Illinois State Water Survey Division WATER QUALITY SECTION

AT PEORIA, ILLINOIS

SWS Contract Report 403

SITE-SPECIFIC PHYTOTOXICITY OF HEAVY METALS

by Wuncheng Wang

Prepared for the U.S. Environmental Protection Agency

September 1986

S i t e - S p e c i f i c P h y t o t o x i c i t y of Heavy M e t a l s

by W u n c h e n g W a n g

W a t e r Q u a l i t y S e c t i o n Illinois State W a t e r S u r v e y

P e o r i a , IL

Grant N o . R 8 1 0 8 3 4 - 0 1 - 0

Project O f f i c e r Clyde B i s h o p

U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y W a s h i n g t o n D.C.

September 1986

A b s t r a c t

The o b j e c t i v e of this study was to d e t e r m i n e the toxicity of heavy m e t a l s in v a r i o u s river and lake w a t e r s . The w a t e r s a m p l e s e n c o m p a s s a w i d e v a r i e t y of w a t e r q u a l i t y , from very soft to very h a r d . T h e r e w e r e 18 s a m p l e s t a t i o n s , 10 in Illinois and 8 in the n e i g h b o r i n g states of I n d i a n a , Iowa, M i s s o u r i , and W i s c o n s i n . T h r e e metal ions w e r e t e s t e d : b a r i u m , c h r o m i u m ( h e x a v a l e n t ) , and n i c k e l . The test o r g a n i s m w a s common d u c k w e e d , Lemna m i n o r .

The results s h o w that among the three m e t a l s , Ba p h y t o t o x i c i t y was the least and w a s influenced the m o s t by the w a t e r q u a l i t y of the test s a m p l e s . Cr p h y t o t o x i c i t y w a s m o d e r a t e and w a s influenced the least by the w a t e r q u a l i t y of the test s a m p l e s . Ni p h y t o t o x i c i t y w a s the g r e a t e s t and w a s m o d e r a t e l y i n f l u e n c e d by the w a t e r q u a l i t y of the test s a m p l e s .

On the b a s i s of these r e s u l t s , it is s u g g e s t e d that w a t e r q u a l i t y c r i t e r i a be m o d i f i e d , w i t h c o n s i d e r a t i o n g i v e n to the s i t e - s p e c i f i c water q u a l i t y and the s p e c i e s of heavy m e t a l s .

CONTENTS

List of Figures v List of Tables vi Glossary vii Acknowledgments vii 1. Introduction 1 2. Conclusions 2 3. Literature Review 3

Introduction 3 Biotic Factors 4 A. Tolerance 4 B. S i z e a n d l i f e s t a g e s . . . 6 C. Species 7 D. Nutrition 9 Abiotic Factors 9 A. Organics 9

i. Synthetic organics 9 ii. Humic substances 12

B pH 13 C. Temperature 14 D. Alkalinity and hardness 15 E. Inorganic ligands 16 F. Interactions 17 G. Sediments 23 H. In situ 24 Summary and Conclusions 26

4. Methods 27 A. Water samples 27 B. Duckweed culture 27 C. Duckweed test specimens 30 D. Test procedure 31 E. Test compounds 32 F. Growth medium 32 G. Statistical analyses 32

5. Results 34 A. Water quality 34 B. Nutrients 34 C. Controls 37 D. Repeatability 37 E. Ba toxicity 41

i. In duckweed growth medium 41 ii. In water samples (nutrient-enriched) 41

F. Cr toxicity . . . 45 i. In duckweed growth medium 45

ii. In Illinois River water (enriched) 45 iii. In all water samples (enriched) 51

Concluded on next page

iii

G. Ni t o x i c i t y , . . . . 56 i . In d u c k w e e d g r o w t h m e d i u m 56

ii. In H a y e s C r e e k and H o r s e s h o e L a k e s a m p l e s ( e n r i c h e d ) 56 i i i . In I l l i n o i s R i v e r w a t e r ( e n r i c h e d ) 56 iv. In all w a t e r s a m p l e s ( e n r i c h e d ) . . 62

6 . D i s c u s s i o n . . . . 6 6 A. I m p o r t a n c e of a q u a t i c v e g e t a t i o n 66 B . S i t e - s p e c i f i c w a t e r q u a l i t y c r i t e r i a 6 7 C. R e f e r e n c e t o x i c a n t 67

R e f e r e n c e s 69

iv

Figures

1. Sample stations 28 2. C o m p a r i s o n of a l k a l i n i t y of s t a t i o n s in 1956-1966

(Harmeson and L a r s o n , 1969) and this s t u d y , in mg/L as C a C O 3 36

3. N e g a t i v e controls 38 4. D u p l i c a t e tests of enriched Skunk River water s a m p l e ,

3/3/86, Ba toxicity 39 5. D u p l i c a t e tests of enriched Skunk River water s a m p l e ,

3/3/86, Cr toxicity 40 6. Ba toxicity in d u c k w e e d growth m e d i u m , 7 tests 42 7. G e n e r a l i z e d e x p o s u r e - e f f e c t c u r v e , showing diameter of

circles as degree of certainty about data p o i n t s . F r o m W h y t e and B u r t o n , 1980, p. 6 4 . R e p r o d u c e d by p e r m i s s i o n of S C O P E , * 15, P a r i s , France 43

8. Ba toxicity in various water samples ( e n r i c h e d ) 46 9. Ba toxicity ( I C 5 0 ' s ) and S O . c o n c e n t r a t i o n r e l a t i o n s h i p . . 48

10. Cr toxicity in d u c k w e e d growth m e d i u m , 10 tests 49 11. Duckweed growth in water c o n t r o l s ( ) and in Illinois

River sample c o n t r o l s ( ) and Cr toxicity in duckweed growth m e d i u m ( ) and in Illinois River s a m p l e s ( ) . . . . 50

12. Cr toxicity in e n r i c h e d Illinois River water s a m p l e s , 13 tests 52

13. D u c k w e e d growth in growth m e d i u m and 10 s u r f a c e w a t e r s ( e n r i c h e d ) c o n t a i n i n g Cr ion 54

14. G e n e r a l i z e d Cr c o n c e n t r a t i o n - e f f e c t r e l a t i o n s h i p in 18 surface water samples ( e n r i c h e d ) 55

15. Ni toxicity in d u c k w e e d growth m e d i u m , 7 tests 57 16. Ni toxicity in e n r i c h e d H a y e s Creek and H o r s e s h o e Lake

s a m p l e s , 4 tests 59 17. D u c k w e e d growth in water c o n t r o l s ( ) and in Illinois

River sample c o n t r o l s ( ) and Ni toxicity in d u c k w e e d growth m e d i u m (A) and in Illinois River s a m p l e s ( ) . . . . 61

18. Ni toxicity in e n r i c h e d Illinois River water s a m p l e s , 12 tests 63

19. Ni c o n c e n t r a t i o n - e f f e c t r e l a t i o n s h i p in 16 s u r f a c e water samples ( e n r i c h e d ) 65

v

T a b l e s

1. I n t e r a c t i o n s of h e a v y m e t a l s in c o n t a c t w i t h a q u a t i c o r g a n i s m s 19

2. M i n e r a l q u a l i t y c h a r a c t e r i s t i c s of s e l e c t e d s t a t i o n s in I l l i n o i s , m e d i a n v a l u e s i n m g / L from 1 9 5 0 - 1 9 6 6 ( f r o m H a r m e s o n and L a r s o n , 1 9 6 9 ) 29

3. N u t r i e n t stock s o l u t i o n s 33 4. W a t e r q u a l i t y of test s a m p l e s 35 5. D u c k w e e d g r o w t h ( n u m b e r of f r o n d s ) in w a t e r s a m p l e s ,

1 1 / 7 / 8 4 , e n r i c h e d w i t h d i f f e r e n t l e v e l s o f p l a n t n u t r i e n t s : 36

6. Ba t o x i c i t y in e n r i c h e d w a t e r s a m p l e s , in IC50's 44 7. C l a s s i f i c a t i o n of 8a t o x i c i t y in v a r i o u s e n r i c h e d w a t e r

s a m p l e s 47 8. Cr t o x i c i t y in e n r i c h e d w a t e r s a m p l e s , in IC50's 53 9. Ni t o x i c i t y in e n r i c h e d H a y e s C r e e k and H o r s e s h o e Lake

s a m p l e s , in p e r c e n t g r o w t h i n h i b i t i o n 58 1 0 . Ni t o x i c i t y in e n r i c h e d I l l i n o i s R i v e r w a t e r s a m p l e s , in

p e r c e n t g r o w t h i n h i b i t i o n 60 1 1 . Ni t o x i c i t y in e n r i c h e d w a t e r s a m p l e s , in IC50's 64 1 2 . C l a s s i f i c a t i o n of Ni t o x i c i t y in v a r i o u s w a t e r s a m p l e s . . 64

vi

G l o s s a r y

A x e n i c c u l t u r e - A c u l t u r e t r e a t e d to r e m o v e life forms o t h e r than the test o r g a n i s m .

C o l o n y - S e v e r a l f r o n d s ( e . g . , of d u c k w e e d ) a t t a c h e d as a u n i t , u s u a l l y c o n s i s t i n g o f m o t h e r and d a u g h t e r f r o n d s .

D u c k w e e d g r o w t h - Net i n c r e a s e of d u c k w e e d frond number d u r i n g an i n c u b a t i o n p e r i o d .

D u c k w e e d g r o w t h m e d i u m - D o u b l e s t r e n g t h of algal g r o w t h m e d i u m ( S t a n d a r d M e t h o d s . 1 9 8 5 ) .

F r o n d - A l e a f - l i k e s t r u c t u r e ; the f u s i n g of leaf and stem of d u c k w e e d p l a n t s .

IC50 - A t o x i c a n t c o n c e n t r a t i o n w h i c h c a u s e s 5 0 % i n h i b i t i o n e f f e c t .

S a m p l e c o n t r o l - A m b i e n t w a t e r s a m p l e s e n r i c h e d w i t h d u c k w e e d g r o w t h mediurn.

W a t e r c o n t r o l - D e i o n i z e d w a t e r s a m p l e s e n r i c h e d w i t h d u c k w e e d g r o w t h mediurn.

A c k n o w l e d g m e n t s

T h i s r e s e a r c h p r o j e c t w a s s u p p o r t e d b y the U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , G r a n t 8 1 0 8 3 4 - 0 1 - 0 , P r o j e c t O f f i c e r M r . C l y d e B i s h o p .

J u d s o n M . W i l l i a m s p r o v i d e d t e c h n i c a l a s s i s t a n c e t h r o u g h o u t this study a s well a s g r a p h i c w o r k and s a m p l e c o l l e c t i o n . D o n a l d H . S c h n e p p e r a s s i s t e d i n c o m p u t e r p r o g r a m m i n g . G a i l T a y l o r e d i t e d the r e p o r t . L i n d a J o h n s o n , Don B l a k l e y , D a n a S h a c k l e f o r d , D a v i s B e u s c h e r , and D a v i d H u l l i n g e r all p a r t i c i p a t e d in w a t e r s a m p l e c o l l e c t i o n . D a v i d H u l l i n g e r and D a n a S h a c k l e f o r d p e r f o r m e d all c h e m i c a l a n a l y s e s . L i n d a J o h n s o n t y p e d the m a n u s c r i p t . D r . P h i l l i p e R o s s r e v i e w e d and c o m m e n t e d o n the l i t e r a t u r e r e v i e w s e c t i o n . T h e i r a s s i s t a n c e i s s i n c e r e l y a p p r e c i a t e d .

vii

1 . I n t r o d u c t i o n

The d e v e l o p m e n t of water q u a l i t y c r i t e r i a is at a c r o s s r o a d . On one h a n d , c r i t e r i a are d e v e l o p e d under laboratory c o n t r o l l e d c o n d i t i o n s w i t h the goal of o b t a i n i n g u n i f o r m and universal r e s u l t s . In other w o r d s , the results are n e c e s s a r i l y r e p e a t a b l e since the e s s e n c e of s c i e n t i f i c m e t h o d is r e p e a t a b i l i t y . M a n y i n t e r - I a b o r a t o r y , r o u n d - t a b l e s t u d i e s have been c o n d u c t e d to test r e p e a t a b i l i t y ( L e m k e , 1 9 8 1 ) .

On the other h a n d , the f u n d a m e n t a l rule of e n v i r o n m e n t a l s c i e n c e is that there are no " s t a n d a r d " e n v i r o n m e n t a l c o n d i t i o n s . It is i n a p p r o p r i a t e to d e r i v e a set of water q u a l i t y c r i t e r i a under laboratory c o n d i t i o n s and then to apply them to v a r i o u s w a t e r s . For e x a m p l e , the N a t i o n a l A c a d e m y of S c i e n c e s - N a t i o n a l A c a d e m y of E n g i n e e r i n g (National R e s e a r c h C o u n t i l , 1972) reviewed the toxicity of lead, z i n c , and copper to r a i n b o w trout and found that the lethal c o n c e n t r a t i o n varied by two o r d e r s of m a g n i t u d e , d e p e n d i n g on the h a r d n e s s of the w a t e r .

C o n s e q u e n t l y the trend in e s t a b l i s h i n g water q u a l i t y c r i t e r i a is to take s i t e - s p e c i f i c w a t e r p r o p e r t i e s into c o n s i d e r a t i o n . In g u i d e l i n e s p r e p a r e d by the U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y for d e r i v i n g s i t e - s p e c i f i c c r i t e r i a , four m e t h o d s are s u g g e s t e d , a l l o w i n g each state to adopt a m e t h o d a p p r o p r i a t e for its local s i t u a t i o n . B a s i c a l l y the d e r i v a t i o n of s i t e - s p e c i f i c c r i t e r i a is based on national water q u a l i t y c r i t e r i a w h i c h are then m o d i f i e d by taking into account s i t e - s p e c i f i c w a t e r q u a l i t y c h a r a c t e r i s t i c s such as resident s p e c i e s , heavy metal s p e c i a t i o n , e t c . C U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , 1 9 8 2 ) .

The o b j e c t i v e of this study w a s to d e t e r m i n e the toxicity of heavy m e t a l s in v a r i o u s river and lake w a t e r s . T h e s e w a t e r s e n c o m p a s s a w i d e variety of w a t e r q u a l i t y , from very soft to very h a r d . T h r e e metal ions w e r e tested: b a r i u m , c h r o m i u m C h e x a v a l e n t ) , and n i c k e l . The test o r g a n i s m w a s common d u c k w e e d , L e m n a m i n o r .

1

2 . C o n c l u s i o n s

The duckweed toxicity test as c o n d u c t e d in this study is economical and s e n s i t i v e . It o f f e r s an a l t e r n a t i v e / c o m p l e m e n t a r y test to general toxicity tests that use the common test s p e c i e s of d a p h n i d s and fathead m i n n o w s . F u t h e r m o r e , it is a much m o r e s u i t a b l e test for a s s e s s i n g the p h y t o t o x i c i t y of herbicidal c o m p o u n d s .

The duckweed c u l t u r e can be m a i n t a i n e d with very little e f f o r t . In a long-term e x p e r i m e n t , the stock c u l t u r e remained v i g o r o u s and test s p e c i m e n s w e r e a v a i l a b l e y e a r - r o u n d .

A m o n g three metal ions tested, Ba had the least p h y t o t o x i c i t y and w a s influenced the most by the water quality of the test s a m p l e s . Cr had m o d e r a t e p h y t o t o x i c i t y and w a s influenced the least by the water quality of the test s a m p l e s . Ni had the greatest p h y t o t o x i c i t y and w a s m o d e r a t e l y influenced by the water quality of the test s a m p l e s .

T h e s e results suggest that the Cr water quality c r i t e r i o n may be a d o p t e d u n i v e r s a l l y for all s u r f a c e w a t e r s . The Ba c r i t e r i o n (5.0 m g / L ) as stated by the State of Illinois ( 1 9 8 6 ) for the p r o t e c t i o n of aquatic o r g a n i s m s can be s u b s t a n t i a l l y m o d e r a t e d a c c o r d i n g to water q u a l i t y . The Ni c r i t e r i o n (1.0 m g / L ) is e x t r e m e l y i n a d e q u a t e . At this c o n c e n t r a t i o n , a 30 p e r c e n t growth inhibition of aquatic v e g e t a t i o n (based on common d u c k w e e d as the test o r g a n i s m ) can be e x p e c t e d in almost all surface w a t e r s (Fig. 19) and 70 percent inhibition can be e x p e c t e d in e x t r e m e l y soft w a t e r s (Fig. 1 6 ) .

The common p r a c t i c e for c o n d u c t i n g biological toxicity tests is to include c o n t r o l s , commonly called n e g a t i v e c o n t r o l s . In this s t u d y , two types of n e g a t i v e c o n t r o l s w e r e used t h r o u g h o u t : water control and sample c o n t r o l . The n e g a t i v e c o n t r o l s m e a s u r e d the test o r g a n i s m s under favorable c o n d i t i o n s . It is highly r e c o m m e n d e d , h o w e v e r , that a p o s i t i v e control also be used to m e a s u r e the response of the test o r g a n i s m s to a u n i v e r s a l , r e f e r e n c e t o x i c a n t . M a n y o r g a n i c and inorganic c o m p o u n d s have been s u g g e s t e d as r e f e r e n c e t o x i c a n t s . The results of this study suggest that Cr is a strong c a n d i d a t e to be the r e f e r e n c e toxicant for s u r f a c e w a t e r s .

2

3. L i t e r a t u r e R e v i e w

This literature review e n c o m p a s s e s a q u a t i c e n v i r o n m e n t a l toxicities of heavy m e t a l s . The e m p h a s i s is on the e f f e c t s of environmental factors on heavy metal toxicity to a q u a t i c o r g a n i s m s . The e f f e c t s of environmental factors on heavy metal uptake are also d i s c u s s e d . An attempt w a s m a d e to cover as m u c h literature as p o s s i b l e , but u n d o u b t e d l y some reports have been missed i n a d v e r t e n t l y .

The literature review is g e n e r a l l y divided into d i s c u s s i o n s of biotic and a b i o t i c f a c t o r s ; each is then further d i v i d e d . The literature shows d i v e r g e n t r e s u l t s . N e v e r t h e l e s s , the results can be summarized into four general laws of e n v i r o n m e n t a l toxicology that relate to q u a n t i t y , o r g a n i s m , e n v i r o n m e n t a l c o n d i t i o n s , and t o l e r a n c e .

Introduction

Heavy metal toxicity to a q u a t i c o r g a n i s m s can be a f f e c t e d by m a n y e n v i r o n m e n t a l factors including a l k a l i n i t y , h a r d n e s s , p H , d i s s o l v e d o x y g e n , t e m p e r a t u r e , t u r b i d i t y , carbon d i o x i d e , m a g n e s i u m s a l t s , p h o s p h a t e s , and c h e l a t i n g a g e n t s (National R e s e a r c h C o u n c i l , 1 9 7 2 ) . The toxicity can be either p o t e n t i a t e d or a t t e n u a t e d by these f a c t o r s . An important effect of these e n v i r o n m e n t a l factors is the change of metal s p e c i a t i o n , w h i c h plays a key role in metal toxicity ( F l o r e n c e , 1 9 8 3 ; B a u d o , 1 9 8 1 ) .

There are m a n y r e v i e w a r t i c l e s d e a l i n g w i t h s p e c i f i c m e t a l s , including their a q u e o u s c h e m i s t r y , t o x i c o l o g y , and e n v i r o n m e n t a l behavior ( T a y l o r , 1 9 8 2 ; T a y l o r , 1983; Spear and P i e r c e , 1979; Taylor and D e m a y o , 1 9 8 0 ; Demayo et a l . , 1 9 8 0 ; Reeder et al., 1 9 7 9 ) . It is well r e c o g n i z e d , h o w e v e r , that a metal p o l l u t a n t rarely e x i s t s a l o n e . In the case of water c o n t a i n i n g m o r e than one t o x i c a n t , water q u a l i t y s t a n d a r d s for s p e c i f i c s u b s t a n c e s might need to be m o d i f i e d . In general the c o n c e n t r a t i o n - a d d i t i o n m e t h o d has been shown to be a p p l i c a b l e in most c i r c u m s t a n c e s . V a r i a t i o n s in uptake of s p e c i f i c s u b s t a n c e s in the p r e s e n c e of other toxicants do not appear to be related to the combined toxic potential of the m i x t u r e ( E I F A C , 1 9 8 0 ) .

Other review a r t i c l e s c o n t a i n i n g relevant information can be found in the Annual R e v i e w series of the W a t e r P o l l u t i o n Control F e d e r a t i o n . An i n t e r e s t i n g area w h i c h should receive greater a t t e n t i o n is the m i c r o b i a l system. Metal toxicity to m i c r o o r g a n i s m s can be m o d e r a t e d or e n h a n c e d by e n v i r o n m e n t a l factors including the p r e s e n c e of other c o m p e t i n g or s y n e r g i s t i c s u b s t a n c e s , formation o f c o m p l e x e s w i t h inorganic a n i o n s , p H , redox p o t e n t i a l , t e m p e r a t u r e , suspended p a r t i c u l a t e o r g a n i c m a t t e r , and d i s s o l v e d o r g a n i c matter ( S t o t z k y , 1 9 8 0 ; B a b i c h and S t o t z k y , 1985; B a b i c h et al., 1 9 8 5 ) . M i c r o o r g a n i s m s are known to

3

d e v e l o p m e c h a n i s m s for r e s i s t i n g m e t a l t o x i c i t y , i n c l u d i n g e n e r g y - d r i v e n e f f l u x p u m p s t o keep toxic m e t a l o u t , o x i d a t i o n , b i o s y n t h e s i s o f i n t r a m o l e c u l a r p o l y m e r s s e r v i n g a s m e t a l t r a p s , b i n d i n g of m e t a l ions to the cell s u r f a c e , p r e c i p i t a t i o n of m e t a l i o n s , and b i o m e t h y l a t i o n and t r a n s p o r t of m e t a l ions t h r o u g h the cell m e m b r a n e (Wood and W a n g , 1 9 8 3 ) . H i g h e r p l a n t s g e n e r a l l y k e e p toxic metal ions away from a c t i v e m e t a b o l i c s i t e s by c h e l a t i o n at the cell w a l l , and the c h e l a t i o n p r o c e s s is h i g h l y m e t a l - s p e c i f i c ( A n t o n o v i c s e t a l . , 1 9 7 1 ) .

The f a c t o r s w h i c h a f f e c t metal t o x i c i t y t o a q u a t i c o r g a n i s m s can g e n e r a l l y be d i v i d e d into two m a j o r c a t e g o r i e s : b i o t i c and a b i o t i c f a c t o r s . E a c h g r o u p can be f u r t h e r d i v i d e d into m a n y s u b c a t e g o r i e s . T h i s l i t e r a t u r e r e v i e w will b e p r e s e n t e d a c c o r d i n g l y .

B i o t i c F a c t o r s

A . T o l e r a n c e

In e n v i r o n m e n t a l t o x i c o l o g y , t o l e r a n c e , a d a p t a t i o n , and a c c l i m a t i o n are used i n t e r c h a n g e a b l y . T o l e r a n c e is an i m p o r t a n t m e c h a n i s m by w h i c h an o r g a n i s m r e a c t s to an a d v e r s e e n v i r o n m e n t . D u n c a n and K l a v e r k a m p ( 1 9 8 3 ) r e p o r t e d that w h i t e s u c k e r s ' ( C a t o s t o m u s c o m m e r s o n i ) t o l e r a n c e of and r e s i s t a n c e to Cd i n c r e a s e d as a c o n s e q u e n c e of p r e v i o u s e x p o s u r e . M e c h a n i s m s w h i c h m i g h t h a v e b e e n r e s p o n s i b l e for the d e c r e a s e d Cd t o x i c i t y included d e c r e a s e d u p t a k e , i n c r e a s e d e x c r e t i o n , r e d i s t r i b u t i o n o f m e t a l s t o less s e n s i t i v e target s i t e s , a n d / o r induced s y n t h e s i s of m e t a l l o t h i o n e i n for p r o t e i n a c e o u s m e t a l c h e l a t i o n . B e n s o n and B i r g e ( 1 9 8 5 ) s h o w e d that m e t a l - c o n t a m i n a t e d fly ash pond m i n n o w s w e r e s i g n i f i c a n t l y m o r e t o l e r a n t of Cd and Cu than w e r e h a t c h e r y m i n n o w s . At the e x p o s u r e c o n c e n t r a t i o n of 6 m g / L Cd in m o d e r a t e l y hard w a t e r , the m e d i a n p e r i o d of survival ( L T 5 0 ) for fly ash pond m i n n o w s w a s 5 0 . 5 h, c o m p a r e d w i t h 6.8 h for h a t c h e r y m i n n o w s . The L T 5 0 v a l u e s for ash pond and h a t c h e r y fish e x p o s e d to 0.5 m g / L Cu in m o d e r a t e l y hard w a t e r w e r e 17.0 and 4.5 h, r e s p e c t i v e l y . A f t e r b e i n g t r a n s f e r r e d to r e c o n s t i t u t e d w a t e r in the l a b o r a t o r y for 7 d, ash pond m i n n o w s s i g n i f i c a n t l y d e c r e a s e d their t o l e r a n c e to Cd and C u . C o n v e r s e l y , t o l e r a n c e w a s i n c r e a s e d in h a t c h e r y m i n n o w s f o l l o w i n g a c c l i m a t i o n to the s u b l e t h a l Cd c o n c e n t r a t i o n . A n o t h e r study showed that f a t h e a d m i n n o w s e x h i b i t e d s i m i l a r induced t o l e r a n c e to the Ag ion. The i n c r e a s e d t o l e r a n c e and r e s i s t a n c e to Ag by a c c l i m a t e d o r g a n i s m s w e r e lost if m i n n o w s w e r e p l a c e d in a control w a t e r , i n d i c a t i n g that the induced t o l e r a n c e w a s not a s u s t a i n e d r e s p o n s e ( B i r g e e t a l . , 1 9 8 4 ) .

H a r r i s o n ( 1 9 8 3 ) e x a m i n e d two m e c h a n i s m s o f m e t a l d e t o x i f i c a t i o n in the c o m m o n m u s s e l : the b i n d i n g of Cu to m e t a l l o t h i o n e i n - l i k e p r o t e i n s and the i n c o r p o r a t i o n of Cu into l y s o s o m e - I i k e v e s i c l e s . C h a n g e s w e r e n o t e d in the k i n d s and q u a n t i t i e s of C u - b i n d i n g p r o t e i n s and in the l a t e n c y of lysosomal e n z y m e s o c c u r r i n g in the d i g e s t i v e g l a n d c e l l s . C h r o n i c e x p o s u r e to Cu r e s u l t e d in Cu c o n c e n t r a t i o n in low m o l e c u l a r w e i g h t m e t a l l o t h i o n e i n - l i k e p r o t e i n s , w h i l e C u c o n c e n t r a t i o n i n h i g h

4

m o l e c u l a r w e i g h t p r o t e i n s w a s d i r e c t l y related to the mussel m o r t a l i t y .

The induced tolerance of fish to a toxic metal m a y also result from a factor other than the m e t a l . Calamari et al. ( 1 9 8 0 ) reported that fish a c c l i m a t e d in 320 m g / L C a C O 3 h a r d n e s s but tested at 20 mg/L h a r d n e s s reacted to m e t a l s in a d i f f e r e n t way from those m a i n t a i n e d and tested at 20 m g / L . In this test p r o c e d u r e , chemical s p e c i a t i o n of Cd was i d e n t i c a l , yet the 48-h LC50 for the h a r d n e s s - a c c l i m a t e d fish w a s a p p r o x i m a t e l y 7 times that for the control fish (0.677 and 0.091 m g / L Cd, r e s p e c t i v e l y ) .

Plant species can a l s o tolerate and adapt to e n v i r o n m e n t a l s t r e s s . Higher plants d e v e l o p m e c h a n i s m s for metal d e t o x i f i c a t i o n including e x c l u s i o n , cell wall b i n d i n g , o r g a n i c acid c o m p l e x a t i o n , e n z y m i c a d a p t a t i o n s , and p e r m e a b i l i t y regulation ( T h u r m a n , 1 9 8 1 ) . Stockner and A n t i a ( 1 9 7 6 ) cited literature e x a m p l e s for algal a d a p t i o n and subsequent growth after e x p o s u r e to p o l l u t a n t s . E x p e r i m e n t a l results showed that it m i g h t take as long as 20 to 40 d of e x p o s u r e to p o l l u t a n t s for algae to a c q u i r e successful a d a p t a t i o n s . P h y t o p l a n k t o n that initially tolerated only a low level of pollutant c o n c e n t r a t i o n could be trained to accept levels s e v e r a l f o l d higher by repeated e x p o s u r e . W a n g ( 1 9 8 6 A ) used algal c o m m u n i t i e s from Peoria and F a r m i n g t o n s o u r c e s (both in I l l i n o i s ) to illustrate their a c c l i m a t i o n to Zn t o x i c i t y . P e o r i a and F a r m i n g t o n algal c o m m u n i t i e s e x h i b i t e d d i f f e r e n t types of r e s p o n s e s to Z n . In the d o s e - r e s p o n s e r e l a t i o n s h i p , one e x h i b i t e d a c o n c a v e - u p response and the o t h e r , c o n c a v e - d o w n . W h e n P e o r i a algae w e r e a c c l i m a t e d to the e n v i r o n m e n t of the F a r m i n g t o n s a m p l e , the response to Zn t r a n s f o r m e d into the same type as for the F a r m i n g t o n a l g a e , and v i c e v e r s a . After a d d i t i o n a l e x p e r i m e n t s , it w a s concluded that the P e o r i a algal c o m m u n i t y w a s a c e l i m a t e d to an e l e v a t e d Zn c o n c e n t r a t i o n , w h i l e the F a r m i n g t o n c o m m u n i t y w a s used to a low Zn e n v i r o n m e n t .

The tolerance to m e t a l toxicity has also been studied in s i t u . Jones and M c L e a n ( 1 9 7 5 ) examined the a q u a t i c flora of the rivers Y s t w y t h and C l a r a c h , W a l e s , both of w h i c h are p o l l u t e d by old lead m i n e s . The r e s u l t s showed that H a r m i d i u m spp. w e r e the most tolerant f i l a m e n t o u s green algae and that the b r y o p h y t e S c a p a n i a u n d u l a t e w a s tolerant of p o l l u t i o n . W h e n S c a p a n i a w a s t r a n s p l a n t e d from its natural polluted site to a less p o l l u t e d area there was no m a r k e d c h a n g e in its metal c o n t e n t , but w h e n the less tolerant F o n t i n a l i s s q u a m o s a w a s t r a n s p l a n t e d to p o l l u t e d s i t e s , its contents of P b , C u , Z n , and Mn increased w i t h i n 6 w e e k s , and p l a n t s s t a r t e d to die and decay after 18 w e e k s . M c N a u g h t o n et al. ( 1 9 7 4 ) s i m i l a r l y studied c l o n e s of the b r o a d - l e a v e d cattail and soil samples from a r e a s near a Zn smelter and a control location. They reported that the s p e c i e s w a s able to c o l o n i z e in soils c o n t a i n i n g heavy m e t a l s , yet there w a s no e v i d e n c e to indicate the e v o l u t i o n of s p e c i e s tolerant to heavy m e t a l s . Ernst ( 1 9 7 5 ) indicated that plant s p e c i e s d e v e l o p e d a metal tolerance s p e c i f i c only to those m e t a l s a b u n d a n t in their h a b i t a t . The m e c h a n i s m of r e s i s t a n c e w a s p r o b a b l y not by the e x c l u s i o n p r o c e s s b e c a u s e of the high metal content in resistant p l a n t s .

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S t o k e s et al. ( 1 9 7 3 ) r e p o r t e d that the lake w a t e r s in the S u d b u r y s m e l t i n g a r e a , O n t a r i o , c o n t a i n e d a b n o r m a l l y high l e v e l s o f m e t a l s , e s p e c i a l l y C u and N i . Two algal s p e c i e s w e r e i s o l a t e d , S c e n e d e s m u s and C h l o r e l l a . In c o m p a r i s o n w i t h l a b o r a t o r y s t r a i n s , the isolated s p e c i e s w e r e found to be t o l e r a n t of the heavy m e t a l s Ni ( S c e n e d e s m u s ) and Cu ( S c e n e d e s m u s and C h l o r e l l a ) . S t o k e s ( 1 9 7 5 A ) further r e p o r t e d that Cu and Ni t o l e r a n c e in the same o r g a n i s m s could be s e p a r a t e d and that the e x p r e s s i o n of e a c h w a s i n d e p e n d e n t . G r e g o r y and B r a d s h a w ( 1 9 6 5 ) c o n c l u d e d that the t o l e r a n c e m e c h a n i s m o f A g r o s t i s t e n u i s w a s a l m o s t a l w a y s metal - s p e c i f i c ; i o i e r a n c e to one m e t a l did not a u t o m a t i c a l l y confer t o l e r a n c e to a n o t h e r .

The t o l e r a n c e of C h i r o n o m u a t e n t a n s larvae to v a r y i n g levels o f heavy m e t a l s (Cd, C r , and Z n ) w a s s t u d i e d ( L i n d e s t r o m , 1 9 8 0 ) . L a r v a e f r o m s e d i m e n t low in heavy m e t a l s s h o w e d a lower rate of s u r v i v a l w h e n e x p o s e d to a s e d i m e n t high in heavy m e t a l s than larvae c o l l e c t e d from s e d i m e n t s high in heavy m e t a l s e x p o s e d to s e d i m e n t low in h e a v y m e t a l s .

In s u m m a r y , an o r g a n i s m s u b j e c t e d to e n v i r o n m e n t a l metal t o x i c i t y will e i t h e r s u r v i v e or d i e . The s u r v i v i n g o r g a n i s m is the one w h i c h p o s s e s s e s m e c h a n i s m s to t o l e r a t e the m e t a l t o x i c i t y . The a c q u i r e d t o l e r a n c e is likely t e m p o r a r y and m e t a l - s p e c i f i c .

B. Size and life s t a g e s

The size and m o r e i m p o r t a n t l y the life stage of an o r g a n i s m have an i m p o r t a n t e f f e c t on its s e n s i t i v i t y to metal t o x i c i t y . It is known that the early life s t a g e of an o r g a n i s m is m o r e s e n s i t i v e to t o x i c i t y than the j u v e n i l e and the a d u l t . N e b e k e r et a l . ( 1 9 8 5 ) d e t e r m i n e d the n o - o b s e r v e d - e f f e c t c o n c e n t r a t i o n of Ni to r a i n b o w trout and r e p o r t e d that the e a r l y life s t a g e s w e r e m o s t s e n s i t i v e w h e n n e w l y f e r t i l i z e d e g g s w e r e i n i t i a l l y e x p o s e d , f o l l o w e d in s e n s i t i v i t y by eyed e g g s , larval f i s h , and j u v e n i l e f i s h . R e i s h et al. ( 1 9 7 6 ) tested two p o l y c h a e t e s and a r r i v e d at the same c o n c l u s i o n . E a t o n ( 1 9 7 4 ) o b s e r v e d that a d u l t b l u e g i l l s p a w n e d at 2 3 9 ug/L Cd s o l u t i o n , but that m o s t larvae w e r e s e v e r e l y c r i p p l e d 6 d after h a t c h i n g at that c o n c e n t r a t i o n .

U s i n g a q u a t i c insects a s test o r g a n i s m s . Smock ( 1 9 8 3 B ) found that the m e t a l c o n c e n t r a t i o n s of C o , C r , F e , S b , and Sc in o r g a n i s m s d e c r e a s e d w i t h i n c r e a s i n g b o d y s i z e , i n d i c a t i n g that s u r f a c e a d s o r p t i o n w a s an i m p o r t a n t m e t h o d of a c c u m u l a t i o n for these m e t a l s . W h i l e little o r n o c o r r e l a t i o n b e t w e e n c o n c e n t r a t i o n and body size w a s o b s e r v e d for K , M n , and N a , S y k o r a et al. ( 1 9 7 2 ) t e s t e d the effect of l i m e - n e u t r a l i z e d f e r r i c h y d r o x i d e on y o u n g b r o o k t r o u t . The r e s u l t s s h o w e d a trend t o w a r d s s m a l l e r fish and lower w e i g h t w i t h i n c r e a s i n g c o n c e n t r a t i o n of s u s p e n d e d ferric h y d r o x i d e . R a i n b o w trout of 10-g body w e i g h t w e r e 2.5 times m o r e r e s i s t a n t to Cu l e t h a l i t y than the 0.7-g size ( H o w a r t h and S p r a g u e , 1 9 7 8 ) .

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In microbial toxicity t e s t s , the size of the inoculum can also influence the toxicity results for heavy m e t a l s such as Cd (Truhaut e t a l . 1 9 8 0 ) .

C. Species

The toxicity of heavy m e t a l s is greatly influenced by the species of test o r g a n i s m s . Thorp and Lake (1974) determined Cd toxicity in soft water to selected freshwater i n v e r t e b r a t e s . LC50 values varied from 0.04 m g / L for a m p h i p o d s , 0.06 m g / L for s h r i m p , and 0.84 m g / L for nymphs to well over 2000 mg/L for T r i c h o t e r a n larvae. Studying a wide range of aquatic insects. Smock (1983A) reported that metal c o n c e n t r a t i o n s were highest in b u r r o w i n g m a y f l i e s and some C h i r o n o m i d a e that ingest sediments along with d e t r i t u s . F i l t e r - f e e d e r s had the next highest c o n c e n t r a t i o n s , while carnivores and s u r f a c e - f e e d e r s had the lowest. R o d g e r s et al. (1978) observed that duckweed was the only abundantly occurring m a c r o p h y t e inhabiting a stream-swamp d r a i n a g e system that received high q u a n t i t i e s of coal ash and thermal d i s c h a r g e s from a fossil fuel power p l a n t , indicating that duckweed w a s hardy and/or adaptive in c o m p a r i s o n with other s p e c i e s . Duckweed from unpolluted a r e a s , h o w e v e r , was found to be sensitive to heavy metal toxicity (Wang, 1 9 8 6 B ) .

Other plant species also exhibit widely different responses to toxicity. For e x a m p l e , Ditvlum underwent osmotic d i s t u r b a n c e s in 10 and 10 mol/L C u , with swelling of cell c o n t e n t s , while P h a e o d a c t v l u m grew in c o n t i n u o u s culture., surviving single doses of up to 10 mol/L Cu without diminution in growth with c o n s i d e r a b l e uptake of Cu (Bentley-Mowat and R e i d , 1 9 7 7 ) . The uptake of the heavy m e t a l s Z n , C u , P b , Cd, M n , and Fe by aquatic plants clearly was species d e p e n d e n t . In g e n e r a l , submerged taxa had greater metal content than floating ones (Aulio and Salin, 1982; Van der Werff and P r u y t , 1 9 8 2 ) .

W o n g and Beaver ( 1 9 8 0 ) proposed an interesting two-species m e t h o d for toxicity t e s t s . One s p e c i e s , C h l o r e l l a fusca. was commonly found in lakes with high metal c o n c e n t r a t i o n s , while Ank i s t rodesmus was very sensitive to m e t a l s . By d e t e r m i n i n g the m a x i m u m yield ratio between A n k i s t r o d e s m u s and C h l o r e l l a . it was possible to compare toxic strength of harmful m e t a l s .

Using a m u l t i - s p e c i e s community a p p r o a c h , Ruthven and Cairns (1973) found protozoa to be more resistant than Daphnia to p h e n o l , K C r O . , and C u . Some protozoan s p e c i e s , h o w e v e r , w e r e more sensitive than Daohnia to Z n , nitric acid, and H C l . The results suggested that there w a s no uniformity of toxic response to various toxic s u b s t a n c e s by different o r g a n i s m s . The m u l t i - s p e c i e s approach has been advocated and discussed ( C a i r n s , 1 9 8 5 ) . M u l t i - s p e c i e s toxicity tests o b v i o u s l y offer m o r e than single species toxicity tests can. Following are three e x a m p l e s .

F i r s t , Hutchinson and Czyrska (1975) reported that when both Lemna valdiviana and S a l v i n i a natans were p r e s e n t , the addition of 0.01-0.05 mg/L Cd resulted in greater toxicity to Lemna than when the same amount of Cd was added to Lemna a l o n e . In c o n t r a s t ,

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S a l v i n i a g r e w a good deal b e t t e r w i t h Lemna in the p r e s e n c e of 0 . 0 1 - 0 . 0 5 m g / L Cd than it did w h e n it g r e w by i t s e l f . The p o s s i b l e reason for this d i s c r e p a n c y is the s p e c i e s c o m p e t i t i o n . H u t c h i n s o n and C z y r s k a o b s e r v e d that "under the s t r e s s of c o m p e t i t i o n L e m n a grows less well and c a d m i u m levels are m a r k e d l y increased in the t i s s u e s , w h i l e S a l v i n i a g r o w s better and the c a d m i u m c o n c e n t r a t i o n s are c o r r e s p o n d i n g l y r e d u c e d . " The i n t e r - s p e c i e s i n t e r a c t i o n c l e a r l y cannot be o b t a i n e d by using a single s p e c i e s t e s t . A n o t h e r e x a m p l e is food c h a i n s . V i n o t and Larpent ( 1 9 8 4 ) studied water p o l l u t i o n caused by u r a n i u m ore treatment w o r k s . They found that in c e r t a i n s t r e a m s , the fauna and flora d i s a p p e a r e d c o m p l e t e l y . The study showed that ore tailings did not d i r e c t l y affect fish or c r u s t a c e a n s but only a l g a e , the g r o w t h of w h i c h w a s either p a r t l y or c o m p l e t e l y i n h i b i t e d , d e p e n d i n g on the d i l u t i o n p r o v i d e d by the w a t e r c o u r s e . The toxic s u b s t a n c e s w e r e found to be Cu and Z n , w h i c h c a u s e d the d i s a p p e a r a n c e of the m i c r o a l g a e and w h i c h in t u r n , through the food c h a i n s , r e s u l t e d in the d i s a p p e a r a n c e of the higher t r o p h i c s p e c i e s leading to f i s h . The third e x a m p l e involves two freshwater o l i g o c h a e t e s p . ( C h a p m a n e t a l . , 1 9 8 2 A ) . C o m p a r i s o n o f data b e t w e e n m i x e d s p e c i e s tests and individual s p e c i e s tests indicated that o r g a n i s m s in the m i x e d s p e c i e s tests w e r e s i g n i f i c a n t l y m o r e tolerant of the t o x i c a n t s C d , H g , and s o d i u m p e n t a c h l o r o p h e n o l .

S u g i u r a e t a l . ( 1 9 8 2 ) used a q u a t i c m i c r o c o s m s c o n t a i n i n g a l g a e , p r o t o z o a , r o t i f e r s , o l i g o c h a e t e s , and b a c t e r i a . The effect of Cu s t r e s s w a s found to . be g r e a t e r in the early s t a g e of h e t e r o t r o p h i c s u c c e s s i o n than at later s t a g e s . At the later s t a g e , it w a s a s s u m e d that the m i c r o c o s m w a s m o r e e s t a b l i s h e d and thus m o r e r e s i l i e n t to the s t r e s s .

L e B l a n c ( 1 9 8 4 ) d i v i d e d toxic s u b s t a n c e s into n o n p e s t i c i d e o r g a n i c s , p e s t i c i d e s , and m e t a l s and c o m p a r e d their a c u t e t o x i c i t i e s to a q u a t i c o r g a n i s m s . He found that all s p e c i e s a n a l y z e d - fresh and s a l t w a t e r f i s h , i n v e r t e b r a t e s , and a l g a e r e s p o n d e d s i m i l a r l y to the toxicity of n o n p e s t i c i d e o r g a n i c s , w h i l e d i f f e r e n t g r o u p s of o r g a n i s m s r e s p o n d e d d i f f e r e n t l y to the p e s t i c i d e s . D i f f e r e n t s p e c i e s of fish of the same family r e s p o n d e d almost i d e n t i c a l l y to the toxicity of p e s t i c i d e s , w h e r e a s fishes of d i f f e r e n t f a m i l i e s reacted s i m i l a r l y , but to a lesser d e g r e e . No relation e x i s t e d b e t w e e n the a c u t e s e n s i t i v i t i e s of fish and i n v e r t e b r a t e s . A s i g n i f i c a n t c o r r e l a t i o n w a s d e t e r m i n e d in a c u t e s e n s i t i v i t i e s to m e t a l s b e t w e e n bluegill and fathead m i n n o w , and b e t w e e n b l u e g i l l and D a o h n i a m a g n a . T h e m o d e of t o x i c i t y of m e t a l s s e e m s to be the same among s p e c i e s , a l t h o u g h the d e g r e e of toxicity may v a r y .

It can be c o n c l u d e d that the test s p e c i e s is an important p a r a m e t e r for c o n d u c t i n g t o x i c i t y t e s t s . In e n v i r o n m e n t a l t o x i c o l o g y , it is a d v i s a b l e to c o n s i d e r a b a t t e r y of o r g a n i s m s of d i f f e r e n t trophic l e v e l s , including a l g a e , higher p l a n t s , i n v e r t e b r a t e s , and fish to e n c o m p a s s the e n t i r e food c h a i n .

8

D. N u t r i t i o n N u t r i t i o n of a test o r g a n i s m has a s t r o n g i n f l u e n c e on its

state of health a n d , c o n s e q u e n t l y , a d i r e c t impact on its s e n s i t i v i t y to e n v i r o n m e n t a l t o x i c i t y . S e t o et al. ( 1 9 7 9 ) e x a m i n e d the e f f e c t s of Cd on the d u c k w e e d L e m n a gibba _ They found that the r a t i o of c h l o r o t i c f r o n d s to total f r o n d s w a s g r e a t l y i n f l u e n c e d by the d i f f e r e n c e in c o n c e n t r a t i o n of m i n e r a l n u t r i e n t s . W h e n c u l t u r e d in a h i g h - n u t r i e n t c o n c e n t r a t i o n , the c h l o r o t i c fronds did not appear in spite of high Cd c o n c e n t r a t i o n in the frond. On the other h a n d , w h e n c u l t u r e d in s o l u t i o n s w i t h low c o n c e n t r a t i o n s of n u t r i e n t s , the c h l o r o t i c f r o n d s a p p e a r e d in spite of low Cd c o n t e n t in the frond.

J o h n e l s et al. ( 1 9 6 7 ) studied Hg a c c u m u l a t i o n by pike CEsoc l u c i u s ) and other f i s h e s from S w e d i s h l a k e s . They found higher a c c u m u l a t i o n rates in o l i g o t r o p h i c lakes than in m o r e e u t r o p h i c ones w i t h the same d e g r e e of Hg p o l l u t i o n . The Hg a c c u m u l a t i o n w a s a p p a r e n t l y a f u n c t i o n of m a n y e n v i r o n m e n t a l f a c t o r s i n c l u d i n g the role of o r g a n i c m a t t e r in s o r p t i o n and/or c h e l a t i o n , and a c t i v i t i e s of m i c r o o r g a n i s m s r e s p o n s i b l e for Hg m e t h y l a t i o n .

Hart and C a i r n s ( 1 9 8 4 ) d e f i n e d a q u a t i c e c o s y s t e m a s s i m i l a t i o n c a p a c i t y as the a b i l i t y of an e c o s y s t e m to a s s i m i l a t e a s u b s t a n c e or stress w i t h o u t d e g r a d i n g or d a m a g i n g its e c o l o g i c a l i n t e g r i t y , n a m e l y the m a i n t e n a n c e of s t r u c t u r a l and f u n c t i o n a l c h a r a c t e r i s t i c s . T h e y c o m p a r e d two types of l a k e s , one at the U n i v e r s i t y of M i c h i g a n ( e u t r o p h i c ) and the other at Smith M o u n t a i n , V i r g i n i a ( o l i g o t r o p h i c ) . It w a s found that e u t r o p h i c c o m m u n i t i e s had a g r e a t e r structural a s s i m i l a t i v e c a p a c i t y than did o l i g o t r o p h i c c o m m u n i t i e s . The e u t r o p h i c c o m m u n i t i e s had a g r e a t e r p r o p o r t i o n of s p e c i e s that w e r e tolerant of low c o n c e n t r a t i o n s of C u . T h i s is i n t e r e s t i n g in light of the fact that e u t r o p h i c a t i o n d e c r e a s e s s p e c i e s d i v e r s i t y ( H o o p e r , 1 9 6 9 ) .

The rates of metal u p t a k e by o r g a n i s m s m a y vary among m e t a l s . Rudd and Turner ( 1 9 8 3 A ) found that s t i m u l a t i o n of p r i m a r y p r o d u c t i o n by a d d i t i o n of n u t r i e n t s tended to i n c r e a s e Hg u p t a k e and reduce the c o n c e n t r a t i o n of Se in the food c h a i n , p r o b a b l y as a result of d i l u t i o n of Se by g r e a t e r g r o w t h of v a r i o u s a q u a t i c o r g a n i s m s .

In s u m m a r y , it can be c o n c l u d e d that the state of test o r g a n i s m s has an important effect on their s u s c e p t i b i l i t y to metal t o x i c i t y . G e n e r a l l y , o r g a n i s m s w h i c h are m o r e m a t u r e , h e a l t h i e r , and p r e - c o n d i t i o n e d to the s t r e s s can be e x p e c t e d to fare better than the y o u n g , less n o u r i s h e d , and u n - c o n d i t i o n e d .

A b i o t i c F a c t o r s

A . O r o a n i c a i. S y n t h e t i c o r g a n i c s . Many s t u d i e s have tested the e f f e c t s

of o r g a n i c c o m p o u n d s on metal toxicity and u p t a k e . The m a j o r i t y of these test c o m p o u n d s are c h e l a t i n g s u b s t a n c e s . M u r a m o t o ( 1 9 8 0 ) s t u d i e d the e f f e c t s of c h e l a t i n g a g e n t s EDTA ( e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d ) , NTA ( n i t r i l o t r i a c e t a t e ) , and

9

other agents on the toxicity of Cd, C u , Z n , and Pb to c a r p . He reported that fish exposed to m e t a l s plus chelating agents (1:3 molar ratio) contained lower metal c o n c e n t r a t i o n s in gills and other parts than did fish exposed to m e t a l s a l o n e . The c h e l a t o r s apparently inhibited metal a c c u m u l a t i o n . Nasu et al. ( 1 9 8 3 ) indicated that only 30 uM EDTA was s u f f i c i e n t to prevent the absorption of Cu by Lemna p a u c i c o s t a t a at a c o n c e n t r a t i o n of 5-10 uM, w h i l e 400 uM of EDTA w a s required to prevent the a b s o r p t i o n of Cd at 5-10 uM. The growth of Lemna was inhibited in p r o p o r t i o n to the amount of Cu and Cd a b s o r b e d .

M o r r i s and Russel ( 1 9 7 3 ) studied Cu toxicity to the brown alga Ectocarpus and found that the plant growth rate dropped rapidly with increasing Cu c o n c e n t r a t i o n , and stopped at a c o n c e n t r a t i o n of 0.45 m g / L . The presence of EDTA increased this critical c o n c e n t r a t i o n and the alga continued to grow up to a level of over 0.85 m g / L . Stokes and H u t c h i n s o n (1976) reported that Cu bound with EDTA w a s not toxic and that the strong chelator prevented Cu uptake by algal c e l l s , w h i l e acetate had little effect on Cu toxicity. N i s h i k a w a and T a b a t a (1969) used EDTA to e l i m i n a t e the toxicity of wastewater from Cu m i n e s , using Daohnia and dace as test o r g a n i s m s .

The infaunal amphipod crustaceans R h e p o x v n i u s a b r o n i u s and E o h a u s t e r i u s s e n c i l l u s are c h a r a c t e r i s t i c of n e a r s h o r e sandy bottoms on the C a l i f o r n i a coast. They are s e n s i t i v e to m o d e r a t e levels of heavy m e t a l s . In laboratory e x p e r i m e n t s , EDTA increased survival of the a m p h i p o d s in sediments c o n t a i n i n g lethal levels of Cd (8.5 u g / g ) (Oakden et al., 1 9 8 4 ) .

A n d r e w (1976) c o n c l u d e d that acute Cu toxicity to fathead m i n n o w s (Pimephales p r o m e l a s ) and Daphnia m a g n a was much less when NTA was added. S i m i l a r l y , Cu p r e c i p i t a t e s w e r e not b i o l o g i c a l l y active or toxic. S p r a g u e (1968) found that NTA selectively chelated Cu f i r s t , then Zn a f t e r w a r d s , and proposed using NTA as an agent to detoxify heavy m e t a l s . T e s t i n g with D a p h n i a m a g n a . Biesinger et al. (1974) reported that Cu and Zn toxicities were c o n s i d e r a b l y less when NTA was added. H o n g v e et al. ( 1 9 8 0 ) found that NTA had a superior d e t o x i f i c a t i o n capacity for C u , Cd, Z n , and Pb because of a strong m e t a l - N T A complex w h i c h did not affect the p h o t o s y n t h e t i c rate of natural p h y t o p l a n k t o n . Hg-NTA c o m p l e x , on the c o n t r a r y , was m o r e toxic than the ionic form of H g . In a metal m i x t u r e of C u - C d - Z n - P b - H g , they observed that NTA d r a m a t i c a l l y enhanced the toxicity of the m i x t u r e to algal g r o w t h , possibly due to a s y n e r g i s t i c e f f e c t . Sunda et al. ( 1 9 7 8 ) found that after 96 h e x p o s u r e to a given c o n c e n t r a t i o n of C d , grass shrimp m o r t a l i t y d e c r e a s e d with increasing c o n c e n t r a t i o n s of NTA and increasing s a l i n i t y . They a t t r i b u t e d the p r o t e c t i v e effect of high salinity and NTA to the complexation of Cd. C h y n o w e t h et al. (1976) tested v a r i e t i e s of organic c o m p o u n d s , g l y c i n e , c y s t e i n e , EDTA, N T A , citric acid, a l b u m i n , humic a c i d , and secondary sewage e f f l u e n t . The results indicated that unbound Cu was more toxic to guppies than bound Cu.

The effects of other synthetic c o m p o u n d s on metal toxicity have also been studied. Calamari and M a r c h e t t i (1973) reported

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that the toxicity of Cu and Hg and a n i o n i c s u r f a c t a n t s to r a i n b o w trout was "more than a d d i t i v e , " w h i l e for the m i x t u r e of n o n - i o n i c detergent and m e t a l , the toxic effect w a s "less than a d d i t i v e . " M u r a m o t o and Oki (1984) investigated the effect of a surfactant SDS (sodium dodecyl s u l f a t e ) on Cd and Ni uptake by water h y a c i n t h s . E x p o s u r e to Cd and Ni alone caused a decrease of b i o m a s s . S i m u l t a n e o u s a d m i n i s t r a t i o n of SOS did not alter this p a t t e r n . The addition of SOS decreased Cd uptake by p l a n t s , w h i t e it did not s i g n i f i c a n t l y affect Ni u p t a k e . B o r g m a n n and C h a r l t o n (1984) examined the toxicity of Cu to D a p h n i a m a g n a in an artificial m e d i u m , m e d i u m plus a l g a e , and water samples from H a m i l t o n H a r b o r and Lake O n t a r i o . Cu toxicity was greatest in the inorganic m e d i u m and lowest in the inorganic m e d i u m plus a l g a e . After Tris a d d i t i o n , h o w e v e r , Cu toxicity w a s greatest in lake water and lowest in inorganic m e d i u m . The uptake of both Cu and Pb by the a l g a , N o s t o c . decreased when c i t r a t e w a s added (Schecher and D r i s c o l l , 1 9 8 5 ) . C u - a m i n o acid c o m p l e x e s w e r e found to be less toxic than free Cu ions (Borgmann and R a l p h , 1 9 8 3 ) .

The o r g a n i c c o m p o u n d s , h o w e v e r , do not always reduce metal uptake and s u b s e q u e n t l y toxicity to a q u a t i c o r g a n i s m s . Piccardi and Clauser ( 1 9 8 3 ) reported that iris p o s s e s s e d a high a f f i n i t y for a b s o r p t i o n of heavy m e t a l s . Tests w i t h Cu solutions indicated that a high amount of absorbed metal ( 8 2 ± 8 % ) was in the r o o t s . High c o n c e n t r a t i o n s of s u r f a c t a n t s (both a n i o n i c and n o n i o n i c ) had little effect on metal u p t a k e . Triton X-100 did not affect the specific growth rate of C h l o r e l l a c u l t u r e s w h e n it w a s added to a culture m e d i u m (Wong, 1 9 8 5 ) . The c o m p o u n d , h o w e v e r , was found to be capable of e n h a n c i n g Cd t o x i c i t y , p a r t i c u l a r l y at the low c o n c e n t r a t i o n range. For C u , the compound enhanced the metal toxicity only when Cu reached a high c o n c e n t r a t i o n . C a n t e r f o r d and C a n t e r f o r d (1980) investigated the e f f e c t s of chelation on the toxicity of C u , Z n , C d , P b , H g , A g , and Ti to a m a r i n e d i a t o m , Ditylum. and indicated that the p r e s e n c e of EDTA in the c u l t u r e m e d i u m did not lower the toxicity of all heavy m e t a l s . A m o n g these m e t a l s , the free metal c o n c e n t r a t i o n s of H g , A g , and Ti w e r e independent of EDTA c o n c e n t r a t i o n , s u g g e s t i n g little effect of EDTA on their toxicity. A h s a n u l l a h and F l o r e n c e (1984) tested o r g a n o c o p p e r toxicity to the adult m a r i n e a m p h i p o d , A l l o r c h e s t e s . They reported that water s o l u b l e ligands N T A , 8 - h y d r o x y q u i n a I i n e - 5 - s u I f o n i c a c i d , and tannic acid a m e l i o r a t e d Cu toxicity by d e c r e a s i n g the c o n c e n t r a t i o n of the free Cu ion, w h i l e lipid s o l u b l e ligands such as oxins and p o t a s s i u m e t h y l x a n t h o g e n a t e increased Cu t o x i c i t y . p r e s u m a b l y as a result of increased p e r m e a b i l i t y through the cell m e m b r a n e .

Some interesting results were reported by Nasu and K u g i m o t o (1981) showing that the reproduction rate of the duckweed Lemna p a u c i c o s t a t a increased two- to threefold if 1 percent sucrose w a s added to the growth m e d i u m . The toxic effect of Cd and Cu on the r e p r o d u c t i o n rate w a s more n o t i c e a b l e w h e n sucrose was a d d e d , w h i l e that of Cr (VI) was not affected by the sucrose a d d i t i o n .

F e r r i c iron is generally not c o n s i d e r e d highly toxic. Its effect on algal a c t i v i t y , i n t e r e s t i n g l y , could be expressed on an equi-molar basis with EDTA (Wang, 1 9 8 3 ) . G e o r g e and Coombs ( 1 9 7 7 )

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found that Fe ( I I I ) c o m p l e x e s of c i t r a t e , E D T A , and 1 , 1 0 - p h e n a n t h r o l i n e i n c r e a s e d the rate of Fe u p t a k e , w h e r e a s f e r r i c h r o m e b and Fe ( I I I ) c o m p l e x e s of a c e t o - and b e n z o - h y d r o x a m i c a c i d s c a u s e d a d e c r e a s e .

ii. H u m i c s u b s t a n c e s . N a t u r a l w a t e r c o n t a i n s v a r i o u s a m o u n t s o f d i s s o l v e d and s u s p e n d e d o r g a n i c m a t t e r . B r o w n e t a l . ( 1 9 7 4 ) found that Cu t o x i c i t y to r a i n b o w trout w a s q u a n t i t a t i v e l y r e d u c e d w h e n h u m i c s u b s t a n c e s and s u s p e n d e d o r g a n i c m a t t e r w e r e p r e s e n t .

Z i t k o et al. ( 1 9 7 3 ) used i n c i p i e n t lethal levels ( I L L , a c o n c e n t r a t i o n level b e y o n d w h i c h 50 p e r c e n t of the p o p u l a t i o n c a n n o t s u r v i v e i n d e f i n i t e l y ) to e x p r e s s the e f f e c t of h u m i c s u b s t a n c e s on Cu t o x i c i t y to j u v e n i l e A t l a n t i c s a l m o n . The e f f e c t w a s very p r o n o u n c e d . The ILL'S of C u , Cu + 5 m g / L h u m i c a c i d , and Cu + 10 m g / L h u m i c acid w e r e 2 5 , 9 0 , and 165 u g / L , r e s p e c t i v e l y . In the case of f u l v i c a c i d , the ILL's w e r e 110 and 2 4 0 ug/L w i t h the t r e a t m e n t of 5 and 10 m g / L f u l v i c a c i d , r e s p e c t i v e l y . The r e s u l t s of other s t u d i e s a l s o i n d i c a t e d that Cu t o x i c i t y w a s r e d u c e d b y natural o r g a n i c s u b s t a n c e s ( B r o w n e t a l . 1 9 7 4 ; S u n d a and G u i l l a r d , 1 9 7 6 ; S u n d a and L e w i s , 1 9 7 8 ) .

The e f f e c t of h u m i c s u b s t a n c e s on m e t a l t o x i c i t y , h o w e v e r , is not a l w a y s n e g a t i v e . W i n n e r ( 1 9 8 4 ) r e p o r t e d that the a d d i t i o n of h u m i c acid to test w a t e r d e c r e a s e d the a c u t e and c h r o n i c t o x i c i t y of C u , but i n c r e a s e d the a c u t e and c h r o n i c t o x i c i t y of C d , w h i l e h u m i c acid had no e f f e c t on b i o a c c u m u l a t i o n of e i t h e r m e t a l . L a e g r e i d et al. ( 1 9 8 3 ) a l s o o b s e r v e d that Cd in s o m e lake w a t e r w a s m o r e toxic to S e l e n a s t r u m c a p r i c o r n u t u m than that in a g r o w t h m e d i u m . They s u g g e s t e d the p o s s i b i l i t y o f d i s s o l v e d o r g a n i c - i n d u c e d t o x i c i t y e n h a n c e m e n t .

The c o m p l e x i n g c a p a c i t y of a w a t e r s a m p l e can be m e a s u r e d by s e l e c t i v e ion e l e c t r o d e s . B u c k l e y ( 1 9 8 3 ) r e p o r t e d that the Cu c o m p l e x i n g c a p a c i t y of a s e w a g e t r e a t m e n t p l a n t e f f l u e n t a v e r a g e d 0.3 m g / L . S i x t y - s e v e n p e r c e n t of the c o m p l e x a t i o n w a s due to c o m p o u n d s o f less than 1 0 , 0 0 0 m o l e c u l a r w e i g h t u n i t s . H e a l s o found that o r g a n i c c o m p o u n d s in the e f f l u e n t r e m o v a b l e by a c t i v a t e d c a r b o n c o m p o s e d 88 p e r c e n t of the total o r g a n i c c a r b o n and w e r e r e s p o n s i b l e for 87 p e r c e n t of the c o m p l e x a t i o n . S i m i l a r l y , V a r i n i et al ( 1 9 8 1 ) found that Cu t o x i c i t y to D a p h n i a m a g n a w a s m a i n l y d u e to the f r a c t i o n of low m o l e c u l a r w e i g h t c o m p o u n d s . W i l s o n ( 1 9 7 2 ) o b s e r v e d that a d d i n g spent s u l f i t e liquor to Cu s o l u t i o n i n c r e a s e d the s u r v i v a l rate of A t l a n t i c s a l m o n . G e l a t i n and s i l i c a gel a s g e l l i n g a g e n t s for m i c r o b i a l m e d i a w e r e found to r e d u c e the a p p a r e n t t o x i c i t y of i n o r g a n i c Sn ( I V ) , w h e r e a s s e r i n e and hyd r o x y f l a v o n e e n h a n c e d it ( H a l l a s et a l . , 1 9 8 2 ) .

In s u m m a r y , the e v i d e n c e g e n e r a l l y i n d i c a t e s that o r g a n i c s u b s t a n c e s can r e d u c e the t o x i c i t y of m e t a l s such as C u . It r e m a i n s to be seen w h e t h e r other m e t a l s can be a f f e c t e d s i m i l a r l y .

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B . pH The e f f e c t of pH on heavy m e t a l t o x i c i t y is very c o m p l e x : the

e f f e c t is p r i m a r i l y d e p e n d e n t on the m e t a l s p e c i e s . N a s u et al. ( 1 9 8 3 ) d e m o n s t r a t e d that both Cd and Cu i n h i b i t e d the g r o w t h of L e m n a p a u c i c o s t a t a m o r e at pH 5.1 than at pH 4.1. They a l s o o b s e r v e d that Cd a b s o r p t i o n w a s g r e a t e r w h e n initial pH w a s h i g h e r , but Cu a b s o r p t i o n w a s not s i g n i f i c a n t l y a f f e c t e d by an initial pH b e t w e e n 3.6 and 5 . 1 . H a r g r a v e s and W h i t t o n ( 1 9 7 6 ) isolated a c o m m o n s t r e a m a l g a , H o r m i d i u m . from an acid m i n e d r a i n a g e and found that its o p t i m u m range for g r o w t h w a s pH 3 . 5 - 4 . 0 . The Zn t o x i c i t y to this a l g a i n c r e a s e d at h i g h e r pH v a l u e s . The b i n d i n g c a p a c i t y of the h e a v y m e t a l s C u , C d , and Zn on the b l u e - g r e e n a l g a , C h r o o c o c c u s p a r i s , w a s found to i n c r e a s e as pH i n c r e a s e d from 4 to 7 (Les and W a l k e r , 1 9 8 4 ) . A c c u m u l a t i o n of Cd and Zn by the a q u a t i c l i v e r w o r t S c a o a n i a a l s o i n c r e a s e d as pH i n c r e a s e d ( W h i t t o n et al., 1 9 8 2 ) . The i n c r e a s i n g t o x i c i t y of Cu at high pH w a s a t t r i b u t e d to the i n t e r a c t i o n s of the m e t a l ion w i t h s u l f h y d r y l - c o n t a i n i n g p r o t e i n s o r e n z y m e s ( A n d r e w , 1 9 7 6 ) .

A l t e r n a t i v e l y , there are m a n y s t u d i e s s u g g e s t i n g that metal t o x i c i t y can be r e d u c e d at h i g h e r pH v a l u e s . V e r m a et al ( 1 9 8 5 ) o b s e r v e d that Hg (II) t o x i c i t y to fish d e c r e a s e d as pH i n c r e a s e d . R o b i n s o n and Deano ( 1 9 8 5 ) s h o w e d that Al and a c i d i t y had s y n e r g i s t i c toxic e f f e c t s . M i c h n o w i c z and W e a k s ( 1 9 8 4 ) r e p o r t e d that the a l g a S e l e n a s t r u m c a p r i c o r n u t u m P r i n t z g r e w s i g n i f i c a n t l y b e t t e r in a test s o l u t i o n c o n t a i n i n g 0.2 m g / L As w h e n it had p r e v i o u s l y been c u l t u r e d at pH 8 than w h e n c u l t u r e d at pH 4. B a b i c h and S t o t z k y ( 1 9 8 2 A ) i n v e s t i g a t e d the e f f e c t of pH on Ni t o x i c i t y using a b r o a d s p e c t r u m of m i c r o o r g a n i s m s , i n c l u d i n g b a c t e r i a , a c t i n o m y c e t e s , y e a s t s , and f u n g i . They found that Ni t o x i c i t y w a s p o t e n t i a t e d as pH w a s d e c r e a s e d to a c i d i c l e v e l s . The r e s u l t s , t h e r e f o r e , implied that acid p r e c i p i t a t i o n could c a u s e d i r e c t i n j u r i e s as well as i n d i r e c t e f f e c t s such as t o x i c i t y e n h a n c e m e n t o f heavy m e t a l s . A n i n t e r e s t i n g , a l t h o u g h s e e m i n g l y c o n t r a d i c t o r y , o b s e r v a t i o n w a s that Ni a c c u m u l a t i o n by a l g a e w a s h i g h e s t at pH 8 (Wang and W o o d , 1 9 8 4 ) . U s i n g C h l o r e l l a for Hg t o x i c i t y t e s t s , Rai and K h a t o n i a r ( 1 9 8 0 ) found that a c i d i c pH e n h a n c e d Hg t o x i c i t y , w h i l e a l k a l i n e pH r e d u c e d it. Pb t o x i c i t y to fungi w a s p o t e n t i a t e d under a c i d i c c o n d i t i o n s at pH 5 and 6 ( B a b i c h and S t o t z k y , 1 9 7 9 ) , w h i l e a s i g n i f i c a n t i n c r e a s e of Pb u p t a k e at lower pH v a l u e s w a s a l s o r e p o r t e d (Merlani and P o z z i , 1 9 7 7 ; L e w i s and M c i n t o s h , 1 9 8 4 ) .

U s i n g z e b r a f i s h egg h a t c h i n g and larvae survival as a test p r o c e d u r e , D a v e ( 1 9 8 5 ) d e t e r m i n e d the p H e f f e c t o n A l , C d , and F e t o x i c i t y . He r e p o r t e d that Al and Cd w e r e m o r e toxic at h i g h pH levels w h i l e Fe w a s m o r e toxic at low pH l e v e l s . S t e n d a h l and S p r a g u e ( 1 9 8 2 ) s t u d i e d the t o x i c i t y of v a n a d i u m p e n t a o x i d e at pH 5 . 5 , 6.6, 7 . 7 , and 8.8. The V t o x i c i t y to 25-g trout w a s g r e a t e s t at pH 7 . 7 , at w h i c h level the p r e d o m i n a t i n g ion H 2 V O 4 w a s a p p a r e n t l y the m o s t toxic form. HVO 4 p r e v a i l e d at high pH and w a s c a l c u l a t e d t o b e 6 0 p e r c e n t a s toxic a s H 2 V O 4 ' w h i l e the m e t a l p r e s e n t as d e c a v a n a d a t e s at pH 6.6 and 5.5 w a s 50 p e r c e n t as toxic a s H 2 V O 4 . O ' K e e f e e t a l . ( 1 9 8 4 ) r e p o r t e d that C d u p t a k e b y the w a t e r h y a c i n t h i n c r e a s e d as the pH i n c r e a s e d from 2 to 5, w h i l e in H o a g l a n d ' s s o l u t i o n , h i g h e r pH v a l u e s d e c r e a s e d the u p t a k e r a t e .

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In s u m m a r y , the e v i d e n c e s u g g e s t s that pH e f f e c t on metal t o x i c i t y is d e p e n d e n t on the m e t a l s p e c i e s and is w i t h o u t a set p a t t e r n .

C . T e m p e r a t u r e

An e x t e n s i v e , l i t e r a t u r e r e v i e w on t e m p e r a t u r e e f f e c t upon c h e m i c a l t o x i c i t y has b e e n r e p o r t e d ( C a i r n s e t a l . , 1 9 7 5 A ) . The t e m p e r a t u r e e f f e c t on m e t a l t o x i c i t y can g e n e r a l l y be seen by two o p p o s i n g r e s u l t s . On one h a n d , it w a s r e p o r t e d that t e m p e r a t u r e b e t w e e n 15 and 28 C had no s i g n i f i c a n t e f f e c t on lethal c o n c e n t r a t i o n s o f C d , C r , C u , H g , N i , and Z n ( R e h w o l d t e t a l . , 1 9 7 2 ) . S i m i l a r l y , t e m p e r a t u r e w a s found to have no e f f e c t on the rate of Hg u p t a k e or e l i m i n a t i o n by f r e s h w a t e r c l a m s ( S m i t h et al., 1 9 7 5 ) . On the o t h e r h a n d , the m a j o r i t y of r e p o r t s have i n d i c a t e d that t e m p e r a t u r e had an i m p o r t a n t e f f e c t on metal t ox i c i t y .

B r y a n t et al. ( 1 9 8 4 ) s t u d i e d t e m p e r a t u r e e f f e c t s on Cr (VI) t o x i c i t y to i n v e r t e b r a t e s . In g e n e r a l , they found that Cr t o x i c i t y i n c r e a s e d w i t h i n c r e a s i n g t e m p e r a t u r e . M a c L e o d and P e s s a h ( 1 9 7 3 ) r e p o r t e d that the L C 5 0 v a l u e s for H g C L 2 to r a i n b o w trout at 5, 1 0 , and 20 C w e r e 0 . 4 , 0 . 2 8 , and 0.22 m g / l , r e s p e c t i v e l y , and the v e l o c i t y of m o r t a l i t y (the r e c i p r o c a l of time to d e a t h in h) w a s l i n e a r l y r e l a t e d to t e m p e r a t u r e , V = 0.002 + 0 . 0 0 2 3 t , w h e r e t = t e m p e r a t u r e . I n c r e a s e d t e m p e r a t u r e also a p p e a r e d to i n c r e a s e Zn t o x i c i t y to m a r i n e fish ( N e g i l s k i , 1 9 7 6 ) , Zn t o x i c i t y to L e m n a p a u c i c o s t a t a ( N a s u and K u g i m o t o , 1 9 8 1 ) , and Cd t o x i c i t y to larval fish ( M i d d a u g h et al., 1 9 7 5 ) . O t h e r reports i n d i c a t e d that i n c r e a s e d t e m p e r a t u r e r e s u l t e d in i n c r e a s e d u p t a k e o f h e a v y m e t a l s ( J a c k i m e t a l . , 1 9 7 7 ; F r a z i e r , 1 9 7 6 ; M a c L e o d and P e s s a h , 1 9 7 3 ) . The i n c r e a s e in t e m p e r a t u r e a l s o a f f e c t e d the t u r n o v e r of m e t a l s in o r g a n i s m s . The b i o l o g i c a l h a l f - l i v e s of the h e a v y m e t a l s Co 60 and Zn 65 in s h r i m p s w e r e r e d u c e d by a factor of about 2 for an i n c r e a s e of 10 C (Van W e e r s , 1 9 7 5 ) . T h e r e is a d d i t i o n a l e v i d e n c e i n d i c a t i n g that t e m p e r a t u r e has an important i n f l u e n c e on m e t a l t o x i c i t y .

B r a g i n s k i i and S h c h e r b a n ( 1 9 7 8 ) found no a c u t e t o x i c i t y of C d , C u , M n , N i , and Zn to six s p e c i e s of f r e s h w a t e r i n v e r t e b r a t e s in the t e m p e r a t u r e r a n g e 1 0 - 1 5 C, but the t o x i c i t i e s i n c r e a s e d s h a r p l y at 2 5 - 3 0 C. It is a p p a r e n t that t e m p e r a t u r e - m e d i a t e d m e t a b o l i c rate has an i m p o r t a n t role in metal t o x i c i t y . This v i e w p o i n t is further s u b s t a n t i a t e d by R o c h and M a l y ' s e x p e r i m e n t s ( 1 9 7 9 ) . T h e y o b s e r v e d that c o l d - a c c l i m a t e d ( 6 C ) r a i n b o w t r o u t , e x p o s e d to lethal c o n c e n t r a t i o n s of C d , s u r v i v e d longer than w a r m - a c c l i m a t e d (12 and 18 C) t r o u t . The c o l d - a c c l i m a t e d trout s u r v i v e d higher C d c o n c e n t r a t i o n s . S i m i l a r l y , the c o l d - a c c l i m a t e d A t l a n t i c salmon s u r v i v e d longer than w a r m - a c c l i m a t e d salmon ( H o d s o n and S p r a g u e , 1 9 7 5 ) . The s u r v i v a l time of fish in toxic c o n c e n t r a t i o n s of a m m o n i a ( a l b e i t a m m o n i a is not a m e t a l s p e c i e s ) a l s o d e c r e a s e d m a r k e d l y a s t e m p e r a t u r e i n c r e a s e d ( C a i r n s e t a l . , 1 9 7 5 B ) . N e v e r t h e l e s s , it s h o u l d be p o i n t e d out that o r g a n i s m s

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might recover better from metal toxicity at higher t e m p e r a t u r e than at lower t e m p e r a t u r e (Remacle et al., 1 9 8 2 ) .

It can g e n e r a l l y be c o n c l u d e d that seasonal t e m p e r a t u r e v a r i a t i o n can affect metal toxicity to aquatic o r g a n i s m s . The ecological implication is that water q u a l i t y criteria should take this into account and that the c r i t e r i a should be m o r e s t r i n g e n t in summer than in w i n t e r .

D. A l k a l i n i t y and h a r d n e s s

A l k a l i n i t y and h a r d n e s s of a water sample n o r m a l l y go h a n d - i n - h a n d and thus they are d i s c u s s e d here t o g e t h e r .

There are many reports indicating that a l k a l i n i t y and h a r d n e s s can g e n e r a l l y lead to d e t o x i f i c a t i o n of heavy m e t a l s . B r k o v i c - P o p o v i c and P o p o v i c C 1 9 7 7 ) indicated that the toxicity of the heavy m e t a l s Cd, C r , C u , H g , N i , and Zn to Tubifex w a s dependent on a l k a l i n i t y and h a r d n e s s , except that Hg was m u c h less a f f e c t e d than the other m e t a l s . S l o n i m and S l o n i m (1973) r e p o r t e d that the lethal c o n c e n t r a t i o n of Be to g u p p i e s (96h L C 5 0 ' s ) w a s a linear function of water h a r d n e s s , b e t w e e n 10-400 mg/L (as C a C O 3 ) . H o w a r t h and S p r a g u e ( 1 9 7 8 ) reported that Cu toxicity to r a i n b o w trout involved a complex response over a w i d e range of h a r d n e s s and pH. The trend, h o w e v e r , can be g e n e r a l i z e d by stating that high h a r d n e s s decreased Cu toxicity at any pH.

Rai and K h a t o n i a r ( 1 9 8 0 ) o b s e r v e d that 1 m g / L Hg ( H g C l 2 ) w a s highly toxic to the alga C h l o r e l l a . and that up to 20 m g / L Ca c o u n t e r a c t e d the Hg toxicity and s t i m u l a t e d the g r o w t h of C h l o r e l l a . S i m i l a r l y , Cd toxicity to oak leaf could be m i n i m i z e d by Ca ( F u h r e r , 1 9 8 3 ) . W e i s ( 1 9 8 0 ) showed that Zn retarded limb r e g e n e r a t i o n in the fiddler c r a b , Uca pergilator, and the e f f e c t s of Zn (1-5 m g / L ) were p o t e n t i a t e d under c o n d i t i o n s of d e c r e a s e d salinity (7-8 0/00 as o p p o s e d to 30 0 / 0 0 ) , possibly o w i n g to a lower Ca c o n c e n t r a t i o n .

S t e p h e n s o n (1983) e x a m i n e d the e f f e c t s of water h a r d n e s s , t e m p e r a t u r e , and the size of freshwater shrimp on the s u s c e p t i b i l i t y to Cu t o x i c i t y . Among these v a r i a b l e s , he found that only water h a r d n e s s had s t a t i s t i c a l l y significant e f f e c t s on LC50 v a l u e s . Cu was 4-6 times m o r e toxic in soft water than in hard w a t e r . Ajmal and Khan ( 1 9 8 4 ) reported that Cd toxicity to s a p r o p h y t i c and n i t r i f y i n g b a c t e r i a d e c r e a s e d as h a r d n e s s i n c r e a s e d , w i t h a m i n i m u m at 320 mg/L (as C a C O 3 ) h a r d n e s s for n i t r i f y i n g b a c t e r i a and 400 m g / L for s a p r o p h y t i c b a c t e r i a . P o s t o n et al. ( 1 9 8 4 ) reported that U ( V I ) acute toxicity (48h L C 5 0 ) to D a p h n i a m a g n a d i m i n i s h e d by a factor of 7.5 as mean water h a r d n e s s and a l k a l i n i t y increased from 70 and 57 m g / L to 195 and 130 m g / L , r e s p e c t i v e l y .

There are two schools of thought for interpreting the e f f e c t s of metal d e t o x i f i c a t i o n by a l k a l i n i t y and h a r d n e s s . The first h y p o t h e s i s involves a b i o l o g i c a l m e c h a n i s m for the o b s e r v e d e f f e c t s . As m e n t i o n e d in the section on t o l e r a n c e , Calamari et al. ( 1 9 8 0 ) reported that Cd toxicity to salmon w a s vastly

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d i f f e r e n t if the fish w a s a c c l i m a t e d in hard w a t e r , even though it was tested in soft w a t e r . A p p a r e n t l y a biological m e c h a n i s m may be involved in the d e t o x i f i c a t i o n p r o c e s s e s . A d d i t i o n a l e v i d e n c e of a b i o l o g i c a l m e c h a n i s m is that the fecundity of D a o h n i a magna w a s c o r r e l a t e d with C a S O . c o n t e n t , in the range 9 1 - 2 1 0 0 mg/L (LeBlanc and S u r p r e n a n t , 1 9 8 4 ) . It is likely that when a metal toxicity test involving i n v e r t e b r a t e s is p e r f o r m e d , the varying a m o u n t s of Ca can influence the test results a c c o r d i n g l y . On the other h a n d , there is e v i d e n c e p o i n t i n g to a chemical m e c h a n i s m as the cause of metal d e t o x i f i c a t i o n to o r g a n i s m s .

S l o n i m and S l o n i m ( 1 9 7 3 ) a t t r i b u t e d the d e t o x i f i c a t i o n of Be by w a t e r h a r d n e s s partly to the b u f f e r i n g c a p a c i t y and Ca a n t a g o n i s m s in hard water s o l u t i o n . There may be a c o m p e t i t i v e uptake between toxic m e t a l s and Ca in hard w a t e r . For e x a m p l e , K i n k a d e and Erdman (1975) found that the initial uptake of Cd in a s i m u l a t e d freshwater e c o s y s t e m was faster in hard water than in soft w a t e r . H o w e v e r , the total c o n c e n t r a t i o n of Cd w a s greater in the o r g a n i s m s cultured in soft water than in those in hard w a t e r . S i m i l a r l y , C h a p m a n and D u n l o p ( 1 9 8 1 ) reported that Zn uptake by freshwater p r o t o z o a ( T e t r a h v m e n a p v r i f o r m i s ) w a s reduced from 0.5 ug/L to 0.1 ug/L per 1 m i l l i o n cells by adding 500 m g / L Ca alone or 500 m g / L Ca + M g . Mg alone w a s about half as e f f e c t i v e in reducing Cd toxicity. The m e c h a n i s m is p o s s i b l y due to c o m p e t i t i o n at the cell m e m b r a n e in the p r e s e n c e of Ca and Mg. P o s t o n et al. (1984) a t t r i b u t e d the loss of U ( V I ) toxicity to the c o m p l e x a t i o n of the U ion with the c a r b o n a t e ion.

T h e r e is also indirect e v i d e n c e s u g g e s t i n g the e f f e c t s of a l k a l i n i t y and hardness on metal uptake by p l a n t s . Franzin and M c F a r l a n e (1980) investigated the a q u a t i c e c o s y s t e m in the v i c i n i t y of a base-metal smelter located at Flin F l o n , M a n i t o b a . They reported that metal c o n t e n t s in plant material w e r e different from what w e r e expected on the basis of metal c o n c e n t r a t i o n s in the water s a m p l e s . V a r y i n g Ca c o n t e n t s in lake water were s p e c u l a t e d to be the cause of the d i s c r e p a n c y .

In s u m m a r y , a l k a l i n i t y and h a r d n e s s can be c o n s i d e r e d as a " b u f f e r " in natural water for m o d e r a t i n g heavy metal t o x i c i t y .

E. Inorganic Iigands

Inorganic ligands such a s C l - , O H - , P O 43 - , e t c . , have been

shown to influence heavy metal toxicity to aquatic o r g a n i s m s . The results are c o m p l e x . For e x a m p l e , B a b i c h and S t o t z k y ( 1 9 7 8 ) found Zn t o x i c i t y to f u n g i , b a c t e r i a , and c o l i p h a g e s w a s u n a f f e c t e d , l e s s e n e d , and increased, r e s p e c t i v e l y , by the a d d i t i o n of high c o n c e n t r a t i o n s of N a C l . They reported that the increased Zn toxicity in the p r e s e n c e of NaCl was not due to s y n e r g i s m between Zn and Cl and elevated o s m o t i c p r e s s u r e , but to the formation of a complex Zn-Cl s p e c i e s . Babich and Stotzky ( 1 9 8 3 ) further reported that Ni toxicity to m i c r o b e s ( b a c t e r i a , a c t i n o m y c e t e s , and y e a s t s ) in m a r i n e s y s t e m s w a s reduced by increasing s a l i n i t y .

In g e n e r a l , C r ( V I ) toxicity to i n v e r t e b r a t e s d e c r e a s e d with increasing s a l i n i t y . For C o r o o h i u m v o l u t a t o r . h o w e v e r , there was

16

a d e c r e a s e in toxicity w i t h increasing salinity over the range 5-30 0/00, and increased toxicity caused by further salinity increases up to 40 0/00 (Bryant et al. , 1 9 8 4 ) . Hallas et al. ( 1 9 8 2 ) found that replacement of SO 4

2 - w i t h NO 3 did not change Sn toxicity to e s t u a r i n e m i c r o o r g a n i s m s , w h i l e the replacement of Cl with NO 3

- decreased Sn toxicity. Piccardi and Clauser ( 1 9 8 3 ) observed that Iris p s e u d a c o r u s survived 3 m g / L Cu in the p r e s e n c e of N a C l , w i t h o u t showing signs of s t r e s s . This was likely due to d e c r e a s i n g Cu a b s o r p t i o n by the plant in the p r e s e n c e of N a C I . Hg ions did not exhibit s y n e r g i s t i c effects with reduced salinity and m e r e l y acted a d d i t i v e l y (Gray, 1 9 7 6 ) . Frank and R o b e r t s o n (1979) reported that the m e d i a n lethal c o n c e n t r a t i o n (96h L C S 0 ) of Cr to the blue c r a b , C a l l i n e c t e s s a p i d u s . was 89 and 98 mg/L at 15 and 35 0/00 s a l i n i t y , r e s p e c t i v e l y . In c o m p a r i s o n , the LC50's of Cd w e r e 4.7 and 11.6 m g / L at the respective s a l i n i t i e s . Jones et al. ( 1 9 7 6 ) found that both high and low s a l i n i t i e s increased the toxicity of Cu to the p o l y c h e a t e N e r e i s d i v e r s i c o l o r . They suggested that the cause was s y n e r g i s t i c a c t i o n , instead of increased b i o a c c u m u l a t i o n of C u . Jackim et al. (1977) o b s e r v e d that a decrease in salinity depressed Cd a c c u m u l a t i o n in m a r i n e b i v a l v e s .

PO 43 - is w i d e l y d i s t r i b u t e d in natural water and may have

some effects on metal toxicity. S a n d e r s ( 1 9 7 9 ) , using algal cultures low in p h o s p h o r u s , found that As toxicity to algal growth could be suppressed by increasing the p h o s p h o r u s c o n c e n t r a t i o n s . S i m i l a r l y , Cd uptake by C h l o r e l l a was o b s e r v e d to d e c r e a s e as PO 4

3 - increased (Khummongkol et al., 1 9 8 2 ) , and Zn-P interactions w e r e significant in influencing Selenast rum cell yield results ( K u w a b a r a , 1 9 8 5 ) . It is likely that PO4

3-. c o m p e t e s at the sorption sites against As and also forms n o n - d i s s o I v e d chemical species such as C d - P O 4 or Z n - P O 4 , resulting in a d e c r e a s e of the metal b i o a v a i l a b i l i t y .

Other ligands have also been e x a m i n e d . A n d r e w et al. ( 1 9 7 7 ) studied effects of the ligands c a r b o n a t e - b i c a r b o n a t e , o r t h o p h o s p h a t e , and p y r o p h o s p h a t e on Cu toxicity to D a p h n i a at constant pH and total h a r d n e s s . They found that m o r t a l i t y rates and reciprocal survival times w e r e d i r e c t l y correlated w i t h Cu and Cu hydroxy [Cu(OH)n] ion a c t i v i t i e s . Cu toxicity w a s n e g a t i v e l y correlated with a c t i v i t i e s of soluble CuCO3. and other c o m p l e x e s . Riedel ( 1 9 8 4 ) d e t e r m i n e d the acute toxicity of C r ( V I ) to the d i a t o m T h a l a s s i o s i r a in artificial m e d i a containing 3.2 and 0.32 0/00 salinity and varying SO.4

- c o n c e n t r a t i o n s . The increase of salinity was found to d e c r e a s e C r ( V I ) t o x i c i t y . F u r t h e r m o r e , C r ( V I ) inhibition w a s a function of the ratio of C r ( V I ) to S O 4

± . Inhibition occurred w h e n the ratio exceeded about 5 0 0 : 1 . It w a s s p e c u l a t e d that SO 4

= and C r O 4= compete at the same s i t e s ,

resulting in reduced C r ( V I ) toxicity.

F. I n t e r a c t i o n s

One strength of biological toxicity tests is that the tests reflect the combined integrated effects of all toxic c o n s t i t u e n t s present in a test s a m p l e . W a s t e w a t e r s d i s c h a r g i n g into natural w a t e r s frequently carry m o r e than one toxic or p o t e n t i a l l y toxic

17

s u b s t a n c e . The combined toxic e f f e c t s of all c o n s t i t u e n t s are often not e q u i v a l e n t to the summation of all individual effects -they are s o m e t i m e s g r e a t e r , other times l e s s .

The i n t e r a c t i o n s between t o x i c a n t s , h o w e v e r , are very c o m p l e x . There are many variables influencing the i n t e r a c t i o n s , such as metal s p e c i e s , test o r g a n i s m s , and lethal versus sublethal c o n c e n t r a t i o n . The joint a c t i o n s are c o m m o n l y d e s c r i b e d as 1) s y n e r g i s m , w h e r e the combined toxic effect of a m i x t u r e is greater than the sum of the individual t o x i c i t i e s , 2) a n t a g o n i s m , w h e r e the combined toxic effect of a m i x t u r e is less than the sum of the individual t o x i c i t i e s , and 3) n o n - i n t e r a c t i v e , or a d d i t i v e , w h e r e the combined effect is identical to the sum of the individual t o x i c i t i e s . All three types of interaction w e r e found b e t w e e n Fe and Zn in different sources of algal s a m p l e s (Wang, 1 9 8 5 ) .

The interactions b e t w e e n heavy m e t a l s appear to be w i t h o u t a set p a t t e r n . For e x a m p l e , interactions between the most studied metal p a i r , Cd and Z n , varied from a n t a g o n i s t i c to s y n e r g i s t i c as o b s e r v e d by v a r i o u s r e s e a r c h e r s using d i f f e r e n t test o r g a n i s m s (Table 1 ) . Thorp and Lake ( 1 9 7 4 ) reported that Cd and Zn interaction w a s d e p e n d e n t on the q u a n t i t y of toxic m e t a l s . S y n e r g i s t i c interaction between Cd and Zn w a s o b s e r v e d by H u t c h i n s o n and C z y r s k a ( 1 9 7 5 ) and Marshall e t a l . ( 1 9 8 1 ) , w h i l e a n t a g o n i s t i c interaction w a s reported by Spehar et al. ( 1 9 7 8 ) , W e i s ( 1 9 8 0 ) , Attar and Maly ( 1 9 8 2 ) , Singh and Y a d a v a ( 1 9 8 4 ) , and O'Keefe et al. ( 1 9 8 4 ) . H u t c h i n s o n and C z y r s k a ( 1 9 7 5 ) found that Zn at 0.05 and 0.08 m g / L had a s t i m u l a t o r y effect on both Lemna and S a l v i n i a . The s t i m u l a t o r y effect of Zn w a s s u p p r e s s e d by the p r e s e n c e of Cd at 0.01 to 0.03 m g / L . The m i x t u r e of Cd and Zn showed a n o t i c e a b l e s y n e r g i s t i c e f f e c t . Spehar et al. ( 1 9 7 8 ) indicated that survival of flagfish larvae exposed as embryos was s i g n i f i c a n t l y d e c r e a s e d in both individual Cd and Zn c o n c e n t r a t i o n s . They further noted that the m i x t u r e of these m e t a l s did not further suppress the s u r v i v a l , s u g g e s t i n g a n t a g o n i s m between these m e t a l s . W e i s ( 1 9 8 0 ) used limb r e g e n e r a t i o n in the fiddler crab as the indicator and found an a n t a g o n i s m between Cd and Z n .

Other binary metal m i x t u r e s have a l s o been s t u d i e d . Stauber and F l o r e n c e ( 1 9 8 5 ) reported that the m a r i n e d i a t o m N i t z s c h i a closterium w a s m o r e tolerant to Cu toxicity when cultured w i t h normal Fe levels (0.79 m g / L ) than when c u l t u r e d in an F e - d e f i c i e n t m e d i u m (0.079 m g / L ) . They speculated that colloidal F e ( O H ) 3 binding to the d i a t o m cell m e m b r a n e sorbed Cu and prevented it from p e n e t r a t i n g into the c e l l . Stokes ( 1 9 7 5 B ) observed that Cu and Ni had strong s y n e r g i s t i c effects on algae isolated from S u d b u r y , O n t a r i o . The results of uptake and toxicity of Ni in the p r e s e n c e of Cu s u p p o r t e d the theory that Cu increased the p e r m e a b i l i t y of the c e l l . Cu once inside the cell w a s firmly bound. On the other h a n d , A n d e r s o n and W e b e r ( 1 9 7 6 , 1977) e x p e r i m e n t e d w i t h m a t u r e m a l e guppies and found that the binary solution c o n t a i n i n g Cu and Ni e x h i b i t e d strict additive i n t e r a c t i o n . It is p o s s i b l e that these m e t a l s acted with a common m o d e of lethal a c t i o n .

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Table 1. Interactions of Heavy M e t a l s in Contact with A q u a t i c O r g a n i s m s

T o x i c a n t s Binary Test oraani sms Interact ions R e f e r e n c e s Cd-Zn freshwater shrimp Less than a d d i t i v e Thorp and Lake ( 1 9 7 4 )

when less than 1 toxic unit A d d i t i v e when >1 toxic unit

Cd-Zn aquatic plants s y n e r g i s t i c H u t c h i n s o n and C z y r s k a C 1 9 7 5 )

Cd-Zn freshwater fish a n t a g o n i s t i c Spehar et al. (1978)

Cd-Zn fiddler crab a n t a g o n i s t i c W e i s ( 1 9 8 0 )

Cd-Zn freshwater z o o p l a n k t o n s y n e r g i s t i c M a r s h a l l et al. ( 1 9 8 1 )

Cd-Zn i n v e r t e b r a t e a n t a g o n i s t i c Attar and M a l y ( 1 9 8 2 )

Cd-Zn freshwater algae a n t a g o n i s t i c Singh and Y a d a v a ( 1 9 8 4 )

Cd-Zn a q u a t i c plants a n t a g o n i s t i c O ' K e e f e e t a l . ( 1 9 8 4 )

Cd-Ni freshwater algae s y n e r g i s t i c Prasad and Prasad ( 1 9 8 2 )

Cd-Pb freshwater algae s y n e r g i s t i c Prasad and Prasad ( 1 9 8 2 )

Cd-Fe freshwater algae a n t a g o n i s t i c G i p p s and Coller ( 1 9 8 2 )

Cd-Cu freshwater algae a n t a g o n i s t i c B a r t l e t t et al. ( 1 9 7 4 )

Cu-Fe m a r i n e algae a n t a g o n i s t i c Stauber and F l o r e n c e (1985)

Cu-Mn freshwater algae s y n e r g i s t i c C h r i s t e n s e n et al. (1979)

Cu-Ni freshwater algae s y n e r g i s t i c Stokes ( 1 9 7 5 B )

Cu-Ni freshwater algae s y n e r g i s t i c H u t c h i n s o n and S t o k e s ( 1 9 7 5 )

Continued on next page

T a b l e 1. C o n t i n u e d

Cu-Ni f r e s h w a t e r fish n o n - i n t e r a c t i v e A n d e r s o n and W e b e r ( 1 9 7 6 )

C u - P b freshwater fish a n t a g o n i s t i c Ozoh ( 1 9 7 9 )

C u - Z n f r e s h w a t e r fish s y n e r g i s t i c A n d e r s o n and. W e b e r ( 1 9 7 7 )

Cu-Zn m a r i n e p h y t o p l a n k t o n s y n e r g i s t i c Braek e t a l . ( 1 9 7 6 ) a n t a g o n i s t i c ( d e p e n d i n g on s p e c i e s )

Fe-Zn f r e s h w a t e r algae n o n - i n t e r a c t i v e W a n g ( 1 9 8 5 ) an tagon i s t i c s y n e r g i s t i c ( d e p e n d i n g on algal

s o u r c e s )

H g - S e freshwater fish s y n e r g i s t i c H u c k a b e e and G r i f f i t h ( 1 9 7 4 ) egg

H g - S e freshwater c o m m u n i t y a n t a g o n i s t i c Rudd and Turner ( 1 9 8 3 B )

N i - H g fungus a n t a g o n i s t i c B a b i c h and S t o t z k y ( 1 9 8 2 B )

Ni-Zn fungus a n t a g o n i s t i c " "

N i - P b fungus a n t a g o n i s t i c " "

N i - P b freshwater algae n o n - i n t e r a c t i v e Prasad and P r a s a d ( 1 9 8 2 )

C d . C u . Z n C h i n o o k salmon n o n - i n t e r a c t i v e F i n l a y s o n and V e r r u e ( 1 9 8 2 ) f r e s h w a t e r fish or a n t a g o n i s t i c

C d . C u . H g . P b a q u a t i c p l a n t s s y n e r g i s t i c Jana and C h o u d h u r i ( 1 9 8 4 )

A s , C d , C u , H g , P b , Z n freshwater z o o p l a n k t o n s y n e r g i s t i c B o r g m a n n ( 1 9 8 0 )

Table 1. Concluded

Mixture Cd.Cu.Zn freshwater fish synergistic Eaton (1973)

Cd.Cu.Pb.Zn aquatic plants synergistic Nakada et al. (1979)

As.Cd.Cr.Cu freshwater algae synergistic Wong et al. (1978) Fe.Hg.Ni ,Pb Se , Zn

The m e t a l i n t e r a c t i o n , f u r t h e r m o r e , is c o m p l i c a t e d by the s p e c i e s of test o r g a n i s m s . Braek et al. ( 1 9 7 6 ) s t u d i e d the c o m b i n e d e f f e c t s of Cu and Zn on three m a r i n e d i a t o m s and one d i n o f l a g e l l a t e . They found that the m o d e of i n t e r a c t i o n was u n p r e d i c t a b l e . For e x a m p l e , w i t h A. carteri and T. p s e u d o m a n a the metal i n t e r a c t i o n s showed a clear case of s y n e r g i s m , w h i l e the m e t a l s e x h i b i t e d a n t a g o n i s m in the case of the d i a t o m P. t r i c o r n u m t u m . W a n g (1985) found that Zn was m u c h m o r e toxic than Fe to the natural algal c o m m u n i t y . The binary s o l u t i o n of Zn and Fe acted d i f f e r e n t l y a c c o r d i n g to the sources of algal p o p u l a t i o n . T h r e e types of i n t e r a c t i o n - s y n e r g i s t i c , a n t a g o n i s t i c , and n o n - i n t e r a c t i v e - w e r e all o b s e r v e d .

T h e r e are other s e e m i n g l y c o n t r a d i c t o r y r e s u l t s in the l i t e r a t u r e . The m i x t u r e of Hg and Se showed a g r e a t e r d e p r e s s i o n of h a t c h a b i l i t y of the eggs of c a r p , C y p r i n u s c a r p i o . than of eggs e x p o s e d to these m e t a l s s i n g l y , s u g g e s t i n g s y n e r g i s t i c toxic e f f e c t s ( H u c k a b e e and G r i f f i t h , 1 9 7 4 ) . The b i o a c c u m u l a t i o n of Hg and S e , h o w e v e r , indicated that the p r e s e n c e of Se reduced the u p t a k e of H g . The r e d u c t i o n a p p e a r e d to d e p e n d on the p o s i t i o n of the o r g a n i s m in the food c h a i n . For fish the r e d u c t i o n was p r o p o r t i o n a l to the amount of Se a c c u m u l a t e d (Rudd and T u r n e r , 1 9 8 3 B ) .

B o r g m a n n ( 1 9 8 0 ) e x p e r i m e n t e d w i t h binary m i x t u r e s from the toxic metal ions A s , C d , C u , H g , P b , and Zn u s i n g b i o m a s s p r o d u c t i o n k i n e t i c s of f r e s h w a t e r c o p e p o d s . The a v e r a g e o b s e r v e d g r o w t h time i n c r e a s e d , s u g g e s t i n g s i g n i f i c a n t s y n e r g i s m . S i m i l a r l y , Jana and C h o u d h u r i ( 1 9 8 4 ) indicated p o t e n t i a t e d toxicity in metal c o m b i n a t i o n s among C d , C u , H g , and Pb c o m p a r e d w i t h individual m e t a l s toward m a t u r e leaves of P o t a m o g e t o n . V a l l i s n e r i a . and H v d r i l l a . F i n l a y s o n and V e r r u e ( 1 9 8 2 ) , h o w e v e r , found that the binary s o l u t i o n s of C d , C u , and Zn w e r e either a d d i t i v e or a n t a g o n i s t i c in their lethality to C h i n o o k s a l m o n .

The c o m p l e x r e s p o n s e s of v a r i o u s test o r g a n i s m s to binary metal s o l u t i o n s , h o w e v e r , has not d i s c o u r a g e d r e s e a r c h e r s from s t u d y i n g m i x t u r e s c o n t a i n i n g three o r m o r e m e t a l s . E a t o n ( 1 9 7 3 ) found that C d , C u , and Zn m i x t u r e s at sublethal levels acted s y n e r g i s t i c a l l y toward the fathead m i n n o w . U s i n g a m i x t u r e c o n t a i n i n g C d , C u , P b , and Z n , N a k a d a e t a l . ( 1 9 7 9 ) showed s y n e r g i s t i c i n t e r a c t i o n in the case of E l o d e a . W o n g et al. ( 1 9 7 8 ) indicated that 1 0 m e t a l s ( A s , C d , C r , C u , F e , H g , N i , P b , S e , and Z n ) w e r e not toxic to a l g a e w h e n p r e s e n t e d i n d i v i d u a l l y at the levels e s t a b l i s h e d by the G r e a t Lakes water q u a l i t y c r i t e r i a . The m i x t u r e of these m e t a l s at the same c o n c e n t r a t i o n , h o w e v e r , was s t r o n g l y i n h i b i t i v e .

C h e r r y et al. ( 1 9 7 7 ) o b s e r v e d that the a d d i t i o n of Hg at 40 ng/L a p p e a r e d to induce g r e a t e r increase of heavy m e t a l u p t a k e by b a c t e r i a than did the a d d i t i o n of Cu at 2 m g / L in b r a c k i s h and ash basin w a t e r . In fresh w a t e r , h o w e v e r , the r e v e r s e w a s f o u n d .

In s u m m a r y , toxicity tests of complex e f f l u e n t s r e p r e s e n t a great c h a l l e n g e to a q u a t i c t o x i c o l o g i s t s , r e g u l a t o r y a g e n c i e s , c o n s u l t i n g e n g i n e e r s , and industrial c o n c e r n s . The c o n f l i c t i n g

22

results in the literature are a p p a r e n t l y because of the use of different test o r g a n i s m s , test m e t h o d s , toxic s u b s t a n c e s , and the like. M o r e studies on toxicity interaction are required before a degree of predictability can be a c h i e v e d .

G. Sediments

Natural waters contain various qualities and q u a n t i t i e s of suspended s e d i m e n t s . These sediments have important e f f e c t s on metal toxicity to and b i o a c c u m u l a t i o n by aquatic o r g a n i s m s . Pesch and M o r g a n (1978) exposed the adult m a l e p o l y c h a e t e , N e a n t h e s a r enaceden t a t e . to Cu w.i t h and without clean sand. They observed that Cu toxicity was lower w i t h sand than without sand: the 28-d LC50's w e r e 0.1 and 0.044 mg/L C u , r e s p e c t i v e l y . A possible e x p l a n a t i o n was the a d s o r p t i o n of Cu by sand, causing the loss of Cu c o n c e n t r a t i o n . Cd bound w i t h b e n t o n i t e was absorbed by the soft-shell clam, Mya a r e n a r i a . as fast as that in the chloride form, w h i l e Cd bound in humic acid, a l b u m i n , and e s t u a r i n e sediment showed much slower uptake by the clams ( P h e l p s , 1 9 7 9 ) . Pesch (1979) also e x p e r i m e n t e d with adult m a l e w o r m s exposed to 0.1 ± 0.015 mg/L Cu in seawater in c o n t i n u o u s - f l o w b i o a s s a y . The different sediment types s i g n i f i c a n t l y influenced the C u - i n d u c e d m o r t a l i t y . The time of 50 percent m o r t a l i t y was 7.8 days witho u t s e d i m e n t , 36.5 days w i t h sand, 54.5 days with the sand-mud m i x t u r e , and 50.0 days w i t h m u d . S i m i l a r l y , Graney et al. (1984) studied the accumulation of Cd in the Asian clam, C o r b i c u l a f l u m i n e a . using different s u b s t r a t e s . The greatest tissue a c c u m u l a t i o n of Cd occurred in the no-substrate e n v i r o n m e n t , w h i l e the lowest occurred in an environment containing a m i x t u r e of sand, c l a y , and organic m a t t e r . The environment w i t h a m i x t u r e of sand, s i l t , and clay e x h i b i t e d a m e d i a n e f f e c t . Nay and Van Hassel (1983) investigated v a r i a t i o n s of Pb, N i , Cd, and Zn c o n c e n t r a t i o n s among six species of fish from a h i g h w a y - c o n t a m i n a t e d s t r e a m and reported that a s s o c i a t i o n with stream sediment appeared to influence w h o l e - b o d y metal a c c u m u l a t i o n , while the metal contents in m u s c l e w e r e not c o r r e l a t e d .

There are many studies indicating that sediment s u b s t a n c e s e f f e c t i v e l y reduce metal toxicity. Brinkhurst et al. (1982) showed that the tolerance of o l i g o c h a e t e s to Cd and Hg was increased by the presence of s e d i m e n t . Hongve et al. (1980) reported that the addition of 7.6 mg/L lake sediment at 0.64 n e p h e l o m e t r i c turbidity units c o n s i s t e n t l y reduced toxic effects of C d , C u , Hg, Pb, and Zn to the natural p h y t o p l a n k t o n p h o t o s y n t h e s i s . Hardy et al. (1981) observed that the clam P r o t o t h a c a showed a linear uptake of Cd. W h e n 3.6 g/L washed sediment was added, h o w e v e r , the uptake was reduced to 17 percent of that in the s e d i m e n t - f r e e s a m p l e . Phelps (1979) found that the uptake of the clam Mya arenaria exposed to 10 mg/L total Cd was reduced to 33 percent of the uptake of the control when 0.1 g/L of e s t u a r i n e sediment was a d d e d . S e d i m e n t s also e f f e c t i v e l y reduced Ni toxicity to b a c t e r i a , a c t i n o m y c e t e s , and yeasts (Babich and S t o t z k y , 1 9 8 3 ) , Hg toxicity to and a c c u m u l a t i o n by fish and other aquatic organisms (Turner and Rudd, 1 9 8 3 ) , and pollutant effects on aquatic oligochaetes (Chapman et al., 1982B). Ahsanullah et al.

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al. ( 1 9 8 4 ) exposed the b u r r o w i n g s h r i m p , C a l l i a n a s s a a u s t r a l i e n s i s . to C d - c o n t a m i n a t e d water and s e d i m e n t . They found that the shrimp a c c u m u l a t e d Cd from water at a rate c o m m e n s u r a t e with the Cd c o n c e n t r a t i o n in the water and the d u r a t i o n of e x p o s u r e . S e d i m e n t s e f f e c t i v e l y removed Cd from the a q u e o u s phase and thus kept Cd from being b i o a v a i l a b l e to the s h r i m p . In c o n t r a s t , H a r t m a n and M a r t i n ( 1 9 8 4 ) showed that s u s p e n d e d s e d i m e n t s increased the s h o r t - t e r m toxicity of g l y p h o s a t e to d a p h n i d s at all c o n c e n t r a t i o n l e v e l s .

D i f f e r e n t types of lake s e d i m e n t s can also affect metal toxicity and a v a i l a b i l i t y . S c h i e r u p and Larsen (1981) reported that e u t r o p h i c , s e w a g e - p o l l u t e d lake s e d i m e n t s contained up to 80 times the heavy metal c o n c e n t r a t i o n s of o I i g o t r o p h i c , n o n - p o l l u t e d lake s e d i m e n t s . The metal a v a i l a b i l i t y , h o w e v e r , w a s greater in the latter than the former.

A q u a t i c m a c r o p h y t e s e x h i b i t e d further c o m p l i c a t e d p a t t e r n s . M a y e s et al. ( 1 9 7 7 ) d e t e r m i n e d the u p t a k e of Cd and Pb by Elodea c a n a d e n s i s . They found that p l a n t s grown in the same water but in s e d i m e n t s from different sources had s i g n i f i c a n t l y d i f f e r e n t c o n c e n t r a t i o n s of Cd and Pb. A d d i t i o n a l l y E l o d e a samples rooted in the sediment from the same sources but grown in different w a t e r s with d i f f e r e n t levels of m e t a l s also a c c u m u l a t e d s i g n i f i c a n t l y d i f f e r e n t a m o u n t s of Pb and Cd. The results indicated that both water and sediment were important s o u r c e s of metal intake for rooted a q u a t i c v e g e t a t i o n . W e l s h and Denny ( 1 9 8 0 ) s u g g e s t e d two p a t h w a y s for the transfer of m e t a l s from s e d i m e n t s to the s h o o t s . Pb a c c u m u l a t i o n in the shoots w a s c o n s i d e r e d the result of a d s o r p t i o n from the w a t e r . Cu a c c u m u l a t i o n was suggested to be due m a i n l y to a b s o r p t i o n by the roots and t r a n s l o c a t i o n w i t h i n the plant to the s h o o t s .

H. In situ

As indicated p r e v i o u s l y , m a n y s t u d i e s showed that metal toxicity to a q u a t i c o r g a n i s m s is greatly a f f e c t e d by e n v i r o n m e n t a l f a c t o r s . The results of in situ s t u d i e s , in contrast to c o n t r o l l e d laboratory s t u d i e s , c o n s e q u e n t l y b e c o m e even m o r e c o m p l e x . The seasonal cycle alone may cause v a r i a t i o n in the r e s u l t s . B r u n g e et al. (1976) reported that the nominal total Cu 96-h LC50 v a l u e s to fathead m i n n o w s ranged from 1.6 to 21 m g / L , a p p a r e n t l y influenced by the source of d i l u t i o n w a t e r : a natural stream d o w n s t r e a m from a sewage treatment p l a n t . S t o r c h (1977) used a natural community of Lake Erie p h y t o p l a n k t o n for d e t e r m i n i n g the e f f e c t s of F e ( l l l ) . The results showed that F e ( l l l ) m i g h t either s t i m u l a t e or inhibit algal p h o t o s y n t h e s i s d e p e n d i n g on the c o n c e n t r a t i o n and the time of y e a r . The s t i m u l a t i o n , 10 to 18 p e r c e n t , o c c u r r e d in late summer w h e n 0.05 mg/L was a d d e d . E r i c k s o n (1972) found that the Cu inhibition on the growth of T h a l a s s i o s i r a p s e u d o n a n a in u n e n r i c h e d seawater samples varied w i t h season and location of c o l l e c t i o n . Fe toxicity to fresh and m a r i n e water p h y t o p l a n k t o n w a s v a r i a b l e over time ( W a n g , 1983; W i l s o n and F r e e b e r g , 1 9 8 0 ) .

The d e g r e e of d i s s o l v e d oxygen s a t u r a t i o n varies a c c o r d i n g to

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s e a s o n . E x p e r i m e n t a l r e s u l t s have shown that the toxicity of Cd to a q u a t i c insects increased as d i s s o l v e d o x y g e n c o n c e n t r a t i o n increased (Clubb et al., 1 9 7 5 ) . This finding can be e x p l a i n e d by the increase in the a m o u n t of Cd found in the insects as d i s s o l v e d oxygen i n c r e a s e d . They a l s o reported that oxygen c o n s u m p t i o n increased as d i s s o l v e d o x y g e n i n c r e a s e d . S i m i l a r l y , Karbe et al. (1975) reported that a c o r r e l a t i o n e x i s t e d b e t w e e n heavy metal a c c u m u l a t i o n in m u s s e l s and o x y g e n s a t u r a t i o n .

Taylor and C r o w d e r ( 1 9 8 3 ) a n a l y z e d s o i l - s e d i m e n t s a m p l e s and plant t i s s u e s . They found no c o r r e l a t i o n between metal c o n c e n t r a t i o n in tissues or leaves and Z n , M g , or Ca in s o i l - s e d i m e n t , but a high d e g r e e of c o r r e l a t i o n b e t w e e n C u , N i , F e , and Mn c o n c e n t r a t i o n s in the root zone and the r e p r o d u c t i v e tissues and s o i l - s e d i m e n t . A c c u m u l a t i o n of Fe and Mn in all plant tissues w a s c o r r e l a t e d w i t h c o n c e n t r a t i o n s in the s o i l - s e d i m e n t m a t e r i a l .

The c o m b i n a t i o n of Cu c o n c e n t r a t i o n and sediment p a r t i c l e size had an e f f e c t on b e n t h i c m a c r o i n v e r t e b r a t e s , a l t h o u g h the sediment p a r t i c l e size itself a p p e a r e d to have no effect on m i c r o i n v e r t e b r a t e d i s t r i b u t i o n (Kraft and S y p n i e w s k i , 1 9 8 1 ) . Foster ( 1 9 8 2 ) studied algal flora at several s t a t i o n s on s t r e a m s drained through m i n i n g r e g i o n s . Both the total algal a b u n d a n c e and the number of s p e c i e s w e r e d e p r e s s e d at sites of high metal c o n c e n t r a t i o n s . It w a s found that the d e g r e e of metal p o l l u t i o n , rather than the p o l l u t i n g metal c o n c e n t r a t i o n per s e , d e t e r m i n e d the s p e c i e s p r e s e n t .

Spehar and C a r l s o n ( 1 9 8 4 ) c o m p a r e d Cd t o x i c i t y in in-site and laboratory w a t e r . Their results of a c u t e tests with several species showed that Cd w a s less toxic in site water than in laboratory w a t e r . F u r t h e r m o r e , acute tests c o n d u c t e d m o n t h l y in site w a t e r showed that Cd toxicity varied by m o r e than a factor of 3 over the y e a r . W a n g ( 1 9 8 6 C ) e x a m i n e d the effect of river water on the toxicity of B a , C d , and C r ( V I ) to c o m m o n d u c k w e e d , Lemna m i n o r . The results indicated that Cd t o x i c i t y was s l i g h t l y r e d u c e d , Ba toxicity w a s almost n i l , and C r ( V I ) toxicity changed very l i t t l e . The removal of Ba toxicity w a s due to b a r i u m s u l f a t e p r e c i p i t a t i o n .

An i n t e r e s t i n g a l t h o u g h little s t u d i e d effect is that of light i l l u m i n a t i o n on metal toxicity ( D o n g m a n n and Nuernberg, 1 9 8 2 ) . They found that the toxicity t h r e s h o l d s of Cd and Ni on the m a r i n e d i a t o m T h a l a s s i o s i r a rotula d e c r e a s e d w i t h i n c r e a s i n g i l l u m i n a t i o n . The results can be e x p l a i n e d by the greater algal m e t a b o l i c rates due to the a v a i l a b i l i t y of light e n e r g y .

Engler e t a l . ( 1 9 8 2 ) conducted s o l i d - p h a s e plant b i o a s s a y s testing the a v a i l a b i l i t y of heavy m e t a l s . They reported influencing f a c t o r s such as redox p o t e n t i a l , o r g a n i c m a t t e r c o n t e n t , total sulfur c o n t e n t , and pH. They o b s e r v e d that the a v a i l a b i l i t y of Cd and Zn for plant u p t a k e increased in upland c o n d i t i o n s , w h i l e As increased under flooded c o n d i t i o n s .

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S u m m a r y and C o n c l u s i o n s

In s u m m a r y , the a q u a t i c e n v i r o n m e n t a l t o x i c o l o g y of heavy m e t a l s is very c o m p l e x . M a n y d i s c o v e r i e s have been r e p o r t e d only r e c e n t l y , and u n d o u b t e d l y m a n y m o r e will be reported in the f u t u r e . On the basis of this l i t e r a t u r e p r e s e n t a t i o n , there appear to be four basic laws of e n v i r o n m e n t a l t o x i c o l o g y :

First Law ( Q u a n t i t y ) -Any Chemical Can Be T o x i c ; The Q u e s t i o n is Q u a n t i t y .

Second Law ( O r g a n i s m ) -T o x i c i t y Is Not a C o n s t a n t ; It D e p e n d s on the Individual O r g a n i s m .

Third Law ( E n v i r o n m e n t a l C o n d i t i o n s ) -T o x i c i t y Is Not a C o n s t a n t ; It Can Be P o t e n t i a t e d or A t t e n u a t e d

under V a r i o u s E n v i r o n m e n t a l C o n d i t i o n s .

F o u r t h Law ( T o l e r a n c e ) -T o x i c i t y Is Not a C o n s t a n t ; O r g a n i s m s May P o s s e s s Repair M e c h a n i s m s and

D e v e l o p a T o l e r a n c e T o w a r d It.

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4. M e t h o d s

A. W a t e r samples

During the 19-month study period (September 1984 to April 1 9 8 6 ) , m a n y ambient water samples w e r e taken. Fig. 1 d e p i c t s the locations of the 18 sample s t a t i o n s , of w h i c h 10 w e r e in the state of Illinois and 8 were in the neighbor ing states of Indiana, Iowa, M i s s o u r i , and W i s c o n s i n . The s e l e c t i o n of the ten in-state stations was based on 1) g e o g r a p h i c a l d i s t r i b u t i o n and 2) water quality c o n s i d e r a t i o n s . A c c o r d i n g to a 10-year, m o n t h l y m o n i t o r i n g p r o g r a m , these w a t e r s e n c o m p a s s e d a w i d e s p e c t r u m of water q u a l i t y , from low to high a l k a l i n i t y , s o f t n e s s to h a r d n e s s , and low to high amounts of total dissolved s o l i d s , Table 2 (Harmeson and L a r s o n , 1 9 6 9 ) .

All the in-state stations w e r e sampled during the first year of the study. Two in-state stations (at the Illinois River and Lake M i c h i g a n ) and all eight o u t - o f - s t a t e stations w e r e sampled for the remaining m o n t h s of the study. The o u t - o f - s t a t e s t a t i o n s w e r e at Lake G e n e v a , W i s c o n s i n , and seven major r i v e r s , all in areas b o r d e r i n g the state of I l l i n o i s . These w a t e r s are c o n s i d e r e d typical m i d w e s t e r n streams draining through intensive farming a r e a s .

The water samples were collected by using the surface grab m e t h o d . All stations were visited at least t w i c e , and one station (Illinois River at P e o r i a ) was visited 13 t i m e s . Water q u a l i t y p a r a m e t e r s w e r e analyzed a c c o r d i n g to Standard M e t h o d s ( 1 9 8 5 ) .

B. D u c k w e e d culture

The source of duckweed (Lemna m i n o r ) stock c u l t u r e w a s a g r o u n d - w a t e r recharge pit located inside the p r o p e r t y of the Illinois State Water S u r v e y . This water body is in an e n c l o s e d area and no p o l l u t a n t s are known to enter it. The. d u c k w e e d stock culture is the same as that used in p r e v i o u s studies (Wang, 1986B, 1 9 8 6 C ) . F u r t h e r m o r e , this stock culture has been offered as a reference species for duckweed toxicity tests as a part of the A m e r i c a n Society for Testing and M a t e r i a l s standard d e v e l o p m e n t protocol ( W a n g , 1 9 8 6 D ) .

The culture was m a i n t a i n e d in the laboratory in a p o l y p r o p y l e n e tub, 20-L c a p a c i t y . A p p r o x i m a t e l y 15 L was m a i n t a i n e d by adding cold tap water w e e k l y to c o m p e n s a t e for e v a p o r a t i o n loss. Plant n u t r i e n t s w e r e added in a q u a n t i t y twice the c o n c e n t r a t i o n (2X) recommended by Standard M e t h o d s for algal growth m e d i u m (Standard M e t h o d s . 1 9 8 5 ) . The plants w e r e illuminated w i t h c o n t i n u o u s c o o t - w h i t e fluorescent light at 3300 lux. R o o m t e m p e r a t u r e was m a i n t a i n e d at 2 5 - 2 7 C.

Under these favorable c o n d i t i o n s , duckweed grew v i g o r o u s l y and p r o v i d e d sufficient test s p e c i m e n s y e a r - r o u n d . The only time a d i f f i c u l t y was e n c o u n t e r e d w a s w h e n insect infestation reached

27

Fig. 1. Sample s ta t ions.

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Table 2. Mineral Quality Characteristics of Selected Stations in Illinois

Median values in mg/L from 1956-1966 (from Harmeson and Larson, 1969)

Station no. Station Alk Hardness TDS

1 Beaucoup Creek @ Rte. 127 80 585 995 2 Embarras River @ Camargo 205 375 350 3 Fox River @ Algonquin 252 318 400 4 Hayes Creek @ Glendale 45 60 110 5 Horseshoe Lake @ Miller City NA NA NA 6 Illinois River @ Peoria 160 260 400 7 LaMoine River @ Colmar 205 250 320 8 Lake Michigan @ Glencoe 118 130 165 9 Rend Lake @ Ina 60 135 260 10 Sangamon River § Oakford 240 300 380

NA - No t available TDS - Total dissolved solids

29

such p r o p o r t i o n s that the d u c k w e e d stock w a s damaged to a n o t i c e a b l e d e g r e e . N e v e r t h e l e s s , the d u c k w e e d g r e w fast and w a s never in short s u p p l y . The insect p r o b l e m was solved c o m p l e t e l y with only one spray of a b r o a d - s p e c t r u m c o m m e r c i a l l y a v a i l a b l e i n s e c t i c i d e . The d u c k w e e d was not a f f e c t e d by the s p r a y .

M a n y snails w e r e p r e s e n t in the stock c u l t u r e . They c o n s u m e d a n e g l i g i b l e amount of d u c k w e e d p l a n t s c o n s i d e r i n g the d u c k w e e d biomass and the rate of multiplication. No attempt was made to e l i m i n a t e the snail p o p u l a t i o n . T h e r e w e r e a l g a e p r e s e n t in the tub. S i n c e d u c k w e e d formed a dense m a t at the water s u r f a c e , algae had no c h a n c e to reach b l o o m p r o p o r t i o n s .

C. D u c k w e e d test s p e c i m e n s

T w e n t y - f o u r hours b e f o r e the t o x i c i t y e x p e r i m e n t s w e r e i n i t i a t e d , d u c k w e e d s p e c i m e n s w e r e s e l e c t e d for t e s t s . A scoopful of d u c k w e e d from the stock was d i s p e r s e d in a small tub c o n t a i n i n g cold tap w a t e r . The s e l e c t i o n c r i t e r i a for test s p e c i m e n s w e r e that the c o l o n i e s be h e a l t h y - l o o k i n g and u n i f o r m , w i t h two f r o n d s of a p p r o x i m a t e l y equal size per c o l o n y . P l a n t s w h i c h w e r e o v e r - s i z e d , u n d e r - s i z e d , irregularly s h a p e d , d i s c o l o r e d , insect b i t t e n , and the like w e r e not used. P l a s t i c f o r k s , instead of f o r c e p s , w e r e used in order to prevent injuries to the d u c k w e e d p l a n t s . The s e l e c t e d s p e c i m e n s w e r e kept away from l i g h t .

The a d v i s a b i l i t y of using t w o - f r o n d c o l o n i e s instead of t h r e e - and f o u r - f r o n d c o l o n i e s has been q u e s t i o n e d b e c a u s e some d u c k w e e d s p e c i e s ( p a r t i c u l a r l y Lemna g i b b a ) . have very few t w o - f r o n d c o l o n i e s . L. m i n o r w a s p r e f e r r e d over other s p e c i e s for two r e a s o n s . F i r s t , L . m i n o r is a n a t i v e North A m e r i c a n s p e c i e s that is w i d e l y d i s t r i b u t e d , w h i l e L. gibba is m u c h less c o m m o n . The s e l e c t i o n of L . m i n o r as a test s p e c i e s for e n v i r o n m e n t a l p r o t e c t i o n thus m a k e s m o r e sense than the s e l e c t i o n of L. g i b b a . S e c o n d , the s e l e c t i o n of two-frond c o l o n i e s p e r m i t s use of a n a r r o w age g r o u p , w h e r e a s t h r e e - and f o u r - f r o n d c o l o n i e s c o n s i s t of two or m o r e g e n e r a t i o n s .

A n o t h e r i n t e r e s t i n g point has been raised c o n c e r n i n g using an a x e n i c c u l t u r e (a c u l t u r e treated to remove other life f o r m s ) for d u c k w e e d toxicity t e s t s . A x e n i c c u l t u r e s of d u c k w e e d have been reported in the l i t e r a t u r e . H o w e v e r , this w a s not a t t e m p t e d in this s t u d y . The reason is that the m e t h o d s used to a c h i e v e an a x e n i c c u l t u r e ( m e m b r a n e f i l t r a t i o n , chemical t r e a t m e n t , and a u t o c l a v i n g ) invariably alter the w a t e r q u a l i t y of a m b i e n t w a t e r s a m p l e s . For e x a m p l e , a u t o c l a v i n g w o u l d result in c a l c i u m c a r b o n a t e p r e c i p i t a t i o n , a l k a l i n i t y and h a r d n e s s r e d u c t i o n , and p H e l e v a t i o n . M e m b r a n e f i l t r a t i o n w o u l d r e m o v e p a r t i c u l a t e m a t t e r , which is a part of the a m b i e n t water q u a l i t y , p o s s i b l y a f f e c t i n g heavy metal t o x i c i t y . T h e s e t r e a t m e n t s w o u l d defeat the p u r p o s e of this s t u d y .

It may be asked w h e t h e r there is any i n t e r f e r e n c e to the e x p e r i m e n t s from other life f o r m s , e s p e c i a l l y a l g a e . The d u c k w e e d

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plants w e r e u s u a l l y a t t a c h e d w i t h a l g a e ; also the lake and s t r e a m w a t e r s c o n t a i n e d v a r y i n g algal p o p u l a t i o n s . D u c k w e e d p l a n t s , being f l o a t i n g o r g a n i s m s , are not a f f e c t e d to' a large e x t e n t by a l g a e . This is e s p e c i a l l y true for a 96-h e x p o s u r e p e r i o d . During this short t i m e , a l g a e w e r e r e l a t i v e l y few. Any tests of longer d u r a t i o n m i g h t result in algal b l o o m s , m a k i n g i n t e r p r e t a t i o n of d u c k w e e d toxicity test results d i f f i c u l t .

D . Test p r o c e d u r e

The toxicity test e x p e r i m e n t s w e r e p e r f o r m e d by using 2 3 6 - m L fruit jars. For toxicity s t u d i e s , a s e r i e s of metal s o l u t i o n s w e r e p r e p a r e d using a 45 percent d i l u t i o n s c a l e . F r o m a 1-L solution (either d e i o n i z e d water or a m b i e n t water s a m p l e ) c o n t a i n i n g the highest metal c o n c e n t r a t i o n and duckweed g r o w t h m e d i u m (double s t r e n g t h of algal g r o w t h m e d i u m . S t a n d a r d M e t h o d s . 1 9 8 5 ) , a 450-mL p o r t i o n w a s w i t h d r a w n w h i c h served as the h i g h e s t metal c o n c e n t r a t i o n s o l u t i o n . The r e m a i n i n g s o l u t i o n w a s r e p l e n i s h e d w i t h plant n u t r i e n t s to the original c o n c e n t r a t i o n , diluted w i t h the same w a t e r , and m i x e d v i g o r o u s l y . A second 450-mL p o r t i o n w a s w i t h d r a w n , w h i c h served as the s e c o n d - h i g h e s t solution (55 percent metal c o n c e n t r a t i o n of the first s o l u t i o n ) . The p r o c e d u r e w a s repeated until there w e r e seven test s o l u t i o n s , in c o n c e n t r a t i o n series of 1 0 0 , 5 5 , 3 0 , . . . 2.8 p r o p o r t i o n . Each 450-mL p o r t i o n w a s d i v i d e d into three equal p o r t i o n s , w h i c h were placed in three fruit jars. These w e r e c o n s i d e r e d t r i p l i c a t e s .

Two kinds of c o n t r o l s w e r e p r e p a r e d . For the water c o n t r o l s , duckweed growth was tested in a s o l u t i o n c o n t a i n i n g plant n u t r i e n t s in d e i o n i z e d w a t e r ( i . e . , d u c k w e e d growth m e d i u m ) . It is also of interest to d e t e r m i n e d u c k w e e d growth in the a m b i e n t water s a m p l e s c o n t a i n i n g plant n u t r i e n t s . This is called s a m p l e c o n t r o l . W a t e r control and sample control tests w e r e c o n d u c t e d throughout this study along w i t h the toxicity t e s t s .

Immediately before the selected d u c k w e e d s p e c i m e n s w e r e tested, they w e r e e x a m i n e d again for any i r r e g u l a r i t y or m u l t i p l i c a t i o n . Only the p l a n t s w h i c h met the original c r i t e r i a were used. The d u c k w e e d p l a n t s w e r e added at the rate of 30 f r o n d s , or 15 c o l o n i e s , to each jar. A c o n s t a n t i l l u m i n a t i o n w a s p r o v i d e d by c o o l - w h i t e f l u o r e s c e n t b u l b s at an intensity of 6456 lux. Each jar w a s c o v e r e d w i t h a w a t c h g l a s s to prevent e x c e s s i v e e v a p o r a t i o n loss and to keep foreign o b j e c t s , d e b r i s , i n s e c t s , and the like from falling into the jars. T e m p e r a t u r e w a s m a i n t a i n e d at 2 5 - 2 9 C. On three o c c a s i o n s t e m p e r a t u r e e x c e e d e d this range and the e x p e r i m e n t s w e r e r e p e a t e d . The incubation time w a s 96 h.

At the end of i n c u b a t i o n , the number of fronds in each jar was counted w i t h the aid of a lighted m a g n i f y i n g g l a s s . Each r e c o g n i z a b l e , p r o t r u d i n g bud was c o u n t e d . The net increase in frond number w a s indicative of d u c k w e e d growth and the r e s p o n s e to toxicity.

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E. Test c o m p o u n d s

Three reagent grade chemical c o m p o u n d s w e r e tested, BaCl 2 2 H 2 O , K 2 C r O 4 , and NiCl 2 6 H 2 O . Three stock solutions were p r e p a r e d , c o n t a i n i n g 4 0 , 0 0 0 mg/L B a , 10,000 m g / L C r , and 2,000 m g / L N i . The m a x i m u m metal c o n c e n t r a t i o n s in all tests using ambient water samples w e r e 4 0 0 , 100, and 20 m g / L for B a , C r , and N i , r e s p e c t i v e l y . In other w o r d s , a 10-mL metal stock solution w a s added to 1 L water' to m a k e the highest metal c o n c e n t r a t i o n . Toxicity tests using d u c k w e e d growth m e d i u m w e r e also c o n d u c t e d . In this c a s e , the m a x i m u m metal c o n c e n t r a t i o n w a s d e c r e a s e d to 200 m g / L B a , 100 mg/L C r , and 5 m g / L Ni in order to obtain the proper d o s e - r e s p o n s e range.

The chemical assay of the stock s o l u t i o n s showed that the d e t e r m i n e d c o n c e n t r a t i o n s of B a , C r , and Ni w e r e 9 5 , 1 0 0 , and 95 percent of c a l c u l a t e d c o n c e n t r a t i o n s , r e s p e c t i v e l y .

F. G r o w t h m e d i u m

At least six d u c k w e e d growth m e d i a are d i s c u s s e d in the l i t e r a t u r e . It would be a major task to c o m p a r e them. For this s t u d y , it w a s decided to adopt the algal g r o w t h m e d i u m (Standard M e t h o d s , 1985) for the d u c k w e e d culture and test s o l u t i o n s . The reasons are that this m e d i u m is w i d e l y a c c e p t e d , even though there are r e s e r v a t i o n s about it p r i m a r i l y b e c a u s e of the m i c r o n u t r i e n t s tin and s e l e n i u m , and that algae and d u c k w e e d are both plant s p e c i e s . The a d v a n t a g e s are o b v i o u s in terms of c o m p a r i s o n , d e v e l o p m e n t , and s t a n d a r d i z a t i o n of these t e s t s .

The duckweed growth m e d i u m used in this s t u d y , as m e n t i o n e d e a r l i e r , was double the s t r e n g t h (2X) of the algal growth m e d i u m . The reason is given later (see " R e s u l t s " s e c t i o n , " N u t r i e n t s " s u b s e c t i o n ) . The algal m e d i u m outlined in S t a n d a r d M e t h o d s is composed of 6 stock s o l u t i o n s , each of w h i c h c o n t a i n s one chemical c o m p o u n d , and 1 solution c o n t a i n i n g a m i x t u r e of m i c r o n u t r i e n t s . B e c a u s e some c o m p o u n d s are c o m p a t i b l e and together are unlikely to p r o d u c e chemical c h a n g e s , it w a s decided to c o m b i n e a few of them in order to reduce the number of nutrient stock s o l u t i o n s . The s o l u t i o n s are shown in T a b l e 3. It can be seen that in this m a n n e r , only 3 s o l u t i o n s (A, B, and C) are used r e g u l a r l y , instead of the 7 s e p a r a t e stock s o l u t i o n s o u t l i n e d in S t a n d a r d M e t h o d s .

G. Statistical a n a l y s e s

IC50 values (the c o n c e n t r a t i o n w h i c h causes 50 percent inhibition in c o m p a r i s o n with the control s a m p l e ) and 95 percent c o n f i d e n c e limits w e r e c a l c u l a t e d by the binomial m e t h o d ( S t e p h e n , 1 9 7 7 ) , using a personal c o m p u t e r . This m e t h o d p r o d u c e d more realistic values than either the m o v i n g a v e r a g e or probit m e t h o d . Linear regressional a n a l y s i s w a s also used. The t-test was p e r f o r m e d to d e t e r m i n e any significant d i f f e r e n c e b e t w e e n mean values of test g r o u p s .

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Table 3. Nutrient Stock Solutions

Solution A NaNO3 25.5 g/L NaHCO3 15 K2HPO4 1.04

Solution B MgCl2 5.7 CaCl 22H 20 4.41

2 2 Solution C MgSO4 7H20 14.7 H3BO3 0.186 MnSO4 H3O 0.264 1 mL of Solution 0, as follows:

ZnCl2 3.27 mg/L CoCl2 0.78 CuCl2 0.009 NaMoO4 2H2O 7.26 FeCl2 96

Two mL each of solutions A, B, and C are used to make 1 L solution of duckweed growth medium

The methodology described in this section was largely used as the basis of the "Proposed New Standard Guide for Conducting Static Toxicity Tests with Duckweed," for the American Society for Testing and Materials, currently in draft #6 (Wang, 1986D). Interested readers can contact the author for a copy.

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5. R e s u l t s

A. W a t e r quality

W a t e r q u a l i t y c h a r a c t e r i s t i c s of the test samples are given in Table 4. The station n u m b e r s in this table c o r r e s p o n d to the locations in F i g . 1. A c o m p a r i s o n of T a b l e s 2 and 4 indicates t h a t t h e w a t e r q u a l i t y o f t h e i n - s t n t e s t a t i o n s d u r i n g t h i s s t u d y was similar to that during 1 9 5 6 - 1 9 6 6 (also see Fig. 2 ) .

The pH v a l u e s of s a m p l e s from all 18 s t a t i o n s w e r e in the range of 7-8 (Table 4), except for a sample from H a y e s C r e e k , pH 6.32. This station is located inside S h a w n e e N a t i o n a l P a r k . Other water q u a l i t y c h a r a c t e r i s t i c s of this station are e x t r e m e l y low a l k a l i n i t y (10-15 m g / L ) and h a r d n e s s (37-38 m g / L ) . The station w i t h the hardest water is B e a u c o u p C r e e k . This s t a t i o n is p o s s i b l y under the influence of oil e x t r a c t i o n a c t i v i t i e s . It has a very high level of total d i s s o l v e d s o l i d s , 995 m g / L (Table 2 ) .

The s a m p l e s from two glacial l a k e s . Lake M i c h i g a n and Lake G e n e v a , w e r e m o d e r a t e l y a l k a l i n e and m o d e r a t e l y hard. S u r p r i s i n g l y , Rend L a k e , an a r t i f i c i a l i m p o u n d m e n t , w a s less a l k a l i n e and less hard than Lake M i c h i g a n .

B. Nutr ients

The m e t h o d s used in this study w e r e similar to those in p r e v i o u s s t u d i e s (Wang, 1986B, 1 9 8 6 C ) . Early in this s t u d y , it was d e c i d e d to test the effect of n u t r i e n t content on d u c k w e e d g r o w t h . Two samples w e r e u s e d , from the Fox River and Lake M i c h i g a n . Of these two s a m p l e s , the Fox River c o n t a i n e d c o n s i d e r a b l y m o r e plant n u t r i e n t s than Lake M i c h i g a n . For e x a m p l e , the total p h o s p h o r u s and n i t r a t e - n i t r o g e n w e r e 0.12 and 1.58 m g / L for the Fox River s a m p l e , and 0.01 and 0.13 m g / L for the Lake M i c h i g a n s a m p l e . The e x p e r i m e n t w a s c o n d u c t e d by adding d i f f e r e n t levels of plant n u t r i e n t s -- 1X, 2 X , and 3X -- a c c o r d i n g to the level r e c o m m e n d e d in S t a n d a r d M e t h o d s ( 1 9 8 5 ) . Control s a m p l e s , w i t h n o n u t r i e n t s a d d e d , w e r e also used.

The r e s u l t s in Table 5 c l e a r l y s h o w that in the case of the Lake M i c h i g a n s a m p l e , d u c k w e e d g r o w t h w a s s i g n i f i c a n t l y g r e a t e r (P < 0.05) w i t h regular ( 1 X ) s t r e n g t h plant n u t r i e n t s than w i t h o u t n u t r i e n t s (control s a m p l e ) . In the case of the Fox River s a m p l e , d u c k w e e d g r o w t h was s i g n i f i c a n t l y higher than in the control s a m p l e only at d o u b l e s t r e n g t h ( 2 X ) plant n u t r i e n t s . At triple s t r e n g t h ( 3 X ) , there w a s no s i g n i f i c a n t l y greater g r o w t h of d u c k w e e d . C o n s e q u e n t l y it w a s d e c i d e d to use d o u b l e s t r e n g t h of algal g r o w t h m e d i u m in all t e s t s . This was called the d u c k w e e d growth m e d i u m .

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T a b l e 4. W a t e r Q u a l i t y of Test S a m p l e s

No. of Range Station No. Station samples pH Alk* Hard* Turb+

1 Beaucoup Creek @ Rte. 127 2 7.64-7.77 89-116 274-400 50-114 2 Embarras River @ Camargo 2 8.00-8.10 136-214 245-320 6-70 3 Fox River @ Algonquin 2 8.13-8.29 214-227 290-318 17-26 4 Hayes Creek @ Glendale 2 6.32-7.25 10-15 37-38 3-11 5 Horseshoe Lake @ Miller City 2 6.92-7.63 46-73 54-78 11-21 6 Illinois River @ Peoria 13 7.85-8.16 158-236 232-364 33-105 7 LaMoine River @ Col mar 2 7.80-8.06 168-177 252-257 14-16 8 Lake Michigan @ Glencoe 5 7.95-8.12 108-115 140-146 0-137 9 Rend Lake @ Ina 3 7.32-7.78 40-53 84-87 29-54 10 Sangamon River @ Oakford 2 7.95-8.08 186-210 185-298 54 11 Lake Geneva @ Williams Bay. Wl 3 8.08-8.26 186-194 237-242 3-4 12 Kankakee River @ Schneider, IN 3 7.85-8.15 182-207 308-311 13-16 13 Mississippi River @ Fort Madison, IA 3 7.57-8.09 50-160 82-256 70-109 14 Missouri River @ St. Charles, MO 3 7.95-8.12 164-188 225-265 38-258 15 Rock River @ Janesville, Wl 3 8.05-8.32 114-236 160-310 8-28 16 Salt River @ Rte. 79, MO 3 7.30-7.96 50-95 80-129 16-246 17 Skunk River @ Rte. 61, IA 3 7.48-8.27 90-227 126-314 57-414 18 Wabash River @ Perryville, IN 3 7.82-8.34 156-224 244-325 26-156

Total 59 samples * in mg/L as CaCO3 + in NTU

Fig. 2. Comparison of alkalinity of stations in 1956-1966 (Harmeson

and Larson, 1969) and this study, in mg/L as CaCO,-

Table 5. Duckweed Growth (Number of Fronds) in Water Samples, 11/7/84, Enriched with Different Levels of Plant Nutrients

Fox River sample Lake Michigan sample Mean t-test Mean t-test

Control 51, 50, 55 52 38, 36, 38 37 1X 56, 55. 60 57 2.32 64, 55, 67 62 6.73* 2X 58, 66, 59 61 3.06* 58, 70, 61 63 7.00* 3X 63, 59, 67 63 3.97* 81, 62, 85 76 5.43*

* 95% significance level (degrees of freedom, 4)

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C. C o n t r o l s

In view of the long study p e r i o d , a fundamental question can be asked about the health and/or the r e p r o d u c i b i l i t y of the test s p e c i m e n s over time.

The health of the test s p e c i m e n s can be indicated by using results for the control s a m p l e s . If the controls show e x t r e m e v a r i a t i o n , the choice of the test s p e c i m e n s will be q u e s t i o n a b l e . In this s t u d y , both water c o n t r o l s and sample c o n t r o l s w e r e prepared in every test. Equal a m o u n t s of n u t r i e n t s w e r e added to b o t h . Each experiment was conducted w i t h t r i p l i c a t e s for water control and 12 replicates for sample c o n t r o l . F i g . 3 p o o l s all the r e s u l t s .

The m e a n number of duckweed fronds ± standard d e v i a t i o n s for water c o n t r o l s and sample c o n t r o l s w e r e 65 ± 1 2 , and 73 ± 10. The t-test showed that the d i f f e r e n c e in m e a n values between these two c o n t r o l s w a s highly s i g n i f i c a n t CP < 0 . 0 1 ) . O b v i o u s l y duckweed grew better in the sample c o n t r o l s than in the water c o n t r o l s . This is interesting in light of the fact that a total of 59 w a t e r s a m p l e s w e r e c o l l e c t e d , e n c o m p a s s i n g a w i d e v a r i a t i o n in water q u a l i t y . A l t h o u g h equal a m o u n t s of plant n u t r i e n t s w e r e added to both types of c o n t r o l s , it is likely that the sample c o n t r o l s c o n t a i n e d additional plant n u t r i e n t s and/or unknown s t i m u l a n t s w h i c h p r o m o t e d the growth of d u c k w e e d p l a n t s .

The results of this 19-month study p r o v i d e g u i d e l i n e s for d e c i d i n g w h e t h e r results of future d u c k w e e d e x p e r i m e n t s should be a c c e p t e d or rejected. As m e n t i o n e d e a r l i e r , the number of fronds in the water and sample c o n t r o l s w e r e 65 ± 12 and 73 ± 1 0 , r e s p e c t i v e l y . The numbers of r e s p e c t i v e tests w e r e 61 and 5 8 . Taking 2 as the a p p r o x i m a t e t-value (degrees of freedom 6 0 ) , the 95 percent c o n f i d e n c e limits are r e s p e c t i v e l y 41-89 and 5 3 - 9 3 . In other w o r d s , any control test that gives values that are o u t l i e r s of these ranges will be a cause of c o n c e r n . The health of test s p e c i m e n s , e n v i r o n m e n t a l c o n d i t i o n s , c o n t a m i n a t i o n , e t c . , all r e q u i r e close e x a m i n a t i o n . D u r i n g this s t u d y , there was only one instance w h e r e duckweed growth in water controls e x c e e d e d the upper limit C90 in c o m p a r i s o n w i t h 8 9 ) . There w e r e three instances w h e r e duckweed growth in sample controls e x c e e d e d the upper limit ( 9 5 , 9 6 , and 97 in c o m p a r i s o n with 9 3 ) . It is likely that additional plant n u t r i e n t s w e r e present in the w a t e r s a m p l e s in these c a s e s . There was no instance w h e r e the growth fell b e l o w the Iower limit.

B e c a u s e of the d i s c r e p a n c y between duckweed growth in water c o n t r o l s and sample c o n t r o l s , metal toxicity data w e r e c a l c u l a t e d only on the basis of sample control v a l u e s .

D. R e p e a tability

The Skunk River sample c o l l e c t e d on March 3, 1 9 8 6 , w a s tested twice for b a r i u m and c h r o m i u m t o x i c i t y . The results in terms of p e r c e n t inhibition of duckweed growth are shown in F i g s . 4 and 5. The m e a n n u m b e r s of duckweed fronds ± standard d e v i a t i o n s for the

37

Fig. 3. Negative controls.

Fig. 4. Duplicate tests of enriched Skunk River water sample, 3/3/86,

Ba toxicity.

39

Fig. 5. Duplicate tests of enriched Skunk River water sample, 3/3/86,

Cr toxicity.

40

water c o n t r o l s in Tests I and II w e r e 63 ±. 6 and 75 ± 3 (replication 3 ) , r e s p e c t i v e l y , w h i l e for the sample controls the mean numbers of duckweed fronds w e r e 83 ± 7 and 74 ± 4, r e s p e c t i v e l y . The results in F i g s . 4 and 5 indicated that there was some v a r i a t i o n between the two t e s t s ; h o w e v e r , the v a r i a t i o n was relatively m i n o r .

E. Ba toxicity

i. In duckweed growth m e d i u m

Between January 1985 and January 1986, a total of seven tests w e r e conducted to determine Ba toxicity to duckweed in the duckweed growth m e d i u m . The results of these tests are s u m m a r i z e d in Fig. 6.

The m a x i m u m Ba c o n c e n t r a t i o n was 200 m g / L . W i t h seven test solutions in a 45 percent dilution s c a l e , the semi-log d o s e - r e s p o n s e relationship is a sigmoid c u r v e . The point of tangent is situated at a p p r o x i m a t e l y 55 percent inhibition of duckweed g r o w t h . The IC50 value was 25 m g / L w i t h a 95 percent confidence limit of 18-33 m g / L .

A c c o r d i n g to the literature (Fig. 7 ) , the certainty of dose-effect r e l a t i o n s h i p s is greater at the extreme high and extreme low c o n c e n t r a t i o n r a n g e s , and lower in the m i d d l e range (Whyte and B u r t o n , 1 9 8 0 ) . The empirical results in Fig. 6 suggest a higher degree of c e r t a i n t y , as suggested by the lower coefficient of v a r i a t i o n , at the extreme high c o n c e n t r a t i o n range. For e x a m p l e , at 200 mg/L Ba c o n c e n t r a t i o n , the coefficient of variation w a s 3 p e r c e n t . This is m u c h smaller than the values of 48 and 21 percent at 0 and 33 mg/L c o n c e n t r a t i o n s , r e s p e c t i v e l y .

ii. In water samples ( n u t r i e n t - e n r i c h e d )

Ba toxicity results in enriched water samples are expressed as IC50 v a l u e s , in m g / L , in Table 6. The mean values range from 102 and 107 m g / L for Hayes Creek and H o r s e s h o e Lake to values out of range in the test for Beaucoup C r e e k . In fact, the B e a u c o u p Creek s a m p l e s taken on December 4, 1984 and March 7, 1985 exhibited 0 and 6 percent duckweed growth inhibition when 400 m g / L Ba was a d d e d , attesting to the strong d e t o x i f i c a t i o n capacity at this s t a t i o n .

The most extensive testing was conducted using the Illinois River s a m p l e s . A total of 13 samples w e r e tested and the IC50 results can be seen in Table 6. In six, or nearly h a l f , of these samples 400 mg/L Ba did not cause over 50 percent inhibition, so that IC50's are given as >400 m g / L . The p e r c e n t a g e s of inhibition in these six samples were only 4 6 , 3 7 , 2 9 , 2 5 , 4 5 , and 4 6 .

The results in Table 6 indicate that, in g e n e r a l , lake water samples show a m o r e consistent response to Ba toxicity than do river water s a m p l e s . The IC50's in water samples from Horseshoe'

41

Fig. 6. Ba toxicity in duckweed growth medium, 7 tests.

42

Fig. 7. Generalized exposure-effect curve, showing diameter of circles

as degree of certainty about data points. From Whyte and Burton, 1980, p. 64. Reproduced by permission of SCOPE, #15, Paris, France.

43

Table 6. Ba Texicity in Enriched Water Samples, in IC50's

IC50, mg/L Mean

1. Beaucoup Creek >400 >400 2. Embarras River 300 328 314 3. Fox R. 155 337 246 4. Hayes Cr. 73 130 102 5. Horseshoe Lake 155 58 107 6. Illinois R. 280 >400 M O O >400 >400

>400 333 265 400 300 M O O 232 326

7. LaMoine R. 310 M O O 8. L. Michigan 121 135 104 118 120 9. Rend L. 189 194 143 175 10. Sangamon R. 330 M O O 11. L. Geneva 148 149 113 137 12. Kankakee R. 328 365 372 355 13. Mississippi R. 151 52 173 125 14. Missouri R. 386 331 M O O 15. Rock R. 156 113 135 16. Salt R. 61 104 143 103 17. Skunk R. 245 152 18. Wabash R. 345 220 230 265

44

L a k e , Lake M i c h i g a n , Rend L a k e , and Lake G e n e v a all fell in a r e l a t i v e l y n a r r o w range w i t h m e a n IC50's of 1 0 7 , 1 2 0 , 1 7 5 , and 137 m g / L , r e s p e c t i v e l y . In the river w a t e r s a m p l e s , the mean IC50's varied from lows of 102 and 103 m g / L for H a y e s Creek and Salt River to well over 400 m g / L for B e a u c o u p C r e e k .

The d o s e - r e s p o n s e r e l a t i o n s h i p s for typical s a m p l e s are d e p i c t e d in F i g . 8. The r e l a t i o n s h i p for the d u c k w e e d growth m e d i u m is a l s o included for c o m p a r i s o n . O b v i o u s l y Ba toxicity in v a r i o u s natural w a t e r s fell between the e x t r e m e s of that in H a y e s Creek or H o r s e s h o e Lake and that in B e a u c o u p C r e e k .

The water s a m p l e s i n v e s t i g a t e d in this study can be c l a s s i f i e d into three g r o u p s on the basis of Ba toxicity r e s u l t s . One p o s s i b i l i t y is to d i v i d e Ba IC50's into < 1 5 0 , 1 5 1 - 3 0 0 , and >300 m g / L (Table 7 ) . A c c o r d i n g t o this s c h e m e , seven sample s t a t i o n s w e r e in the highly s e n s i t i v e to Ba toxicity c a t e g o r y , seven w e r e in the less s e n s i t i v e c a t e g o r y , and the remaining four w e r e in the m o d e r a t e l y s e n s i t i v e c a t e g o r y . It is i n t e r e s t i n g to note that the s t a t i o n s at the M i s s i s s i p p i River at Fort M a d i s o n , Iowa, and Salt River at R o u t e 7 9 , M i s s o u r i , both had water s a m p l e s that w e r e highly s e n s i t i v e to Ba t o x i c i t y , w h i l e the samples from the M i s s o u r i River at S t . C h a r l e s , M i s s o u r i , w e r e less s e n s i t i v e to Ba t o x i c i t y .

A p r e v i o u s study ( W a n g , 1 9 8 6 C ) showed that Ba t o x i c i t y in Illinois River w a t e r w a s c o n t r o l l e d by the s u l f a t e content of the water s a m p l e . Other w a t e r quality p a r a m e t e r s such as s u s p e n d e d solids had little e f f e c t . C o n s e q u e n t l y , an a t t e m p t w a s m a d e to c o r r e l a t e s u l f a t e c o n c e n t r a t i o n and Ba IC50 v a l u e s for all s a m p l e s . All r e s u l t s , e x c l u d i n g those w i t h IC50's greater than 400 m g / L , are pooled in F i g . 9. A linear r e l a t i o n s h i p w i t h a r e l a t i v e l y high c o e f f i c i e n t of d e t e r m i n a t i o n w a s o b t a i n e d , R = 0.68. This indicates that Ba toxicity in a w a t e r s a m p l e is c o n t r o l l e d by s u l f a t e c o n c e n t r a t i o n to a large e x t e n t . F a c t o r s other than s u l f a t e c o n t e n t a f f e c t e d only 32 p e r c e n t v a r i a t i o n of Ba t o x i c i t y .

F. Cr toxicity

i. In d u c k w e e d growth m e d i u m

Cr toxicity in the duckweed g r o w t h m e d i u m w a s tested ten t i m e s . The results are p l o t t e d in F i g . 10. The s e m i - l o g plot of the d o s e - r e s p o n s e relationship, is linear w i t h near p e r f e c t c o r r e l a t i o n of d e t e r m i n a t i o n , R = 0.99. The IC50 value w a s 16 m g / L and the 95 p e r c e n t c o n f i d e n c e limit w a s 9.2-17 m g / L .

i i . In Illinois River water ( e n r i c h e d )

T h i r t e e n Illinois River water s a m p l e s w e r e taken b e t w e e n O c t o b e r 1984 and F e b r u a r y 1986 and tested for Cr t o x i c i t y . The results are d e p i c t e d in F i g . 11.

45

Fig. 8. Ba toxicity in various water samples (enriched).

Table 7. Classification of Ba Toxicity in Various Enriched Water Samples

Ba IC50 Stations mg/L

Highly <150 Hayes C r e e k , H o r s e s h o e L a k e , L. M i c h i g a n sensitive L. G e n e v a , Mississippi R i v e r , Rock R.,

Salt R.

M o d e r a t e l y 151-300 Fox R., Rend L., Skunk R., W a b a s h R. sensitive

Less >300 Beaucoup C., Embarras R., Illinois R., sensitive LaMoine R., Sangamon R., Kankakee R.,

Missouri R.

47

Fig. 9. Ba toxicity (IC50's) and SO4, concentration relationship.

48

Fig. 10. Cr toxicity in duckweed growth medium, 10 tests.

49

Fig. 11. Duckweed growth in water controls ( ) and in Illinois River sample controls ( ) and Cr toxicity in duckweed growth medium ( and in Illinois River samples ( ).

In this f i g u r e , the IC50's of individual samples are given as open c i r c l e s , w h i l e the d u c k w e e d net g r o w t h values of the s a m p l e controls are given as solid c i r c l e s . The results of three Cr toxicity tests during this period that used duckweed growth m e d i u m are also shown for c o m p a r i s o n . (The remaining tests using duckweed g r o w t h m e d i u m w e r e c o n d u c t e d o u t s i d e of this time frame and are shown in F i g . 1 3 ) . The IC50 v a l u e s for the d u c k w e e d growth m e d i u m and the d u c k w e e d net growth in the water c o n t r o l s are shown as open t r i a n g l e s and solid t r i a n g l e s , r e s p e c t i v e l y . The mean and standard d e v i a t i o n of the Cr ICSO's in the Illinois River samples are 12 ± 3 m g / L . This m e a n value of 13 s a m p l e s compares f a v o r a b l y w i t h that in the d u c k w e e d growth m e d i u m , 16 m g / L , from 10 t e s t s .

There a p p e a r s to be no r e l a t i o n s h i p b e t w e e n IC50 v a l u e s and duckweed g r o w t h .

From the combined results of all Illinois River w a t e r s a m p l e s , a d o s e - r e s p o n s e r e l a t i o n s h i p can be shown (Fig. 1 2 ) . As in F i g . 1 0 , a linear s e m i - l o g r e l a t i o n s h i p was o b t a i n e d . The regressional a n a l y s i s indicated that it is highly c o r r e l a t e d , w i t h R2 = 0.99. The r e g r e s s i o n e q u a t i o n s are Y = -5 + 45.2 log X for the d u c k w e e d g r o w t h m e d i u m (Fig. 10) and Y = -4 + 50.6 log X for the Illinois River water s a m p l e s . They are s t r i k i n g l y s i m i l a r .

i i i . In all water samples ( e n r i c h e d )

Table 8 s u m m a r i z e s Cr toxicity v a l u e s in terms of IC50's in all water s a m p l e s . The results show that they are in a r e l a t i v e l y n a r r o w r a n g e , 3.5 - 29 m g / L , w i t h a v a r i a t i o n of less than one order of m a g n i t u d e . The m e a n IC50 value is 12 mg/L and the standard d e v i a t i o n is 4.7 m g / L . The c o e f f i c i e n t of v a r i a t i o n is 4 1 p e r c e n t .

Many c o n c u r r e n t tests w e r e c o n d u c t e d using seven Cr c o n c e n t r a t i o n s in d u c k w e e d growth m e d i u m and in water s a m p l e s . The water s a m p l e s w e r e from ten s t a t i o n s : stations 11 through 18 (Table 4 ) , Lake M i c h i g a n , and the Illinois R i v e r . The results are p r e s e n t e d in F i g . 13. With the d i v e r s e water quality in the samples used in this s t u d y , it is interesting to note that a linear r e l a t i o n s h i p existed b e t w e e n the Cr toxicity in the a m b i e n t w a t e r s and in the growth m e d i u m . The c o r r e l a t i o n of d e t e r m i n a t i o n was very h i g h , R = 0.88.

When the results for all the water s a m p l e s are c o m b i n e d , the g e n e r a l i z e d d o s e - r e s p o n s e r e l a t i o n s h i p of Cr toxicity to d u c k w e e d growth can be d e p i c t e d as in F i g . 14. This d i a g r a m can be considered the general r e l a t i o n s h i p for Cr toxicity to d u c k w e e d p l a n t s . The m e a n IC50 v a l u e w a s 11 m g / L and the 95 p e r c e n t c o n f i d e n c e limit w a s 9.2-17 m g / L .

51

Fig. 12. Cr toxicity in enriched Illinois River water samples, 13 tests.

52

Table 8. Cr Toxicity in Enriched Water Samples, in IC50's

IC50, mg/L Mean, mg/L

1. Beaucoup Creek 16 13 15 2. Embarras River 7.3 6.4 6.9 3. Fox R. 13 12 13 4. Hayes C. 10 11 11 5. Horseshoe Lake 14 9.2 12 6. Illinois R. 12 14 13 11 15

14 5.5 8.3 8.4 11 9.6 16 13 12

7. LaMoi ne R. 9.6 10 10 8. L. Michigan 6.8 9.2 16 23 11 13 9. Rend L. 29 21 13 21 10. Sangamon R. 8.7 7.5 8 11. L. Geneva 14 7.9 16 13 12. Kankakee R. 16 6.3 14 12 13. Mississippi R. 10 5.5 6.5 7.3 14. Missouri R. 8.9 4.5 13 8.3 15. Rock R. 12 8.8 19 13 16. Salt R. 13 3.5 8.2 8.2 17. Skunk R. 6.8 7.5 7.2 18. Wabash R. 18 8.3 12 13

53

Fig. 13. Duckweed growth in growth medium and 10 surface waters (enriched) containing Cr ion.

54

Fig. 14. Generalized Cr concentration-effect relationship in 18 surface waters (enriched).

55

G. N i toxicity

i . In duckweed growth m e d i u m

Ni toxicity to duckweed using the duckweed growth m e d i u m was tested seven times. The results are s u m m a r i z e d in Fig. 15. The d o s e - r e s p o n s e relationship a p p e a r s to be a sigmoid p a t t e r n , with the point of tangent situated at a p p r o x i m a t e l y 0.46 mg/L Ni c o n c e n t r a t i o n and 60 percent growth i n h i b i t i o n . The highest Ni c o n c e n t r a t i o n used in the growth m e d i u m tests w a s 5 m g / L . In c o m p a r i s o n , 20 mg/L Ni was the m a x i m u m c o n c e n t r a t i o n used for the water s a m p l e s . The c o m p o s i t e Ni toxicity data give an IC50 value of 0.33 mg/L Ni with a 95 percent c o n f i d e n c e limit of 0.25 to 0.46 m g / L .

i i . In Hayes Creek and H o r s e s h o e Lake s a m p l e s ( e n r i c h e d )

Among all the ambient water s a m p l e s , H a y e s Creek and H o r s e s h o e Lake showed the most s e n s i t i v e response to Ni toxicity. For e x a m p l e , the IC50's w e r e 0.36 and 0.21 m g / L for these two s t a t i o n s , r e s p e c t i v e l y , w h i l e the other 16 stations pooled together showed mean and standard d e v i a t i o n values of 2.5 ± 0.83 m g / L . C o n s e q u e n t l y , results for these two stations w e r e treated s e p a r a t e l y .

The results for four water samples from these two stations are given in Table 9. The highest Ni c o n c e n t r a t i o n was set at 5 m g / L , the same as that used in the growth m e d i u m e x p e r i m e n t s . An e x c e p t i o n was the Hayes Creek sample taken December 4, 1 9 8 4 , in which a m a x i m u m c o n c e n t r a t i o n of 20 m g / L was tested. Because these two stations behaved strikingly s i m i l a r l y , the results were c o m b i n e d .

The d o s e - r e s p o n s e r e l a t i o n s h i p is nearly linear in the semi-log plot in Fig. 16. This feature is somewhat different from that of using the growth m e d i u m (Fig. 1 5 ) . An interesting point is that at 0.14 mg/L Ni c o n c e n t r a t i o n , the percent duckweed growth inhibition was 29 ± 10 percent and 40 ± 8 percent respectively for the growth m e d i u m and the H a y e s C r e e k - H o r s e s h o e Lake s a m p l e s . A t-test indicated, h o w e v e r , that the d i f f e r e n c e was i n s i g n i f i c a n t .

i i i . In Illinois River water ( e n r i c h e d )

There w e r e a total of 12 Illinois River water samples tested for the effect- of Ni t o x i c i t y . The samples w e r e taken from O c t o b e r 1984 until F e b r u a r y 1986. The r e s u l t s of the Ni toxicity tests are given in Table 10. These results indicate that the Ni toxicity to duckweed w a s in a relatively n a r r o w r a n g e . The m a x i m u m and m i n i m u m IC50 v a l u e s w e r e 4.4 and 0.97 m g / L , r e s p e c t i v e l y . The mean value of the IC50's was 2.8 with a standard d e v i a t i o n of 0.97 m g / L .

56

Fig. 15. Ni toxicity in duckweed growth medium, 7 tests.

57

Table 9. Ni Toxicity in Enriched Hayes Creek and Horseshoe Lake Samples, in Percent Growth Inhibition

Ni concentration, mg/L 0.14 0.25 0.46 0.83 1.51 2.75 5

Hayes Creek 12/4/84 - - 49 60 76 88 99 3/7/85 49 58 69 77 80 90 95

Hor seshoe Lake 3/19/85 32 52 55 68 74 83 86 8/13/85 40 60 78 84 95 98 100

Mean 40 57 63 72 81 90 95 SD 8.5 4 13 10 10 6 6

58

Fig. 16. Ni toxicity in enriched Hayes Creek and Horseshoe Lake samples, 4 tests.

59

Table 10. Ni Toxicity in Enriched Illinois River Water Samples, in Percent Growth Inhibition

Sample Ni concentration. mg/L dates 0.55 1.0 1.8 3.3 6.1 11 20 IC50 95% C.L.

mg/L 10/8/84 17 12 22 43 53 78 95 3.5 3.3-6.1 1/21/85 15 24 23 38 64 85 91 4.4 3.3-6.1 2/4 15 15 37 51 68 79 87 3.2 1.8-3.3 2/18 25 30 45 58 70 77 87 2.3 1.8-3.3 7/1 17 19 30 53 73 87 94 2.8 1.8-3.3 7/15 33 23 29 41 65 80 97 3.9 3.3-6.1 7/29 35 38 48 66 75 89 89 1.9 1.8-3.3 9/16 31 51 54 72 79 95 100 0.97 0.55-1.0 10/7 14 12 30 66 84 91 98 2.4 1.8-3.3. 1/13/86 4 4 27 43 67 77 80 3.7 3.3-6.1 2/3 14 24 35 48 80 92 97 3.1 3.3-6.1 2/17 30 30 47 61 82 85 94 2.0 1.8-3.3

60

The Ni toxicity results are plotted in F i g . 17, along with the duckweed net growth results for the control tests. As w i t h the Cr results (Fig. 1 1 ) , the IC50's w e r e independent of the duckweed growth in the sample c o n t r o l s . U n l i k e with the Cr r e s u l t s , Ni toxicity to duckweed was very m u c h reduced in the Illinois River water samples in comparison with- that in the growth m e d i u m . The former was 2.8 mg/L expressed as the Ni IC50 v a l u e , while the latter Was 0.33 m g / L . The a t t e n u a t i o n of Ni toxicity in the Illinois River was thus greater than 8-fold.

The d o s e - r e s p o n s e relationship of the Ni ion in the Illinois River water samples is presented in Fig. 18. A typical sigmoid curve is o b t a i n e d . The point of tangent was situated at a p p r o x i m a t e l y the 3.8 mg/L Ni c o n c e n t r a t i o n where duckweed growth inhibition was 55 p e r c e n t .

iv. In all water samples ( e n r i c h e d )

Ni toxicity in all the water samples is summarized in Table 11. The results show that Ni toxicity in all water samples is in a very n a r r o w range. E x c l u d i n g the results for Hayes Creek and H o r s e s h o e Lake s a m p l e s , the mean value for the' remaining 16 stations was 2.5 mg/L w i t h a standard d e v i a t i o n of 0.83 m g / L . If these two stations are also included, the c o r r e s p o n d i n g overall values w e r e 2.2 and 1.0 m g / L .

Even though Ni toxicity results appeared to be in a relatively n a r r o w r a n g e , the sample can be c l a s s i f i e d into three groups as in Table 12. The samples w h i c h are highly s e n s i t i v e to Ni toxicity include those from Hayes Creek and H o r s e s h o e L a k e . These s a m p l e s showed a toxic response nearly identical to that of the growth m e d i u m , with Ni IC50 values s u b s t a n t i a l l y below 1 m g / L . The Illinois R i v e r , Missouri R i v e r , Rock R i v e r , and W a b a s h River all a t t e n u a t e d Ni toxicity greatly (IC50's > 3 m g / L ) , causing Ni toxicity m u c h less than in the growth m e d i u m . The samples from the great m a j o r i t y of stations (IC50's 1-3 m g / L ) m o d e r a t e l y attenuated Ni toxicity.

C o m b i n i n g Ni toxicity results from the 16 stations y i e l d s a g e n e r a l i z e d d o s e - r e s p o n s e relationship as presented in F i g . 19. This r e l a t i o n s h i p is useful as an e s t i m a t e of Ni toxicity in ambient w a t e r s , except in the extremely s o f t , c l e a n , forested s t r e a m s , for w h i c h the relationship in Fig. 16 is m o r e a p p r o p r i a t e .

61

Fig. 17. Duckweed growth in water controls ( ) and in Illinois River sample controls ( ) and Ni toxicity in duckweed growth medium ( and in Illinois River samples ( ).

Fig. 18. Ni toxicity in enriched Illinois River water samples, 12 tests.

63

Table 11. Ni Toxicity in Enriched Water Samples, in IC50's

IC50. mg/L Mean, mg/L

1. Beaucoup Creek 3.8 1.8 2.8 2. Embarras River 2.6 2.3 2.5 3. Fox R. 2.5 2.0 2.3 4. Hayes C. 0.56 0.15 0.36 5. Horseshoe Lake 0.24 0.18 0.21 6. Illinois R. 3.5 4.4 3.2 2.3 3.9

5.7 3.7 3.1 5.0 2.8 1.9 0.97 2.4 3.1

7. LaMo ine R. 2.9 1.3 2.1 8. L.Michigan 1.3 1.2 0.77 1.1 1.1 1.1 9. Rend L. 2.1 1 . 2 0 . 6 4 1.3 10. Sangamon R. 3.8 1.7 2.7 11. L. Geneva 1.8 0.85 4.6 2.4 12. Kankakee R. 1.9 1.6 3.7 2.4 13. Mississippi R. 1.9 0.79 2.2 1.6 14. Missouri R. 2.5 1.5 8.1 4.0 15. Rock R. 3.8 2.6 4.7 3.7 16. Salt R. 1.6 1.5 1.7 1.6 17. Skunk R. 3.1 1.5 2.3 18. Wabash R. 1.9 2.5 5.7 3.4

(16 stations) C18 stations) 2 . 5 1 ± 0.83 2.2 ± 1.0

Table 12. Classification of Ni Toxicity in Various Water Samples

Ni IC50 mg/L

Highly <1 Hayes Creek, Horseshoe Lake sensitive

Moderately 1-3 Beaucoup C, Embarras R., Fox R., LaMoine R., sensitive L. Michigan, Rend L., Sangamon R., L. Geneva,

Kankakee R., Mississippi R., Salt R., Skunk R.

Less >3 Illinois R., Missouri R., Rock R., Wabash R. sensitive

64

Fig. 19. Ni concentration-effect relationship in 16 surface water samples (enriched).

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6. D i s c u s s i o n

C u r r e n t water q u a l i t y c r i t e r i a rely p r i m a r i l y on values d e r i v e d from laboratory s t u d i e s . For e x a m p l e , the R u l e s and R e g u l a t ions. State of I l l i n o i s , listed the s t a n d a r d s for the chemical c o n s t i t u e n t s B a , Cr ( V I ) , and Ni for s e c o n d a r y contact and indigenous a q u a t i c o r g a n i s m s as 5, 0.3 and 1.0 m g / L , r e s p e c t i v e l y , all in total form (State of I l l i n o i s , 1 9 8 6 ) . A catch-all s e c t i o n is p r o v i d e d w h i c h s t a t e s that "Any s u b s t a n c e toxic to a q u a t i c life not listed in S e c t i o n 3 0 2 . 4 0 7 shall not e x c e e d one half of the 9 6 - h o u r m e d i a n t o l e r a n c e limit (96-hour T L m ) for n a t i v e fish and essential fish food o r g a n i s m s . " T h e r e are three important issues h e r e .

A. Importance of a q u a t i c v e o e t a t i o n

A healthy e n v i r o n m e n t is one in w h i c h o r g a n i s m s are in a b a l a n c e d and d y n a m i c s t a t e . In that s t a t e , plant life as a p r o d u c e r p r o v i d e s o x y g e n and o r g a n i c s u b s t a n c e s , w h i l e a n i m a l s and m i c r o o r g a n i s m s c o n s u m e , d e c o m p o s e , and recycle these s u b s t a n c e s back to plant life, m a k i n g a c o m p l e t e c y c l e . In this t r i p a r t i t e e c o s y s t e m , each part ( p l a n t s , a n i m a l s , and m i c r o o r g a n i s m s ) has an e s s e n t i a l role to p l a y . An e m p h a s i s on any one part w i t h o u t a d e q u a t e l y a d d r e s s i n g the other p a r t ( s ) is u n l i k e l y to lead to a h e a l t h y e n v i r o n m e n t ( W a n g , 1 9 8 4 ) . The Illinois River o f f e r s an i n t e r e s t i n g e x a m p l e .

The Illinois River and its t r i b u t a r i e s are an important asset to the State of Illinois as a major water r e s o u r c e . The annual p e r - a c r e yield of fish from the river d e c l i n e d d r a s t i c a l l y in the Illinois River b e t w e e n the 1950's and 1 9 7 0 ' s , w h i l e that in the Upper M i s s i s s i p p i River a c t u a l l y increased s l i g h t l y (Sparks and S a n d u s k y , 1 9 8 1 ) :

I l l i n o i s River lbs/acre Upper M i s s i s s i p p i River Ibs/acre 1 9 5 0 - 1 9 5 9 38.1 1 9 5 3 - 1 9 6 2 26.3 1 9 7 3 - 1 9 8 0 3.7 1 9 7 3 - 1 9 7 7 2 9 . 2

The d e c l i n e has been s p e c u l a t e d to be due to fish food d e c l i n e . In the m e a n t i m e , B e l l r o s e et al. ( 1 9 7 9 ) noted a general d e c l i n e of a q u a t i c v e g e t a t i o n in the b o t t o m lakes of the r i v e r . They p o i n t e d out four important factors a f f e c t i n g the food sources of w a t e r f o w l : 1) f l u c t u a t i n g w a t e r l e v e l s , 2) t u r b i d i t y , 3) water d e p t h , and 4) c o m p e t i t i o n by other p l a n t s .

In a recent news story (Peoria Journal S t a r , April 2 3 , 1 9 8 6 , page D 1 0 ) , large fish kills involving s m a l l m o u t h b a s s , green s u n f i s h , and other s p e c i e s w e r e r e p o r t e d . The s u s p e c t e d pollutant was a h e r b i c i d e , S o n a l a n . B e c a u s e h e r b i c i d e s are c o m m o n l y 3 to 4 o r d e r s of m a g n i t u d e m o r e toxic to p l a n t s than to a n i m a l s (Bishop and P e r r y , 1 9 8 1 ) , is it not logical to suspect that the p h y t o t o x i c i t y has done untold d a m a g e s to a q u a t i c v e g e t a t i o n ? W i t h the m a g n i t u d e s of h e r b i c i d e s ( m i l l i o n s of p o u n d s a n n u a l l y ) applied by farmers and h o m e o w n e r s , is it u n l i k e l y that the a q u a t i c

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v e g e t a t i o n d e c l i n e s in the face of this c h r o n i c h e r b i c i d e e x p o s u r e ? It is not likely that the loss of a q u a t i c v e g e t a t i o n by i t s e l f , and m o r e likely in c o m b i n a t i o n w i t h other f a c t o r s , c a u s e s fish d e c l i n e , w a t e r f o w l d e c r e a s e , and w a t e r q u a l i t y d e t e r i o r a t i o n m a n i f e s t e d by e r o s i o n , s i l t a t i o n , and other ill e f f e c t s ? On the basis of this d e d u c t i o n , is it not an i n a d e q u a t e a p p r o a c h to p r o t e c t the a q u a t i c e n v i r o n m e n t by p r o t e c t i n g only "fish and e s s e n t i a l fish food o r g a n i s m s " ?

B . S i t e - s p e c i f i c w a t e r q u a l i t y c r i t e r i a

T h e current water q u a l i t y c r i t e r i a , a s m e n t i o n e d p r e v i o u s l y , rely on single v a l u e s (State of I l l i n o i s , 1 9 8 6 ) or m a x i m u m and a v e r a g e c o n c e n t r a t i o n s ( U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , 1 9 8 2 ) . The results of this r e s e a r c h p r o j e c t , h o w e v e r , point out a n o t h e r area of c o n c e r n .

W a t e r q u a l i t y v a r i e s from one s t a t i o n to a n o t h e r and from one time to a n o t h e r . Under these d y n a m i c c i r c u m s t a n c e s , the t o x i c i t y of a s u b s t a n c e may be m o d i f i e d and a l t e r e d a c c o r d i n g l y . The m o d i f i c a t i o n of t o x i c i t y , f u r t h e r m o r e , is t o x i c a n t - s p e c i f i c . On the b a s i s of the e x p e r i m e n t a l r e s u l t s , B a , C r , and Ni t o x i c i t i e s in a m b i e n t w a t e r s can be s u m m a r i z e d as f o l l o w s :

Ba - Its p h y t o t o x i c i t y is the least among the three m e t a l s and is influenced the m o s t by the w a t e r q u a l i t y of the test s a m p l e s .

Cr - Its p h y t o t o x i c i t y is m o d e r a t e among the three m e t a l s and is influenced the least by the w a t e r q u a l i t y of the test s a m p l e s .

Ni - Its p h y t o t o x i c i t y is the g r e a t e s t a m o n g the three m e t a l s and is i n f l u e n c e d m o d e r a t e l y by the w a t e r q u a l i t y of the test s a m p l e s .

C o n s e q u e n t l y , the Cr w a t e r q u a l i t y c r i t e r i o n m a y be a d o p t e d u n i v e r s a l l y for all s u r f a c e w a t e r s . The Ba c r i t e r i o n C5.0 m g / L ) can be s u b s t a n t i a l l y m o d e r a t e d a c c o r d i n g to w a t e r q u a l i t y . The Ni c r i t e r i o n C1.0 m g / L ) as stated in the R u l e s and R e g u l a t i o n s (State of I l l i n o i s , 1986) is e x t r e m e l y i n a d e q u a t e . At this c o n c e n t r a t i o n , a 30 p e r c e n t g r o w t h i n h i b i t i o n of a q u a t i c v e g e t a t i o n (based on d u c k w e e d as the test o r g a n i s m ) can be e x p e c t e d in almost all s u r f a c e w a t e r s ( F i g . 1 9 ) , and 70 p e r c e n t i n h i b i t i o n can be e x p e c t e d in e x t r e m e l y soft w a t e r s (Fig. 1 6 ) .

C . R e f e r e n c e t o x i c a n t

For c o n d u c t i n g b i o l o g i c a l t o x i c i t y t e s t s , it is e s s e n t i a l to a s c e r t a i n the health and s u i t a b i l i t y of test o r g a n i s m s . The common p r a c t i c e is to include a control test in w h i c h the e x p e r i m e n t a l c o n d i t i o n is identical to the test c o n d i t i o n , except that the test s u b s t a n c e ( s ) is a b s e n t . T h i s control is a l s o called n e g a t i v e c o n t r o l . In this s t u d y , both w a t e r c o n t r o l s and sample

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c o n t r o l s w e r e u s e d . H o w e v e r , the n e g a t i v e c o n t r o l , m e a s u r i n g only the test o r g a n i s m under f a v o r a b l e c o n d i t i o n s , is not s u f f i c i e n t . A c o m p l e t e a p p r o a c h is to add a second control (also known as p o s i t i v e c o n t r o l ) so that the test o r g a n i s m can also be o b s e r v e d under u n f a v o r a b l e c o n d i t i o n s .

Both o r g a n i c and inorganic c o m p o u n d s have been s t u d i e d as p o t e n t i a l r e f e r e n c e t o x i c a n t s . They include p e n t a c h l o r o p h e n o I , G u t h i o n - R , p h e n o l , p i c l o r a m , s o d i u m dodecyl s u l f a t e , d e h y d r o a b i e t i c a c i d , s o d i u m c h l o r i d e , c a d m i u m , and c h r o m a t e (Davis and H o o s , 1 9 7 5 ; A d e l m a n and S m i t h , 1 9 7 6 ; F o g e l s and S p r a g u e , 1 9 7 7 ; T h r e a d e r and H o u s t o n , 1 9 8 3 ; L e w i s and W e b e r , 1 9 8 5 ; Jop et al. in p r e s s ) . Jop et al. (in p r e s s ) m e n t i o n the a d v a n t a g e s of using the c h r o m a t e ion as high p u r i t y , a c c u r a c y and ease of d e t e r m i n a t i o n , and the like.

A factor w h i c h has r e c e i v e d little a t t e n t i o n is the s u i t a b i l i t y of a r e f e r e n c e c o m p o u n d . This factor b e c o m e s important w h e n the c o m p o u n d is intended for a m b i e n t w a t e r s or c o m p l e x m i x t u r e s . For e x a m p l e , a n t i b i o t i c s and s o d i u m c h l o r i d e w e r e e f f e c t i v e in inhibiting o x y g e n u p t a k e in s e d i m e n t s a m p l e s . They lost e f f e c t i v e n e s s , h o w e v e r , in 30 to 40 h ( W a n g , 1 9 8 0 ) . As another e x a m p l e , when d u c k w e e d w a s used as a test s p e c i m e n it w a s found that Cd toxicity in a river w a t e r s a m p l e w a s a m e l i o r a t e d by both d i s s o l v e d and p a r t i c u l a t e f r a c t i o n s in river w a t e r ( W a n g , 1 9 8 6 C ) . The Ba ion, on the other h a n d , w a s a f f e c t e d only by the d i s s o l v e d f r a c t i o n of the river w a t e r s a m p l e s . In other w o r d s , the toxic r e s p o n s e s of Ba and Cd ions varied a c c o r d i n g to the water q u a l i t y of a given s a m p l e . In the same s t u d y , the Cr ion was found to be nearly identical in the g r o w t h m e d i u m and the river w a t e r .

The a d d i t i o n a l r e s u l t s as p r e s e n t e d in F i g s . 10 through 14 support the c o n t e n t i o n that Cr can be the universal r e f e r e n c e toxicant for b i o l o g i c a l toxicity tests u s i n g s u r f a c e w a t e r s .

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