l i v e r and s o i l standards were run to test the accuracy of the methods. Some of the re su l t s are given i n Tables 3 and 4. (Carter , w r i t t e n communication). These data ind ica te that both methods are r e l i a b l e for determining Cd, Cu and Zn concentrations and that Pb determinations are v a r i a b l e and inaccura te . Lead concentrations may have been near the detect ion l i m i t of the instrument. Selected chemical and p h y s i c a l propert ies were determined for the bulk Ap s o i l sample. The s o i l pH was determined by a pH meter for the - 27 -Table 3 Analyses of N .B.S. Orchard Leaf Standard Method sample dry wt. Hg/gm dry wt. mg Cd Cu Pb Zn Block Digestor 92.5 0.07 not done 22.2 96.0 0.07 not done 23.4 188 0.02 not done 19.4 99.8 0.07 not done 24 53.1 0.09 not done 19 25 48.9 0.10 not done 20 23 Hot Pla te 103.3 0 12 36 211.5 0 10 26 206.0 not done 8 24 230.0 not done not done 23 200.0 0.05 11 33 Expected Value 0.1 12 ± 2 44 28 Table 4 Analyses of N .B.S. L i v e r Standard Method sample dry wt. Hg/gm dry wet mg Cd Cu Pb Zn Block Digestor 9.0 0.56 161 not done 117 10.6 0.47 149 not done 132 47.1 0.37 not done 0.27 130 48.5 0.36 144 0.12 119 Expected Value 0.27 193 0.34 130 s o i l mixed 1:2 with 0.01 M CaCl 2 . T o t a l carbon (%C) was determined using the Leco Analyzer . T o t a l n i t rogen (%N) i was determined by the auto analyzer a f ter d iges t ion i n s a l i c y l i c - i s u l f u r i c ac id using the Technican Block Diges tor , Model BD-20. The cat ion exchange capaci ty (CEC) was determined by NHi+ displacement , and Semi-Mlcrokjeldahl a n a l y s i s . A por t ion of the extract from the CEC determination was used to determine - 28 -the amount of exchangeable Ca, Mg, K and Na using an atomic absorption spectrophotometer. P a r t i c l e s ize ana lys i s using the hydrometer method was used to determine % sand, s i l t and c l a y . The methods for a l l these a n a l y t i c a l procedures are described i n d e t a i l i n Methods Manual (Dept. S o i l Science, 1977). The data for metal concentrations were then analyzed s t a t i s t i c a l l y using the UBC Genl in programme for ana lys i s of variance for repeated measures (Greig and B j e r r i n g , 1977). Duncan's and Scheffe ' s M u l t i p l e Range tests were a lso used. The data were also analyzed using the Mann-Whitney test s t a t i s t i c . ( S i e g e l , 1956). Re la t ionsh ips between earthworm body weights and metal content (ug) were determined for cadmium, copper, lead and z i n c , using the stepwise regress ion a n a y l s i s , UBC TRP programme. (Le and T e n i s c i , 1978). 2.2 STATISTICAL ANALYSIS The experimental design u t i l i z e d was the randomized block design having repeated measures. This design requires that more than one measurement i s made on the same experimental u n i t . The experimental u n i t s i n t h i s study were the flowerpots f i l l e d with the various s o i l - m i l o r g a n i t e a p p l i c a t i o n s . For each experimental u n i t , the metal concentrat ions for three taxa of earthworms (up to ten i n d i v i d u a l earthworms were analyzed) were determined. This i s the repeated measures aspect of the design. This i s a more useful and more powerful design than the regular randomized block des ign. I f the repeated measures design was not used, then i n order to test the concentrat ions of metals i n the three taxa at s ix d i f f e rent mi lorgani te app l i ca t ions wi th f ive r e p l i c a t e s of each a p p l i c a t i o n would require n i n i t y experimental uni t s instead of t h i r t y . The growth chamber used i n the experiment held a maximum of t h i r t y f lowerpots . A l s o , each taxon w i t h i n a given experimental uni t i s subjected to the same condit ions which decreases the sources of v a r i a b i l i t y . I f the randomized block design, and, there fore , n inety experimental uni t s were used the p o s s i b i l i t y of increased experimental v a r i a b i l i t y i s greater . The repeated measure design i s , therefore , a more powerful experimental design as measurements from a s ing le experimental uni t should be more homogeneous than measurements from s i m i l a r experimental units (F i sher and McDonald, 1978; Lee, 1975; Winer, 1971). L i t t l e and H i l l s (1972) suggest that from four to e ight r e p l i c a t i o n s are required to obtain reasonable p r e c i s i o n for f i e l d and - 30 -vegetable crop research. In t h i s study each a p p l i c a t i o n of mi lorgani te was repeated f ive time. The number of i n d i v i d u a l earthworms analyzed for each treatment var ied with the taxon and the s u r v i v a l of the earthworms. The lowest number of Lumbricus rube l lus i n d i v i d u a l s was f i f t e e n for the 100 gm/kg treatment. The lowest number of Aporrectodea spp. i n d i v i d u a l s was eighteen for the 100 gm/kg treatment. For the 0 gm/kg treatment only seven speciments of Octo las ion cyaneum were analyzed. The suggested approach to determine di f ferences between treatments i s to use the ana lys i s of variance F t e s t . This test w i l l determine i f there were s i g n i f i c a n t treatment d i f f e rences , but i f more than two treatments are invo lved , the experimenter w i l l not know which treatments are d i f f e r e n t . In order to determine which treatments are s i g n i f i c a n t l y d i f f e r e n t , mul t ip le comparison or mul t ip le range tests are needed to compare each treatment mean with every other treatment mean. Duncan's mul t ip le range test i s most commonly used ( L a r k i n , 1977; L i t t l e and H i l l s , 1972; Mendenhall , 1968; S tee l and T o r r i e , 1960) although the Scheffe ' s test i s more r igorous . Scheffe ' s test s t a t i s t i c i s a more conservative method with regards to type I errors which re jec t H Q when i n fact i t i s true (Wilner , 1976). The UBC Genl in ana lys i s of variance programme u t i l i z e d the F test and i f the F value i s s i g n i f i c a n t for the tes ted terra, the mul t ip le range tests are done (Greig and B j e r r i n g , 1977). The Mann Whitney U test was a lso used to detect d i f ferences among treatment means (milorganite a p p l i c a t i o n ) for each taxon. The Mann - 31 -Whitney U test i s a non-parametric test which i s a ranking t e s t . It i s a powerful non-parametric te s t , having 95% of the power of the parametric t t e s t , and i t i s often used as an a l t e r n a t i v e to the t test when the assumptions for the t test are not met. The non-parametric t e s t s do not require that the samples come from normally d i s t r i b u t e d populations and also that the populations are not required to have the same var iance . For the Mann Whitney U t e s t , the n u l l hypothesis i s that the two populations from which the samples are drawn have the same d i s t r i b u t i o n . This implies the n u l l hypothesis that the two populations have the same means ( L a r k i n , 1977; S i e g e l , 1956). In the present study, treatment d i f ferences were determined with Duncan's mul t ip le range te s t , Scheffe ' s mul t ip le range test and the Mann Whitney U t e s t . The s i g n i f i c a n t d i f ferences detected were only s l i g h t l y d i f f e ren t for the body concentrations of cadmium for a given taxon. Although the F test value for the a d d i t i o n of mi lorgani te was s i g n i f i c a n t , for each taxon Ducan's and Scheffe ' s mul t ip le range te s t detected no s i g n i f i c a n t d i f ferences i n the body t i s sue copper and z i n c for each mi lograni te a p p l i c a t i o n . The Mann Whitney U test did f ind some s i g n i f i c a n t d i f ferences i n body concentrations of z inc and copper. The s i g n i f i c a n t d i f ferences detected by the 3 s t a t i s t i c a l tes t s were d i f f e r e n t for the metal concentrat ions i n the faeces. The Mann Whitney U test tended to detect more s i g n i f i c a n t d i f ferences i n the faeces concentrations of metals among the mi lorgani te a p p l i c a t i o n s . The 3 s t a t i s t i c a l tests may not produce the same s i g n i f i c a n t groupings of treatments for evera l reasons. Both Duncan's and Scheffe ' s - 32 -m u l t i p l e range tests assume that the treatments being tested are uncorrelated and for unbalanced cases th i s assumption i s v i o l a t e d to some extent for the predic ted means (Greig and B j e r r i n g , 1977). The Mann Whitney U test allows for unbalanced comparisons. Secondly, the assumptions that the observations being tested have the same variance and normal d i s t r i b u t i o n may be i n v a l i d and, therefore , the non-parametric tes t would be the tes t to use. The majority of studies i n v o l v i n g the determination of s i g n i f i c a n t di f ferences i n metal concentrations i n earthworms from d i f f e r e n t treatments ( s o i l s with d i f f e r e n t metal concentrat ions) use parametric s t a t i s t i c a l t e s t s . Student 's t test has been used by Andersen (1979, 19800, Ash and Lee (1980), I re land (1975, 1976), I re land and Richards (1977, 1981). Duncan's mul t ip le range test was used by Gish and Christensen (1973) and I re land (1979). I re land (1977) and I re land and Wooton (1976) used Scheffe ' s M u l t i p l e range t e s t , and two way ana lys i s of var iance . Roberts and Johnson (1978) used the Mann Whitney U t e s t . To determine the r e l a t ionsh ip s between metal content (ug) and body weights a step wise l i n e a r regress ion analys i s was used. Al so 2 ca lcu la ted were the r values for the equations. Boyden (1977) and Bryan and Hummerstone (1973) used regress ion ana lys i s to determine r e l a t i o n s h i p s between metal content and body weights for a number of spec ies . Regression ana lys i s was a lso used by Har tens te in , et a l . (1980) to determine re l a t ionsh ip s between metal concentrations and body weights for the earthworm; Eisena foe t ida . Parker , et a l . (1980) used - 33 -covariance analysis to determine the r e l a t i o n s h i p s between body concentrations of metals and body weights of the polychaete, Arenicola marina. 3 RESULTS AND DISCUSSION 3.1 Metal Concentrations In Milorganite, Soil, Earthworms and Faeces Concentrat ion of metals i n mi lorgani te are given i n Table 5. These data are i n agreement with those obtained by John and van Laerhoven, 1976; Carson, (wri t ten communication). Table 5 Metal Concentrat ion (|j.g/gm a i r dry weight) i n M i l o r g a n i t e . (values are the mean of 30 determinations) Meta l x (ISD) Cd 99 (31) Cu 316 (36) Ni 67 (7) Pb 573 (78) Zn 792 (91) Meta l concentrations i n the bulk Ap sample are given i n Table 6. Selected chemical and phys i ca l data for the Ap Crescent S o i l Ser ies (Luttmerding, 1981) and the bulk Ap sample used i n th i s experiment are given i n Table 6. Metal concentrations i n the bulk Ap sample are given i n Table 7. Table 6 Metal Concentrations (|ig/gm a i r dry weight) i n bulk Ap Sample. (Sample Size = 3 0 ) Meta l x (ISD) Cd 0.6 ± 0.4 Cu 32 ± 1.8 Ni 38 ± 4.5 Pb 12 ± 11 Zn 72 ± 5.2 Table 7 Selected Chemical and Physical Properties of Ap Samples (Cresent Series adapted from Lutmerding, 1981b) Exchangeable Bases (NH4OAC) - Particle Size Depth pH %C CEC % Sand % Silt % Clay Sample (cm) (0.01 (organic) %N Ca Mg K Na neg/lOOg MCaCl2 Soil Ap Crescent 0-28 4.3 3.07 0.20 8.44 1.13 0.51 0.35 23.5 8.00 66.6 25.4 Series Ap bulk 0-20 4.1 2.30 0.27 5.55 0.97 0.16 0.12 22.8 4.50 66.05 29.45 sample Table 8 Cadmium Concentration (ug/gm dry weight) in Soil, Body Tissue and Faeces of Three Earthworm Taxa from Six Milorganite Application Rates. (Values ± 1 S.D., number of samples in brackets). Milorganite Application Kate (gm ndlorganite/kg Soil) 0 5 10 25 50 100 Soil Cd 0.8 ± 0.40 (5) 1.5 + 0.76 (5) 2.5 ± 0.94 (5) 3.8 ± 0.94 (5) 7.3 + 4.75 (5) 12 + 3.49 (5) Taxa Body tissue Lumbricus rubelus 11.0 ± 5.14 18.0 ± 4.23 21.4 + 6.07 32.4 + 7.63 38.0 ± 7.97 38.8 ± 9.06 (18) (19) (18) (16) (18) (15) Aporectodea spp. 8.4 + 2.45 (20) 13.7 ± 2.79 (20) 13.4 + 2.79 (20) 19.0 + 5.48 (20) 27.1 + 6.72 (19) 31.4 ± 6.28 (18) Octolosion cyaneum 1.24 + 4.39 (7) 18.5 ± 4.17 (8) 21.0 + 5.42 (8) 27.2 ± 3.17 (8) 43.8 + 9.68 (9) 41.0 ± 5.67 (8) Faeces Lumbricus rubelus 0.4 ± 0.20 (10) 1.4 ± 0.47 (10) 2.7 + 2.21 (8) 6.3 ± 4.47 (10) 12.7 ± 3.67 (10) 9.0 ± 5.74 (9) Aporectodea spp. 0.5 ± 0.47 (11) 1.8 ± 1.26 (10) 2.0 + 1.05 (8) 4.0 ± 1.81 (9) 5.4 + 3.67 (9) 5.1 ± 2.34 (8) Octolasion cyaneum 0.7 ± 0.64 (8) 1.1 ± 0.44 (8) 2.9 ± 1.17 (7) 3.7 + 1.06 (9) 6.4 ± 2.29 (9) 13.6 ± 14.74 (8) Table 9. Copper Concentration (p,g/gm dry weight) in Soil, Body Tissue and Faeces of Three Earthworm Taxa from Six Milorganite Application Rates. (Values ± 1 S.D., number of samples in brackets). Milorganite Application Rate (ga milorganlte/kg s o i l ) 10 25 50 100 Soil Cu 37 ± 2.10 37 ± 2.51 40 ± 2.10 44 ± 2.11 52 + 5.60 65 + 3.11 (5) (5) (5) (5) (5) (5) > ro Taxa Body tissue I : Lumbricus rubelus 15.2 + 6.11 16.2 ± 3.67 17.3 + 4.53 19.4 + 5.64 21.5 ± 5.34 20.2 + 5.64 (18) (19) (18) (16) (18) (15) Aporectodea spp. 10.5 + 3.04 14.2 ± 3.31 18.4 + 4.51 17.0 + 11.07 16.3 + 19.77 18.5 ± 19.19 (20) (20) (20) (20) (19) (18) Octolosion cyaneum 24.7 + 3.90 24.9 ± 4.55 25.4 ± 4.39 26.6 ± 4.55 28.4 ± 2.90 29.6 + 5.48 (8) (8) (8) (8) (9) (8) Faeces Lumbricus rubelus 29.2 + 6.75 38.1 ± 9.94 46.7 ± 14.3 58.0 ± 24.03 85.6 ± 21.11 61.9 ± 17.87 (10) (10) (10) (8) (10) (9) Aporectodea spp. 29.6 + 9.32 38.2 ± 4.57 39.3 ± 5.23 44.3 ± 7.57 52.2 + 11.12 45.5 ± 9.14 (11) (9) (10) (8) (9) (8) Octlasin cyaneum 320 ± 56 71 + 536 427 + 870 54 + 4.43 6.4  8.51 3.7 ± 85.45 8) 8 8) 7Table 10. Lead Concentration (|ig/gm dry weight) in Soil, Body Tissue and Faeces of Three Earthworm Taxa from Six Milorganite Application Rates. (Values ± 1 S.D., number of samples in brackets). Milorganite Application Kate (gm milorganite/kg soil) Soil Pb 10 25 21 + 2.91 (5) 21 ± 2.20 (5) 27 + 5.02 (5) 33 + 3.11 (5) 50 100 51 ± 11.31 (5) 76 ± 8.31 (5) Taxa Body tissue Lumbricus rubelus 1.2 ± 1.12 (18) 0.7 ± 0.48 (19) 1.3 ± 1.65 (18) 1.5 ± 1.38 (16) 4.0 ± 4.59 (18) 2.1 ± 1.53 (15) Aporectodea spp. 1.2 ± 1.09 (20) 2.2 ± 2.05 (20) 3.6 ± 4.93 (20) 1.6 ± 2.00 (20) 1.5 ± 1.54 (19) 2.5 ± 4.40 (18) Octoloslon cyaneum 5.0 + 4.68 4.0 ± 5.44 5.7 ± 6.72 2.8 ± 3.20 3.0 + 2.63 5.8 ± 7.80 (7) (8) (8) (8) (8) (8) Faeces Lumbricus rubelus 12.8 ± 11.41 24.7 ± 13.32 28.5 + 22.57 51.9 + 43.50 113.3 + 44.81 72.0 + 46.84 (10) . (10) (10) (8) (10) (9) Aporectodea spp. 20.5 ± 20.66 17.4 ± 7.77 17.4 ± 9.74 52.7 ± 33.04 51.4 + 22.91 54.3 ± 31.38 (11) (9) (10) (8) (9) (8) Octolasion cyaneum 7.0 + 2.47 10.3 ± 5.65 18.4 + 6.62 25.2 ± 12.56 44.2 + 21.72 116.2 ± 138.02 (8) (8) (8) (7) (9) Table 11. Zinc Concentration (p.g/gm dry weight) in Soil, Body Tissue and Faeces of Three Earthworm Taxa from Six Milorganite Application Rates. (Values ± 1 S.D., number of samples in brackets). Milorganite Application Rate (gm mllorganlte/kg soli) 10 25 50 100 Soil Zn 77 ± 7.78 74 ± 7.38 80 + 1.90 95 + 6.89 116 ± 7.58 161 ± 21.87 (5) (5) (5) (5) (5) (5) Taxa Body tissue Lumbricus rubelus 388.6 ± 122.51 317.2 ± 61.36 321.6 ± 89.34 293.5 ± 112.42 281.4 + 63.78 307.2 + 86.23 (18) (19) (18) (16) (18) (15) Aporectodea spp. 378.2 ± 114.29 334.7 ± 120.52 332.9 ± 74.67 332.9 + 87.30 343.1 ± 75.05 392.7 ± 77.75 (20) (20) (20) (20) (19) (18) Octolosion cyaneum 180.3 ± 46.94 184.8 ± 34.14 207.7 + 46.55 217.0 ± 47.15 236.6 + 24.34 225.7 + 26.07 (7) • (8) (8) (8) (9) (8) Faeces Lumbricus rubelus 71.9 ± 28.47 65.7 + 21.42 89.7 ± 35.73 106.4 + 41.40 160.5 ± 34.57 135.8 ± 49.20 (10) (10) (10) (8) (10) (9) Aporectodea spp. 67.5 + 13.22 79.0 ± 15.40 89.1 ± 20.67 92.2 + 23.09 101.8 ± 17.58 98.2 + 15.94 (ID (9) (10) (9) (9) (8) Octolasion cyaneum 72.4 ± 7.5  5.5 + 1.5 91.9 ± 3.80 7.4 ± 17.48 24.9 + .1 136.0 ± 60.32 8) 8 ) Figure 1, E f f e c t s of sewage sludge applications on cadmium l e v e l s i n tissue and faeces of mature adults of Lumbricus rubellus. 501 40 CADMIUM IN EARTHWORM TISSUE AND FAECES 30 i 20 IO-EARTHWORM TISSUE" EARTHWORM FA£C£S X±5D T *6> .-CO") Rates of sludge a p p l i c a t i o n (gm/kg a i r dried s o i l ) i n parentheses. >C(25) - 1 — 2 4 X (50 • 3£WAG£ -6 —r-8 CADM/UM IN60IL C^Lg/ r^w cty .vt.) io 6 o o ) — i — Figure 2. E f f e c t s of sewage sludge applications on copper l e v e l s i n tissue and faeces of mature adults of Lumbricus rubellus. Rates of sludge a p p l i c a t i o n (gm/kg a i r dried s o i l ) i n parentheses. C O P P E R IN EARTHWORM TISSUE AND FAECES ^45$m dry wi-) l o o -80 H 601 4<?4 ZO-\ \ EARTHWORM V3SUE, -jc±SD EARTHWORM FA£C£S, ^±SD 1(10) (25) CO) T X 1 •»C50: S£WAQ£ T X 1 4 Ooo) 1 30 -1 1 1 1 1 1 1 1 1 1 1 40 50 COPPER IN SOIL tjjgfet* dry wt.) 60 10 - 42 -500 ~5 4 0 0 1 3 300| 8 ZD % zoo cc s M 1 0 0 (0) (lO) EARTHWORM TISSUE X ± SD I EARTHWORM FAECES X ± SD (25) (50) (lOfy SEWAGE / k g S O I L ) 60 100 140 180 Figure 3. E f f e c t s of sewage sludge applications on zinc l e v e l s i n tissue and faeces of mature adults of Lumbricus rubellus. Rates of sludge a p p l i c a t i o n (gm/kg a i r dried s o i l ) i n parentheses. Table 12 - ANOVA Table for Cadmium in Earthworm Body Tissue (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 4804.0 141.91 0.00000 Pots (Milorganite) 24 32.271 0.95327 0.52951 Taxa 2 1985.9 58.664 0.00000 Milorganite * Taxa 10 106.42 3.1435 0.00087 Residual 227 33.853 Total 268 Table 13 - ANOVA Table for Cadmium in Earthworm Faeces i (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 348.46 141.91 0.00000 Pots (Milorganite) 23 22.742 1.4831 0.08940 Taxa 2 66.174 4.3155 0.01551 Milorganite * Taxa 10 45.264 2.9519 0.00239 Residual 119 15.334 Total 159 Table 14 - ANOVA Table for Copper ln Earthworm Body Tissue (5% probability level) Source DF Mean Square F Ratio Probability Milorganite Pots (Milorganite) Taxa Milorganite * Taxa Residual Total 5 24 2 10 227 268 258.51 80.626 1900.6 34.607 78.798 3.2063 1.0232 24.120 0.43919 0.00701 0.43741 0.00000 0.92608 Table 15 - ANOVA Table for Copper in Earthworm Faeces (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 5369.2 12.347 0.0000 Pots (Milorganite) 23 783.62 1.8020 0.02210 Taxa 2 1703.3 3.9169 0.02252 Milorganite * Taxa 10 884.70 2.0345 0.03548 Residual 119 434.86 Total 159 Table 16 - ANOVA Table for Lead In Earthworm Body Tissue (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 16.49 1.6040 0.15996 Pots (Milorganite) 24 13.992 1.3669 0.12470 Taxa 2 115.51 11.284 0.00002 Milorganite * Taxa 10 18.140 1.7721 0.06678 Residual 226 10.236 Total 267 Table 17 - ANOVA Table for Copper in Earthworm Faeces (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 2200.5 18.739 0.00000 Pots (Milorganite) 23 3454.5 2.9417 0.00007 Taxa 2 3544.2 3.0181 0.05265 Milorganite * Taxa 10 3806.3 3.2413 0.00100 Residual 119 1174.6 Total 159 Table 18 - ANOVA Table for Zinc ln Earthworm Body Tissue (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 19239.0 2.7889 0.01820 Pots (Milorganite) 24 12391.0 1.7889 0.01590 Taxa 2 0.33977xl06 49.255 0.00000 Milorganite * Taxa 10 10170.0 1.4743 0.15003 Residual 227 6898.3 Total 268 Table 19 - ANOVA Table for Zinc in Earthworm Faeces (5% probability level) Source DF Mean Square F Ratio Probability Milorganite 5 16963.0 21.155 0.00000 Pots (Milorganite) 23 871.64 1.0870 0.36954 Taxa 2 4160.9 5.1891 0.00691 Milorganite * Taxa 10 2004.4 2.4997 0.00925 Residual 119 801.85 Total 159 Table 20 - S i gn i f i c an t Groupings (5% p r o b a b i l i t y l e v e l ) of Mi lorgan i te A p p l i c a t i o n Rates (gm/kg) for Cd Concentrations i n 3 E arthworm Taxa Body Tis sue and Faeces (App l i ca t ion Rates Within a Taxon Followed by a Common L e t t e r are not S i g n i f i c a n t l y Di f ferent at the 5% L e v e l ) . Taxon (Body Tissue) Lumbricus rubel lus  Aporrectodea spp.  Octo la s i an cyaneum Taxon (Faeces) Lumbricus rubel lus  Aporrectodea spp.  Octo la s ion cyaneum Duncan's Oa 5b, 10b 25c 50d lOOd Oa 5b 10b 25c 50d lOOe Oa 5ab 10bc25c 50d lOOd Oa 5a 10ab25bd50c lOOd Oa 5ab 10ab25ab50b 100b Oa 5a 10ab25ab50b 100c M u l t i p l e Range Test Schef fe ' s Oa 5ab 10b 25c 50c 100c 0a 5ab 10ab25abc50bcl00c 0a 5a 10a 25ad 50c lOOcd 0a 5a 10a 25ab50b lOOab 0a 5a 10a 25a 50a 100a 0a 5a 10a 25ab50abl00b Mann Whitney 0a 5b 10b 25c 50c 100c 0a 5b 10b 25c 50d lOOd 0a 5b 10b 25c 50d lOOd 0a 5b 10c 25c 50d lOOd Oa 5b 10b 25c 50c 100c Oa 5a 10b 25b 50c 100c Table 21 - S i g n i f i c a n t Groupings (5% p r o b a b i l i t y l e v e l ) of M i l o r g a n i t e A p p l i c a t i o n Rates (gm/kg) for Cu Concentrations i n 3 Earthworm Taxa Body Tissue and Faeces (Appl i ca t ion Rates Within a Taxon Followed by a Common Le t t e r are not S i g n i f i c a n t l y Di f ferent at the 5% L e v e l ) . Taxon (Body Tissue) M u l t i p l e Range Test Duncan's Schef fe ' s Mann Whitney Lumbricus rubel lus  Aporrectodea spp.  Octo las ian cyaneum Taxon (Faeces) Lumbricus rubel lus  Aporrectodea spp.  Octo las ion cyaneum Oa 5a, 10a 25a 50a 100a 0a 5a, 10a 25a 50a 100a 0a 5a 10a 25b 50b 100b 0a 5a, 10a 25a 50a 100a 0a 5a, 10a 25a 50a 100a 0a 5b, 10c 25c 50c 100c 0a 5a, 10a 25a 50a 100a 0a 5a, 10a 25a 50a 100a 0a 5a, 10a 25a 50a 100a 0a 5ab 10abd25b 50c lOOd 0a 5ab 10ab25ab50b lOOab 0a 5b lObc 25ce50d lOOe 0a 5ab lOab 25ab50b lOOab 0a 5a 10a 25a 50a 100a 0a 5ab 10b 25ab50b lOOab 0a 5ab lOab 25ab50b 100c Oa 5a 10a 25a 50a 100a Oa 5ab 10b 25b 50c 100c 4> 00 Table 22 - S i gn i f i c an t Groupings (5% p r o b a b i l i t y l e v e l ) of M i l o r g a n i t e A p p l i c a t i o n Rates (gm/kg) for Pb Concentrations i n 3 Earthworm Taxa Body Tissue and Faeces (App l i ca t ion Rates Within a Taxon Followed by a Common L e t t e r are not S i g n i f i c a n t l y Di f ferent at the 5% L e v e l ) . M u l t i p l e Range Test Taxon (Body Tissue) Duncan's Schef fe ' s Mann Whitney Lumbricus rubel lus  Aporrectodea spp.  Octo la s i an cyaneum Taxon (Faeces) Lumbricus rubel lus  Aporrectodea spp.  Octo la s ion cyaneum Oa 5a 10a 25a 50a 100a Oa 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5abl0ab25bd50c lOOd 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100b 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10ab25ab50abl00b 0a 5a 10a 25a 50a 100a 0a 5a 10a 25ab50abl00b 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5b 10bc25ce50del00e 0a 5a 10a 25b 50abl00b 0a 5a 10b 25b 50b 100b Table 23 - S i gn i f i c an t Groupings (5% p r o b a b i l i t y l e v e l ) of M i l o r g a n i t e A p p l i c a t i o n Rates (gm/kg) for Zn Concentrations i n 3 Earthworm Taxa Body Tissue and Faeces (Appl i ca t ion Rates Within a Taxon Followed by a Common L e t t e r are not S i g n i f i c a n t l y Di f ferent at the 5% L e v e l ) . Taxon (Body Tissue) M u l t i p l e Range Test Duncan's Scheffe ' s Mann Whitney Lumbricus rubel lus  Aporrectodea spp.  Octo la s i an cyaneum Taxon (Faeces) Lumbricus rubel lus  Aporrectodea spp.  Octo la s ion cyaneum Oa 5a 10a 25a 50a 100a Oa 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10a 25ab50c lOOd 0a 5abl0a 25ab50b 100b 0a 5a 10a 25a 50bcl00c 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10a 25a 50a 100a 0a 5a 10a 25ab50b 100b 0a 5a 10a 25a 50a 100a 0a 5a 10a 25ab50b 100b 0a 5b 10b 25b 50b 100b 0a 5a 10a 25a 50abl00b 0a 5a 10ab25ab50b 100b 0a 5a 10b 25bd50c lOOcd 0a 5a 10ab25ab50b 100b 0a 5a 10bd25bd50c lOOcd o - 51 -The Cd, Cu, Pb and Zn concentrations i n earthworm body t i s s u e , casts and s o i l - m i l o r g a n i t e media from the treatments are given i n Tables 8 through 11. The body t i s s u e metal concentrations f o r O c t o l a s i o n  cyaneum should be treated w i t h c a u t i o n , as t h i s species d i d not e f f e c t i v e l y c l e a r I t s guts, and the body t i s s u e would be contaminated w i t h faeces. As the concentrations of Cd and Zn i n faeces are lower than body t i s s u e l e v e l s , the contamination by casts may not be s e r i o u s , whereas incomplete clearance of the gut would c o n t r i b u t e to the body t i s s u e Cu and Pb l e v e l s . Faecal concentrations of these two metals exceeded body t i s s u e l e v e l s . Figures 1-3 present the Cd, Cu, and Zn concentrations i n the body t i s s u e and faeces of Lumbricus r u b e l l u s . ANOVA tab l e s f o r the 4 metals and the treatments are presented i n Tables 12 through 19. Tables 20 through 23 present the s i g n i f i c a n t groupings of the m i l o r g a n i t e a p p l i c a t i o n s w i t h regards to the metal concentrations w i t h i n the earthworm taxa. The highest s u r v i v a l percentage was achieved by the Aporrectodea Spp. whereas O c t o l a s i o n cyaneum had the lowest. 3.2 Cadmium Cadmium was accumulated by the body t i s s u e s of a l l 3 earthworms taxa studied (Table 8 ) . Tissue concentrations i n a l l 3 taxa a l s o exceeded those i n the s o i l - m i l o r g a n i t e mixture (the earthworm's food). There was a h i g h l y s i g n i f i c a n t e f f e c t on earthworm t i s s u e Cd concentrations r e s u l t i n g from the a d d i t i o n of m i l o r g a n i t e (Table 12). Cadmium l e v e l s i n t i s s u e increased s t e a d i l y as m i l o r g a n i t e a p p l i c a t i o n s increased u n t i l s o i l concentrations exceeded 7 ug/gm Cd. I n c r e a s i n g the - 52 -mi lorgani te a p p l i c a t i o n rate from 50 gm/kg to 100 gm/kg did not re su l t i n s i g n i f i c a n t increases i n the Cd concentrat ions i n the body t i s sues (Table 20). Figure 1 i l l u s t r a t e s the e f fects of mi lorgani te app l i ca t ions on Cd concentrations i n the body t i s sue and faeces of L . r u b e l l u s . The above re su l t s are i n agreement with those from other s tud ies . Helmke et a l . (1979) determined that cadmium was accumulated by A . tubercu la ta . With increas ing sewage sludge a p p l i c a t i o n s , the mean cadmium concentrations i n the earthworm increased for 7 to 18 [xg/gm for the contro l s to over 100 p.g/gm for the highest a p p l i c a t i o n rate (60 tonnes/ha) . The i r lowest Cd value for the contro l s i s s i m i l a r to the c o n t r o l values for Aporrectodes spp. i n th i s study, whereas the concentrat ion for the highest mi lorgani te a p p l i c a t i o n i n the present study (equivalent to 225 tonnes/ha) was 31 |ag/gm. There are severa l reasons why t h i s d i f ference may have occurred. In the present study, Aporrectodea spp . , cons i s t s of A . tuberculata and A. trapezoides with pos s ib le d i f ferences between the species . The sewage sludge used by Helmke et a l . may have had more ava i l ab le Cd, although the t o t a l Cd was s i m i l a r to the t o t a l Cd i n the mi lorgan i te . The study conducted by Helmke et a l . was a f i e l d experiment conducted over a f ive year term, whereas the present study was a very short term laboratory study l a s t i n g for ten days. The earthworms i n Helmke's study may have accumulated Cd i n a step wise manner. Thei r a c t i v i t y may have decreased due to unfavourable temperatures and/or metal t o x i c i t y a l lowing the earthworms to reduce t h e i r body burden of cadmium. Feeding would again resume when condit ions became favourable (Carter et a l . 1981). - 53 -Hartenste in et a l . (1980) found that E . foe t ida accumulated cadmium above l eve l s i n s o i l ammended with sewage sludge. They observed a maximum concentrat ion of 50 ng/gm for t h e i r f i e l d s tudies . Higher concentrat ions , up to 170 ng/gm were found i n earthworms ra i sed i n sewage sludge spiked with soluble CdSO^. Andersen (1979) found that severa l species of earthworms s t rongly concentrated Cd over the l e v e l s i n the s o i l . He found that the earthworms with the higher Cd concentrations were sampled from f i e l d s r ece iv ing sewage sludge with the higher concentrations of cadmium. Andersen a l so found that cadmium l eve l s were highest i n L . t e r r e s t r i s sampled from s o i l s near busy roads where s o i l cadmium l e v e l s were greatest . In a l l cases, the earthworms had greater Cd concentrations than the s o i l . I re land (1979) reported that _L. rube l lus concentrated cadmium over the l e v e l s i n the s o i l . His values are s i m i l a r to those for t h i s study. Wright and St inger (1980) determined the Cd concentrations i n var ious species of earthworms sampled from s o i l s downwind from a l ead-z inc smelter . The i r r e s u l t s again ind ica ted that earthworms concentrated Cd above s o i l l e v e l s . The higher Cd body concentrations were i n specimens sampled from s i te s with higher s o i l cadmium. The r e s u l t s quoted were s i m i l a r to the r e s u l t s obtained i n the present study. Gish and Christensen (1973), Czarnowska and Japkiewiez (1978) and Ash and Lee (1980) studying s o i l s and earthworms near busy roads, a l l found that earthworms concentrated Cd above s o i l l e v e l s and accumulated the metal i n response to increas ing s o i l cadmium l e v e l s . The ANOVA given i n Table 13 ind ica te s that the a p p l i c a t i o n of - 54 -mi lorgani te had a h ighly s i g n i f i c a n t ef fect on the faeca l Cd concentrat ions . The data i n Table 8 show that the Cd concentrations i n the casts increased with increas ing mi lorgani te a p p l i c a t i o n u n t i l the s o i l Cd l eve l s exceeded 7 ug/gm and then dropped for L . rubel lus and l e v e l l e d out for Aporrectodea spp. In contra s t , f aeca l Cd concentrations for 0. cyaneum continued to increase with the 100 gm/kg a p p l i c a t i o n . However, the d i f ferences i n Cd concentrat ions between casts from the 2 highest a p p l i c a t i o n rates are not s i g n i f i c a n t (Table 20). The re su l t s for Cd concentrations i n earthworm casts obtained i n t h i s study are comparable to those reported i n the l i t e r a t u r e . Czarnowska and Jopkiewicz (1978) found much lower Cd concentrations i n the casts of L. t e r r e s t r i s than i n the body t i s sue s , as d id Helmke et a l . (1979) for A. tubercula ta . In both s tudies , the Cd concentrat ions i n the faeces were s i m i l a r to s o i l Cd concentrations and i n the l a t t e r study the Cd concentrations i n the casts increased with increa s ing sewage sludge a p p l c i a t i o n . The same pattern was also noted by Ash and Lee (1980). Lumbricus t e r r e s t r i s and Aporrectodea c h l o r o t i c a had higher amounts of Cd i n t h e i r casts when sampled from s o i l s with higher Cd l e v e l s . Cadmium i n the faeces was much lower than the concentrai ton i n the body t i s sue of these two species . S i m i l a r re su l t s were obtained for Al lo lobophora longa i n Denmark (Andersen, 1980). The casts had much lower Cd concentrations than did the earthworm body t i s sue and less than the s o i l when the s o i l Cd concentrat ion was less than 0.65 ug/gm. The data, obtained i n t h i s study suggest that at the two highest - 55 -a p p l i c a t i o n rates of mi lorgan i te , feeding and excretory a c t i v i t i e s of the 3 taxa slowed down perhaps i n a tox ic response to l eve l s of metals i n the s o i l - m i l o r g a n i t e medium. The Cd concentrations i n the faeces were much lower than the concentrat ion i n the s o i l mi lorgani te media. These r e su l t s suggest that the ingested cadmium was e f f e c t i v e l y concentrated i n the body t i s sue and the excre t ion of Cd i s very slow. F r i b e r g et a l . 91979) ind ica te s that for humans the excret ion of cadmium i s from 0.005 to 0.01% of the t o t a l body burden per day. Van Hook and Yates (1975) determined that c r i cke t s a s s imi la ted 17% of ingested 1 0 9 C d 109 and that t i s sue e l imina t ion of a s s imi la ted Cd was 0.09-0.11% per day and that gut e l imina t ion was 0.8-0.9% per day. The e l i m i n a t i o n of 1 0 9 C d by l y c o z i d spiders was 0.007% per day. There i s a h ighly s i g n i f i c a n t d i f ference i n the body t i s sue cadmium concentrat ion of a l l three taxa (Table 12). Aporrectodea spp. contained s i g n i f i c a n t l y lower concentrations of Cd i n t h e i r body t i s sue than did the other two taxa s tudied . (Duncan's and Scheffe ' s m u l t i p l e range t e s t s , p<0.05). There was no s i g n i f i c a n t di f ferences between the Cd concentrations i n the body t i s sue of L . rubel lus and 0. cyaneum. There i s a s i g n i f i c a n t d i f ference (P<0.05) between the Cd concentrat ion i n the casts of the d i f f e ren t taxa (Table 13). The casts of Aporrectodea spp. had s i g n i f i c a n t l y lower Cd concentrations than the faeces of L . r u b e l l u s . The faeces of 0_. cyaneum had Cd concentrat ions that were not s i g n i f i c a n t l y d i f f e ren t from the other two taxa. Dif ferences between the taxa might have been due to di f ferences i n the absorpt ion of cadmium from the d ie t or r e f l e c t di f ferences i n feeding - 56 -and excretory a c t i v i t i e s . Lumbricus rubellus i s a shallow burrowing species and a mixed feeder whereas the other two taxa are deep burrowers and may feed more on mineral s o i l . S i g n i f i c a n t differences in cadmium concentration among d i f f e r e n t earthworm species have been reported i n the l i t e r a t u r e . Ireland (1979) reported than Dendrobaena veneta contained s i g n i f i c a n t l y higher amounts of Cd than L. rubellus and E i s e n i e l b a tetraedra. The l a t t e r two species were not s i g n i f i c a n t l y d i f f e r e n t from each other i n Cd content. L. rubellus and L. t e r r e s t r i s did not d i f f e r s i g n i f i c a n t l y i n t h e i r Cd l e v e l s but were higher than Cd l e v e l s i n A. c h l o r o t i c a (Ash and Lee 1980). Wright and Stinger (1980) found that Allolobophora c a l i g i n o s a had s i g n i f i c a n t l y higher Cd concentrations than did the f i v e other species studied, which had no s i g n i f i c a n t differences among them. 3.3 Copper Copper concentrations i n the body tissue of a l l three taxa showed a s l i g h t increase wth increasing a p p l i c a t i o n rates of milorganite, although at any a p p l i c a t i o n rate, copper concentrations i n the earthworms did not exceed s o i l copper concentrations (Table 9). There was a s i g n i f i c a n t e f f e c t due to the a p p l i c a t i o n of milorganite (Table 14). However, there were no s i g n i f i c a n t miloganite x taxa i n t e r a c t i o n s (ANOVA Table 14). The Mann Whitney U test did not f i n d any s i g n i f i c a n t differences (p>0.05) among the milorganite applications for Octolasion  cyaneum. There were s i g n i f i c a n t differences (p<0.05) among the applications of milorganite, using the Mann Whitney U test, for Lumbricus rubellus and Aporrectodea spp. (Table 21). The high mean body - 57 -t i s sue copper concentrations for Aporrectodea spp. for the 50 gm/kg and 100 gm/kg are the re su l t of several i n d i v i d u a l values which are much higher than the remaining i n d i v i d u a l determinations. These high values (92.9 [ig/gm Cu for one i n d i v i d u a l from the 50 gm/kg treatment the next highest concentrat ion for t h i s a p p l i c a t i o n was 26.8 8 [ig/gm Cu; 45.3 |j.g/gm Cu, 53.7 [ig/gm Cu, 74.4 |ig/gm Cu were the three highest concentrat ions obtained for i n d i v i d u a l Aporrectodea spp. specimens from the 100 gm/kg a p p l i c a t i o n , while the next highest concentrat ion was 16.4 |j.g/gm Cu) may be o u t l i e r s and a r e su l t of contamination. With these high values d i scarded, the mean body t i ssue copper concentrations for the 50 gm/kg and 100 gm/kg app l i ca t ions are 12.0 ± 7.28 p.g/gm and 10.7 ± 4.30 [ig/gm r e s p e c t i v e l y . The data i n Table 9 compare favourably with the re su l t s from other s tudies . Helmke, et a l . (1979) determined copper concentrat ion i n Aporrectodea tuberculata sampled from f i e l d s r e c e i v i n g increa s ing amounts of sewage sludge. There were s l i g h t increases i n copper concentrat ions i n the earthworms as the sewage sludge a p p l i c a t i o n increased , although no s t a t i s t i c a l evaluat ions of the data were done. The concentrations that Helmke, et a l . reported were s i m i l a r to the copper concentations obtained for the Aporrectodea spp. i n the present study. Har tens te in , et a l . (1979) found that copper concentrations i n the earthworm, E i s e n i a f o e t i d a , feed gra in or a manure-sewage sludge mixture were much higher i n the earthworms feed the l a t t e r medium. E i s e n i a f o e t i d a , ra i sed i n sewage sludge, var ied s l i g h t l y i n t h e i r copper concentrat ions , increa s ing with increased l eve l s i n the sludge, - 58 -although the copper l e v e l s i n the sewage sludge exceeded those i n the earthworm. The copper l eve l s reported by Har tens te in , et a l . (1979) for E_. foetIda suggest there i s only s l i g h t v a r i a t i o n i n t i s sue concentrations while there i s a greater v a r i a t i o n i n the sludge copper l e v e l s . In I r e l and ' s study (1979), copper concentrations i n Lumbricus  r u b e l l u s , sampled from s o i l s with d i f f e r i n g copper concentrat ions , were not s i g n i f i c a n t l y d i f f e r e n t . The copper concentrations i n the earthworms were lower than the s o i l copper concentrat ions . His data for copper concentrat ion i n L . rubel lus were lower than those obtained i n t h i s study. I re land and Richards (1977) compared the copper concentrat ions i n L . rube l lus and Dendrobaena rubida sampled from a sewage f i l t e r bed and s o i l inf luenced by a lead z inc mine s p o i l , although the s o i l concentrations were d i f f e ren t at each s i t e , there were no s i g n i f i c a n t d i f ferences between the earthworm concentrat ions from each s i t e for a given species . Van Rhee (1977) reported that the copper content i n a mixed sample of Al lo lobophora spp. and L . r u b e l l u s , from s o i l s treated with copper fung ic ides , increased with increas ing s o i l copper concentrat ions . There was a s i g n i f i c a n t c o r r e l a t i o n between s o i l l e v e l s and earthworm l e v e l s . The re su l t s reported by Ash and Lee (1980) suggest that copper was accumulated by Lumbricus t e r r e s t r i s , _L. r u b e l l u s , and Allobophora  c h l o r o t i c a sampled close to roads with varying t r a f f i c d e n s i t i e s . They found that Lumbricus t e r r e s t r i s sampled from the s i t e with the lowest s o i l copper concentrat ion had s i g n i f i c a n t l y lower body t i s sue l eve l s of - 59 -copper. The re su l t s obtained for L . t e r r e s t r i s for the other s i t e s were not s i g n i f i c a n t l y d i f f e rent from each other . There were a lso s i g n i f i c a n t di f ferences i n the copper body l eve l s of A . c h l o r o t i c a among the s i t e s . Earthworms c o l l e c t e d from s o i l s , near Warsaw roads, with higher copper l eve l s were found to have higher body t i s sue concentrat ions of copper. Copper was accumulated by L . t e r r e s t r i s but not concentrated as s o i l copper l e v e l s exceeded those i n the earthworms (Czarnowska and Japkiewicz , 1978). The present study also ind ica te s that copper concentrations i n the faeces of a l l three taxa increases with increas ing miloganite a p p l i c a t i o n . Mean copper l eve l s i n the faeces of L . rube l lus and Aporrectodea spp. decl ined once the s o i l sewage sludge mixture concentrat ion of copper exceeded 52 ng/gm (Figure 4 and Table 9 ) . Octo la s ion cyaneum's faeces continued to have higher mean copper concentrat ions at the highest mi lorgani te a p p l i c a t i o n . This high mean value re su l t s from two exceedingly high values (276.4 and 137.9 |j.g/gm). When these two values are included i n the mean for the 100 gm/kg a p p l i c a t i o n , the value i s 78.5 ± 81.45 [ig/gm Cu. Without these two values , the mean Cu concentrat ion i s 41.74 ± 12.36 ng/gm. These two high values are most l i k e l y the re su l t of contamination and probably should be disregarded. When these values are disregarded, the copper concentrat ion i n the faeces of Octo la s ion cyaneum from the highest mi lorgan i te a p p l i c a t i o n a lso shows the same dec l ine i n copper concentrat ions demonstrated for the other two taxa s tudied . The dec l ine i n copper concentrat ion i n the faeces at the highest a p p l i c a t i o n of - 60 -mi lorgan i te may ind ica te a drop i n the feeding or excret ion a c t i v i t i e s of these earthworms. At the highest a p p l i c a t i o n rate of m i l o r g a n i t e , the metal l e v e l s i n the s o i l mi lorgani te mixture may have been t o x i c to the earthworms. Copper l eve l s i n the faeces of a l l three taxa exceeded the copper l eve l s i n the body t i s sue and suggests that copper i s e f f e c t i v e l y excreted by the earthworms. Copper l eve l s i n the faeces were s i g n i f i c a n t l y (p<0.05) af fected as mi lorgani te a p p l i c a t i o n rates increased (Table 15). The Cu concentations i n the faeces of L . rube l lus (Figure 2) and Aporrectodea spp. increased s t ead i ly and then dec l ined once s o i l Cu l eve l s exceeded 52 ug/gm Cu. The casts of 0. cyaneum showed an increase i n Cu concentrat ion with the 100 gm/kg mi lorgani te a p p l i c a t i o n ra te , although t h i s increase was not s i g n i f i c a n t (Scheffe ' s and Mann Whitney t e s t s , Table 21). The decrease i n Cu concentrat ion i n the casts of L . rube l lu s from the 100 gm/kg treatment was s i g n i f i c a n t by Duncan's and Mann Whitney range tests (Table 21). Helmke, et a l . (1979) ind ica ted that the copper concentrations i n the casts of Aporrectodea tubercula ta were higher than the concentrat ions for the earthworm body t i s sues . The copper content i n the casts a lso increased wth increas ing sewage sludge a p p l i c a t i o n s . They did not observe a drop i n the copper content i n the casts from the highest a p p l i c a t i o n as was the case for the Aporrectodea spp. i n the present study, although the decrease was not s i g n i f i c a n t . However, the highest a p p l i c a t i o n of sewage sludge was 60 tonnes/hectare, whereas i n the present study, the highest a p p l i c a t i o n of mi lorgani te i s equivalent - 61 -to 225 tonnes/hectare. In contras t , to the present study and that of Helmke, et a l . (1979), Ash and Lee (1980) reported that t i s sue samples of Lumbricus rubel lus obtained from s o i l s watered with a 1% copper sa l t so lu t ion had twice as much copper as did the faeca l samples. The body t i s sue samples of Ii. t e r r e s t r i s , L . r u b e l l u s , and Allobophora c h l o r a t i c a obtained from t h e i r roadside study were a lso higher i n copper than the f aeca l samples. These studies ind ica te that the chemical form of the element i s important. There was a s i g n i f i c a n t d i f ference among taxa i n the copper concentrations of earthworm body t i s sue (Table 14). The o v e r a l l mean for earthworm body t i s sue copper concentrations i n 0. cyaneum were s i g n i f i c a n t l y higher than the concentrations i n L . rubel lus and Aporrectodea spp . , whereas the concentrations i n the l a t t e r two taxa were not s i g n i f i c a n t l y d i f f e rent from each other (Duncan's and Schef fe ' s m u l t i p l e range t e s t s , p<0.05). Copper i s an e s s e n t i a l metal and _0. cyaneum may have e i ther a higher b i o l o g i c a l requirement for copper or may have a higher tolerance for copper than the other two taxa s tudied . There was a taxa d i f ference i n the copper concentrations of the faeces (Table 15). The o v e r a l l mean for copper concentrations i n earthworm casts was s i g n i f i c a n t l y higher i n L. rubel lus than i n Aporrectodea spp. Octo las lan cyaneum did not s i g n i f i c a n t l y d i f f e r i n the copper concentrat ion of i t s casts from those of e i ther Aporrectodea spp. and _L. r u b e l l u s . This suggests that L . rube l lus may be more e f f i c i e n t at excret ing copper than the Aporectodea spp . , as the body t i s sue copper concentrations of these two taxa were not s i g n i f i c a n t l y - 62 -d i f f e r e n t (Duncan's and Scheffe 's M u l t i p l e range t e s t s , p<0.05). The lack of s i g n i f i a n t increase i n the body t i s sue copper concentrat ions of Octo las ian cyaneum with increas ing mi lorgani te app l i ca t ions (Table 21) suggest that th i s species i s regu la t ing i t s body copper l e v e l s . The copper concentrat ions i n the body t i s sue of Aporrectodea spp. from the 10 gm/kg, 25 gm/kg, 50 gm/kg and 100 gm/km were not s i g n i f i c a n t l y d i f f e ren t (Mann Whitney t e s t , Table 19) suggesting that th i s taxon i s regu la t ing copper concentrations once a c e r t a i n threshold concentrat ion i s obtained. For Lumbrius r u b e l l u s , the 0 gm/kg, 5 gm/kg and 10 gm/kg app l i ca t ions were not s i g n i f i c a n t l y d i f f e r e n t and there were no s i g n i f i c a n t d i f ferences among the 25 gm/kg, 50 gm/kg and 100 gm/kg a p p l i c a t i o n s . (Mann Whitney t e s t . Table 21). The increa s ing copper l eve l s i n the casts of the earthworm taxa with increa s ing s o i l mi lorgani te copper concentrations (Figure 2 i l l u s t r a t e s the data for L . r u b e l l u s ) , i n d i c a t i n g that the earthworms were inges t ing the copper and excret ing the excess copper, although as Table 21 ind ica te s not a l l the increases i n f aeca l copper were s i g n i f i c a n t . The s i g n i f i c a n t dec l ine i n the faeca l copper concentrations of L. rube l lus from the 100 gm/kg a p p l i c a t i o n compared with the 50 gm/kg and the lack of s i gn i f i c ance between the faeca l copper concentrations of Aporrectodea spp. from the 25 gm/kg, 50 gm/kg and 100 gm/kg and also the casts of 0. cyaneum from the 50 gm/kg and 100 gm/kg treatments (Table 21) suggests that the feeding and/or excretory a c t i v i t i e s of these earthworms decreased, perhaps due to tox i c e f fec t s of the metals i n the m i l o r g a n i t e . Ireland (1979) also observed no s i g n i f i c a n t increase i n the copper concentrations of _L. rubellus sampled from s o i l s with increasing concentrations of copper. The same re s u l t s were obtained for L. rubellus and Dendrobaena rubida (Ireland and Richards, 1977). Both Ireland (19790 and Ireland and Richards (1977) suggest that copper concentrations are regulated by Lumbricus rubellus and Dendrobaena  rubida. In vertebrates, the absorption of ingested copper i s about 50 per cent and i s normally regulated by homeo-static mechanisms (Piscat o r , 1979). In contrast to these studies, Van Rhee (1963, 1975, 1977) found decreased earthworm populations in orchard s o i l s where copper fungicides such as copper sulphate had been used and i n pasture s o i l s where pig s l u r r y containing high amounts of copper had been applied. High concentrations of copper were found to reduce earthworm reproductivity capacity as well as body growth. Copper l e v e l s i n a mixed species sample of earthworms showed increasing l e v e l s of copper i n the body tissue with increasing s o i l l e v e l s although the l e v e l s i n the earthworms did not exceed that of the s o i l . 3.4 Lead Lead concentrations were extremely variable i n the body tissue of a l l three taxa (Table 10), and no pattern of increasing concentrations with increasing milorganite a p p l i c a t i o n could be established. Lead l e v e l s i n the earthworms were lower than s o i l l e v e l s . Addition of milorganite had no s i g n i f i c a n t e f f e c t on the lead l e v e l s of the body tisues of any of the three taxa studied (Table 16). As mentioned previously (Tables 3 and 4) the Pb analysis may not be accurate and the data suspect. - 64 -The published l i t e r a t u r e on lead concentrations i n earthworms i s v a r i a b l e . Van Hook (1974) found that a sample of mixed earthworm genera ranged i n lead concentrat ion from 4.0 to 5.5 ng/gm Pb which was lower r e l a t i v e to s o i l concentrations which ranged from 15 to 50 |ig/gm Pb. Earthworms from s o i l s with higher lead concentrations did not cons i s t en t ly have higher t i s sue lead l e v e l s . Andersen (1979) determined lead concentrat ion i n severa l species of Al lo lobophora sampled from f i e l d s treated with sewage sludges with d i f f e r i n g lead concentrat ions . Lead l eve l s i n s o i l and earthworms were comparable to those obtained i n the present study. In Andersen's study, lead concentrations i n the earthworms did not exceed the s o i l lead concentrat ions . There was a lso no s i g n i f i c a n t di f ferences i n lead content of the earthworms from the contro l s and from the two sewage sludges. These r e s u l t s are very consis tent with the observations made i n the present study. E i s e n i a foe t ida could accumulate lead (up to 325 \ig/gm) but t i s sue l eve l s did not exceed the l eve l s i n the food source: sewage sludge spiked with lead acetate (Hartenste in , et a l . , 1980). Various studied report that there i s a s i g n i f i c a n t increase i n lead content i n earthworms sampled adjacent to busy roads (Glsh and Chri s tensen, 1973; Czarnowska and Japkiewicz , 1978; Andersen, 1979; Ash and Lee, 1980). In these four studies the s o i l lead concentrations were greater than those i n the earthworms. Lead concentrations i n the earthworms were greater than those recorded i n the present study. Robert and Johnson (1978) found that there was a s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between s o i l lead l e v e l s and l eve l s of lead i n Lumbricus t e r r e s t r i s , sampled from s i te s - 65 -contaminated by a l ead-z inc smelter, with higher lead l eve l s i n the organisms c o r r e l a t i n g with higher s o i l l e v e l s . Lumbricus t e r r e s t r i s and var ious species of Al lo lobophora had s i g n i f i c a n t l y higher lead concentrations i n s o i l s with higher lead l eve l s although earthworm lead content was less than the s o i l l eve l s (Wright and S t r i n g e r , 1980). The values reported for Al lo lobophora tuberculata are much greater than the l e v e l s for the Aporrectodea spp. used i n t h i s study (Al lolobophora  tuberculata = Aporrectodes tubercu la ta , Edwards and L o f t y , 1977). However, the s o i l lead l eve l s i n Wright and S t r i n g e r ' s (1980) study were greater than those i n the present study. Al so t h e i r area was contaminated by f a l l o u t from a l ead-z inc smelter and the s o i l lead may have been more ava i l ab l e to the earthworm. In s o i l s contaminated by mine wastes, which had lead concentrat ions of 1314 ug/gm, Dendrobaena rubida and L . rube l lus had lead concentrations as high as 7592 ug/gm and 3529 ug/gm r e s p e c t i v e l y , whereas i n s o i l s where the s o i l lead concentrat ion was 629 ug/gm, the lead concentrat ion i n L . rubel lus was only 9 ug/gm ( I re land , 1979; I re land and Richards , 1977). In another study, I re land (1975) again demonstrated large concentrations of lead i n D. rubida . From s o i l s with lead concentrations of 127 ug/gm and 1713 ug/gm, the lead concentrations i n th i s earthworm were 100 ug/gm and 4160 ug/gm r e s p e c t i v e l y . A d d i t i o n of mi lorgani te had a s i g n i f i c a n t (p<0.05) ef fect on the concentrat ion of Pb i n the casts (Table 17). The faeca l lead concentrat ions for 0. cyaneum increased with increas ing mi lorgani te a p p l i c a t i o n (Table 10) suggesting that th i s species i s e f f e c t i v e l y - 66 -excre t ing ingested lead . The lead l eve l s i n the casts of L . rubel lus increased with increas ing mi lorgani te a p p l i c a t i o n , u n t i l the s o i l lead concentrat ion was 50 ng/gm. The lead content i n the faeces of t h i s species dropped with the 100 gm/kg mi lorgani te a p p l i c a t i o n . This dec l ine a lso suggests that the feeding and/or excretory a c t i v i t i e s of t h i s species may be d e c l i n i n g due to metal poisoning. When the lead concentrat ion i n the s o i l was between 20 and 30 |ag/gm, the concentrat ion i n Aporrectodea spp. was f a i r l y constant, between 17 and 20 |j,g/gm. As the s o i l lead concentrations increased from 30 (ig/gm to over 70 p.g/gm with increas ing mi lorgani te a p p l i c a t i o n s , the faeca l lead concentrat ions for Aporrectodea remained f a i r l y constant, around 52 ng/gm. This suggests that th i s taxon i s not excret ing lead e f f i c i e n t l y , or has slowed down i t s feeding and/or excretory a c t i v i t i e s . However, the data i s extremely va r i ab le and may not be accurate . There are not many s i g n i f i c a n t d i f ferences i n the Pb concentrations of the faeces of a given taxon from the d i f f e rent a p p l i c a t i o n rates of mi lorgani te (Table 22). For a l l three taxa, the l eve l s of lead i n the faeces exceeded the concentrat ions i n the body t i s sue and were i n most cases lower than or s i m i l a r to the s o i l l e v e l s . These re su l t s agree with those i n the l i t e r a t u r e . Andersen (1979, 1981) found that the lead concentrat ion i n the faeces of Al lo lobophora longa was higher i n the casts than i n the earthworm body t i s sue . The l eve l s i n the casts were a lso higher than the s o i l l e v e l s . Czarnowska and Jopkiewicz (1978) found that the lead content i n the casts of IJ. t e r r e s t r i s increased with increas ing s o i l Pb - 67 -l e v e l s . They a lso found, that the worm casts had lead l eve l s s i m i l a r to the s o i l lead content but far greater than the lead l eve l s i n the body t i s s u e . Contras t ing re su l t s were obtained by Ash and Lee (1980) for L . t e r r e s t r i s , A. c h l o r o t i c a and L . r u b e l l u s . They found high lead l eve l s i n the earthworm body t i s sue , lead l e v e l s i n the worm casts d i f f e r e d with d i f f e r i n g s o i l lead l e v e l s . There was a s i g n i f i c a n t d i f ference i n the lead concentrat ion of the three taxa body t i ssues (Table 16). Octo la s ion cyaneum contained s i g n i f i c a n t l y more lead than did the body t i s sue of _L. rubel lus and Aporectodea spp. (Duncan's and Scheffe ' s mul t ip le range tests at p<0.05). The l a t t e r two taxa were not s i g n i f i c a n t l y d i f f e rent from each other . These f indings suggest that 0. cyaneum i s e i t h e r more e f f i c i e n t at absorbing lead from the d i e t or has a higher tolerance to lead body burdens or has a d i f f e rent feeding and excretory a c t i v i t y . There Is no s i g n i f i c a n t d i f ferences among the taxa i n faeca l lead content (Table 17, and t h i s suggests that these taxa had s i m i l a r excret ion e f f i c i e n c e s for l e ad . Species d i f ferences i n the body l e v e l s of Pb have been recorded i n the l i t e r a t u r e . I re land (1979) found that L . rube l lus contained s i g n i f i c a n t l y lower l eve l s of lead i n i t s body t i s sue than d id Dendrobaena venta and E i s e n i e l l a te t raedra . The l a t t e r two species were not s i g n i f i c a n t l y d i f f e rent from each other . Dendrobaena rubida had s i g n i f i c a n t l y higher amounts of lead than did _L. rube l lus ( Ire land and Richards , 1977). Ash and Lee (1980) found higher lead l eve l s i n Al lo lobophora c h l o r a t i c a than i n L_. t e r r e s t r i s . L . t e r r e s t r i s was not - 68 -significantly different from L. rubellus. The lack of significant differences in the lead content of the body tissue with increasing milorganite application indicates that these three taxa of earthworms may be regulating the absorption of lead at the s o i l lead levels encountered in this study. This has not been reported for earthworms previously in the literature, although the data of Andersen (1979, 1980), and Van Hook (1974) suggest regulation of body tissue lead content. Lead levels in the worm casts exceeded the levels in the body tissues and also were similar to the s o i l levels. Lead regulation has been reported in marine annelids. Packer, et a l . (1980) indicated that lead regulation by Arenicola marine occurred in sediments with high lead concentrations. Lead concentrations in the polychaete, Nereis diversicolor were extremely variable, especially where the sediments were highly contaminated by lead. (Bryan and Hummerstone, 1977). 3.5 Zinc The data in Table 11 indicate that zinc concentrations in the body tissues of Lumbricus rubellus individuals treated with milorganite were f a i r l y constant, even as the milorganite applications and s o i l zinc levels increased. The zinc concentrations were lower for the individuals kept in milorganite treated s o i l than were the concentrations of zinc in the body tissue of Lumbricus rubellus kept in the control s o i l without milorganite (Figure 3). The zinc concentrations in the body tissues of Aporrectodea spp. remained f a i r l y constant, and the concentations of zinc in Octolasion cyaneum increased - 69 -s l i g h t l y with increas ing mi lorgani te app l i ca t ions and l e v e l l e d off once the s o i l z inc concentrat ion exceeded 116 ug/g (Table 11). The body t i s sue z inc concentrations are greater than the z i n c concentrations of the s o i l - m i l o r g a n i t e mixture. These r e s u l t s suggest that the three taxa s tudied concentrated z inc over and above the s o i l l e v e l s , but d id not accumulate z inc i n response to increas ing environmental z inc l e v e l s . There was a s i g n i f i c a n t (p<0.05) e f fect on the z inc concentrat ion of earthworms due to the a p p l i c a t i o n of mi lorgani te (Table 18). Although there i s no s i g n i f i c a n t species x sludge i n t e r a c t i o n , the Mann Whitney test d id detect a few s i g n i f i c a n t d i f ferences among the treatments Zn concentrations i n the body t i s sues of the 3 taxa (Table 23). For Lumbricus r u b e l l u s , the highest concentrations of z inc occurred i n the i n d i v i d u a l s from the c o n t r o l . There were no s i g n i f i c a n t d i f fe rences i n the body z i n c concentrat ions of t h i s species kept i n the mi lorgani te treated s o i l . This r e su l t suggests that z inc regu la t ion may be occurr ing i n the i n d i v i d u a l s l i v i n g i n s o i l s po l lu ted with z i n c . The decrease i n the z inc concentrat ion of L . rubel lus from the c o n t r o l and the mi lorgani te treated s o i l , may re su l t from cadmium rep lac ing some of the required z inc i n the earthworm body t i s s u e . Cadmium and z inc have s i m i l a r chemical propert ies and are i n the same p e r i o d i c group, and the presence of cadmium can cause changes i n z inc metabolism and d i s t r i b u t i o n i f cadmium i s subst i tuted for z inc i n some enzymes or other e s s e n t i a l b i o l o g i c a l systems ( F r i b e r g , et a l . , 1979; Nordberg, et a l . , 1979). The data (Table 23) a lso suggest that Aporrectodea spp. and 0_. - 70 -cyaneum may be regulating t h e i r body Zn concentrations. These data agree favourably with those found i n the l i t e r a t u r e . Van Hook (1974) determined that a mixed sample of earthworm taxa concentrated zinc above s o i l l e v e l s by a factor ranging from 4 to 13. The concentration of zinc i n the earthworms were s i m i l a r to those determined i n t h i s study. Helmke, et a l . (1979) observed that the zinc concentrations i n A. tuberculata exceeded s o i l zinc concentrations and increased with increasing sewage a p p l i c a t i o n s . The zinc concentrations for the earthworms were within the range obtained i n the present study. The compost worm, E i s e n i a foetida did not accumulate zinc from zinc acetate spiked sewage sludge (Hartstein, et a l . , 1980). When Zinc concentrations i n the species raised on grain were compared with those fed on manure-sludge mixture, concentrations were considerably higher i n earthworms fed on the l a t t e r medium. There were not s i g n i f i c a n t d ifferenes i n the tissue zinc concentrations i n L. rubellus and I). rubida sampled from two s o i l s with d i f f e r e n t zinc concentrations (Ireland and Richards, 1977). Ireland and Wooton (1976) determined the zinc concentrations i n D. rubida sampled from s o i l s with three d i f f e r e n t zinc concentrations: 172, 286, and 880 p.g/gm. The zinc concentrations i n earthworms from the s i t e with the lowest zinc l e v e l were s i g n i f i c a n t l y lower than those for the earthworms sampled from the two si t e s with higher s o i l zinc l e v e l s . Earthworms from the l a t t e r two s i t e s did not d i f f e r s i g n i f i c a n t l y i n t h e i r zinc concentrations. Lumbricus rubellus sampled from s o i l s with Zn concentrations ranging from 100 ng/gm to 992 [ig/gm did not d i f f e r s i g n i f i c a n t l y i n t h e i r body - 71 -t i s sue Zn concentrat ion which ranged from 416 ug/gm ( I re land , 1979). I re l and (1975) found contras t ing re su l t s for D. r u b i d i a which has s i g n i f i c a n t l y higher z inc t i s sue concentrations when sampled from s o i l s wi th low pH and exceedingly high z inc l eve l s r e l a t i v e to the cont ro l s i t e . In roadside s o i l s , Roberts and Johnson (1978) found that there was no s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between s o i l z inc l e v e l s and _L. t e r r e s t r i s body z inc l e v e l s . However, earthworms sampled from the h igh ly contaminated s i t e s had s i g n i f i c a n t l y more (twice as much) z inc than earthworms from the c o n t r o l s i t e s . Wright and S t r inger (1980) compared the conentrations of z inc i n severa l species of earthworms sampled from s o i l s contaminated by a l ead-z inc smelter . The s o i l s and earthworms sampled c lose to the smelter (4 km) had s i g n i f i c a n t l y higher concentrat ions than the s o i l s and earthworms from the c o n t r o l s i t e 9 km away from the smelter. A s i m i l a r pat tern was found i n s o i l and earthworms sampled from increas ing distance from severa l busy higheways (Gish and Chri s tensen, 1973). Of the three highways s tudied , they found a s i g n i f i c a n t (p<0.05) decrease i n concentrat ion i n both s o i l and earthworms with increas ing distance from two of the three highways s tudied . In one study s i t e , the z i n c concentrations i n the earthwoms from a l l distances were not s i g n i f i c n t l y d i f f e ren t although s o i l z inc l e v e l s changed. In a l l cases the earthworm z inc concentrat ions were greater than those for the s o i l . The r e s u l t s from another roadside study (Czarnowska d Japkiewicz , 1978) ind ica te s that earthworms had higher z inc concentrations than the s o i l . Zinc concentrations i n the - 72 -earthworms tended to be higher for those sampled from s o i l s with higher s o i l z inc concentrat ions . The data i n Table 11 and Figure 3, ind ica te that there were s l i g h t increases i n z inc concentrations i n the faeces as s o i l z inc concentrat ions and rates of mi lorgani te a p p l i c a t i o n increased. A d d i t i o n of mi lorgani te had a s i g n i f i c a n t ef fect (p<0.05) on the Zn concentrat ions i n the casts (Table 19). Within each taxon, there were s i g n i f i c a n t di f ferences i n Zn l eve l s i n the faeces, as the mi lorgani te a p p l i c a t i o n rates incrased (Table 23). The Zn l eve l s i n the casts of L . rube l lus s i g n i f i c a n t l y dec l ined at the highest a p p l i c a t i o n rate and the Zn concentrations i n the faeces of the other 2 taxa l e v e l l e d o f f . The lack of s i gn i f i cance between the concentrat ion of z inc i n the casts of a l l three taxa from the 50 gm/kg and 100 gm/kg and 25 gm/kg and 100 gm/kg suggests that the excre t ion and/or feeding a c t i v i t i e s of the earthworms decreased at the highest a p p l i c a t i o n of mi lorgani te perhaps due to metal po i soning . Z inc l eve l s i n the faeces of a l l three taxa were lower than the z inc l eve l s i n the body t i s s u e . The l eve l s of z inc i n the faeces were s i m i l a r to the l eve l s of z i n c recorded for the s o i l mi lorgani te mixture suggesting that the earthworms were excret ing most of the ingested z i n c . Helmke, et a l . (1979) observed s i m i l a r r e s u l t s : z inc concentrat ion i n faeces increased with increa s ing sewage sludge a p p l i c a t i o n . The l eve l s i n faeces were a lso lower than the z inc concentrat ions i n the body t i s s u e . Czarnowska and Jopkiewicz (1978) a l so reported that the z inc concentrat ion i n earthworm faeces were lower - 73 -than the z inc concentrat ion i n the body t i s sues . The faeces z inc concentrat ion was va r i ab le but tended to increase with increas ing s o i l z i n c concentrat ions . The apparent regula t ion of z inc observed i n th i s study has been observed i n other earthworm genera. I re land (1979) observed z inc regula t ion by L . rube l lus and i n D. rubida ( I re l and , 1975; Ire land and Richards , 1977). Bryan and Hummerstone (1973) determined that z inc was regulated by the marine annel id Nereis d i e r s i c o l o r . In vertebrates absorpt ion of ingested z inc i s h igh ly v a r i a b l e , ranging from 10 to 90 per cent (E l inder and P i s c a t o r , 1979). Zinc i s regulated by homeostatic mechanisms (Roberts and Johnson, 1978; E l i n d e r and P i s c a t o r , 1979). Zinc concentrations i n the three taxa were s i g n i f i c a n t l y d i f f e r e n t (Table 18). Aporrectodea spp. had greater body concentrat ions of z inc than the other two taxa. Lumbricus rube l lus had greater concentrat ions than Octolas ion cyaneum (Duncan's and Scheffe ' s mul t ip le range tests p<0.05). These taxa d i f ferences suggest a d i f ference i n the taxa ' s requirement for z inc and/or di f ferences i n body absorption e f f i c i e n c y of to lerances . Lumbricus rube l lus i s a shallow burrower and mixed feeder, whereas the other two taxa are deep burrowers and, there fore , some di f ferences may be due to feeding a c t i v i t i e s or food in take . Dif ferences In body concentrations of z inc between earthworm species have been observed by other researchers . Wright and S t r inger (1980) found s i g n i f i c a n t di f ferences i n z inc content of various species of A l lo lobophora . I re land (1979) found that L . rubel lus had a - 74 -s i g n i f i c a n t l y higher Zn concentrations than did E i s e n i e l l a t e t raedra , which he a t t r ibuted mainly to di f ferences i n feeding hab i t s . The species d i f ferences i n Zn t i s sue concentrations fo r L . rubel lus and^D. rubida was thought to be more re la ted to d i f f e r e n t i a l absorption of z i n c than d i f f e r e n t i a l feeding ( Ire land and Richards , 1977). There was also a s i g n i f i c a n t d i f ference i n the faeca l l eve l s of the d i f f e ren t taxa (Table 19). Duncan's mul t ip le range test (p<0.05) ind ica te s that Aporrectodea spp. had s i g n i f i c a n t l y lower concentrat ions of Zn i n i t s faeces than did the other two taxa s tudied . Levels of Zn i n the casts of L . rubel lus and 0. cyaneum were not s i g n i f i c a n t l y d i f f e r e n t from each other. As Aporrectodea spp. had s i g n i f i c a n t l y higher body z inc concentrations than did the other two taxa, the low concentrat ion of z inc i n the faeces r e f l e c t s greater e f f i c i e n c y i n absorbing z inc from the diet or that Aporrectodea spp. has a higher body requirement for z i n c . - 75 -4 GENERAL RESULTS AND DISCUSSION 4.1 Soil Metal Concentrations The metal concentrations of the s o i l increased with increa s ing a p p l i c a t i o n of mi lorgani te (Tables 8-11). The s o i l cadmium concentrat ions increased appreciably with each increase i n mi lorgani te a p p l i c a t i o n . For copper the increases i n s o i l copper concentrations were small and the cont ro l and lowest mi lorgani te a p p l i c a t i o n had the same s o i l copper concentrat ions . Increases i n the lead concentrat ions i n the s o i l were s l i g h t u n t i l 50 gm/kg and 100 gm/kg mi lorgani te were added to the s o i l . The 5 mg/kg mi lorgani te a p p l i c a t i o n d id not increase the s o i l lead l eve l s over those of the c o n t r o l . The z inc concentrations i n the s o i l Increased appreciably as the mi lorgani te a p p l i c a t i o n rates increased with the exception that the c o n t r o l and the lowest a p l i c a t i o n rate of mi lorgani te did not have d i f f e rent s o i l z inc concentrat ions . In order to help prevent the bui ld-up of metal concentrations to tox ic l e v e l s , various government agencies i n a number of i n d u s t r i a l c o u n t r i e s , i n c l u d i n g the Ontario M i n i s t r i e s of A g r i c u l t u r e and Food and, Environment have proposed guide l ines for the recommended maximum metal concentrat ions i n a g r i c u l t u r a l s o i l s (Freedman and Hurchinson, 1981; Ontario M i n i s t r i e s Environment and A g r i c u l t u r e 1978). These gu ide l ines are reproduced i n Table 24. The s o i l Cd l eve l s for a l l Mi lorgan i te treatments exceeded the proposed Ontario guidel ines for metal c r i t e r i a for sewage sludge a p p l i c a t i o n . The s o i l Cu and Zn l eve l s were wi th in the proposed g u i d e l i n e s . The s o i l Pb l e v e l s at the 100 gm/kg a p p l i c a t i o n exceeded - 76 -Table 24 Proposed Ontario Guidel ines for Maximum Metal Concentrations i n A g r i c u l t u r a l S o i l s and Maximum Metal Addi t ions i n Sewage Sludges (after Freedman and Hutchinson, 1981; Ontario M i n i s t r i e s Environment and A g r i c u l t u r e and Food, 1978). Meta l Mean Concentrat ion Maximum Recommended Maximum Recommended i n Uncontaminated Meta l Concentrat ion A d d i t i o n to S o i l Ontario S o i l s i n A g r i c u l t u r a l S o i l (kg/ha) (fig/gm dw) ((ig/gm dw) Arsen ic 6.5 13.0 15.0 Cadmium 0.7 1.4 1.6 Cobolt 4.5 18.0 30.0 Chromium 14.0 112.0 220.0 Copper 25.0 100.0 168.0 Mercury 0.08 0.5 0.9 Moly bdenum 0.4 1.6 2.7 N i c k e l 16.0 32.0 36.0 Lead 14.0 56.0 94.0 Selenium 0.4 1.6 2.7 Z inc 54.0 216.0 363.0 - 77 -the proposed gu ide l ine . These re su l t s ind ica te that the cadmium concentrat ion i n mi lorgani te i s s u f f i c i e n t l y high enough to make t h i s sewage sludge unsuitable for a g r i c u l t u r a l use. 4.2 Soil Metal Concentrations - Comparisons With Other Studies The data from the present study and other studies using sewage sludge spiked with soluble metal s a l t s and data from s i t e s po l lu ted by automobile exhaust or mining wastes may not be comparable. B e i j e r and Jernelou (1979) ind ica te that the type of chemical species present g r e a t l y inf luences the uptake and accumulation of metal by an organism. The t o t a l l eve l s of metals i n a given medium may not ind ica te the amount of metal i n a form ava i l ab l e to the organism. The major i ty of lead found i n s o i l s along roadsides w i l l be i n the form of the so luble h a l i d e , lead chlorobromide (PbCIBr) (de Haan & Zwerman 1976). These soluble forms of lead may be more ava i l ab l e to earthworms as Hartens te in et a l . (1980) found exceedingly high concentrations of lead i n E i s e n i a  foe t ida kept i n sewage sludge spiked with lead acetate . The lead l e v e l s i n earthworms sampled close to busy roadways were s u b s t a n t i a l l y higher than the lead l eve l s observed i n the earthworms used i n th i s study. (Gish & Chr i s tensen , 1973; Czarnauska and Japkiewicz , 1978; Ash & Lee , 1980). The studies reported by I re land (1975, 1979) ind ica ted high l e v e l s of lead i n earthworms and the s o i l s sampled from s i t e s contaminated by l ead-z inc mine s p o i l s . The lead i n these s o i l s may have been i n an highly ava i l ab l e form to the earthworms. In earthworms sampled from f i e l d s contaminated by p ig manure earthworms were found to accumulate copper i n response to increa s ing s o i l coper l e v e l s . (van Rhee 1977). The copper i n p ig manure i s i n the form of soluble s a l t s - 78 -(van Rhee 1975). Various studies (Ash & Lee, 1980; Hartenstein et a l . , 1980; and Ireland and Richards, 1981) demonstrated that earthworms kept i n media treated with solutions of soluble metal s a l t s had excessive concentrations of cadmium, copper, lead and zinc i n the body ti s s u e s . The r e s u l t s of Ireland and Richards (1981) are quoted as an example. Lumbricus rubellus kept for 26 days on f i l t e r paper saturated with 5 Hg/gm CdCl2 had a body tissue concentration of 416 p.g/gm Cd, compared to 6 p-g/gm for the controls. In sewage sludge metals are l i k e l y to be complexed with organic matter and less a v a i l a b l e to organisms. Differences i n the r e s u l t s obtained by the various authors i n the l i t e r a t u r e may also r e f l e c t differences i n s o i l properties. The amount of extractable metals i n the s o i l i s influenced by the pH, organic matter and clay content (Machean and L a n g i l l e , 1976). Cadmium (Chubin and Street, 1981), copper and lead (Haster, 1979), and zinc (Sehumas, 1975) adsorption i s greater i n s o i l s with higher CEC, exchangeable bases, organic matter and clay content. The clay mineralogy and presence of i r o n and aluminum hydroxides also influences the adsorption of these metals. (Schuman, 1976; Haster, 1979; and Chubin & Street, 1981). Both Cavalloro & McBridge (1978) and Kuo & Baker (1980) indi c a t e that pH influences Cd, Cu, and Zn adsorption by s o i l s . The adsorption capacity of a s o i l are lower at lower pH. Care must be exercised when comparing the r e s u l t s of the present study with f i e l d studies. In t h i s study the milorganite was well mixed with the s o i l , a condition u n l i k e l y to be obtained i n a f i e l d s i t u a t i o n . In a f i e l d study p r e f e r e n t i a l feeding by earthworms may lead - 79 -to more v a r i a b i l i t y i n the metal content i n the earthworms. Al so sewage sludge under f i e l d condit ions may undergo more extensive p h y s i c a l , chemical and b i o l o g i c a l changes under the inf luence of na tura l weather condit ions over time (Hartenstein et a l . 1980). Van Rhee (1965) ind ica te s that general ly earthworms do not l i k e container condit ions and a f ter severa l months w i l l e i ther die or ae s t i va te . In the present study i n severa l of the pots , some of the i n d i v i d u a l s were a e s t i v a t i n g and some had d ied ; De V r i e s and T i l l e r (1978) compared leve l s of metals i n vegetables grown i n sewage sludge i n greenhouse containers and i n the f i e l d and found higher metal l eve l s i n the t i s sue from the greenhouse study. They warn that flowerpot studies may give t o t a l l y d i f f e r e n t responses from f i e l d s tudies , as temperature, l i g h t , humidity and other environmental condit ions w i l l most l i k e l y between f i e l d and greenhouse s i t u a t i o n s . 4.3 Body Weights The v a r i a b i l i t y obtained i n th i s experiment may, i n par t , be due to the natura l b i o l o g i c a l v a r i a b i l i t y wi th in i n d i v i d u a l s . In order to minimize the v a r i a b i l i t y due to r e p r o d u c t i v i t y stage, a l l the earthworms used i n th i s study were sexual ly mature, c l i t e l l a t e i n d i v i d u a l s . Carter et a l . (1980) demonstrated that j u v e n i l l e n o n - c l i e t l l a t e Lumbricus  rube l lus i n d i v i d u a l s had lower l eve l s of Cd than mature c l i t e l l a t e specimens of the same species . Whole earthworms were analyzed to overcome v a r i a b i l i t y due to sampling p a r t i c u l a r body par t s . I re l and (1974) found that the highest concentrat ion of lead and z inc i n Dendribaeba rubida was i n the pos te r io r al imentary t r a c t . Andersen (1980) reported that i n the pos te r io r most 10 segments of Al lo lobophora Table 25 Regression Equations and r Values R e l a t i n g Body Dry Weights (mg) and Element Contents (ug) for Lumbricus rube l lus for s ix M i l o r g a n i t e Treatments (5% p r o b a b i l i t y l eve l ) gm M i l o r g a n i t e / kg s o i l Body Weight (mg) x 1SD ugCd ugCu = ugPb = ugZn = 73.18 ± 18.48 .011 body wt .310 0.015 body wt .307 .084 27.59 10 90.30 ± 26.02 .018 body wt .516 .009 body wt .804 .0008 body wt .262 .327 body wt .700 .00007 body wt 84.72 ± 28.41 .021 body wt .590 .017 body wt .657 .094 oo o .309 body wt .665 i 25 93.11 ± 40.48 3.59+160 body wt .818 .020 body wt .760 .134 .0014 body wt 2 378x10 body wt 10.99 + .155 .487 body wt 50 97.20 ± 26.07 .039 body wt .597 .021 body wt .482 .378 .269 body wt .384 100 91.51 ± 23.71 .037 body wt .317 .020 body wt .437 .172 .288 body wt .242 Table 26 Regression Equations and r Values Re la t ing Body Dry Weights (mg) and Metal Content (fig) for Aporrectodea spp. for s ix M i l o r g a n i t e Treatments (5% p r o b a b i l i t y l e v e l ) . gm M i l o r g a n i t e / kg s o i l Body Weight mg x ISD (igCu = ligPb = (igZn = 135.91 ± 31.89 .229 .011 body wt .571 .168 .369 body wt .246 10 166.07 ± 47.81 .666 1.40+0.22 .785 .259 body wt 169.62 ± 33.67 .518 1.97+.030 .746 .662 body wt 21.37+.214 .406 body wt .327 body wt .342 I oo 25 171.21 ± 40.09 .404 .016 body wt .109 .282 .311 body wt .295 50 155.43 ± 34.27 .272 .016 body wt .049 .0032 body wt+ .457 .322 body wt .476 .00003 body wt 100 140.91 ± 37.52 .506 .239 .360 .402 body wt .604 Table 27 Regression Equations and r Values Re la t ing Body Weights and Element Contents for Octolos ion cyaneum from Six Mi lorgan i te Treatments (5% p r o b a b i l i t y l e v e l ) . gm M i l o r g a n i t e / kg s o i l Body Weight (mg) X + 1 SD Cd r 2 HgCu = Cu r HgPb= Pb r HgZn= Zn r 10 25 50 100 2 3 6 . 5 7 ± .325 .025 body wt 40.04 2 2 5 . 8 5 ± .467 .025 body wt 41.82 2 5 2 . 0 7 ± .769 .027 body wt 124.82 227.851 .806 .027 body wt 62.86 187.271 0.0 22.66 .028 body wt .807 1.057 .659 0.0 0.0 .010 148.501 .849 .031 body wt 36.16 .963 .0062 body wt .450 .872 .0031 body wt .270 .513 .543 .012 .854 .802 .193 body wt. .190 body wt. .190 body wt. .396 body wt. , .00076 body wt' .239 body wt. .038 .233 body wt. .302 .376 .804 .026 .299 .814 - 83 -longa kept i n sewage sludge amended s o i l , the coelomic sacs were f i l l e d with waste bodies containing higher l eve l s of Pb and Cd than the gut w a l l . In each of the treatments earthworms ranged i n whole body weights . The r e l a t i o n s h i p between body weight and metal content for Cd, Cu, Pb and Zn for the three taxa was determined using step-wise regres s ion a n a l y s i s . The re su l t s are given i n Tables 25, 26 and 27. The cadmium content (ug) i n a l l three taxa increased with increas ing body s i z e . The slopes of the equations increase with increa ing mi lorgani te a p p l i c a t i o n rate and, therefore , increas ing s o i l Cd l e v e l s . Thi s ind ica tes that the earthworms at higher s o i l Cd l eve l s had higher body burdens of cadmium. When the data i s converted from absolute metal content (ug) to concentrat ion (ug/gm or ppm) by d i v i d i n g through by the i n d i v i d u a l body weights, the slope becomes zero . Concentrat ion of Cd i n a l l three taxa i s , therefore , independent of body weight. The cadmium equations for Octo la s ion cyaneum 50 gm/kg treatment and for Lumbricus  rube l lu s 25 gm/kg treatment do not conform to the trend es tab l i shed by the other treatments and may ar i se from random v a r i a t i o n . Copper content (ug) for a l l three taxa was a lso re la ted to body weight. Unl ike the trend exhib i ted by Cd, the slope for a given taxon for Cu content i s r e l a t i v e l y constant. For Lumbricus r u b e l l u s , the average slope i s 0.019 and for Octolasium cyaneum the average slope i s 0.027. The re su l t s for Aporectoedea spp. were more v a r i a b l e . Three of the treatments re l a te copper content to body weight and for these the average slope i s 0.014. A constant slope for the d i f f e rent mi lorgani te a p p l i c a t i o n ind ica tes that the copper l eve l s i n body t i s sue are - 84 -regula ted . Converting the data to concentrat ion , the slope r e l a t i n g body weight to copper concentrat ion i s zero i n d i c a t i n g that copper concentrat ion i s independent of body weight. Zinc content was a lso re l a ted d i r e c t l y to body weight for a l l three taxa. As for copper, the slope for a given taxa was r e l a t i v e l y constant o v e r a l l mi lorgani te app l i ca t ions i n d i c a t i n g regula t ion of z i n c by the earthworms. Z inc concentrat ion i s independent of body s i z e . The 25 gm/kg treatment for 0. cyaneum, the 5 gm/kg treatment for Aporrectodea spp. and the 0 gm/kg and 25 gm/kg treatments for L . rube l lus did not f i t the trend es tab l i shed by the remaining treatments and may be the re su l t of random v a r i a t i o n . The lead data were v a r i a b l e , but for a l l three taxa the lead content was constant for a given a p p l i c a t i o n of mi lorgani te with the fo l lowing exceptions : the 50 gm/kg treatment for Aporectodea spp . , the 5 gm/kg treatment for Lumbricus rube l lus and the 10 gm/kg and 25 gm/kg treatments for Octo la s ion cyaneum. These four r e s u l t s may be a r e s u l t of random v a r i a t i o n . Lead concentrat ion i s , therefore , dependent upon body s ize with the higher concentrations i n smaller i n d i v i d u a l s . Body s ize could s i g n i f i c a n t l y af fect the Pb re su l t s but the lead content i n the body t i s sue i s extremely var iab le making in te rpre t a t ions d i f f i c u l t (Table 10). Also the lead data may be suspect and inaccurate . Boyden (1977) reviewed metal content and s ize r e l a t i o n s h i p for severa l species of s h e l l f i s h . For the oyster , Ostrea e d u l i s , z i n c concentrat ion (ppm) was independent of body s ize for oysters from metal - 85 -(1980). They ind ica ted that the concentrat ion of Mg, Fe, A l , Nn, and Zn were greatest i n the smaller i n d i v i d u a l s . In contrast the l a rger i n d i v i d u a l s had higher concentrations (ug/gm) of copper. The r e l a t i o n s h i p between body weight and metal concentrat ions va r i e s from emtal to metal and from species to species . The r e l a t i o n s h i p s determined i n t h i s experiment were not incons i s tent with r e l a t i o n s h i p determined for other species . Boyden (1977) a lso reported that the slope of the regress ion l i n e r e l a t i n g cadmium content (ug) to body weight of the l impet , P o t e l l a , increased with increa s ing Cd concentrat ion i n the environment. The same trend was observed for a l l the earthworm taxa studied i n t h i s experiment. Earthworms as Biological Indicators of Metal Contamination The second ob ject ive of t h i s study was to determine i f the earthworm taxa studied could be used as b i o l o g i c a l monitors of metal contamination of s o i l . There are severa l c r i t e r i a that a s o i l organism should meet before i t can be considered useful as an i n d i c a t o r of s o i l e t a l concentrat ion: 1) contain the metal i n proport ion to i t s concentation i n the s o i l , i . e . , the concentrat ion of metal i n the organism should change pred ic tab ly with the s o i l concentrat ions , 2) the organism should accumulate the metal but not regulate the metal , 3) be of reasonable s ize to provide s u f f i c i e n t t i s sue for a n a l y s i s , and 4) be easy to sample and i d e n t i f y and be able to survive In the laboratory , to a l low for defecat ion before analyses , and for laboratory studies of metal uptake (Bryan and Hummerstone, 1977; P h i l i p s , 1978; Wieser, et - 86 -a l . , 1976). Helmke, et a l . (1979), Car ter , et a l . (1980), Czarnowska and Jopkiewicz (1978) and Van Hook (1974) mention that earthworms may be use fu l i n d i c a t o r s of metal p o l l u t i o n i n t he s o i l . Feeding and burrowing a c t i v i t i e s of earthworms expose them to the s o i l . I n d i v i d u a l earthworms conta in enough t i s sue to al low for analyses . They are ubiquitous and are r e l a t i v e l y easy to c o l l e c t . Adult c l i t e l l a t e earthworms can be i d e n t i f i e d using morphological c h a r a c t e r i s t i c s without d i s s e c t i o n , for example using the key produced by Reynolds (1977). The concentrat ion r a t i o s of the four metals for the three taxa are given i n Table 28. The concentrat ion r a t i o i s defined as the r a t i o of ug/gm element i n the consumer's organism t i s sue ( in t h i s case, earthworm) to the ug/gm element i n i t s food items ( s o i l ) (Van Hook, 1974). My re su l t s are i n general agreement with those reported by Van Hook (1974), Mart in and Coughtrey (1976), and Wright and Str inger (1980) and those ca lcu la ted from the data of Gish and Christensen (1973), I re land (1975, 1979) and Andersen (1979, 1980). The re su l t s i n Table 28 v e r i f y that lead and copper are not concentrated by the 3 earthworm taxa, above s o i l l eve l s for these two elements. As copper s o i l concentrat ions increased from 37 to 52 ug/gm, the concentrat ion r a t i o for copper i n Lumbricus rube l lus remained f a i r l y constant i n d i c a t i n g that there was a near l i n e a r r e l a t i o n s h i p between increas ing s o i l copper and earthworm body t i s sue concentrat ions . However, the increases i n s o i l copper were very s l i g h t and there were very few s i g n i f i c a n t d i f ferences i n the earthworm body t i s sue copper concentrat ions . When copper concentrations increased to 65 ug/gm the concentrat ion factor for Table 28 S o i l Metal Concentrations and Concentrat ion Factors for Cd, Cu, Pb, Zn, for Three Taxa of Earthworms Lumbricus rubel lus Aporrectodea spp. Octo la s ion cyanium Cadmium: Soil Cd ug/gm 0.8 13.8 10.5 15.5 1.5 12.0 9.1 12.3 2.5 8.6 5.4 8.4 3.8 8.5 5.0 7.2 7.3 5.2 3.7 6.0 12 _ 3.2 _ 2.6 _ 3.4 x 8.5 ± 4.0 x 6.0 ± 3.1 x 8.8 ± 4.4 Copper: Soil Cu ug/gm 37 0.41 0.28 0.67 37 0.44 0.38 0.67 40 0.43 0.46 0.64 44 0.44 0.39 0.60 52 0.41 0.31 0.55 65 _ 0.31 _ 0.28 _ 0.46 x 0.41 ± 0.05 x 0.35 ± 0.07 x 0.60 ± 0.08 Lead: Soil Pb ug/gm 21 0.06 0.06 0.24 21 0.03 0.10 0.19 27 0.05 0.13 0.21 33 0.05 0.05 0.08 51 0.08 0.03 0.06 76 _ 0.03 _ 0.03 _ 0.08 x 0.05 ± 0.02 x 0.07 + 0.04 x 0.14 ± 0.08 Zinc: Soil Zn ug/gm 77 5.0 4.9 2.3 74 4.3 4.5 2.5 80 4.0 4.2 2.6 95 3.1 3.5 2.3 116 2.4 3.0 2.0 161 1.9 2.4 1.4 x 6.0 ± 3.1 x 3.8 ± 0.95 x 2.2 ± 0.44 - 88 -concentration factor for Lumbricus rubellus decreased the copper concentration factor for Aporrectodea spp. was variable but tended to decrease as s o i l concentrations increased suggesting copper regulation. The copper concentration factors for Octolasion cyaneum decreased as s o i l concentrations increased. This i s consistent with the hypothesis that t h i s species i s regulating copper concentrations i n i t s body. The zinc concentration factors for a l l three taxa decreased as s o i l concentrations increased, again suggesting that zinc i s regulated. The concentration factors for zinc are greater than unity, an i n d i c a t i o n that body tissue zinc concentrations are greater than the concentrations i n the s o i l . The lead concentration factors are variable but tend to decrease with increasing s o i l lead concentrations supporting the hypothesis that lead i s regulated by the three taxa. Cadmium was concentrated by a l l three taxa to a much greater extent than zinc. Even though the cadmium concentrations i n a l l three taxa continued to increase as s o i l cadmium concentrations increased from 0.8 ng/gm to 7.3 |j.g/gm, the concentration factors decreased. This suggests that the s o i l cadmium concentrations are increasing to a greater degree r e l a t i v e to the earthworm body tissue concentrations, as the rates of milorganite a p p l i c a t i o n increase. The re l a t i o n s h i p between s o i l cadmium concentrations and earthworm tissue concentrations i s not l i n e a r , even though the soil - m i l o r g a n i t e mixture i s the source of cadmium. The s o i l cadmium concentration values used i n the concentration factor c a l c u l a t i o n were determined using a t o t a l s o i l cadmium concentration and may not be i n d i c a t i v e of the actual amount of cadmium available to the - 89 -earthworm. At the highest s o i l cadmium concentrat ion It was suggested that earthworm feeding and or excretory a c t i v i t i e s had slowed down perhaps i n response to metal poi soning . The data suggest that i f s o i l cadmium concentrations are to be predicted using the earthworm body t i s sue concentrations and the average concentrat ion factor the s o i l cadmium concentrat ion could sometimes be underestimated or over est imated. For example, us ing the average concentrat ion factor of 8.5 for L . rubel lus would tend to underestimate s o i l Cd l eve l s i n s o i l s wi th cadmium l e v e l s greater than 4 ug/gm and over estimate the s o i l Cd concentrat ion where s o i l concentrat ion i s less than 2 ug/gm. Thi s requires further work to determine the Cd l eve l s i n one of the given taxa and use the quoted concentrat ion factors to pred ic t s o i l Cd va lues . Comparing predic ted s o i l Cd values with actua l values determined by r e l i a b l e ana lys i s would ind ica te the usefulness of earthworms as b i o l o g i c a l monitors of s o i l contamination by Cd. The data i n Table 28 a lso suggest that there are taxa di f ferences among the concentrat ion r a t i o s which r e f l e c t s the taxa di f ferences i n metal concentrat ions discussed p r e v i o u s l y . The re su l t s ind ica te that thse three taxa of earthworms would not be an appropriate b i o l o g i c a l monitor for Cu, Pb and Zn as these three elements appear to be regulated by the organism. These taxa, therefore , f a i l to meet c r i t e r i o n #1, for these three metals . As the lead and copper concentrations are much lower i n the earthworms r e l a t i v e to s o i l l eve l s i t would be eas ier to detect s o i l Pb and Cu contamination by analyzing the s o i l . The earthworm taxa s tudied may be used to ind ica te Cd contamination i n the s o i l but there - 90 -was not a l i n e a r r e l a t i o n s h i p . I f earthworms are to be used to indicate s o i l Cd l e v e l s , then d i f f e r e n t taxa should not be pooled. - 91 -5 CONCLUSIONS For a l l three taxa of earthworms the tissue concentration |J.g/gm of Cd, Cu, and Zn was independent of body weight. Lead concentration data were extremely variable. However, the data indicated lead concentrations were higher in smaller individuals. The body tissue concentrations of copper, zinc and lead appear to be regulated in a l l three taxa studied. In contrast, cadmium concentrations in the body tissue increased with Increasing s o i l Cd concentrations. The concentrations of Cd, Cu, Pb and Zn increased in the casts with increasing rates of milorganite application. In L. rubellus for a l l four metals studied, at the highest milorganite application rate, there was a decrease in the faecal metal concentrations suggesting a slow down in feeding and excreting act i v i t i e s perhaps the result of metal poisoning. For Aporrectodea spp. the concentrations of metals in the casts tended to level out at the highest application rate of milorganite, suggesting that activities were starting to slow down in response to metal loading of the s o i l . In contrast, the Cd and Zn concentrations in the faeces of 0. cyaneum continued to increase at the highest milorganite application rate while the Cu and Pb concentrations levelled out supporting differences in metal tolerances. There were significant differences (p<0.05) in the metal concentrations in the faeces and body tissue among the taxa. There was no consistent pattern for the uptake either for a given metal or given taxon. As the pattern of metal uptake is not consistent, these - 92 -differences may r e f l e c t d i f f e r e n t excretory a c t i v i t i e s and d i f f e r e n t tolerances and requirements for metals rather than s e l e c t i v e feeding. The r e s u l t s suggest that the earthworm taxa studied would not be useful b i o l o g i c a l i n d i c a t o r organisms for s o i l Cu, Pb and Zn concentrations. Earthworms might be useful to predict cadmium s o i l concentrations but further work would be required to validate t h i s conclusion. This study indicates that r e s u l t s from d i f f e r e n t taxa should not be pooled as species differences i n metal concentrations did occur. - 93 -6 REFERENCES Andersen, C. 1979. Cadmium Lead and Calcium Content, Number and Biomass, i n Earthworms (Lumbricidae) from Sewage Sludge Treated S o i l . Pedobio log ia . 19: 309-319. Andersen, C . 1980. Lead and Cadmium Content i n Earthworms (Lumbricidae) from Sewage Sludge Amended Arable S o i l . In D i n d a l , D . L . ( e d . ) , S o i l Biology as Related to Land Use P r a c t i c e s , Proceedings VII Internat ions S o i l Zoology Colloquium. EPA 560/13-80-038, Washington pg. 148-156. Ash , C . P . J , and D . L . Lee. 1980. Lead, Cadmium, Copper and Iron i n Earthworms From Roadside S i t e s . E n v i r o n . P o l l u t . Ser. A . 22: 59-67. B e i j e r , K. and A . Jerne lov . 1979. General Aspects and S p e c i f i c Data on E c o l o g i c a l E f fec t s of Metals i n F r i b e r g L . et a l . (ed) Handbook on the Toxicology of Metals E l s e v i e r / N o r t h - H o l l a n d Biomedical Press , pp. 197-209. Boyden, C R . 1977. E f f e c t of Size upon Meta l Content of S h e l l f i s h . J . Mar. Ass . U .K. 57: 675-714. Bryan, G.W., and L . G . Hummerstone. 1971. Adaptation of the Polychaete Nereis d i v e r s i c o l o r to Estuar ine Sediments Containing High Concentrations of Heavy Metals I . General Observations and Adaption to Copper. J . Mar. Ass . U.K. 51: 845-863. Bryan, G .W. , and L . G . Hummerstone, 1973. Adaptat ion of the Polychaete Nereis d i v e r s i c o l o r to Estuar ine Sediments Containing High Concentrat ions of Z inc and Cadmium. J . Mar. Ass . U.K. 53: 839-857. Bryan, G .W. , and L . G . Hummerstone. 1973. Adaptat ion of the Polychaete Nereis d i v e r s i c o l o r to Manganese i n Estuar ine Sediments. J . Mar. Ass . U.K. 53: 859-872. Bryan, G .W. , and L . G . Hummerstone. 1977. Indicators of Heavy-Metal Contamination i n the Looe Estuary (Cornwall) with P a r t i c u l a r Regard to S i l v e r and Lead. J . Mar. Ass . U .K. 57: 75-92. C a r t e r , A . C , E . A . Kenney, and T . F . Guthr ie . 1980. Earthworms as B i o l o g i c a l Monitors of Changes i n Heavy Metal Level s i n an A g r i c u l t u r a l S o i l i n B r i t i s h Columbia. In D i n d a l , D . L . (ed) , S o i l Biology as Related to Land Use P r a c t i c e s , Proceedings VII In te rna t iona l S o i l Zoology Colloquium. EPA 560/13-80-038, Washington, pp. 344-357. Car te r , A . C , E . A . Kenney, and T . F . Guthr i e . 1983. Heavy Metals i n Earthworms i n Non-Contaminated and Contaminated A g r i c u l t u r a l S o i l From Near Vancouver. Proceedings Darwin Symposium (In Pre s s ) . - 94 -C a v a l l a r o , N . , and M.B. McBride. 1978. Copper and Cadmium Adsorpt ion . C h a r a c t e r i s t i c s of Selected A c i d and Calcareous S o i l s . S o i l S c i . Soc. Am. Proc . V42: 550-556. Chubin, R . G . , and J . J . S t reet . 1981. Absorpt ion of Cadmium on S o i l Const i tuents i n the Presence of Complexlng Ligands . J . E n v i r o n . Qual. 10: 225-228. Cros s l ey , D .A. , D . E . Re i ch le , and C A . Edwards. 1971. Intake and Turnover of Radioact ive Cesium by Earthworms (Lumbricidae) . Pedobio log ia . 11: 71-76. Czarrowska, K . , and K. Jopkiewicz . 1978. Heavy Metals i n Earthworms as an index of S o i l Contamination. P o l i s h Journal of S o i l Science. 11: 57-62. de Haan, F . A . M . , and P . J . Zwerman. 1976. P o l l u t i o n of S o i l i n S o i l Chemistry A Basic Elements. G . H . Bo l t and M.G.M. Bruggenwert. (eds) E l s e v i e r S c i e n t i f i c Pub l i sh ing Company, New York, pp 192-271. de V r i e s , M . P . C , and K . G . T i l l e r . 1978. Sewage Sludge as a S o i l Amendment with Spec ia l Reference to Cd, Cu, Mn, N i , Pb and Zn -Comparison of Results from Experiments Conducted Inside and Outside a Glasshouse. Env i ron . P o l l u t . 16: 231-240. Edwards, C A . and J .R . L o f t y . 1977. Biology of Earthworms. Chapman and H a l l , London, second e d i t i o n , 333 pps. E l i n d e r , C . - G . , and M. P i s c a t o r . 1979. Z inc i n Handbook on the Toxicology of Meta l s . F r i b e r g , L . et a l . (eds) E l s e v i e r / N o r t h -Hol land Biomedical Press , pp. 675-685. F i s h e r , L . , and J . McDonald. 1978. Fixed E f fec t s Analys i s of Variance Academic Press , New York. Freedman, B . , and T . C . Hutchinson. 1981. Sources of Metal and Elemental Contamination of T e r r e s t r i a l Contamination i n E f f ec t of Heavy Metal P o l l u t i o n on Plants Volume 2 Metals i n the Environment. Lepp, N.W. (ed) Appl ied Science Pub l i sher s , New Jersey, pp. 35-94. F r i b e r g , L . , T . K j e l l s t r o m , G. Nordberg, and M. P i s c a t o r . 1979. Cadmium In Handbook on the Toxicology of Meta l s . F r ibe rg L . et a l . (eds) E l s e v i e r / N o r t h - H o l l a n d Biomedical Press , pp. 355-381. G i s h , C D . , and R . E . Chris tensen. 1973. Cadmium, N i c k e l , Lead and Z inc i n Earthworms from Roadside S o i l . Env i ron . S c i . Technol . 7: 1060-1062. G r e i g , M . , and J . B j e r r i n g . 1977. UBC Genl in A General Least Squares Ana lys i s of Variance Program Revised August 1980 Computing Centre U n i v e r s i t y of B r i t i s h Columbia, Vancouver 55 pp. - 95 -Har tens te in , R . , A . L . Leaf , E . F . Neuhauser, and D.H. Bickelhaugt . 1980. Composition of the Earthworm E i s e n i a foet ida (Savigny) and A s s i m i l a t i o n of 15 Elements from Sludge during Growth. Comp. Biochem. P h y s i o l . 66: 187-192. Har tens te in , R . , and E . F . Neuhauser, J . C o l l i e r . 1980. Accumulation of Heavy Metals i n the Earthworm E i s e n i a Foet ida . J . Env i ron . Qual. 9: 23-26. H a r t e r , R .D. 1979. Adsorpt ion of Copper and Lead by Ap and B 2 Horizons of Several North Eastern United States . S o i l S c i . Soc. Am. Proc. 43: 679-683. Helmke, P . A . , W.P. Robarge, R . L . Koroter , and P . J . Schomberg. 1979. E f fec t s of S o i l - A p p l i e d Sewage Sludge on Concentrations of Elements i n Earthworms. J . E n v i r o n . Qual. 8: 322-327. Hughes, M . K . , N.W. Lepp, and D.A. Phipps. 1980. A e r i a l Heavy Meta l P o l l u t i o n and T e r r e s t r i a l Ecosystems In Advances i n E c o l o g i c a l Research 11 A . MacFadyen (ed) pp. 217-327. I r e l a n d , M.P . 1975. D i s t r i b u t i o n of Lead, Z inc and Calcium i n Dendrobaena rubida (Oligochaeta) L i v i n g i n S o i l Contaminated by Base Metal Mining i n Wales. Comp. Biochem. P h y s i o l . 52B: 551-555. I r e l a n d , M.P. 1975. Metal Content of Dendrobaena rubida (Oligochaeta) i n a Base Metal Mining Area Oikos. 26: 74-79. I r e l a n d , M.P. 1975. The E f fec t of the Earthworm Dendrobaena rubida on the S o l u b i l i t y of Lead, Z i n c , and Calcium i n Heavy Metal Contaminated S o i l i n Wales. J . S o i l Sc. 26: 313-318. I r e l a n d , M.P. 1976. Excre t ion of Lead, Zinc and Calcium by the Earthworm Dendrobaena rubida L i v i n g i n S o i l Contaminated with Z inc and Lead. S o i l B i o l . Biochem. 8: 347-350. I r e l a n d , M.P. 1977. Lead Retention i n Toads, Xenopus l a e v i s Fed Increas ing Levels of Lead-Contaminated Earthworms Env i ron . P o l l u t . 12: 85-92. I r e l a n d , M.P. 1979. Metal Accumulation by the Earthworms Lumbricus r u b e l l u s , Dendrobaena Veneta and E i s e n i e l l a te traedra L i v i n g i n Heavy Meta l Po l lu ted S i t e s . Env i ron . P o l l u t . 19: 201-206. I r e l a n d , M.P. , and K . S . R ichards . 1977. The Occurrence and L o c a l i s a t i o n of Heavy Metals and Glycogen i n the Earthworms Lumbricus  rube l lu s and Dendrobaena rubida from a Heavy Metal S i t e . His tochemis t ry . 51: 153-166. I r e l a n d , M . P . , and K .S . R ichards . 1981. Meta l Content, Af ter exposure to Cadmium, of Two Species of Earthworms of known D i f f e r i n g Calcium Metabol ic A c t i v i t y . Env i ron . P o l l u t . Ser. A. 26: 69-78. - 96 -I r e l a n d , M . P . , and R . J . Wooton. 1976. V a r i a t i o n s i n the Lead, Z inc and Calcium Content of Dendrobaena rubida (Oligochaeta) i n a Base Metal Mining Area . Env i ron . P o l l u t . 10: 201-208. K o i r t y Shawn, S .R . , G. Wallace , and E . Hinderberger. 1976. Mult i-element Ana lys i s of Drosophi la for Environmental Monitor ing Purposes Using Carbon Furnace Atomic Absorpt ion . Can. J . Spectrosc. 21: 61-64. Kuo, S. , and A . S . Baker. 1980. Sorption of Copper, Zinc and Cadmium by Some Ac id S o i l s . S o i l S c i . Soc. Am. Proc . 44: 969-974. L a r k i n , P . A . 1977. A Handbook of Elementary S t a t i s t i c a l Tests Designed for Use i n Biology Zoo (Biometrics) U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 161 pp. L e , C , T . T e n i s c i . 1978. UBC TRP Tr iangular Regression Package. Computing Centre, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 197 pp. Lee, W. 1975. Experimental Design and A n a l y s i s . W.H. Freeman and Company, San Franc i sco . L i t t l e , T . M . , and F . J . H i l l s . 1972. S t a t i s t i c a l Methods i n A g r i c u l t u r a l Research. U n i v e r s i t y of C a l i f o r n i a , Dav i s . 242 pp. Luttmerding, H .A. 1980. S o i l s of the Langley-Vancouver Map Area . V o l . 1. S o i l Map Moscucs and Legend, Lower Fraser V a l l e y , (Scale 1:25,000) R . A . B . B u l l e t i n 18, Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . Luttmerding, H . A . 1981a. S o i l s of the Langley-Vancouver Map Area . V o l . 3 Desc r ip t ion of the S o i l s . R . A . B . B u l l e t i n 18. Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . Luttmerding, H .A. 1981b. So i l s of the Langley-Vancouver Map Area V o l . 6. Technica l Data-So i l P r o f i l e Descr ip t ions and A n a l y t i c a l Data. R . A . B . B u l l e t i n 18. Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . MacLean, K . S . , and W.M. L a n g i l l e . 1976. The Extractable Trace Element Content of A c i d So i l s and the Influence of pH, Organic Matter , and Clay Content. Comm. i n S o i l Sc. Plant A n a l . 7: 777-785. M a r t i n , M . H . , and P . J . Coughtrey, 1975. Pre l iminary Observations on the Levels of Cadmium i n a Contaminated Environment Chemosphere 3: 155-160. M a r t i n , M . H . , and P . J . Coughtrey. 1976. Comparisons between the l e v e l s of Lead, Zinc and Cadmium w i t h i n a contaminated environment. Chemosphere 1: 15-20. - 97 -McBride, M . B . , L . D . T y l e r , and D.A. Houde. 1981. Cadmium Adsorpt ion by S o i l s and Uptake by Plants as Affected by S o i l Chemical P r o p e r t i e s . S o i l Sc. Soc. Am. Proc . 45: 739-744. Mendenhall , W. 1968. Introduct ion to L inear Models and the Design and Analyses of Experiments. Duxbury Press , A D i v i s i o n of Wadsworth P u b l i s h i n g Company, Inc . Belmont, C a l i f o r n i a . 465 pp. Nordberg, G . F . , J . Par izek , and M. P i s c a t o r . 1979. Factors Inf luencing E f fec t s and Dose-Response Re la t ionsh ip of Meta l s . In Handbook on the Toxicology of Metals F r i b e r g , L . et a l . (eds) E l s v i e r / N o r t h - H o l l a n d Biomedical Press , pp. 143-157. Oak Ridge Nat iona l Laboratory. 1974. Environmental Monitor ing of Toxic Mater i a l s i n Ecosystems i n Ecology and Ana lys i s of Trace Contaminants. Progress Report January 1973-September 1973. ORNL-NSF-EATC-6 pp 95-139. Ontar io M i n i s t r y of the Environment. 1978. Guide l ines for Sewage Sludge U t l i z a t i o n on A g r i c u l t u r a l Lands. 28 pp. Packer, D . M . , M.P. and I r e l and , R . J . Wootton. 1980. Cadmium, Copper, Lead, Zinc and Manganese i n the Polychaete A r e n i c o l a marina from Sediments around the Coast of Wales. E n v i r o n . P o l l u t . Ser. A . 22: 309-321. Peredelsky, A . A . , N .A. Poryadkova, and L . Z . Rodionova. 1957. The Role of Earthworms i n C lea r ing S o i l of Contamination by Radioact ive Isotopes. Doklady-Bio. Sc. Sec. 115: 638-641. P h i l l i p s , D . J . H . 1978. Use of B i o l o g i c a l Indica tor Organisms to Quantitate Organochlorine Po l lu tant s i n Aquatic Environments - A Review. Env i ron . P o l l u t . 16: 167-229. P i s c a t o r , M. 1979. Copper In Handbook on the Toxicology of Metals F r i b e r g , L . et a l . (eds) . E l s e v i e r / N o r t h - H o l l a n d Biomedical Press , pp. 411-420. R e i c h l e , D . E . , O.A. Cros s ley , C A . Edward^, J . F . McBrayer, and P. S o l l i n s . 1971. Organic Matter and Cs Turnover i n Forest S o i l by Earthworm Populat ions : A p p l i c a t i o n of Bioenerget ic models to Radionucl ide Transport . In . Radionucl ides i n Ecosystems Proceedings of the T h i r d Nat iona l Symposium on Radioecology. Nelson, D . J . (ed) . US: A . E . C . Conf.-71051, pp 240-246. Reynolds, J .W. 1977. The Earthworms (Lumbricidae and Sparganophil idae) of Ontar io . Royal Ontario Museum L i f e S c i . M i s c . Pub . , Toronto. 141 pp. Roberts , R . D . , and M.S. Johnson. 1978. D i s p e r s a l of Heavy Metals from Abandoned Mine Workings and t h e i r Transference Through T e r r e s t r i a l Food Chains . Env i ron . P o l l u t . 16: 293-310. - 98 -S a t c h e l l , J . E . 1967. Lumbricidae i n S o i l B io logy , Burgess, A , F . Raw (eds) Academic Press , London, pp 259-322. Schuman, L . M . 1975. The E f fec t of S o i l Propert ies on Zinc Adsorpt ion by S o i l s . S o i l Sc. Soc. Am. Proc . 39: 454-458. Schuman, L . M . 1976. Zinc Adsorpt ion Isotherms for S o i l Clays with and without Iron oxides Removed. S o i l Sc. Am. Proc . 40: 349-352. S i e g e l , S. 1956. Nonparametric S t a t i s t i c s for the Behavioral Sciences McGraw-Hil l Book Company, Toronto. S t e e l , R . G . D . , and J . H . T o r r i e . 1960. P r i n c i p l e s and Procedures of S t a t i s t i c s with Spec ia l Reference to the B i o l o g i c a l Sciences . McGraw-Hil l Book Company, Inc. Toronto. Van Hook, R . I . 1974. Cadmium, Lead and Zinc D i s t r i b u t i o n s Between Earthworms and S o i l s : Po ten t i a l s for B i o l o g i c a l Accumulation B u l l . E n v i r o n . Contam. T o x i c a l . 12: 509-511. Van Hook, R . I . , and A . J . Yates . 1975. Trans ient Behavior of Cadmium i n a Grassland Antropod Food Chain. Env i ron . Res. 9: 76-83. Van Loon, J . C , and T. Lichwa. 1973. A study of the Atomic Absorpt ion Determination of Some Important Heavy Metals i n F e r t i l i z e r s and Domestic Sewage Plant Sludges. Env i ron . L e t t . 4: 1-8. Van Rhee, J . A . 1963. Earthworm A c t i v i t i e s and the Breakdown of Organic Matter i n A g r i c u l t u r a l S o i l s . In S o i l Organisms, Doeksen, J . , J . vander D r i f t , (ed). North-Holland Pub. C o . , Amsterdam, pp 55-59. Van Rhee, J . A . 1965. Earthworm A c t i v i t y and Plant Growth i n A r t i f i c i a l Cu l ture s . Plant and S o i l . 22: 45-49. Van Rhee, J . A . 1975. Copper Contamination E f fec t s on Earthworms by Disposa l of P ig Waste i n Pastures . In Progress i n S o i l Zoology, Proceedings of the 5th In te rna t iona l Colloquium on S o i l Zoology. Academia, Praque. pp. 451-456. Van Rhee, J . A . 1977. E f fec t s of S o i l P o l l u t i o n on Earthworms. Pedobio log ia . 17: 201-208. Wieser, W. , G. Busch . , and L . Buchel . 1976. Isopods as Indicators of the Copper Content of S o i l and L i t t e r . Oecologia . 23: 107-114. Winer, B . J . 1971. S t a t i s t i c a l P r i n c i p a l s i n Experimental Design, Second e d i t i o n . McGraw H i l l Book Company, New York. Wright , M . A . , and A . S t r inger . 1980. Lead, Zinc and Cadmium Content of Earthworms from Pasture i n the v i c i n i t y of an I n d u s t r i a l Smelting Complex. Env i ron . P o l l u t . Ser. A. 23: 313-321. - 99 -PART 2 The uptake of Cadmium, Copper, Lead, Nickel and Zinc by Lettuce (Lactuca sativa L.) Grown in Soil Amended with Milorganite and in the Presence of Earthworms (Lumbricidae) - 100 -1. INTRODUCTION AND LITERATURE REVIEW Sewage sludges have been appl ied to a g r i c u l t u r a l lands as a means of waste d i sposa l and u t i l i z i n g the nutr ient s i n the sludges to promote plant growth. Many sludges contain metals such as cadmium, copper, n i c k e l and z inc at concentrations which may be tox ic to plants and t h e i r consumers. The uptake of metals by plants from sewage sludges has been reviewed by Page (1974) and more recent ly by Webber (1979, 1980). The uptake of metals by le t tuce grown i n sludge amended s o i l s has been s tudied by a number of researchers , i n p a t i c u l a r l y MacLean (1974, 1976), MacLean and Dekker (1978), John (1975), John and van Larhoven (1972, 1976, 1976) and Singh (1981). In genera l , sewage sludge promotes plant growth and re su l t s i n higher concentrat ion of cadmium, copper, n i c k e l and z inc i n plant t i s sues . Lead concentrations i n plant t i s sue tend to be unaffected by sewage sludge add i t ions . Plant species respond d i f f e r e n t l y to metal concentrations i n the s o i l . In genera l , l ea fy vegetables such as l e t t u c e , tend to have higher metal concentrations i n t h e i r t i s sues than do other vegetables . The amount of metals taken up by plants i s inf luenced by a number of factors i n c l u d i n g s o i l p r o p e r t i e s , propert ies of the metals and plant species . Soon and Yates (1982) ind ica ted that there are f ive chemical pools for metal cat ions i n the s o i l : (1) water so lub le , (2) e a s i l y exchangeable, (3) complexed or adsorbed and exchangeable only by other cat ions with higher a f f i n i t i e s for the sorpt ion/complexat ion s i t e s , (4) occluded by or coprec ip i ta ted with metal oxides , carbonates of phosphates, and other secondary minerals (5) metals held i n primary - 101 -minera l s . The f i r s t three pools are genera l ly thought to be a v a i l a b l e to p l a n t s , while the l a s t two pools are considered to be unavai lable to p l a n t s . T o t a l metal concentrations i n s o i l s are often determined by d ige s t ion with concentrated ac id s . Although the r e l a t i o n s h i p between t o t a l metal concentrations and plant uptake have been questioned (MacLean and L a n g i l l e , Symeonides and MacRae, 1977, 1976) gu ide l ines for sewage sludge d i sposa l on to land are based on t o t a l metal concentrat ions i n the sludges. (Freedman and Hutchinson, 1981). T o t a l metal concentrat ion i n s o i l ind ica te s the degree of p o l l u t i o n of s o i l by metals but does not neces sar i ly ind ica te the amount that plants can u t i l i z e . Many ex t rac t ing so lut ions which have been u t l i z e d to determine the amount of ava i l ab le metals i n s o i l . In many B r i t i s h l a b o r a t o r i e s , the concentrat ion of metals extracted by 0.5 N HOAC (ace t i c acid) i s considered to ind ica te the amount of metals i n s o i l or sludges that i s a v a i l a b l e for plant uptake (Page, 1974). In the United States and Canada, 0.1 N HCI has been used as an ex t rac t ing agent. (Brown et a l . , 1971; MacLean and L a n g i l l e , 1976). More r e c e n t l y , the amount of metals extracted by DTPA (0.005 M diethylenetr iaminepentaacet ic a c i d , pH 7.3) has been used to determine plant ava i l ab l e metals with good c o r r e l a t i o n between extracted metal concentrations and plant t i s sue concentrat ions . (Lindsay and N o r v e l l , 1978; Karim et a l . , 1976; Korcak and Fanning, 1978. Karim et a l . (1976) ind ica ted that ac id ex t rac t ing so lut ions may overestimate the ava i l ab le metals and that amount of metals extracted by the che la t ing agent, DTPA was a more r e l i a b l e estimate of plant - 102 -a v a i l a b l e metals In the s o i l . The amount of extractable metals i n the s o i l or sewage sludge i s inf luenced by a number of propert ies such as pH, organic matter content, metal species e tc . (Webber and Corneau, 1975; S i l v i e r a and Sommers, 1977; Korcak and Fanning, 1978; MacLean and Dekker, 1978; MacLean, 1974; John, 1972; Haq et a l . , 1980). Many researchers have ind ica ted that earthworms can increase p lant y i e l d s although th i s conclus ion i s often reached on the basis of f aeca l ana lys i s (see reviews by Bar ley , 1961; Ghabbour, 1966; and Edwards and L o f t y , 1977). Few researchers have experimental ly studied metal uptake by plants grown i n the presence of earthworms, or the e f fec t of earthworm a c t i v i t y ( inges t ion and excret ion) on the ava i l ab le pool of metals . Lunt and Jacobson (1944) found that earthworm faeces contained higher amounts of t o t a l N and n i t r a t e N, organic matter, t o t a l and exchangeable Ca, exchangeable K and Mg and ava i l ab l e P, than did the surrounding s o i l . T o t a l Mg i n the earthworm faeces was s i m i l a r to s o i l l e v e l s . I re land (1975) determined that a c e t i c ac id extractable Zn and Ca had higher concentrations i n earthworm casts than the surrounding s o i l s , whereas extractable Pb was greater i n the s o i l than earthworm cast s . P u t r i f i e d earthworm t i s sue had higher concentrations of extrac tab le Pb, Zn and Ca than did the surrounding s o i l . T o t a l concentrat ions of Pb, Zn and Ca were higher i n the decomposing earthworm t i s sue but equal i n the faeces and s o i l . Edwards and Lofty (1977) ind ica ted that earthworms increased the amount of plant ava i l ab l e Mo i n pasture s o i l s . - 103 -In laboratory s tudies , concentrations of N, Mg, Cu and Zn were s i m i l a r i n sludge amended s o i l s with and without earthworms but there were higher concentrations of d i l u t e ac id extractable P, K, Ca, Fe and Mn i n the sludge treated s o i l s with earthworms than without earthworms (Kirkman, 1979). She also found higher Mn concentrat ions i n the roots of wheat grown i n the presence of earthworms than i n t h e i r absence. The concentrat ions of Fe , Cu and Zn i n wheat were unaffected by earthworms. P r e v i o u s l y , P e r e d e l ' s k i i et a l . (1957) reported that C o 6 0 uptake by a number of plant species was grea t ly increased when grown i n the presence of earthworms. In the present study, l e t tuce (Lactuca sa t iva L . C V . ear ly cur led Simpson ( l e a f ) . ) was grown i n the presence and absence of earthworms (Lumbricus rube l lus Hoffmeister , Aporrectodea spp. e i ther Aporrectodea trapezoides Duges or Aporrectodea tuberculata E i s e n , or a combination of these 2 spec ies , and Octo las ion cyaneum Savigny) and i n the presence and absence of the sewage sludge, mi lorgani te which i s h igh i n metal concentrat ions . The ob ject ives of th i s study were to determine: 1) i f Cd, Cu, NI, Pb and Zn, uptake by le t tuce would be af fected when grown i n sludge amended s o i l and i n the presence of earthworms. 2) If y i e l d s of l e t tuce would be increased by the a d d i t i o n of the mi lorgani te and earthworms to the s o i l ; 3) i f DTPA extractable Cd, Cu, N i , Pb and Zn would be inf luenced by the presence of earthworms. - 104 -2. MATERIALS AND METHODS 2.1 Experimental Design and Preparation Bulk s o i l samples were c o l l e c t e d from the Ap horizon of a Cresent-Westham Is land s o i l complex located on Mr. Hugh Reynold's farm on Westham Is land i n the Fraser Del ta of B r i t i s h Columbia. S o i l p r o f i l e descr ip t ions and s o i l maps are given i n Luttmerding (1980, 1981 a, b ) . The s o i l was a i r - d r i e d , crushed with a wooden r o l l e r and passed through a 6 mm screen. For the sludge treatments, the s o i l (6313 gm) was thoroughly mixed with 158 gm of mi lorgani te (50 gm/kg which i s equivalent to 56 T / h a ) . Twelve flower pots (21 cm d x 21 cm h) were f i l l e d with the s o i l - m i l o r g a n i t e mixture and 12 were f i l l e d with s o i l 3 (6313 gm) and a l l pots were packed to a bulk density of 1200 kg/m . A f t e r f i l l i n g , the pots were watered with a f e r t i l i z e r so lu t ion conta ining 100 pg/gm each of n i t rogen , phosphorus and potassium to ensure adequate nutr ient supply. Each pot was watered with 2300 mis of d i s t i l l e d water, to approximate f i e l d capaaci ty . The pots were weighed and the i n i t i a l weights recorded. There were four treatments: 1) s o i l with no earthworms or mi lorgan i te added; 2) s o i l with only earthworms added; 3) s o i l with only mi lorgani te added; 4) s o i l with both earthworms and mi lorgani te added. Each treatment was r e p l i c a t e d s ix times u t i l i z i n g a randomized block design. For treatments 2 and 4, ten mature, c l i t e l l a t e earthworms were placed on top of the s o i l medium and allowed to burrow. Four Lumbricus - 105 -rube l lu s Hoffmeister , four Aporrectodea spp. (a combination of A . trapezoides Duges and A . tuberculata Esien) and two Octo la s ion cyaneum Savigny were used i n the earthworm treatments (approximate density In 1 2 m i s 263 earthworms). The earthworms were obtained by digging on Westham Is land near the f i e l d where the s o i l was obtained. The bottom of the pots were covered with nylon mesh i n order to prevent the earthworms escaping. F ive seeds of l ea f l e t tuce (Lactuca sa t iva L . cv. ea r ly cur led Simpson) were planted i n each pot . There was a s ixteen hour photoperiod provided by a mix of incandescent and f luorescent l i g h t i n g . This l i g h t i n g regime i s recommended for le t tuce (Bickford and Dunn, 1972). Temperature and r e l a t i v e humidity were monitered by a hydrothermograph (Weather Measure Corporat ion , Model H311). The day temperature during most of the experimental per iod (December 11, 1978 to February 9, 1979) was 2 1 ° C ± 2 ° C and the night temperature was 1 9 ° C ± 2 ° C . During the l a s t week of December and f i r s t week of January the night temperatures were mainly 1 0 ° C and the day temperatures were mainly 1 5 ° C . Re la t ive humidity f luctuated between 40% and 60%. D i s t i l l e d water was added d a i l y to br ing the flower pots back to the i n i t i a l weight. 2.2 Sample Preparation and Chemical Analysis A f t e r two months, the plants were harvested. The l ea f mater ia l from each pot was pooled and fresh weights were obtained. The l e t tuce leaves were then oven dr ied at 8 0 ° C for three days. Af te r weighing, the samples were ground i n a s t a in le s s s tee l Wiley m i l l . The main roots from each pot were c o l l e c t e d , washed with deionized water, oven dr ied at - 106 -8 0 ° C as above and weighed on an e l e c t r o n i c microbalance. One gram samples of oven dr ied ( 8 0 ° C ) leaf mater ia l were dry ashed overnight at 4 8 0 ° C i n a muffle furnace. The cooled ash was d i s so lved i n 5 mL of 2N HCI and evaporated to dryness on a ho tp la te . The residue was then d i s so lved i n 5 ml of 2N HCI, f i l t e r e d through Whatman #42 f i l t e r paper and brought to 25 ml volume (Dept. S o i l S c . , U . B . C . , 1977). Cadmium, copper, l ead , n i c k e l and z inc i n the lea f extract were determined by atomic absorption spectrophotometry using a Perkin Elmer Model 306 atomic absorption spectrophotometer with an a i r acetylene flame and the fo l lowing wavelengths: 288.8 nm for Cd; 324.7 mn for Cu, 283.3 nm for Pb, 232.0 nm for Ni and 213.9 nm for Zn. The instrument was equipped with background c o r r e c t i o n . The roots were weighed, placed i n ac id washed' Kimax glass v i a l s (70 x 20 mm O.D.) and digested i n n i t r i c ac id using a block digestor set at 1 5 0 ° C as described previous ly i n part 1 for earthworms under 100 mg dry weight. This method was used as i n s u f f i c i e n t mater ia l was obtained to use the same method as the leaves . These 2 methods give comparable r e s u l t s (Car ter , personal communication). Lead and n i c k e l concentrations i n the roots were determined by flameless atomic absorpt ion spectroscopy, using the Perkin Elmer Hollow Graphite Analyzer (HGA) Model 2100 equipped with deuterium arc background c o r r e c t i o n . The furnace operating parameter, wavelengths, and s l i t set t ings for the HGA are given i n Table 2 of Part 1. - 107 -The s o i l and s o i l - m i l o r g a n i t e mixtures were a i r d r i e d , crushed with a wooden r o l l e r and passed through a 2 mm s t a in le s s s t e e l s i eve . Three r e p l i c a t e s of the medium from each flower pot were prepared and analyzed for cadmium, copper, l ead , n i c k e l and z i n c . Samples weighing 0.5 gm were placed i n an ac id washed glass t a l l f o r m 100 ml beakers and digested with 9 mis of concentrated HC1 and 3 mis of concentrated HNO3 (aqua regia) on a hot plate set at medium for at leas t ha l f hour. The beakers were covered with ac id washed watch g lasses . The samples were f i l t e r e d Whatman #42 f i l t e r paper and brought to 25 ml volume (Van Loon and Lichwa, 1973). Cadmium, copper, l ead , n i c k e l and z inc were determined using flame atomic absorption spectroscopy as described p r e v i o u s l y , i n Part 1. Extractab le metals i n the s o i l and s o i l - m i l o r g a n i t e mixture were determined using the d iethylenetr iaminepentaacet ic ac id (DTPA) ex t rac tant . Ten grams of a i r d r i e d s o i l were weighed i n t o 125 ml wide mouth p l a s t i c bot t les with 20 mis of DTPA ex t rac t ing so lu t ion and shaken on an Eberback two-speed h o r i z o n t a l shaker with an 8.0 cm stroke and a low speed of 12- cyc le s /min . for 2 hours at 2 5 ° C . The samples were then f i l t e r e d through Whatman #42 f i l t e r paper and the f i l t r a t e analyzed for Cd, Cu, N i , Pb and Zn by flame atomic absorption spectroscopy as descr ibed p r e v i o u s l y . (Department S o i l Science, 1977). The DTPA extractant was chosen over the 0.5 N HOAc ( ace t i c ac id) extractant as described by Webber and Corneau (1975) and the 0.1 NHC1 extractant as described by Department of S o i l Sc ience, U . B . C . (1977). In a separate study le t tuce (Lactuca sa t iva L . cv ear ly cur led Simpson) - 108 -was grown in the soil-milorganite mixture (20 gm milorganite/kg soil) as described previously. The extractable Cd, Cu, Ni, Pb and Zn in the soil-sludge medium were determined using DTPA, 0.5 N HOAc and 0.1 N HC1. Total metal levels in the soil-milorganite mixture and lettuce leaf tissue were also determined by the methods described previously. The DTPA extract of s o i l had the best correlation of the three extractants with copper, nickel and zinc concentrations determined for the leaf tissue. 2.3 St a t i s t i c a l Analysis The data were s t a t i s t i c a l l y analyzed using the UBC-MFAV programme for analysis of variance. The data for zinc concentrations in lettuce leaves and roots were s t a t i s t i c a l l y analyzed using the UBC-BMD programme for the analysis of variance, as this programme can handle missing data. The tissue from one replicate was contaminated with zinc during the analytical procedures. Paired-difference tests were used to determine i f metal concentrations in the root tissue. The paired-difference test was used instead of the t-test to determine differences between two means, as the t test requires the two samples be independent and random. The lettuce leaf and root tissue from the same plant would not be independent. The paired difference test eliminates the effect of the flowerpot to flowerpot variability and yields more information on the mean difference in the metal concentrations of the different tissue types. (Mendenhall, 1971). - 109 -3. RESULTS 3.1 Survival and Activity of the Earthworms There was l i m i t e d a c t i v i t y of the earthworms i n the flower pots. The majority of earthworms (82%) survived the experiment but a l l the Aporrectodea spp. and Octolasion cyaneum i n d i v i d u a l s which were recovered were aestivating (see plate 1), and were located near the bottom or centre of the pots. The surviving Lumbricus rubellus i n d i v i d u a l s were a l l recovered near the surface and most were a c t i v e . Lumbricus rubellus had the poorest s u r v i v a l record (58%) of the three taxa used while 92% of the Octolasion syaneum and 96% of the Aporrectodea spp. survived. In some of the earthworm treated flower pots, there was no evidence of earthworm a c t i v i t y (no burrows or casts present). The s o i l i n which earthworm a c t i v i t y was evident also tended to be more crumbly than s o i l with no earthworm a c t i v i t y . 3.2 Yield of Lettuce Tissue The fresh and dry weights of harvested lettuce leaf tissue are given i n Table 1. Analysis of variance indicated that the addition of milorganite was highly s i g n i f i c a n t (p<0.05). The addition of milorganite resulted i n an increase i n the lettuce leaf t i s s u e . Earthworms had no e f f e c t on the production of t i s s u e . 3.3 Metal Concentrations i n Milorganite Metal concentrations i n miloganite are presented i n Table 2. These data are s i m i l a r to the r e s u l t s presented by John and van Laerhoven (1976) and Carson (written communication). - 110 -Plate 1 Aestivating earthworms. The taxon, on the l e f t i s Aporrectodea spp. and Octolasion cyaneum i s on the r i g h t . There i s some evidence of burrowing a c t i v i t y to the l e f t and above the Octolasion  cyaneum i n d i v i d u a l . - I l l -P l a t e 2 An example o f t he s o i l f r o m t r e a t m e n t w i t h e a r t h w o r m s , t o i l l u s t r a t e t h e b u r r o w i n g a c t i v i t y . - 112 -Plate 3 An example of the s o i l from treatment with no earthworms. - 113 -Table 1 Fresh and Dry Weight ( 8 0 ° C ) and Standard Deviat ions for Lettuce Leaf Tissue Harvested Af te r Two Months Exposure to Treatment. Treatment Grams Fresh Weight Grams Dry Weight No earthworms, no mi lorgani te 10.902 ± 5.079 0.703 ± 0.383 Earthworms, no mi lorgani te 11.082 ± 1.371 0.710 ± 0.136 No earthworms, 50 gm 64.059 ± 11.781 5.039 ± 1.063 mi lorgan i te /kg s o i l Earthworm, 50 gm mi lorgan i te / 59.851 ± 11.548 4.734 ± 0.949 kg s o i l Table 2 T o t a l Cadmium, Copper, Lead, N i c k e l and Zinc Concentrat ions (Hg/gm) i n Lettuce Leaf Tissue Grown i n S o i l Amended with 20 gm M i l o r g a n i t e / k g s o i l , the * M i l o r g a n i t e and S o i l . Cd Cu Pb Ni Zn Lettuce 2 1 . 7 ± 3.35 9 . 6 ± 2.33 7± 2.7 8 ± 2 . 1 6 7 . 2 ± 1 2 . 5 0 M i l o r g a n i t e 99 ± 3 1 3 1 6 . 0 ± 3 6 5 7 3 ± 7 8 6 7 ± 7 792 ±91 S o i l 0 . 6 ± 0.4 3 2 . 0 ± 1.8 1 2 ± 1 1 3 8 ± 4 . 5 72 ± 5.2 *The mean and standard dev ia t ion for mi lorgani te and s o i l based on 30 determinat ions . Table 3 Total and Extractable Soil Cadmium, Copper, Lead, Nickel and Zinc Concentrations (ug/gm ± 1 Std. dev.) Extractable Metals Also Expressed as Percentage of Total Soil Metal Cadmium Extractant Ug/m Cone. HCI & HN03 3.0 ± 0.45 (Total Soil Metal) DTPA 0.1 HCI 0.5 HoAc 2.3 ± 0.16 2.6 ± 0.15 1.6 ± 0.26 % Total Copper Lead ug/m % Total ug/m Total Nickel ug/m 100 25.7 + 3.24 77 10.0 ± 0.93 88 9.9 + 0.49 60 2.0 ± 0.45 100 39 39 2.8 + 5.2 3.0 ± 0.51 3.8 + 0.42 Zinc Total ug/m % Total 8 nonedetectable 100 5.0 + 8.3 11 3.2 ± 0.31 13 4.4 + 0.26 nonedetectable 100 123.2 + 27.51 100 6 11.8 + 1.21 10 9 18.7 + 1.29 15 11.7 ± 5.5 9 Table 4 Correlation Coefficients1 Between Different Soil Extractants and Metal Levels in Letuce Leaves, Grown in Soil Amended with 20 gm Milorganite/kg soil. Extractant Cadmium Copper Lead Nickel Zinc Cone. HCI & HNO3 -0.08 0.57 -0.77* 0.79** 0.68* (Total Soil Metal) DTPA 0.13 0.82** -0.18 0.95** -0.91** 0.1 N HCI -0.21 0.99** 0.64* 0.43 -0.93** 0.5 N HoAc -0.62* -1.00** - -. -0.95** Coefficients not significant unless otherwise stated * • sig. p<0.05 ** = highly sig. p<0.01 - 115 -3.4 Metal Extractablllty and Correlations with Lettuce Metal Contents The t o t a l metal concentrat ions determined by concentrated n i t r i c and hydroch lor i c ac id and extractable metal concentrations determined by DTPA, 0.1 NHC1, and 0.5 HOAc i n the s o i l mixed with 20 gm mi lorgani te /kg s o i l are given i n Table 3. The metal concentrations i n the le t tuce lea f t i s sue are given i n Table 2. In Table 4, the c o r r e l a t i o n c o e f f i c i e n t s (r values) obtained between the extractable metals and metal concentrat ions i n the l e t tuce lea f t i s sue are presented. The r e s u l t s ind ica te that at the 20 gm/kg a p p l i c a t i o n rate of mi logani te , the copper, l ead , n i c k e l and z inc concentrations i n the l e t tuce were cor re l a ted with the t o t a l concentrations of copper, l ead , n i c k e l and z i n c i n the s o i l . The c o r r e l a t i o n c o e f f i c i e n t s between the extractable metals and le t tuce concentrations v a r i e d . The DTPA extractable copper, n i c k e l and z inc was h ighly s i g n i f i c a n t l y corre la ted with l e t tuce l ea f concentrations for these three metals , which are the metals often considered to be of most concern i n s o i l metal p o l l u t i o n . The three extractants had h ighly s i g n i f i c a n t c o r r e l a t i o n with lea f copper and z inc concentra t ions . Extractable cadmium determined by 0.5 NHOAc was s i g n i f i c a n t l y corre la ted with le t tuce cadmium concentrat ions . The a c e t i c a c i d , however, f a i l e d to extract any n i c k e l or lead from the s o i l - m i l o g a n i t e mixture. As DTPA extractable copper, n i c k e l and z i n c c o r r e l a t e d s i g n i f i c a n t l y wi th l e t tuce concentrations for these metals , i t was chosen as the extractant to be used i n the second experiment. DTPA extracted s l i g h t l y less cadmium, l ead , n i c k e l and z inc and equal amounts of copper than did 0.1 N HCI, and more metals than 0.5 N Table 5 DTPA Extractable Metals (ug/gm dry weight ± I.S.D.) ln Soil After Two Months Exposure to Treatment. Treatment Cd Cu Ni Pb Zn No earthworms, 0.12 ± 0.04 8.3 ± 0.14 4 ± 0.4 1 ± 0.45 1.9 + 0.26 no milorganite Earthworms, 0.12 ± 0.04 8.4 ±0.16 4 ± 0.0 2 ± 1.6 1.8 ± 0.72 no milorganite No earthworms 2.3 + 0.12 10.8 ± 0.27 5+0.5 2 ± 0.41 10.3 ± 0.20 50 gm milorganite/kg soil Earthworms, 50 gm 2.3 ± 0.25 10.7 ± 0.52 5 + 0.5 2 ± 0.63 9.9 + 1.98 milorganite/kg soil Table 6 Correlation Coefficients (r values) for Metal Concentration in Letuce Leaves, Total Soil Metal Concentration and DTPA Extractable Metals, With and Without Milorganite Added to the Soil. Cd Cu Ni Pb Zn Treatment Total DTPA Total DTPA Total DTPA Total DTPA Total DTPA Treatments without -0.52 0.29 -0.33 -0.21 -0.25 0.36 0.03 -0.33 -0.22 0.50 milorganite Treatments with -0.43 -0.26 -0.09 -0.15 -0.26 -0.24 0.48 -0.65* 0.83** 0.79** 50 gm milorganite/ kg soil For explanation of significance levels, see footnote Table 4. - 117 -HOAc, with the exception that DTPA and 0.5 N HOAc extracted equal amounts of z i n c . The ace t i c acid extractant did not extract any lead or n i c k e l . Much of the t o t a l cadmium present i n the mi lorgani te amended s o i l was extracted by the three extractants (76%). Based on the per-cent of the t o t a l metals extracted , the o v e r a l l e f fect iveness of DTPA and 0.1 N HC1, was i n the order cadmium > copper > z inc = lead > n i c k e l ; whereas for 0.5 N HOAc the order of e f fect iveness was cadmium > copper = z i n c . Table 5 gives the concentrations of DTPA extractable metals i n s o i l without and amended with 50 gm mi lorgani te /kg s o i l . The a d d i t i o n of mi lorgani te s i g n i f i c a n t l y (p < 0.05) increased the amount of DTPA extractable metals present i n the s o i l . At least 40% of the z inc and cadmium present i n the mi lorgani te was DTPA ext rac tab le . The presence of earthworms did not s i g n i f i c a n t l y ef fect the amount of DTPA extractable metals. Table 6 presents the c o r r e l a t i o n c o e f f i c i e n t s for metal concentrat ions i n le t tuce leaves and t o t a l s o i l metal concentrations and DTPA extractable metals . As earthworms had no s i g n i f i c a n t ef fect on the DTPA extractable metals, the c o r r e l a t i o n c o e f f i c i e n t s were ca lcu la ted pool ing the treatments without mi lorgani te and with m i l o r g a n i t e . There i s much v a r i a t i o n i n the c o r r e l a t i o n c o e f f i c i e n t s . In the mi lorgani te treatments the DTPA extractable z inc had c o r r e l a t i o n c o e f f i c i e n t s which were s i g n i f i c a n t . The c o r r e l a t i o n c o e f f i c i e n t s presented i n Table 6 and Table 4 also are i n c o n s i s t e n t , and th i s may r e f l e c t the d i f f e rent a p p l i c a t i o n rates of mi lorgan i te , and subsequent Table 7 Metal Concentrations (ng/gm dry weight + S.D.) in Letuce Leaves After Two Months Exposure to Treatment. Treatment Cd Cu Ni Pb Zn No earthworms, no milorganite 5.9 ± 2.02 12.4 ± 2.95 19.0 ± 4.3 8.5 + 5.6 58.0 ± 15.79 Earthworms, no milorganite 5.9 ± 1.40 12.2 + 1.70 19.8 + 1.8 11.3 + 2.8 65.4 ± 12.52 No earthworms 50 gm milorganite/kg soil 50.7 + 6.41 10.2 ± 1.25 9.0 + 2.1 24.5 + 14.0 89.9 ± 24.29 Earthworms, 50 gm milorganite/kg soil 55.2 ± 4.01 10.9 + 0.39 11.3 + 1.5 24.7 + 14.3 93.1 ± 9.69 Table 8 Metal Concentrations (|ig/gm dry weight + I.S.D.) in Letuce Roots After Two Months Exposure to Treatment. Treatment Cd Cu Ni Pb Zn No earthworms, no milorganite 4.5 + 2.65 49.6 + 8.28 95.2 + 39.3 18.8 + 8.5 87.4 + 25.76 Earthworms, no milorganite 4.3 + 1.85 39.5 + 5.72 67.3 + 24.0 9.3 + 3.4 75.4 ± 29.16 No earthworms 50 gm milorganite/kg soil 48.4 ± 6.21 54.7 + 16.43 41.5 + 15.7 12.7 ± 1.9 148.1 + 14.74 Earthworms, 50 gm milorganite/kg soil 56.0 ± 12.13 45.2 + 12.52 39.7 + 10.5 10.2 + 1.2 129.5 ± 39.51 - 119 -d i f f e r e n c e s i n t o t a l and DTPA e x t r a c t a b l e metals i n the s o i l and d i f f e r e n c e s i n metal concentrations In the t i s s u e between the two m i l o r g a n i t e a p p l i c a t i o n s . Comparison of Tables 3 and 5 i n d i c a t e s that the 20 gm/kg and 50 gm/kg a p p l i c a t i o n s of m i l o r g a n i t e d i d not r e s u l t i n d i f f e r e n c e s i n the amounts of DTPA e x t r a c t a b l e cadmium copper, lead and z i n c . E x t r a c t a b l e n i c k e l was s l i g h t l y higher i n the 50 gm/kg a p p l i c a t i o n . 3.5 Metal Concentrations ln Lettuce Tissues The concentrations of cadmium, copper, n i c k e l , lead and z i n c i n the l e t t u c e l e a f and root t i s s u e s are given i n tab l e s 7 and 8 r e s p e c t i v e l y . The a d d i t i o n of m i l o r g a n i t e s i g n i f i c a n t l y (p < 0.05) increased cadmium and z i n c concentrations i n the l e a f and root t i s s u e s , m i l o r g a n i t e had not s i g n i f i c a n t e f f e c t on the copper or lead concentrations i n l e t t u c e leaves and r o o t s . The average lead c o n c e n t r a t i o n i n the l e a f t i s s u e from the m i l o r g a n i t e t r e a t e d s o i l was greater than that from the non-treated s o i l . There was a large standard d e v i a t i o n a s s o c i a t e d w i t h the m i l o r g a n i t e t r e a t e d p l a n t s and t h i s may e x p l a i n the la c k of s i g n i f i c a n c e due to the a d d i t i o n of the sludge. N i c k e l concentrations i n the leaves and roots were s i g n i f i c a n t l y lower (p < 0.05) when m i l o r g a n i t e was added to the s o i l . There was only one s i g n i f i c a n t e f f e c t due to the presence of earthworms. When earthworms were present, the lead concentration i n the l e t t u c e roots was s i g n i f i c a n t l y lower (p < 0.05) than the lead c o n c e n t r a t i o n i n the roots grown without earthworms present. P a i r e d d i f f e r e n c e t e s t s between the cadmium concentrations i n the Table 9 T o t a l Metal Concentrations ((ig/gm dry weight ± S .D.) i n S o i l Af te r Two Months Exposure to Treatment. Treatment Cd Cu Ni Pb Zn No earthworms, 1.6 ± 0.16 34.8 ± 0.82 37 ± 3.4 19 ± 1.8 69.0 ± 1.32 no mi lorgani te Earthworms, 1.7 ± 0.15 34.8 ± 0.84 37 ± 6.2 22 ± 3.0 65.8 ± 2.20 no mi lorgani te No earthworms 4.7 ± 0.47 41.1 ± 1.30 36 ± 2.9 38 ± 2.6 88.4 ± 2.46 50 gm mi lorgani te /kg s o i l Earthworms, 50 gm 4.4 ± 0.36 40.9 ± 1.47 35 ± 3.1 35 ± 5.7 86.5 ± 3.43 mi lorgan l te /kg s o i l - 121 -leaves and roots indicate that there was no s i g n i f i c a n t difference between the two tissue types. Copper and n i c k e l concentrations i n the roots were s i g n i f i c a n t l y higher than the copper and n i c k e l concentrations In the lettuce leaf tissue. The lead concentrations i n the tissues were variable and the paired difference test (p < 0.05) detected s i g n i f i c a n t differences between the lead concentrations i n the le a f and root tissues i n 2 of the 4 treatments Zinc concentrations i n the root tisues from three of the four treatments were s i g n i f i c a n t l y higher than the leaf zinc concentrations. The zinc concentrations i n the root tissue from the treatment with earthworms and no milorganite on average were greater than the leaf tissue but the difference were not s i g n i f i c a n t . 3.6 M e t a l Concen t ra t ions i n the S o i l Table 9 presents the metal concentrations i n the s o i l a f t e r two months exposure to treatment. The addition of milorganite s i g n i f i c a n t l y increased the t o t a l concentrations of cadmium, copper, lead and zinc and did not s i g n i f i c a n t l y increase the n i c k e l concentration (p < 0.05). The concentration of n i c k e l i n milorganite i s low r e l a t i v e to the concentrations of the other four metals studied (Table 2). The addi t i o n of earthworms to the s o i l did not a f f e c t the t o t a l metal concentrations i n the s o i l . Comparison of the t o t a l metal concentrations i n the s o i l with 50 gm/kg milorganite a p p l i c a t i o n (Table 9) with the s o i l amended with 20 gm/kg milorganite (Table 2) indicates that the increased a p p l i c a t i o n of milorganite resulted i n s l i g h t l y higher cadmium, copper, and lead - 122 -concentrat ions . N i c k e l and z inc concentrations were higher i n the s o i l from the 20 gm/kg mi lorgani te a p p l i c a t i o n . A l s o , the copper concentrat ions from the c o n t r o l (no added mi lo rgan i t e , Table 9) were higher than the copper concentrations than the s o i l treated with 20 gm/kg mi lo rgan i te . This suggests that the two a p p l i c a t i o n rates may not have resu l ted i n d i f f e rent t o t a l metal loadings of the s o i l and may r e f l e c t the v a r i a b i l i t y i n metal l eve l s i n the miloganite and i n the s o i l (Table 2 ) . 3.7 Soil Properties Selected p h y s i c a l and chemical propert ies of the s o i l used i n the present study are presented i n Table 10. For complete descr ip t ions of the Westham and Crescent s o i l s the reader i s re fe r red to Luttmerding, 1981. Table 10 - Selected S o i l Propert ies of the Buk Ap S o i l Samples and of an Ap Horizon from the Crescent S o i l Series (Luttmerding, 1981b). Treatment Depth (cm) pH (CaCl 2 ) % C % N Exchangeable Bases (NH^ OAc) meq/100 gm s o i l Ca Mg K Na CEC meg/100 m S o i l % Sand P a r t i c l e % S i l t Size % Clay Crescent - Ap 0.28 4.3 3.07 0.20 8.44 1.13 0.51 0.35 23.5 8.00 66.60 25.40 S o i l Ser ies Bulk Sample - Ap 0.20 3.9 2.36 0.18 4.71 0.82 0.17 0.15 20.3 3.12 68.38 28.50 I - 124 -4. DISCUSSION 4.1 Yield The re su l t s (Table 1) suggest that the a d d i t i o n of the f e r t i l i z e r s o l u t i o n containing n i t rogen , phosphorus and potassium was inadequate and that the add i t ion of mi lorgani te increased the supply of nut r i ent s ava i l ab le to the l e t t u c e . Mi lorgan i te contains 1.3% t o t a l P, 6.6% t o t a l N (360 mg/kg NH3 -N and 3.0 mg/kg N0 3 -N) and 0.63% t o t a l K (Carson, wr i t t en communication). Anderson (1959) reported s i m i l a r nutr ient a n a l y s i s : 5.96% t o t a l N, 3.96% t o t a l P 2 0 5 ; 0.41% ac id soluable K 2 0 . Increases i n crop y i e l d as a re su l t of sewage sludge addi t ions have been reported by various workers. From a review of the l i t e r a t u r e , Webber (1979) reported that i n near ly a l l the studies reviewed, sewage sludge increased the y i e l d s of cerea l crops. John and Laerhoven (1976) reported an increase i n l e t tuce y i e l d grown i n s o i l amended with 5 gm/kg and 10 gm/kg app l i ca t ions of mi lorgani te (a greenhouse experiment). An a p p l i c a t i o n rate of 100 gm/kg resu l ted i n y i e l d s s i g n i f i c a n t l y lower than the c o n t r o l . The y i e l d obtained for the cont ro l In the present greenhouse study was s u b s t a n t i a l l y lower than the y i e l d s obtained by John and Laerhoven (1976). The earthworms were i n a c t i v e i n th i s experiment and consequently there was no s i g n i f i c a n t e f fect on y i e l d due to t h e i r presence. The greenhouse condit ions may not have been su i tab le for earthworm a c t i v i t y . Other researchers have reported b e n e f i c i a l e f fect s of earthworms on plant growth. Kirkham (1978) reported that the dry weight y i e l d s of wheat grown i n s o i l s amended with sewage sludge and without sludge were - 125 -increased when the earthworm, E i s e n i a f o e t i d a , was added to the s o i l . Root growth of wheat was unaffected by the presence of earthworms, the increase i n wheat y i e l d was a t t r i b u t e d to the earthworms increa s ing the water use e f f i c i e n c y of the wheat. A t l a v i n y t e and Vanagas (1973) reported the growth of bar ley was increased when earthworms (Al lo lobophora ca l ig inosa ) were present. They found that the earthworm a c t i v i t y increased the amounts of plant ava i l ab l e phosphorus and potassium In the s o i l and that t h i s accounted for the increased bar ley y i e l d when earthworms were present . A t l a v i n y t e , et a l . (1968) reported increased barley y i e l d s when grown i n flower pots , i n the presence of earthworms. Y i e l d s were greater where there were greater numbers of ac t ive earthworms. They also report that Lumbricus rube l lus had a poor s u r v i v a l record (18-30% s u r v i v i n g ) , the earthworm, Al lo lobophora  c a l i g i n o s a did not re su l t i n increased y i e l d s of spr ing wheat and peas grown i n c lay s o i l s packed in to flower pots . Van Rhee (1965) a lso reported that the earthworms did not l i k e flower pot cond i t ions , and a f te r seven months most of the earthworms were miss ing or dead. Those that survived were a e s t i v a t i n g . Van Rhee (1965) also determined the e f fec t s of four earthworm species on the y i e l d s of grass , spr ing wheat, peas, berseem and Egyptian c l o v e r , u t i l i z i n g a r t i f i c i a l c u l t u r e s . Earthworms resul ted i n subs tan t i a l dry matter y i e l d increases over the contro l s for grass (287%), wheat (111%) and c lover (877%) and a 39% decrease i n the y i e l d of peas. Barley (1961) reviewed a number of po t -cu l ture experiments that tested the ef fects of earthworms on plant growth, and concludes that earthworms can s i g n i f i c a n t l y increase y i e l d s - 126 -of p lants grown i n po t -cu l ture s , and that the increases are not merely the r e s u l t of nutr ient s released from dead earthworms. Earthworm dens i t i e s s i m i l a r to the densi ty used i n t h i s present study re su l ted i n increased y i e l d s of barley grown i n flower pots (At l av iny te et a l . , 1969) as w e l l as increased a v a i l a b i l i t y of phosphorus and potassium (At l av iny te and Vanagas, 1973). These workers ind ica ted that increa s ing the densi ty of earthworms i n the flower pots increased the y i e l d s of bar ley and ava i l ab le phosphorus and potassium. In the present study, had the earthworms been a c t i v e , the densi ty should not have been l i m i t i n g . 4.2 Extractable Metals As expected, the amounts of metals extracted by aqua reg ia were greater than the amounts of metals extracted by the 3 ex t rac t ing agents. The concentrations determined by ex t rac t ion with the concentrated acids are considered to be the t o t a l metal concentrat ions i n the s o i l ( E l l i s et a l . , 1976). As noted i n Table 3, DTPA extracted s l i g h t l y less cadmium, l e a d , n i c k e l and z i n c and equal amounts of copper than did 0.1 N HCI, and more metals than 0.5 N HOAc, with the exception that DTPA and 0.5 N HOAc extracted equal amounts of z i n c . A c e t i c ac id d id not extract any lead or n i c k e l . Much of the t o t a l cadmium present i n the mi lorgani te amended s o i l was extracted by the three extractants (76%). Based on the per cent of the t o t a l metals extracted , the o v e r a l l e f fect iveness of DTPA and 0.1 N HCI, was i n the order cadmium > copper > z inc = lead > n i c k e l ; whereas for 0.5 N HOAc the order of e f fect iveness was cadmium > copper = z i n c . - 127 -A c e t i c ac id i s a weak ac id and i s only p a r t i a l l y d i s soc ia ted whereas hydrochlor ic ac id i s a strong ac id and d i s soc ia te s completely i n water. The hyrdochlor i c ac id extractant , there fore , has more hydrogen ions which can replace metal cations on the s o i l exchange complex, than does a c e t i c a c i d . This accounts for the higher concentrations of metals extracted by the 0.1 N HCI. Symeonides and MacRae (1977) and Karim, et a l . (1976) ind ica ted that hydroch lor i c ac id and other ac id ex t rac t ing so lut ions may also d i s so lve metals from the l a b i l e s o l i d phases. DTPA i s a che la t ing agent which combines with the free metal ions i n the s o i l s o l u t i o n to form soluble complexes which reduces the a c t i v i t y of the free metals ions i n s o l u t i o n . To r e p l e n i s h the free metal ions i n s o l u t i o n , metal ions desorb from the s o i l surfaces or d i s so lve from the l a b i l e s o l i d phases (Lindsay and N o r v e l l , 1978). The concentrated acids not only provide hydrogen ions to replace metal cat ions on the exchange s i t e , d i s so lve l a b i l e s o l i d phases, but also d i s so lve metal p r e c i p i t a t e s such as the oxides , carbonates and phosphates (Soon and Yates , 1982). The metal p r e c i p i t a t e s are not considered important for plant uptake. The re su l t s from the present study are comparable to the r e s u l t s reported i n the l i t e r a t u r e . Symeonides and MacRae (1977) determined that 1 N HCI extracted 100% of the t o t a l cadmium i n s o i l , whereas 5% N HOAc extracted , on average, 50% of the t o t a l s o i l cadmium. Webber and Corneau (1975) reported that 0.5 N HOAc was not e f f ec t ive i n ex t rac t ing n i c k e l from s o i l s mixed with sewage sludge. They also reported that DTPA extracted less z inc from s o i l s than did the ace t i c ac ids , whereas i n the present study, DTPA and 0.5 N HOAc extracted equal amounts of - 128 -z i n c . Korcak and Fanning (1978) found for sludge treated s o i l s , that the average percentages of extractable metals was re la ted to the t o t a l metal concentrations i n the sludges. The increase i n DTPA extractable metals from s o i l s amended with sludges was a lso observed by S i l v i e r a and Sommers (1977). Th i s was a lso true for the present study. Earthworms did not s i g n i f i c a n t l y (at the 5% p r o b a b i l i t y l e v e l ) a f f ec t the l e v e l of DTPA extractable metals i n the s o i l (Table 5 ) . As determined prev ious ly , i n Part I the t o t a l concentrat ions of cadmium, copper, lead and z inc i n the earthworm faeces were s i m i l a r to the t o t a l concentrat ions of metals i n the s o i l . In the present study, the DTPA extrac tab le metals for the s o i l with earthworms was determined for s o i l plus any faeces present. The extractable metals were not determined for the faeces. I n s u f f i c i e n t faeca l mater ia l could be c o l l e c t e d for ana lys i s of extractable metals , due to the i n a c t i v i t y of the earthworms. I f there was indeed any increase i n extractable metals i n the casts , the increase would l i k e l y be d i l u t e d by the metals from the s o i l . As the earthworm body t i s sue concentrated cadmium and z i n c above s o i l l e v e l s , there may be poss ib le a f fects on ava i l ab le cadmium and z inc when these earthworms die and decompose. 4.3 Correlations Between Extractable Metals and Lettuce Metal Concentrations Table 6 gives the c o r r e l a t i o n c o e f f i c i e n t s obtained between metal concentrat ions i n le t tuce lea f t i s sue grown i n s o i l amended with 50 gm/kg mi lorgani te a p p l i c a t i o n r a t e , and t o t a l and DTPA extractable metals i n the so i l - s ludge mixture. The c o r r e l a t i o n c o e f f i c i e n t s between - 129 -t o t a l metals and plant concentrations ind ica te poor c o r r e l a t i o n . This i s i n agreement with what i s expected, as i t i s genera l ly accepted that t o t a l metals overestimate the amounts that plants can a c t u a l l y u t i l i z e (Karim, et a l . , 1976). T o t a l cadmium for the cont ro l was not s i g n i f i c a n t l y corre la ted ( p>0.05) with plant cadmium. This accounts for only 27% of the v a r i a b i l i t y i n the le t tuce cadmium, however. There was also poor c o r r e l a t i o n between DTPA extractable metals and the concentrations i n the p l an t . Only DTPA extractable z inc from the sludge treated s o i l was s i g n i f i c a n t l y corre la ted (p<0.01) with l e t tuce z i n c . These re su l t s were surpr i s ing as DTPA extractable copper, n i c k e l and z i n c from the s o i l treated with 20 gm mi lorgani te /kg s o i l were s i g n i f i c a n t l y corre la ted with le t tuce t i s sue metal concentrat ions . However, comparing the concentrations of metals i n the le t tuce t i s sue from the 20 gm/kg mi lorgani te a p p l i c a t i o n with the l e t tuce concentrat ions from the second experiment, ind ica te s that the higher a p p l i c a t i o n of mi lorgani te re su l ted i n higher concentrat ion of cadmium, l e a d , n i c k e l and z inc and only s l i g h t l y higher copper concentrations i n the l e t t u c e . As the concentrat ions of DTPA extractable metals i n the two m i l o r g a n i t e - s o i l mixtures were approximately equal and the concentrat ions of metals i n the l e t tuce t i s sue were higher i n the p lant s from the 50 gm/kg mi lorgani te a p p l i c a t i o n , i t i s no longer su rpr i s ing that the c o r e l a t i o n c o e f f i c i e n t s are not equa l . The re su l t s suggest that at the higher a p p l i c a t i o n rate of mi lorgani te the plants are taking up metals from other pools not measured by DTPA. The c o r r e l a t i o n c o e f f i c i e n t s between DTPA extractable metals and - 130 -plant t i s sue from the 50 gm/kg mi lorgani te a p p l i c a t i o n were low and accounted for only a small amount of the v a r i a b i l i t y i n metal concentrat ions i n the l e t tuce t i s s u e . Although DTPA and other extractants have been widely used and discussed i n the l i t e r a t u r e (see review Lindsay and N o r v e l l , 1978) wi th vary ing degree of c o r r e l a t i o n with metal uptake by p lant s , DTPA was i n i t i a l l y developed by Lindsay and N o r v e l l as a tes t to access copper and z inc d e f i c i e n c i e s i n calcareous s o i l s . In a c i d i c s o i l s with large concentrations of metals due to the a d d i t i o n of sewage sludges, the use of DTPA may be inappropr ia te and th i s would be re f l ec ted by poor c o r r e l a t i o n with plant uptake. S i l v i e r a and Sommers (1977) warn that although DTPA extractable metals may cor re l a te with plant uptake, the add i t ion of sewage sludge may re su l t i n a large increase i n DTPA extractable metals and consequently the concentrat ion of DTPA used i n the ex t rac t ion may not be appropriate for s o i l t reated with sewage sludge. 4.4 Lettuce Cadmium Concentrations Lettuce l ea f t i s sue concentrated cadmium above s o i l l e v e l s and a lso accumulated Cd with increas ing s o i l Cd concentrat ions . Cadmium concentrat ions i n the l ea f t i s sue from the contro l s exceeded the s o i l concentrat ions by a factor of 3.6 and for the 50 gm/kg a p p l i c a t i o n of mi lorgan i te the concentrat ion fac tor was 11.7. L i s k (1972) reported that the r a t i o of cadmium concentrat ion i n plant t i s sue to s o i l concentrat ion i s genera l ly around 10 to 1. The increase i n cadmium concentrat ion i n the le t tuce t i s sue with the a p p l i c a t i o n of mi lorgani te and hence increased s o i l cadmium - 131 -concentrat ions i s consis tent with the l i t e r a t u r e . From his review of the l i t e r a t u r e , Webber (1979) ind ica te s that the concentrations of cadmium i n vegetable crops grown i n ac id s o i l s amended with sludges wi th high cadmium concentrations can be quite l a rge . The concentrations of cadmium for the cont ro l plants are higher than those reported by Page et a l . (1981) for le t tuce grown i n uncontaminated s o i l . However, the s o i l cadmium concentrat ion of the c o n t r o l (1.6 ug/gm) i s above the mean of 0.88 ug/gm Cd for noncontaminated s o i l s i n the Lower Fraser V a l l e y (John, 1978), and may ind ica te contamination, perhaps by superphosphate f e r t i l i z e r s . John (1973) reported le t tuce had cadmium concentrations of 4.8 ug/gm i n l e t tuce lea f t i s sue and 2.8 ug/gm i n root t i sue when grown i n non-contaminated s o i l c o l l e c t e d from the Ap horizon of a Hazelwood, s i l t loam. He a l so reported l e t tuce lea f cadmium concentrations of 7.1 ug/gm and root concentrations of 4.8 ug/gm for plants grown i n s o i l from the Ap of a Hjor th s i l t y c l ay loam with a pH of 4.04 and a cadmium concentrat ion of 0.67 ug/gm determined by IN HNO3 (John, 1972). The values for h i s controls are s i m i l a r to the values for the contro l s determined i n the present study. John (1972) a l so reported a concentrat ion of 50.8 ug/gm cadmium i n the l e t tuce l ea f t i s sue of plants grown i n the Hjor th s o i l amended with 5 ug/gm cadmium c h l o r i d e . However the magnitude of the cadmium concentrat ions In the plant t i s sues determined i n my study are s i m i l a r only to the concentrations of l e t tuce t i s s u e i n s tudies us ing cadmium sa l t s or sewage sludges spiked with cadmium s a l t s . John and van Laerhoven (1976) grew le t tuce i n s o i l from the Ap of a Monroe s i l t loam amended with various a p p l i c a t i o n rates of - 132 -m i l o r g a n i t e . They report plant concentrations much less than those obtained i n the present study and also reported that increases i n mi lorgan i te a p p l i c a t i o n re su l ted i n decreased cadmium concentrations which was associated with decreased y i e l d s at app l i ca t ions exceeding 10 gm/kg. The increase i n organic matter associated with increased a p p l i c a t i o n s of sludge was credi ted with reducing the amount of cadmium a v a i l a b l e for plant uptake. Lettuce cadmium concentrations increased three fo ld when sewage sludge app l i ca t ions were increased from the c o n t r o l to 220 t /ha (Zwarich and M i l l s , 1982). However the sludge used i n t h e i r study had a cadmium concentrat ion of only .22 ug/gm and p lant concentrat ions increased from 1.4 |!g/gm when no sludge was u t i l i z e d to 4.3 ug/gm when 220 t /ha sludge was app l i ed . Singh (1982) determined cadmium concentrations i n le t tuce which was grown i n sludge amended s o i l ( s i l t - l o a m , pH 6.7 and sludge from Sarnia with 99 ug Cd/gm and s o i l amended with various inorganic cadmium s a l t s ) . The concentrat ions i n l e t tuce grown i n the cadmium sa l t amended s o i l s were s i m i l a r to the concentrat ions i n l e t tuce grown i n mi lorgan i te , whereas the cadmium concentrat ions i n the l e t tuce grown i n the s o i l amended with the Sarnia sludge were much lower than those obtained i n the present study. M i t c h e l l , et a l . (1978) reported a cadmium concentrat ion of 43 ug/gm for l e t tuce harvested from a calcaeous, s i l t loam amended with a cadmium s a l t enriched sewage sludge to produce a cadmium concentrat ion of 5 ug/gm. These researchers reported increased cadmium concentrations i n l e t tuce t i s sue (up to 413 ug/gm) as s o i l cadmium concentrat ions increased up to 320 ug/gm. - 133 -In the current study, the paired d i f ference test at the 5% p r o b a b i l i t y l e v e l did not detect a s i g n i f i c a n t d i f ference between the cadmium concentrations i n the l e t tuce l ea f and root t i s s u e . Webber (1980) reports that cadmium concentrations i n leafy vegetables tend to be higher i n the edib le parts of the plants rather than the root s . John (1972) reported s i g n i f i c a n t l y greater cadmium concentrations i n the above ground parts of l e t tuce and rad i sh plants than the roots from both contro l s and cadmium amended s o i l . However, John (1973) found that although the c o n t r o l plants had higher cadmium concentrat ions i n the lea f t i s sue r e l a t i v e to the root s , the reverse was the case for l e t tuce grown i n cadmium amended s o i l . These r e s u l t s suggest that the l e t tuce plants vary i n t h e i r a b i l i t y to immobilize cadmium i n the root s . 4.5 Lettuce Copper Concentrations The r e s u l t s suggest that copper concentrations may be regulated by the plant or that copper uptake i s reduced when the concentrat ion of other metals i s increased, or that a d d i t i o n a l copper i n the mi lorgani te i s not u t i l i z e d by the l e t tuce p l an t s . Copper In the sewage sludge may be immobilized by forming stable organic copper complexes. Several other authors also report that coper concentrations i n l e t t u c e t i s sues d id not increase with inc rea s ing s o i l copper concentrat ions . M i t c h e l l , et a l . (1978) reported that copper concentrat ions i n l e t tuce remained unchanged at an average concentrat ion of 7.1 ug/gm copper even though the s o i l copper concentrations increased from 0 to 320 ug/gm. When the s o i l copper Increased to 640 ug/gm, there was a s i g n i f i c a n t increase of l e t tuce concentrations to 9.3 ug Cu/gm. - 134 -M i l o r g a n i t e addi t ions to a Monroe s i l t loam, re su l ted i n increas ing concentrat ions of 1.0 N HNO3 extractable copper, but did not re su l t i n inc rea s ing copper concentrations i n l e t tuce lea f t i s s u e . The re su l t s reported by John and van Laerhoven (1976) report a higher copper concentrat ion for l e t tuce (19.9 ug/gm for plants grown i n s o i l amended with 25 gm/kg mi lorgani te ) than the copper concentrat ion i n the plants of the present study. MacLean and Dekker (1978) determined copper concentrations i n le t tuce grown i n s o i l amended with sewage sludge spiked with varying concentrations i n le t tuce remained f a i r l y constant as the s o i l concentrat ion increased . Zwarich and M i l l s (1982) a l so app l ied sewage sludge to s o i l i n increa s ing a p p l i c a t i o n rates and determined copper concentrations i n l e t t u c e . They report an increase i n l e t tuce copper with increased sludge a p p l i c a t i o n . However, the concentrat ion of copper from two sludge treatments, 55 t /ha and 110 t / h a were not s i g n i f i c a n t l y d i f f e rent e i t h e r but were from the cont ro l and the 220 t /ha a p p l i c a t i o n . In the current study, the concentrat ion of copper i n the root t i s sues was s i g n i f i c a n t l y higher (p<0.05) than the copper concentrat ions of the l ea f t i s sue . This agrees with Webber (1980) who concluded that leafy vegetables tended to have greater concentrations of copper i n t h e i r roots than i n t h e i r leaves . M i t c h e l l , et a l . (1978) found higher concentrations of copper i n the roots of wheat than i n wheat gra in and wheat leaves . They suggest that copper once taken-up by the roots remains bound up by amino acids and forms stable organic complexes which are not t rans located to the l ea f t i s sue . Czuba and Hutchinson (1980) - 135 -a l so found higher copper concentrations i n l e t tuce roots than i n the l e t tuce leaves . 4.6 Lettuce Nickel Concentrations A d d i t i o n of mi lorgani te re su l ted i n s i g n i f i c a n t decreases i n the n i c k e l concentrations i n the l e t tuce l ea f t i s sue (Table 7) and l e t tuce root t i s sue (Table 8 ) , even though the t o t a l concentrat ion of n i c k e l i n the s o i l from the contro l treatments and mi lorgani te treatments were unaf fected . These r e s u l t s suggest that the organic matter added i n mi lorgani te may be binding with the n i c k e l although there was a s l i g h t but s i g n i f i c a n t increase i n DTPA extractable n i c k e l when mi lorgani te was added to the s o i l . Halstead et a l . (1969) determined that Niconcentrat ions i n a l f a l f a and oats grown i n Nicontaminated s o i l s were reduced when the s o i l organic matter content was increased . This was a t t r i b u t e d to the formation of s table Ni-organic matter complexes which were unavai lable to p l ant s . Hutchinson (1981) ind ica tes that the a d d i t i o n of organic matter decreased ammonium acetate extractable n i c k e l i n s o i l s , while Hag, et a l . (1980) ind ica ted that DTPA extractable n i c k e l concentrat ion i n s o i l was p o s i t i v e l y corre la ted with organic matter and that n i c k e l concentrat ion extracted by a c e t i c ac id was decreased by increased organic matter content. The increased concentrat ions of the other metals present i n the s o i l mi lorgani te mixture may re su l t i n the in ter ference with n i c k e l uptake by the p l a n t . Hughes, et a l . (1980) ind ica ted that n i c k e l uptake by soybean seedlings was reduced when the concentrations of copper and z inc were increased . - 136 -The add i t ion of sewage sludge to s o i l genera l ly r e su l t s i n increased n i c k e l concentrations i n lea f and root t i s sues of a wide range of crop plants (Page, 1974; Webber, 1979; Hutchinson, 1981). N i c k e l concentrat ions i n plant t i s sues do not exceed s o i l concentrations and u sua l ly are less than 10 ug/gm for plants grown i n s o i l uncontaminated by n i c k e l (Hutchinson, 1981). MacLean and Dekker (1978) determined n i c k e l concentrations i n corn and le t tuce which had been grown i n s o i l s amended with increas ing amounts of n i c k e l sulphate s a l t s and to which sewage sludge had also been added. They found that n i c k e l concentrat ions i n the plant t i s sues increased with corresponding increases i n s o i l concentrat ions . However, the plants grown i n s o i l to which the sludge had also been added had lower n i c k e l concentrat ions than plants grown i n s o i l s amended only with the inorganic s a l t . M i t c h e l l , et a l . (1978) grew le t tuce and wheat i n s o i l amended with metal spiked sewage sludge and determined that n i c k e l concentrat ions i n the plants increased as s o i l n i c k e l concentrations Increased. N i c k e l concentrat ions i n le t tuce and onions grown i n s o i l amended with sewage sludge increased for plants grown i n pot cul tures i n a greenhouse but d i d not increase i n plants grown i n the f i e l d , amended with equivalent a p p l i c a t i o n rates of sludge (de V r i e s and T i l l e r , 1978). John and van Laerhoven (1976) reported that the add i t ion of mi lorgani te to unlimed s o i l s d id not re su l t i n increases i n the n i c k e l concentrat ion of l e t tuce t i s sue or beet tubers but d id r e su l t i n increased concentrations of n i c k e l i n the beet tops. The add i t ion of Iona sludge to unlimed s o i l lead to s i g n i f i c a n t increases i n the n i c k e l concentrations of l e t t u c e , - 137 -d i d not a f fect the concentrat ion i n beet tops, and reduced the n i c k e l concentrations i n the beet tubers (John and van Laerhoven, 1976). Pa i red d i f ference te s t s at the 5% p r o b a b i l i t y l e v e l ind ica ted that l e t tuce roots had s i g n i f i c a n t l y higher concentrations of n i c k e l than did the l ea f t i s sues , i n t h i s study. M i t c h e l l , et a l . (1978) found higher n i c k e l concentrations i n the root t i s sues of wheat than In the l ea f and gra in t i s sues . They a t t r i b u t e the higher root concentrations to e f f ec t ive binding of n i c k e l by organic molecules wi th in the root , and that the root re ta ins these n i c k e l - o r g a n i c complexes against t ranso loca t ion to the leaf t i s sue (same as copper) . 4.7 Lettuce Lead Concentrations Lead concentrations i n the l ea f and root t i s sues of l e t tuce were not s i g n i f i c a n t l y affected by the add i t ion of mi lorgani te to the s o i l . The average lead value for the l ea f t i s sue from the mi lorgani te treatment was much greater than the value for the c o n t r o l . However, there were large standard deviat ions associated with both values and, therefore , the di f ferences were not s i g n i f i c a n t . Page (1974), de V r i e s and T i l l e r (1978), Zwarich and M i l l s (1982) ind ica ted that the add i t ion of sewage sludge to s o i l did not lead to s i g n i f i c a n t increases i n lead i n a v a r i e t y of crop plants i n c l u d i n g l e t t u c e . John and van Laerhoven (1976) ind ica te that although the a d d i t i o n of mi lorgani te s i g n i f i c a n t l y increased s o i l lead l e v e l s , the concentrat ions of lead i n le t tuce t i s sues remained unchanged. Adding 1000 ug/gm of lead as inorganic lead s a l t s to s o i l d id r e su l t i n s i g n i f i c a n t increases i n le t tuce concentrations (John and van Laerhoven, - 138 -1972), there fore , maybe the form of the lead i s important. There was s i g n i f i c a n t l y higher concentrations of lead i n the roots than i n the lea f t i s sue for two of the four treatments, (the treatments were treatment 1) no earthworms and no mi lo rgan i t e , and treatment 4) earthworms and mi lo rgan i t e , pa i red d i f ference tests at the 5% p r o b a b i l i t y l e v e l ) . Webber (1980) reported that leafy vegetables tend to have higher lead l e v e l s i n roots r e l a t i v e to lea f t i s sue . Czuba and Hutchinson (1980) found that i n t h e i r study the lead concentrat ions i n l e t tuce root t i s sue were not cons i s t en t ly higher than lea f t i s sue . The l i t e r a t u r e on lead uptake by plants was reviewed recent ly by Koeppe (1981). He reports that the roots of p lants grown i n lead contaminated s o i l s always had more lead than the above ground plant par t s . Koeppe (1981) ind ica ted that lead was adsorbed by the c e l l wal l s of roots of some plants and i n others lead was p r e c i p i t a t e d by c e l l o rgane l le s . Lettuce Zinc Concentrations The add i t ion of mi lorgani te to the s o i l re su l ted i n s i g n i f i c a n t (p < 0.05) increases i n the z inc concentrations of both l e t tuce t i s sue types . S o i l t o t a l z inc concentrations were s i m i l a r to or s l i g h t l y le s s than the l ea f t i s sue z inc concentrations whereas the concentrat ions i n the roots exceeded the concentrations i n the s o i l . Other workers a lso report increased z inc concentrations i n p l a n t s , i n c l u d i n g l e t t u c e , when sewage sludge i s added to the s o i l (Page, 1974; M i t c h e l l et a l . , 1978; Webber, 1979; Schauer et a l . , 1980). Zwarich and M i l l s (1982) reported that there was a l i n e a r r e l a t i o n s h i p between le t tuce z inc concentrations and the s o i l z inc - 139 -concentra t ions . John and van Laerhoven (1976) reported that although addi t ions of mi lorgani te increased s o i l z inc concentrat ions , l e t tuce grown i n unlimed s o i l had a s i g n i f i c a n t decrease i n t i s sue z inc concentrat ions . They a t t r i b u t e t h i s to z inc being bound by the increa s ing addi t ions of organic matter. For th i s study, paired d i f ference tests at the 5% p r o b a b i l i t y l e v e l ind ica ted that the roots had s i g n i f i c a n t l y higher z inc concentrat ions than the lea f t i s sue d id i n three of the four treatments. The v a r i a b i l i t y i n z inc concentrat ions i n the roots of the l e t tuce grown without mi lorgani te i n the presence of earthworms was too large to detect any di f ferences between the l ea f and root z inc concentrat ions . Webber (1980) ind ica ted that leafy vegetables tended to have higher concentrations of z i n c i n above ground t i s sues rather than i n the root s . Zinc i n wheat roots was higher than i n wheat leaves and g ra in ( M i t c h e l l et a l . , 1978). Lindsay (1972) reported that when plants were grown i n s o i l s with high ava i l ab le z inc concentrations i n plant tops tended to be less than the l eve l s i n the root s , and that i f the supply of s o i l z inc i s adequate luxury consumption of z inc by roots often occurs . 4.9 Effects of Earthworms on Lettuce Metal Concentrations The decrease i n lead concentrat ion i n roots i n the presence of IS earthworms >f not due to a reduct ion i n the lead a v a i l a b i l i t y as K earthwornf* had no e f fect on the concentrat ion of DTPA extractable lead . K Earthworms are not accumulating the lead i n t h e i r own t i s s u e . ( re fer to previous experiment Part 1) and thereby reducing the amount ava i l ab l e to - 140 -the p l ant s . However, DTPA extractable lead was only weakly corre la ted (r = -0.33) with plant lead when no mi lorgani te was present and when mi lorgani te was present the c o r r e l a t i o n was increased (r = -0 .65) . DTPA extractable lead only accounts for 11% of the v a r i a b i l i t y of lead t i s sue concentrat ions for plants without mi lorgani te treatment and for only 42% of the v a r i a b i l i t y i n plant t i s sue lead concentrat ion for p lants t reated wi th mi lorgan i te . It i s poss ib le that earthworms have t i e d up lead that i s not measured by DTPA but i s i n a form that would be used by the p l a n t s . I re land (1975) ind ica ted that earthworms did not increase a c e t i c ac id extrac t rab le lead . Although not very act ive i n the s o i l , i t i s poss ib le that there was a s l i g h t increase i n the aerat ion of the s o i l due to burrowing a c t i v i t y , r e s u l t i n g i n a decrease i n the a v a i l a b i l i t y of l ead . Metals genera l ly are less ava i l ab l e when the supply of oxygen i s increased . Only two reports i n the l i t e r a t u r e ex i s t concerning metal uptake by plants i n the presence of earthworms. Peredelsky et a l . (1957) reported that when mustard was grown i n the presence of earthworms there was a f i v e f o l d increase i n the cobalt concentrat ion i n plant t i s sue s . Earthworms also resu l ted i n an increased cobalt concentrat ion i n severa l ce rea l s , legumes, hemp and buckwheat. When wheat was grown i n sludge amended s o i l s i n the presence of the earthworm, E i s e n i a foet ida there was a s i g n i f i c a n t increase i n the concentrat ion of manganese In the r o o t s . Earthworms had no ef fect on the copper and z inc concentrations i n the wheat roots or shoots (Kirkham, 1979). - 141 -5. GENERAL DISCUSSION The re su l t s obtained i n th i s experiment are i n general agreement with those reported i n the l i t e r a t u r e , although the metal concentrat ions i n the l e t tuce t i s sue tended to be higher than those determined by other researchers . There are severa l reasons for th i s v a r i a t i o n between s tudies and involve di f ferences due to sludge v a r i a t i o n , d i f ferences i n s o i l p roper t i e s , and di f ferences i n metal uptake by plant v a r i e t i e s . Sewage sludges from d i f f e rent treatment plants vary not only i n t o t a l metal concentrations but also i n amounts of ava i l ab le metals , metal spec ies , pH and organic matter content. (Page, 1974). As sludges conta in d i f f e r e n t amounts of ava i l ab l e metals i t i s reasonable that p lants grown i n d i f f e rent sludges would have v a r i a t i o n s i n t i s sue metal cont ra t ions . A l s o , many of the studies reported i n the l i t e r a t u r e invo lve growing plants i n s o i l s amended with sewage sludge spiked with inorganic metal s a l t s or growing plants i n s o i l s amended with inorganic metal s a l t s o l u t i o n s . Webber (1980) concluded from his l i t e r a t u r e review that studies using inorganic sa l t s at high concentrations to simulate sludges with high metal content should be viewed with caut ion . High concentrations of metal s a l t s tend to r e su l t i n decreased y i e l d s and high plant metal concentrat ions . ( M i t c h e l l et a l . , 1978; Korcak and Fanning, 1978; Zwarich and M i l l s , 1982). Singh (1981) reported that the Cd concentrat ion was 5 times higher i n l e t tuce grown i n s o i l t reated with Cd sa l t s than i n l e t tuce grown i n the same s o i l treated with sewage sludges with comparable Cd concentrat ions . These studies demonstrate that metals i n sewage sludges are less ava i l ab l e than metals i n - 142 -inorganic s a l t s . S o i l propert ies such as pH, organic matter content, ca t ion exchange capaci ty and c lay content inf luence the amount of metal taken up by plants (Webber, 1980). Liming s o i l s amended with sewage sludges or inorganic metal s a l t s reduced the Cd, Cu, N i , Pb and Zn concentrat ions i n le t tuce (John and van Laerhoven, 1972, 1976; MacLean, 1974; 1976; Mahler et a l . , 1982). MacLean (1989) reported that the add i t ion of organic matter to s o i l s decreased Pb uptake by p l a n t s . Organic matter add i t ion decreased Cd and increased Zn uptake by le t tuce (MacLean, 1974, 1976). Gaynor and Halstead (1976) ind ica te that the concentrat ions of Cd, Cu, Pb and Zn i n letuce were le s s i n p lants grown i n f i n e r textured s o i l s . General ly plants grown i n s o i l s with low c a t i o n exchange capac i t i e s tend to have higher metal concentrat ions than do plants grown i n s o i l s with high cat ion exchange capac i t i e s (Webber, 1980). Care must be exercised when comparing the re su l t s of the present study with f i e l d s tudies . In t h i s study the mi lorgani te was w e l l mixed with the s o i l , a condi t ion u n l i k e l y to be obtained i n a f i e l d s i t u a t i o n . A l s o , sewage sludge under f i e ld condit ions may undergo more extensive p h y s i c a l , chemical and b i o l o g i c a l changes under the inf luence of na tura l weather condit ions over time (Hartenstein et a l . , 1980). De V r i e s and T i l l e r (1978) and Page et a l . , (1981) compared l eve l s of metals i n vegetables grown i n s o i l amended with sewage sludge i n greenhouse containers and i n the f i e l d and found higher metal concentrat ions i n the t i s sues from the greenhouse study. Page et a l . , - 143 -(1981) ind ica ted that container-grown plants root e n t i r e l y wi th in a contaminated s o i l whereas plants grown i n the f i e l d may extend roots below the contaminated l ayer . A l s o , the v a r i a t i o n s i n l i g h t , temperature, humidity, moisture and other environmental condit ions may be more pronounced i n a f i e l d experiment compared to one i n a greenhouse which may af fect metal uptake by plants (de Vr ie s and T i l l e r , 1978). Not only do d i f f e ren t plant species d i f f e r i n t h e i r a b i l i t y to take up metals but there are also di f ferences among plant v a r i e t i e s (Page, 1974; Webber, 1980; Page et a l . , 1981). John and van Laerhoven (1976) determined the Cd concentrat ion i n 9 v a r i e t i e s of l e t tuce (6 head, 2 l e a f , iromaine) grown i n s o l u t i o n cul ture with C d C l 2 . There was s i g n i f i c a n t d i f ferences among the v a r i e t i e s i n t h e i r tolerances to Cd p h y t o t o x i c i t y and Cd concentrat ion. The highest and lowest Cd concentrations were recorded for head le t tuce v a r i e t i e s . The data of Giordano et a l . , (1979) a lso suggest d i f ferences i n Cd and Zn concentrat ions among d i f f e rent l e t tuce c u l t i v a r s . In the present study, earthworms did not inf luence plant growth which i s contrary to most re su l t s reported i n the l i t e r a t u r e . (Bar ley , 1961; A t l a v i n y t e et a l . , 1968). Van Rhee (1965) ind ica ted that genera l ly earthworms do not l i k e container condit ions and af ter severa l months w i l l e i ther die or ae s t iva te . The surv iv ing earthworms i n the present study were ae s t iva t ing and some had d i ed . A l s o , the taxa used i n th i s study were d i f fe rent from the species and commonly used i n the l i t e r a t u r e , although the density of earthworms used was s i m i l a r to those used i n the l i t e r a t u r e . - 144 -6. C O N C L U S I O N S The concentrations of t o t a l s o i l Cd, Cu, Pb and Zn but not Ni and the concentrations of DTPA extractable Cd, Cu, Ni, Pb and Zn were increased when milorganite was added to the s o i l . The concentrations of both t o t a l and DTPA extractable metals were unaffected by the presence of earthworms. The concentrations of Cd and Zn i n both leaves and roots were Increased when milorganite was added to the s o i l . Nickel concentrations decreased i n both tissue types and Cu and Pb concentrations were unaffected by milorganite amendments to the s o i l . Only the Pb concentrations i n lettuce roots were affected by the presence of earthworms. With earthworms present i n s o i l both with and without milorganite the Pb concentrations i n the roots were lower than when the earthworms were absent. The concentrations of Cu, Ni and Zn i n lettuce leaf tissue were s i g n i f i c a n t l y correlated with the concentrations of DTPA extractable metals when milorganite was added to the s o i l at the rate of 20 gm/kg. Only Zn concentrations i n le a f tissues were s i g n i f i c a n t l y but negatively correlated with DTPA extractable Zn when milorganite was added at the rate of 50 gm/kg. These r e s u l t s demonstrated that at increased l e v e l s of sludge a p p l i c a t i o n the plants are u t i l i z i n g metals from s o i l metal pools not measured by DTPA. Yields of lettuce were increased when milorganite was added to the s o i l . The a d d i t i o n a l N, P, and K added i n the sludge resulted i n increased y i e l d s . However, the Cd concentrations i n the s o i l a f t e r the - 145 -a d d i t i o n of mi lorgani te exceed the proposed Ontario guide l ines for sludge addi t ions to l and . (OME, OMAF, 1978). Mi lo rgan i t e i s not recommended as a f e r t i l i z e r . Earthworms were not very act ive i n the flower pots and most recovered i n r e s t i n g p o s i t i o n s . The presence of earthworms did not a f fect plant y i e l d s . - 146 -REFERENCES Anderson, M.S. 1959. F e r t i l i z i n g c h a r a c t e r i s t i c s of sewage sludge. Sewage and I n d u s t r i a l Wastes. 31: 678-682. A t l a v i n y t e , 0 . , Z . Bagdonaviciene, and I . Budavicieno. 1968. The e f fec t of Lumbricidae on the bar ley crops i n various s o i l s . Pedobio log ia . 8: 415-423. A t l a v i n y t e , 0. and J . Vanagas. 1973. M o b i l i t y of n u t r i t i v e substances i n r e l a t i o n to earthworm numbers i n the s o i l . Pedobio log ia . 13: 344-352. Bar ley , K . P . 1961. The abundance of earthworms i n a g r i c u l t u r a l land and t h e i r poss ib le s i gn i f i c ance i n a g r i c u l t u r e . Advan. Agron. 13: 249-268. B i c k f o r d , E . D . and S. Dunn. 1972. L i g h t i n g for p lant growth. Kent State U n i v e r s i t y Press . 221 p. B j e r r i n g , J . H . , M. Gre igh , and J . Halm. 1975. UBC BMD lOv general l i n e a r hypothesis . Computing Centre , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 31 p. Brown, A . L . , J . Quick, and J . . Eddings. 1971. A comparison of a n a l y t i c a l methods for s o i l z i n c . S o i l S c i . Soc. Am. Proc . 35: 105-107. Czuba, M. and T . C . Hutchinson. 1980. Copper and lead l e v e l s i n crops and s o i l s of the Holland Marsh area - Ontar io . J . E n v i r o n . Qual. 9: 566-575. DeVr ie s , M . P . C . and K . G . T i l l e r . 1978. Sewage sludge as a s o i l ammendment, with spec i a l reference to Cd, Cu, Mn, N i , Pb and Zn -comparisons of re su l t s from experiments conducted ins ide and outside a glasshouse. Env i ron . P o l l u t . 16: 231-240. Department of S o i l Science. 1977. Methods manual, pedology l abora tory . U n i v e r s i t y of B r i t i s h Columbia, Vancouver. 224 p. Edwards, C A . and J .R . L o f t y . 1977. Biology of earthworms. 2nd ed. Chapman and H a l l , London. 333 p. E l l i s , R . , J . J . Hanway, G. Holmgren, D.R. Keeney. 1976. Sampling and ana lys i s of s o i l s , p l an t s , waste waters, and sludge suggested s tandardizat ion and methodology. Subcommittee of NC-118, A g r i c u l t u r a l Experiment S t a t i o n , Kansas State U n i v e r s i t y . 20 p. Freedman, B. and T . C . Hutchinson. 1981. Sources of metal and elemental contamination of t e r r e s t r i a l environments. In : E f fec t of Heavy Meta l P o l l u t i o n on Plants V o l . 2. Metals i n the Environment. (N.W. Lepp(ed . ) ) , Appl ied Science P u b l i s h e r s , London, pp. 35-94. - 147 -Gaynor, J . D . , and R . L . Halstead. 1976. Chemical and plant e s t r a c t a b i l i t y of metals and plant growth on s o i l s amended with s ludge. Can. J . Soil S c i . 56: 1-8. Ghabbour, S . I . 1966. Earthworms i n a g r i c u l t u r e : a modern e v a l u a t i o n . Rev. E c o l . B i o l . S o l . I l l : 259-271. Giordano, P . M . , D .A. Mays, and A . D . Behel , J r . 1979. S o i l temperature e f fec t s on uptake of cadmiumand z inc by vegetables grown on sludge-amended s o i l . J . Env i ron . Qual. 8: 233-236. Hal s tead , R . L . , B . J . F i n n , and A . J . MacLean. 1969. E x t r a c t a b i l i t y of n i c k e l added to s o i l s and i t s concentrat ion i n p l an t s . Can. J . S o i l S c i . 49: 335-342. Har tens te in , R . , E . F . Neuhauser, and J . C o l l i e r . 1980. Accumulation of heavy metals i n the earthworm EiBenia f o e t i d a . J . Env i ron . Qual. 9: 23-26. Haq, A . U . , T . E . Bates, and Y . K . Soon. 1980. Comparison of extractants for plant ava i l ab le z i n c , cadmium, n i c k e l , and copper i n contaminated s o i l s . S o i l S c i . Soc. Am. J . 44: 772-777. Hughes, M . K . , N.W. Lepp, and D.A. Phipps . 1980. A e r i a l heavy metal p o l l u t i o n and t e r r e s t r i a l ecoslystems. Adv. E c o l . Res. 11: 217-327. Hutchinson, T . C . 1981. N i c k e l In: E f fec t of Heavy Metal P o l l u t i o n on P lan t s . V o l . I, E f fec t s of Trace Metals on Plant Func t ion . (N.W. Lepp ( e d . ) ) , Appl ied Science P u b l i s h e r s , London, pp. 171-211. I r e l a n d , M.P. 1975. The e f fect of the earthworm Dendrobaena rubida on the s o l u b i l i t y of l ead , z i n c , and calcium i n heavy metal contaminated s o i l s i n Wales. J . S o i l S c i . 26: 313-318. John, M.K. 1972a. E f fec t of lime on s o i l ex t rac t ion and on a v a i l a b i l i t y of s o i l appl ied cadmium to rad i sh and lea f l e t tue p l a n t s . S c i . T o t a l Env i ron . 1: 303-308. John, M.K. 1972b. Lead a v a i l a b i l i t y re l a ted to s o i l propert ies and extractable l ead . J . Env i ron . Qual. 1: 295-298. John, M.K. 1972c. Influence of s o i l propert ies and extractable z inc on z inc a v a i l a b i l i t y . S o i l S c i . 113: 222-227. John, M.K. 1973. Cadmium uptake by eight food crops as inf luenced by various so i l eve l s of cadmium. Env i ron . P o l l u t . 4: 7-15. John, M.K. 1975. Transfer of heavy metals from s o i l s to p l an t s . In : In tern . Conf. on Heavy Metals i n the Environment, v o l . 2, Pathways and c y c l i n g . ( T . C . Hutchinson ( e d . ) ) , I n s t i t u t e for Environmental S tudies , U n i v e r s i t y of Toronto, pp. 365-377. - 148 -John, M . K . , and C. van Laerhoven. 1972. Lead uptake by l e t tuce and oats as af fected by l ime, n i t rogen , and sources of l ead . J . E n v i r o n . Qual. 1: 169-171. John, M . K . , and C. van Laerhoven. 1976a. E f f ec t s of sewage sludge composit ion, a p p l i c a t i o n ra te , and lime regime on plant a v a i l a b i l i t y of heavy metals . J . Env i ron . Qual. 5: 246-251. John, M . K . , and C. van Laerhoven. 1976b. D i f f e r e n t i a l e f fec t s of cadmium on le t tuce v a r i e t i e s . Env i ron . P o l l u t . 10: 163-173. Karim, H . , J . E . Sedberry, and B . J . M i l l e r . 1976. The p r o f i l e d i s t r i b u t i o n of t o t a l and DTPA-extractable copper i n se lected s o i l s i n L o u i s i a n a . Commun. S o i l S c i . and Plant ana l . 7: 437-452. Kirkham, M.B. 1979. A v a i l a b i l i t y to wheat of elements i n sludge treated s o i l with earthworms. In : U t i l i z a t i o n of S o i l Organisms i n Sludge Management, Conference Proceedings. (R. Hartenste in ( e d . ) ) , Syracuse, Nat ional Science Foundation, pp 103-121. Koeppe, D . E . 1981. Lead: understanding the minimal t o x i c i t y of lead i n p l an t s . In : E f fec t of Heavy Metal P o l l u t i o n on P l a n t s . V o l . 1, E f f ec t s of Trace Metals on Plant Funct ion . (N.W. Lepp(ed . ) ) , Appl ied Science Pub l i sher s , London, pp. 55-76. Korcak, R . F . , and D.S. Fanning. 1978. E x t r a c t a b i l i t y of cadmium, copper, n i c k e l , and z inc by double ac id versus DTPA and plant content at excessive s o i l l e v e l s . J . Env i ron . Qual. 7: 506-512. Le , C D . 1980. UBC MFAV ana lys i s of var iance /covar iance . Computing Centre , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 64 p . L indsay , W.L. 1972. Zinc i n s o i l s and plant n u t r i t i o n . Advan. Agron. 24: 147-186. L indsay , W . L . , and W.A. N o r v e l l . 1978. Development of a DTPA s o i l tes t for z i n c , i r o n , manganese, and copper. S o i l S c i . Soc. An. J . 42: 421-428. L i s k , D . J . 1972. Trace metals i n s o i l s , p l a n t s , and animals. Advan. Agron. 24: 267-325. Lunt , H . A . , and H.G.M Jacobson. 1944. The chemical composition of earthworm cast s . S o i l S c i . 58: 367-375. Luttmerding, H .A. 1980. S o i l s of the Langley-Vancouver map area. V o l 1. S o i l map mosaics and legend, Lower Fraser V a l l e y . (Scale 1: 25,000) R . A . B . B u l l . 18. Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . Luttmerding, H .A. 1981a. So i l s of the Langley-Vancouver map area . V o l . 3. D e s c r i p t i o n of the s o i l s . R . A . B . B u l l . 18, Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . - 149 -Luttmerding, H .A. 1981b. S o i l s of the Langley-Vancouver map area, V o l . 6, t e c h n i c a l d a t a - s o i l p r o f i l e descr ip t ions and a n a l y t i c a l data . R . A . B . B u l l . 18 Province of B r i t i s h Columbia, M i n i s t r y of Environment, Assessment and Planning D i v i s i o n . MacLean, A . J . 1974. E f fec t s of s o i l propert ies and amendments on the a v a i l a b i l i t y of z inc i n s o i l s . Can. J . S o i l S c i . 54: 369-378. MacLean, A . J . 1976. Cadmium i n d i f f e r e n t plant species and i t s a v a i l a b i l i t y i n s o i l s as inf luenced by organic matter and addi t ions of l ime, P, Cd and Zn. Can. J . S o i l S c i . 56: 129-138. MacLean, A . J . , and A . J . Dekker. 1978. A v a i l a b i l i t y of z i n c , copper, and n i c k e l to plants grown i n sewage-treated s o i l s . Can. J . S o i l S c i . 58: 381-389. MacLean, A . J . , R . L . Hals tead, and B . J . F i n n . 1969. E x t r a c t a b i l i t y of added lead i n s o i l s and i t s concentrat ion i n p l an t s . Can. J . S o i l . S c i . 49: 327-334. MacLean, K. S . , and W.M. L a n g i l l e . 1976. The extractable trace element content of ac id s o i l s and the inf luence of pH, organic matter and c lay content, commun. S o i l S c i . and Plant A n a l . 7: 777-785. Mahler , R . J . , F . T . Bingham, and A . L . Page. 1978. Cadmium-enriched sewage sludge a p p l i c a t i o n to ac id and calcareous s o i l s : e f fect on y i e l d and cadmium uptake by l e t tuce and chard. J . Env i ron . Qual. 7: 274-281. Mahler , R . J . , F . T . Bingham, A . L . Page, and J . A . Ryan. 1982. Cadmium-enriched sewage sludge a p p l i c a t i o n to ac id and calcareous s o i l s : e f fect on s o i l and n u t r i t i o n of l e t t u c e , corn , tomatoe, and swiss chard. J . Env i ron . Qual. 11: 694-700. Mendenhall , W. 1971. Introduct ion to p r o b a b i l i t y and s t a t i s t i c s . 3rd ed. Wadsworth Pub l i sh ing Company, Inc. Belmont, C a l i f o r n i a . 466 p. M i t c h e l l , G . A . , F . T . Bingham, and A . L . Page. 1978. Y i e l d and metal composition of l e t tuce and wheat grown on s o i l s amended with sewage sludge enriched with cadmium, copper, n i c k e l , and z i n c . J . E n v i r o n . Qual. 7: 165-171. Ontar io M i n i s t r y Environment. 1978. Guidel ines for sewage sludge u t i l i z a t i o n on a g r i c u l t u r a l lands , ad Hoc Jo in t Committee of Ontario M i n i s t r y of A g r i c u l t u r e and Food - Ontario M i n i s t r y of the Environment. Page, A . L . 1974. Fate and ef fects of trace elements i n sewage sludge when appl ied to a g r i c u l t u r a l lands a l i t e r a t u r e review study. EPA-670/2-74-005. Environmental Pro tec t ion Agency, C i n c i n n a t i , Ohio 45268, 98 p. - 150 -Page, A . L . , F . T . Bingham, and A . C . Chang. 1981. Cadmium In : E f fec t of Heavy Metal P o l l u t i o n on P lan t s . V o l . 1, E f fec t s of Trace metals on Plant Funct ion . (N.W. Lepp(ed . ) ) , Appl ied Science Pub l i sher s , London, pp. 77-109. Peredelsky, A . A . , N.A. Poryadkova, and L . Z . Rodionova. 1957. The r o l e of earthworms i n c l e a r i n g s o i l of contamination by rad ioac t ive i s to topes . Dak lady-B io log ica l Sciences Sections 115: 638-641. Schauer, P . S . , W.R. Wright , and J . Pe lchat . 1980. Sludge-borne heavy metal a v a i l a b i l i t y and uptake by vegetable crops under f i e l d cond i t ions . J . Env i ron . Qual. 9: 69-73. S i l v i e r a , D . J . , and L . E . Sommers. 1977. E x t r a c t a b i l i t y of copper, z inc cadmium, and lead i n s o i l s incubated with sewage sludge. J . E n v i r o n . Qual. 6: 47-52. Singh, S.S. 1981. Uptake of cadmium by le t tuce (Lactuca sa t iva) as inf luenced by i t s add i t ion to a s o i l as inorganic forms or i n sewage sludge. Can. J . S o i l S c i . 61: 19-28. Soon, Y . K . , and T . E . Bates. 1982. Chemical pools of cadmium, n i c k e l , and z inc i n po l lu ted s o i l s and some pre l iminary i n d i c a t i o n s of t h e i r a v a i l a b i l i t y to p l a t s . J . S o i l S c i . 33: 477-488. Symeonides, C , and S.G. McRae. 1977. The assessment of p lant a v a i l a b l e cadmium i n s o i l s . J . env i ron . Qual. 6: 120-123. Van Loon, J . C , and T. Lichwa, 1973. A study of the atomic absorption determination of some important heavy metals i n f e r t i l i z e r s and domestic sewage plant s ludges. E n v i r o n . L e t t . 4: 1-8. Van Rhee, J . A . 1965. Earthworm a c t i v i t y and plant growth i n a r t i f i c i a l c u l t u r e s . Plant and S o i l . 22: 45-49. Webber, J . 1979. The e f fects of metals i n sewage sludge on crop growth and composition. In: u t i l i s a t i o n of Sewage Sludge on land . Papers and Proceedings of a Water Research Centre Conference. (A.M. Bruce ( e d . ) ) , Oxford. Webber, J . 1980. Metals i n Sewage sludge appl ied to the land and t h e i r e f fec t on crops. In : Inorganic P o l l u t i o n and A g r i c u l t u r e , Proceedings of a Converence organised by the A g r i c u l t u r a l Development and Advisory Serv ice , A p r i l , 1977. M . A . F . F . Ref. Book 326. H . M . S . O . , London pp. 222-234. Webber, M . D . , and D.G.M. Corneau. 1975. Metal e x t r a c t a b i l i t y from s l u d g e - s o i l mixtures . In : In tern . Conf. on Heavy Metals i n the Environment, v o l . 1 ( T . C . Hutchinson ( e d . ) ) , I n s t i t u t e for Environmental Studies , U n i v e r s i t y of Toronto, pp. 205-225. Zwarich, M . A . , and J . G . M i l l s . 1982. Heavy metal accumulation by some vegetable crops on sewage-sludge-amended s o i l s . Can. J . S o i l S c i . 62: 243-247. 

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